Fig 2: Effects of ATP6V1A mutations on endo-lysosomal markers and v-ATPase recruitment to autophagosomes. (A) Left: Representative images of HEK cells transfected with wild-type ATP6V1A (w.t.), Asp349Asn ATP6V1A (Asp349Asn) or Asp100Tyr ATP6V1A (Asp100Tyr) variants and incubated with LysoTracker® (200 nM, 1 h). Right: LysoTracker® fluorescence intensity was quantified in 38 (wild-type), 41 (Asp349Asn) and 45 (Asp100Tyr) cells from three independent experiments. Individual data and means ± SEM are shown. *P < 0.05, ****P < 0.0001 versus wild-type; Kruskall-Wallis/Dunn’s tests. (B) Representative images and densitometric quantification from HEK cells transfected as above and immunolabelled with LAMP1 (left) or EEA1 (right). Histograms show quantification of signal intensity. Data are means ± SEM of 24–33 cells per experimental condition, from three independent experiments. **P < 0.001 versus wild-type Kruskall-Wallis/Dunn’s tests. (C) Representative images from HEK cells transfected as above and immunolabelled with ATP6V1B2 and LC3B under control conditions or after starvation for 2 h. Graph shows the quantification of ATP6V1B2 and LC3B co-localization using ImageJ software to determine the Pearson’s correlation coefficient. Data are means ± SEM of 30 cells per experimental condition. *P < 0.05 versus respective control; #P < 0.001 versus non-starved wild-type; §P < 0.001 versus starved wild-type. Data were analysed by two-way ANOVA/Bonferroni’s tests.

Fig 3: Impact of ATP6V1A mutations on protein expression and stability. (A) Representative images of HEK cells transfected with vectors coding wild-type ATP6V1A (w.t.), Asp349Asn ATP6V1A (Asp349Asn) or Asp100Tyr ATP6V1A (Asp100Tyr) variants. ATP6V1A immunolabelling, DAPI nuclear stain and Cherry reporter fluorescence are shown. Scale bar = 10 µm. (B) Representative western blot (left) from HEK cells transfected as above and lysed 24 h after transfection. ATP6V1A and ATP6V1B2 intensities were quantified by densitometric analysis with respect to GAPDH intensity (right). Data are means ± standard error of the mean (SEM) from five independent experiments. *P < 0.05 versus wild-type; Kruskall-Wallis/Dunn’s tests. (C) Left: Representative western blots of HEK cell lysates stained with anti-ATP6V1A antibody and anti-GADPH as loading control. Cells were transfected with wild-type, Asp349Asn or Asp100Tyr ATP6V1A variants and incubated with cycloheximide for 2, 4, 8, 24 h or vehicle (DMSO; 24 h) as a control (–). Right: Densitometric analysis of ATP6V1A intensity with respect to GADPH expressed in percent of control samples without cycloheximide. Data are means ± SEM from four independent experiments. The areas under the respective curves were compared using the Kruskal-Wallis/Dunn’s tests. *P < 0.05 versus wild-type. (D) Representative western blot from lymphoblast lysates (30 µg) of patient affected by the Asp349Asn mutation (proband) and the healthy mother. (E) Representative western blot from lymphoblast lysates (30 µg) of patient affected by the Asp100Tyr mutation (proband) and the healthy mother. In D and E, ATP6V1A and ATP6V1B2 intensity were quantified by densitometric analysis with respect to GADPH. Data are means ± SEM from four independent experiments. *P < 0.05; Mann Whitney U-test.

Fig 4: Cellular localization and abnormal lysosomal acidification of ATP6V01A1 mutants.a Stable HEK293FT cell lines expressing ATP6V0A1-3×HA were stained with antibodies against HA (green), ATP6V1A (red), and a lysosomal marker, Lamp2 (purple), showing partial colocalization of ATP6V0A1, ATP6V1A, and Lamp2. Magnified inserts showed colocalization of HA-ATP6V0A1, ATP6V1A, and Lamp2. Scale bars, 10 µm. b Quantification of lysosomal pH in the cell lines expressing wild-type (WT) and three different mutants of ATP6V0A1-3×HA using LysoSensor. All three ATP6V0A1 mutants caused elevated pH compared with wild-type ATP6V0A1. Data represented as mean values ± standard deviation of six independent calculations. c Wild-type and mutant HA-ATP6V0A1 expressing stable neuro2a cell lines were stained with an anti-HA antibody (green) and LysoTracker fluorescence dye (red). Scale bars, 10 µm. d Quantification of the LysoTracker intensity of untransfected neuro2a cells, HA-ATP6V0A1 wild-type, and mutant expressing stable cell lines. The intensity was significantly reduced in all the mutant expressing cells compared with untransfected control. Data represented as mean values ± standard deviation of five measurements. Ordinary one-way ANOVA with Dunnett’s multiple comparison test was used for comparing among ATP6V0A1 mutants (b, d). Cell image data in a and c are representative of more than three independent experiments. Source data are provided as a Source data file.

Fig 5: Abnormal cortical and cerebellar layers and decreased Atp6v0a1 protein expression in Atp6v0a1A512P/A512P pups.a The dorsal view of Atp6v0a1+/+ (WT) and Atp6v0a1A512P/A512P (Homo) brains at postnatal 10 day (P10), showing the smaller size of the Atp6v0a1A512P/A512P brain. Scale bars, 2 mm. b Nissl-stained coronal sections of the cortex (CTX) and hippocampus (Hip) in Atp6v0a1+/+ and Atp6v0a1A512P/A512P pups at P10 showed no gross structural abnormalities. Scale bars, 200 µm. c Immunostaining of the cortical layer V marker calbindin (green) and the neuronal marker NeuN (red) in coronal sections of Atp6v0a1+/+ and Atp6v0a1A512P/A512P cortexes at P10. Nuclei were stained with DAPI (blue). Scale bars, 50 µm. d The number of calbindin-positive cells (left chart) and the ratios of calbindin-positive/NeuN-positive cells and of calbindin-positive/DAPI-positive cells within a 200-µm-wide bin (right chart) were decreased in the cortical layer of Atp6v0a1A512P/A512P pups at P10. Data represented as mean values ± standard deviation of eight or more counts. e Sagittal sections of the cerebellums in Atp6v0a1+/+ and Atp6v0a1A512P/A512P pups at P10 stained with antibodies against calbindin as a Purkinje cell marker (green) and NeuN (red). Scale bars, 50 µm. f Comparison of layer volumes of external granule layer (EGL), molecular layer (ML), internal granule layer (IGL), and white matter (WM). Reduced ML volume was evident, suggesting that dendrite development of Purkinje cells would be impaired. Data represented as mean values ± standard deviation of seven or more measurements. g Immunoblot analysis of Atp6v0a1, Atp6v1a, and GAPDH loading control in the CTX, Hip, and cerebellum (CB) of Atp6v0a1+/+ and Atp6v0a1A512P/A512P pups at P10. h, i Quantification of the band intensity of the immunoblot, which shows reduced levels of Atp6v0a1 protein in all the three regions of Atp6v0a1A512P/A512P brains (h) and reduced level of Atp6v1a in cerebellum (i). Data represented as mean values ± standard deviation of three independent experiments. Paired t test (d, left), two way ANOVA with Sidak’s multiple comparisons test (d, right), or two way ANOVA with Bonferroni’s multiple comparisons test (f, h, i) was used. Data in a–c, e, and g are representative of more than three independent experiments. Source data are provided as a Source data file.