Mechanisms of a delayed calcium uptake in mouse ischemic model

我们报道了钙调神经磷酸酶和嗜红细胞蛋白D的信号转导通路是开发抗缺血性药物的新靶点。为了进一步分析这两种酶的作用，我们在使用或不使用免疫抑制剂cydosporin A或FK506进行缺氧和低糖刺激后，测量了海马锥体神经元细胞内钙的含量。结果表明，钙调磷酸酶活性可能通过存在于细胞膜上的未知钙通道调节钙的摄取。此外，我们还发现了钙调磷酸酶与缺氧反应因子（HIF）之间的新关系，HIF是血管生成、糖酵解代谢、细胞凋亡和红细胞增生的关键诱导因子。缺氧因子在缺血性脑损伤和帕金森病、阿尔茨海默病等退行性疾病中发挥重要作用。在我们的实验中，钙调磷酸酶可以稳定hif的表达，激活hif的转录。通过酵母双杂交，我们发现了抑制HIF2转录活性和表达的新阳性克隆。其中一个已被发现的基因是Int6/eIF3e，它与小鼠哺乳动物肿瘤病毒（MMTV）引起的乳腺癌有关。MMTV在多个整合位点被整合到基因组中，并破坏了目标基因。Int6是靶基因之一。利用重组Int6突变体进一步分析表明，Int6通过直接结合HIF2并降解而成为肿瘤抑制因子。我们证明了注射特异性抑制HIF2的si RNA后，小鼠皮肤有强大的血管生成能力。根据这些结果，我们开始使用转染了Int6显性阴性突变体或siRNA表达载体的小鼠进行分析。根据本研究计划的具体目的，我们尝试建立小鼠缺血模型（前脑小鼠缺血模型）。有两个因素至关重要，一个是维持体温。我们在显微镜周围铺上丙烯板，保持37℃。另一个影响因素是颈动脉结扎后缺血小鼠的选择。多普勒血流仪观察到约20%的手术小鼠有足够的血供。遗憾的是，我们无法对PLC D1 KO小鼠和NCX2 KO小鼠进行评估，但我们计划在完成这个项目后，对Int6 siRNA或显性阴性Tg小鼠进行评估。少

We have reported that signal transduction pathway involving calcineurin and cydophilin D is a novel target for developing anti-ischemic drugs. For further analysis to clarify the role of both enzymes, we measured the intracellular calcium in pyramidal neuronal cells in hippocampal shoes after stimulation of hypoxia and low glucose with or without immunosuppressants cydosporin A or FK506. The results suggested that calcineurin activity might regulate the uptake of calcium through unknown calcium channel existed on plasma membranes.In addition, we found the new relation between calcineurin and hypoxia responsive factor (HIF), which was reported to be critical in inducing factors in angiogenesis, glycolytic metabolism, apoptosis, and erythrocytosis. This hypoxic factors deeply involved in mechanisms by which hypoxia played an important role in ischemic brain damage and degenerative diseases such as Parkinson disease and Alzheimer disease. In our experiments, calcineurin can stabilize the … More expression and activate the transcription of HIFs. We performed yeast two hybrid to discover novel factors regulating HIF and found novel positive clones which suppressed HIF2 transcription activity as well as expression. One of fished done identified is Int6/eIF3e, which involed in breast cancer caused by mouse mammalian tumor virus (MMTV). MMTV was integrated into genome at several integration sites and damaged the target genes. Int6 was one of the target gene. Further analysis using recombinant Int6 mutants showed that Int6 was a tumor suppressor by direct binding to HIF2 and the degradation. We demonstrated the potent angiogenesis in mouse skin after injection of si RNA which specifically suppressed HIF2. Under these results, we started analysis using mice transgened with a Int6 dominant negative mutant or siRNA expression vector.According to our specific aims in this research plan, we tried to establish a mouse ischemic model (forebrain mouse ischemic model). Two factors were critical One was the maintenance of body temperature. We equipped acryl bords surrounding microscope to keep 37℃. Another factor was selection of ischemic mouse after ligation of carotid arteries. Around 20 % of operated mice was observed to have enough blood supply through co-lateral blood flow by Doppler flow meter. It was regret that we could not evaluate PLC D1 KO mice nor NCX2 KO mice, but we plan to perform evaluation of Int6 siRNA or dominant negative Tg mice even after finishing this project. Less