作者要感谢郑晖博士（美国贝勒医学院）的APP基因。这个工作是由美国国立卫生研究院资助R21AG031948（DB，JMB），F30AG030878（ESS），R01DK073394（AFO），约翰·西利纪念基金会生物医学研究基金（AFO），并在Jean C.和威廉·D·威利斯神经研究基金（ESS）。 ，JMB是一个学者的翻译研究学者项目和大学的德克萨斯州医学科克劳德·E.辣椒年长美国人独立中心的成员（由美国国立卫生研究院资助UL1RR029876和P30-AG-024832，分别支持）。

<html> 1。表达的重组APP胞内结构域（AICD） <ol><li>变换E. coli菌株BL21与人类AICD（残基649-695的APP，神经元亚型编号）克隆到载体pGEX-4T-1（GE Healthcare公司）。该载体将快递AICD作为谷胱甘肽-S-转移酶（GST）的融合蛋白的C-末端基团。该载体也编码凝血酶裂解序列的GST部分以有利于去除。 AICD的克隆到pGEX-4T-1的详细信息可以在我们以前出版4。 </li><li>从单菌落，接种用氨苄青霉素（100微克/毫升）的10毫升的LB肉汤培养基中，并在37℃下剧烈振荡培养过夜。 </li><li>第二天早晨，接种400毫升LB /氨苄青霉素在一个1升的烧瓶中，用5ml的过夜培养。 </li><li>孵育在37℃下剧烈振荡培养，直至在600 nm处的光密度达到0.4到0.6。这通常需要2到2.5小时。 </li></ol>内容"&gt;在这一点上，它可能是可取的取出一个小样本（〜10微升）遵循纯化，通过SDS-PAGE（参见图1A，B），每个后续的纯化步骤也应该有一个小的等分试样进行分析除去通过SDS-PAGE。 <ol start="5"><li>加入0.4毫米的异丙基-β-D-半乳糖苷（IPTG）诱导表达的GST-AICD。剧烈摇动5小时，在37℃下孵育。 </li><li>离心细胞在4℃下以6,000 xg离心15分钟将细胞沉淀在-80℃下，可以存储数个月，在这一点上的程序。 </li></ol> 2。纯化GST-AICD的<ol><li>准备50 ml裂解缓冲液中，如下所示:1mM的乙二胺四乙酸（EDTA），1mM的二硫苏糖醇，0.1％的Triton-X100，1mM苯甲基磺酰氟（PMSF），完全蛋白酶抑制剂鸡尾酒（罗氏应用科学），和磷酸盐缓冲盐水。冰浴。 </li><li>解冻后细菌沉淀（如果先前冻结）冰。 </li></ol>在这一点上，这是至关重要的所有步骤都在4℃下进行，以限制的蛋白水解。所有的试管，缓冲剂，和其他试剂应该是预冷。如果使用法国媒体或乳化剂的溶解，这些都应该是预冷。 <ol start="3"><li>彻底细菌沉淀悬浮在20毫升的裂解缓冲液中。用10ml移液管在冰上的涡流和研磨的组合将确保沉淀完全重悬。 </li><li>通过通过乳化剂的再悬浮的细菌细胞（ 例如 ，Avestin EmulsiFlex-C 3）或法国的压力盒，预先冷却至4℃下以30,000磅。通过溶胞产物通过乳化剂的第二时间。成预先冷却的50毫升的试管中收集的溶胞产物。 </li></ol>这两个工具的主要优点是，它们最大限度地减少样品的加热。常用的技术的超声产生显着的声处理探针的尖端处的热量，并可以导致重组蛋白质的聚集。 <ol start="5"><li>离心裂解液以15,000 xg离心30分钟。分离并保持成预先冷却的50毫升的试管上清液。 </li><li>加入500μl的谷胱甘肽 - 琼脂糖（Sigma-Aldrich公司）的50％的淤浆。旋转30分钟，在4℃下当样品旋转，使新鲜的洗脱缓冲液:50mM的Tris 7.8，0.1％的Triton-X100，0.5 mM的二硫苏糖醇，10mM还原型谷胱甘肽。 </li></ol>的唯一步骤的协议，它是在室温下进行的洗脱步骤。因此，洗脱缓冲液在室温下保持。洗脱缓冲液应新鲜配制。 <ol start="7"><li>的溶胞产物和淤浆倒入到5"一次性聚苯乙烯色谱柱与粗（90-130微米）过滤器（常绿科学）。洗净塔5次，用3毫升的磷酸盐缓冲盐水。 </li><li>盖上的列的底部和室温洗脱缓冲液中加入500μl。盖上的顶部列和5分钟，在室温下旋转。冰鲜1.5 ml离心管，收集洗脱液。重复洗脱2次以上（总共需要1.5毫升的洗脱液）。 </li><li>透析的洗脱液在10,000分子量截止透析膜（ 例如 ，Thermo Scientific的幻灯片-A仪透析盒），在4升的磷酸盐缓冲盐水中，在4℃下过夜透析对额外的4升的磷酸盐缓冲盐水中，在4℃下1小时，翌日。 </li><li>除去蛋白质从透析盒进行蛋白定量测定，以确定产量的GST-AICD。从400毫升培养一个典型的纯化将导致产率&gt; 20毫克的纯化蛋白的浓度为10-20毫克/毫升。等分试样的蛋白质成200μl的等份并冷冻在-80℃下</li></ol> 3。凝血酶切割GST-AICD和纯化AICD <ol><li>解冻200μl的纯化GST-AICD，加入20μl1 U /μl的血栓形成英寸，37℃孵育过夜。 </li></ol>制造商之间的凝血酶的质量和纯度有很大的差别，并且可以是一个重大的污染来源。我们使用凝血酶GE医疗（见表的试剂）。过夜培养后，&gt; 95％应裂解的融合蛋白（ 图1B）。 <ol start="2"><li>与凝血酶裂解过夜后，它很可能是一小部分被解放的AICD聚集和解决方案可能出现混浊。删除的聚合材料通过离心分离30分钟，在4℃下以20,000 xg离心到预冷冻的微量离心管中，除去上清液。 </li><li>加入50μl的50％谷胱甘肽 - 琼脂糖浆料和50μl50％对 - 氨基苯甲脒 - 琼脂糖浆料以除去GST和凝血酶，分别。孵育5分钟，在室温下的旋转。 </li><li>短暂离心沉淀琼脂糖珠，去除上清到一个新的管。提取吨他GST与谷胱甘肽 - 琼脂糖四次。与p-氨基苯甲脒-琼脂糖是一个单一的提取足以除去20 U的凝血酶。 </li><li> 5微升的纯化AICD执行蛋白定量测定。从400毫升培养物的典型产率在0.2至0.5毫克/毫升的浓度为50〜100微克。较高的收益率是可能的裂解更大量的GST-AICD。 </li></ol>纯净的AICD应新鲜配制，生物化学/生物物理特性。未使用的材料应被丢弃。它也可以通过冷冻干燥的样品，并重新悬浮在一个更小的体积的缓冲液6集中AICD。如果检测到的AICD需要杂交后（如过滤器陷阱或斑点杂交实验），应进行描述由Pimplikar和Suryanarayana 12细胞膜上的抗原检索。 4。 AICD聚合原子力显微镜（AFM） <ol><li>稀释新鲜烹制的AICD在phosphate缓冲盐水中的浓度为0.1毫克/毫升的最终体积为15微升。在这一点上，各种化合物和/或蛋白可加入，以确定它们是否抑制AICD聚集。例如，我们已经表明，在该测定4等摩尔浓度的ubiquilin-1防止聚集AICD。 </li></ol>另外，也可以聚合反应体积较大的生化实验，如过滤器捕集阱检测和光散射4。我们已经成功地执行过滤器捕集阱检测反应混合物的酸稀释到浓度低至1μM为400μL。 <ol start="2"><li>诱导热聚集在Eppendorf恒温在800 rpm的转速下振荡48小时，在43℃下温育样品。在该温度下根据的伴侣化验，检查热聚集的模型伴侣基板柠檬酸合成酶4,13选择。 </li></ol><ol start="3"><li>在去离子水和点2μL20倍稀释样品新鲜剥离的云母片上。在干燥器中干燥样品过夜。这将是必要的经验确定的最佳稀释度进行成像，从而几个稀释度范围从10 - 100倍。将审慎。 </li><li>可视化的聚合的AICD使用AFM轻敲模式工作在14。悬臂使用的是镀金微锋利的氮化物的杆（MSNL，布鲁克的名义尖端半径为2 nm）。典型的攻振幅过程中在悬臂的共振频率（30-50千赫）ING成像是10-20纳米。 </li></ol>我们发现，不同的蛋白质聚集体的图像质量和对比度依赖于所施加的力和实验的持续时间。为了尽量减少样品由尖端的扰动，我们保持所施加的力相对低的保持的一组点的比例在95％以上，和限制每个样品的扫描5-10分钟。与WSxM软件（Nanotec的）进行图像处理。标准的图像处理包括平面减法和扁平化。我们的实验室使用"自制的"原子力显微镜连接到商业扫描探针显微镜控制系统（NANOTEC）15。 5。代表性的成果示出在图1A中的表达及相对富集的GST-AICD。裂解后，大部分的融合蛋白是存在于可溶组分，并因此从inclusi萃取对机构不是必需的。从谷胱甘肽琼脂糖柱洗脱的材料，纯度&gt; 95％。在制备图1中所示，从柱中洗脱的蛋白质浓度为14.5毫克/毫升，且产率为22毫克（400毫升）从起始的培养。 200μl的此制备用20 U的凝血酶过夜的裂解导致裂解的融合蛋白（ 图1B）几乎为100％。所用的凝血酶的量是足够低，以不被察觉，考马斯亮蓝染色（参见"劈开"车道， 图1B）。凝血酶和GST的去除导致丧失一些的材料，然而它是&gt; 90％纯的浓度为0.35毫克/毫升的收率在此准备70微克AICD在图1C中示出的流程图，用于聚集AICD和随后的分析。 AICD的聚集体通过AFM成像通常球体/无定形的，尺寸范围从50至100纳米（ 图1D，F）。汇总材料是不可检测的，当等摩尔量的分子伴侣ubiquilin-1被添加到的聚集反应（ 图1E）4。 <img alt="图1" src="/files/ftp_upload/4204/4204fig1.jpg" /> 图1。纯化和聚集AICD A）考马斯亮蓝染色的纯化过程中的每个步骤，在采集的样本。对于未诱导的和诱导的样品中，20微升的总细菌凝胶上运行。上清液部分，5μl的溶胞产物（从总体积为20ml），在凝胶上运行。对于沉淀级分中，将沉淀物重新悬浮于20 ml裂解缓冲液，5微升这种材料，超声处理30分钟，在水浴中超声波发生器，在4℃下前装载到凝胶。对于流出液，5微升，在凝胶上运行。加载到凝胶洗脱液的量表示。 B）考马斯亮蓝染色的凝胶的未裂解的和被裂解的GST-AICD（2微升），1和5＆米U;升纯化AICD。 C）的AICD的纯化，聚合和分析的流程。 D，E）代表的AFM图像的热AICD中的情况下（D）和存在的分子伴侣ubiquilin-1（E）的聚合。 F）AICD的聚集体的粒度分布的直方图。

