Unexpected mitochondrial matrix localization of Parkinson's disease-related DJ-1 mutants but not wild-type DJ-1.

DJ-1 has been identified as a gene responsible for recessive familial Parkinson's disease (familial Parkinsonism), which is caused by a mutation in the PARK7 locus. Consistent with the inferred correlation between Parkinson's disease and mitochondrial impairment, mitochondrial localization of DJ-1 and its implied role in mitochondrial quality control have been reported. However, the mechanism by which DJ-1 affects mitochondrial function remains poorly defined, and the mitochondrial localization of DJ-1 is still controversial. Here, we show the mitochondrial matrix localization of various pathogenic and artificial DJ-1 mutants by multiple independent experimental approaches including cellular fractionation, proteinase K protection assays, and specific immunocytochemistry. Localization of various DJ-1 mutants to the matrix is dependent on the membrane potential and translocase activity in both the outer and the inner membranes. Nevertheless, DJ-1 possesses neither an amino-terminal alpha-helix nor a predictable matrix-targeting signal, and a post-translocation processing-derived molecular weight change is not observed. In fact, wild-type DJ-1 does not show any evidence of mitochondrial localization at all. Such a mode of matrix localization of DJ-1 is difficult to explain by conventional mechanisms and implies a unique matrix import mechanism for DJ-1 mutants.

Parkin-catalyzed ubiquitin-ester transfer is triggered by PINK1-dependent phosphorylation.

PINK1 and PARKIN are causal genes for autosomal recessive familial Parkinsonism. PINK1 is a mitochondrial Ser/Thr kinase, whereas Parkin functions as an E3 ubiquitin ligase. Under steady-state conditions, Parkin localizes to the cytoplasm where its E3 activity is repressed. A decrease in mitochondrial membrane potential triggers Parkin E3 activity and recruits it to depolarized mitochondria for ubiquitylation of mitochondrial substrates. The molecular basis for how the E3 activity of Parkin is re-established by mitochondrial damage has yet to be determined. Here we provide in vitro biochemical evidence for ubiquitin-thioester formation on Cys-431 of recombinant Parkin. We also report that Parkin forms a ubiquitin-ester following a decrease in mitochondrial membrane potential in cells, and that this event is essential for substrate ubiquitylation. Importantly, the Parkin RING2 domain acts as a transthiolation or acyl-transferring domain rather than an E2-recruiting domain. Furthermore, formation of the ubiquitin-ester depends on PINK1 phosphorylation of Parkin Ser-65. A phosphorylation-deficient mutation completely inhibited formation of the Parkin ubiquitin-ester intermediate, whereas phosphorylation mimics, such as Ser to Glu substitution, enabled partial formation of the intermediate irrespective of Ser-65 phosphorylation. We propose that PINK1-dependent phosphorylation of Parkin leads to the ubiquitin-ester transfer reaction of the RING2 domain, and that this is an essential step in Parkin activation.

Esophageal cancer-related gene 4 is a secreted inducer of cell senescence expressed by aged CNS precursor cells.

Mammalian aging is thought to be partially caused by the diminished capacity of stem/precursor cells to undergo self-renewing divisions. Although many cell-cycle regulators are involved in this process, it is unknown to what extent cell senescence, first identified as irreversible growth arrest in vitro, contributes to the aging process. Here, using a serum-induced mouse oligodendrocyte precursor cell (mOPC) senescence model, we identified esophageal cancer-related gene 4 (Ecrg4) as a senescence inducer with implications for the senescence-like state of postmitotic cells in the aging brain. Although mOPCs could proliferate indefinitely when cultured using the appropriate medium (OPC medium), they became senescent in the presence of serum and maintained their senescent phenotype even when the serum was subsequently replaced by OPC medium. We show that Ecrg4 was up-regulated in the senescent OPCs, its overexpression in OPCs induced senescence by accelerating the proteasome-dependent degradation of cyclins D1 and D3, and that its knockdown by a specific short hairpin RNA prevented these phenotypes. We also show that senescent OPCs secreted Ecrg4 and that recombinant Ecrg4 induced OPC senescence in culture. Moreover, increased Ecrg4 expression was observed in the OPCs and neural precursor cells in the aged mouse brain; this was accompanied by the expression of senescence-associated beta-galactosidase activity, indicating the cells' entrance into senescence. These results suggest that Ecrg4 is a factor linking neural-cell senescence and aging.

Clinical and laboratory features of in-patients with multiple sclerosis in a University Hospital in Tokyo from 1988-2002.

OBJECTIVE: The aim of this study was to analyze the clinical and laboratory features of each subtype of multiple sclerosis (MS) (relapsing-remitting, primary progressive, and secondary progressive) in the Tokyo metropolitan area. METHODS AND PATIENTS: We retrospectively analyzed the medical records of 104 consecutive patients with a diagnosis of MS, who had been admitted to our university hospital from 1988 to 2002. They all met criteria for definite MS, by clinical or laboratory standards. RESULTS: Eighty-four (80.8%) patients were classified as having relapsing-remitting MS, while 8 patients (7.7%) and 12 patients (11.5%) were classified as having primary progressive MS and secondary progressive MS, respectively. A significant female predominance existed in the relapse-remitting MS (female : male=2.4 : 1) cohort, but this ratio was 1 : 1 in both primary progressive and secondary progressive MS. The age at onset was older in the primary progressive MS (36.6+/-17.1 years of old) population than in either the relapsing-remitting MS (27.9+/-11.1) or the secondary progressive MS (27.8+/-11.5) subjects. Although the duration of illness was similar among the three types of MS, the number of exacerbations in the secondary progressive (5.9+/-4.6) cohort was significantly higher than that in the relapsing-remitting MS subjects (3.2+/-2.6). Patients with primary progressive MS showed a significantly higher rate of gait disturbance (87.5%) as the initial symptom than those with relapsing-remitting MS (23.8%), and this was thought to be due to the higher incidence of brainstem and spinal cord lesions. Visual disturbance as the initial symptom was frequently noted in those with secondary progressive MS (50.0%), while it was noted only in 29.8% and 12.5% in the relapsing-remitting and primary progressive patients, respectively. Primary progressive MS subjects had a higher propensity to be wheelchair-bound (75.0%) than those suffering from relapsing-remitting MS (1.2%). Increased total protein in the cerebrospinal fluid (CSF) of the secondary progressive cohort was statistically significant compared to the relapsing-remitting cohort. The frequency of oligoclonal IgG bands was rather low in each type of MS (17.1-33.3%). Gadolinium enhancement of plaques on MRI was more frequently present in secondary progressive MS (66.7%) than in either relapsing-remitting MS (32.1%) or primary progressive MS (50.0%). Of note, the opticospinal form was found in only 16.3% of the total MS patients, a proportion less than that in previous reports from southern Japan. CONCLUSION: The present study confirms that while the clinical and laboratory features of the MS patients in the Tokyo metropolitan area are similar to those in Western countries in most regards, features such as proportionally fewer primary and secondary progressive MS patients as well as less oligoclonal IgG bands on CSF analysis are different from those in Western countries.