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A cell-autonomous role for border-associated macrophages in ApoE4 neurovascular dysfunction and susceptibility to white matter injury

Nature Neuroscience volume 27, pages 2138–2151 (2024)Cite this article

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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 (BAMs), myeloid cells closely apposed to neocortical microvessels, are both sources and effectors of ApoE4 mediating the neurovascular dysfunction through reactive oxygen species. ApoE4 in BAMs 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.

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Fig. 1: The rApoE4 or lipidated rApoE4 alters functional hyperemia and endothelial vasoactivity.
Fig. 2: BAMs mediate the deleterious cerebrovascular effects of ApoE4 through NOX-derived ROS.
Fig. 3: ApoE4 induces Ca2+ currents in BAMs, leading to ROS production.
Fig. 4: BAM depletion prevents the neurovascular dysfunction induced by ApoE4.
Fig. 5: Deletion of ApoE4 selectively in BAMs restores neurovascular function.
Fig. 6: ApoE4 in BAMs induces CBF dysfunction in ApoE3-TR mice, whereas ApoE3 in BAMs reverses the dysfunction in ApoE4-TR mice.
Fig. 7: In a model of cerebral hypoperfusion ApoE4 in BAMs worsens CBF reduction and white matter damage in ApoE3-TR mice, whereas ApoE3 in BAMs ameliorates the phenotype.
Fig. 8: In a model of cerebral hypoperfusion, ApoE4 in BAMs worsens cognitive deficits in ApoE3-TR mice, whereas ApoE3 in BAMs ameliorates the cognitive phenotype.

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Data availability

The authors declare that the data supporting the findings of the present study are available within the article and Extended Data Figures. The dataset with GEO accession no. GSE174574 can be accessed at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE174574. Source data are provided with this paper.

Code availability

No customized software codes were used in the paper.

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Acknowledgements

The present study was supported by NIH grant nos. R01-NS126467 (to C.I.), R01-NS097805 (to L.P.) and K22-NS123507 (to M.M.S.) and the BrightFocus Foundation (to A.A.). We thank the Feil Family Foundation for their support.

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Authors and Affiliations

  1. Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY, USA

    Antoine Anfray, Samantha Schaeffer, Yorito Hattori, Monica M. Santisteban, Nicole Casey, Gang Wang, Ping Zhou, Josef Anrather, Laibaik Park & Costantino Iadecola

  2. Department of Neurology, Hope Center for Neurological Disorders, Charles F. and Joanne Knight Alzheimer’s Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA

    Michael Strickland & David M. Holtzman

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Contributions

A.A., L.P., S.S., M.M.S., N.C. and Y.H. conducted the experiments and performed the data analysis. G.W. performed the ROS measurement in BAMs ex vivo. N.C. performed the RNAscope and histology experiments and mouse genotyping. P.Z. assisted in mouse breeding. M.S. and D.M.H. provided and validated lipidated ApoE3 and ApoE4 and edited the manuscript. L.P., J.A. and C.I. supervised the research. L.P. and C.I. provided funding and wrote the manuscript.

Corresponding authors

Correspondence to Laibaik Park or Costantino Iadecola.

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Competing interests

D.M.H. is on the scientific advisory board of C2N diagnostics and has equity. He is also on the scientific advisory board of Denali Therapeutics, Genentech and Cajal Therapeutics and consults for Asteroid. C.I. is on the scientific advisory board of Broadview Ventures. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Effect of rApoE4 in ApoE−/− or CD36−/− mice, and of VLDL receptor antibodies and sex in ApoE4-TR mice.

a. Topical application of rApoE4 (10 µg/ml), but not rApoE3 (10 µg/ml), leads to neurovascular dysfunction in ApoE−/− mice, prevented by CLO. b. Superfusion with VLDL receptor blocking antibodies (anti-VLDLr; 500 nM) fails to prevent the neurovascular dysfunction in ApoE4-TR mice, while RAP superfusion (200 nM) is effective. c. rApoE4 induces neurovascular dysfunction in mice lacking CD36. d. The detrimental neurovascular effects of ApoE4 are not sexually dimorphic. Data were analyzed using one-way or two-way ANOVA with Tukey’s or paired two-tailed t-test and are presented as mean±SEM. N = 5/group.

