Cell autonomous role of border associated macrophages in ApoE4 neurovascular dysfunction and susceptibility to white matter injury

Abstract

Apolipoprotein-E4 (ApoE4), the strongest genetic risk factor for sporadic Alzheimer's disease, is also a risk factor for microvascular pathologies leading to cognitive impairment, particularly subcortical white matter injury. These effects have been attributed to alterations in the regulation of the brain blood supply, but the cellular source of ApoE4 and the underlying mechanisms remain unclear. In mice expressing human ApoE3 or ApoE4 we report that border associated macrophages (BAM), myeloid cells closely apposed to neocortical microvessels, are both the source and the target of the ApoE4 mediating the neurovascular dysfunction through reactive oxygen species. ApoE4 in BAM is solely responsible for the increased susceptibility to oligemic white matter damage in ApoE4 mice and is sufficient to enhance damage in ApoE3 mice. The data unveil a new aspect of BAM pathobiology and highlight a previously unrecognized cell autonomous role of BAM in the neurovascular dysfunction of ApoE4 with potential therapeutic implications.

Figure 1. Recombinant ApoE4 (rApoE4) or lipidated rApoE4 alters functional hyperemia and endothelial vasoactivity.

A. Neocortical superfusion of recombinant ApoE4 (rApoE4) or lipidated rApoE4 attenuates functional hyperemia produced by whisker stimulation and the increase of CBF produced by neocortical superfusion of the endothelium-dependent vasodilator acetylcholine. The increase in CBF produced by neocortical superfusion of the smooth muscle relaxant adenosine (smooth muscle vasoreactivity), is not impaired. B. Neocortical superfusion of lipidated rApoE3 does not affect functional hyperemia, endothelial or smooth muscle vasoactivity. C. Neocortical pretreatment with the ApoE receptors inhibitor receptor-associated protein (RAP) (200nM) prevents rApoE4 from altering functional hyperemia and endothelial vasoactivity but does not affect smooth muscle vasoreactivity. N=5/group; one-way ANOVA with Tukey’s test; data presented as mean±SEM.

Figure 2. BAM mediate deleterious cerebrovascular effects of ApoE4 through NADPH oxidase-derived ROS.

A. Neocortical superfusion of the NADPH oxidase peptide inhibitor gp91ds, but not its scrambled control (sgp91ds), rescues functional hyperemia and endothelial vasoactivity in ApoE4-TR mice. The peptide does affect CBF regulation in ApoE3-TR mice. B. Pretreatment with gp91ds, but not sgp91ds, prevents the attenuation of functional hyperemia and endothelial vasoactivity induced by neocortical superfusion of rApoE4 in WT mice (10μg/ml). CBF responses are not attenuated by rApoE3 and the peptides have no effect. N=5/mice group. C. In vivo ROS measurement by 2-photon microscopy. Left panel: WT mice received intracerebroventricular (i.c.v.) injection of dextran (10 kDa) to label border-associated macrophages (BAM, blue) and, 24 hrs later, intravenous infusion of the ROS marker dihydroethidium (DHE, green). Then, WT mice were equipped with an open cranial window and blood vessels were labeled with i.v. dextran (70 kDa, magenta). Middle panel: representative images illustrating that superfusion of rApoE3 (top) does not increase ROS (green), but superfusion with rApoE4 (bottom) increases the ROS signal. Right panel: quantification of ROS production in BAM; N=5 mice/group; 3–6 cells/mouse. D. rApoE4 (10μg/ml), but not rApoE3, increases ROS production in BAM and microglia, but not in endothelial cells (ECs) from WT mice. ROS were measured ex vivo by flow cytometry. E. ROS production is higher in BAM of ApoE4-TR mice than in ApoE3-TR mice but not in microglia and ECs. Scale bars in C: 50 μm in left upper and lower panels and 10 μm in the enlarged images in the right panels; One-way ANOVA and Tukey’s test; data presented as mean±SEM.

Figure 3. BAM depletion prevents the neurovascular dysfunction induced by ApoE4.

A. BAM depletion by clodronate in WT mice. Mice were injected i.c.v. with PBS liposomes (vehicle) or clodronate and depletion was assessed 7 days later. BAM (green) surround blood vessels labeled by DiO (red; top panel). BAM numbers do not differ in WT, ApoE3-TR, and ApoE4-TR mice injected with vehicle. Clodronate depleted BAM equally in all groups (quantification in right panel). B. BAM depletion prevents the attenuation in functional hyperemia and endothelial vasoactivity in ApoE4-TR mice compared to WT and ApoE3-TR mice. C. BAM depletion counteracts the deleterious vascular effects of rApoE4 in WT mice. One-way ANOVA and Tukey’s test; N=5/group; data presented as mean±SEM.

Figure 4. Deletion of ApoE4 selectively in BAM restores neurovascular function.

