Abnormalities in the SOD1 protein are a common factor in all types of motor neuron diseases

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Researchers have found an abnormal protein usually linked to a rare inherited form of motor neuron disease is present in all types of motor neuron disease, suggesting a common link between the different forms of the disease.

The study, published in the neuroscience journal Brain, is the first to confirm toxic changes to the protein in individuals with genetic or non-genetic forms of motor neuron disease.

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease. Ten percent of ALS cases are hereditary, with remaining cases lacking an apparent genetic cause.

“The results suggest this abnormal protein contributes to cell death in many forms of motor neuron disease, not just rare genetic cases of motor neuron disease,” says senior author Professor Kay Double from the Brain and Mind Center, Faculty of Medicine and Health.

“It is a big step in advancing our understanding of motor neuron disease. Our findings will direct further research and could ultimately lead to more effective treatments.”

Normally, the protein superoxide dismutase 1 (SOD1) protects cells, but a mutation in its gene is thought to make the protein “toxic”; this toxic protein form is associated with hereditary forms of ALS. Abnormal mutant SOD1 is only found in regions of the spinal cord where nerve cells die, implicating this abnormal protein in cell death.

Previous investigations into the role of toxic forms of SOD1 protein largely focused on mutant forms of the protein and were primarily conducted using animal and cellular models of ALS.

The study, led by a team from the University of Sydney’s Brain and Mind Center, advances our understanding of the causes of motor neuron disease by studying this abnormal protein in post-mortem tissues from patients with ALS.

“We have shown for the first time that mechanisms of disease long hypothesized to occur in animal and cellular models are present in patients with motor neuron disease,” says lead author Dr. Benjamin Trist from the Brain and Mind Center, Faculty of Medicine and Health.

“This is a significant milestone in our understanding of ALS and motor neuron disease more broadly.”

In related experiments, Professor Double and her team are also currently studying how abnormal SOD1 interacts with other disease-linked proteins in motor neuron disease. This work is in press and will be published in Acta Neuropathologica Communications.


From over 185 SOD1 mutations identified to date, only some (e.g., H46R, D90A, and R115G) cause ALS with a defined clinical phenotype, including characteristic age of onset, survival time and/or site of onset (lower limbs in D90A and H46R). Other mutations present a more varied course or have only been identified in individual patients/families, making a comprehensive analysis of the genotype–phenotype relation difficult.

The frequency of SOD1 mutations in fALS patients varies between populations from 13 to 20% and in sALS patients from 1 to 2%28–30. Thus the mutation frequency in the present study (21.1% in fALS, 2.3% in sALS) is relatively high. The L144S and K3E variants were the most frequent among the Polish ALS patients (but not in other populations, except for L144S among Brazilian patients31–33. Contrary, D90A, the most frequent European SOD1 mutation, was only present in 0.4% of cases (4.1% of fALS).

From the mutations with the highest number of representing individuals the L144S was associated with the earliest disease onset and the slowest progression, while the G41S was characterized by a particularly aggressive progression and a short survival, comparable with the phenotype reported for A4V in North American population34.

Although the number of affected individuals were not sufficient for statistical analysis, based on the clinical observation, the individuals with G37R, N86S, homozygous D90A or G93C mutations presented with a relatively less severe, while A4V, G72S, and S105L with more severe phenotypes (based on time for reaching clinical end-points), similarly to previous reports from other populations 9,35–38.

The L126* mutation, previously described as aggressive, showed a highly variable survival in our study, ranging from 36 to 228 months39. As for the clinical phenotypes, the statistical analysis showed that N139D mutation was linked to the most homogenous clinical presentation of PMA, as opposed to the K3E characterized by the prevalence of classic ALS.

From the clinical observations: beside the N139 D, the isolated lower motor neuron involvement (PMA) was observed in the G72S, N86S, G93C, S105L and L126, while the classic phenotype prevailed among patients with the K3E, G41S, D90A, and L144S mutations, and was also shown in A4V, W32 and G37R represented by single individuals.

The D90A homozygotes shared the classic phenotype with prevalent lower motor neuron involvement and lower limbs onset, whereas one of the patients heterozygous for D90A had a bulbar onset and a short survival of 25 months. We cannot exclude that the MSA-P in the patient with the D109Y mutation was an accompanying condition, as the mutation had previously been described in classic ALS with prevalent UMN involvement, bulbar onset and long survival40. In contrast to other studies we found a highly infrequent onset in the upper limbs among patients harboring SOD1 mutations.

