Susceptibility of Mutant SOD1 to Form a Destabilized Monomer Predicts Cellular Aggregation and Toxicity but Not In vitro Aggregation Propensity

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the rapid and progressive degeneration of upper and lower motor neurons in the spinal cord, brain stem and motor cortex. The first gene linked to ALS was the gene encoding the free radical scavenging enzyme superoxide dismutase-1 (SOD1) that currently has over 180, mostly missense, ALS-associated mutations identified. SOD1-associated fALS patients show remarkably broad mean survival times (~17 years death post-diagnosis) that are mutation dependent. 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 destabilization and/or oxidative damage leading to the misfolding and aggregation of SOD1 into a neurotoxic species. Here we aim to understand the relationship between SOD1 variant toxicity, structural stability, and aggregation propensity using a combination of cell culture and purified protein assays. Cell based assays indicated that aggregation of SOD1 variants correlate closely to cellular toxicity. However, the relationship between cellular toxicity and disease severity was less clear. We next utilized mass spectrometry to interrogate the structural consequences of metal loss and disulfide reduction on fALS-associated SOD1 variant structure. All variants showed evidence of unfolded, intermediate, and compact conformations, with SOD1G37R, SOD1G93A and SOD1V148G having the greatest abundance of intermediate and unfolded SOD1. SOD1G37R was an informative outlier as it had a high propensity to unfold and form oligomeric aggregates, but it did not aggregate to the same extent as SOD1G93A and SOD1V148G in in vitro aggregation assays. Furthermore, seeding the aggregation of DTT/EDTA-treated SOD1G37R with preformed SOD1G93A fibrils elicited minimal aggregation response, suggesting that the arginine substitution at position-37 blocks the templating of SOD1 onto preformed fibrils. We propose that this difference may be explained by multiple strains of SOD1 aggregate and this may also help explain the slow disease progression observed in patients with SOD1G37R

Susceptibility of mutant SOD1 to form a destabilized monomer predicts cellular aggregation and toxicity but not in vitro aggregation propensity

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the rapid and progressive degeneration of upper and lower motor neurons in the spinal cord, brain stem and motor cortex. The first gene linked to ALS was the gene encoding the free radical scavenging enzyme superoxide dismutase-1 (SOD1) that currently has over 180, mostly missense, ALS-associated mutations identified. SOD1-associated fALS patients show remarkably broad mean survival times (~17 years death post-diagnosis) that are mutation dependent. 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 destabilization and/or oxidative damage leading to the misfolding and aggregation of SOD1 into a neurotoxic species. Here we aim to understand the relationship between SOD1 variant toxicity, structural stability, and aggregation propensity using a combination of cell culture and purified protein assays. Cell based assays indicated that aggregation of SOD1 variants correlate closely to cellular toxicity. However, the relationship between cellular toxicity and disease severity was less clear. We next utilized mass spectrometry to interrogate the structural consequences of metal loss and disulfide reduction on fALS-associated SOD1 variant structure. All variants showed evidence of unfolded, intermediate, and compact conformations, with SOD1G37R, SOD1G93A and SOD1V148G having the greatest abundance of intermediate and unfolded SOD1. SOD1G37R was an informative outlier as it had a high propensity to unfold and form oligomeric aggregates, but it did not aggregate to the same extent as SOD1G93A and SOD1V148G in in vitro aggregation assays. Furthermore, seeding the aggregation of DTT/EDTA-treated SOD1G37R with preformed SOD1G93A fibrils elicited minimal aggregation response, suggesting that the arginine substitution at position-37 blocks the templating of SOD1 onto preformed fibrils. We propose that this difference may be explained by multiple strains of SOD1 aggregate and this may also help explain the slow disease progression observed in patients with SOD1G37R

Strategies to promote the maturation of ALS-associated SOD1 mutants: small molecules return to the fold

In summary, new small molecules on the horizon show some promise for treating sufferers of SOD1-fALS and should be pursued for translation into the clinic

