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: 8048986
• 负责人: STEFAN STRACK
• 金额: $ 31.75万
• 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 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8048986
• 关键词: A kinase anchoring protein; Abbreviations; Adult; Alternative Splicing; Antibodies; Apoptosis; Apoptotic; Attention; Behavioral; Biological Assay; Brain; Brain Injuries; CAG repeat; Calcium; Cell Death; Cells; Collaborations; Complex; Confocal Microscopy; Corpus striatum structure; Cyclic AMP-Dependent Protein Kinases; Cytochromes; Data; Development; Dominant-Negative Mutation; Event; Free Radicals; Functional RNA; Galactosidase; Genes; Genotype; Glucose; Glutamates; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hippocampus (Brain); Holoenzymes; Homeostasis; Hydrolysis; Hypoglycemia; Hypoxia; IGFBP2 gene; In Vitro; Injection of therapeutic agent; Injury; Intervention; Ischemia; Ischemic Stroke; Knock-out; Knockout Mice; Label; Length; Mediating; Metabolic; Middle Cerebral Artery Occlusion; Mitochondria; Mitochondrial Membrane Protein; Mitochondrial Proteins; Modeling; Molecular; Morphology; Mus; Mutagenesis; Nerve Degeneration; Neurodegenerative Disorders; Neuronal Injury; Neurons; Neuroprotective Agents; Outer Mitochondrial Membrane; Oxygen; PC12 Cells; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Plasmids; Predisposition; Process; Protein phosphatase; Proteins; RNA Interference; RNA Splicing; Rattus; Recombinant Proteins; Recombinants; Regulation; Reporter; Research; Research Infrastructure; Research Personnel; Resistance; Role; Rotenone; Shapes; Signal Transduction; Slice; Spinocerebellar Ataxias; Staging; Stream; Stroke; Subfamily lentivirinae; Synapses; Testing; Therapeutic; Toxic effect; Transient Cerebral Ischemia; Variant; Virus; brain cell; density; deprivation; follow-up; in vivo; inhibitor/antagonist; killings; loss of function; neglect; neuron apoptosis; neuronal survival; neuroprotection; overexpression; prevent; pro-apoptotic protein; programs; protein phosphatase 2A regulatory subunit 65 kDa; response; scaffold; small hairpin RNA; stroke therapy

DESCRIPTION (provided by PI): Ischemic stroke kills brain cells by causing massive depolarization and glutamate release, which in turn disrupts calcium homeostasis and generates free radicals. Mitochondria are central to these events, as well as to the wave of delayed cell death that follows the ischemic injury. Attention has focused on neuroprotective drugs that prevent the initial calcium overload, while interventions into later stages of neuronal damage have been largely neglected despite the promise of a wider treatment window. Protein phosphatase 2A (PP2A) is an essential and ubiquitous Ser/Thr phosphatase that predominately exists as a heterotrimer of two core subunits (a catalytic and a scaffolding subunit) complexed to a third, regulatory subunit Bp2 is an alternative splice variant of a PP2A regulatory subunit gene that is expressed only in the adult brain. A non-coding CAG repeat expansion in the B(3 gene causes the neurodegenerative disorder spinocerebellar ataxia type 12. The alternative N terminus of B(32 promotes translocation of PP2A to the outer mitochondrial membrane (OMM) to induce apoptosis in PC 12 cells (JBC 278:24976; JBC 280:27375), whereas interfering with endogenous mitochondrial PP2A protects neurons against ischemic insults in vitro. The proposal is centered on the observation that mitochondrial shape transitions underlie the pro- apoptotic activity of PP2A/Bp2, with overexpression of Bp2 fragmenting mitochondria and Bp2 silencing causing mitochondrial elongation in hippocampal neurons. Opposing the fission activity of the phosphatase is protein kinase A (PKA) anchored to the OMM via A-kinase anchoring protein D-AKAP1. Aim 1 test the hypothesis that OMM-localized PP2A and PKA control neuronal survival by reciprocally modulating mitochondrial fission and fusion, which in turn alters free radical and calcium homeostasis. Aim 2 explores the fission GTPase Drp1 as an effector substrate for outer-mitochondrial PP2A and PKA. In Aim 3, in vivo delivery of viruses expressing three kinds of PP2A/Bp2 antagonists is intended to provide proof-of-concept evidence that this neuron-specific pro-apoptotic protein is a promising target for stroke therapy. Finally, Aim 4 characterizes Bp2 null mice for mitochondrial changes and resistance to ischemic injury.

描述（PI提供）：缺血性卒中通过引起大量去极化和谷氨酸释放杀死脑细胞，进而破坏钙稳态并产生自由基。线粒体是这些事件的核心，也是缺血性损伤后延迟细胞死亡的核心。人们的注意力集中在防止初始钙超载的神经保护药物上，而对神经元损伤后期阶段的干预在很大程度上被忽视，尽管有希望提供更宽的治疗窗口。蛋白磷酸酶2A（PP 2A）是一种必需的普遍存在的丝氨酸/苏氨酸磷酸酶，主要以两个核心亚基（催化亚基和支架亚基）与第三个调节亚基复合的异源三聚体形式存在，Bp 2是PP 2A调节亚基基因的选择性剪接变体，仅在成人脑中表达。B β基因中的非编码CAG重复扩增导致神经退行性疾病脊髓小脑共济失调12型。B β 2的替代N末端促进PP 2A易位至线粒体外膜（OMM）以诱导PC 12细胞中的细胞凋亡（JBC 278：24976; JBC 280：27375），而干扰内源线粒体PP 2A在体外保护神经元免受缺血性损伤。该提议集中于观察到线粒体形状转变是PP 2A/Bp 2的促凋亡活性的基础，其中Bp 2的过表达使线粒体片段化，Bp 2沉默引起海马神经元中的线粒体延长。与磷酸酶的分裂活性相反的是通过A-激酶锚定蛋白D-AKAP 1锚定至OMM的蛋白激酶A（PKA）。目的1验证OMM定位的PP 2A和PKA通过调节线粒体的分裂和融合，进而改变自由基和钙稳态来控制神经元存活的假设。目的2探索分裂GTdsp Drp 1作为外线粒体PP 2A和PKA的效应底物。在目的3中，表达三种PP 2A/Bp 2拮抗剂的病毒的体内递送旨在提供概念验证证据，证明这种神经元特异性促凋亡蛋白是中风治疗的有希望的靶标。最后，目的4表征Bp 2缺失小鼠的线粒体变化和对缺血性损伤的抗性。