Effects of TDP-43 Pathology on Innate Antiviral Mechanisms in Neurodegenerative Disease

项目总结/摘要 反式激活反应区DNA结合蛋白43（TDP-43）的调节异常与许多 神经退行性疾病，包括额颞叶痴呆、肌萎缩侧索硬化和阿尔茨海默氏症 疾病（AD）。TDP-43具有与其RNA结合基序相关的多种功能，包括调节 转录、剪接和RNA转运。沿着这些作用，TDP-43改变干扰素（IFN）的表达， 抗病毒反应所必需的相关和其他免疫基因。病毒性病原体与 TDP-43是双向的，因为暴露于模拟病毒病原体的poly（I：C），可以促进亚细胞 TDP-43的错误定位。病毒病原体，如TDP-43失调，与AD和其他痴呆症有关; AD与病毒病原体如单纯疱疹病毒1型（HSV-1）的存在增加有关， 干扰素相关信号和神经免疫级联反应的改变。我们的实验室发现，像神经元TDP-43一样， 星形胶质细胞TDP-43可错误定位于AD的细胞质。小鼠星形胶质细胞TDP-43的调节异常 模型引起神经缺陷和抗病毒和IFN诱导因子的细胞自主变化。此外，本发明还 TDP-43失调增加星形胶质细胞对HSV-1的易感性。星形胶质细胞对HSV-1的易感性相关 通过阻断人TDP-43结合RNA的能力，降低了人TDP-43的过表达。 以前的研究也表明TDP-43上的RNA结合结构域是其与其他疾病相关的必需的。 方面的影响.基于这些证据，我将检验痴呆相关TDP-43功能障碍影响 通过改变TDP-43与宿主RNA的结合，增加神经对HSV-1的易感性， 在异常宿主抗病毒和免疫基因表达和受损的先天抗病毒信号传导中。我建议使用 各种细胞和分子技术，以检查体外（目标1.1）和体内（目标1.2）对 在TDP-43变体的细胞特异性表达后的HSV-1，所述TDP-43变体维持核定位、错误定位 不能与RNA结合，或者两者都不能。我还将确定细胞特异性分子机制， 通过单细胞RNA测序（Aim 2.1）促进抗病毒途径的差异，并进行靶向 选择性剪接（ScISOr-Seq）、转座因子表达（TE转录本）和蛋白质分析 蛋白质印迹法（Western blotting）。最后，我将研究感兴趣的差异基因的生理功能 目标2.1中使用遗传学和药理学方法鉴定的。揭示了 将TDP-43失调与抗病毒途径联系起来，病毒易感性可能定义新的病理生物学 预防神经退行性疾病发作和进展的机制和治疗靶点。

Project Summary/Abstract Dysregulation of transactivating response region DNA-binding protein-43 (TDP-43) has been linked to many neurodegenerative diseases, including frontotemporal dementia, amyotrophic lateral sclerosis, and Alzheimer’s disease (AD). TDP-43 has a variety of functions linked to its RNA-binding motif, including regulation of transcription, splicing, and RNA transport. Along with these effects, TDP-43 alters expression of interferon (IFN)- related and other immune genes essential for antiviral responses. The relationship between viral pathogens and TDP-43 is bidirectional, as exposure to poly(I:C), which simulates viral pathogens, can promote subcellular mislocalization of TDP-43. Viral pathogens, like TDP-43 dysregulation, are linked to AD and other dementias; AD has been associated with increased presence of viral pathogens, like herpes simplex virus 1 (HSV-1), and altered IFN-related signaling and neuroimmune cascades. Our laboratory found that, like neuronal TDP-43, astrocytic TDP-43 can be mislocalized to the cytoplasm in AD. Dysregulation of astrocytic TDP-43 in mouse models caused neural deficits and cell-autonomous changes in antiviral and IFN-inducible factors. Further, dysregulated TDP-43 increased astrocytic susceptibility to HSV-1. Astrocytic susceptibility to HSV-1 associated with overexpression of human TDP-43 was reduced by blocking the ability of human TDP-43 to bind RNA. Previous studies also show that the RNA-binding domain on TDP-43 is necessary for its other disease-linked effects. Based on this evidence, I will test the hypothesis that dementia-related TDP-43 dysfunction affects antiviral pathways and increases neural susceptibility to HSV-1 by altering TDP-43 binding to host RNA, resulting in aberrant host antiviral and immune gene expression and impaired innate antiviral signaling. I propose to use a variety of cellular and molecular techniques to examine in vitro (Aim 1.1) and in vivo (Aim 1.2) susceptibility to HSV-1 following cell-specific expression of TDP-43 variants that either maintain nuclear localization, mislocalize to the cytoplasm, cannot bind to RNA, or both. I will also determine cell-specific molecular mechanisms that promote differences in antiviral pathways via single-cell RNA sequencing (Aim 2.1), and conduct targeted analysis of alternative splicing (ScISOr-Seq), transposable element expression (TEtranscripts), and protein levels (Western blotting). Finally, I will examine the physiological functions of differential genes of interest identified in Aim 2.1 using genetic and pharmacological approaches. Uncovering the mechanistic links that connect TDP-43 dysregulation to antiviral pathways and viral susceptibility may define new pathobiological mechanisms and therapeutic targets to prevent neurodegenerative disease onset and progression.