Disulfide cross-linked protein represents a significant fraction of ALS-associated Cu, Zn-superoxide dismutase aggregates in spinal cords of model mice

Abstract

Point mutations in Cu, Zn-superoxide dismutase (SOD1) cause a familial form of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Aggregates of mutant SOD1 proteins are observed in histopathology and are invoked in several proposed mechanisms for motor neuronal death; however, the significant stability and activity of the mature mutant proteins are not readily explained in such models. Recent biochemical studies suggest that it is the immature disulfide-reduced forms of the familial ALS mutant SOD1 proteins that play a critical role; these forms tend to misfold, oligomerize, and readily undergo incorrect disulfide formation upon mild oxidative stress in vitro. Here we provide physiological support for this mechanism of aggregate formation and show that a significant fraction of the insoluble SOD1 aggregates in spinal cord of the ALS-model transgenic mice contain multimers cross-linked via intermolecular disulfide bonds. These insoluble disulfide-linked SOD1 multimers are found only in the spinal cord of symptomatic transgenic animals, are not observed in unafflicted tissue such as brain cortex and liver, and can incorporate WT SOD1 protein. The findings provide a biochemical basis for a pathological hallmark of this disease; namely, incorrect disulfide cross-linking of the immature, misfolded mutant proteins leads to insoluble aggregates. disulfide bond ͉ protein aggregation ͉ oxidative stress ͉ neurodegnerative disease A myotrophic lateral sclerosis (ALS) is one of the most common adult-onset neurodegenerative diseases, and, in 1993, several mutations in the Cu, Zn-superoxide dismutase (SOD1) gene were identified as a cause of a subset of familial ALS (fALS) (1, 2). Since that time, Ͼ100 types of SOD1 mutations have been linked with fALS; however, its molecular mechanism remains unresolved. SOD1, which is a homodimer with a copper and zinc ion in each subunit, functions as an antioxidant enzyme by converting superoxide radical to oxygen and hydrogen peroxide (3). Some of the SOD1 mutant proteins have comparable levels of dismutase activity, and the SOD1-knockout mouse does not develop ALS-like motor neuron symptoms (4), leading to the idea that SOD1-mediated toxicity in fALS is due to a new activity acquired by mutations in the SOD1 gene. One of the proposed toxic functions in the SOD1 mutant proteins is an aberrant copper chemistry. A subset of ALS mutations in SOD1 can lead to catalytic nitration of tyrosine residues and peroxide or superoxide production at the copper site (5); however, the copper-toxicity model is difficult to reconcile with the fact that ALS symptoms still are observed in the transgenic mouse expressing SOD1 proteins in which all four copper ligands are mutated (6). Although some copper chelators can reduce the toxicity of mutant proteins in transgenic ALS model mice (7), a role for aberrant copper chemistry in the disease has not been widely addressed.Another hypothesis for a gain of toxic function is that the mutations in SOD1 facilitate protein aggregation (8). An enzyma...

Common molecular signature in SOD1 for both sporadic and familial amyotrophic lateral sclerosis

“…30-32. More recently, studies from transgenic mouse models of FALS have suggested that the intermolecular disulfide-linked SOD1 aggregates are associated with disease toxicity (8,23). One study used immunoprecipitation to conclude an apparent SOD1 species of Ϸ35 kDa was a complex between SOD1 and Bcl-2 (19), albeit our mass spectrometric analysis offered no support for any linkage to Bcl-2, at least in SALS.…”

“…Many studies have investigated the possibility of structural differences in SOD1 caused by mutations as a basis for such toxic effects (4,5). Although several common features of mutant SOD1 proteins such as instability, higher sensitivity to denaturing conditions, and propensity to form aggregates are found to be associated with mutant SOD1-linked FALS disease course and severity (6)(7)(8)(9)(10)(11), no single biochemical feature has been found to be shared by all identified mutant SOD1s, nor has there been a single molecular feature of SOD1 identified to be associated with SALS.…”

Soluble misfolded subfractions of mutant superoxide dismutase-1s are enriched in spinal cords throughout life in murine ALS models

“…In the model that was most closely examined, G93A, these forms appeared to predominate, and were present throughout life. The hSOD1 that is present as insoluble aggregates in terminal spinal cords is partially composed of disulfidelinked dimers and multimers (23,24). Because SOD1 is mainly present in the reducing cytosol, the presence of the oxidized species is surprising.…”

“…In the latter, they accumulate in the final phase concomitantly with the appearance of overt symptoms, and are commonly suggested to cause the disease (24,26). Their appearance probably reflects a terminal disruption of the protein surveillance and degrading systems.…”

Metal-free superoxide dismutase forms soluble oligomers under physiological conditions: A possible general mechanism for familial ALS

“…Although the exact cellular sites and mechanisms of toxicity are unknown, aberrant SOD1 protein oligomerization has been strongly implicated in disease causation (7,8). Several recent publications have presented compelling evidence that abnormal disulfide cross-linking of ALS-mutant SOD1 plays a role in this oligomerization, and disulfide-linked SOD1 multimers have been detected in neural tissues of SOD1-ALS transgenic mice that are presumed to be components of higher-molecular-weight species or intermediates in their formation (7,(9)(10)(11).…”