Molecular Pathogenesis of DYT1 Dystonia Público
Giles, Lisa Mariko (2008)
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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