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Preparation of Crude Synaptosomal Fractions from Mouse Brains and Spinal Cords
利用小鼠脑和脊髓粗制突触体组分   

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Molecular Neurobiology
Jan 2017

Abstract

The current protocol describes the preparation of crude synaptosomal fractions from mouse brain or spinal cord samples. In detail, a sequential protocol yielding crude synaptosomal and light membrane fractions is provided. This fast and easy method might be sufficient to assess the amount of synaptic proteins in down-steam applications like Western-blot or ELISA in e.g., mouse models of Alzheimer’s disease or other neurodegenerative conditions.

Keywords: Brain (脑), Synaptosome (突触体), Fractionation (分离), Homogenization (匀化), Extraction (萃取), Synapse (突触)

Background

Analyzing synaptosomes, representing isolated synaptic terminals from neurons, can yield valuable information on synaptic integrity in diverse neurological diseases. They contain membrane-bound compartments that detach from axon terminals after brain homogenization under certain conditions. The current protocol describes a fast and easy method for the enrichment of crude synaptosomal fractions (see Figure 1). These can be either used for quantification of synaptic proteins by Western-blot or can be further purified using density gradient centrifugation to yield highly purified synaptosome subfractions (Gurd et al., 1974). The preparation of crude synaptosomal fractions might be sufficient to assess e.g., the amount of pre- and post-synaptic proteins like SNAP25 or post-synaptic density protein 95 (PSD95) in e.g., mouse models with a neurodegenerative phenotype (Breyhan et al., 2009; Saul and Wirths, 2017).


Figure 1. Flow-chart describing the sequential extraction procedure

Materials and Reagents

  1. Pipette tips
    10 µl pipette tips (SARSTEDT, catalog number: 70.1130 )
    200 µl pipette tips (SARSTEDT, catalog number: 70.760.002 )
    1,000 µl pipette tips (SARSTEDT, catalog number: 70.762 )
  2. Reaction tubes
    1.5 ml tubes (SARSTEDT, catalog number: 72.706 )
    2 ml tubes (SARSTEDT, catalog number: 72.691 )
  3. Mice (Protocol has been tested with male and female C57Bl/6 mice of 8-52 weeks)
  4. Sucrose (≥ 99.5%) (Sigma-Aldrich, catalog number: S0389 )
  5. HEPES (≥ 99.5%) (Sigma-Aldrich, catalog number: H3375 )
  6. Protease inhibitor cocktail (cOmpleteTM Mini EDTA-free EasyPack) (Roche Diagnostics, catalog number: 04693159001 )
  7. Phosphatase inhibitor cocktail 3 (Sigma-Aldrich, catalog number: P0044 )
  8. Sodium chloride (NaCl) (≥ 99.5%) (Carl Roth, catalog number: 3957 )
  9. Potassium chloride (KCl) (≥ 99%) (Carl Roth, catalog number: P017 )
  10. Di-Sodium hydrogen phosphate (Na2HPO4) (≥ 98%) (Carl Roth, catalog number: P030 )
  11. Potassium di-hydrogen phosphate (KH2PO4) (≥ 99%) (Carl Roth, catalog number: 3904 )
  12. Lysis buffer (pH 7.5) (see Recipes)
  13. Phosphate-buffered saline (PBS) (see Recipes)

Equipment

  1. Pipettes (Research 0.5-10 µl, 10-100 µl, 100-1,000 µl) (Eppendorf)
  2. Heraeus Biofuge Stratos ( Rotor) (Heraeus Holding, catalog number: 3332 )
  3. Ultrasound sonicator (Emerson Electric, BRANSON, model: 150 )
  4. Stirring device (Ingenieurbüro CAT M. Zipperer, model: R 50D )
  5. Glass homogenizer (2 ml) (Carl Roth, catalog number: TT57.1 )
  6. Teflon pestle (Carl Roth, catalog number: TT63.1 )

