Phosphorylation of N-terminal Mutant Huntingtin is Related to its Preferential Nuclear Accumulation in Striatal Neurons Pubblico

Abstract

Abstract

Phosphorylation of N-terminal Mutant Huntingtin is Related to its Preferential Nuclear Accumulation in Striatal Neurons

By Lauren Smith Havel

Huntington's disease (HD) is a fatal, late-onset neurodegenerative disease caused by a CAG repeat expansion of more than 37 in exon 1 of the gene encoding the huntingtin (htt) protein. Htt is a large 350 kDa protein that is normally localized to the cytoplasm. Cleavage of htt releases N-terminal fragments that can diffuse into the nucleus. In an HD KI mouse model, the striatum, which is the most profoundly affected brain region, shows preferential accumulation of N-terminal mutant htt. The nuclear localization of mutant N-terminal fragments leads to transcriptional dysregulation due to the binding of soluble mutant htt to various transcription factors. Compared to non-expanded N-terminal htt, mutant htt shows decreased binding to the nuclear pore proteins leading to nuclear accumulation. In this study, we found a negative correlation between fragment length and nuclear accumulation as well as toxicity. Recently, the first 17 amino acids (N17) of htt have been implicated in regulation of the subcellular distribution of htt. In this study, we found that compared to serine 13 (S13), serine 16 (S16) is more important in regulating the nuclear accumulation of htt. We generated phosphomimetic (S16D) and loss-of-phosphorylation (S16A) mutants, expressed them in HEK 293 cells and primary striatal neurons and found that phosphorylation increases nuclear accumulation and aggregation. We also found that compared to the cortex and cerebellum, the striatum is enriched for phosphorylated N-terminal htt. More specifically, the nuclei of the striatum appear to be enriched for phosphorylation activity when compared to the cytoplasm of striatal neurons. Mechanistically, our results show that phosphorylation of N-terminal mutant htt inhibits the interaction between htt and the nuclear pore in a fragment length dependent manner and that this interaction is weaker in the striatum than the cortex or cerebellum. Thus we have provided a novel mechanism for the preferential accumulation of N-terminal mutant htt in the striatal nuclei that will be important for the future development of HD therapeutics.

Table of Contents

TABLE OF CONTENTS

Chapter 1: General Introduction 1 1.1 The Polyglutamine Diseases 2

1.2 Nuclear Accumulation of Mutant huntingtin 5

1.3 N-terminal huntingtin 8

1.4 Transcriptional Dysregulation 10

1.5 Misfolding and Aggregation of PolyQ proteins 15

1.6 Huntingtin Post-translational Modifications 19

Chapter 2: The size of N-terminal huntingtin plays a role in the nuclear accumulation of mutant huntingtin. 29

2.1 Abstract 30

2.2 Introduction 30

2.3 Materials and Methods 33

2.4 Results 36

2.5 Discussion 40

Chapter 3: Phosphorylation of serine 16 of huntingtin regulates its nuclear accumulation and toxicity 56

3.1 Abstract 57

3.2 Introduction 57

3.3 Materials and Methods 59

3.4 Results 65

3.5 Discussion 70

Chapter 4: Mutant huntingtin phosphorylated at S16 is enriched in the nuclei of the striatum 98

4.1 Abstract 99

4.2 Introduction 99

4.3 Materials and Methods 101

4.4 Results 106

4.5 Discussion 114

Chapter 5: Conclusions and Future Directions 143

5.1 Summary 144
5.2 Remaining questions and future directions 146

5.3 Conclusions 152

References 156

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Genetics and Molecular Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Biology, Neuroscience Biology, Molecular
Parola chiave	Triple repeat disorder Polyglutamine repeat disorder Phosphorylation Huntington's Disease
Committee Chair / Thesis Advisor	Li, Xiao-Jiang, Emory University
Committee Members	Fridovich-Keil, Judith, Emory University Corbett, Anita, Emory University Peng, Junmin, Emory University Chan, Anthony, Emory University

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