Many hereditary and sporadic neurodegenerative disorders are characterized by the accumulation of aberrant proteins. In sporadic Parkinson's disease, representing the most prevalent movement disorder, oxidative and nitrosative stress are believed to contribute to disease pathogenesis, but the exact molecular basis for protein aggregation remains unclear. In the case of autosomal recessive-juvenile Parkinsonism, mutation in the E3 ubiquitin ligase protein parkin is linked to death of dopaminergic neurons. Here we show both in vitro and in vivo that nitrosative stress leads to S -nitrosylation of wild-type parkin and, initially, to a dramatic increase followed by a decrease in the E3 ligase-ubiquitin-proteasome degradative pathway. The initial increase in parkin's E3 ubiquitin ligase activity leads to autoubiquitination of parkin and subsequent inhibition of its activity, which would impair ubiquitination and clearance of parkin substrates. These findings may thus provide a molecular link between free radical toxicity and protein accumulation in sporadic Parkinson's disease.

We isolated and identified a stress protein that is up-regulated in response to hypoxia in primary-cultured glial cells. Protein-disulfide isomerase (PDI) was up-regulated not only by hypoxia in glia in vitro, but also by transient forebrain ischemia in rats in vivo. To determine whether newly synthesized PDI is involved in tolerance to ischemic stress, we carried out two procedures to induce PDI gene expression in human neuroblastoma SK-N-MC cells, as well as intrahippocampal injection following electroporation of an expression vector capable of overexpressing PDI in rats. Overexpression of this gene resulted in attenuation of the loss of cell viability induced by hypoxia in neuroblastoma SK-N-MC cells and a reduction in the number of DNA-fragmented cells in the CA1 area of the hippocampus in brain ischemic rats, respectively. These findings suggest that up-regulated PDI may play a critical role in resistance to ischemic damage, and that the elevation of levels of this protein in the brain may have beneficial effects against brain stroke.

Endoplasmic reticulum (ER) stress is defined as an accumulation of unfolded proteins in the endoplasmic reticulum. 4‐phenylbutyrate (4‐PBA) has been demonstrated to promote the normal trafficking of the ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) mutant from the ER to the plasma membrane and to restore activity. We have reported that 4‐PBA protected against cerebral ischemic injury and ER stress‐induced neuronal cell death. In this study, we revealed that 4‐PBA possesses chemical chaperone activityin vitro, which prevents the aggregation of denatured α‐lactalbumin and bovine serum albumin (BSA). Furthermore, we investigated the effects of 4‐PBA on the accumulation of Parkin‐associated endothelin receptor‐like receptor (Pael‐R) pathologically relevant to the loss of dopaminergic neurons in autosomal recessive juvenile parkinsonism (AR‐JP). Interestingly, 4‐PBA restored the normal expression of Pael‐R protein and suppressed ER stress induced by the overexpression of Pael‐R. In addition, we showed that 4‐PBA attenuated the activation of ER stress‐induced signal transduction pathways and subsequent neuronal cell death. Moreover, 4‐PBA restored the viability of yeasts that fail to induce an ER stress response under ER stress conditions. These results suggest that 4‐PBA suppresses ER stress by directly reducing the amount of misfolded protein, including Pael‐R accumulated in the ER.

Nitric oxide (NO) physiologically regulates numerous cellular responses through S -nitrosylation of protein cysteine residues. We performed antibody-array screening in conjunction with biotin-switch assays to look for S -nitrosylated proteins. Using this combination of techniques, we found that phosphatase with sequence homology to tensin (PTEN) is selectively S -nitrosylated by low concentrations of NO at a specific cysteine residue (Cys-83). S -nitrosylation of PTEN (forming SNO-PTEN) inhibits enzymatic activity and consequently stimulates the downstream Akt cascade, indicating that Cys-83 is a critical site for redox regulation of PTEN function. In ischemic mouse brain, we observed SNO-PTEN in the core and penumbra regions but found SNO-Akt, which is known to inhibit Akt activity, only in the ischemic core. These findings suggest that low concentrations of NO, as found in the penumbra, preferentially S -nitrosylate PTEN, whereas higher concentrations of NO, known to exist in the ischemic core, also S -nitrosylate Akt. In the penumbra, inhibition of PTEN (but not Akt) activity by S -nitrosylation would be expected to contribute to cell survival by means of enhanced Akt signaling. In contrast, in the ischemic core, SNO-Akt formation would inhibit this neuroprotective pathway. In vitro model systems support this notion. Thus, we identify unique sites of PTEN and Akt regulation by means of S -nitrosylation, resulting in an “on–off” pattern of control of Akt signaling.