Kinase/Phosphatase-mediated Mitochondrial Restructuring in Neuroprotection

描述（由PI提供）：缺血性中风通过引起大量去极化和谷氨酸释放来杀死脑细胞，从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浪潮的核心。人们的注意力集中在预防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了，尽管有更广泛的治疗窗口。蛋白磷酸酶2A （PP2A）是一种必不可少的、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Bp2的异源三聚体的形式存在，Bp2是PP2A调节亚基基因的另一种剪接变体，仅在成人大脑中表达。B(3基因中的非编码CAG重复扩增导致12型脊髓小脑性共济失调神经退行性疾病。B(32的替代N端促进PP2A向线粒体外膜（OMM）易位，诱导pc12细胞凋亡（JBC 278:24976; JBC 280:27375），而干扰内源性线粒体PP2A在体外可保护神经元免受缺血性损伤。该提议的中心是观察到线粒体形状的转变是PP2A/Bp2促凋亡活性的基础，Bp2的过表达使线粒体断裂，Bp2沉默导致海马神经元线粒体伸长。与磷酸酶的裂变活性相反的是通过A激酶锚定蛋白D-AKAP1锚定在OMM上的蛋白激酶A （PKA）。目的1验证omm定位的PP2A和PKA通过相互调节线粒体裂变和融合来控制神经元存活的假设，而线粒体裂变和融合反过来改变自由基和钙稳态。Aim 2探讨了裂变GTPase Drp1作为线粒体外PP2A和PKA的效应底物。在Aim 3中，体内递送表达三种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卒中治疗的有希望的靶点。最后，Aim 4表征了Bp2缺失小鼠的线粒体变化和对缺血性损伤的抵抗。

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.