Research ArticleALZHEIMER’S DISEASE

Autoregulated paracellular clearance of amyloid-β across the blood-brain barrier

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Science Advances  18 Sep 2015:
Vol. 1, no. 8, e1500472
DOI: 10.1126/sciadv.1500472
  • Fig. 1 Down-regulation of claudin-5 and occludin (C/O) in mouse brain endothelial cells allows size-selective movement of Aβ(1–40) peptides across cell monolayers.

    (A) Western blot of claudin-5 and occludin protein levels 72 hours after siRNA transfection (C/O, claudin-5 and occludin siRNAs). (B) Densitometric analysis of relative protein levels normalized to a NT siRNA sample at each time point (unpaired Student’s t test: **P ≤ 0.01; data are means ± SEM, n = 3 separate cell transfections). (C) TEER measurements across brain endothelial cell monolayers after treatment with NT siRNA, claudin-5 siRNA, occludin siRNA, or C/O siRNAs [one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test for multiple comparisons: *P ≤ 0.05 compared to siNT treatment; 95% confidence interval; data are means ± SEM, n = 3 separate cell transfections]. (D) Schematic diagram of FITC–Aβ(1–40) application to the apical chamber of a Transwell plate and its movement across brain endothelial cell monolayers (left). FITC–Aβ(1–40) peptide was applied to the apical chamber of a Transwell plate 72 hours after siRNA treatment of brain endothelial cells, and its movement across cell monolayers was monitored by fluorescence spectrophotometry over the course of 2 hours (middle). The apparent permeability coefficient (Papp) was also measured for all siRNA treatments (one-way ANOVA followed by Bonferroni’s post hoc test for multiple comparisons: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, all compared to siNT treatment; 95% confidence interval; data are means ± SEM, n = 3 separate cell transfections) (right). (E) Schematic diagram of FITC–Aβ(1–40) application to the basolateral chamber (left), movement of FITC–Aβ(1–40) from the basolateral to the apical chamber (middle), and Papp for movement of FITC–Aβ(1–40) from the basolateral to the apical chamber (right). (F) Comparison of apparent permeability coefficients (Papp) for synthetic Aβ(1–40)F19P monomer and Aβ(1–40)DiY dimer after apical application and movement across brain endothelial cell monolayers.

  • Fig. 2 In vivo cosuppression of claudin-5 and occludin and assessment of BBB permeability in claudin-5 and occludin (C/O) siRNA–treated mice.

    (A) Transcript levels of claudin-5 and occludin as measured by RT-PCR in brain capillary fractions isolated from animals treated with nontargeting (NT) siRNA or C/O siRNAs (n = 3 to 4 animals per treatment; data are means ± SEM). (B) Western blot of claudin-5 and occludin protein levels in brain capillary fractions isolated from NT siRNA– or C/O siRNA–treated animals. (C) Densitometric analysis of claudin-5 and occludin protein levels in brain capillary fractions isolated from NT siRNA– or C/O siRNA–treated animals (unpaired Student’s t test: **P ≤ 0.01; data are means ± SEM, n = 4 to 5 animals per treatment). (D) Contrast-enhanced MRI and bolus chase analysis of clearance rates of Gd-DTPA (742 daltons) in hippocampal regions after tail-vein injection of the contrast agent in animals treated with NT siRNA or C/O siRNAs (unpaired Student’s t test: **P ≤ 0.01; data are means ± SD, n = 4 animals per treatment). (E) Top: Animals treated with NT siRNA, claudin-5 siRNA, occludin siRNA, or C/O siRNAs were given a transcardial perfusion of 3-kD (left) or 10-kD (right) biotin-dextran, and brain sections were stained with isolectin IB4 and streptavidin-Cy3 (scale bars, 50 μm). Bottom: Streptavidin-Cy3 fluorescence measurements relative to NT siRNA control (one-way ANOVA followed by Bonferroni’s post hoc test for multiple comparisons: *P < 0.05, siC/O versus siNT; 95% confidence interval; data are means ± SEM, n = 3 to 4 animals per treatment).

  • Fig. 3 Systemic administration of claudin-5 and occludin (C/O) siRNAs enhances the paracellular movement of amyloid-β (Aβ) 1–40 from brain to blood in Tg2576 transgenic mice.

    (A) Top: Immunohistochemical analysis of occludin and claudin-5 levels in brain microvessels after injection of siRNAs in Tg2576 mice (scale bar, 100 μm). Bottom: Quantification of claudin-5– and occludin-positive vessels in siRNA-treated Tg2576 mice (unpaired Student’s t test: *P = 0.0406 for claudin-5, **P = 0.0036 for occludin; data are means ± SEM, n = 4 to 6 animals per experimental group). (B) ELISA of plasma Aβ(1–40) levels in Tg2576 mice after intravenous administration of nontargeting (NT) siRNA or C/O siRNAs (unpaired Student’s t test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; data are means ± SEM, n = 4 to 6 animals per experimental group). (C) T-maze assessment of hippocampal-linked spatial memory as measured by percentage alternation rates in NT siRNA– or C/O siRNA–treated Tg2576 mice at 6 months of age (unpaired Student’s t test: *P = 0.0403; n = 8 to 9 animals per experimental group). (D) ELISA of brain/plasma Aβ(1–40) ratios [pg/g soluble brain Aβ(1–40) per pg/ml plasma Aβ(1–40)] in NT siRNA– or C/O siRNA–treated Tg2576 mice (unpaired Student’s t test: *P = 0.0159; n = 9 animals per group).

