Proteins That Bind to Misfolded Mutant Superoxide Dismutase-1 in Spinal Cords from Transgenic Amyotrophic Lateral Sclerosis (ALS) Model Mice

Abstract

Mutant superoxide dismutase-1 (SOD1) has an unidentified toxic property that provokes ALS. Several ALS-linked SOD1 mutations cause long C-terminal truncations, which suggests that common cytotoxic SOD1 conformational species should be misfolded and that the C-terminal end cannot be involved. The cytotoxicity may arise from interaction of cellular proteins with misfolded SOD1 species. Here we specifically immunocaptured misfolded SOD1 by the C-terminal end, from extracts of spinal cords from transgenic ALS model mice. Associated proteins were identified with proteomic techniques. Two transgenic models expressing SOD1s with contrasting molecular properties were examined: the stable G93A mutant, which is abundant in the spinal cord with only a tiny subfraction misfolded, and the scarce disordered truncation mutant G127insTGGG. For comparison, proteins in spinal cord extracts with affinity for immobilized apo G93A mutant SOD1 were determined. Two-dimensional gel patterns with a limited number of bound proteins were found, which were similar for the two SOD1 mutants. Apart from neurofilament light, the proteins identified were all chaperones and by far most abundant was Hsc70. The immobilized apo G93A SOD1, which would populate a variety of conformations, was found to bind to a considerable number of additional proteins. A substantial proportion of the misfolded SOD1 in the spinal cord extracts appeared to be chaperone-associated. Still, only about 1% of the Hsc70 appeared to be associated with misfolded SOD1. The results argue against the notion that chaperone depletion is involved in ALS pathogenesis in the transgenic models and in humans carrying SOD1 mutations.Amyotrophic lateral sclerosis (ALS) 2 is characterized by degeneration of motor neurons in the motor cortex, the brainstem, and the spinal cord. This results in progressive muscular atrophy and the patients usually succumb to respiratory failure within a few years. About 10% of ALS cases cluster in families (1), and in some of these the disease is linked to mutations in the gene of the antioxidant enzyme superoxide dismutase-1 (SOD1).Overall, about 6% of all cases with ALS show SOD1 mutations, and more than 160 such mutations have been identified (2). The mutations confer a cytotoxic gain of function of unknown character to the enzyme (3-4). The ALS-linked mutant SOD1s, including nine with long C-terminal truncations, are likely to cause ALS by essentially the same mechanism. In the shortest of these the native sequence ends with Leu-117 followed by four novel amino acids and a premature stop codon. The truncated mutants lack the stabilizing Cys-57-Cys-146 disulfide bond and ␤-strand 8 of the ␤-barrel core of the protein, and should show limited native folding. Thus, any cytotoxic conformational species common to the ALSlinked SOD1 mutations should be misfolded. Furthermore, the C-terminal end cannot be involved in the cytotoxicity. One conceivable mechanism by which such misfolded SOD1 species might provoke the disease is through interaction with essential...

Macrophage Migration Inhibitory Factor as a Chaperone Inhibiting Accumulation of Misfolded SOD1

“…Inhibition of misfolded SOD1 association with mitochondria was specific to MIF, as other chaperones tested, including Hsp27, Hsc70, aB-crystallin [previously reported to interact with mutant and misfolded SOD1 and modulate its aggregation (Karch and Borchelt, 2010; Krishnan et al, 2008; Wang et al, 2009; Wang et al, 2005; Yerbury et al, 2013; Zetterstrom et al, 2011b)], cyclophilin-A [one of our 7 candidate proteins and previously proposed to act as a chaperone (Freskgard et al, 1992)], or glutathione peroxidase [a protein with thiol-oxidoreductase activity, an activity that has been proposed to be crucial for the association of mutant SOD1 proteins with mitochondria (Cozzolino et al, 2008; Ferri et al, 2006)], had no effect on mutant SOD1 association with normal mitochondria (Figure 3F, G). …”

Post-transcriptional Inhibition of Hsc70-4/HSPA8 Expression Leads to Synaptic Vesicle Cycling Defects in Multiple Models of ALS

“…Given the general role of Hsc70-4 in clearance of misfolded or aggregated proteins (Liu et al, 2012; Stricher et al, 2013; Zetterstrom et al, 2011), we hypothesized that Hsc70-4 OE may influence TDP-43 aggregation. To examine this, we performed subcellular fractionations from Drosophila larvae co-overexpressing wild-type or mutant TDP-43 with Hsc70-4 in motor neurons and quantified the amount of TDP-43 in the soluble (low salt, LS), sarkosyl (Sark), and urea (Urea) fractions.…”

Molecular chaperone Hsp110 rescues a vesicle transport defect produced by an ALS-associated mutant SOD1 protein in squid axoplasm

“…5C). The misfolded mutant protein apparently exposes epitopes that are sufficiently extended and hydrophobic to recruit endogenous Hsp(c)70 in axoplasm, as occurs in vivo in mouse spinal cord (Table S1; 28,37,38). Notably, Hsc70 is the most abundant chaperone in mouse motor neurons, as revealed by laser capture microdissection and proteolysis/MS.…”

“…Blocks Slowing. Earlier studies have indicated that the molecular chaperone Hsc70 interacts with mutant human SOD1 protein in the spinal cord of transgenic mice that develop ALS (37,38). This interaction also occurred in mouse strains transgenic for G85R SOD1YFP (28).…”