我们感谢 Kopito, 罗和皮尔斯实验室的成员在这些方法开发过程中的许多有益的讨论。我们还感谢布赖恩 Temsamrit 对这篇手稿的批判性阅读。这项工作得到了科学大学和水史密斯慈善信托基金的资助。

1. 在果蝇中耦合 Gal4 和 Htt 的转基因表达
<ol><li>收集和/或生成含有组织特定 Gal4 或资历 "驱动程序" 的转基因果蝇线, 以及包含 Gal4-UAS38或 Htt QUAS39转基因下游的野生型或突变体的线。确保从这些转基因表达的蛋白质被融合到荧光蛋白或表位标记, 以允许分化的突变和野生型 Htt 转基因产品在同一苍蝇。请参见图 1。 注意: 我们通常使用的外显子1片段的人类 Htt 基因27。然而, 转基因苍蝇可以生成, 以表达较长的 Htt 片段或其他基因, 如果需要的话。</li>
	<li>计划基因策略, 如突变体和野生型 Htt 转基因将表达在不同的, 非重叠的细胞群体。<ol>
<li>如果任何表达重叠发生, 突变体和野生型 Htt 蛋白合成在同一细胞将共同聚集和防止检测朊病毒样的事件。为避免这种情况, 请分别使用 Gal4 和阻遏、Gal80 和 QS40(图 1)。选择发育控制的 Gal4 和/或有资历的驱动程序可以帮助限制突变体的表达 Htt 后有丝分裂细胞, 消除了细胞分裂可能有助于细胞对细胞聚集扩散的可能性。</li>
	</ol>
</li>
	<li>使用标准培养条件, 交配苍蝇产生的后代, 通过 Htt (或 Gal4) 在一个 "捐献者" 细胞种群和野生类型的 Htt 通过 Gal4 (或资历) 在 "接受" 细胞群体中表达突变体。有关显示可能的遗传组合的示意图, 请参见图 1 。 注意: 用于生成图 1-4和我们以前的出版物27中显示的数据的资历和 Gal4 驱动程序包括 DA1 嗅受体神经元 (ORN) 驱动程序、Or67d-QF 和泛胶质驱动程序 repo-Gal4。</li>
	<li>生成控制苍蝇平行, 表达野生型 Htt 在 Gal4-labeled 细胞种群。</li>
	<li>收集所需基因型的后代, 并适当地将动物年龄。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56906/56906fig1.jpg"> 图 1.利用 QUAS 和 Gal4-UAS 二元表达系统对突变体和野生型 Htt 转基因进行耦合表达的遗传方法.在 "细胞 A" 中, 一种含有致病长度 polyQ 拉伸 (Q91) 的 mCherry 标记突变 Htt 蛋白, 使用位于组织特定启动子 A ("PA") 下游的一个资历驱动程序表示。在 "细胞 B" 中, 含有正常 polyQ 拉伸 (Q25) 的 YFP 标记野生型 Htt 通过由组织特定启动子 b ("PB") 控制的 Gal4 驱动程序表示。在图 2-4中, Or67d-QF 用于在 DA1 ORNs 中驱动 QUAS-HttQ91-mCherry 表达式, repo-Gal4 用于在所有胶质细胞27中表示 UAS-HttQ25-YFP。重要的是, HttQ91-mCherry 只是表达了在表达的细胞, 凭借 QUAS 序列放置在上游的转基因。同样, HttQ25-YFP 只通过 Gal4 来表示, 它专门识别无人使用。如果检测到 Gal4 驱动程序的组织分布有任何重叠, 则可以介绍 Gal4-expressing 细胞中的 QS 转基因编码和 Gal80 的表达。将荧光蛋白标签添加到野生型和突变体 Htt, 可以在成像过程中对两种蛋白质进行分化, 并能够测量适当的供者/受体对 (例如、CFP/YFP 或 YFP/mCherry) 之间的焦虑。<a href="//cloudflare.jove.com/files/ftp_upload/56906/56906fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
