Abstract
Genome-wide association studies have identified many gene polymorphisms associated with an increased risk of developing late-onset Alzheimer’s disease (LOAD). Many of these LOAD risk-associated alleles alter disease pathogenesis by influencing innate immune responses and lipid metabolism of microglia (MG). Here we show that boosting the expression of angiotensin-converting enzyme (ACE), a genome-wide association study LOAD risk-associated gene product, specifically in MG, reduces amyloid-β (Aβ) plaque load, preserves vulnerable neurons and excitatory synapses, and significantly reduces learning and memory abnormalities in the 5xFAD amyloid mouse model of AD. ACE-expressing MG surround plaques more frequently and they have increased Aβ phagocytosis, endolysosomal trafficking and spleen tyrosine kinase activation downstream of the major Aβ receptors, triggering receptor expressed on myeloid cells 2 (Trem2) and C-type lectin ___domain family 7 member A (Clec7a). These findings establish a role for ACE in enhancing microglial immune function and they identify a potential use for ACE-expressing MG as a cell-based therapy to augment endogenous microglial responses to Aβ in AD.
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Data availability
The snRNA-seq and GeoMx data have been deposited with the Gene Expression Omnibus under accession nos. GSE288410 and GSE288280, respectively. The RNA-seq data used the mouse genome sequence GRCm38 (mm10) for reference (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000001635.20/). Source data are provided with this paper. Any other data reported in this paper are available from the corresponding author upon reasonable request.
Code availability
No custom code was used for any analysis in these studies.
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Acknowledgements
This study was supported by NIH grant nos. RF1-AG074365 and R01-AG074365 to W.G.T., NIH grant no. R01-AI164519 to K.E.B. and a Cedars-Sinai Goldrich Alzheimer’s Center grant to W.G.T. and K.E.B. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We thank L. Karman for technical assistance and A. Moser for reviewing the paper.
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W.G.T. conceived the project and designed the studies. A.R.G., S.W., H.R.B., A.K.M.G.M, L.L., A.M., J.I. and J.C. performed the experiments. W.G.T., A.R.G., S.W., H.R.B., A.K.M.G.M, K.E.B., L.L. and R.A. analyzed the data. W.G.T. and A.R.G. wrote the paper.
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Extended data
Extended Data Fig. 1 ACE expression in microglia from wildtype, 5xFAD and ACE10/10 mice.
Representative immunofluorescence images from 6-month-old brain cut in the coronal plane at -2mm Bregma from wildtype (WT), homozygous 5xFAD (5xFADTg/Tg) and homozygous ACE10/10 mice using antibodies to Iba1 to label microglia (green) and ACE (magenta). Representative images depicted were derived from analysis of n = 3 mice of each genotype and 2 brain sections per mouse with consistent results. (scale, 100 µm).
Extended Data Fig. 2 Generating and characterizing knock-in mice with ACE expression restricted to microglia.
a, Rosa-hACE-flox mice were generated by targeting the Gt(Rosa) safe harbor locus in mouse embryonic stem cells. The targeting construct contains a loxP-site flanked transcriptional stop sequence upstream of a bicistronic TurboGFP and c-terminal flag-tagged human ACE (hACE-3xF) expression unit that is expressed after loxP site recombination in the genome. Embryonic stem cell clones were screened by PCR (3′ screen) to identify positive clones that generated a 4,355 bp PCR amplicon. Secondary screening was used to detect 5’ homologous recombination by Southern blotting on MfeI restricted genomic DNA to identify a 10.3 kb fragment indicative of heterozygous homologous recombination and germline heterozygous ACE-flox (Rosa+/hACE-flox) mice were generated. b, ACE-flox mice were mated to Cx3Cr1+/CreERT2 mice to generate RACE- (Cx3Cr1+/CreERT2; Rosa+/+) and RACE+ (Cx3Cr1+/CreERT2; Rosa+/hACE-flox) mice. c, RACE- and RACE+ mice were injected with Tamoxifen (TMX; 125 mg/kg, IP) for four consecutive days to activate Cre-recombinase in R- and R+ myeloid-derived cells. d, Myeloid cells isolated from brain, spleen and blood were gated on DAPI, CD11b and Cx3Cr1 and then sub-gated on GFP to detect transgene expression, 7, 21 and 180 days after TMX injection. e, Transgene expression persisted in myeloid cells in the brain and became undetectable in the blood and spleen by 3 weeks after TMX injection (data are plotted as mean ± SEM).
Extended Data Fig. 3 ACE expression in the brain of R+ mice.
Representative immunofluorescence images from 9-month-old R+ mouse brain cut in the coronal plane at -2mm Bregma and two representative brain regions shown (Parietal Association Cortex - PtA and hippocampus CA1, stratum radiatum – CA1:SR). Labeling for ACE expression in microglia (green) does not co-localize with a, neurons (NeuN; magenta), b, astrocytes (GFAP; magenta) or c, oligodendroglia cells (Sox10; magenta). Experiments depicted in representative images were derived from analysis of n = 2; R+ mice and 2 sections per mouse with consistent results in all areas of the brain. (scale, 50 µm).
