Kinase/Phosphatase-mediated Mitochondrial Restructuring in Neuroprotection

描述(由PI提供)：缺血性中风通过引起大量的去极化和谷氨酸释放而杀死脑细胞，这反过来又破坏钙稳态并产生自由基。线粒体是这些事件的中心，也是缺血性损伤后延迟性细胞死亡浪潮的核心。人们的注意力集中在防止最初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了，尽管有望提供更广泛的治疗窗口。蛋白磷酸酶2A(PP2A)是一种普遍存在的丝氨酸/苏氨酸磷酸酶，主要以两个核心亚单位(催化亚基和支架亚基)的异源三聚体形式存在，第三个调控亚基BP2是PP2A调节亚单位基因的选择性剪接变异体，仅在成人脑中表达。B(3)基因的一个非编码CAG重复序列的扩展导致12型脊髓小脑型共济失调。B(32)的另一个N末端促进PP2A易位到线粒体膜外，从而诱导PC 12细胞的凋亡(JBC278：24976；JBC280：27375)，而干扰内源性线粒体PP2A则在体外保护神经元免受缺血损伤。该建议的中心是观察到线粒体形状转变是PP2A/BP2促凋亡活性的基础，BP2的过度表达使线粒体碎裂，BP2的沉默导致海马神经元中线粒体的延长。与磷酸酶分裂活性相反的是蛋白激酶A(PKA)，它通过A-激酶锚定蛋白D-AKAP1锚定到OMM上。目的1验证OMM定位的PP2A和PKA通过相互调节线粒体的分裂和融合来控制神经元存活，进而改变自由基和钙稳态的假设。目的2探索裂解GTP酶Drp1作为线粒体外PP2A和PKA效应底物的作用。在目标3中，体内传递表达三种PP2A/BP2拮抗剂的病毒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前途的靶点。最后，Aim 4研究了BP2基因缺失小鼠的线粒体变化和对缺血损伤的抵抗力。

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.