Abstract

Vascular contributions to cognitive impairment are increasingly recognized1,2,3,4,5 as shown by neuropathological6,7, neuroimaging4,8,9,10,11, and cerebrospinal fluid biomarker4,12 studies. Moreover, small vessel disease of the brain has been estimated to contribute to approximately 50% of all dementias worldwide, including those caused by Alzheimer’s disease (AD)3,4,13. Vascular changes in AD have been typically attributed to the vasoactive and/or vasculotoxic effects of amyloid-β (Aβ)3,11,14, and more recently tau15. Animal studies suggest that Aβ and tau lead to blood vessel abnormalities and blood–brain barrier (BBB) breakdown14,15,16. Although neurovascular dysfunction3,11 and BBB breakdown develop early in AD1,4,5,8,9,10,12,13, how they relate to changes in the AD classical biomarkers Aβ and tau, which also develop before dementia17, remains unknown. To address this question, we studied brain capillary damage using a novel cerebrospinal fluid biomarker of BBB-associated capillary mural cell pericyte, soluble platelet-derived growth factor receptor-β8,18, and regional BBB permeability using dynamic contrast-enhanced magnetic resonance imaging8,9,10. Our data show that individuals with early cognitive dysfunction develop brain capillary damage and BBB breakdown in the hippocampus irrespective of Alzheimer’s Aβ and/or tau biomarker changes, suggesting that BBB breakdown is an early biomarker of human cognitive dysfunction independent of Aβ and tau.

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Data availability

All data included in this study are available in Supplementary Tables containing detailed statistical analyses and in the accompanying Source Data. The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

The work of B.V.Z. is supported by the National Institutes of Health (NIH) grant nos. R01AG023084, R01NS090904, R01NS034467, R01AG039452, 1R01NS100459, 5P01AG052350, and 5P50AG005142 in addition to the Alzheimer’s Association strategic 509279 grant, Cure Alzheimer’s Fund, and the Foundation Leducq Transatlantic Network of Excellence for the Study of Perivascular Spaces in Small Vessel Disease reference no. 16 CVD 05. D.B and M.G.H. are supported by the L.K. Whittier Foundation, grant nos. P01AG052350, RO1AG054434, and R01AG055770. D.N. is supported by the NIH grant no. R21AG055034 and Alzheimer’s Association grant no. AARG-17–532905. Enrollment of participants into the Washington University Knight ADRC is supported by NIH grant nos. P50AG05681 (J.C.M.), P01AG03991 (J.C.M.), and P01AG026276 (J.C.M.). Enrollment of participants into the USC ADRC is supported by NIH grant no. 5P50AG005142 (H.C.C.).

Author information

Author notes

  1. These authors contributed equally: Daniel A. Nation, Melanie D. Sweeney, Axel Montagne.

Affiliations

  1. Department of Physiology and Neuroscience, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    • Daniel A. Nation
    • , Melanie D. Sweeney
    • , Axel Montagne
    • , Abhay P. Sagare
    • , Maricarmen Pachicano
    • , Amy R. Nelson
    •  & Berislav V. Zlokovic
  2. Alzheimer’s Disease Research Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    • Daniel A. Nation
    • , Lina M. D’Orazio
    • , John M. Ringman
    • , Lon S. Schneider
    • , Helena C. Chui
    • , Meng Law
    • , Arthur W. Toga
    •  & Berislav V. Zlokovic
  3. Department of Psychology, University of Southern California, Los Angeles, CA, USA

    • Daniel A. Nation
  4. Department of Neurology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    • Lina M. D’Orazio
    • , John M. Ringman
    • , Lon S. Schneider
    •  & Helena C. Chui
  5. Laboratory of Neuro Imaging, USC Stevens Neuroimaging and Informatics Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    • Farshid Sepehrband
    • , Meng Law
    •  & Arthur W. Toga
  6. Huntington Medical Research Institutes, Pasadena, CA, USA

    • David P. Buennagel
    •  & Michael G. Harrington
  7. Department of Radiology, Washington University School of Medicine, St. Louis, MO, USA

    • Tammie L. S. Benzinger
  8. The Hope Center for Neurodegenerative Disorders, Washington University School of Medicine, St. Louis, MO, USA

    • Tammie L. S. Benzinger
    • , Anne M. Fagan
    •  & John C. Morris
  9. Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA

