Fig 2: Cell Surface Proteomics Reveals Targets for SorCS2-Dependent Sorting(A) Workflow of surface proteome analysis in primary neurons.(B) Comparison of the surface proteomes of WT and S2KO primary neurons (mixed cortical-hippocampal cultures). n = 3 biological replicates/group (two technical replicates per biological replicate). Threshold applied: log2ratio(S2KO/WT) = ± 0.5; false discovery rate (FDR) = 0.15. See also Tables S2 and S3.(C and D) JWA levels in brain lysates from WT (S2+) and S2KO (S2-) mice as shown by western blot analysis (C) and densitometric quantification thereof (D). N-cadherin served as loading control. Mean ± SEM. n = 6 mice per genotype. *p < 0.05 (unpaired t test). See also Figure S2.(E and F) JWA mRNA levels in cortex (E) and hippocampus (hipp.) (F) of WT and S2KO mice as assessed by qRT-PCR (relative to actin). Mean ± SEM. n = 9 mice per genotype.(G) SorCS2 (red) and EAAT3 (green) immunostainings on mouse brain sections (single confocal z planes). Sections are counterstained with DAPI. Insets show higher magnification of the overview pictures of cortex, dentate gyrus (DG), and CA2. Scale bars, 50 µm. See also Figure S3.(H) SorCS2 (green) and EAAT3 (red) immunostainings in mouse hippocampal neurons (left) and C6 cells (right) (single confocal z planes). Cells are counterstained with DAPI. Higher magnification insets are shown. Scale bars, 10 µm.(I) Co-immunoprecipitation (coIP) of EAAT3 with SorCS2-GFP (S2), but not ß-galactosidase-GFP (ßG), overexpressed in CHO cells. This experiment was replicated three times.(J) coIP of EAAT3 with SorCS2 from mouse hippocampal lysates. IgG, non-specific IgG; IP, immunoprecipitation. This experiment was replicated three times.

Fig 3: Immunoblot of SLC1A1/2/3 transporters from whole-cell lysates for scrambled siRNA control cells in the quiescent state (SSC_Q), siRNA control cells in the proliferating state (SSC_P), Mfn2 knockdown cells in the quiescent state (Mfn2KD_Q), and Mfn2 knockdown cells in the proliferating state (Mfn2KD_P).

Fig 4: Validation of the human BNB transcriptome. Merged digital photomicrographs of sural nerve endoneurial microvessels (UEA-1 FITC-positive, green) in situ show BNB endothelial cell expression (yellow-green, yellow or orange co-localization dependent of the relative fluorescent intensity of protein marker in red) of transporters (ABCA8, ABCB1, AQP1, SLC1A1, SLC2A1, SLC3A2, SLC5A6, SLC16A1 and SLC19A2), cytoskeletal proteins (ACTG1, CALD1, FLNA, MYO10), cell membrane proteins (CAV1, CD44, CD63, ESAM), junctional complex proteins (CDH5, CDH6, CLDN4, CLDN5, CTNNA1, GJA1, LIN7A, MPDZ, PCDH1, TJP1, VEZT, ZYX), vascular endothelial cell secreted protein CTGF, and chemokine receptor CXCR4. Positive expression of AQP1, CALD1, CAV1, CD44, CD63, FLNA, MYO10 and SLC2A1 (red) by cells that surround and are in direct contact with endothelial cells (most likely pericytes), and ABCA8 by cells close to but not in direct contact with endothelial microvessels (most likely Schwann cells) are also observed. Nuclei are identified in blue. Original magnification 400X.

Fig 5: Increased α-synuclein aggregation in cortical neurons of hSyn/EAAT3−/− mice. (A) Immunostaining for phosphoserine-129 α-synuclein (pSyn; green), with neuronal nuclei identified by NeuN (red). Nuclei of all cells are counterstained with DAPI (blue) in the merged images. (B) α-synuclein aggregates are identified by proximity ligation assay (PLA). Red squares demark areas shown in magnified view. Examples of single dots are shown with arrows, and multiple dots are shown with arrowheads. All scale bars = 20 μm. (C) Quantification of pSyn immunostaining. * p < 0.05, n = 8–11. (D) Quantification of single-puncta PLA signals; * p < 0.05, n = 5–7. (E) Quantification of multi-puncta PLA signals. For all quantifications, data from each mouse are expressed relative to signal measured in hSyn mice sections prepared and analyzed in parallel.