Tau-Spliceosome Interactions in Alzheimer's Disease

信使RNA前体(Pre-mRNA)剪接的保真度对于保障神经元转录程序至关重要，剪接因子与神经系统发育和退化都有关。剪接体是由剪接体介导的，剪接体是一个动态而复杂的核蛋白机器，包括大约200个核心蛋白、5个小核RNA和一系列辅助因子。在引人注目的初步研究中，我们通过对人类临床病理队列的综合分析和果蝇黑腹果蝇的实验，将剪接体中断与阿尔茨海默病(AD)的发病机制联系起来。在患有AD神经病理的人脑中，剪接体U1小核核糖核蛋白颗粒(SnRNP)的许多成分在不溶的皮质组分中异常丰富。我们进一步证明，广泛的内含子保留在mRNA转录本中，与剪接体功能障碍一致，导致全球剪接失败。死后脑组织的免疫组织化学显示U1SnRNP蛋白在神经元中的胞浆错误定位以及与Tau神经原纤维缠结的共聚集，Tau神经原纤维缠结是AD的定义病理之一。在果蝇中，人Tau的转基因表达诱导了年龄相关的异常定位和U1 SnRNP组分水平的急剧下降，同时伴随着编码必要突触蛋白的候选转录本的内含子保留率增加。剪接体基因的功能丧失或获得增强和抑制了Tau诱导的神经变性，而编码核心剪接体蛋白的SMB突变导致了不依赖Tau的进行性神经退行性改变。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神经变性。我们将首先(目标1)对500个人脑尸检样本的不可溶蛋白质组中的U1、其他剪接体SnRNPs和相关因素进行量化，在一个子集中，我们还将描述潜在的神经元核耗竭。最有希望的结果将通过死后组织的免疫组织化学和免疫荧光研究来证实。下一步(目标2)，利用果蝇可获得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退行性变的影响。生物化学和共聚焦显微镜的结合将被用来检查剪接体因子的改变的溶解性和聚集性。最后(目标3)，我们将把现有的人类皮质转录数据与从Tau转基因果蝇的大脑中产生的新的参考转录组进行整合，以准确地定位剪接错误与神经退化的因果作用。影响：通过将人脑的蛋白质组学和转录学研究与果蝇的靶向功能研究相结合，我们将阐明Tau神经病理负担如何导致剪接体蛋白的破坏，并剖析导致神经退化的因果链。

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.