作者赞赏以前的实验室成员 Mackett, 桑德拉 Slusher 和杰陈谁工作的优化方法的努力。作者还感谢国立卫生研究院, 迈克尔 j ·福克斯基金会, 德克萨斯理工大学健康科学中心埃尔帕索学术活动研究项目 (SARP), Lizanell 和科尔伯特科德韦尔基金会, 多系统萎缩联盟,霍伊的家庭研究, 和安娜。

1. 缓冲器和试剂的制备
<ol><li>
对硝基苯基磷酸酯 (pNPP) 缓冲液的制备
	<ol>
<li>准备50毫升的 pNPP 缓冲器, 如下所示。</li>
		<li>称量出0.30285 克的三基色 (见材料表), 并将其溶解在25毫升的超纯去离子水中。<ol>
<li>添加41.6 µL 120 mM CaCl2。调整到 pH 值7.0 与 1 M HCl 和带来最后的解决方案体积到50毫升与超纯去离子水。</li>
			<li>确保最终浓度是50毫米的三盐酸, 100 毫米 CaCl2, pH 7.0。</li>
			<li>将 pNPP 缓冲区存储在 4 oC 处。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
对于孔雀石绿解决方案, 请准备以下内容。
	<ol>
<li>准备解决方案 A , 将32.8 毫升浓缩 hcl 添加到67.2 毫升的超纯去离子水中, 以创建4米盐酸溶液。 注意: 孔雀石溶液是在通风柜中使用一次性手套、面罩和安全护目镜制备的。</li>
		<li>通过称量4.2 克钼酸铵并将其添加到95.8 毫升溶液 A 中, 准备溶液 B 。</li>
		<li>通过称量0.045 克孔雀石绿草酸盐和添加超纯去离子水来制备解决方案 C , 使最终的总容量为100毫升。</li>
		<li>通过将解决方案 B 和 C 在 1:3 (v/v) 比混合, 在室温下搅拌1小时, 准备解决方案 D 。</li>
		<li>通过使用50毫升注射器的0.22 µm 孔径硝化纤维膜过滤装置过滤解决方案 D 。</li>
		<li>存储准备的孔雀石溶液在 4 oC 保护免受光照。</li>
	</ol>
</li>
	<li>
活化剂的制备
	<ol>
<li>要准备一个1% 吐温20溶液, 吸管10µL 的吐温20成990µL 的超纯去离子水 (v/v) 然后漩涡, 直到补间20完全溶解。</li>
	</ol>
</li>
	<li>
孔雀石绿吐温 20 () 工作液的制备。
	<ol>
<li>将激活器 (步骤 1.3) 与孔雀石绿解决方案 (解决方案 D, 步骤 1.2.5) 的比例为 1:100 (例如1 µL 活化剂到99µL 孔雀石绿溶液)。</li>
	</ol>
</li>
	<li>
苏氨酸酪 (KRpTIRR) (pT) 基质的制备。
	<ol>
<li>准备一个2毫米的股票, 在冰上放置一个1毫克酪瓶, 添加550µL 的超纯去离子水, 和漩涡轻轻地溶解。<ol>
<li>使〜10等分和存储 pT 在-20oC。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
核制剂
	<ol>
<li>使用一次性手套、面罩和安全护目镜准备 synucleins 在通风罩中。</li>
		<li>重1毫克的重组核在1毫升超纯去离子水中, 最终浓度为1毫克/毫升。</li>
		<li>轻轻的涡旋以溶解和放置在冰上。</li>
		<li>准备50µL 等分并存储在-80 oC 上。</li>
	</ol>
</li>
	<li>
磷酸酯标准的制备
	<ol>
<li>准备0.1 毫米磷酸盐库存溶液如下。<ol>
<li>称0.1361 克2PO4 , 并在烧杯中放置80毫升超纯去离子水。加入搅拌棒和混合溶解。把所有的解决方案转移到一个毕业的气缸, 并带来最终的总体积为100毫升, 超纯去离子水。</li>
