Tomohiro Omura

A ubiquitin ligase HRD1 promotes the degradation of Pael receptor, a substrate of Parkin

It has been proposed that in autosomal recessive juvenile parkinsonism (AR‐JP), a ubiquitin ligase (E3) Parkin, which is involved in endoplasmic reticulum‐associated degradation (ERAD), lacks E3 activity. The resulting accumulation of Parkin‐associated endothelin receptor‐like receptor (Pael‐R), a substrate of Parkin, leads to endoplasmic reticulum stress, causing neuronal death. We previously reported that human E3 HRD1 in the endoplasmic reticulum protects against endoplasmic reticulum stress‐induced apoptosis. This study shows that HRD1 was expressed in substantia nigra pars compacta (SNC) dopaminergic neurons and interacted with Pael‐R through the HRD1 proline‐rich region, promoting the ubiquitylation and degradation of Pael‐R. Furthermore, the disruption of endogenous HRD1 by small interfering RNA (siRNA) induced Pael‐R accumulation and caspase‐3 activation. We also found that ATF6 overexpression, which induced HRD1, accelerated and caused Pael‐R degradation; the suppression of HRD1 expression by siRNA partially prevents this degradation. These results suggest that in addition to Parkin, HRD1 is also involved in the degradation of Pael‐R.

A different pathway in the endoplasmic reticulum stress-induced expression of human HRD1 and SEL1 genes

Human HRD1 and SEL1 are components of endoplasmic reticulum‐associated degradation (ERAD), which is a retrograde transport mechanism from the ER to the cytosol for removing unfolded proteins. The expression of HRD1 and SEL1 was induced by ER stress‐inducing agents and overexpression of both ER stress‐responsive transcription factors, ATF6 and XBP1. Inhibition of IRE1 and ATF6 revealed that ER stress‐induced HRD1 and SEL1 expressions are mediated by IRE1‐XBP1‐ and ATF6‐dependent pathways, respectively. These results suggest that the ER stress‐induced ERAD gene expressions are mediated by different pathways, which are attributed to the differences in the promoter regions.

Endoplasmic Reticulum Stress and Parkinson’s Disease: The Role of HRD1 in Averting Apoptosis in Neurodegenerative Disease

Endoplasmic reticulum (ER) stress has been known to be involved in the pathogenesis of various diseases, particularly neurodegenerative disorders such as Parkinsons disease (PD). We previously identified the human ubiquitin ligase HRD1 that is associated with protection against ER stress and its associated apoptosis. HRD1 promotes the ubiquitination and degradation of Parkin-associated endothelin receptor-like receptor (Pael-R), an ER stress inducer and causative factor of familial PD, thereby preventing Pael-R-induced neuronal cell death. Moreover, upregulation of HRD1 by the antiepileptic drug zonisamide suppresses 6-hydroxydopamine-induced neuronal cell death. We review recent progress in the studies on the mechanism of ER stress-induced neuronal death related to PD, particularly focusing on the involvement of HRD1 in the prevention of neuronal death as well as a potential therapeutic approach for PD based on the upregulation of HRD1.

Meloxicam protects cell damage from 1-methyl-4-phenyl pyridinium toxicity via the phosphatidylinositol 3-kinase/Akt pathway in human dopaminergic neuroblastoma SH-SY5Y cells

Parkinsons disease (PD) is a common neurodegenerative disorder characterized by dopaminergic neuronal death in the substantia nigra pars compacta. There is growing interest in the effects of nonsteroidal antiinflammatory drugs (NSAIDs) against PD progression. In this study, we investigated the neuroprotective effect of NSAIDs on neuronal damage induced by 1-methyl-4-phenyl pyridinium (MPP(+)) in human dopaminergic SH-SY5Y neuroblastoma cells. Of the NSAIDs tested, only meloxicam indicated protective effect on MPP(+)-induced neurotoxicity in SH-SY5Y cells, although such an effect was not established with indomethacin, ibuprofen and cyclooxygenase (COX)-2 selective inhibitors (NS-398 and CAY-10404). The neuroprotective effect of meloxicam against MPP(+) toxicity was specific, as toxicities induced by other cytotoxic agents (such as rotenone, MG-132, tunicamycin and ethacrynic acid) were not attenuated by meloxicam. The neuroprotective effect of meloxicam on MPP(+)-induced apoptosis was abolished by a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, but not by a MEK inhibitor, PD98059. The Akt phosphorylation levels were predominantly suppressed 4h after MPP(+) incubation (i.e. when the cell toxicity was not apparently observed yet). Meloxicam completely prevented the Akt phosphorylation suppression caused by MPP(+) exposure, while meloxicam per se did not promote the Akt phosphorylation. These results strongly suggest that the neuroprotective effect of meloxicam is mediated by the maintenance of cell survival signaling in the PI3K/Akt pathway, but not by COX-2 inhibition. Therefore, meloxicam may have therapeutic potential in preventing development or delaying progress of PD.

