Pathomechanistic characterization of DMT1 mediated manganese cytotoxicity implications in neurodegeneration

PATHOMECHANISTIC CHARACTERIZATION OF DMT1-MEDIATED MANGANESE CYTOTOXICITY: IMPLICATIONS IN NEURODEGENERATION TAI YEE KIT B. Sci (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2013 Declaration DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Tai Yee Kit 05 January 2013 I Acknowledgements ACKNOWLEDGEMENTS This thesis is the work of many hands. I wrote it myself, indeed, but never quite alone. So many others, the past and the present, by providence or design, helped shaped the work that I’ve done for the past four years. I could not have come thus far if it’s not for these people. This is my modest attempt to thank a few. To A/P Soong Tuck Wah, for being a great mentor, the one who made this journey possible. To A/P Lim Kah Leong, Dr.Katherine Chew, Dr.Ang Eng-Tat, Dr.Sharon Thio, Dr.Nupur Nag, Dr.Loh Kok Poh and Dr.Calvin Yeo, your discerning comments have been instrumental in making my thesis stronger. To Bryce Tan, Zhi Rong, Sophia Yang, Tan Fong, Pey Rou, Mui Cheng and many others at the National Neuroscience Institute and National University of Singapore, your support made a lot of this journey possible. To my family, who supported and believed in me. To my classmates, running mates, teachers and friends, who encouraged and challenged me to become the best of myself in every aspect of life. Creator God, You are indeed the intelligent designer. Thank you for life and life abundant. TAI YEE KIT II Table of Contents TABLE OF CONTENTS I II III VI VIII X Declaration Acknowledgements Table of Contents List of Figures and Tables Abbreviations Summary Chapter 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10 1.11 1.12 Introduction Overview Neurodegenerative Diseases Metals in Neurodegenerative Diseases Basal Ganglia and Movement Disorders Basal Ganglia and Metal Ion Toxicity Manganism Parkinson’s Disease (PD) Iron 1.8.1 Mechanism of Iron Transport and Cellular Metabolism 1.8.2 Transferrin-Mediated Iron Transport 1.8.3 Transport of Non-Transferrin Bound Iron (NTBI) 1.8.4 Divalent Metal Ion Transporter (DMT1) 1.8.5 DMT1 and Neurodegenerative Disease 1.8.6 Cellular Iron Storage - Ferritin 1.8.7 Labile Iron Pool (LIP) and Cellular Iron Regulation by IRP/IRE System 1.8.8 Degradation of Iron-Regulatory Proteins 1.8.9 Mechanism of Cellular Iron Toxicity Manganese 1.9.1 Mechanism of Manganese Transport and Cellular Metabolism 1.9.2 Mechanism of Cellular Manganese Toxicity c-jun-N-Terminal Kinase (JNK) and Cell Death Neuroprotection and Management of Iron and Manganese Toxicity Rational and Objective Chapter Materials and Methods 2.1 Materials 2.1.1 cDNAs 2.1.2 Antibodies 2.1.3 Reagents 2.2 Methods 2.2.1 Cell Culture and Western Blot 2.2.2 Calcein-Am Quenching Assay 2.2.3 MTT Cell Viability Assay III 1 12 16 18 19 21 22 23 24 30 31 31 35 38 41 43 45 47 48 50 52 56 56 56 57 57 58 58 58 59 Table of Contents 2.2.4 RNA Extraction and RT-PCR 2.2.5 Immunocytochemistry and Confocal Microscopy for Lysosomal and LC3 Puncta Staining 2.2.6 Intracellular ROS and Flow Cytometry 2.2.7 Densitometric and Statistical Analysis 2.2.8 Tail Digestion, DNA Purification and PCR 2.2.9 Brain Digestion and Western Blot 2.2.10 T2-Weighted Magnetic Resonance Imaging (MRI) 2.2.11 Proton-Induced X-ray Emission (PIXE) and Nuclear Magnetic Resonance 2.2.12 Prussian Perls Iron Staining 2.2.13 Immunohistochemistry and Immunofluorescence of Brain Sections 2.2.14 Rotarod Test 2.2.15 Fe55 Uptake Assay 2.2.16 Biotin-Switch Assay Chapter Results 3.1 Overview 3.2 Enhanced JNK Activation and Cellular Iron Depletion Mediated by Mn2+ Toxicity via DMT1 3.2.1 Expression of DMT1 and Fe2+ uptake in DMT1 overexpressing cells 3.2.2 DMT1-mediated Fe2+ and Mn2+ uptake, cytoplasmic accumulation and reduction in cell viability 3.2.3 Effect of Mn2+ and Fe2+ on MAP kinase pathway 3.2.4 Effect of Mn2+on autophagy pathway 3.2.5 Role of autophagy in Mn2+toxicity 3.2.6. Effect of Mn2+on iron storage ferritin protein 3.2.7 Involvement of ubiquitin-proteasome and autophagylysosomal pathways in Mn2+-mediated ferritin degradation 3.2.8 Effect of Fe2+ repletion (pre-treatment) on Mn2+-mediated JNK phosphorylation 3.2.9 Effect of JNK inhibition on Mn2+-mediated ferritin degradation 3.2.10 Effect of ferritin overexpression on Mn2+-mediated JNK phosphorylation 3.2.11 Effect of JNK inhibition on Mn2+-mediated autophagy activation 3.2.12 Intracellular ROS formation in Fe2+ and Mn2+-treated cells 3.2.13 Effect of lysosomal inhibition on Mn2+-mediated JNK activation 3.2.14 Effect of thioredoxin overexpression on Mn2+-mediated JNK phosphorylation IV 59 60 60 61 61 62 62 62 63 63 64 65 65 65 67 67 70 71 74 80 88 93 97 98 103 107 108 110 116 119 Table of Contents 3.3 Characterization of Transgenic Mouse Model of Divalent Metal Transporter (DMT1) 3.3.1 Generation of MoPrP-DMT1B-myc transgene and transgenic founder 3.3.2 Brain expression of DMT1B-myc 3.3.3 Brain iron content in DMT1_Tg measured using T2weighted magnetic resonance imaging (T2-MRI) 3.3.4 Brain iron content in DMT1_Tg measured using protoninduced X-ray emission (PIXE) and histological Perls iron staining 3.3.5 DMT1 and iron-mediated microglial activation 124 124 126 129 131 134 3.4 The Role of Nitric Oxide (NO) on DMT1 Function 139 3.4.1 NO increases DMT1-mediated Fe2+ influx 3.4.2 NO-mediated S-nitrosylation of DMT1 139 140 Chapter Discussion and Conclusions 4.1 Part One 4.2 Part Two and Part Three 4.3 Future Works 143 144 152 159 162 References Appendix Effect of Dietary Manganese and Iron on Rotarod Performance of DMT1_Tg A.1 Overview A.2 Materials and Methods A.3 Results and Discussions 207 207 207 208 211 Publication V List of Figures and Tables LIST OF FIGURES AND TABLES 1.1 1.1 1.2 1.3 1.4 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10 3.11 3.12 3.13 3.14 3.15 3.16 3.17 3.18 3.19 3.20 Introduction The nigrostriatal system and basal ganglia pathologies. Basal ganglia diseases with metals accumulation. DMT1-mediated transferrin dependent and independent iron uptake. Putative DMT1 topology. The IRP/IRE Regulatory System. Results: Part One Expression of DMT1 and Fe2+ uptake. Expression of exogenous GFP-DMT1 localized to the plasma and acidic lysosomal membrane. Cytoplasmic accumulation of labile Mn2+. Loss of cell viability in S-DMT1 cells treated with Mn2+, but Fe2+treated cells showed resistance. JNK MAP kinase activation in Mn2+-treated S-DMT1 cells. JNK inhibition and Fe2+ treatment rescued Mn2+-mediated cell viability loss. Mn2+-mediated increase in autophagy reversed with Fe2+ treatment. Mn2+ increased LC3 puncta formation in mRFP-GFP-LC3 transfected cells. Chemical inhibition of autophagy using unspecific PI3K Class III inhibitors, 3MA and WM did not rescue Mn2+-mediated cell viability reduction. Cell viability reduction in autophagy-deficient MEF cells treated with Mn2+. Mn2+-mediated downregulation of cytoplasmic ferritin. Cytoplasmic ferritin loss was due to enhanced protein degradation provoked by Mn2+. Fe2+ repletion (pre-treatment) and Fe2+ co-incubation diminished Mn2+mediated JNK phosphorylation. Mn2+-mediated ferritin degradation is independent of JNK phosphorylation. Ferritin overexpression does not affect Mn2+-mediated JNK phosphorylation. Mn2+-mediated autophagy activation independent of JNK activation. Reduction in intracellular ROS with Mn2+ treatment. Lysosomal inhibition enhanced disruption to Fe2+ homeostasis mediated by Mn2+. Effect of thioredoxin overexpression on Mn2+-mediated