Development of novel neuroprotective drugs targeting for neurovascular unit therapy.

我们在缺血性神经退行性疾病中引入了新的神经保护药物，包括钙调素拮抗剂DY-9760e和蛋白激酶B （Akt）激活剂VO （OPT）。然而，这些化合物对神经血管单位的保护机制尚不清楚。我们发现DY-9760e及其活性代谢物DY-9836是体内钙/钙调素依赖性一氧化氮合酶（NOS）的有效抑制剂。我们在此定义了在苯肾上腺素（PE）诱导的肥厚心肌细胞中，通过解偶联eNOS产生超氧化物的病理生理作用和机制。在pe诱导的肥厚心肌细胞中，超氧化物的产生与eNOS解偶联状态的增加有关。因此，eNOS的解偶联解释了长时间PE暴露产生的超氧化物，从而诱导凋亡细胞死亡。此外，dy9836的心脏保护作用与通过抑制eNOS活性抑制异常超氧化物的产生密切相关。dy9836治疗还能更好地保护心肌细胞免受小窝蛋白-3/肌营养不良蛋白的破坏，这是心肌细胞小窝中支架eNOS的主要成分。同样地，DY-9836治疗也减少了血管内皮细胞中不偶联的eNOS在脑缺血后产生的超氧化物。因此，dy9760e /DY9836对超氧化物产生的抑制对缺血诱导的神经变性中神经血管单位的保护至关重要。在海马的亚颗粒区（SGZ）有充分的证据表明神经发生。特别是在海马神经发生中，神经发生在学习和记忆形成中的基本作用已经得到解决。在此，我们利用小鼠短暂性大脑中动脉闭塞（MCAO）模型，评估了磷脂酰肌醇3-激酶(PI3K)/Akt和细胞外信号调节激酶（ERK）通路的刺激剂VO （OPT）是否促进脑缺血后的神经发生。VO （OPT）是一种有效的蛋白酪氨酸磷酸酶（PTPs）抑制剂，腹腔注射VO （OPT）刺激脑缺血后成人齿状回（DG）的神经发生。VO （OPT）导致PI3K/Akt和ERK通路的激活，这两个通路分别被特异性抑制剂wortmannin或U-0126抑制。这两种治疗方法均显著抑制了脑缺血后VO （OPT）诱导的神经发生，表明这两种途径都需要诱导VO （OPT）诱导的神经发生。在一些行为学研究中，我们发现VO （OPT）诱导的神经发生可以改善脑缺血后的记忆缺陷。这些结果表明，VO （OPT）通过激活PI3K/Akt和ERK来刺激脑缺血后的神经发生。此外，Akt-和erk诱导的神经发生对脑缺血后记忆缺陷的改善至关重要。少

We have introduced novel neuroprotective drugs including calmodulin antagonist DY-9760e and protein kinase B (Akt) activator VO (OPT) in the ischemic neurodegeneration. However, the mechanisms underlying neurovascular unit protection by these compounds remain unclear. We found that DY-9760e and its active metabolite, DY-9836 are potent inhibitors of calcium/calmodulin dependent nitric oxide synthase (NOS) in vivo. We here defined pathophysiological role and mechanism of superoxide generation through uncoupled eNOS in phenylephrine (PE)-induced hypertrophic cardiomyocytes. In PE-induced hypertrophic cardiomyocytes, the superoxide generation was associated with increased uncoupling state of eNOS. Thus, uncoupling of eNOS accounts for superoxide generation by prolonged PE exposure, thereby inducing apoptotic cell death. Furthermore, cardioprotective effects of DY-9836 well correlated with inhibition of aberrant superoxide generation by suppression of eNOS activity. DY-9836 treatment also … More protected cardiomyocytes from breakdown of caveolin-3/dystrophin, which are major components to scaffold eNOS in cardiomyocyte caveolae. Similarly, DY-9836 treatment also attenuated superoxide generation following brain ischemia by uncoupled eNOS in vascular endothelial cells. Thus the inhibition of superoxide production by DY-9760e/DY9836 is critical for protection of neurovascular units in ischemia-induced neurodegenaration.Neurogenesis is well documented in the subgranular zone (SGZ) of hippocampus. Especially, in the hippocampal neurogenesis, fundamental role of neurogenesis in learning and memory formation has been addressed. We here assessed whether VO (OPT), a stimulator of phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal regulated kinase (ERK) pathways promotes neurogenesis following brain ischemia using a mouse transient middle cerebral artery occlusion (MCAO) model. Intraperitoneal administrations of VO (OPT), which is a potent inhibitor for protein tyrosine phosphatases (PTPs), stimulated neurogenesis in the adult dentate gyrus (DG) following brain ischemia. VO (OPT) led to activation of PI3K/Akt and ERK pathways, which are inhibited by treatment with specific inhibitor, wortmannin or U-0126, respectively. Each treatment of them significantly inhibited the neurogenesis, suggesting that both pathways need to elicit VO (OPT)-induced neurogenesis following brain ischemia. In some behavioral studies, we showed that VO (OPT)-induced neurogenesis accounts for improvement of memory deficits following brain ischemia. These results suggest that intraperitoneal administrations of VO (OPT) stimulate neurogenesis following brain ischemia through PI3K/Akt and ERK activation. Moreover, Akt- and ERK-induced neurogenesis is critical for improvement of memory deficits following brain ischemia. Less