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Extended Data Fig. 2 Smooth muscle vasoactivity, BBB, Iba1+ cell, or BAM engraftment in the mice studied.

a. The NOX peptide inhibitor gp91ds-tat or its scrambled control sgp91ds-tat does not alter the CBF increase induced by neocortical application of adenosine (smooth muscle vasoactivity) in WT, ApoE3-TR, and ApoE4-TR mice, or in WT mice treated with vehicle, rApoE4 or rApoE3. b. BBB permeability to 3kD dextran was not altered in ApoE4-TR mice, with or without i.c.v. administration of CLO. c. CLO does not reduce the number of Iba1+ cells. d. Smooth muscle vasoactivity in ApoE3- or 4-TR mice with or without CLO treatment. e. Smooth muscle vasoactivity in Mrc1Cre+, Mrc1Cre+/ApoE4fl/fl, or Mrc1Cre+/ApoE3fl/fl treated with tamoxifen alone or with rApoE4 (10 µg/ml). f. Numbers of GFP+, CD206+, and GFP-CD206 double-positive cells in WT, ApoE3-TR, ApoE4-TR mice transplanted with GFP+ BM. g. Smooth muscle vasoactivity in ApoE3, ApoE4, WT BM chimeras. Data were analyzed using one-way or two-way ANOVA with Tukey’s test and are presented as mean±SEM. N = 4-5/group.

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Extended Data Fig. 3 Targeting vector, BAM targeting selectivity, and efficiency of ApoE deletion in Mrc1Cre+ mice crossed with R26tdT, ApoE3fl/fl, or ApoE4fl/fl mice.

a. DNA construct used to generate Mrc1Cre+ mice. Tamoxifen (TAM) treatment of Mrc1Cre+/R26tdT crosses induces recombinase activity in over 80–90% of cells expressing the BAM marker CD206 but not in Iba1+ microglia or CD31+ cerebral endothelial cells. N = 5 mice/group; represented images selected from N = 5 mice/group; 2-3 sections analyzed in neocortex per mouse; unpaired t-test; mean±SEM. b–e. 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 showing expression of ApoE in Mrc1+ cells in Mrc1Cre+/ApoE4fl/fl (b) and Mrc1Cre+/ApoE3fl/fl (c) mice treated with vehicle (corn oil). However, in Mrc1Cre+/ApoE4fl/fl (d) and Mrc1Cre+/ApoE3fl/fl (e) mice treated with TAM ApoE levels in Mrc1+ cells are markedly reduced. Scale bars = 20 µm in a–e. f–g. The number of Mrc1+ cells (f) and their Mrc1 puncta (g) is comparable in vehicle- and TAM-treated Mrc1Cre+/ApoE4fl/fl and Mrc1Cre+/ApoE3fl/fl mice. N = 5 mice/group; 1-2 sections/mouse; 5–12 cells analyzed in neocortex per section; two-way ANOVA with Tukey’s test; mean±SEM.

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Extended Data Fig. 4 ApoE in GFAP+ cells and ApoE levels in CSF and plasma in Mrc1Cre+/ApoE4 fl/fl and Mrc1Cre+/ApoE3fl/fl mice.