All the cds mutations identified in our study group were classed as disease-causing according to the Human SOD1 non synonymous SNP analysis (http://bioinfogroup.com/sod1/snp/), which is consistent with our prioritization results41. However, according to the SNP analysis database none of the mutations was predicted to influence the aggregation tendency or amyloid propensity.

Our protein modeling results (Table ​(Table3,3, Supplementary Figs. S1–S15) in which we compared WT and mutated SOD1 structures after the all-atom MD simulations of a SOD1 dimer in a water environment imitating mammalian cytosol conditions42 showed that only two mutations, D109Y and C111Y, had an increased aggregation potential.

However, these mutations were not considered aggregation-prone by SNP analysis, Aggrescan, TANGO or Aggrescan3d (Supplementary Table S5). This is concordant with previous observations that SOD1 in fALS has a reduced propensity to form aggregates, while soluble heterodimers and trimeric SOD1 complexes may be more toxic as compared to large aggregates43,44.

A slightly higher aggregation potential was predicted for the A4V mutant. The movement of loops adjacent to residue after the A > V mutation is considerable (Supplementary Fig. S3), which can facilitate aggregation. After the mutation, the valine side chain is directed towards the center of the b-barrel rather than exposed but it still could promote movements of adjacent fragments. Ours result appears to support the hypothesis that folding intermediates of SOD1 are an important source of cytotoxic conformations in ALS pathology6.

Indeed, it was previously proposed that A4V mutation destabilizes SOD1 monomer and weaken the dimer interface45. Recently Brasil et al. observed low levels of SOD1 monomers in cells co-expressing WT and A4V SOD1, and the predominant formation of heteromeric species46. Based on this they suggested that WT SOD1 might exist primarily as unfolded monomeric intermediates and then fully active dimers. On the other hand, unfolded and misfolded monomers might be the predominant mutant SOD1 form.

We found L126* to be the most damaging to the protein structure. The truncation of a substantial fragment of the polypeptide (loop 126–141 and β-thread 142–151) rearranges both the dimer interface and the active site. The reported differences between the results concerning SOD1 protein stability obtained with different methods (bioinformatics, molecular modeling, in vitro, in vivo) suggest the existence of as yet unidentified factors involved in the formation of pathogenic SOD1 conformations in vivo43,47. SOD1 gene variants undergoing alternative splicing have already been described in fALS patients 48,49.

In the present study potential for alternative splicing due to the cds mutations was disputable, as only some of programs used indicated a small probability of such an effect. Nevertheless, the effects of two of those mutations, W32* and S105L seems sufficiently likely to deserve an experimental verification. Potentially altered splicing predicted with HSF 3.0 software was significantly associated with bulbar involvemenhuman spt in sALS patients and respiratory insufficiency in familial and sporadic ALS patients.

We observed that the least evolutionarily conserved positions in SOD1 (D90 and L144) were also the most frequently mutated in our study group, and the D90A and L144S carriers suffered from a slowly progressing ALS. Mutations of conserved residues of SOD1 were significantly associated with shorter survival times and shorter time between the disease onset and respiratory failure in ALS patients.

Similarly, the PredictSNP (a consensus classifier for prediction of disease-related amino acid mutations) classified the K3E, D90A, D109Y, and L144S mutations as neutral and their carriers’ symptoms were relatively less severe in terms of age of onset and survival times.

The pathogenicity of at least some ALS-related SOD1 mutations seems to involve the formation of amyloid-like aggregates. However, Aggrescan classified WT SOD1 and nearly all its mutated versions studied here as of low aggregation propensity (highly negative Na4vSS scores); still, specific nucleation points from which an ordered fibrillary structure could spread under certain conditions would nevertheless make the mutated protein amyloidogenic.

Notably, the K3E, L144S, and N139D variants were predicted to be even less aggregate-prone than the WT SOD1, while A4V was the only mutant with a markedly enhanced aggregation potential. Algorithms that have been derived from and used to predict amyloid fibril formation in the absence of other biological factors also offer a considerable degree of accuracy for predicting amyloid-aggregation propensity in vivo remains to be improve to extend this prediction to disease manifestation and pathology 50.