  • Fig. 4 RNAi-mediated cosuppression of claudin-5 and occludin (C/O) in Tg2576 mice does not affect the levels of major Aβ receptors and transporters.

    (A) Western blot (left) and densitometric analysis (right) of claudin-5, occludin, LRP1, and RAGE in brain vascular protein fractions isolated from NT siRNA– or C/O siRNA–treated Tg2576 mice (unpaired Student’s t test: *P ≤ 0.05, **P ≤ 0.01; data are means ± SEM, n = 3 animals per treatment). (B) Western blot (left) and densitometric analysis (right) of APP, LRP1, RAGE, and ApoE in total brain protein fractions isolated from siRNA-treated Tg2576 mice (data are means ± SEM). (C) Western blot (left) and densitometric analysis (right) of APP, LRP1, RAGE, and ApoE in liver protein fractions isolated from siRNA-treated Tg2576 mice (data are means ± SEM).

  • Fig. 5 Claudin-5 and occludin are reduced in CAA-affected microvessels of aged Tg2576 mouse brains and in response to Aβ(1–40) monomers and dimers in mouse brain endothelial cells.

    (A) TEER measurements across confluent monolayers of brain endothelial cells isolated from aged Tg2576 mice and age-matched wild-type (WT) controls (unpaired Student’s t test: ***P = 0.0005; data are means ± SD, n = 16 Transwells per group). (B) Western blot analysis of tight junction proteins (claudin-5, occludin, ZO-1, and tricellulin) and Aβ transcellular receptors (LRP1 and RAGE) in brain endothelial cells isolated from aged (20 months) Tg2576 mice and age-matched WT controls (n = 5 animals per group). (C) Immunohistochemical analysis of claudin-5 and occludin in microvessels of young (3 months) and aged (20 months) WT mice and aged (20 months) Tg2576 mice (scale bar, 50 μm). (D) Top: Western blot (left) and densitometric analysis (right) of tight junction protein levels 12 hours after treatment of brain endothelial cells with increasing concentrations of scrambled Aβ(1–40) peptide, Aβ(1–40)F19P monomer, or Aβ(1–40)DiY dimer. Bottom: Western blot (left) and densitometric analysis (right) of tight junction protein levels over the course of 48 hours after treatment of brain endothelial cells with 1 μM of Aβ(1–40)F19P monomer or Aβ(1–40)DiY dimer (data are means ± SEM). (E) Immunocytochemical analysis of claudin-5, occludin, and ZO-1 in response to treatment of brain endothelial cells with 1 μM of Aβ(1–40)F19P monomer (top) or Aβ(1–40)DiY dimer (bottom) (images are representative of three separate cell transfections per time point; scale bars, 40 μm). (F) RT-PCR analysis of tight junction mRNA levels over the course of 48 hours in response to 1 μM of Aβ(1–40)F19P monomer (top) or Aβ(1–40)DiY dimer (bottom) (data are means ± SEM).

  • Fig. 6 Decreased levels of claudin-5 and occludin at the BBB segregate with Aβ/CAA in human AD brains.

    (A) Characterization of claudin-5 and occludin in brain microvessels and assessment of amyloid pathology (using Congo red and anti-Aβ immunostaining) in paraffin-embedded brain sections from a cohort of patients with neurological disease. Representative images from a nonneurodegenerative case [traumatic brain injury (TBI); contralateral region from injury site], a non-AD neurodegenerative case [amyotrophic lateral sclerosis (ALS)], and early-onset (EO) familial AD (APP-Iowa mutation) and late-onset (LO) sporadic AD cases (scale bar in all images, 50 μm). (B) Quantification of claudin-5– and occludin-positive vessels (per square millimeter) in donor brains from a cohort of patients with neurological disease (PSP, progressive supranuclear palsy; LBD, Lewy body dementia; one-way ANOVA followed by Bonferroni’s post hoc test for multiple comparisons: **P ≤ 0.01 for claudin-5 in AD versus PSP, ALS, and LBD; ***P ≤ 0.001 for claudin-5 in AD/CAA versus PSP, ALS, and LBD; ***P ≤ 0.001 for occludin in AD/CAA versus PSP, ALS, and LBD; data are means ± SD). (C) Strong microvascular claudin-5 and occludin immunoreactivity in microvessels close to amyloid plaques in AD brains (scale bar, 50 μm).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/8/e1500472/DC1

    Fig. S1. Western blot of ZO-1 levels 24 to 96 hours after siRNA transfection (top panel; NT, nontargeting siRNA; C/O, claudin-5 and occludin siRNAs) and densitometric analysis of relative ZO-1 protein levels normalized to NT siRNA sample at each time point (bottom panel; data are means ± SEM).

