Identification of SARM1 Activation Patterns and Mechanisms

SARM1 激活模式和机制的鉴定

• 批准号: 9302554
• 负责人: Daniel A Gibson
• 金额: $ 5.92万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 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/9302554
• 关键词: Address; Affect; Alzheimer' s Disease; Animal Model; Axon; Back; Bioinformatics; Biological Assay; Biological Preservation; Biosensor; Characteristics; Chemotherapy-induced peripheral neuropathy; Clinical; Development; Disease; Disease Progression; Disease model; Distal; Dyes; Enzymes; Fluorescence Resonance Energy Transfer; Future; Genetic; Genetic Screening; Goals; Health; Hot Spot; Human; Image; Imagery; Injury; Ischemic Brain Injury; Knowledge; Lead; Length; Location; MAPK8 gene; Maps; Mediating; Mediator of activation protein; Membrane; Methods; Microscopy; Mitochondria; Molecular; Morphology; Motor; Multiple Sclerosis; Mutate; Nerve Degeneration; Nervous System Trauma; Neurodegenerative Disorders; Neurological Models; Neuronal Injury; Neurons; Parkinson Disease; Pathway interactions; Pattern; Peripheral Nervous System Diseases; Pharmacology; Positioning Attribute; Post-Translational Protein Processing; Process; Proteins; Proteomics; Quality of life; Role; Sensory; Site; Stroke; Swelling; Technology; Testing; Therapeutic Intervention; Time; Toll-like receptors; Traumatic Nerve Injury; Visual; axon injury; axonal degeneration; chronic pain; disabling symptom; genetic approach; genome-wide; in vivo; inhibitor/antagonist; injured; knock-down; molecular targeted therapies; nervous system disorder; new therapeutic target; novel; overexpression; prevent; programs; public health relevance; ratiometric; response; response to injury; sensor; small hairpin RNA; spatiotemporal; tool

 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（由适用提供）：轴突变性（AXD）是多种神经退行性疾病的标志性特征，例如阿尔茨海默氏病和帕金森氏病，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内源性变性程序的存在，该程序在激活后会导致轴突自我毁灭。 AXD在疾病和损伤状态下的宽度出现表明，保护轴突免受退化的治疗干预措施可能会对人类健康和生活质量产生深远的影响。尽管AXD具有临床重要性，但关于将变性程序激活的机制的知识存在关键的差距，以及程序成分的激活与AXD的进展如何相关。最近对SARM1作为该程序的重​​要组成部分的识别表明，进一步了解我们对SARM1的理解可能对我们对AXD机制和进步的更广泛理解具有深远的影响。我已经开发了一种SARM1生物传感器，该生物传感器允许使用比率FRET显微镜在轴突中可视化损伤诱导的SARM1激活。在AIM 1中，该生物传感器将被优化，并用于映射轴突中的损伤诱导的SARM1激活，并通过使用Ca2+成像，线粒体染料和形态分析将其与重要的AXD标记相关联，以确定与SARM1激活相关的AXD特征的发作和进展。生物传感器还将用于测试受伤后近端轴突段是否保存是由于差异引起的 SARM1激活。 AIM 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。 SARM1的固有调节特征将通过在生物传感器中突变的电阻来鉴定，该电阻预测会进行翻译后修饰（PTM），并测试受伤和未加热的轴突中对生物传感器激活和AXD的影响。预计将使用SHRNA和药物抑制剂测试被介导为重要的PTM的酶。同时，将测试已知的AXD调节剂，以确定它们是否通过调节SARM1激活来起作用。最后，将使用生物传感器筛选一个先前由我们实验室鉴定为保护AXD的富集shRNA靶标，以鉴定介导损伤诱导的SARM1激活的新型上游分子成分。这些目标将填补有关AXD程序激活机制及其与AXD进展的关系的关键空白。通过1）定义SARM1激活与AXD进展之间的关系，以及2）确定损伤引起的SARM1激活的机制，该建议将显着增强我们对AXD的基本理解，并可能确定用于治疗新功能疾病和损伤的治疗性干预的新分子靶标。