Procedure

Notes:

  1. The following protocol has been used to extract crude synaptosomal and free mitochondria fractions from mouse brain samples from mouse models of Alzheimer’s disease (see Breyhan et al. [2009] and Saul and Wirths [2017]). The user might need to modify the amount of solutions (e.g., lysis buffer) depending on the particulate downstream application (e.g., Western blotting or ELISA). For Western blots, we recommend to use 20 to 50 µg of total protein per well. This procedure will yield crude synaptosomal fractions with concentrations of approx. 7-10 µg/µl and microsomal and soluble enzyme fractions of approx. 4-6 µg/µl.
  2.  For mouse brain preparation, animals were anesthetized by carbon dioxide (CO2) and sacrificed by cervical dislocation. Tissues were carefully dissected and either directly used or kept at -80 °C until further use. No differences were noted in yield or quality of the protein fractions if fresh or frozen tissue has been used.
  1. Mechanically homogenize fresh or deep-frozen brain hemispheres in a weight:volume ratio of 1:10 using cold lysis buffer (1,000 µl buffer per 100 mg tissue, see Recipes) with 10 strokes in a glass-Teflon homogenizer at 800 rpm (Figure 2).
    Note: Our lab uses one brain hemisphere with cerebellum and olfactory bulb removed and flash frozen on a plate of dry ice, however the presence of cerebellum or olfactory bulb does not compromise the procedure. For convenience, we do not remove the meninges upon harvesting the tissue.


    Figure 2. Motor-driven homogenizer

  2. Transfer the homogenate to 1.5 ml reaction tubes.
  3. Spin the samples at 1,000 x g for 10 min at 4 °C to separate nuclei (Pellet P1).
    Note: Centrifuge should be pre-chilled to 4 °C.
  4. Remove the supernatant (S1) and transfer to a new 1.5 ml reaction tube. The pellet (P1) can be discarded.
  5. Spin the supernatant at 12,000 x g for 10 min at 4 °C and transfer the resulting supernatant (S2) containing the light membrane fraction and soluble enzymes to a new 1.5 ml reaction tube.
  6. Resuspend the pellet (P2) containing crude synaptosomes and mitochondria in 500 µl PBS (see Recipes).
  7. Homogenates can be stored at -80 °C until further use.

Data analysis

The protein fractions obtained by using this protocol can be used in down-stream applications like Western-blot. Successful enrichment of crude synaptosomes and mitochondria can be e.g., demonstrated by Western blot analysis using marker proteins like post-synaptic density protein 95 (PSD95) or cytochrome c oxidase (CoxIV) (Saul and Wirths, 2017) (Figure 3).


Figure 3. Exemplary Western blot using antibodies against PSD95 and COXIV with β-actin as loading control

Recipes

  1. Lysis buffer (pH 7.5)
    0.32 M sucrose
    5 mM HEPES
    cOmpleteTM protease inhibitor cocktail (1 tablet per 10 ml)
    Phosphatase inhibitor cocktail 3 (use at 1:100 dilution)
    Note: Can be stored at 4 °C for 1 month or at -20 °C for up to 3 months.
  2. PBS (1x working solution pH 7.4)
    137 mM NaCl
    2.7 mM KCl
    8 mM Na2HPO4
    2 mM KH2PO4

Acknowledgments

Financial support of the Alzheimer Forschung Initiative e.V. is gratefully acknowledged. This protocol was adapted from Gurd et al. (1974) with modifications.

References

  1. Breyhan, H., Wirths, O., Duan, K., Marcello, A., Rettig, J. and Bayer, T. A. (2009). APP/PS1KI bigenic mice develop early synaptic deficits and hippocampal atrophy. Acta Neuropathol 117(6): 677-685.
  2. Gurd, J. W., Jones, L. R., Mahler, H. R. and Moore, W. J. (1974). Isolation and partial characterization of rat brain synaptic plasma membranes. J Neurochem 22(2): 281-290.
  3. Saul, A. and Wirths, O. (2017). Endogenous Apolipoprotein E (ApoE) fragmentation is linked to amyloid pathology in transgenic mouse models of Alzheimer’s disease. Mol Neurobiol 54(1): 319-327.

