Parkin gene mutations have been implicated in autosomal-recessive early-onset parkinsonism and lead to specific degeneration of dopaminergic neurons in midbrain. To investigate the role of Parkin in neuronal cell death, we overproduced this protein in PC12 cells in an inducible manner. In this cell line, neuronally differentiated by nerve growth factor, Parkin overproduction protected against cell death mediated by ceramide, but not by a variety of other cell death inducers (H(2)O(2), 4-hydroxynonenal, rotenone, 6-OHDA, tunicamycin, 2-mercaptoethanol and staurosporine). Protection was abrogated by the proteasome inhibitor epoxomicin and disease-causing variants, indicating that it was mediated by the E3 ubiquitin ligase activity of Parkin. Interestingly, Parkin acted by delaying mitochondrial swelling and subsequent cytochrome c release and caspase-3 activation observed in ceramide-mediated cell death. Subcellular fractionation demonstrated enrichment of Parkin in the mitochondrial fraction and its association with the outer mitochondrial membrane. Together, these results suggest that Parkin may promote the degradation of substrates localized in mitochondria and involved in the late mitochondrial phase of ceramide-mediated cell death. Loss of this function may underlie the degeneration of nigral dopaminergic neurons in patients with Parkin mutations.

Mutations in the gene encoding parkin cause an autosomal recessive juvenile-onset form of Parkinson's disease. Parkin functions as a RING-type E3 ubiquitin-ligase, coordinating the transfer of ubiquitin to substrate proteins and thereby targeting them for degradation by the proteasome. We now report that the extreme C terminus of parkin, which is selectively truncated by a Parkinson's disease-causing mutation, functions as a class II PDZ-binding motif that binds CASK, the mammalian homolog of Caenorhabditis elegans Lin-2, but not other PDZ proteins in brain extracts. Importantly, parkin co-localizes with CASK at synapses in cultured cortical neurons as well as in postsynaptic densities and lipid rafts in brain. Further, parkin associates not only with CASK but also with other postsynaptic proteins in the N-methyl D-aspartate (NMDA) receptor-signaling complex, in rat brain in vivo. Finally, despite exhibiting E2-dependent ubiquitin ligase activity, rat brain parkin does not ubiquitinate CASK, suggesting that CASK may function in targeting or scaffolding parkin within the postsynaptic complex rather than as a direct substrate for parkin-mediated ubiquitination. These data implicate for the first time a PDZ-mediated interaction between parkin and CASK in neurodegeneration and possibly in ubiquitination of proteins involved in synaptic transmission and plasticity.Parkinson's disease (PD) 1 involves the selective degeneration of midbrain dopamine neurons, resulting in motor abnormalities and progressive disability. Recently, three genes responsible for inherited forms of PD have been identified. Mutations in the genes encoding ␣-synuclein and ubiquitin C-terminal hydrolase L1 (UCH-L1) each cause rare autosomal dominant forms of PD (1, 2). In contrast, mutations in the gene encoding parkin cause an autosomal recessive, juvenile-onset form of PD and account for more cases of PD than all other familial causes combined (3, 4). Further, Lewy bodies, the cytoplasmic inclusions that constitute the pathological hallmark of the disease, contain ␣-synuclein, UCH-L1 and parkin deposits even in sporadic cases, suggesting a broader role for these three genes in PD (2, 5, 6). Interestingly however, Lewy bodies do not occur in the brains of patients with familial PD caused by parkin mutations, implicating parkin in the biogenesis of these inclusions (3).Another major component of Lewy bodies is ubiquitin (Ub), a small protein that can be covalently attached to other proteins (7). Conjugation of Ub onto proteins requires the concerted activity of three enzymes, an E1 Ub-activating enzyme, an E2 Ub-conjugating enzyme, and an E3 Ub-ligase. Ubiquitinated proteins are then targeted for degradation by the 26 S proteasome. The ubiquitin proteasome pathway (UPP) is one of the main pathways for protein degradation and has been implicated in a number of important cellular regulatory processes (8). There is considerable evidence that parkin also functions in the UPP. Indeed, the N terminus of parkin shares homology with ubiquitin (Ub-like dom...

Evidence that hepatitis E is zoonotic is accumulating. Serum samples were collected from pigs, cattle, and goats from various regions of China to determine whether they had been infected with hepatitis E virus (HEV). An in-house enzyme immunoassay (EIA) and reverse transcriptase-polymerase chain reaction (RT-PCR) with primers from open reading frame (ORF) 2 were used to detect anti-HEV antibodies and HEV RNA. The mean positivity rates of anti-HEV antibody for pigs and cattle were 78.8% and 6.3% but none of the goat sera were positive. Pigs may be more susceptible to infection with HEV than cattle or goats. Five of 263 pig sera were positive for HEV RNA and four of these five were also positive for anti-HEV. The PCR products (nt 6007-6354) were cloned and sequenced and compared to other HEV sequences in the nucleotide databases. The five sequences shared 83-93% identity to each other at the nucleotide level and 74-79%, 73-74%, 73-78%, and 83-99% identity to HEV genotypes 1, 2, 3, and 4, respectively. They were closely related to human isolates of HEV genotype 4. Phylogenetic analyses also place these swine sequences in HEV genotype 4, resembling most closely viruses isolated from Chinese patients with acute hepatitis. These data support the hypothesis that sporadic hepatitis E in China is zoonotic.

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