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

描述(申请人提供)：tau病是一类神经退行性疾病，其特征是微管相关蛋白tau在人脑中病理性积聚。在许多疾病中都观察到tau的聚集，如阿尔茨海默病和额颞叶变性。在疾病的早期阶段，tau的病理通常局限于大脑的离散和陈旧的区域。然而，随着疾病的发展，病理变化通常会根据特定的解剖模式扩散到整个神经系统。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可以作为疾病传播剂的概念一致。然而，tau聚集体进入细胞并随后进入细胞质以诱导天然tau蛋白错误折叠的机制仍不清楚。该提案中概述的实验旨在增加对这些过程的有限了解，并为可能减轻神经退行性疾病负担的治疗进展提供信息。在目标1中，将测试硫酸乙酰肝素蛋白多糖(HSPGs)作为tau聚集结合和摄取的细胞介体的作用。药物HSPG抑制剂，如肝素、肝素酶III、氯酸钠、可溶性多糖和肝素模拟物将被评估以减少细胞结合和tau聚集体的内化。利用缺乏糖胺多聚糖合成的突变细胞系的遗传方法以及shRNA敲除技术将被用来进一步确定HSPGs对tau聚合体内化的作用。MTBR聚集结合和摄取的定量将使用流式细胞术和自动分析显微镜进行检测。目标2包括测试外源性的tau聚集体，一旦通过巨噬细胞吞噬作用内化，是否可以逃脱囊泡腔与细胞质接触。神经母细胞瘤细胞系将暴露在重组tau纤维中，然后通过超速离心法进行分离。将使用生化方法来探测所产生的组分中是否存在tau聚集体。这些研究将有助于描绘tau聚集体将自然折叠的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.