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Cited by 281Alzheimer's disease research and treatmentsBarrier Structure and Function StudiesCerebrospinal fluid and hydrocephalus

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Blood-brain barrier-associated pericytes internalize and clear aggregated amyloid-β42 by LRP1-dependent apolipoprotein E isoform-specific mechanism

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Ma, Qingyi, Zhao, Zhen, Sagare, Abhay P, Wu, Yingxi, Wang, Min, Owens, Nelly Chuqui, Verghese, Philip B, Herz, Joachim, Holtzman, David M, Zlokovic, Berislav V·2018·Molecular Neurodegeneration
DOI 10.1186/s13024-018-0286-0PMC PMC6194676
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Abstract

Abstract Background Clearance at the blood-brain barrier (BBB) plays an important role in removal of Alzheimer’s amyloid-β (Aβ) toxin from brain both in humans and animal models. Apolipoprotein E (apoE), the major genetic risk factor for AD, disrupts Aβ clearance at the BBB. The cellular and molecular mechanisms, however, still remain unclear, particularly whether the BBB-associated brain capillary pericytes can contribute to removal of aggregated Aβ from brain capillaries, and whether removal of Aβ aggregates by pericytes requires apoE, and if so, is Aβ clearance on pericytes apoE isoform-specific. Methods We performed immunostaining for Aβ and pericyte biomarkers on brain capillaries (< 6 μm in diameter) on tissue sections derived from AD patients and age-matched controls, and APPSwe/0 mice and littermate controls. Human Cy3-Aβ42 uptake by pericytes was studied on freshly isolated brain slices from control mice, pericyte LRP1-deficient mice (Lrplox/lox;Cspg4-Cre) and littermate controls. Clearance of aggregated Aβ42 by mouse pericytes was studied on multi-spot glass slides under different experimental conditions including pharmacologic and/or genetic inhibition of the low density lipoprotein receptor related protein 1 (LRP1), an apoE receptor, and/or silencing mouse endogenous Apoe in the presence and absence of human astrocyte-derived lipidated apoE3 or apoE4. Student’s t-test and one-way ANOVA followed by Bonferroni's post-hoc test were used for statistical analysis. Results First, we found that 35% and 60% of brain capillary pericytes accumulate Aβ in AD patients and 8.5-month-old APPSw/0 mice, respectively, compared to negligible uptake in controls. Cy3-Aβ42 species were abundantly taken up by pericytes on cultured mouse brain slices via LRP1, as shown by both pharmacologic and genetic inhibition of LRP1 in pericytes. Mouse pericytes vigorously cleared aggregated Cy3-Aβ42 from multi-spot glass slides via LRP1, which was inhibited by pharmacologic and/or genetic knockdown of mouse endogenous apoE. Human astrocyte-derived lipidated apoE3, but not apoE4, normalized Aβ42 clearance by mouse pericytes with silenced mouse apoE. Conclusions Our data suggest that BBB-associated pericytes clear Aβ aggregates via an LRP1/apoE isoform-specific mechanism. These data support the role of LRP1/apoE interactions on pericytes as a potential therapeutic target for controlling Aβ clearance in AD.