JNK phosphorylation. Effect of thioredoxin overexpression on Mn2+-treated N2A cholinergic cells and cell viability of Mn2+-treated S-DMT1 cells. VI 10 13 22 29 37 71 73 75 79 82 86 90 92 94 96 98 101 104 108 110 112 115 117 122 123 List of Figures and Tables 3.21 3.22 3.23 3.24 3.25 3.26 3.27 Results: Part Two Genotyping strategy and expression of DMT1B-myc. Brain regional expression of the transgene. Magnetic resonance imaging (MRI) and iron deposition in the brain. Proton-induced X-ray emission (PIXE). Histological Perls Prussian blue iron staining. TH neurons and microglial activation. Microglial activation associated with nitrosative stress. 125 127 130 132 133 135 137 3.28 3.29 Results: Part Three Nitric oxide increases DMT1-mediated iron uptake. S-nitrosylation of DMT1. 140 142 4.1 Discussions and Conclusions A proposed model of cellular Mn2+ toxicity via DMT1. 145 A1 A2 Appendix Dietary manipulation strategy and weight of mice. Rotarod performance of mice. 208 210 VII Abbreviations ABBREVIATIONS Biotin-HPDP pyridyldithiol-biotin DAB DMEM DMSO DPX 3,3'-diaminobenzidine Dulbecco modified Eagle's minimal essential medium dimethyl sulfoxide di-N-butyle phthalate in xylene Cd Co cadmium cobolt ECL EDTA EGTA ER enhanced chemoluminescence ethylenediaminetetraacetic acid ethylene glycol tetraacetic acid endoplasmic reticulum FBS fetal bovine serum GSH glutathione H2DCFDA HEPES HIF 2',7'-dichlorodihydrofluorescein diacetate 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid hypoxia-inducible factor LRRK2 leucine-rich repeat kinase I.P. I.V. intraperitoneal intravenous MES MMTS MPTP MTT methyl methanethiosulfonate 4-morpholinoethanesulfonic acid 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine dimethyl thiazolyl diphenyl tetrazolium salt Ni NOC-18 nickel 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene PBS PFA PINK-1 PVDF phosphate buffered saline paraformaldehyde PTEN-induced putative kinase polyvinylidene difluoride RIPA radioimmunoprecipitation assay SDS-PAGE SIN-1 SNAP STEAP sodium dodecyl sulfate polyacrylamide gel electrophoresis 3-(4-Morpholinyl)sydnonimine, hydrochloride s-nitroso-n- acetylpenicillamine six-transmembrane epithelial antigen of the prostate VIII Abbreviations TBST Tris-base saline with tween VO vanadium IX References Zhou, B., S. K. Westaway, B. Levinson, M. A. Johnson, J. Gitschier and S. J. Hayflick (2001). "A novel pantothenate kinase gene (PANK2) is defective in Hallervorden-Spatz syndrome." Nat Genet 28(4): 345-349. Zhou, Z. D., Y. H. Lan, E. K. Tan and T. M. Lim (2010). "Iron species-mediated dopamine oxidation, proteasome inhibition, and dopaminergic cell demise: implications for iron-related dopaminergic neuron degeneration." Free Radic Biol Med 49(12): 1856-1871. Zhu, W., X. Li, W. Xie, F. Luo, D. Kaur, J. K. Andersen, J. Jankovic and W. Le (2010). "Genetic iron chelation protects against proteasome inhibition-induced dopamine neuron degeneration." Neurobiol Dis 37(2): 307-313. Zorzi, G., F. Zibordi, L. Chiapparini, E. Bertini, L. Russo, A. Piga, F. Longo, B. Garavaglia, D. Aquino, M. Savoiardo, A. Solari and N. Nardocci (2011). "Iron-related MRI images in patients with pantothenate kinase-associated neurodegeneration (PKAN) treated with deferiprone: results of a phase II pilot trial." Mov Disord 26(9): 1756-1759. Zucca, F. A., C. Bellei, S. Giannelli, M. R. Terreni, M. Gallorini, E. Rizzio, G. Pezzoli, A. Albertini and L. Zecca (2006). "Neuromelanin and iron in human locus coeruleus and substantia nigra during aging: consequences for vulnerability." J Neural Transm 113(6): 757-767. 