2. 成人果蝇脑的显微解剖和固定
注意: 此解剖过程已从以前的出版物44中进行了修改, 可用于为 Htt 荧光蛋白融合的成像直接荧光信号准备大脑。下一节将讨论对染色大脑的过程进行的修改。
<ol><li>收集下列材料和放置在冰上: 磷酸盐缓冲盐水含有0.03% 海卫 X-100 (PBS/T);离心管含有970µL 的4% 多聚甲醛 (粉煤灰) 固定液, 加入200µL 20% 粉煤灰至770µL PBS/T;含有良好的透明玻璃碟;一次性转移吸管;两个解剖钳 (一3和一 5)。</li>
	<li>麻醉使用 CO2的成人苍蝇, 并将它们转移到冰上的玻璃盘中的一个井中。</li>
	<li>使用转移吸管, 增加一个小数量 (~ 500 µL) 冷 PBS/T 对井包含苍蝇并且到空井。避免引入太多的气泡, 因为它们可以干扰解剖。</li>
	<li>将玻璃盘子放在解剖显微镜下的平坦表面上, 调整放大倍数, 直到飞体填充视野并聚焦。</li>
	<li>放置一对鹅颈光源, 使光线照亮玻璃盘子的两面。在玻璃盘子下锚定一个折叠的实验室组织, 在解剖过程中丢弃身体部位/角质层。</li>
	<li>在非显性手上使用3钳, 将一只苍蝇转移到含有 PBS/t 的井中, 通过抓住腹部与腹侧朝上, 将苍蝇固定在飞行中。</li>
	<li>保持苍蝇完全淹没在 PBS/T, 删除飞头与5钳在主手。在苍蝇头一侧的喙旁边的小空间中, 将这个钳的一个尖端插入到角质层下。通过捏紧钳子抓住两个侧面的眼睛, 以确保头部的抓地力。</li>
	<li>将两个钳拆开, 将飞头从其体内取出。在附近的实验室组织中处理飞行物体。保持主手钳的压力, 使飞头不会丢失。</li>
	<li>将3钳的一个尖端放在非显性的手里, 在喙另一侧的角质层下的同一小空间内。一旦定位, 捏钳, 抓住眼睛在同一位置两侧的头部。</li>
	<li>一旦对角质层的抓地力是安全的, 轻轻地拉钳在180°分开。这一行动将打破头部角质层, 而不损害大脑。在实验室组织中丢弃角质层残留物。<ol>
<li>虽然理想, 头部角质层可能不会完全删除一步。在这种情况下, 取出角质层, 直到大脑完全暴露。注意不要通过避免与镊子直接接触而损害大脑。</li>
	</ol>
</li>
	<li>从 PBS/T 中取出解剖过的大脑, 或者通过抓住连接的气管, 或者通过毛细管动作将大脑吸进钳尖之间的空间。 注: 气管切除是可选的, 因为它往往不会掩盖我们通常想象的大脑前表面的触角裂片。然而, 如果气管干扰成像, 仔细地删除它们, 这样大脑就不会被这种操作所破坏。</li>
	<li>将飞入的大脑转移到含有冰上固定液的离心管中。确保大脑与镊子分离, 并浸入固定液中。</li>
	<li>一旦所有的大脑被解剖, 将其置于一个 "短修复" 的 nutator, 在室温下将闭合的管子放置在黑暗中5分钟。 注意: 这个短的固定步骤确保大脑在随后的步骤中更容易处理, 不坚持离心管的两侧。</li>
	<li>用 P1000 吸管除去大多数固定液, 然后丢弃。<ol>
<li>避免在这段期间和任何随后的洗涤步骤中从管子中吸出大脑。将 P1000 设置为较小的卷 (例如, 650 µL), 并在两个步骤中移除上清。另外, 将离心管与光源 (如架空灯) 保持一致, 同时吸气以更好地可视化大脑。</li>