			<li>通过使用50毫升注射器的0.22 µm 孔径硝化纤维膜过滤器来过滤整个溶液;最后的浓度是10毫米。</li>
			<li>稀释溶液 (10 mM) 1:100;最终浓度将是0.1 毫米磷酸盐 (PO4) 用于化验标准。</li>
			<li>将磷酸盐库存解决方案存储在 4 oC 上。</li>
		</ol>
</li>
		<li>有关限制稀释的表 1, 用于制定 PO4标准, 最终总容量为1250年µL。按表1添加适量的磷酸盐和超纯去离子水到1.5 毫升管。<ol>
<li>在-20 oC 处存储准备好的 PO4标准。</li>
		</ol>
</li>
	</ol>
</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="7">磷酸盐标准</td>
		</tr>
<tr>
<td>0.1 mM PO4解决方案 (mL)</td>
			<td>0</td>
			<td>75</td>
			<td>150</td>
			<td>300</td>
			<td>600</td>
			<td>1200</td>
		</tr>
<tr>
<td>超纯水去离子水 (毫升)</td>
			<td>1250</td>
			<td>1175</td>
			<td>1100</td>
			<td>950</td>
			<td>650</td>
			<td>50</td>
		</tr>
<tr>
<td>PO4的最终浓度 [pmol]</td>
			<td>0</td>
			<td>150</td>
			<td>300</td>
			<td>600</td>
			<td>1200</td>
			<td>2400</td>
		</tr>
</tbody></table>表 1: 磷酸盐标准。
2. PP2A 测定对重组 Synucleins 的反应
注: 每个反应的最终总体积为60µL, 因此实验者必须适当调整 pNPP 缓冲器的初始容积, 以容纳每个样品的 synucleins 量。在大多数情况下, pNPP 缓冲区的容量将介于40和44µL 之间。
<ol><li>
准备每 PP2Ac 反应如下。
	<ol>
<li>在1.5 毫升的管, 增加39µL 的 pNPP 缓冲和地方在冰上。</li>
		<li>添加1µL (105 ng/µL 或 0.0007 U/µL) 的人类重组 PP2Ac (rPP2Ac)。</li>
		<li>增加4µL 0, 1.5, 7.5, 15 或30µM synucleins 到 rPP2Ac 在 pNPP 缓冲器为最后的浓度 0, 0.1, 0.5, 1.0 和2.0 µM 和孵育在4° c 为30分钟的旋转单位;最后一卷将是60µL。</li>
		<li>30分钟后, 将样品移动到冰上, 加入16µL 2 mM 的铂基板。</li>
		<li>在间歇晃动的水浴中孵育每 PP2A 化验混合物10分钟, 30 ° c。</li>
		<li>对平底96井板, 在从低到高的重复井中添加25µL 的每个 PO4标准 (0、150、300、600、1200和 2400 pmol)。</li>
		<li>接下来添加25µL 的每一个实验样品 (PP2A 化验±核), 准备在步骤 2.1.1 2.1.6, 以重复井在同一 96-井板。</li>
		<li>接下来, 在每个包含标准和样品的井中添加75µL 的工作解决方案。</li>
		<li>室温下留板10分钟。</li>
		<li>观察在磷酸盐水平上有可测量的差异的样品的颜色变化。</li>
	</ol>
</li>
</ol>3. 光度分析
<ol><li>用波长设置为 630 nm29的分光光度板阅读器分析包含标准和样品的96井板。</li>
	<li>绘制产生的吸收曲线 (在 630 nm 上读取的样本), 并注意它保持线性 2400 pmol 浓度的 PO4。</li>
</ol>

