Identification of molecular mechanisms and suppressors of TDP-43 pathology Public

Abstract

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders with overlapping clinical, pathologic and genetic features. Cytoplasmic inclusions that contain the TAR DNA-binding protein 43 (TDP-43) are found as a common pathological hallmark in the brain and spinal cord of individuals with ALS and FTD. TDP-43 is an RNA-binding protein that predominantly resides in the nucleus. TDP-43 pathology is characterized by loss of this protein from the nucleus and the accumulation of insoluble TDP-43 aggregates in the cytoplasm. The reported global impairments in RNA and protein homeostasis may be caused by the sequestration of its interacting proteins into the TDP-43-positive inclusions. The exact cellular mechanisms for TDP-43 pathology remain unclear, which is also due to the limited understanding of the components of TDP-43 aggregates. Here, I demonstrate that proximity-dependent biotin identification (BioID) allows for successful interactome mapping of insoluble TDP-43 aggregates. I have identified proteins involved in nucleocytoplasmic transport, such as nucleoporins and transport factors, as its major components. Aggregated and mutant form of TDP-43 triggered the cytoplasmic aggregation of several nucleoporins and transport factors, disrupted nuclear pore complex and nuclear membrane structure, and disturbed nuclear transport of protein and RNA. I also found Nup205-positive inclusions as well as a widespread loss of Nup205 immunoreactivity in the motor and frontal cortex of sporadic ALS cases and those with genetic mutations in TARDBP and C9orf72. Furthermore, I report poly(A)-binding protein nuclear 1 (PABPN1) as a novel TDP-43 interaction partner that acts as a potent suppressor of TDP-43 toxicity. Overexpression of PABPN1 significantly reduced TDP-43-mediated neurotoxicity and protein aggregates in an ubiquitin proteasome-dependent manner. PABPN1 also restored the nuclear localization of TDP-43 and proper stress responses that were compromised by TDP-43 pathology. Taken together, these findings advance our understanding of the nucleocytoplasmic transport defects as a common disease mechanism in ALS/FTD and PABPN1 as a novel target to halt or even reverse the progression of TDP-43 pathology.

Table of Contents

Abbreviations..................................................................................................................... 1

Chapter 1............................................................................................................................ 4

General Introduction........................................................................................................ 4

1.1 Overview of neurodegenerative diseases with TDP-43 pathology..................... 5

1.1.1 Amyotrophic lateral sclerosis (ALS).................................................................... 6

1.1.2 Frontotemporal dementia (FTD)....................................................................... 12

1.1.3 Other TDP-43 proteinopathy: Alzheimer's disease (AD)................................... 15

1.2 TAR DNA-binding protein 43 (TDP-43)............................................................... 17

1.2.1 Structure............................................................................................................ 17

1.2.2 Normal TDP-43 function.................................................................................. 19

1.2.3 TDP-43 mislocalization and aggregation............................................................ 20

1.3 Models of TDP-43 aggregation............................................................................. 24

1.3.1 Aberrant stress granule (SG) formation............................................................... 24

1.3.2 Inhibiting nucleocytoplasmic transport.............................................................. 27

1.4 Hypothesis and Objective..................................................................................... 33

1.5 Figures.................................................................................................................... 35

1.6 Table........................................................................................................................ 42

Chapter 2.......................................................................................................................... 44

Materials and Methods................................................................................................... 44

2.1 Yeast two hybrid assay.......................................................................................... 45

2.2 Yeast spotting assays............................................................................................ 45

2.3 Constructs.............................................................................................................. 46

2.4 Drosophila genetics................................................................................................ 48

2.5 Adult Eye Imaging................................................................................................. 48

2.6 Larval Turning Assays.......................................................................................... 48

2.7 Primary neuron and mammalian cell culture and transfection..................... 49

2.8 Immunofluorescence and image acquisition and analysis.............................. 50

2.9 Fluorescence in situ hybridization (FISH) and immunofluorescence.............. 52

2.10 Metabolic labeling of newly synthesized proteins.......................................... 53

2.11 Electron microscopy of nuclear envelope......................................................... 53

2.12 Immunohistochemistry...................................................................................... 54

2.13 Cell death assay.................................................................................................. 55

2.14 Co-immunoprecipitation of FLAG-tagged constructs from mammalian cells 55

2.15 Co-immunoprecipitation of endogenous protein from mouse brain tissue. 56

2.16 Histidine pulldown assay................................................................................... 56

2.17 Affinity pulldown of biotinylated proteins and immunoblotting.................. 57

2.18 Sample digestion for mass spectrometry analysis.......................................... 58

2.19 LC-MS/MS analysis............................................................................................. 58

2.20 Database search................................................................................................... 59