    • Anne M. Fagan
    •  & John C. Morris
  10. The Knight Alzheimer’s Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA

    • Anne M. Fagan
  11. Department of Psychiatry and Behavioral Sciences, University of Southern California, Los Angeles, CA, USA

    • Lon S. Schneider
  12. Department of Radiology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    • Meng Law

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Contributions

D.A.N., M.D.S., A.M., A.P.S., and B.V.Z. designed the research study and analyzed and interpreted the data. M.D.S., A.M., A.P.S., L.M.D., M.P., F.S., and D.P.B. performed the experiments and analyzed the data. L.M.D. and A.R.N. prepared and submitted the study to the IRB. M.G.H., T.L.S.B., A.M.F., J.M.R., L.S.S., J.C.M., H.C.C., M.L., and A.W.T. recruited the participants and performed and provided the imaging scans. A.P.S., M.G.H., T.L.S.B., A.M.F., J.M.R., L.S.S., J.C.M., H.C.C., M.L., and A.W.T. provided critical reading of the manuscript. D.A.N., M.D.S., and A.M. contributed to manuscript writing and B.V.Z. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Berislav V. Zlokovic.

Extended data

  1. Extended Data Fig. 1 ADAM10 mediates sPDGFRβ shedding in human brain pericytes in vitro.

    a, Primary human brain vascular SMCs and pericytes were subjected to treatment with ionomycin (2.5 µM), a calcium ionophore that activates ADAM10, or control treatment (media only); media was immunoprecipitated to measure sPDGFRβ by quantitative western blot. Compared to pericytes, SMCs shed extremely low levels of sPDGFRβ, which was not significantly increased by ionomycin. Pericytes shed high basal levels of sPDGFRβ that was significantly increased fivefold by treatment with ionomycin, which activated ADAM10. To further determine the involvement of ADAM10, ionomycin treatment was conducted in the presence of the pharmacological inhibition of ADAM10 with marimastat (4 µM), which inhibits ADAM10 by binding to active-site zinc, and genetic small interfering RNA (siRNA) knockdown of ADAM10. Both pharmacological (marimastat) and genetic (siRNA) inhibition of ADAM10 significantly reduced sPDGFRβ shedding activated by ionomycin by >90 and 75%, respectively. b, The siRNA ADAM10 knockdown efficiency in this study was 85%, as shown by the western blot analysis. Data was generated from n = 3–6 independent culture experiments and plotted as means ± s.e.m. a, SMC data by two-tailed Student’s t-test; pericyte data by ANOVA with Tukey post-hoc test. b, Two-tailed Student’s t-test. Significance set at α = 0.05 for all analyses.

  2. Extended Data Fig. 2 CSF sPDGFRβ increases with CDR impairment, independent of Aβ and tau, and reflects BBB breakdown.

    ab, Site-specific analysis of CSF sPDGFRβ and the standard AD biomarkers, Aβ42 and pTau, indicates an early increase in sPDGFRβ with increasing CDR at both independent clinical sites, USC (a) and Washington University (b). There were no changes in Aβ42 and pTau at the USC site (a), whereas Aβ42, but not pTau, was altered at the Washington University site; supports Fig. 1a–c. cd, Site-specific analysis of CSF sPDGFRβ increases with CDR, independent of CSF Aβ42 and pTau status at two independent sites, USC (c) and Washington University (d); supports Fig. 1d–f. ef, CSF sPDGFRβ is associated with BBB breakdown. CSF sPDGFRβ positively correlates with the conventional biochemical biomarkers of BBB breakdown including the CSF/plasma albumin ratio (Qalb) (e) and CSF fibrinogen (f); supports Figs. 1 and 3. g, CSF sPDGFRβ is increased with CDR, independent of amyloid positivity by 11C-PiB-PET; supports Fig. 1d–f. hi, No differences were observed in CSF Aβ (h) and tau (i) oligomer levels in individuals with CDR 0 versus CDR 0.5; supports Fig. 1d–f. jk, Increases in CSF sPDGFRβ (j) and regional BBB Ktrans in the HC and PHG (k) of individuals with CDR 0.5 versus CDR 0 remain significant after statistically controlling for the impact of CSF tau oligomers; supports Fig. 1d–f. ad, gi, The box and whisker plot lines indicate the median values, the boxes indicate the IQR, and the whiskers indicate the minimum and maximum values. ad, g, Significance tests from ANCOVAs. ef, Statistical significance determined by Pearson correlation coefficient (r). hi, Significance by two-tailed Student’s t-test at α = 0.05. jk, ANCOVA models representing estimated marginal means ± s.e.m. The brackets denote the sample size (n) in each analysis.

