Mitochondrial regulation in health and neurodegenerative disease Pubblico
Shaw, Dana Val (2014)
Abstract
Mitochondria are responsible for energy production, calcium buffering, and the regulation of cell death. Mitochondrial dysfunction due to genetic mutations or environmental insults results in the inability to respond to energy needs, accumulation of oxidative stress, cell dysfunction, cell death, and disease. In this study, we examined the role of two proteins, PINK1 and Mgrn1 and their roles in neuroprotection. Using structured illumination microscopy (SIM), we determined that PINK1 was dual targeted to separate regions of the mitochondria responding to mitochondrial health. We also observed Parkinson's disease linked mutants of PINK1 have aberrant submitochondrial targeting and fail to recruit parkin to damaged mitochondria in neurons. We determined that loss of Mgrn1 might regulate mitochondrial dynamics. Our data show that loss of Mgrn1-mediated ubiquitination results in mitochondrial fragmentation, accumulation of damaged mitochondria, and increased susceptibility to oxidative stress induced cell death, which may contribute to pathogenesis of spongiform neurodegeneration. Together the findings presented in this dissertation reveal novel insights into the regulation of mitochondrial dynamics and health related to neurodegenerative diseases. We determined that Mgrn1 may regulate mitochondrial dynamics in response to age-related stresses and PINK1 functions as a molecular switch to trigger two separate signaling cascades related to protection from mitochondrial dysfunction.
Table of Contents
CHAPTER I 1
INTRODUCTION AND BACKGROUND 1
OPENING REMARKS 2
MITOCHONDRIA 3
Structure 4
FUNCTIONS OF THE MITOCHONDRIA 5
Cellular respiration 5
Calcium buffering 6
Apoptosis 7
CONSEQUENCES OF MITOCHONDRIAL DYSFUNCTION 8
The role of oxidative stress in aging and disease 10
The role of mitochondrial DNA in aging and disease 12
MITOCHONDRIAL DYNAMICS 14
Fusion 15
Fission 17
Posttranslational modifications regulate fission and fusion protein activity 18
Mitophagy 19
NEURODEGENERATIVE DISEASES LINKED TO MITOCHONDRIAL DYSFUNCTION 20
Parkinson's disease (PD) 21
PD genetics linked to mitochondrial dysfunction 22
1. PINK1 22
2. Parkin 24
3. a-synuclein 24
4. DJ-1 25
Environmental causes of PD 25
Human spongiform neurodegenerative disorders 27
1. Alzheimer's disease (AD) 28
2. Prion diseases 30
3. Lysosomal storage disorders 32
Animal models of spongiform neurodegeneration 33
1. Mahogunin RING finger 1 (Mgrn1) mutant mice 33
2. Attractin (Atrn) mutant mice and rats 35
3. Manganese superoxide dismutase (SOD2 or MnSOD) mutant mice 36
DIAGNOSTICS AND THERAPEUTICS 36
Therapeutics in PD 37
Therapeutics in spongiform neurodegenerative disorders 39
Mitochondrial targets for the development of novel therapeutics against neurodegenerative diseases 40
HYPOTHESES AND OVERVIEW 42
CHAPTER II 70
SUBMITOCHONDRIAL SITES OF PINK1 ACTION REVEALED BY 3D-SIM SUPER-RESOLUTION MICROSCOPY 70
Abstract 72
Introduction 73
Results 75
Dual color 3D-SIM super- resolution imaging analysis enables visualization of protein submitochondrial localization 75
PINK1 resides in the cristae membrane/intracristae space of healthy mitochondria and translocates to the OMM upon mitochondrial depolarization 76
PINK1 colocalizes with TRAP1 in the IMM/IMS of healthy mitochondria and colocalizes with parkin on the OMM of dysfunctional mitochondria 78
Mitochondrial depolarization-induced PINK1 translocation to the OMM is independent of new PINK1 protein synthesis 79
The ability of PINK1 to translocate to the OMM is impaired by PD-linked PINK1 C92F and W437X mutations 80