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Figures

Fig. 1 Aβ accumulation in brain pericytes in AD patients and 8.5-month-old APPSw/0 mice. a-f Representative confocal microscopy images showing Aβ colocalization with CD13+ (a-b) and PDGFRβ+ (d-e) pericytes in brain cortical sections from AD patients compared to negligible levels in age-matched controls, and quantification of Aβ + area in pericytes expressed as the percentage of Aβ + area occupying CD13+ (c) or PDGFRβ+ (f) pericyte capillary profiles. N = 6 per group; mean ± s.d., p < 0.01 by Student’s t-test. Orthogonal views shown on the right in A, B, D and E are from 10 μm Z-stacks. Scale bar, 25 μm. g-i Representative confocal microscopy images showing Aβ colocalization with CD13+ pericytes (g) in brain sections from 8.5-month old APPSw/0 mice and age-matched littermate control, and quantification of Aβ + area within CD13+ pericyte profiles in APPSw/0 mice and controls (h). N = 3 mice per group; mean ± SD, p < 0.001 by Student’s t-test. Orthogonal views from 10 μm Z-stacks. Scale bar, 25 μm. i Representative images showing TUNEL staining (green) in CD13+ brain pericytes in 8.5-month old APPSw/0 mice. Lectin (blue), labels brain endothelium. Aβ (white), shows perivascular and intracellular accumulation. Scale bar: 10 μm. Asterisk shows Aβ deposit; Arrowheads show TUNEL+ CD13+ pericytes. j Quantification of TUNEL+ CD13+ pericytes in the cortex of 3- and 8.5-month old APPSw/0 mice compared to 8.5-month old littermate controls. N = 3 mice per group; mean ± s.e.m.; p < 0.05 by Student’s t-test
Fig. 1 Aβ accumulation in brain pericytes in AD patients and 8.5-month-old APPSw/0 mice. a-f Representative confocal microscopy images showing Aβ colocalization with CD13+ (a-b) and PDGFRβ+ (d-e) pericytes in brain cortical sections from AD patients compared to negligible levels in age-matched controls, and quantification of Aβ + area in pericytes expressed as the percentage of Aβ + area occupying CD13+ (c) or PDGFRβ+ (f) pericyte capillary profiles. N = 6 per group; mean ± s.d., p < 0.01 by Student’s t-test. Orthogonal views shown on the right in A, B, D and E are from 10 μm Z-stacks. Scale bar, 25 μm. g-i Representative confocal microscopy images showing Aβ colocalization with CD13+ pericytes (g) in brain sections from 8.5-month old APPSw/0 mice and age-matched littermate control, and quantification of Aβ + area within CD13+ pericyte profiles in APPSw/0 mice and controls (h). N = 3 mice per group; mean ± SD, p < 0.001 by Student’s t-test. Orthogonal views from 10 μm Z-stacks. Scale bar, 25 μm. i Representative images showing TUNEL staining (green) in CD13+ brain pericytes in 8.5-month old APPSw/0 mice. Lectin (blue), labels brain endothelium. Aβ (white), shows perivascular and intracellular accumulation. Scale bar: 10 μm. Asterisk shows Aβ deposit; Arrowheads show TUNEL+ CD13+ pericytes. j Quantification of TUNEL+ CD13+ pericytes in the cortex of 3- and 8.5-month old APPSw/0 mice compared to 8.5-month old littermate controls. N = 3 mice per group; mean ± s.e.m.; p < 0.05 by Student’s t-testCC BY · PubMed Central
Fig. 2 LRP1-dependent Cy3-Aβ42 uptake by pericytes in mouse brain slices. a A diagram illustrating the experimental procedure in cultured mouse brain slices used to determine Cy3-Aβ42 uptake by pericytes. Brain slices were first cultured in transwell inserts with oxygenated aCSF (see method) for 4 h before adding Cy3-Aβ42 (1 μM). b-c Representative low-magnification images (b) and quantification (c) of cellular uptake of Cy3-Aβ42 by CD13+ pericytes in brain slices at 30 min and 2 h after the addition of Cy3-Aβ42. Scale bar: 25 μm. d Representative high magnification images showing Cy3-Aβ42 internalization by CD13-positive pericytes in brain slices in 2 h, in the presence of NI-IgG, anti-LRP1 or RAP. Scale bar: 20 μm. Orthogonal views on the right show Cy3-Aβ42 accumulation in CD13+ pericytes; scale bar: 5 μm. e Quantification of Cy3-Aβ42 uptake by CD13+ pericytes in mouse brain slices with and without NI-IgG, anti-LRP1, and RAP. N = 4 independent cultures; mean ± s.e.m.; p < 0.05 by One-way ANOVA followed by Bonferroni post-hoc test. Asterisks show colocalization of Cy3-Aβ42 and CD13 signals in (b) and (d)