A. BAM express ApoE at levels comparable to those of astrocytes but higher than microglia, endothelial cells, and cells of the vascular wall (MC, mural cells). B. Dual RNAScope in situ hybridization with mRNA probes for Mrc1 (green) and ApoE (magenta), combined with DAPI nuclear staining (blue) and the basement membrane marker laminin (yellow). Representative images illustrating abundant expression of ApoE in Mrc1⁺ cells in Mrc1^Cre+/ApoE4^fl/fl and Mrc1^Cre+/ApoE3^fl/fl mice treated with vehicle (corn oil). However, in Mrc1^Cre+/ApoE4^fl/fl and Mrc1^Cre+/ApoE3^fl/fl mice treated with tamoxifen (TAM) ApoE levels in Mrc1⁺ cells are markedly reduced. C. ApoE puncta quantification in Mrc1⁺ cells; N=4–5 mice/group; 1–2-sections/mice; 5–12 cells/section. In B, larger images on the left were reconstructed using Imaris software and smaller images on the right cropped from confocal photographs (see Extended Data Fig. 2B-E). Scale bars = 20 μm. D. Brain ApoE levels, quantified by MSD, are comparable in vehicle and TAM-treated Mrc1^Cre+/ApoE4^fl/fl and Mrc1^Cre+/ApoE3^fl/fl mice (N=5/group). E-F. BAM-specific deletion of ApoE restores functional hyperemia and endothelial vasoactivity in TAM-treated Mrc1^Cre+/ApoE4^fl/fl mice (N=5/group) (E), but does not alter CBF responses in TAM-treated Mrc1^Cre+/ApoE3^fl/fl mice (N=5/group) (F). rApoE4 markedly attenuates functional hyperemia and endothelial vasoactivity in TAM-treated Mrc1^Cre+/ApoE4^fl/fl and Mrc1^Cre+/ApoE3^fl/fl mice (N=5/group), attesting to the integrity of ApoE4 signaling pathways leading to neurovascular dysfunction despite BAM ApoE deletion. Data in C-F were analyzed using two-way ANOVA with Tukey’s test and are presented as mean±SEM.

Figure 5. ApoE4 in BAM induces CBF dysfunction in ApoE3-TR mice, while ApoE3 in BAM reverses the dysfunction in ApoE4-TR mice.

A. Mice received bone marrow transplantation (BMT) at 2.5 months of age and were studied 12 weeks later. B-C. In WT mice transplanted with WT bone marrow (WT→WT) CBF responses are comparable to those in of naïve WT mice (see Fig. 1–3), whereas in ApoE4-TR transplanted with ApoE4 bone marrow (E4→E4) CBF responses are attenuated as in ApoE4-TR mice (see Fig. 2A). Remarkably, transplant of ApoE4 bone marrow into WT (E4→WT) or ApoE3-TR (E4→E3) mice, attenuates neurovascular responses as in ApoE4-TR mice, and, conversely, transplant of E3 bone marrow into ApoE4-TR mice (E3→E4) normalizes neurovascular function. Data in B-C were analyzed using one-way ANOVA with Tukey’s test and are presented as mean±SEM; N=5/group.

Figure 6. In a model of cerebral hypoperfusion ApoE4 in BAM worsens CBF reduction and white matter damage in ApoE3-TR mice, while ApoE3 in BAM ameliorates the phenotype.

A. Mice were transplanted as in Fig. 5A. Twelve weeks later, forebrain hypoperfusion was induced by bilateral common carotid artery stenosis (BCAS). B. The reduction in neocortical CBF assessed by laser speckle flowmetry was worse in E4→E4 than in E3→E3 and WT→WT chimeras. However, E3→E4 BMT ameliorates the CBF reduction while E4→E3 BMT worsened it. N=5/group. Representative laser speckle images were shown from N=5/group. Scale bar = 2 mm. C. Klüver-Barrera (KB) white matter stain of the corpus callosum in the same groups of mice in which CBF was assessed showing increased white matter damage in E4→E3 compared to E3→E3 chimeras, and reduced white matter damage in E3→E4 compared to E4^®E4 chimeras. N=5 mice/group; scale bar = 100 μm. D. Double-labeling immunofluorescence of myelin basic protein (MBP, green) and the pan-axonal neurofilament marker SMI312 (red) after BCAS illustrating a worsening of myelin integrity in E4→E3 chimeras, and improvement in E3→E4 chimeras. N=5 mice/group; scale bar = 100 μm. E. Immuno fluorescence stain of MBP (green) and the oligodendrocyte marker Olig2 (red) illustrating a worse oligodendrocyte depletion in E4→E3 compared to E3→E3 chimeras, and an improvement in E3→E4 compared to E4→E4 chimeras. N=5 mice/group; scale bar = 100 μm. F. Immunofluorescence stain of the nodal Nav1.6 channels (red) and the paranodal protein Caspr (green) showing increase nodal exposure in E4→E3 compared to E3→E3 chimeras, and an improvement in E3→E4 compared to E4→E4 chimeras. N=5/group; scale bar = 3 μm. In C-F, representative images are shown on the left and related quantification on the right; representative images for each group are selected from 10 sections (2 sections per mouse) on which quantification was done. Data in B were analyzed with one-way ANOVA and Tukey’s test at each time point; data in B-Fanalyzed using two-way ANOVA with Tukey’s test; data are presented as mean±SEM.

Figure 7. In a model of cerebral hypoperfusion ApoE4 in BAM worsens cognitive deficits in ApoE3-TR mice, while ApoE3 in BAM ameliorates the cognitive phenotype.

In agreement with the CBF and WM damage data, E4→E3 chimeras exhibit worse cognitive deficits than E3→E3 chimeras at the novel object recognition (A) and Y-maze (B) tests, while E3→E4 chimeras exhibit cognitive improvement compared to E4→E4 chimeras. Indices of locomotor activity, recorded during the novel object recognition test (distance traveled) or the Y-maze test (number of arm entries), do not differ among groups. Data in A and B were analyzed with two-way ANOVA and Tukey’s test and are presented as mean±SEM. N=10–12/group.