Protein aggregate accumulation is a pathological hallmark of several neurodegenerative disorders. Autophagy is critical for clearance of aggregate-prone proteins. In this study, we identify a novel role of the multifunctional glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in clearance of intracellular protein aggregates. Previously, it has been reported that though clearance of wild-type huntingtin protein is mediated by chaperone-mediated autophagy (CMA), however, degradation of mutant huntingtin (mHtt with numerous poly Q repeats) remains impaired by this route as mutant Htt binds with high affinity to Hsc70 and LAMP-2A. This delays delivery of misfolded protein to lysosomes and results in accumulation of intracellular aggregates which are degraded only by macroautophagy. Earlier investigations also suggest that mHtt causes inactivation of mTOR signaling, causing upregulation of autophagy. GAPDH had earlier been reported to interact with mHtt resulting in cellular toxicity. Utilizing a cell culture model of mHtt aggregates coupled with modulation of GAPDH expression, we analyzed the formation of intracellular aggregates and correlated this with autophagy induction. We observed that GAPDH knockdown cells transfected with N-terminal mutant huntingtin (103 poly Q residues) aggregate-prone protein exhibit diminished autophagy. GAPDH was found to regulate autophagy via the mTOR pathway. Significantly more and larger-sized huntingtin protein aggregates were observed in GAPDH knockdown cells compared to empty vector–transfected control cells. This correlated with the observed decrease in autophagy. Overexpression of GAPDH had a protective effect on cells resulting in a decreased load of aggregates. Our results demonstrate that GAPDH assists in the clearance of protein aggregates by autophagy induction. These findings provide a new insight in understanding the mechanism of mutant huntingtin aggregate clearance. By studying the molecular mechanism of protein aggregate clearance via GAPDH, we hope to provide a new approach in targeting and understanding several neurodegenerative disorders.

蛋白质聚集体积累是几种神经退行性疾病的病理标志。自噬对于清除易聚集的蛋白质至关重要。在这项研究中，我们确定了多功能糖酵解酶甘油醛-3-磷酸脱氢酶 (GAPDH) 在清除细胞内蛋白质聚集体中的新作用。以前，据报道，虽然野生型亨廷顿蛋白的清除是由伴侣介导的自噬 (CMA) 介导的，但是突变亨廷顿蛋白（具有许多多 Q 重复序列的 mHtt）的降解仍然受到这种途径的损害，因为突变体 Htt 与对 Hsc70 和 LAMP-2A 具有高亲和力。这延迟了错误折叠的蛋白质向溶酶体的传递，并导致细胞内聚集体的积累，这些聚集体只能被巨自噬降解。早期的研究还表明，mHtt 导致 mTOR 信号失活，导致自噬上调。GAPDH 早先曾被报道与 mHtt 相互作用导致细胞毒性。利用 mHtt 聚集体的细胞培养模型以及 GAPDH 表达的调节，我们分析了细胞内聚集体的形成并将其与自噬诱导相关联。我们观察到转染 N 端突变亨廷顿蛋白（103 个聚 Q 残基）聚集倾向蛋白的 GAPDH 敲低细胞表现出自噬减少。发现 GAPDH 通过 mTOR 途径调节自噬。与空载体转染的对照细胞相比，在 GAPDH 敲低细胞中观察到显着更多和更大尺寸的亨廷顿蛋白聚集体。这与观察到的自噬减少有关。GAPDH 的过表达对细胞具有保护作用，导致聚集体负荷减少。我们的结果表明，GAPDH 通过自噬诱导帮助清除蛋白质聚集体。这些发现为理解突变亨廷顿蛋白聚集体清除的机制提供了新的见解。通过研究通过 GAPDH 清除蛋白质聚集体的分子机制，我们希望为靶向和了解几种神经退行性疾病提供一种新方法。