Kinase/phosphatase-mediated Mitochondrial Restructuring in Neuroprotection

描述（由申请人提供）：目前脑卒中治疗的安全性和治疗窗口亟待改进。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。线粒体结构由对立的裂变和融合反应决定，最近被认为是神经元和非神经元细胞存活的关键决定因素。线粒体分裂由机械酶动力蛋白相关蛋白1 （Drp1）催化，促进细胞色素C的释放和细胞凋亡。此外，中风期间线粒体片段和病理性Drp1激活发生在神经退行性疾病中。另一方面，我们和其他人已经表明，线粒体融合成一个相互连接的网络具有神经保护作用，这可能涉及增加能量产生、活性氧和钙封存，以及使细胞器免于自噬降解。尽管线粒体动力学的疾病相关性得到广泛认可，但我们对控制线粒体结构的调节机制的理解仍处于起步阶段。在之前的融资周期中，我们确定了一个关键的磷酸化位点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，导致线粒体的无对抗融合。与PKA相反的是钙依赖性蛋白磷酸酶钙调神经磷酸酶（CaN），它使S656去磷酸化，促进线粒体断裂。Phospho-Drp1保护PC12细胞免于凋亡，而dephospho-Drp1使PC12细胞致敏。我们还发现线粒体定位的a激酶锚定蛋白1 （AKAP1）在海马神经元中具有有效的神经保护活性，这是由Drp1在S656位点的磷酸化和线粒体网络的稳定介导的。有趣的是，一个缺乏PKA结合的AKAP1突变体会使线粒体破碎，这表明一些与AKAP1相关的信号分子可能会反对PKA对线粒体的稳定作用。我们建议在三个具体目标（SA）中继续进行这一调查。

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.