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

Abstract

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.

Keywords: UBQLN2; amyotrophic lateral sclerosis; autophagy; ubiquilin; vacuolar ATPase pump.

Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.

UBQLN2 mutations induce pathologic disturbances in autophagy in humans and the P497S mouse model of ALS/FTD. (A) Immunoblots of hippocampus and SC lysates of three independent mice for the different mouse lines probed with the antibodies shown. (B) Quantification of the immunoreactivity for the proteins shown in A. *P < 0.05, **P < 0.01, ***P < 0.001. (C) Confocal microscopy (1× and corresponding 2 or 5× zoomed) images of the merged UBQLN2, p62, and DAPI staining of the dentate gyrus (DG) of the hippocampus (a–f) and ventral horn of the SC (g–l) for the three mouse genotypes at 52 wk of age. (D) Confocal images of the molecular layer (ML), DG, and polymorphic layer (PML) brain region (large image) of a human P497H UBQLN2 patient. Zoomed images demonstrate colocalization between UBQLN2 inclusions (green) and p62 (red) in the molecular layer. Bar sizes shown in this and all subsequent figures.

Fig. 2.

Alteration of autophagic proteins in UBQLN2 KO HeLa cells and mouse lines. (A) Immunoblots of three independent lysates from the parental HeLa lines and CRISPR/cas9 UBQLN2 KO8 and KO12 lines for the proteins shown. Detection of the different UBQLN isoforms (second panel) with an antibody that detects all UBQLNs. (B) Quantification of total UBQLN protein expression in the parental and KO HeLa lines. *P < 0.05. (C) Immunoblots of total brain lysates (three independent animals) from non-Tg and UBQLN2 KO animals of 6 and 12 mo of age probed for the proteins shown. (D) Quantification of the changes in the different proteins in the KO relative to the 6-mo-old non-Tg (WT) animals. *P < 0.05.

Fig. 3.

Visual detection of UBQLN2 in autophagosomes. (A) Fluorescent (YFP) images of HeLa cells transfected with NVenus-UBQLN2 WT and CVenus (a). YFP-fluorescence of HeLa cells transfected with NVenus-UBQLN2 WT and CVenus-LC3B (b), DAPI image (c), and the combined YFP and DAPI images (d). (e–h) Cells transfected with NVenus-UBQLN2 WT and CVenus and counterstained for LC3A/B. Arrows show colocalization of the staining in puncta. (B) Images of HeLa cells cotransfected with CVenus-mCherry-LC3B with NVenus-UBQLN2 WT and left untreated (a–d) or treated for 4 h with Bafilomycin A1 (e–h).

Fig. 4.

Defect in rescue of autophagosome acidification by ALS/FTD mutant UBQLN2 proteins. (A) Representative images of the combined YFP, mCherry, and DAPI fluorescent signal in HeLa UBQLN2 KO8 cells cotransfected with CVenus-mCherry-LC3B and either WT or ALS mutant NVenus-UBQLN2 constructs. (B) Quantification of autophagosome acidification. ****P < 0.0001.

Fig. 5.

Reduction of ATP6v1g1 in P497S mutant mice and UBQLN2 KO cells. (A) Immunoblots of hippocampus and SC lysates of 8-mo-old mice for the three mouse genotypes (three independent mice for each genotype) and probed for the proteins shown. (B) Quantification of protein changes of two different V-ATPase subunits from the blots shown in B. *P < 0.05. (C and D) Immunoblots of HeLa and NSC34 parental and UBQLN2 KO lines for the proteins shown and (E and F) corresponding quantification of the protein levels for the two V-ATPase subunits in the cell lines. *P < 0.05, **P < 0.01.

Fig. 6.

Function and interaction of ATP6v1g1 and UBQLN2. (A) Immunoprecipitation of endogenous proteins from NSC34 cells with either an anti-UBQLN2 antibody or IgG control antibody, and subsequent immunoblots for the proteins shown. (B and C) Immunoprecipitation analysis of proteins from HeLa cells transfected with the constructs shown on the top with either anti-HA (B) or anti-Myc (C) antibodies. The arrowheads show the IgG bands and the arrows indicate the respective proteins that were immunoprecipitated (Upper) and coimmunoprecipitated (Lower). (D) Demonstration that UBQLN2 stimulates biogenesis of ATP6v1g1. HeLa cell cultures were cotransfected with identical amounts of total plasmid cDNAs (μg) shown on the top. The next day lysates were prepared from the cultures and immunoblotted for the proteins shown. (E) Quantification of the expressed proteins shows ATPv1g1 expression is increased by UBQLN2 in a dose-dependent manner. (F) Immunoblots of cultures after transfection for different days to knockdown ATP6v1g1. (G) Coverslips showing the different number of autolysosomes (red) collected from the same cultures shown in F. (H) Quantification of autophagososme acidification in the three transfected cultures. ****P < 0.0001. (I) Fluorescent images of HeLa UBQLN2 KO8 cells transfected with GFP-mCherry-LC3B alone (a–d) or cotransfected with ATP6v1g1-myc cDNA (e–h). (J) Quantification showing acidification of HeLa KO8 cells is increased in cells transfected with ATP6v1g1-myc cDNA. **P < 0.01.