206 neuronal Appendix APPENDIX Effect of Dietary Manganese and Iron on Rotarod Performance of DMT1_Tg A.1 Overview The data presented in this short chapter is an extended preliminary result of an ongoing experiment to investigate the effect of manganese and iron-enriched diet on DMT1_Tg. The data was included in this thesis to extend the understanding of the role of DMT1 in mediating possible harmful effects of manganese in affecting motor performance of DMT1_Tg. A.2 Materials and Methods Dietary manipulation of 1000mg/kg carbonyl iron and 1000mg/kg carbonyl manganese on normal feed AIN93M as shown in Figure A1 (A), was started earlier at months of age as oppose to months in the initial experiment presented in Chapter 3, part two. Feeding was started earlier to reduce the likely death to mice due to aging. The weight of the mice were measured fortnightly as a gross determination of general health. Rotarod test was performed fortnightly on an accelerating protocol. Briefly, at every test, mice were first acclimatised to the procedure room for 30 at the same time of the day. Mice were made to move on the rotating beam at rpm for 30 sec. Once the mice were stable without any random falls, the test was started. Acceleration speed was ramped up from to 40 rpm in 240 sec. Mice were given three trials with at least 15 rest between trials. Results were analysed as latency to fall in seconds. Statistics were performed using Microsoft Office Excel Student’s t-test (two-tailed distribution) with two-sample unequal variance. 207 Appendix A.3 Results and Discussion Weights of both non-Tg and DMT1_Tg mice on dietary manipulation up to 22 weeks did not show any significant change as compared to normal feed as shown in Figure A1 (B) and (C). While DMT1_Tg on iron supplementation showed a borderline decrease in body weight as compared to normal feed, however iron supplementation did not impair growth in the mice, Figure A1 (C). Figure A1. Dietary manipulation strategy and weight of mice. (A) DMT1_Tg and non-Tg were started on normal, iron or manganese-enriched diets 90 days post-weaning for 22 weeks and beyond. Weight and rotarod performance were determined fortnightly. Weight of mice (in grams) over 22 weeks are shown in (B) non-Tg (WT), (C) DMT1_Tg (DMT1), with n: representing the number of mice per group. No significant weight change was observed in either non-Tg or DMT1_Tg with dietary manipulations. Even as dietary manipulation did not affect the weight of mice, we hypothesized that high dietary iron or manganese in the presence of brain DMT1 overexpression may 208 Appendix result in motor phenotype. Thus, we closely monitored the mice using a rotarod for every two weeks. Motor performance of DMT1_Tg on normal diet (n=9) was not significantly different from non-Tg (n=8). In addition, motor performance of DMT1_Tg (n=7) was also similar to non-Tg (n=7) both on iron diet. Interestingly, DMT1_Tg was shown to be vulnerable to manganese diet (n=8) showing significant (p[...]... Wilson’s disease (Doraiswamy and Finefrock 2004, Lorincz 2010) Interestingly, zinc ion (Zn2+) is a biologically stable species and does not readily undergo redox cycling Zinc is important for its function in many enzymatic proteins and is especially crucial for its role in maintaining structural stability of many proteins, through the formation of zinc-finger motif Additionally, zinc ion can act as an anti-oxidant... proteins, the deficiency or overload of metal ions may have complicated implications 6 Introduction on cellular functions While many investigations revolve around the study of the effects of metal ions individually, the study of the effects of uptake of two or more metal ions and their interactions at the point of entry or their signalling pathways are still lacking Specifically, investigations at the intracellular... neurons, are a group of heterogeneous cells in the brain which contain tyrosine hydroxylase (TH) for the