SOD1 activity threshold and TOR signalling modulate VAP(P58S) aggregation via reactive oxygen species-induced proteasomal degradation in a Drosophila model of amyotrophic lateral sclerosis
SOD1 activity threshold and TOR signalling modulate VAP(P58S) aggregation via reactive oxygen species-induced proteasomal degradation in a Drosophila model of amyotrophic lateral sclerosis
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DOI：
10.1242/dmm.033803
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发表时间：
2019-02-01
影响因子：
4.3
通讯作者：
Ratnaparkhi, Girish S.
中科院分区：
文献类型：
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作者：
Chaplot, Kriti;Pimpale, Lokesh;Ratnaparkhi, Girish S.
家族性肌萎缩侧索硬化症 (ALS) 是一种无法治愈的迟发性运动神经元疾病，与多种致病基因位点密切相关。 ALS8 编码 VAMP 相关蛋白 B (VAPB) 中的错义突变 P56S，导致该蛋白错误折叠并形成细胞聚集体。揭示影响聚集动力学的基因和机制将极大地帮助我们增加对这种疾病的了解并带来潜在的治疗方法。我们开发了一种定量高通量果蝇 S2R+ 基于细胞的动力学测定法，结合荧光显微镜，对参与与 GFP 融合的果蝇直系同源物 VAP(P58S) 聚集体调节的基因进行评分。针对 900 个基因的靶向 RNA 干扰筛选确定了 150 个改变聚集的命中，包括 ALS 位点 Sod1 和 TDP43（也称为 TBPH），以及属于 mTOR 途径的基因。此外，开发了一种测量果蝇幼虫大脑中 VAP(P58S) 聚集程度的系统，以验证基于细胞的筛选的结果。在幼虫大脑中，我们发现 SOD1 水平的降低或 mTOR 信号传导的减少会减少聚集，可能是通过增加细胞活性氧 (ROS) 的水平来实现的。聚集体清除的机制主要是蛋白酶体降解，这似乎是由 ROS 增加触发的。因此，我们发现了 SOD1、ROS 和 mTOR 信号之间有趣的相互作用，调节 VAP 聚集的动态。这种细胞调节网络的机制过程将有助于更好地理解 ALS 的发生和进展。
Familial amyotrophic lateral sclerosis (ALS) is an incurable, late-onset motor neuron disease, linked strongly to various causative genetic loci. ALS8codes fora missense mutation, P56S, in VAMP-associated protein B(VAPB) that causes the protein to misfold and form cellular aggregates. Uncovering genes and mechanisms that affect aggregation dynamics would greatly help increase our understanding of the disease and lead to potential therapeutics. We developed a quantitative high-throughput Drosophila S2R+ cell-based kinetic assay coupled with fluorescent microscopy to score for genes involved in the modulation of aggregates of the fly orthologue, VAP(P58S), fused with GFP. A targeted RNA interference screen against 900 genes identified 150 hits that modify aggregation, including the ALS loci Sod1 and TDP43 (also known as TBPH), as well as genes belonging to the mTOR pathway. Further, a system to measure the extent of VAP(P58S) aggregation in the Drosophila larval brain was developed in order to validate the hits from the cell-based screen. In the larval brain, we find that reduction of SOD1 levels or decreased mTOR signalling reduces aggregation, presumably by increasing the levels of cellular reactive oxygen species (ROS). The mechanism of aggregate clearance is, primarily, proteasomal degradation, which appears to be triggered by an increase in ROS. We have thus uncovered an interesting interplay between SOD1, ROS and mTOR signalling that regulates the dynamics of VAP aggregation. Mechanistic processes underlying such cellular regulatory networks will lead to better understanding of the initiation and progression of ALS.