Immunohistochemical localization of a ubiquitin ligase HRD1 in murine brain

HRD1 is an E3 ubiquitin ligase and plays an important role in endoplasmic reticulum‐associated degradation (ERAD). Parkin‐associated endothelin receptor‐like receptor (Pael‐R) is a substrate of the E3 ubiquitin ligase parkin, which has been implicated in ER stress‐induced cell death in dopamine neurons in autosomal recessive juvenile parkinsonism (AR‐JP). Recently, we demonstrated that endogenous HRD1 interacts with Pael‐R, and that HRD1 promotes the degradation of Pael‐R and protects cell death caused by the accumulation of Pael‐R. Another group recently reported that HRD1 suppresses the toxicity of polyglutamine‐expanded huntingtin. However, the topographical localization of HRD1 protein in the brain, especially related to neurodegenerative disease, is unclear. In this study, we used immunohistochemistry to investigate the topographical localization of HRD1 in the brain and demonstrated that HRD1 immunoreactivity was expressed widely in the substantia nigra pars compacta (SNC) containing dopaminergic neurons and was expressed in the cerebral cortex, hippocampus, dentate gyrus, striatum, globus pallidus, and Purkinje cells of the cerebellar cortex. Furthermore, HRD1 immunoreactivity was detected in the neuronal cells but not in the glial cells. These results suggest that HRD1 may play an important role in maintaining higher brain function, including motor function or learning and memory. In addition, HRD1 may have substrates other than Pael‐R that are implicated in neurodegenerative disorders.

Sodium tauroursodeoxycholate prevents paraquat-induced cell death by suppressing endoplasmic reticulum stress responses in human lung epithelial A549 cells.

Paraquat is a commonly used herbicide; however, it is highly toxic to humans and animals. Exposure to paraquat causes severe lung damage, leading to pulmonary fibrosis. However, it has not been well clarified as how paraquat causes cellular damage, and there is no established standard therapy for paraquat poisoning. Meanwhile, endoplasmic reticulum stress (ERS) is reported to be one of the causative factors in many diseases, although mammalian cells have a defense mechanism against ERS-induced apoptosis (unfolded protein response). Here, we demonstrated that paraquat changed the expression levels of unfolded protein response-related molecules, resulting in ERS-related cell death in human lung epithelial A549 cells. Moreover, treatment with sodium tauroursodeoxycholate (TUDCA), a chemical chaperone, crucially rescued cells from death caused by exposure to paraquat. These results indicate that paraquat toxicity may be associated with ERS-related molecules/events. Through chemical chaperone activity, treatment with TUDCA reduced paraquat-induced ERS and mildly suppressed cell death. Our findings also suggest that TUDCA treatment represses the onset of pulmonary fibrosis caused by paraquat, and therefore chemical chaperones may have novel therapeutic potential for the treatment of paraquat poisoning.

Meloxicam ameliorates motor dysfunction and dopaminergic neurodegeneration by maintaining Akt-signaling in a mouse Parkinson's disease model.

A series of oxicam non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to be neuroprotective against 1-methyl-4-phenyl pyridinium in human neuroblastoma SH-SY5Y cells via the phosphatidylinositol 3-kinase (PI3K)/Akt pathway independent of cyclooxygenase (COX) inhibition. The present study endeavored to establish this novel effect of meloxicam (MLX), an oxicam NSAID, in a mouse Parkinsons disease (PD) model using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Male C57BL/6 mice, which received MPTP (30 mg/kg/day; s.c.) for 5 consecutive days (chronic model) with 10-day follow-up saline administrations, showed significant motor dysfunction in the pole test due to reduced tyrosine hydroxylase (TH) protein levels in the brain on day 16 after MPTP/saline treatment. Daily coadministrations of MLX (10mg/kg/day; i.p.) and MPTP for the first 5 days and follow-up 10 days with MLX administrations alone (MPTP/MLX treatment) significantly ameliorated MPTP-induced behavioral abnormalities in mice. Concomitant decreases of TH protein levels in the striatum and midbrain of MPTP/MLX-treated mice were not only significantly (p<0.01 and p<0.05, respectively) ameliorated but phosphorylated Akt (pAkt473) expression in the midbrain was also significantly (p<0.01) increased in the midbrain when compared with MPTP/saline-treated mice. These results suggest that MLX, an oxicam NSAID, attenuated dopaminergic neuronal death in the experimental MPTP-PD model by maintenance of the Akt-signaling. Oxicam NSAIDs may serve as potential drugs for PD treatment via a novel mechanism of action.

