Fig 2: VPS26b redistributes to recycling endosomes during neuronal stimulation(A and B) Subcellular distribution of both VPS26 paralogs was performed using confocal and ultracryomicrotomy analyses.(A) Confocal microscopy quantifications based on Pearson’s correlation coefficient were obtained by analysis of 15–27 cells per group/condition. Kruskal-Wallis test with a Dunn’s post hoc test was used for the statistical analysis. Note that while VPS26a is broadly detected in all different compartments, VPS26b is highly enriched in early (EEA1) and recycling (Syntaxin13 and pulse-chase transferrin) endosomes, with less presence in the trans-Golgi network (Golgin97) and late endosomes (Rab7). Data expressed as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.(B) Immunogold labeling quantifications (percetange of gold particles) for VPS26b and VPS26a on ultrathin cryosections shown in Figure S3B. Localization of an equivalent number of gold particles in each sample was assessed relative to the indicated cell compartment. Note that whereas the bulk of VPS26b localizes to tubular-vesicular structures found at the vicinity of endosomes, VPS26a is broadly distributed among the different compartments.(C) Primary hippocampal neurons were stimulated with glycine for 5 min to induce cLTP, and the subcellular distribution of VPS26b and VPS26a was assessed by confocal microscopy using markers of early (EEA1) and recycling (Syntaxin13 and pulse-chase transferrin) endosomes. Compared to basal conditions, cLTP caused VPS26b to increase its co-localization with markers of the recycling endosomes (in an ANOVA analysis; pulse-chase transferrin: F[1,33] = 20.7, p = 6.9E-5; Syntaxin13: F[1,36] = 38.8, p = 3.5E-7) and caused VPS26a to increase its co-localization with a marker of early endosomes (EEA1: F[1,33] = 24.5, p = 2.1E-5), as illustrated in the representative confocal images of dendritic segments. Arrows indicate sites of co-localization. Scale bar, 10µm (top panel).(D) Co-localization studies based on Pearson’s correlation coefficient were used to generate the line graphs (n = 31–41 cells per condition, from four independent experiments) (left panels). An illustration of the changes in distribution observed post-stimulation for both VPS26 paralogs is shown (right panel).See also Figures S3 and S4.

Fig 3: VPS26b mediates Alzheimer’s-related pathologies(A) Western blotting analysis of different subregions of the entorhinal cortex from healthy controls and patients with AD immunoblotted for SORL1 protein is shown. TEC, trans-EC; LEC; lateral EC; IEC, intermediate EC; MEC, medial EC (left panel). An ANOVA analysis revealed that, compared with age-matched healthy controls (n = 9), AD brains (n = 8) showed a significant and reliable reduction of SORL1 in the TEC region compared with other EC subregions (F[1,16] = 12.32, p = 0.003, as summarized in the brain graphs showing normalized means). Stippled lines represent the mean of the healthy controls (right panel).(B) Quantitative analysis of SORL1 immunoblots from 7- to 9-month-old mice showing a reduction of SORL1 in the entorhinal cortex of Vps26b KO compared with Vps26b WT mice (total SORL1: p = 0.0160, in an unpaired two-sided Student’s t test; mature SORL1: p = 0.0185, in a on parametric Mann Whitney test; immature SORL1: p = 0.1167, in an unpaired two-sided Student’s t test).(C) Biotinylated cell-surface proteins were immunoprecipitated from total lysates of cortical neurons depleted for VPS26b (n = 8–9 biological replicates) or VPS26a (n = 7 biological replicates). Western blot analysis revealed that only VPS26b (p = 0.0274, in a two-tailed non-parametric Mann Whitney test), but not VPS26a (p = 0.9755, in a two-tailed non-parametric Mann Whitney test), depletion results in a decrease in SORL1 cell-surface levels.(D) Bar graph showing the levels of Aß40 and Aß42 levels measured by ELISA from entorhinal cortex homogenates harvested from Vps26b KO mice and control littermates at 6–7 months (Aß40 measurements: Vps26b WT [n = 9] versus Vps26b KO [n = 9], p = 0.0002 in a two-tailed non-parametric Mann Whitney test; for Aß42 measurements: Vps26b WT [n = 8] versus Vps26b KO [n = 10], p = 0.0013). Data expressed as mean ± SEM from 2–4 independent experiments (left panel). Bar graph showing the levels of entorhinal Aß40 and Aß42 measured by ELISA harvested from Vps26b mice (Aß40 measurements: Vps26b WT [n = 12] versus Vps26b HET [n = 16], p = 0.0011; Aß42 measurements: Vps26b WT [n = 16] versus Vps26b HET [n = 20], p = 0.1203). Vps26a mice (Aß40 measurements: Vps26a WT [n = 20] versus Vps26a HET [n = 19], p = 0.8886; Aß42 measurements: Vps26a WT [n = 15] versus Vps26a HET [n = 15], p = 0.8059). Two-tailed unpaired Student’s t test was used for the statistical analysis (right panel).