The Role of Neuronal DNA Double Strand Breaks in Neuroinflammation

项目摘要 对细胞遗传物质的损伤可导致组织功能障碍。神经系统尤其 易受基因组不稳定性和神经元DNA损伤的累积，特别是DNA双链 在阿尔茨海默病患者中观察到的早期病理学事件是DSB。CK-p25小鼠 在严重神经变性的模型中，DSB的积累先于所有其他DSB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， 神经毒性。已知病理水平的DSB诱导I型干扰素和抗病毒药物。 反应目前尚不清楚DSB是否启动了神经元中的先天免疫信号，以及这是否在标志性免疫反应中起作用。 与神经退行性疾病相关的神经炎症级联反应。本提案的目的是测试 标志性的神经炎性活动是由神经元中的DSB诱导的先天免疫的结果引发的。 我们首先建立了DSB足以驱动I型干扰素和抗病毒基因表达， 神经元培养，这是依赖于Stat 1活性，转录因子驱动的抗病毒药物， Jak/Stat信号通路。我们使用荧光激活的细胞核分选法分离CK-p25中的神经元， 皮质γ H2 AX免疫反应性增加，这是DSB的一个可靠标志物。转录组学分析显示 “γ H2 AX阳性”神经元富含先天免疫和抗病毒途径。我们假设这些 γ H2 AX阳性神经元使用先天免疫信号传导在神经胶质细胞类型中启动神经炎症反应。 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显示，小胶质细胞具有 在该时间点最强的神经胶质炎症反应。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窘迫信号。 本提案的第一个目的是检验DSB介导的I型干扰素和抗病毒药物 来自神经元的活动发出小胶质细胞炎症的信号，并且这依赖于神经元Jak/Stat 发信号。这一目的将通过在原代神经元培养物中进行DNA DSB诱导，然后进行共诱导来实现。 与小胶质细胞培养。该提案的第二个目标将确定是否先天免疫信号来源于 γ H2 AX阳性神经元激活体内小胶质细胞中的炎症信号。这一目标将通过以下方式实现： 病毒敲低CK-p25前脑神经元中的Stat 1。我将利用细胞核分选来评估细胞类型特异性 炎症基因在γ H2 AX阳性神经元和小胶质细胞中的表达。要验证 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。这 一项提案将评估DSB诱导的先天免疫信号在神经炎症启动中的影响 与神经变性相关，可能为治疗干预提供新的途径。

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.