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

追踪错误折叠 Tau 蛋白的传播：细胞摄取和运输的研究

• 批准号: 8453718
• 负责人: Brandon Blake Holmes
• 金额: $ 2.85万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8453718
• 关键词: Alzheimer' s Disease; Binding; Biochemical; Biological Assay; Brain; Brain region; Cell Fractionation; Cell Line; Cells; Coculture Techniques; Cultured Cells; Cytoplasm; Cytosol; Data; Disease; Disease Progression; Dot Immunoblotting; Environment; Event; Extracellular Space; Flow Cytometry; Frontotemporal Lobar Degenerations; Genetic; Glycosaminoglycans; Goals; Heparan Sulfate Proteoglycan; Heparin; Heparitin Sulfate; Human; Inherited; Lead; Mediating; Mediator of activation protein; Methods; Microscopy; Microtubules; Mus; Mutation; Nerve Degeneration; Nervous system structure; Neuraxis; Neurodegenerative Disorders; Neurons; Pathogenesis; Pathology; Pathway interactions; Pattern; Polysaccharides; Prions; Process; Publications; Recombinants; Research; Research Project Grants; Role; Seeds; Staging; Stereotyping; Tauopathies; Technology; Testing; Therapeutic; Ultracentrifugation; Western Blotting; Work; design; heparinase III; inhibitor/antagonist; mimetics; monomer; mutant; neuroblastoma cell; protein folding; protein misfolding; research study; retinal rods; small hairpin RNA; sodium chlorate; sulfation; tau Proteins; tau aggregation; trafficking; transmission process; uptake

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.

描述（由申请人提供）：tauopathies是一类神经退行性疾病，其特征是微管相关蛋白Tau在人脑中的病理积累。在许多疾病中观察到tau骨料积累，例如阿尔茨海默氏病和额颞叶变性。在疾病的早期，TAU病理通常仅限于大脑的离散和定型区域。然而，随着疾病进展，病理变化通常根据特定的解剖学模式通过神经系统传播。新兴的证据表明，tau聚集体能够跨细胞扩散到共培养的细胞上，这与聚集的tau可以作为疾病传播的药物的概念一致。但是，tau聚集物进入细胞并随后进入细胞质以诱导天然tau蛋白折叠的机制尚不清楚。该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，将测试乙酰硫酸盐蛋白聚糖（HSPG）作为TAU聚集物结合和摄取的细胞介质的作用。将评估药理学HSPG抑制剂，例如肝素，肝素III，氯酸钠，可溶性聚糖和乙酰肝素模拟物，以减少TAU聚集体的细胞结合和内在化。然后将采用遗传方法利用糖胺聚糖合成中缺乏突变细胞系以及SHRNA敲低技术，以进一步确定HSPG在TAU骨料内部化中的作用。使用流式细胞术和自动分析显微镜测定MTBR聚集结合和摄取的定量。 AIM 2包括测试是否通过大型细胞增多症进行内化的外源衍生的tau聚集体可以逃脱囊泡腔以接触细胞质。神经母细胞瘤细胞系将暴露于重组tau原纤维，然后通过超速离心分离。将使用生化方法探测所得的级分。这些研究将有助于描绘tau聚集体将折叠tau转化为聚集的纤维状形式的细胞环境。