自噬抑制改善果蝇线粒体Hsc70缺失引起的结构和功能缺陷

Authors: Jun-yi Zhu, Shabab B. Hannan, Nina M. Dräger, et al., Tobias M. Rasse (corresponding author)
School: Research Group Synaptic Plasticity, Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany; Center for Genetic Medicine Research, Children’s National Medical Center, Washington, DC USA; Schaller Research Group at the University of Heidelberg and DKFZ, German Cancer Research Center, Heidelberg, Germany; Department of Biology, University of York, York, UK; Scientific Service Group Microscopy, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
Date: 2021
Journal: Autophagy

Title: Autophagy inhibition rescues structural and functional defects caused by the loss of mitochondrial chaperone Hsc70-5 in Drosophila

Abstract:
The study investigates whether the loss of the mitochondrial chaperone Hsc70-5 in Drosophila is sufficient to induce pathological changes commonly seen in Parkinson's disease (PD). The research found no effects on terminal size, bouton size or number, or synapse size or number at the larval neuromuscular junctions, suggesting an early stage of pathogenesis. Defects in synaptic vesicle proteins, active zone components, delayed synapse maturation, reduced evoked and spontaneous excitatory junctional potentials, increased synaptic fatigue, and cytoskeleton rearrangements were observed. Adult models displayed ATP depletion, altered body posture, and susceptibility to heat-induced paralysis. The phenotypes could be suppressed by knocking down components related to PD, and the knockdown of autophagy components or overexpression of human HSPA9 largely rescued larval and adult phenotypes. However, disease-associated HSPA9 variants did not rescue, and overexpression of Pink1 or promotion of autophagy exacerbated defects.

Materials:
Transgenic fly stocks were obtained from the Indiana University Stock Center or Vienna Drosophila RNAi center, including w1118 (BDSC3605) and OAMB RNAi (BS31171). VDRC lines included Octβ1R RNAi (v47895), Octβ2R RNAi (v8486), and Octβ3R RNAi (v101189).

Methods:

1. Flies were split into control and experimental groups and treated with ethanol vehicle or mifepristone (RU486).
2. An exercise device induced a repetitive negative geotaxis response.
3. Climbing endurance was measured using the Power Tower exercise machine.
4. Drosophila were tested weekly for 4-5 weeks to assess the decline in negative geotaxis with age.
5. Flies were ejected into a flight tube to record flight performance.
6. Electrical pacing was applied to 25-day old flies.
7. Adult fly fat bodies were stained with Lysotracker.
8. Western blotting was performed using an anti-dAtg8a antibody.
9. qRT PCR was conducted on RNA from adult fly hearts, indirect flight muscle, or fat body.

Results:

1. OAMB, Octβ1R, Octβ2R, and Octβ3R were detected in adult muscle, with Octβ3R also found in the adult fat body.
2. OAMB is required for improved climbing speed, endurance, and cardioprotection with exercise.
3. The Octopamine β1 receptor is specifically required in muscle for the adaptive response to chronic exercise.
4. Octopamine β2 and β3 receptor expression in muscle coordinates exercise adaptations.
5. The Octopamine β3 receptor is important for fat body homeostasis.
6. Octopamine feeding can rescue some, but not all, octopamine receptor knockdown phenotypes.
7. Some receptors are specifically required for particular exercise adaptations, indicating not all OA receptors are interchangeable.

Background:
Exercise and octopamine have been shown to increase autophagy and lipolysis in flying insects. The study provides insights into the role of Hsc70-5 in PD pathology, suggesting a complex interplay between mitochondrial function, autophagy, and synaptic integrity.

