Effects of apolipoprotein e on n methyl d aspartate receptor signalling the roles of ageing and chicken extract

... 2000) There are 14 Chapter 1: Introduction isoform-specific neurodegeneration and as age-dependent human effects apoE3 of prevents human kainic apoE on acid-induced neurodegeneration in comparison... regions more intensely than non-carriers and exhibit compensatory neural recruitment (Han and Bondi, 2008) This phenomenon occurs even in non-demented older adults suggesting ε4 carriers undergo... than age-matched apoE3 counterparts The NR2B expression levels show an unexpected decrease in the hippocampus at middle age which extend into the cortex at old age The age-dependent and region-specific

EFFECTS OF APOLIPOPROTEIN E ISOFORMS ON N-METHYL-D-ASPARTATE RECEPTOR SIGNALLING: THE ROLES OF AGEING AND CHICKEN EXTRACT YONG SHAN MAY (B.Sc. (Hons), UM) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entity. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has not been submitted for any degree in any university previously. __________________________ YONG SHAN MAY 23rd January 2014 ACKNOWLEDGEMENTS I would like to express my gratitude to my supervisor, Dr. Wong Boon Seng for his guidance and supervision throughout my years of PhD study. He has taught me to think critically and helped me to grow as an individual. I am truly grateful to my TAC team members, Dr. Lim Kah Leong and Dr. Ramani, for the guidance provided in my project. I also would like to thank Dr. Low Chian Ming for his advice to overcome several obstacles that I experienced. I extent my gratitude to Dr. Paramjeet Singh (Cerebros Pacific Limited) for providing the CE powder for my project. I sincerely thank my previous and current fellow lab mates, Ching Ching, Li Min, Hong Heng, Ray, Shiau Chen, Mei Li, Ira, Elizabeth, Cynthia, Pei Ling, Ket Yin, Bei En and past FYP students for making my lab experience a memorable and an enjoyable one. Special thanks to Irwin, Alvin, Francis, Vanessa and Li Ren for their camaraderie and support. All of them have been great mentors and friends, offering me advice and encouragement that helped me to pull through the difficult times. I will always remember the time that we have spent together and thank you for making a difference in my life. I express my heartfelt gratitude to my family members for their support and unconditional love. Last but not least, I want to thank my boyfriend, Chee Wai, for being so understanding and supportive during my last two years of study. i TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................ i TABLE OF CONTENTS ...................................................................................ii SUMMARY ...................................................................................................... vi LIST OF TABLES ......................................................................................... viii LIST OF FIGURES .......................................................................................... ix ABBREVATIONS ............................................................................................ xi LIST OF PUBLICATION...………………………………………………...xiv Chapter 1: Introduction ...................................................................................... 1 1.1. Apolipoprotein E (ApoE) ....................................................................... 1 1.1.1 Characteristics of ApoE ................................................................ 1 1.1.2. Expression and functions of apoE in CNS: astrocytic vs neuronal apoE........................................................................................................ 2 1.1.3. Differences between apoE isoforms............................................. 4 1.1.4. ApoE isoforms in synaptic plasticity ........................................... 5 1.1.5. ApoE receptors ............................................................................. 7 1.2. Alzheimer’s disease (AD) ...................................................................... 9 1.2.1. AD pathogenesis and progression ................................................ 9 1.2.2. Hippocampus in synaptic plasticity and memory formation...... 10 1.2.3. Ageing and synaptic plasticity ................................................... 12 1.2.4. Genetic risk factor for sporadic AD: apolipoprotein E (apoE) variants ................................................................................................. 13 1.3. N-methyl-D-aspartate receptor (NMDAR): a key player in learning and memory ...................................................................................................... 15 1.3.1. Characteristics and expression of NMDAR ............................... 16 1.3.2. Functions of NMDAR ................................................................ 19 1.3.3. Modulation of NMDARs by phosphorylation and dephosphorylation mechanisms ........................................................... 20 1.3.4. Opposing roles of NMDAR subunits in synaptic plasticity ....... 21 ii 1.3.5. NMDAR in ageing ..................................................................... 22 1.3.6. NMDAR in AD .......................................................................... 23 1.3.7. NMDAR in excitotoxity............................................................. 24 1.4. α-amino-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPAR) .... 25 Chapter 2: Materials And Methods .................................................................. 28 2.1. Common reagents and materials .......................................................... 28 2.2. Animal model ....................................................................................... 28 2.2.1. Tissue preparation for protein analysis ...................................... 29 2.2.2. Preparation of brain homogenates .............................................. 29 2.2.3. Protein quantitation of brain lysates ........................................... 30 2.2.4. Western blotting ......................................................................... 30 2.2.5. Tissue preparation for immunofluorescence .............................. 32 2.2.6. Immunofluorescence .................................................................. 32 2.3. Cell culture ........................................................................................... 35 2.3.1. Immortalization and transfection of apoE-knockout neuronal cells ...................................................................................................... 35 2.3.2. CE treatment............................................................................... 36 2.3.3. Cell lysis ..................................................................................... 37 2.3.4. Protein quantitation of cell lysates ............................................. 37 2.3.5. Western blotting ......................................................................... 37 2.3.6. Calcium assay............................................................................. 39 2.4. Statistical analysis ................................................................................ 40 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice . 41 3.1. Introduction .......................................................................................... 41 3.1.1. Human apoE gene-knockin (apoE-KI) mice model for investigating effects of apoE isoforms on NMDAR changes during ageing. .................................................................................................. 41 3.1.2. Interactions between apoE and N-methyl-D-aspartate receptor (NMDAR) ............................................................................................ 47 3.1.3. Postsynaptic density (PSD) proteins/ NMDAR-associated proteins (NAPs) .................................................................................... 48 3.1.3.2. PSD95 ...................................................................................... 50 3.1.3.2. Calcium/calmodulin-dependent protein kinase II (CaMKII) ... 51 3.1.4. Signalling pathways coupled to NMDAR ................................. 53 3.1.4.1. Protein kinase C (PKC) pathway ............................................. 53 iii 3.1.4.2. Protein kinase A (PKA) pathway............................................. 55 3.1.4.3. Ras/mitogen-activated protein kinase (MAPK) pathway ........ 56 3.1.5. CREB in learning and memory .................................................. 59 3.2. Objectives of study ............................................................................... 61 3.3. Results .................................................................................................. 63 3.3.1. Expression of human apoE in brains of huApoE-knockin (apoEKI) mice across three time points......................................................... 65 3.3.2. Expression of apoE receptor and PSD95 in brains of huApoE-KI mice across three time points ............................................................... 66 3.3.3. NMDAR subunits phosphorylation profile in brains of huApoEKI mice across three time points .......................................................... 68 3.3.4. PKA and PKC signalling profile in brains of huApoE-KI mice across three time points ........................................................................ 71 3.3.5. GluR1 and αCaMKII expression profile in brains of huApoE-KI mice across three time points ............................................................... 73 3.3.6. ERK-CREB signalling pathway in brains of huApoE-KI mice across three time points ........................................................................ 76 3.3.7. Expression of human apoE in neurons and astrocytes of huApoE- KI mice brains across three time points ............................... 79 3.3.8. Protein expression level of NMDAR subunits in brains of huApoE-KI mice across three time points ........................................... 85 3.4. Discussion ............................................................................................ 89 3.4.1. ApoE4 isoform downregulates expression level of total apoE but upregulates neuronal apoE production with increasing age ................. 89 3.4.2. ApoE4-isoforms decreases expression of apoE receptor i.e. LRP1 and postsynaptic protein PSD95. ............................................... 95 3.4.3. ApoE4 spatio-temporally regulate NMDAR expression and phosphorylation profiles during ageing. .............................................. 99 3.4.4. Modulation of ERK and CREB activity in apoE4-KI mice is mediated via PKC but not PKA signalling pathway. ......................... 107 Chapter 4: Impacts of apoE isoforms in cellular responses to chicken extract (CE) treatment ................................................................................................ 119 4.1. Introduction ........................................................................................ 119 4.1.1. Cyclic nucleotide phosphodiesterase (PDE) and CREB .......... 119 4.1.1. Chicken extract (CE): potential PDE inhibitor ........................ 120 4.1.2. Beneficial effects of CE to mental health ................................ 121 4.2. Objective of study .............................................................................. 124 4.3. Results ................................................................................................ 125 4.3.1. Chronic expression of apoE in huApoE stable cells ................ 125 iv 4.3.2. Effects of CE treatment on expression of human apoE in huApoE stable cells ............................................................................ 126 4.3.3. Effects of CE treatment on basal intracellular calcium level in huApoE stable cells ............................................................................ 127 4.3.4. Effects of CE treatment on phosphorylation of NR1 subunit in huApoE stable cells ............................................................................ 129 4.3.5. Effects of CE treatment on PKA and PKC signalling profile in huApoE stable cells ............................................................................ 131 4.3.6. Effects of CE treatment on GluR1 and αCaMKII expression profile in huApoE stable cells ............................................................ 134 4.3.7. Effects of CE treatment on ERK-CREB signalling in huApoE stable cells .......................................................................................... 136 4.4. Discussion .......................................................................................... 138 4.4.1. Different intracellular calcium responses in huApoE-transfected neurons and downregulation of apoE4 expression upon CE treatment ............................................................................................................ 138 4.4.2. Upregulation of PKC pathway in huApoE3-transfected neurons and downregulation of PKA pathway in huApoE4-transfected neurons upon CE treatment.............................................................................. 141 4.4.3. ERK-CREB signalling in CE-treated huApoE3- and huApoE4transfected neurons............................................................................. 145 4.3 Conclusion........................................................................................... 152 4.4 Future Directions ................................................................................. 154 BIBLIOGRAPHY .......................................................................................... 162 Appendix ........................................................................................................ 225 v SUMMARY Apolipoprotein E4 (apoE4) isoform has been shown to accelerate cognitive decline in human and mouse models during ageing compared to apoE3 isoform. Mice expressing human apoE4 display impaired learning and memory and glutamatergic neurotransmission. Despite the ongoing studies to look for preventive measures and therapeutic strategies, researchers have yet to unravel the complex underlying mechanisms and rectify the pathological effect of apoE4 in learning and memory. My study shows that apoE4 regulates expression of NMDAR subunits and its activity in a temporal and region-specific manner during ageing when compared with apoE3-knock in (apoE3-KI) mice. Western blotting analyses show an increased phosphorylation of NR1 subunit particularly at Ser896 in young apoE4-KI mice. In contrast, this phosphorylation is downregulated in old apoE4-KI mice when compared with apoE3-KI mice. The tyrosine phosphorylation of NR2A of apoE4-KI mice is reduced regardless of age whereas there is no difference in NR2B activity between apoE3- and apoE4KI mice across all time-points. Immunofluorescence studies demonstrate an increase in NR1 signal intensity in the hippocampus and cortex at week 12 followed by downregulation of its total expression at week 72 in the hippocampus. Similarly, NR2A subunit expression levels in most hippocampal subregions and cortex of apoE4-KI mice are always lower than age-matched apoE3 counterparts. The NR2B expression levels show an unexpected decrease in the hippocampus at middle age which extend into the cortex at old age. The age-dependent and region-specific modulation of the NMDAR subunits is correlated to the source of apoE4 production. This suggests that the increasing neuronal apoE4 production at old age particularly in the CA3 and cortical area, exerts a detrimental effect on NMDAR expression due to the neurotoxicity of apoE4 fragments. Hence, the downregulated phosphorylation and region-specific expression of NMDAR of apoE4-KI mice may partially explain their impaired behavioural performances at old age compared to agematched apoE3 counterparts as observed by others. vi Analysis on NMDAR-associated proteins and the signalling pathways coupled to NMDAR activity in apoE4-KI mice show that the postsynaptic density protein PSD95 and apoE receptor, LRP1, are downregulated in apoE4-KI mice at all ages. This suggests that a reduced NMDAR functionality mediated via these proteins. Immunoblotting of Ca2+-sensitive kinases including αCaMKII, PKCα and PKA-Cα demonstrate an increased αCaMKII and PKCα activation with expected elevation in their common downstream target, GluR1 phosphoS831 without affecting PKA-Cα in young apoE4-KI mice. In contrast, phosphorylation of αCaMKII, PKCα and GluR1 Ser831 are downregulated at old age. The correspondence between signalling profiles of ERK-CREB versus αCaMKII and ERK-CREB versus PKCα strongly suggest their key roles played in facilitating ERK and CREB activation. Second part of my study investigated the effects of chicken extract (CE) on the NMDAR and its downstream signalling cascades in the context of apoE isoforms in vitro as this dietary supplement has been shown to improve cognition in human. Howver, the underlying mechanisms are unclear. Data from CE treatment of apoE-transfected neurons demonstrate the differential effects of apoE isoforms on intracellular Ca2+ responses and triggering of the Ca2+-dependent signalling pathways. In particular, PKA-Cα pathway is downregulated whilst PKCα pathway is upregulated in CE-treated apoE4 and apoE3 neurons respectively. Moreover, the basal intracellular Ca2+ level, αCaMKII and GluR1 S831 are increased in apoE3 neurons whereas the opposite occurs for mock and apoE4-transfected neurons after treatment. These might have led to enhanced ERK1/2 and CREB activity in apoE3 neurons but reduced ERK-CREB signalling in mock as well as apoE4 neurons. (564 words) vii LIST OF TABLES Table 2.1. List of primary antibodies used for immunofluorescence. The source and the dilution factor used are as shown. ............................................ 34 Table 2.2. Composition of CE compound ....................................................... 37 Table 2.3. List of primary antibodies used for immunoblotting. The source and the dilution factor used are as shown. .............................................................. 38 Table 2.4. List of secondary antibodies used throughout the study. The purpose, source and dilution factor used are as shown. ................................... 39 Table 3.1. Samples, brain regions of interest and findings of reviewed articles .......................................................................................................................... 46 Table 3.2. Recapitulative table on all significant comparisons between huApoE3 and huApoE4 mouse lines. ............................................................ 117 Table 4.1. Recapitulative table on the findings of CE treatment on mock, huApoE3 and huApoE4 stable cell lines. ...................................................... 150 viii LIST OF FIGURES Figure 1.1. Amino acid sequence of different apoE isoforms .......................... 2 Figure 1.2. Structure of NMDAR subunit ...................................................... 18 Figure 1.3. Model of bidirectional plasticity in AMPAR phosphorylation and dephosphorylation ............................................................................................ 27 Figure 3.1. Schematic diagram of the interactions between apoE and NAPs, and NMDAR-coupled signalling cascades. ..................................................... 61 Figure 3.2. Expression level of huApoE in brains of apoE-KI mice models across three time points i.e. 12, 32 and 72 weeks. ........................................... 65 Figure 3.3. Protein expression level of LRP1 and PSD95 in brains of apoE-KI mice models across three time points i.e. 12, 32 and 72 weeks. ...................... 66 Figure 3.5. Protein expression level of PKA-Cα and PKCα in brains of apoEKI mice models across three time points i.e. 12, 32 and 72 weeks. ................ 71 Figure 3.6. Protein expression level of GluR1 and αCaMKII in brains of apoEKI mice models across three time points i.e. 12, 32 and 72 weeks. ................ 73 Figure 3.7. Protein expression level of ERK and CREB in brains of apoE-KI mice models across three time points i.e. 12, 32 and 72 weeks. ...................... 76 Figure 3.8. Cellular expression levels of huApoE in brains of apoE-KI mice models across three time points i.e. 12, 32 and 72 weeks. .............................. 82 Figure 3.9. Expression level of total NMDAR subunit proteins in brains of apoE-KI mice models across three time points i.e. 12, 32 and 72 weeks. ....... 87 Figure 3.10. Model of age-dependent regulation of intracellular signalling pathways by apoE4. ....................................................................................... 118 Figure 4.1. Protein expression level of apoE in mock and apoE-transfected neurons. .......................................................................................................... 125 ix Figure 4.2. Protein expression level of apoE in mock and apoE-transfected neurons. .......................................................................................................... 126 Figure 4.3. Basal intracellular calcium ion concentration in mock and apoEtransfected neurons. ....................................................................................... 127 Figure 4.4. Phosphorylation level of NR1 subunit in mock and apoEtransfected neurons. ....................................................................................... 129 Figure 4.5. Protein expression level of PKA-Cα and PKCα in mock and apoEtransfected neurons. ....................................................................................... 131 Figure 4.6. Protein expression level of GluR1 subunit and αCaMKII in mock and apoE-transfected neurons. ....................................................................... 134 Figure 4.7. Protein expression level of ERK and CREB in mock and apoEtransfected neurons. ....................................................................................... 136 Figure 4.8. Model of differential regulation of cellular responses to CE treatment by apoE isoforms. .......................................................................... 151 Supplementary Figure 1. Protein expression levels of GFAP and NeuN in brains of apoE-KI mice models across three time points i.e. 12, 32 and 72 weeks.............................................................................................................. 225 Supplementary Figure 2. Protein expression level of LP1 in mock and apoEtransfected neurons. ....................................................................................... 225 x ABBREVIATIONS AC Adenylyl cyclase AD Alzheimer’s disease AMPAR α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor ApoE Apolipoprotein E ApoER2 ApoE receptor 2 APP Amyloid precursor protein ATP Adenosine triphosphate Bcl2 B-cell lymphoma 2 BDNF Brain-derived neurotrophic factor CA Cornus ammoni CaMK Calcium-calmodulin dependent kinase cAMP Cyclic adenosine monophosphate CaN Calcineurin CE Chicken extract CNS Central nervous system CREB cAMP-responsive element-binding protein CSF Cerebrospinal fluid DAG Diacylglycerol DG Dentate gyrus EDTA Ethylenediaminetetraacetic acid EPSC Excitatory postsynaptic current ERK Extracellular-signal regulated kinase FDG-PET Fluorodeoxyglucose positron emission tomography GFAP Glial fibrillary acidic protein GLUT Glutamate transporter GRF Guanine nucleotide Releasing Factor GTP Guanosine triphosphate HDL High density lipoprotein HFS High frequency stimulation ICD Intracellular domain iGluR Ionotropic glutamate receptor Ins(1,4,5)P3 Inositol-1,4,5-triphosphate JNK Jun-N-terminal kinase xi KI Knockin KO Knockout LDLR Low density lipoprotein receptor LFS Low frequency stimulation LRP1 LDL-related protein 1 LTD Long-term depression LTP Long-term potentiation mAchR Muscarinic acetylcholine receptor MAGUK Membrane-associated guanylate kinase MAP2 Microtubule-associated protein 2 MAPK Mitogen activated protein kinase MCI Mild cognitive impairment MF Mossy fibre mGluR Metabotropic glutamate receptor MK801 [5R,10S]-[+]-5-methyl-10,11-dihydro-5H-dibenxo [a,d]cyclohepten-5,10-imine mRNA Messenger ribonucleic acid MWM Morris water maze NMDAR N-methyl-D-aspartate receptor NO Nitric oxide NRHyper NMDAR hyperactivity NRHypo NMDAR hypoactivity NSE Neuron-specific-enolase Oxy-Hb Oxyhemoglobin PDE Phosphodiesterase PDZ PSD95, disc large, zona occludens-1 PI3K Phosphatidylinositol 3-kinases PKA-C Protein kinase A catalytic subunit PKC Protein kinase C PMA Phorbol 12-myristate 13-acetate PP1/2A Phosphatases 1,2A PSD95 Postsynaptic density protein 95 RSK Ribosomal protein S6 kinase SAP Synapse-associated-protein SC Schaeffer collateral SDS Sodium dodecyl sulfate xii tPA Tissue plasminogen activator TR Targeted replacement U0126 dicyano-1,4-bis[2-aminophenylthio]butadiene α2M Alpha-2 marcoglobulin xiii LIST OF PUBLICATION S. Yong, Q. Ong, B. Siew and B. B. Wong, Food Funct., 2014, DOI: 10.1039/C4FO00428K. xiv Chapter 1: Introduction Chapter 1: Introduction 1.1. Apolipoprotein E (ApoE) 1.1.1 Characteristics of ApoE ApoE is a 34 kilodalton (kDa) protein bearing 299 amino acids in sequence. ApoE is characterized by a single nucleotide polymorphism (SNP) on the gene located at chromosome 19. The majority of the human population is homozygous for apoE3 with an allelic frequency of 78% among Europeans. ApoE4 makes up about 14% of the population and the rest are apoE2 (8%). In a study of apoE polymorphisms and lipid profiles in three ethnic groups i.e. Malays, Chinese and Indians in the Singapore population, ε3 allele is the most common (82%) followed by ε4 (10%) and ε2 (8%) (Tan et al., 2003). The three major isoforms predominantly expressed in the human population are apoE2, apoE3 and apoE4. These isoforms differ by two amino acids at position 112 and 158 in which apoE2 has two cysteines (Cys), apoE3 has Cys112 and Arg-158 (arginine) whereas apoE4 has Arg-112 and Arg-158 (Rall et al., 1982; Weisgraber, 1994). In general, apoE has two structural domains including a 22kDa N-terminal domain which binds to its receptor and a 10kDa C-terminal domain which is the lipid-binding site that can interact with other extracelullar proteins such as Aβ (Mahley and Rall, 2000). In the CNS, apoE exists in the form of discoidal-shaped high-density lipoprotein (HDL)-like particles containing phospholipids and cholesterol which is distinct from those in the peripheral system (DeMattos et al., 2001a; Pitas et al., 1987). It is the major lipoprotein in the CNS among other apolipoproteins such as apoA, apoC, apoD and apoJ (also known as clusterin) whilst apoB is absent (Holtzman et al., 2012). Concentration of apoE in the cerebrospinal fluid (CSF) is 100-200 nM (Riemenschneider et al., 2002) and total concentration in brain extract is approximately 5 µg/mL (Haass and Selkoe, 2007). 1 Chapter 1: Introduction Figure 1.1. Amino acid sequence of different apoE isoforms The three major apoE isoforms differ in their amino acids at positions 112 and 158 which give rise to their distinct properties. Each comprises two structural domains that bind to apoE receptors at the N-terminal and lipid particles such as HDL at the C-terminal. 1.1.2. Expression and functions of apoE in CNS: astrocytic vs neuronal apoE Under normal conditions, apoE is mainly synthesized by astrocytes but can also be found in oligodendrocytes, neurons, smooth muscle cells and choroid plexus in the CNS (Boyles et al., 1985; Herz and Beffert, 2000; Xu et al., 2006). Astrocyte-derived apoE is involved in the transport of cholesterol into neurons to regulate synaptic plasticity through lipid homeostasis (Gong et al., 2002; Mauch et al., 2001). It is antioxidative in nature and helps in the clearance of Aβ (DeMattos, 2004; Lomnitski et al., 1999; Ye et al., 2005). Besides that, apoE mediates neurite outgrowth and stabilization of microtubules. These structural changes affect synapse formation and hence influence synaptic plasticity (Nathan et al 2002). 2 Chapter 1: Introduction Under pathological conditions, excitotoxic injury in CNS such as kainic acid treatment, oxidative stress and even ageing induces neurons to increase apoE production to initiate the repair process and mediate protection from these insults (Boschert et al., 1999; Huang, 2010; Mahley, 1988; Roses, 1997; Xu et al., 2006). However, proteolytical processing of neuronal apoE4 produces harmful fragments that induce neurodegeneration especially in ε4 carriers (Brecht et al., 2004; Huang, 2010; Huang et al., 2004; Mahley et al., 2006; Roses, 1997) which may underlie its correlation to the earlier onset of Alzheimer’s disease (AD) (Boschert et al., 1999). Both in vitro and in vivo studies have shown that neuronal apoE4 is more susceptible to cleavage by a neuron-specific, chymotrypsin-like serine protease generating neurotoxic fragments which are detrimental to the neurological repair or maintenance process compared to apoE3 (Huang et al., 2001; Tolar et al., 1999; Tolar et al., 1997). The effect of the ε4 allele on the CNS apoE protein levels has been studied in both AD patient CNS and animal models, reporting controversial results showing either reduced levels (Beffert et al., 1999; Ong et al., 2014; Poirier, 2005; Riddell et al., 2008), no change (Fryer et al., 2005; Sullivan et al., 2004) or increases (Fukumoto et al., 2003) in apoE expression compared to that of apoE4 individuals. The reduced brain apoE level in ε4 carriers (Farrer et al., 1997; Poirier, 2005) and their predisposition to AD implies that apoE is required to sustain a certain level of cognitive function perhaps by maintaining synaptic integrity. It is suggested that adequate level of apoE may be crucial to regulate brain homeostasis during ageing as apoE expression increases in the liver in an agedependent manner (Gee et al., 2005). However, it is unclear whether apoE expression changes in the brain with ageing in human whereas conflicting observation have been made from animal studies. In rodents, apoE expression level decreases more than five-fold in the cortex and hypothalamus (Jiang et al., 2001) but increases in the hippocampus (Terao et al., 2002) of aged mice. Aged rats also demonstrate elevated glial apoE expression in basal ganglia and corpus callosum (Morgan et al., 1999). In contrast, a recent study reported 3 Chapter 1: Introduction there is no alteration in apoE mRNA and protein expression level in the cortex, hippomcapus and striatum of aging rat brains (Gee et al., 2006). 1.1.3. Differences between apoE isoforms The replacement of Arg in apoE4 at position 112 has an impact on its structure and functions making it the least stable and a more pathological isoform compared to the other two. Arg-112 mediates the interaction between Arg-61 at the N-terminus and Glu-255 (glutamine) at the C-terminus causing the whole molecule to exist as a partially folded intermediate or molten globule (Dong et al., 1994; Weisgraber, 1990). This unstable structure denatures at a lower temperature making it less concentrated in the CNS. In fact there is a lower level of apoE in brain and serum of AD subjects whereby 40 to 80% of them have at least one ε4 allele (Farrer et al., 1997). ApoE4 has a lower cholesterol transport capacity and Aβ clearance ability compared to apoE2 and apoE3 leading to increased Aβ production (Dodart et al., 2005; Holtzman et al., 2000). Furthermore, apoE4 mediates some of the detrimental effects such as tau1 phosphorylation (Mandelkow and Mandelkow 1994), lysosomal leakage, mitochondrial dysfunction, neurodegeneration and cognitive deficits in AD (Buttini et al., 2002; Chang et al., 2005; Huang et al., 2001; Ji et al., 2002; Raber et al., 2002; Risner et al., 2006). ApoE4 has been shown to cause cytotoxicity in a dose- and time-dependent manner. Significant neurotoxicity was detected 24 hours after apoE4 treatment and the percentage of cell death plateaus at 72 hours (Qiu et al., 2003). Generation of neurotoxic fragments such as apoE4 (Δ1-272, Δ272-299, Δ127242) are harmful to neurons and Neuro-2a cell lines derived from mouse neuroblastoma. For instance, apoE4 1 (Δ1-272) is associated with Tau is one of the microtubule-associated proteins that plays a role in the stabilization of neuronal microtubules and provides the tracks for intracellular transport. However, when tau undergoes modifications such as phosphorylation, hyperphosphorylated tau will aggregate into paired helical fragments that coalesce into neurofibrillary tangles. 4 Chapter 1: Introduction neurofibrillary tangle-like structures in cortical and hippocampal neurons in transgenic mice (Harris et al., 2003; Huang et al., 2001). ApoE4 (Δ272-299) which is present in AD brains peaks at 6-7 month old in mice and impairs spatial learning and memory (Brecht et al., 2004; Harris et al., 2003). Furthermore, AD patients also have much higher levels of apoE fragments compared to non-demented controls (Harris et al., 2003; Huang et al., 2001). Thus under pathological conditions, it is worse for neurons to express apoE4 than to express no apoE at all, as opposed to apoE2 and apoE3 which are more stable and more effective in maintenance of neurons (Mahley, 1988; Morrow et al., 2002). In other words, apoE4 has an adverse gain-of-function compared to the other two isoforms (Buttini et al., 2000; Huang, 2006; Raber et al., 2000). 1.1.4. ApoE isoforms in synaptic plasticity ApoE isoforms differentially modulate synaptic plasticity and learning and memory. ApoE-targeted replacement mice (huApoE-TR) or human apoEknockin (huApoE-KI) expressing human apoE isoforms have shown that huApoE3 mice have enhanced LTP compared to huApoE4 mice (Grootendorst et al., 2005; Trommer et al., 2004). ApoE4 has been postulated to impair synaptic plasticity by reducing NMDAR-dependent Ca2+ influx mediated by apoE receptors in a dose-dependent manner. Furthermore, apoE4 may impede apoE receptor recycling by sequestering the receptors intracellularly such that they are unable to be expressed on neuronal surfaces and trigger downstream signalling pathways upon ligand binding (Chen et al., 2010). Exogenously added apoE4 has been shown to reduce surface expression of apoE receptor 2 (apoER2) in primary neurons which functions as a dual receptor for apoE and reelin, a ligand that modulates NMDAR functions both in vitro and in vivo (Beffert et al., 2005). ApoE3 and apoE4 differ from each other in their influences on neurite extension (DeMattos et al., 1998; DeMattos et al., 2001b; Fagan et al., 1996; Handelmann et al., 1992; Nathan et al., 1995). In cultured dorsal root ganglion neurons and Neuro-2a cells, apoE3 together with βVLDL stimulates neurite branching and extension as opposed to apoE4 5 Chapter 1: Introduction (Holtzman et al., 1995; Nathan et al., 1994). This stimulatory effect on neurite outgrowth is also seen in rat hippocampal neurons mediated by astrocytederived apoE3 but not apoE4 (Sun et al., 1998). In organotypic hippocampal slice culture isolated from huApoE-TR mice model, apoE3 can stimulate neuronal sprouting which is inhibited by apoE4 (Teter et al., 2002). This contrasting effect of apoE4 is probably due to its facilitation of tubulin depolymerization in neuronal cells leading to microtubule instability (Nathan et al., 1994). In addition, in vivo and in vitro studies show apoE4 impedes synaptogenesis as apoE4 transgenic mice have reduced number of synapses per neuron (Cambon et al 2000), and lower density of dendritic spines than apoE3 mice (Dumanis et al., 2009; Ji et al., 2003). Similarly, exogenously added apoE4 or its proteolytic fragments decreases spine density in primary cortical neuronal cultures (Brodbeck et al., 2008). These structural deformities in apoE4 mice perhaps contribute to learning and memory deterioration. In spite of all these, young apoE4-KI mice display enhanced plasticity compared to apoE3 mice but this effect disappears with age (Kitamura et al., 2004). Furthermore, this enhanced LTP may be mediated by postsynaptic properties as there is no difference in presynaptic release mechanisms between apoE3 and apoE4-KI animals. Similarly, young human ε4 carriers exhibit higher intelligence and event-related potential (Yu et al., 2000) suggesting that apoE isoform modification of LTP may be age-dependent. Studies that support the beneficial effect of apoE4 in early life show that school children with ε4 allele score better in verbal fluency scores and outperform ε2 peers in Rey Complex Figure Test (RCFT) copy trial (Alexander et al., 2007; Bloss et al., 2010). One of the interesting observations is that ε4 carriers tend to recruit task-related regions more intensely than non-carriers and exhibit compensatory neural recruitment (Han and Bondi, 2008). This phenomenon occurs even in non-demented older adults suggesting ε4 carriers undergo compensatory changes in order to achieve the same performance as noncarriers (Kukolja et al., 2010; Tuminello and Han, 2011). However, the beneficial role of apoE4 in early life remains controversial as there are contradictory findings indicating that 5 to 7-year-old ε4 carriers with sleep apnoea show impaired cognition and middle-aged adults do not differ in 6 Chapter 1: Introduction cognitive performance (Dennis et al., 2010; Filbey et al., 2010; Gozal et al., 2007). Furthermore, additional environmental risk factors such as family history of AD and gender susceptibility may interact with ε4 allele to affect cognition as 7 to 10 year-old girls have lower spatial memory retention and lower visual recall scores on Family Pictures test (Acevedo et al., 2010; Bloss et al., 2008). Perhaps the potential benefit of ε4 allele may be confined to a very narrow window in early life span and as the detrimental effects of apoE4 surfaces, the compensatory mechanism cannot sustain the premorbid cognitive performance levels and hence the condition worsens with age. 1.1.5. ApoE receptors ApoE binds to members of low density lipoprotein receptor (LDLR) family including very low density lipoprotein receptor (VLDLR), apoER2, LDLrelated protein 1 (LRP1), LDL-related protein-1B (LRP-1B), multiple EGF repeat-containing protein-7 (MEGF7) and megalin (Brecht et al., 2004; Holtzman et al., 2012). These receptors have one or more ligand-binding domains, epidermal growth factor (EGF) and a cytoplasmic tail containing a consensus amino acid NPxY motif which acts as an interaction site for intracellular adaptor proteins that are further coupled to downstream transduction pathways (Beffert et al., 2005; Gotthardt et al., 2000; Hoe et al., 2006; Trommsdorff et al., 1998). Some of the functions of apoE receptors are utilization of mitogen-activated protein kinases (MAPK), tyrosine kinases, lipid kinases and ligand-gated ion channels such as glutamate receptors (Beffert et al., 2005; Bock and Herz, 2003). LRP1 is one of the major apoE receptors that regulates CNS apoE levels. It is a 600 kDa receptor dimer consisting of an extracellular and intracellular domain which is 515 kDa and 85 kDa respectively. It is expressed ubiquitously by hepatocytes, neurons, vascular smooth muscle, lung, macrophages and embryonic tissues (Bu et al., 1994; Ishiguro et al., 1995; Moestrup et al., 1992). Other than apoE, ligands that bind to LRP1 are activated α2-macroglobulin (α2M*), amyloid precursor protein (APP), tissue plasminogen activator (tPA), protease/protease 7 inhibitor complexes, Chapter 1: Introduction lipoprotein lipase and platelet-derived growth factor (PDGF). It plays dual role in endocytosis and regulation of signal transduction involving calcium currents, PDGF receptor signalling, phagocytosis of apoptotic cells and embryonic development (Boucher et al., 2003; Herz and Strickland, 2001; May et al., 2002; Wang et al., 2003; Yepes et al., 2003). Genetic deletion of LRP1 causes embryonic lethality and hence tissue-specific LRP1 knockout animals have been generated which exhibit pronounced tremor, ataxia, hyperactivity, dystonia and premature death without structural abnormalities in these animals (Herz et al., 1992; May et al., 2004). They also show impaired brain lipid metabolism, age-dependent synaptic loss and neurodegeneration (Liu et al., 2010). This highlights the crucial role of LRP1 in modulation and turnover of synaptic proteins that contributes to the anomalies in morphology and phenotype in the absence of LRP1. As an endocytic receptor, LRP1 facilitates uptake and clearance of neural proteases such as neuroserpin from synaptic regions, as increased level of proteases will disrupt the balance between proteases and protease inhibitors leading to synaptic dysfunction (Makarova et al., 2003). LRP1 has been linked to AD due to its binding to and clearance of Aβ as well as APP metabolism (Beffert et al., 1999; Rebeck et al., 1993; VázquezHiguera et al., 2009), mediated via adaptor protein FE65 that binds to NPxY motif of LPR1 and APP (Kinoshita et al., 2001; Pietrzik et al., 2004; Trommsdorff et al., 1998). It is observed that decreased LRP1 expression in amyloid mouse model and brain capillaries of AD brain may contribute to impaired Aβ clearance (Deane et al., 2004; Van Uden et al., 2002). LRP1 undergoes proteolytic processing whereby the extracellular domain can be cleaved by β-secretase, BACE1, which also cleaves APP and generates the Aβ N-terminus, close to the transmembrane region resulting in the shedding of the extracellular domain (Quinn et al., 1999). The intracellular domain is then released from the plasma membrane by cleavage involving γ-secretase (May et al., 2002). This allows the free C-tail to translocate together with its adaptor proteins to other subcellular localizations. Moreover, the circulating LRP1 may also serve as a peripheral 'sink' for Aβ clearance (Sagare et al., 2007). 