UBQLN2 undergoes a reversible temperature-induced conformational switch that regulates binding with HSPA1B: ALS/FTD mutations cripple the switch but do not destroy HSPA1B binding.

Here we present evidence, based on alterations of its intrinsic tryptophan fluorescence, that UBQLN2 protein undergoes a conformational switch when the temperature is raised from 37 °C to 42 °C. The switch is reset on restoration of the temperature. We speculate that the switch regulates UBQLN2 function in the heat shock response because elevation of the temperature from 37 °C to 42 °C dramatically increased in vitro binding between UBQLN2 and HSPA1B. Furthermore, restoration of the temperature to 37 °C decreased HSPA1B binding. By comparison to wild type (WT) UBQLN2, we found that all five ALS/FTD mutant UBQLN2 proteins we examined had attenuated alterations in tryptophan fluorescence when shifted to 42 °C, suggesting that the conformational switch is crippled in the mutants. Paradoxically, all five mutants bound similar amounts of HSPA1B compared to WT UBQLN2 protein at 42 °C, suggesting that either the conformational switch is not instrumental for HSPA1B binding, or that, although damaged, it is still functional. Comparison of the poly-ubiquitin chain binding revealed that WT UBQLN2 binds more avidly with K63 than with K48 chains. The avidity may explain the involvement of UBQLN2 in autophagy and cell signaling. Consistent with its function in autophagy, we found UBQLN2 binds directly with LC3, the autophagosomal-specific membrane-tethered protein. Finally, we provide evidence that WT UBQLN2 can homodimerize, and heterodimerize with WT UBQLN1. We show that ALS mutant P497S-UBQLN2 protein can oligomerize with either WT UBQLN1 or 2, providing a possible mechanism for how mutant UBQLN2 proteins could bind and inactivate UBQLN proteins, causing loss of function.

Overexpression of UBQLN1 reduces neuropathology in the P497S UBQLN2 mouse model of 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.

ALS/FTD mutations in UBQLN2 impede autophagy by reducing autophagosome acidification through loss of function.

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.