|Budget Amount *help
¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 1998 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 1997 : ¥2,100,000 (Direct Cost : ¥2,100,000)
In this study, we investigated the mechanisms of attenuation of apoptosis of cerebellar granule cells (CGCs), which is controlled by Ca^<2+> influx into neurons, in terms of Ca^<2+> responsive gene expression and mitochondrial participation in apoptosis.
1. Survey for novel Ca^<2+> responsive genes (CaRGs) whose products are responsible for attenuating neuronal apoptosis.
Besides brain-derived neurotrophic factor (BDNF), we identified the secretogranin II and PACAP (pituitary adenylate cyclase polypeptide) genes whose expression can be controlled by Ca^<2+> suignals, and found that their processing, secretory products, secretoneurin and PACAP38, respectively, have the.attenuating effects on apoptosis of CGCs.
2. Establishment of DNA transfection for promoter analysis in primary neuronal cell culture.
Using calcium/phosphate-DNA precipitation method, we developed a convenient and reproducible conditions for DNA transfection in primary neuronal cell culture. Using this optimum condition, we have found that there could be multiple Ca^<2+> signaling pathways to control the expression of CaRGs in neurons.
3. Involvement of mitochondrial damage in Ca^<2+> deprivation-induced apoptosis of CGCs.
Upon a deprivation of Ca^<2+> influx into CGCs, inactivation of aconitase, a redox- sensitive TCA cycle enzyme, was induced with apoptosis. In a good correspondence to aconitase inactivation, the expression of manganese (Mn)-supcroxide dismutase gene decreased, indicating that a generation of superoxide radicals leads to mitochondrial damage. Upon recovery of Ca^<2+> influx, however, aconitase inactivation was attenuated in concert with attenuation of apoptosis.