Tracking the Propagation of Misfolded Tau: A Study of Cellular Uptake and Traffic

描述（由申请人提供）：tau病是一类神经退行性疾病，其特征是人脑中微管相关蛋白tau的病理积累。在许多疾病中，如阿尔茨海默病和额颞叶变性，都观察到Tau聚集性积累。在疾病的早期阶段，tau病理通常局限于大脑的离散和刻板区域。然而，随着疾病的进展，病理改变通常根据特定的解剖模式扩散到神经系统。新出现的证据表明，tau聚集体能够跨细胞传播到共培养的细胞，这与聚集的tau可以作为疾病传播媒介的概念一致。然而，tau聚集物进入细胞并随后进入细胞质诱导天然tau蛋白错误折叠的机制尚不清楚。本提案中概述的实验旨在增加对这些过程的有限理解，并为可能减轻神经退行性疾病负担的治疗进展提供信息。在Aim 1中，将测试硫酸肝素蛋白聚糖（HSPGs）作为tau聚集体结合和摄取的细胞介质的作用。药理HSPG抑制剂，如肝素、肝素酶III、氯酸钠、可溶性聚糖和肝素模拟物将被评估为减少细胞结合和tau聚集体内化。利用缺乏糖胺聚糖合成的突变细胞系以及shRNA敲低技术的遗传学方法将被用于进一步确定HSPGs在tau聚集体内化中的作用。将使用流式细胞术和自动分析显微镜对MTBR聚合体结合和摄取进行定量分析。目的2包括测试外源来源的tau聚集体，一旦通过巨噬细胞作用内化，是否可以逃离泡腔接触细胞质。神经母细胞瘤细胞系将暴露于重组tau原纤维中，随后通过超离心分离。所产生的分数将被探测的tau聚集体使用生化方法的存在。这些研究将有助于描述tau聚集物将天然折叠tau转化为聚集的纤维状形式的细胞环境。

DESCRIPTION (provided by applicant): Tauopathies are a class of neurodegenerative disorders characterized by the pathological accumulation of microtubule-associated protein tau in the human brain. Tau aggregate accumulation is observed in many diseases, such as Alzheimer's disease and Frontotemporal Lobar Degeneration. In the early stage of disease, the tau pathology is often restricted to discrete and stereotyped regions of the brain. With disease progression, however, the pathological changes typically spread through the nervous system according to specific anatomical patterns. Emerging evidence demonstrates that tau aggregates are capable of transcellular spread to co-cultured cells, consistent with the notion that aggregated tau can serve as an agent of disease propagation. However, the mechanisms by which tau aggregates enter cells and subsequently access the cytoplasm to induce misfolding of native tau protein remain unknown. The experiments outlined in this proposal are designed to add to the limited understanding of these processes and to inform therapeutic advances that may reduce the burden of neurodegenerative disease. In Aim 1, the role of heparan sulfate proteoglycans (HSPGs) as a cellular mediator of tau aggregate binding and uptake will be tested. Pharmacological HSPG inhibitors such as heparin, heparinase III, sodium chlorate, soluble glycans and heparan mimetics will be evaluated for reduced cellular binding and internalization of tau aggregates. Genetic approaches exploiting mutant cell lines deficient in glycosaminoglycan synthesis as well as shRNA knockdown technology will then be employed to further determine the role of HSPGs on tau aggregate internalization. Quantification of MTBR aggregate binding and uptake will be assayed using flow cytometry and automated analysis microscopy. Aim 2 consists of testing if exogenously derived tau aggregates, once internalized via macropinocytosis, can escape the vesicular lumen to contact the cytoplasm. Neuroblastoma cell lines will be exposed to recombinant tau fibrils and subsequently fractionated via ultracentrifugation. The resulting fractions will be probed for the presence of tau aggregates using biochemical approaches. These studies will help delineate the cellular environment in which tau aggregates convert natively folded tau into an aggregated, fibrillar form. PUBLIC HEALTH RELEVANCE: Recent evidence suggests that transfer of misfolded tau protein from cell to cell may underlie the progression of many neurodegenerative diseases, such as Alzheimer's disease. A clear understanding of how the misfolded tau enters cells and subsequently corrupts the normal tau protein does not yet exist. This research project will allow us to mechanistically understand these processes so that disease progression may be targeted by new therapies for neurodegeneration.