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Chapter 18, (2010).</li><li>Anwar, S., 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=Functional+alterations+to+the+nigrostriatal+system+in+mice+lacking+all+three+members+of+the+synuclein+family.">Functional alterations to the nigrostriatal system in mice lacking all three members of the synuclein family.</a> J Neurosci. 31 (20), 7264-7274 (2011).</li><li>Vargas-Medrano, J., 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=Novel+FTY720-based+compounds+stimulate+neurotrophin+expression+and+phosphatase+activity+in+dopaminergic+cells.">Novel FTY720-based compounds stimulate neurotrophin expression and phosphatase activity in dopaminergic cells.</a> ACS Med Chem Lett. 5 (7), 782-786 (2014).</li><li>Swingle, M., Ni, L., Honkanen, R. E., Moorhead, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3,+Small-molecule+inhibitors+of+Ser/Thr+protein+phosphatases.">Chapter 3, Small-molecule inhibitors of Ser/Thr protein phosphatases.</a> Methods in Molecular Biology. 365, Protein Phosphatase Protocols, 23-38 (2007).</li></ol>

Dr. 佩雷斯是1999年至2011在匹兹堡大学医学院神经学系的成员。

采用 pT 的孔雀石绿法测定 PP2Ac 活性, 因为它比使用32P 标记基板的经典方法更容易执行。这种分析的另一个优点是, 放射性同位素会带来一些风险, 而且成本高昂, 特别是32P 标记的分子, 它们的半衰期很短, 这就限制了在试剂到期前可以执行的化验数量。使用孔雀石绿而不是放射性, 也消除了处理32P 废料的需要。在测定丝氨酸/苏氨酸磷酸的活性时, 孔雀石绿测定法与使用基体 p-硝基苯 (pNPP) 的另一种色度分析方法相比, 也相当敏感, 这是非选择性的, 因为它可以化由广泛的酵素32。
商业孔雀石绿包测量 PP2A 的活动已经有一段时间, 并在实验室以前使用。然而, 当做许多孔雀石绿的化验, 这种套件变得令人望而却步的昂贵。此外, 用于 PP2A 活动的商用套件只稳定一年, 同时具有粉状和适当贮存的试剂, 可提供在结构上较长时间内稳定的现成的化验组件。
在测试 bSyn 和 gSyn 之前, 异步电动机被用作一种积极的控制来刺激 PP2Ac 的活动, 并确定化验所需的 PP2Ac 量, 还要确认所产生的数据与标准曲线保持比例 (150-2400 pmol 的 PO4).值得注意的是, 如果异步电动机刺激 PP2Ac 太强烈, 虽然反应通常是线性的, 数据将脱落的规模和化学品在该方案的沉淀, 使它无法测量准确数量的发布自由 PO4与一个板块读者.
鉴于异步电动机有助于 PP2Ac 活动在体内和在体外12,19,21,23,25, 所有 synucleins 有相当的同源性, 它已经假设所有核家族成员都可以在细胞自由测定中刺激 PP2Ac。使用这里描述的比色法, 发现事实上, 所有三重组 synucleins (异步电动机, bSyn, gSyn) 刺激 PP2Ac 活动, 提高的可能性, 所有三 synucleins 可能执行类似的功能在神经系统。然而, 大脑从帕金森氏症或痴呆与路易体体病例有较少的 PP2Ac 活动的组织中含有高度聚集的异步电动机23。鼠组织窝藏路易体体样病变也表现为减少 PP2Ac 活动25。这一点, 以及图 3中的新数据以及来自异步电动机击倒鼠标12的先前数据, 强烈建议 bSyn 和 gSyn 都不能补偿大脑中可溶性异步电动机的丢失, 或者说那些核同系物可能不与 PP2Ac 相关联, 如异步电动机所示 (图 3)。艾伦脑图谱显示定位的所有三核基因在许多脑区, 虽然三核淘汰赛小鼠已生成9,33, PP2Ac 活动尚未评估在该模型。有趣的是, 异步电动机磷酸化在 Ser129 上的马球样激酶 2, 是不太能够激活 PP2Ac12, 但这里显示的证据 bSyn 表明 bSyn 磷酸化不太可能影响 PP2Ac 的活动, 因为很少 bSyn 下来PP2Ac 在鼠标的大脑中 (图 3)。同时, 数据表明, 内源性 PP2Ac 活性调节剂, 如异步电动机, 可能有助于健康和疾病。
这里概述的协议是一个简单而强大的技术, 以确定 PP2Ac dephosphorylate 的能力, 以响应各种处理的酪基板。例如, 同样的方法可以用来测试其他 PP2Ac 激活剂, 如神经和潜在的新疗法34, 或评估 PP2Ac 抑制剂的影响,体外。同样重要的是要指出, 类似的化验可以测量 PP2Ac 的活动, 已被1D6 沉淀从细胞提取物或身体组织, 如先前所描述的作者12,19, 25. 孔雀石绿检测的未来应用存在于测量 PP2Ac 活性之外, 因为 ATP 活性也可以通过这种检测来确定。
注意, 每一次独立的 PP2Ac 化验都必须生成标准曲线。这是强制性的, 以确保所有使用的试剂是无磷酸盐, 由于自由磷酸盐人工增加背景信号和产生错误的结果。在化验的当天, 必须准备足够的新鲜的解决方案, 所有样品都要化验。此外, 公司是唯一的好, 它是做的一天, 往往在几个小时后沉淀。从供应商购买的 PP2Ac 必须 aliquotted, 并在收到后立即保存, 以防止冻结解冻循环减少其活动。由于其他磷酸可以 dephosphorylate pT 衬底 (例如PP1 和 PP535), 因此在分析 PP2Ac 未被分离的细胞或组织提取物时, 必须通过柱上的样品来除去游离磷酸盐。和特定的磷酸抑制剂必须用于确认磷酸酶的特异性, 如前所述18。

