Name: modeling neuronal death and degeneration in mouse primary cerebellar granule neurons
Uploaded: 2017-11-06T16:30:00
Description: Watch this Scientific Journal Video about Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons at JoVE.com

这项工作得到加拿大自然科学和工程研究理事会以及加拿大卫生研究院的资助。

此协议基于先前描述的过程的修改 18 、 24 、 25 , 26 , 27 . 本议定书由麦吉尔大学动物保育委员会批准. 
 1. 实验准备 注意: 可以准备和维护以下库存解决方案直到使用. 
<ol> <li> 解剖解决方案 <ol> <li> 溶解3.62 克?...

<ol>
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T., Puttfarcken, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress,+glutamate,+and+neurodegenerative+disorders.">Oxidative stress, glutamate, and neurodegenerative disorders.</a> Science. 262 (5134), 689-695 (1993).</li><li>Cole, K., Perez-Polo, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+trauma+model:+in+search+of+Thanatos.">Neuronal trauma model: in search of Thanatos.</a> Int J Dev Neurosci. 22 (7), 485-496 (2004).</li><li>Cheung, E. C., McBride, H. M., Slack, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dynamics+in+the+regulation+of+neuronal+cell+death.">Mitochondrial dynamics in the regulation of neuronal cell death.</a> Apoptosis. 12 (5), 979-992 (2007).</li><li>Jahani-Asl, 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=Mitofusin+2+protects+cerebellar+granule+neurons+against+injury-induced+cell+death.">Mitofusin 2 protects cerebellar granule neurons against injury-induced cell death.</a> J Biol Chem. 282 (33), 23788-23798 (2007).</li><li>Gallo, V., Kingsbury, A., Balazs, R., Jorgensen, O. 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R., Galli, C., Ciotti, T., Calissano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+apoptosis+in+cerebellar+granule+neurons+by+low+potassium:+inhibition+of+death+by+insulin-like+growth+factor+I+and+cAMP.">Induction of apoptosis in cerebellar granule neurons by low potassium: inhibition of death by insulin-like growth factor I and cAMP.</a> Proc Natl Acad Sci U S A. 90 (23), 10989-10993 (1993).</li><li>Gallo, V., Ciotti, M. T., Coletti, A., Aloisi, F., Levi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+release+of+glutamate+from+cerebellar+granule+cells+differentiating+in+culture.">Selective release of glutamate from cerebellar granule cells differentiating in culture.</a> Proc Natl Acad Sci U S A. 79 (24), 7919-7923 (1982).</li><li>Messer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+maintenance+and+identification+of+mouse+cerebellar+granule+cells+in+monolayer+culture.">The maintenance and identification of mouse cerebellar granule cells in monolayer culture.</a> Brain Res. 130 (1), 1-12 (1977).</li><li>Jahani-Asl, 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=CDK5+phosphorylates+DRP1+and+drives+mitochondrial+defects+in+NMDA-induced+neuronal+death.">CDK5 phosphorylates DRP1 and drives mitochondrial defects in NMDA-induced neuronal death.</a> Hum Mol Genet. 24 (16), 4573-4583 (2015).</li><li>Jahani-Asl, 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=The+mitochondrial+inner+membrane+GTPase,+optic+atrophy+1+(Opa1),+restores+mitochondrial+morphology+and+promotes+neuronal+survival+following+excitotoxicity.">The mitochondrial inner membrane GTPase, optic atrophy 1 (Opa1), restores mitochondrial morphology and promotes neuronal survival following excitotoxicity.</a> J Biol Chem. 286 (6), 4772-4782 (2011).</li><li>Li, M., Husic, N., Lin, Y., Snider, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+lentiviral+vectors+for+transducing+cells+from+the+central+nervous+system.">Production of lentiviral vectors for transducing cells from the central nervous system.</a> J Vis Exp. (63), e4031 (2012).</li><li>Wang, X., McManus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+production.">Lentivirus production.</a> J Vis Exp. (32), (2009).</li><li>Lee, H. Y., Greene, L. A., Mason, C. A., Manzini, M. 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在这里, 我们提供了一个简单的方法, 培养小鼠小脑颗粒神经元 (CGNs), 失去和增益的功能研究, 并建模不同的机制, 细胞死亡。有几个因素影响的重复性的结果使用这个程序, 需要密切监测。这些包括文化的纯净, 包括在文化中消除胶质细胞, 文化的汇合, 以及维持健康的细胞。在这些因素中引入变异性会对结果产生偏见, 并挑战重复性。下面我们将描述如何最小化影响结果的因素。
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小脑颗粒神经元 (CGNs) 具有良好的文化特征, 并已成为研究神经元死亡和发育的有效模型 1,2,3,4,56. CGN 培养的 n-甲基-d-天门冬氨酸 (nmda) 受体在体外的早期表达, 使其成为研究 NMDA 诱导信号的有吸引力的模型。这些受体的激活与 NMDA 结合膜去极化是用来模拟生理神经元活动的刺激, 并允许研究突触可塑性的机制7,8。相反, 这些受体的过度的 NMDA 配体可用于模型兴奋, 一个主要机制的神经元丧失的急性脑损伤和神经退行性疾病9。一个机制为诱导兴奋是通过 ATP 饥饿与减少的氧气, 如看见与急性神经元损伤。这导致膜的去极化和高水平的谷氨酸释放在突触。随后刺激的 NMDA 受体的高活性谷氨酸导致过量的 ca2 +通过这些受体的流入, 反过来又激活了包括 ca2 +激活的蛋白酶, 磷脂和酶, 导致严重的细胞成分和细胞死亡失控的退化。此外, 高胞内钙2 +导致氧自由基和线粒体损伤的生成10,11。
当 NMDA 诱导的神经元兴奋的大部分神经元丢失是由于钙的流入和 Bax/Bak 的独立, 其他机制的细胞死亡不能排除在此模型。兴奋的坏死和凋亡样细胞死亡的出现, 部分原因是由于高胞内 Ca2级级别12导致的活性氧 (ROS) 和 DNA 损伤的产生。DNA 损伤通过凋亡机制导致神经元死亡, 与凋亡细胞死亡的特征相关, 如染色质肿块和凋亡体的出现。诱导细胞凋亡是介导的细胞色素 c 从线粒体释放, 并已证明是依赖于 Bax/Bak 齐聚13。Bax/Bak 齐聚促进外线粒体膜的孔隙形成, 导致细胞色素 c 释放和亡调节剂的活化, 见轻度缺血性损伤14。
ROS 的产生是一个重要的问题, 在大脑中由于低内源性的抗氧化剂, 加上大的氧气需求的神经元功能的15。当暴露于缺血性事件时, 一氧化氮合酶上调, 产生一氧化氮和增加活性氧的种类14。氧自由基浓度的增加会导致 DNA 损伤, 并间接导致能量的饥饿。高水平的 DNA 双断裂是通过活化的聚 adp-核糖 polymerase-1 (PARP-1), 一个真核染色质结合蛋白负责催化转移的 ADP 核糖单位从和+, 一个过程积分到DNA 修复16。然而, 由于氧化应激造成的过度损害, PARP-1 活化可能导致能量饥饿, 由于增加的流失, 在和+, 一个必要的基质 ATP 生产通过氧化磷酸化。最终, 氧化应激将触发凋亡的 Bax/Bak 的依赖方式导致线粒体细胞色素 c 释放, 并已证明诱导线粒体重塑在 CGNs 17。
最后, CGN 培养基中氯化钾 (氯化钾) 浓度的变化可以用来模拟低钾/去极化介导的凋亡18,19,20。当暴露于低水平的 K+时, CGNs 经历了明显的生理变化, 导致线粒体呼吸和糖酵解减少, 这归因于细胞需求减少21, 以及减少核因子-nf-(NFκB), 它调节活动, 包括炎症和突触传递22。该模型对神经细胞发育过程中细胞死亡的研究具有特别的意义。低 K+环境更接近于生理条件, 并导致神经元发育过程中出现的细胞死亡特征23。
总之, CGNs 提供了一个长期的模型来研究神经元死亡和变性的基本分子机制。下面的协议将允许分离和培养 CGNs, 表达或抑制特定的基因通路使用病毒和诱导神经元死亡通过不同的机制, 代表神经元损伤和变性。

