Mutations inUBQLN2cause 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 ALSUBQLN2patients 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 treatUBQLN2-linked ALS/FTD.

Missense mutations in UBQLN2 cause X-linked dominant inheritance of amyotrophic lateral sclerosis with frontotemporal dementia (ALS/FTD). UBQLN2 belongs to a family of four highly homologous proteins expressed in humans that play diverse roles in maintaining proteostasis, but whether one isoform can substitute for another is not known. Here, we tested whether overexpression of UBQLN1 can alleviate disease in the P497S UBQLN2 mouse model of ALS/FTD by crossing transgenic (Tg) mouse lines expressing the two proteins and characterizing the resulting genotypes using a battery of pathologic and behavioral tests. The pathologic findings revealed UBQLN1 overexpression dramatically reduced the burden of UBQLN2 inclusions, neuronal loss and disturbances in proteostasis in double Tg mice compared to single P497S Tg mice. The beneficial effects of UBQLN1 overexpression were primarily confirmed by behavioral improvements seen in rotarod performance and grip strength in male, but not female mice. Paradoxically, although UBQLN1 overexpression reduced pathologic signatures of disease in P497S Tg mice, female mice had larger percentage of body weight loss than males, and this correlated with a corresponding lack of behavioral improvements in the females. These findings lead us to speculate that methods to upregulate UBQLN1 expression may reduce pathogenicity caused by UBQLN2 mutations, but may also lead to gender-specific outcomes that will have to be carefully weighed with the therapeutic benefits of UBQLN1 upregulation.

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