Fig. 2 LRP1-dependent Cy3-Aβ42 uptake by pericytes in mouse brain slices. a A diagram illustrating the experimental procedure in cultured mouse brain slices used to determine Cy3-Aβ42 uptake by pericytes. Brain slices were first cultured in transwell inserts with oxygenated aCSF (see method) for 4 h before adding Cy3-Aβ42 (1 μM). b-c Representative low-magnification images (b) and quantification (c) of cellular uptake of Cy3-Aβ42 by CD13+ pericytes in brain slices at 30 min and 2 h after the addition of Cy3-Aβ42. Scale bar: 25 μm. d Representative high magnification images showing Cy3-Aβ42 internalization by CD13-positive pericytes in brain slices in 2 h, in the presence of NI-IgG, anti-LRP1 or RAP. Scale bar: 20 μm. Orthogonal views on the right show Cy3-Aβ42 accumulation in CD13+ pericytes; scale bar: 5 μm. e Quantification of Cy3-Aβ42 uptake by CD13+ pericytes in mouse brain slices with and without NI-IgG, anti-LRP1, and RAP. N = 4 independent cultures; mean ± s.e.m.; p < 0.05 by One-way ANOVA followed by Bonferroni post-hoc test. Asterisks show colocalization of Cy3-Aβ42 and CD13 signals in (b) and (d)CC BY · PubMed Central
Fig. 3 LRP1 genetic deletion from pericytes inhibits Cy3-Aβ42 uptake by pericytes in mouse brain slices. a Diagram illustrating generation of Lrp1lox/lox; Cspg4-Cre mice by crossing Lrp1lox/lox mice with Cspg4-Cre mice. b Representative high magnification images showing LRP1 expression in CD13+ pericytes on isolated murine brain capillaries from control Lrp1lox/lox mice, but not Lrp1lox/lox; Cspg4-Cre mice with LRP1 deletion from pericytes. Asterisks show LRP1 immunostaining in CD13+ pericytes in control Lrp1lox/lox mice; arrow shows loss of LRP1 immunoreactivity in Lrp1lox/lox; Cspg4-Cre mice. Scale bar: 5 μm. c Representative images showing Cy3-Aβ42 internalization by CD13+ pericytes in brain slices from control Lrp1lox/lox mice, and a substantial loss of Cy3-Aβ42 uptake by pericytes in Lrp1lox/lox; Cspg4-Cre mice. Asterisks show colocalization between Cy3-Aβ42 and CD13 signals. Scale bar: 25 μm. d High magnification images showing greatly reduced Cy3-Aβ42 internalization by a CD13+ pericyte on brain slices from Lrp1lox/lox; Cspg4-Cre mice, in contrast to Aβ uptake by lectin-positive endothelial cells. Arrow points to pericyte lacking Cy3-Aβ42 signal. Scale bar: 5 μm. e, f Quantification of Cy3-Aβ42 cellular uptake by CD13+ pericytes (e) compared to all other CD13- (negative) brain cells (f) in brain slices from control Lrp1 lox/lox and Lrp1 lox/lox; Cspg4-Cre mice. N = 3 mice per group; mean ± s.e.m.; NS, not significant, p < 0.05 by Student’s t-test
Fig. 3 LRP1 genetic deletion from pericytes inhibits Cy3-Aβ42 uptake by pericytes in mouse brain slices. a Diagram illustrating generation of Lrp1lox/lox; Cspg4-Cre mice by crossing Lrp1lox/lox mice with Cspg4-Cre mice. b Representative high magnification images showing LRP1 expression in CD13+ pericytes on isolated murine brain capillaries from control Lrp1lox/lox mice, but not Lrp1lox/lox; Cspg4-Cre mice with LRP1 deletion from pericytes. Asterisks show LRP1 immunostaining in CD13+ pericytes in control Lrp1lox/lox mice; arrow shows loss of LRP1 immunoreactivity in Lrp1lox/lox; Cspg4-Cre mice. Scale bar: 5 μm. c Representative images showing Cy3-Aβ42 internalization by CD13+ pericytes in brain slices