Distribution and role of calcium independent phospholipase a2 in the brain

DISTRIBUTION AND ROLE OF PHOSPHOLIPASE A2 IN THE BRAIN LEE LI YEN (B.Sc), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ANATOMY NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgements ACKNOWLEDGEMENTS I wish to express my deepest appreciation and heartfelt thanks to my supervisor, Associate Professor Ong Wei Yi, Department of Anatomy, National University of Singapore, for suggesting this study topic and his constant and patient guidance and encouragement throughout the course of the study. During my postgraduate study at National University of Singapore, he has not only introduced me to a new research field but has also been a role model for hardwork and commitment to research. His deep and sustained interest and stimulating discussion have been most invaluable in the accomplishment of this thesis. I am very grateful to Professor Ling Eng Ang, former Head of Department of Anatomy, National University of Singapore, for giving me the opportunity to my postgraduate study at National University of Singapore. I am grateful to Professor Bay Boon Huat, Head of Department of Anatomy, for his full support in using the excellent research facilities. My deep indebtedness goes to Associate Professor Gavin Stewart Dawe, Department of Pharmacology, National University of Singapore, who gives me guidance and comment on my study and kindly offered help in teaching me the use of acoustic startle reflex apparatus. I am greatly indebted to Associate Professor Go Mei Lin, Department of Pharmacy, National University of Singapore, for their valuable suggestions and friendly help during this study. I Acknowledgements I am also acknowledging my gratitude to Mrs Ng Geok Lan and Mrs Yong Eng Siang for their excellent technical assistance; Miss Chan Yee Gek, and Mdm Yu Ya Jun for Electron Microscopy work; Mr Yick Tuck Yong for his constant assistance in computer work; Mdm Ang Lye Gek Carolyne and Mdm Teo Li Ching Violet for their secretarial assistance. I would like to thank all other staff members and my fellow postgraduate students and vital friends in Histology Lab, Neurobiology Programme, Centre for Life Science, National University of Singapore: Pan Ning, Lim Seok Wei, Jinatta Jittiwat, Tang Ning, Lee Hui Wen Lynette, Kim Ji Hyun, Ma May Thu, Poh Kay Wee, and Chia Wan Jie, for their help and support in many ways. It was a joyful experience working with all of them. I would like to take this opportunity to express my heartfelt thanks to my parents and sister for their full and endless support for my study. I am very grateful to Mr Chow Kum Seng Bernard and Mdm Cheong Hoon Moy Jean for providing generous financial support. Finally, I am greatly indebted to my husband, Mr Xu Congyuan for his understanding and encouragement during this study. II Table of Contents TABLE OF CONTENTS ACKNOWLEDGEMENTS …………………………………… …… .………………. I TABLE OF CONTENTS………………………………….……………… ………… III SUMMARY…………………………………………………….…………… … .VIII LIST OF TABLES………………………………………………………………… .XI LIST OF FIGURES…………………………………………………………… … XII ABBREVIATIONS…………………………………………………… … ………….XIII PUBLICATIONS……………………………….…………… ……….…… .…….XVI Chapter I INTRODUCTION 1.General introduction . 1.1. sPLA2 isoforms . 1.2. cPLA2 isoforms . 1.3. iPLA2 isoforms . 2.cPLA2 in normal brain . 11 2.1. Molecular genotype 11 2.2. Localization and distribution of cPLA2 . 13 2.3. cPLA2 functions . 13 2.4. Fatty acid released by cPLA2 . 14 2.5. cPLA2 inhibitors . 16 3.iPLA2 in normal brain 17 3.1. Molecular genotype 18 3.2. Localization and distribution of iPLA2 19 III Table of Contents 3.3. iPLA2 functions 20 3.4. Fatty acid released by iPLA2 . 22 3.5. iPLA2 inhibitors 23 4.cPLA2 in pathological diseases . 