An erratum was issued for: <a href="https://www.jove.com/video/55361/recombinant-synucleins-stimulate-protein-phosphatase-2a-catalytic">Recombinant &#945;- &#946;- and &#947;-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays</a>
One of the authors&#39; names was corrected from:
Brian Marcus
to:
Brian S. Marcus

An erratum was issued for: <a href="https://www.jove.com/video/55361/recombinant-synucleins-stimulate-protein-phosphatase-2a-catalytic">Recombinant &#945;- &#946;- and &#947;-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays</a>

Erratum: Recombinant &#945;- &#946;- and &#947;-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays

synucleins 在脑和脊髓中的分布的研究表明, 所有三 synucleins 是丰富的神经组织, 虽然不同的本地化模式已经报告了1。例如, 异步电动机是最丰富的在前部位的大脑皮层和基底节2,3, bSyn 在大脑和脊髓中有更均匀的分布4,5, 而 gSyn 更丰富的脊髓和周边神经节6。虽然所有 synucleins 的一些胚胎表达可能发生, 三同系物在新生儿期间变得更加丰富4,5,6,7,8。值得注意的是, 来自核三联击倒小鼠的数据表明, 所有三 synucleins 的丢失导致年龄开始的神经元功能障碍9, 支持重要的核功能在中枢神经系统 (CNS)10,11. 目前尚不清楚核三联击的小鼠是否显示 PP2Ac 分布或活动的变化。来自单个核击倒小鼠的数据表明, 单异步电动机的丢失能够显著减少脑部12的 PP2Ac 活动。除了中枢神经系统, 所有的 synucleins 本地化到其他组织的整个身体13,14,15。
由于其与神经16,17的成熟的遗传链接, 异步电动机是最好的研究三 synucleins。佩雷斯实验室长期以来一直致力于定义异步电动机的正常功能活动, 特别是在 CNS11、12、18、19、20、21中, 22,23和最近的外围组织24,25。其中, 发现异步电动机在刺激 PP2Ac 活动中起着关键的调节作用19。PP2A 活动对正常的脑功能有广泛的贡献, 包括对多巴胺生产的调节26。PP2A 全包括三独立亚基, 催化 C 亚基, PP2Ac, 脚手架 a 亚基, 和几个不同的管理/目标 B 亚基, 帮助本地化 PP2A 特定亚细胞微, 从而提供 PP2A功能多样性27。证据显示, 异步电动机与 PP2Ac 的相互作用对其磷酸酶活性有显著的贡献在体外和在体内12,19,21,23, 25。当异步电动机积累在蛋白质聚集物, 因为它做当路易体身体形成在帕金森氏症的神经元和老年痴呆与路易体身体 (DLB), 组织与聚集的异步电动机减少了 PP2Ac 活动23,25, 当可溶性异步电动机水平降低。鉴于所有三 synucleins 具有显著的同源性28 (异步电动机与 bSyn 和56% 与 gSyn 共享66% 身份), 并且异步电动机有助于激活 PP2Ac, 因此假设核蛋白家族的所有成员都可能能够增加 PP2Ac 活动, 至少是体外。为了评估这一点, PP2Ac 的活动是为了响应重组异步电动机, bSyn 和 gSyn 使用一个简单的色度测定, 仿照 Fathi 的方法et al.29. 本文详细介绍了该方法和新的研究结果。