8 Chapter 1: Introduction 1.2. Alzheimer’s disease (AD) AD is the most common cause of dementia among the elderly (>65 years) and accounts for up to 75 percent of all dementia cases. Global prevalence of dementia is estimated to be 3.9 % in people aged 60 years and above, and affects more than 25 million people in the world (Brookmeyer et al., 2007; Ferri et al., 2005; Wimo et al., 2003). Population ageing has become a global phenomenon. In both developed and developing countries, the United Nation Ageing Program and the United States Centers for Disease Control and Prevention have projected that the aged population (65 years or more) in the world is expected to rise from 420 million in 2000 to nearly 1 billion by 2030, which is an increase from 7 to 12 percent of world population (Centers for Disease Control and Prevention, 2003). Similarly, a recent study reported the worldwide total number of 26.6 million AD patients will quadruple by the year 2050 (Brookmeyer et al., 2007). With the incidence rate of 5 million cases per year and the age-specific prevalence of AD doubling every five years after the age of 65 years, Alzheimer's disease has rapidly become a major public health crisis by imposing financial and societal burden as 43% of AD patients require high level of care such as nursing homes and institutions. The societal cost of dementia worldwide was estimated to be more than US$315 billion in 2005 (Wimo et al., 2007). Hence, much effort has been made to seek for effective therapeutic and preventive strategies as even a 1year delay in the onset of and progression of AD will significantly reduce the global burden of this disease (Brookmeyer et al., 1998; Brookmeyer et al., 2007). 1.2.1. AD pathogenesis and progression AD is a multifactorial disease which is subtyped into two groups i.e. familial making up 5% of the cases, and sporadic, for the rest. The former, also known as early-onset AD, is caused by rare autosomal inherited mutations involving presenillin 1 and 2 (PS1 and PS2), and the amyloid precursor protein gene (APP), which typically occurs before age 65. The age of onset for the latter is 9 Chapter 1: Introduction usually after 65 years old and the aetiology is a complex interplay between other genetic risk factors such as apolipoprotein E4 (apoE4), family history and environmental factors. AD is characterized by several hallmarks including neuronal loss, synaptic damage, deposition of beta-amyloid (Aβ) and neurofibrillary tangles, loss of cholinergic activity and elevated oxidative stress which is detrimental to messenger ribonucleic acid (mRNA) as well as protein synthesis (Ding et al., 2005; Keller et al., 2005). AD progression has been characterized in different Braak stages I to VI (Braak and Braak, 1994) depending on the brain structures affected. The related pathology is propagated in a typical topological pattern, originating in subcortical structures such as locus coeruleus (Braak and Del Tredici, 2011), and extends into entorhinal cortex at Braak stages I and II. It subsequently progresses to the hippocampus and amygdala via the perforant pathway characterized as Braak stages III and IV, and eventually spreads into other cortical regions at Braak stages V and VI. Memory deficits are often displayed in mild cognitive impairment (MCI) before it progresses into dementia which is the main symptom of AD patients (Petersen, 2004). One of the methods to evaluate cognitive function is the investigation of spatial memory in hippocampus as it plays a crucial role in spatial memory performance (Clarke RE, 2007; Morris et al., 1982; Rolls, 2000). The hippocampus is important in synaptic plasticity, being the cellular basis for learning and memory and is particularly vulnerable to environmental insults (Sultana et al., 2010). 1.2.2. Hippocampus in synaptic plasticity and memory formation Synaptic plasticity refers to activity-induced changes at appropriate synapses during memory formation and is necessary and sufficient for the information storage mediated by the brain area in which that plasticity is observed. It occurs mainly in hippocampal sub-regions namely dentate gyrus (DG), cornus ammoni (CA1) and CA3 neurons. There are three major circuits in the hippocampus to assist in formation of declarative memories which are the 10 Chapter 1: Introduction perforant, mossy fibre (MF) and Schaeffer collateral (SC) pathway. The first pathway extends from the entorhinal cortex to granule cells of DG; the second comprises of axonal projections from granular cells in DG to pyramidal cells in hippocampal CA3; and the third pathway originates from CA3 and culminates at CA1. High frequency stimulation (HFS) of the perforant pathway induces long-lasting potentiation of synaptic transmission between perforant path fibres and dentate granule cells (Bliss and Lomo, 1973). DG is a region capable of neurogenesis as it consists of newly formed neurons involved in learning and memory circuits (Zhao et al., 2006). The SC pathway involves formation of excitatory synapses between axons of CA3 pyramidal cells and dendrites of CA1 pyramidal cells and damage in the CA1 area is sufficient to prevent the formation of new memories (Auer et al., 1989; ZolaMorgan et al., 1986). This pathway displays a form of long-term potentiation (LTP) that requires Ca2+ influx via N-methyl-D-aspartate receptor (NMDAR), a glutamate receptor that plays a key role in synaptic plasticity (Bliss and Collingridge, 1993; Collingridge et al., 1983; Lynch et al., 1983; Malenka and Nicoll, 1999). In contrast, LTP induced via the MF pathway can occur independently of NMDAR activation (Harris and Cotman, 1986; Johnston et al., 1992; Weisskopf and Nicoll, 1995; Zalutsky and Nicoll, 1990). There are two forms of synaptic plasticity: LTP and long-term depression (LTD) (Norris et al., 1998). LTP is one of the functional indices of synaptic plasticity (Quan et al., 2010) and serves as a link between structural and behavioural outcome measures. For instance, it is used to study the effects of apoE4 allele in increasing susceptibility to cognitive impairment at the level of dynamic functional changes in synaptic transmission (Trommer et al., 2004). LTP is divided into two temporal phases: early- (E-LTP) and longer lasting late-LTP (L-LTP) (Andersen et al., 1980; Frey et al., 1988; Huang and Kandel, 1994; Reymann et al., 1988). E-LTP is known as the induction phase which involves the release of neurotransmitters such as L-glutamate and excitation of glutamate receptors to allow calcium ion (Ca2+) influx. Formation of Ca2+ and calmodulin (Ca2+/CaM) complex further activates glutamate receptors in a positive feedback loop to increase efficiency of ion fluxes. Another source of Ca2+ may come from intracellular stores released after the activation of 11 Chapter 1: Introduction metabotropic receptors such as group 1 metabotropic glutamate receptors (mGluRs), adenosine 2A receptors (A2ARs) and muscarinic acetylcholine receptors (mAChRs). On the other hand, L-LTP is distinguished from the early phase by the events of protein synthesis. Activation of the second messenger, cyclic adenosine monophosphate (cAMP), by Ca2+/CaM further phosphorylates protein kinase A (PKA) which then translocates into the nucleus to initiate gene transcriptions. As mentioned before, synaptic plasticity is bidirectional and is determined by the intracellular Ca2+ concentration. Under basal conditions, Ca2+ concentration is 50 to 100 nanomolar (nM) which can increase to 10 to 100 micromolar (μM) upon stimulation. When given a HFS, more Ca2+ influx is elicited and as intracellular Ca2+ level exceeds 5 μM, protein kinases are activated to phosphorylate synaptic proteins producing LTP. On the other hand, a low-frequency stimulation (LFS) triggers less Ca2+ influx and a concentration of Ca2+ less than 1 μM will activate protein phosphatases leading to dephosphorylation of synaptic proteins and hence yields LTD. However, electrophysiological studies of cellular models of learning and memory do not always correlate with behavioural and cognitive deficits. An example of inverse correlation is the mutation of postsynaptic density protein 95 (PSD95) which enhances LTP but reduces spatial learning and memory (Migaud et al., 1998). In such cases there seem to be no relationship between synaptic plasticity and behavioural changes in cognition. 1.2.3. Ageing and synaptic plasticity Younger or immature brains are more 'plastic' or receptive to external stimulation than the mature central nervous system (CNS) during induction of synaptic plasticity. The underlying reason for this phenomenon is that there is a critical period during postnatal development whereby the activity of restructuring and re-organization of synaptic connectivity is at its peak (Keuroghlian and Knudsen, 2007). This has been demonstrated by the ability to induce a change in receptive field of young animals by merely exposing 12 Chapter 1: Introduction them to pure tones (de Villers-Sidani et al., 2007). On the other hand, adults need to be subjected to behavioural learning or exposed to plasticity-related neuromodulators such as acetylcholine for plasticity to occur (Kilgard and Merzenich, 1998). Besides that, rodents experience a reduction in plasticity with maturation in primary auditory cortex (A1) and primary visual cortex (V1) as there is a decreased susceptibility to the induction of NMDA-dependent LTP with increasing age (Sato and Stryker, 2008). Furthermore, Jang and colleagues showed that cortical circuits of adults are more resistant to express changes in synaptic connectivity (Jang et al., 2009). It is inevitable that synaptic plasticity and cognition declines with age (Brayne, 2007; Salthouse, 2009; Singh-Manoux et al., 2012). In rodents, cognitive dysfunction particularly in the spatial aspect of the learning and memory is an age-related process that affects both sexes (Benice et al., 2006; Zhao et al., 2009) and all apoE genotypes (Siegel et al., 2012). However, there is a genderbiased susceptibility as female mice performed worse in passive avoidance tasks (Benice et al., 2006). Furthermore, apoE genotype has an additional influence on the cognitive impairment which will be discussed in details in chapter three. 1.2.4. Genetic risk factor for sporadic AD: apolipoprotein E (apoE) variants There has been much evidence that ApoE4 poses as a genetic risk factor for late-onset AD (Corder et al., 1993; Farrer et al., 1997; Strittmatter et al., 1993). Overall, apoE4 individuals have a 20% risk of getting AD compared to the other two isoforms by the age of 85. ε4 allele increases the risk of getting AD in a gene-dose dependent manner as ε4 carriers have a three-fold higher chance of getting AD and the risk is twelve-fold higher for homozygotes when comparing within the group (Holtzman et al., 2012). In fact, this isoform accounts for 65 to 80% of sporadic AD cases (Farrer et al., 1997). The differential effect of apoE4 is also gender-specific as women with ε4 allele have a higher risk of developing AD compared to men (Acevedo et al., 2010; Breitner et al., 1988; Lautenschlager et al., 1996; Rao et al., 1994; van Duijn et 13 Chapter 1: Introduction al., 1993). Fluorodeoxyglucose positron emission tomography (FDG-PET) studies indicate that there is less glucose utilization in normal and AD patients carrying ε4 allele compared to ε3 individuals (Reiman et al., 2004; Small et al., 1995). Not only are the old-aged ε4 carriers affected but middle-aged ones are also more susceptible to impaired glucose metabolism as well. This may be due to the disruption of mitochondrial functions by neurotoxic apoE4 fragments through perturbing mitochondrial trafficking of organelles and promoting mitochondrial apoptotic pathways (Ji et al., 2002; Reynolds and Rintoul, 2004). The areas affected were mainly hippocampus and cortex which strikingly resembles AD pathogenesis (Reiman et al., 2001; Reiman et al., 2004). Taken together, apoE4 may exert its effect before the onset of AD and the modulation is region-specific. ApoE4 has been correlated to many hallmarks of AD including neurodegeneration. ApoE4 produced by injured neurons is able to disrupt mitochondrial electropotential which in turn halts synaptogenesis and causes loss of synapto-dendritic connections in apoE mice models (Buttini et al., 1999; Li et al., 2004). In human brains, apoE4 dose is inversely proportional to dendritic spine density in AD and aged normal controls (Ji et al., 2003). Another infamous hallmark of AD is the accumulation of Aβ and many studies have been done to dissect the role of apoE4 in Aβ metabolism. ApoE4 can be found in amyloid plaques and neurofibrillary tangles (Riddell et al., 2008) and a combination of apoE4 and Aβ42 (the amyloidogenic fragment) treatment causes neurotoxicity in primary culture and impairs long-term potentiation (LTP) in hippocampal slices (Trommer et al., 2005). Furthermore, apoE4 mice have lower rates of Aβ clearance compared to apoE3 mice (Dodart et al., 2005; Holtzman et al., 2000). This supports the observations of increased Aβ deposition in apoE4 transgenic mice and humans (Fryer et al., 2005; Fryer et al., 2003; Rebeck et al., 1993). Notably, apoE4 can also contribute to AD pathogenesis independent of Aβ. Transgenic mice expressing apoE4 with or without expression of human βamyloid precursor protein (hAPP), a precursor of Aβ, have decreased presynaptic terminals (Buttini et al., 2002; Holtzman et al., 2000). There are 14 Chapter 1: Introduction isoform-specific neurodegeneration and as age-dependent human effects apoE3 of prevents human kainic apoE on acid-induced neurodegeneration in comparison to Apoe-null and apoE4 mice which were not protective (Buttini et al., 1999). These morphological changes are further translated into behavioural deficits as other models of transgenic mice which express human apoE4 specifically in neurons or astrocytes showed impaired working memory. Not only are these effects age- and isoform dependent, there is also an influence of gender as female neuron-specific-enolase (NSE)-apoE4 mice lacking mouse apoE and expressing human apoE4 in neurons demonstrated impairment in water maze and vertical exploratory test (Buttini et al., 1999; Hartman et al., 2001; Raber et al., 1998). Notably, there is no accumulation of Aβ in these transgenic mouse lines suggesting that apoE4 causes abnormal morphological and behavioural changes independently of Aβ. In other words, apoE4 is sufficient to impair synaptic plasticity per se which accounts for the learning and memory decline in AD. 1.3. N-methyl-D-aspartate receptor (NMDAR): a key player in learning and memory NMDAR-dependent activity is an eminent mechanism underlying LTP induction which is fundamental to formation of learning and memory (Bear and Malenka, 1994; Bliss and Collingridge, 1993). It has been widely implicated in excitatory synaptic transmission and plasticity, neuronal survival, maturation and migration (Balazs et al., 1989; Komuro and Rakic, 1993). NMDAR is a ligand- and voltage-gated glutamate receptor that is highly permeable to calcium ions, at least four to eight times higher compared to other ionotropic glutamate receptors (iGluRs). As activation of NMDAR requires not only binding of glutamate, an excitatory neurotransmitter, but also α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)induced postsynaptic membrane depolarization (Chen and Lipton, 2006; Kuryatov et al., 1994), these two glutamate receptors work in cooperation to facilitate the induction of synaptic plasticity. 15 Chapter 1: Introduction There are several signalling cascades implicated in synaptic plasticity that are regulated by Ca2+ influx through NMDAR channels (Madison et al., 1991; Micheau and Riedel, 1999; Soderling and Derkach, 2000). Some of the kinases such as PKC, PKA, cyclin-dependent-kinase 5 (cdk5), and Src-family tyrosine kinases (SFKs) are involved in phosphorylation of NMDARs and these interactions are coupled to the downstream extracellular signal-regulated kinase (ERK/MAPK) pathway which culminates in CREB-mediated gene transcription to influence neuronal survival and plasticity (Hardingham and Bading, 2010; Salter and Kalia, 2004; Sanz-Clemente et al., 2013). In the brain, 10 to 70 percent of NR1 and NR2 subunits are phosphorylated by PKC and PKA contributing to functional heterogeneity of NMDARs (Leonard and Hell, 1997). 1.3.1. Characteristics and expression of NMDAR NMDAR is subjected to alternative splicing and mRNA editing giving rise to subunit diversity with at least eight isoforms in existence. Functional NMDAR is a heteromultimer (Wollmuth and Sobolevsky, 2004) comprising NR1, NR2A-2D and sometimes NR3A and 3B (Figure 1A). NMDAR is activated upon binding of glutamate to NR2 subunit and glycine, a co-activator, to NR1 subunit. Under resting state, opening of NMDAR channel is inhibited by magnesium (Mg2+) block which is relieved when the membrane is depolarized to threshold level and further triggers Ca2+ influx. NMDA channel has slow kinetics due to delayed unbinding of glutamate and is subject to various modulations due to its sensitivity to extracellular microenvironment (CullCandy and Leszkiewicz, 2004; Paoletti, 2011; Traynelis et al., 2010). In adult CNS, functional NMDARs are essentially made up of two NR1 and two NR2 subunits in which NR2A/NR2B are the two predominant ones coexisting with the mandatory NR1 subunit as the key players in synaptic plasticity (Watanabe et al., 1992; Wenzel et al., 1997a). This gives rise to formation of major diheteromeric receptor complexes with either NR1/NR2A or NR1/NR2B combination, and also triheteromeric receptor complexes with 16 Chapter 1: Introduction NR1/NR2A/NR2B subunits concentrated in the forebrain (Hatton and Paoletti, 2005), representing from 15% to less than half of the total receptor population (Al-Hallaq et al., 2007; Gray et al., 2011; Rauner and Köhr, 2011). The NR2 subunits dictate the characteristics of a receptor-ion channel complex as they influence the channel conductance, gating properties such as channel open probability and deactivation kinetics (Cull-Candy and Leszkiewicz, 2004; Paoletti, 2011; Traynelis et al., 2010). For instance, diheteromeric NR2A and NR2B-containing receptors are highly permeable to Ca2+ and generate highconductance channel openings compared to NR2C and NR2D-contaning diheteromeric receptors. NR1/NR2A receptors also have a higher open probability and faster deactivation compared to other NR2 subunits (Chen et al., 1999; Erreger et al., 2005). In contrast, incorporating NR3 subunit results in low single channel conductance, Ca2+ permeability and Mg2+ block (Sasaki et al., 2002). Hence, subunit composition of NMDARs affects the channel properties which in turn modulate neuronal functions. In situ hybridization studies demonstrated that mRNAs for NMDAR subunits are differentially distributed throughout the brain. NR1 and NR2A mRNAs and proteins are ubiquitously expressed throughout the brain (Paoletti et al., 2013), whilst NR2B mRNA is mostly found in forebrain regions such as the cortex and hippocampus. On the other hand, NR2C mRNA is usually expressed in the cerebellum and mid- and hindbrains of mammals (Akazawa et al., 1994; Monyer et al., 1994). NR1 subunit mRNA is further spliced into 8 isoforms: NR1-1a-4a and NR1-1b-4b which differ by the presence of three domains namely C1, C2 and C2’ (Dingledine et al., 1999). All NR1 molecules contain either C2 or C2’ domain while C1 domain is only present in NR1-1 and NR1-3 isoforms (Figure 1B). NR1 variants are differentially distributed as NR1-1 and NR1-4 have corresponding distribution, of which the former is found abundantly in cortex and hippocampus whereas NR1-2 is widely distributed (Paoletti, 2011). Protein expression pattern also differs developmentally and spatially for NR2 subunits as major changes occur during the first two postnatal weeks. Initially NR2B and NR2D predominate in the neonatal brain but they are gradually replaced by mostly NR2A and NR2C subunits. NR2A subunit expression in rodents begins at postnatal day 6 (P6) 17 Chapter 1: Introduction and the ratio of NR2A to NR2B increases progressively with corresponding changes occurring at mRNA level (Hoffmann et al., 2000; Liu et al., 2004; Nase et al., 1999). NR2A is eventually distributed throughout the brain whereas NR2B is restricted to the forebrain (Monyer et al., 1994; Sheng et al., 1994). This can be inferred from speeding decay of NMDAR-induced excitatory postsynaptic current (EPSC) as NR2A-mediated EPSC is characterized by a faster deactivation compared with that of NR2B (Erreger et al., 2005). Furthermore sensitivity to NR2B antagonist is diminished at P7. NR2C appears at P10 and is found mainly in cerebellum as well as olfactory bulb while expression of NR2D is limited to brainstem (Paoletti et al., 2013). Figure 1.2. Structure of NMDAR subunit (A) The N-terminal domain (NTD) in the extracellular region consists of a tandem of large globular bi-lobed domains which is involved in allosteric modulation; and is linked to the agonist-binding domain (ABD) formed by two segments (S1 and S2) which binds glycine in NR1 and NR3 subunits and glutamate in NR2 subunits. The transmembrane domain (TMD) is made of up three helices (M1, M2, M4) and a re-entrant loop (M2 or known as P element) that lines the ionic selectivity filter; whereas the intracellular C-terminal domain (CTD) is important for receptor trafficking, anchoring and coupling to downstream signalling molecules. (B) Alternative splicing of exons 5, 21 and 22 in NR1 subunit gives rise to eight NR1 variants containing cassettes N1, C1, C2 and C2’. (adapted from (Dingledine et al., 1999; Paoletti et al., 2013) 18 Chapter 1: Introduction 1.3.2. Functions of NMDAR Since deletion of NR1, NR2A and NR2B causes neonatal lethality (Bach et al., 1995; Forrest et al., 1994; Kutsuwada et al., 1996), genetic manipulations of these subunits are made in regions of interest rather than the entire CNS in order to study their functions. NR1 is the obligatory subunit in a functional NMDA channel and plays a crucial role in induction of LTP. Pharmacological and animal studies using CA1-restricted NR1 knockout mice show that these animals lack NMDAR-mediated synaptic currents and LTP, which is further translated into impaired spatial memory (Nakazawa et al., 2003; Tsien et al., 1996). This effect is also seen with treatment of NMDAR antagonists. Similarly, genetic perturbation of NR2A activation in CA1 cells attenuates LTP (Sakimura et al., 1995) and causes deficits in a series of behavioural studies such as operant discrimination, reversal learning and spatial working memory (Bannerman et al., 2008; Brigman et al., 2008). Likewise, forebrainand corticohippocampal-deletion of NR2B as well as treatment with NR2B antagonist in rodents impair their performance in object recognition, lever pressing tasks, Morris water maze (MWM) and attentional tasks (Brigman et al., 2008; Duffy and Nguyen, 2003; Higgins et al., 2003). Both NR1 and NR2A subunits contribute to neurite outgrowth (Le Greves et al., 2002). Postnatal modifications in NR2 subunit composition is implicated in synaptic plasticity during development (Bear, 2003). NR2A is deemed more stable compared to NR2B subunit and thus may help to stabilize synapses while making structural and functional changes more difficult (Lavezzari et al., 2004; Petralia et al., 2005). In contrast NR2B is crucial to formation and retraction of dendritic spines and hence NR2B dominant synapses are more plastic (Ling et al., 2012) due to its increased mobility observed in cultured neurons (Groc et al., 2006). Site-directed mutagenesis and targeted truncation of NR2A and NR2B C-terminal tails support their important roles in synaptic localization and NMDAR clustering as their C-termini contain PDZ (PSD95, disc large, zona occludens-1) binding motif that is associated with scaffolding protein (Lin et al., 2004; Steigerwald et al., 2000). Taken together NMDAR 19 Chapter 1: Introduction plays a major role in the maintenance of structure and functions of the synapses. 1.3.3. Modulation of NMDARs dephosphorylation mechanisms by phosphorylation and Studies on LTP induction at the SC-CA1 synapse (Bashir et al., 1991; Bliss and Lomo, 1973; Madison et al., 1991) and LTD at the cerebellar parallel fibre-Purkinje cell synapse (Ito, 1989; Linden and Connor, 1993) have illustrated the functional consequences of protein phosphorylation on glutamate receptors. NR1 subunit is subjected to phosphorylation at either serine (Ser) or threonine (Thr) residues while NR2A and NR2B subunits are phosphorylated at tyrosine residues. Activation of serine residues on Cterminal C1 cassette of NR1 positively modulates trafficking of the subunit to the membrane surface and increases synaptic transmission (Tingley et al., 1997; Zou et al., 2000). Chiu and colleagues have shown that cocaine- and amphetamine-regulated transcript peptide (CARTp) increases phosphorylation of serine residues at position 896 and 897 which subsequently activates extracellular-regulated kinase (ERK) via protein kinase C (PKC) and protein kinase A (PKA) pathway respectively (Chiu et al., 2009). Whilst phosphorylation of these two residues increases NR1 surface expression, another serine residue at position 890 which is also phosphorylated by PKC plays a role in receptor clustering (Tingley et al., 1997). Tyrosine phosphorylation of NR2 subunits increases ion gating and reduces endocytosis of NMDARs (Salter and Kalia, 2004; Snyder et al., 2005). This results in overall enhancement of NMDAR function and synaptic strength. Mice with truncated NR2A C-terminus without phosphorylation sites shared similar phenotype as the NR2A-knockout mice. Their impaired CA1-LTP and contextual learning highlight the critical functions of C-terminus and its phosphorylation sites (Sprengel et al., 1998). Most of the studies are done on tyrosine phosphorylation of NR2B at residue 1472 as it is the major phosphorylation site involved in synaptic plasticity (Jiang et al., 2011a). This phosphorylation limits the clathrin-mediated endocytosis of NR2B subtypes 20 Chapter 1: Introduction and localizes NR2B-containing NMDARs at postsynaptic density (Prybylowski et al., 2005). On the other hand, the activation of NMDARs can be suppressed by dephosphorylation through serine and threonine phosphatases 1, 2A (PP1/PP2A), or 2B (calcineurin) (Lieberman and Mody, 1994; Wang et al., 1994). Tyrosine phosphatases may also downregulate NMDA channel opening probability as application of tyrosine phosphatase inhibitor increases channel opening rates in rat spinal neurons (Wang et al., 1996). Taken together, phosphorylation and dephosphorylation mechanisms modulate clustering and interactions of NMDAR with other intracellular proteins. In addition to these mechanisms, NMDAR functionality is also influenced by mGluRs or by cAMP concentration which is activated by adenylate cyclase (Bleakman et al., 1992; Courtney and Nicholls, 1992; Koh et al., 1991; Martin et al., 1997). 1.3.4. Opposing roles of NMDAR subunits in synaptic plasticity Activation of NMDARs facilitates formation of both LTP and LTD (Bear and Malenka, 1994; Bliss and Collingridge, 1993) and there are several hypotheses regarding the roles of NMDAR in bidirectional plasticity. Earlier studies have shown that the degree of NMDAR activation which is predetermined by the degree of stimulus, and subsequently the level of postsynaptic calcium elevation, governs the direction of NMDAR-induced synaptic plasticity (Cummings et al., 1996; Nishiyama et al., 2000). Recently, there has been increasing evidence that NR2 subtypes may also determine the polarity of synaptic plasticity. When LFS is used to trigger LTD, there is a larger total charge transfer facilitated by NR1/NR2B receptors compared to NR1/NR2A receptors and vice versa when HFS is used to induce LTP (Erreger et al., 2005). Furthermore, in hippocampal slice preparations, inhibition of NR2Acontaining NMDARs abolishes LTP whereas blockade or loss of NR2B subunits prevents formation of LTD but not LTP (Brigman et al., 2008; Liu et al., 2004). With the postnatal developmental switch from NR2B to NR2A subunit (Monyer et al., 1994; Mutel et al., 1998) and the increasing difficulty to induce LTD at these synapses (Errington et al., 1995; Kemp et al., 2000), as 21 Chapter 1: Introduction well as impaired LTP in NR2A-deficient mice (Köhr et al., 2003; Sakimura et al., 1995), all of these suggest the dominant role of NR2A-containing NMDARs in LTP. However, conflict arises as some studies argue that NR2B is also required for LTP (Barria and Malinow, 2005; Berberich et al., 2005; Clayton et al., 2002; Tang et al., 1999; Weitlauf et al., 2005). Application of NR2B-specific antagonist, ifenprodil, attenuates LTP in immature hippocampal slices and anterior cingulate cortex of juvenile mice ranging from few days to few weeks of age (Barria and Malinow, 2005; Lu et al., 2001; Zhao et al., 2005). On the other hand, overexpression of NR2B subunit in 4- to 6-month-old transgenic mice enhances hippocampal LTP (Tang et al., 1999). Discrepancies may be caused by differences in developmental and regional NMDAR subunit expression, LTP and LTD induction protocols, or poor specificity of selective antagonists. 1.3.5. NMDAR in ageing Ageing has been negatively associated with NMDAR binding densities and functionality (Barnes, 2003; Foster, 2012; Magnusson et al., 2010). In fact, NMDAR is more susceptible to the deleterious effects of ageing than other glutamate receptors and the degree of vulnerability is different for specific NMDAR subunits (Magnusson et al., 2010). In rodents, there is a general deterioration of NR1 and NR2B subunit while NR2A subunit is relatively being spared during ageing (Magnusson, 2000; Magnusson et al., 2002). Particularly, protein expression of NR1 and NR2B subunit in the hippocampus, cerebral cortex (Clayton and Browning, 2001; Eckles-Smith et al., 2000; Magnusson et al., 2002) and crude synaptosomes 2 from frontal cortex (Magnusson et al., 2007) decline with age. However, few contrasting studies reported that only NR2B in the frontal cortex of mice (Ontl et al., 2004); or NR2A and NR2B but not the NR1 subunit in the hippocampus and cortex (Sonntag et al., 2000; Zhao et al., 2009); show age-related decrement. It is clear that NR2B is the most vulnerable subunit to the effects of ageing than the rest (Magnusson, 2000; Magnusson et al., 2007; Ontl et al., 2004; Zhao et al., 2 sealed presynaptic structures 22 Chapter 1: Introduction 2009) with a greater reduction within the synaptic membrane as compared to the whole homogenate of the frontal lobe (Zhao et al., 2009). This suggests that ageing process may also modulate synaptic localization of the NR2B subunit. 1.3.6. NMDAR in AD NMDAR dysfunction or hypofunction has been linked to AD pathology and loss of NMDAR is not only subunit- but also brain region-specific. Constant administration of NMDA antagonist over a period of time in rats renders NMDAR to a chronic hypofunctional state that causes neuronal death in cerebrocortical and limbic regions. This damage particularly affects pyramidal and multipolar neurons in hippocampus, cortex and amygdala. Neurodegeneration involving excitotoxic retraction of dendritic spines leads to synaptic loss which is most strongly correlated with cognitive dysfunction in AD (Corso et al., 1997). In NMDAR hypoactivity (NRHypo) animal model, there is no deposition of amyloid plaque suggesting that this mechanism is capable of generating neuropathological symptoms of AD independently of Aβ (Olney et al., 1997). In postmortem human AD brain, there is reduced NR1 in frontal cortex and hippocampus but not amygdala (Amada et al., 2005); and reduced NR2A and NR2B (Bi and Sze, 2002) in hippocampus whereas NR2C and NR2D do not seem to be affected (Hynd et al., 2004). Furthermore, there is a differential and subunit-specific decrease of NR1 and NR2B with increasing AD severity (Mishizen-Eberz et al., 2004). In the hippocampi of schizophrenic patients there is also downregulation of NR1 mRNA level and these patients exhibit impairment in verbal memory as well as executive functioning and attention (Gao et al., 2000). This suggests that loss of NR1 may correlate with cognitive deficits in AD. Several factors may contribute to NRHypo in AD including age and NMDAR hyperactivity (NRHyper) during early stages of the disease (Olney et al., 1997). Normal ageing has been negatively associated with NMDAR functionality across species such as mice, rats, monkeys and human. Advancing age may also interact with other genetic risk factors such as apoE 23 Chapter 1: Introduction genotype that can exacerbate NRHypo state preceding neurodegeneration in AD brain. 1.3.7. NMDAR in excitotoxity It is proposed that chronic NRHyper state can lead to excitotoxic insults on synapses and deletion of NMDARs upon neuronal death thus plummeting into NRHypo state (Olney et al., 1997). Under normal circumstances, NMDAR channels should open only for brief periods of time to allow mainly influxes of Ca2+ and other cations. However, hyperactivity of glutamate receptor or prolonged exposure to elevated glutamate concentrations relieves the Mg2+ block and causes excessive amounts of Ca2+ influx into neurons. Some of the factors that sensitize NMDAR to glutamate are oxidative stress and impaired energy metabolism of which the latter causes a partial membrane depolarization that eliminates Mg2+ inhibition. In such cases, even normal levels of glutamate will be able to drive abnormal currents. In cultured neurons, nitric oxide (NO) pathway generates free radicals that disrupt glycolytic metabolism and triggers NRHyper via the same mechanism as dysregulated energy metabolism. Persistent intracellular Ca2+ overload affects mitochondrial metabolism and leads to production of free radicals, resulting in a positive feedback loop in sensitization of NMDAR, activation of degrading enzymes such as caspases and release of apoptotic factors (Olney, 1994; Rothman and Olney, 1995). These events destroy cellular components and ultimately lead to synaptic damage and cell death (Dawson et al., 1991; Naskar et al., 1999) promoting NRHypo state. This ultimately escalates to a variety of neurological diseases such as AD, Huntington’s disease, Parkinson’s disease, amylotrophic lateral sclerosis (ALS), etc on a long-term basis (Choi, 1992; Cull-Candy et al., 2001; Olney, 1971, 1994). Hence, tight regulation of NMDAR function is critical for optimal activity in response to external stimulation, but at the same time to avoid excessive activation as excitotoxicity may contribute to neurodegeneration. 24 Chapter 1: Introduction 1.4. α-amino-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPAR) AMPAR is another iGluR that allows ion fluxes particularly sodium (Na+) through the channels leading to development of LTP and LTD. These receptors are found alongside NMDARs at postsynaptic terminals to trigger synaptic transmission when presynaptic terminals release L-glutamate which binds to AMPARs and NMDARs at the surface membrane of postsynaptic terminal. Like NMDAR, AMPAR genes undergo post-transcriptional modification by RNA editing generating four different subunits which are GluR1, GluR2 (short and long isoforms exist), GluR3 and GluR4/4c. These subunits combine in different ratios to form functional receptors (Dingledine et al., 1999; Hollmann and Heinemann, 1994). GluR1 and GluR2 subunits are heavily expressed in the hippocampus and deletion of the former prevents LTP production which can be rescued by exogenous expression of GluR1 (Mack et al., 2001). The majority (80%) of functional AMPARs in CA1 synapses comprises GluR1 subunit, highlighting the importance of this subunit (Lu et al., 2009b). The C-terminus of GluR1 contains sites for specific phosphorylation to modulate synaptic plasticity as serine residues 831 (S831) can be phosphorylated by protein kinase C (PKC) or calcium/calmodulindependent protein kinase II (CaMKII) and 845 (S845) by PKA (Roche et al., 1996). Phosphorylation of GluR1 S845 by PKA increases channel open probability and stabilizes AMPARs recruited to synapses via CaMKII-driven exocytosis (Banke et al., 2000; Esteban et al., 2003; Hayashi et al., 2000). On the other hand, CaMKII phosphorylation of GluR1 S831 enhances channel conductance during LTP (Bredt and Nicoll, 2003; Malenka, 2003; Song and Huganir, 2002). It is proposed that the two different phosphorylation sites of GluR1 determine the opposing polarities of synaptic plasticity. HFS given to naïve synapses stimulates phosphorylation of GluR1 S831 by CaMKII or PKC and favours LTP. Conversely, LTD induction by LFS in naïve synapses causes dephosphorylation of GluR1 S845 by protein phosphatases (PP1/PP2A) and calcineurin (CaN) (Lee et al., 2000; Lee et al., 1998; Soderling and Derkach, 2000). These phosphorylation and dephosphorylation mechanisms elicit 25 Chapter 1: Introduction addition and endocytic removal of synaptic AMPARs respectively to regulate AMPAR trafficking during synaptic plasticity (Carroll et al., 1999; Hayashi et al., 2000; Man et al., 2000; Shi et al., 1999). Other than that, both NMDAR activity and protein phosphatases are needed for AMPAR internalisation followed by PKA-dependent recycling of the receptors. In contrast, AMPAR activation without NMDAR will target the former for degradation in lysosomes (Ehlers, 2000). Activation of AMPARs allows Na+ influx which depolarizes the postsynaptic membrane to the threshold that would remove the Mg2+ block of NMDAR. As NMDA channels open to allow Ca2+ influx, an increase in Ca2+/CaM complex causes phosphorylation of AMPARs and new AMPARs are rapidly inserted into the postsynaptic sites in a process known as 'AMPAfication'. This is followed by NMDAR insertion which can also occur per se (Bashir et al., 1991; Grosshans et al., 2002; Watt et al., 2004) to induce LTP. It is observed that AMPAR activity plays a critical role in maturation of thalamocortical networks by facilitating conversion of silent or dormant synapses to functional ones (Feldman et al., 1999). Notably, LTP elicited by NMDAR and AMPAR differs in the expression mechanisms but may influence each other during synaptic plasticity (Watt et al., 2004). In particular, NMDAR-dependent Ca2+ signalling affects synaptic strength through kinases and phosphatases to modulate AMPAR channel properties and localization in CA3-CA1 synapses (Malenka and Bear, 2004). However, NMDARs can also undergo plasticity independently of AMPARs at adult synapses such as mossy fibre-CA3 synapses and in midbrain neurons (Kwon and Castillo, 2008; Rebola et al., 2008). 