<ol>
	<li>De Strooper, B., Vassar, R., Golde, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secretases:+enzymes+with+therapeutic+potential+in+Alzheimer+disease.">The secretases: enzymes with therapeutic potential in Alzheimer disease.</a> Nature reviews. Neurology. 6, 99-107 (2010).</li><li>Chang, K. A., Suh, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+roles+of+amyloid+intracellular+domain+of+amyloid+precursor+protein.">Possible roles of amyloid intracellular domain of amyloid precursor protein.</a> BMB reports. 43, 656-663 (2010).</li><li>McLoughlin, D. M., Miller, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FE65+proteins+and+Alzheimer's+disease.">The FE65 proteins and Alzheimer's disease.</a> J. Neurosci. Res. 86, 744-754 (2008).</li><li>Stieren, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquilin-1+is+a+molecular+chaperone+for+the+amyloid+precursor+protein.">Ubiquilin-1 is a molecular chaperone for the amyloid precursor protein.</a> J. Biol. Chem. 286, 35689-35698 (2011).</li><li>Ramelot, T. A., Nicholson, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation-induced+structural+changes+in+the+amyloid+precursor+protein+cytoplasmic+tail+detected+by+NMR.">Phosphorylation-induced structural changes in the amyloid precursor protein cytoplasmic tail detected by NMR.</a> J. Mol. Biol. 307, 871-884 (2001).</li><li>Ramelot, T. A., Gentile, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+structure+of+the+amyloid+precursor+protein+cytoplasmic+tail+indicates+preordering+of+structure+for+binding+to+cytosolic+factors.">Transient structure of the amyloid precursor protein cytoplasmic tail indicates preordering of structure for binding to cytosolic factors.</a> Biochem. 39, 2714-2725 (2000).</li><li>Radzimanowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+intracellular+domain+of+the+amyloid+precursor+protein+in+complex+with+Fe65-PTB2.">Structure of the intracellular domain of the amyloid precursor protein in complex with Fe65-PTB2.</a> EMBO Rep. 9, 1134-1140 (2008).</li><li>Ohkawara, T., Nagase, H., Koh, C. S., Nakayama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+precursor+protein+intracellular+domain+alters+gene+expression+and+induces+neuron-specific+apoptosis.">The amyloid precursor protein intracellular domain alters gene expression and induces neuron-specific apoptosis.</a> Gene. 475, 1-9 (2011).</li><li>von Rotz, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+APP+intracellular+domain+forms+nuclear+multiprotein+complexes+and+regulates+the+transcription+of+its+own+precursor.">The APP intracellular domain forms nuclear multiprotein complexes and regulates the transcription of its own precursor.</a> J. Cell Sci. 117, 4435-4448 (2004).</li><li>Hamid, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+precursor+protein+intracellular+domain+modulates+cellular+calcium+homeostasis+and+ATP+content.">Amyloid precursor protein intracellular domain modulates cellular calcium homeostasis and ATP content.</a> J. Neurochem. 102, 1264-1275 (2007).</li><li>Ghosal, K., Stathopoulos, A., Pimplikar, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APP+intracellular+domain+impairs+adult+neurogenesis+in+transgenic+mice+by+inducing+neuroinflammation.">APP intracellular domain impairs adult neurogenesis in transgenic mice by inducing neuroinflammation.</a> PLoS ONE. 5, e11866 (2010).</li><li>Pimplikar, S. W., Suryanarayana, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+APP+intracellular+domain+in+brain+tissue.">Detection of APP intracellular domain in brain tissue.</a> Met. Molecul. Biol. 670, 85-91 (2011).</li><li>Buchner, J., Grallert, H., Jakob, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+chaperone+function+using+citrate+synthase+as+nonnative+substrate+protein.">Analysis of chaperone function using citrate synthase as nonnative substrate protein.</a> Met. Enzymol. 290, 323-338 (1998).</li><li>Hansma, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+atomic+force+microscopy+of+DNA.">Recent advances in atomic force microscopy of DNA.</a> Scanning. 15, 296-299 (1993).</li><li>Valbuena, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quasi-simultaneous+imaging/pulling+analysis+of+single+polyprotein+molecules+by+atomic+force+microscopy.">Quasi-simultaneous imaging/pulling analysis of single polyprotein molecules by atomic force microscopy.</a> Rev. Sci. Instrum. 78, 113707 (2007).</li><li>Radzimanowski, J., Beyreuther, K., Sinning, I., Wild, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overproduction,+purification,+crystallization+and+preliminary+X-ray+analysis+of+human+Fe65-PTB2+in+complex+with+the+amyloid+precursor+protein+intracellular+domain.">Overproduction, purification, crystallization and preliminary X-ray analysis of human Fe65-PTB2 in complex with the amyloid precursor protein intracellular domain.</a> Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 64, 409-412 (2008).</li><li>Chen, T. Y., Liu, P. H., Ruan, C. T., Chiu, L., Kung, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intracellular+domain+of+amyloid+precursor+protein+interacts+with+flotillin-1,+a+lipid+raft+protein.">The intracellular domain of amyloid precursor protein interacts with flotillin-1, a lipid raft protein.</a> Biochem. Biophys. Res. Commun. 342, 266-272 (2006).</li><li>Kim, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Notch1+signaling+by+the+APP+intracellular+domain+facilitates+degradation+of+the+Notch1+intracellular+domain+and+RBP-Jk.">Regulation of Notch1 signaling by the APP intracellular domain facilitates degradation of the Notch1 intracellular domain and RBP-Jk.</a> J. Cell Sci. 124, 1831-1843 (2011).</li><li>Lazarov, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport,+amyloid+precursor+protein,+kinesin-1,+and+the+processing+apparatus:+revisited.">Axonal transport, amyloid precursor protein, kinesin-1, and the processing apparatus: revisited.</a> J. Neurosci. 25, 2386-2395 (2005).</li><li>Kakuda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equimolar+production+of+amyloid+beta-protein+and+amyloid+precursor+protein+intracellular+domain+from+beta-carboxyl-terminal+fragment+by+gamma-secretase.">Equimolar production of amyloid beta-protein and amyloid precursor protein intracellular domain from beta-carboxyl-terminal fragment by gamma-secretase.</a> J Biol. Chem. 281, 14776-14786 (2006).</li><li>Gosal, W. S., Myers, S. L., Radford, S. E., Thomson, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+under+the+atomic+force+microscope.">Amyloid under the atomic force microscope.</a> Protein Pept. Lett. 13, 261-270 (2006).</li></ol>