Statistical analyses indicate some ALS clinically relevant end-points (bulbar involvement, wheelchair-bound, respiratory insufficiency, survival time) with increase aggregation propensity but also neutral by TANGO software prediction. To sum up, while the prediction programs and molecular modeling could not define consistently the pathogenicity of each and every SOD1 mutation, there was a good agreement between those predictions and the disease severity for the both ends of the ALS spectrum, i.e., the least and the most severe cases.

A caveat of our study is that the only experimentally-derived SOD1 structure available is a dimer of the mature molecule, whereas the prion-like ALS pathomechanism is most probably associated with an immature or misfolded protein6. For instance it was shown recently that both WT and mutant SOD1 form dimers and oligomers, but only mutant SOD1 aggregates and form intracellular inclusions. Moreover, co-expression of WT and mutant SOD1 in various cell models resulted in the formation of a larger number of inclusions, as compared to cells expressing WT or mutated SOD1 separately51.

Taking into consideration the dysfunction of numerous cellular pathways observed in ALS, aggregation of SOD1 does not seem to be the only cause of ALS. According to the multistep hypothesis of ALS52, single SOD1 mutations may influence more than one step leading to ALS onset.

The major effect of SOD1 mutations in ALS is linked to the protein aggregation and a prion-like propagation of misfolded molecules. These mutations may also lead to a loss of function of SOD1 by affecting its structure and/or interactions pattern. The loss of function involves not only the dismutase enzymatic activity, e.g., associated with the N86S mutation53, but may also involve a loss of the nuclear function where SOD1 acts as a transcription factor 54.

In one sporadic ALS patient we identified a nonsense mutation at codon 32 (p.W32), which was absent from the whole exome/genome databases (1000 GP, gnomAD)55. Since the W32 was also found in the patient’s asymptomatic mother, age > 70, we were not able to prove the mutation was pathogenic. We further found that SOD1 W32* was associated with a dismutation activity in erythrocytes reduced by half53, which might point at the loss of SOD1 function 56,57. The premature stop codon could result in the shortest reported truncated SOD1 protein, but most likely the nonsense-mediated mRNA decay prevents the synthesis of such abnormal protein58.

A putative loss of SOD1 function in ALS was reported in previous studies. For instance, the mutation V30Dfs859 should produce a very short, non-functional truncated SOD1 protein. G28_P29del caused by alternative splicing of exon 2 of SOD1 leads to reduced transcription and a low level of SOD1 protein in the mutation carriers 60.

A similar result was reported for mutation S108Lfs1561, as the authors observed ca. 50% reduction of the SOD1 protein level and could not detect the truncated SOD1 (a protein with the predicted molecular weight) by Western blotting. The above mentioned SOD1 mutations, as well as other pathogenic variants including D90A, G41S, and I112M62,63, showed a reduced penetrance.

Interestingly, a recent in vitro study has shown that the tryptophan residue at position 32 (W32) is necessary for the formation of a competent seed for aggregation allowing the prion-like propagation of SOD1 misfolding from cell to cell, and the W32S substitution blocked this phenomenon64. Also a study on SOD1 single copy/knock-in models of ALS in C. elegans suggests an involvement of both the loss and gain of function of SOD1 in ALS development65.

The contribution of the loss and gain of function mechanisms vary in different neuronal populations. In the studied model, a glutamatergic neuron degeneration was induced by oxidative stress due to the loss of SOD1 function, a phenomenon also observed in a significant fraction of ALS patients. Also recent reports on children with a homozygous truncation mutation (p.C112Wfs*11) with no SOD1 activity and severe symptoms during infancy suggest that the loss of SOD1 enzymatic activity contributes to motor neuron disorders 66,67.

To sum up, the SOD1 haploinsufficiency with all its consequences might be one of the factors in an oligogenic etiology of ALS. It is most likely that many cases of ALS are due to the presence of multiple gene variants with different pathogenicity. Understanding the input of such variants to the development of neurodegeneration and their interactions with diverse environmental factors (e.g., toxins or the microbiome) is critical for the development of efficient therapies, especially in regards to potential gene therapy4,68.

reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8742055/


Original Research: Open access.
Altered SOD1 maturation and post-translational modification in amyotrophic lateral sclerosis spinal cord” by Benjamin G Trist et al. Brain

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