    Fig. S2. FITC–Aβ(1–42) peptide was applied to the apical chamber of a Transwell plate 72 hours after siRNA treatment of Bend.3 cells, and its movement across cell monolayers was monitored by fluorescence spectrophotometry over the course of 2 hours (left panel).

    Fig. S3. FITC-inulin was applied to the basolateral chamber of a Transwell plate 72 hours after siRNA treatment of Bend.3 cells, and its movement across cell monolayers was monitored by fluorescence spectrophotometry over the course of 2 hours.

    Fig. S4. Down-regulation of claudin-5, occludin, and LRP1 in primary mouse brain endothelial cells and analysis of FITC–Aβ(1–40) movement.

    Fig. S5. FITC–Aβ(1–40) (10 nM) was applied to the basolateral chamber of a Transwell plate 72 hours after siRNA treatment of Bend.3 cells, and its movement across cell monolayers was assessed by ELISA over the course of 2 hours (left panel).

    Fig. S6. Relative purity of mouse brain capillary fractions isolated using a dispase/dextran method.

    Fig. S7. Occludin expression and BBB permeability in APPP/PS1 mice post suppression of tight junctions.

    Fig. S8. Analysis of peripheral organ permeability post suppression of claudin-5 and occludin.

    Fig. S9. Increased plasma levels of Aβ in APP/PS1 mice post suppression of claudin-5 and occludin.

    Fig. S10. Aβ in retinas of 8-month-old siRNA-treated DBA/2J mice.

    Fig. S11. Analysis of BBB integrity to endogenous immunoglobulin G (IgG) after chronic intravenous administration of nontargeting (NT) siRNA or claudin-5 and occludin (C/O) siRNAs in Tg2576 mice (scale bar, 100 μm; n = 5 to 6 animals per experimental group; data are means ± SEM).

    Fig. S12. Hematoxylin and eosin (H&E) staining of liver, lung, heart, spleen, and kidney paraffin-embedded sections from nontargeting (NT) siRNA–treated and claudin-5 and occludin (C/O) siRNA–treated APP (Tg2576) transgenic mice at 12 months of age (n = 4 to 6 animals per experimental group; scale bar for all images, 200 μm).

    Fig. S13. Quantification of claudin-5– and occludin-positive vessels in young WT, aged WT, and aged Tg2576 animals (n = 4 animals per group; data are means ± SEM).

    Fig. S14. Western blot analysis of tight junction protein levels in Bend.3 cells 12 hours after treatment with increasing concentrations (0.1 to 1 μM) of recombinant unmodified Aβ(1–40) monomer peptides (n = 3 separate cell transfections per concentration).

    Fig. S15. MTS-based cell viability assay measured 12 hours after treatment of Bend.3 cells with increasing concentrations (0.5 to 5 μM) of Aβ(1–40)F19P monomer or Aβ(1–40)DiY dimer.

    Fig. S16. Schematic model of paracellular Aβ movement across CVECs of the BBB.

    Table S1. Brain regions, pathological diagnoses, age, gender, and postmortem (PM) interval for the neurological disease patient cohort examined in this study.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Western blot of ZO-1 levels 24 to 96 hours after siRNA transfection (top panel; NT, nontargeting siRNA; C/O, claudin-5 and occludin siRNAs) and densitometric analysis of relative ZO-1 protein levels normalized to NT siRNA sample at each time point (bottom panel; data are means ± SEM).
    • Fig. S2. FITC–Aβ(1–42) peptide was applied to the apical chamber of a Transwell plate 72 hours after siRNA treatment of Bend.3 cells, and its movement across cell monolayers was monitored by fluorescence spectrophotometry over the course of 2 hours (left panel).
    • Fig. S3. FITC-inulin was applied to the basolateral chamber of a Transwell plate 72 hours after siRNA treatment of Bend.3 cells, and its movement across cell monolayers was monitored by fluorescence spectrophotometry over the course of 2 hours.
    • Fig. S4. Down-regulation of claudin-5, occludin, and LRP1 in primary mouse brain endothelial cells and analysis of FITC–Aβ(1–40) movement.
    • Fig. S5. FITC–Aβ(1–40) (10 nM) was applied to the basolateral chamber of a Transwell plate 72 hours after siRNA treatment of Bend.3 cells, and its movement across cell monolayers was assessed by ELISA over the course of 2 hours (left panel).
    • Fig. S6. Relative purity of mouse brain capillary fractions isolated using a dispase/dextran method.
    • Fig. S7. Occludin expression and BBB permeability in APPP/PS1 mice post suppression of tight junctions.

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