Research Article

Amyloid β oligomers constrict human capillaries in Alzheimer’s disease via signaling to pericytes

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Science  19 Jul 2019:
Vol. 365, Issue 6450, eaav9518
DOI: 10.1126/science.aav9518
  • Live human and rodent brain capillaries become constricted in Alzheimer’s disease.

    Tissue from humans and rodents (left) that were healthy or developing Alzheimer’s disease (AD) was imaged in vivo and as brain slices (center), revealing that pericytes constrict brain capillaries early in AD via a mechanism involving ROS generation and release of endothelin-1, which activates ETA receptors (right).

  • Fig. 1 Oligomeric Aβ acts on pericytes to constrict capillaries in human brain slices.

    (A) IB4-labeled capillary in a human cortical slice, with two pericyte somata (white arrowheads) outlined by their basement membrane. Nuclei are stained with DAPI (blue). (B) Pericyte labeled with antibody to PDGFRβ. (C and D) Arteriole (C) and pericyte (D) labeled with IB4 and antibody to α smooth muscle actin (α-SMA, localized in processes originating from the pericyte soma). (E) Images of a capillary (red lines between yellow arrowheads indicate diameter) and pericyte soma (white arrowheads) in a live human brain slice before drug application, in the presence of 2 μM superfused noradrenaline (+NA), with 2 μM NA and 500 μM glutamate superfused (+NA +Glu), and after stopping drug superfusion (washout). Graph shows time course of capillary diameter at red line throughout the experiment. (F) Mean (± SEM) glutamate-evoked dilation and noradrenaline-evoked constriction in experiments as in (E) (numbers of pericytes on bars; change in diameter was quantified relative to that before application of each drug; relative to the pre-noradrenaline diameter, the glutamate-evoked dilation was 26.8 ± 7.7%). (G) Silver staining of an SDS-PAGE gel for Aβ solutions prepared as in materials and methods. (H) Images of a human capillary before and after superfusion of 72 nM Aβ1–42, showing a region (red line) being constricted by a pericyte (arrowheads). Graph shows mean (±SEM) diameter change at four pericyte locations from four slices treated with Aβ and three pericyte locations from three slices superfused with aCSF lacking Aβ (significantly reduced at 40 min in Aβ, P = 0.01).

  • Fig. 2 Aβ acts via ROS and ETA receptors.

    (A and B) Bright-field images (A) and two-photon–evoked IB4 fluorescence (B) of capillaries in rat cortical slices in aCSF and after applying 72 nM Aβ1–42, showing constriction (yellow arrowheads and red lines) near pericytes (white arrowheads, compare with figs. S2 and S3). (C) Mean (± SEM) time course of capillary diameter during superfusion with aCSF (n = 51 vessels), 109 nM scrambled Aβ1–42 (n = 32), 72 nM Aβ1–42 (n = 20), or 100 nM Aβ1–40 (n = 6). (D) Constriction evoked after 1 hour by different concentrations of Aβ1–42 (0 nM, n = 51; 2.9 nM, n = 11; 14 nM, n = 10; 57 nM, n = 19; 72 nM, n = 20). Curve is a Michaelis-Menten relation with a Km of 4.7 nM and a maximum of 16.1%. (E to J) Time course of diameter when applying the following agents (experiments in each panel were interleaved; blockers were present for 5 to 15 min before Aβ). (E) 57 nM Aβ1–42 alone (n = 19) or in the presence of SOD1 (150 units/ml, n = 19) or the ETA blocker BQ-123 (1 μM, n = 14). (F) 72 nM Aβ1–42 alone (n = 7) or in the presence of the NOS blocker L-NNA (100 μM, n = 6), the NADPH oxidase blocker DPI (10 μM, n = 5), or the NOX4 blocker GKT137831 (0.45 μM, n = 7). (G) Constriction produced at 60 min for (C) to (F). (H) Effect of aCSF (n = 10), ET alone (10 nM, n = 10), or ET in the presence of the ETA blocker BQ-123 (1 μM, n = 10) or the ETB blocker BQ-788 (1 μM, n = 12). (I) aCSF or ET (5 nM) in the absence (n = 12) or presence of SOD1 (150 units/ml, n = 8). (J) aCSF or the ROS generator H2O2 (1 mM, n = 9, which evokes constriction: P = 1.1 × 10–5 at 20 min) or H2O2 with the ETA blocker BQ-123 (1 μM, n = 11, constriction is reduced, P = 0.009). (K) Two-photon image of mouse cortical pericyte expressing GCaMP5G (green), before and while applying ET (10 nM), which raises [Ca2+]i (increase in green intensity) in pericyte soma (arrowhead; dashed line shows ROI analyzed) and processes, and constricts the capillary (see white line on image of the tdTomato reporter of GCaMP5G expression, red). (L) Mean [Ca2+]i time course in eight pericyte somata in response to ET (significantly elevated, P = 0.0014) and in seven somata in aCSF (no significant change, P = 0.74). (M) Incubating rat brain slices (numbers on bars) with Aβ1–42 oligomers (1.4 μM) or ET (100 nM) for 3 hours does not increase pericyte death.

