Analysis of transgenic mice expressing familial amyotrophic lateral sclerosis (ALS)-linked mutations in the enzyme superoxide dismutase (SOD1) have shown that motor neuron death arises from a mutant-mediated toxic property or properties. In testing the disease mechanism, both elimination and elevation of wild-type SOD1 were found to have no effect on mutant-mediated disease, which demonstrates that the use of SOD mimetics is unlikely to be an effective therapy and raises the question of whether toxicity arises from superoxide-mediated oxidative stress. Aggregates containing SOD1 were common to disease caused by different mutants, implying that coaggregation of an unidentified essential component or components or aberrant catalysis by misfolded mutants underlies a portion of mutant-mediated toxicity.

Copper-zinc superoxide dismutase (SOD1) plays a protective role against oxidative stress. On the other hand, recent studies suggest that SOD1 itself is a major target of oxidative damage and has its own pathogenicity in various neurodegenerative diseases, including familial amyotrophic lateral sclerosis. Only human and great ape SOD1s among mammals have the highly reactive free cysteine residue, Cys 111 , at the surface of the SOD1 molecule. The purpose of this study was to investigate the role of Cys 111 in the oxidative damage of the SOD1 protein, by comparing the oxidative susceptibility of recombinant human SOD1 modified with 2-mercaptoethanol at Cys 111 (2-ME-SOD1) to wild-type SOD1. Wild-type SOD1 was more sensitive to oxidation by hydrogen peroxide-generating fragments, oligomers, and charge isomers compared with 2-ME-SOD1. Moreover, wild-type SOD1, but not 2-ME-SOD1, generated an upper shifted band in reducing SDS-PAGE even by air oxidation. Using mass spectrometry and limited proteolysis, this upper band was identified as an oxidized subunit of SOD1; the sulfhydryl group (Cys-SH) of Cys 111 was selectively oxidized to cysteine sulfinic acid (Cys-SO 2 H) and to cysteine sulfonic acid (Cys-SO 3 H). (2). Moreover, incubation with excess H 2 O 2 caused oxidation of almost all histidine and cysteine residues (3), fragmentation (4, 5) and aggregation (6, 7) of SOD1 itself. Co-incubation with bicarbonate and H 2 O 2 also induced bicarbonate radical anion formation, resulting in oligomerization of human SOD1 (8).The familial form of amyotrophic lateral sclerosis (ALS) is associated with specific mutations in the SOD1 gene (SOD1) that encodes 153 amino acids (9, 10). To date, more than 110 familial ALS (FALS)-causing mutations in SOD1 have been identified (available on the World Wide Web); however, the mechanism by which SOD1 mutants induce ALS remains unknown. The presence of intracellular aggregates that contain SOD1 in spinal cord motor neurons is thought to be a pathological hallmark of ALS. In particular, FALS-linked mutant SOD1s are prone to misfolding and aggregation (11,12). Recently, Ezzi et al. (7) reported that even wild-type SOD1 results in aggregation after oxidation, and the oxidized wildtype SOD1 gains properties like FALS mutant SOD1s. In addition to ALS, oxidative damaged SOD1 proteins were detected in the brains of patients with Alzheimer and Parkinson diseases (13). These findings suggest that oxidized SOD1 plays a role in the pathophysiology of various neurodegenerative diseases.

Approximately 20% of familial amyotrophic lateral sclerosis (FALS) arises from germ-line mutations in the superoxide dismutase-1 (SOD1) gene. However, the molecular mechanisms underlying the process have been elusive. Here, we show that a neuronal homologous to E6AP carboxyl terminus (HECT)-type ubiquitin-protein isopeptide ligase (NEDL1) physically binds translocon-associated protein-␦ and also binds and ubiquitinates mutant (but not wild-type) SOD1 proportionately to the disease severity caused by that particular mutant. Immunohistochemically, NEDL1 is present in the central region of the Lewy body-like hyaline inclusions in the spinal cord ventral horn motor neurons of both FALS patients and mutant SOD1 transgenic mice. Two-hybrid screening for the physiological targets of NEDL1 has identified Dishevelled-1, one of the key transducers in the Wnt signaling pathway. Mutant SOD1 also interacted with Dishevelled-1 in the presence of NEDL1 and caused its dysfunction. Thus, our results suggest that an adverse interaction among misfolded SOD1, NEDL1, translocon-associated protein-␦, and Dishevelled-1 forms a ubiquitinated protein complex that is included in potentially cytotoxic protein aggregates and that mutually affects their functions, leading to motor neuron death in FALS.Amyotrophic lateral sclerosis (ALS) 1 is a progressive, fatal, neurodegenerative disease that is characterized by selective loss of motor neurons in the spinal cord, brain stem, and motor cortex. The sporadic and familial forms of the disease have similar clinical and pathological features. About 10% of ALS cases are familial, and mutation of superoxide dismutase-1 (SOD1) is found in 20% of familial ALS (FALS) patients (1, 2). Mice that express mutant SOD1 transgenes develop an age-dependent ALS phenotype independent of levels of dismutase activity, suggesting that FALS pathology is because of a toxic gain of function in SOD1 and that the abnormal protein structure of mutant SOD1 is critical in the pathogenesis of motor neuron death (3-6). Recently, proteasome expression and activity have been reported to decrease with age in the spinal cord (7,8). Furthermore, mutant SOD1 turns over more rapidly than wild-type SOD1, and an inhibitor of proteasome action inhibits this turnover and thus selectively increases the steadystate level of mutant SOD1 (8). These results suggest the involvement of the ubiquitin-proteasome function in the cause of FALS. However, the biochemical nature of this gain-of-function mutation in SOD1 and the mechanism by which SOD1 mutations cause the degeneration of motor neurons have remained elusive.We show here the identification of a novel HECT-type ubiquitin-protein isopeptide ligase (E3), NEDL1, which is expressed in neuronal tissues, including the spinal cord, and selectively binds to and ubiquitinates mutant (but not wildtype) SOD1. NEDL1 is physically associated with transloconassociated protein-␦ (TRAP-␦), one of the endoplasmic reticulum (ER) translocon components that has previously been reported to bind mutant SOD...

