Toxicity and solubility of mutant superoxide dismutase 1 in amyotrophic lateral sclerosis Open Access
Brotherton, Terrell Eileen (2012)
Published
Abstract
Abstract
Toxicity and solubility of mutant superoxide dismutase 1 in
amyotrophic lateral
sclerosis
Mutations in the gene encoding superoxide dismutase 1 (SOD1)
account for
approximately 20% of cases of familial amyotrophic lateral
sclerosis. It is not known how the
mutant protein causes disease, nor why only a subset of cell types
(i.e . motor neurons) are
targeted. The propensity of mutant SOD1 to form aggregates
selectively in pathologically
affected tissue has implicated these poorly soluble protein
aggregates as being instrumental
to the disease process. However, because aggregates are composed of
misfolded SOD1, it
has been difficult to distinguish the effects of aggregates and
their misfolded, soluble
precursors. Instead of being cytotoxic, aggregates may exist as a
protective compensatory
mechanism to sequester misfolded soluble protein, thereby
decreasing aberrant cytosolic
interactions. Here, I present data supporting the hypothesis that
misfolded soluble, not
insoluble aggregated, SOD1 is cytotoxic.
In this dissertation, I investigate several aspects of mutant SOD1
as it relates to
cellular toxicity and the pathogenesis of ALS. I first present
clinical data on a novel SOD1
mutation in familial ALS patients. This mutation, SOD1C6S, presents
with a mixed phenotype
that includes an unusual characteristic of reduced disease
penetrance. I then characterize an
antibody, the C4F6 antibody that specifically marks "toxic" mutant
SOD1. This antibody
distinguishes pathologically affected and healthy tissue both in
humans and in rodent disease
models. Interestingly, C4F6 preferentially recognizes easily
soluble, not insoluble, mutant
SOD1 suggesting that toxic mutant SOD1 is soluble.
Finally, I investigate the relative solubility and cytotoxicity of
separate SOD1
mutants. Through manipulating protein quality control pathways, the
effects of aggregates
and misfolded, soluble aggregate precursors are distinguished. I
demonstrate that increased
solubility is associated with increased toxicity, and furthermore,
that decreasing the presence
of misfolded, soluble SOD1 ameliorates this toxicity regardless of
the presence of insoluble
SOD1. Additionally, I demonstrate that increasing mutant SOD1
solubility exacerbates
mutant SOD1-associated cytotoxicity. These findings implicate
easily soluble, misfolded
SOD1 as being toxic to the cell, and support the hypothesis that
reducing solubility of
mutant SOD1 proteins through aggregation may occur as a
self-protective response in the
cell.
Table of Contents
Chapter 1: Introduction and background……………………………………….1
Introduction to Amyotrophic Lateral
Sclerosis……………………………..2
1.1: Aggregation across neurodegenerative
diseases………………………...3
1.1.2: Protein misfolding, degradation, and quality
control………………….5
1.2. Amyotrophic lateral sclerosis and superoxide dismutase
1…………….14
1.2.1: Protein aggregation in
ALS………………………………………….15
1.2.2: Superoxide dismutase 1
structure…………………………………....18
1.2.3: Mechanisms of SOD1
misfolding…………………………………...21
1.2.4: Quality control mechanisms of misfolded
SOD1………………...….25
1.2.5: Relationship between SOD1 misfolding and
cytotoxicity…………....30
1.2.6: Does soluble, misfolded SOD1 transfer between
cells?.........................33
1.2.7: Can we identify "toxic" misfolded
SOD1?...............................................38
1.2.8: Conclusions and
hypothesis………………………………………....40
Chapter 2: A novel ALS SOD1 C6S mutation with implications
for
aggregation-related
toxicity………………………………………..43
Abstract…………………………………………………………………...44
2.1:
Introduction…………………………………………………………..45
2.2: Materials and
Methods………………………………………………..46
SOD1
genotyping…………………………………………………46
Nomenclature……………………………………………………..46
Western Immunoblots and SOD1 activity
analysis………………...47
2.3:
Results………………………………………………………………...48
Proband clinical
examination……………………………………...48
Family
history……………………………………………………..50
SOD1
activity……………………………………………………..52
2.4: Discussion……………………………………………………………54
Chapter 3: Tracking SOD1-associated toxicity: localization of a
subset of fALS
SOD1 protein to pathologically affected
tissues…………………..57
Abstract…………………………………………………………………...58
3.1:
Introduction…………………………………………………………..59
3.2: Materials and
Methods………………………………………………..61
Animals…………………………………………………………....61
Cell Culture and
Transfections……………………………………61
Immunohistochemistry……………………………………………62
Immunocytochemistry…………………………………………….63
Differential Extraction of SOD1 protein from
tissue……………...63
Immunoblotting…………………………………………………..64
Hydrophobic Interaction
Chromatography………………………..65
Classifying C4F6
Immunoreactivity……………………………….66
3.3:
Results………………………………………………………………...66
C4F6 immunoreactivity in SOD1G93A
tissues……………………....66
Solubility of C4F6 immunoreactive
protein……………………….72
Hydrophobicity of C4F6 immunoreactive
protein………………...74
C4F6 immunoreactivity of non-G93A SOD1
mutants……………76
C4F6 immunoreactivity in human
tissue…………………………..78
3.4:
Discussion……………………………………………………………81
Chapter 4: Cellular toxicity of mutant SOD1 protein is linked to
an easily
soluble, non-aggregated
form……………………………………...91
Abstract…………………………………………………………...92
4.1:
Introduction…………………………………………………..93
4.2: Materials and
Methods………………………………………..94
Cell
Culture……………………………………………….94
Pharmacological
Administration…………………………..95
Cytotoxicity……………………………………………….95
Solubility
Fractionation…………………………...…….…95
Proteasome
Activity………………………………………96
Heat
Shock………………………………………………..96
Statistical
Analysis………………………………………....96
4.3:
Results………………………………………………………..97
Solubility differs among hSOD1
mutants…………….......100
An increase in soluble mutant hSOD1 correlates with increase in
toxicity...……………………………………...103
Refolding soluble, misfolded SOD1 decreases
toxicity…...106
Increasing mutant SOD1 solubility increases
toxicity…….109
4.4:
Discussion……………………………………………..…….115
Chapter 5: Discussion and Future
Directions………………………………..129
5.1: Identifying characteristics of toxic SOD1
………………......131
5.2: Future
directions………….………………………..……….136
5.3: Therapeutic
Implications……….…………………………....140
5.4:
Conclusion..…………………………………………………141
References………………………………………………………………………142
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