The Role of Neuronal DNA Double Strand Breaks in Neuroinflammation

神经元 DNA 双链断裂在神经炎症中的作用

基本信息

批准号：
10159750
负责人：
Gwyneth Margaret Welch
金额：
$ 4.6万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10159750
关键词：
Address Age Aging Alzheimer&apos s Disease Alzheimer&apos s disease patient Antiviral Agents Antiviral Response Appearance Astrocytes Autopsy Behavioral Bioinformatics Biological Brain Capsid Cause of Death Cell Nucleus Cells Coculture Techniques DNA Damage DNA Double Strand Break Dependence Development Disease Distress Double Strand Break Repair Etoposide Event Fluorescence Functional disorder Future Gamma-H2AX Gene Expression Gene Expression Profile Gene Silencing Genes Genetic Materials Genetic Transcription Genomic Instability Goals HDAC1 gene Immune Immune signaling Immunohistochemistry Impaired cognition In Vitro Individual Inflammation Inflammatory Inflammatory Response Interferon Type I Learning Lesion Link Mediating Memory Microglia Morphology Mus Natural Immunity Nerve Degeneration Nervous system structure Neurodegenerative Disorders Neuroglia Neurons Oligodendroglia Pathologic Pathology Pathway interactions Patients Peripheral Pharmacology Pharmacotherapy Phenocopy Phenotype Play Prosencephalon Protein Fragment Proxy Reporting Research Role SIRT1 gene STAT1 gene Sampling Science Scientist Signal Pathway Signal Transduction Sorting - Cell Movement Symptoms System Testing Therapeutic Intervention Time Tissues Topoisomerase-II Inhibitor Training United States Viral aging brain calmodulin-dependent protein kinase II cell type effective therapy experience experimental study human old age (65+)immunoreactivity improved in vivo inhibitor/antagonist knock-down mild cognitive impairment mouse model neuroinflammation neuron loss neurotoxic neurotoxicity new therapeutic target novel overexpression programs response skills small hairpin RNA stem therapeutic development training opportunity transcription factor transcriptome sequencing transcriptomics

项目摘要

Project Summary Damage to the genetic material of cells can result in tissue dysfunction. The nervous system is particularly vulnerable to genomic instability, and the accumulation of neuronal DNA damage, particularly DNA double strand breaks (DSBs), is an early pathological event observed in Alzheimer’s Disease patients. In the CK-p25 mouse model of severe neurodegeneration, the accumulation of DSBs precedes the appearance of all other pathologies, including neuronal loss and cognitive decline. This suggests DSBs are an initiating lesion of neurotoxicity in these mice. Pathological levels of DSBs are known to induce type I interferon and antiviral response. It is unclear if DSBs initiate innate immune signaling in neurons, and if this plays a role in the hallmark neuroinflammatory cascade associated with neurodegenerative disease. The goal of this proposal is to test if hallmark neuroinflammatory activity is initiated by consequence of DSB-induced innate immunity in neurons. We first established DSBs are sufficient to drive type I interferon and antiviral gene expression in primary neuronal culture, and that this is dependent upon Stat1 activity, a transcription factor that drives the antiviral Jak/Stat signaling pathway. We used fluorescence-activated nuclei sorting to isolate neurons in the CK-p25 cortex with increased immunoreactivity for γH2AX, a robust marker for DSBs. Transcriptomic analysis revealed “γH2AX-positive” neurons are enriched for innate immune and antiviral pathways. We hypothesized these γH2AX-positive neurons use innate immune signaling to initiate a neuroinflammatory response in glial cell types. Cell type specific transcriptional analysis of astrocytes, microglia, and oligodendrocytes revealed microglia have the strongest glial inflammatory response at this time-point. This suggests neurons harboring many DSBs may be signaling innate immune distress signals to microglia during the early stages of neurodegenerative disease. The first aim of this proposal will test the hypothesis that DSB-mediated type I interferon and antiviral activity from neurons signals microglial inflammation, and that this is dependent upon neuronal Jak/Stat signaling. This aim will be achieved through DNA DSB induction in primary neuronal culture, followed by co- culture with microglia. The second aim of this proposal will determine if innate immune signaling stemming from γH2AX-positive neurons activate inflammatory signaling in microglia in vivo. This aim will be achieved through viral knock down of Stat1 in neurons in the CK-p25 forebrain. I will utilize nuclei sorting to assess cell type specific expression of inflammatory genes in both γH2AX-positive neurons and microglia. To validate changes in the microglial inflammatory response, I will use immunohistochemistry to assess microglial morphology. This proposal will evaluate the impact of DSB-induced innate immune signaling in the initiation of neuroinflammation associated with neurodegeneration, potentially providing new avenues for therapeutic intervention.

项目摘要细胞遗传物质的损害会导致组织功能障碍。神经系统特别是容易受到基因组不稳定性的影响，以及神经元DNA损伤的积累，尤其是DNA双链断裂（DSB）是在阿尔茨海默氏病患者中观察到的早期病理事件。在CK-P25鼠标中严重神经变性的模型，DSB的积累在所有其他的外观之前病理，包括神经元丧失和认知能力下降。这表明DSB是启动的病变这些小鼠的神经毒性。已知DSB的病理水平诱导I型干扰素和抗病毒回复。目前尚不清楚DSB是否启动神经元中的先天免疫信号传导，并且这是否在标志中发挥作用与神经退行性疾病相关的神经炎症级联。该建议的目的是测试是否标志性神经炎性活性是由于DSB诱导的神经元的先天免疫而引发的。我们首先建立的DSB足以驱动I型干扰素和抗病毒基因表达神经元培养 jak/stat信号通路。我们使用荧光激活的核分子分离CK-P25中的神经元皮质具有γH2AX免疫反应性的增加，γH2AX是DSB的可靠标记。转录组分析显示 “γH2AX阳性”神经元富含先天免疫和抗病毒途径。我们假设这些 γH2AX阳性神经元使用先天免疫信号传导来启动神经胶质细胞类型中的神经炎症反应。细胞类型的星形胶质细胞，小胶质细胞和少突胶质细胞的转录分析表明小胶质细胞具有在此时间点，强烈的神经胶质炎症反应。这表明携带许多DSB的神经元可能在神经退行性疾病的早期阶段向小胶质细胞发出先天免疫信号。该提案的第一个目的是检验DSB介导的I型干扰素和抗病毒的假设神经元的活性信号小胶质细胞注射，这取决于神经元JAK/STAT 信号。该目标将通过原发性神经元培养中的DNA DSB诱导来实现，然后共同小胶质细胞培养。该提案的第二个目的将确定先天免疫信号是否源于 γH2AX阳性神经元在体内激活小胶质细胞中的炎症信号传导。这个目标将通过 CK-P25前脑神经元中Stat1的病毒敲低。我将利用核分子来评估特定细胞类型的特定 γH2AX阳性神经元和小胶质细胞中炎症基因的表达。验证变化小胶质细胞炎症反应，我将使用免疫组织化学评估小胶质细胞形态。这提案将评估DSB诱导的先天免疫信号传导在神经炎症的主动性中的影响与神经变性有关，有可能为治疗干预提供新的途径。