Brain Perivascular Macrophages Initiate the Neurovascular Dysfunction of Alzheimer Aβ Peptides

Abstract

Rationale: Increasing evidence indicates that alterations of the cerebral microcirculation may play a role in Alzheimer disease, the leading cause of late-life dementia. The amyloid-β peptide (Aβ), a key pathogenic factor in Alzheimer disease, induces profound alterations in neurovascular regulation through the innate immunity receptor CD36 (cluster of differentiation 36), which, in turn, activates a Nox2-containing NADPH oxidase, leading to cerebrovascular oxidative stress. Brain perivascular macrophages (PVM) located in the perivascular space, a major site of brain Aβ collection and clearance, are juxtaposed to the wall of intracerebral resistance vessels and are a powerful source of reactive oxygen species.

Objective: We tested the hypothesis that PVM are the main source of reactive oxygen species responsible for the cerebrovascular actions of Aβ and that CD36 and Nox2 in PVM are the molecular substrates of the effect.

Methods and results: Selective depletion of PVM using intracerebroventricular injection of clodronate abrogates the reactive oxygen species production and cerebrovascular dysfunction induced by Aβ applied directly to the cerebral cortex, administered intravascularly, or overproduced in the brain of transgenic mice expressing mutated forms of the amyloid precursor protein (Tg2576 mice). In addition, using bone marrow chimeras, we demonstrate that PVM are the cells expressing CD36 and Nox2 responsible for the dysfunction. Thus, deletion of CD36 or Nox2 from PVM abrogates the deleterious vascular effects of Aβ, whereas wild-type PVM reconstitute the vascular dysfunction in CD36-null mice.

Conclusions: The data identify PVM as a previously unrecognized effector of the damaging neurovascular actions of Aβ and unveil a new mechanism by which brain-resident innate immune cells and their receptors may contribute to the pathobiology of Alzheimer disease.

Keywords: Alzheimer disease; brain; cerebrovascular disease; endothelium; oxidative stress.

Figure 1. PVM, juxtaposed to penetrating cerebral blood vessels, are depleted by clodronate

In WT mice (non-transgenic littermates) injected with PSB-liposome (vehicle), PVM identified by phagocytized dextran (green) are closely apposed to penetrating neocortical vessels, identified by the lipophilic dye DiO (red) (A). PVM in Tg2576 mice injected with vehicle have numbers and distributions similar to WT mice (B). Intracerebroventricular (icv) administration of clodronate-containing liposomes depletes PVM both in WT and Tg2576 mice 5 days later (C,D). Quantification of the number of PVM in WT and tg2576 mice after icv injection of liposomes containing PBS or clodronate (E). No differences between WT and Tg2576 mice are observed. *p<0.05 from PBS; n=5/group; Analysis of variance and Tukey’s test.

Figure 2. PVM depletion prevents the cerebrovascular effects of neocortical superfusion of Aβ_1-40 in WT mice

In WT mice treated with PBS liposomes, superfusion of the somatosensory cortex with Aβ_1-40 (5μM) attenuates resting CBF (A), and the increase in CBF evoked by whisker stimulation (B), or by neocortical application of ACh (10μM) (C), but not adenosine (400μM) (D). Treatment with clodronate does not affect baseline cerebrovascular responses, but rescues the attenuation in resting CBF and CBF responses to whiskers stimulation and ACh induced by Aβ_1-40 superfusion. Furthermore, clodronate suppresses the Aβ-induced increase in cerebrovascular ROS in the somatosensory cortex, assessed by DHE microfluorography (E). LDU, arbitrary laser-Doppler perfusion units; RFU, relative fluorescence units; *p<0.05 from vehicle, ANOVA and Tukey’s test; n=5/group.

Figure 3. PVM depletion counteracts the neurovascular dysfunction and vascular oxidative stress in Tg2576 mice

PVM depletion by clodronate counteracts the attenuation in the CBF response to whisker stimulation (A) or neocortical application of ACh (B), as well as the increase in cerebrovascular ROS (C) in Tg2576 mice. Clodronate does not alter brain concentrations of SDS-soluble Aβ_1-40 and Aβ_1-42 in tg2576 mice. RFU, relative fluorescence units; *p<0.05 from WT, ANOVA and Tukey’s test; n=5–6/group.