  • Fig. 3 Aβ evokes ROS generation in pericytes.

    (A) Fluorescence images of dihydroethidium (DHE)–loaded rat cortical slices incubated in control aCSF or aCSF containing Aβ1–42 (72 nM) or Aβ1–42 + SOD1 (150 units/ml) for 40 min, showing that Aβ increases ROS level and that this is inhibited by SOD1. (B) Fluorescence (normalized to value in aCSF, mean ± SEM) of slices incubated in aCSF (n = 6), Aβ1–42 (n = 7), or Aβ1–42 + SOD1 (n = 6). (C) Left: Image of a cortical slice showing that the brightest DHE-labeled cells are located on IB4-labeled blood vessels (arrowhead). Right: Immunolabeling shows that these cells colocalize with NG2 but not with Iba1, implying that they are pericytes rather than microglia or perivascular macrophages. (D) Soma DHE fluorescence [arbitrary units (a.u.), mean ± SEM] from the population of pericytes, or of Iba1-labeled cells, after 40 min in the absence or presence of Aβ1–42. Numbers on bars are slices (fluorescence was averaged across three image stacks for each slice).

  • Fig. 4 Pericyte-mediated capillary constriction occurs in humans with Aβ deposits.

    (A and B) Specimen images of human cortical biopsies, labeled for PDGFRβ (brown in top panels) to show pericytes (arrows), from patients lacking (A) or exhibiting (B) Aβ deposits (brown in bottom panels, hematoxylin counterstain in blue). Red lines indicate capillary diameter. (C) Mean (± SEM) diameter of capillaries in patients lacking (3921 diameters measured) or exhibiting (5121 diameters measured) Aβ deposits (numbers of images analyzed shown on bars). (D) Dependence of capillary diameter on distance from a visible pericyte soma (in 5-μm bins from 0 to 20 μm, plotted at the mean distance for each bin) for patients lacking or exhibiting Aβ deposits (moderate and severe Aβ deposition pooled together). P values assess whether slope of regression line is significantly different from zero. (E) Examples of Aβ labeling assessed by the neuropathologist as absent, moderate, or severe. (F) Slope of regression lines as in (D) plotted as a function of neuropathologist-rated parenchymal Aβ load for each biopsy (n = 6 biopsies for none, n = 3 for moderate, n = 4 for severe). P value compares slope of line with zero. (G) Slope of regression lines as in (D) plotted as a function of severity of Aβ deposition measured optically for each biopsy, with subjects grouped by color [defined in (F)] as classified by the neuropathologist. (H) Dependence of extrapolated diameter at soma [as in (D)] on severity of Aβ deposition measured optically for each biopsy, with subject points colored as classified by the neuropathologist [defined in (F)]. Lines through data in (F) to (H) show the trends in the data.

