Regulation of apoptosis and degeneration after spinal cord injury

描述(申请人提供)：线粒体功能障碍是存在于各种神经退行性疾病和神经病理状况中最普遍和最具破坏性的过程之一。在暴露在紫外线胁迫下的培养物中，墨水途径已被证明在调节线粒体功能方面发挥关键作用。在墨水异构体中，JNK3是独一无二的，在神经系统中富含，并在兴奋性毒性介导的细胞凋亡中发挥关键作用。然而，JNK3是否在病理条件下被激活，以及JNK3激活是否/如何在体内传递到线粒体仍是未知的。我们的初步数据表明，JNK3在脊髓损伤后被选择性地激活，并调节损伤介导的细胞色素C(CytC)的释放和少突胶质细胞的凋亡。因此，这项建议的目的是确定JNK3在体内执行细胞色素C释放的关键靶点，并了解JNK3调节这些分子启动细胞色素C释放的机制。我们已经确定Mcl-1是促进生存的Bcl-2成员，在体内是JNK3的底物。这种磷酸化的结果是促进泛素介导的Mcl-1的降解，而Mcl-1通常被Pinl结合抑制。PIN1是一种丙基异构酶，在底物被磷酸化后与MAPK底物结合，从而调节底物的降解或活性。当Mcl-1发生突变，使其独立于JNK3的磷酸化时，Pin1在应激状态下不能与Mcl-1解离，Mcl-1的降解被延迟。这些结果表明，依赖于JNK3的Mel-1的磷酸化是取代Pin-1所必需的，从而使Mcl-1泛素化并被降解，从而释放CytC。最近发现Mule是一种BH3-E3泛素连接酶，负责Mcl-1的泛素化和降解。我们的数据表明，Mule在体内与Mcl-1结合，Pin1可能调节其结合特性。因此，我们假设JNK3和Pin1相反地调节Mule对Mcl-1的泛素化，进而调节Mcl-1的降解和随后脊髓损伤后细胞色素C的释放。在这一假设下，我们提出了以下三个具体目标。目的一是通过分析Mcl-1的条件缺失来确定Mcl-1是否是脊髓损伤后细胞色素C释放所必需的；二是通过损伤Pinl“‘”和Pinr^JNKS“’小鼠来确定PIN1是否对Mcl-1的稳定是必需的，以及JNK3是否参与了Pin1的作用；以及三是确定JNK3调节Mcl-1泛素化的分子机制。我们相信，这些研究将大大提高我们对JNK3在病理条件下的作用的理解，并可能为退行性疾病提供新的治疗靶点。

DESCRIPTION (provided by applicant): Mitochondrial dysfunction is one of the most pervasive and damaging processes that exist in various neurodegenerative diseases and neuropathological conditions. In cultures that were exposed to UV as a stress, the INK pathway has been shown to play critical roles in regulating mitochondrial function. Of the INK isoforms, JNK3 is unique, being enriched in the nervous system and playing critical roles in excitotoxicity- mediated apoptosis. Whether JNK3 is activated under pathological conditions and whether/how JNK3 activation is relayed to mitochondria in vivo, however, have remained unknown. Our preliminary data indicate that JNK3 is selectively activated after spinal cord injury, and regulates injury-mediated cytochrome C (cytC) release and oligodendrocyte apoptosis. The goal of this proposal is therefore to identify critical in vivo targets of JNK3 that execute cytC release, and understand the mechanisms by which JNK3 regulates these molecules to initiate cytC release. We have identified that Mcl-1, a pro- survival Bcl-2 member, is a substrate for JNK3 in vivo. The outcome of this phosphorylation is to facilitate ubiquitin-mediated degradation of Mcl-1, which is normally inhibited by Pinl binding. Pinl, a propyl- isomerase, binds MAP kinase substrates after they are phosphorylated, thereby regulating either the degradation or the activities of the substrates. When Mcl-1 is mutated to render it independent of JNK3 phosphorylation, Pinl fails to dissociate from Mcl-1 under stress and Mcl-1 degradation is delayed. These results together suggest that JNK3-dependent phosphorylation of Mel-1 is necessary for displacing Pin 1, allowing Mcl-1 to be ubiquitinated and degraded so that cytC can be released. Recently, Mule, a BH3-only E3 ubiquitin ligase, was identified being responsible for Mcl-1 ubiquitination and degradation. Our data suggest that Mule binds Mcl-1 in vivo and Pinl may regulate its binding characteristics. We therefore hypothesize that JNK3 and Pinl oppositely regulate ubiquitination of Mcl-1 by Mule, in turn regulating degradation of Mcl-1 and subsequent cytC release after spinal cord injury. Under the hypothesis, we propose the following three specific aims. Aim I is to determine whether Mcl-1 is necessary for cytC release after spinal cord injury by analyzing the conditional null of Mcl-1; Aim II is to determine whether Pinl is necessary for Mcl-1 stabilization and whether JNK3 is involved in Pinl action after spinal cord injury by subjecting Pinl"'" and Pinr^JNKS"'" mice to injury; and Aim III is to determine the molecular mechanisms by which JNK3 regulates Mcl-1 ubiquitination by Mule. We believe these studies will lead to a significant advancement in our understanding of JNK3 action under pathological conditions, and likely to provide novel therapeutic targets for degenerative conditions.