Molecular mechanisms and therapeutic approach for ischemic brain damages

本研究旨在阐明线粒体特异表达的钙调神经磷酸酶和亲环素D在大鼠前脑缺血模型CA1区迟发性神经细胞死亡中的作用机制。该模型缺血10min再灌流的一系列实验结果表明，免疫抑制剂环孢素A对缺血性脑损伤有明显的神经保护作用。这些结果表明，环孢素A对缺血性脑损伤的神经保护机制是通过抑制线粒体基质中钙调神经磷酸酶的活性和亲环素D的表达来实现的。CsA对亲环素D的抑制抑制了线粒体通透性转换(MPT)孔的组装，细胞死亡诱导剂如细胞色素c和半胱氨酸氨基转移酶(Caspase)通过该孔释放。据报道，…孔由三个组分组成：电压依赖阴离子通道(VDAC)、腺核苷酸转位酶(ANT)和亲环素D。进一步证明亲环素D通过亲环素D亲和柱直接与ANT和/或VDAC结合。在此基础上，我们与藤泽制药合作开发了抗脑缺血新药FR901459。公司有限公司，日本。这种新药表现出较低的抗免疫抑制作用和较强的抗异构酶活性，是一种理想的抗缺血药物。我们正在开发这种药物用于临床。此外，我们专注于缺血性损伤中缺氧或缺氧的关键事件，发现缺氧诱导因子HIF是一种转录因子，参与代谢、血管生成和红细胞生成途径，在调节神经细胞死亡方面发挥关键作用。首先，我们证明了HIF家族的一种亚型HIF3的mRNA在缺血24小时后主要在海马区的CA1表达，尽管HIF1的mRNA是有反应的，但表达水平远低于HIF3。在培养细胞中，钙调神经磷酸酶的活性在其去磷酸化的HIF1调节中起重要作用。这些结果促使我们研究涉及HIF的低氧途径以及钙调神经磷酸酶/免疫亲和素的作用。我们计划证实这一假设，即HIF家族成员将是缺血事件中钙/钙调素途径中的关键因素。较少

In this research project, we pursued clarifying mechanisms by which calcineurin and cyclophilin D, specifically expressed in mitochondria, playes an important role in delayed neuronal cell death of CA1 sector of rat forebrain ischemic model. The series of results from this model with 10 min ischemia and reperfusion showed the drastic neuroprotective effect of an immunosuppressant cyclosporin A. These results clearly demonstrated that the mechams of the neuroprotection of cyclosporin A to ischemic brain damages were due to the inhibition of both calcineurin activity and cyclophilin D specifically expressed in mitochondrial matrix. The inhibition of cyclophilin D by CsA suppressed the assembly of MPT (mitochondrial permeability transition) pores through which cell death inducers such as cytochrome c and caspases were released. MPT pores were reported to be consisted of three components ; VDAC (voltage dependent anion channel), ANT (adenine nucleotide translocase), and cyclophilin D. We d … More emonstrated that cyclophilin D directly bind to ANT and/or VDAC using affinity colomn bound to cyclophilin D.Under these information, we succeeded in development of the new anti-ischemic drug, FR901459 in collabaration with Fujisawa Pharm. CO. LTD., Japan. This new drug shows ideal characterization as an anti-ischemic drug, such as lower anti-immunosuppressive effect and a strong anti-isomerase activity. We are now developing this drug for a clinical use.Furthermore, we focused on the critical event of hypoxia or anoxia during ischemic insult, and found hypoxic inducible factor HIF, which was a transcription factor and involved in the metabolic, angiogenetic, and erythropoietic pathway, played an critical role in regulatingneuronal cell deatrh. First, we have demonstrated that mRNA of HIF3, a subtype of the HIF family, mainly expressed in CA1 of hippocumpus 24 hours after ischemia, eventhough mRNA of HIF1 was responsive but much lower expressed than that of HIF3. In culture cells, calcineurin activity was suggested to be important in the HIF1 regulation by its dephosphorylatio.These results prompt us to investigate the role of hypoxic pathway involving HIF as well as calcineurin/immunophilins. We plane to cralify the hypothesis that HIF family members would be critical factors in the pathway under calcium/calcineuin in ischemic events. Less