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Neuroscience

用于评估啮齿动物和人类脑组织中行为素聚合状态的基于时间的荧光光谱分析

Published: June 03, 2021
doi:
10.3791/62268
,
1Department of Anatomy, School of Biomedical Sciences,University of Otago

Summary

我们报告了一种简单、省时和高通量荧光光谱分析，用于从啮齿动物和人类受试者脑组织 中对 活体细丝进行定量。

Abstract

Actin 是细胞骨骼的主要成分，在维持神经元结构和功能方面起着至关重要的作用。在生理状态下，行为素以两种形式以平衡形式发生：单体球状（G-actin）和聚合丝（F-actin）。在突触终端，作用细胞细胞细胞形成关键突触前和突触后功能的基础。此外，作用素聚合状态的动态变化（球状和细丝形式的作用之间的转换）与突触结构和功能的可塑性相关变化密切相关。我们在这里报告一种基于荧光的修改方法，以评估 前体内 环境中作用素的聚合状态。该检测采用荧光标签的法洛丁，一种法洛托辛，专门结合到作用丝（F-actin），提供聚合丝活性蛋白的直接测量。作为原则的证明，我们为在啮齿动物和死后人类脑组织同质化中进行检测的适用性提供了证据。使用拉特伦库林A（一种使肌细丝脱聚的药物），我们确认该检测在监测F-actin水平变化方面的作用。此外，我们扩展检测到分离突触终端的生化部分，其中我们确认通过高细胞外 K+去极化刺激刺激时增加作用素聚合。

Introduction

细胞骨质蛋白作用素涉及多种细胞功能，包括结构支持、细胞传输、细胞动力和分裂。Actin 以两种形式以均衡形式发生：单体球状作用素 （G-actin） 和聚合丝作用素 （F-actin）。actin的聚合状态（其G-和F-形式之间的互通）的快速变化导致快速的丝组装和拆卸，并成为其细胞生理学调控作用的基础。Actin是神经元细胞骨结构的主要组成部分，影响广泛的神经元功能1，2。值得注意的是，细胞球菌的作用是突触终端结构平台的组成部分。因此，它是突触形态形成和生理学的主要决定因素，在控制突触3、4、5的大小、数量和形态方面起着基础性作用。特别是，动态作用聚合-去聚合是与记忆和学习过程背后的突触可塑性相关的突触重塑的关键决定因素。事实上，前突性（如神经递质释放6，7，8，9，10）和后突体功能（可塑性相关的动态改造11，12，13，14）都严重依赖于在细胞细胞细胞顿的聚合状态的动态变化。

在生理条件下，F-actin水平通过多式联运途径进行动态和严格调节，包括后翻译修饰4、15、16以及活性结合蛋白（ABPs）4、17。ABP 可以影响多个级别的活性动力学（如启动或抑制聚合、诱导丝分枝、将细丝分离到较小的碎片、促进去聚合和防止脱聚），并依次在对各种细胞外信号敏感的严格调制控制下18、19、20。这种多层次的监管检查要求对突触细胞细胞细胞的活性动力学进行严格监管，在基础和活动诱发状态下对神经元生理学的前期和后突触方面进行微调。

鉴于行为素在神经元生理学中的重要作用，一些研究提供了证据，证明行为动力学的改变与各种神经系统疾病有关，包括神经退化、心理疾病以及神经发育疾病3、21、22、23、24、25、26、27等。然而，尽管大量的研究数据表明作用在神经元生理学和病理生理学中的关键作用，但在对行为动力学的理解上，特别是在突触细胞细胞细胞学方面，仍然存在着重大差距。需要更多的研究来更好地了解神经元作用及其在病理条件下的改变。在这方面，一个主要的重点领域是评估作用物聚合状态。有西方印迹为基础的商业套件（G-Actin/F-Actin在体内测定生化套件;赛托斯凯尔顿SKO BK03728，29）和自制的检测评估F-actin水平6。然而，由于这些需要F-actin和G-actin的生化分离，并且由于它们随后的定量基于免疫凝固协议，因此它们可能非常耗时。我们这里报告一个荧光光谱为基础的检测，改编自先前的研究30与修改，可用于评估F-actin的基础水平，以及其组装拆解的动态变化。值得注意的是，我们已经有效地修改了原来的协议，要求样品适合1 mL的cuvette到目前的96井板格式。因此，修改后的方案显著减少了检测所需的组织/样本量。此外，我们提供证据表明，该协议不仅适用于脑组织同质化，还适用于细胞下部分，如分离突触终端（突触体和突触体）。最后，该检测可用于刚解剖的啮齿动物脑组织和长期储存的死后人类大脑样本。值得注意的是，虽然测定是在神经元环境中呈现的，但它可以适当地扩展到与之相关的其他细胞类型和生理过程。