Amyotrophic Lateral Sclerosis: Proteins, Proteostasis, Prions, and Promises

Copyright 2020 McAlary, Chew, Lum, Geraghty, Yerbury and Cashman. Amyotrophic lateral sclerosis (ALS) is characterized by the progressive degeneration of the motor neurons that innervate muscle, resulting in gradual paralysis and culminating in the inability to breathe or swallow. This neuronal degeneration occurs in a spatiotemporal manner from a point of onset in the central nervous system (CNS), suggesting that there is a molecule that spreads from cell-to-cell. There is strong evidence that the onset and progression of ALS pathology is a consequence of protein misfolding and aggregation. In line with this, a hallmark pathology of ALS is protein deposition and inclusion formation within motor neurons and surrounding glia of the proteins TAR DNA-binding protein 43, superoxide dismutase-1, or fused in sarcoma. Collectively, the observed protein aggregation, in conjunction with the spatiotemporal spread of symptoms, strongly suggests a prion-like propagation of protein aggregation occurs in ALS. In this review, we discuss the role of protein aggregation in ALS concerning protein homeostasis (proteostasis) mechanisms and prion-like propagation. Furthermore, we examine the experimental models used to investigate these processes, including in vitro assays, cultured cells, invertebrate models, and murine models. Finally, we evaluate the therapeutics that may best prevent the onset or spread of pathology in ALS and discuss what lies on the horizon for treating this currently incurable disease

Evaluating protein cross-linking as a therapeutic strategy to stabilize SOD1 variants in a mouse model of familial ALS

Mutations in the gene encoding Cu-Zn superoxide dismutase 1 (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS) cases. A shared effect of these mutations is that SOD1, which is normally a stable dimer, dissociates into toxic monomers that seed toxic aggregates. Considerable research effort has been devoted to developing compounds that stabilize the dimer of fALS SOD1 variants, but unfortunately, this has not yet resulted in a treatment. We hypothesized that cyclic thiosulfinate cross-linkers, which selectively target a rare, 2 cysteine-containing motif, can stabilize fALS-causing SOD1 variants in vivo. We created a library of chemically diverse cyclic thiosulfinates and determined structure-cross-linking-activity relationships. A pre-lead compound, “S-XL6,” was selected based upon its cross-linking rate and drug-like properties. Co-crystallographic structure clearly establishes the binding of S-XL6 at Cys 111 bridging the monomers and stabilizing the SOD1 dimer. Biophysical studies reveal that the degree of stabilization afforded by S-XL6 (up to 24°C) is unprecedented for fALS, and to our knowledge, for any protein target of any kinetic stabilizer. Gene silencing and protein degrading therapeutic approaches require careful dose titration to balance the benefit of diminished fALS SOD1 expression with the toxic loss-of-enzymatic function. We show that S-XL6 does not share this liability because it rescues the activity of fALS SOD1 variants. No pharmacological agent has been proven to bind to SOD1 in vivo. Here, using a fALS mouse model, we demonstrate oral bioavailability; rapid engagement of SOD1G93A by S-XL6 that increases SOD1G93A’s in vivo half-life; and that S-XL6 crosses the blood–brain barrier. S-XL6 demonstrated a degree of selectivity by avoiding off-target binding to plasma proteins. Taken together, our results indicate that cyclic thiosulfinate-mediated SOD1 stabilization should receive further attention as a potential therapeutic approach for fALS

Investigations into the Unfolding, Misfolding, and Aggregation of Superoxide Dismutase-1 using Native Mass Spectrometry

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 (\u3c 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

Glutathionylation potentiates benign superoxide dismutase 1 variants to the toxic forms associated with amyotrophic lateral sclerosis

Dissociation of superoxide dismutase 1 dimers is enhanced by glutathionylation, although the dissociation constants reported to date are imprecise. We have quantified the discreet dissociation constants for wild-type superoxide dismutase 1 and six naturally occurring sequence variants, in their unmodified and glutathionylated forms, at the ratios expressed. Unmodified superoxide dismutase 1 variants that shared similar dissociation constants with SOD1WT had disproportionately increased dissociation constants when glutathionylated. This defines a key role for glutathionylation in superoxide dismutase 1 associated familial amyotrophic lateral sclerosis