26 Chapter 1: Introduction Figure 1.3. Model of bidirectional plasticity in AMPAR phosphorylation and dephosphorylation A model explaining phosphorylation and dephosphorylation mechanisms of GluR1 subunit of AMPAR that govern the direction of synaptic plasticity. High frequency stimulation (HFS) given to ‘depressed’ synapses (without phosphorylated residues) will activate PKA to phosphorylate GluR1 at serine 845 (S845) and further phosphorylation of naïve synapses at GluR1 serine 831 (S831) by PKC or CaMKII gives rise to LTP induction. Low frequency stimulation (LFS) will activate phosphatases (PP1/2A) or calcineurin (CaN) to dephosphorylate the potentiated synapses (with both phosphorylated serine residues) at GluR1 S831 and further dephosphorylation of naïve synapses at S845 shifts the direction of plasticity towards LTD formation. 27 Chapter 2: Materials And Methods Chapter 2: Materials And Methods 2.1. Common reagents and materials Dulbecco’s Modified Eagles Medium (DMEM) culture medium (Gibco) supplemented with 10% Fetal Bovine Serum (FBS, Gibco), 100 mM sodium pyruvate (Gibco), antibiotic-antimycotic (Gibco) and 2 µg/mL blasticidin S (Invitrogen) was used for culture and maintenance of apoE-transfected neuronal cell lines. Freezing media was prepared using the same DMEM supplemented with 40% volume/volume (v/v) FBS, 100 mM sodium pyruvate, antibiotic-antimycotic and 10% v/v sterile dimethyl sulfoxide (DMSO). 0.5% Trypsin- Ethylenediaminetetraacetic acid (EDTA) (Gibco) was diluted with DMEM to 0.02% and used for detaching cells. Sterile phosphate buffered saline (PBS) used for cell culture was prepared from 10X concentrated solutions (1st Base Asia, Singapore) and autoclaved. Non-sterile PBS (pH7.4) was prepared as a 10X stock from salts and used for non-cell culture work. Running buffer (pH8.3) for immunoblotting was prepared as 10X stock using Tris base, glycine and sodium dodecyl sulfate (SDS). 1X Transfer buffer was diluted from 25X Novex® Tris-Glycine Transfer Buffer with methanol and distilled water and then kept at 4°C. Washing buffer PBST was prepared using PBS with 0.1% v/v Tween 20 added. 2.2. Animal model The animal experimentation in this study was approved by the Institutional Animal Care and Use Committees (IACUC) at the National University of Singapore under the protocol #009/10. ApoE-targeted replacement (apoE-TR) or apoE-knockin (apoE-KI) mice model, B6.129P2-Apoetm3 mice and B6.129P2-Apoetm4 (APOE*4) Mae (APOE*3) Mae N8 N8 mice with defined C57BL6/J background (Knouff et al., 1999), were bought from Taconic Farms, Inc (Germantown, NY, USA). Mice were back-crossed to wild type C57BL/6J 28 Chapter 2: Materials And Methods (Harlan 2BL/610) for eight generations (N8) and were homozygous for human apoE3 (3/3) or apoE4 (4/4) alleles. The colony is maintained through breeding of homozygotes and animals were housed on a 12-hour light/12-hour dark cycle in vivarium supplemented with normal feed and water ad libitum. Only female mice were used in this study based on the rationale that female apoETR mice are more susceptible to learning and memory deficits (Bour et al., 2008; Raber et al., 2000). 2.2.1. Tissue preparation for protein analysis Upon reaching the time-points designated i.e. 12, 32 and 72 weeks, they were fasted one hour prior to euthanization in accordance with guidelines approved by the Institutional Animal Care and Use Committees (IACUC) at the National University of Singapore. Brains were harvested and rinsed with sterile phosphate buffered saline (PBS). The left and right hemispheres were separated and kept in -80°C till further use. 2.2.2. Preparation of brain homogenates Brains were weighed and lysed in Radioimmunoprecipitation assay (RIPA) buffer (Cell Signalling Technology, Danvers, USA) which is composed of 20 mM Tris-HCl (pH 7.5), 150 mM sodium chloride, 1 mM sodium EDTA, 1 mM ethylene glycol tetraacetic acid (EGTA), 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate and 1 µg/mL leupeptin with protease and phosphatase inhibitor cocktail tablets, PhosSTOP (Roche Molecular Biochemicals, Indianapolis, IN, USA) at 20% weight/volume (w/v) ratio. Tissues were homogenized with a handheld homogenizer and further lysed with 18G needles attached to syringe to ensure complete lysis. Tissue lysates were placed on ice for one hour before centrifugation at 20 000g for 30 minutes at 4°C. The supernatant comprising of the soluble portions of lysates were aliquoted to avoid freeze-thaw and stored at -80°C. 29 Chapter 2: Materials And Methods 2.2.3. Protein quantitation of brain lysates Pierce™ MicroBicinchoninic acid (BCA) Assay kit (Thermofisher Scientific, Waltham, USA) was used to quantify the protein concentration of brain lysates. Lysates were diluted 100 fold with PBS and 25 μL of diluted samples were pipetted into microplate wells together with the BSA standards in duplicates within the same plate. 200 μL of working reagent was added to each well and incubated for 30 minutes at 37°C before absorbance was read at 562 nm with a Tecan microplate reader. Protein concentrations of samples were then calculated based on the standard curve constructed from BSA standards. 2.2.4. Western blotting Protein lysates were loaded at 60 to 100 µg except for one of the targets, PKCα whereby the protein load was 30 µg per lane. Final volume was standardized by adding PBS and 6x loading dye containing β-mercaptoethanol were added. The mixtures were heated at 95°C for 5 minutes and loaded onto a hand-cast 7.5 or 10% Tris-glycine polyacrylamide gel with Precision Plus ProteinTM KaleidoscopeTM Standards (Bio-Rad Laboratories, Hercules, California USA) as a molecular weight marker that ran alongside with samples. Gel resolution was performed in either Mini-PROTEAN Tetra electrophoretic system (Bio-Rad Laboratories). The stacking gel was subjected to 70 or 75 volt (V) for 30 minutes, after which the resolving gel was subjected to 110 to 120V until the dye front had reached the bottom of the gel. Proteins were then transferred onto a nitrocellulose membrane of pore size 0.45 µm using Mini Trans-Blot cell (Bio-Rad Laboratories) at 100V for one to two hours depending on the size of the protein targets. The transfer efficiency was verified with Ponceau S (Sigma-Aldrich, St-Louis, USA) staining and rinsed with PBS with 0.1% v/v Tween 20 (PBST) to wash off the stain. The membrane was blocked with 5% w/v non-fat milk in PBST for 30 minutes with constant rocking and rinsed three times with PBST for 5 minutes each 30 Chapter 2: Materials And Methods with agitation. Primary antibody of interest diluted in either 5% w/v non-fat milk or 5% w/v BSA in PBST was added onto the blot which was then sandwiched between two pieces of parafilm and incubated overnight. After rinsing the blot three times with PBST, it was incubated with secondary antibody dissolved in 3% w/v non-fat milk in PBST for one hour with gentle rocking. The blot was then rinsed with PBST three times and protein bands were visualized using KODAK Image Station 4000R (Carestream Health Inc, New York, USA) with either SuperSignal West Dura or SuperSignal West Femto (ThermoFisher Scientific, Waltham, USA). The blot was stripped with Restore Western Blot stripping buffer (ThermoFisher Scientific, Waltham, USA) and blocked with 5% non-fat milk w/v in PBST prior to incubation with either another primary antibody of interest or anti-actin primary antibody followed by the corresponding secondary antibody. The blot could only be stripped for a maximum of two times. Western blots were analyzed using ImageJ software which is available online at http://rsb.info.nih.gov/ij/. The method of quantitative analysis of the protein bands was featured under http://lukemiller.org/index.php/2010/11/analyzinggels-and-western-blots-with-image-j/. Briefly, the file opened in ImageJ must contain the experimental blot (bands of interest) and the total protein or actin (loading control) blot side by side. A rectangular box is drawn around the band of interest in the first lane such that the box is wide enough to cover the largest band and but will not overlap with a band in the next lane. This lane is selected as the first lane and the size of the rectangular box is standardized for the rest of the lanes. Lane profiles were plotted and a straight horizontal line was drawn defining the x-axis (the background) for each lane plotted. The line must touch both sides of the curve. A vertical line was drawn from the top of the curve (usually at the peak) down to the horizontal line. The area under the curve was measured by clicking inside the shape on left or right side defined by the line and the curve using the wand tool. 31 Chapter 2: Materials And Methods 2.2.5. Tissue preparation for immunofluorescence Transcardial perfusion with 4% (w/v) paraformaldehyde (PF) was utilized to fix the brain tissue for immunofluorescence studies. 4% phosphate-buffered PF (pH7.4) was prepared and stirred overnight to ensure complete dissolution of the powder. Mice were fasted one hour before being injected intraperitoneally with mice anaethesia consisting of ketamine 7.5 mg/ml and medetomidine 0.1 mg/ml at a dosage of 0.1ml/10g of body weight. After ensuring the mice were unconscious, blood was flushed out of the system with Ringer’s solution before being perfused with 4% w/v PF for at least half an hour or more. Brains were removed and postfixed in 4% w/v PF overnight at 4°C. This was followed by immersion of tissues in a gradient of sucrose in PBS starting from 10%, 20% to 30% w/v for 24 hours each to allow the brain to sink to the bottom. The right and left hemispheres were snapped frozen separately in isopentane cooled by liquid nitrogen and stored frozen till further use. 2.2.6. Immunofluorescence Brain sections were cut using cryostat into 16 µm thick slices at an angle of 4 degrees. The temperature of the cryostat was preset to -20°C for chamber temperature and -18°C for operating temperature. Sections were mounted onto polysine slides (ThermoFisher Scientific) and smoothed with PBS then left to dry overnight at room temperature. To ensure a fair comparison of the protein-of-interest between apoE3 and apoE4 brain sections, all the sections under various conditions within the same experiment were stained together as a batch. The exposure time for both genotypes are standardized within the same experiment but may differ among batches of staining. Hence, only the apoE3 and apoE4 brain sections of the same time-point were compared against each other and the signal intensities for apoE4 were normalized against apoE3 for the individual time-point. 32 Chapter 2: Materials And Methods Prior to staining, a water-repellent barrier was drawn around the brain section with ImmEdge hydroprobic barrier pen (Vector Laboratories, Inc., Burlingame, USA) to contain the reagent within the region of interest during incubation. After drying, each section was permeabilized with 50 µL of 0.3% v/v Triton-X 100 in PBS for 10 minutes. This was followed by washing the sections with PBS twice, 5 minutes each wash and then blocking with 3% w/v bovine serum albumin (BSA) in PBS for 30 minutes at room temperature. After tapping off excess blocking reagent, sections were incubated with primary antibody diluted with the same blocking buffer for 48 hours at 4°C in a humidified chamber. Sections were washed four times with PBS for 5 minutes each and incubated with secondary antibody conjugated with Alexa Fluor (AF) fluorescence dye (Invitrogen) for one hour in dark at room temperature before repeating the washing step. Excess PBS was wiped away and 10 µL of Prolong Gold® Antifade Reagent with DAPI (Invitrogen) was added to each section before being mounted gently with cover slip. Sections were sealed with nail polish and dried overnight in dark at room temperature. For co-localization analysis, the reagents and protocol used were slightly modified as the blocking reagent used was 0.3% w/v bovine serum albumin (BSA) in PBS. Incubation condition for primary antibody diluted with the same blocking buffer was 24 hours at room temperature in a humidified chamber. After secondary antibody incubation and washing, blocking step was repeated and the subsequent primary antibody of interest was incubated overnight at room temperature. Secondary antibody against the primary antibody was incubated for 90 minutes in dark and then sections were washed for five times with PBS. The subsequent mounting steps were same as above. Dried slides were visualized using a motorized inverted fluorescence microscope IX81 (Olympus Corporation, Japan). Images of negative controls which are sections stained in the absence of the primary antibody were first taken and the samples were subsequently imaged under the same settings. Images were captured using the 20X objective and zoomed up to 40X where necessary and subsequently exported to TIFF format as raw data containing multichannel information. 33 Chapter 2: Materials And Methods Antibody NR1 NR2A NR2B ApoE MAP2 GFAP Source Goat polyclonal Rabbit polyclonal Rabbit polyclonal Mouse monoclonal Rabbit polyclonal Rabbit polyclonal Company Santa Cruz Chemicon Upstate Santa Cruz Santa Cruz Sigma Aldrich Dilution Factor 1: 25 1: 100 1: 25 1: 25 1: 25 1: 100 Table 2.1. List of primary antibodies used for immunofluorescence. The source and the dilution factor used are as shown. Images were processed and analysed using ImageJ software. Briefly, the images were converted into 8 bit (black and white) prior to analysis, while a threshold was determined based on the signal intensity to minimize the background noise. This threshold value was standardized for all sections of the same target. The scale bar was calibrated by a standard micrometer to the individual pixel size of the image and this scale bar is applied to all images taken under the 20X objective. Region-of-interest (ROIs) were demarcated to exclude the unusually bright staining (artefacts) at the periphery of the sections. The total area of staining over the threshold within the ROIs was computed by the software and this total area of staining is used as the measurement to quantitate fluorescence intensity for each section. For cortex, three fields of images were acquired and the total area of staining was averaged for each mouse. The colocalized images of apoE with microtubule microtubule-associated protein 2 (MAP2) and apoE with glial fibrillary acidic protein (GFAP) were first separated into different colour channels which would be automatically converted into 8 bit images. A threshold value was individually set for MAP2 and GFAP to minimize the background noise and this threshold was standardized for all colocalized images of the same target. Images of apoE were then overlapped and recombined with MAP2 or GFAP and colocalized area was computed by ImageJ software as the integrated density of the two different stainings. Integrated density is the density of the signal of the second image that was segmented (MAP2 or GFAP) according to the first image (apoE). Hence, the standard threshold values were only set for MAP2 and GFAP but the apoE signal intensity remains unaltered. 34 Chapter 2: Materials And Methods 2.3. Cell culture 2.3.1. Immortalization and transfection of apoE-knockout neuronal cells ApoE-transfected neuronal cell lines were generated in our lab previously. Briefly, cortical neurons were isolated from P1 mouse pups of B6.129P2ApoetmlUnc/J (APOE null) background and seeded on poly-L-lysine (Sigma Aldrich, USA) coated flasks. Primary neurons were cultured in neuronal enrichment media supplemented with Minimum Essential Medium (MEM, Gibco) supplemented with 10% v/v FBS, 10% v/v horse serum, 0.01% v/v non-essential amino acid (100x) (Gibco), 200 mM L-glutamine (Gibco), 100 mM sodium pyruvate, antibiotic-antimycotic and 0.5g D-glucose for two days. 10 μM of cytosine arabinoside (Ara-C, Sigma Aldrich) was prepared fresh and added to the freshly changed medium. Cells were cultured for another three days until it reaches about 80% confluency and Ara-C was removed from the medium. Simian virus 40 (SV40) T antigen is used to induce immortalization of primary neuronal cells. Human apoE3 and apoE4 gene constructs were kindly provided by Dr. Katherine Youmans and Dr. Mary Jo LaDu (University of Illinois, Chicago, USA) and were cloned into pcDNA6.2 vectors using the Gateway pENTR system (Invitrogen). These plasmids were stored as bacterial gylcerol stocks and cultured when desired. The plasmid DNA was purified using QIAprep Spin Miniprep kit (Qiagen) and the DNA concentration was quantified with NanoDrop spectrophotometer (ThermoScientific). 2ug plasmid DNA carrying the different isoforms of human apoE gene was transfected into immortalized neurons using Amaxa® cell line Nucleofector® kit V (Lonza) and subjected to electroporation with the appropriate programme (T-024 for highest transfection efficiency). An empty vector was used as a control to generate mock cell lines. The transfected cells were gently transferred to a 6well plate and allowed to stabilize overnight in a humidified incubator at 37°C supplied with 5% CO2. Positively-transfected clones were selected by various concentrations of blasticidin S (Invitrogen, Eugene, OR, USA) and finally maintained with 2 µg/mL blasticidin S. 35 Chapter 2: Materials And Methods 2.3.2. CE treatment Cells were cultured in T75 flasks and kept in a humidified incubator at 37°C supplied with 5% CO2. Media was changed every three days. CE stock 100 mg/mL was prepared by diluting CE powder provided by Dr. Paramjeet Singh (Cerebros Pacific Limited) in PBS and stored in aliquots at -80°C. When cells reached about 80% confluency, they were rinsed once with sterile PBS and fresh media containing CE of final concentration 100 µg/mL was incubated with cells for 24 hours in a humidified incubator at 37°C supplied with 5% CO2. This CE concentration was previously determined in our lab based on CellTiter 96® Aqueous One Solution Cell Proliferation (MTS) assay (Promega, USA) (Siew, 2011). Ingredient Amount −1 Protein (Peptide) (mg mL ) 83.0 Free amino acid (mg mL−1) 3.1 −1 L-anserine (mg mL ) 2.3 −1 L-carnosine (mg mL ) 0.8 Taurine (mg mL−1) 0.7 −1 Hexose (mg mL ) 0.8 Phosphatidyl choline (mg mL−1) 0.4 Minerals (μg mL−1) Calcium 26 Iron 1 Zinc 2 Magnesium 32 Potassium 1740 Sodium 550 Chlorine 1340 Phosphorus 480 Sulfur 500 Copper 2 Manganese 5 Selenium 0.05 Vitamins (μg mL−1) Vitamin B2 1.0 36 Chapter 2: Materials And Methods Vitamin B6 0.37 Vitamin B12 0.002 Niacin 6.4 Falacin 0.15 Vitamin C 15 Table 2.2. Composition of CE compound (adapted from Zhai et al., 2012) 2.3.3. Cell lysis Media was removed from the flask and cells were rinsed once with PBS. The cells were then mechanically scraped in PBS using a cell scraper (SPL Life Sciences, Korea). Cells were collected in eppendorf tubes and centrifuged at 600 g for 5 minutes. Supernatant is discarded and cell pellets were resuspended in 200 µL of RIPA buffer. Cell pellet was lysed with a 27G needle until a homogenous solution was obtained. Lysate was then incubated on ice for one hour and centrifuged at 14 000 rpm for 10 minutes at 4°C. Supernatants containing the soluble portion of lysates were stored as aliquots in –80°C till further use. 2.3.4. Protein quantitation of cell lysates Procedure is the same as those stated in section 2.1.3 except that cell lysates were diluted 30 fold with PBS. 2.3.5. Western blotting Refer to section 2.1. Antibody Source 37 Company Dilution Chapter 2: Materials And Methods ApoE GFAP NeuN LRP1 PSD95 Phospho NR1 (Ser890) Phospho NR1 (Ser896) Phospho NR1 (Ser897) NR1 Phospho NR2A (Tyr1246) NR2A Phospho NR2B (Tyr1472) NR2B Phospho GluR1 (Ser831) Phospho GluR1 (Ser845) GluR1 Phospho PKA Cα (Thr197) PKA-Cα Phospho PKCα (Thr497) PKCα Phopho αCaMKII (Thr286) αCaMKII Phospho ERK 1/2 (Thr185/Tyr187) ERK1/2 Phospho CREB (Ser133) CREB Goat polyclonal Rabbit polyclonal Mouse monoclonal Goat polyclonal Mouse monoclonal Rabbit polyclonal Rabbit polyclonal Rabbit polyclonal Rabbit monoclonal Rabbit polyclonal Chemicon Sigma Millipore Factor 1: 500 1: 500 1: 500 Santa Cruz Pierce 1: 200 1: 500 Cell Signaling Cell Signaling Cell Signaling Cell Signaling 1: 500 1: 500 1: 500 1: 500 Cell Signaling 1: 500 Rabbit polyclonal Rabbit polyclonal Chemicon Cell Signaling 1: 500 1: 500 Rabbit polyclonal Rabbit polyclonal Rabbit monoclonal Rabbit polyclonal Rabbit monoclonal Rabbit monoclonal Rabbit monoclonal Rabbit polyclonal Rabbit polyclonal Cell Signaling Santa Cruz Cell Signaling 1: 500 1: 500 1: 500 Cell Signaling Cell Signaling 1: 500 1: 500 Cell Signaling 1: 500 Abcam 1:1000 Abcam Cell Signaling 1:1000 1: 500 Rabbit polyclonal Rabbit polyclonal Cell Signaling Invitrogen 1: 500 1: 500 Rabbit polyclonal Rabbit polyclonal Rabbit monoclonal Cell Signaling Cell Signaling Cell Signaling 1: 1000 1: 500 1: 500 Table 2.3. List of primary antibodies used for immunoblotting. The source and the dilution factor used are as shown. 38 Chapter 2: Materials And Methods Secondary antibody Company Goat anti Mouse IgG HRP Millipore Dilution Factor 1: 10 000 Purpose Goat anti Rabbit IgG HRP Rabbit anti Goat IgG HRP Goat anti Rabbit IgG AF488 Goat anti Rabbit IgG AF568 Goat anti Mouse IgG AF568 Donkey anti Goat IgG AF568 Millipore 1: 10 000 Western blot Millipore 1: 10 000 Western blot Invitrogen 1: 200 Immunofluorescence Invitrogen 1: 200 Immunofluorescence Invitrogen 1: 200 Immunofluorescence Invitrogen 1: 200 Immunofluorescence Western blot Table 2.4. List of secondary antibodies used throughout the study. The purpose, source and dilution factor used are as shown. 2.3.6. Calcium assay Fluo-4 DirectTM Calcium Assay Kit (Invitrogen) was used to measure the basal intracellular calcium ion concentration. Cells were seeded in sterile black-walled clear bottom 96-well plate (Corning, USA) at a density of 2000 cells/well and allowed to attach overnight. On the subsequent day, cells were rinsed once with sterile PBS and treated with CE 100 ug/mL for 24 hours in a humidified incubator at 37°C supplied with 5% CO2. Reagents for assay were prepared fresh on the day of experiment according to the manual provided by the supplier. Briefly, 1mL of Fluo-4 DirectTM Calcium Assay buffer was added to 77 mg vial of water-soluble probenecid to prepare 250 mM stock probenecid. Probenecid inhibits the extrusion of calcium indicators out of the cells by organic anion transporters. This prevents the extracellular source of fluorescence that may interfere with the readings and reduces baseline signal. Mixture was vortexed and allowed to sit for 5 minutes to ensure the reagent was completely dissolved and then vortexed again. The excess assay buffer was aliquoted and together with stock probenecid was stored at -20°C. 2X Fluo-4 DirectTM Calcium reagent loading solution was prepared with final 39 Chapter 2: Materials And Methods concentration of probenecid 5 mM. Mixture was vortexed to ensure the reagent was comptely dissolved before loading cells. Cells which were near confluent had their media removed and rinsed once with assay buffer. Cells were then incubated in 50 µL assay buffer and 50 µL of 2X Fluo-4 DirectTM Calcium reagent loading solution at 37°C for 30 minutes and then at room temperature for another 30 minutes. Fluorescence was measured with Tecan plate reader for excitation at 495 nm and emission at 516 nm. The readings from 7 wells for each cell type either treated or non-treated were taken within the same plate and averaged. Assays were repeated five times (n=5) under the same conditions and data is expressed as mean± standard error of mean (sem). 2.4. Statistical analysis Data is expressed as mean+ standard error of mean. Statistical comparison of mean was performed by Student’s T-test. 40 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice 3.1. Introduction 3.1.1. Human apoE gene-knockin (apoE-KI) mice model for investigating effects of apoE isoforms on NMDAR changes during ageing. The apoE-knockin (apoE-KI) mouse model has been extensively studied to characterize their phenotypes especially in terms of behavioural performances, electrophysiology, excitatory neurotransmission and molecular changes in the brain, as summarized in the table 3.1. It has been reported that glutamatergic synapses of apoE4-KI mice are negatively affected which impairs hippocampal LTP (Trommer et al., 2004). Furthermore, altered composition of glutamatergic nerve terminals including reduced glutamatergic vesicular transporter (VGlut) and glutamate production have also been observed in apoE4-KI mice by others (Dumanis et al., 2013; Liraz et al., 2013). As a result, they exhibit abnormal behavioural phenotype including poor spatial memory retention although the rate of learning is unaffected (Bour et al., 2008; Grootendorst et al., 2005). Although this animal model does not develop hallmarks of AD including the plaques and tangles (Huang, 2011; Sullivan et al., 1997; Wang et al., 2005a), apoE4 has been associated with subtle neurological impairments without manifestation of overt neurological diseases (Greenwood et al., 2000; Reiman et al., 2001). ApoE4-KI mice have either simpler or altered neuronal structures in the hippocampus, cortex and amygdala (Andrews-Zwilling et al., 2010; Dumanis et al., 2009; Wang et al., 2005a). However, a recent study reported contrasting observation as Aβ accumulation could be detected at a higher level especially in the CA3 region of 2- to 4-month-old apoE4-KI mice (Liraz et al., 2013). 41 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice References Gender Age group Brain regions studied Findings Grootendorst et al. (2005) - Male Female - 4 to 5 months - NA - ApoE4 females showed impaired spatial recognition memory compared to apoE3 females, whereas males showed intact spatial recognition performance regardless of genotype. Leung et al. (2012) - Male Female - 16 months - NA - Aged apoE4 female mice showed spatial learning and (longterm) memory deficits, but male mice did not. Raber et al. (2000) - Male Female - 6 and 18 months - NA 18-month old female apoE4 mice (without hAPP/Aβ) showed impaired spatial memory retention and learning to locate the hidden platform - 6- and 18-month-old male apoE4 mice in (without hAPP/Aβ) did not differ significantly from age-matched male mice expressing apoE3, even in the more difficult water maze test. - spatial memory in female apoE4-KI mice was impaired based on their poor performances in i. the probe test of the water-maze reference memory task, ii. the water-maze working memory task and iii. an active avoidance Y-maze task. 1. Bour et al. (2008) - Male Female - 15 to 18 months - NA 42 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice AndrewsZwilling et al. 2010 - Female - 12- 21 months Siegel et al 2012 - Female - 6 to 8 months (young) 10 to 13 months (middleaged) 14 to 22 months (aged) - 1 and 4 months - - - Liraz et al. 2013 - Male - - Deficits in spatial learning and memory retention in apoE4 mice apparent at 16 months onwards. Cortex Hippocampus Amygdala - Spatial memory retention declines with age regardless of genotype. - NA Young apoE4 mice showed enhanced task learning in Morris water maze and passive avoidance test - Increased measures of anxiety in the elevated maze in young and middle-aged apoE4 mice. - ApoE expression level i. Higher in amygdala but not in hippocampus or cortex of young apoE3 compared to apoE4 mice. ii. Aged apoE3 mice have higher apoE expression level in all three regions examined compared to age-matched apoE4 mice. iii. Within genotype, apoE expression level decreases with age only in the amygdala of apoE3 mice. iv. ApoE level decreases with age in all three brain regions of apoE4 mice. Impairments in spatial memory of 4-month-old apoE4-KI mice. Hippocampus - Lower level of VGlut in the CA1, CA3 and DG of 4-month-old apoE4-KI mice than those of apoE3 mice. 43 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice - Mitochondrial abnormalities with increment in mitochondrial markers such as COX1 and Tom40 in CA3 neurons already apparent at 1 month of age in apoE4-TR mice. - ApoE expression level i. Increases with age but apoE level is always lower in apoE4-TR mice compared to apoE3 mice at 1 and 4 months of age. ii. Trommer et al 2004 Kitamura et al. 2004 - - Male Male - - 2 to 4 months 2 months 6 to 7 months - - Hippocampal DG Hippocampal CA1 Neuronal apoE expression increases with age in CA3 in both apoE3 and apoE4 mice. - ApoE isoforms do not alter baseline synaptic transmission - Reduced DG-LTP (perforant pathway) in apoE4-KI mice compared to apoE3 mice. Enhanced CA1-LTP (Schaeffer collateral pathway) in 2-month-old apoE4-KI mice compared to apoE3 mice but this effect disappears at 6 to 7 months. - No difference in distribution and density of GluR1-4 and NR1, NR2A/2B subunits in hippocampus of apoE3 and apoE4 mice. - 44 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice Korwel et a. 2009 - NA - 3 months - 1 year - - Hippocampal CA1 - 3 months i. Increased CA1-LTP induction in apoE4-KI mice. ii. Reduced NMDAR currents in apoE4-TR mice compared to apoE3 mice. iii. No difference in tyrosine phosphorylation and total expression of NR2A and NR2B between apoE3 and apoE4 mice. iv. Increased ERK1/2 phosphorylation in apoE4-KI mice. - 1 year i. No difference in apoER2 expression level in the hippocampal homogenate between apoE3 and apoE4 mice. Wang et al 2005 - Male - 7 months - Amygdala - ApoE4 mice displayed i. Reduced excitatory transmission. ii. Reduced dendritic arborization and length. iii. No sign of gliosis, amyloid deposition and neurofibrillary tangles. Dumanis et al. 2013 - Male - 4 to 5 months 1 year - Whole brain - Reduced soluble fraction of presynaptic VGlut but increased membrane-bound VGlut in apoE4 mice compared to apoE3 mice. - Altered composition of glutamatergic presynaptic nerve terminals in 4 to 5-month-old apoE4-TR mice with reduced - 45 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice glutaminase levels and glutamate production. Dumanis et al 2009 - NA - 1 month 3 months 1 year - Cortex Hippocampus - Age dependent effects of apoE4 on spine density in cortex but not hippocampus i. ApoE4 mice had significantly reduced spine density at all time-points and shorter spines at 1 year compared with apoE3 mice. Buttini et al 2002 - Male Female - 6 to 7 months 12 to 15 months 19 to 24 months - Neocortex Hippocampal DG - Age-related modulation of synaptic and cholinergic deficits by apoE4 isoform i. apoE4 mice showed reduced synaptophysinimmunoreactive (SYN-IR) presynaptic terminals and choline acetyltransferase (ChAT)-positive fibres in both neocortex and hippocampus compared to apoE3 mice. - ii. Table 3.1. Samples, brain regions of interest and findings of reviewed articles 46 loss of ChAT activity in apoE4 mice. Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice 3.1.2. Interactions between apoE and N-methyl-D-aspartate receptor (NMDAR) It has been discovered that unfavourable apoE genotype may promote an NMDAR hypofunctional state seen in both sporadic and familial AD (Olney et al., 1997). ApoE can modulate NMDAR functionality in vivo and in vitro. Primary neuronal-glial cultures and pluripotent embryonic carcinoma (P19) neuronal cell lines expressing NMDARs are sensitive to NMDA-induced excitotoxicity (Grant et al., 2001). Exogenous application of apoE is able to protect the neurons from NMDA-induced excitotoxicity partially resembling the action of NMDAR antagonist, [5R,10S]-[+]-5-methyl-10,11-dihydro-5Hdibenxo[a,d]cyclohepten-5,10-imine (MK801). In fact, this effect was observed either in the absence or presence of glia suggesting that apoE acts directly on neurons to exert neuroprotective effect (Aono et al., 2002). It has been hypothesized that apoE interacts with its receptors expressed on neuronal surface to modulate Ca2+ influx though NMDARs (Bacskai et al., 2000; Martin et al., 2008; May et al., 2004). Binding of apoE to its receptors facilitates NMDAR maturation (Sinagra et al., 2005) which increases NMDAR currents and activates downstream signalling pathways coupled to NMDARs (Qiu et al., 2006; Rebeck et al., 2006). Hence, apoE receptors can trigger neuronal signal transduction which further modulates synaptic plasticity, spine growth and survival as well as learning and memory (Beffert et al., 2006; Herz and Chen, 2006; Niu et al., 2008). These apoE receptors are able to affect the trafficking and processing of NMDARs via adaptor proteins such as disabled-protein 1 (Dab1) and PSD95 (Hoe et al., 2006; Martin et al., 2008). For instance, LRP1 regulates Ca2+ influx upon NMDA treatment in neurons (Qiu et al., 2002). Another apoE receptor, apoER2 has been shown to recruit Dab1 which stimulates Src family tyrosine kinases (SFKs) to phosphorylate tyrosine residues on NR2A and NR2B subunits of NMDAR (Bock and Herz, 2003). VLDLR- and apoER2knockout mice as well as mice with mutated PSD95 exhibit LTP and memory impairment (Mattar et al., 2005; Migaud et al., 1998; Weeber et al., 2002). Given that apoE4 mice have reduced LTP, apoE isoforms potentially modulate 47 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice NMDAR-induced LTP via interactions of apoE receptors and the intracellular adaptor proteins with NMDARs. On the other hand, apoE receptors can also act as co-receptors interacting with NMDAR through extracellular domain thus modulating biological activities of the latter (Beffert et al., 2005; Hoe et al., 2006; May et al., 2004). Interestingly, co-immunoprecipitation and immunohistochemistry studies show co-localization of LRP1, PSD95 and NMDAR (Martin et al., 2008; May et al., 2004). Hence, it is postulated that LRP1 is one of the components of the large postsynaptic density complex to enable its modulation of ion channel conductance. LRP1 is implicated in L-LTP due to its high affinity for Ca2+ which is crucial for receptor conformation and ligand recognition and degradation (Strickland et al., 1991; Williams et al., 1998). LRP1 binds to scaffolding PSD95 which also interacts with NMDAR thereby indicating modulation of NMDA-dependent Ca2+ influx by this receptor (Martin et al., 2008; May et al., 2004). LRP1 also regulates neurite outgrowth and migration by modulating neuronal calcium homeostasis and MAPK activation which in turn promotes cyclic AMP-responsive element-binding protein (CREB)mediated gene transcription (Postuma et al., 1998; Qiu et al., 2004). Indeed, elevated LRP1 expression can sustain ERK1/2 activity in neonatal dorsal root ganglion neurons (Yamauchi et al., 2013). Primary cortical neurons that are LRP1-deficient show decreased basal NMDA-induced phosphorylation of CREB, NMDA target gene transcription, and internalization of AMPAR which may lead to reduced dendritic branching (Nakajima et al., 2013). 3.1.3. Postsynaptic density (PSD) proteins/ NMDAR-associated proteins (NAPs) At the postsynaptic membranes of excitatory synapses, PDZ domaincontaining scaffold proteins such as PSD95, glutamate receptor interacting protein (GRIP), Shank and PICK1, combine with other adhesion molecules and cytoskeletal proteins that are structurally organized and spatially restricted in a large macromolecular signalling complex called postsynaptic density (PSD) (Kennedy, 1997; Kim and Sheng, 2004). NMDAR and AMPAR are 48 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice linked to actin cytoskeleton and intracellular signalling molecules in dendritic spines through this network of PSD proteins. All of these components orchestrate together to alter the synaptic structure during development, regulate receptor activity and localization, synaptic plasticity in learning and memory and neuronal dysfunction and death in neuropathology (Garner et al., 2000; Scannevin and Huganir, 2000; Sheng, 2001). NMDARs reside in synaptic, extrasynaptic and perisynaptic sites in neurons. They can be differentiated via immunohistochemistry and electrophysiological studies as synaptic NMDARs are activated by glutamate during low-frequency synaptic events whereas extrasynaptic NMDARs are not (Chen and Diamond, 2002; Clark and Cull-Candy, 2002; Harris and Pettit, 2007; Scimemi et al., 2004). Synaptic NMDARs are clustered in the PSD compartment of dendrites such that it is in closest proximity to the presynaptic terminals of axons to facilitate excitatory transmission. Some of the locations where extrasynaptic receptors reside are cell body, dendritic shaft and the neck of dendritic spine, while perisynaptic NMDARs are found at the area adjacent to PSD (Petralia et al 2010). Notably, NMDAR subtypes vary according to subcellular localization. In the adult CNS, diheteromeric NR1/NR2A and triheteromeric NR1/NR2A/NR2B receptors are abundant in synaptic sites whilst peri- and extrasynaptic sites mostly consist of NR2B-containing NMDARs (Gladding and Raymond, 2011; Hardingham and Bading, 2010). In neuronal cultures, there is a decrease in exchange between synaptic and extrasynaptic NMDARs during maturation as NR2A-containing NMDARs are less mobile than NR2Bcontaining NMDARs resulting in a decrease in the synaptic dwell time of NR2B subunits (Groc et al., 2006; Harris and Pettit, 2007; Köhr, 2007). However, other studies propose that both NR2A and NR2B subunits exist at extrasynaptic sites and the ratio of the two subtypes is similar to that of synaptic NMDARs (Mohrmann et al., 2000; Thomas et al., 2006). The C-terminal tails of NR2 subunits contain specific motifs that allow interactions with other cytoplasmic molecules which are implicated in receptor trafficking and signalling (Martel et al., 2012; Sanz-Clemente et al., 2013; Sprengel et al., 1998). For instance, both NR2A and NR2B subunits possess 49 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice PDZ-binding motifs that allow them to interact with membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins to couple NMDARs to intracellular signalling pathways (Kennedy, 2000). 3.1.3.2. PSD95 One of the more well-studied scaffolding proteins is PSD95 or PSD95synapse-associated-protein 90 (SAP90) which has multidomains that bind to glutamate receptors. This protein belongs to the MAGUK family and is highly expressed in excitatory synapses. Other members of the PSD-95 family include SAP97, PSD93/chapsyn-110 and SAP 102 (Kennedy, 1997). PSD95 has three PDZ or Discs-large homologous region (PDZ/DHR) domains namely PSD-95, disc-large (Dlg), zona occludens-1 (ZO-1); one src homology domain (SH3) and one guanylate kinase (GK) domain. The GK domain binds guanosine monophosphate (GMP) but has no kinase activity. Of all three PDZ domains, the second Dlg domain has the highest affinity for NR2 subunits whereas the third ZO-1 domain binds to neurolignin which recruits NMDARs (Irie et al., 1997). In vitro studies have demonstrated that NMDAR and PSD95 co-cluster in transfected cells and primary neurons (Kornau et al., 1995) to localize NMDARs to synapses in fibroblasts (Buller and Monaghan, 1997; Hoe et al., 2006). PSD95 modulates NMDAR trafficking to neuronal cell surface (Ling et al., 2012) and inhibits NR2-mediated internalization (Lavezzari et al., 2004; Roche et al., 2001) by linking apoER2 to NR2 subunits to regulate Ca2+ influx and LTP. The first PDZ (PDZ1) domain is associated with the intracellular domain of apoER2 whilst both PDZ1 and PDZ2 domains are able to interact with the C-termini of NR2 subunits (Niethammer et al., 1996) which is critical to the action of PSD95 on NMDAR clustering at synapses (Kim et al., 1996; Lin et al., 2004). Other functions of PSD95 include promoting maturation of excitatory synapses, reducing NMDAR desensitization and membrane targeting (El-Husseini et al., 2000; Feyissa et al., 2009). Co-immunoprecipitation of PSD95 with NR1, NR2B and α-actinin implicates its structural role in anchoring of NMDAR in synapses (Wyszynski et al., 1997). In addition, PSD95 and guanylate kinase-associated proteins (GKAPs) are clustered at postsynaptic sites opposite presynaptic 50 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice terminals to provide the scaffold before recruitment of NMDARs and AMPARs to the synapse (Rao et al., 1998). Disruption of the interaction between PSD95 and NMDAR or knocking down this protein will uncouple synaptic NMDARs from downstream signalling molecules such as neuronal nitric oxide synthase (nNOS) (Sattler et al., 1999). Collectively, PSD95 serves as an important link to facilitate the interaction between apoE receptors and NMDARs. 