Protocol

所有实验程序均按照奥塔哥大学保护和使用实验室动物道德委员会的规定进行（道德议定书第1号）AUP95/18 和 AUP80/17）和新西兰立法机构。人脑组织是从西班牙巴塞罗那的Clénic-IDIBAPS生物库的神经组织库获得的。所有组织收集协议均得到巴塞罗那克莱尼奇医院道德委员会的批准，并征得家属的知情同意。 1. 准备缓冲器和试剂 为脑组织的同质化和合成终端的丰富部分的?…

Representative Results

F-actin 水平评估的检测的线性首先，确定了亚历克萨荧光647法洛丁荧光线性增加的标准曲线，并重复了每组实验（图1）。为了调查检测的线性范围，处理了不同数量的大脑同质性，从啮齿动物（图2A和2B）和死后人类受试者（图3A和3B）。检测结果发现，该检测在50-200微克蛋白质范围内是线性的，通?…

Discussion

这里描述的检测，基本上改编自先前的研究30与修改，采用了一种法洛托辛，法洛丁标签与荧光标签。荧光法洛丁类比被认为是固定组织47、48、49染色蛋白丝的黄金标准。事实上，它们是专门识别行为细丝50的最古老的工具，并且仍然是检测行为细丝的最广泛使用的工具，特别是用于随后的?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了新西兰神经学基金会（1835-PG）、新西兰健康研究理事会（#16-597）和新西兰奥塔哥大学解剖学系的支持。我们感谢HCB-IDIBAPS生物库（西班牙）的神经组织库用于人体脑组织。我们感谢张嘉贤在录制和编辑视频方面所做的帮助。

Materials

3.5 mL, open-top thickwall polycarbonate tube Beckman Coulter 349622 For gradient centrifugation (synaptosome prep)
Alexa Fluor 647 Phalloidin Thermo Fisher Scientific A22287 F-actin specific ligand
Antibody against  b-actin Santa Cruz Biotechnology Sc-47778 For evaluation of total actin levels by immunoblotting
Antibody against GAPDH Abcam Ab181602 For evaluation of GAPDH levels by immunoblotting
Bio-Rad Protein Assay Dye Reagent Concentrate Bio-Rad 5000006 Bradford based protein estimation
Calcium chloride dihydrate (CaCl2·2H2O) Sigma-Aldrich C3306 Krebs buffer component
cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Sigma-Aldrich 4693159001 For inhibition of endogenous protease activity during sample preparation
Corning 96-well Clear Flat Bottom Polystyrene Corning 3596 For light-scattering measurements
D-(+)-Glucose Sigma-Aldrich G8270 Krebs buffer component
Dimethyl sulfoxide Sigma-Aldrich D5879 Solvent for phalloidin and latrunculin A
Fluorescent flatbed scanner (Odyssey Infrared Scanner) Li-Cor Biosciences For detection of immunoreactive signals on immunoblots
Glutaraldehyde solution (25% in water) Grade II Sigma-Aldrich G6257 Fixative
HEPES Sigma-Aldrich H3375 Buffer ingredient for sample preparation and Krebs buffer component
Latrunculin A Sigma-Aldrich L5163 Depolymerizer of actin filaments
Magnesium chloride hexahydrate (MgCl2·6H2O) Sigma-Aldrich M2670 Krebs buffer component
Microplates
Mitex membrane filter 5 mm Millipore LSWP01300 Preparation of synaptoneurosomes
Nunc F96 MicroWell Black Plate Thermo Fisher Scientific 237105 For fluorometric measurements
Nylon net filter 100 mm Millipore NY1H02500 Preparation of synaptoneurosomes
Phosphatase Inhibitor Cocktail IV Abcam ab201115 For inhibition of endogenous phosphatase activity during sample preparation
Potassium chloride (KCl) Sigma-Aldrich P9541 Krebs buffer component and for depolarization of synaptic terminals
Potassium phosphate monobasic ((KH2PO4) Sigma-Aldrich P9791 Krebs buffer component
Sodium borohydride (NaBH4) Sigma-Aldrich 71320 Component of Permeabilization buffer
Sodium chloride (NaCl) LabServ (Thermo Fisher Scientific) BSPSL944 Krebs buffer component
Sodium hydrogen carbonate (NaHCO3) LabServ (Thermo Fisher Scientific) BSPSL900 Krebs buffer component
SpectraMax i3x Molecular Devices For fluorometric measurements
Sucrose Fisher Chemical S/8600/60 Buffer ingredient for sample preparation
Swimnex Filter Holder Millipore Sx0001300 Preparation of synaptoneurosomes
Tissue grinder 5 mL Potter-Elvehjem Duran Wheaton Kimble 358034 For tissue homogenization
Triton X-100 Sigma-Aldrich X100 Component of Permeabilization buffer
Trizma base Sigma-Aldrich T6066 Buffer ingredient for sample preparation
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Tags

ActinCytoskeletonF-actinG-actinPolymerizationFluorescence SpectroscopyPhalloidinSynaptic TerminalsSynaptosomesSynaptoneurosomesNeuropathologyNeuronal Physiology
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Ahmad, F., Liu, P. A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues. J. Vis. Exp. (172), e62268, doi:10.3791/62268 (2021).
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