  3. Extended Data Fig. 3 sPDGFRβ increases with CDR independent of VRFs, and no change in other neurovascular unit biomarkers.

    ac, CSF sPDGFRβ is increased with CDR, independent of VRF burden in the combined site analysis (a) and at two independent clinical sites from USC (b) and Washington University (c). VRFs 0–1: no or 1 VRF. VRFs 2+: 2 or more VRFs. See Supplementary Table 1 for the list of VRFs; supports Fig. 1a–f. The box and whisker plot lines indicate the median values, the boxes indicate the IQR, and the whiskers indicate the minimum and maximum values. Significance tests from ANCOVAs. The brackets denote the sample size (n) in each analysis.

  4. Extended Data Fig. 4 Other CSF biomarkers of the neurovascular unit are not altered with CDR cognitive impairment.

    ac, CSF markers of glial, inflammatory, or neuronal injury exhibited no significant differences between unimpaired and impaired individuals on CDR, including S100B, IL-6, TNFα, or NSE in the combined site analysis (a) and similarly in the site-specific analysis of individuals from USC (b) and Washington University (c); supports Fig. 1a–c. The box and whisker plot lines indicate the median values, the boxes indicate the IQR, and the whiskers indicate the minimum and maximum values. Significance tests from ANCOVAs. The brackets denote the sample size (n) in each analysis.

  5. Extended Data Fig. 5 Regional BBB breakdown Ktrans increases with CDR independent of CSF Aβ, tau, and VRFs, and relates to sPDGFRβ only in hippocampal gray matter regions.

    ab, An increase in Ktrans values in the HC, PHG, and the CA1, CA3, and dentate gyrus HC subfields, with increasing CDR (a), but not in other brain regions including the superior frontal cortical gyrus and inferior temporal cortical gyrus, white matter regions including the subcortical white matter fibers, corpus callosum, and internal capsule, and deep gray matter regions including the thalamus, caudate nucleus, and striatum (b). cd, Additional brain regions showed no significant differences in Ktrans BBB permeability values in individuals with CDR 0 and CDR 0.5, regardless of CSF Aβ42 (c) or pTau (d) status. ef, VRF burden does not influence an increase in the Ktrans BBB permeability values with increasing CDR in the HC, PHG, and HC subfields (that is, CA1, CA3, and dentate gyrus) (e), and no change in the Ktrans BBB permeability values in other brain regions (f). See Supplementary Table 1 for the list of VRFs. af, Supports Fig. 1g–k. gj, CSF sPDGFRβ is associated with BBB breakdown measured by neuroimaging in hippocampal gray matter regions (gh), but not in white matter regions (ij); supports Figs. 1 and 3. af, The box and whisker plot lines indicate the median values, the boxes indicate the IQR, and the whiskers indicate the minimum and maximum values. Significance tests after FDR correction from ANCOVAs. gj, Statistical significance determined by Pearson correlation coefficient (r) . The brackets denote the sample size (n) in each analysis; applies to all regions within each panel.

  6. Extended Data Fig. 6 CSF sPDGFRβ increases with CDR impairment, independent of Aβ, tau, and VRFs.

    ab, Site-specific analysis of CSF sPDGFRβ and the standard AD biomarkers, Aβ42 and pTau, indicates an early increase in sPDGFRβ with increasing impaired domains at both independent clinical sites, USC (a) and Washington University (b); supports Fig. 3a–c. cd, Site-specific analysis of CSF sPDGFRβ indicates increases with the number of impaired cognitive domains, independent of CSF Aβ42 and pTau status at two independent sites, USC (c) and Washington University (d); supports Fig. 3d–f. eg, CSF sPDGFRβ is increased with the increasing number of impaired cognitive domains, independent of VRF burden in the combined site analysis (e) and at two independent clinical sites, USC (f) and Washington University (g). VRFs 0–1: no or 1 VRF. VRFs 2+: 2 or more VRFs. See Supplementary Table 2 for the list of VRFs. Supports Fig. 3a–f. ag, The box and whisker plot lines indicate the median values, the boxes indicate the IQR, and the whiskers indicate the minimum and maximum values. Significance tests from ANCOVAs. The brackets denote the sample size (n) in each analysis.