a-f. Triple or dual RNAScope in situ hybridization with Mrc1 mRNA probes (green in A–B), GFAP (gray in A–B and green in C-D), and ApoE (magenta), DAPI (blue) and the basal lamina marker laminin (gray in C-D). a, c. In vehicle-treated Mrc1Cre+/AoE4fl/fl (A) and Mrc1Cre+/AoE3fl/fl (C) mice, ApoE expression is found both in Mrc1+ (green, A) and GFAP+ (gray in A and green in C) cells. In tamoxifen (TAM)-treated Mrc1Cre+/AoE4fl/fl (B) and Mrc1Cre+/AoE3fl/f (D) mice, ApoE is deleted in Mrc1+ cells (B), but its expression is not affected in GFAP+ cells (B, D). Representative confocal images (A-D) from N = 3–5 mice/group. Scale bars = 20 μm. E–F. The numbers of GFAP+ and ApoE+GFAP+ cells are comparable in vehicle- and TAM-treated Mrc1Cre+/ApoE4fl/fl and Mrc1Cre+/ApoE3fl/fl mice (N = 5 mice/group; 2-3 sections/mice). G–H. ApoE levels in CSF (G) and plasma (H), quantified by MSD, are comparable in vehicle- and tamoxifen-treated Mrc1Cre+/ApoE4fl/fl and Mrc1Cre+/ApoE3fl/fl mice. N = 5/group. Data in E-H were analyzed using two-way ANOVA with Tukey’s test and are presented as mean±SEM. N = 5/group.

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Extended Data Fig. 5 Tamoxifen treatment of Mrc1Cre+/ApoE4fl/fl mice reduces ApoE immunoreactivity in CD206+ cells, but not in CD206− cells.

Quadruple labeling with Dapi (nuclei: blue), CD206 (BAM: green), human ApoE (magenta), and CD31 (endothelium: gray) in WT (A, B) or MrcCre+/ApoE4fl/fl (C, F) mice treated with corn oil (A, C) or tamoxifen (B, D). No ApoE immunoreactivity is observed in WT mice, attesting to the specificity of the human ApoE antibody (A, B). In corn oil-treated MrcCre+/ApoE4fl/fl mice strong ApoE immunoreactivity is observed both in CD206+ and CD206− cells (C). However, tamoxifen treatment of MrcCre+/ApoE4fl/fl mice suppresses ApoE immunoreactivity in CD206+ but not in CD206− cells (D) (Scale bar = 100 µm in A-D). The number of CD206+ cells is comparable in corn oil- or tamoxifen-treated WT or MrcCre+/ApoE4fl/fl mice (E). The number of CD206+ApoE+ cells (F), but not CD206−ApoE+ cells (G), is markedly reduced in tamoxifen-treated MrcCre+/ApoE4fl/fl mice. Data in E-G were analyzed using two-way ANOVA with Tukey’s test and are presented as mean±SEM. N = 5/group.

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Extended Data Fig. 6 Tamoxifen treatment of Mrc1Cre+/ApoE4fl/fl mice does not alter CD206, TMEM119 or Iba-1 immunoreactivity.

Quadruple labeling with Dapi (nuclei: blue), CD206 (BAM: green), human ApoE (magenta), and CD31 (endothelium: gray) in WT (A, B)or MrcCre+/ApoE4fl/fl (C, D) mice treated with corn oil (A,C) or tamoxifen (B, D). Tamoxifen treatment of WT or MrcCre+/ApoE4fl/fl mice does not alter the number and % area of CD206+ (E) TMEM119+ (F) and Iba1+ cells (G). Scale bars = 100 µm in A-D. N = 5/group. Data in E-G were analyzed using two-way ANOVA with Tukey’s test and are presented as mean±SEM. N = 5/group.