25 5.iPLA2 in pathological diseases 26 6. Roles of PLA2 in neurodegeneration 27 6.1. Cerebral ischemia 28 6.2. Alzheimer’s disease . 28 6.3. Parkinson’s disease . 30 7. Roles of iPLA2 in neuropsychiatric disorders 31 7.1. Schizophrenia . 31 7.2. Depression . 32 8. Animal models of neuropsychiatric disorders 33 8.1. Acoustic startle reflex . 33 8.2. Vacuous chewing movement 36 8.2.1. Dopamine receptors and antipsychotic side effects 38 8.2.2. Dopamine D2 receptor occupancy 39 8.2.3. Other factors affecting VCM 40 9. Roles of PLA2 in mitochondrial diseases . 41 Chapter II AIM OF STUDY . 43 Chapter III EXPERIMENTAL STUDIES . 46 Chapter 3.1 Distribution of Calcium-Independent Phospholipase A2 in the Rat Brain . 47 1. Introduction . 48 IV Table of Contents 2. Materials and methods 49 2.1. Animals . 49 2.2. Immunocytochemistry 49 2.3. Electro microscopy . 51 3. Results . 52 3.1. Light microscopy . 52 3.1.1. Distribution of iPLA2 in the normal forebrain of Wistar rat 52 3.1.2. Distribution of iPLA2 in the basal ganglia and cerebellum of Wistar rat brain 54 3.2. Electron microscopy . 56 4. Discussion . 58 Chapter 3.2 Role of Calcium-Independent Phospholipase A2 in Cortex Striatum Thalamus Cortex Circuitry-Enzyme Inhibition Causes Vacuous Chewing Movements in Rats 1. Introduction . 62 2. Materials and methods 64 2.1. Chemicals 64 2.2. Antisense oligonucleotides . 64 2.3. Rats and treatment . 68 2.4. Stereotaxic injections . 70 2.5. Behavioral assessment . 73 2.6. Western blot analysis . 73 3. Results . 74 3.1. Rats treated with BEL 74 3.2. Rats treated with MAFP 77 V Table of Contents 3.3. Rats treated with iPLA2 inhibitors plus benztropine 79 3.4. Rats treated with iPLA2 antisense oligonucleotides 81 4. Discussion . 83 Chapter 3.2 Role of Calcium-Independent Phospholipase A2 in Cortex Striatum Thalamus Cortex Circuitry-Enzyme Inhibition Causes Vacuous Chewing Movements in Rats 61 Chapter 3.3 Role of Phospholipase A2 in Prepulse Inhibition of the Auditory Startle Reflex in Rats . 89 1. Introduction . 90 2. Materials and methods 90 2.1. Rats and treatment . 90 2.2. Antisense oligonucleotides . 91 2.3. Acoustic startle reflex recordings . 92 2.4. Statistical analysis 95 3. Results . 96 3.1. Acoustic startle response 96 3.2. Prepulse intensity . 97 4. Discussion . 99 Chapter 3.4 Effects of Calcium-Independent Phospholipase A2 on Exocytosis in Rat PC12 Cells . 103 1. Introduction . 104 2. Materials and methods 105 2.1. Cell culture . 105 2.2. Patch clamp and capacitance measurements . 105 2.3. PC12 cell treatment 107 VI Table of Contents 2.4. Mitochondrial membrane potential assay . 108 3. Results . 109 3.1. Effects of BEL and factors affecting BEL-induced exocytosis on capacitance measurements in PC12 cells . 110 3.2. Effects of BEL on mitochondrial membrane potential 111 4. Discussion . 112 Chapter IV CONCLUSION 116 Chapter V REFERENCE . 121 VII Summary SUMMARY One class of phospholipase A2 (PLA2) that does not require calcium for its activity is the cytosolic calcium-independent PLA2 (iPLA2). In brain tissues, the basal expression and activity of iPLA2 is higher than either cytosolic calciumdependent PLA2 (cPLA2) or secretory calcium-dependent PLA2 (sPLA2) (Molloy et al. 1998; Farooqui et al. 1999), and its protein expression decreases during aging (Aid and Bosetti, 2007). iPLA2 is not only responsible for regulation of membrane phospholipid homeostasis (“housekeeping”) in cells (Balsinde et al. 1995), but also plays important roles in intracellular signal transduction. The present study aims to elucidate the role and distribution of iPLA2 in the brain. iPLA2 immunoreactivity was observed in structures derived from the telencephalon, whereas structures