Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation

Sleep deprivation reduces the dextran radial distribution and 125I-apoE inflow from CSF into brain. A-B) Representative images of cascade blue dextran (CB) in mice on normal sleep cycle (A) and in mice during sleep deprivation (SD) (B). Cascade blue dextran (10 kDa) was injected into cisterna magna and the mice perfusion fixed (PFA) at 15 min. The vasculature was outline by lectin (green). Scale bars 100 μm (A-B). C) 125I-ApoE2 (yellow column), 125I-apoE3 (red column) and 125I-apoE4 (orange column) inflow into brain from the CSF were reduced in SD mice. D) 14C-inulin inflow into brain from the CSF was reduced with SD and not affected by apoE isoforms. 125I-ApoE (10 nM) and 14C-inulin were intracisternally injected and the brain analyzed for radioactivity. Values are mean ± SEM. N = 6 mice per group. (EPS 15099 kb

Additional file 11: Figure S11. of Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation

Cultured primary mouse choroid plexus epithelial cells secrete apoE. A) Representative apoE images from Western blots of the conditional medium from choroidal epithelial cells and astrocytes. B) Primary cultured choroidal epithelial cells (CP) produced similar levels of apoE as astrocytes (astro). Values are mean ± SEM, N = 4. C) Representative image of AQP1, a marker of choroid plexus epithelial cell, expression in the cultured primary mouse choroid plexus. Lack of significant immunolabeling of NeuN (neurons; D) and GFAP (astrocytes; E) in the cultured CP epithelial cells. F) Expression of GFAP in the cultured primary mouse astrocytes. DAPI (blue). (EPS 15343 kb

Additional file 2: Figure S2. of Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation

ApoE4 radial distribution around arteries is shorter than that of dextran. A) Representative images of radial distribution around arteries but not veins for dextran-cascade blue (inert reference molecule, CB) and apoE4-Alexa647 (apoE-647, purple) after 10 min post-injection in NG2Ds-Red (NG2, red) reporter mice. The vasculature was outlined by intravascular labeling with lectin (green). Scale bar = 100 μm. Arteries (yellow arrow head). Veins (blue arrows). B) A custom made image J plugin was used to generate the mean radial distribution of cascade blue labeled dextran (Dextran-CB, blue) and apoE4-Alexa647 (colored red) from the lectin stained vessel wall (green). Distribution from the vessel wall was determined at 1000 intensity units and maximum intensity indicate saturation at 4096 intensity units (212 – 12 bit image depth). (EPS 32092 kb

Additional file 1: Figure S1. of Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation

More GFAP-positive astrocytes on the arterial wall than that on veins. a-e) Representative images of GFAP-positive astrocytes on the wall and around arteries and veins. The vasculature was outline by lectin (green) in NG2dsRed reporter mice and perfusion fixed (PFA) at 15 min. A) Astrocytes (GFAP, blue), B) Vessels (lectin-FITC, green), C) arteries (NG2-dsRed, red), D) endogenous apoE (purple) and E) Merged images. Images of GFAP-positive astrocytes around an artery (identified by the expression of NG2-DsRed and vasculature staining with lectin) as shown in the yellow box in C (F) and a vein (identified by the lack of NG2-DsRed expression but with the vasculature stained with lectin) as shown in the white box in C (G). Scale bars: 100 μm (A-E), 50 μm (F-G). Arrow heads (panel F) identify some astrocytic endogenous apoE. (EPS 7646 kb

Additional file 9: Figure S9. of Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation

Endogenous apoE expression in the choroid plexus. Immunolabeling of mouse endogenous apoE in the choroid plexus. Note the high apoE expression level in choroid plexus. Blue, DAPI; green, apoE; red, NG2-dsRed; gray, Lectin. (EPS 824 kb

Additional file 10: Figure S10. of Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation

Lenti-apoE3 transduced the choroid plexus but not the parenchyma cells. A-H) Representative images showing transduction in the choroidal cells of lenti-apoE3 and lenti-EGFP at 4 weeks after intraventricular delivery. A) Merged image showing choroid plexus (CP; scale bar, 100 μm). B) EGFP (green); C) apoE3 (magenta); D) AQP1 (red); E) merged images of B, C and D (scale bar 50 μm). Arrows show apoE3 in choroidal cells in C and E. F-H) cortex (Cx) showing no transduction (no EGFP) (F, scale bar 200 μm) but the presence of apoE3 (G-H). H, scale bar 50 μm. Lenti-apoE3/lenti-EGFP mixture was delivered intraventicularly (3 μL lenti-human apoE (4.06 × 108 TU/ml) and lenti-EGFP (1012 TU/ml) and after 4 weeks the brains were perfusion fixed followed by immunolabeling. (EPS 41288 kb

Additional file 8: Figure S8. of Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation

Lenti-EGFP effectively transduced the choroid plexus. A) Lenti-EGFP unilateral injected into the lateral ventricle preferentially transduced the choroid plexus and ependymal layer but not brain parenchyma at 1 week. DAPI (blue); EGFP (green). B-E) Lenti-EGFP expression in the choroid plexus at 1 week. Images are DAPI (blue; B), AQP1 (red; C); EGFP (green; D) and merged images (E). Scale bar 50 μm. Intraventricular injection of 3 μL lenti-EGFP (1012 TU/ml). N = 5. (EPS 75360 kb

Additional file 4: Figure S4. of Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation

ApoE isoforms does not affect cascade blue-dextran radial distribution. Similar radial distribution distances of dextran-cascade blue (CB) in the presence of apoE2 (E2), apoE3 (E3) and apoE4 (E4) at 15 min after their intracisternal injection. Values are mean ± SEM, N = 5-9. (EPS 564 kb