<table border="0" cellpadding="0" cellspacing="0" width="804">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Reagent</td>
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		<tr height="20">
			<td height="20" style="height:20px;">1 M Trizma Base</td>
			<td>Sigma-Aldrich</td>
			<td>T15003</td>
			<td></td>
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		<tr height="24">
			<td height="24" style="height:24px;">CaCl2 (calcium chloride)</td>
			<td>Fisher Scientific</td>
			<td>C70-500</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Concentrated HCl&#160;&#160;</td>
			<td>JT Baker</td>
			<td>9535-33</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Ammonium molybdate</td>
			<td>Sigma-Aldrich</td>
			<td>A1343</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Malachite green oxalate salt</td>
			<td>Sigma-Aldrich</td>
			<td>M6880</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416</td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Threonine phosphopeptide (KRpTIRR)</td>
			<td>New England peptide LLC</td>
			<td>BP12-011</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">KH2PO4 (potassium phosphate)</td>
			<td>Sigma-Aldrich</td>
			<td>795488</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recombinant human PP2A C subunit</td>
			<td>Cayman Chemical&#160;</td>
			<td>10011237</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recombinant human &#945;-synuclein</td>
			<td>rPeptide</td>
			<td>S-1001-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recombinant human &#946;-synuclein</td>
			<td>rPeptide</td>
			<td>S-1003-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recombinant human &#947;-synuclein</td>
			<td>rPeptide</td>
			<td>S-1007-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">PP2Ac 1D6 antibody</td>
			<td style="width:200px;">Millipore</td>
			<td>05-421</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PP2Ac antibody 1D6</td>
			<td>Novus Biologicals</td>
			<td>NB100-92217</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">aSyn antibody C-20</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-7011-R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">bSyn antibody EP1537Y</td>
			<td>Novus Biologicals</td>
			<td>NB100-79903</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">gSyn antibody E-20</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-10698</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zeba Spin Desalting Columns, 7K MWCO, 0.5 mL</td>
			<td>Thermo-Fisher Scientific</td>
			<td>89882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Name</td>
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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Multiskan Spectrum Plate Reader</td>
			<td style="width:200px;">Thermo Fisher Scientific&#160;</td>
			<td>51118650</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Prism Software</td>
			<td style="width:200px;">GraphPad, San Diego, CA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">pH Meter&#160;</td>
			<td style="width:200px;">Fisher Scientific&#160;</td>
			<td>13-636-AB15P</td>
			<td></td>
		</tr>
	</tbody>
</table>

recombinant &#945;- &#946;- and &#947;-synucleins stimulate protein phosphatase 2a catalytic subunit activity in cell free assays

α-核 (异步电动机), β-核 (bSyn), 和γ-核 (gSyn) 是一个保守的家庭的成员类似的伴侣的蛋白质, 是高度表达的脊椎动物神经组织。在三 synucleins, 只有异步电动机有强烈的牵连神经退行性疾病, 如帕金森氏病, 痴呆与路易体体, 和多系统萎缩。在研究正常异步电动机功能时, 数据表明异步电动机刺激了大量表达的 dephosphorylating 酶的催化亚单位的活性, PP2Ac体外和体内。先前的数据表明, 异步电动机聚集在人脑减少 PP2Ac 活动在路易体体病理, 其中可溶性异步电动机已成为不溶性。然而, 由于所有三 synucleins 在氨基酸序列中有相当的同源性, 实验被设计来测试是否都能调节 PP2Ac 活性。采用重组 synucleins 和重组 PP2Ac 蛋白, 采用孔雀石绿比色法测定活性。数据显示, 所有三重组 synucleins 刺激 PP2Ac 活性在无细胞的检测, 提高的可能性, 保守的同源性之间的 synucleins 可能赋予所有三同系物的能力绑定和激活 PP2Ac。然而, 沉淀数据表明, PP2Ac 调制可能发生在异步电动机和 PP2Ac 之间的内生相互作用的在体内。