	</ol>
</li>
	<li>增加1毫升的新鲜 PBS/T 的大脑。在黑暗中迅速洗涤 (< 1 分钟), 从大脑中吸取上清, 然后丢弃。</li>
	<li>根据以下时间表在黑暗中重复洗涤与 PBS/T 在室温下: 2 快速 (< 1 分钟) 洗涤, 1 x 5 分钟, 3 X 20 分钟和 1 X 1 h 洗涤。在离心管上安全顶部, 并将管子放在洗涤间的 nutator 上。<ol>
<li>如果需要 DAPI 染色, 请用 1 x 30 分钟的孵化 DAPI 在 PBS/T 中稀释1小时, 然后用 2 x 快速和 1 x 20 分钟洗涤。</li>
	</ol>
</li>
	<li>最后洗完后, 取出大部分洗涤缓冲器上清, 注意不要扰乱大脑, 并添加30µL 甘油基 antifade 试剂给大脑。在黑暗中孵化4摄氏度, 不运动至少1小时, 最多24小时。</li>
</ol>3. 对成人大脑染色2节的修改
注: 使用本协议用于成像非荧光蛋白或荧光蛋白融合弱荧光。
<ol><li>每步增加10倍 (pbs + 0.3% 海卫 X-100) 在 PBS/T 中 X-100 的浓度。这确保了足够的洗涤剂存在 permeabilize 细胞膜和允许抗体进入细胞内的空间。</li>
	<li>在室温下将4% 只粉煤灰中的大脑固定在 PBS/T 中, 20 分钟。</li>
	<li>在完成所有的洗涤后, 孵化大脑在新鲜准备的阻断缓冲 (PBS/T + 5% 正常山羊血清 ()) 在室温下在黑暗中30分钟。</li>
	<li>删除并丢弃阻塞缓冲区。添加0.5 毫升的主抗体溶液每管准备作为一个主混合, 稀释原抗体适当的新鲜堵塞缓冲区 (见材料表, 用于常用抗体和稀释)。在 nutator 的主要抗体上孵育大脑至少24小时, 在4摄氏度的黑暗中。</li>
	<li>用 P1000 和储备在新鲜的试管中去除大脑中的主要抗体溶液。添加1毫升的 PBS/T 洗脑。 注意: 保留的主要抗体溶液可以储存在4摄氏度, 长达四周。在随后的实验中, 我们成功地将原抗体回收了两次。</li>
	<li>快速冲洗大脑 (< 1 分钟), 从大脑中吸取上清, 然后丢弃。再次重复此快速清洗。</li>
	<li>在室温下继续以1毫升的 PBS/T 洗涤, 按照以下时间表: 1 x 5 分钟, 3 x 20 分钟, 1 X 1 小时清洗。在离心管上安全顶部, 在洗涤过程中将管子放在 nutator 上。</li>
	<li>最后一次清洗后, 除去大多数的 PBS/T 上清, 并添加0.5 毫升的二级抗体解决方案, 作为一个主混合, 稀释的抗体适当的新鲜堵塞缓冲区 (见材料表常用抗体和稀释)。在黑暗中, 在4摄氏度的24小时内孵化大脑的二级抗体。</li>
	<li>在3.5、3.6 和3.7 步骤中重复清洗步骤, 然后再用二级抗体进行孵化。</li>
	<li>最后洗完后, 取出大部分的洗涤缓冲器, 确保所有的大脑都位于管子的底部。给大脑添加30µL antifade 试剂。在黑暗中孵化4摄氏度, 不运动16-24 小时。</li>
</ol>4. 全脑安装
<ol><li>将玻璃显微镜幻灯片放在解剖显微镜下, 用标识信息进行标签。 注: 或者, 大脑可以直接安装在盖玻璃上, 以便在成像过程中访问大脑的两侧。描述此安装过程的协议以前已发布45。</li>
	<li>使用钝吸管尖 (用剃刀刀片将底部 ~ 1 厘米从1-200 µL 尖上取下) 取出每个离心管的大脑, 并将它们转移到幻灯片中间。尽可能少的用大脑 antifade 试剂。</li>
