. 2025 Jan 2;32(1):86-104.e7.

doi: 10.1016/j.stem.2024.10.005. Epub 2024 Nov 4.

Microglia depletion reduces human neuronal APOE4-related pathologies in a chimeric Alzheimer's disease model

Affiliations

¹ Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, CA, USA; Developmental and Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
² Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, CA, USA; Gladstone Center for Translational Advancement, Gladstone Institutes, San Francisco, CA, USA.
³ Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, CA, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
⁴ Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, CA, USA; Neuroscience Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
⁵ Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, CA, USA.
⁶ Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, CA, USA; Developmental and Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, CA, USA; Gladstone Center for Translational Advancement, Gladstone Institutes, San Francisco, CA, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA; Neuroscience Graduate Program, University of California, San Francisco, San Francisco, CA, USA; Department of Neurology, University of California, San Francisco, San Francisco, CA, USA; Department of Pathology, University of California, San Francisco, San Francisco, CA, USA. Electronic address: yadong.huang@gladstone.ucsf.edu.

PMID: 39500314
PMCID: PMC11701721
DOI: 10.1016/j.stem.2024.10.005

Microglia depletion reduces human neuronal APOE4-related pathologies in a chimeric Alzheimer's disease model

Antara Rao et al. Cell Stem Cell. 2025.

. 2025 Jan 2;32(1):86-104.e7.

doi: 10.1016/j.stem.2024.10.005. Epub 2024 Nov 4.

Authors

Affiliations

¹ Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, CA, USA; Developmental and Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
² Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, CA, USA; Gladstone Center for Translational Advancement, Gladstone Institutes, San Francisco, CA, USA.
³ Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, CA, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
⁴ Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, CA, USA; Neuroscience Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
⁵ Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, CA, USA.
⁶ Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, CA, USA; Developmental and Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, CA, USA; Gladstone Center for Translational Advancement, Gladstone Institutes, San Francisco, CA, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA; Neuroscience Graduate Program, University of California, San Francisco, San Francisco, CA, USA; Department of Neurology, University of California, San Francisco, San Francisco, CA, USA; Department of Pathology, University of California, San Francisco, San Francisco, CA, USA. Electronic address: yadong.huang@gladstone.ucsf.edu.

PMID: 39500314
PMCID: PMC11701721
DOI: 10.1016/j.stem.2024.10.005

Abstract

Despite strong evidence supporting the important roles of both apolipoprotein E4 (APOE4) and microglia in Alzheimer's disease (AD) pathogenesis, the effects of microglia on neuronal APOE4-related AD pathogenesis remain elusive. To examine such effects, we utilized microglial depletion in a chimeric model with induced pluripotent stem cell (iPSC)-derived human neurons in mouse hippocampus. Specifically, we transplanted homozygous APOE4, isogenic APOE3, and APOE-knockout (APOE-KO) iPSC-derived human neurons into the hippocampus of human APOE3 or APOE4 knockin mice and then depleted microglia in half of the chimeric mice. We found that both neuronal APOE and microglial presence were important for the formation of Aβ and tau pathologies in an APOE isoform-dependent manner (APOE4 > APOE3). Single-cell RNA sequencing analysis identified two pro-inflammatory microglial subtypes with elevated MHC-II gene expression enriched in chimeric mice with human APOE4 neuron transplants. These findings highlight the concerted roles of neuronal APOE, especially APOE4, and microglia in AD pathogenesis.

Keywords: APOE4; Alzheimer’s disease; RNA-seq; amyloid; chimeric disease model; iPSC; microglia; tau; transplantation.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests Y. Huang is a cofounder and scientific advisory board member of GABAeron, Inc.

