Background: DJ-1 is a cytoprotective protein that is activated in response to various pathological stimuli. The ability of DJ-1 to perform its cellular actions is dependent on a conformational change, as evidenced by previous findings that it is cleaved by oxidative stress and that this cleavage activates its cytoprotective function. Previously, we demonstrated that the loss of functional DJ-1 lead to the enhancement of myocardial infarction and exacerbation of cardiac dysfunction following the onset of acute myocardial ischemia-reperfusion (I/R) injury. Currently, it is not known if the gain of functional DJ-1 has cardioprotective effects. Here, we investigated if the delivery of the cleaved form of DJ-1 using a gene therapy approach protected against the development of ischemic-induced heart failure. Methods and Results: We generated a viral vector to express the active form of DJ-1 (AAV9-DJ1WTΔC). To test the efficiency, specificity, and dose-dependence of AAV9-mediated transduction, we injected increasing doses of AAV9-GFP into the femoral vein of mice. A dose-dependent increase in GFP levels was observed in the heart and our analysis found that the 2x10 11 vg/mL dose achieved maximal delivery to the heart with minimal delivery to other tissue. GFP-stained sections of heart indicated that a substantial proportion of cardiomyocytes expressed the transduced gene. Next, we evaluated the protein expression of the full-length and active form of DJ-1 2 weeks after the administration of AAV9-DJ1WTΔC. No changes in the expression of the full-length form of DJ-1 were observed, but a 2-fold increase in the active form of DJ-1 was observed. Finally, mice administered AAV9-DJ1WTΔC displayed significant less left ventricular dilatation, left ventricular dysfunction, and hypertrophy compared to the AAV9-Empty treated following mice 60 minutes of myocardial ischemia and 4 weeks of reperfusion. Conclusion: These results indicated that DJ-1 is an endogenous cytoprotective protein that protects against the development of ischemic heart failure. Importantly, these results demonstrate the feasibility of using a gene therapy approach to deliver the cleaved form of DJ-1 to the heart as a therapeutic strategy to protect against ischemic heart disease.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a key neuronal deubiquitinating enzyme which is mutated in Parkinson disease (PD) and in childhood-onset neurodegenerative disorder with optic atrophy. Furthermore, reduced UCH-L1 protein levels are associated with a number of neurodegenerative diseases, whereas up-regulation of UCH-L1 protein expression is found in multiple types of cancer. However, very little is known about how UCH-L1 protein level is regulated in cells. Here, we report that UCH-L1 is a novel interactor and substrate of PD-linked E3 ubiquitin-protein ligase parkin. We find that parkin mediates K63-linked polyubiquitination of UCH-L1 in cooperation with the Ubc13/Uev1a E2 ubiquitin-conjugating enzyme complex and promotes UCH-L1 degradation by the autophagy-lysosome pathway. Targeted disruption of parkin gene expression in mice causes a significant decrease in UCH-L1 ubiquitination with a concomitant increase in UCH-L1 protein level in brain, supporting an in vivo role of parkin in regulating UCH-L1 ubiquitination and degradation. Our findings reveal a direct link between parkin-mediated ubiquitin signaling and UCH-L1 regulation, and they have important implications for understanding the roles of these two proteins in health and disease.

Mutations in Cu/Zn superoxide dismutase (SOD1) cause autosomal dominant amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease with no effective treatment. Despite ample evidence indicating involvement of mutation-induced SOD1 protein misfolding and aggregation in ALS pathogenesis, the molecular mechanisms that control cellular management of misfolded, aggregation-prone SOD1 mutant proteins remain unclear. Here, we report that parkin, an E3 ubiquitin-protein ligase which is linked to Parkinson's disease, is a novel regulator of cellular defense against toxicity induced by ALS-associated SOD1 mutant proteins. We find that parkin mediates K63-linked polyubiquitination of SOD1 mutants in cooperation with the UbcH13/Uev1a E2 enzyme and promotes degradation of these misfolded SOD1 proteins by the autophagy-lysosome system. In response to strong proteotoxic stress associated with proteasome impairment, parkin promotes sequestration of misfolded and aggregated SOD1 proteins to form perinuclear aggresomes, regulates positioning of lysosomes around misfolded SOD1 aggresomes, and facilitates aggresome clearance by autophagy. Our findings reveal parkin-mediated cytoprotective mechanisms against misfolded SOD1 toxicity and suggest that enhancing parkin-mediated cytoprotection may provide a novel therapeutic strategy for treating ALS.

Charcot-Marie-Tooth disease type 1C (CMT1C) is a dominantly inherited motor and sensory neuropathy. Despite human genetic evidence linking missense mutations in SIMPLE to CMT1C, the in vivo role of CMT1C-linked SIMPLE mutations remains undetermined. To investigate the molecular mechanism underlying CMT1C pathogenesis, we generated transgenic mice expressing either wild-type or CMT1C-linked W116G human SIMPLE. Mice expressing mutant, but not wild type, SIMPLE develop a late-onset motor and sensory neuropathy that recapitulates key clinical features of CMT1C disease. SIMPLE mutant mice exhibit motor and sensory behavioral impairments accompanied by decreased motor and sensory nerve conduction velocity and reduced compound muscle action potential amplitude. This neuropathy phenotype is associated with focally infolded myelin loops that protrude into the axons at paranodal regions and near Schmidt-Lanterman incisures of peripheral nerves. We find that myelin infolding is often linked to constricted axons with signs of impaired axonal transport and to paranodal defects and abnormal organization of the node of Ranvier. Our findings support that SIMPLE mutation disrupts myelin homeostasis and causes peripheral neuropathy via a combination of toxic gain-of-function and dominant-negative mechanisms. The results from this study suggest that myelin infolding and paranodal damage may represent pathogenic precursors preceding demyelination and axonal degeneration in CMT1C patients.

