Name: an in vitro model for studying tau aggregation using lentiviral-mediated transduction of human neurons
Uploaded: 2019-05-23T14:00:00
Description: Watch this Scientific Journal Video about An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons at JoVE.com

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。

1. 介质和试剂的制备
<ol><li>在4°C 时解冻培养板的基底膜基质涂层 (不允许基底膜基质升温或固化)。制作1毫升的等价物, 并将其存放在-20°c 或-70°c。</li>
	<li>在 10Μg/ml 的无菌磷酸盐缓冲盐水 (PBS) 中重新构建碱性成纤维细胞生长因子 (bfgf), 并制成 10μl aliquots。将它们存放在4°c。</li>
	<li>在一个新的, 未开封的, 500 毫升瓶 DMM/F12 与谷氨酰胺, 加入 B27 (10 毫升), N2 (5 毫升), 青霉素链霉素 (5 毫升)。将这?...

<ol>
	<li>Rojas, J. C., Boxer, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegenerative+disease+in+2015:+Targeting+tauopathies+for+therapeutic+translation.">Neurodegenerative disease in 2015: Targeting tauopathies for therapeutic translation.</a> Nature Reviews Neurology. 12 (2), 74-76 (2016).</li><li>Kadavath, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+stabilizes+microtubules+by+binding+at+the+interface+between+tubulin+heterodimers.">Tau stabilizes microtubules by binding at the interface between tubulin heterodimers.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (24), 7501-7506 (2015).</li><li>Wang, Y., Mandelkow, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+in+physiology+and+pathology.">Tau in physiology and pathology.</a> Nature Reviews Neurology. 17 (1), 5-21 (2016).</li><li>Gomez-Ramos, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+and+consequences+of+muscarinic+receptor+activation+by+tau+protein.">Characteristics and consequences of muscarinic receptor activation by tau protein.</a> European Neuropsychopharmacology. 19 (10), 708-717 (2009).</li><li>Warmus, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau-mediated+NMDA+receptor+impairment+underlies+dysfunction+of+a+selectively+vulnerable+network+in+a+mouse+model+of+frontotemporal+dementia.">Tau-mediated NMDA receptor impairment underlies dysfunction of a selectively vulnerable network in a mouse model of frontotemporal dementia.</a> Journal of Neuroscience. 34 (49), 16482-16495 (2014).</li><li>DeVos, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+reduction+in+the+presence+of+amyloid-beta+prevents+tau+pathology+and+neuronal+death+in+vivo.">Tau reduction in the presence of amyloid-beta prevents tau pathology and neuronal death in vivo.</a> Brain. 141 (7), 2194-2212 (2018).</li><li>Cheng, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+reduction+diminishes+spatial+learning+and+memory+deficits+after+mild+repetitive+traumatic+brain+injury+in+mice.">Tau reduction diminishes spatial learning and memory deficits after mild repetitive traumatic brain injury in mice.</a> PLOS ONE. 9 (12), e115765 (2014).</li><li>Ando, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+human+mutant+tau+aggregation+by+knocking+out+murine+tau+in+a+transgenic+mouse+model.">Accelerated human mutant tau aggregation by knocking out murine tau in a transgenic mouse model.</a> The American Journal of Pathology. 178 (2), 803-816 (2011).</li><li>Reilly, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+human+neuronal+tau+model+exhibiting+neurofibrillary+tangles+and+transcellular+propagation.">Novel human neuronal tau model exhibiting neurofibrillary tangles and transcellular propagation.</a> Neurobiology of Disease. 106, 222-234 (2017).</li><li>Kfoury, N., Holmes, B. B., Jiang, H., Holtzman, D. M., Diamond, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-cellular+Propagation+of+Tau+Aggregation+by+Fibrillar+Species.">Trans-cellular Propagation of Tau Aggregation by Fibrillar Species.</a> The Journal of Biological Chemistry. 287 (23), 19440-19451 (2012).</li><li>Yuan, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-surface+marker+signatures+for+the+isolation+of+neural+stem+cells,+glia+and+neurons+derived+from+human+pluripotent+stem+cells.">Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells.</a> PLOS ONE. 6 (3), e17540 (2011).</li><li>Marchenko, S., Flanagan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passaging+human+neural+stem+cells.">Passaging human neural stem cells.</a> Journal of Visualized Experiments. (7), e263 (2007).</li><li>Lathuiliere, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motifs+in+the+tau+protein+that+control+binding+to+microtubules+and+aggregation+determine+pathological+effects.">Motifs in the tau protein that control binding to microtubules and aggregation determine pathological effects.</a> Scientific Reports. 7 (1), 13556 (2017).</li><li>Asai, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+microglia+and+inhibition+of+exosome+synthesis+halt+tau+propagation.">Depletion of microglia and inhibition of exosome synthesis halt tau propagation.</a> Nature Neuroscience. 18 (11), 1584-1593 (2015).</li></ol>

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?...