Strategies to promote the maturation of ALS-associated SOD1 mutants: Small molecules return to the fold

In summary, new small molecules on the horizon show some promise for treating sufferers of SOD1-fALS and should be pursued for translation into the clinic

Propensity of mutant SOD1 to form a destabilized monomer predicts cellular aggregation and toxicity but not in vitro aggregation propensity

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the rapid and progressive degeneration of upper and lower motor neurons in the spinal cord, brain stem and motor cortex. The first gene linked to ALS was the gene encoding the free radical scavenging enzyme superoxide dismutase-1 (SOD1) that currently has over 180, mostly missense, ALS-associated mutations identified. SOD1-associated fALS patients show remarkably broad mean survival times (< 1 year to ~17 years death post-diagnosis) that are mutation dependent. 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 and/or oxidative damage leading to the misfolding and aggregation of SOD1 into a neurotoxic species. Here we aim to understand the relationship between SOD1 variant toxicity, structural stability, and aggregation propensity using a combination of cell culture and purified protein assays. Cell based assays indicated that aggregation of SOD1 variants correlate closely to cellular toxicity. However, the relationship between cellular toxicity and disease severity was less clear. We next utilised mass spectrometry to interrogate the structural consequences of metal loss and disulfide reduction on fALS-associated SOD1 variant structure. All variants showed evidence of unfolded, intermediate, and compact conformations, with SOD1G37R, SOD1G93A and SOD1V148G having the greatest abundance of intermediate and unfolded SOD1. SOD1G37R was an informative outlier as it had a high propensity to unfold and form oligomeric aggregates, but it did not aggregate to the same extent as SOD1G93A and SOD1V148G in in vitro aggregation assays. Furthermore, seeding the aggregation of DTT/EDTA-treated SOD1G37R with preformed SOD1G93A fibrils elicited minimal aggregation response, suggesting that the arginine substitution at position-37 blocks the templating of SOD1 onto preformed fibrils. We propose that this difference may be explained by multiple strains of SOD1 aggregate and this may also help explain the slow disease progression observed in patients with SOD1G37R

CuATSM Protects Against the in Vitro Cytotoxicity of Wild-Type-Like Copper-Zinc Superoxide Dismutase Mutants but not Mutants That Disrupt Metal Binding

Mutations in the SOD1 gene are associated with some forms of familial amyotrophic lateral sclerosis (fALS). There are more than 150 different mutations in the SOD1 gene that have various effects on the copper-zinc superoxide dismutase (SOD1) enzyme structure, including the loss of metal binding and a decrease in dimer affinity. The copper-based therapeutic CuATSM has been proven to be effective at rescuing neuronal cells from SOD1 mutant toxicity and has also increased the life expectancy of mice expressing the human transgenes SOD1G93A and SOD1G37R. Furthermore, CuATSM is currently the subject of a phase I/II clinical trial in Australia as a treatment for ALS. To determine if CuATSM protects against a broad variety of SOD1 mutations, we used a well-established cell culture model of SOD1-fALS. NSC-34 cells expressing SOD1-EGFP constructs were treated with CuATSM and examined by time-lapse microscopy. Our results show a concentration-dependent protection of cells expressing mutant SOD1A4V over the experimental time period. We tested the efficacy of CuATSM on 10 SOD1-fALS mutants and found that while protection was observed in cells expressing pathogenic wild-type-like mutants, cells expressing a truncation mutant or metal binding region mutants were not. We also show that CuATSM rescue is associated with an increase in human SOD1 activity and a decrease in the level of SOD1 aggregation in vitro. In conclusion, CuATSM has shown to be a promising therapeutic for SOD1-associated ALS; however, our in vitro results suggest that the protection afforded varies depending on the SOD1 variant, including negligible protection to mutants with deficient copper binding