Kinase/phosphatase-mediated Mitochondrial Restructuring in Neuroprotection

激酶/磷酸酶介导的线粒体重构在神经保护中的作用

• 批准号: 8619667
• 负责人: STEFAN STRACK
• 金额: $ 32.7万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8619667
• 关键词: A kinase anchoring protein; Address; Apoptosis; Architecture; Binding; Bioenergetics; Biological Assay; Brain; Brain region; Buffers; Calcineurin; Calcium; Cessation of life; Complex; Cyclic AMP-Dependent Protein Kinases; Cytochromes; Cytosol; Disease; Docking; Dynamin; Enzymes; Funding; Hippocampus (Brain); Injury; Ischemia; Ischemic Brain Injury; Ischemic Stroke; Knockout Mice; Lead; Maps; Mediating; Metabolic; Metabolism; Middle Cerebral Artery Occlusion; Mitochondria; Molecular; Morphology; Mus; Nerve Degeneration; Neurodegenerative Disorders; Neuroglia; Neurons; Organelles; Outer Mitochondrial Membrane; PC12 Cells; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Production; Property; Protein Dephosphorylation; Protein phosphatase; Proteins; Reaction; Recruitment Activity; Regulation; Reporting; Role; Safety; Severities; Signal Transduction; Signaling Molecule; Site; Staging; Stroke; Testing; improved; in vivo; in vivo Model; infancy; mutant; neuronal survival; neuroprotection; public health relevance; scaffold; stroke therapy

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.

描述（由申请人提供）：迫切需要改善当前中风疗法的安全和治疗窗口。线粒体在缺血性脑损伤的早期（几分钟）和晚期（几天）阶段起关键作用，因此被认为是神经保护疗法的有希望的靶标。通过相反的裂变和融合反应确定的线粒体结构最近已成为神经元和非神经元细胞生存的关键决定因素。通过机械酶动力蛋白相关蛋白1（DRP1）催化的线粒体裂变促进了细胞色素C的释放和凋亡。此外，在神经退行性疾病中，中风和病理DRP1激活期间的线粒体片段发生。另一方面，我们和其他人表明，将线粒体融合到相互联系的网络中具有神经保护作用，这可能涉及增加能量产生，ROS和钙隔离以及从自噬降解中对细胞器进行保留。尽管线粒体动力学具有广泛欣赏的疾病相关性，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。在上一个融资周期中，我们确定了一个关键的磷酸化位点I DRP1。 S656在所有后生动物中保守，蛋白激酶A（PKA）磷酸化以抑制裂变酶，从而导致线粒体无反对的融合。对面PKA是钙依赖性的蛋白质磷酸酶钙调蛋白（CAN），它使S656脱磷酸化以促进线粒体片段化。 Phospho-DRP1可保护，而Dephospho-DRP1使PC12细胞对凋亡敏感。我们还发现了在海马神经元中线粒体 - 链酶锚定蛋白1（AKAP1）的有效神经保护活性，该激酶锚定蛋白1（AKAP1）是由S656时DRP1磷酸化介导的，并稳定了线粒体网络。有趣的是，PKA结合缺陷的AKAP1突变体碎裂线粒体，这表明一些据报道与AKAP1相关的信号分子可能与PKA相反的线粒体稳定。我们建议在三个特定目标（SA）中继续进行此询问。 在SA1中，我们将表征AKAP1基因敲除小鼠，以改变局灶性缺血后线粒体形态，生物能，DRP1磷酸化和损伤严重程度的变化。 SA2检查了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建议在钙介导的线粒体裂变和缺血性死亡中阐明罐头募集到DRP1的分子机制。该提案解决了总体假设，即AKAP1在线粒体外膜上组装信号体，该膜整合了来自细胞质和线粒体内的死亡和生存信号，以通过S656的DRP1的可逆磷酸化来重组细胞器。拟议的研究将提高我们对线粒体碎片化的机械理解及其通过神经元中可逆磷酸化的调节，这可能会导致对中风和疾病中神经变性的更好疗法。