2.21 Identification of the TDP-43 and TDP-CTF interactome................................. 59

2.22 Bioinformatics analysis...................................................................................... 60

2.23 Protein extract, subcellular fractionation and western blots........................ 61

2.24 Cycloheximide chase assay................................................................................ 62

2.25 Time-lapse live-cell imaging.............................................................................. 63

2.26 Stress granule formation assay......................................................................... 64

2.27 Statistical analyses............................................................................................. 64

Chapter 3.......................................................................................................................... 65

TDP-43 pathology disrupts nuclear pore complexes and nucleocytoplasmic transport in ALS/FTD......... 65

3.1 Abstract................................................................................................................... 66

3.2 Introduction........................................................................................................... 66

3.3 Results.................................................................................................................... 68

3.3.1 Cytoplasmic TDP-43 aggregates are enriched for components of nucleocytoplasmic transport pathway 68

3.3.2 TDP-43 pathology causes the cytoplasmic aggregation and mislocalization of nucleoporins (Nups) and transport factors (TFs) 70

3.3.3 FG-Nups contain prion-like domains (PrLDs) that mediate cytoplasmic co-aggregation with TDP-CTF 71

3.3.4 TDP-43 pathology disrupts the morphology of the nuclear membrane (NM) and nuclear pore complexes (NPCs) 72

3.3.5 TDP-43 pathology disrupts nuclear import of proteins and export of mRNA.... 74

3.3.6 PABPN1, a suppressor of TDP-43 toxicity, rescues nucleocytoplasmic transport defects 75

3.3.7 Nucleoporin genes act as modifiers of TDP-43 pathology in Drosophila models for ALS 76

3.3.8 Nuclear pore pathology is common in brain tissue of ALS cases with TDP-43 inclusions 77

3.4 Discussion.............................................................................................................. 77

3.5 Figures.................................................................................................................... 82

3.6 Supplementary Figures........................................................................................ 94

3.7 Supplementary Tables........................................................................................ 113

Chapter 4........................................................................................................................ 118

PABPN1 suppresses TDP-43 toxicity in ALS disease models.................................. 118

4.1 Abstract................................................................................................................. 119

4.2 Introduction......................................................................................................... 119

4.3 Results.................................................................................................................. 123

4.3.1 TDP-43 is a novel interaction partner of PABPN1......................................... 123

4.3.2 PABPN1 modulates TDP-43 toxicity in yeast and primary neuron models...... 124

4.3.3 PABPN1 loss of function enhances TDP-43 toxicity in Drosophila models.... 127

4.3.4 PABPN1 specifically reduces protein levels of pathological TDP-43............... 128

4.3.5 PABPN1 facilitates the clearance of pre-formed pathological TDP-43 aggregates 130

4.3.6 PABPN1 facilitates the clearance of pathological TDP-43 via the ubiquitin-proteasome mechanism .............. 131

4.3.7 PABPN1 levels regulates nuclear localization of endogenous TDP-43.............. 132

4.3.8 PABPN1 overexpression rescues defects in SGs caused by TDP-43 pathology.. 134

4.4 Discussion............................................................................................................ 136

4.5 Figures.................................................................................................................. 146

4.6 Supplementary Figures...................................................................................... 165

4.7 Table...................................................................................................................... 169

4.8 Supplementary Table.......................................................................................... 170

Chapter 5........................................................................................................................ 171

Conclusions and Future Directions............................................................................ 171

5.1 Summary.............................................................................................................. 172

5.2 Remaining Questions and Future Directions.................................................. 176

5.2.1 Mapping PABPN1 protein functional domains................................................ 176

5.2.2 PABPN1 rescues TDP-43 toxicity mediated by gain-of-function but not loss-of-function 177

5.2.3 Effects of PABPN1 truncation mutants on TDP-CTF levels........................... 178

5.2.4 Effects of PABPN1 on the degradation of aggregate-prone proteins................ 179

5.2.5 Effects of PABPN1 on ubiquitin-proteasomal degradation............................... 180

5.2.6 Effects of PABPN1 on proteasome activity.................................................... 183

5.2.7 Future directions.............................................................................................. 184

5.3 Conclusions......................................................................................................... 185

5.4 Figures.................................................................................................................. 187

References.....................................................................................................197

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience Biology, Molecular Biology, Cell
Mot-clé	TDP-43 Amyotrophic lateral sclerosis Aggregate Frontotemporal dementia
Committee Chair / Thesis Advisor	Bassell, Gary, Emory University Rossoll, Wilfried O, Mayo Clinic
Committee Members	Seyfried, Nicholas, Emory University Kukar, Thomas, Emory University Li, Xiao-Jiang,

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