蛋白质的功能和活性可以改变后的翻译修改, 如磷酸化。许多蛋白质可以磷酸化的激酶, 或化的磷酸酶。在蛋白质磷酸酶 2A (PP2A) 的情况下, 这种 multisubunit 磷酸酶促进磷酸的丝氨酸和苏氨酸残留在广泛的磷酸。异常的 PP2A 活动可能导致失调的蛋白质功能发生在许多疾病, 包括帕金森氏症的异步电动机可以成为 hyperphosphorylated, 并在阿尔茨海默氏症的地方 tau 蛋白可以成为 hyperphosphorylated。这为优化 in-house 程序以快速评估 PP2Ac 活动提供了依据, 并确定异常 PP2Ac 活动是否可能导致细胞功能障碍。
使用这种无细胞比色法测定, PP2Ac 活性是通过量化的自由 po4从 pT 衬底 (图 1A) 切割的数量, 并与标准曲线中的 po4的已知 picomolar 量进行比较 (图1B).PP2Ac 活动也在响应0.0、0.1、0.5、1.0 和2.0 µM synucleins (图 1A) 时进行测量, 与在没有添加核 (位于图 1A中的大箭头) 中的基线 PP2Ac 活动相比。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55361/55361fig1.jpg"> 图 1: PP2A 活化的重组 synucleins, 是衡量相对于已知水平的磷酸盐在标准.在 (a) 中, 异步电动机、bSyn 和 gSyn 在 0-2 µM 蛋白质浓度范围内进行了评估, 以便与 0-2400 pmol PO4标准进行比较。(B) 生成孔雀石绿 PP2Ac 试验的标准曲线, 以提供已知数量的磷酸盐, 以计算在实验条件下获得的值。在所有情况下, 当 PP2Ac 活动增加时, 从 pT 衬底中切割出来的自由 PO4的数量会增加并产生较深的绿色。<a href="//cloudflare.jove.com/files/ftp_upload/55361/55361fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
结果表明, 在初始孵化步骤中加入 synucleins PP2Ac 可以增加自由 PO4的量, 从而显示出增加的 PP2Ac 活动。经过多次重复的统计分析表明, 所有三重组 synucleins (异步电动机、bSyn、gSyn) 显著增加了 PP2Ac 活动 (图 2)。这些结果表明, 可溶性细胞 synucleins 量的变化可以改变 PP2Ac 活性;因此可能影响细胞稳态。然而, 不知道, 如果所有三 synucleins 贡献激活 PP2Ac在体内, 虽然数据在图 3建议异步电动机可能有助于 PP2Ac 活动在体内。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55361/55361fig2.jpg"> 图 2: PP2Ac 活动由所有三 synucleins 激发.图示的 PP2Ac 活化反应不同浓度的重组异步电动机, bSyn 或 gSyn 显示。所有三重组 synucleins 刺激 PP2Ac 活动。异步电动机在大多数实验中似乎是稍微更有力, 虽然 ANOVAs 执行使用统计软件是非常相似的整体。数据代表4-9 独立实验的平均± SEM。ns, 不显着;* p < 0.05;** p < 0.01;p < 0.0001。<a href="//cloudflare.jove.com/files/ftp_upload/55361/55361fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
为了评估哪些核 (s) 可以与大脑中的 PP2Ac 相互作用, co-immunoprecipitation (co-IP) 进行了使用野生型鼠脑和 PP2Ac-specific-antibody, 1D6。然后用 SDS 页分离蛋白质, 用已建立的方法19探测 PP2Ac、异步电动机、bSyn 和 gSyn。重组核控制显示异步电动机, bSyn 和 gSyn 抗体特异性 (图 3A)。联合 ip 数据显示, 与 PP2Ac 在脑中的主要核是异步电动机的, 更少的 bSyn 在 co ip (图 3B) 中下来, 并且没有 gSyn 在 co ip 中出现 (数据没有显示)。这表明内源性异步电动机在调节脑 PP2Ac 中具有独特的作用。它也提高了一种可能性, PP2Ac 调制与 bSyn-based 疗法可能有可能恢复 PP2A 活动在那些与 synucleinopathy, bSyn 是已知的 nonamyloidogenic, 但在这里显示可以刺激 PP2Ac30, 31。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55361/55361fig3v2.jpg"> 图 3:PP2Ac 与内源性 synucleins 在小鼠脑中的相互作用.(A) 重组异步电动机、bSyn 和 gSyn 的核特异抗体进行了探讨, 如下所述。(B) 使用小鼠脑匀浆联合 IP 进行抗体 1D6, 然后对 immunoblots 进行样品分析。数据显示为 PP2Ac, 异步电动机和 bSyn, 但不是为 gSyn 与 PP2Ac 没有共同知识产权。开始样品显示相对水平 PP2Ac (车道 1), 异步电动机 (车道 4) 和 bSyn (车道 7) 在最初的匀。结束样品显示剩余的水平 PP2Ac (车道 2), 异步电动机 (车道 5) 和 bSyn (车道 8) 在匀以后 1D6 PP2Ac Co IP。重要水平的 PP2Ac 是 immunoprecipitated 使用1D6 抗体 (车道 3), 这也带来了大量的异步电动机 (车道 6, 在箭头), 但很少 bSyn (车道 9, 强烈曝光显示微弱的 bSyn 信号, 在箭头)。* 星号和括号标记在墨迹上的非特定信号。有关所用抗体的详细信息, 请参阅材料目录。<a href="//cloudflare.jove.com/files/ftp_upload/55361/55361fig3v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Recombinant Î±- Î²- and Î³-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays at JoVE.com