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that primarily involves the motor neuron system. Approximately 5-10% of ALS is familial. Superoxide dismutase 1 (SOD1) gene mutations are shown to be associated with about 20% of familial ALS (FALS) patients. The neuronal Lewy-body-like hyaline inclusion (LBHI) and astrocytic hyaline inclusion (Ast-HI) are morphological hallmarks of certain SOD1-linked FALS patients with SOD1 gene mutant and transgenic mice expressing human SOD1 with G85R mutation. From the detailed immunohistochemical analyses, the essential common protein of both inclusions is SOD1. Ultrastructurally, both inclusions consist of granule-coated fibrils 15-25 nm in diameter. Based on the immuno-electron microscopical finding that these abnormal granule-coated fibrils are positive for SOD1, the formation (or aggregation) of the abnormal fibrils containing SOD1 would be essential evidence in diseases caused by various SOD1 mutations. The granule-coated fibrils are also modified by advanced glycation end products (AGEs). The AGEs themselves are insoluble molecules with direct toxic effects on cells. AGE formation of SOD1 composing the granule-coated fibrils (probable AGE-modified mutant SOD1) may amplify their aggregation and produce a more marked toxicity.

SummaryOver 100 mutants in superoxide dismutase 1 (SOD1) are reported in familial amyotrophic lateral sclerosis (ALS). However, the precise mechanism by which they are degraded through a ubiquitin-proteasomal pathway (UPP) remains unclear. Here, we report that heat-shock protein (Hsp) or heatshock cognate (Hsc)70, and the carboxyl terminus of the Hsc70-interacting protein (CHIP), are involved in proteasomal degradation of mutant SOD1. Only mutant SOD1 interacted with Hsp/Hsc70 in vivo, and in vitro experiments revealed that Hsp/Hsc70 preferentially interacted with apo-SOD1 or dithiothreitol (DTT)-treated holo-SOD1, compared with metallated or oxidized forms. CHIP, a binding partner of Hsp/Hsc70, interacted only with mutant SOD1 and promoted its degradation. Both Hsp70 and CHIP promoted polyubiquitination of mutant SOD1-associated molecules, but not of mutant SOD1, indicating that mutant SOD1 is not a substrate of CHIP. Moreover, mutant SOD1-associated Hsp/Hsc70, a known substrate of CHIP, was polyubiquitinated in vivo, and polyubiquitinated Hsc70 by CHIP interacted with the S5a subunit of the 26S proteasome in vitro. Furthermore, CHIP was predominantly expressed in spinal neurons, and ubiquitinated inclusions in the spinal motor neurons of hSOD1 G93A transgenic mice were CHIP-immunoreactive. Taken together, we propose a novel pathway in which ubiquitinated Hsp/Hsc70 might deliver mutant SOD1 to, and facilitate its degradation, at the proteasome.

No abstract

Infantile neuroaxonal dystrophy (INAD) is a fatal neurodegenerative disease characterized by the widespread presence of axonal swellings (spheroids) in the CNS and PNS and is caused by gene abnormality in PLA2G6[calcium-independent phospholipase A 2 ␤ (iPLA 2 ␤)], which is essential for remodeling of membrane phospholipids. To clarify the pathomechanism of INAD, we pathologically analyzed the spinal cords and sciatic nerves of iPLA 2 ␤ knock-out (KO) mice, a model of INAD. At 15 weeks (preclinical stage), periodic acid-Schiff (PAS)-positive granules were frequently observed in proximal axons and the perinuclear space of large neurons, and these were strongly positive for a marker of the mitochondrial outer membrane and negative for a marker of the inner membrane. By 100 weeks (late clinical stage), PAS-positive granules and spheroids had increased significantly in the distal parts of axons, and ultrastructural examination revealed that these granules were, in fact, mitochondria with degenerative inner membranes. Collapse of mitochondria in axons was accompanied by focal disappearance of the cytoskeleton. Partial membrane loss at axon terminals was also evident, accompanied by degenerative membranes in the same areas. Imaging mass spectrometry showed a prominent increase of docosahexaenoic acidcontaining phosphatidylcholine in the gray matter, suggesting insufficient membrane remodeling in the presence of iPLA 2 ␤ deficiency. Prominent axonal degeneration in neuroaxonal dystrophy might be explained by the collapse of abnormal mitochondria after axonal transportation. Insufficient remodeling and degeneration of mitochondrial inner membranes and presynaptic membranes appear to be the cause of the neuroaxonal dystrophy in iPLA 2 ␤-KO mice.

No abstract