没有利益冲突的声明。

在这个协议中，我们提出的结构，生物物理和生物化学分析，获得高纯度的AICD的程序。此过程不需要复杂的色谱仪，因此大多数实验室访问。其他团体纯化的AICD 5-7,16，包括GST-AICD 17日至19日 ，生化/结构的分析。到先前的协议的缺点包括AICD 16的溶解性差，小于理想纯度17，和尺寸排阻16的要求，或反相色谱法6,20。因此，这里所描述的协议，极大地?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> PGEX-4T-1 </td><td> GE医疗集团</td><td> 28-9545-49 </td><td></td></tr><tr><td>凝血酶</td><td> GE医疗集团</td><td> 27-0846-01 </td><td></td></tr><tr><td>氨苄青霉素</td><td> Fisher Scientific则</td><td> BP1760 </td><td></td></tr><tr><td> Bradford蛋白测定试剂</td><td> Bio-Rad公司</td><td> 500-0002 </td><td></td></tr><tr><td>考马斯亮蓝</td><td> Bio-Rad公司</td><td> 161-0786 </td><td></td></tr><tr><td> IPTG（异丙基-β-D硫代半乳糖苷） </td><td> Sigma-Aldrich公司</td><td> I6758 </td><td></td></tr><tr><td>谷胱甘肽 - 琼脂糖</td><td> Sigma-Aldrich公司</td><td> G4510 </td><td></td></tr><tR&gt; <td>对氨基苯甲脒琼脂糖</td><td> Sigma-Aldrich公司</td><td> A7155 </td><td></td></tr><tr><td>完整的蛋白酶抑制剂混合物</td><td>罗氏公司</td><td> 11836170001 </td><td></td></tr><tr><td>幻灯片-A仪透析磁带的</td><td> Thermo Scientific的</td><td> 66380 </td><td></td></tr><tr><td>层析柱</td><td>长荣科学</td><td> 208-3367-050 </td><td></td></tr><tr><td>乳化剂</td><td> Avestin公司</td><td> EmulsiFlex-C3 </td><td>强烈推荐</td></tr><tr><td>的Eppendorf恒温</td><td> Eppendorf公司</td><td> 022670107 </td><td></td></tr><tr><td>云母磁盘</td><td>特德·佩拉</td><td> 50-12 </td><td></td></tr><tr><td>原子力显微镜的悬臂</td><td>布鲁克</td><td> MSNL-10 </td><td></td></tr><tr><td> WSxM软件</td><td> NANOTEC </td><td> N / A </td><td>神父EE下载</td></tr></tbody></table>

