Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS

Howard Hughes Medical Institute, Department of Genetics, Section of Comparative Medicine, and Department of Neurosurgery, Yale University School of Medicine, New Haven, CT 06510, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 02/2009; 106(5):1392-7. DOI: 10.1073/pnas.0813045106
Source: PubMed


Recent studies suggest that superoxide dismutase 1 (SOD1)-linked amyotrophic lateral sclerosis results from destabilization and misfolding of mutant forms of this abundant cytosolic enzyme. Here, we have tracked the expression and fate of a misfolding-prone human SOD1, G85R, fused to YFP, in a line of transgenic G85R SOD1-YFP mice. These mice, but not wild-type human SOD1-YFP transgenics, developed lethal paralyzing motor symptoms at 9 months. In situ RNA hybridization of spinal cords revealed predominant expression in motor neurons in spinal cord gray matter in all transgenic animals. Concordantly, G85R SOD-YFP was diffusely fluorescent in motor neurons of animals at 1 and 6 months of age, but at the time of symptoms, punctate aggregates were observed in cell bodies and processes. Biochemical analyses of spinal cord soluble extracts indicated that G85R SOD-YFP behaved as a misfolded monomer at all ages. It became progressively insoluble at 6 and 9 months of age, associated with presence of soluble oligomers observable by gel filtration. Immunoaffinity capture and mass spectrometry revealed association of G85R SOD-YFP, but not WT SOD-YFP, with the cytosolic chaperone Hsc70 at all ages. In addition, 3 Hsp110's, nucleotide exchange factors for Hsp70s, were captured at 6 and 9 months. Despite such chaperone interactions, G85R SOD-YFP formed insoluble inclusions at late times, containing predominantly intermediate filament proteins. We conclude that motor neurons, initially "compensated" to maintain the misfolded protein in a soluble state, become progressively unable to do so.