Figures

**Figure 1.. Transplanted human neuronal progenitors survive and develop into neurons in chimeric mouse hippocampus.**
(A) Experimental design: iPSCs with different APOE genotypes were differentiated into neuronal progenitors and transplanted into human APOE-KI mice. Chimeric mice were aged for 8 months, with half the mice receiving PLX3397 (PLX) for the latter 4 months. All mice were used for histological or transcriptomic analysis. (B) Representative images of human cell transplants in the hippocampus of 12-month-old chimeric mice (8 months post transplantation). Top row: Human Nuclear Antigen (HNA, red) marking human cell transplants; second row: human-preferential MAP2 (gray) marking neurons; third row: composite of HNA and MAP2 images. Scale bar, 100 μm. (C) Quantification of number of HNA+ cells per human transplant. Each dot represents one mouse per condition (hE3-E3KI, n=5; hE3-E3KI-PLX, n=5; hE4-E4KI, n=9; hE4-E4KI-PLX, n=9; hEKO-E4KI, n=4; hEKO-E4KI-PLX, n=6). (D) Quantification of size of human transplants per condition, as percent of hippocampus. Each dot represents one mouse (hE3-E3KI, n=5; hE3-E3KI-PLX, n=6; hE4-E4KI, n=9; hE4-E4KI-PLX, n=8; hEKO-E4KI, n=4; hEKO-E4KI-PLX, n=6). (E–F) Representative immunohistochemical images of transplanted human cells expressing mature neuronal (NEUN and MAP2) and synaptic (synaptophysin) markers. Scale bar, 25 μm. (G,H) Whole-cell current-clamp recordings of transplanted E4/4 (top) and iE3/3 (bottom) iPSC-derived neurons in *ex vivo* slices demonstrating the ability to fire action potentials (G, the membrane potential responses to a 1-s, 50-pA depolarizing current injection) and the ability to receive spontaneous excitatory post-synaptic currents (H, the membrane current traces recorded at a holding potential of −70mV). For quantifications, all data are expressed as mean ± S.E.M. Differences between groups were determined by two-way ANOVA with Benjamini’s post hoc test for multiple comparisons

**Figure 2.. PLX depletes microglia, but does not affect astrocytes, in chimeric mouse hippocampus.**
(A) Representative images of microglia (Iba1, red) in the hippocampus of chimeric mice of each condition. Scale bar, 500 μm. (B) Quantification of number/mm² of Iba1⁺ microglia in the hippocampus. Each dot represents one mouse (hE3-E3KI, n=5; hE3-E3KI-PLX, n=7; hE4-E4KI, n=9; hE4-E4KI-PLX, n=8; hEKO-E4KI, n=4; hEKO-E4KI-PLX, n=6). (C) Representative images of astrocytes (GFAP, red) in the hippocampus of chimeric mice of each condition. Scale bar, 500 μm. (D) Quantification of number/mm² of GFAP⁺ astrocytes in the hippocampus. Each dot represents one mouse (hE3-E3KI, n=5; hE3-E3KI-PLX, n=7; hE4-E4KI, n=9; hE4-E4KI-PLX, n=8; hEKO-E4KI, n=4; hEKO-E4KI-PLX, n=6). All data are expressed as mean ± S.E.M. Differences between groups were determined by two-way ANOVA with Benjamini’s post hoc test for multiple comparisons. Adjusted p-values (q-values) are displayed.

