Superoxide dismutase‐1 and other proteins in inclusions from transgenic amyotrophic lateral sclerosis model mice

Abstract

J. Neurochem. (2010) 114, 408–418. Abstract Mutant superoxide dismutase‐1 (SOD1) causes amyotrophic lateral sclerosis (ALS) through a cytotoxic mechanism of unknown nature. A hallmark in ALS patients and transgenic mouse models carrying human SOD1 (hSOD1) mutations are hSOD1‐immunoreactive inclusions in spinal cord ventral horns. The hSOD1 inclusions may block essential cellular functions or cause toxicity through sequestering of other proteins. Inclusions from four different transgenic mouse models were examined after density gradient ultracentrifugation. The inclusions are complex structures with heterogeneous densities and are disrupted by detergents. The aggregated hSOD1 was mainly composed of subunits that lacked the native stabilizing intra‐subunit disulfide bond. A proportion of subunits formed hSOD1 oligomers or was bound to other proteins through disulfide bonds. Dense inclusions could be isolated and the protein composition was analyzed using proteomic techniques. Mutant hSOD1 accounted for half of the protein. Ten other proteins were identified. Two were cytoplasmic chaperones, four were cytoskeletal proteins, and 4 were proteins that normally reside in the endoplasmic reticulum (ER). The presence of ER proteins in inclusions containing the primarily cytosolic hSOD1 further supports the notion that ER stress is involved in ALS.

The small heat shock proteins αB-crystallin and Hsp27 suppress SOD1 aggregation in vitro

“…Such a mechanism is consistent with recent work in which αB-crystallin has been reported to be capable of binding to preformed fibrils and, in doing so, suppressing further fibril formation (Shammas et al 2011;Waudby et al 2010). Moreover, in both humans and mouse models of ALS, αB-crystallin is primarily associated with the insoluble inclusions (Basso et al 2009;Bergemalm et al 2010;Kato et al 1997). Thus, we directly tested whether αB-crystallin is capable of binding to mature SOD1 aggregates using a dot blot assay (Fig.…”

“…The accumulation of this misfolded protein signifies a breakdown in the quality control system that prevents protein aggregation or an inability of these systems to cope with the increased load brought about by mutant SOD1 expression in ALS (Wang et al 2009). There are many other proteins associated with SOD1 deposits in vivo including molecular chaperones HSP70 and αB-crystallin and structural proteins such as vimentin, neurofilament heavy chain and tubulin (Bergemalm et al 2010). The reason why these proteins are present in these deposits is unknown; however, it has been proposed that, at least in the case of chaperones, it may signify their failed attempt to keep the aggregating protein soluble in solution (Muchowski and Wacker 2005).…”

“…αB-crystallin and Hsp25 have also been identified as components of inclusions from mutant SOD1 (G127X, G93A, D90A and G37R) mice using proteomics techniques on isolated inclusions (Bergemalm et al 2010) and detergent-insoluble fractions (Basso et al 2009). Moreover, SOD1 can be immunoprecipitated using αB-crystallin antibodies in cell models of SOD1-associated ALS (Shinder et al 2001).…”

Reduced Calreticulin Levels Link Endoplasmic Reticulum Stress and Fas-Triggered Cell Death in Motoneurons Vulnerable to ALS

“…Mislocalization of CRT constitutes another hypothesis. Indeed, mass spectrometry-liquid chromatography analysis of protein aggregates from end-stage mSOD1 mouse spinal cords revealed the presence of CRT together with mSOD1, indicating a sequestration of CRT outside the ER (Bergemalm et al, 2010). Our own preliminary immunoprecipitation experiments confirm the CRT affinity to mSOD1 both in NSC34 cells transfected with either SOD1…”

Análise in vitro da esclerose lateral amiotrófica tipo 8 e estudo genético da paraplegia espástica 4

“…This is highlighted specially in situations where the affected protein is expressed since the pluripotent stage, as it is the case of VAPB (this work) and for the SMN protein, responsible for Spinal Muscular Atrophy (Ebert et al, 2008). Intracytoplasmic aggregates comprise a hallmark in different ALS forms, SOD1 (ALS1) (Brujin et al, 1997;Bergemalm et al, 2010), TDP-43 (ALS10) (Gitcho et al, 2008;, FUS/TLS (ALS6) and VAPB itself Chai et al, 2008;Ratnaparkhi et al, 2008;Suzuki et al, 2009;. TDP-43 immunoreactive aggregates have a highlighted role in those inclusions, since they are found in most ALS cases that are SOD1-negative (LagierTourenne and Cleveland, 2009) and in VAPB-P56S transgenice mice (Tudor et al, 2010).…”