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Neuroscienze

遗传性痉挛性截瘫中人类诱导多能干细胞衍生神经元的线粒体迁移与形态分析

Published: February 09, 2020
doi:
10.3791/60548
, , , ,
1Department of Biomedical Sciences,University of Illinois College of Medicine Rockford, 2Department of Bioengineering,University of Illinois at Chicago, 3MD Program,University of Illinois College of Medicine Rockford

Summary

受损的线粒体迁移和形态学涉及各种神经退行性疾病。提出的协议使用诱导多能干细胞衍生前脑神经元来评估线粒体迁移和遗传性痉挛性截瘫的形态。该协议允许沿斧子对线粒体贩运进行表征，并分析其形态，这将有助于神经退行性疾病的研究。

Abstract

神经元对高能量有强烈的需求，以支持其功能。在人类神经元中观察到沿着斧子的线粒体传输受损，这可能导致各种疾病状态的神经退化。虽然研究活人类神经中的线粒体动力学具有挑战性，但这种模式对于研究线粒体在神经退化中的作用至关重要。本文介绍一个协议，用于分析前脑神经元的线粒体迁移和线粒体形态学，该词从人类诱导多能干细胞（iPSCs）中提取。iPSCs 使用成熟的方法分化为端脑谷氨酸神经元。神经元的线粒体被MitoTracker CMXRos染色，并且使用装有细胞培养箱的活细胞成像显微镜捕获斧子内的线粒体运动。使用带有”多Kymograph”、”生物格式导入器”和”宏”插件的软件对延时图像进行分析。生成线粒体传输的线粒体图，并从测速仪中读取反逆向线粒体的平均线粒体速度。在线粒体形态分析方面，利用ImageJ获得线粒体长度、面积和纵横比。总之，该协议允许沿斧子对线粒体贩运进行表征，并分析其形态，以促进神经退行性疾病的研究。

Introduction

线粒体运动和分布在满足极化神经元的可变和专门能量需求方面发挥着至关重要的作用。神经元可以延长极长的斧子，通过形成突触与目标连接，而突触对Ca2+缓冲和电电流需要高水平的能量。线粒体从索马到斧子的传输对于支持神经元的斧突和突触功能至关重要。空间和时空动态线粒体运动是通过快速的辅助体传输以每秒几微米的速度进行。

具体来说，运动或适配器蛋白，如激酶和dynein，参与沿着微管的快速细胞器传输，以控制线粒体2，3的运动。正常的神经元活动需要将新组装的线粒体从神经元索马正确传输到远端斧子（异端等向等同体传输），并将线粒体从远端斧子反向运输回细胞体（逆向传输）。最近的研究表明，不正确的线粒体分配与神经元缺陷和运动神经元退行性疾病4，5密切相关。因此，要剖析线粒体在神经退化中的作用，必须建立研究活文化中沿斧突运动的线粒体运动的方法。

在检查和分析线粒体的跟踪时，有两个主要挑战：（1） 从每个帧的背景识别线粒体，（2） 分析和生成每个帧之间的连接。在解决第一个挑战时，广泛使用荧光标记方法区分线粒体与背景，如MitoTracker染料或荧光融合线粒体靶向蛋白（例如mito-GFP）6、7、8的转染。为了分析帧之间的关联，在前面的研究9中描述了几种算法和软件工具。在最近的一篇论文中，研究人员比较了四种不同的自动化工具（例如，沃洛蒂、伊马里斯、wrMTrck和差异跟踪器），以量化线粒体传输。结果表明，尽管轨道长度、线粒体位移、运动持续时间和速度存在差异，但这些自动化工具适用于治疗后的运输差异。除了这些工具，一个集成插件”宏”为ImageJ（由Rietdorf和Seitz编写）已广泛用于分析线粒体传输11。此方法生成可用于分析线粒体运动（包括逆向和逆行方向的速度）的图形。

线粒体是高度动态的细胞器，在数量和形态上不断变化，以响应生理和病理条件。线粒体裂变和融合严格调节线粒体形态和平衡。线粒体裂变和融合之间的不平衡可诱发极短或长线粒体网络，从而损害线粒体功能，导致神经元异常活动和神经退化。受损的线粒体传输和形态涉及各种神经退行性疾病，如阿尔茨海默病、帕金森病、亨廷顿舞蹈症和遗传性痉挛性截瘫（HSP）12、13、14、15。HSP是一个异质的遗传性神经系统疾病组，其特征是皮质脊柱退化，随后未能控制下肢肌肉16、17。在这项研究中，iPSC衍生前脑神经元用于评估HSP中的线粒体迁移和形态。该方法为检查活培养体中神经元斧子的线粒体动力学提供了独特的范例学范式。

Protocol

1. 从iPSC生成端脑谷氨酸神经元 注：用于维护iPSC及其分化为端脑谷氨酸神经元的详细协议与前面描述的18相似。在这里，介绍并强调了人类多能干细胞分化过程中的关键过程。 人类胚胎干细胞（hESC）培养基细胞（MEF）的小鼠胚胎成纤维细胞（MEF）分供器的培养iPSC，辅以成纤维细胞生长因子（bFGF，4纳克/mL）。 用消炎（1mg/mL）孵育3分钟后，将i…

