Deciphering how N-terminal phosphorylation alters fused in sarcoma (FUS) function: Implications for FTD/ALS pathogenesis Open Access

Johnson, Michelle (Summer 2021)

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Fused in sarcoma (FUS) is an RNA/DNA binding protein that shuttles between the nucleus and cytoplasm to accomplish its various cellular functions. Genetic and nongenetic factors can trigger FUS to accumulate into toxic cytoplasmic aggregates. These aggregates occur in ~1% of amyotrophic lateral sclerosis (ALS) cases and ~10% of frontotemporal dementia (FTD) cases. While over 70 mutations in FUS are cause either FTD or ALS pathology, most cases with FUS pathology are not caused by genetic mutation. Identifying triggers of FUS aggregation independent of FUS genetic mutations is imperative to understanding FUS pathology. Certain post-translational modifications (PTMs) can shift a proteins cellular localization. Phosphorylation is the most common PTM used to regulate protein function in the cell. FUS can be phosphorylated at multiple N- and C-terminal residues. Specifically, our lab discovered that double strand DNA breaks (DSBs) induces DNA-PK to phosphorylate FUS at 12 N-terminal residues. Phosphorylated FUS then accumulates into cytoplasmic punctate. Nonetheless, it remains unclear 1) what mechanism mediates phosphorylated FUS to accumulate in the cytoplasm and 2) whether accumulation of phosphorylated FUS affects cellular function. Therefore, the goal of this dissertation was to investigate the role of N-terminal phosphorylation in shaping FUS function. In this dissertation, we established that mouse-derived cells do not phosphorylate or re-localize FUS following calicheamicin γ1 (CLM) induced DSBs. This finding suggests that mouse cells may not be a good model of DSB induced FUS pathology. Next, we performed proximity labeling via ascorbate peroxidase 2 (APEX2) paired with mass spectrometry to investigate whether phosphorylation shifts the FUS interactome and protein function. We found that expression of a mimetic of N-terminal phosphorylation, phosphomimetic FUS, shifted the FUS proteome towards regulating mRNA translation and metabolism. Overall, these studies indicate that phosphorylation of FUS is a primate specific response that may be an important regulator of FUS function and pathology.

Table of Contents

Chapter 1: Introduction 1

1.1 Overview 2

1.2 Frontotemporal Dementia (FTD) 3

1.2.1 A brief history of FTD 3

1.2.2 Epidemiology of FTD 3

1.2.3 Clinical subtypes of FTD 4

1.2.4 Neuropathology of frontotemporal lobar degeneration (FTLD) 6

1.2.4 Current treatments for FTD 8

1.3 Amyotrophic Lateral Sclerosis (ALS) 8

1.3.1 A brief history of ALS 8

1.3.2 Epidemiology of ALS 9

1.3.3 Clinical presentation of ALS 10

1.3.5 Neuropathology of ALS 11

1.3.4 Current treatments for ALS 13

1.4 Evidence of FTD/ALS spectrum 14

1.4.1 The overlapping clinical profiles of FTD and ALS 14

1.4.2 The overlapping neuropathology of FTLD and ALS 15

1.4.3 The genetic interplay of FTD and ALS 17

1.4.4 FUS on the spectrum of FTD and ALS 18

1.5 FUS-mediated pathology 19

1.5.1 Identification of FUS as a ubiquitously expressed, multifunctional protein 19

1.5.2 Current understanding of the FUS gene and protein structure 23

1.5.3 Homeostatic FUS functions 27 Nuclear functions 28 Cytoplasmic functions 33

1.5.4 Role of genetic mutations in FUS-mediated ALS/FTD pathology 34

1.5.5 Role of nongenetic factors in FUS mediated ALS/FTS pathology 36

1.6 Summary and goals of dissertation 40

Chapter 2: Divergent FUS phosphorylation in primate and mouse cells following double-strand DNA damage 48

2.1 Abstract 49

2.2 Introduction 50

2.3 Materials and Methods 52

2.4 Results 58

2.5 Discussion 82

2.6 Acknowledgements 87

Chapter 3: Quantitative proteomics reveals that DNA damage-induced N-terminal phosphorylation of fused in sarcoma (FUS) leads to distinct changes in the FUS proteome 88

3.1 Abstract 89

3.2 Introduction 89

3.3 Materials and Methods 93

3.4 Results 103

3.5 Discussion 126

3.6 Acknowledgments 135

Chapter 4: Conclusions and future directions 136

4.1 Outline 137

4.2 Summary of findings 137

4.3 Inbred mice may be an insufficient model for DNA damage induced FUS pathology 138

4.4 N-terminal phosphorylation in phase separation 141

4.5 Proposed role of N-terminal phosphorylation in cellular function 146

4.6 Future Directions 150

4.7 Final Conclusions 155

References 156

Appendix A: Supplemental Figures for Chapter 2 211

Appendix B: Supplemental Figures for Chapter 3 217

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