  7. Extended Data Fig. 7 BBB breakdown is independent of amyloid and tau oligomers.

    a, CSF sPDGFRβ is increased with impaired cognitive domains, independent of amyloid positivity by PiB-PET; supports Fig. 3d–f. bc, No differences were observed in CSF Aβ (b) and tau (c) oligomer levels in individuals with 0 or 1+ impaired cognitive domains. de, Increases in CSF sPDGFRβ (d) and regional BBB Ktrans in the HC and PHG (e) of individuals with 1+ versus 0 cognitive domain impairment remain significant after statistically controlling for the impact of CSF tau oligomers; supports Fig. 3d–f. ac, The box and whisker plot lines indicate the median values, the boxes indicate the IQR, and the whiskers indicate the minimum and maximum values. a, Significance tests from ANCOVAs. bc, Significance by two-tailed Student’s t-test at α = 0.05. de, ANCOVA models representing the estimated marginal means ± s.e.m. The brackets denote the sample size (n) in each analysis.

  8. Extended Data Fig. 8 Other CSF biomarkers of the neurovascular unit are not altered with cognitive domain impairment.

    ac, CSF markers of glial, inflammatory, or neuronal injury exhibited no significant differences between unimpaired and impaired individuals on neuropsychological exams, including S100B, IL-6*, TNFα, or NSEin the combined site analysis (a) or in the site-specific analysis of individuals from USC (b) or from Washington University (c). ac, The box and whisker plot lines indicate the median values, the boxes indicate the IQR, and the whiskers indicate the minimum and maximum values. Significance tests after FDR correction from ANCOVAs with post-hoc Bonferroni comparisons. The brackets denote the sample size (n) in each analysis. aAnalysis did not survive significance after FDR correction. bIndividual group comparison P values reported because the omnibus test was P < 0.05 but the post-hoc group comparisons were null. Supports Fig. 3a–c.

  9. Extended Data Fig. 9 Regional BBB breakdown Ktrans increases with cognitive domain impairment, independent of CSF Aβ, tau, and VRFs.

    ab, An increase in Ktrans values in the HC, PHG, and the CA1, CA3, and dentate gyrus HC subfields with increasing cognitive impairment measured by the number of impaired cognitive domains (a), but not in other brain regions, including the superior frontal cortical gyrus and the inferior temporal cortical gyrus, white matter regions including the subcortical white matter fibers, corpus callosum, and internal capsule, and deep gray matter regions including thalamus, caudate nucleus, and striatum (b). cd, Additional brain regions showed no significant difference in Ktrans BBB permeability in individuals with 0 and 1+ impaired cognitive domains, regardless of CSF Aβ42 (c) and pTau (d) status. ef, Ktrans BBB permeability is increased with increasing cognitive domain impairment in the HC, PHG, and HC subfields (that is, CA1, CA3, and dentate gyrus), independent of VRF burden (e), but not in other brain regions (f). VRFs 0–1: no or 1 VRF; VRFs 2+: 2 or more VRFs. See Supplementary Table 2 for the list of VRFs. af, The box and whisker plot lines indicate the median values, the boxes indicate the IQR, and the whiskers indicate the minimum and maximum values. Significance tests after FDR correction from ANCOVAs. The brackets denote the sample size (n) in each analysis; applies to all regions within each panel. Supports Fig. 3g–k.

  10. Extended Data Fig. 10 CSF sPDGFRβ and medial temporal BBB permeability Ktrans values are not correlated with age, indicating that changes in CSF sPDGFRβ and Ktrans capture processes relating to cognitive impairment are independent of normal aging.

    In CDR 0 individuals, age does not correlate with CSF sPDGFRβ (a) or regional Ktrans in the HC (c) and PHG (e). Similarly, in CDR 0.5 individuals, age does not correlate with CSF sPDGFRβ (a) or regional Ktrans in the HC (c) and PHG (e). Statistical significance determined by Pearson correlation coefficient (r); the brackets denote the sample size (n) in each analysis. Supports Figs. 1 and 3.

Supplementary information

  1. Supplementary Table

    Supplementary Tables 1–10

  2. Reporting Summary

Source data

  1. Source Data

    Full annotated Western blot gels

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DOI

https://doi.org/10.1038/s41591-018-0297-y