Fig 2: Significant cell death is observed in the CNS of zebrafish vps11(plt) mutants.TUNEL assay was performed on cryosectioned CNS tissue from vps11(plt) mutants and wild-type siblings at 3, 5, and 7 days post-fertilization (dpf). (A-C) Minimal apoptotic TUNEL+ cells were observed in the hindbrain of vps11(plt) mutants at 3, 5, and 7 dpf, respectively. (D-E) Tissue section of the midbrain of a wild-type control animal at 7 dpf showing minimal cell death (panel E, arrow). (F-G) Tissue section of the midbrain of a vps11(plt) mutant at 7 dpf showing extensive cell death. (H) Quantification of the average number of TUNEL+ cells observed in the hindbrain in vps11(plt) mutants and wild-type siblings at 3, 5, and 7 dpf. No significant differences were observed. (I) Quantification of the average number of TUNEL+ cells observed in the midbrain in vps11(plt) mutants and wild-type siblings at 3, 5, and 7 dpf. Asterisk indicates significantly different from control.

Fig 3: The VPS C846G mutation decreases the stability of VPS11 protein.HeLa cells were transfected with 0.5 ug DNA of empty vector (Ctrl), FLAG-tagged VPS11-WT or VPS11-C846G. (A) Expression of transfected plasmids was examined at 48 hrs post-transfection by Western blot analysis. (B) The chase started 24 hrs post-transfection and was allowed for the indicated time. Blots are representatives of four independent experiments. IB: Immunoblot. (C) The percentage of protein decay was graphically reported and the difference of the in cellulo half-life between the WT and C846 mutant of VPS11 was analyzed in (D). The statistical significance was determined by an unpaired student t test. Results are the mean±SEM of four independent experiments. (E) Images were prepared with PyMOL (www.pymol.org) using coordinates from the Protein Model Portal (VPS11_Q9H270, PDB 1iymA). (F) Far-UV CD spectra of the purified WT and C846G RING domain fragment proteins (amino acids 821–860). Results are the mean of three independent experiments. (G) HeLa cells were transfected with 2.0 ug DNA of empty vector, 1.0 ug of FLAG-tagged VPS11-WT with 1.0 ug of empty vector or 2.0 ug of FLAG-VPS11-C846G. Immunoprecipitations (IPs) were performed 48 hrs post-transfection and followed by Western blot analysis. Blots are representatives of four independent experiments. (H-I) The percentage of VPS11 ubiquitination was calculated and the difference of the in cellulo total ubiquitination profile was verified. The statistical significance was determined by an unpaired student t test. Results are the mean±SEM of four independent experiments. Data were considered significant when P values were <0.05 (*), <0.01(**) or <0.001 (***).

Fig 4: The p.C846G mutation impairs autophagy.(A) HeLa cells were transfected with empty vector (Ctrl), FLAG-tagged VPS11-WT or VPS11-C846G. Immunoprecipitations were performed 48 hrs post-transfection and followed by Western blot analysis. Blots are representatives of three independent experiments. (B) The densitometry values of co-immunoprecipitated endogenous VPS18 were normalized to the amount of immunoprecipitated FLAG-VPS11 WT or C846G and reported as the mean±SEM of three independent experiments. The statistical significance was determined using an unpaired student t test. (C) Cells were transfected with scrambled siRNA control (siCtrl) or siVPS11. Blot is representative of three independent experiments. (D) The densitometry values of endogenous p62 and LC3-II were normalized to the amount of actin and reported as the mean±SEM of three independent experiments. The statistical significance was determined using a Two-way ANOVA test followed by Sidak’s multiple comparisons test. (E-F) Cells were transfected with scrambled siRNA control (siCtrl) or siVPS11. Quantification of autophagosomes (yellow puncta) and autolysosomes (red puncta) was performed using Green and Red Puncta Colocalization Macro for Image J (D. J. Swiwarski modified by R.K. Dagda). Scale bars: 20 μm. Data are representatives of two independent experiments. Results are the mean±SEM of at least fifteen images. The statistical significance was determined using a Two-way ANOVA test followed by Tukey’s multiple comparisons test. Data were considered significant when P values were <0.05 (*), <0.01(**) or <0.001 (***).

Fig 5: Brain MRI demonstrated white matter abnormality suggesting leukoencephalopathy in a patient homozygous for VPS11: c.2536T>G (p.C846G).In patient B at nine months of age, thin corpus callosum (A), T2 diffuse hyperintensity in the peri-Rolandic white matter (B) and posterior occipital white matter region (C) were seen; FLAIR diffuse hyperintensity in the white matter region was also seen (D). At five years of age, diminutive corpus callosum (E), T2 diffuse hyperintensity signal in peri-Rolandic areas (F) and supratentorial white matter, most pronounced within the bilateral parieto-occipital regions (G) were seen; FLAIR diffuse hyperintensity in the white matter region with a mild increase of myelination was seen(H).