Toxicity of Familial ALS-Linked SOD1 Mutants from Selective Recruitment to Spinal Mitochondria

196

186

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...

Section: Discussionmentioning

confidence: 98%

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

Furukawa¹,

Deng

et al. 2006

196

186

“…Cytosolic proteins from the autopsy samples were prepared as described in ref. 33. Protein concentrations were measured by using the BCA method (Pierce Chemical, Rockford, IL).…”

Section: Discussionmentioning

confidence: 99%

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

Gruzman

Wood

Alpert

et al. 2007

Self Cite

173

136

“…Gold particles within the ER and the cytosol were counted. Using a similar reasoning as Liu et al (33), the cytosol was defined as the area within 30 nm of either side of the ER membrane.…”

Section: Methodsmentioning

confidence: 99%

Spinal cord endoplasmic reticulum stress associated with a microsomal accumulation of mutant superoxide dismutase-1 in an ALS model

Kikuchi

Almer

Yamashita

et al. 2006

281

233

Mutation in superoxide dismutase-1 (SOD1), which is a cause of ALS, alters the folding patterns of this protein. Accumulation of misfolded mutant SOD1 might activate endoplasmic reticulum (ER) stress pathways. Here we show that transgenic mice expressing ALS-linked SOD1 mutants exhibit molecular alterations indicative of a recruitment of ER's signaling machinery. We demonstrate by biochemical and morphological methods that mutant SOD1 accumulates inside the ER, where it forms insoluble high molecular weight species and interacts with the ER chaperone immunoglobulin-binding protein. These alterations are age-and regionspecific, because they develop over the course of the disease and occur in the affected spinal cord but not in the nonaffected cerebellum in transgenic mutant SOD1 mice. Our results suggest a toxic mechanism for mutant SOD1 by which this ubiquitously expressed pathogenic protein could affect motor neuron survival and contribute to the selective motor neuronal degeneration in ALS.neurodegeneration ͉ protein misfolding A LS is the most common adult-onset paralytic disease characterized by a loss of motor neurons in the cerebral cortex, brainstem, and spinal cord. Insights into the neurodegenerative mechanisms followed the discovery that dominant mutations in the gene for superoxide dismutase-1 (SOD1) cause familial ALS. Overexpression of SOD1 mutants in rodents recapitulates ALS clinical and pathological hallmarks through a toxic gain of function (1). Many mutations in SOD1 decrease its stability and increase its unfolding rates and propensity to aggregate (2). High molecular weight complexes of SOD1 are observed in mammalian cells and spinal cords of transgenic mice expressing this mutant protein (3). In these animals, intracellular ubiquitin-positive proteinaceous inclusions are also often seen in spinal cord motor neurons and, in some cases, in neighboring astrocytes (3-5). These findings posit that accumulation of misfolded mutant SOD1 could contribute to the demise of motor neurons.Endoplasmic reticulum (ER) stress signaling, otherwise known as the unfolded protein response (UPR), is triggered by an increased load of misfolded proteins in the organelle (6). Herein, we show that transgenic mice expressing mutant SOD1 exhibit age-and region-specific molecular alterations indicative of a broad recruitment of ER signaling pathways, including caspase-12, a prototypical ER cell death effector (7). We also show that mutant SOD1, and to a lesser extent wild-type SOD1 (SOD1 WT ), do accumulate in the ER. Within this organelle, mutant, but not SOD1 WT , forms high molecular weight species and interacts with the ER chaperone immunoglobulin-binding protein (BiP), which is a key component of the ER misfolded protein recognition machinery (6). The preferential accumulation of mutant SOD1 in the ER in the spinal cord cells and the ensuing stress response may represent novel aspects of motor neuron degeneration in this ALS model.