No abstract

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.

Mutant Cu/Zn-superoxide dismutase (SOD1) protein aggregation has been suggested as responsible for amyotrophic lateral sclerosis (ALS), although the operative mediating factors are as yet unestablished. To evaluate the contribution of motoneuronal Ca2+-permeable (GluR2 subunit-lacking) alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors to SOD1-related motoneuronal death, we generated chat-GluR2 transgenic mice with significantly reduced Ca2+-permeability of these receptors in spinal motoneurons. Crossbreeding of the hSOD1G93A transgenic mouse model of ALS with chat-GluR2 mice led to marked delay of disease onset (19.5%), mortality (14.3%) and the pathological hallmarks such as release of cytochrome c from mitochondria, induction of cox2 and astrogliosis. Subcellular fractionation analysis revealed that unusual SOD1 species first accumulated in two fractions dense with neurofilaments/glial fibrillary acidic protein/nuclei and mitochondria long time before disease onset, and then concentrated into the former fraction by disease onset. All these processes for unusual SOD1 accumulation were considerably delayed by GluR2 overexpression. Ca2+-influx through atypical motoneuronal AMPA receptors thus promotes a misfolding of mutant SOD1 protein and eventual death of these neurons.

Vaccinations targeting extracellular superoxide dismutase 1 (SOD1) mutants are beneficial in mouse models of amyotrophic lateral sclerosis (ALS). Because of its misfolded nature, wild-type nonmetallated SOD1 protein (WT-apo) may have therapeutic application for vaccination of various SOD1 mutants. We compared the effects of WT-apo to those of a G93A SOD1 vaccine in low-copy G93A SOD1 transgenic mice. Both SOD1 vaccines induced antibody against G93A SOD1 and significantly delayed disease onset compared with saline/adjuvant controls. WT-apo SOD1 significantly extended the life span of vaccinated mice. The vaccines potentiated TH2 deviation in the spinal cord as determined by the ratio of interleukin-4 to interferon-γ (IFNγ) or tumor necrosis factor and induced C1q deposition around motor neurons. Transgenic mice had abundant microglial expression of signal transducers and activators of transcription 4, an activator of transcription of IFNγ, in the spinal cord implicating IFNγ in the pathogenesis. On the other hand, the sera from G93A SOD1-vaccinated mice showed higher IFNγ or tumor necrosis factor and yielded a lower IgG1/IgG2c ratio than the sera from WT-apo-vaccinated mice. These results indicate that the TH1/TH2 milieu is affected by specific vaccinations and that antigenicity might counteract beneficial effects by enhancing TH1 immunity. Thus, because of its lower TH1 induction, WT-apo may be a therapeutic option and have broader application in ALS associated with diverse SOD1 mutations.