3.1.3.2. Calcium/calmodulin-dependent protein kinase II (CaMKII) The second messenger effects of Ca2+ are mostly mediated via Ca2+-sensing protein calmodulin (CaM) which undergoes a conformational change upon saturation with Ca2+ so that it can bind to target enzymes and trigger a variety of cellular responses (Hoeflich and Ikura, 2002). Although initial activation of CaM-kinases is dependent on binding of Ca2+/CaM, some can sustain their own activation either independently or via additional modifications such as phosphorylation by regulators. CaM-kinases have both catalytic and regulatory domains in which the latter is subdivided into autoinhibitory and CaM-binding domains. At basal Ca2+ levels, CaM-kinases remain dormant by autoinihibition which is relieved upon binding of Ca2+/CaM following an increase in intracellular Ca2+ concentration. CaM-kinases are classified as Ser/Thr kinases as the targeted phosphorylation sites of their substrates contain either a serine or threonine. The kinases that have multiple targets include CaMKK, CaMKI, CaMKII and CaMKIV; whilst others like CaMKIII and myosin light chain kinase only act upon a specific substrate. Among all the CaMK-kinases, CaMKII targets itself via a feedback mechanism involving NMDARs, AMPARs and L-type Ca2+ channels with its physiological role in regulating synaptic plasticity, spatial memory, ion channels and gene transcription (Giese et al., 1998; Swulius and Waxham, 2008). Alternative splicing gives rise to four isoforms of CaMKII: α, β, γ and δ (Hudmon and Schulman, 2002). The α and β isoforms are brain-specific and constitute up to 1% of total brain protein and 2% of hippocampal protein whilst the γ and δ isoforms are ubiquitously expressed in the body at a lower concentration (Erondu and Kennedy, 1985; Tobimatsu and Fujisawa, 1989). 51 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice NMDAR is one of the αCaMKII docking proteins first discovered (Gardoni et al., 1998; Strack and Colbran, 1998) and later a direct association is identified between αCaMKII and both NR2A and NR2B-containing NMDARs (Yan et al., 2011), though CaMKII has a higher affinity for NR2B compared to NR2A subunit (Gardoni et al., 1999; Leonard et al., 1999; Strack and Colbran, 1998; Strack et al., 2000). This indicates that αCaMKII plays a central role in mediating NMDAR-dependent synaptic plasticity as NMDAR activation causes extracellular Ca2+ influx and increases Ca2+/CaM which binds to αCaMKII. Subsequently, αCaMKII undergoes autophosporylation at Thr286 and translocates to postynaptic membrane to interact with NMDAR (Lisman et al., 2002; Merrill et al., 2005; Shen and Meyer, 1999). Autophosphorylation also increases the affinity of αCaMKII for Ca2+/CaM and triggers autonomous activity that allows the kinase to act beyond the period of transient Ca2+ influx (Meyer et al., 1992; Miller and Kennedy, 1986). Hence, αCaMKII is deemed the 'memory molecule' as autophosphorylation mechanism is required for learning and memory (Giese et al., 1998; Lisman and Zhabotinsky, 2001; Mullasseril et al., 2007). Prolonged activation of αCaMKII allows interaction with other substrates in close proximity especially AMPAR that is crucial for synapse maturation. Phosphorylation of GluR1 subunit by αCaMKII increases its channel conductance and further upregulates NMDAR-dependent Ca2+ influx (Benke et al., 1998; Derkach et al., 1999). In addition, CaMKII facilitates AMPAR accumulation at the synapse by incorporating new channels into the postsynaptic membrane (Hayashi et al., 2000; Li et al., 2004; Wang et al., 2011). It is proposed that CaMKII also plays a structural role by linking AMPAR to NMDAR via synapse-associated protein 97 (SAP97), band 4.1, F-actin and actinin (Lisman et al., 2002). Homozygous αCaMKIIknockout (αCaMKII-/-) mice have demonstrated LTD instead of LTP in controls when given a train of HFS. These transgenic mice also have unstable hippocampal place cells and deficient learning and retention of memory (Bach et al., 1995; Mayford et al., 1995). Similarly, mutation in autophosphorylation site of αCaMKII T286A leads to disrupted CA1-LTP and profound spatial learning deficits although the mutants neurotransmitter release (Giese et al., 1998). 52 have unaltered presynaptic Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice 3.1.4. Signalling pathways coupled to NMDAR 3.1.4.1. Protein kinase C (PKC) pathway PKCs exist in eleven homologous isozymes which are sub-classified into three subtypes: conventional PKCs (cPKCs: α, βI, βII and γ), novel PKCs (nPKCs: δ, ε, θ and η), and atypical PKCs (aPKCs: ζ, ι/λ) (Coussens et al., 1986; Ono et al., 1988; Ono et al., 1987; Parker et al., 1986). In general, PKCs consist of a regulatory domain at N-terminal and a catalytic domain at C-terminal end. The former contains an autoinhibitory domain (pseudosubstrate) and two membrane-targeting domains (C1 and C2), whilst the latter contains ATP and magnesium-binding motif (C3) as well as substrate-binding site (C4) (Freeley et al., 2011; Reyland, 2009; Rosse et al., 2010; Steinberg, 2008). The feature that distinguishes cPKCs from others is the presence of the calcium-binding C2 domain in the regulatory region. Hence cPKCs require calcium for activation in addition to diacylglycerol (DAG) and inositol-1,4,5-triphosphate (Ins(1,4,5)P3) which are second messengers generated by phospholipase C (PLC) upon simulation by activators such as neurotransmitters, growth factors, hormones, etc (Cole et al., 2008). The role of Ins(1,4,5)P3 is to trigger Ca2+ release from internal stores to increase the intracellular calcium concentration. Under certain circumstances, PKC activation does not require external stimulators as high cytosolic calcium level can directly activate PLC. Phosphorylation of the activation loop rapidly leads to autophosphorylation of the turn motif and hydrophobic site in the catalytic domain of PKCs, resulting in addition of a phosphate group from ATP to the Ser/Thr residue of target proteins (Chou et al., 1998; Le Good et al., 1998; Sonnenburg et al., 2001). Activated PKC is translocated from the cytosol to the plasma membrane and other organelles (Mochly-Rosen, 1995). The selective anchoring of the isozymes to different subcellular locations enables specific phosphorylations of nearby substrates by activated PKCs triggering numerous downstream signalling pathways. 53 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice PKC isozymes play diverse roles in both normal and diseased states including regulation of cell proliferation and cell death, increased gene transcription and translation, and modulation of receptors and ion channels (Chen et al., 2001; Murriel and Mochly-Rosen, 2003). Notably, even the same isozyme can have contrasting effects in the same cell depending on the context of stimulation (Basu and Pal, 2010). PKC activators have been found to have neuroprotective effects in animals as they can induce synaptic maturation, increase neurotrophin levels, reduce beta-amyloid levels and improve learning (Alkon et al., 2007; Bonini et al., 2007; Hongpaisan and Alkon, 2007); whereas PKC inhibition causes reduced spatial learning (Jiang et al., 2011b). In fact, abnormal changes in PKC signalling cascade have been observed in AD patients during initial stages of the disease (Favit et al., 1998). The ability of PKC in induction of LTP comes from its modulation of cellular localization of NMDARs. PKCα isozyme phosphorylates the serine residue of NR1 subunit at position 896 (Sánchez-Pérez and Felipo, 2005) to influence NMDAR functionality (Scott et al., 2001; Tingley et al., 1997). In addition, PKC promotes NMDAR trafficking to the cell surface via interaction with NAPs such as PSD95 and CaMKII. PKC enhances Src-dependent phosphorylation of NR2 subunits which strengthens the binding of PSD95 to C-terminal tails of NMDAR and stabilizes the docking of the receptors at postsynaptic sites (Lan et al., 2001; Lu et al., 1999; Rong et al., 2001; Salter and Kalia, 2004; Zheng et al., 2006). Activated PKC also indirectly potentiates CaMKII which increases its association with NR2 subunits followed by cotranslocation in the form of NR2-CaMKII complex and ultimately NMDA channel insertion into postsynaptic membrane (Yan et al., 2011). Besides interaction with NMDARs, PKC can also induce LTP mediated via AMPAR activity possibly by phosphorylating GluR1 subunit of AMPAR. In fact, this interaction is abolished by application of CaMKII antagonist suggesting a complex interplay between NMDARs, AMPARs and CaMKII during LTP event (Yan et al., 2011). Furthermore, PKC interacts with either Ras or Raf-1, a MAPK kinase kinase (MAPKKK), both of which are upstream 54 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice regulators of ERK signalling cascade (Sweatt, 2001). These are all involved in memory and synaptic plasticity as mentioned previously (§ 1.3.3). 3.1.4.2. Protein kinase A (PKA) pathway The PKA family expressed in the brain consists of four regulatory subunits: RIα, RIβ, RIIα, and RIIβ; and three catalytic subunits: Cα, Cβ, and Cγ (Cadd and McKnight, 1989; McKnight et al., 1988). PKA activation is initiated by Ca2+ entry through NMDAR (Roberson and Sweatt, 1996) causing an increase in intracellular Ca2+/CaM. This in turn stimulates Ca2+/CaM sensitive adenylyl cyclases (ACs) particularly AC1 and AC8 in the hippocampus (Wong et al., 1999) which act as a second messenger to produce cAMP (Eliot et al., 1989). This results in the dissociation of PKA catalytic subunit (PKA-C) from the regulatory subunit (PKA-R) (Gibbs et al., 1992; Taylor et al., 1990; Wang et al., 1991). Activated PKA-C further phosphorylates serine and threonine residues of several substrates including NMDAR, AMPAR and CREB to modulate the functions of these receptors (Feliciello et al., 1999; Snyder et al., 2005; Snyder et al., 1998; Tingley et al., 1997; Westphal et al., 1999). In addition, PKA stimulates B-Raf which is a MAPKKK that triggers downstream ERK pathway (Vossler et al., 1997). In vitro work shows that GluR1 subunit of AMPAR can be phosphorylated by PKA-Cα at Ser845 (Blackstone et al., 1994; Mammen et al., 1997; Pearson and Kemp, 1991; Roche et al., 1996) resulting in potentiation of AMPA channels (Keller et al., 1992); Roche et al., 1996) by modifying the current amplitude, number of active channels, receptor desensitization and channel opening rate (Banke et al., 2000). There is evidence that the PKA pathway is required for formation of L-LTP and long-term memory. In vitro activation of PKA by Sp-cAMPS, a phosphodiesterase-resistant activator, directly enhances excitatory synaptic currents in hippocampal neurons. Application of PKA-C also potentiates AMPA whole-cell currents in neurons and increases the opening frequency of these channels (Greengard et al., 1991; Wang et al., 1991). AMPAR synthesis and incorporation into synapses are 55 mediated via PKA-dependent Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice phosphorylation of GluR1 (Esteban et al., 2003; Nayak et al., 1998). Furthermore, in vivo overexpression of dominant negative mutant of PKA-RIα, Rα (AB), which acts as an inhibitor of PKA-C in mice (Clegg et al., 1987; Ginty et al., 1991), causes a 40 percent decrease in PKA activity and subsequently decreases L-LTP without affecting E-LTP (Abel et al., 1997). Other consequences include severely impaired long-term memory and long term place cell stability (Rotenberg et al., 2000; Woo et al., 2000; Woo and Nguyen, 2002). Double mutant mice lacking both AC1 and AC8 (ΔAC1/ΔAC8) exhibit profound L-LTP and long term memory deficits which can be rescued by forskolin, an activator of all ACs, suggesting that the downstream signalling cascade of L-LTP is still intact in these mutants (Wong et al., 1999). Clinical studies have demonstrated a downregulation of AC/cAMP/PKA signalling pathway in AD brains which may be implicated in the aetiology of neurofibrillary tangles and hyperphosphorylation of tau (Bonkale et al., 1999; García-Jiménez et al., 1999; Hanger et al., 2009; Liang et al., 2007; Shi et al., 2011). This further decreases the activity of one of its downstream target, CREB which may contribute to the impaired synaptic plasticity and memory decline in AD (Scott Bitner, 2012). Expression of PKA-Cα is ubiquitous (Cadd and McKnight, 1989) and infusion of this subunit into CA1 pyramidal neurons induces persistent synaptic potentiation (Duffy and Nguyen, 2003). However, phosphorylation of postsynaptic proteins by PKA-Cα alone is not sufficient to produce L-LTP suggesting that additional events not necessarily linked to cAMP/PKA signalling pathway may be required for formation of electrically induced LLTP (Nguyen and Woo, 2003). 3.1.4.3. Ras/mitogen-activated protein kinase (MAPK) pathway MAPK/ERK pathway starts with Ras signalling which is regulated by Guanine nucleotide Releasing Factors (GRFs) found primarily in PSD (Shou et al., 1992). GRFs promote the active GTP-bound Ras state and couple the latter to 56 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice Ca2+/CaM, cAMP, PKC, G protein and receptor tyrosine kinase signalling. This triggers the downstream activation of MAPKKKs, B-Raf or Raf-1, found abundantly in the hippocampus (Barnier et al., 1995; Kim et al., 1990; Morice et al., 1999), and subsequently MAPK/ERK kinase (MEK) which in turn phosphorylates MAPK (Takai et al., 2001). This signalling cascade has been demonstrated in neuronal culture and hippocampal slices (Grewal et al., 2000; Morozov et al., 2003) and signifies a point of convergence for cell surface signals regulating cell adhesion, migration, survival, differentiation, metabolism, proliferation, transcription and cell cycle progression (Chang and Karin, 2001; Roskoski, 2012). ERK1 and ERK2 are members of the MAPK family that are heavily expressed in brain especially in post-mitotic neurons (Boulton and Cobb, 1991; Ortiz et al., 1995) and both isoforms share similar functions (Lefloch et al., 2009; Lloyd, 2006). Phosphorylation of human ERK1/2 by dual specificity MEK1/2 at tyrosine (Tyr204/187) followed by threonine (Thr202/185) residues are essential for enzyme activation (Ferrell and Bhatt, 1997). Ablation of Erk2 gene is embryonic lethal (Yao et al., 2003) and although ERK1 is dispensable for development, it has been shown that the latter is required for formation of synaptic plasticity and learning and memory (Mazzucchelli et al., 2002). Using homozygous ERK1-/- mice, dopaminergic stimulation could increase phosphorylation of ERK2 and enhance LTP and memory in active passive avoidance test. Erk gene dosage is crucial for mouse survival (Lefloch et al., 2008) and expression of ERK2 exceeds that of ERK1. Hence, the severe effect of knocking out ERK2 is due to the diminished total ERK content rather than its unique biological function. The involvement of Ras/ERK cascade in LTP and hippocampal-dependent learning is accentuated when ERK is phosphorylated following LTP-inducing stimuli, contextual fear conditioning and water maze training (Davis et al., 2000; Dudek and Fields, 2002). Increased nuclear translocation of activated ERK is also observed in stimulated cells and dendrites concurrently with LTP induction (Patterson et al., 2001; Rosenblum et al., 2002; Yuan et al., 2002). Conversely, Ras-GRF1 knockout mice exhibit impaired learning (Giese et al., 57 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice 2001) and MEK inhibitors which block ERK phosphorylation and memory recall after water maze learning suggesting that MAPK activation is necessary for memory consolidation (Atkins et al., 1998; Berman et al., 1998; Davis et al., 2000; English and Sweatt, 1997; Sgambato et al., 1998). These effects of Ras/ERK signalling on learning and memory are largely due to its influences on neuronal gene expression, particularly activation of CREB transcription factor (Adams and Sweatt, 2002; Impey et al., 2002; Lonze and Ginty, 2002; Xia et al., 1996). In addition, sustained ERK1/2 signalling is required for neuronal differentiation (Desbarats et al., 2003; Traverse et al., 1994) which may contribute to synaptic plasticity. Classical LTP occurring at CA3 to CA1 synapses require NMDAR activation to allow Ca2+ entry which in turn phosphorylates ERK1/2 (Thomas and Huganir, 2004; Xia et al., 1996). This hypothesis is supported by in vitro studies showing that activation of ERK2 in cortical and hippocampal neuronal culture can be mediated via NMDAR (Bading and Greenberg, 1991; Fiore et al., 1993; Kurino et al., 1995). NMDA application to hippocampal slices isolated from wild type mice also increases ERK phosphorylation by threefold (Tian et al., 2004). In vivo, ERK phosphorylation are coupled to Ca2+dependent upstream activators including PKC and PKA in hippocampus and mouse dorsal horn (Ji et al., 2009). Notably, Ca2+ influx through other calcium permeable channels such as AMPARs and voltage-gated calcium channels are also capable of activating ERK1/2. Exogenous AMPA application in cultured neurons activated MAPK which can be inhibited by lack of extracellular calcium (Wang and Durkin, 1995). This is possibly mediated via phosphatidylinositol 3-kinases (PI3K) and CaMK which are Ca2+-sensitive protein kinases (Perkinton et al., 2002; Perkinton et al., 1999). Other regulators of ERK activation which trigger intracellular Ca2+ release from endoplasmic reticulum are mGluRs, mAChRs, dopaminergic receptors and adrenergic receptors. 58 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice 3.1.5. CREB in learning and memory Gene transcription and protein synthesis are the two major events that occur during formation of L-LTP (Frey et al., 1988; Nguyen et al., 1994; Stanton and Sarvey, 1984). Signalling pathways activated by Ca2+ influx via NMDAR channels including αCaMKII, PKA, PKC and Ras/MAPK pathways, all converge in a common downstream target which is cAMP-responsive element binding protein (CREB) (Ahn et al., 2000; Mao et al., 2007; Matynia et al., 2002). CREB is a nuclear transcription factor that promotes transcription of genes containing the cAMP responsive elements (CRE) in the promoter region (Berkowitz et al., 1989; Sheng et al., 1990) some of which are c-fos, B-cell lymphoma 2 (Bcl2), brain-derived neurotrophic factor (BDNF), etc (Riccio et al., 1999; Shieh et al., 1998; Tao et al., 1998). It regulates cell division and proliferation, confers neuroprotective effect and increases spinogenesis (Mantamadiotis et al., 2002; Marie et al., 2005; Vitolo et al., 2002). In response to elevated Ca2+ and cAMP level, CREB is phosphorylated at serine 133 (S133) which results in binding of transcription coactivators CREBbinding protein and p300 to CREB, thus initiating CRE-mediated transcription. Although CREB can be phosphorylated at other sites such as Ser129, Ser142 and Ser143 (Finkbeiner, 2001), the function of CREB is regulated predominantly by its phosphorylation at Ser133 where multiple kinases can bind (Bollen and Prickaerts, 2012; Meyer and Habener, 1993; Montminy et al., 1990). CREB has been shown to be activated during behavioural training and memory consolidation (Hall et al., 2001). Studies in Aplysia and Drosophila have provided evidence that CREB facilitates long-lasting forms of synaptic plasticity and long-term memory (Bartsch et al., 1995; Dash et al., 1990; Martin et al., 1997; Tully, 1991; Yin et al., 1994). Mice with deleted specific CREB isoforms exhibit impaired CA1-LTP which correlates with poor longterm spatial memory and contextual fear conditioning (Bourtchuladze et al., 1994; Hummler et al., 1994). This may be caused by disruption in maintenance of AMPARs in PSD and lowered AMPAR-mediated miniature EPSC (mEPSC) frequency (Middei et al., 2013). In contrast, overexpression of 59 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice CREB in mice enhance plasticity and learning implying that the level of this transcription factor is positively related to synaptic changes (Barco et al., 2002). This is probably due to increased NMDAR transmission and the surface levels of NMDAR subunits (Huang et al., 2008; Marie et al., 2005). ERK, one of the upstream regulators of CREB, is required for sustaining its activation although an initial increase in CREB phosphorylation is mediated by CaMKIV (Hardingham et al., 2001; Wu et al., 2001). Application of MEK inhibitor to hippocampal slices can attenuate CREB phosphorylation (Roberson et al., 1999) supporting the view of the crucial role MAPK has in driving CREB signalling which in turn facilitates expression of hippocampal L-LTP (English and Sweatt, 1996; Impey et al., 1998; Kanterewicz et al., 2000). This regulation of CREB by ERK is mediated indirectly via ribosomal protein S6 kinases (RSKs) 1-3 (Sweatt, 2001). 60 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice Figure 3.1. Schematic diagram of the interactions between apoE and NAPs, and NMDAR-coupled signalling cascades. A model that shows the link between apoE and NMDAR; NMDAR and AMPAR; and NMDAR-coupled signalling cascades. ApoE binds to apoE receptor, LRP1, and acts indirectly to influence the NMDAR functionality via NAP such as PSD95. LRP1 also regulates NMDAR-mediated Ca2+ influx. NMDAR and AMPAR are able to influence each other at the postsynaptic membrane as AMPAR-mediated Na+ influx helps to relieve NMDA channel of its Mg2+ block. Enhanced NMDAR functionality results in activation of Ca2+/CaM and the ‘memory’ molecule, αCaMKII autophosphorylation. Increased Ca2+ influx also stimulates other Ca2+-sensitive protein kinases such as PKC and PKA and their downstream targets. PKC and αCaMKII shares a common phosphorylation site on GluR1 of AMPA channel which increases the ionic conductance of AMPA channel. PKA signalling involves phosphorylation of serine residues on NR1 and GluR1 subunit by PKA. This in turn also enhances NMDAR and AMPAR functionality. 3.2. Objectives of study It is well-known that ageing induces memory decline caused by impaired synaptic plasticity. One of the underlying mechanisms is altered activity and/or expression of NMDARs which is more vulnerable to ageing and insults than other glutamate receptors. ApoE is one of the most prominent genetic risk 61 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice factors for sporadic AD cases affecting cognition. To date, most of the studies that associate apoE with AD pathology involve amyloidopathy and tau phosphorylation. However, not much is known about the Aβ-independent effects of apoE isoforms on NMDAR functionality during ageing. We hypothesize that specific apoE isoforms alter NMDAR signalling by modifying its expression and activation in a subregion-specific manner. As NMDAR-dependent Ca2+ influx activates PKA and PKC pathway, we postulate that apoE exerts isoform-specific effects on NMDAR signalling via PKC and PKA to further regulate the ERK-CREB pathway in ageing animals. To address this issue, I utilized human apoE-targeted replacement (huApoETR) or apoE-knockin (huApoE-KI) mice model which has been known to manifest the detrimental effects of apoE4 particularly in learning and memory. The aims and rationales for this study are as follows: Aim 1: To investigate the differential effects of human apoE isoforms on the region-specific expression of NMDAR and its activities in ageing animals. Controversial findings have been observed in human apoE4-knockin (huApoE4-KI) mice with respect to synaptic plasticity and their performance in learning and memory. ApoE4 modification of NMDAR-induced LTP and cognition is found to be age-dependent as young apoE4-KI mice display enhanced LTP and learning (Kitamura et al., 2004; Korwek et al., 2009; Siegel et al., 2012). However, this effect disappears as they age (Bour et al., 2008; Grootendorst et al., 2005; Kitamura et al., 2004). Similarly, human studies have shown some beneficial effects of apoE4 in early life which are not sustained at old age (Tuminello and Han, 2011). It is unclear whether this effect is due to predisposition of apoE4 carriers to altered NMDAR signalling especially at old age. In either case, the molecular pathway accountable for these electrophysiological and behavioural differences has not been deciphered. Hence, we would like to determine in vivo if apoE4 downregulates NR1, NR2A and NR2B subunit activity and expression level during ageing. Aim 2: To elucidate the molecular mechanism by which apoE signalling alters NMDAR transduction. 62 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice One of the signalling pathways mediated by apoE is the extracellular signalregulated kinase 1/2 (ERK1/2) signalling cascade upon binding of apoE to LRP1 receptor. In vitro, NMDAR antagonist, MK801, could block ERK activation implying that regulation of ERK1/2 activity is dependent on Ca2+ influx through NMDAR. This eventually leads to CREB activation which is required for neurite outgrowth, initiation and extension (Hoe et al., 2005). Due to its high Ca2+ permeability, NMDAR activity influences calcium-sensitive protein kinases such as CaMKII, PKC and PKA which ultimately converge in ERK-CREB signalling. Hence, we seek to dissect the changes in the molecules involved downstream of these signalling pathways. 3.3. Results My study utilizes apoE-knockin (apoE-KI) mice in which murine apoE gene has been replaced by human apoE gene leaving the regulatory loop intact such that they express human apoE isoforms under the control of the endogenous murine promoter (Wang et al., 2005a). This gives an expression pattern that recapitulates that of non-demented humans in a temporal and spatial manner (Sullivan et al., 2004) compared to NSE- or GFAP-apoE mice models which specifically express apoE in neurons and astrocytes respectively. There is also a high degree of conservation between murine and human apoE receptors (Kim et al., 1996) which facilitates binding of human apoE to endogenous receptors. It is noticeable that mouse apoE is structurally and functionally similar to human apoE3 isoform (Raffai et al., 2001; Weisgraber, 1994). This can be observed at the morphological, electrophysiological and behavioural levels whereby the effects of knocking in human apoE3 gene into murine apoEknockout mice model help to preserve the neuronal sprouting, LTP and performances in Morris water maze such that these phenotypes are similar to wild type controls (Andrews-Zwilling et al., 2010; Kitamura et al., 2004; Trommer et al., 2004; Veinbergs et al., 1999). Although another apoE isoform, apoE2, is protective against the effects of ageing on cognitive impairment 63 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice (Corder et al., 1994; Corder et al., 1993; Strittmatter et al., 1993), the allelic frequency in the human population is the lowest among the three apoE variants (Hallman et al., 1991). On the other hand, apoE3 is the most common isoform in human population that gives a normal phenotype as it can only delay the age of onset of cognitive impairment but does not further exacerbate the neurological defects of ageing (Davignon et al., 1988; Mahley, 1988; Poirier et al., 1993; Saunders et al., 1993). Hence, I decided to compare the apoE4-KI mice against age-matched apoE3-KI mice as my controls to investigate the pathological effects of apoE4 after the normalization of ageing effect. When comparisons were made between singly transgenic apoE-KI mice and bigenic mice carrying additional hAPP mutation which produces Aβ, it was discovered that apoE4 isoform alone is sufficient to induce impaired spatial learning and memory in 18-month-old female apoE4 mice (without hAPP/Aβ) per se but not in singly transgenic 6- and 18-month-old male apoE4 mice (Raber et al., 2000). The gender-specific influence of apoE4 isoform on cognition may have started early as 4 to 5-month-old females showed impaired spatial recognition memory retention compared to age-matched apoE3 females whereas males have intact spatial recognition regardless of genetic modification (Grootendorst et al., 2005). In addition, aged (16-monthold) female rather than male apoE4-KI mice made more errors in avoidanceconditioning tasks and water maze test (Leung et al., 2012). Moreover, there has been an interesting report on abnormal cortical electroencephalogram (EEG) and seizure phenotype demonstrated by apoE4-KI mice (Hunter et al., 2012). Taken together, these studies strongly support the idea that apoE isoforms affect brain structures and functions even in the absence of conventional AD pathologies, and the effects of apoE4 on cognitive function and behaviour are modulated by gender as well as age. In view of these reports, I decided to focus on effects of apoE3 and apoE4 isoforms in female mice at three time points i.e. week 12 (3-month-old, young), 32 (8-month-old, middleaged) and 72 (18-month-old, aged), to determine whether the detrimental effects of apoE4 on NMDAR-induced signalling pathways occurred even 64 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice before the appearance of abnormal behavioural phenotypes such as impaired memory in 4-month-old female apoE4-TR mice (Grootendorst et al., 2005). 3.3.1. Expression of human apoE in brains of huApoE-knockin (apoE-KI) mice across three time points Figure 3.2. Expression level of huApoE in brains of apoE-KI mice models across three time points i.e. 12, 32 and 72 weeks. Western blot analysis of human apoE expression in huApoE3 (white bar) and huApoE4 (grey bar) mice with β-actin as loading control. Densitometry of huApoE relative to β-actin was performed using NIH ImageJ software. Quantitation of the results was performed using NIH ImageJ software and the relative value for apoE4-KI mice was normalized against age-matched apoE3KI mice. Each value for apoE4-KI mice represents mean value ± SEM relative to apoE3-KI mice for individual mouse brain sample (n=5) at each time point. For statistical analysis, Student's t-test was used to test for significance (** p < 0.01, *** p < 0.001). The first thing I looked at was the apoE expression level in the brains of huApoE-KI mice models as there has been controversial findings about the apoE expression level in these mice whereby some studies indicate a difference between the apoE expression level between apoE3- and apoE4-KI mice (Ramaswamy et al., 2005; Riddell et al., 2008) whereas others do not (Siegel et al., 2012; Sullivan et al., 2004). Overall, figure 3.2 shows that there is a significantly lower level of huApoE expression level for female apoE4-KI mice at week 12 (-37%), 32 (-29%) and 72 (-24%). The marked decreased expression level of apoE4 at all time-points 65 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice suggest the basal difference in these two mice models that can contribute to the subsequent observations. This indicates that there may be insufficient apoE expressed in the apoE4-KI mice to regulate cholesterol transport into neurons for maintenance of neuronal health. 3.3.2. Expression of apoE receptor and PSD95 in brains of huApoE-KI mice across three time points Figure 3.3. Protein expression level of LRP1 and PSD95 in brains of apoE-KI mice models across three time points i.e. 12, 32 and 72 weeks. Western blot analysis of (A) LRP1 and (B) PSD95 expression in huApoE3 (white bar) and huApoE4 (grey bar) mice with β-actin as loading control. Densitometry of the respective protein relative to β-actin is shown on the right panel. Quantitation of the results was performed using NIH ImageJ software and the relative value for apoE4-KI mice was normalized against age-matched apoE3-KI mice. Each value for apoE4-KI mice represents mean value ± SEM relative to apoE3-KI mice for individual mouse brain sample (n=5) at each time point. For statistical analysis, Student's t-test was used to test for significance (* p < 0.05, ** p < 0.01, *** p < 0.001). 66 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice As apoE binds to its major receptor, LRP1, to induce neuronal signalling cascades, I proceeded to look at this receptor to see whether there is a similar change in the expression level of LRP1 in apoE-KI mice. Figure 3.3A shows a reduced apoE receptor, LRP1, expression level in apoE4KI mice at all time-points i.e. week 12 (-27%), 32 (-27%) and 72 (-36%). This implies that there may be less LRP1 available for binding to apoE to trigger downstream signalling pathways in apoE4-KI mice. One of the functions of LRP1 is to support synaptic integrity (Yoon et al., 2013), thus I also looked the postsynaptic marker, PSD95, to see whether there is any synaptic alteration in apoE4-KI mice. Immunoblotting shows that the reduction of PSD95 level in apoE4-KI mice mirrors the downregulation of activity of NR2A at all ages. There is a significantly lower level of PSD95 expression at week 12 (-23%), 32 (-38%) and 72 (-31%) (Figure 3.3B). This implies that there is indeed synaptic alterations since PSD95 is a scaffolding protein that helps to stabilise synapses (Kennedy, 1997). In addition, this may pose a detrimental effect upon glutamatergic synapses of apoE4-KI mice as PSD95 is required for intracellular trafficking and clustering of NMDARs at synapses (Kim et al., 1996; Lin et al., 2004; Ling et al., 2012). 67 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice 3.3.3. NMDAR subunits phosphorylation profile in brains of huApoE-KI mice across three time points Figure 3.4. Phosphorylation status of NMDAR subunits in brains of apoE-KI mice models across three time points i.e. 12, 32 and 72 weeks. Western blot analysis of (A) phosphorylated NR1 at serine residues (p-NR1 S896 and p-NR1 S897) and NR1; (B) phosphorylated NR2A (p-NR2A) and 68 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice NR2A; (C) phosphorylated NR2B (p-NR2B) expression in huApoE3 (white bar) and huApoE4 (grey bar) mice with β-actin as loading control. Densitometry of p-NR1 relative to NR1, p-NR2A relative to NR2A and pNR2B relative to NR2B shown on the right panel was performed using NIH ImageJ software. The relative value for apoE4-KI mice was normalized against age-matched apoE3-KI mice. Each value for apoE4-KI mice represents mean value ± SEM relative to apoE3-KI mice for individual mouse brain sample (n=3-5) at each time point. For statistical analysis, Student's t-test was used to test for significance (* p < 0.05, ** p < 0.01, *** p < 0.001). NMDAR is enriched at postsynaptic site and has been found to interact with PSD95 (Kornau et al., 1995; Wyszynski et al., 1997). Thus I wanted to know whether the NMDAR signalling corresponds with PSD95 expression level in apoE4-KI mice. As NMDAR signalling is largely regulated by phosphorylation mechanism (Bashir et al., 1991; Bliss and Lomo, 1973; Madison et al., 1991), and apoE4 isoform has been found to interfere with phosphorylation of glutamate receptor to influence the synaptic stability (Chen et al., 2010), I went on to examine the phosphorylation status of NMDAR in apoE-KI mice as it is one of the main postsynaptic glutamate receptors. Surprisingly, not all the subunits exhibit a similar phosphorylation profile in apoE4-KI mice suggesting that the modification of the NMDAR is subunitspecific and may be regulated by different upstream mediators. There is a significant 2.5 fold increase in phosphorylation of NR1 subunit at residue serine 896 (S896) in young apoE4-KI mice at week 12 compared to agematched apoE3-KI mice. This is unexpected as phosphorylation of NR1 at serine residues increases NMDAR surface expression and synaptic transmission (Tingley et al., 1997; Zou et al., 2000). However, no difference is observed between both mouse lines at week 32. In contrast, there is a significant reduction in phosphorylation of NR1 subunit (-40%) at old age i.e. week 72 (Figure 3.4A). On the other hand, there is no difference in the phosphorylation of NR1 subunit at serine residue 897 implying that there are different factors modulating the phosphorylation of serine residues even if they are on the same subunit. Taken together, this enhanced phosphorylation of NR1 S896 at week 12 suggests a possible beneficial effect of apoE4 at early stage of life but this effect disappears with ageing. 69 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice Figure 3.4B shows that phosphorylation of NR2A subunit is significantly reduced in apoE4-KI mice at all three time points i.e. week 12 (-65%), 32 (32%) and 72 (-51%). On the other hand, activity of NR2B remains relatively constant between the two mouse models during ageing (Figure 3.4C). Hence, apoE4-KI mice may have less NMDAR surface expression as tyrosine phosphorylation of NR2A reduces endocytosis of NMDARs and enhances ion channel open probability. In conclusion, only NR2A phosphorylation subunit seems to mirror that of apoE, LRP1 and PSD95. Although NR2A is not a direct substrate of these proteins, these proteins may modulate the phosphorylation of NR2A via other intracellular signalling molecules. 70 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice 3.3.4. PKA and PKC signalling profile in brains of huApoE-KI mice across three time points Figure 3.5. Protein expression level of PKA-Cα and PKCα in brains of apoE-KI mice models across three time points i.e. 12, 32 and 72 weeks. Western blot analysis of (A) phosphorylated PKA-Cα (p-PKA-Cα) and PKACα; (B) phosphorylated PKCα (p-PKCα) and PKCα in huApoE3 (white bar) and huApoE4 (grey bar) mice with β-actin as loading control. Densitometry of (A) p-PKA-Cα relative to PKA-Cα; (B) p-PKCα relative to PKCα and (C) pαCaMKII relative to αCaMKII was performed using NIH ImageJ software and the relative value for apoE4-KI mice was normalized against age-matched apoE3-KI mice. Each value for apoE4-KI mice represents mean value ± SEM relative to apoE3-KI mice for individual mouse brain sample (n=3-5) at each time point. For statistical analysis, Student's t-test was used to test for significance (* p < 0.05, *** p < 0.001). NMDAR activity is coupled to intracellular calcium-sensitive kinases such as PKC and PKA (Roberson and Sweatt, 1996). The activation of these kinases results in a positive mechanism whereby they enhance the NMDARdependent Ca2+ influx (Scott et al., 2001; Tingley et al., 1997) which leads to synaptic plasticity and the formation of learning and memory (Alkon et al., 2007; Hongpaisan and Alkon, 2007). In particular, PKC and PKA have been 71 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice shown to phosphorylate NR1 at serine 896 and 897 respectively. To find out the upstream signalling molecules that is responsible for NR1 phosphorylation, I did some immunoblotting experiments to determine their level of expression and activity status. There is no difference between the total brain lysates from two different mouse models in PKA-Cα activity at all three time-points (Figure 3.5A). In contrast, there is a significant increase in PKCα phosphorylation in apoE4-KI mice at week 12 (49%) but no difference compared to apoE3-KI mice at week 32, followed by a significant decrease at week 72 (-28%) (Figure 3.5B). This coincides with the earlier results that NR1 S896, downstream of PKCα, is also upregulated and then downregulated in apoE4-KI mice at young and old age respectively (Figure 3.5A). Similarly, there is also no change in NR1 S897 which is a downstream target of PKA-Cα (Figure 3.5A, lower panel). The corresponding phosphorylation profile between NR1 and the protein kinases strongly suggests that phosphorylation of the serine residues on NR1 subunit is regulated by their respective protein kinases. Taken together, NR1 and PKC phosphorylation is differentially regulated by apoE isoforms at different ages as opposite trends are observed in the two mouse lines at week 12 and 72. Since both molecules are implicated in synaptic plasticity (Matynia et al., 2002), the downregulation of NR1 and PKC phosphorylation may underlie the cognitive deficits demonstrated by apoE4KI mice especially at old age (Grootendorst et al., 2005; Leung et al., 2012; Raber et al., 2000). On the other hand, the unchanged PKA phosphorylation suggests that this kinase is not implicated in the pathogenic effects of apoE4. 