	<li>使用镊子将大脑定位在所需的方向上 (例如, 背向指向幻灯片顶部和朝向触角叶成像的前表面)。在定向大脑时, 考虑显微镜的光路, 用于成像它们。</li>
	<li>使用折叠的实验室组织的尖角, 不干扰大脑, 从幻灯片中取出多余的 antifade 试剂。让幻灯片在黑暗中坐5-10 分钟, 让大脑坚持幻灯片。</li>
	<li>周围的苍蝇大脑与四小块的破碎盖玻璃放置在大脑的每一侧, 形成一个正方形, 是 ~ 19 毫米 x 19 毫米. 将 22 mm x 22 毫米1.5 盖玻璃的一个边放在这些小玻璃片的外面, 并轻轻地降低盖玻片的大脑, 以完成桥安装。</li>
	<li>慢慢地分配新鲜的 antifade 试剂填充盖玻片下的表面, 小心不要扰乱盖玻片或大脑。用折叠的实验室组织的尖角擦拭任何多余的 antifade, 而不直接接触盖玻片。</li>
	<li>添加一滴清晰, 快速干燥指甲油四角的盖玻片。允许干燥10分钟。然后, 用指甲油封住盖玻片的四边缘, 完全包围大脑。</li>
	<li>图像大脑立即, 或存储在4°c, 直到准备就绪。<ol>
<li>如果从 Htt 荧光蛋白融合成像内在荧光, 图像的大脑在24小时内的最佳信号。</li>
	</ol>
</li>
</ol>5. 影像学和量化的聚集体病毒样传播
<ol><li>使用装有40X 或 60/63 x 油的共焦显微镜图像安装的大脑目的是通过大脑区域收集 z 切片图像 (图 2a, B), 这些 Gal4 和资历驱动程序被表示出来。<ol>
<li>激发荧光蛋白融合或 immunolabeled 蛋白质使用适当的激光器 (例如, 488 毫微米为 GFP/YFP/FITC 或552毫微米为 mCherry/Cy3)。设置检测窗口, 捕捉最大的荧光信号, 同时消除通道交叉交谈使用的光谱检测系统或带/长通行证发射过滤器特定的每个荧光。 注意: 成像蛋白聚集时要细心。在每个聚合体中心的像素很容易变得饱和, 特别是在较大的骨料中。尝试尽量减少图像中的饱和度, 但要知道从较小的聚合种中丢失信号的风险 (图 2B、C、D)。过多的饱和度会增加图像的背景, 从而更难识别孤立的 puncta。在拍摄每个数据集的最终图像之前, 测试不同的成像设置以进行优化。</li>
	</ol></li>
	<li>通过通过单独的 z 切片 (例如、 图 2和图 4) 或在3维度中呈现共焦切片 (图 3 ) 后, 通过手动量化单个 puncta 来分析数据。A、B)。 注意: 这可以在图像 J 或其他图像处理软件中完成。<ol>
<li>如果聚合体分离良好并且有极小的背景荧光, 则使用图像分析软件系统地识别、量化和分析共焦栈3D 重建中的不同 "对象" 或 "曲面" (图 3B)。 注意: 所描述的软件操作特定于此处使用的仪器和软件 (请参阅材料表)。<ol>
<li>在3D 查看模式下可视化共焦 z 系列。使用 "分析" 向导标识选定通道中的各个点 (例如图 3中 HttQ91-mCherry 聚合的红色通道)。调整阈值和筛选器, 以准确地将所有异种地大小的聚合作为图像中的单个对象来表示。</li>
			<li>在 "二进制处理预筛选" 下启用 "拆分对象", 以分离与软件算法合并的 aberrantly 紧密关联的聚合。请注意, 在 "测量" 下报告的对象总数及其相关度量值。 注: 这种量化突变 Htt 骨料的方法不适合染色协议, 因为淀粉样体聚集的中心, 对抗体是坚不可摧的。结果表明, 聚集体在显微镜下作为环形结构出现, 图像分析软件无法准确识别和区分这些个体的 "斑点"。</li>