**Figure 3.. Human neuronal APOE isoform and microglial depletion affect Aβ pathology in chimeric mouse hippocampus.**
(A) Representative images of Aβ aggregates within and immediately surrounding human neuronal transplants. Top row: 3D6⁺ Aβ aggregates (green); second row: composite of 3D6⁺ Aβ aggregates (green) and MAP2⁺ (gray) human neuron transplants. Scale bar, 50 μm. (B) Quantification of number of 3D6⁺ Aβ aggregates/μm² within a 100 μm perimeter per transplant area (hE3-E3KI, n=5; hE3-E3KI-PLX, n=6; hE4-E4KI, n=9; hE4-E4-KI-PLX, n=9; hEKO-E4KI, n=4; hEKO-E4KI-PLX, n=6). Unadjusted p-value for comparison between hE4-E4KI and hE4-E4KI-PLX (p = 0.0426) is significant. (C) Representative images of Thioflavin-S⁺ dense-core deposits within and immediately surrounding human neuronal transplants. Top row: Thioflavin-S⁺ dense-core deposits (green); second row: composite of Thioflavin-S⁺ dense-core deposits (green) and MAP2⁺ (gray) human neuron transplants. Scale bar, 50 μm. (D) Quantification of number of Thioflavin-S⁺ dense-core deposits/μm² within a 100 μm perimeter per transplant area (hE3-E3KI, n=5; hE3-E3KI-PLX, n=7; hE4-E4KI, n=9; hE4-E4KI-PLX, n=9; hEKO-E4KI, n=4; hEKO-E4KI-PLX, n=6). Unadjusted p-value for comparison between hE4-E4KI and hE4-E4KI-PLX (p = 0.0318) is significant. (E) Representative images of 3D6⁺ Aβ aggregates (magenta) and Thioflavin-S⁺ dense-core deposits (green) within human neuron transplants. Diffuse Aβ deposits were defined as 3D6⁺/Thioflavin-S⁻ deposits. Scale bar, 100 μm. (F) Representative magnified images of 3D6⁺ Aβ aggregates (magenta, first column), Thioflavin-S⁺ dense-core deposits (green, second column), and composite of Aβ and Thioflavin-S images (third column) showing both dense-core and diffuse Aβ deposits within human neuron transplants. Top row: hE4-E4KI; bottom row: hEKO-E4KI. Scale bar, 20 μm. (G) Quantification of number of diffuse (3D6⁺/Thioflavin-S⁻) Aβ deposits/μm² within a 100 μm perimeter per transplant area (hE3-E3KI, n=5; hE3-E3KI-PLX, n=7; hE4-E4KI, n=9; hE4-E4KI-PLX, n=9; hEKO-E4KI, n=4; hEKO-E4KI-PLX, n=6). Unadjusted p-value for comparison between hEKO-E4KI-PLX and hE4-E4KI-PLX (p = 0.0238) is significant. For all quantifications, values are normalized to the hE4-E4KI condition, and each dot represents one mouse. All data are expressed as mean ± S.E.M. Differences between groups were determined by two-way ANOVA with Benjamini’s post hoc test for multiple comparisons. Adjusted p-values (q-values) are displayed.

**Figure 4.. Human neuronal APOE isoform and microglial depletion affect p-tau deposits in chimeric mouse hippocampus.**
(A) Representative images of p-tau deposits within and immediately surrounding human neuronal transplants. Top row: AT8⁺ p-tau deposits (green); second row: composite of AT8⁺ p-tau deposits (green) and MAP2⁺ (gray) human neuron transplants. Scale bar, 50 μm. (B) Many AT8⁺ p-tau deposits are present within transplanted MAP2⁺ human neurons. Each image represents the same field of view, stained for p-tau (AT8, green, first column), human neuronal transplants (MAP2, gray, second column), and a composite of AT8 and MAP2 (third column). Scale bar, 20 μm. (C) Quantification of number of AT8⁺ p-tau deposits/μm² within a 100 μm perimeter per transplant area (hE3-E3KI, n=4; hE3-E3KI-PLX, n=7; hE4-E4KI, n=8; hE4-E4KI-PLX, n=9; hEKO-E4KI, n=4; hEKO-E4KI-PLX, n=5). Identical adjusted p-values (q-values) displayed in this graph represent true data, with the following unadjusted p-values: hE4-E4KI versus hE3-E3KI (p = 0.0150), hE4-E4KI versus hEKO-E4KI (p = 0.0176), hE4-E4KI versus hE4-E4KI-PLX (p = 0.0068). (D) Representative images of p-tau deposits within and immediately surrounding human neuronal transplants. Top row: PHF1⁺ p-tau deposits (green); second row: composite of PHF1⁺ p-tau deposits (green) and MAP2⁺ (gray) human neuron transplants. Scale bar, 25 μm. (E) Representative magnified image of p-tau deposits (PHF1, green, first column) within human neuronal. Transplants (MAP2, gray, second column), with a composite of PHF1 and MAP2 (third column). Scale bar, 20 μm. (F) Quantification of number of PHF1⁺ p-tau deposits/μm² within a 100 μm perimeter per transplant area (hE3-E3KI, n=5; hE3-E3KI-PLX, n=6; hE4-E4KI, n=9; hE4-E4KI-PLX, n=9; hEKO-E4KI, n=4; hEKO-E4KI-PLX, n=6). Unadjusted p-value for comparison between hE4-E4KI and hE3-E3KI (p = 0.0243) and between hE4-E4KI and hEKO-E4KI (p = 0.0278) are significant. For all quantifications, values are normalized to the hE4-E4KI condition, and each dot represents one mouse. All data are expressed as mean ± S.E.M. Differences between groups were determined by two-way ANOVA with Benjamini’s post hoc test for multiple comparisons. Adjusted p-values (q-values) are displayed.

