DISSECTION OF SARM1-INDUCED AXON DEGENERATION AND CELL DEATH

描述(申请人提供)：神经退行性疾病以及脑的创伤性和缺血性损伤的特征是神经细胞死亡和轴突退化。程序性细胞死亡和轴突变性是截然不同的自毁程序，它们被用来消除受损的细胞和/或轴突；然而，在许多神经疾病中，它们被不适当地激活。虽然导致细胞死亡的途径的组成部分相对较好地描述了，但涉及轴突变性的机制在很大程度上是未知的。对wlds小鼠的研究表明，过度表达合成NAD的nmnat酶可以防止轴突退化，最近，一种名为Sarm1的Toll样受体适配器蛋白被发现是内在轴突退化计划的重要组成部分。包括我们自己的实验室在内的其他研究表明，Sarm1还可以促进神经元和其他细胞的细胞死亡。事实上，Sarm1的缺失可以保护神经元免受代谢压力和线粒体功能障碍的影响。总而言之，这些突破表明，Sarm1驱动着一种我们称之为sarmdesis的一般细胞破坏程序。我们对Sarm1的分子分析表明，SAM结构域是其多聚化所必需的，而TIR结构域是其激活细胞死亡和轴突变性所必需的。为了研究sarmosis的分子方面，我们开发了各种工具，包括可调节的Sarm1 TIR结构域二聚系统，该系统允许我们触发细胞死亡，或使用分隔的小室，以受控的方式触发轴突变性。我们的目标是了解腋窝下垂，以便设计出治疗药物来阻止这一过程，因为这可能是治疗许多神经疾病的有用方法。在这项提案中，我们概述了利用这些试剂来定义Sarm1参与的促进细胞破坏的分子途径的实验。首先，我们将确定Sarm1 TIR结构域中激活细胞破坏的结构基序。我们将通过分析一系列TIR结构域的定点突变来鉴定这些关键的功能残基。其次，我们将确定参与介导Sarm1激活后NAD耗竭的酶(S)。第三，我们将使用抑制子筛选来确定在这一破坏性途径下游发挥作用的蛋白质。将对已识别的抑制物进行机制研究，以确定它们在肩周炎中的作用。

DESCRIPTION (provided by applicant): Neurodegenerative disorders as well as traumatic and ischemic injuries to the brain are characterized by neuronal cell death and axonal degeneration. Programmed cell death and axonal degenerative are distinct self-destructive programs that are invoked to eliminate damaged cells and/or axons; however, they are activated inappropriately in many neurological disorders. While the components of pathways leading to cell death are relatively well characterized, the mechanisms involved in axonal degeneration are largely unknown. Studies of the wlds mouse revealed that overexpression of Nmnat enzymes, which synthesize NAD, can prevent axonal degeneration and, more recently, a toll-like receptor adaptor protein called Sarm1 was discovered to be an important component of the intrinsic axon degeneration program. Additional studies, including from our own labs, show that Sarm1 can also promote cell death in neurons and other cells. Indeed, loss of Sarm1 protects neurons from metabolic stress and mitochondrial dysfunction. Together, these breakthroughs show that Sarm1 drives a general cell destruction program that we term sarmoptosis. Our molecular analysis of Sarm1 shows that the SAM domains are necessary for its multimerization, whereas the TIR domain is required for its ability to activate cell death and axonal degeneration. To study molecular aspects of sarmoptosis, we have developed a variety of tools including a regulable Sarm1 TIR domain dimerization system that allows us to trigger cell death or, using compartmentalized chambers, axonal degeneration in a controlled fashion. It is our goal to understand sarmoptosis so that therapeutic agents can be devised to block this process, as this could be a useful method for treating many neurological disorders. In this proposal, we outline experiments that utilize these reagents to define the molecular pathways engaged by Sarm1 to promote cell destruction. First, we will identify structural motifs in the Sarm1 TIR domain that activate cell destruction. We will identify these key functional residues by analyzing a series of site-directed TIR domain mutants. Second, we will identify the enzyme(s) involved in mediating the NAD depletion that occurs after Sarm1 activation. Third, we will identify proteins that function downstream in this destructive pathway using a suppressor screen. Mechanistic studies of identified suppressors will be performed to characterize their role in sarmoptosis.