Name: measuring glucose uptake in drosophila models of tdp-43 proteinopathy
Uploaded: 2021-08-03T15:00:00
Description: Watch this Scientific Journal Video about Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy at JoVE.com

我们感谢Stefanie Schirmeier和Takeshi Iwatsubo提供 果蝇 菌株。我们还感谢Patricia Jansma协助亚利桑那大学Marley Imaging Core的成像工作。这项工作由美国国立卫生研究院NIH NS091299，NS115514（到DCZ），HHMI Gilliam奖学金（到EM）和本科生生物学研究计划（到HB）资助。

UAS FLII12Pglu-700μδ6转基因苍蝇在Volkenhoff等人10 中报道，并由S. Schirmeier博士提供。UAS TDP-43G298S 转基因系由T. Iwatsubo13博士提供。在Zarnescu实验室使用标准遗传方法产生了含有UAS FLII12Pglu-700μδ6和UAS TDP-43转基因的重组 果蝇 系，并在Manzo等人中报道。D42 GAL4用于单独或与TDP-43G298S 一起驱动运动神经元中葡萄糖传感器的表达。所?...

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这里详细描述的技术可以应用于使用FLII12Pglu-700μδ6测量活果蝇感兴趣的特定细胞类型的葡萄糖摄取，FLII12Pglu-700μδ6是基于FRET的传感器，可以检测葡萄糖水平的变化到毫摩尔范围10，11，12。该传感器以前曾与UAS-GAL4系统结合使用，以将其表达靶向特定的细胞类型，包括神经元9，10?...

肌萎缩性侧索硬化症（ALS）是一种进行性神经退行性疾病，目前无法治愈。ALS 影响上下运动神经元，导致运动协调丧失、不可逆性瘫痪、呼吸衰竭，并在诊断后 2-5 年内最终死亡1。ALS与代谢缺陷有关，例如体重减轻，血脂异常和代谢亢进（第2篇综述）;然而，目前尚不清楚这些代谢变化与运动神经元变性的关系。ALS和相关神经退行性疾病的一个共同点是TDP-43，这是一种参与RNA处理3，4，5的几个步骤的核酸结合蛋白。虽然TDP-43的突变仅影响3%-5%的患者，但在>97%的ALS病例的细胞质聚集体中发现了野生型TDP-43蛋白（在6中回顾）。这种病理学是通过运动神经元中人类野生型或突变TDP-43（G298S）的过表达在果蝇中建模的，这概括了ALS的多个方面，包括细胞质包涵体，运动功能障碍和寿命缩短7，8。使用这些模型，最近报道TDP-43蛋白病导致丙酮酸水平和磷酸果糖糖激酶（PFK）mRNA（糖酵解9的限速酶）显着增加。在患者来源的运动神经元和脊髓中发现了PFK转录本的类似增加，这表明在TDP-43蛋白病的背景下糖酵解被上调。有趣的是，使用饮食和遗传操作进一步增加糖酵解减轻了几种ALS表型，如运动功能障碍和TDP-43蛋白病苍蝇模型中寿命的增加，与退行性运动神经元的补偿性神经保护机制一致。
为了进一步探索糖酵解的变化并测量TDP-43蛋白病果蝇模型中的葡萄糖摄取，先前报道的基于FRET的遗传编码传感器FLII12Pglu-700μδ610专门使用UAS-GAL4表达系统在运动神经元中表达。FLII12Pglu-700μδ6葡萄糖传感器使用两种绿色荧光蛋白变体（青色和黄色荧光蛋白（CFP和YFP）之间的共振能量转移来检测细胞水平的葡萄糖。它由来自大肠杆菌MglB基因的细菌葡萄糖结合结构域组成，该基因融合到分子两端的CFP和YFP。当与葡萄糖分子结合时，传感器经历构象变化，使CFP和YFP更紧密地结合在一起并允许FRET发生，然后可用于量化细胞内葡萄糖水平10，11，12（图1）。在这里，我们展示了FLII12Pglu-700μδ6传感器如何用于确定由运动神经元中TDP-43蛋白病引起的葡萄糖摄取变化。这里描述的实验表明，与对照组相比，运动神经元中ALS相关突变体TDP-43G298S的过表达会导致葡萄糖摄取显着增加。这种方法可用于其他类型的ALS（例如，SOD1，C9orf72等）和/或其他细胞类型（例如，神经胶质细胞，肌肉），以确定与神经变性相关的葡萄糖摄取的变化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm tissue culture dishes</td>
			<td>Sigma Aldrich</td>
			<td>CLS430165</td>
			<td></td>
		</tr>
		<tr>
			<td>40X water immersion lens</td>
			<td>Zeiss</td>
			<td>440090</td>
			<td>dippable, N.A. 0.8</td>
		</tr>
		<tr>
			<td>dissection scissors</td>
			<td>Roboz</td>
			<td>RS-5618</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 forceps</td>
			<td>VWR</td>
			<td>100189-236</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #55 forceps</td>
			<td>VWR</td>
			<td>100189-244</td>
			<td></td>
		</tr>
		<tr>
			<td>Minutien pins</td>
			<td>Fine Science tools</td>
			<td>26002-10</td>
			<td>used for dissections</td>
		</tr>
		<tr>
			<td>SYLGARD 184 Silicone Elastomer Kit</td>
			<td>Dow</td>
			<td>1317318</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM880 NLO upright multiphoton/confocal microscope</td>
			<td>Zeiss</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring glucose uptake in drosophila models of tdp-43 proteinopathy

