Mitochondrial regulation in health and neurodegenerative disease Open Access

Shaw, Dana Val (2014)

Permanent URL: https://etd.library.emory.edu/concern/etds/4f16c3349?locale=en
Published

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

About this Dissertation

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
School
Department
Subfield / Discipline
Degree
Submission
Language
  • English
Research field
Keyword
Committee Chair / Thesis Advisor
Committee Members
Last modified

Primary PDF

Supplemental Files