purification and aggregation of the amyloid precursor protein intracellular domain

淀粉样前体蛋白（APP）是与阿尔茨海默氏病（AD）的发病机制相关联的I型跨膜蛋白。 APP是其特征在于由一个大的胞外结构域和一个短的胞质域称为APP胞内结构域（AICD）。在成熟过程中通过分泌途径，APP可以由蛋白酶被称为α，β，和γ-分泌1裂解。顺序的蛋白水解切割APP的β和γ-分泌导致一个小的蛋白水解肽，称为Aβ是淀粉样蛋白形成和老年斑的核心成分的生产。 AICD是从膜中解放出来后分泌处理，并通过FE65和TIP60的相互作用，可转位到细胞核内参与多个靶基因的转录调节2,3。蛋白质 - 蛋白质相互作用的AICD可能会影响贩运，加工，细胞功能的全息APP和C-termin人的片段。最近，我们已经表明，AICD可以聚集在体外 ，而这个过程被抑制的AD-牵连分子伴侣ubiquilin的，1 4。与这些结果一致，AICD暴露出疏水区，并有内在的紊乱体外 5,6，但它获得稳定的二级结构时，势必FE65 7。我们已经提出，ubiquilin-1防止AICD不适当间和分子内相互作用，防止聚集在体外和在完整细胞4。虽然大多数研究专注于APP在AD的发病机制中的作用，AICD在这个过程中的作用目前尚不清楚的。已被证明的表达AICD诱导细胞凋亡8，调制信号通路9，调节钙信号10。 AICD和FE65过度表达的转基因小鼠模型中诱导阿尔茨海默氏症样病变11日 ，并于最近AICD中发现了胸罩在裂解液免疫印迹当使用合适的抗原修复技术12。为了方便的AICD的结构，生化和生物物理研究，我们开发了一个程序，以重组产生大量的高纯度AICD蛋白。我们进一步对诱导在体外热聚集，的​​AICD并分析利用原子力显微镜的描述方法。描述的方法是有用的生物化学，生物物理与结构表征，AICD的影响AICD聚集的分子伴侣。

Watch this Scientific Journal Video about Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain at JoVE.com

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

APP胞内结构域（AICD）用于大规模纯化的方法，进行说明。我们也将介绍的方法来诱导<em在体外</emAICD聚合和原子力显微镜的可视化。所描述的方法是有用的生化/结构表征AICD和聚集在其分子伴侣的影响。

淀粉样前体蛋白胞内结构域的分离纯化和聚集

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

purification-aggregation-amyloid-precursor-protein-intracellular

Research

JoVE Journal

Medicine

11.8K 次观看.  University of Texas Medical Branch. APP胞内结构域（AICD）用于大规模纯化的方法，进行说明。我们也将介绍的方法来诱导

视频: Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Performing and Processing FNA of Anterior Fat Pad for Amyloid