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Extended Data Fig. 7 Effect of ApoE4 on neurovascular function in Mrc1Cre+, ApoE4fl/fl, or Mrc1Cre+/ApoE4fl/fl mice and on ROS in Mrc1Cre+ and Mrc1Cre+/ApoE4fl/fl brain slices.

a. In Mrc1Cre+ mouse, neocortical superfusion of rApoE4 attenuates functional hyperemia and endothelial vasoactivity compared to vehicle (corn oil), but it has no effect on smooth muscle vasoactivity. b. In ApoE4fl/fl mice, neocortical superfusion with RAP (200 nM) reverses the neurovascular dysfunction. c. In vehicle-treated Mrc1Cre+/ApoE4fl/fl mice, functional hyperemia and endothelial vasoactivity, but not smooth muscle vasoactivity, are attenuated as found in ApoE4-TR mice (see Fig. 2a, Extended Data Fig. 2a). In A-C, N = 5/group. d–e. Baseline ROS production in BAM, assessed by DHE in cortical brain slices in which BAM were labeled by i.c.v. injection of Alexa Fluor 680-labeled dextran (10 kDa). ROS production is higher in BAM of MrcCre+/ApoE4fl/fl mice compared to Mrc1Cre+ mice (D) and is reduced by treatment with tamoxifen (E); N = 3-4 mice per group, 1-2 brain slices/mouse, and 3–7 cells/slice; two-tailed t-test. Data are expressed as mean±SEM. Scale bar = 50 µm.

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Extended Data Fig. 8 Validation steps of preparing the lipidated recombinant ApoE using POPC and cholesterol.

a. Technical replicates of recombinant ApoE3 or ApoE4 lipidated with POPC and cholesterol on an SDS-PAGE gel. ApoE was lipidated at a 1:50:10 molar ratio of ApoE:POPC:Cholesterol. SDS-PAGE was performed under nonreducing (NR) or reducing (R) conditions. Reducing conditions were performed with the addition of 50 mM DTT. One µg of protein was loaded in each well. SDS-PAGE was run on a 4–12% bis-tris gel with MES buffer. Gel was stained with coomassie blue showing only the presence of the ApoE protein. The y-axis represents molecular weight in kDA. Shown are three independent replicates of the samples. b. Native PAGE of technical replicates of recombinant ApoE3 and ApoE4 lipidated with POPC and cholesterol. ApoE was lipidated at a 1:50:10 molar ratio of ApoE:POPC:Cholesterol. One µg of protein was loaded in each well. High molecular weight ladder (Cytiva, 17044501) was detected using PonceauS staining. ApoE was detected by western blot using an anti-ApoE antibody (Academy Bio-Medical, 50A-G1b). Native gel shows the presence of lipidated ApoE. Shown are three independent replicates of the samples. c. FPLC curve of recombinant ApoE3 and ApoE4 lipidated with POPC and cholesterol. Lipidated ApoE was purified from fractions 15−20 and verified by negative stain TEM. Samples were run on a Superose 6 Increase 10/300 GL column (Cytiva, 29091596) at 0.5 mL/min in 20 mM phosphate buffer, 50 mM NaCl, pH 7.4. The x axis represents elution volume. d. Negative stain TEM of size exclusion chromatography fractions of recombinant ApoE3 and ApoE4 lipidated with POPC and cholesterol. Negative stain TEM imaging shows the presence of discoidal lipoprotein. Micrographs (N = 1/sample) were taken on a JEOL JEM-1400 at 120 kV at 80,000× magnification (0.138889 nm/pixel).

Extended Data Fig. 9 Putative pathway by which ApoE4 in BAM induces vascular oxidative stress, neurovascular dysfunction and enhanced white matter injury.

(1) ApoE4 in BAM acts on ApoE receptors, for example, LRP1 as a candidate receptor, to increase intracellular Ca2+ in BAM in a cell autonomous manner resulting in NOX activation, (2) vascular oxidative stress, and (3) neurovascular dysfunction and reduced cerebral perfusion, which, in turn, leads to (4) enhanced white matter damage and cognitive impairment.

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Anfray, A., Schaeffer, S., Hattori, Y. et al. A cell-autonomous role for border-associated macrophages in ApoE4 neurovascular dysfunction and susceptibility to white matter injury. Nat Neurosci 27, 2138–2151 (2024). https://doi.org/10.1038/s41593-024-01757-6

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