PINK1 translocates to the OMM to recruit parkin upon mitochondrial depolarization in neurons and the translocation requires combined action of PINK1 transmembrane and C-terminal domains 82
Discussion 83
Materials and methods 86
Expression constructs and antibodies 86
Cell transfection and treatment 87
PINK1 knockout mice and primary neuronal culture 88
Parkin recruitment in neurons 88
3D-Structured Illumination Microscopy 89
Image analysis 89
Quantification of colocalization 90
Statistical analysis 90
Acknowledgments 91
Abbreviations list 91
CHAPTER III 111
SPONGIFORM NEURODEGENERATION LINKED E3 LIGASE MGRN1 IS LOCALIZATED TO MITOCHONDRIA WHERE IT REGULATES MITOCHONDRIAL MORPHOLOGY 111
Abstract 112
Introduction 113
Results 116
Mgrn1 mutant mice suffer from spongiform neurodegeneration 116
Mgrn1 is primary localized to mitochondria 116
The putative N-myristoylation motif is required for mitochondrial localization of Mgrn1 118
Mgrn1 regulates mitochondrial morphology 120
Mgrn1 is required for mitochondrial health 122
Mitochondria from aged Mgrn1 mutant mice exhibit ultrastructural defects as shown by electron microscopy 123
Mgrn1 is cytoprotective against mitochondrial dysfunction 124
Discussion 125
Materials and Methods 128
Expression constructs and antibodies 128
Cell transfection and treatment 129
Mgrn1 mutant mice and primary cell cultures 129
Hematoxylin and eosin staining 130
Immunofluorescence confocal microscopy 130
3D-Structured illumination microscopy 131
Purification of mitochondria 131
Live imaging of mitochondria 132
Perfusion of mice and electron microscopy 133
Cell viability and apoptosis assays 133
Image analysis 134
Quantification of colocalization 134
Mitochondrial Length 134
Mitochondrial membrane potential 135
Statistical analysis 135
Acknowledgments 136
CHAPTER IV 152
DISCUSSION AND FUTURE DIRECTIONS 152
Summary of Findings 153
PINK1 DISCUSSION AND FUTURE DIRECTIONS 154
3D-SIM analyses provide insights into PINK1 spatiotemporal dynamics 154
3D-SIM can differentiate submitochondrial compartments 154
PINK1 is dual targeted depending on the health of mitochondria 158
PINK1 differentially colocalizes with its substrates depending on mitochondrial health 159
Reduced OMM-translocation of PINK1 as a novel mechanism of PD pathogenesis 161
PD-linked mutants of PINK1 are not translocated to the OMM following loss of membrane potential 161
Translocation-defective PINK1 mutants are unable to recruit parkin to damaged mitochondria 162
PINK1 Future Directions 164
Determine the structural determinants of PINK1 dual targeting 164
What are the physiological triggers of PINK1 translocation? 165
How does PINK1 translocate? 166
Determine the effect of translocation defective PINK1 mutants in cell death and neurodegeneration 167
MGRN1 DISCUSSION 169
Mgrn1 is targeted to the outer mitochondrial membrane via N-myristoylation 169
Mgrn1 is primarily localized to mitochondria 169
N-myristoylation targets Mgrn1 to the OMM 170
Mgrn1 maintains mitochondrial health 171
Loss of Mgrn1 results in mitochondrial fragmentation 171
Loss of Mgrn1 results in mitochondrial dysfunction 172
Ubiquitination of Mgrn1 mitochondrial substrate(s) may confer cytoprotection 173
Mgrn1 Future Directions 174
Identification of mitochondrial substrates of Mgrn1-mediated ubiquitination 174
Does Mgrn1 inhibit mitochondrial fission or promote mitochondrial fusion? 175
How do the mitochondrial and endosomal roles of Mgrn1 contribute to neuronal health? 176
What is the link between Mgrn1 deficiency and spongiform neurodegenerative diseases? 177
FINAL WORDS 178
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