		</ol>
</li>
		<li>通过手动移动 z 堆栈和评分野生类型的 Htt 聚合来量化野生类型的 Htt 聚合, 确保如果它们出现在多个切片 (图 4A) 中, 则不会对聚合进行双重评分。 注意: 如果每个共焦堆栈中的聚合数合理 (例如20-50), 则可以使用此手动量化方法。当图像分析软件无法将单个 puncta 与周围的信号 (例如) 区分开来时, 它也很有用 (图 2B, C和图 4A). 对野生型 Htt 骨料进行量化也很有挑战性, 因为大脑中的许多正常结构出现点状 (例如、细胞过程和突触)。野生型和突变 Htt 信号的协同定位可以作为选择标准;然而, 某些突变体的 Htt "种子" 可能低于共焦检测的极限。不在接收单元 (例如、GFP) 中聚合的非 Htt 的蛋白质可用于标记大脑中不相关的点状结构。</li>
	</ol></li>
	<li>使用图像分析软件对单个骨料进行进一步的表征。例如, 确定聚合的大小分布 (图 3C)、突变体和野生型 Htt 蛋白之间的协定位百分比 (图 4A), 或使用烦恼直接测量蛋白质-蛋白质相互作用 (图 4B)。<ol>
<li>使用现场或表面检测算法确定单个 puncta 的大小分布, 以便准确地识别特定通道中的所有可见聚合。使用图像分析软件对点或曲面进行相关测量, 例如, 在步骤5.2.1 中描述的软件 "分析" 向导中, 从 "测量" 中获取 "总直径" (图 3C)、"体积" 或 "强度" 信息。 注意: 在图 3C中, 我们报告了在单个 DA1 肾小球中确定的所有 HttQ91-mCherry puncta 的直径分布。</li>
		<li>通过通过共聚焦 z 堆栈 (最精确的方法) 手动移动切片, 确定 HttQ25-YFP 和 HttQ91-mCherry 聚合之间的百分比。但是, 注意不要数两次聚合。为防止这种情况发生, 仅当焦点的平面通过聚合的中心 (图 4a) 时, 才计算特定 z 切片中的聚合。</li>
		<li>采用受体漂白法计算 colocalized HttQ91-mCherry 信号诱发 HttQ25-YFP 集料的烦恼效率。<ol>
<li>首先, 在 YFP 和 mCherry 通道之间消除潜在的交叉对话, 方法是建立检测窗口, 以便在其他信道中不产生信号的 mCherry 和仅 YFP 信号。使用这些设置, 请在单个 HttQ25-YFP puncta 及其关联的 HttQ91-mCherry 信号 (图 4B) 中采用 "前映像"。</li>
			<li>然后, 通过调整红色激光器 (例如, 552 nm) 到100% 的强度和扫描直到信号消失, photobleach mCherry 信号。还原为 "前映像" 所用的设置, 并在同一 puncta 中采取 "后映像" (图 4B)。</li>
			<li>用图像分析软件计算漂白前后各泪的荧光强度测量。</li>
			<li>通过在 "图像后" (YFPfinal) 中, 从 YFP 捐赠者荧光中减去在 "图像前" (YFP初始) 中测量的 YFP 捐赠者荧光来计算烦恼效率。通过 YFPfinal除以此值, 并乘以100。 注意: 对于每个 HttQ25-YFP 聚合 (图 4B), 可以按像素或整体计算 "烦恼" 效率。受体漂白是一个特别有用的技术, 以计算苦恼效率时, mCherry 信号与每个 HttQ25-YFP 泪是足够高, 以产生可检测 YFP dequenching 后, mCherry。</li>
		</ol>
</li>
	</ol></li>
</ol>