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modeling neuronal death and degeneration in mouse primary cerebellar granule neurons

小脑颗粒神经元 (CGNs) 是一种常用的神经元模型, 在小脑中形成了丰富的均质种群。根据其产后发育、丰度和可及性, CGNs 是研究神经元过程的理想模型, 包括神经元发育、神经元迁移和生理神经元活动刺激。此外, CGN 文化为研究不同的细胞死亡模式, 包括兴奋和细胞凋亡提供了一个很好的模型。在一个星期内的文化, CGNs 表达 n-甲基-d-天门冬氨酸 (NMDA) 受体, 一个特定的离子谷氨酸受体与许多关键功能的神经元健康和疾病。在 CGN 培养基中加入低浓度的 nmda 与膜去极化剂结合, 用于模拟生理性神经元活动刺激, 同时增加高浓度的 nmda 可用于模型兴奋神经元损伤。本文介绍了6天幼崽 CGNs 的分离培养方法以及腺和慢对 CGNs 的遗传操作。我们还提出了优化的协议, 如何刺激 NMDA 诱导的兴奋, 低诱导的凋亡, 氧化应激和 DNA 损伤后, 这些神经元的转导。

仔细解剖后, 完整的大脑应该被删除, 最小的伤害, 如图 1A-b所示。应努力减少对大脑的损伤, 特别是对小脑的损伤。损害小脑, 使更困难的识别和完全清除脑膜, 并增加了可能的污染的神经元文化。一旦脑膜被切除, 小脑可以从剩余的组织中解剖, 如图 1C所示, 并准备分离。
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Watch this Scientific Journal Video about Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons at JoVE.com

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

本协议描述了一种简单的方法, 分离和培养的小鼠脑颗粒神经元 (CGNs) 从6-7 天的老幼崽, 有效的转导 CGNs 的损失和增益的功能研究, 并建模 NMDA 诱导神经元兴奋,低诱导的细胞死亡, DNA 损伤, 和氧化应激使用相同的培养模式。

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

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