production of dopamine As dopaminergic neurons lack the two downstream enzymes for the production of norepinephrine (dopamine β-hydroxylase) and epinephrine (phenylethanolamine N-methyltransferase), consequently dopamine is the main neurotransmitter utilized by these neurons Dopamine is synthesized... thus halting redox cycling The displacement of Fe2+ by Zn2+ may have a dual role in determining the fate of the cell, depending on the cellular redox status Intuitively, if excessive Fe2+ is catalyzing the formation of ROS, then the displacement of Fe2+ by Zn2+ is favourable However, if the formation of ROS is 5 Introduction required for cellular signalling, then the competitive inhibition of Fe2+ by... example, insertion mutations to ferritin light chain gene are associated with autosomal dominant neuroferritinopathy The disease is characterized by progressive degeneration of neurons leading to motor and cognitive impairment Brain T2-magnetic resonance imaging (MRI) shows hypointensity in the basal ganglia suggesting iron deposition As the mutation results in the insufficiency of ferritin to store... to store iron, ferritin inclusions are often found in neurons and glia cells, suggesting that neuroferritinopathy results in the impairment in cellular iron storage (Barbeito, Garringer et al 2009, McNeill and Chinnery 2012) In addition, inherited autosomal recessive loss of ceruloplasmin function called aceruloplasminemia, is characterized by progressive neuronal degeneration in the basal ganglia... circulating plasma (via TfR and DMT1) and may undergo the same basolateral iron processing analogous to the crypt of the intestinal cells involving IREG1 IREG1, also known ferroportin is expressed in the endothelial cells of the BBB, neurons and neuroglia, hence supporting the role of the transferrin cycle in iron brain uptake (Wu, Leenders et al 2004, Zecca, Youdim et al 2004) 1.8.3 Transport of Non-Transferrin... neurotransmitter in the presynaptic terminal, transported into synaptic vesicles and released into the synaptic cleft upon arrival of electrical signals that trigger the neurotransmission process The initial step to its production involves the hydroxylation of the amino acid tyrosine into L-3,4-dihydroxyphenylalanine (LDOPA) via the enzyme tyrosine hydroxylase The enzymatic conversion of tyrosine into L-DOPA... presence of such quinine in pathology remains unanswered The interaction among oxygen tension, pH, the balance between antioxidants and prooxidants and the presence of physiological iron chelators are a few considerations which may significantly affect the outcome of dopamine oxidation in- vivo Nonetheless, these studies provided great insights into the potential of unbound iron in mediating toxicity in the... the iron in the process of RBCs regeneration Hence, Tf iron pool is maintained by the turnover of RBCs and is less dependent on newly absorbed dietary iron 21 Introduction Figure 1.2 DMT1- mediated transferrin dependent and independent iron uptake Simplified schematic illustrating the uptake of Fe2+ and Fe3+ into the cell (1) Fe3+-bound transferrin (Tf) at the cell surface binds to the transferrin receptor . many enzymatic proteins and is especially crucial for its role in maintaining structural stability of many proteins, through the formation of zinc-finger motif. Additionally, zinc ion can act as. Dopamine-producing neurons, also known as dopaminergic neurons, are a group of heterogeneous cells in the brain which contain tyrosine hydroxylase (TH) for the production of dopamine. As dopaminergic. effects of metal ions individually, the study of the effects of uptake of two or more metal ions and their interactions at the point of entry or their signalling pathways are still lacking. Specifically,