Kinase/phosphatase-mediated Mitochondrial Restructuring in Neuroprotection

描述(申请人提供)：迫切需要改进目前中风疗法的安全性和治疗窗口。线粒体在缺血性脑损伤的早期(几分钟)和晚期(几天)都起着关键作用，因此被认为是神经保护治疗的有希望的靶点。线粒体结构由相反的分裂和融合反应决定，最近已成为神经元和非神经元细胞生存的关键决定因素。机械酶动力蛋白相关蛋白1(Drp1)催化的线粒体分裂促进细胞色素C的释放和细胞凋亡。此外，中风时会出现线粒体碎片，在神经退行性疾病中会出现病理性的Drp1激活。另一方面，我们和其他人已经证明，线粒体融合到一个相互连接的网络中具有神经保护作用，这可能涉及增加能量产生、ROS和钙隔离，以及防止细胞器自噬降解。尽管线粒体动力学与疾病的相关性得到了广泛的认可，但我们对控制线粒体结构的调控机制的理解仍处于初级阶段。在之前的资金周期中，我们确定了一个关键的磷酸化位点I Drp1。S656在所有后生动物中都是保守的，它被蛋白激酶A(PKA)磷酸化以抑制裂变酶，导致线粒体的非对抗性融合。与PKA相对的是钙依赖的蛋白磷酸酶钙调神经磷酸酶(CaN)，它使S656去磷酸化，促进线粒体碎裂。磷酸化的Drp1对PC12细胞具有保护作用，而去磷酸化的Drp1则使PC12细胞对凋亡敏感。我们还发现了线粒体定位的A激酶锚定蛋白1(AKAP1)在海马神经元中具有强大的神经保护活性，该活性是由S656处的Drp1磷酸化和线粒体网络的稳定所介导的。有趣的是，一个PKA结合缺陷的AKAP1突变体使线粒体碎片化，这表明一些被报道与AKAP1相关的信号分子可能反对PKA对线粒体的稳定。我们建议在三个具体目标(SA)中继续进行这一调查。 在SA1中，我们将研究AKAP1基因敲除小鼠在局灶性缺血后线粒体形态、生物能量学、Drp1磷酸化和损伤严重程度的变化。SA2研究了AKAP1相互作用蛋白磷酸酶(PP1，CaN)在线粒体重塑和神经元存活中的作用。最后，SA3建议阐明钙介导的线粒体分裂和缺血性死亡中CaN向Drp1募集的分子机制。该提议解决了AKAP1在线粒体膜外膜组装信号小体的总体假设，该信号小体整合了来自细胞质和线粒体内的死亡和生存信号，通过S656处Drp1的可逆磷酸化来重组细胞器。拟议的研究将增加我们对线粒体断裂及其在神经元中可逆磷酸化调节的机制的理解，这可能会导致更好的治疗中风和疾病的神经退行性变。

DESCRIPTION (provided by applicant): There is an urgent need to improve upon the safety and treatment window of current stroke therapies. Mitochondria play key roles during both early (minutes) and late (days) stages of ischemic brain injury, and are thus recognized as promising targets for neuroprotective therapy. Mitochondrial architecture, as determined by opposing fission and fusion reactions, has recently emerged as a critical determinant for survival of both neuronal and non-neuronal cells. Mitochondrial fission catalyzed by the mechanoenzyme dynamin-related protein 1 (Drp1) facilitates cytochrome C release and apoptosis. In addition, mitochondria fragment during stroke and pathological Drp1 activation occurs in neurodegenerative disorders. On the other hand, we and others have shown that fusion of mitochondria into an interconnected network has a neuroprotective effect, which may involve increased energy production, ROS and calcium sequestration, and sparing of the organelle from autophagic degradation. Despite the widely appreciated disease relevance of mitochondrial dynamics, our understanding of regulatory mechanisms controlling mitochondrial architecture is still in its infancy. In the previous funding cycle, we identified a pivotal phosphorylation site i Drp1. Conserved in all metazoans, S656 is phosphorylated by protein kinase A (PKA) to inhibit the fission enzyme, leading to unopposed fusion of mitochondria. Opposite PKA is the calcium-dependent protein phosphatase calcineurin (CaN), which dephosphorylates S656 to promote mitochondrial fragmentation. Phospho-Drp1 protects from, while dephospho-Drp1 sensitizes PC12 cells to apoptosis. We also uncovered a potent neuroprotective activity of mitochondria-localized A kinase anchoring protein 1 (AKAP1) in hippocampal neurons, which is mediated by Drp1 phosphorylation at S656 and stabilization of the mitochondrial network. Intriguingly, a PKA binding- deficient AKAP1 mutant fragmented mitochondria, suggesting that some of the signaling molecules reported to also associate with AKAP1 may oppose mitochondrial stabilization by PKA. We propose to continue with this line of inquiry in three specific aims (SA). In SA1, we will characterize AKAP1 knockout mice for changes in mitochondrial morphology, bioenergetics, Drp1 phosphorylation, and injury severity following focal ischemia. SA2 examines the role of AKAP1-interacting protein phosphatases (PP1, CaN) in mitochondrial remodeling and neuronal survival. Finally, SA3 proposes to elucidate molecular mechanisms of CaN recruitment to Drp1 in calcium-mediated mitochondrial fission and ischemic death. The proposal addresses the overall hypothesis that AKAP1 assembles a signalosome at the outer mitochondrial membrane, which integrates death and survival signals from the cytosol and from within mitochondria to restructure the organelle via reversible phosphorylation of Drp1 at S656. The proposed studies will increase our mechanistic understanding of mitochondrial fragmentation and its regulation by reversible phosphorylation in neurons, which may lead to better therapies for neurodegeneration in stroke and disease.