Identification of SARM1 Activation Patterns and Mechanisms

SARM1 激活模式和机制的鉴定

基本信息

批准号：
8909981
负责人：
Daniel A Gibson
金额：
$ 5.24万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8909981
关键词：
Address Affect Alzheimer&apos s Disease Animal Model Axon Back Bioinformatics Biological Assay Biological Preservation Biosensor Characteristics Chemotherapy-induced peripheral neuropathy Clinical Development Disease Disease Progression Dyes Enzymes Fluorescence Resonance Energy Transfer Future Genetic Genetic Screening Goals Health Hot Spot Human Image Imagery Injury Ischemic Brain Injury Knowledge Lead Length Life Location MAPK8 gene Maps Mediating Mediator of activation protein Membrane Methods Microscopy Mitochondria Modeling Molecular Molecular Target Motor Multiple Sclerosis Mutate Nerve Degeneration Nervous System Trauma Neurodegenerative Disorders Neuronal Injury Neurons Parkinson Disease Pathway interactions Pattern Peripheral Nervous System Diseases Positioning Attribute Post-Translational Protein Processing Process Proteins Proteomics Quality of life Role Sensory Site Stroke Swelling Symptoms Technology Testing Therapeutic Intervention Time Toll-like receptors Traumatic Nerve Injury Visual axon injury axonal degeneration chronic pain feeding genetic approach genome-wide in vivo inhibitor/antagonist injured knock-down nervous system disorder novel overexpression prevent programs public health relevance ratiometric response response to injury sensor small hairpin RNA spatiotemporal tool

项目摘要

DESCRIPTION (provided by applicant): Axon degeneration (AxD) is a hallmark feature of a variety of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, as well as various neuronal injuries, such as traumatic and ischemic brain injuries, and peripheral neuropathies. A major component of AxD is the presence of an endogenous degeneration program that, when activated, results in axonal self-destruction. The widespread occurrence of AxD in both disease and injury states suggests that therapeutic interventions that protect axons from degeneration could have a profound impact on human health and quality of life. In spite of the clinical importance of AxD, there is a critical gap in knowledge regarding th mechanisms by which the degeneration program is activated and how activation of program components relates to the progression of AxD. The recent identification of Sarm1 as an essential component of this program suggests that furthering our understanding of Sarm1 may have profound implications for our broader understanding of AxD mechanisms and progression. I have developed a Sarm1 biosensor that allows visualization of injury-induced Sarm1 activation in axons using ratiometric FRET microscopy. In Aim 1 this biosensor will be optimized and used to map injury-induced Sarm1 activation in axons and correlate it with important AxD markers by using Ca2+-imaging, mitochondrial dyes, and morphological analysis to determine how the onset and progression of AxD features relates to Sarm1 activation. The biosensor will also be used to test if the preservation of the proximal axonal segment after injury is due to differential Sarm1 activation. Aim 2 will use the Sarm1 biosensor to identify mechanisms regulating injury-induced Sarm1 activation. Intrinsic regulatory features of Sarm1 will be identified by mutating residues in the biosensor that are predicted to undergo posttranslational modification (PTM) and testing the effect on biosensor activation and AxD in both injured and uninjured axons. Enzymes predicted to mediate PTMs identified as being important will be tested using shRNAs and pharmacological inhibitors. In parallel, known modulators of AxD will be tested to determine if they act by modulating Sarm1 activation. Lastly, an enriched pool of shRNA targets previously identified by our lab as protecting against AxD will be screened using the biosensor to identify novel upstream molecular components mediating injury-induced Sarm1 activation. These aims will fill a critical gap in knowledge regarding the mechanisms of AxD program activation and its relationship to AxD progression. By 1) defining the relationship between Sarm1 activation and AxD progression and 2) identifying the mechanism(s) of injury-induced Sarm1 activation, this proposal will significantly enhance our fundamental understanding of AxD and may identify novel molecular targets for therapeutic intervention in the treatment of neurological disease and injury.

描述（由申请人提供）：轴突变性（AxD）是多种神经退行性疾病的标志性特征，例如阿尔茨海默病和帕金森病，以及各种神经元损伤，例如创伤性和缺血性脑损伤以及周围神经病。 AxD 的一个主要组成部分是存在内源性变性程序，该程序激活后会导致轴突自我毁灭，AxD 在疾病和损伤状态下的广泛发生表明。保护轴突免于变性的治疗干预措施可能对人类健康和生活质量产生深远影响，尽管 AxD 具有临床重要性，但关于变性程序的激活机制以及如何激活的知识仍存在重大差距。最近确定 Sarm1 作为该程序的重要组成部分，这表明进一步了解 Sarm1 可能对我们更广泛地了解 AxD 机制和进展产生深远的影响。允许使用比率 FRET 显微镜可视化轴突中损伤诱导的 Sarm1 激活。在 Aim 1 中，该生物传感器将被优化并用于绘制轴突中损伤诱导的 Sarm1 激活图，并通过使用 Ca2+ 成像、线粒体染料将其与重要的 AxD 标记相关联。和形态学分析以确定 AxD 特征的发生和进展如何与 Sarm1 激活相关。生物传感器还将用于测试近端是否保存。损伤后的轴突段是由于差异 Sarm1 激活。目标 2 将使用 Sarm1 生物传感器来识别调节损伤诱导的 Sarm1 激活的机制。通过突变生物传感器中预计会发生翻译后修饰 (PTM) 的残基并测试对生物传感器的影响来识别 Sarm1 的内在调节特征。预计介导被认为重要的 PTM 的酶将使用 shRNA 和 AxD 进行测试。与此同时，我们将测试已知的 AxD 调节剂，以确定它们是否通过调节 Sarm1 激活来发挥作用。最后，我们实验室先前确定的可预防 AxD 的丰富 shRNA 靶点将使用生物传感器进行筛选，以识别新的上游。介导损伤诱导的 Sarm1 激活的分子成分通过 1) 定义 Sarm1 激活和 AxD 之间的关系，将填补有关 AxD 程序激活机制及其与 AxD 进展关系的关键知识空白。 2）确定损伤诱导的 Sarm1 激活的机制进展，该提案将显着增强我们对 AxD 的基本理解，并可能确定神经疾病和损伤治疗干预的新分子靶点。