(E) Bar graph showing tau CSF levels measured by Simoa technology from Vps26b KO mice and control littermates at 3–4 months: Vps26b WT (n = 10) versus Vps26b KO (n = 10), p = 0.0226, in a two-tailed unpaired Student’s t test; 6–7 months: Vps26b WT (n = 9) versus Vps26b KO (n = 9), in a two-tailed unpaired Student’s t test, p = 0.0056, in a non-parametric Mann Whitney test; and 12–14 months: Vps26b WT (n = 9) versus Vps26b KO (n = 9), **p = 0.0012, in a two-tailed unpaired Student’s t test (left panel). (F) Bar graph showing tau CSF levels measured by Simoa technology from Vps26 HET mice at 9–12 months. Vps26b mice (Vps26b WT[n = 7] versus Vps26b HET [n = 10], p = 0.0270) and Vps26a mice (Vps26a WT [n = 13] versus Vps26a HET [n = 10], p = 0.4169). Two-tailed unpaired Student’s t test was used for all statistical analysis (right panel). Data expressed as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig 4: Cognitive profiling supports VPS26b’s regional association(A and B) Vps26b mice were tested in the novel object recognition (NOR) and object-context recognition (OCR) tasks at 3 time points, as described in STAR Methods. Data are expressed as mean ± SEM. A two-way ANOVA with Bonferroni’s post hoc test was used for the analysis.(A) In the NOR task targeting the perirhinal cortex (n = 10 animals for each of the 6 independent groups), a defect in memory performance was observed only in 12- to 14-month-old mice.(B) In the entorhinal-sensitive OCR task (n = 10–14 for each of the 6 independent groups), a two-way ANOVA with Bonferroni’s post hoc corrections revealed a significant genotype X age interaction that was driven by age-related worsening in the Vps26b KO mice: F(2,67) = 13.92, p < 0.001.(C) OCR defects were also found in 12- to 14-month-old Vps26b HET mice versus their WT littermates (n = 9–14 per independent group; p = 0.0016, in unpaired non-parametric Mann-Whitney t test) but not in 12- to 14-month-old Vps26a HET mice versus their WT littermates (n = 8 per group; p = 0.5077, in unpaired two-sided Student’s t test).(D) Schematic representation of the mouse brain showing the injection site in the lateral recess of the lateral ventricle (image 77, http://atlas.brain-map.org/atlas?atlas=602630314#atlas=602630314&structure=549&resolution=10.26&x=5700&y=4000&zoom=-3&plate=576989860) (left panel). VPS26b overexpression in the EC of WT mice (C57BL6/J): mice were injected at 3 months of age with AAV9-VPS26b at different doses and volume and harvested at 4 months of age (top right panel). VPS26b rescue in EC of Vps26b KO mice: mice were injected at ~3.7 months of age, and brains were harvested at ~7.7 months (bottom right panel).(E) Vps26b mice were tested in the OCR task. Data are expressed as mean ± SEM. As expected, a defect in memory performance was observed in the Vps26b KO mice compared with the Vps26b WT controls, both injected with AAV9-GFP (n = 4–6 per group, p = 0.0028, in unpaired two-sided Student’s t test). An OCR behavioral rescue was observed in Vps26b KO mice injected with AAV9-VPS26b compared with Vps26b KO mice injected with AAV9-GFP alone (n = 4–6 animals/group, p = 0.0044, in a one-way ANOVA with Tukey’s post hoc test [two-sided]). *p < 0.05, **p < 0.01, and ***p < 0.001.(F) Mean fEPSP slopes expressed as a percentage of baseline measured before and after high-frequency stimulation in the TEC region of Vps26b WT– AAV9-GFP (n = 5), Vps26b KO – AAV9-GFP (n = 6), and Vps26b KO – AAV9-VPS26b (n = 6) mice. In a two-way repeated measured ANOVA test with a Dunnett’s post hoc, a rescue of LTP defects is observed in Vps26b KO mice injected with AAV9-VPS26b-GFP compared with Vps26b KO mice injected with AAV9-GFP (Vps26b KO – AAV9-VPS26b-GFP: 151.6% ± 0.40% versus Vps26b KO – AAV9-GFP: 103.6% ± 0.95% versus Vps26b WT – AAV9-GFP: 178.3% ± 1.08%, F[2,14] = 6.427, p = 0.0105).See also Figure S7.

Fig 5: VPS26b mediates glutamate receptor trafficking from the recycling endosome(A) Biotinylated cell-surface proteins were immunoprecipitated from total lysates of cortical neurons depleted for VPS26b (n = 11 biological replicates) or VPS26a (n = 5 biological replicates). Western blot analysis revealed that only VPS26b depletion results in a decrease in GluA1 cell-surface levels.(B) Representative confocal images of Vps26b KO neurons and Vps26b WT littermates subjected to pulse-chase transferrin uptake and stained for GluA1 and additional endosomal markers. Arrows indicate sites of co-localization. Scale bar, 10 µm (left panel). Quantitative colocalization analysis of GluA1 with early and recycling markers based on Pearson’s correlation coefficients revealed that VPS26b depletion results in an accumulation of GluA1 in recycling endosomes (transferrin: p < 0.0001; Syntaxin13: p < 0.0001) and to a lesser extent in early endosomes (EEA1: p = 0.032) in a non-parametric Kruskal-Wallis with Dunn’s post hoc test (n = 27–37 cells per group/condition, from three separate cultures) (right panel).(C) Cell-surface GluA1 levels were assessed by confocal microscopy in non-permeabilized cultured neurons incubated with an N-terminal GluA1 antibody in three conditions: Vps26b WT neurons infected with lentivirus expressing GFP alone (“Vps26b WT + GFP vector”), Vps26b KO neurons infected with lentivirus expressing GFP alone (“Vps26b KO + GFP vector”); and Vps26b KO neurons infected with lentivirus expressing VPS26b-GFP (“Vps26b KO + VPS26b-GFP”) (left panel). Scale bar, 10 µm (left panel). Mean fluorescence intensity values revealed that VPS26b repletion fully restored GluA1 surface localization in Vps26b KO neurons, as summarized in the bar graph (p < 0.0001), in a non-parametric Kruskal-Wallis with Dunn’s post hoc test (n = 25–26 neurons/condition, from four independent experiments) (right panel). Data expressed as mean ± SEM., *p < 0.05, **p < 0.01, and ***p < 0.001.See also Figure S6.