Fig 2: Retromer complex proteins are reduced in cortices of young and aged subjects with Down syndrome (DS) subjects. (A) Representative Western blot of retromer recognition core proteins (VPS35, VPS26, VPS26B, and VPS29) and cargo receptors (cation‐independent mannose 6‐phosphate receptors [CI‐MPR] and Sorla) in cortices from young subjects with DS and matched control (CTR) subjects (15–40 years old). (B) Representative Western blot of retromer recognition core proteins (VPS35, VPS26, VPS26B, and VPS29) and cargo receptors (CI‐MPR and Sorla) in cortices from aged subjects with DS and matched control subjects (40–65 years old). (C) Densitometry analysis of Western blots shown in panel A (CTR, n = 11; DS, n = 11). (D) Densitometry analysis of Western blots shown in panel B (CTR, n = 18; DS, n = 18). Values represent mean ± standard error of the mean (*p < 0.05, #p < 0.10). GAPDH, glyceraldehyde 3‐phosphate dehydrogenase.

Fig 3: Retromer complex proteins are reduced in hippocampi of young and aged subjects with Down syndrome (DS). (A) Representative Western blot of retromer recognition core proteins (VPS35, VPS26, VPS26B, and VPS29) and cargo receptors (cation‐independent mannose 6‐phosphate receptors [CI‐MPRs] and Sorla) in hippocampus from young subjects with DS and matched control (CTR) subjects (15–40 years old). (B) Representative Western blot of retromer complex core proteins (VPS35, VPS26, VPS26B, and VPS29) and cargo receptors (CI‐MPR and Sorla) in hippocampus from old subjects with DS and CTR subjects (40–65 years old). (C) Densitometry analysis of Western blots shown in panel A (CTR, n = 5; DS, n = 4). (D) Densitometry analysis of Western blots shown in panel B (CTR, n = 14; DS, n = 10). Values represent mean ± standard error of the mean (*p < 0.05, #p < 0.10). GAPDH, glyceraldehyde 3‐phosphate dehydrogenase.

Fig 4: ERBB4, SORL1, and their GO enrichment. a Protein expressions of ERBB4 in NSCs, OPCs, and OLs as confirmed by immunofluorescence assay; scale bar = 200 μm, 20 μm, and 100 μm. b Protein expressions of SORL1 in NSCs, OPCs, and OLs as confirmed by immunofluorescence assay; scale bar = 200 μm, 20 μm, and 100 μm. c Representative Western blots and d Statistical analyses of ERBB4 and SORL1 of relative protein level in NSCs, OPCs and OLs. Means ± SD from three independent experiments represented. e Interaction network of ERBB4, SORL1, and GO enrichment terms that may be related to differentiation and myelination, ERBB4 and SORL1 may affect NSC differentiation and OL maturation through olfactory bulb interneurons and by regulating the development of the CNS, respectively. f Heatmap of ERBB4 and SORL1 expressed in NSCs, OPCs, and OL. g ERBB4 and SORL1 levels of NSC, OPC, and OL markers as detected by qPCR. Means ± SD from three independent experiments represented. ERBB4 and SORL1 gradually increased during NSC-OL differentiation

Fig 5: 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.