72 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice 3.3.5. GluR1 and αCaMKII expression profile in brains of huApoE-KI mice across three time points Figure 3.6. Protein expression level of GluR1 and αCaMKII in brains of apoE-KI mice models across three time points i.e. 12, 32 and 72 weeks. Western blot analysis of phosphorylated serine residues of GluR1 subunit. (A) Phosphorylated GluR1 S845 (p-GluR1 S845), (B) phosphorylated GluR1 S831 (p-GluR1 S831) and (C) phosphorylated αCaMKII (p-αCaMKII) and αCaMKII of huApoE3 (white bar) and huApoE4 (grey bar) mice with β-actin as loading control. Densitometry of (A) p-GluR1 S845 relative to GluR1 and (B) p-GluR1 S831 relative to GluR1 performed using NIH ImageJ software is shown on the right panel. The relative value for apoE4-KI mice was normalized against age-matched apoE3-KI mice. Each value for apoE4-KI mice represents mean value ± SEM relative to apoE3-KI mice for individual mouse brain sample (n=5) at each time point. For statistical analysis, Student's t-test was used to test for significance (* p < 0.05, ** p < 0.01). 73 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice AMPAR is another ionotropic glutamate receptor which is tightly associated with NMDAR at the postsynaptic membrane as its activation enhances NMDAR-dependent Ca2+ influx (Bashir et al., 1991; Grosshans et al., 2002; Watt et al., 2004). GluR1 subunit of AMPAR is the major subunit being expressed in functional AMPAR which also influences the channel activation by phosphorylation mechanism (Lu et al., 2009b). Two major phosphorylation sites on GluR1 subunit i.e. serine 831 and 845 are regulated by PKC and PKA respectively to enhance the channel functionality (Banke et al., 2000; Esteban et al., 2003; Hayashi et al., 2000; Roche et al., 1996). In particular, phosphorylation of these serine residues leads to increased channel open probability and stabilization of AMPARs at the glutamatergic synapses. αCaMKII is another Ca2+-sensitive kinase that shares a common target as PKC i.e. GluR1 S831 to enhance the AMPA channel conductance (Bredt and Nicoll, 2003). Hence, I wanted to investigate whether there is a differential effect on GluR1 subunit and αCaMKII phosphorylation in apoE-KI mice that may explain the genotypic difference in susceptibility to cognitive defects. As expected, the GluR1 S831 phosphorylation profile mirrors that of its upstream PKC, with a significant upregulation (44%) and followed by downregulation (-25%) in apoE4-KI mice at week 12 and 72 respectively, as well as a comparable activity in both cohorts at middle age (Figure 3.6B). However, there is no difference in phosphorylation of GluR1 subunit at serine residue 845 at all ages (Figure 3.6A) which reflects the trend of its upstream PKA. In figure 3.6C, αCaMKII activity in apoE4-KI mice depicts a similar trend as that of GluR1 S831 with an increase at week 12 (40%) and a reduction at week 72 (-20%). This shows that other than PKC, αCaMKII may also contribute to the phosphorylation of GluR1 S831. It is also possible that CaMKII acts synergistically with PKC to stabilise AMPA channels at glutamatergic synapses. However, the higher level of phosphorylation of these intracellular signalling molecules in apoE4-KI mice could only be seen at young age. This trend was not sustained at middle age and levels of 74 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice phosphorylated proteins were even lower than that of apoE3-KI mice at old age. Hence, there may be a deleterious effect of apoE4 compared to apoE3 at a later stage of life. 75 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice 3.3.6. ERK-CREB signalling pathway in brains of huApoE-KI mice across three time points Figure 3.7. Protein expression level of ERK and CREB in brains of apoEKI mice models across three time points i.e. 12, 32 and 72 weeks. Western blot analysis of (A) phosphorylated ERK1/2 (p-ERK1 and pERK2) and ERK1/2 and (B) phosphorylated CREB (p-CREB) and CREB expression in huApoE3 (white bar) and huApoE4 (grey bar) mice with β-actin as loading control. Densitometry of (A) p-ERK1 relative to ERK1, p-ERK2 relative to ERK2 and (B) p-CREB relative to CREB performed using NIH ImageJ software is shown on the right panel. The relative value for apoE4-KI mice was normalized against age-matched apoE3-KI mice. Each value for apoE4KI mice represents mean value ± SEM relative to apoE3-KI mice for individual mouse brain sample (n=5) at each time point. For statistical analysis, Student's t-test was used to test for significance (* p < 0.05, *** p < 0.001). 76 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice As discussed earlier, ERK1/2 and CREB are intracellular proteins that regulate crucial biological functions such as cell viability, differentiation and metabolism as well as neurite outgrowth (Chang and Karin, 2001; Marie et al., 2005; Roskoski, 2012; Vitolo et al., 2002). ERK activation has shown to be mediated via NMDAR-dependent Ca2+ influx (Kurino et al., 1995; Ohkubo et al., 2001; Xia et al., 1996) which subsequently phosphorylates CREB (Hardingham et al., 2001; Hardingham and Bading, 2002; Shaywitz and Greenberg, 1999) in hippocampal neuronal culture. Thus I wanted to examine whether differences in NMDAR signalling profiles in apoE4-KI mice at different ages could contribute to differences in the activity of ERK and CREB signalling pathways in vivo. Figure 3.7A illustrates that at week 12, phosphorylated ERK1/2 for apoE4-KI mice significantly increased by two-fold. There is no difference between both mouse knock-in models at week 32 but at old age, activity of ERK1/2 is notably down-regulated with a 25% and 31% reduction for phosphorylated ERK1 and ERK2 respectively. One of the downstream targets of ERK1/2, CREB, has a similar activation profile as that of ERK1/2. ApoE4-KI mice showed a significant 1.5-fold increase in phosphorylated CREB at week 12 but there is little difference at week 32 compared to apoE3-KI mice. There is again a significant loss of CREB activity (-30%) at week 72 for apoE4-KI mice (Figure 3.7B). The higher ERK and CREB phosphorylation in young apoE4 mice may confer a neuroprotective effect by upregulating physiological processes such as cell proliferation, differentiation and cell cycle progression. However, these beneficial effects of apoE4 was not observed at middle age. The lower ERKCREB signalling observed in aged apoE4-KI mice compared to apoE3 counterparts implies a dysregulation of the cellular functions. In conclusion, the apoE expression level in apoE4-KI mice is downregulated compared to that of apoE3-KI mice at all ages which is mirrored in the expression levels of apoE receptor, LRP1 and the LRP1- and NMDAR77 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice associated scaffolding protein, PSD95. As I observed a similar reduction in phosphorylated NR2A subunit in apoE4-KI mice, this could be due to loss of function of apoE with less LRP1 binding to its ligand to mediate signal transduction of the intracellular pathways. However, this decreased apoE4 expression does not seem to affect the NMDAR-coupled signalling involving PKC, PKA, αCaMKII, ERK and CREB. Hence, there may be alternative mechanisms that account for the differential regulation of these proteins by apoE4 isoform. 78 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice 3.3.7. Expression of human apoE in neurons and astrocytes of huApoEKI mice brains across three time points Region-specific colocalization of apoE with GFAP Region-specific colocalization of apoE with MAP2 79 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice 80 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice 81 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice Figure 3.8. Cellular expression levels of huApoE in brains of apoE-KI mice models across three time points i.e. 12, 32 and 72 weeks. (A) Co-localization analysis of human apoE expression by glial cells in huApoE3 (white bar) and huApoE4 (grey bar) mice. (B) Co-localization analysis of human apoE expression by neurons in huApoE3 (white bar) and huApoE4 (grey bar) mice. Fluorescence intensity is expressed as integrated density of apoE co-localised with (A) GFAP and (B) MAP2 in three hippocampal subfields and cortex. Fluorescence microscopy utilising apoE (red) and (C) glial maker GFAP (green) and (D) neuronal maker MAP2 (green) antibodies. A representative section (X20 magnification) of the individual stainings and their superposition is presented over three time-points. Scale = 200 μm. (E) Representative images (X40 magnification) of CA3 subfield in apoE3-KI mice showing the expression of apoE (red), GFAP (green, upper panel), MAP2 (green, lower panel) and the co-expression of GFAP or MAP2 with apoE (yellow) as indicated by white arrows. Scale = 30 μm. The relative value for apoE4-KI mice was normalized against age-matched apoE3-KI mice. Each value for apoE4-KI mice represents mean value ± SEM relative to apoE3-KI mice for individual mouse brain sample (n=3) at each time point. For statistical analysis, Student's t-test was used to test for significance (* p < 0.05, ** p < 0.01, *** p < 0.001). It has been postulated that pathogenic effects of apoE4 may depend on its source of production rather than the total amount of apoE in brain. Astrocytic apoE4 confers protection from excitotoxicity but not neuronal apoE4 (Buttini et al., 2010), as the latter undergoes proteolysis to generate neurotoxic fragments (Brecht et al., 2004; Huang, 2010; Huang et al., 2004; Mahley et al., 2006; Roses, 1997). NSE-apoE4 which specifically express apoE in neurons demonstrated an age-dependent accumulation of C-terminal truncated apoE 82 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice fragments and phosphorylated tau, but all of these was not detected in GFAPapoE4 mice which express apoE in astrocytes only. The pattern of apoE fragmentation in NSE-apoE4 mice resembled that of AD brains, with a regionspecific accumulation in neocortex and hippocampus which are tightly associated with cognition (Brecht et al., 2004). As there is no previous studies that examine the level of apoE produced by different sources i.e. neurons and glial cells in apoE-KI mice, I wanted to compare these parameters in the neocortex and hippocampus of the two animal lines by utilising immunofluorescence study that shows colocalization of apoE with either neuronal (MAP2) or glial (GFAP) marker. To verify whether there is a basal difference in the total population of neurons and astrocytes between apoE3- and apoE4-KI mice, immunoblotting of whole brain lysate was done using neuronal and glial cell markers which showed no genotypic differences in the basal amount of neurons and astrocytes during ageing (supplementary figure 1). At 12 weeks of age, there is a significant decrease in apoE4 produced by glial cells in CA1 (-47%) and CA3 (-52%) as demonstrated in figure 3.8A. At week 32, the amount of apoE4 colocalized with glial cells reduces in DG (27%) which persists till week 72. In addition to DG area, there is also less astrocyte-derived apoE4 in hippocampal CA3 (-57%) and cortex (-32%) at week 72. On the other hand, apoE4 expressed by neurons significantly decreases at 12 week in all hippocampal sub-fields i.e. CA1 (-65%), CA3 (-61%) and DG (54%) compared to apoE3 expression (Figure 3.8B). This trend extends to cortex at week 32 with the reduction of apoE4 expression in CA1 (-64%), DG (-54%) and cortex (-66%). However, there is a significant enhancement in neuronal-derived apoE4 at 72 weeks with a 2.5 fold and 3.6 fold increase in CA3 and cortex respectively. Since there is significant lower expression of apoE4 by both neurons and glial cells in hippocampus at week 12, there is neither protective nor detrimental 83 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice effect of apoE4. In the cortex, there are comparable levels of glial and neuronal apoE4 expression and this may help to balance the neurotoxic effect of apoE4. Overall, this may favour neuronal survival and prevent the overt signs of neurological defects. However, the significantly higher level of neuronal apoE expression in CA3 and cortical regions of apoE4-KI mice than that of apoE3-KI mice at week 72 suggest that they may be more vulnerable to external insults which activate endogenous synthesis of apoE in neurons to initiate repair process (Boschert et al., 1999; Huang, 2010; Xu et al., 2006). 84 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice 3.3.8. Protein expression level of NMDAR subunits in brains of huApoEKI mice across three time points 85 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice 86 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice Figure 3.9. Expression level of total NMDAR subunit proteins in brains of apoE-KI mice models across three time points i.e. 12, 32 and 72 weeks. 87 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice Immunofluorescence studies utilising different NMDAR antibodies (red) were performed to show expression level in different brain regions across three time-points. Quantification of (A) NR1, (C) NR2A and (E) NR2B expression in hippocampus and cortex of huApoE3 (white bar) and huApoE4 (grey bar) mice. Representative images (X20 magnification) of the indicated hippocampal subfields and cortex are presented in (B) NR1, (D) NR2A and (F) NR2B. Scale = 50 µm. The relative value for apoE4-KI mice was normalized against age-matched apoE3-KI mice. Each value for apoE4-KI mice represents mean value ± SEM relative to apoE3-KI mice for individual mouse brain sample (n=3) at each time point. For statistical analysis, Student's t-test was used to test for significance (* p < 0.05, ** p < 0.01, *** p < 0.001). I hypothesize that increased apoE4 expression by neurons may exert a negative impact on NMDAR expression level in vivo since neuronal apoE4 poses a toxic effect in vitro (Jordan et al., 1998; Tolar et al., 1999). Therefore, I examined the intensity of immunostaining of total NMDAR subunits in neocortex and hippocampus of apoE-KI mice at different ages. Sub-regional analysis of NMDAR subunit immunofluorescence indicates a significant increase in NR1 subunit expression in all the brain areas examined of apoE4-KI mice with an 8 fold enhancement in CA1 and cortex, as well as 3 fold and 5.5 fold increase in CA3 and DG respectively at week 12 (Figure 3.9A). There is no significant difference between the two animal models in most of the regions except for DG with almost 5 fold increment in NR1 subunit expression at week 32. At old age, the expression level deteriorates in all the regions with a significant reduction in CA1 (-63%), CA3 (-62%) and DG (-44%). Figure 3.9C shows a significant drop in NR2A expression level in CA1 (72%), CA3 (-52%) and cortex (-51%) of 12-week-old apoE4-KI mice compared to age-matched apoE3 counterparts. This trend persists at week 32 with a reduction in the same brain regions i.e. CA1 (-39%), CA3 (-33%) and cortex (-61%). At old age, the decline in expression level extends into DG area (-85%) in addition to a greater decrease in CA1 (-94%), CA3 (-90%) and cortex (-94%). 88 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice In figure 3.9E, there is no genotypic differences between the two lines in NR2B expression level at week 12, but apoE4-KI mice start to show a significant reduction in NR2B subunit expression at week 32 in CA1 (-88%), CA3 (-80%) and DG (-78%). This deterioration further extends to cortex (76%) with a similar trend in hippocampal sub-fields i.e. CA1 (-87%), CA3 (93%) and DG (-82%) at week 72. Overall, apoE4-KI mice have a higher contents of NMDAR subunits particularly NR1 and NR2B at young age which may compensate for the lower levels of NR2A subunits in apoE4-KI mice at all ages. This also suggest that majority of glutamatergic receptors in young apoE4-KI mice may comprise NR1/NR2B receptors. However, the higher expression levels of total NR1 and NR2B subunits in apoE4-KI mice became lower than that of apoE3-KI mice at old and middle age respectively. This may reduce the availability of NMDAR subunits to assemble into functional receptors at the synapses. In conclusion, although apoE4 may play a favourable role at young age, this effect seems to disappear with age. 3.4. Discussion 3.4.1. ApoE4 isoform downregulates expression level of total apoE but upregulates neuronal apoE production with increasing age ApoE level is significantly downregulated in apoE4-KI female mice compared to apoE3 cohorts regardless of age (Figure 3.2). This is consistent with the observation by Riddell and colleagues of reduced brain apoE levels in apoE4KI mice compared with apoE3 mice (Riddell et al., 2008) and a lower apoE level in the hippocampus and cortex of young (5-month-old) and old (14- to 22-month-old) female apoE4 compared to apoE3-KI mice (Ramaswamy et al., 2005). In apoE-KI mice model, the replacement of murine apoE with human apoE gene eliminates the mouse Thr61 and introduces human Arg61 residue. As a result, Arg112 in human apoE4 can mediate domain interaction between 89 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice Arg61 and Glu255 enabling this molecule to assume the confirmation of unstable ‘molten globule’ (Hatters et al., 2006; Ramaswamy et al., 2005). This conformation is highly unstable and more susceptible to denaturation. The increased proteolytic cleavage of human apoE4 may contribute to the lowered protein expression detected in the apoE4- compared to apoE3-KI mice at all time-points. However, some groups have shown a lack of genotypic differences within a given brain region particularly in hippocampus, frontal cortex and cerebellum of young male (3- to 5-month-old) and female (6- to 8month-old) (Siegel et al., 2012; Sullivan et al., 2004) and in hippocampus of 12-month-old apoE-KI mice (Korwek et al., 2009). The discrepancy in apoE levels observed may arise from the different methods used to extract and measure apoE in tissues such as the conventional SDS or modified guanidine extraction procedures. In addition, differences in age and gender of the mice as well as environmental factors may influence apoE transcriptional and translational processes which contribute to these divergent findings. I utilised the soluble fraction of the brain homogenate and it is thought that astrocyte-secreted apoE is generally more soluble compared to insoluble form of apoE associated with amyloidogenic proteins (Näslund et al., 1995). Hence, my observation of lowered total apoE4 level in soluble fraction could indicate less production of apoE by astrocytes in apoE4-KI mice. Indeed, I have demonstrated a decreased astrocyte-derived apoE4 in some of the brain regions examined which may contribute to the reduced total apoE level and will be discussed below. In line with my findings, human studies also demonstrated lower level of soluble apoE in apoE4 carriers compared to apoE3 individuals (Beffert et al., 1998; Bertrand et al., 1995) which was attributed to increased deposition of apoE4 in plaques and tangles. However, it is unlikely that the lowered apoE4 level in apoE4-KI mice is due to the increased association with plaques and tangles as these symptoms have not been observed in the same mice model by others (Huang, 2011; Sullivan et al., 1997; Wang et al., 2005a). There is no observable change in apoE expression within genotype during ageing. This is consistent with our previous lab findings that did no detect any 90 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice difference in apoE expression level between 32-week-old and 72-week-old female apoE-KI mice (Ong et al., 2014). However, another group reported an age-dependent decrease in apoE expression in cortex, hippocampus, and amygdala in female apoE4 mice which was observed only in amygdala of apoE3-KI mice (Siegel et al., 2012). In contrast, Liraz and colleagues noticed an increasing apoE expression level in hippocampal CA3 region of both apoE3- and apoE4-KI mice when comparing between two ages i.e. at one and four months of age. Notably, they used male instead of female mice and this suggests a differential gender influence on apoE expression level during ageing. Furthermore, the difference in age groups of the samples may also affect the observations as the oldest mice used in Liraz’z group was 4 months of age whereas mice from 8 months onwards were used in my study and by others. In my study, this lowered apoE expression level in apoE4-KI mice at all ages is mirrored in reduced expression levels of apoE receptor, LRP1, and the scaffolding protein PSD95 that can interact with both LRP1 and NR2 subunits of NMDAR. Whether this results in less interactions between these proteins in the NMDAR signalling complex remains to be determined in follow-up studies. Notably, the NMDAR-induced signalling pathways particularly involving CaMKII, PKC, ERK and CREB do not have a similar trend as that of apoE expression level and this strongly supports that the downstream observations are not due to the loss-of-function of apoE4. AD brains demonstrate a more pronounced proliferation and activation of astrocytes, a process known as astrogliosis (Eddleston and Mucke, 1993; Hol et al., 2003; Nichols et al., 1993). It has been suggested that astrocytes secrete factors that can modulate neuronal apoE expression possibly via the ERK pathway (Harris et al., 2004). Transcription of APOE is regulated by tissuespecific cis-acting regulatory elements spanning across the 20-kilobase region of the APOE locus (Artiga et al., 1998; Grehan et al., 2001; Shih et al., 2000; Simonet et al., 1991; Smith et al., 1988). Astrocytosis leading to increased secretion of astroglial factors triggers the ERK pathway and activates regulatory elements that may be modified by APOE promoter polymorphism. 91 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice However, my findings of an enhanced neuronal apoE expression (Figure 3.8B) but a decreased ERK signalling (Figure 3.7A) in aged apoE4-KI mice suggest that there are alternative pathways that regulate neuronal apoE expression. I have shown a decreased glial apoE expression in most of the brain areas examined including CA3, DG and cortex of 72-week-old apoE4-KI mice. This could be due to a feedback mechanism in astrocytic expression of apoE stimulated by factors released through injured neurons (Yin et al., 2012). According to their report, ERK1/2 pathway is implicated astrocytic apoE expression modulated either by neurons or in an autocrine manner. Hence, the reduced ERK1/2 activity in apoE4-KI mice at week 72 could have hindered astrocyte-derived apoE expression and secretion regulated per se and/or by neuronal signalling. This is supported by Yin’s study that observed an inhibition of ERK activity by U0126 in astrocytes could abolish the enhancement of apoE expression by astrocytes. Furthermore, astrocytic apoE synthesis can be regulated in an autocrine fashion by fibroblast growth factor (FGF-1), which is produced after cryo-injury of mouse to promote healing (Lu et al., 2009a; Tada et al., 2004). FGF-1 stimulates ERK to increase 25hydroxyholesterol biosynthesis (Ito et al., 2007) which activates liver X receptor α (LXRα) in cultured rat astrocytes. Enhanced binding of LXRα to apoE promoter elevates apoE transcription and the apoE-HDL is then secreted via PI3K/Akt pathway (Lu et al., 2009a). Taken together, ERK1/2 pathway may play a role in mediating astrocytic apoE expression only at old but not at young age due to simultaneous decreases in both molecules. It is certain that in vitro models only partially imitates the condition in vivo, the mechanisms for regulation of apoE level in brain may be different and are possibly more complicated than that in vitro. There may be other unknown signalling pathways that modulate apoE expression between neurons and astrocytes under physiological and pathological conditions. I have first demonstrated that the aged apoE4-KI mice express a significantly higher amount of neuronal apoE compared to apoE3-KI mice in hippocampal CA3 and cortex (Figure 3.8B). While another study only examined the neuronal apoE production in hippocampus only, they found that both apoE392 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice KI and apoE4-KI mice showed similar increase in neuronal-derived apoE in CA3 region with age (Liraz et al., 2013). This discrepancy may be due to different age group used by Liraz and colleagues as their oldest mice were 4month-old only whereas my study observers up till 18 months of age. ApoE4KI mice may be more susceptible to environmental stress and brain injury that triggers neuronal apoE synthesis under pathological conditions (Boschert et al., 1999; Huang, 2010; Mahley, 1988; Roses, 1997; Xu et al., 2006). This is supported by Siegel and colleagues which reported a higher stress level evaluated by elevated zero maze test in female apoE4-KI mice compared to their apoE3 counterparts (Siegel et al., 2012). In line with apoE4-KI mice, other animal models of neurodegeneration and neurological patients have demonstrated high levels of neuronal apoE which is due to endogenous synthesis rather than extracellular uptake (Han et al., 1994a; Han et al., 1994b; Ishimaru et al., 1996). I propose that the brains of apoE4-KI mice may experience an NRHyper state during young age which predisposes them to excitotoxicity and lead to increased neuronal apoE4 expression particularly in CA3 and cortex with increasing age (Figure 3.8B). This is supported by Liraz and colleagues who reported an effect of apoE4 on neuronal mitochondrial dysfunctions in CA3 thus increasing susceptibility of neurons to external insults in this region (Liraz et al., 2013). Moreover, synapses in CA3 is deduced to be less stable than CA1 (Ling et al., 2012) thus sensitizing the pyramidal neurons in CA3 to environmental stimuli such as stress and traumatic brain injury (Christian et al., 2011; Deng and Xu, 2011). In this study, less glial apoE4 is produced especially in CA3 and DG thus conferring less neuroprotective effects (Figure 3.8A) which may exacerbate the deteriorating consequences of neuronal apoE4 at old age. This imbalance between astrocytic and neuronal apoE4 expression may increase susceptibility to neurological diseases involving excitotoxic mechanisms. Increased neuronal expression in aged apoE4-KI mice may facilitate formation of neurotoxic fragments as apoE4 is more susceptible to proteolysis (Brecht et al., 2004; Huang et al., 2001). The presence of apoE fragments which are more 93 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice abundant in brains of AD patients and NSE-apoE4 mice compared to NSEapoE3 mice (Brecht et al., 2004) remains to be confirmed in apoE4-KI mice model. This is because immunoblotting with apoE antibody against the fulllength apoE (34 kDa) and truncated apoE fragment of lower molecular weight (29-30 kDa) could not detect the C-terminal-truncated apoE fragment in apoE4-KI mice (Brecht et al., 2004; Huang et al., 2001) and may require techniques that offers a better resolution of the protein fragments such as mass spectrometry. Notably, apoE proteolysis is an age-dependent phenomenon that follows a region-specific distribution as the ratio of truncated apoE4 to full lengthapoE4 was significantly higher in the neocortex and hippocampus than in the cerebellum of NSE-apoE4 mice. It is possible that the C-terminally truncated apoE4 causes loss of NMDAR subunits expression in old apoE4-KI mice as it is toxic to cultured neurons and do not support the maintenance of neurites and neuronal growth (Jordan et al., 1998; Tolar et al., 1999; Tolar et al., 1997). This induces NRHypo state in aged apoE4 mice which may be due to the lower density of dendritic spines in the cortex and neurodegeneration observed in apoE4-KI mice compared to apoE3-KI cohorts (Dumanis et al., 2009; Harris et al., 2003; Huang et al., 2001). I have indeed shown a downregulation in phosphorylated NR1 S896 (Figure 3.4A) and levels of NMDAR subunits in hippocampus and cortex of apoE4-KI mice at old age (Figure 3.9). The NRHypo state does not seem to affect NR1 S897 possibly because phosphorylation of this site does not necessarily require NMDAR activation. In fact, Llansola and colleagues reported that blocking NMDAR with MK801 could not prevent phosphorylation of NR1 S897 implying this phenomenon may occur before channel activation, thus supporting our observations (Llansola et al., 2004). 94 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice 3.4.2. ApoE4-isoforms decreases expression of apoE receptor i.e. LRP1 and postsynaptic protein PSD95. In parallel to total apoE expression profile, I observed a decreased LRP1 in apoE4-KI mice at all ages (Figure 3.3A). LRP1 is a major apoE receptor primarily expressed on neurons that influences apoE catabolism (Bu, 2009; Li et al., 2000; Liu et al., 2007). Although previous findings reported that deletion of the Lrp1 gene in forebrain neurons increases apoE levels (Liu et al., 2007) whereas overexpression of LRP1 receptor in mouse brain decreases brain apoE level which may be caused by increased uptake and metabolism of apoE (Zerbinatti et al., 2004), this study suggests otherwise as the decreased apoE expression is reflected by decreased LRP1 in apoE4-KI mice at all timepoints. This could be due to accelerated neurodegeneration in apoE4-KI mice as LRP1 is mainly expressed in neurons and to a lesser degree in glia as suggested by others (Rapp et al., 2006; Rebeck et al., 1993). Even though LRP1 is a major neuronal apoE receptor, there is evidence that LRP1 activation is important for glial survival as well (Campana et al., 2006; Fuentealba et al., 2009). Thus, reduced LRP1 in apoE4-KI mice may indicate a decrease in glial viability which is a major source of apoE. This is followed by a decrease in apoE expression in apoE4-KI mice. Notably, apoE-KI mice share many characteristics with conditional LRP1knockout (LRP1-KO) mice model which are deficient in LRP1 in forebrain neurons. Apart from the common feature i.e. reduced LRP1 expression in brain, apoE4-KI mice has shown reduced expression of NR1, LPR1 and PSD95 at 18 months of age (week 72) (Figures 3.9A and 3.3). These strongly agree with the age-dependent synaptic loss accompanied by reduced levels of NR1 and GluR1 subunits and PSD95 exhibited by age-matched LRP1- deficient mice as reported by Liu and colleagues (Liu et al., 2010). As these molecular changes ultimately lead to neurodegeneration and memory decline in LRP1-deficient mice, apoE4-KI and conditional LRP1-KO mice models may share a similar molecular mechanism that underlies the abnormal behavioural phenotype. The reduction in basal PSD95 level in apoE4-KI mice suggests abnormal synaptic alterations as LRP1 supports neuronal function 95 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice and synaptic integrity (Yoon et al., 2013). In addition, decreased LRP1 in apoE4-KI mice may result in dysregulation of AMPAR trafficking, abnormal synaptic transmission and impaired LTP as part of the underlying mechanism for their cognitive defects as LRP1 is also involved in endocytosis of GluR1 subunit of AMPAR (Nakajima et al., 2013) and modulates LTP in hippocampal slices (Liu et al., 2010). One of the limitations of this study is the LTP was not measured in apoE-KI mice and hence should be followed up in future studies. Collectively, reduced LRP1 in apoE4-KI mice has a negative impact on composition of postsynaptic density complexes comprising PSD95, NR1 and GluR1; increases susceptibility to neuronal loss and worsens the burden of apoE4 isoform on neurodegeneration. A plausible explanation for decreased LRP1 observed in apoE4-KI mice is the antibody utilised in this study is against the short-lived free intracellular domain (LRP1-ICD) generated by proteolytical processing. Decreased LRP1 in apoE4-KI mice may reflect less availability of LRP1-ICD to activate intracellular signalling pathway since LRP1-ICD can directly trigger downstream signalling during synaptic transmission. Furthermore, there may be a compromised NMDAR-dependent Ca2+ influx due to the ability of LRP1 to interact with NMDAR (Martin et al., 2008) and modulate NMDAdependent Ca2+ currents in vitro (Qiu et al., 2002). Interestingly, apoE4 was found to regulate transcriptional activity of CREB via intracellular Ca2+ signalling mediated by LRP1 (Ohkubo et al., 2001). However, this phenomenon can only be seen in aged apoE4-KI mice but not the young ones implying that increased ERK and CREB activation may be mediated via other pathways that can surpass LRP1-dependent NMDA signalling at younger ages. The possibility of other LRP1-binding ligands such as α2M* and tPA mediating the ERK-CREB signalling pathway in apoE4-KI mice cannot be excluded as LRP1 also modulates NMDAR function through tPA that involves the second NMxY motif in LRP1 (Martin et al., 2008). Although there is a possible role of LRP1 in mediating ERK-CREB signalling in apoE4-KI mice especially at old age, there have been findings that suggest 96 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice otherwise. Nakajima's group proposed that CREB was activated by LRP1 via the fast CaMKIV-mediated pathway in LRP1-deficient primary neurons as ERK phosphorylation was not affected despite a reduction in NMDAdependent phosphorylation of CREB (Nakajima et al., 2013). However, it is evident that ERK activity profile resembles that of CREB in apoE4-KI mice throughout this study which strongly suggests that ERK-mediated signalling is required to maintain CREB activity in vivo. The intracellular signalling triggered by LRP1 in apoE4-KI mice may not be as robust as that of apoE3-KI counterparts due to the differential effects of apoE isoforms in receptor binding as proposed by Ruiz and colleagues. By using surface plasma resonance (SPR) measurements, they found that apoE3 but not apoE4 tend to form dimers via disulphide bridges that potentially bind to two LRP1 molecules (Ruiz et al., 2005). The similarities between apoE3 and another ligand of LRP1, α2M*, which can form tetramers upon receptorbinding, on potentiation of calcium signalling and neuroprotection against the detrimental effects of apoE4 (Hashimoto et al., 2000) suggest that the differences in apoE isoforms binding to LRP1 may exacerbate the consequence of decreased LRP1 expression in apoE4-KI mice. This study is the first to show a decreased PSD95 expression in apoE4-KI mice at all ages (Figure 3.3B). This may lead to less surface expression and hence reduced activity of NR2A subunit but not that of NR2B subunit, as many other groups have reported that PSD95 differentially interacts with NR2A and NR2B (Lin et al., 2004; Niethammer et al., 1996; Prybylowski et al., 2005; Sans et al., 2003). In particular, NR2A binds to PSD95 with higher affinity compared to NR2B and hence PSD95 preferentially stabilizes NR1/NR2A over NR1/NR2B receptor complexes (Losi et al., 2003; Sans et al., 2003; Townsend et al., 2003). This may be because NR2A is able to associate with two PDZ domains (i.e. PDZ1 and PDZ2) with equal strength whereas NR2B preferentially binds to PDZ2 domain only (Hoe et al., 2006; Kornau et al., 1995; Niethammer et al., 1996). Hence, the hypothesis that reduced surface expression of NR2A-contaning NMDARs due to decreased PSD95 level in apoE4-KI mice is in line with evidence from several studies that 97 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice showed promotion of synaptic insertion and prevention of internalization of NR2A-containing NMDARs by PSD95 (Lin et al., 2004; Losi et al., 2003). A reduction in PSD95 level in apoE4 mice may indicate less functional NMDA channels to facilitate Ca2+ influx and activate downstream signalling cascades compared to apoE3 counterparts because PSD95 modulates NMDA channel gating and surface expression by increasing number of functional channels at the cell surface. Specifically, PSD95 increases the rate of channel insertion and decreases the rate of internalization (Lin et al., 2004). Both in vitro and in vivo studies have demonstrated the role of PSD95 in potentiating NMDAR functionality. For instance, co-expression of PSD95 in oocytes increases NMDA-elicited whole-cell currents (Iwamoto et al., 2004; Yamada et al., 1999). My findings on loss of NMDAR activity and expression in apoE4-KI mice correspond well with reduced NMDAR currents in CA1 region in the same mouse model in a previous study (Korwek et al., 2009). However, they did not find any difference in hippocampal expression of another apoE receptor, apoER2, between 1-year-old apoE3- and apoE4-KI mice. Hence, their findings on the decreased NMDAR currents may be due to my observations of reduced LRP1 and PSD95 expression in apoE4-KI mice instead. Although PSD95 is mainly found in CA1 and CA3 of adult wild type rat (Ling et al., 2012), it is possible that there is a differential distribution of LRP1 and PSD95 in hippocampal subregions in CA1 area apoE4-KI mice that is responsible for the compromised NMDAR currents. This warrants future investigations to look into region-specific expression of LRP1 and PSD95 in apoE-KI mouse model. In addition, the reduction in PSD95 level may contribute to the aberrant behavioural outcome of the apoE4-KI mice as decreased PSD95 expression is associated with impaired spatial learning and memory as well as defective locomotion in mice shown by others (Migaud et al., 1998; Nyffeler et al., 2007). This phenomenon can be extrapolated to human studies as AD brains also demonstrated decreased PSD95 (Gylys et al., 2004; Love et al., 2006; Proctor et al., 2010). Based on these findings, reduced PSD95 level in apoE4KI mice render NMDAR less functional resulting in impaired LTP and 98 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice cognition. As a follow-up, electrophysiological studies should be used to verify the role of PSD95 in facilitating the NMDAR functionality and currents as one of the underlying mechanisms for LTP formation in apoE-KI mice during ageing. This will be able to address the limitation of this study which is the lack of functional readout of NMDA channel activity. 