derived from the diencephalon were lightly labeled. The midbrain, vestibular, trigeminal and inferior olivary nuclei, and the cerebellar cortex were densely labeled. Immunoreactivity was observed on the nuclear envelope of neurons, dendrites and axon terminals using electron microscopy. Drug-induced tremulous oral movements have been linked to human Parkinsonian tremor (Salamore et al. 1998). Inhibitors of iPLA2 could induce vacuous chewing movements (VCM) in rats. Striatal injections of iPLA2 inhibitor, bromoenol lactone (BEL), resulted in significantly increased VCM in Wistar rats from to days after injection. Significantly increased VCM was also observed after intrathalamic or intracortical injections of BEL. In contrast, no significant effect was observed after BEL injection into the cerebellum. The effects of BEL VIII Summary were replicable using another PLA2 inhibitor, methyl arachidonyl fluorophosphonate (MAFP). These findings suggest that increased VCM after MAFP injection was because of inhibition of iPLA2. The observations with BEL and MAFP point to a role for inhibition of PLA2 enzymatic activity in VCM. Prepulse inhibition (PPI) of the acoustic startle reflex has been widely used as a model of sensory information processing and sensorimotor gating (Graham, 1975; Kumari and Sharma, 2002). Our results demonstrate that systemic administration of the non-specific PLA2 inhibitor, quinacrine, resulted in significantly decreased PPI of the auditory startle reflex at 76, 80, and 84 decibel (dB), compared to saline injected controls. Rats that received intrastriatal injection of antisense oligonucleotide to iPLA2 also showed significant reduction in PPI at prepulse intensities of 76 and 84 dB compared to scrambled sense injected controls. iPLA2 inhibition apparently has the same effect as increased dopamine receptor stimulation, in terms of its effects on PPI. These findings appear consistent with the previous findings that iPLA2 inhibition causes VCM. We have shown that mitochondrial permeability transition pore (MPTP) is essential in meditating the effect of BEL on exocytosis in PC12 cells. Blocking of MPTP with the inhibitors bongkrekic acid (BKA) and cyclosporine A (CsA) resulted in reduced exocytosis after intracellular addition of BEL. ptrifluromethoxy carbonyl cyanide phenyl hydrazone (FCCP) which depolarizes mitochondria transition potential and deplete mitochondrial calcium did not have any effects on exocytosis after addition of BEL. 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(2010) Protection of pancreatic beta-cells by group VIA phospholipase A2mediated repair of mitochondrial membrane peroxidation. Endocrinology 151:3038-3048 Zoratti M, Szabo I. (1995) The mitochondrial permeability transition. Biochim Biophys Acta 1241:139-176 174 [...]... mutation in exon 3 This mutation terminates out of frame in exon 4, resulting in a disruption of the calcium binding domain and loss of both activity domains encoded by exons 4 and 5 The identification of this mutation has important implications in mouse inflammatory models, which serve to define the physiological role of sPLA2 and ascertain its contribution in the inflammatory process 1.2 cPLA2 isoforms The. .. I Introduction documented that quinacrine might exert certain anti-inflammatory properties by acting as a phospholipase inhibitor (Horrobin et al 1977; Al Moutaery and Tariq, 1997) Quinacrine is one of the non-specific inhibitors of PLA2, inhibiting cPLA2 and iPLA2 activities from several sources (Ginsburg et al 1993; Hope et al 1993) Quinacrine appears in monkey brain 24 h after intrauterine administration... 