  • Fig. 5 Capillaries, but not arterioles or venules, are constricted in AD mice.

    (A) Specimen images (taken through the dura) of blood vessels in the somatosensory neocortex of wild-type (WT) and homozygous AD (APPNL-G-F) NG2-DsRed mice, with FITC-albumin (green) in the blood (pericytes are labeled red). (B) Examples of single neocortical capillaries and pericytes, showing a larger diameter at the pericyte soma in a WT mouse and constriction of a capillary at the pericyte soma in an AD mouse. (C) Images of neocortex labeled for nuclei (DAPI, blue) and for amyloid plaques (green, 82E1 antibody). (D) Mean (± SEM) capillary diameter in neocortical layers I to IV in three WT mice (2131 diameters measured; measurements on same capillary were averaged) and four AD mice (1403 diameters measured). Numbers of capillaries are shown on bars. (E) Mean neocortical capillary diameter at pericyte somata in three WT and four AD mice (numbers of pericytes on bars). (F) Plot of neocortical capillary diameter as a function of distance from pericyte somata shows a smaller diameter at the soma in AD mice and a larger diameter in WT mice (compare with xref ref-type="fig" rid="F4">Fig. 4D; each WT mouse studied showed a negative slope for this relationship, and each AD mouse showed a positive slope). (G) Plots as in (F) but for the cerebellum, which lacks amyloid plaques, show no constriction near the pericyte somata in the AD mice (regression line is a fit to all data from three WT and three APP mice). (H) Mean diameter of neocortical penetrating arterioles and venules in WT and AD mice. Numbers of vessels are shown on bars. Diameters were assessed at depths that did not differ significantly: 158.4 ± 6.7 μm and 131.9 ± 5.0 μm (P = 0.23, Mann-Whitney test) for neocortical capillaries, 142 ± 26 μm and 137 ± 21 μm (P = 0.88) for arterioles, and 85 ± 15 μm and 89 ± 9 μm (P = 0.81) for venules, in WT and AD mice, respectively.

  • Fig. 6 Aβ effects on capillaries may amplify the onset of AD and are reversible.

    (A) Applying GKT137831 (0.45 μM) to block NOX4 and BQ-123 (1 nM) to block ETA receptors, or applying C-type natriuretic peptide (CNP, 100 nM; see fig. S8), significantly reduced the constriction evoked by Aβ (72 nM, P = 0.027 and 0.029, respectively, corrected for multiple comparisons; data are means ± SEM). (B) Summary of our results and their implications. Our data reveal the pathway within the yellow dashed box. Aβ oligomers activate NOX4 in pericytes to generate ROS. These in turn release, or potentiate the constricting effects of, endothelin-1, which acts via ETA receptors on pericytes on capillaries—the locus (16) of the largest component of vascular resistance within the brain parenchyma. Capillary constriction decreases cerebral blood flow and hence the supply of oxygen and glucose to the brain. Green arrows at the left show that this increases the production of Aβ, in part by up-regulating (13, 14) the expression of BACE1, thus forming an amplifying positive feedback loop. Blue arrows at the right show that a rise in Aβ concentration (directly, via downstream tau production, or via the decrease in oxygen and glucose supply) leads to the loss of synapses and neurons. Potential sites for therapeutic intervention are highlighted at the stages of ROS production by NOX4 (GKT), endothelin receptors (BQ-123), and CNP receptors (see also fig. S8).

Supplementary Materials

  • Amyloid β oligomers constrict human capillaries in Alzheimer's disease via signaling to pericytes

    Ross Nortley, Nils Korte, Pablo Izquierdo, Chanawee Hirunpattarasilp, Anusha Mishra, Zane Jaunmuktane, Vasiliki Kyrargyri, Thomas Pfeiffer, Lila Khennouf, Christian Madry, Hui Gong, Angela Richard-Loendt, Wenhui Huang, Takashi Saito, Takaomi C. Saido, Sebastian Brandner, Huma Sethi, David Attwell

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Figs. S1 to S8
    • References

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