3.4.3. ApoE4 spatio-temporally regulate NMDAR expression and phosphorylation profiles during ageing. My study demonstrates that NMDAR phosphorylation and expression especially that of NR1 subunit, differs between genotypes during ageing. In particular, the phosphorylation levels of the protein at residue serine 896 of apoE4-KI mice show opposite trends at week 12 and 72. Young female apoE4-KI mice exhibit a significant 2.5-fold increase in NR1 phosphorylation (Figure 3.4A) which is reflected in a ten-fold-increase in total NR1 expression in all hippocampal subregions (CA1, CA3, DG) and cortex compared to agematched apoE3 counterparts (Figure 3.9A). Although immunoblotting of total NR1 expression level does not show a significant increase in 12-week-old apoE4 mice as detected in the total NR1 signal intensity by immunofluorescence, the immunofluorescence studies only encompass the hippocampus and cortex whereas immunoblotting utilises whole brain lysate. Since NR21 is ubiquitously expressed throughout the brain (Paoletti et al., 2013), perhaps there is a lower expression level in the other brain regions such as cerebellum that accounts for the different observations by immunoblotting and immunofluorescence studies. My foremost mechanistic observation of elevated NR1 S896 phosphorylation can be correlated to another functional study using the same animal model demonstrating an increased NMDAR-dependent CA1-LTP in young (8 to 20 weeks) apoE4-KI mice compared to age-matched apoE3 counterparts (Kitamura et al., 2004). This is based on the report by Tingley’s group that activation of the mandatory NR1 subunit by its phosphorylation potentiates NMDAR functions and increases synaptic efficacy (Tingley et al., 1997). A 99 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice similar observation by Moriguchi’s group also showed increased NR1 phosphorylation in ddY mice given a high tetanic stimulation that leads to enhanced LTP (Moriguchi et al., 2006). In spite of the enhanced LTP observed in young apoE4-KI mice, Kitamura and colleagues were not able to detect any differences in distribution and density of hippocampal NR1 subunit between the apoE3- and apoE4-KI mice. This may be because they used male instead of female mice, where the latter is more susceptible to damaging consequences of apoE4 as discussed above. Another study by Trommer & colleagues (2004) observed a decreased DG-LTP in 8- to 12-week-old apoE4-KI mice compared to apoE3 counterparts. As both studies examined different hippocampal subregions, modulation of synaptic plasticity in the perforant pathway (DG-LTP) may differ from that of SC pathway (CA1-LTP). This notion supports my observation that the initial dysregulation of NR1 and NR2A subunits was seen in the CA1 and CA3 but not the DG regions of hippocampus which will be discussed later. Discrepancy in both of the studies mentioned above may arise from the differences in hippocampal slice preparations and stimulation protocols. Due to the different types of LTP exhibited in different brain regions, regionspecific studies of LTP is not a clear indication of overall synaptic plasticity in brain and behavioural outcome. My study indicates changes in global NR1 phosphorylation is accompanied by increased expression in the hippocampus as well as cortex. A relevant behavioural study also observed better task learning in MWM and passive avoidance test exhibited by young apoE4-KI female mice although they have a higher stress level (Siegel et al., 2012). However, only my study has sought to identify the molecular mechanisms that may underlie this enhanced behavioural phenotype. In particular, phosphorylation of NR1 S896 by PKC activates ERK pathway which will be discussed later. It is possible that apoE4 not only differentially modulates the NR1 subunit changes but also the neuronal structures during ageing. It was reported by several groups that 1- to 2-year-old GFAP-apoE4 and apoE4-KI mice have a lower spine density and apical arborisation than apoE3 mice which were not observed in younger (3-week-old) mice (Dumanis et al., 2009; Ji et al., 2003). Hence, it is evident that the detrimental effects of apoE4 only 100 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice surfaces at later stages of life and may even be beneficial at younger ages. This age-dependent difference of apoE4 on NR1 subunit expression and phosphorylation, neuronal integrity and behaviours can be correlated to the increased risk of AD observed in aged apoE4 carriers. In human, some behavioural studies reported that young ε4 carriers may comprise an intellectual supergroup (Han and Bondi, 2008; Mondadori et al., 2007) with higher intelligent quotients (IQs) particularly among females (Yu et al., 2000); and higher educational achievement (Hubacek et al., 2001). 16to 30-year-old ε4 individuals showed better decision making, prospective memory performance and verbal fluency. As post-mortem studies on younger human brains are limited, apoE4-KI mice provide a relevant model to address this limitation in revealing the isoform-specific effects on molecular alterations especially at younger ages. I postulate that the enhanced NR1 phosphorylation and expression in apoE4-KI mice may be responsible for the superior phenotype at younger age. However, as mentioned by Kitamura and colleagues, modification of LTP in apoE4-KI mice is age-dependent as the enhanced LTP diminishes in adult mice of 24 weeks and above (Kitamura et al., 2004). This phenomenon is reflected in our apoE4-KI mice as well since there is no difference in NR1 phosphorylation and expression level (except in DG) between the two mice strains during middle age. As seen in Figures 3.4A and 3.9A, there is a decreased phosphorylation and expression level of NR1 in aged female apoE4 mice (week 72), in contrast to the two earlier time-points. As deletion of NR1 in CA1 region in animals results in impaired NMDAR-mediated LTP and LTD which then translates into spatial memory deficit (Tsien et al., 1996), loss of NR1 expression and phosphorylation in old apoE4-KI mice may have a similar impact on their behavioural outcome. Indeed, impaired learning and memory were observed in apoE4-KI female mice in many behavioural studies (Andrews-Zwilling et al., 2010; Bour et al., 2008; Grootendorst et al., 2005; Leung et al., 2012). This phenomenon can be extrapolated to human studies whereby AD brains also show a decrease in NR1 level (Amada et al., 2005; Ulas and Cotman, 1997). Amada and colleagues (2005) have shown that there is a downregulation of 101 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice NR1 subunit in AD subjects of which 50% are ε4 carriers and 85% of them are females. This suggests that apoE4 downregulates the expression of NR1 during ageing which may underlie the memory deficits in AD and this effect is gender-specific. Few clinical studies demonstrated that the NR1 subunit expression level deteriorated in AD whether it involves the whole hippocampus (Del Bel and Slater, 1991; Mishizen-Eberz et al., 2004; Sze et al., 2001) or the hippocampal subregions such as CA1. These findings mentioned above are consistent with apoE4-KI mice which showed a decreased NR1 level in all hippocampal subfields examined at old age. Collectively, NR1 subunit is more susceptible to loss in hippocampus and perhaps contribute to NMDAR dysfunction at glutamatergic synapses. My study is the first to show a genotypic difference in NR2A subunit phosphorylation and expression level among the apoE-KI female mice during ageing. There is a reduced NR2A phosphorylation at all three time-points (Figure 3.4B) which is accompanied by a reduced expression in hippocampus (except DG at week 12 and 32) and cortex (Figure 3.9C). The immunoblotting of total NR2A expression level does not show a significant reduction in apoE4 mice as displayed in the total NR2A immunofluorescence in hippocampus and cortex. This may be because NR2A is ubiquitously expressed throughout the brain (Monyer et al., 1994; Sheng et al., 1994) and higher expression levels in the other parts of the brain are able to enhance the signal intensities observed in immunoblotting. In contrast, Korwek and colleagues who did not find any difference in NR2A tyrosine phosphorylation in CA1 region of young apoE4KI mice (Korwek et al., 2009). But they did not specify the gender of mice used in their study, which is one of the important factors in modulating the effects of apoE4 on learning and memory decline. The variation in observation of NR2A activity between Korwek’s study and mine may arise due to the different brain areas being examined, as they used hippocampal CA1 homogenate to correlate to LTP in this region. This is in contrast to my study whereby whole brain lysate was utilised to investigate how changes in NMDAR activity as a whole can affect its downstream pathways. A separate study by Kitamura and colleagues shows no difference in hippocampal NR2A 102 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice expression level between apoE3- and apoE4-KI male mice (Kitamura et al., 2004). Hence, loss of NR2A phosphorylation and expression observed in my study seems to be restricted to female mice which supports the gender susceptibility to cognitive dysfunction in AD. The reduced tyrosine phosphorylation and lower level of NR2A in CA1, CA3 and cortical region of apoE4-KI mice compared to apoE3 age-matched counterparts at all ages could contribute to impaired LTP which also negatively affects their learning and memory. This has been proven by several groups that NMDAR-dependent LTP can be modified by tyrosine phosphorylation of NR2A (Caputi et al., 1999; Izquierdo, 1991; Linden and Routtenberg, 1989) and is strongly correlated to NR2A content at CA1 and CA3 synapses (Peng et al., 2010; Sprengel et al., 1998). As apoE4-KI mice often show impaired spatial learning and memory (Andrews-Zwilling et al., 2010; Bour et al., 2008; Grootendorst et al., 2005; Leung et al., 2012; Raber et al., 2000), loss of NR2A-containing NMDARs may be one of the main reasons for such behavioural phenotype because NR2A subtypes play a larger role in spatial working memory compared to NR2B-containing receptors (Boyce-Rustay and Holmes, 2006; Jacobs and Tsien, 2012; von Engelhardt et al., 2008). In line with animal studies, human ε4 carriers also have poorer spatial memory and performed less well in maze test and word recall than nonε4 peers but not in other aspects of cognition such as verbal fluency and prospective memory. (Alexander et al., 2007; Marchant et al., 2010). This accentuates that apoE4 is especially damaging to spatial learning and memory by downregulating NR2A expression in hippocampus that is important for spatial memory (Abrahams et al., 1997; Astur et al., 2002; Rosenbaum et al., 2000; Teng and Squire, 1999). In relation to human AD studies, MCI and AD patients have also shown reduced NR2A mRNA and protein levels in their brain (Bi and Sze, 2002; Sultana et al., 2010). In their study, loss of NR2A was particularly significant in entorhinal cortex as phosphorylated and total NR2A subunit were reduced by 40 and 30 percent respectively compared to healthy controls. However, they noticed that protein expression was unchanged in hippocampus which is 103 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice consistent with a corresponding study by Mishizen-Eberz and colleagues that did not find any change in NR2A expression in AD (Mishizen-Eberz et al., 2004). Notably, only half of the AD cohorts were female in both studies and there was no clear indication of genetic background which could limit the observation of apoE isoform effects on human. My novel findings in young apoE4-KI mice suggest that apoE4 individuals may actually experience early loss of NR2A subunit phosphorylation and expression which can only be revealed in old AD patients due to limited molecular studies in younger human brains. This may be one of the underlying factors that predispose them to deterioration of memory in the progression of AD. Although I focus on NR2A expression in neocortex of apoE-KI mice instead of the entorhinal cortex in the human study mentioned above, the neocortex represents more than 90 percent of the total cortical area and hence encompasses a wider area of the cerebral cortex which is responsible for higher intellectual functions of brain. I did not find any change in the level of phosphorylated NR2B (Figure 3.4C) but noticed a decreased NR2B expression in hippocampus at week 32 which further extends into the cortical area at week 72 (Figure 3.9E). I noticed an unchanged total NR2B expression in the immunoblot despite the reduced NR2B expression level in hippocampus and cortex at week 72. Since the NR2B subunit expression is restricted to the forebrain (Monyer et al., 1994; Sheng et al., 1994), the total expression level in the forebrain may not be similar to the region-specific expression level especially in hippocampus and cortex which may be more vulnerable to loss of NR2B subunit. The trend at young age is supported by Korwek and colleagues as they did not detect a change in both NR2B activity and expression in CA1 region of three to fivemonth-old apoE4-KI mice. One of the explanations for the unchanged phosphorylation despite loss of NR2B subunit in hippocampus of middle-aged apoE4-KI mice in my results is that the remaining cortical NR2B may be extensively phosphorylated to compensate for the decreased hippocampal expression level. I have shown a downregulation of NR2B expression level at old age in both hippocampal and cortical region which are parts of the forebrain where the majority of NR2B subunit population resides. The plausible explanation is that there may be higher NR2B subunit expression in 104 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice other regions of forebrain such as thalamus and amygdala to sustain its activity. As my study focuses on the phosphorylation site at tyrosine 1472 (Y1472) in NR2B which is enriched in synaptic site (Goebel-Goody et al., 2009), perhaps most of the changes in activity occur in extrasynaptic sites since NR2Bcontaining NMDARs predominates in this area compared to synaptic sites. It is evident that Y1472 is the most commonly studied and the major phosphorylation site implicated in synaptic plasticity (Jiang et al., 2011a). Although as many as 25 multiple tyrosine residues have been identified so far (Nakazawa et al., 2001; Wang et al., 2006), little is known about their physiological functions. Hence, the possibility of other phosphorylated residues that may contribute to NMDAR activity in apoE4-KI mice is not ruled out. In contrast to my observation of downregulated NR2A subunit phosphorylation and expression in the apoE4-KI mice, most behavioural studies have not been able to detect significant phenotypic differences between younger apoE3- and apoE4-KI mice. In fact, only a few of the studies have described an enhanced LTP and learning in apoE4-KI mice at young age (Kitamura et al., 2004; Siegel et al., 2012). This may be due to the distinct mechanism underlying LTP events in animals at an early developmental stage compared to adult animals (Grosshans et al., 2002). As NR2B subunit is crucial in facilitating synaptic plasticity especially in juvenile animals (Barria and Malinow, 2005; Lu et al., 2001; Zhao et al., 2005; Zhou et al., 2007), young apoE4-KI mice may be able to utilise NR2B-containing receptors to mediate LTP formation. This hypothesis is in line with the inability of genetic perturbation of NR2A to eliminate LTP in P28 mice indicating that NR1/NR2B diheteromeric receptors are sufficient to induce LTP (Berberich et al., 2005; Weitlauf et al., 2005). However, the developmental switch involving the gradual replacement of NR2B by NR2A with increasing age signifies the increase in dependence of LTP induction on NR2A-containing receptors. Hence, the downregulation of NR2A expression in aged apoE4-KI mice may increase their susceptibility to impaired learning and memory. 105 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice Similar to the aged apoE4-KI mice, human AD brains have shown a reduction in region-specific NR2B expression particularly in hippocampus and entorhinal cortex (Sze et al., 2001). Since reduced NR2A and NR2B expressions impair LTP (Ito et al., 1997), loss of these subunits in apoE4-KI mice may contribute to defects in synaptic plasticity. These NMDAR anomalies in apoE4-KI mice are probably more complicated and do not simply reflect neuronal loss. If these changes are merely a consequence of neuronal loss then the deterioration will be non-selective in the brain regions examined i.e. decreased NR1 and NR2 subunits altogether. Hence, it is prominent that apoE4 isoform differentially modulates NMDAR subunits that may have distinct consequences at different stages of life. Losses of NMDAR subunits level and activity may be correlated with presynaptic defects as such phenomenon was seen in early human AD brains (Sze et al., 2000; Sze et al., 1997). Indeed, recent advances have shown that apoE4 exerts negative impacts on pre-synaptic terminal composition as both young and old apoE4-KI mice have reduced glutamate and glutaminase, an enzyme that synthesizes glutamate from glutamine (Dumanis et al., 2013), and reduced vesicular glutamate transporter 1 (VGLUT1) in hippocampus suggesting an impairment in glutamatergic neurons (Liraz et al., 2013). This supports the notion that the observed changes in NMDAR activity in my study may be due to the presynaptic deformities which affect NMDAR-dependent excitatory neurotransmission. In relation to levels of neuronal- and astrocyte-derived apoE expression, the relatively low levels of neuronal apoE expression in young apoE4-KI mice (figure 3.8B) may prevent the pathological effects of apoE from surfacing and help to maintain a higher or comparable NMDAR expression particularly NR1 and NR2B subunits in hippocampus and cortex. However, NR2A subunit expression does not seem to be affected by changes in regional and cellular apoE expression as NR2A expression remains low in most of the brain regions of apoE4-KI mice at all three-time-points. At week 32, similar level of astrocyte-derived apoE3 and apoE4 production (figure 3.8A) may help to retain the NR1 and NR2B expression in cortex of apoE4-KI mice at a level 106 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice similar to that of apoE3 lines (figures 3.9A and B). Furthermore, lower amounts of neuronal apoE4 being expressed in most areas except in CA3 sustains its toxicity. I have first shown a downregulated expression of a few molecules critical for LTP induction and learning and memory including NR1 and NR2 subunits as well as their interacting proteins, LRP1 and PSD95, regardless of ages in apoE4 KI mice. This is in line with many studies that have also found an influence of apoE4 isoform on healthy younger apoE4 carriers and even neonates (Conejero-Goldberg et al., 2011; Heise et al., 2011; Knickmeyer et al., 2013; Marchant et al., 2010; O'Dwyer et al., 2012; Scarmeas et al., 2005; Valla et al., 2010) although this isoform is strongly associated with the aberrations in brains of aged population as well. (Riley et al., 2000; Small et al., 2000). In fact, my findings on the downregulation of NR2A, apoE receptor and PSD95 in 12-week-old apoE4-KI mice are consistent with recent reports which focused on neuropathology (Dumanis et al., 2013; Liraz et al., 2013), glial activation and vasculature (Bell et al., 2012; Zhu et al., 2012) of young apoE4 mice. All of these suggest that the pathological effects of apoE4 are already apparent in young developing brain and may contribute to induction of disease later in life. 3.4.4. Modulation of ERK and CREB activity in apoE4-KI mice is mediated via PKC but not PKA signalling pathway. There is a total correspondence between the phosphorylation profiles of ERK and CREB throughout the three time-points in apoE4-KI mice. Both molecules show a higher and lower phosphorylation than apoE3-KI mice at week 12 and 72 respectively (Figures 3.7A and B). This is in agreement with many other studies showing that ERK1/2 targets CREB indirectly to maintain its phosphorylation although CREB is not a direct substrate of the former (Hardingham et al., 2001; Sweatt, 2001). CREB in turn triggers transcription of BDNF which also activates MAPK pathway upon binding to tyrosinekinase B (TrkB) receptor (Meyer-Franke et al., 1998). Activation of CREB may form a positive feedback loop via BDNF to enhance ERK-CREB 107 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice signalling in young apoE4-KI mice. The involvement of BDNF in the upregulation of ERK and CREB awaits verification in future studies. The trends in ERK and CREB phosphorylation also correspond to the pattern of NR1 activity particularly phosphorylation of serine residue 896 (Figure 3.4A). There is possibly an increased NMDAR activity in apoE4-KI mice at young age that is coupled to ERK-CREB signalling. This mode of action of NMDAR is supported by the observation that Ras-GRFs, upstream activators of MAPK signalling cascade, can sustain CREB phosphorylation through positive regulation of ERK activity after NMDA stimulation (Tian et al., 2004). Both in vitro and in vivo studies have demonstrated that the obligatory NR1 subunit possesses all properties of the NMDA receptor-channel complex and NR2A subunits only serve to modulate NR1 activity (Nakanishi et al., 1994; Sze et al., 2001). Although there is a decrease in NR2A phosphorylation regardless of age in apoE4-KI mice, this does not seem to affect the downstream molecules suggesting that perhaps the constant NR2B activation is able to sustain overall NMDAR activity that may compensate for the downregulation of NR2A phosphorylation. While NR1/NR2B diheteromeric receptors may have lower peak currents and open probability compared to NR1/NR2A channels, Ca2+ imaging studies demonstrated they can carry about two-fold more Ca2+ charge for a single synaptic event and also deactivate slower (Erreger et al., 2005; Sobczyk et al., 2005). Thus the possible role of triheteromeric receptor complexes in mediating Ca2+ influx and downstream signalling cannot be excluded which further complicates the situation. The elevated ERK1/2 phosphorylation in young apoE4-KI mice that I see is consistent with a previous report that observed an increase in phosphorylation of ERK1/2 in 12- to 20-week-old apoE4-KI mice (Korwek et al., 2009). However, there has been controversial in vitro findings on effects of different apoE isoforms in ERK signalling. Ohkubo and colleagues have demonstrated that exogenous application of apoE4 but not apoE3 could stimulate ERK and CREB in primary hippocampal neurons (Ohkubo et al., 2001). Contrastingly, apoE3 was found to activate ERK1/2 in neural stem cells instead of apoE4 108 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice (Gan et al., 2011). It is possible that the different types of cell cultures give rise to the divergent cellular responses induced by apoE isoforms. Hence, the investigation of the ERK-CREB signalling profile in vivo is more relevant as I examine the effects of chronic exposure to apoE4 isoform on this signalling cascade in apoE4-KI mice model. In contrast to young mice, I observed a decreased phosphorylation in ERK pathway of aged apoE4-KI mice with an expected decrease in its target, CREB. One of the plausible explanations is that decreased CREB phosphorylation in old apoE4-KI mice is caused by upregulated CREB phosphatase in mature neurons which could inhibit NMDA activation of the transcription factor described by others (Hardingham and Bading, 2002; Sala et al., 2000). Another possibility for the opposite trend in CREB phosphorylation observed in apoE4-KI mice during ageing may be due to increased activation of synaptic and extrasynaptic NMDARs at week 12 and 72 respectively. In support of synaptic and extrasynaptic NMDARs playing distinct roles in synaptic plasticity, coupling to intracellular signalling pathways, neurotoxicity and cell death (Hardingham et al., 2002; Liu et al., 2004; Massey et al., 2004), it has been found that extrasynaptic NMDARs preferably induce cell death by triggering CREB shut-off pathway and blocking BDNF expression, whilst synaptic receptors mediate induction of synaptic plasticity and exert a neuroprotective effect (Hardingham and Bading, 2010; Hardingham et al., 2002). I postulate that increased activation of synaptic NMDARs promotes CREB activation of young apoE4-KI mice whereas developmental switch that leads to enhanced activity of extrasynaptic NMDARs downregulates CREB and induce neuronal death by inhibiting CREB-driven BDNF expression. Since CREB is a transcription factor that facilitates neuronal growth and differentiation, downregulation of CREB activity may lead to decreased spine density and neuronal development which increases susceptibility to neurodegeneration and impaired plasticity in aged apoE4-KI mice. NMDAR-dependent Ca2+ influx is coupled to multiple downstream signalling pathways that mainly involve Ca2+-sensitive protein kinases such as PKC, PKA and CaMKII. I have demonstrated that ERK1/2 and CREB 109 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice phosphorylation stauts in apoE4-KI mice correspond to that of PKCα and αCaMKII but not PKA-Cα during ageing (Figures 3.5A and B). In fact, there is no change in phosphorylation of PKA-Cα and its downstream targets i.e. NR1 S897 (Figure 3.4A) and GluR1 S845 (Figure 3.6A) throughout all ages. Notably, activation of ERK is coupled to Ca2+-dependent upstream activators including PKC and PKA in hippocampus and mouse dorsal horn (Ji et al., 2009). Similarly, phosphorylation of CREB at Ser133 can be induced by protein kinases including PKC, PKA and CaMKII, via Ca2+-dependent mechanism (Ginty et al., 1994; Hagiwara et al., 1993; Mao et al., 2007; Sheng et al., 1991; Sun et al., 1994). Based on these findings, the phosphorylation of ERK and CREB are more likely to be mediated via PKCα and αCaMKII rather than PKA-Cα. The higher phosphorylation of PKCα at week 12 in apoE4-KI mice led to increased phosphorylation of its substrates, NR1 S896 (Figure 3.4A) and GluR1 S831 (Figure 3.6B), with a concomitant increase in ERK-CREB signalling. This finding is in agreement with many other in vitro and in vivo studies showing that PKCα can directly phosphorylate Raf-1 and regulate downstream ERK pathway (Kolch et al., 1993; Lenormand et al., 1998; Novitskaya et al., 2000; Sakai et al., 1999; Zhao and Brinton, 2003). PKC also assists in nuclear translocation of the active MAPK/ERK (Cai et al., 1997; Stadheim and Kucera, 1998) which is tightly linked to the activation of nuclear CREB transcription factor. Moreover, phosphorylation of serine site at NR1 by PKC prevents calmodulin binding to the NR1 subunit and thus inhibits the inactivation of NMDARs by calmodulin, which in turn enhances NMDAR functionality (Hisatsune et al., 1997). Hence, I hypothesize that the greater amount of NMDA-dependent Ca2+ influx activates PKCα which in turn phosphorylates NR1 subunit and enhances channel activation in a positive feedback mechanism. This also leads to activation and translocation of ERK to nucleus to stimulate CREB-mediated gene transcription. At week 32, there is no difference between apoE3 and apoE4-KI mice in all the molecules involved in both pathways implying that perhaps this is a transition period before the pathogenic effects of apoE4 surface at old age. In 110 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice contrast, the significant drop in phosphorylation of PKCα and its substrates, NR1 S896 and GluR1 S831, in apoE4-KI mice at week 72 implies that there is possibly a reduced amount of NMDA-dependent Ca2+ influx. As expected, there is a simultaneous decrease in downstream ERK and CREB phosphorylation. All of these suggest strongly that modulation of ERK-CREB signalling in apoE4-KI mice is mediated via PKC instead of PKA pathway during ageing. PKCα isozyme is enriched in the post-synaptic region and plays a critical role in synaptogenesis (Kamphuis et al., 1995), hence reduced PKC phosphorylation in aged apoE4-KI mice may indicate synaptic loss which is one of the hallmarks of AD. Our observation of downregulation of phosphorylation of PKCα, and αCaMKII, NR1 S896, GluR1 S831 in aged apoE4-KI mice concurs with the signalling profiles of these molecules in the hippocampal CA1 region of olfactory bulbectomized (OBX) mice (Moriguchi et al., 2006). These mice also exhibited impaired LTP induction and learning disability which support the notion that PKCα regulates synaptic plasticity via phosphorylation of NR1 subunit at serine residue 896, and eventually aids in receptor trafficking to the membrane surface (Scott et al., 2001; Tingley et al., 1997; Zheng et al., 1999). Hence, I hypothesize that NRHypo elicits less Ca2+ influx which causes a reduction in αCaMKII and PKCα activation thereby diminishing phosphorylation of downstream NR1S896 and GluR1 S831. This serves as a potential underlying mechanism for the learning impairment in apoE4-KI mice during ageing. Other than NMDA-mediated Ca2+ influx which may activate PKCα, extracellular signals acting through G-coupled-protein receptors (GPCRs) may also stimulate PKC to phosphorylate Raf-1 suggesting an alternative mediator of PKCα activation that subsequently regulates ERK pathway in apoE4-KI mice. It is intriguing that GPCR ligands such as muscarine and lysophosphatidic acid also enhance NMDA-evoked currents via PKC pathway implying a feedback mechanism of PKC to modulate NMDA functionality (Lu et al., 1999). Furthermore, treatment of hippocampal CA1 slices with PKC activator, phorbol 12-myristate 13-acetate (PMA), increases tyrosine 111 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice phosphorylation of NR2A and NR2B subunits (Grosshans and Browning, 2001). Although I have found a difference in tyrosine phosphorylation of NR2A subunit between apoE3- and apoE4-KI mice which may alter the overall NMDA channel activity and downstream signalling cascades, this raises further questions on the exact PKC isoform that targets the tyrosine residues on NR2A subunit. In primary neuronal cultures, modulation of ERK and CREB by PKC has been proposed to be mediated via apoE receptor, LRP1, which alters Ca2+ signal transduction coupled to NMDAR (Qiu et al., 2004). However, I did not find an increase in LRP1 expression along with PKCα activation at week 12 suggesting that perhaps stimulatory effect of apoE4 on PKC and ERK-CREB pathway is mediated via other apoE receptors such as LDLR. Besides PKCα, another upstream regulator of GluR1 S831 is αCaMKII which also shares a similar trend with PKCα in the activation profile (Figure 3.5C). Phosphorylation of both serine residues at residue 831 and 845 of GluR1 is required for synaptic incorporation of GluR1 (Esteban et al., 2003). According to the model of bidirectional synaptic plasticity, dephosphorylated GluR1 of ‘depressed’ synapses favours formation of LTD whereas phosphorylation of GluR1 S845 by PKA-Cα converts the depressed synapses into ‘naïve’ ones. Additional phosphorylation of GluR1 S831 of naïve synapses by PKCα and αCaMKII increases ionic conductance of AMPA channel (Lledo et al., 1995; Mammen et al., 1997; Roche et al., 1996) and facilitates LTP induction (Lee et al., 2000). In my study, increased phosphorylation of GluR1 S831 of naïve synapses by PKCα and αCaMKII in young apoE4-KI mice may potentiate AMPAR functionality and enhance LTP as observed by others (Kitamura et al., 2004). This is also in line with other studies that show phosphorylation and trafficking of AMPARs to potentiated synapses in conjunction with activation of intracellular signalling pathways stabilizes the synaptic changes and increase synaptic strength in hippocampus (Cingolani and Goda, 2008; Lu and Roche, 2012) 112 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice In contrast, the downregulation of GluR1 S831 together with both of its upstream regulators in apoE4-KI mice at week 72 imply that there may be reduced insertion of AMPARs and hence less number of potentiated synapses or increased naïve synapses. However, protein phosphatases also controls LTD by downregulating AMPAR functionality (Mulkey et al., 1994; Yan et al., 1999). Hence, this does not rule out the possibility that there is a parallel decrease and increase in dephosphorylation of the potentiated synapses by serine phosphatases (PP1/PP2A) at week 12 and 72 respectively. It has been shown that CREB aids in trafficking of GluR1 subunit to PSD and AMPAR-mediated synaptic transmission in CA1 pyramidal neurons under basal conditions and also during learning-induced insertion of GluR1 into synapses (Middei et al., 2013). CREB does not directly control GluR1 gene expression as the total level of GluR1 subunit did not change in hippocampal homogenates of mice expressing mutant form of CREB (mCREB) compared to wild type. Instead, CREB regulates expression of other proteins modulating AMPAR trafficking such as BDNF (Caldeira et al., 2007; Shieh et al., 1998) and immediate early gene Arc (Bramham et al., 2008; Caldeira et al., 2007; Kawashima et al., 2009). Similar to the aged apoE4-KI mice in this study, decreased phosphorylation of GluR1 S831 and GuR1 S845 were observed by others in mCREB mice which are essential for hippocampal LTP (Lee et al., 2003; Middei et al., 2013). The dominant negative mutation is from serine 133 to alanine 133 suggesting the crucial role of phosphorylation of CREB at S133. This further strengthens my hypothesis that reduced CREB S133 phosphorylation in apoE4-KI mice at week 72 may account for the compromised GluR1 subunit phosphorylation. Although PKA can activate CREB which may result in an unknown positive feedback mechanism that upregulates PKA-dependent phosphorylation of GluR1 S845, the apoE4-KI mice model do not exhibit any changes in PKA pathway but demonstrate CREB activation profile which reflects that of GluR1 S831 in my study. This suggests that CREB may indirectly alter the proteins that regulate phosphorylation of GluR1 S831 in a positive feedback loop. 113 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice αCaMKII is a brain-specific isoform mainly found in neurons. Its expression is more concentrated in hippocampus (Erondu and Kennedy, 1985; Tobimatsu and Fujisawa, 1989) indicating that this ‘memory molecule’ plays a crucial role in hippocampal synaptic plasticity. The contrasting αCaMKII phosphorylation profile in brains of apoE4-KI mice at young and old age strongly suggests that loss of αCaMKII activity contributes to increased susceptibility of apoE4 to memory impairment in mice. This is based on the human studies that AD brains also demonstrate selective loss of αCaMKIIcontaining neurons in hippocampal CA1 region of (Wang et al., 2005b). On the other hand, the elevated αCaMKII Thr286 autophosphorylation at week 12 suggests its possible role in improving learning and memory in young apoE4KI mice (Siegel et al., 2012). One of the many ways whereby increased activity of αCaMKII can contribute to enhanced synaptic plasticity is by potentially acting as a scaffolding protein to organize the signalling complexes at postsynaptic site through direct binding to NMDAR, densin-180 which is a trans-synaptic protein and α-actinin, an actin binding protein (Shen and Meyer, 1999; Walikonis et al., 2001). Like PSD95, CaMKII also interacts with cytosolic tails of the NR2A and NR2B subunits (Gardoni et al., 1999; Leonard et al., 1999; Strack and Colbran, 1998) and perhaps play a similar role in stabilising the channels at the synapse. Notably, both αCaMKII and PSD95 co-immunoprecpitate with NMDA receptor complex under basal conditions (Gardoni et al., 2001b) demonstrating the interactions between these scaffolding proteins. Although NRHyper and increased αCaMKII autophosphorylation in young apoE4-KI mice could lead to enhanced plasticity, these phenomena could also perturb the interaction between NR2A and PSD95 because glutamate stimulation disrupts these interactions and significantly reduces coprecipitation of PSD95 with NR2A subunit. αCaMKII Thr286 phosphorylates PSD95 at serine residue 73 which influences NR2A but not NR2B binding to PSD95 (Gardoni et al., 2006). While I have demonstrated a reduction in PSD95 and NR2A expression level in apoE4-KI mice, the extent of interaction between these two molecules under the influence of αCaMKII Thr286 remains to be explored further. Interestingly, these alterations do not affect NMDAR subunits localization at 114 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice synapses suggesting that PSD95 phosphorylation represents a molecular mechanism modulating which signalling cascade will be recruited to NR2Acontaining NMDARs rather than the availability of receptors at synaptic sites (Gardoni et al., 2003; Gardoni et al., 2006; Meng et al., 2004; Picconi et al., 2004). It is therefore possible that αCaMKII phosphorylation modulates interactions between PSD95 and NR2A which are coupled to specific downstream pathway such as PKC signalling cascade in apoE4-KI mice. The similarities between the effects of PKC and PSD95 on NMDAR gating and insertion suggest that PKC acts in conjunction with this scaffolding protein to regulate NMDAR trafficking and functionality (Lan et al., 2001; Rong et al., 2001; Salter and Kalia, 2004; Zheng et al., 2006). I have shown an increased phosphorylation of both PKC and αCaMKII in young apoE4-KI mice and thus propose that PKC activation promotes αCaMKII autophosphorylation and its subsequent interaction with NMDAR. This ultimately leads to functional NMDAR insertion as suggested by Yan and colleagues. These NMDAR potentiation and PKC-induced synaptic plasticity are abolished by CaMKII antagonist or interfering with NR2-CaMKII association (Yan et al., 2011) highlighting the importance of αCaMKII and its binding to NR2 subunits in mediating the action of PKC. However, it is unknown whether the reduced NMDAR subunits and PKC phosphorylation in aged apoE4-KI mice is due to less interaction between NR2 subunits and αCaMKII. Furthermore, PKC and CaMKII share a few common substrates such as GluR1 subunit of AMPAR and Ins(1,4,5)P3 receptors suggesting a common signalling pathway in the modulation of NMDAR and LTP induction, possibly activating each other by triggering intracellular Ca2+ release (Bultynck et al., 2003). Hence, downregulation of PKC pathway in aged apoE4-KI mice may indicate the reduction of both NMDA and AMPA channel activity, less interaction of NAPs such as CaMKII and PSD95 with NMDARs leading to impaired synaptic plasticity and learning deficits. In conclusion, the increased NMDAR phosphorylation may be coupled to ERK and CREB activation, which is possibly mediated through PKC pathway in young apoE4-KI mice. All of these perhaps compensate for the intrinsic 115 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice dowregulation of apoE receptor and PSD95 thus suppressing the development of behavioural abnormalities. There is not much difference in many of the intracellular signalling cascades studied between apoE3- and apoE4-KI mice at middle age which could signify a transition period that precedes the surfacing of anomalies. At old age, NRHypo with a corresponding loss of expression in many brain regions of apoE4-KI mice may have led to reduced activity of NMDAR-coupled signalling pathways. One of the possible contributing factors may be the increased neuronal apoE4 expression which poses a detrimental effect that predisposes apoE4-KI mice to neurological diseases. Week Total expression levels of apoE, LRP1 and PSD95 Tyrosine phosphorylation of NR2A Phosphorylation of NR1 S896, GluR1 S831, PKCα and αCaMKII Phosphorylation of ERK1/2 and CREB 12 huApoE4 lower than huApoE3 32 huApoE4 lower than huApoE3 72 huApoE4 lower than huApoE3 huApoE4 lower than huApoE3 huApoE4 lower than huApoE3 huApoE4 lower than huApoE3 huApoE4 higher than huApoE3 huApoE4 lower than huApoE3 Glial apoE expression level huApoE4 lower than huApoE3 in: - CA1 - CA3 No difference between huApoE3 and huApoE4 No difference between huApoE3 and huApoE4 huApoE4 lower than huApoE3 in: - DG Neuronal apoE expression level huApoE4 lower than huApoE3 in: - CA1 - CA3 - DG huApoE4 higher than huApoE3 116 huApoE4 lower than huApoE3 in: - CA1 - DG - Cortex huApoE4 lower than huApoE3 huApoE4 lower than huApoE3 in: - CA3 - DG - Cortex huApoE4 lower than huApoE3 in: - DG huApoE4 higher than huApoE3 in: - CA3 - Cortex Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice Regional expression level of total NR1 subunit Regional expression level of total NR2A subunit Regional expression level of total NR2B subunit huApoE4 higher than huApoE3 in: - CA1 - CA3 - DG - Cortex huApoE4 lower than huApoE3 in: - CA1 - CA3 - Cortex huApoE4 higher than huApoE3 in: - DG huApoE4 lower than huApoE3 in: - CA1 - CA3 - DG huApoE4 lower than huApoE3 in: - CA1 - CA3 - Cortex huApoE4 lower than huApoE3 in: - CA1 - CA3 - DG - Cortex No difference between huApoE3 and huApoE4 in all regions huApoE4 lower than huApoE3 in: - CA1 - CA3 - DG huApoE4 lower than huApoE3 in: - CA1 - CA3 - DG - Cortex Table 3.2. Recapitulative table on all significant comparisons between huApoE3 and huApoE4 mouse lines. 117 Chapter 3: Impaired NMDAR-induced Signalling In Aged ApoE4-KI Mice Figure 3.10. Model of age-dependent regulation of intracellular signalling pathways by apoE4. ApoE4 binds to LRP1 receptor and modulates NMDAR functionality possibly mediated via PSD95 interacting intracellularly with LRP1 and NR2 subunits. There is a differential regulation of PKC pathway by apoE4 during ageing. At young age (week 12), AMPAR and NMDAR may be highly activated resulting in increased Ca2+ influx and subsequently upregulation of Ca2+sensitive kinases such as αCaMKII and PKC. This further increases ERK1/2 and CREB activation. In contrast, there is a downregulation of NMDAR and AMPAR activity with a concomitant decrease in αCaMKII, PKC, ERK1/2 and CREB activation at old age (week 72). 