The etiology of motor neuron degeneration in amyotrophic lateral sclerosis (ALS) remains to be better understood. Based on the studies from ALS patients and transgenic animal models, it is believed that ALS is likely to be a multifactorial and multisystem disease. Many mechanisms have been postulated to be involved in the pathology of ALS, such as oxidative stress, glutamate excitotoxicity, mitochondrial damage, defective axonal transport, glia cell pathology and aberrant RNA metabolism. Mitochondria, which play crucial roles in excitotoxicity, apoptosis and cell survival, have shown to be an early target in ALS pathogenesis and contribute to the disease progression. Morphological and functional defects in mitochondria were found in both human patients and ALS mice overexpressing mutant SOD1. Mutant SOD1 was found to be preferentially associated with mitochondria and subsequently impair mitochondrial function. Recent studies suggest that axonal transport of mitochondria along microtubules and mitochondrial dynamics may also be disrupted in ALS. These results also illustrate the critical importance of maintaining proper mitochondrial function in axons and neuromuscular junctions, supporting the emerging “dying-back” axonopathy model of ALS. In this review, we will discuss how mitochondrial dysfunction has been linked to the ALS variants of SOD1 and the mechanisms by which mitochondrial damage contributes to the disease etiology.

, tetrameric red fluorescent protein; FL, full-length; GFP, green fluorescent protein; HA, hemagglutinin; HEK293, human embryonic kidney cells; KO, knockout; LC3, microtubule-associated protein 1 light chain 3B; LIR, LC3 interaction region; MEF, mouse embryonic fibroblast; PB1, Phox and Bem1; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SMIR, SOD1 mutant interaction region; SOD, superoxide dismutase; UBA, ubiquitin association; WT, wild-type. AbstractThe p62/sequestosome 1 protein has been identified as a component of pathological protein inclusions in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). P62 has also been implicated in autophagy, a process of mass degradation of intracellular proteins and organelles. Autophagy is a critical pathway for degrading misfolded and/or damaged proteins, including the copper-zinc superoxide dismutase (SOD1) mutants linked to familial ALS. We previously reported that p62 interacted with ALS mutants of SOD1 and that the ubiquitin-association domain of p62 was dispensable for the interaction. In this study, we identified two distinct regions of p62 that were essential to its binding to mutant SOD1: the N-terminal Phox and Bem1 (PB1) domain (residues 1-104) and a separate internal region (residues 178-224) termed here as SOD1 mutant interaction region (SMIR). The PB1 domain is required for appropriate oligomeric status of p62 and the SMIR is the actual region interacting with mutant SOD1. Within the SMIR, the conserved W184, H190 and positively charged R183, R186, K187, and K189 residues are critical to the p62-mutant SOD1 interaction as substitution of these residues with alanine resulted in significantly abolished binding. In addition, SMIR and the p62 sequence responsible for the interaction with LC3, a protein essential for autophagy activation, are independent of each other. In cells lacking p62, the existence of mutant SOD1 in acidic autolysosomes decreased, suggesting that p62 can function as an adaptor between mutant SOD1 and the autophagy machinery. This study provides a novel molecular mechanism by which mutant SOD1 can be recognized by p62 in an ubiquitin-independent fashion and targeted for the autophagy-lysosome degradation pathway. Keywords: autophagy, familial amyotrophic lateral sclerosis, mutant superoxide dismutase 1, sequestosome 1/p62.

Scaffold proteins play a critical role in controlling the activity of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. Shoc2 is a leucine-rich repeat scaffold protein that acts as a positive modulator of ERK1/2 signaling. However, the precise mechanism by which Shoc2 modulates the activity of the ERK1/2 pathway is unclear. Here we report the identification of the E3 ubiquitin ligase HUWE1 as a binding partner and regulator of Shoc2 function. HUWE1 mediates ubiquitination and, consequently, the levels of Shoc2. Additionally, we show that both Shoc2 and HUWE1 are necessary to control the levels and ubiquitination of the Shoc2 signaling partner, RAF-1. Depletion of HUWE1 abolishes RAF-1 ubiquitination, with corresponding changes in ERK1/2 pathway activity occurring. Our results indicate that the HUWE1-mediated ubiquitination of Shoc2 is the switch that regulates the transition from an active to an inactive state of the RAF-1 kinase. Taken together, our results demonstrate that HUWE1 is a novel player involved in regulating ERK1/2 signal transmission through the Shoc2 scaffold complex.

Although transplantation of human mesenchymal stem cells (MSCs) derived from amnion (hAMSCs), bone marrow (hBMSCs) and adipose tissues (hADSCs) has been shown to aid in the repair of cutaneous wounds in mouse models, little information is available regarding the relative efficacy of MSCs from different sources. In this study, we compared their therapeutic potentials by transplanting equal numbers of hAMSCs, hBMSCs or hADSCs in a mouse model of cutaneous wounds. The results suggested that an hADSC injection has the most pronounced effect on wound closure. Histological evaluation showed enhanced re-epithelialization in the hADSC group compared with the hBMSC and hAMSC groups. Although there was a slight improvement in wound healing in the hAMSC and hBMSC groups, the differences between the groups were statistically insignificant. In a trans-well coculture model, wound healing migration and transwell migration assays showed that hADSCs were superior to hAMSCs and hBMSCs at promoting human dermal fibroblast (hDF) migration. However, there was no significant difference in fibroblast proliferation between the hAMSC, hBMSC and hADSC groups, as measured by WST assay. Our results also indicated that hDFs cocultured with hADSCs for 48 h significantly upregulated their mRNA expression of the cytokine vascular endothelial growth factor, basic fibroblast growth factor, keratinocyte growth factor and transforming growth factor-β and increased the mRNA and protein levels of type I collagen. Collectively, these data suggest that hADSCs are a potential source of MSCs for therapeutic healing in cutaneous wounds in terms of efficacy, accessibility and availability.