Fig 2: 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.

Fig 3: The trans-entorhinal cortex differentially depends on VPS26b(A) Functional MRI. Cerebral blood volume (CBV) fMRI maps were generated from the whole brain of 3- to 4-month-old Vps26b KO and their control littermates (n = 10 per group) and 12- to 14-month-old Vps26b KO mice and their control littermates (n = 9–10 per group) (top left panel). A voxel-based analysis across the whole brain revealed a significant genotype X age focal defect (voxel-wise p < 0.005; cluster-wise p < 0.05; cluster size >25 voxels) (top right panel), which upon magnification is found to localize to the TEC (bottom left panel) (color bar represents t values of the interaction; PRC, perirhinal cortex; EC, entorhinal cortex, HIP, hippocampus; red arrow indicates the TEC). A region of interest (ROI) analysis of the relative CBV (rCBV) at the TEC between Vps26b WT and Vps26b KO revealed a significant age-dependent worsening of rCBV in the TEC region (in an ANOVA analysis of the genotype × age interaction: F[3,38] = 16.08, p = 0.0003; n = 9–10 animals/genotype) (top right panel).(B) Electrophysiology. An example of an acute ex vivo brain slice whose precise anatomical coordinates were matched to the neuroimaging defects, illustrating electrode placement (top left panel) (TEC, red arrow; MEC, blue arrow). Mean fEPSP slopes, expressed as the percentage of baseline measured before and after high-frequency stimulation in the TEC (top right panel) and in the MEC (bottom left panel), showed that 12- to 14-month-old Vps26b KO mice, compared with control littermates (n = 6 per group), have LTP defects in the TEC (F(1,14) = 69.2; p < 0.001) but normal LTP in the MEC (F(1,14) = 0.93; p = 0.365) in a repeated-measures ANOVA post hoc Tukey test. Abnormal LTP was also found in the TEC of 18-month-old Vps26b HET mice but not in 18-month-old Vps26a HET mice (Vps26b HET versus WT: F(2,11) = 3.27; p = 0.005; Vps26a HET versus WT: p = 0.42) (post hoc Tukey) (bottom right panel).(C) By comparing co-registered brain slices of GluA1 immunostainings from 14-month-old Vps26b WT mice (n = 5) (left panel) and Vps26b KO mice (n = 5) (middle panel), a pixel-based analysis (“Vps26b WT versus KO”) showed that Vps26b KO mice have focal TEC reductions in GluA1 immunostaining levels (indicated by the red arrow) (right panel) in a two-sample t test; pixel-wise p < 0.001; color bar represents t values. Scale bar, 500 µm.See also Figure S5.

Fig 4: 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 5: VPS26b and VPS26a define distinct retromer cores(A) An illustration of the proposed hypothesis for how the VPS26 paralogs form two separate retromer cores (top panel). Co-immunoprecipitation analysis of retromer proteins extracted from primary neuronal cultures using VPS35 (10 µg; left panel), VPS26a (10 µg; middle panel), and VPS26b (10µg; right panel) as baits (bottom panel) support this hypothesis.(B) A representative confocal image showing partial co-localization of both VPS26 paralogs (left panel), while quantitative colocalization studies, based on Pearson’s correlation, reveal that VPS35 shows a higher percentage of co-localization with each VPS26 paralog (n = 28–29 cells, from three independent experiments) (right panel). Scale bar, 10 µm.(C) Support for VPS26b forming a distinct retromer core is shown by how a primary depletion of VPS26b, in primary cultures derived from Vps26b heterozygous (HET) mice (n = 6) or Vps26b KO mice (n = 6) compared with Vps26b WT mice (n = 8), has no effect on VPS26a but causes a secondary reduction in VPS29 and VPS35, as summarized in the bar graphs and illustrated by representative immunoblots (in a one-way ANOVA with Tukey’s post hoc test’s two-sided analysis).(D) Support for VPS26a forming a distinct retromer core is shown by how a primary depletion of VPS26a, induced by infecting neurons from Vps26aflox/flox mice with a lentivirus-expressing Cre recombinase (Cre, n = 11), compared with neurons infected with a lentivirus expressing a catalytically dead Cre recombinase (?Cre, n = 12), has no effect on VPS26b (p = 0.1480, in an unpaired t test with Welch’s correction) but causes a secondary reduction in VPS29 and VPS35, as summarized in the bar graphs and illustrated with representative immunoblots. Statistical analyses were performed using either unpaired two-sided Student’s t test, with Welch’s correction when required, or a non-parametric Mann-Whitney t test.(E) Western blots of hippocampus homogenates from Vps26a and Vps26b HET mice (Vps26a WT, n = 4; Vps26a HET, n = 11; Vps26b WT, n = 7; Vps26b HET, n = 7). Quantitative analysis of the western blots probed for retromer proteins shows that a primary deficiency in VPS26a (p = 0.0015) results in a secondary reduction VPS35 (p = 0.0037) and VPS29 (p = 0.0061) but not VPS26b (p = 0.8101), while a primary deficiency in VPS26b (p < 0.0001) results in a secondary reduction in VPS35 (p = 0.0004) and VPS29 (p = 0.0009) but not VPS26a (p = 0.6373), arguing in favor of separate retromer cores. All statistical analysis were performed using two-sided Student’s t test except for VPS26a in the Vps26a WT versus Vps26a HET analysis. Values denote mean ± SEM, where *p < 0.05, **p < 0.01, and ***p < 0.001.See also Figures S1 and S2.