Molecular Pathogenesis of DYT1 Dystonia Open Access

Giles, Lisa Mariko (2008)

Permanent URL: https://etd.library.emory.edu/concern/etds/41687h77v?locale=en
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Abstract

Abstract

Molecular Pathogenesis of DYT1 Dystonia
By Lisa Mariko Giles
Early onset-generalized torsion dystonia (DYT1) has been linked to two mutations in the
C-terminal tail of the protein torsinA. The most common mutation is a 3-bp in-frame
deletion that results in the loss of one of a pair of glutamate residues at position 302 or
303 (torsinA ΔE). The second mutation, identified in a single family, is an 18-bp in-
frame deletion that results in the loss of six amino acids from position 323-328 (torsinA
Δ323-8). It is unclear why torsinA mutations result in a neuronal phenotype despite
widespread expression in multiple tissues.
Here we report a neuronal cell-type specific nuclear envelope (NE) preference for
torsinA. Further, ATP-bound and dystonia-associated mutant torsinA display an
enhanced NE preference compared to WT and ATP-unbound torsinA. We find that the
N-terminal portion of torsinA is sufficient for oligomerization, and that dystonia-
associated mutations do not disrupt oligomerization. We also demonstrate that, while
torsinA WT is a long-lived protein that is processed through the autophagy-lysosomal
pathway, both dystonia-associated mutations destabilize torsinA protein and result in
premature degradation through the ubiquitin proteasome pathway and the autophagy-
lysosome pathway.
We conducted a yeast-two hybrid screen for torsinA-interacting proteins and
identified a novel protein, which we named printor (protein interactor of torsinA).
Printor co-distributes with torsinA in brain and other tissues, and exists in both cytosolic
and membrane-associated pools. Printor co-localizes with torsinA at the endoplasmic
reticulum (ER), however, unlike torsinA, printor shows a distinct ER preference. Printor
shows reduced co-localization with ATP-bound and dystonia-associated mutant torsinA,
and does not interact with ATP-bound torsinA or torsinA ΔE. Together, our findings

demonstrate a neuronal cell-type specific phenotype for torsinA and implicates premature
degradation as a possible mechanism for mutant torsinA loss of function. Further, our
findings suggest that printor is a novel component of the DYT1 pathogenic pathway.

Table of Contents

TABLE OF CONTENTS

CHAPTER I. INTRODUCTION AND BACKGROUND

Opening Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Dystonia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Primary dystonia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Dystonia-plus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Secondary dystonia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

Heredodegenerative dystonia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

Treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

DYT1 and the Identification of TorsinA . . . . . . . . . . . . . . . . . . . . . . . . 6

TorsinA is Localized to the ER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

TorsinA Belongs to the AAA+ Superfamily and has Molecular

Chaperone Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

TorsinA is involved in neurite extension and plays a role

in the secretory pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

TorsinA transgenic animals reveal loss of function phenotype. . . 15

Torsin Family Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

Human torsinA family members. . . . . . . . . . . . . . . . . . . . . . . . . . .17

TorsinA orthologues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18

TorsinA interacting proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19

Kinesin light chain1 (KLC1). . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19

Dopamine Transporter (DAT). . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Lamina Associated Polypeptide 1 (LAP1)/Lumenal Domain

Like Lap 1 (LULL1). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Vimentin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Snapin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

e -Sarcoglycan. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Mechanisms of Protein Quality Control in the ER . . . . . . . . . . . . . . . . 22

Unfolded Protein Response (UPR). . . . . . . . . . . . . . . . . . . . . . . . .22

ER-Associated Degradation (ERAD). . . . . . . . . . . . . . . . . . . . . . . 24

Ubiquitin Proteasome System. . . . . . . . . . . . . . . . . . . . . . . . . . . . .24

ERAD II. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Autophagy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27

Hypotheses and Organizational Overview . . . . . . . . . . . . . . . . . . . . . . .31

CHAPTER II. Dystonia-associated mutations cause premature degradation of torsinA protein and cell type-specific mislocalization to the nuclear envelope

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Experimental Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..42

Expression constructs and antibodies. . . . . . . . . . . . . . . . . . . . . . 42

Cell transfections and co-immunoprecipitation. . . . . . . . . . . . . . . 42

Primary cell culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Immunofluorescence confocal microscopy. . . . . . . . . . . . . . . . . . .43

Quantitative analysis of the NE/ER distribution. . . . . . . . . . . . . . 43

[35S]Methionine pulse-chase analysis. . . . . . . . . . . . . . . . . . . . . . 44

Treatment of cells with proteasome, autophagy, and

lysosome inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Analysis of torsinA localization reveals neuronal cell

type-specific enrichment in the nuclear envelope. . . . . . . . . . . . . .46

Dystonia-associated mutations cause neuronal cell

type-specific translocation of torsinA from the ER

to nuclear envelope. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48

Dystonia-associated mutations do not disrupt torsinA

Oligomerization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

TorsinA oligomerization does not require its C-terminal

region. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Dystonia-associated mutations destabilize torsinA protein. . . . . .50

Dystonia-associated mutations promote the degradation of

torsinA by both the proteasome and autophagy-lysosome

pathways. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .50

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .57

CHAPTER III. PRINTOR, A NOVEL TORSINA-INTERACTING PROTEIN IMPLICATED IN DYSTONIA PATHOGENESIS

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Experimental Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Expression constructs and antibodies. . . . . . . . . . . . . . . . . . . . . . 75

