国立情報学研究所で電子化したコンテンツを使用している。

The present study was undertaken to investigate the effects of the T-type calcium channel blocker, Mibefradil, on white adipocytes. Unexpected for a T-type channel blocker, Mibefradil was found to increase intracellular calcium levels, cause lipid droplet fusion, and result in cell death. Calcium imaging of white adipocytes showed an increase of calcium concentration by Mibefradil at concentrations ranging from10-50 µM. The elevation in calcium by Mibefradil was significantly reduced by pretreatment of cells with Thapsigargin, an endoplasmic reticulum (ER) specific Ca ATPase inhibitor. Additionally, lipid droplet fusion and cell death were also attenuated by Thapsigargin pretreatment in white adipocytes. We conclude that Mibefradil elevated intracellular calcium levels, induced lipid droplet fusion and cell death in white adipocytes via mobilizing intracellular calcium stores from the ER. These results describe novel effects of Mibefradil on white adipocytes and may provide new insight into how this drug might be repurposed in obesity research.

The number of children and young adolescents diagnosed with cancer is increasing, leading to a need for new therapeutic strategies with diminished neurodegenerative side- effects. This report presents preliminary observations on glioma-initiating cells (GICs) in the way to develop a strategy that induces cell-cycle arrest or quiescence in neural stem cells (NSCs). To test how changes in membrane potential due to pharmacological treatments have effects on localization and levels of REV-ERBα protein, proneural (PN) and mesenchymal (MES) cells were treated with varying concentrations of REV-ERBα agonist SR9009 drug and T-type calcium channel blocker mibefradil. Treatments showed that both drugs do not relocalize REV-ERBα to the nucleus. However, SR9009 decreases the levels of REV-ERBα protein, whereas mibefradil does not have a similar effect.  Our preliminary data on mouse NSCs showed they engage with REV-ERBα protein while going into contact inhibition. Therefore, we investigated whether high confluency put PN and MES GICs into quiescence and the role of the main molecular clock protein REV-ERBα in this process. Cells were grown up to certain confluency, and following qPCR gene expression analysis revealed PN cells go into contact inhibition whereas MES cells continue proliferating even after they are grown to confluency. Moreover, REV-ERBα protein does not have any role in both outcomes.

Heterozygous and recently identified homozygous mutations in the SPTBN2 gene, encoding b-III spectrin, are implicated in spinocerebellar ataxia type 5 (SCA5) and spectrin-associated autosomal recessive cerebellar ataxia type 1 (SPARCA1), respectively. Our mouse model, lacking b-III spectrin (KO), mimics the progressive human phenotype displaying motor deficiencies as well as reduced Purkinje cell firing frequency followed by dendritic tree degeneration and cell death. The aims of this study were to evaluate progression of Purkinje cell degeneration following loss of b-III spectrin function and determine whether the reintroduction of C-terminus (C-trm) of b-III spectrin to the cerebellum is enough to halt, alleviate or reverse the disease phenotype. Additionally, this study investigated whether the abnormal electrophysiological and morphological phenotypes of Purkinje cells from KO mice are re-capitulated in a primary cerebellar culture and if so, whether they could be rescued by modulating calcium signaling. Morphological and histological analyses revealed that Purkinje cell degeneration is not uniform throughout the cerebellum of KO mice with Purkinje cells from posterior cerebellar regions possessing significantly smaller dendritic trees when compared to anterior cerebellum (p=0.0003, N=4-6, n=11-29). Similarly, significant reduction in Purkinje cell density was observed in posterior, not anterior regions of KO mice when compared to WT animals (p=0.014, N=3) and reduced tonic firing is most significant in Purkinje cells from the posterior cerebellum compared to WT mice (p=0.0328, N=3-6, n=11-29), with posterior KO PCs appearing to have elevated input resistance. Two-week expression of C-trm b-III spectrin in 3-month old KO animals significantly reduced Purkinje cell input resistance when compared to non-transduced cells (p=0.0139, N=4-5, n=15), but no effect was seen 9 months after viral injection. In contrast, a difference in cell surface area was no longer detected between WT and KO animals at 12 months of age following 9-months of viral expression. Nevertheless, using the elevated beam test motor deterioration was still observed 5 months after surgery (p=0.0023, N=4). In contrast, earlier stereotaxic injections at 6-weeks of age had a positive effect on mice motor performance with no deterioration in performance detected 5 months after the surgery. Latency to stay on the rotarod at 3 rpm was also significantly extended 6 months after stereotaxic injections at 6-weeks of age with slower motor deterioration (p=0.0348, N=6). In primary cerebellar cultures, Purkinje cells from KO animals exhibit an abnormal morphology with significantly more dendritic branches (p < 0.0001, N=4-7, n=35-69) and a larger total dendritic length (p=0.0079). Chronic application of 2 μM mibefradil, a T-type calcium channel blocker, was observed to reduce total dendritic length and branching in KO animal cultures bringing these morphological measurements closer to WT Purkinje cell levels. Finally although after 14 days in vitro 40% of Purkinje cells were found to be spontaneously firing, no significant difference in firing frequency (p=0.9434) or input resistance (p=0.8434, N=4, n=6-10) was detectable between WT and KO cultures. In summary, Purkinje cells in posterior cerebellar regions of KO mice were found to be more susceptible to dendritic degeneration and cellular death than cells in the anterior cerebellum. Expression of C-trm b-III spectrin at 3 months of age had an immediate effect on cell input resistance and a modest effect on Purkinje cell morphology but no effect on motor decline. Viral injections at 6-weeks of age, however, significantly slowed motor decline. Although an abnormal KO cell morphology could be successfully recapitulated in primary cell culture, it was not possible to discern any differences in electrophysiological properties. Nevertheless, the abnormal cell morphology was successfully modified in vitro by manipulating calcium signaling via T-type calcium channels.