作者没有什么可透露的。

随着神经退行性疾病患者的数量继续增加, 迫切需要增加对这些疾病分子发病机制的了解, 以便更好地开发治疗方法。在这里, 我们描述的方法, 可以监测病毒样的传播致病蛋白聚集在不同的细胞类型之间的模型有机体,果蝇。我们最近使用这种方法来演示突变体 Htt 集的病毒样传播在体内, 并确定一种吞噬性受体, 介导这些聚合体从神经元扩散到胶质细胞27。我们的方?...

朊病毒假说指出, 负责传染性海绵状性脑病(例如, 人克雅氏病, 羊瘙痒病绵羊, 鹿和麋鹿的慢性浪费疾病, 和牛的疯牛病) 的传染性剂) 完全由蛋白质组成, 缺乏核酸1。在朊病毒疾病中, 细胞朊蛋白 (prpC) 假设一个非本机、稳定的折叠 (prpSc), 它具有高度 beta 版, 可以通过将单体 PrPC分子转换和招募到稳定的淀粉样中进行自我传播。聚合.PrPSc聚合使用此自复制机制在有机体中的不同单元格之间进行传播, 甚至在单个有机体之间进行扩展2。
蛋白质错误折叠和聚集也是大多数神经退行性疾病 (阿尔茨海默病 (AD)、帕金森病 (PD)、亨廷顿氏病 (HD) 和肌萎缩侧索硬化症 (ALS)) 的中心特征 3.这些疾病中或超细胞内聚集蛋白的形成与细胞毒性4密切相关, 随着时间的推移, 通过大脑的高度可重现性和疾病特定的路径进展5,6. 这些传播模式表明, 与这些疾病相关的致病性聚集物具有类似朊病毒的性质。现在有强有力的支持, 因为朊病毒样的传播与 AD、PD、HD 和 ALS 有关的聚合体-它们从细胞到细胞和模板在以前未受影响的细胞中单体形式的构象变化7, 8。
迄今为止, 大多数研究都是利用哺乳动物细胞培养模型进行的, 调查蛋白质聚集蛋白样的扩散, 从细胞外空间或从另一细胞中转移到天真细胞的细胞质中。细胞质9,10,11,12,13,14,15, 或将含骨料材料注入鼠标大脑和监测聚合外观在注入站点的外部16,17,18,19,20,21,22, 23. 最近, 转基因动物被用来证明胞内聚集物扩散到完整大脑中的其他细胞中24,25,26,27, 28,29,30。在这里, 我们描述了一个方法, 直接可视化的总转移之间的单个细胞在完整的大脑中的果蝇。果蝇模型的 HD/polyglutamine (polyQ) 疾病的首次开发近两年前的31,32 , 并提供了许多宝贵的洞察力的致病机制, 这些疾病的根源33. HD 是一种遗传性神经退行性疾病, 由常染色体显性突变引起, 该基因的编码为蛋白杭丁顿蛋白 (Htt) 34.这种突变导致在 Htt 的 N 终点附近的 polyQ 伸展扩展超过 37 glutamines 的致病阈值, 导致蛋白质 misfold 和聚合35,36。野生型 Htt 蛋白含有 < 37 glutamines 在这个拉伸达到他们的本机褶皱, 但可以诱导聚合后, 直接物理接触 Htt 聚合 "种子"12,27,37。我们利用这一着, 有核聚集的野生型 Htt 作为一个读数的朊病毒样转移和细胞质进入的突变 Htt 聚合体起源于其他细胞。
确定像朊病毒样的聚集物在细胞间穿梭的机制, 可以为无法治愈的神经退行性疾病确定新的治疗靶点。我们利用快速生命周期, 易用性和基因驯良的果蝇, 以确定分子机制的细胞对细胞扩散的突变 Htt 集。我们的实验策略采用了两个二进制表达系统, 在果蝇, 建立良好的 Gal4-specific 上游激活序列 (Gal4-UAS) 系统38和最近开发的 QUAS 系统39。耦合这两个独立的系统允许限制突变体和野生型 Htt 转基因在同一飞行中的不同细胞群的表达40。利用这一方法, 我们通过监测胞质野生型 Htt 从其通常弥漫性、可溶性状态到聚集状态的分布, 对突变体的 Htt 的传播进行检查, 这是与预先形成的突变体物理接触的直接后果 Htt聚合 "种子"。利用突变体 Htt 的生化或生物物理技术, 可以证实野生型 Htt 的转化, 如荧光共振能量转移 (烦恼)9,27,41.
重要的是, 我们还可以访问大量的基因工具在果蝇, 以确定基因和/或途径, 介导朊病毒样的传播蛋白质聚集。最近, 我们使用这种方法来揭开细胞表面清道夫受体, 德雷柏42,43的关键作用, 将突变 Htt 聚合体从神经元轴突转移到果蝇中心附近的吞噬细胞中。神经系统 (中枢神经系统)27。因此, 我们在这里描述的基于遗传和影像学的方法可以揭示在简单使用但功能强大的模型有机体 (果蝇) 中有关疾病相关现象的重要基本生物学信息。

