Biomedical subjects

Tomohiro Kabuta

Publications and source records attributed to Tomohiro Kabuta.

5 recordsLinked to original sources

PubMed2006

Dopaminergic neuronal loss in transgenic mice expressing the Parkinson's disease-associated UCH-L1 I93M mutant.

The I93M mutation in ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) was reported in one German family with autosomal dominant Parkinson's disease (PD). The causative role of the mutation has, however, been questioned. We generated transgenic (Tg) mice carrying human UCHL1 under control of the PDGF-B promoter; two independent lines were generated with the I93M mutation (a high- and low-expressing line) and one line with wild-type human UCH-L1. We found a significant reduction in the dopaminergic neurons in the substantia nigra and the dopamine content in the striatum in the high-expressing I93M Tg mice as compared with non-Tg mice at 20 weeks of age. Although these changes were absent in the low-expressing I93M Tg mice, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment profoundly reduced dopaminergic neurons in this line as compared with wild-type Tg or non-Tg mice. Abnormal neuropathologies were also observed, such as silver staining-positive argyrophilic grains in the perikarya of degenerating dopaminergic neurons, in I93M Tg mice. The midbrains of I93M Tg mice contained increased amounts of insoluble UCH-L1 as compared with those of non-Tg mice, perhaps resulting in a toxic gain of function. Collectively, our data represent in vivo evidence that expression of UCHL1(I93M) leads to the degeneration of dopaminergic neurons.

Animals↗

PubMed2006

Degradation of amyotrophic lateral sclerosis-linked mutant Cu,Zn-superoxide dismutase proteins by macroautophagy and the proteasome.

Mutations in the Cu,Zn-superoxide dismutase (SOD1) gene cause approximately 20% of familial cases of amyotrophic lateral sclerosis (fALS). Accumulating evidence indicates that a gain of toxic function of mutant SOD1 proteins is the cause of the disease. It has also been shown that the ubiquitin-proteasome pathway plays a role in the clearance and toxicity of mutant SOD1. In this study, we investigated the degradation pathways of wild-type and mutant SOD1 in neuronal and nonneuronal cells. We provide here the first evidence that wild-type and mutant SOD1 are degraded by macroautophagy as well as by the proteasome. Based on experiments with inhibitors of these degradation pathways, the contribution of macroautophagy to mutant SOD1 clearance is comparable with that of the proteasome pathway. Using assays that measure cell viability and cell death, we observed that under conditions where expression of mutant SOD1 alone does not induce toxicity, macroautophagy inhibition induced mutant SOD1-mediated cell death, indicating that macroautophagy reduces the toxicity of mutant SOD1 proteins. We therefore propose that both macroautophagy and the proteasome are important for the reduction of mutant SOD1-mediated neurotoxicity in fALS. Inhibition of macroautophagy also increased SOD1 levels in detergent-soluble and -insoluble fractions, suggesting that both detergent-soluble and -insoluble SOD1 are degraded by macroautophagy. These findings may provide further insights into the mechanisms of pathogenesis of fALS.

Amyotrophic Lateral Sclerosis↗

PubMed2005

Does proteasome [corrected] inhibition decrease or accelerate toxin-induced dopaminergic neurodegeneration?

Parkinson's disease (PD) is pathologically characterized by dopaminergic (DA) cell death and the presence of Lewy bodies (LB) in the brain. alpha-Synuclein (alpha-syn) and ubiquitin (Ub) are the major components of LB, however, the process of their accumulation and their relationship to DA cell loss has not yet been resolved. Now, in this journal, Inden et al. showed the protective effect of proteasome inhibitors (PSI) on DA cell death in the rat PD model using 6-hydroxyl dopamine (6-OHDA). Co-administration of PSI, lactacystin, or MG-132 significantly prevented the nigral degeneration and apomorphine-induced rotational asymmetry of the model with increased appearance of alpha-syn- and Ub-positive inclusions in the substantia nigra. This study indicates that in their model, accelerated formation of inclusions via proteasome inhibition protects against DA cell death. Previous literature linked the impairments or inhibitions of the ubiquitin-proteasome system (UPS) and DA cell death. However, this report implies that the relationship between the UPS and the pathogenesis of PD may be more complex than we thought.