118 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment Chapter 4: Impacts of apoE isoforms in cellular responses to chicken extract (CE) treatment 4.1. Introduction 4.1.1. Cyclic nucleotide phosphodiesterase (PDE) and CREB Cyclic nucleotides influence neuronal viability either by targeting upon CREB directly (Lu and Hawkins, 2002; Merz et al., 2011) or via MAPK signalling that also triggers activation of CREB and other anti-apoptotic factors such as bcl-2 (Bonni et al., 1999; Boucher et al., 2000; Boucher et al., 2003; Doronzo et al., 2011). Cyclic nucleotide phosphodiesterases (PDEs) catalyse hydrolysis of cAMP and cyclic guanosine monophosphate (cGMP) and hence are involved in regulating cyclic nucleotide-mediated synaptic plasticity and neuroprotection (Cui and So, 2004). Levels of these cyclic nucleotides are more dependent on the breakdown by PDEs rather than their own synthesis by ACs. As such, PDE inhibitors have been widely studied as therapeutic targets for neurodegenerative disorders including AD. PDE exists in different isozymes (PDE1-PDE11) and is encoded by 21 identified genes, some of which may have multiple splice variants such as PDE4A-PDE4D, making up to more than 100 different PDE proteins. The subfamilies of PDEs are classified according to their affinity for specific substrates. The cAMP-specific enzymes are PDE4, 7 and 8; PDE5, 6 and 9 have high affinity for cGMP; and the dual-substrate PDEs which can act on both cyclic nucleotides are PDE1, 2, 3, 10 and 11. Most of the PDE isoforms are expressed in brain (PDE1, PDE2, PDE3, PDE4, PDE5A, PDE7A, PDE7B, PDE8B, PDE9A, PDE10A, and PDE11A) and their abundance in neurons suggest an important role in modulating cellular signalling cascades even though their distribution may vary between different brain regions and subsets of neurons (García-Osta et al., 2012). AD is associated with changes in PDE4, PDE7 and PDE8 expression in the brain (Bollen and Prickaerts, 2012). In particular, the majority of PDE4D 119 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment isozymes except the short isoform PDE4D1 is reduced in AD brains (McLachlan et al., 2007). Another study reported that PDE7A transcripts are reduced whereas PDE8B mRNA level is significantly increased in hippocampus and entorhinal cortex at an advanced stage of the disease (Braak stages III-VI) (Pérez-Torres et al., 2003). However, whether these changes are a cause or consequence of AD is still unknown and remains to be elucidated. 4.1.1. Chicken extract (CE): potential PDE inhibitor Previously our lab tested the effects of chicken extract (CE) on huApoEtransfected neurons and found an inhibitory effect of the supplement similar to phosphodiesterase (PDE) inhibitors. This supplement is produced following the concept of traditional culinary method using water extraction of chicken meat extract under high pressure and temperature, to be taken as a supplement for health and vitality reasons (Azhar and Mohsin, 2003; Konagai et al., 2013). We have shown that CE treatment downregulates PDE8A and PDE8B mRNA level in apoE4 and apoE3 neurons respectively (Siew, 2011). This suggests that CE potentially acts as a PDE8 inhibitor to elevate cAMP/PKA signalling pathway which in turn promotes CREB-mediated gene-transcription. The cAMP-specific PDE8 subfamily has two splice variants, PDE8A and PDE8B, of which the former is ubiquitously expressed whereas the latter is confined to the brain and thyroid (Bender and Beavo, 2006). In CNS, the highest levels of PDE8 are found in hippocampus, cortex and caudate. Despite the abnormal PDE8B expression in brain region associated with memory deficits in severe AD cases, no data regarding the effects of PDE8 inhibitors have been reported so far. Other PDE subtypes such as PDE1B, PDE2A, PDE4, PDE5, PDE9 and PDE10 have been constantly targeted due to the availability of optimal inhibitors and the efficacy of these compounds in AD mouse models (GarcíaOsta et al., 2012). Since CE is considered a health supplement and can be taken daily, there is a potential that it can be utilised as an adjuvant therapy for rectifying memory loss in AD without concerns of adverse effects observed with current PDE inhibitors in clinical use. 120 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment 4.1.2. Beneficial effects of CE to mental health Numerous studies have been performed to evaluate the functions of CE to different aspects of health including cognition, metabolism and energy utilization, immunity, cardiovascular system, physical performance, lactation, iron absorption and utilization, renal function and regulation of circadian rhythm (Chao et al., 2004; Fujii et al., 2003; Geissler et al., 1996; Man et al., 2005; Sim, 2001; Wu et al., 2011; Xu and Sim, 1997; Yamano et al., 2013; Yamano et al., 2001). Beneficial effects of CE have been demonstrated as it stimulates hematopoiesis, increases thermic response, promotes lactation and positively influences composition of breast milk, accelerates the clearance of post-exercise lactate and ammonia (Chao et al., 2004; Geissler et al., 1989; Lo et al., 2005; Williams and Schey, 1993). To date, human studies on cognitive functions encompass the effects of CE on stress and recovery from fatigue. It has been shown that a period of CE consumption significantly improved attention and working memory during mental task performance in stressed individuals (Azhar and Mohsin, 2003) and reduced fatigue (Nagai et al., 1996; Yamano et al., 2013). CE brought the cortisol level back to pre-workload level in a shorter time resulting in a smaller error rate for mental tasks compared to that of placebo (Nagai et al., 1996). It also improved selective attention of healthy males assessed by Stroop trials (Nagai et al., 1996; Yamano et al., 2013) possibly by activating prefrontal cortex and anterior cingulate cortex, as these two brain regions have been associated with fatigue (Botvinick et al., 1999; Caseras et al., 2008; Danckert et al., 2000; de Lange et al., 2008; Lorist et al., 2005). In addition, CE helped to improve anxiety scores in patients diagnosed with mild to moderate anxiety by reducing their pulse rates and systolic blood pressures when administered as an adjunct to psychotherapy (Azhar et al., 2001). In rats, CE significantly increased 5-hydroxyindoleacetic acid (5-HIAA) which is a metabolite of serotonin in CSF indicating that there may be an increase in brain serotonin activity (Xu and Sim, 1997). This suggests that CE has the potential to regulate mood and may even have an antidepressant effect. 121 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment A recent cross-over study revealed that oxyhemoglobin (oxy-Hb) concentration in prefrontal areas of brains increased during working memory task in the elderly who consumed CE for a week compared to placebo (Konagai et al., 2013). Hemoglobin (Hb) oxygenation is one of the functional indices of brain activation assessed by near-infrared spectroscopy (NIRS). The outcome i.e. oxy-Hb and total Hb has been positively associated with cognition and working memory (Matsui et al., 2007; Molteni et al., 2008; Schreppel et al., 2008; Watanabe et al., 2003). It is influenced by Ageing as the elderly showed lower increase than younger subjects in both parameters in frontal cortex during cognitive tasks (Herrmann et al., 2006; Hock et al., 1995; Sakatani et al., 1999). In contrast to previous findings that showed stressrelieving effect of CE, Konagai and colleagues (2013) did not find any significant changes on stress reduction in the elderly who consumed CE. This may be due to the age differences of the subjects as earlier studies involved younger cohorts. Another possible explanation is the fact that anxiety level is reduced during normal ageing in human and mice (Jorm, 2000; Siegel et al., 2012). In animal studies, CE protected against restraint stress-induced liver damage and alleviated glucose metabolic dysfunction by elevating insulin level (Kurihara et al., 2006; Zhai et al., 2012). CE could diminish oxidative stress by lowering malonaldehyde (MDA), an oxidative stress marker; and upregulating two enzymes i.e. superoxide dismutase (SOD) and glutathione peroxidase (GPX) which are scavengers of reactive oxygen species (ROS) (Zhai et al., 2012). In the context of glucose metabolism, CE treatment improved glucose utilization and activated glycogen synthesis in liver compared to restrained mice treated with water (Kurihara et al., 2006). Other than that, CE exerted hypoglycemic action in diabetic rodents (Sim et al., 2009). CE has been shown to attenuate hyperglycemia in type II diabetic KKay mice and GK rats without affecting plasma insulin levels and the magnitude of the hypoglycemic responses are equivalent to those obtained with other hypoglycemic agents in the same models (Kitahara et al., 2002). Type II diabetic mellitus (T2DM) is characterized by plasma insulin resistance. CE could mitigate insulin resistance by increasing insulin-induced 122 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment translocation of glucose transporter type 4 (GLUT4) to surface membrane and elevating upstream tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) (Sim et al., 2009). Notably, insulin signalling pathway is impaired in AD brain (Frölich et al., 1998; Reger et al., 2008; Rivera et al., 2005) and higher levels of insulin resistance markers are associated with poor episodic and working memory in AD and MCI cases (Ma et al., 2009). In addition, intranasal insulin administration could improve episodic and working memory in older women (Krug et al., 2010). Hence, there is a potential role of CE in enhancing insulin sensitivity and downstream signalling pathways which further affects cognition. As CE composition comprises different types of proteins, peptides and amino acids, it is difficult to identify the active compound that accounts for its beneficial effects in the various studies mentioned. So far, the two most promising compounds identified are carnosine (β-alanyl-L-histidine) and its derivative anserine (β-alanyl-1-methyl-L-histidine) as they exist in high concentration in human brain and confer neuroprotection (Artun et al., 2010; Boldyrev et al., 2004; Kang et al., 2000; Kohen et al., 1988). These imidazole dipeptides are natural antioxidants in meat extract and have the capability to inhibit tissue damage (MacFarlane et al., 1991; Nagai et al., 1990). Carnosine crosses the blood brain barrier (BBB) and acts as a precursor to histamine which is a neurotransmitter implicated in combating stress (Shen et al., 2010). It elevates DG-LTP in rats (Suer et al., 2011) and enhances performance in water maze test in aged rats (Acosta et al., 2010). Furthermore, it has an antidepressant-like effect similar to CE (Tomonaga et al., 2008) and accelerates metabolism of the stress hormone cortisol (Nagai et al., 1990). In spite of these, CE is a complex extract and is likely to have other active compounds that can possibly work synergistically to produce these beneficial effects. 123 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment 4.2. Objective of study Although CE has been shown to improve mental performance in human, the underlying mechanisms still remain unclear. As I have shown that the signaling molecules involved in learning and memory such as NMDAR and the downstream mediators particularly PKC, CaMKII, ERK and CREB, can be downregulated by apoE4 isoform in vivo, I wanted to investigate the effects of CE on the NMDAR-induced signaling cascades using cellular models. In this context, I utilize apoE-transfected neuronal cell lines to examine the cellular responses to the CE treatment and hypothesize that this compound may upregulate NMDAR-coupled downstream signalling in vitro particularly in apoE4-expressing stable cell lines. The aim and rationale for this study are as follows: Aim 1: To investigate the effects of CE on cellular responses differentially regulated by apoE isoforms CE has long been taken as a supplement in boosting mental health. Furthermore, CE has been linked to insulin signalling pathway that affects cognition. However, its effects in conjunction with apoE isoforms have not been studied yet. Thus, we seek to identify novel molecular mechanisms that may underlie its potential role in cognition. Given that our previous studies showed that CE inhibits PDE in hApoE-transfected cell lines, we seek to investigate the PKA pathway downstream of PDE and correlate it to NMDAR activity. In addition, we also explore the possible role of PKC in CE-induced cellular responses regulated by apoE isoforms. 124 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment 4.3. Results To investigate the differences between apoE isoforms in response to CE treatment, I harness the proliferative capabilities of the stably apoE-transfected neuronal cell lines derived from apoE-null mice. Although primary neuronal cultures will be more reflective of the in vivo events, the yield is lower and the preparation is more time-consuming. Hence, I wanted to verify the effects of CE on the signalling proteins that are involved using the cell lines before the primary cultures are utilised for future studies. 4.3.1. Chronic expression of apoE in huApoE stable cells Figure 4.1. Protein expression level of apoE in mock and apoEtransfected neurons. Western blot analysis of apoE in mock, huApoE3 and huApoE4 neuronal cells with β-actin as loading control. Densitometry of apoE relative to β-actin and was performed using NIH ImageJ software. Each value represents mean value ± SEM for individual sample (n=4). For statistical analysis, Student's t-test was used to test for significance (* p < 0.05). Before I looked at the effects of CE treatment on the cells, I first determined the apoE expression in the stable cells to see if they constantly express apoE that recapitulates the chronic apoE expression in apoE-KI mice. Although the apoE expression in the stable cell lines remains constant over time, the apoE expression in apoE4 neurons is reduced by 36%. Hence, this cellular model is able to recapitulate the level of apoE expression in vivo since apoE4-KI mice also express apoE at a lower level compared to apoE3-KI mice. Mock 125 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment neuronal cell line which does not express any apoE protein is used as a negative control to determine whether the cellular responses are due to loss-offunction or gain-of-adverse functions. The treatment of the apoE stable neuronal cells with CE at 100 µg/mL overnight was previously determined by MTS assay as it maintained the cell viability of all three cell lines at 100% (Siew, 2011). As the basal level of apoE expression between apoE3- and apoE4 transfected neurons are already different with the latter expressing lower amount of apoE even before CE treatment, comparisons in intracellular signalling are made between nontreated and treated cultures within the same cell lines. 4.3.2. Effects of CE treatment on expression of human apoE in huApoE stable cells Figure 4.2. Protein expression level of apoE in mock and apoEtransfected neurons. Western blot analysis of apoE expression in mock, huApoE3 and huApoE4 neuronal cells before (white bar) and after (grey bar) CE treatment with βactin as loading control. Densitometry of apoE relative to β-actin was performed using NIH ImageJ software. Relative value for treated cells was normalized against untreated cells and each value for post-treatment cells represents mean value ± SEM relative to pre-treatment cells for individual sample (n=4). For statistical analysis, Student's t-test was used to test for significance (* p < 0.05). After CE treatment, I examined the apoE expression level in the cells again to see if the expression levels change with treatment. Immunoblotting confirms 126 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment there is still undetectable level of human apoE expression in mock neurons regardless of CE treatment. Surprisingly, although basal apoE expression level in apoE4 neurons are already lower than that of apoE3 neurons, the apoE4 expression reduced further by 56% compared to expression level before treatment whereas apoE3 expression level remains the same after treatment. This may help us to interpret the subsequent observations whether CE treatment aggravates the conditions due to loss-of-functions in apoE4 neurons or helps to diminish the gain-of-adverse functions in apoE4 neurons. 4.3.3. Effects of CE treatment on basal intracellular calcium level in huApoE stable cells Figure 4.3. Basal intracellular calcium ion concentration in mock and apoE-transfected neurons. Intracellular Ca2+ level measured in mock, huApoE3 and huApoE4 neuronal cells before (white bar) and after (grey bar) CE treatment. Basal intracellular Ca2+ concentration is expressed as absolute fluorescence unit measured at excitation at 495 nm and emission at 516 nm. Each value for pre- and posttreatment cells represents mean value ± SEM of five repeated assays (N=5). For statistical analysis, Student's t-test was used to test for significance (** p < 0.01). 127 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment I hypothesized that CE treatment may help to regulate signalling pathways involved in learning and memory particularly NMDAR-induced downstream signalling. Since NMDAR plays a role in modulating intracellular Ca2+ ion concentration, I wanted to measure the intracellular Ca2+ ion level before and after CE treatment using direct calcium assay. There is a significant decrease in basal intracellular Ca2+ concentration after CE treatment in both mock (-10%) and huApoE4-transfected neurons (-11%). In contrast, basal intracellular level Ca2+ level increased by 13% in CE-treated huApoE3-transfected neurons. This implies that CE treatment may affect Ca2+ homeostasis in the stable cells per se as even the intracellular Ca2+ concentration in mock neurons changes after treatment. These changes may subsequently lead to alterations in the Ca2+-sensitive kinases such as PKA, PKC, αCAMKII that are coupled 128 to NMDAR-dependent activity. Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment 4.3.4. Effects of CE treatment on phosphorylation of NR1 subunit in huApoE stable cells Figure 4.4. Phosphorylation level of NR1 subunit in mock and apoEtransfected neurons. Western blot analysis of phosphorylated NR1 at serine residues (A) p-NR1 S896 and (B) p-NR1 S897 and NR1expression in mock, huApoE3 and huApoE4 neuronal cells before (white bar) and after (grey bar) CE treatment with β-actin as loading control. Densitometry of p-NR1 at different serine residues relative to NR1 was performed using NIH ImageJ software. Relative value for treated cells was normalized against untreated cells and each value for post-treatment cells represents mean value ± SEM relative to pre-treatment cells for individual sample (n=4). For statistical analysis, Student's t-test was used to test for significance (** p < 0.01, *** p < 0.001). Before I went on to investigate the underlying mechanisms upon CE treatment, I examined the phosphorylation profile of the mandatory NR1 subunit after CE treatment as NMDAR-induced Ca2+ influx is one of the main source of Ca2+ ion. I find a significant increase in phosphorylation of NR1 subunit at residue serine 896 by 33% in CE-treated apoE3-tranfected neurons (Figure 4.4A). Unexpectedly, there is a significant drop in phosphorylation of NR1 129 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment S897 (-70%) in apoE4-transfected neurons upon treatment (Figure 4.4B). Since the mock neurons are not affected, this implies that the differential regulation of the phosphorylation of NR1 S896 and S897 by CE is mediated via apoE in apoE3 and apoE4 stable cells. This also suggests that there could be a beneficial effect of CE on apoE3 neurons as upregulation of NR1 S896 increases NR1 subunit surface expression and enhance synaptic transmission (Tingley et al., 1997; Zou et al., 2000). However, the downregulation of NR1 S897 in apoE4 neurons may have an opposite effect on the receptor expression. 130 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment 4.3.5. Effects of CE treatment on PKA and PKC signalling profile in huApoE stable cells Figure 4.5. Protein expression level of PKA-Cα and PKCα in mock and apoE-transfected neurons. Western blot analysis of (A) phosphorylated PKA-Cα (p-PKA-Cα) and PKACα; (B) phosphorylated PKCα (p-PKCα) and PKCα in mock, huApoE3 and huApoE4 neuronal cells before (white bar) and after (grey bar) CE treatment with β-actin as loading control. Densitometry of (A) p-PKA-Cα relative to PKA-Cα; (B) p-PKCα relative to PKCα was performed using NIH ImageJ software. Relative value for treated cells was normalized against untreated cells and each value for post-treatment cells represents mean value ± SEM relative to pre-treatment cells for individual sample (n=4). For statistical analysis, Student's t-test was used to test for significance (* p < 0.05, ** p < 0.01). I next examined the upstream Ca2+-sensitive kinases that phosphorylate NR1 S896 and S897 i.e. PKC and PKA respectively to see if these molecules correspond to their downstream targets and also the intracellular Ca2+ ion after treatment. There is a small but significant decrease (-11%) in PKA-Cα activity in CE-treated apoE4-transfected neurons (Figure 4.5A) whilst PKCα activity 131 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment increases by 18% in apoE3-transfected neurons (Figure 4.5B). These observations resembles phosphorylation of NR1 serine residues as expected. This also implies that CE treatment is able to enhance PKC signalling in apoE3-KI mice but may not be able to rectify the decreased PKC phosphorylation as seen in apoE4-KI mice at old age. 132 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment 4.3.6. Effects of CE treatment on GluR1 and αCaMKII expression profile in huApoE stable cells Figure 4.6. Protein expression level of GluR1 subunit and αCaMKII in mock and apoE-transfected neurons. Western blot analysis of phosphorylated serine residues of GluR1 subunit. (A) Phosphorylated GluR1 S845 (p-GluR1 S845) and (B) phosphorylated GluR1 S831 (p-GluR1 S831) and (C) phosphorylated αCaMKII (p-αCaMKII) and αCaMKII of mock, huApoE3 and huApoE4 neuronal cells before (white bar) and after (grey bar) CE treatment with β-actin as loading control. Densitometry of (A) p-GluR1 S845 relative to GluR1 and (B) p-GluR1 S831 relative to GluR1 and (C) p-αCaMKII relative to αCaMKII was performed using NIH ImageJ software is shown on the right panel. Relative value for treated cells was normalized against untreated cells and each value for posttreatment cells represents mean value ± SEM relative to pre-treatment cells for 134 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment individual sample (n=4). For statistical analysis, Student's t-test was used to test for significance (* p < 0.05, ** p < 0.01, *** p < 0.001). As phosphorylation of GluR1 subunit and αCAMKII was shown to be affected in apoE4-KI mice, I wanted to see whether CE treatment can reverse the downregulation of GluR1 subunit and αCAMKII phosphorylation in apoE4 neurons. As shown by western blot analysis, level of phosphorylated GluR1 S845 and GluR1 S831 decreases significantly in apoE4-transfected neurons by 49% and 40% upon CE treatment respectively (Figures 4.6A and B). However, there is also an unexpected 25% reduction in treated mock neurons in GluR1 S831 phosphorylation (Figure 4.6B). By contrast, phosphorylation of GluR1 S831 is significantly enhanced (37%) in apoE3-transfected neurons (Figure 4.6B) compared to untreated cells. On the other hand, αCaMKII Thr286 autophosphorylation demonstrates a corresponding reduction after treatment in mock (-49%) suggesting that αCaMKII is the upstream regulator of GluR1 S831 since there was no change observed in PKC phosphorylation after CE treatment. There is also decrease in αCaMKII phosphorylation of apoE4transfected neurons (-26%) whereas apoE3-transfected neurons show a 36% increment in phosphorylated αCaMKII 135 level (Figure 4.6C). Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment 4.3.7. Effects of CE treatment on ERK-CREB signalling in huApoE stable cells Figure 4.7. Protein expression level of ERK and CREB in mock and apoE-transfected neurons. Western blot analysis of (A) phosphorylated ERK1/2 (p-ERK1 and pERK2) and ERK1/2 and (B) phosphorylated CREB (p-CREB) and CREB expression in mock, huApoE3 and huApoE4 neuronal cells before (white bar) and after (grey bar) CE treatment with β-actin as loading control. Densitometry of (A) p-ERK1 relative to ERK1, p-ERK2 relative to ERK2 and (B) p-CREB relative to CREB performed using NIH ImageJ software is shown on the right panel. Relative value for treated cells was normalized against untreated cells and each value for post-treatment cells represents mean value ± SEM relative to pre-treatment cells for individual sample (n=4). For statistical analysis, Student's t-test was used to test for significance (* p < 0.05, ** p < 0.01, *** p < 0.001). 136 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment It has been shown that PKC and PKA activity is coupled to ERK1/2 phosphorylation in vivo (Ji et al., 2009). Since the NMDAR-coupled signalling pathways involving the kinases examined all converge in the transcription factor, CREB (Mao et al., 2007; Matynia et al., 2002), I further verify the phosphorylation of CREB in the cells after CE treatment. ERK1/2 and CREB activities show similar trends in all three cell lines after CE treatment. There is a significant decrease in phosphorylated ERK1 (-37%), ERK2 (-26%) and CREB (-20%) in mock neurons. ApoE4-transfected neurons also demonstrate a significant reduction in phosphorylated ERK1 (-38%), ERK2 (-25%) and CREB (-22%) while apoE3-trasnfected neurons show enhanced ERK1 (20%), ERK2 (27%) and CREB (53%) phosphorylation (Figures 4.7A and B). In conclusion, CE treatment upregulates the phosphorylation of proteins implicated in synaptic plasticity including essential subunits of glutamatergic receptors, PKC, αCaMKII, ERK and CREB in apoE3 neurons. This suggests that CE may benefit apoE3 carriers which account for the majority of human population. Although CE treatment decreases phosphorylation of PKA, αCaMKII, ERK and CREB in apoE4 neurons, it remains to be verified whether this is detrimental or beneficial to the cells as apoE4 has been known to cause neurotoxicity and cell death via dysregulation of Ca2+ homeostasis (Tolar et al., 1999; Veinbergs et al., 2002). Hence, CE treatment may be able to temper some unfavourable aspects of apoE4 such as excitotoxicity by decreasing intracellular Ca2+ concentration. 137 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment 4.4. Discussion 4.4.1. Different intracellular calcium responses in huApoE-transfected neurons and downregulation of apoE4 expression upon CE treatment Basal intracellular Ca2+ ion concentration in apoE3-transfected neurons increases significantly but decreases in mock and apoE4-transfected neurons after CE treatment. Intracellular calcium level plays a key role in neurite outgrowth as primary cultures treated with BAPTA which promotes Ca2+ influx demonstrated increased axon extension. In contrast, treatment with BayK, a chemical that chelates intracellular calcium had the opposite effect (Qiu et al., 2004). It was found that in Neuro-2a cells transfected with apoE3 and supplemented with β-VLDL or HDL from CSF, the neurons exhibited greater sprouting compared to the same cell line transfected with apoE4 (Bellosta et al., 1995). It is possible that the neuronal sprouting is mediated by regulation of intracellular Ca2+ level. Based on these observations, upregulation of Ca2+ influx in apoE3-transfected neurons by CE may promote neuronal growth and trigger Ca2+-dependent signalling pathways whereas the opposite occurs in mock and apoE4-transfected neurons. There is a concurrent decrease in basal intracellular Ca2+ concentration (Figure 4.3) along with apoE4 expression level (Figure 4.2). It is tempting to propose that the decline in basal intracellular Ca2+ ion concentration is due to loss-offunction and that certain amount of apoE expression is required to mediate the Ca2+ influx or release from intracellular calcium stores. However, the basal intracellular Ca2+ levels in huApoE4-transfected and mock neurons are comparable before treatment thus refuting this hypothesis. Therefore, this seems to be the sole impact of CE treatment on both cell lines. It is possible that CE compound is able to regulate Ca2+ homeostasis in both apoE-independent (mock neurons) and apoE-dependent manners (apoE expressing neurons). Ca2+ homeostasis is one of the eminent mechanisms that prolongs neuronal health and synaptic function (Bezprozvanny, 2009; Kumar et al., 2009; Wang et al., 2013). It mainly involves the balance between the 138 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment CaMKII activity in phosphorylating the postsynaptic proteins to strengthen synapses and CaN (phosphatase) that does the otherwise (Bezprozvanny and Hiesinger, 2013). The steady-state levels of CaMKII activity is predominantly maintained by extracellular source of Ca2+ influx via postsynaptic AMPA and NMDA channels (Kavalali et al., 2011), and alternatively via the neuronal store-operated Ca2+ entry (nSOC) pathway (Putney, 2003). On the other hand, CaN is activated by increased Ca2+ release from intracellular stores via 1,4,5triphosphate receptors (InsP3R) and ryanodine receptors (RyanR), increased Ltype VGCC-mediated or reduced NMDAR-dependent Ca2+ influxes (Gant et al., 2006; Kumar et al., 2009; Toescu and Verkhratsky, 2007). Generally, the changes in the phosphorylation status of the protein molecules in CE-treated cells indicate an unbalanced kinase-phosphatase activities due to a change in the source of Ca2+ supply. The reduced phosphorylation of αCaMKII, GluR1 subunit and ERK in CE-treated mock and apoE4 neurons may be caused by a shift in the balance from kinases to phosphatases which can be attributed to reduced NMDAR-mediated Ca2+ influx or increased Ca2+ release from intracellular stores (Gant et al., 2006; Kumar et al., 2009; Toescu and Verkhratsky, 2007). Although it is possible there is less NMDA channel activation and hence less Ca2+ influx, it is unlikely that there is increased Ca2+ release from endoplasmic reticulum (ER) because I observed a concurrent downregulation of intracellular Ca2+ level after CE treatment. The only plausible explanation is there may be alternative system that is suppressing the ion release from the calcium stores. In the presence of apoE3, CE treatment shifts the balance in Ca2+ regulation by increasing extracellular ion influx to activate αCaMKII while downregulating intracellular Ca2+ release and hence CaN. The role of CaN in modulating Ca2+ homeostasis in CE-treated cells awaits verification in future investigations. ApoE4 has been associated with dysregulation of Ca2+ homeostasis to exert neurotoxicity effect in vitro (Tolar et al., 1999; Veinbergs et al., 2002; Wang and Gruenstein, 1997). The potentiation of apoE4 on the rise in the cytosolic calcium and cell death in a dose-dependent manner seems to be mediated via extracellular Ca2+ influx through plasma membrane calcium channels and was 139 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment not influenced by inhibitors of intracellular Ca2+ reserves (Veinbergs et al., 2002). More importantly, this calcium elevation can be blocked by pretreating the cells with LRP-associated protein 1 (RAP), an antagonist of LRP1. This strongly suggests that apoE binds to its receptors to trigger intracellular signalling events that induce Ca2+ influx (Tolar et al., 1997; Wang and Gruenstein, 1997). In this study, CE compound downregulates expression levels of apoE4 and its receptor, LRP1 (supplementary figure 2) in apoE4 cells which may in turn helps to diminish the neurotoxic effects of apoE4. Taken together, CE treatment may modulate Ca2+ homeostasis by directly affecting the Ca2+ influx via calcium channels (apoE-independent) and also in an apoEdependent manner by reducing the triggering of Ca2+ influx via binding of apoE4 to LRP1. As aforementioned, neuronal apoE4 is excitotoxic compared to apoE3. ApoE4, but not apoE3 significantly increases resting calcium, calcium response to NMDA and causes cell damage via NMDAR-dependent calcium influx which could be abolished by antagonist MK801 (Qiu et al., 2003). In mock and apoE4-transfected cell lines, there is an inherently higher level of basal intracellular Ca2+ ion than that of apoE3 which could signify chronic exposure of neurons to elevated Ca2+ ion concentration and increased sensitivity to excitotoxicity. It is possible that CE treatment can reduce this sensitization to excitotoxicity by reducing the basal intracellular Ca2+ level in these neurons. Excitatory neurotransmission is a subtle process involving optimal influx of Ca2+ ions without causing excitotoxicity, as persistent intracellular Ca2+ overload leads to mitochondrial dysfunction and cell death (Dawson et al., 1991; Naskar et al., 1999). Since Ca2+ ion also acts as a second messenger that triggers multiple pathways, it is important to tightly regulate the amount of Ca2+ influx such that the normal signal transmission is not perturbed and yet does not cause excessive activation. 140 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment 4.4.2. Upregulation of PKC pathway in huApoE3-transfected neurons and downregulation of PKA pathway in huApoE4-transfected neurons upon CE treatment. This study on CE treatment is an extension of previous findings in our laboratory whereby changes in PDE mRNA level were observed (Siew, 2011). In this study, CE treatment induces a decrease in PKA-Cα phosphorylation (Figure 4.5A) which could imply that there is a decrease in cAMP level due to increased PDE activity in huApoE4-transfected cell lines. This was unexpected as previous findings saw a downregulation of PDE8A mRNA level in apoE4 neurons after treatment. Since there was no change in mRNA level of PDE4 which is also cAMP-specific in all the three cell lines, it is unlikely that this PDE subtype is implicated in CE treatment. However, the possibility of another cAMP-specific PDE i.e. PDE7 and other dual-substrate PDEs that may contribute to changes in PKA activity in apoE4-transfected neurons are not ruled out. Furthermore, it is suspected that the changes in PDE expression is more of a consequence rather than a causative factor as some PDE subtypes, for example PDE4, are downregulated along with their upstream cAMP and PKA in AD brains (Bender and Beavo, 2006; Sette and Conti, 1996). Hence, the PDE mRNA changes observed may be downstream of the perturbation in cAMP/PKA signalling instead of upstream. Decreased synthesis of cAMP from ATP by ACs may also affect cAMP level and subsequently PKA activity. This suggests the possible roles of other cAMPspecific PDEs and dual-substrate PDEs in regulating CE-induced PKA signalling in apoE-transfected neurons. It is unlikely that downregulation of PKA pathway in huApoE4 neurons is due to the loss-of-function with respect to the reduced apoE expression after CE treatment. This is because there is no change in PKA phosphorylation and its downstream signalling in mock neurons which do not express apoE at all. The unchanged PKA-Cα and PKCα activity (Figures 4.5A and 4.5B) in mock neurons after treatment allows us to postulate that the upregulation of PKCα and PKA-Cα pathway in CE-treated huApoE3- and huApoE4-transfected 141 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment neurons respectively are due to differential effects of apoE isoforms in these cell lines. As expected, there is a concomitant decrease in phosphorylation of two targets of PKA-Cα (Figure 4.5A), NR1 S897 (Figure 4.4B) and GluR1 S845 (Figure 4.6A) in CE-treated apoE4 neurons. This phenomenon can also be mediated via phosphatase CaN which opposes actions of PKA by dephosphorylating its substrates and therefore suppressing NMDAR activity. Notably, the magnitude of decrease in phosphorylations of both NR1 S897 and GluR1 S845 are greater than that of PKA-Cα itself. It is tempting to propose that PKA-Cα acts as a sole molecular switch that dictates the phosphorylation of NR1 S897 such that a minor alteration in PKA-Cα activity has a huge effect on its donwnstream target. However, there may be other modulators that contribute to the downregulation of NR1 phospho-S897 as described by others. In cerebellar neurons, it has been observed that pituitary adenylate cyclaseactivating polypeptide (PACAP), a neuropeptide that activates AC and increases concentration of cAMP upon binding to its receptors (Yaka et al., 2003), also increases phosphorylation of NR1 S897 (Llansola et al., 2004). In addition, another target of cAMP, Exchange Protein Activated by cAMP (Epac), activates Akt which in turn phosphorylates NR1 S897 (de Rooij et al., 1998; Mei et al., 2002). Hence, the greatly reduced phosphorylated NR1 S897 seen in this study could be due to decreased activation of other upstream regulators such as PACAP and Akt. Since PKA also phosphorylates and activates CREB, perhaps CREB activity is involved in PKA-dependent regulation of GluR1 S845 phosphorylation. This is based on a recent study that reported CREB, besides PKA-Cα and PKCα, indirectly affects phosphorylation of GluR1 S845 and GluR1 S831 via its downstream effectors such as BDNF (Middei et al., 2013). In my study, there is a similar percentage of reduction (20%) in CREB activity in both CE-treated mock and apoE4-transfected neurons (Figure 4.7B) but phosphorylation of PKA-Cα and GluR1 S845 remains unchanged in the former. This supports the 142 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment notion that CREB-mediated phosphorylation of GluR1 S845 requires and may be a result of positive feedback of PKA activity. Hence, a slight decrease in PKA-Cα activity (-17%) (Figure 4.5A) is able to cause a reduction in GluR1 S845 phosphorylation almost by half in apoE4-transfected neurons after treatment (Figure 4.6A). Notably, the decreased phopshorylation of GluR1 S831 (Figure 4.6B) mimics that of αCaMKII (Figure 4.6C) but not of PKCα (Figure 4.5B) in CE-treated mock and huApoE4-transfected neurons even though GluR1 subunit can be phosphorylated at S831 by both upstream regulators PKCα and αCaMKII. Thus, changes in GluR1 S831 phosphorylation are mediated via αCaMKII instead of PKCα activity. In other words, PKCα pathway may not be implicated in CE treatment of mock and huApoE4-transfected neurons. Although both cell lines respond with a similar decrease in intracellular Ca2+ ion upon treatment, the percentage of decrease in the Ca2+-sensitive αCaMKII of mock is almost double that of apoE4 neurons (Figure 4.6C). The difference between these two cell lines is that there is a total absence of apoE in mock whereas apoE4 is still expressed albeit in minute amount (50% of non-treated apoE4 neurons) after treatment (Figure 4.2). Perhaps interaction between CE and apoE is necessary for mediating the binding of Ca2+/CaM to αCaMKII to activate the kinase and/or for sustaining the autophosphorylated form of αCaMKII. This implies that perturbation in αCaMKII activity is due to the loss-of-function of apoE in both mock and apoE4-transfected neurons. However, apoE3 expression level remains unchanged even though αCaMKII activation increases in treated apoE3 neurons. In this context, the extent of interaction between CE and apoE may not be the limiting factor in modifying αCaMKII activity as it may already have been at a saturated level with the amount of apoE expressed. Hence, the limiting factor in apoE3-transfected neurons would be intracellular Ca2+ level instead. On the other hand, the percentage of reduction in phosphorylation of GluR1 S831 in CE-treated apoE4-transfected neurons is almost twice that of treated 143 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment mock neurons (Figure 4.6B). It is tempting to speculate that the extensive decrease in phosphorylation of GluR1 S831 in apoE4 neurons is due to the additive effect of reduced activation of CREB and αCaMKII, both of which are upstream regulators of GluR1 S831. However, mock neurons also exhibit a similar decrease in CREB activity and the magnitude of reduction in αCaMKII Thr286 autophosphorylation is even 2-fold higher compared to that of apoE4 neurons after treatment. Hence, it is unlikely that CREB and αCaMKII are the main contributors in downregulating GluR1 S831 in apoE4 neurons. One of the explanations is that apoE4 which is still produced at a low level in apoE4-transfected neurons but completely absent in mock neurons after treatment may pose a detrimental effect in activating phosphatases (PP1/PP2) and CaN that can dephosphorylate GluR1 at S831 in conjunction with the decreased phosphorylation by its activators. The gain-of-adversefunction of apoE4 in increasing the susceptibility to dephosphorylation of GluR1 at S831 under the influence of CE remains to be investigated in the future. PKA and CaMKII phosphorylate GluR1 subunit of AMPAR simultaneously to facilitate its insertion into synapses during LTP (Esteban et al., 2003). In huApoE3-transfected neurons, there is an increased phosphorylation of PKCα, αCaMKII and their common target i.e. GluR1 S831 with no change in activity of PKA-Cα and its substrate GluR1 S845 after CE treatment (Figures 4.5 and 4.6). Phosphorylation of GluR1 S831 at naïve synapses induces LTP implying that CE could enhance synaptic plasticity in huApoE3 neurons via PKC and αCaMKII as both phosphorylates GluR1 S831 to enhance unitary conductance of GluR1 subunit and ultimately AMPA channel conductance (Barria et al., 1997; Benke et al., 1998; Derkach et al., 1999). Contrastingly, CE treatment causes a downregulation of phosphorylated αCaMKII and GluR1 S831 as well as phosphorylated PKA-Cα and GluR1 S845 in huApoE4-transfected neurons. This indicates a decrease in AMPA channel opening rate and single-channel conductance since phosphorylation of GluR1 S845 by PKA increases opening frequency. In addition, L-LTP induction increases AMPAR synthesis which is impeded by PKA inhibitor implying that this is one of the means of PKA in 144 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment modulating L-LTP (Nayak et al., 1998). Less phosphorylation by αCaMKII and/or increased dephosphorylation of GluR1 S831 at potentiated synapses followed by reduced phosphorylation by PKA-Cα and/or further dephosphorylation of GluR1 S845 in naïve synapses shifts the direction of synaptic plasticity towards LTD formation in CE-treated huApoE4-transfected neurons. Taken together, cellular responses to CE treatment are differentially regulated by apoE isoforms. 