from control Lrp1lox/lox mice, and a substantial loss of Cy3-Aβ42 uptake by pericytes in Lrp1lox/lox; Cspg4-Cre mice. Asterisks show colocalization between Cy3-Aβ42 and CD13 signals. Scale bar: 25 μm. d High magnification images showing greatly reduced Cy3-Aβ42 internalization by a CD13+ pericyte on brain slices from Lrp1lox/lox; Cspg4-Cre mice, in contrast to Aβ uptake by lectin-positive endothelial cells. Arrow points to pericyte lacking Cy3-Aβ42 signal. Scale bar: 5 μm. e, f Quantification of Cy3-Aβ42 cellular uptake by CD13+ pericytes (e) compared to all other CD13- (negative) brain cells (f) in brain slices from control Lrp1 lox/lox and Lrp1 lox/lox; Cspg4-Cre mice. N = 3 mice per group; mean ± s.e.m.; NS, not significant, p < 0.05 by Student’s t-testCC BY · PubMed Central
Fig. 4 LRP1 mediates clearance of aggregated Cy3-Aβ42 by mouse pericytes. a-b Multiphoton/confocal laser scanning microscopy of multi-spot glass slides coated with Cy3-Aβ42 without cells (a), and with primary mouse brain pericytes cultured for 5 days in the presence of NI-IgG or anti-LRP1, after si.Lrp1 silencing compared to scrambled si.Control, and with RAP or vehicle (b). Scale bar, 50 μm. c Quantification of Cy3-Aβ42 relative signal intensity on multi-spot slides after 5 days without cells (open bar on the left) and with pericytes in the presence of vehicle (control), NI-IgG and anti-LRP1, after silencing with scrambled si.Control or si.Lrp1, and in the presence of RAP. N = 4 independent cultures (biological replicates, see Methods); mean ± s.e.m.; p < 0.05 by One-way ANOVA followed by Bonferroni post-hoc test. d Quantification of TUNEL+ pericyte cell death at 3 and 7 days after seeding on multi-spot glass slides coated with Cy3-Aβ42 in the presence and absence of NI-IgG and anti-LRP1, and after si.Lrp1 silencing or si.Ctrl as in (b). N = 3 independent cultures per group; mean ± s.e.m.; p < 0.05 by One-way ANOVA followed by Bonferroni post-hoc test
Fig. 4 LRP1 mediates clearance of aggregated Cy3-Aβ42 by mouse pericytes. a-b Multiphoton/confocal laser scanning microscopy of multi-spot glass slides coated with Cy3-Aβ42 without cells (a), and with primary mouse brain pericytes cultured for 5 days in the presence of NI-IgG or anti-LRP1, after si.Lrp1 silencing compared to scrambled si.Control, and with RAP or vehicle (b). Scale bar, 50 μm. c Quantification of Cy3-Aβ42 relative signal intensity on multi-spot slides after 5 days without cells (open bar on the left) and with pericytes in the presence of vehicle (control), NI-IgG and anti-LRP1, after silencing with scrambled si.Control or si.Lrp1, and in the presence of RAP. N = 4 independent cultures (biological replicates, see Methods); mean ± s.e.m.; p < 0.05 by One-way ANOVA followed by Bonferroni post-hoc test. d Quantification of TUNEL+ pericyte cell death at 3 and 7 days after seeding on multi-spot glass slides coated with Cy3-Aβ42 in the presence and absence of NI-IgG and anti-LRP1, and after si.Lrp1 silencing or si.Ctrl as in (b). N = 3 independent cultures per group; mean ± s.e.m.; p < 0.05 by One-way ANOVA followed by Bonferroni post-hoc testCC BY · PubMed Central
Fig. 5 Apolipoprotein E-dependent and isoform-specific effect on LRP1-mediated clearance of aggregated Cy3-Aβ42 by mouse pericytes. a Multiphoton/confocal laser scanning microscopy of multi-spot glass slides coated with Cy3-Aβ42 with primary mouse brain pericytes cultured for 5 days in the presence of mouse apoE-specific blocking antibody (anti-apoE), after silencing mouse endogenous apoE (si.Apoe) compared to scrambled si.Control, and after silencing mouse apoE (si.Apoe) in the presence of astrocyte-derived lipidated human apoE3 or apoE4 (40 nM) with and without anti-LRP1 antibody. Scale bar, 50 μm. b Time-dependent Cy3-Aβ42 