Abstract
Mutations in Cu/Zn superoxide dismutase 1 (SOD1) associated with familial amyotrophic lateral sclerosis cause the protein to aggregate via a prion-like process in which soluble molecules are recruited to aggregates by conformational templating. These misfolded SOD1 proteins can propagate aggregation-inducing conformations across cellular membranes. Prior studies demonstrated that mutation of a Trp (W) residue at position 32 to Ser (S) suppresses the propagation of misfolded conformations between cells, whereas other studies have shown that mutation of Trp 32 to Phe (F), or Cys 111 to Ser, can act in cis to attenuate aggregation of mutant SOD1. By expressing mutant SOD1 fused with yellow fluorescent protein (YFP), we compared the relative ability of these mutations to modulate the formation of inclusions by ALS-mutant SOD1 (G93A and G85R). Only mutation of Trp 32 to Ser persistently reduced the formation of the amorphous inclusions that form in these cells, consistent with the idea that a Ser at position 32 inhibits templated propagation of aggregation prone conformations. To further test this idea, we produced aggregated fibrils of recombinant SOD1-W32S in vitro and injected them into the spinal cords of newborn mice expressing G85R-SOD1: YFP. The injected mice developed an earlier onset paralysis with a frequency similar to mice injected with WT SOD1 fibrils, generating a strain of misfolded SOD1 that produced highly fibrillar inclusion pathology. These findings suggest that the effect of Trp 32 in modulating the propagation of misfolded SOD1 conformations may be dependent upon the “strain” of the conformer that is propagating.
Highlights
10–20% of familial amyotrophic lateral sclerosis cases are associated with mutations in the ubiquitously expressed superoxide scavenging cytosolic enzyme Cu-Zn superoxide dismutase (SOD1) [1,2,3]
To standardize for the amount of fibrils produced in the assay, we assessed the levels of large aggregates by filter trap assay and observed a ~5 fold greater level WT-superoxide dismutase 1 (SOD1) fibrils than W32S-SOD1 fibrils in our preparations (Fig 1a)
We performed a second passage by injecting a naïve litter of P0 G85R-SOD1: yellow fluorescent protein (YFP) mice with spinal cord homogenate prepared from paralyzed mice that had received the W32S-SOD1 injections
Summary
10–20% of familial amyotrophic lateral sclerosis (fALS) cases are associated with mutations in the ubiquitously expressed superoxide scavenging cytosolic enzyme Cu-Zn superoxide dismutase (SOD1) [1,2,3]. A subset of mutations result in early translation termination, yielding truncated proteins that lack a portion or all of the residues encoded in the 5th and last coding exon. These early truncation mutations are clearly catastrophic for enzymatic activity and protein stability, the effect of disease associated point mutations is more variable with some mutations having minimal impact on activity or protein half-life [7,8,9,10]. There is considerable evidence that the misfolding and aggregation of mutant SOD1 is a key event in the toxic processes that produce motor neuron disease (reviewed in [36])
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