<table><tbody><tr><td>Phosphate buffered saline (PBS), 10X, pH 7.4</td><td>ThermoFisher Scientific</td><td>AM9625</td><td>Dilute to 1X</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284-1L</td><td></td></tr><tr><td>Kimwipes</td><td>Thomas Scientific</td><td>2904F24</td><td></td></tr><tr><td>20% paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences</td><td>15713-S</td><td></td></tr><tr><td>Normal Goat Serum (NGS), filtered</td><td>Lampire Biological Laboratories</td><td>7332500</td><td>Aliquot and freeze upon receipt</td></tr><tr><td>Chicken anti-GFP</td><td>Aves Labs</td><td>GFP-1020</td><td>Use at 1:500 dilution</td></tr><tr><td>Rabbit anti-DsRed</td><td>Clontech</td><td>632496</td><td>Use at 1:2000 dilution; can recognize DsRed-based fluorescent proteins (e.g. mCherry, mStrawberry, tdTomato, etc.)</td></tr><tr><td>Mouse anti-Bruchpilot</td><td>Developmental Studies Hybridoma Bank</td><td>nc82</td><td>Use at 1:100 dilution; will label active pre-synaptic structures thoughout the fly brain</td></tr><tr><td>FITC anti-chicken</td><td>ThermoFisher Scientific</td><td>SA1-7200</td><td>Use at 1:250 dilution</td></tr><tr><td>Alexa Fluor 568 anti-rabbit</td><td>Life Technologies</td><td>A11011</td><td>Use at 1:250 dilution</td></tr><tr><td>Alexa Fluor 647 anti-mouse antibody</td><td>Life Technologies</td><td>A21235</td><td>Use at 1:250 dilution</td></tr><tr><td>Slowfade Gold Antifade Reagent</td><td>Life Technologies</td><td>S36936</td><td></td></tr><tr><td>Microscope Slides (25 x 75 x 1.0 mm)</td><td>Fisher Scientific</td><td>12-550-143</td><td></td></tr><tr><td>Cover Glass (22 x 22 mm)</td><td>Globe Scientific</td><td>1404-15</td><td></td></tr><tr><td>Dumont Biology Grade Forceps, Style 3</td><td>Ted Pella</td><td>503</td><td>use in non-dominant hand</td></tr><tr><td>Dumont Biology Grade Forceps, Style 5</td><td>Ted Pella</td><td>505</td><td>use in dominant hand</td></tr><tr><td>LAS X image analysis software</td><td>Leica</td><td></td><td></td></tr><tr><td>Imaris image analysis software</td><td>Bitplane</td><td></td><td></td></tr></tbody></table>

monitoring cell-to-cell transmission of prion-like protein aggregates in drosophila melanogaster

蛋白质聚集是大多数神经退行性疾病的中心特征, 包括阿尔茨海默病 (AD)、帕金森病 (PD)、亨廷顿氏病 (HD) 和肌萎缩侧索硬化症 (ALS)。蛋白质团聚体与这些疾病中的神经病理学密切相关, 尽管不知道异常蛋白质聚集扰乱正常细胞稳态的确切机制。新出现的数据有力地支持了这种假说, 即 AD、PD、HD 和 ALS 中的致病性聚集物与普利子有许多相似之处, 它们是负责传染性海绵状性脑病的仅蛋白感染性药物。普利子自复制通过模板化同一蛋白质本机折叠的版本的转换, 导致聚集表型的传播。在 AD、PD、HD 和 ALS 中, 普利子和朊病毒样蛋白是如何从一个细胞移动到另一单元的, 目前是一个激烈调查的领域。在这里, 一个果蝇模型, 允许监测朊病毒样, 细胞对细胞的传播与 HD 相关的突变杭丁顿蛋白 (Htt) 聚集。该模型利用强大的工具来处理许多不同的果蝇组织中的转基因表达, 并利用荧光标记的细胞质蛋白直接报告突变 Htt 聚合体朊病毒样转移。重要的是, 我们在这里描述的方法可以用来确定新的基因和途径, 在不同的细胞类型之间, 介导蛋白质聚合体的传播在体内。从这些研究中获得的信息将扩大对神经退行性疾病的致病机制的有限理解, 并揭示治疗干预的新机会。

这里描述的方法可以产生强健的数据, 表明在完整的飞行中枢神经系统中, Htt 蛋白聚集物从一个细胞种群转移到另一个体细胞。在捐赠者 ORNs 中 HttQ91-mCherry 表达式 (图 2a-c和图 4a, B) 中, Htt 的 YFP 融合蛋白的直接荧光观察了野生型的从弥漫到点的转化。准确报告这两个细胞之间的朊病毒转移事件需要仔细?...

