DISSECTION OF SARM1-INDUCED AXON DEGENERATION AND CELL DEATH

描述（由申请人提供）：神经退行性疾病以及脑外伤和缺血性损伤的特征是神经元细胞死亡和轴突变性。程序性细胞死亡和轴突变性是两种不同的自我毁灭程序，用于消除受损细胞和/或轴突；然而，它们在许多神经系统疾病中被不恰当地激活。虽然导致细胞死亡的途径的组成部分已经比较清楚地描述了，但轴突变性的机制在很大程度上是未知的。对野生小鼠的研究表明，合成NAD的Nmnat酶的过表达可以防止轴突变性，最近，一种叫做Sarm1的toll样受体衔接蛋白被发现是内在轴突变性程序的重要组成部分。包括我们自己实验室的其他研究表明，Sarm1也可以促进神经元和其他细胞的细胞死亡。事实上，失去Sarm1可以保护神经元免受代谢应激和线粒体功能障碍的影响。总之，这些突破表明，Sarm1驱动了一个一般的细胞破坏程序，我们称之为sarmoptosis。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结构域则是其激活细胞死亡和轴突变性的能力所必需的。为了研究sarmoptosis的分子方面，我们开发了多种工具，包括可调节的Sarm1 TIR结构域二聚化系统，该系统允许我们以受控的方式触发细胞死亡或使用区室，轴突变性。我们的目标是了解臂膀下垂，以便设计出治疗药物来阻断这一过程，因为这可能是治疗许多神经系统疾病的有效方法。在本提案中，我们概述了利用这些试剂来定义Sarm1参与促进细胞破坏的分子途径的实验。首先，我们将确定激活细胞破坏的Sarm1 TIR结构域的结构基序。我们将通过分析一系列位点导向的TIR结构域突变来识别这些关键的功能残基。其次，我们将确定参与介导Sarm1激活后发生的NAD消耗的酶。第三，我们将使用抑制筛选确定在这一破坏性途径下游发挥作用的蛋白质。将对已识别的抑制因子进行机制研究，以表征它们在臂膀下垂中的作用。

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.