clearance by mouse pericytes cultured for 1, 3 and 5 days after silencing mouse endogenous apoE (si.Apoe) compared to si.Control, and in the presence of human apoE3 or apoE4. Dashed line indicates Cy3-Aβ42 signal in the absence of cells (control without cells). c Quantification analysis of Cy3-Aβ42 relative signal intensity on multi-spot slides after 5 days of culture with pericytes under different experimental conditions as indicated. Gray bar shows Cy3-Aβ42 signal in the absence of cells (control without cells). N = 3 independent cultures; mean ± s.e.m.; p < 0.05 by one-way ANOVA followed by Bonferroni post-hoc test
Fig. 5 Apolipoprotein E-dependent and isoform-specific effect on LRP1-mediated clearance of aggregated Cy3-Aβ42 by mouse pericytes. a Multiphoton/confocal laser scanning microscopy of multi-spot glass slides coated with Cy3-Aβ42 with primary mouse brain pericytes cultured for 5 days in the presence of mouse apoE-specific blocking antibody (anti-apoE), after silencing mouse endogenous apoE (si.Apoe) compared to scrambled si.Control, and after silencing mouse apoE (si.Apoe) in the presence of astrocyte-derived lipidated human apoE3 or apoE4 (40 nM) with and without anti-LRP1 antibody. Scale bar, 50 μm. b Time-dependent Cy3-Aβ42 clearance by mouse pericytes cultured for 1, 3 and 5 days after silencing mouse endogenous apoE (si.Apoe) compared to si.Control, and in the presence of human apoE3 or apoE4. Dashed line indicates Cy3-Aβ42 signal in the absence of cells (control without cells). c Quantification analysis of Cy3-Aβ42 relative signal intensity on multi-spot slides after 5 days of culture with pericytes under different experimental conditions as indicated. Gray bar shows Cy3-Aβ42 signal in the absence of cells (control without cells). N = 3 independent cultures; mean ± s.e.m.; p < 0.05 by one-way ANOVA followed by Bonferroni post-hoc testCC BY · PubMed Central

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Metadata

Authors
Ma, Q., Zhao, Z., Sagare, A. P., Wu, Y., Wang, M., Owens, N. C., Verghese, P. B., Herz, J., Holtzman, D. M., & Zlokovic, B. V.
Affiliations
  • University of Southern California (US)
  • C2N Diagnostics (United States) (US)
  • The University of Texas Southwestern Medical Center (US)
  • Washington University in St. Louis (US)
ORCID
Venue
Molecular Neurodegeneration
Publisher
Springer Science and Business Media LLC
Published
2018-10-19
Volume
13
Issue
1
Pages
57-57
ISSN
1750-1326 (ISSN-L)
DOI
10.1186/s13024-018-0286-0
PMC
PMC6194676
OpenAlex
W2897433925
Cited by
281
References
71
Citations by year
2026: 24 · 2025: 54 · 2024: 40 · 2023: 41 · 2022: 35 · 2021: 30 · 2020: 36 · 2019: 21
FWCI
14.91
Type
journal article
Language
en
Update notices
Open access
Licence
Locations
  • Molecular Neurodegeneration (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Digital Commons@Becker (Washington University School of Medicine) (submittedVersion, cc-by)
  • The Scholars Repository - LLU (Loma Linda University) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
National Institutes of Health (R01NS034467, R01AG023084, R01AG039452)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
  • Health Sciences › Medicine › Clinical Neurology › Cerebrospinal fluid and hydrocephalus
Keywords
blood-brain barrier, amyloid-beta 42, LRP1, apolipoprotein E isoforms, APOE3, APOE ε4, Alzheimer's disease, amyloid-beta clearance, receptor-mediated uptake, pericyte dysfunction, APP overexpression
MeSH terms
Animals, Apolipoproteins E, Biological Transport, Blood-Brain Barrier, Brain, Humans, Mice, Transgenic, Peptide Fragments, Receptors, LDL, Amyloid beta-Peptides, Pericytes, Tumor Suppressor Proteins, Low Density Lipoprotein Receptor-Related Protein-1

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