Brain mitochondria and glycogen synthase kinase-3beta

摘要 NADH：泛醌氧化还原酶(Complex-I)是氧化磷酸化相关疾病中最常见的损伤部位。这些疾病包括一些神经发育和神经退行性综合征，如利综合征、MELAS和某些形式的帕金森-S病。虽然Complex-I缺乏症的临床症状已经确定，但对线粒体中调节Complex-I功能的信号通路知之甚少。最近，我们的团队发现，未受调控的糖原合成酶激酶-3β(GSK3β)活性抑制Complex-I功能，增加活性氧的产生，使线粒体碎裂，并增加细胞对Complex-I毒素的敏感性。GSK3beta是一种信号蛋白，已知会影响代谢途径和大脑发育。它的调控是多层次的，这意味着它的作用可以通过其丝氨酸-9位点的磷酸化、蛋白质-蛋白质的相互作用和细胞内的定位来控制。有趣的是，还没有人完全阐明它在大脑中的细胞内分布。在初步结果中，我们发现GSK3β的显著部分存在于脑线粒体中。此外，我们现在发现了以前未发现的GSK3β在其他细胞间隔区的表达。这项建议的第一个具体目标是通过电子显微镜完全确定GSK3β在小鼠和人脑中的神经解剖超微结构分布。此前还没有进行过这种彻底的调查。内源性GSK3β信号在线粒体中的作用尚不完全清楚。在具体目标2中，目标是检验内源性GSK3β调节Complex-I功能的假设。内源性线粒体GSK3β活性通过分子和药理学方法进行调控，以评估GSK3β信号对Complex-I的影响。STAT3和GRIM19蛋白在线粒体中结合在一起，两者都参与Complex-I的功能。我们还知道，GSK3β与这两种蛋白都有关联。我们的假设是，GSK3β信号通过由GSK3β、STAT3和GRIM19组成的三方蛋白复合体来调节Complex-I的活性。通过这个项目，我们希望阐明线粒体GSK3β信号及其对Complex-I的影响，并有望利用我们的发现发现缓解Complex-I疾病的措施。

Abstract NADH:ubiquinone oxidoreductase (complex-I) is the most common site of impairment of the oxidative phosphorylation-related disorders. These disorders include some neurodevelopmental and neurodegenerative syndromes such as Leigh syndrome, MELAS, and some forms of Parkinson¿s disease. While the clinical symptoms of complex-I deficiency are defined, signaling pathways regulating complex-I function in the mitochondria are less understood. Recently, our group found that unregulated glycogen synthase kinase-3beta (GSK3beta) activity inhibits complex-I function, increases reactive oxygen species production, fragments mitochondria, and increases the cell's sensitivity to complex-I toxins. GSK3beta is a signaling protein that is known to affect metabolic pathways and brain development. Its regulation is multi-tiered meaning that its actions can be controlled by phosphorylation at its serine-9 site, by protein-protein interactions, and by its intracellular localization. Interestingly, no one has fully elucidated its intracellular distribution in the brain. In preliminary results, we have found that a salient fraction of GSK3beta exists in brain mitochondria. Furthermore, we have now found previously undiscovered pockets of GSK3beta expression in other cellular compartments. The first specific aim of this proposal is to fully determine the neuroanatomic ultrastructural distribution of GSK3beta in the mouse and human brain by electron microscopy. A thorough investigation of this sort has not been conducted previously. The actions of endogenous GSK3beta signaling in the mitochondria are not fully known. In specific aim 2, the goal is to test the hypothesis that endogenous GSK3beta modulates complex-I functions. Endogenous mitochondrial GSK3beta activity is manipulated by molecular and pharmacological methods to assess the affects of GSK3beta signaling on complex-I. The proteins STAT3 and GRIM19 are bound together in the mitochondria and both are involved in complex-I function. It is also known that GSK3beta associates with both these proteins. Our hypothesis is that GSK3beta signaling regulates complex-I activity through a tripartite protein composite consisting of GSK3beta, STAT3, and GRIM19. With this project we hope to elucidate mitochondrial GSK3beta signaling and its affects on complex-I with the prospect that our findings could be used to discover measures to alleviate complex-I disorders.