Degree Name

Doctor of Philosophy


School of Biological Sciences


Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by the rapid and progressive degeneration of upper and lower motor neurons in the spinal cord, brain stem and motor cortex. Approximately 90% of ALS cases are sporadic (sALS) in nature, and the remaining 10% are termed familial (fALS), being associated with mutations in a broad set of genes including SOD1, TDP-43, OPTN, C9orf72, ALS2, FUS, and UBQLN2. The first of these genes linked to ALS was the gene encoding the ubiquitous free radical scavenging enzyme superoxide dismutase-1 (SOD1), which currently has over 180, mostly missense, ALS-associated mutations identified. Curiously, SOD1-associated fALS patients show remarkably broad mean survival times (< 1 year to ~17 years death post-diagnosis) which are mutation dependent, indicating that mutation may govern disease severity. In its native fold, SOD1 is a 32 kDa homodimer where each 153 amino acid subunit contains an intramolecular disulfide, as well as zinc and copper cofactors; the latter of which catalyses the conversion of oxygen radicals to either molecular oxygen or hydrogen peroxide, and all of which confer significant thermal and kinetic stability. Early research, using knockout animal models, determined that a loss of enzymatic activity is not the cause of SOD1-associated fALS pathology, suggesting that mutation is inducing a gain of cytotoxic function. A hallmark of SOD1-associated ALS is the deposition of SOD1 into large insoluble aggregates in motor neurons. This is thought to be a consequence of mutation induced structural destabilisation (dimer dissociation, metal binding disruption, and disulfide reduction) and/or oxidative damage leading to the misfolding of SOD1 into a neurotoxic species. Recent work has emphasised the ability of SOD1 to transmit misfolding molecularly, intercellularly, and from organism-to-organism in a prion-like manner, providing some rationale for the spatiotemporal spread of pathology observed in ALS. In this study we investigate the effects v of fALS-associated mutations and their consequences on protein structure and aggregation using recombinant protein and cultured cell models. Using native mass spectrometry (MS) to direct the dissociation and unfolding of purified SOD1 variant dimers in vacuo, we determined that the SOD1G37R variant had significantly altered charactersitics comparative to the other variants examined, where it presented a more asymmetric partitioning of charge yet did not dissociate more readily. Following from this we observed that our SOD1 variants were modified at Cys111 by glutathione, which the data present here suggested alters the dimer dissociation constant (KD) of SOD1. MS analysis determined the extent of glutathionylation, as well as the dimer KD’s of several SOD1 variants in their unmodified (uSOD1) and glutathionylated (gsSOD1) forms, finding that glutathionylation increased the dimer KD’s differentially, where specific wild-type-like mutants were significantly augmented (uSOD1G93A = 12 ± 1 nM, gsSOD1G93A = 160 ± 32 nM) compared to SOD1WT (uSOD1WT = 9 ± 1 nM, gsSOD1WT = 34 ± 5 nM). These data suggest that glutathionylation, and potentially other modifications, of Cys111 in SOD1 may contribute to its misfolding and subsequent aggregation, and highlights the necessity of identifying post-translational modifications prior to biophysical analysis.

Owing to the ability of native MS to resolve differentially modified protein species, provide information on oligomeric distribution, as well as information on protein conformation, we utilised MS to interrogate the structural consequences of metal loss and disulfide reduction on fALS-associated SOD1 variants. We determined that, after DTT/EDTA-treatment, the most abundant SOD1 species was reduced apo-SOD1 for all variants, and that the conformational state of this species was dependent on mutation. All variants showed evidence of unfolded, intermediate, and compact conformations (as determined by Gaussian distributions of massto- charge ratios), with SOD1G37R, SOD1G93A and SOD1V148G having the greatest abundance of intermediate and unfolded SOD1. SOD1G37R was a curious outlier as it did not aggregate to vi the same extent (measured by thioflavin T) as SOD1G93A and SOD1V148G in aggregation assays. Furthermore, seeding the aggregation of DTT/EDTA-treated SOD1G37R with preformed SOD1G93A fibrils elicited minimal response, indicating that the arginine substitution at position-37 blocks the templating of SOD1 onto preformed fibrils. Position-37 is encompassed by a sequence segment (30KVWGSIKGL38) identified as having a high aggregation propensity and being involved in the intermolecular transmission of misfolding. Within this sequence segment is Trp32 which has previously been identified as a contributor to SOD1 misfolding and aggregation. We investigated the effect of Trp32 on aggregation by generating SOD1 variants with a W32S mutation. Our analysis established that the W32S substitution decreased the stability of the reduced apo-form of each SOD1 variant, but remarkably decreased their aggregation propensity both in isolated protein assays and in cell culture. We found that SOD1G93A-W32S had a decreased ability to template onto preformed SOD1G93A aggregates, suggesting that Trp32 is an aggregation modulating residue.

This study clearly demonstrates the utility of native mass spectrometry in the study of disordered and modified proteins in the ability to assess both conformational and modification state simultaneously. The data reported here illustrate the complexity involved in unravelling the cause of SOD1-associated fALS, but provides evidence that strongly indicates the role of a sequence segment encompassing Trp32 in aggregation and propagation. It is hoped that by understanding the interplay between mutation effect on SOD1 stability and subsequent aggregation formation may help identify the particular aggregate species responsible for toxicity and provide targets for therapeutic intervention.

FoR codes (2008)

060112 Structural Biology (incl. Macromolecular Modelling), 060109 Proteomics and Intermolecular Interactions (excl. Medical Proteomics), 060199 Biochemistry and Cell Biology not elsewhere classified, 1109 NEUROSCIENCES



Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.