Mutant SOD1 detoxification mechanisms in intact single cells

Abstract

Mutant superoxide dismutase 1 (mtSOD1) causes dominantly inherited amyotrophic lateral sclerosis (ALS). The mechanism for mtSOD1 toxicity remains unknown. Two main hypotheses are the impairment of proteasomal function and chaperone depletion by misfolded mtSOD1. Here, we employed FRET/FLIM and biosensor imaging to quantitatively localize ubiquitination, as well as chaperone binding of mtSOD1, and to assess their effect on proteasomal and protein folding activities. We found large differences in ubiquitination and chaperone interaction levels for wild-type (wt) SOD1 versus mtSOD1 in intact single cells. Moreover, SOD1 ubiquitination levels differ between proteasomal structures and cytoplasmic material. Hsp70 binding and ubiquitination of wt and mtSOD1 species are highly correlated, demonstrating the coupled upregulation of both cellular detoxification mechanisms upon mtSOD1 expression. Biosensor imaging in single cells revealed that mtSOD1 expression alters cellular protein folding activity but not proteasomal function in the neuronal cell line examined. Our results provide the first cellby-cell-analysis of SOD1 ubiquitination and chaperone interaction. Moreover, our study opens new methodological avenues for cell biological research on ALS. Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease caused by the selective loss of motoneurons. 1 While ALS mostly occurs sporadically, 5-10% of cases are hereditary. About 20% of familial and few sporadic cases show point or frameshift mutations within the superoxide dismutase 1 (SOD1) gene. 2 The principle underlying mutant superoxide dismutase 1 (mtSOD1) toxicity seems to be a toxic gain -rather than loss -of function, since symptomatic mtSOD1-transgenic mice still express wild-type (wt), SOD1, some SOD1 mutants retain their enzymatic activity, 2,3 and neither knockout of endogenous SOD1 nor transgenic overexpression of wtSOD1 affect the course of disease in mtSOD1-transgenic mice. 4 Nonetheless, the downstream events in mtSOD1-mediated motoneuron death have not yet been resolved, although several hypotheses have been proposed that are based on properties that are specific for mtSOD1: altered protein folding, decreased solubility, and the propensity to form cytoplasmic aggregates. 5-10 One of the major hypotheses states that misfolded mtSOD1, through binding, depletes neuroprotective chaperones. 5-7 Accordingly, recombinant mtSOD1 inhibited chaperone activity in vitro. 11 Moreover, expression of Hsp70, Hsp27, or Hsp40 decreased aggregation of mtSOD1 and protected against mtSOD1 toxicity in cell lines and primary motoneurons. 6,12 However, overexpression of Hsp70 alone did not improve motoneuron survival in mtSOD1-transgenic mice. 13 This is possibly explained by a requirement, in vivo, for correction of further cellular functions or the combined action of different chaperones in the prevention of motoneuron cell death. Indeed, arimoclomol treatment, known to upregulate both Hsp70 and Hsp90 in the spinal cord, extended the lifespan of SOD1 ...

Destabilizing Protein Polymorphisms in the Genetic Background Direct Phenotypic Expression of Mutant SOD1 Toxicity

“…It is thus possible that SOD1 mutant proteins form different intermediate folding states in vivo depending on the nature of the mutation, and as such may possess different functional interactions with the folding machinery of the cell. Indeed, G85R and G93A proteins were recently shown to have different interactions with HSP70 in cultured cells [41]. On the other hand, the structure and functions of paramyosin, UNC-45, and Ras are diverse (a structural coiled-coil protein, a soluble TPR domain-containing protein and a small GTPase, respectively) and these proteins are not overexpressed as they are expressed from their endogenous chromosomal loci.…”

The proteasomal system

“…The UPS-mediated degradation of the mutant SOD1 in ALS is realized via the Hsp70-CHIP pathway (Choi et al, 2004;Urushitani et al, 2004;Ishigaki et al, 2007). Experiments showed that only mutant SOD1, but not the wild type protein induces the formation of aggregates during proteasome inhibition (Kabashi and Durham, 2006), as well as only the mutant form seems to be polyubiquitinated in neuronal cells (Basso et al, 2006;Ganesan et al, 2008), mediated by the E3 ligases dorfin, CHIP and NEDL1 (Miyazaki et al, 2004;. These results suggest that even the susceptible motor neurons are capable of degrading mutant SOD1, if the efficiency of the UPS is ensured.…”

Protein Homeostasis in Amyotrophic Lateral Sclerosis: Therapeutic Opportunities?

“…In cell based assays and mouse models, ALS mutant SOD1G93A and G85R show increased interaction with a number of chaperones, including Hsp70, suggesting that ALS-associated mutant species may result in a depletion of available chaperones and chaperone activity, therefore leading to cellular toxicity (Tummala et al, 2005; Ganesan et al, 2007). Furthermore, reduced levels of the chaperone alpha-B-crystallin (CRYAB) and increased incorporation of other molecular chaperones, including Hsc70, into the insoluble aggregate fraction are features of a faster progressing phenotype in SOD1G93A transgenic mice (Marino et al, 2015).…”