Regulation of apoptosis and degeneration after spinal cord injury

描述（由申请人提供）：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学病症中的最普遍和最具破坏性的过程之一。在暴露于UV作为应激的培养物中，已显示INK途径在调节线粒体功能中起关键作用。在INK亚型中，JNK 3是独特的，在神经系统中富集并在兴奋性毒性介导的细胞凋亡中发挥关键作用。然而，JNK 3是否在病理条件下被激活以及JNK 3激活是否/如何在体内被传递到线粒体仍然是未知的。我们的初步数据表明，JNK 3是选择性激活脊髓损伤后，并调节损伤介导的细胞色素C（cytC）的释放和少突胶质细胞凋亡。因此，本提案的目标是确定执行cytC释放的JNK 3的关键体内靶点，并了解JNK 3调节这些分子以启动cytC释放的机制。我们已经鉴定了Mcl-1，一种促存活Bcl-2成员，是体内JNK 3的底物。这种磷酸化的结果是促进泛素介导的Mcl-1降解，其通常被Pinl结合抑制。Pinl是一种丙基异构酶，在MAP激酶底物被磷酸化后结合MAP激酶底物，从而调节底物的降解或活性。当Mcl-1被突变以使其不依赖于JNK 3磷酸化时，Pinl在应激下不能与Mcl-1解离，并且Mcl-1降解被延迟。这些结果共同表明，JNK 3依赖的Mel-1磷酸化是取代Pin 1所必需的，允许Mcl-1被泛素化和降解，从而可以释放cytC。最近，Mule，一种仅含BH 3的E3泛素连接酶，被鉴定负责Mcl-1的泛素化和降解。我们的数据表明，Mule结合Mcl-1在体内和Pinl可能调节其结合特性。因此，我们假设JNK 3和Pinl反向调节Mule对Mcl-1的泛素化，进而调节脊髓损伤后Mcl-1的降解和随后的cytC释放。在这一假设下，我们提出了以下三个具体目标。目的I是通过分析Mcl-1的条件无效来确定Mcl-1是否是脊髓损伤后细胞色素C释放所必需的;目的II是通过使Pinl和Pinr_（JNKS）小鼠受到损伤来确定Pinl是否是脊髓损伤后Mcl-1稳定所必需的以及JNK 3是否参与Pinl的作用;目的III是确定JNK 3通过Mule调控Mcl-1泛素化的分子机制。我们相信这些研究将导致我们对JNK 3在病理条件下作用的理解的重大进展，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.