APOE4 leads to phosphosite dysregulation at the BBB. (A) Schematic of brain capillary isolation workflow from mouse cortex for phosphoproteome and proteome study. CD brain is prepared for postsynaptic PSD-95 immunoprecipitation assays. See Materials and methods for details. (B–E) Isolated brain capillaries stained for lectin+-endothelium (B), green; DRAQ5 nuclear stain, pink; bar, 100 µm; Pdgfrβ+-PCs (C); Pdgfrβ, red; lectin+-endothelium, green; DRAQ5, blue; bar, 10 µm; and aquaporin 4 (AQP4)+-astrocyte end feet (D); AQP4, green; lectin+-endothelium, red; DRAQ5, blue; bar, 50 µm; but did not stain for smooth muscle cell marker SMA (E); SMA, red; lectin+-endothelium, green; bar, 50 µm (see also Fig. S3). (F) Immunoblotting of brain capillaries and CD brain for the PC marker Pdgfrβ; endothelial markers CD31, TfR, Glut-1, and Claudin-5; neuronal marker, TuJ1; and astrocyte marker, GFAP. (G) Distribution of functional groups for all nonredundant proteins with differentially regulated phosphosites in brain capillaries from E4F compared with E3F mice at 7 mo of age. Legend shows abundant functional groups with number of proteins with dysregulated phosphosites per functional group indicated. The genes encoding proteins with differentially regulated phosphosites in ECs, PCs, and astrocyte end feet are given in Table S1 H. (H) Distribution of substrate-kinase family pairs among differentially regulated phosphosites in abundant functional groups including cytoskeletal proteins, DNA- and RNA-binding proteins, cell adhesions, and others. Blue, AGC (PKA, PKG, and PKC); orange, CMGC (cyclin-dependent kinases, mitogen-activated protein kinase, glycogen synthase kinase, and CDC-like kinase); yellow, STE (serine/threonine kinases); green, atypical kinases; gray, TKL (tyrosine kinase–like kinases); red, TLK (tousled-like kinase). (I) Heatmap showing hierarchical clustering of single-cell RNA-seq gene expression for all nonredundant proteins found to contain differentially regulated phosphosites in brain capillary ECs and PCs. Proteins showing preferential cell-type enrichment in either ECs or PCs are highlighted by blue brackets. The z-scores of proteins with dysregulated phosphosites in ECs and PCs are reported in Table S1 J. (J) Distribution of functional groups within ECs and PCs assigned to nonredundant proteins found to contain differentially regulated phosphosites. Legend shows abundant functional groups. The number of proteins with dysregulated phosphosites for the most abundant functional groups in the EC and PC pie charts are indicated. Proteins assigned to astrocyte end feet are excluded from analysis. (K–M) Plots showing the percentage of all differentially regulated phosphosites (K), differentially regulated phosphosites within cytoskeletal proteins (L), or within nuclear proteins (M) predicted to be regulated by the indicated kinase family separated by assigned cell type as ECs and PCs. Color code for different kinases as in H, plus turquoise, TK (tyrosine kinase). All data in G–M are from four mice per group. Source data are available for this figure: SourceData F3.
Figure 3.

APOE4 leads to phosphosite dysregulation at the BBB. (A) Schematic of brain capillary isolation workflow from mouse cortex for phosphoproteome and proteome study. CD brain is prepared for postsynaptic PSD-95 immunoprecipitation assays. See Materials and methods for details. (B–E) Isolated brain capillaries stained for lectin+-endothelium (B), green; DRAQ5 nuclear stain, pink; bar, 100 µm; Pdgfrβ+-PCs (C); Pdgfrβ, red; lectin+-endothelium, green; DRAQ5, blue; bar, 10 µm; and aquaporin 4 (AQP4)+-astrocyte end feet (D); AQP4, green; lectin+-endothelium, red; DRAQ5, blue; bar, 50 µm; but did not stain for smooth muscle cell marker SMA (E); SMA, red; lectin+-endothelium, green; bar, 50 µm (see also Fig. S3). (F) Immunoblotting of brain capillaries and CD brain for the PC marker Pdgfrβ; endothelial markers CD31, TfR, Glut-1, and Claudin-5; neuronal marker, TuJ1; and astrocyte marker, GFAP. (G) Distribution of functional groups for all nonredundant proteins with differentially regulated phosphosites in brain capillaries from E4F compared with E3F mice at 7 mo of age. Legend shows abundant functional groups with number of proteins with dysregulated phosphosites per functional group indicated. The genes encoding proteins with differentially regulated phosphosites in ECs, PCs, and astrocyte end feet are given in Table S1 H. (H) Distribution of substrate-kinase family pairs among differentially regulated phosphosites in abundant functional groups including cytoskeletal proteins, DNA- and RNA-binding proteins, cell adhesions, and others. Blue, AGC (PKA, PKG, and PKC); orange, CMGC (cyclin-dependent kinases, mitogen-activated protein kinase, glycogen synthase kinase, and CDC-like kinase); yellow, STE (serine/threonine kinases); green, atypical kinases; gray, TKL (tyrosine kinase–like kinases); red, TLK (tousled-like kinase). (I) Heatmap showing hierarchical clustering of single-cell RNA-seq gene expression for all nonredundant proteins found to contain differentially regulated phosphosites in brain capillary ECs and PCs. Proteins showing preferential cell-type enrichment in either ECs or PCs are highlighted by blue brackets. The z-scores of proteins with dysregulated phosphosites in ECs and PCs are reported in Table S1 J. (J) Distribution of functional groups within ECs and PCs assigned to nonredundant proteins found to contain differentially regulated phosphosites. Legend shows abundant functional groups. The number of proteins with dysregulated phosphosites for the most abundant functional groups in the EC and PC pie charts are indicated. Proteins assigned to astrocyte end feet are excluded from analysis. (K–M) Plots showing the percentage of all differentially regulated phosphosites (K), differentially regulated phosphosites within cytoskeletal proteins (L), or within nuclear proteins (M) predicted to be regulated by the indicated kinase family separated by assigned cell type as ECs and PCs. Color code for different kinases as in H, plus turquoise, TK (tyrosine kinase). All data in G–M are from four mice per group. Source data are available for this figure: SourceData F3.

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