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis. Since the clinical presentation of this disease is so variable and non-specific, the associated risks of these biopsies are too great for the diagnostic yield. Other sites that have a lower biopsy risk, such as skin or gingival, are also relatively invasive and expensive. In addition, these biopsies may not always have sufficient amyloid deposits to establish a diagnosis. Fat pad aspiration has demonstrated good clinical correlation with low cost and minimal morbidity. However, there are no standardized protocols for performing this procedure or processing the aspirated specimen, which leads to variable and nonreproducible results. The most frequently utilized modality for detecting amyloid in tissue is an apple-green birefringence on Congo red stained sections using a polarizing microscope. This technique requires cell block preparation of aspirated material. Unfortunately, patients presenting in early stage of amyloidosis have minimal amounts of amyloid which greatly reduces the sensitivity of Congo red stained cell block sections of fat pad aspirates. Therefore, ultrastructural evaluation of fat pad aspirates by electron microscopy should be utilized, given its increased sensitivity for amyloid detection. This article demonstrates a simple and reproducible procedure for performing anterior fat pad aspiration for the detection of amyloid utilizing both Congo red staining of cell block sections and electron microscopy for ultrastructural identification.

Fat pad aspiration is a preferred, minimally invasive, and low cost approach as compared to other methods to detect amyloid for diagnosis of systemic amyloidosis. This video article demonstrates a procedural outline for performing fat pad aspiration with appropriate processing of the specimen for the optimal diagnostic outcome.

表演和加工为淀粉样蛋白前脂肪垫活检

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

科研

医学

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

MACS纯化光感受器前体细胞的亚视移植到成年小鼠视网膜中

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

在实验颈脊髓损伤协同使用神经前体细胞，并自组装肽的

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

自发交替评估，淀粉样蛋白β和脑神经病理学转基因小鼠模型中的新物体识别和肢体紧扣

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor of Pannexin1 (Panx1) channels.

We describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. As an example we apply this method for studying the aggregation response of human platelets to collagen after a pre-incubation with the Pannexin1 channel inhibitor Brilliant Blue FCF.

比浊法对人洗涤血小板:所述Pannexin1抑制剂的影响亮蓝FCF对胶原诱导的聚集

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or ...

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

肌网钙储备与细胞内舒张钙在心室肌细胞中的分离评价

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

在体内图像的Cx3cr1gfp/gfp 记者光谱域光学相干层析成像和扫描激光眼底

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

鼠心围细胞的分离与纯化

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

Implantation of Combined Telemetric ECG and Blood Pressure Transmitters to Determine Spontaneous Baroreflex Sensitivity in Conscious Mice

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. The baroreflex is a key homeostatic mechanism to counteract acute, short-term changes in arterial BP and to maintain BP in a relatively narrow physiological range. BP is sensed by baroreceptors located in the aortic arch and carotid sinus. When BP changes, signals are transmitted to the central nervous system and are then communicated to the parasympathetic and sympathetic branches of the autonomic nervous system to adjust HR. A rise in BP causes a reflex decrease in HR, a drop in BP causes a reflex increase in HR.
Baroreflex sensitivity (BRS) is the quantitative relationship between changes in arterial BP and corresponding changes in HR. Cardiovascular diseases are often associated with impaired baroreflex function. In various studies reduced BRS has been reported in e.g., heart failure, myocardial infarction, or coronary artery disease. 
Determination of BRS requires information from both BP and HR, which can be recorded simultaneously using telemetric devices. The surgical procedure is described beginning with the insertion of the pressure sensor into the left carotid artery and positioning of its tip in the aortic arch to monitor arterial pressure followed by the subcutaneous placement of the transmitter and ECG electrodes. We also describe postoperative intensive care and analgesic management. After a two-week period of post-surgery recovery long-term ECG and BP recordings are performed in conscious and unrestrained mice. Finally, we include examples of high-quality recordings and the analysis of spontaneous baroreceptor sensitivity using the sequence method.

The baroreflex is a heart-rate regulation mechanism by the autonomic nervous system in response to blood-pressure changes. We describe a surgical technique to implant telemetry transmitters for continuous and simultaneous measurement of electrocardiogram and blood pressure in mice. This can determine spontaneous baroreflex sensitivity, an important prognostic marker for cardiovascular disease.

植入遥测心电图和血压递质组合以确定意识小鼠的自发压力反射敏感性

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. ...