简介

目前的方案描述了从小鼠脑或脊髓样品制备粗突触体部分。 详细地,提供了产生粗突触体和轻质膜部分的顺序方案。 这种快速和容易的方法可能足以在例如阿尔茨海默病或其他神经变性病症的小鼠模型中评估下蒸汽应用中的突触蛋白质的量,例如Western印迹或ELISA。
【背景】分析突触体,代表孤立的突触终端从神经元,可以产生有价值的信息,在不同的神经系统疾病的突触完整性。它们含有在特定条件下脑匀浆后从轴突末端脱离的膜结合区。目前的方案描述了一种快速和简便的浓缩粗突变体级分的方法(见图1)。这些可以用于通过蛋白质印迹定量突触蛋白质,或者可以使用密度梯度离心法进一步纯化以产生高度纯化的突触体亚级分(Gurd等人,1974)。粗突触体部分的制备可能足以评估例如的突触前和突触后蛋白如SNAP25或突触后密度蛋白95(PSD95)的量,例如,具有神经变性表型的小鼠模型(Breyhan等人,2009; Saul and Wirths,2017)。


图1.描述顺序提取过程的流程图

关键字:脑, 突触体, 分离, 匀化, 萃取, 突触

材料和试剂

  1. 移液器提示
    10μl移液器吸头(SARSTEDT,目录号:70.1130)
    200μl移液器吸头(SARSTEDT,目录号:70.760.002)
    1,000μl移液器吸头(SARSTEDT,目录号:70.762)
  2. 反应管
    1.5ml管(SARSTEDT,目录号:72.706)
    2毫升管(SARSTEDT,目录号:72.691)
  3. 小鼠(方案已经测试了雄性和雌性C57Bl / 6小鼠8-52周)
  4. 蔗糖(≥99.5%)(Sigma-Aldrich,目录号:S0389)
  5. HEPES(≥99.5%)(Sigma-Aldrich,目录号:H3375)
  6. 蛋白酶抑制剂混合物(cOmplete TM 不含EDTA的EasyPack)(Roche Diagnostics,目录号:04693159001)
  7. 磷酸酶抑制剂混合物3(Sigma-Aldrich,目录号:P0044)
  8. 氯化钠(NaCl)(≥99.5%)(Carl Roth,目录号:3957)
  9. 氯化钾(KCl)(≥99%)(Carl Roth,目录号:P017)
  10. 磷酸氢二钠(Na 2 HPO 4)(≥98%)(Carl Roth,目录号:P030)
  11. 二氢磷酸氢钾(KH 2 PO 4)(≥99%)(Carl Roth,目录号:3904)
  12. 裂解缓冲液(pH 7.5)(见配方)
  13. 磷酸盐缓冲盐水(PBS)(见食谱)

设备

  1. 移液器(研究0.5-10μl,10-100μl,100-1,000μl)(Eppendorf)
  2. Heraeus Biofuge Stratos(转子)(Heraeus Holding,目录号:3332)
  3. 超声波超声波发生器(艾默生电气,BRANSON,型号:150)
  4. 搅拌装置(IngenieurbüroCAT M.Ziperer,型号:R 50D)
  5. 玻璃均化器(2ml)(Carl Roth,目录号:TT57.1)
  6. 铁氟龙杵(Carl Roth,目录号:TT63.1)

程序

注意:

  1. 已经使用以下方案从阿尔茨海默病小鼠模型的小鼠脑样品中提取粗突触体和游离线粒体部分(参见Breyhan等人[2009]和Saul and Wirths [2017])。用户可能需要根据颗粒下游应用(例如,Western印迹或ELISA)来修饰溶液的量(例如裂解缓冲液)。对于蛋白质印迹,我们建议每孔使用20至50μg的总蛋白。该程序将产生浓度约为的粗突变体级分。 7-10μg/μl,微粒体和可溶性酶组分约为4-6μg/μl。
  2. 对于小鼠脑准备,将动物用二氧化碳(CO 2 >)进行麻醉,并通过颈脱位处死。小心切开组织,直接使用或保存在-80°C直至进一步使用。如果使用新鲜或冷冻的组织,蛋白质组分的产量或质量没有差异。
  1. 使用冷裂解缓冲液(每100毫克组织1,000μl缓冲液,参见食谱),以玻璃 - 特氟龙匀浆器(800 rpm)10次冲洗,以重量:体积比1:10,以机械方式匀浆新鲜或深冻的脑半球(图2 )。
    注意:我们的实验室使用一个脑半球与小脑和嗅球去除,并在一块干冰上快速冷冻,然而小脑或嗅球的存在不会影响手术。为了方便起见,我们不会在收集纸巾时清除脑膜。


    图2.电动均质器

  2. 将匀浆转移到1.5ml反应管中
  3. 在4℃下将样品以1,000×g旋转10分钟以分离核(颗粒P1)。
    注意:离心机应预冷至4°C。
  4. 去除上清液(S1)并转移到新的1.5ml反应管中。颗粒(P1)可以被丢弃。
  5. 在4℃下将上清液以12,000×g旋转10分钟,将含有轻质膜部分和可溶性酶的所得上清液(S2)转移到新的1.5ml反应管中。
  6. 将含有粗突触体和线粒体的沉淀(P2)重悬于500μlPBS(见配方)。
  7. 均质剂可以储存在-80°C直到进一步使用。

数据分析

通过使用该方案获得的蛋白质级分可用于下游应用,如Western印迹。通过使用标记蛋白如突触后密度蛋白95(PSD95)或细胞色素c氧化酶(CoxIV)(Saul和Wirths,2017)的蛋白质印迹分析证明,粗突触体和线粒体的成功富集可以是例如 (图3)

图3.使用具有β-肌动蛋白作为载体对照的PSD95和COXIV的抗体的示例性Western印迹

食谱

  1. 裂解缓冲液(pH 7.5)
    0.32 M蔗糖
    5 mM HEPES
    cOmplete TM 蛋白酶抑制剂混合物(1片每10ml)
    磷酸酶抑制剂混合物3(以1:100稀释使用)
    注意:可以在4°C储存1个月,或在-20°C储存3个月。
  2. PBS(1x工作溶液pH 7.4)
    137 mM NaCl
    2.7 mM KCl
    8mM Na 2 HPO 4
    2mM KH PO 4

致谢

阿尔茨海默氏福音计划e.V.的财政支持非常感谢。该协议改编自Gurd等人(1974)进行修改。

参考

  1. Breyhan,H.,Wirths,O.,Duan,K.,Marcello,A.,Rettig,J.and Bayer,TA(2009)。  APP / PS1KI bigenic小鼠发生早期突触缺陷和海马萎缩。神经酰胺Acta Neuropathol 117(6) :677-685。
  2. Gurd,JW,Jones,LR,Mahler,HR和Moore,WJ(1974)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/4364339 “target =”_ blank“>大鼠脑突触细胞膜的分离和部分表征。 Neurochem 22(2):281-290。
  3. Saul,A.和Wirths,O.(2017)。  内源性载脂蛋白E(ApoE)片段化与阿尔茨海默病转基因小鼠模型中的淀粉样蛋白病理学相关。 Mol Neurobiol 54(1):319-327。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wirths, O. (2017). Preparation of Crude Synaptosomal Fractions from Mouse Brains and Spinal Cords. Bio-protocol 7(15): e2423. DOI: 10.21769/BioProtoc.2423.
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