肌萎缩性侧索硬化症是一种神经退行性疾病，在诊断后 2-5 年内导致进行性肌无力和死亡。临床表现包括体重减轻、血脂异常和代谢亢进。然而，目前尚不清楚这些与运动神经元变性的关系。使用TDP-43蛋白病的 果蝇 模型，该模型概括了ALS的几个特征，包括细胞质包涵体，运动功能障碍和寿命缩短，我们最近发现了广泛的代谢缺陷。其中，糖酵解被发现是上调的，遗传相互作用实验为代偿性神经保护机制提供了证据。事实上，尽管磷酸果糖激酶（糖酵解中的限速酶）的上调，但使用饮食和遗传操作增加糖酵解被证明可以减轻运动功能障碍并延长TDP-43蛋白病苍蝇模型的寿命。为了进一步研究TDP-43蛋白病对运动神经元糖酵解通量的影响，使用了先前报道的遗传编码的基于FRET的传感器FLII12Pglu-700μδ6。该传感器由细菌葡萄糖传感域和青色和黄色荧光蛋白组成，作为FRET对。葡萄糖结合后，传感器发生构象变化，允许发生FRET。使用FLII12Pglu-700μδ6，发现表达TDP-43G298S的运动神经元（一种引起ALS的变异体）的葡萄糖摄取显着增加。在这里，我们展示了如何在TDP-43蛋白病的背景下测量表达葡萄糖传感器FLII12Pglu-700μδ6的幼虫腹侧神经索制剂中的葡萄糖摄取， 离体。这种方法可用于测量葡萄糖摄取并评估不同细胞类型或导致ALS和相关神经退行性疾病的各种突变的糖酵解通量。

腹侧神经索（VNC）中葡萄糖传感器的图像采集， 离体 为了确定基于TDP-43的ALS果蝇模型中葡萄糖摄取的差异，使用了基于FRET的遗传编码葡萄糖传感器。该传感器由CFP和YFP组成，从大肠杆菌MglB基因融合到葡萄糖结合域。葡萄糖结合引发构象变化，这可以通过CFP和YFP 10，11，12之间的荧光共振能量转移...

Watch this Scientific Journal Video about Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy at JoVE.com

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

受 TAR DNA 结合蛋白 （TDP-43） 蛋白病影响 的果蝇 运动神经元中的葡萄糖摄取增加，如基于 FRET 的遗传编码葡萄糖传感器所示。

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 ...