Recombinant &#945;- &#946;- and &#947;-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays

本出版物提供了一项协议, 显示如何测量重组蛋白磷酸酶2A 催化亚基 (PP2Ac) 的活性, 以响应重组α-核, β-核, 或γ核蛋白使用简单的比色法。我们展示了关于α-核特异性向 PP2Ac 在小鼠脑中的发现。

重组αβ-γ-Synucleins 刺激蛋白磷酸酶2A 催化亚基活性在细胞自由测定中的作用

&#945;-Synuclein (aSyn), &#946;-Synuclein (bSyn), and &#947;-Synuclein (gSyn) are members of a conserved family of chaperone-like proteins that are highly ...

recombinant-synucleins-stimulate-protein-phosphatase-2a-catalytic

Research

JoVE Journal

Neuroscience

6.7K 次观看.  Texas Tech University Health Sciences Center El Paso. 本出版物提供了一项协议, 显示如何测量重组蛋白磷酸酶2A 催化亚基 (PP2Ac) 的活性, 以响应重组α-核, β-核, 或γ核蛋白使用简单的比色法。我们展示了关于α-核特异性向 PP2Ac 在小鼠脑中的发现。

视频: Recombinant α- β- and γ-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

本地化的蛋白质三维神经干细胞培养:一个混合的可视化方法

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

科研

神经科学

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

运行轮活动在啮齿类动物的昼夜节律的记录和分析

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Olfactory Assays for Mouse Models of Neurodegenerative Disease

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson&#8217;s disease as well as in testing potential disease-modifying therapies.

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

嗅觉测定的神经退行性疾病的小鼠模型

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

条纹分析来研究蛋白质底物使用游离海马神经元的吸引力或令人反感的活动

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

使用克隆测定神经干细胞的计数。

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize and to study. Therefore, bees have long been a favored model amongst neurobiologists and neuroethologists for studying the neural basis of social and natural behavior. It is important, however, that the experimental techniques used to study bees do not interfere with the behaviors being studied. Because of this, it has been necessary to develop a range of techniques for pharmacological manipulation of honey bees. In this paper we demonstrate methods for treating restrained or free-flying honey bees with a wide range of pharmacological agents. These include both noninvasive methods such as oral and topical treatments, as well as more invasive methods that allow for precise drug delivery in either systemic or localized fashion. Finally, we discuss the advantages and disadvantages of each method and&#160;describe common hurdles and how to best overcome them. We conclude with a discussion on the importance of adapting the experimental method to the biological questions rather than the other way around.

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

约束和自由飞行，蜜蜂的神经药理学操纵，<em&gt;蜜蜂</em

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...

Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not represent the underlying cytoarchitectural and functional complexity of the affected individuals carrying suspected disease variants. Conventional 2-D models remain incomplete representations of in vivo-like structures and do not adequately capture the complexity of the brain. Thus, there is an emerging need for more 3-D hiPSC-based models that can better recapitulate the cellular interactions and functions seen in an in vivo system.
Here we report a protocol to develop a 3-D system from undifferentiated hiPSCs based on the serum free embryoid body (SFEB). This 3-D model mirrors aspects of a developing ventralized neocortex and allows for studies into functions integral to living neural cells and intact tissue such as migration, connectivity, communication, and maturation. Specifically, we demonstrate that the SFEBs using our protocol can be interrogated using physiologically relevant and high-content cell based assays such as calcium imaging, and multi-electrode array (MEA) recordings without cryosectioning. In the case of MEA recordings, we demonstrate that SFEBs increase both spike activity and network-level bursting activity during long-term culturing. This SFEB protocol provides a robust and scalable system for the study of developing network formation in a 3-D model that captures aspects of early cortical development.

Here we report a protocol to develop a three-dimensional (3-D) system from human induced-pluripotent stem cells (hiPSCs) called the serum free embryoid body (SFEB). This 3-D model can be used like an organotypic slice culture to model human cortical development and for the physiological interrogation of developing neural circuits.

开发HiPSC衍生的无血清胚胎体用于使用生理相关测定的3-D干细胞培养物的询问

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not ...

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

监测果蝇中朊病毒样蛋白聚集物的细胞间传输

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

颅内药物治疗和啮齿动物疼痛检测

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

定量自动放射法测定维沃脑蛋白合成区域速率

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...