Figure 4. Effect of transplantation of CD36−/− or Nox2−/− BM in Tg2576 mice on PVM number and ROS production

Vessels are labeled with DiO (red), ROS production is detected with DHE (blue) and PVM are labeled with dextran (green). In WT mice transplanted with WT BM (WT→WT) PVM have numbers and distribution comparable to those of naïve WT mice (A). Similarly, Tg2576 mice transplanted with WT BM (WT→Tg) exhibited number and distribution similar to those of naïve Tg2576 mice (B). However, ROS production in PVM was observed in WT→Tg mice (DHE insert in B). Transplant of CD36−/− (CD36→Tg ) or Nox2−/− (Nox2→Tg) BM in Tg2576 mice does not alter PVM number or distribution, but suppresses PVM ROS production (DHE inserts in C and D). Quantification of the numbers of PVM in the BM chimeras studied (E). Quantification of cerebrovascular ROS in the BM chimeras showing that ROS production is suppressed in CD36→Tg and Nox2→Tg mice (F). RFU, relative fluorescent units; * p<0.05 from WT→WT, CD36→Tg, or Nox2→Tg; ANOVA and Tukey’s test; n=5/group.

Figure 5. Deletion of CD36 or Nox2 in PVM ameliorates the neurovascular dysfunction and vascular oxidative stress induced by Aβ_1-40 superfusion in WT mice

In WT→WT mice, cerebrovascular response to whisker stimulation, ACh, and adenosine do not differ from those of naïve WT mice (A–D). Furthermore, Aβ_1-40 superfusion attenuates resting CBF and CBF responses to whisker stimulation and ACh, but not adenosine (A–D). However, these cerebrovascular effects of Aβ_1-40 are not observed in CD36→Tg and Nox2→Tg chimeras (A–D). Aβ_1-40 increases cerebrovascular ROS production WT→WT, but not in CD36→Tg and Nox2→Tg mice (E). Transplant of WT (CD36^+/+) BM is able to re-establish the neurovascular dysfunction induced by Aβ in CD36^−/− mice (WT→CD36^−/−), an effect counteracted by neocortical superfusion of the ROS scavenger MnTBAP (50μM) (F,G). RFU, relative fluorescence units; *p<0.05 from vehicle (A–D), CD36→WT, or Nox2→WT (F,G); ANOVA and Tukey’s test; n=5–6/group.

Figure 6. Deletion of CD36 or Nox2 in PVM counteracts the neurovascular dysfunction and vascular oxidative stress in Tg2576 mice

Tg2576 mice receiving WT BM (WT→Tg) exhibit attenuation in CBF responses to whisker stimulation (A,B) and ACh (C) similar to those observed in Tg2576 mice not subjected to BM transplantation. Transplant of CD36−/− or Nox2−/− BM rescues the neurovascular dysfunction in Tg2576 mice (A–D), without altering brain Aβ_1-40 and Aβ_1-42 (E,F). *p<0.05 from WT→WT, CD36→Tg, or Nox2→Tg; ANOVA and Tukey’s test; n=5–6/group.

Figure 7. Circulating Aβ_1-40 reaches PVM and mediates oxidative stress and neurovascular dysfunction

In WT mice treated with PBS, Cy5-labelled Aβ_1-40 infused into the carotid artery (Cy5-Aβ_1-40) (1μM, 150 μl/hr) (blue) colocalizes with dextran-labelled PVM (green) surrounding cerebral blood vessels labelled with DiO (red), and resulting in the cyan color in the merged image (A). Orthogonal projections illustrating co-localization of dextran-labelled PVM (green) and Cy5-labelled Aβ (blue) around blood vessels (red) (B). Cy5-labelled Aβ is colocalized with more than 80% of dextran-positive PVM (C). In mice treated with clodronate, the signal from Cy5-labelled Aβ_1-40 is still present in the brain and not different from PBS treated mice (D), but is no longer associated with PVM, which are depleted (Online Figure VIII). ROS are increased in PVM of mice receiving the i.c. infusion of Aβ_1-40 (icAβ_1-40), which is suppressed by clodronate treatment (E). icAβ_1-40 attenuates the increase in CBF induced by whisker stimulation or ACh, an effect reversed by clodronate (F). Resting CBF or the CBF response to adenosine is not affected by icAβ (F). LDU, arbitrary laser-Doppler perfusion units; RFU relative fluorescence units; *p<0.05 from vehicle, ANOVA and Tukey’s test; n=5–6/group.