Extended Data Fig. 4 Amyloid plaque burden in 5xFAD mice with and without expression of ACE in microglia.
Representative images of 9-month-old a, R−; AD+ and b, R+; AD+ brain cut in the coronal plane at -2mm Bregma and stained with Thioflavin S (Thio. S), showing representative regions of the brain where plaque burden is quantified; hippocampus CA1, stratum oriens (CA1:SO), Parietal Association (PtA) cortex and Amygdala (Amg) (scale, 500 µm). c, Quantification of the percent of Thio. S coverage in R+; AD+ normalized to coverage in R−; AD+ brain in the CA1:SO, PtA and Amg. Representative immunofluorescence images of 9-month-old d, R−; AD+ and e, R+; AD+ brain cut in the coronal plane at -2mm Bregma labeled with 6E10 antibody to label amyloid precursor protein and showing representative regions of the CA1:SO, PtA and Amg that are quantified (scale, 500 µm). f, Quantification of the percent of 6E10 coverage in R+; AD+ and R−; AD+ brains in the CA1:SO, PtA and Amg. For quantification in c, f, n = 3-4 males and 4-5 females from each genotype, sexes are combined since no significant differences were detected between males and females, plotted as mean ± SEM normalized to the mean of R−; AD+, two-way analysis of variation (ANOVA) and Sidak’s post-hoc test for multiple comparisons, * = p < 0.05, ** = p < 0.01, and *** = p < 0.001. (male values = square symbol and female values = round symbol).
Extended Data Fig. 5 Cytokine levels in the hippocampus of wild type and 5xFAD mice with and without ACE expression in microglia.
Cytokine levels for a, TNF-α, b, IL-1β, c, CXCL1, d, IFN-γ, e, IL-6, f, IL-10, g, IL-12p70, h, IL-2 and i, IL-5 measured from 100 µg of total soluble hippocampal protein (THP) from 9-month-old, R−; AD-, R+; AD-, R−; AD+ and R+; AD+ mice. For quantification in a-i, n = 3-5 males and n = 3-5 females, sexes are combined since no significant differences were detected between males and females, plotted as mean ± SEM of analyte mass normalized to unit mass of THP, one-way analysis of variation (ANOVA) and Tukey’s post-hoc test for multiple comparisons, ** = p < 0.01 and **** = p < 0.0001 and ns = non-significant. (male values = square symbol and female values = round symbol).
Extended Data Fig. 6 Aβ1-40, Aβ1-42 and ratio of Aβ1-42/Aβ1-40 protein concentrations in the hippocampus from 5xFAD mice with and without ACE expression in microglia.
Quantification of a, Aβ1-40, b, Aβ1-42 and c, Aβ1-42/Aβ1-40 ratio measured from 10 μg of total hippocampal soluble protein (THP) in 9-month-old R−; AD+ and R+; AD+ mice. For quantification, a–c, n = 8-10 males and n = 10 females, sexes are combined since no significant differences were detected between males and females, plotted as mean ± SEM of analyte mass normalized to unit mass of THP, unpaired T-test, * = p < 0.05 and ns = non-significant. (male values = square symbol and female values = round symbol).
Extended Data Fig. 7 Phagocytosis and endolysomal trafficking in primary microglia with and without ACE expression.
a, Representative Incucyte plots from R- (blue) and R+ (red) microglia treated with oAβ1-42 conjugated to pHrodo, which fluoresces after phagocytosis and loading into the acidic intracellular lysosomal compartment. Data are plotted as integrated intensity of pHrodo fluorescence normalized to the confluency of cells and plotted over 120 h in vitro. Each point represents the mean ± SD of values measured in 8 separate regions of each well. b, Quantification of the amount of oAβ1-42 phagocytosis and lysosome loading relative to R- microglia when it is left in the culture media during the first 30 h of incubation (pulse). c, Quantification of the amount of intracellular oAβ1-42 degraded within the cell between 30 and 120 h after it is removed from the media (chase). d, Representative Incucyte plots identical to a but with R- and R+ microglia are treated with Zymosan conjugated to pHrodo, which binds to microglia CLEC7A receptors. e, Quantification of the amount of Zymosan phagocytosis and lysosome loading relative to R- microglia when it is left in the culture media during the first 30 h of incubation (pulse). f, Quantification of the amount of Zymosan degraded between 30 and 120 h after it is removed from the media (chase). For quantification in b, n = 7 R- and n = 6R+ independent microglia cultures; c, n = 7 R- and n = 6R+ independent microglia cultures; e, n = 13 R- and n = 15R+ independent microglia cultures and f, n = 13 R- and n = 15R+ independent microglia cultures, plotted as mean ± SEM and normalized to the mean of R-, two-tailed unpaired t-test, ** = p < 0.01, *** = p < 0.001 and **** = p < 0.0001.