Download full-text


Available from: Cristian Ruse, Oct 10, 2015
14 Reads
  • Source
    • "It is possible that there is some abundant cellular protein that binds SOD1 and produces the electrophoretic migration patterns observed. Numerous SOD1 binding proteins have been described in the literature and among those described there are abundant proteins such as Hsp70 [18], [22], [40]–[43], Hsc70 [22], [42], [43], and the cytoplasmic dynein complex [44] (for a summary of other findings also see [45]). We would not expect the binding of a single protein to mutant SOD1 to produce the high molecular weight smears seen with the A4V or G93A proteins; such smears suggest either very heterogeneous complexes or homogeneous oligomeric structures of very heterogeneous size. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mutations in superoxide dismutase 1 (SOD1) cause familial forms of amyotrophic lateral sclerosis (fALS). Disease causing mutations have diverse consequences on the activity and half-life of the protein, ranging from complete inactivity and short half-life to full activity and long-half-life. Uniformly, disease causing mutations induce the protein to misfold and aggregate and such aggregation tendencies are readily visualized by over-expression of the proteins in cultured cells. In the present study we have investigated the potential of using immunoblotting of proteins separated by Blue-Native gel electrophoresis (BNGE) as a means to identify soluble multimeric forms of mutant protein. We find that over-expressed wild-type human SOD1 (hSOD1) is generally not prone to form soluble high molecular weight entities that can be separated by BNGE. For ALS mutant SOD1, we observe that for all mutants examined (A4V, G37R, G85R, G93A, and L126Z), immunoblots of BN-gels separating protein solubilized by digitonin demonstrated varied amounts of high molecular weight immunoreactive entities. These entities lacked reactivity to ubiquitin and were partially dissociated by reducing agents. With the exception of the G93A mutant, these entities were not reactive to the C4F6 conformational antibody. Collectively, these data demonstrate that BNGE can be used to assess the formation of soluble multimeric assemblies of mutant SOD1.
    PLoS ONE 08/2014; 9(8):e104583. DOI:10.1371/journal.pone.0104583 · 3.23 Impact Factor
  • Source
    • "The size of SOD1-WT-YFP, calculated from the diffusion time, indicated the presence of a dimeric species (~79 kDa, Fig. 3A, lanes 3–7). By contrast, in the absence of MG-132, mutant SOD1-G85R-YFP exhibited a diffusion time intermediate between those of monomers and dimers (~60 kDa, Fig. 3A, lane 8), suggesting that both monomers and dimers were present under these conditions , as previously reported (Johnston et al. 2000; Wang et al. 2009). In the presence of MG-132 treatment , FCS revealed the presence of high molecular weight species (~1000 kDa) of mutant SOD1 (Fig. 3A, lane 10–12). "
    [Show abstract] [Hide abstract]
    ABSTRACT: A hallmark of protein conformational disease, exemplified by neurodegenerative disorders, is the expression of misfolded and aggregated proteins. The relationship between protein aggregation and cellular toxicity is complex, and various models of experimental pathophysiology have often yielded conflicting or controversial results. In this study, we examined the biophysical properties of amyotrophic lateral sclerosis (ALS)-linked mutations of Cu/Zn superoxide dismutase 1 (SOD1) expressed in human tissue culture cells. Fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) analyses revealed that changes in proteasome activity affected both the expression of FCS- and FRET-detected oligomers and cellular toxicity. Under normal conditions, highly aggregation-prone mutant SOD1 exhibited very little toxicity. However, when the activity of the proteasome was transiently inhibited, only upon recovery did we observe the appearance of ordered soluble oligomers, which were closely correlated with cellular toxicity. These results shed light on the importance of balance in proteostasis and suggest that transient shifts of activity in the cellular machinery can alter the course of protein conformational transitions and dysregulate modulation of proteasome activity. In neurodegenerative disorders including ALS, such changes may be a risk factor for pathogenesis.
    Genes to Cells 01/2014; 19(3). DOI:10.1111/gtc.12125 · 2.81 Impact Factor
  • Source
    • "CHIP dysfunction has also been reported in SBMA (and in amyotrophic lateral sclerosis [ALS]) and its overexpression has a beneficial effect in both diseases (Morishima et al., 2008). Similar observations have been reported for the other members of the hetero-complex, Hsc70 (Urushitani et al., 2004; Wang et al., 2009), or Hsp70 (Adachi et al., 2003; Kobayashi et al., 2000; Warrick et al., 1999; Waza et al., 2005, 2006). The same multiheteromeric complex has been found in muscle (Arndt et al., 2010), where it serves for the recognition of proteins damaged (misfolded) during prolonged physical exercise. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Spinal and bulbar muscular atrophy (SBMA) is an X-linked motoneuron disease caused by an abnormal expansion of a tandem CAG repeat in exon 1 of the androgen receptor (AR) gene that results in an abnormally long polyglutamine tract (polyQ) in the AR protein. As a result, the mutant AR (ARpolyQ) misfolds, forming cytoplasmic and nuclear aggregates in the affected neurons. Neurotoxicity only appears to be associated with the formation of nuclear aggregates. Thus, improved ARpolyQ cytoplasmic clearance, which indirectly decreases ARpolyQ nuclear accumulation, has beneficial effects on affected motoneurons. In addition, increased ARpolyQ clearance contributes to maintenance of motoneuron proteostasis and viability, preventing the blockage of the proteasome and autophagy pathways that might play a role in the neuropathy in SBMA. The expression of heat shock protein B8 (HspB8), a member of the small heat shock protein family, is highly induced in surviving motoneurons of patients affected by motoneuron diseases, where it seems to participate in the stress response aimed at cell protection. We report here that HspB8 facilitates the autophagic removal of misfolded aggregating species of ARpolyQ. In addition, though HspB8 does not influence p62 and LC3 (two key autophagic molecules) expression, it does prevent p62 bodies formation, and restores the normal autophagic flux in these cells. Interestingly, trehalose, a well-known autophagy stimulator, induces HspB8 expression, suggesting that HspB8 might act as one of the molecular mediators of the proautophagic activity of trehalose. Collectively, these data support the hypothesis that treatments aimed at restoring a normal autophagic flux that result in the more efficient clearance of mutant ARpolyQ might produce beneficial effects in SBMA patients.
    Neurobiology of aging 06/2013; 34(11). DOI:10.1016/j.neurobiolaging.2013.05.026 · 5.01 Impact Factor
Show more