1998) In the rat, forebrain and midbrain are very lightly stained with cPLA2α antibody; the hindbrain in contrast, contains many densely labeled nuclei The dorsal and ventral cochlear nuclei, the initial portions of the ascending auditory pathway, and the superior olivary nucleus, showed dense staining as well (Farooqui et al 2000b) Purkinje neurons of the cerebellar cortex itself were labeled, and deep... iPLA2ε (adiponutrin), iPLA2ζ (TTS-2.2), and iPLA2η (GS2) (Jenkins et al 2004) Four of the iPLA2 isoforms (iPLA2α, 8 Chapter I Introduction iPLA2β, iPLA2γ and iPLA2δ) are high molecular weight intracellular phospholipases (84 – 146 kDa), but the remaining three iPLA2 isoforms are between 28 – 57 kDa The most extensively studied enzyme for this group is iPLA2β, also known as Group VIA-2 PLA2, using the. .. (1968) reported the presence of PLA1 and PLA2 activities in rat brain slices and homogenates PLA2 activity was then determined in rat brain synaptosomes and in neuronal and glial cell preparations obtained from rabbit brain (Woelk et al 1978) 2.1 Molecular genotype cPLA2 was first characterized in platelets and macrophage cells (Clark et al 1995) The inferred sequence of murine cPLA2 is more than 90%... cPLA2 family consists of six intracellular enzymes, cPLA2α, cPLA2β, cPLA2γ, cPLA2δ, cPLA2ε and cPLA2ζ They are intracellular PLA2 of a high 6 Chapter I Introduction molecular weight (85 – 110 kDa), and play “housekeeping” roles by facilitating phospholipid remodeling and maintaining phosphatidylcholines (Balsinde et al 1996, 1997; Ramanadham et al 1999; Ma et al 2001) Much research has focused on cPLA2α... et al 1998) and this enzyme is activated in vivo by serum (Stewart et al 2002) cPLA2δ was identified from psoriatic skin (Chiba et al 2004) cPLA2δ, together with cPLA2ε and cPLA2ζ, were cloned and characterized from novel murine cPLA2, which forms a gene cluster with cPLA2β (Ohto et al 2005) The role of these paralogs of cPLA2 in brain tissues remains speculative The PLA2 activity of cPLA2α is characterized... studies that overexpression of iPLA2 has a protective effect on the mitochondria It is possible that inhibition of iPLA2 causes damage to the mitochondria and release of calcium resulting in exocytosis The results highlight the importance of normal “housekeeping” phospholipase A2 in maintaining the normal function of neurons X List of Tables LIST OF TABLES Table 1 Treatment group of Wistar rats……………………………………………... treatment protocols of Wistar rats…………………………………….92 Table 3 Summary of four different types of trials………………………………… 96 Table 4 Acoustic startle in baseline and treatment groups……………………… 97 XI List of Figures LIST OF FIGURES Figure 3.1 Distribution of iPLA2 in the normal forebrain of Wistar rat……………53 Figure 3.2 Distribution of iPLA2 in the basal ganglia and cerebellum of Wistar rat brain …………………………………………………………………………………... eicosanoids They play important roles in regulating signal transduction, gene transcription processes and also in inducing and maintaining acute inflammatory responses (Wolfe and Horrocks, 1994; Phillis et al 2006) Inhibition of cPLA2 was found to be favorable for neuronal survival after ischemia (Arai et al 2001) It was shown that the onset of hypoxia leads to a sustained increase in the concentration of intracellular . to the mitochondria and release of calcium resulting in exocytosis. The results highlight the importance of normal “housekeeping” phospholipase A 2 in maintaining the normal function of neurons microscopy 52 3.1.1. Distribution of iPLA 2 in the normal forebrain of Wistar rat 52 3.1.2. Distribution of iPLA 2 in the basal ganglia and cerebellum of Wistar rat brain 54 3.2. Electron. DISTRIBUTION AND ROLE OF PHOSPHOLIPASE A 2 IN THE BRAIN LEE LI YEN (B.Sc), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ANATOMY