4.4.3. ERK-CREB signalling in CE-treated huApoE3- and huApoE4transfected neurons. PKA-induced association of Rap1 and B-Raf which triggers MAPK signalling pathway was first described in PC12 cells (Vossler et al., 1997). I observe a decrease in PKA-Cα phosphorylation of huApoE4-transfected neurons which may result in reduced ERK-CREB signalling (Figure 4.7) as suggested by many other in vivo studies. When inhibitory Rap1 mutant (iRap1) is expressed in mouse forebrain, forskolin or theta-burst stimulation could not elicit PKAdependent LTP response illustrating that PKA signalling intersects with MAPK pathway via Rap1 and B-Raf interaction in expression of synaptic plasticity (Morozov et al., 2003). It is evident that trends in CREB activity follow that of ERK1/2 in all three cell lines after CE treatment suggesting a direct consequence of enhanced phosphorylation of ERK1/2 in sustaining CREB activation. This is in agreement with the findings that MAPK activation is essential for mediating PKA signalling of CREB phosphorylation in CA1 neurons (Roberson et al., 1999) as PKA activity stimulates nuclear cotranslocation of MAPK to phosphorylate CREB at serine-133 (Impey et al., 1998; Patterson et al., 2001; Xing et al., 1996). It is prominent that the alterations in ERK-CREB signalling profile are almost identical in both mock and apoE4-expressing cell lines especially in their magnitude of decrement. The apoE expression in CE-treated apoE4transfected neurons is also reduced to a very low level which is almost comparable to that of mock neurons (Figure 4.2). This implies that apoE may 145 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment be essential in facilitating the activation of ERK-CREB pathway, which explains the increased ERK-CREB signalling observed in treated apoE3transfected neurons as there is sufficient apoE to enhance the ERK and CREB activity under the influence of CE. In other words, downregulation of ERKCREB pathway appears to be loss-of-function of apoE in triggering this signalling cascade. I believe that normal synaptic transmission induces transient glutamate signalling and increases intracellular Ca2+ level to activate downstream ERK1/2 in CE-treated apoE3-transfected neurons. However, over-activation of glutamate receptors is a potential excitotoxic stimulus that also leads to neuronal death via increased ERK1/2 activation which triggers immediate early gene transcription (Bading and Greenberg, 1991; Murray et al., 1998; Xia et al., 1995) and causes seizure of activity-induced hippocampal neuronal death (Murray et al., 1998). In my study, CE treatment reduces intracellular Ca2+ level simultaneously decreasing ERK1/2 phosphorylation in mock and huApoE4-transfected neurons, suggesting that addition of this supplement may be able to temper glutamate receptor activity and protect against excitotoxicity. This is in agreement with many in vivo and in vitro studies which have demonstrated the role of ERK1/2 in excitotoxic injury. For instance, increased MAPK activity has been shown to mediate neuronal injury in animal models of ischemic brain injury which could be halted by specific inhibition of the upstream activators of ERK1/2 (Alessandrini et al., 1999; Yang et al., 1997). Similarly, inhibition of ERK1/2 provided protection against cell death in primary neuronal culture (Bading and Greenberg, 1991; Hu et al., 2013; Murray et al., 1998) and P19 cell lines (Grant et al., 2001). The effects of CE resemble that of the potent inhibitor of MEK upstream of ERK1/2, dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126) as CE affects both intracellular Ca2+ level and ERK phosphorylation. Unlike CE, U0126 did not affect intracellular Ca2+ responses but successfully prevented glutamateinduced cell death when added before or even hours after glutamate application in differentiated P19 cells expressing NMDARs, suggesting that its protective mechanism occurs downstream of NMDAR activation. This mode 146 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment of action implies that perhaps the CE-induced reduction in the ERK phosphoryation may be sufficient to protect the neurons against excitotoxic death per se. Furthermore, the inability of several other classes of protein kinase inhibitors such as KN62 and H-89 which inhibits CaMKII and PKA respectively, in exerting the same protective effect in differentiated P19 cells strongly suggests that the ERK1/2 pathway is relevant to glutamate excitotoxicity (Grant et al., 2001). This strengthens the specificity of ERK1/2 in mediating excitotoxic-induced cell death. It is also possible that CE reduces non-glutamate-receptor-induced oxidative stress by downregulating ERK1/2 phosphorylation as non-receptor-mediated oxidative toxicity could also be blocked by inhibition of the ERK1/2 pathway (Stanciu et al., 2000). CE treatment may promote the activation of phosphatase which dephosphorylates ERK1/2 to stabilize the signalling pathway in the absence or at lowered apoE levels in mock and apoE4 neurons respectively, but not in apoE3 neurons. In other words, none or low apoE expression under the influence of CE decreases the susceptibility of the neurons to excitotoxicinduced cell death by deactivation of ERK pathway. A recent study reported that pretreatment of rat cerebellar granule neuron culture by a candidate drug for AD, bis(propyl)-cognitin, could elicit neuroprotection against glutamateinduced neurotoxicity by concurrently inhibiting MAPK/ERK, NO and activating PI3K/Akt pathway (Hu et al., 2013). This suggests a few alternative pathways that may be implicated in protection against excitotoxic injury which are worthy of further explorations in elucidating the protective effects of CE on apoE4 neurons. Contrastingly, an in vivo study demonstrated that ERK expression was up-regulated in neurons of rat hippocampal CA1 region for survival in response to glutamate-induced excitotoxic injury (Ortuño-Sahagún et al., 2013). This protective neuronal response could be reversed by U0126 which led to cell death when administered together with glutamate. Collectively, ERK1/2 activation appears to be a double-edged sword that induces either cell survival or death in a cell-dependent context. On one side, ERK1/2 contributes to neuroprotection as a cellular response to excitotoxic injury but on the other, this pathway can also be coupled to glutamate excitotoxicity to induce cell death. The mechanisms by which ERK1/2 147 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment mediates cellular functions under in vitro and in vivo conditions should be compared for any differences to fully elucidate the consequence of ERK1/2 activity in different configurations. GRFs upstream of ERK1/2 pathway are responsible for coupling active GTPbound Ras to Ca2+/CaM which in turn binds to and activates αCaMKII. This implies a close association between CaMKII and ERK1/2 activity which is consistent with my hypothesis that increased intracellular Ca2+ concentration enhances Ca2+/CaM level and subsequently αCaMKII activation, simultaneously triggering Ras-ERK signalling in CE-treated huApoE3transfected neurons. In apoE3 neurons, there may be higher amount of NMDAR-dependent Ca2+ influx as there is a concomitant enhancement in PKC-mediated NR1 S896 phosphorylation. Similarly, reduced NMDAR phosphorylation in treated huApoE4-transfected neurons is reflected by downregulation of PKA-dependent NR1 S897 phosphorylation and less Ca2+ influx leading to decreased αCaMKII and ERK1/2 activity. Meanwhile, phosphorylation of NR1 subunit in mock neurons is unchanged despite a reduced intracellular Ca2+ level, αCaMKII and ERK1/2 suggesting that diminished release of Ca2+ ions from intracellular stores may play a dominant role in this context. Otherwise, there may be less Ca2+ influx through other Ca2+- permeable channels such as AMPA since there is a downregulation of GluR1 S831 phosphorylation in mock neurons. The possibility of other ionotropic receptors such as kainate receptor and metabotropic receptors affecting intracellular Ca2+ responses are not excluded as well. Notably, overexpression of CaMKII induces long-term changes in AMPAR activity as activated CaMKII helps to incorporate AMPARs into synapses via exocytosis (Hayashi et al., 2000). However, this action of CaMKII can be blocked by inhibition of ERK by PD 98059 indicating that ERK activity is required for action of CaMKII on AMPAR (Zhu et al., 2002). Although my findings have demonstrated a good correspondence between αCaMKII, ERK1/2 and phosphorylation of GluR1 S831 in treated cell lines, it raises further issues in validation of the role of ERK1/2 in mediating αCaMKII148 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment induced activation of GluR1 subunit. Accumulating evidence has shown that ERK activity facilitates rapid changes in AMPAR transmission in hippocampal slices during synaptic plasticity. However, the key mediators that bridge the ERK-AMPAR signalling have yet to be identified. It is intriguing that PD 98059 also prevents pairing-induced NMDARdependent LTP suggesting that ERK may also play a role in this particular type of synaptic plasticity. RSKs are effectors of ERK that target upon CREB (Sweatt, 2001) and are present in postsynaptic density fractions together with NMDA receptor complexes (Husi et al., 2000; Suzuki et al., 1999). As all RSK isoforms terminate in a sequence that can bind to PDZ domains, ERK signalling may influence NMDA channel activity via interactions between RSK and NMDARs as well as PDZ-containing proteins such as PSD95 (Thomas and Huganir, 2004). I postulate that changes in ERK1/2 activity induced by CE will modulate the interactions between RSKs and the scaffolding protein bearing the PDZ-domains which in turn alters NMDAR functionality. This warrants further investigations to unravel the mechanism and the direct consequence of these interactions. In summary, although CE holds potential as a promising compound that may enhance NMDAR activation and downstream signalling pathways particularly in apoE3-transfected neurons, it seems to have an opposite effect on apoE4transfected neurons by downregulating apoE4 expression and NMDARdependent signalling cascades. Hence, the therapeutic effect of CE on cognition remains to be validated in vitro by using a more relevant cellular model i.e. in primary cultures and in vivo. 149 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment Types of stable cells Total apoE expression level before treatment Total apoE expression level Intracellular calcium concentration Phosphorylation of NR1 S896 Phosphorylation of NR1 S897 Phosphorylation of GluR1 S831 Phosphorylation of GluR1 S845 Phosphorylation of PKCα Phosphorylation of PKA-Cα Phosphorylation of αCaMKII Phosphorylation of ERK1/2 and CREB Mock NA huApoE3 ++ huApoE4 + NA No change Decreases Decreases Increases Decreases No change Increases No change No change No change Decreases Decreases Increases Decreases No change No change Decreases No change Increases No change No change No change Decreases Decreases Increases Decreases Decreases Increases Decreases Table 4.1. Recapitulative table on the findings of CE treatment (except first row) on mock, huApoE3 and huApoE4 stable cell lines. 150 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment Figure 4.8. Model of differential regulation of cellular responses to CE treatment by apoE isoforms. ApoE3 (blue circle) enhances NMDAR functionality upon CE (chicken extract) treatment which increases baseline intracellular Ca2+ level and triggers activation of αCaMKII and PKC. This in turn increases phosphorylation of downstream GluR1 S831, ERK1/2 and CREB. On the other hand, CE treatment is able to diminish NMDAR activity in the presence of apoE4 (red circle) and subsequently reduces basal intracellular Ca2+ concentration. This leads to decreased activity of PKA, αCaMKII, GluR1 subunit of AMPAR, ERK1/2 and CREB. 151 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment 4.3 Conclusion This study has demonstrated apoE4 isoform spatiotemporally modulates NMDAR subunit expression and activity, which may play a role in molecular mechanisms of memory during ageing. The expression profiles of NR1, NR2A and NR2B subunits exhibit age- and region-specific pattern with NR2A being affected the earliest implying that apoE4 increases susceptibility to NR2A subunit loss. When comparing across regions, hippocampal CA1 and CA3 appears to be more vulnerable to abnormal NMDAR expression level. We find a selective and differential downregulation of NMDAR including NR1 and NR2B subunits in old apoE4-KI mice and NR2A functionality at all ages. It is possible that NMDAR subunits interact with synapse-associated proteins such as PSD95 to regulate the distribution and density of glutamate receptor. Interestingly, NR1 expression and activity are upregulated at young age, where the latter is probably mediated via PKC pathway. This may be one of the underlying mechanisms accountable for enhanced LTP and behavioural performance previously observed in young apoE4-KI mice. Although NR1 hyperactivity may produce beneficial effect i.e. activation of molecules involved in learning and memory such as CaMKII, ERK and CREB, this phenomenon may be transient as NRHyper possibly leads to synaptic degeneration and NRHypo at old age (week 72). This subsequently downregulates NMDAR-coupled signalling molecules via PKC pathway resulting in cognitive deficits of aged female apoE4-KI mice. To date, most of the investigations on human AD brain involve post-mortem studies. This only allows the observation of the abnormal changes in NMDAR expression and the relevant signalling cascades at late stage of the disease. Although animal models may not exhibit phenotypes that fully resemble that of human AD, this ageing study utilizes apoE-KI mice and provides an insight into the molecular changes modified by apoE4, a high genetic risk that contributes to the anomalies observed in AD patients. Enhancing NMDARs in the aged individuals has been shown to improve memory, and ε4 carriers may require more emphasis in this aspect to restore their cognition since apoE4-KI mice are more susceptible to loss of NMDARs. This suggests that 152 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment interventions to rectify these aberrations even before the manifestation of AD symptoms may be a beneficial therapeutic strategy especially in ε4 carriers. Preliminary screening of CE compound on huApoE-transfected neurons has demonstrated a differential influence of apoE isoforms on cellular responses to CE treatment. The main pathway affected is αCaMKII signalling as we observe increases in intracellular Ca2+ responses together with αCaMKII, GluR1, ERK and CREB activations in CE-treated hApoE3-transfected neurons, in contrast to decreased Ca2+ level and activities of downstream molecules in both mock and huApoE4-transfected neurons after treatment. This could be due to lower apoE4 expression after CE treatment and possibly nullifying apoE4 function in the neurons as the degree of change is similar between mock and apoE4 transfectants. In addition, PKCα and its substrate NR1 S896 activities are upregulated in apoE3 neurons whereas phosphorylation of PKA-Cα and NR1 S897 is downregulated in apoE4 neurons. These changes are not observed in mock neurons indicating that apoE isoforms differentially modulate PKC and PKA pathways in response to CE treatment. Taken together, CE potentially acts as a compound that can enhance the signalling pathways implicated in synaptic plasticity in the presence of neuronal apoE3 but under the influence of detrimental neuronal apoE4, CE may be able to reduce apoE4 expression, NMDAR over-activation and the sensitivity of huApoE-transfected neurons to glutamate excitotoxicity thereby exerting neuroprotective effect to a certain extent. 153 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment 4.4 Future Directions The higher neuronal expression of apoE4 in aged apoE4-KI mice suggests the increased susceptibility to proteolysis which needs to be further investigated. There are numerous neurotoxic fragments that can be generated from proteolytic cleavage such as apoE4 Δ1-272, Δ272-299, Δ127-242. Two methods that can be utilised to validate the existence of these fragments is by mass spectrometry analysis and immunoprecipitation with anti-full-length apoE antibody and subsequent detection of the fragments. In addition, the apoE bands ranging in between 14 to 30 kDa are more intense in the detergent-solubilized pellets compared to supernatant of AD patients with ε4 allele (Huang et al., 2001). Hence, the insoluble fraction of the apoE4-KI mice can be further homogenized using higher percentage of salt and detergents such as nonidet P40, SDS or sodium deoxycholate for detection of apoE fragments. Region-specific phosphorylation of NMDAR subunits can be examined by isolating the hippocampal sub-regions from brain slices to correlate the temporal changes in total expression level to its activity in this study. One of the possible challenges is that many tissue samples need to be pooled to ensure sufficient protein concentration for a single load. Real-time polymerase chain reaction (PCR) analysis can be done to see if there is any corresponding alteration at mRNA level in relation to the changes in the protein expression during ageing. There are few possible scenarios: (1) there is a concomitant decrease in mRNA level in apoE4-KI mice which signifies that transcription of the proteins is decreased; (2) the mRNA level is upregulated in contrast to downregulated protein expression which implies that the synthesis of the protein may not be affected but may be degraded rapidly by proteases instead. In the second context, further proteasome activity assay can be done to verify if these proteins are more susceptible to proteolytic degradation. Subcellular localizations of NMDARs determine the fate of neurons as activation of synaptic and extrasynaptic NMDARs are coupled to different intracellular signalling pathways which either leads to neuroprotection or 154 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment excitotoxicity. This prompts an investigation of expression of NR2 subunits in different cellular sites by using a subcellular fractionation approach followed by extraction with Triton X-100 to separate the synaptic and extrasynaptic membranes (Goebel-Goody et al., 2009). After homogenization in sucrose buffer and centrifugation to remove the nuclear fraction and tissue debris, the resulting supernatant can be centrifuged again to yield a crude membrane fraction. This portion is further homogenized in sucrose buffer with Triton X100 and centrifuged to separate the synaptic membrane containing Triton X100-insoluble PSD proteins. Validation of this fraction includes probing for synaptic marker such as PSD95. The supernatant comprising detergent soluble proteins is considered as extrasynaptic membrane compartment. Although the majority of NR1 and NR2 subunits are detected in synaptic membrane fraction of mouse cortical tissue, a low level of NR2A is still expressed in the extrasynaptic membrane (Jiang et al., 2011a). There is surprisingly barely detectable amount of NR2B phosphorylation at three tyrosine residues in extrasynaptic membrane fraction of adult brain. This includes NR2B-Y1336 which is associated with extrasynaptic enrichment and is found at a minimum level in extrasynaptic fraction of P7 mice (Goebel-Goody et al., 2009). Nevertheless, this study utilizes cortical region of brain only and the expression of these proteins and their phosphorylated forms are still unknown in the hippocampal subfields of apoE4-KI mice which remains to be explored in future studies. The pathogenesis of AD originates in entorhinal cortex (Braak and Del Tredici, 2011; Velayudhan et al., 2013) and subsequently spreads to hippocampus and other parts of cortex. Although we have examined neocortex which covers most of the cortical area and observed a downregulation of NR2 subunits at old age, it would be interesting to extend the area of investigation into entorhinal cortex especially at younger ages so that early changes in NMDAR expression level can be detected that may contribute to progression of the disease. Since there is an increased NR1 phosphorylation in young apoE4-KI mice which may signify higher NMDA-induced activity, NRHyper activity should 155 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment induce an excessive Ca2+ influx which is not measured in this study. Electrophyisological studies can be deployed to observe if young apoE4-KI mice is sensitized to NMDAR activity at specific hippocampal subregions and measure intracellular Ca2+ concentration to verify that NRHyper possibly contributes to enhanced LTP or excitotoxicity. Interestingly, NR2A activity has been shown to be modulated by a selective antagonist naturally occurring in brain i.e. zinc. This metal ion is able to inhibit NR2A-containing NMDARs even at low (nM) concentrations (Paoletti et al., 1997; Traynelis et al., 1998) and the concentration in brain can exceed 10 to 20 µM when released into synaptic cleft upon stimulation (Vogt et al., 2000) as it is highly found at mossy fibre zone of hippocampus (Wenzel et al., 1997b). One of the intriguing findings is increased zinc concentrations (>300 µM) in AD brains and serum of apoE4 carriers with AD (Danscher et al., 1997; Deibel et al., 1996; González et al., 1999; Lovell et al., 1998) suggesting a synergistic role of apoE4 and zinc in AD pathogenesis. Hence, it is possible that high zinc level in brains of apoE4-KI mice persistently inhibits NR2Acontaining receptors leading to decreased activation of NR2A subunit which perhaps warrant further investigations in a follow-up project. ApoE4-KI mice have demonstrated reduced LRP1 and PSD95 expression levels at all ages. As both proteins are able to influence NMDAR functionality, we can inspect the regional distribution of these proteins to see whether CA1 region is more susceptible to loss of these NMDAR-associated proteins in relation to reduced NMDAR currents in hippocampal CA1 as the functional outcome in young apoE4-KI mice (Korwek et al., 2009). Besides CA1, NMDAR currents in other hippocampal subregions can be determined to encompass the major memory circuits including perforant and SC pathway that traverse DG, CA3 and eventually culminate in CA1, as different subregions such as CA1 and CA3 has been reported to exhibit different types of plasticity (Ling et al., 2012). The parallel decreases in LRP1, PSD95 and NR2A levels in apoE4-KI mice resemble that of MCI and AD brains (Kang et al., 2000; Pritchard et al., 2005; 156 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment Sultana et al., 2010; Vance et al., 2006). Thus immunoprecipitation studies can be done to determine whether there is reduced interactions between these three molecules that contribute to synaptic defects in apoE4-KI mice observed by others. It is intriguing that a pull-down assay with NR1 subunit is able to detect presence of LRP1 in primary cortical neurons indicating that there is binding between LRP1 and NR1 subunit in addition to NR2 subunits and PSD95 (Nakajima et al., 2013). All of these suggest that binding of apoE receptor to NR1 perhaps modulate effects of apoE on NMDAR functionality. However, whether there is less LRP1-NR1 interaction in apoE4-KI mice and its consequences remain to be elucidated. As these target proteins mentioned above are clustered at postsynaptic sites, postsynaptic density (PSD) fractions can be prepared by isolation of synaptoneurosome which is enriched in synaptosome with attached resealed postsynaptic entities (neurosome) (Quinlan et al., 1999; Villasana et al., 2006). Briefly, brain tissue is homogenized in sucrose buffer and then subjected to sucrose gradient centrifugation to obtain the synaptosomal fraction. The synaptosomes can be further solubilized with few detergents of different properties such as Triton X-100 and sarkosyl which are non-denaturing and denaturing detergents respectively and then centrifuged to extract different PSD fractions (Nakajima et al., 2013). Although SDS is another denaturing detergent commonly used in extraction of PSD fractions, tissue lysis has to be done on ice to minimize protein degradation and SDS easily precipitates out of solution at a temperature lower than 25°C. Hence, sarkosyl is more suitable for protein extraction under refrigerated conditions. (Nakajima et al., 2013). The challenges that may be encountered include loss of material during sucrose gradient centrifugation and thus a large quantity of brain tissue is required for the initial step (Villasana et al., 2006). Besides LRP1, αCaMKII also co-immunoprecipitates with PSD95 and NMDARs (Gardoni et al., 2001b). Since increased αCaMKII activity enhances PSD95 phosphorylation and disrupts PSD95-NR2A binding which may potentially affect the downstream transduction pathway, primary neurons isolated from apoE4-KI mice can be tested for its sensitization to NMDA by 157 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment stimulation of the channels and subsequent phosphorylation of PSD95. Interaction between NR2A and PSD95 can then be observed by immunoprecipitation studies. It is also important to determine whether there is an increased PKC activity in apoE4 primary neurons as a consequence of the pertubation in NR2A-PSD95 binding. Similarly, in vivo changes in αCaMKII and PKC signalling profiles of apoE4KI mice in our ageing study can be further explored if these changes affect association between CaMKII and NR2 subunits as well as the synaptic NMDAR availability. Although NR2 subunits modulate NMDAR function by a predominant tyrosine-phosphorylation-dependent mechanism, it has been found that PKC phosphorylates serine sites of NR2 subunits i.e. NR2ASer1416 and NR2B at Ser1303 as well as Ser1323 (Gardoni et al., 2001a; Liao et al., 2001). However, earlier studies either used a combination of PKC isozymes or a general activator of PKC to examine the phosphorylated sites. Hence, the exact PKC isozyme that targets the serine phosphorylation of NR2 subunits remains to be identified in the future. This can be done by inhibition of specific PKC isoforms and examination of changes in level of phosphorylated NR2 serine sites. Functional output in terms of LTP can subsequently be measured to validate the corresponding changes in PKCinduced synaptic plasticity under basal conditions. Since we have identified PKC pathway as one of the signalling cascades involved in differential regulation of NMDAR, AMPAR, ERK1/2 and CREB activity by apoE4, in vitro studies using hippocampal slices or primary culture isolated from the same animal model can be treated with specific PKCα inhibitor such as 1H-pyrrole-2,5-dione,3-(1-(3-(dimethylamino)propyl)-1Hindol-3-yl)-4-(1H-indol-3-yl)-[MESH] (Gö 6850) or introduced with antisense PKCα in order to verify the implication of this pathway in modulation of synaptic plasticity. To validate the key role of NMDAR-dependent Ca2+ influx in triggering the PKC pathway, hippocampal slices can be treated with AMPA channel blocker such as 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) to exclude the possibility of AMPAR in mediating the ion fluxes. Furthermore, the downregulation of PKC signalling in aged animals warrants future 158 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment investigations to determine the ability of PKCα activator in rescuing the activity of PKC and downstream targets in vivo and reversing the abnormal behavioural phenotypes of aged apoE4-KI mice observed by others. Examples of compounds that specifically activate PKCα at low concentration concentrations (1 to 100 nM) are sapintoxin D, mezerein, indolactam V and resiniferatoxin (Way et al., 2000). Although the pathways examined in our study culminate in CREB, this transcription factor further regulates the level of neurotrophic factor, BDNF, which in turn impacts upon MAPK pathway in a positive feedback loop. This results in activation of antiapoptotic factor, Bcl2, and suppression of proapoptotic factor, Bcl2-assoicated death promoter (Bad). Hence, changes in these factors perhaps are relevant to the ERK-CREB signalling that can be included in future studies. Other than ERK1/2 pathway, CaMKII can also regulate p38 and Jun-Nterminal kinase (JNK) activity (Enslen et al., 1996) which are implicated in signal transduction pathways involved in synaptic plasticity and are differentially modulated by apoE isoforms in vitro and in vivo (Hoe et al., 2005). The cytoplasmic domains of apoE receptors such as LRP1 and apoER2 bind to JNK-interacting proteins (JIPs) (Gotthardt et al., 2000) to regulate region-specific neuronal apoptosis in response to environmental stress, cell proliferation and morphogenesis during brain development (Kuan et al., 1999). Interestingly, phosphorylation of JNK is upregulated in hippocampal CA1 homogenate of young (12- to 20-week-old) apoE4-KI mice as opposed to that of apoE3-KI mice (Korwek et al., 2009). Hence, this suggests another candidate molecule that maybe implicated in apoE4 modification of LTP during ageing. However, this independent pathway is beyond the scope of the current thesis and can only be investigated as a new topic on its own. Given that efforts to rectify NMDAR decline such as drug therapy and caloric restriction are able to improve LTP in aged animals (Magnusson et al., 2010), it may be beneficial to introduce similar interventions among apoE4 carriers at 159 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment early stage of life since apoE4-KI mice demonstrated a higher susceptibility to NMDAR dysfunction along with downregulation of NMDAR-coupled signalling cascades particularly PKC and ERK pathway as they aged. In relation to changes in PDE mRNA level of apoE-transfected neurons observed in our previous laboratory findings, PDE protein level can be examined using PDE assay which can be optimized to work with cAMPspecific PDEs to correlate the changes in mRNA and protein expression. In addition, cAMP assay can be done to measure cAMP level which is regulated by PDE. One of the challenges that may be encountered is the low detection level of cAMP in samples. Under such circumstance, more cells need to be harvested and/or samples can be prepared in acetylated conditions for enhanced sensitivity to enable detection of cAMP concentration up to femtomolar per mL of sample. As the roles of apoE receptor in modifying CE-induced cellular responses are unknown, pharmacological approach can be utilised by inhibition of apoE receptors. For instance, antagonists of LDLR family members such as LRPassociated protein 1 (RAP) can be added to identify the apoE receptor implicated in mediating CE-induced changes. Since apoE receptors interact with NMDAR receptor complex to mediate Ca2+ influx and downstream signalling cascades, co-immunoprecipitation studies can be done to validate the effects of CE on association between apoE receptors and NMDARs which further affects Ca2+-mediated responses. CE treatment of apoE4-transfected neurons is able reduce NMDAR activity and intracellular Ca2+ responses to downregulate ERK1/2 pathway which may be implicated in excitotoxicity and cell death. However, whether these posttreatment effects are mediated via glutamate receptors remain to be elucidated. If NRHyper is indeed the underlying mechanism that predisposes apoE4transfected neurons to excitotoxicity, neuroprotective effects of CE against glutamate-induced cytotoxicity can be further validated using cell viability assays. Several in vitro neurotoxicity models involving hypoxia/hpoglycemiainduced and glutamate-induced excitotoxicity have demonstrated that 160 Chapter 4: Impacts of ApoE Isoforms In Cellular Responses To CE Treatment inhibition of PDE confers a neuroprotective effect by suppressing proapoptotic factor caspase-3 activity (Chen et al., 2007). Thus the potential of CE as a PDE inhibitor could exert a similar protective effect which can be tested by stimulation with a submaximal NMDA concentration and treatment with CE before or after NMDA application. Cell viability is then determined using assays such as (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium) (MTS) or caspase assay. It is also essential to verify whether hApoE-transfected neuronal cell lines recapitulate the crucial properties of glutamate excitotoxicity. 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Protein expression level of LP1 in mock and apoE-transfected neurons. Western blot analysis of LRP1 expression in mock, huApoE3 and huApoE4 neuronal cells before (white bar) and after (grey bar) CE treatment with βactin as loading control. Densitometry of LRP1 relative to β-actin was performed using NIH ImageJ software. Relative value for treated cells was normalized against untreated cells and each value for post-treatment cells represents mean value ± SEM relative to pre-treatment cells for individual 225 Appendix sample (n=4). For statistical analysis, Student's t-test was used to test for significance (*** p < 0.001). 226 [...]... apoE receptors in a dose-dependent manner Furthermore, apoE4 may impede apoE receptor recycling by sequestering the receptors intracellularly such that they are unable to be expressed on neuronal surfaces and trigger downstream signalling pathways upon ligand binding (Chen et al., 2010) Exogenously added apoE4 has been shown to reduce surface expression of apoE receptor 2 (apoER2) in primary neurons... morphological and behavioural changes independently of Aβ In other words, apoE4 is sufficient to impair synaptic plasticity per se which accounts for the learning and memory decline in AD 1.3 N- methyl- D- aspartate receptor (NMDAR): a key player in learning and memory NMDAR-dependent activity is an eminent mechanism underlying LTP induction which is fundamental to formation of learning and memory (Bear and Malenka,... percent of all dementia cases Global prevalence of dementia is estimated to be 3.9 % in people aged 60 years and above, and affects more than 25 million people in the world (Brookmeyer et al., 2007; Ferri et al., 2005; Wimo et al., 2003) Population ageing has become a global phenomenon In both developed and developing countries, the United Nation Ageing Program and the United States Centers for Disease... intensely than non-carriers and exhibit compensatory neural recruitment (Han and Bondi, 2008) This phenomenon occurs even in non-demented older adults suggesting ε4 carriers undergo compensatory changes in order to achieve the same performance as noncarriers (Kukolja et al., 2010; Tuminello and Han, 2011) However, the beneficial role of apoE4 in early life remains controversial as there are contradictory... increases in the liver in an agedependent manner (Gee et al., 2005) However, it is unclear whether apoE expression changes in the brain with ageing in human whereas conflicting observation have been made from animal studies In rodents, apoE expression level decreases more than five-fold in the cortex and hypothalamus (Jiang et al., 2001) but increases in the hippocampus (Terao et al., 2002) of aged mice... Structure of NMDAR subunit (A) The N- terminal domain (NTD) in the extracellular region consists of a tandem of large globular bi-lobed domains which is involved in allosteric modulation; and is linked to the agonist-binding domain (ABD) formed by two segments (S1 and S2) which binds glycine in NR1 and NR3 subunits and glutamate in NR2 subunits The transmembrane domain (TMD) is made of up three helices (M1,... conductance, gating properties such as channel open probability and deactivation kinetics (Cull-Candy and Leszkiewicz, 2004; Paoletti, 2011; Traynelis et al., 2010) For instance, diheteromeric NR2A and NR2B-containing receptors are highly permeable to Ca2+ and generate highconductance channel openings compared to NR2C and NR 2D- contaning diheteromeric receptors NR1/NR2A receptors also have a higher open probability... apoE4 may exert its effect before the onset of AD and the modulation is region-specific ApoE4 has been correlated to many hallmarks of AD including neurodegeneration ApoE4 produced by injured neurons is able to disrupt mitochondrial electropotential which in turn halts synaptogenesis and causes loss of synapto-dendritic connections in apoE mice models (Buttini et al., 1999; Li et al., 2004) In human... brains, apoE4 dose is inversely proportional to dendritic spine density in AD and aged normal controls (Ji et al., 2003) Another infamous hallmark of AD is the accumulation of Aβ and many studies have been done to dissect the role of apoE4 in Aβ metabolism ApoE4 can be found in amyloid plaques and neurofibrillary tangles (Riddell et al., 2008) and a combination of apoE4 and Aβ42 (the amyloidogenic... clearance (Deane et al., 2004; Van Uden et al., 2002) LRP1 undergoes proteolytic processing whereby the extracellular domain can be cleaved by β-secretase, BACE1, which also cleaves APP and generates the Aβ N- terminus, close to the transmembrane region resulting in the shedding of the extracellular domain (Quinn et al., 1999) The intracellular domain is then released from the plasma membrane by cleavage