Autosomal recessive loss-of-function mutations within the PARK2 gene functionally inactivate the E3 ubiquitin ligase parkin, resulting in neurodegeneration of catecholaminergic neurons and a familial form of Parkinson disease. Current evidence suggests both a mitochondrial function for parkin and a neuroprotective role, which may in fact be interrelated. The antiapoptotic effects of parkin have been widely reported, and may involve fundamental changes in the threshold for apoptotic cytochrome c release, but the substrate(s) involved in parkin dependent protection had not been identified. Here, we demonstrate the parkin-dependent ubiquitination of endogenous Bax comparing primary cultured neurons from WT and parkin KO mice and using multiple parkin-overexpressing cell culture systems. The direct ubiquitination of purified Bax was also observed in vitro following incubation with recombinant parkin. We found that parkin prevented basal and apoptotic stressinduced translocation of Bax to the mitochondria. Moreover, an engineered ubiquitination-resistant form of Bax retained its apoptotic function, but Bax KO cells complemented with lysine-mutant Bax did not manifest the antiapoptotic effects of parkin that were observed in cells expressing WT Bax. These data suggest that Bax is the primary substrate responsible for the antiapoptotic effects of parkin, and provide mechanistic insight into at least a subset of the mitochondrial effects of parkin.Parkinson's disease | apoptosis | neuroprotection | mitophagy P arkinson disease (PD) is a neurodegenerative disorder that affects 1-3% of the population over the age of 65 years (1). The symptoms include tremor, rigidity, bradykinesia, and postural instability. These physical characteristics are caused by the progressive degeneration of dopaminergic neurons of the substantia nigra pars compacta and, to a lesser extent, the catecholaminergic neurons of the locus coeruleus. Although most cases of PD are sporadic in nature, a small number of genes are responsible for the rare familial forms of PD (2). Loss-of-function mutations within the PARK2 locus, which encodes the protein parkin, are the most common cause of autosomal recessive PD (3).Parkin is a 465-amino acid protein that is expressed in multiple tissues and functions as an E3 ubiquitin ligase (4). Ubiquitination of substrates is a tightly regulated process, requiring the combined activity of three enzymes: an E1 ubiquitin-activating enzyme, an E2 ubiquitin conjugating enzyme, and an E3 ubiquitin ligase (5). E3 ubiquitin ligases are responsible for substrate recognition, and as such contribute the specificity of a ubiquitin reaction. Defects in parkin-mediated ubiquitination may result in the failure to target specific substrates for degradation, leading to accumulation of potentially toxic proteins and consequent cell death (6). Parkin is widely neuroprotective (7); however, many of the putative parkin substrates reported to date are not thought to directly mediate toxicity in such a simple fashion [reviewed elsewhere (8...

Autosomal-recessive mutations in the Parkin gene are the second most common cause of familial Parkinson's disease (PD). Parkin deficiency leads to the premature demise of the catecholaminergic neurons of the ventral midbrain in familial PD. Thus, a better understanding of parkin function may elucidate molecular aspects of their selective vulnerability in idiopathic PD. Numerous lines of evidence suggest a mitochondrial function for parkin and a protective effect of ectopic parkin expression. Since mitochondria play a critical role in cell survival/cell death through regulated cytochrome c release and control of apoptosis, we sought direct evidence of parkin function in this pathway. Mitochondria were isolated from cells expressing either excess levels of human parkin or shRNA directed against endogenous parkin and then treated with peptides corresponding to the active Bcl-2 homology 3 (BH3) domains of pro-apoptotic proteins and the threshold for cytochrome c release was analyzed. Data obtained from both rodent and human neuroblastoma cell lines showed that the expression levels of parkin were inversely correlated with cytochrome c release. Parkin was found associated with isolated mitochondria, but its binding per se was not sufficient to inhibit cytochrome c release. In addition, pathogenic parkin mutants failed to influence cytochrome c release. Furthermore, PINK1 expression had no effect on cytochrome c release, suggesting a divergent function for this autosomal recessive PD-linked gene. In summary, these data demonstrate a specific autonomous effect of parkin on mitochondrial mechanisms governing cytochrome c release and apoptosis, which may be relevant to the selective vulnerability of certain neuronal populations in PD.

Background Prepulse inhibition (PPI) is a cross-species measure of sensorimotor gating. PPI deficits are observed in humans and rats upon acute treatment with dopamine D2-like receptor agonists, and in patients with schizophrenia. Repeated treatment with a D2-like agonist, however, reverses PPI deficits and increases cAMP signaling in the nucleus accumbens (NAc). The present study examined the short and long-term effects on PPI of treatment with quinpirole and ropinirole, dopamine D2/D3 receptor agonists, and the molecular mechanism by which they occur. Methods PPI was assessed in adult male Sprague-Dawley rats following acute and chronic treatment with quinpirole or ropinirole, and 1, 2, 3, and 4 weeks after termination of repeated ropinirole treatment. Finally, the effect of dominant negative mutant CREB overexpression in the NAc on PPI following chronic quinpirole treatment was assessed. Results Acute quinpirole produced dose-dependent PPI deficits, while ropinirole caused consistent PPI reduction at all but the highest dose. Repeated ropinirole treatment significantly increased PPI compared to acute treatment, and increased CREB phosphorylation in NAc neurons. Subsequent ropinirole challenge had no effect as long as 28 days later, at which time NAc CREB phosphorylation had normalized. Overexpression of dominant negative mutant CREB prevented PPI recovery induced by chronic quinpirole treatment. Conclusions Chronic quinpirole or ropinirole treatment produces sustained PPI recovery; CREB activity in the NAc is required to induce PPI recovery, but not to maintain it. The results suggest that transcriptional regulation by CREB mediates long-lasting changes occurring within NAc circuits to promote recovery of sensorimotor gating.