Acetylcysteine↗

PubMed2004

Motility response to insulin-like growth factor-I (IGF-I) in MCF-7 cells is associated with IRS-2 activation and integrin expression.

In MCF-7L cells, insulin-like growth factor-I (IGF-I) stimulates activation of insulin receptor substrate-1 (IRS-1) and enhances cell proliferation. While others have shown that IGF-I enhances cell motility in MCF-7 cells, we have not been able to demonstrate this. To determine if the source of MCF-7 cells account for these reported differences, we examined the MCF-7 cells available from the American Type Culture Collection (MCF-7/ATCC) and compared them to the MCF-7L cells maintained in our laboratory. Both MCF-7L and MCF-7/ATCC grew in response to 5 nM IGF-I and 1 nM estradiol. However, only MCF-7/ATCC demonstrated IGF-I stimulated motility. Immunoprecipitation of IRS substrates followed by anti-phosphotyrosine blotting demonstrated that both IRS-1 and IRS-2 were activated by IGF-I in these cells. However, MCF-7/ATCC cells had greater phosphorylation of IRS-2 compared to MCF-7L. Immunoblots showed that levels of IRS-1 and IRS-2 were comparable between cell lines. We have previously shown that fibronectin-binding integrins are necessary for IGF-stimulated motility. Similar levels of beta1 integrin were detected in both strains of MCF-7. However, low levels of alpha5 and alpha3 were detected in MCF-7L cells whereas high levels of alpha3 and alpha5 integrin were expressed in MCF-7/ATCC cells. Inhibition of integrin function by a blocking antibody or inhibitory peptide diminished IGF-mediated motility in MCF-7/ATCC. In MCF-7/ATCC cells, IGF-I stimulation was associated with a movement of IRS-2 to the leading edge of filopodia. Thus, patterns of integrin expression among breast cancer cell lines may partially explain the different motility behavior of cells in response to IGF-I. IRS-2 activation and integrin occupancy are both required for IGF-stimulated motility.

Breast Neoplasms↗

PubMed2001

Insulin receptor substrate-3 functions as transcriptional activator in the nucleus.

The family of insulin receptor substrates (IRSs) has been reported to play important roles for signal transduction of various hormones. Four members of the IRS family have been described. Each IRS is believed to have different functions; however, the distinct physiological roles of each IRS are unclear. This study was undertaken to determine the intracellular localization of IRS-3. IRS-3 was expressed in COS-7 cells as fusion with a green fluorescent protein (GFP), and subcellular localization of the chimera protein was analyzed by fluorescent microscopy. Surprisingly, GFP-IRS-3 was localized not only adjacent to the plasma membrane but also in the nucleus. We confirmed by immunostaining with anti-IRS-3 antibody that non-fused IRS-3 protein is also localized in the nucleus of COS-7 cells that were transfected with IRS-3 cDNA. In addition, we detected endogenous IRS-3 in the nucleus of isolated rat adipocytes. We then studied subcellular localization of deletion mutants and fragments of IRS-3 fused with GFP. We found that the region corresponding to amino acid residues 192-223 in the phosphotyrosine binding domain played an important role in nuclear localization. This region includes sequences that are unique to IRS-3. We then investigated intracellular localization of other IRSs fused with GFP. GFP-IRS-1, GFP-IRS-2, and GFP-IRS-4 were mainly localized in the cytosol or plasma membranes. Chimeric protein, Gal4 DNA binding domain fused with IRS-3 C-terminal region, increased transcription of the reporter gene containing Gal4 binding site in human embryonic kidney 293 cells. These results suggest that intracellular localization of IRS-3 is determined by a different mechanism from other IRS proteins, and that IRS-3 possesses a transcription-regulating activity.

Adipocytes↗