Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerations. However, the mechanism by which the UBQLN2 mutations cause disease remains unclear. Alterations in proteins involved in autophagy are prominent in neuronal tissue of human ALS UBQLN2 patients and in a transgenic P497S UBQLN2 mouse model of ALS/FTD, suggesting a pathogenic link. Here, we show UBQLN2 functions in autophagy and that ALS/FTD mutant proteins compromise this function. Inactivation of UBQLN2 expression in HeLa cells reduced autophagic flux and autophagosome acidification. The defect in acidification was rescued by reexpression of wild type (WT) UBQLN2 but not by any of the five different UBQLN2 ALS/FTD mutants tested. Proteomic analysis and immunoblot studies revealed P497S mutant mice and UBQLN2 knockout HeLa and NSC34 cells have reduced expression of ATP6v1g1, a critical subunit of the vacuolar ATPase (V-ATPase) pump. Knockout of UBQLN2 expression in HeLa cells decreased turnover of ATP6v1g1, while overexpression of WT UBQLN2 increased biogenesis of ATP6v1g1 compared with P497S mutant UBQLN2 protein. In vitro interaction studies showed that ATP6v1g1 binds more strongly to WT UBQLN2 than to ALS/FTD mutant UBQLN2 proteins. Intriguingly, overexpression of ATP6v1g1 in UBQLN2 knockout HeLa cells increased autophagosome acidification, suggesting a therapeutic approach to overcome the acidification defect. Taken together, our findings suggest that UBQLN2 mutations drive pathogenesis through a dominant-negative loss-of-function mechanism in autophagy and that UBQLN2 functions as an important regulator of the expression and stability of ATP6v1g1. These findings may have important implications for devising therapies to treat UBQLN2-linked ALS/FTD.

UBQLN2突变会导致肌萎缩侧索硬化症 (ALS)、额颞叶痴呆 (FTD) 和其他神经退行性疾病。然而，UBQLN2 突变导致疾病的机制仍不清楚。在人类 ALS UBQLN2患者的神经元组织和 ALS/FTD 转基因 P497S UBQLN2 小鼠模型中，参与自噬的蛋白质的改变很明显，表明存在致病联系。在这里，我们展示了 UBQLN2 在自噬中的功能，并且 ALS/FTD 突变蛋白会损害该功能。 HeLa 细胞中 UBQLN2 表达失活会减少自噬流和自噬体酸化。酸化缺陷可以通过野生型 (WT) UBQLN2 的重新表达来修复，但不能通过测试的五种不同 UBQLN2 ALS/FTD 突变体中的任何一个来修复。蛋白质组学分析和免疫印迹研究表明，P497S 突变小鼠以及 UBQLN2 敲除 HeLa 和 NSC34 细胞的 ATP6v1g1 表达降低，ATP6v1g1 是液泡 ATP 酶 (V-ATPase) 泵的关键亚基。与 P497S 突变体 UBQLN2 蛋白相比，HeLa 细胞中 UBQLN2 表达的敲除降低了 ATP6v1g1 的更新，而 WT UBQLN2 的过表达则增加了 ATP6v1g1 的生物发生。体外相互作用研究表明，ATP6v1g1 与 WT UBQLN2 的结合比与 ALS/FTD 突变体 UBQLN2 蛋白的结合更强。有趣的是，UBQLN2 敲除 HeLa 细胞中 ATP6v1g1 的过度表达增加了自噬体酸化，这表明了一种克服酸化缺陷的治疗方法。综上所述，我们的研究结果表明，UBQLN2 突变通过自噬中的显性失活功能丧失机制驱动发病机制，并且 UBQLN2 作为 ATP6v1g1 表达和稳定性的重要调节因子。 这些发现可能对设计治疗UBQLN2相关的 ALS/FTD 的疗法具有重要意义。