Representative Results

在这里，人类iPSCs被分化为端脑谷氨酸神经元，其特征是用Tbr1和βIII图布林标记进行免疫染色（图1A）。为了检查线粒体的同线体传输，这些细胞被红色荧光染料染色，并进行了延时成像。由于ImageJ是现成的，更容易获得，线粒体传输被进一步分析与”多Kymograph”和”宏”图像J插件，如图1所示。 线粒体运动有三种各自的状?…

Discussion

本文介绍了一种利用红色荧光染料和ImageJ软件分析神经元斧子线粒体迁移和形态的方法，为神经退行性疾病的斧子退化和线粒体形态学研究提供了独特的平台。该协议中有几个关键步骤，包括线粒体的染色、活细胞成像和分析图像。在这种方法中，使用荧光染料染色线粒体。由于人类iPSC衍生的神经元很容易从培养皿中分离，因此在培养皿中保留一些溶液并轻轻添加神经基质介质非常重要。洗涤可…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到了痉挛性截瘫基金会、布雷泽基金会和NIH（R21NS109837）的支持。

Materials

Accutase Cell Detachment Solution Innovative Cell Technologies AT104
Biosafety hood Thermo Scientific 1300 SERIES A2
Bovine serum albumin (BSA) Sigma A-7906
Brain derived neurotrophic factor (BDNF) Peprotech 450-02
Centrifuge Thermo Scientific Sorvall Legend X1R/ 75004261
Coverslips Chemiglass Life Sciences 1760-012
Cyclic AMP (cAMP) Sigma-Aldrich D0627
Dispase Gibco 17105-041
Dorsomorphin Selleckchem S7146
Dulbecco's modified eagle medium with F12 nutrient mixture (DMEM/F12) Corning 10-092-CV
FBS Gibco 16141-002
Fibroblast growth factor 2 (FGF2, bFGF) Peprotech 100-18B
Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix Gibco A1413201
Gem21 NeuroPlex Serum-Free Supplement Gemini 400-160
Glass Bottom Dishes MatTek P35G-0.170-14-C
9'' glass pipetes VWR 14673-043
Glial derived neurotrophic factor (BDNF) Sigma-Aldrich D0627
GlutaMAX-I Gibco 35050-061
Heparin Sigma H3149
Insulin growth factor 1 (IGF1) Invitrogen M7512
Knockout Serum Replacer Gibco A31815
Laminin Sigma L-6274
2-Mercaptoethanol Sigma M3148-100ML
MitoTracker CMXRos Invitrogen M7512
Neurobasal medium Gibco 21103-049
Non Essential Amino Acids Gibco 11140-050
N2 NeuroPle Serum-Free Supplement Gemini 400-163
Olympus microscope IX83 Olympus IX83-ZDC2
PBS Corning 21-031-CV
Phase contrast microscope Olympus CKX41/ IX2-SLP
6 well plates Corning 353046
24 well plates Corning 353047
Poly-L-ornithine hydrobromide (polyornithine)) Sigma-Aldrich P3655
SB431542 Stemgent 04-0010
Sterile 50ml Disposable Vacuum Filtration System 0.22 μm Millipore Express® Plus Membrane Millipore SCGP00525
Stericup 500/1000 ml Durapore 0.22 μM PVDF Millipore SCGVU10RE
Tbr1 antibody (1:2000) Chemicon AB9616
Trypsin inhibitor Gibco 17075029
50 ml tubes Phenix SS-PH50R
15 ml tubes Phenix SS-PH15R
T25 flasks (untreated) VWR 10861-572
Plugins for softwares
Bio-formats Package http://downloads.openmicroscopy.org/bio-formats/5.1.0/
Fiji software https://fiji.sc/
Kymograph Plugin https://www.embl.de/eamnet/html/body_kymograph.html
MultipleKymograph.class https://www.embl.de/eamnet/html/body_kymograph.html
MultipleOverlay.class https://www.embl.de/eamnet/html/body_kymograph.html
WalkingAverage.class https://www.embl.de/eamnet/html/body_kymograph.html
StackDifference.class https://www.embl.de/eamnet/html/body_kymograph.html
Straighten_.jar https://imagej.nih.gov/ij/plugins/straighten.html
tsp050706.txt https://www.embl.de/eamnet/html/body_kymograph.html
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Tags

Mitochondrial TransportMitochondrial MorphologyHuman Induced Pluripotent Stem Cell-derived NeuronsHereditary Spastic ParaplegiaAxonal DegenerationMitochondrial DynamicsNeurological DiseasesLive Cell ImagingMitochondrial TraffickingTherapeutic TargetsNeurodegenerative DiseasesNeurosphere DifferentiationAxon IdentificationMitochondrial StainingTime-lapse ImagingImageJ Analysis

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Mou, Y., Mukte, S., Chai, E., Dein, J., Li, X. Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia. J. Vis. Exp. (156), e60548, doi:10.3791/60548 (2020).
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