Tau-Spliceosome Interactions in Alzheimer's Disease

信使RNA前体（pre-mRNA）剪接的保真度对于保护神经元转录程序至关重要，剪接因子与神经系统的发育和退化有关。Pre-mRNA剪接是由剪接体介导的，剪接体是一个动态和复杂的核糖核蛋白机器，包括大约200个核心蛋白，5个小核rna和一系列辅助因子。在令人信服的初步研究中，我们通过对人类临床病理队列和果蝇实验的综合分析，暗示剪接体破坏与阿尔茨海默病（AD）的发病机制有关。在患有AD神经病理学的人脑中，剪接体U1小核核糖核蛋白颗粒（snRNP）的许多组分在不溶性皮质组分中异常富集。我们进一步证明了mRNA转录物中广泛的内含子保留与剪接体功能障碍一致，导致全局剪接失败。死后脑组织的免疫组织化学显示神经元中U1 snRNP蛋白的胞质错定位以及与Tau神经原纤维缠结的共聚集，这是AD的典型病理之一。在果蝇中，人类Tau基因的转基因表达诱导年龄依赖性异常定位，并显著降低U1 snRNP成分水平，同时增加编码必需突触蛋白的候选转录本的内含子保留。剪接体基因的功能缺失或功能获得增强或抑制Tau诱导的神经变性，编码核心剪接体蛋白的SmB突变导致不依赖Tau的进行性神经退行性变化。我们假设剪接机制的破坏和由此导致的神经元转录组的紊乱介导了tau诱导的AD神经退行性变。我们将首先（AIM 1）量化500个人脑尸检样本的不溶性蛋白质组中的U1，其他剪接体snRNPs和相关因素，并在一个子集中，我们将额外表征潜在的神经元核耗竭。最有希望的结果将由死后组织的免疫组织化学和免疫荧光研究证实。接下来（AIM 2），利用在果蝇中可用的快速和强大的遗传学，我们将阐明tau剪接体相互作用及其对神经变性的影响。结合生物化学和共聚焦显微镜将用于检查改变的溶解度和剪接体因子的聚集。最后（AIM 3），我们将整合现有的人类皮质转录组数据和从Tau转基因果蝇的大脑中产生的新的参考转录组，以确定剪接错误在神经变性中的因果作用。影响：通过将人脑中的蛋白质组学和转录组学研究与果蝇的靶向功能研究相结合，我们将阐明Tau神经病理负荷如何导致剪接体蛋白的破坏，并剖析导致神经退行性变的因果链。

The fidelity of messenger RNA precursor (pre-mRNA) splicing is critical for safeguarding the neuronal transcriptional program, and splicing factors have been linked to both nervous system development and degeneration. Pre-mRNA splicing is mediated by the spliceosome, a dynamic and complex ribonucleoprotein machine including approximately 200 core proteins, 5 small nuclear RNAs, and an array of accessory factors. In compelling preliminary studies, we implicate spliceosomal disruptions in Alzheimer's disease (AD) pathogenesis through integrative analyses of human clinicopathologic cohorts and experimentation in the fruit fly, Drosophila melanogaster. In human brains with AD neuropathology, numerous components of the spliceosomal U1 small nuclear ribonucleoprotein particle (snRNP) are abnormally enriched within insoluble cortical fractions. We further demonstrate widespread intron retention within mRNA transcripts consistent with spliceosome dysfunction resulting in global splicing failure. Immunohistochemistry in postmortem brain tissue reveals cytoplasmic mislocalization of U1 snRNP proteins in neurons as well as co-aggregation with Tau neurofibrillary tangles, one of the defining pathologies of AD. In Drosophila, transgenic expression of human Tau induces age-dependent aberrant localization and sharply reduced U1 snRNP component levels, concomitant with increased intron retention of candidate transcripts encoding essential synaptic proteins. Loss- or gain-of-function in spliceosomal genes enhance and suppress Tau-induced neurodegeneration, and mutation of SmB, encoding a core spliceosomal protein, causes progressive neurodegenerative changes independent of Tau. We hypothesize that disruptions in the splicing machinery and resulting derangements of the neuronal transcriptome mediate Tau-induced neurodegeneration in AD. We will first (AIM 1) quantify U1, other spliceosomal snRNPs, and related factors in the insoluble proteome of 500 human brain autopsy samples, and in a subset, we will additionally characterize potential neuronal nuclear depletion. The most promising results will be confirmed by immunohistochemistry and immunofluorescence studies of postmortem tissue. Next (AIM 2), leveraging the rapid and powerful genetics available in Drosophila, we will elucidate Tau-spliceosome interactions and their impact on neurodegeneration. A combination of biochemistry and confocal microscopy will be utilized to examine for altered solubility and aggregation of spliceosomal factors. Lastly (AIM 3), we will integrate available human cortical transcriptomic data with a new reference transcriptome generated from the brains of Tau transgenic flies in order to pinpoint splicing errors with causal roles in neurodegeneration. IMPACT: By integrating proteomic and transcriptomic studies in human brains with targeted functional studies in Drosophila we will elucidate how Tau neuropathologic burden leads to disruptions in spliceosomal proteins and dissect the resulting causal chain leading to neurodegeneration.