Mutations in PTEN-induced putative kinase 1 (PINK1) or parkin cause autosomal recessive forms of Parkinson disease (PD), but how these mutations trigger neurodegeneration is poorly understood and the exact functional relationship between PINK1 and parkin remains unclear. Here, we report that PINK1 regulates the E3 ubiquitin-protein ligase function of parkin through direct phosphorylation. We find that phosphorylation of parkin by PINK1 activates parkin E3 ligase function for catalyzing K63-linked polyubiquitination and enhances parkin-mediated ubiquitin signaling through the IkappaB kinase/nuclear factor kappaB (NF-kappaB) pathway. Furthermore, the ability of PINK1 to promote parkin phosphorylation and activate parkin-mediated ubiquitin signaling is impaired by PD-linked pathogenic PINK1 mutations. Our findings support a direct link between PINK1-mediated phosphorylation and parkin-mediated ubiquitin signaling and implicate the deregulation of the PINK1/parkin/NF-kappaB neuroprotective signaling pathway in the pathogenesis of PD.

Despite the critical importance of protein ubiquitination in the regulation of diverse cellular processes, the molecular mechanisms by which cells recognize and transmit ubiquitin signals remain poorly understood. The endosomal sorting machinery component hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) contains a ubiquitin-interacting motif (UIM), which is believed to bind ubiquitinated membrane cargo proteins and mediate their sorting to the lysosomal degradation pathway. To gain insight into the role of Hrs UIM-mediated ubiquitin signaling in cells, we performed a proteomic screen for Hrs UIM-interacting ubiquitinated proteins in human brain by using an in vitro expression cloning screening approach. We have identified 48 ubiquitinated proteins that are specifically recognized by the UIM domain of Hrs. Among them, 12 are membrane proteins that are likely to be Hrs cargo proteins, and four are membrane protein-associated adaptor proteins whose ubiquitination may act as a signal to target their associated membrane cargo for Hrs-mediated endosomal sorting. Other classes of the identified proteins include components of the vesicular trafficking machinery, cell signaling molecules, proteins associated with the cytoskeleton and cytoskeleton-dependent transport, and enzymes involved in ubiquitination and metabolism, suggesting the involvement of Hrs UIM-mediated ubiquitin signaling in the regulation of multiple cellular processes. We have characterized the ubiquitination of two identified proteins, Munc18-1 and Hsc70, and their interaction with Hrs UIM, and provided functional evidence supporting a role for Hsc70 in the regulation of Hrs-mediated endosome-to-lysosome trafficking.

Taurine has potent protective function against glutamate-induced neuronal injury presumably through its function in regulation of intracellular free calcium level, [Ca2+]i. In this communication, we report that taurine exerts its protective function through one or more of the following mechanisms: 1. Inhibition of glutamate-induced calcium influx through L-, N- and P/Q-type voltage-gated calcium channels and NMDA receptor calcium channel; 2. Attenuation of glutamate-induced membrane depolarization; 3. Prevention of glutamate-induced apoptosis via preventing glutamate-mediated down-regulation of Bcl-2; 4. Prevention of cleavage of Bcl-2 by calpain. This action of taurine is due to its inhibition on glutamate induced calpain activation. Based on these observations, we propose that taurine protects neurons against glutamate-induced neurotoxicity in part, by preventing glutamate-induced membrane depolarization, elevation of [Ca2+]i, activation of calpain, reduction of Bcl-2 and apoptosis.

Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme for gamma-aminobutyric acid (GABA) biosynthesis. Previously, we reported the presence of truncated forms of GAD in vivo and in vitro. In addition, an unidentified endogenous protease responsible for proteolytic cleavage of full-length GAD (fGAD) to its truncated form (tGAD) was also observed. In this communication, we report that mu-calpain is a good candidate for conversion of fGAD(67) to tGAD(67). This conclusion is based on the following observations: 1. purified recombinant GAD(67) is cleaved by mu-calpain at specific sites; 2. in brain synaptosomal preparation, GAD(67) is cleaved to its truncated form by an endogenous protease which is inhibited by specific calpain inhibitors; 3. in mu-calpain knockout mice, the level of tGAD in the brain is greatly reduced compared with the wild type; 4. when mu-calpain gene is silenced by siRNA, the level of tGAD is also markedly reduced compared to the control group; and 5. mu-calpain is activated by neuronal stimulation and Ca(2+)-influx. The physiological significance of calpain in regulation of GABA synthesis and GABAergic neurotransmission is also discussed.

Human brain glutamate decarboxylase 65 (hGAD65) was found to exist as full-length and truncated forms when the glutathione S-transferase-tagged hGAD65 fusion protein was subjected to factor Xa cleavage. The truncated form is produced by cleavage at arginine 69 based on N-terminal amino acid sequence analysis, and has a molecular weight of 58 kD. It is resistant to further factor Xa cleavage or mild trypsin treatment and is more active and more stable than the full-length form. Both the full-length and truncated forms of GAD are also observed in brain preparations in the presence of protease inhibitors. Furthermore, full-length GAD could be converted to the truncated form by endogenous proteases, suggesting that the conversion of full-length to truncated GAD mediated by endogenous protease may represent an important mechanism in the regulation of GABA biosynthesis in the brain.