Impact of cytochrome P450 3A5 polymorphism in graft livers on the frequency of acute cellular rejection in living-donor liver transplantation

Objective We investigated whether the cytochrome P450 3A5*3 (CYP3A5*3) genotype affects tacrolimus pharmacokinetics and the risk of acute cellular rejection in living-donor liver transplant patients in Japan. Materials and methods Between July 2004 and June 2011, we enrolled 410 living-donor liver transplant patients receiving tacrolimus. Biopsy specimens of intestinal mucosa and graft liver at surgery were obtained to examine the mRNA expression of CYP3A subfamilies as well as the genotyping of CYP3A5*3 polymorphism. Results The CYP3A5 genotype in the native intestine had no significant effect on the occurrence of acute cellular rejection between postoperative days 14 and 23 in cases with identical or compatible ABO blood types (11.5% for the CYP3A5*1 allele vs. 7.4% for CYP3A5*3/*3; P=0.2643), although the concentration/dose ratio of tacrolimus was significantly higher in patients with the intestinal CYP3A5*3/*3 genotype than in those with the CYP3A5*1 allele for 5 post-transplant weeks. However, patients who received a graft liver with the CYP3A5*1 allele showed a higher rate of acute cellular rejection than those who received a graft liver with the CYP3A5*3/*3 genotype (14.5 vs. 5.7%; P=0.0134). The relative risk for acute cellular rejection associated with the CYP3A5*1 liver allele was 2.629 (P=0.018, Cox regression model). Consequently, graft liver CYP3A5*1 genotype might increase the risk for acute cellular rejection after living-donor liver transplantation, possibly by associating with the local hepatic tacrolimus concentration. Conclusions The target level of tacrolimus may be affected by the CYP3A5*3 genotype of the liver, rather than by that of the small intestine, after postoperative day 14.

Functional involvement of RFVT3/SLC52A3 in intestinal riboflavin absorption.

Riboflavin, also known as vitamin B2, is transported across the biological membrane into various organs by transport systems. Riboflavin transporter RFVT3 is expressed in the small intestine and has been suggested to localize in the apical membranes of the intestinal epithelial cells. In this study, we investigated the functional involvement of RFVT3 in riboflavin absorption using intestinal epithelial T84 cells and mouse small intestine. T84 cells expressed RFVT3 and conserved unidirectional riboflavin transport corresponding to intestinal absorption. Apical [(3)H]riboflavin uptake was pH-dependent in T84 cells. This uptake was not affected by Na(+) depletion at apical pH 6.0, although it was significantly decreased at apical pH 7.4. The [(3)H]riboflavin uptake from the apical side of T84 cells was prominently inhibited by the RFVT3 selective inhibitor methylene blue and significantly decreased by transfection of RFVT3-small-interfering RNA. In the gastrointestinal tract, RFVT3 was expressed in the jejunum and ileum. Mouse jejunal and ileal permeabilities of [(3)H]riboflavin were measured by the in situ closed-loop method and were significantly reduced by methylene blue. These results strongly suggest that RFVT3 would functionally be involved in riboflavin absorption in the apical membranes of intestinal epithelial cells.

Genotyping of 38 insertion/deletion polymorphisms for human identification using universal fluorescent PCR

Short insertion/deletion (Indel) polymorphisms of approximately 2-6 bp are useful as biallelic markers for forensic analysis, and the application of Indel genotyping as a supplementary tool would improve human identification accuracy. We examined the allele frequencies of 37 autosomal Indels in the Japanese population and developed a novel dual-color genotyping method for human identification on the basis of universal fluorescent PCR, including the sex-typing amelogenin locus. Target genomic fragment sizes for 38 Indels were 49-143 bp. We analyzed these Indels in 100 Japanese individuals using the M13(-47) sequence as a universal primer. For dual-color genotyping, we designed a novel universal primer with high amplification efficiency and specificity. Using FAM-labeled M13(-47) and HEX-labeled modified M13(-47) primers, fluorescent signals at all loci were clearly distinguished in two independent multiplex PCRs. Average minor allele frequency was 0.39, and accumulated matching probability was 2.12 × 10(-15). Complete profiles were successfully amplified with as little as 0.25 ng of DNA. This method provides robust, sensitive, and cost-effective genotyping for human identification.