利用生命病毒介导的人神经元转导研究头塔聚集的体外模型
Summary
该协议详细介绍了用慢病毒结构编码为突变体人类 tau 转换人类神经元培养的过程。诱导的区域性显示 tau 聚合和相关的病理。
Abstract
蛋白 tau 的异常聚集是病因参与了一些神经退行性疾病, 包括阿尔茨海默氏症 (AD)。尽管小鼠的病状模型为研究聚集 tau 的神经毒性机制提供了宝贵的资源, 但越来越明显的是, 由于神经生理学的物种间差异, 小鼠的大脑不适合模拟人类的状况。细胞培养方法的进步使人类神经元培养物可用于体外实验, 并有助于神经疗法的发展。然而, 尽管人类神经元细胞培养的适应性, 体外模型的人类程度病还没有得到广泛的应用。该协议描述了一种细胞模型的 tau 聚集, 其中人类神经元被移植与生命病毒衍生的载体, 代码的病原性突变 tau 融合到一个黄色荧光蛋白 (YFP) 记者。诱导培养产生对硫代黄素呈阳性染色的 tau 集合体, 并显示出神经毒性标志物, 如轴突长度缩短和溶酶体体积增加。这个程序可能是一个有用的和具有成本效益的模型, 研究人类的泰氏病。
Introduction
微管相关蛋白 tau 的病理聚集是许多神经退行性疾病的一个决定性特征, 包括 AD、额叶痴呆 (FTD)、拾取病和进行性核上麻痹 (PSP)1.在非病变状态下, tau 结合并稳定神经元轴突2 中的微管丝。然而, 与疾病相关的 tau 高磷酸化促进 tau 聚集、与微管的离解和神经元毒性3。聚集 tau 的毒性作用可能包括胆碱能4和谷氨酸受体5的异常激活, 导致细胞内钙的失调, 并最终导致细胞死亡。在动物模型中, 减少脑 tau 改善了 AD小鼠 6和重复轻度创伤性脑损伤的小鼠模型7的病理。
越来越多的证据表明, 鼠源 tau 的结构和结合亲和力与人类衍生的 tau 不同, 并且鼠标 tau 不适合模拟人类的 taudiies8。然而, 人类细胞病模型并没有在商业上广泛提供。这项工作的总体目标是描述一个体外模型的 tau 聚集, 其中人体神经元被转化与生命病毒衍生的载体包含突变的人 tau 构造9。Tau 集料导致 lentiviral 构造编码的 TAU 重复域窝藏 P301L 和 V337M 突变融合到 YFP 记者 (Tau-rdllm-yfp), 而控制构造代码的野生类型 (Wt) tau 重复域融合到 YFP 记者 (Tau-Wt-YFP)。用这种方法转化的神经元培养物表达的 tau 大约是非转换培养体的9倍。虽然 tau 表达的多度在 Tau-rdlm-yfp-和 To-wt-yfp 移植细胞之间大致相等, 但只有用 To-rdlm-yfp 显示聚合而传导的神经元。用 To-rdlm-yfp 染色对硫代黄素进行了积极的培养, 并在轴突长度和突触密度上表现出减少。因此, 这种细胞模型可能是研究牛生聚集的有用工具。
Protocol
Representative Results
Discussion
该协议描述了一个体外模型的人类神经病, 表现为银-静态阳性聚集物和硫代黄素阳性神经纤维缠结 (Nft) 的生成。此外, 转染细胞表现出诱导的疾病, 如形态缺陷, 减少突触发生, 并增加溶酶体体积。该方案的主要优点是, 它提供了一个可获得的和具有成本效益的神经元病模型, 可用于药物筛选研究, 以及牛毒性分析。这种模型满足了神经退行性研究的物质需求, 因为人类的神经病细胞系还没有得到广?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢阿尔伯特·爱因斯坦医学院的彼得·戴维斯博士提供 PHF-1 和 CP13 抗体, 并感谢西南德克萨斯大学的 Marc Diamon 博士提供 tau 结构。这项工作得到了阿尔茨海默氏症协会 (NIRG-14-332164) 向 s. h. y. 提供的赠款和来自加州再生医学研究所 (TB1-01193) 到 p. r. 的资助。
Materials
| 10 cm culture dishes | Thermofisher | 12556002 | |
| 15 ml tubes | Biopioneer | CNT-15 | |
| 16% paraformaldehyde | Thermofisher | 50-980-487 | |
| 24 well culture plates | Thermofisher | 930186 | |
| 50ml tubes | Biopioneer | CNT-50 | |
| 70% ethanol in spray bottle | Various sources | NA | |
| B27 supplement | Thermofisher | 17504044 | |
| Basement membrane matrix (Matrigel) | Corning | 356231 | |
| Basic FGF | Biopioneer | HRP-0011 | |
| Bovine serum albumin | Sigma | A7906 | |
| Cell culture incubator | Various sources | NA | |
| Centrifuge | Various sources | NA | |
| DMEM-F12 culture media with glutamine | Thermofisher | 10565042 | |
| Ethanol (50% concentration or higher) | Various sources | NA | |
| Flourescently labeled secondary antibodies | Various Sources, experiment dependent | NA | |
| Fluorescent microscope | Various sources | NA | |
| Glass coverslips | Thermofisher | 1254581 | |
| Glass slides | Thermofisher | 12-550-15 | |
| Human neural stem cells | Various sources | NA | |
| Lentiviral vectors | Various sources | custom order | |
| Mounting media | Thermofisher | P36934 | |
| N2 supplement | Thermofisher | 17502048 | |
| Penicillin-Streptomycin | Thermofisher | 15140122 | |
| Phosphate buffered saline | Thermofisher | 14190250 | |
| Primary antibodies | Various Sources, experiment dependent | NA | |
| Rocking or rotating platform | Various sources | NA | |
| Sterile cell culture hood | Various sources | NA | |
| Thioflavin S | Sigma | T1892-25G | |
| Triton X-100 | Thermofisher | BP151-100 | |
| Water bath | Various sources | NA |
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