Watch this Scientific Journal Video about Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster at JoVE.com

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

积累的证据支持这种观点, 即与神经退行性疾病相关的致病蛋白聚集物在细胞间扩散, 与朊病毒类似。在这里, 我们描述了一种方法, 使可视化的细胞到细胞传播的朊病毒样聚集在模型有机体,果蝇。

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), ...

monitoring-cell-to-cell-transmission-prion-like-protein-aggregates

Research

JoVE Journal

Neuroscience

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

7.9K 次观看.  University of the Sciences. 积累的证据支持这种观点, 即与神经退行性疾病相关的致病蛋白聚集物在细胞间扩散, 与朊病毒类似。在这里, 我们描述了一种方法, 使可视化的细胞到细胞传播的朊病毒样聚集在模型有机体,果蝇。

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

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie 1-3. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection 4-6. In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10, although their role in trafficking remains unclear 11-16. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17. These prior studies also suffer from the problem of differentiating between endogenous PrPC and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

监测后腹腔感染的免疫细胞贩运荧光朊病毒棒时间

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

科研

免疫与感染

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

评估神经退行性表型<em&gt;果蝇</em&gt;爬坡试验和全脑多巴胺能神经元免疫组化染色

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

神经科学

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

一种测定果蝇运动活性的简单技术

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

行为学

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

大众组织学量化在神经退行性变<em&gt;果蝇</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy that recapitulates several features of ALS including cytoplasmic inclusions, locomotor dysfunction, and reduced lifespan, we recently identified broad ranging metabolic deficits. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700&#181;&#948;6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700&#181;&#948;6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43G298S, an ALS causing variant. Here, we show how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700&#181;&#948;6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Glucose uptake is increased in Drosophila motor neurons affected by TAR DNA binding protein (TDP-43) proteinopathy, as indicated by a FRET-based, genetically encoded glucose sensor.

测量TDP-43蛋白病果 蝇 模型中的葡萄糖摄取

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

调查的传播和毒性朊病毒样蛋白使用后生动物模式生物<em&gt;温度。线虫</em

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

生物学

通过钙成像监测 果蝇 的癫痫样事件

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals worldwide. Despite the clinical importance of epilepsy, the functional analysis of neural activity in the central nervous system is still to be developed. Recent advancements in imaging technology, in combination with stable expression of genetically encoded calcium indicators, such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels. Drosophila melanogaster has emerged as a tool for investigating the molecular and cellular mechanisms underlying epilepsy due to its sophisticated molecular genetics and behavioral assays. In this study, we present a novel and efficient protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The whole brain is prepared from cac, a well-known epilepsy gene, knockdown flies for calcium imaging with a confocal microscope to identify the neural activity as a follow-up to the bang-sensitive seizure-like behavior assay. The cac knockdown flies showed a higher rate of seizure-like behavior and abnormal calcium activities, including more large spikes and fewer small spikes than wild-type flies. The calcium activities were correlated to seizure-like behavior. This methodology serves as an efficient methodology in screening the pathogenic genes for epilepsy and exploring the potential mechanism of epilepsy at the cellular level.

作者聚焦:对癫痫研究最新进展的技术和发现的见解 

Here, we present a protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The protocol provides a valuable tool for investigating ictal events in adult Drosophila through ex vivo calcium imaging, allowing for exploration of the potential mechanisms of epilepsy at the cellular levels.

离体 癫痫 果蝇 模型的钙成像

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals ...

监测果蝇中朊病毒样蛋白聚集物的细胞间传输

摘要

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此视频中的章节

监测后腹腔感染的免疫细胞贩运荧光朊病毒棒时间

评估神经退行性表型

一种测定果蝇运动活性的简单技术

大众组织学量化在神经退行性变

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

测量TDP-43蛋白病果蝇模型中的葡萄糖摄取

调查的传播和毒性朊病毒样蛋白使用后生动物模式生物

离体癫痫果蝇模型的钙成像

离体癫痫果蝇模型的钙成像

离体癫痫果蝇模型的钙成像

监测果蝇中朊病毒样蛋白聚集物的细胞间传输

摘要

探索更多视频

此视频中的章节

监测后腹腔感染的免疫细胞贩运荧光朊病毒棒时间

评估神经退行性表型

一种测定果蝇运动活性的简单技术

大众组织学量化在神经退行性变

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

测量TDP-43蛋白病果 蝇 模型中的葡萄糖摄取

调查的传播和毒性朊病毒样蛋白使用后生动物模式生物

离体 癫痫 果蝇 模型的钙成像

离体 癫痫 果蝇 模型的钙成像

离体 癫痫 果蝇 模型的钙成像

测量TDP-43蛋白病果蝇模型中的葡萄糖摄取

离体癫痫果蝇模型的钙成像

离体癫痫果蝇模型的钙成像

离体癫痫果蝇模型的钙成像