Yeast two-hybrid screen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Western blot analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Immunofluorescence microscopy. . . . . . . . . . . . . . . . . . . . . . . . . 76

Immunohistochemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77

Co-immunoprecipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77

Subcellular fractionation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Opti-Prep gradient fractionation. . . . . . . . . . . . . . . . . . . . . . . . . . 79

Quantitative analysis of the NE/ER distribution. . . . . . . . . . . . . 79

Quantitative analysis of co-localization. . . . . . . . . . . . . . . . . . . . . 80

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Identification of printor, a torsinA interacting protein. . . . . . . . . . 81

Printor co-distributes with torsinA in brain as well as

other tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Printor interacts and co-localizes with torsinA in cells. . . . . . . . . 85

Printor exists in both cytosolic and membrane-associated

pools and is localized to ER. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

Printor co-localizes with torsinA in the ER but not the NE. . . . . . 87

Printor shows reduced co-localization with ATP-bound

form of torsinA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Printor co-localization with torsinA is reduced by

dystonia-associated mutation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

Printor does not interact with torsinA ΔE or torsinA E171Q. . . . .91

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

CHAPTER IV. DISCUSSION AND CONCLUSIONS

Summary of Findings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

Neuronal Cell-Type Specific NE Localization of TorsinA. . . . . . . . . 113

TorsinA Protein Degradation Pathways . . . . . . . . . . . . . . . . . . . . . . . 115

Printor, a Novel TorsinA-Interacting Protein May Belong

to the BBK Superfamily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .118

BTB/POZ domain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .119

BACK domain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

Kelch domain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

BBK protein superfamily. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

Printor Does Not Interact with ΔE TorsinA or ATP-Bound

TorsinA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

Future Directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Discerning the functional role of torsinA. . . . . . . . . . . . . . . . . . . 127

How do the dystonia-associated mutations disrupt

torsinA function?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Discerning the functional role of printor. . . . . . . . . . . . . . . . . . . 133

Final Words. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143


LIST OF FIGURES

CHAPTER I. INTRODUCTION AND BACKGROUND

Figure II-1 Schematic of the ubiquitin proteasome system. . . . . . . . . . . . . . . .35

CHAPTER II, DYSTONIA-ASSOCIATED MUTATIONS CAUSE PREMATURE DEGRADATION OF TORSINA PROTEIN AND CELL-TYPE SPECIFIC MISLOCATION TO THE NUCLEAR ENVELOPE

Figure II-1. TorsinA is enriched in the nuclear envelope in SH-SY5Y

cells but not in HeLa cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Figure II-2. Endogenous torsinA shows preferential NE localization

in primary cortical neurons compared to fibroblasts. . . . . . . . . . . 60

Figure II-3. Dystonia-associated mutations cause torsinA translocation to

the nuclear envelope in SH-SY5Y cells but not in HeLa cells. . . .62

Figure II-4. Dystonia-associated mutations have no effect on torsinA

oligomerization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Figure II-5. The N-terminal region of torsinA is sufficient for torsinA

oligomerization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Figure II-6. Dystonia-associated mutations cause premature degradation

of torsinA protein. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67

Figure II-7. Effects of proteasome, autophagy, and lysosome inhibition

on wild-type and mutant torsinA levels. . . . . . . . . . . . . . . . . . . . .68

Figure II-8. Degradation of torsinA mutants by both the proteasome

and lysosome pathways. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

CHAPTER III. PRINTOR, A NOVEL TORSINA-INTERACTING PROTEIN IMPLICATED IN DYSTONIA PATHOGENESIS

Figure III-1. Isolation of rat printor as a torsinA interacting protein

from yeast-two hybrid screen. . . . . . . . . . . . . . . . . . . . . . . . . . . . .98

Figure III-2. Printor co-distributes with torsinA in multiple tissues

and brain regions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

Figure III-3. Immunohistochemical analysis of printor protein

distribution in mouse brain. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . 101

Figure III-4. Printor and torsinA interact in vivo. . . . . . . . . . . . . . . . . . . . . . . 103

Figure III-5. Printor is found in both cytosolic and membrane-associated

fractions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

Figure III-6. Co-localization of printor and torsinA in the ER but not

the NE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .106

Figure III-7. Printor displays ER preference in both HeLa and

SH-SY5Y cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

Figure III-8. Printor interaction and co-localization is disrupted by D E

and E171Q torsinA mutation. . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Figure III-9. ATP-bound torsinA displays enhanced NE preference. . . . . . . . 110

CHAPTER IV. DISCUSSION AND CONCLUSIONS

Figure IV-1 A model of torsinA degradation. . . . . . . . . . . . . . . . . . . . . . . . . . 137

Figure IV-2 BTB domain alignment demonstrates printor P/Q

insertion coincides with RhoBTB2 insertion. . . . . . . . . . . . . . . . 138

Figure IV-3 Printor is capable of self-interaction. . . . . . . . . . . . . . . . . . . . . . 139

Figure IV-4 Printor interacts with cullin-3. . . . . . . . . . . . . . . . . . . . . . . . . . . 140

Figure IV-5 WT and dystonia-associated mutant torsinA can be

ubiquitinated. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Figure IV-6 Printor increases the turnover of WT but not dystonia-

associated mutant torsinA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

LIST OF TABLES

CHAPTER I. INTRODUCTION AND BACKGROUND

Table I-1 Identified genes and proteins associated with dystonia disease. . . 34

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