Cite:
Jun-yi Zhu, Shabab B. Hannan, Nina M. Dräger, Natalia Vereshchagina, Ann-Christin Krahl, Yulong Fu, Christopher J.H. Elliott, Zhe Han, Thomas R. Jahn & Tobias M. Rasse (2021) Autophagy inhibition rescues structural and functional defects caused by the loss of mitochondrial chaperone Hsc70-5 in Drosophila, Autophagy, 17:10, 3160-3174, DOI: 10.1080/15548627.2020.1871211

作者： 朱俊义，Shabab B. Hannan，Nina M. Dräger，等，Tobias M. Rasse（通讯作者）
学校： 德国图宾根大学赫蒂临床脑研究所突触可塑性研究小组；美国华盛顿特区国家儿童医疗中心遗传医学研究中心；德国海德堡大学和德国癌症研究中心Schaller研究小组；英国约克大学生物学系；德国巴特瑙海姆马克斯普朗克心肺研究所显微服务小组
日期： 2021年
期刊： Autophagy

标题： 自噬抑制挽救了果蝇线粒体分子伴侣Hsc70-5缺失引起的结构和功能缺陷

摘要：
本研究探讨了果蝇中线粒体分子伴侣Hsc70-5的缺失是否足以引起帕金森病(PD)中常见的病理变化。研究发现在幼虫神经肌肉接头处，并未观察到终端大小、突触前末梢大小或数量、突触大小或数量的影响，这表明我们研究的是一个早期病理阶段。在这一阶段，注意到突触囊泡蛋白和活性区域成分的丢失、突触成熟延迟、减少的诱发性和自发性兴奋性突触后电位、突触疲劳增加以及细胞骨架重排。成体模型显示出ATP耗竭、身体姿势改变和对热诱导的瘫痪的敏感性。通过敲低与PD相关的成分，可以抑制成体表型，而自噬组分的敲低或人类HSPA9的过表达广泛挽救了幼虫和成体表型。然而，与疾病相关的HSPA9变体并未挽救，并且Pink1的过表达或自噬的促进加剧了缺陷。

材料：
转基因果蝇品系来自印第安纳大学昆虫中心或维也纳果蝇RNAi中心，包括w1118 (BDSC3605)和OAMB RNAi (BS31171)。VDRC品系包括Octβ1R RNAi (v47895)、Octβ2R RNAi (v8486)和Octβ3R RNAi (v101189)。

方法：

1. 将果蝇分为对照组和实验组，并用乙醇载体或米非司酮(RU486)处理。
2. 使用运动装置诱导重复的负向重力感应反应。
3. 使用Power Tower运动机测量攀爬耐力。
4. 每周对果蝇进行测试，连续4-5周以评估负向重力感应随年龄的下降。
5. 将果蝇群体释放到飞行管中以记录飞行表现。
6. 对25天大的果蝇进行电刺激。
7. 使用Lysotracker对成年果蝇的脂肪体进行染色。
8. 使用抗-dAtg8a抗体进行WB。
9. 对果蝇成虫的心脏、间接飞行肌或脂肪体的RNA进行qRT-PCR。

结果：

1. 在果蝇成年肌肉中检测到OAMB、Octβ1R、Octβ2R和Octβ3R，Octβ3R在果蝇成虫脂肪体中也有发现。
2. OAMB对于提高攀爬速度、耐力和运动心脏保护是必需的。
3. 章鱼胺β1受体在肌肉中特别需要以适应慢性运动。
4. 肌肉中的章鱼胺β2和β3受体表达分别协调运动适应性。
5. 章鱼胺β3受体对脂肪体稳态很重要。
6. 章鱼胺喂养可以挽救一些（但不是全部）章鱼胺受体敲低的表型。
7. 一些受体特别需要特定的运动适应性，表明并非所有的OA受体都是可互相转换的。

背景：
运动和章鱼胺都已经显示出可以增加各种飞行昆虫的自噬和脂肪分解。该研究为Hsc70-5在PD病理中的作用提供了见解，表明线粒体功能、自噬和突触完整性之间存在复杂的相互作用。

引用：
Jun-yi Zhu, Shabab B. Hannan, Nina M. Dräger, Natalia Vereshchagina, Ann-Christin Krahl, Yulong Fu, Christopher J.H. Elliott, Zhe Han, Thomas R. Jahn & Tobias M. Rasse (2021) Autophagy inhibition rescues structural and functional defects caused by the loss of mitochondrial chaperone Hsc70-5 in Drosophila, Autophagy, 17:10, 3160-3174, DOI: 10.1080/15548627.2020.187121