Extended Data Fig. 8 Experimental design for RNAseq analysis of single nuclei isolated from cortex and hippocampus of wild type and 5xFAD mice with and without ACE expression in microglia.
a, Single nuclei were isolated from cortex and hippocampus of 9-month-old mice, and pooled into samples representing two mice from each genotype and sex (SN1-8). The samples were divided into two groups (Group 1 and Group 2) containing 4 samples that were each labeled with a unique hash-tag antibody. b, The samples were sorted using fluorescence activated nuclear sorting to capture nuclei in the P1 gate that were enriched for microglia by rejecting many neurons (NeuN+), oligodendroglia cells (Sox10+) and astrocytes (Sox9+). c, cDNA libraries were generated from Group 1 and Group 2 samples and the sequences were demultiplexed into samples according to the antibody hash-tag that was used to label the nuclear samples (SN1-SN8). Nuclei that were labeled by more than a single hash-tag antibody (cell colored red) were removed from the datasets within each sample. The TotalSeq hash-tag antibody used and the number of single labeled nuclei per sample are shown.
Extended Data Fig. 9 Quality control of snRNAseq data from 9-month-old wild type and 5xFAD cortex and hippocampus with and without ACE expression in microglia.
a, The number of cells per cluster. b, Violin plot of the number of genes expressed per cell in each cluster. Each dot represents the number of genes detected in a single cell. c, Violin plot of the nUMI per cell in each cluster. Each dot represents the nUMI detected in a single cell. d, Violin plot of the percent of mitochondrial genes per cell in each cluster. Each dot represents the percent of mitochondrial genes detected in a single cell. For a-d, the cluster legend correlates the informatically generated cluster number with their cellular identity. e, Violin plot of the number of genes per cell in each sample (SN1-SN8). Each dot represents the number of genes detected in a single cell. f, Violin plot of nUMI per cell in each sample. Each dot represents the nUMI detected in a single cell. (See Methods for detailed quality control measures applied.)
Extended Data Fig. 10 Distinct cell types identified by snRNAseq from 9-month-old wildtype and 5xFAD cortex and hippocampus with and without expression of ACE in microglia.
a, UMAP principal component analysis of 10 clusters and the relative percent of nuclei within each cluster from SN1-SN8 (cluster number shown in parentheses). b, Cell lineage restricted gene expression defines cluster cellular identity. c, C1qa, Hexb and P2ry12, genes expressed relatively specifically in microglia, map to clusters annotated as microglia (all cell nuclei shown in grey).
Supplementary information
Supplementary Information
Supplementary key resources, methods, data figures and data source information, data tables and references.
Supplementary Table 1
Volcano plot data showing association between DAM-A R+; AD+ and R− AD− gene expression in Fig. 6c.
Supplementary Table 2
Ingenuity Pathway Analysis (IPA) showing all pathways highlighted from genes differentially expressed in from R+; AD+ relative to R− AD+ DMA-A as abbreviated in Fig. 6d.
Supplementary Table 3
Average gene expression in CLEAR signaling, phagosome maturation and production of ROS pathways highlighted by IPA analysis in R−; AD+ and R+; AD+ DAM-A and shown in Fig. 6e.
Supplementary Table 4
Average gene expression in mTOR signaling, PI3K/Akt signaling and oxidative phosphorylation pathways highlighted by IPA analysis in R−; AD+ and R+; AD+ DAM-A and shown in Fig. 6f.
Supplementary Table 5
Average gene expression of DAM-A genes highlighted in IPA function Movement of Cells in R− ; AD+ and R+; AD+ as shown in Supplementary Data Fig. 4.
Supplementary Table 6
Gene expression for volcano plot of GeoMx gene expression of PAM in R+; AD+ versus R−; AD+ shown in Fig. 7c.
Supplementary Table 7
Complete list of Reactome pathways identified from GeoMx expression analysis of R+; AD+ versus R−; AD+ PAM. Highlighted pathways are color coated the same as in Fig. 7d.
Supplementary Table 8
Average gene expression in C-lectin receptor signaling and Clec7a signaling pathways from Reactome analysis in R+; AD+ versus R−; AD+ PAM and shown in Fig. 7e.
Supplementary Table 9
Average gene expression in NF-κB signaling and IL-1 family signaling pathways from Reactome analysis in R+; AD+ versus R−; AD+ PAM and shown in Fig. 7f.
Supplementary Table 10
Average gene expression in translation signaling and rRNA synthesis signaling pathways from Reactome analysis in R+; AD+ versus R−; AD+ PAM and shown in Fig. 7g.
Supplementary Table 11
Volcano plot data of GeoMx gene expression of PAM from R−; AD+ PAM versus nPAM shown in Supplementary Data Fig. 5a.
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Gomez, A.R., Byun, H.R., Wu, S. et al. Boosting angiotensin-converting enzyme (ACE) in microglia protects against Alzheimer’s disease in 5xFAD mice. Nat Aging (2025). https://doi.org/10.1038/s43587-025-00879-1
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DOI: https://doi.org/10.1038/s43587-025-00879-1