Defining the pathogenic relationship of TDP-43 inclusions and cytoplasmic double stranded RNA in AD and FTD

摘要 TDP-43（TAR DNA结合蛋白43）的细胞质内含物在许多阿尔茨海默氏症患者中发现。 疾病（AD）。TDP-43夹杂物的存在预示着在控制了 其他病理。TDP-43胞浆包涵体也存在于> 45%的额颞叶病例中。 痴呆（FTD），包括与C9 ORF 72重复扩增相关的所有病例，这是最常见的 导致FTD的基因突变。假设TDP-43的细胞质聚集体隔离了 蛋白质从生理目标，从而模仿功能丧失突变的TARDBP，也导致 FTD。TDP-43在人细胞、小鼠、果蝇和C. elegans导致 能够形成双链RNA（dsRNA）的内源性反向重复序列的去阻遏。 在初步研究中，我们发现AD和FTD人脑中的TDP-43细胞质 包涵体具有更多的胞质dsRNA（cdsRNA），其是I型干扰素（IFN-I）信号传导的已知激活剂。 我们现在建议通过以下方法研究TDP-43细胞质内含物和cdsRNA之间的关系： 将我们的分析扩展到其他AD和FTD大脑。我们将使用一种方法来实现这一点， 多重显微镜（基于组织的循环免疫荧光; t-CyCIF）由我们的团队开发， 最近扩展到对尸检大脑的分析。在目标1中，我们将使用t-CyCIF来确定是否 TDP-43包涵体和cdsRNA在同一细胞中是一致的，并且它们是否与其他细胞相关， 疾病特征。我们将描述星形胶质细胞，小胶质细胞和神经元的炎症特征， 具有TDP-43内含物和cdsRNA的细胞附近。在目标2中，将从人AD中分离cdsRNA 和FTD脑区域用于无偏的下一代测序以定义每个区域的dsRNAome。dsRNA 将使用新的方法将序列映射到基因组，以确定哪些dsRNA来自 内源性重复序列、基因组损伤和/或病毒序列，特别是内源性重复序列的成员。 疱疹家族。在目标3中，我们将使用以下方法研究连接TDP-43细胞质内含物与cdsRNA的途径： 从致病性TDP-43突变患者以及AD和FTD患者中培养的人iPS源性神经元 目标1中分析的患者。我们还将阐明TDP-43 / cdsRNA介导的细胞凋亡的机制。 在表达TDP-43突变和基因组编码的cdsRNA的两种小鼠模型中， 并使用FDA批准的JAK抑制剂探测IFN-1活化在神经元死亡中的作用。通过定义 胞质TDP-43、dsRNA包涵体、相关炎症表型和 神经元死亡，我们的研究将提供新的见解触发TDP-43夹杂物的神经炎症， AD和FTD。我们还旨在提名炎症特征来识别患有cdsRNA的活体患者- 在药物再利用临床试验中可能受益于JAK抑制剂的AD和FTD相关病理患者。

Abstract Cytoplasmic inclusions of TDP-43 (TAR DNA-binding protein 43) are found in many patients with Alzheimer's disease (AD). The presence of TDP-43 inclusions predicts a steeper cognitive decline after controlling for other pathologies. TDP-43 cytoplasmic inclusions are also present in > 45% of cases of frontotemporal dementia (FTD), including all cases associated with C9ORF72 repeat expansion, which is the most common genetic mutation causing of FTD. Cytoplasmic aggregates of TDP-43 are hypothesized to sequester the protein from physiological targets, thereby mimicking loss of function mutations in TARDBP that also lead to FTD. Loss of function perturbations of TDP-43 in human cells, mice, Drosophila and C. elegans lead to derepression of endogenous inverted repeat sequences capable of forming double stranded RNA (dsRNA). In preliminary studies, we have found that both AD and FTD human brains with TDP-43 cytoplasmic inclusions have more cytoplasmic dsRNA (cdsRNA), a known activator of type I interferon (IFN-I) signaling. We now propose to investigate the relationship between TDP-43 cytoplasmic inclusions and cdsRNA by extending our analysis to additional AD and FTD brains. We will accomplish this using a method for highly multiplexed microscopy (tissue-based cyclic immunofluorescence; t-CyCIF) developed by our team and recently extended to the analysis of autopsied brains. In Aim 1 we will use t-CyCIF to determine whether TDP-43 inclusions and cdsRNA are coincident in the same cells, and whether they associated with other disease features. We will characterize the inflammatory signatures of astrocytes, microglia, and neurons in the vicinity of cells with TDP-43 inclusions and cdsRNAs. In Aim 2 cdsRNA will be isolated from human AD and FTD brain regions for unbiased next generation sequencing to define each region's dsRNAome. dsRNA sequences will be mapped to the genome using novel methods to determine which dsRNAs arise from endogenous repeat sequences, genomic lesions, and / or viral sequences, particularly members of the Herpes family. In Aim 3 we will study the pathway linking TDP-43 cytoplasmic inclusions to cdsRNA using cultured human iPS- derived neurons from patients with pathogenic TDP-43 mutations and from AD and FTD patients analyzed in Aim 1. We will also elucidate mechanisms of TDP-43 / cdsRNA- mediated neurodegeneration in two mouse models that express TDP-43 mutations and genomically-encoded cdsRNA, and probe the role of IFN-I activation in neuronal death using an FDA-approved JAK inhibitor. By defining the relationship between cytoplasmic TDP-43, dsRNA inclusions, associated inflammatory phenotypes, and neuronal death, our studies will provide new insight into triggers of neuroinflammation of TDP-43 inclusions in AD and FTD. We also aim to nominate inflammatory signatures to identify living patients with cdsRNA- associated AD and FTD pathology who might benefit from a JAK inhibitor in a drug repurposing clinical trial.