Regulation of apoptosis and degeneration after spinal cord injury

脊髓损伤后细胞凋亡和变性的调控

• 批准号: 7575119
• 负责人: SUNG OK YOON
• 金额: $ 32.81万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7575119
• 关键词: A Mouse; Affect; Apical; Apoptosis; Apoptotic; BH3 Domain; Binding; Binding Sites; Caspase; Cells; Characteristics; Cytochromes; Cytosol; Data; Degenerative Disorder; Equine mule; Goals; Homeostasis; Injury; Ink; Isomerase; Lead; Life; MAPK10 gene; MAPK8 gene; MCL1 protein; Mediating; Mitochondria; Mitogen-Activated Protein Kinases; Molecular; Mus; Mutate; Nervous system structure; Neurodegenerative Disorders; Oligodendroglia; Outcome; Pathway interactions; Phosphorylation; Play; Positioning Attribute; Process; Protein Isoforms; Regulation; Research Personnel; Role; Spinal cord injury; Stress; Testing; Ubiquitin; Ubiquitination; excitotoxicity; in vivo; member; mitochondrial dysfunction; new therapeutic target; overexpression; programs; protein degradation; ubiquitin-protein ligase

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.

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条件中最普遍和最具破坏性的过程之一。在暴露于紫外线作为应激的培养中，INK途径已被证明在调节线粒体功能方面发挥关键作用。在INK亚型中，JNK3是独特的，在神经系统中富集，并在兴奋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的激活是否/如何在体内传递到线粒体，目前尚不清楚。我们的初步数据表明，JNK3在脊髓损伤后被选择性激活，并调节损伤介导的细胞色素C （cytC）释放和少突胶质细胞凋亡。因此，本提案的目标是确定JNK3执行cytC释放的关键体内靶点，并了解JNK3调节这些分子启动cytC释放的机制。我们已经确定Mcl-1，一个促生存的Bcl-2成员，是JNK3在体内的底物。这种磷酸化的结果是促进泛素介导的Mcl-1降解，而这通常被Pinl结合抑制。Pinl是一种丙基异构酶，在MAP激酶底物磷酸化后与底物结合，从而调节底物的降解或活性。当Mcl-1突变使其不依赖JNK3磷酸化时，Pinl在应激下不能与Mcl-1分离，Mcl-1的降解被延迟。这些结果共同表明，jnk3依赖性的Mel-1磷酸化对于取代Pin -1是必要的，允许Mcl-1被泛素化和降解，从而释放cytC。最近，Mule，一个仅bh3的E3泛素连接酶，被发现负责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，而Pinl可能调节其结合特性。因此，我们假设JNK3和Pinl通过Mule反向调节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。在此假设下，我们提出以下三个具体目标。目的一：通过分析Mcl-1的条件null，确定脊髓损伤后cytC释放是否需要Mcl-1；目的II：通过Pinl“’”和Pinr^JNKS“’”损伤小鼠，确定脊髓损伤后Pinl对Mcl-1的稳定是否必需，以及JNK3是否参与Pinl的作用；目的III是确定JNK3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病理条件下JNK3作用的理解取得重大进展，并可能为退行性疾病提供新的治疗靶点。