这种技术很重要，因为它可以测量活果蝇组织中特定细胞的葡萄糖摄取。基于FRET的传感器可以直接测量细胞内葡萄糖水平的变化。这项技术使研究人员能够实时评估ALS模型与对照组的活运动神经元内葡萄糖水平的变化。一个具有挑战性的方面是在改变缓冲液后对同一组神经元进行成像。将样品留在显微镜上有助于确保VMC尽可能少地移动。在开始解剖之前，打开成像显微镜和激光。然后收集游荡的三龄幼虫，用双蒸馏水冲洗，并将其滴在先前制备的弹性体衬里皿上的HL3缓冲液中。接下来在解剖显微镜下，使用一对镊子将幼虫的前后端，背侧朝上，用昆虫针小心地将幼虫纵向拉伸。使用一把倾斜的虹膜剪刀，在后针上方做一个切口。然后从切口向幼虫的前端垂直切割。如果需要，在添加几滴HL3缓冲液后，切除气管和其余浮动器官，而不会干扰中枢神经系统。然后固定皮瓣，拉伸体壁以暴露中枢神经系统，同时保持神经肌肉系统完整。对于图像采集，请使用带有40倍水浸透镜的正置共聚焦显微镜。然后选择405纳米激光器来激发CFP。接下来，优化采集参数，如扫描速度、平均值、物镜、变焦针孔大小和空间分辨率。调整增益，使信号处于最佳动态范围，并对所有基因型使用相同的参数。要对腹侧神经索内的运动神经元进行成像，请将带有解剖样品的硅胶培养皿放在晶状体下方并降低晶状体，使其与海藻糖蔗糖HL3缓冲液接触。确保镜头完全浸没在缓冲液中，并在需要时添加更多缓冲液。下一个。使用CFP和FRET通道，手动选择由至少六个运动神经元组成的光学选择，这些神经元的焦点位于腹侧神经索中线。然后每 10 秒采集一次图像，持续 10 分钟。这些图像表示基线。对于葡萄糖刺激，使用巴斯德移液管除去含有HL3缓冲液的海藻糖蔗糖，并加入五毫摩尔葡萄糖补充的HL3缓冲液，而不移动腹侧神经索。然后每隔 10 秒采集一次图像，再持续 10 分钟。这些图像代表刺激阶段。将图像保存为CZI文件或成像软件支持的任何文件类型，文件名包括日期，遗传背景，实验条件和使用的通道。重用参数对所有基因型进行成像。要进行图像处理，请打开Fiji-ImageJ成像软件，然后打开CZI文件，选择具有超堆栈的视图堆栈并将颜色模式设置为灰度，然后选择将通道拆分到单独的窗口中。下一个。使用漂移校正插件、turbo reg 和堆栈 reg 处理图像。通过选择插件下的堆栈 reg，然后选择转换为转换，可以单独更正每个通道。通过追踪在整个时间序列中保持焦点的所有运动神经元，手动选择感兴趣的区域或ROI。然后使用 ROI 管理器保存每个运动神经元轮廓。使用多重测量函数来测量每个时间点每个 ROI 中的平均灰度值。然后将从第一通道跟踪的ROI复制到第二通道，以获得预刺激图像。对于数据分析，请使用 FRET 和 CFP 通道的平均灰度值按 CFP 比率计算 FRET。CFP比率引起的FRET变化反映了细胞内葡萄糖浓度的变化。然后按 CFP 比率绘制每个 ROI 和时间点与时间的关系图。基于遗传编码的基于FRET的葡萄糖传感器用于测量运动神经元中的葡萄糖摄取。当葡萄糖不结合到传感器时，CFP激发仅导致CFP发射。当葡萄糖结合时，由于CFP和YFP之间发生的FRET，可以检测到YFP信号。这里显示的是基线和刺激条件下TDP-43突变神经元中葡萄糖传感器对照中的FRET和CFP信号的图像。每个图像中都概述了一个具有代表性的运动神经元，作为ROI的示例。在葡萄糖刺激下，表达葡萄糖传感器的控制运动神经元表现出葡萄糖摄取的小幅增加，而表达TDP-43的神经元表现出显着更高的葡萄糖摄取。进一步的FRET通过CFP比率将TDP-43突变运动神经元在每个时间点归一化为葡萄糖传感器对照。在基线条件下，基因型之间没有差异。然而，在葡萄糖刺激下，与对照组相比，TDP-43突变体观察到葡萄糖摄取的快速显着增加。在条形图表示中，表达葡萄糖传感器的神经元中的葡萄糖刺激表明，与基线相比，通过CFP比率计算，FRET增加了很小但显着。相比之下，与基线相比，表达TDP-43的神经元在刺激时的比率显着增加。表达TDP-43的运动神经元和葡萄糖传感器之间CFP比率的FRET净差异约为10.9%，时间管理对于该过程至关重要，因此神经元不会在成像过程中死亡。在解剖后立即成像以避免这种情况很重要。这项技术强调了葡萄糖代谢在神经元中的重要性，并促使我们的小组进一步研究了糖酵解在运动神经元再生中的作用。

Research

JoVE Journal

Neuroscience

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

含量的运动能力，学习和记忆的赤字果蝇</em神经退行性疾病>模型

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

科研

神经科学

Measuring Motor Coordination in Mice

测量小鼠运动协调

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Visualization of Proprioceptors in Drosophila Larvae and Pupae

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

一种廉价的，可扩展的行为分析测量乙醇镇静灵敏度和快速耐受<em&gt;果蝇</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Measuring and Altering Mating Drive in Male Drosophila melanogaster

测量并改变配套驱动马累<em&gt;果蝇</em

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

海马神经元中突触囊泡吞的测量

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

高效液相色谱分析和触多巴胺摄取对小鼠多巴胺能稳态的评价

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

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

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

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 ...

Alternate Immersion in Glucose to Produce Prolonged Hyperglycemia in Zebrafish

交替浸入葡萄糖中，在斑马鱼中产生长期血糖超血症

Zebrafish (Danio rerio) are an excellent model to investigate the effects of chronic hyperglycemia, a hallmark of Type II Diabetes Mellitus (T2DM). This ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

使用自动图像分析对啮齿动物模型的亚斯坦蒂娅尼格拉多巴明神经元密度进行半定量测定

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

测量TDP-43蛋白病果 蝇 模型中的葡萄糖摄取

测量TDP-43蛋白病果蝇模型中的葡萄糖摄取