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。

<table>
	<tbody>
		<tr>
			<td>10 cm culture dishes</td>
			<td>Thermofisher</td>
			<td>12556002</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>Biopioneer</td>
			<td>CNT-15</td>
			<td></td>
		</tr>
		<tr>
			<td>16% paraformaldehyde</td>
			<td>Thermofisher</td>
			<td>50-980-487</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well culture plates</td>
			<td>Thermofisher</td>
			<td>930186</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml tubes</td>
			<td>Biopioneer</td>
			<td>CNT-50</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol in spray bottle</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Thermofisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Basement membrane matrix (Matrigel)</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic FGF</td>
			<td>Biopioneer</td>
			<td>HRP-0011</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12 culture media with glutamine</td>
			<td>Thermofisher</td>
			<td>10565042</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (50% concentration or higher)</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Flourescently labeled secondary antibodies</td>
			<td>Various Sources, experiment dependent</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Thermofisher</td>
			<td>1254581</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Thermofisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Human neural stem cells</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentiviral vectors</td>
			<td>Various sources</td>
			<td>custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media</td>
			<td>Thermofisher</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Thermofisher</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermofisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Thermofisher</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td>Various Sources, experiment dependent</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocking or rotating platform</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cell culture hood</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioflavin S</td>
			<td>Sigma</td>
			<td>T1892-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Thermofisher</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Various sources</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro model for studying tau aggregation using lentiviral-mediated transduction of human neurons

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。

用 YFP 荧光标记了 to-rdlm-yfp 诱导神经元, rdlm 诱导培养物在转导后显示出聚集物。这些内含物染色对硫代黄素呈阳性 (图 1)。如图 1所示, 该协议产生的神经元培养显示硫代黄素阳性 tau 集合体。对于初始实验, 建议通过在培养物中对神经元特异性标记Β-tubulin III 进行免疫标记来确认神经元分化。重要的是, 荧光标记的二级抗体应具有与 YPF (例如 Cy3) 不重叠...

Watch this Scientific Journal Video about An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons at JoVE.com

An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

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

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。

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). ...

在该协议中，我们详细介绍了人类肌病的体外模型。这种方法填补了对药物筛选和疾病机制研究有用的材料需求。与动物研究相比，这种技术的主要优点是细胞培养范式能够更快速地评估tau毒性和潜在的tau疗法，开始这个程序在四摄氏度下解冻培养板的基底膜基质涂层。制作一毫升的等分，并储存在零下20或零下70摄氏度。接下来，以每毫升10微克的浓度重组无菌PBS中的基本成纤维细胞生长因子。制作 10 微升等分，并将其储存在 4 摄氏度。然后拿出一个新的未开封的500毫升瓶DMEM-F12与谷氨酰胺。加入10毫升B27、5毫升N2和5毫升青霉素链霉素，以准备神经干细胞培养基。将 50 毫升的 NSC 介质放入锥形管中，并加入 10 微升准备好的 bFGF。将此 NSC 加 bFGF 介质存放在摄氏四度。首先，从冰柜中取出细胞培养板的冷冻基底膜基质涂层，并在四摄氏度下解冻。将解冻的地下室膜基质涂层的385微升加入含有青霉素链霉素的5毫升DMEM-F12介质中。如果细胞从冷冻的NSC库存中解冻，从冷冻室中取出一小瓶冷冻细胞，在水浴中加热至37摄氏度，在水中来回移动小瓶。在此之后，用70%乙醇喷洒小瓶。然后在细胞培养罩下，将细胞转移到含有青霉素链霉素的DMEM-F12介质中约10毫升。在室温下在1000次g下离心5分钟。然后吸气介质，并在 NSC 介质中重新暂停细胞。稀释NSC培养基中的细胞，以获得用于细胞培养容器的适当播种密度。添加足够的悬浮在 NSC 介质中的细胞，为地下室膜基质涂层培养皿播种。在37摄氏度下用5%的二氧化碳生长细胞，直到它们75至80%的汇合，确保每隔一天更换一次介质。要用扁病毒进行神经元的转导，每个细胞使用34万个可转接单位的滴度计数。将细胞培养培养基中的可变性单位稀释到必要的浓度，并将其添加到细胞中。添加扁病毒两天后，用不含bFGF的新鲜介质清洗细胞一次。在37摄氏度下继续培养细胞，在没有bFGF的介质中培养5%的二氧化碳。转导后保持细胞约八周，确保每隔一天改变细胞培养培养。使用光学显微镜对细胞进行常规可视化，确保其生存能力。转导后，从培养箱中取出培养皿，确保盖子保持，以保持培养物无菌。使用能够活细胞成像的荧光显微镜，以 514 纳米的激发和 527 纳米的发射过滤器在 10 倍目标下可视化细胞。在这项研究中，用tau-RDLM-YFP扁病毒转导的神经元被荧光标记与YFP。RDLM 转导培养物在转导后显示聚合。这些内含物对硫黄素有阳性的污渍。神经元分化通过神经元特异性标记β-图布林III在培养物中的免疫标记得到确认。重要的是要记住，荧光标记的二级抗体应具有不与YFP重叠的激发发射光谱。虽然在没有染色的情况下，黄色荧光tau聚集物清晰可见，但硫黄素也应用于成像，以确认荧光信号是聚合tau，而不是细胞碎片。在产生tau过度表达细胞后，可以进行一些神经元健康和tau聚合的评估。例如，我们发现这些细胞显示斧神酮退化。除了细胞培养研究外，我们还使用这种方法来检测致病性外源tau。

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Neuroscience

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

科研

神经科学

Studying the Integration of Adult-born Neurons

成人出生的神经元的整合研究

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

嗅觉感官神经元的慢病毒介导的遗传操作和可视化在体内</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

一<em&gt;在体外</em&gt;模型细胞病理生理学的球细胞脑​​白质营养不良的研究

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

一<em&gt;在体外</em&gt;成年小鼠肌肉，神经准备留学肌肉传入放电的放电特性

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

背根神经节神经元和区分脂肪干细胞：一种<em&gt;在体外</em&gt;共培养模型来研究周围神经再生

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

使用荧光探针定量培养的神经元中的内体和溶酶体运动

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

在体外陶氏蛋白聚集与药物筛选研究方法

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other ...

Studying Brain Function in Children Using Magnetoencephalography

脑磁图在儿童脑功能研究中的应用

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the ...