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.

项目概要细胞遗传物质的损伤尤其会导致组织功能障碍。容易受到基因组不稳定的影响，以及神经元 DNA 损伤的积累，特别是 DNA 双链断裂（DSB）是在 CK-p25 小鼠中观察到的阿尔茨海默病患者的早期病理事件。在严重神经退行性变模型中，DSB 的积累先于所有其他物质的出现病理学，包括神经元损失和认知能力下降，这表明 DSB 是一种起始病变。已知 DSB 的病理水平会诱导 I 型干扰素和抗病毒药物的产生。目前尚不清楚 DSB 是否在神经元中启动先天免疫信号传导，以及这是否在该标志中发挥作用。该提案的目的是测试是否与神经退行性疾病相关。标志性的神经炎症活动是由 DSB 诱导的神经元先天免疫引起的。我们首先建立的 DSB 足以在原代细胞中驱动 I 型干扰素和抗病毒基因表达神经培养物，这取决于 Stat1 活性，这是一种驱动抗病毒药物的转录因子我们使用荧光激活的细胞核分选来分离 CK-p25 中的神经元。 γH2AX 的免疫反应性增加，这是 DSB 的有力标记。 “γH2AX 阳性”神经元富含先天免疫和抗病毒途径。 γH2AX 阳性神经元利用先天免疫信号传导来启动神经胶质细胞类型的神经炎症反应。对星形胶质细胞、小胶质细胞和少突胶质细胞的细胞类型特异性转录分析表明，小胶质细胞具有在这个时间点神经胶质炎症反应最强，这表明含有许多 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应激信号。该提案的第一个目标是检验 DSB 介导的 I 型干扰素和抗病毒药物的假设神经元的活动发出小胶质细胞炎症信号，这取决于神经元 Jak/Stat 这一目标将通过在原代神经培养物中诱导 DNA DSB 来实现，然后进行共-信号传导。该提案的第二个目标是确定先天免疫信号是否源于小胶质细胞。 γH2AX 阳性神经元激活体内小胶质细胞的炎症信号传导，这一目标将通过以下方式实现。 CK-p25 前脑神经元中 Stat1 的病毒敲低我将利用细胞核分选来评估细胞类型特异性。 γH2AX 阳性神经元和小胶质细胞中炎症基因的表达，以验证炎症基因的变化。小胶质细胞炎症反应，我将使用免疫组织化学来评估小胶质细胞形态。该提案将评估 DSB 诱导的先天免疫信号在神经炎症引发中的影响与神经退行性变相关，可能为治疗干预提供新途径。