**Figure 5.. Microglial depletion increases APOE levels within human neuronal transplants.**
(A) Representative images of APOE staining within human neuronal transplants. Each column represents the same field of view, with a composite of APOE (green) with Iba1⁺ microglia (magenta, top row), MAP2⁺ human neuron transplants (magenta, second row), and GFAP⁺ astrocytes (magenta, third row, with magnified image insets showing APOE and GFAP overlap). Scale bar, 50 μm. Scale bar for magnified insets, 25 μm. (B) Quantification of average APOE fluorescence intensity within the human neuron transplants (hE3-E3KI, n=5; hE3-E3KI-PLX, n=7; hE4-E4KI, n=9; hE4-E4KI-PLX, n=8; hEKO-E4KI, n=4; hEKO-E4KI-PLX, n=6). All data are expressed as mean ± S.E.M. Differences between groups were determined by two-way ANOVA with Benjamini’s post hoc test for multiple comparisons. (C–H) Correlations for each condition between % ApoE⁺ transplant area and size of transplants (hE3-E3KI, n=5; hE3-E3KI-PLX, n=7; hE4-E4KI, n=9; hE4-E4KI-PLX, n=8; hEKO-E4KI, n=4; hEKO-E4KI-PLX, n=6). Pearson’s correlation analyses (two-sided). For quantifications, each dot represents one mouse.

**Figure 6.. Pro-inflammatory microglia subpopulations enriched in hE4-E4KI mouse hippocampus.**
(A–E) Feature plots highlighting cells in microglial clusters 1 (pink), 4 (gold), and 12 (blue). (F) Quantification of fraction of cells per condition for clusters 1, 4, and 12. (G–I) Volcano plot of the DEGs between cluster 1 (G), cluster 4 (H), or cluster 12 (I) and all other clusters. Dashed lines represent log₂ fold change threshold of 0.5 and p-value threshold of 10 x 10⁻²⁰. NS, not significant. (J) Dot plot of normalized average expression of selected marker genes for clusters 1, 4, and 12. Dot size is proportional to percentage of cells expressing a given gene.

**Figure 7.. Increased homeostatic microglia in hEKO-E4KI mouse hippocampus and increased pro-inflammatory microglia in hE4-E4KI mouse hippocampus.**
(A) Representative images of homeostatic microglial marker P2ry12 staining within the hippocampus of chimeric mice. First column: P2ry12 (green); second column: microglial marker Iba1 (magenta); third column: composite of P2RY12 and Iba1 images. Scale bar, 100 μm. (B) Quantification of P2ry12, as percent area of hippocampus (hE3-E3KI, n=5; hE4-E4KI, n=9; hEKO-E4KI, n=4). Identical adjusted p-values (q-values) displayed in this graph represent true data, with the following unadjusted p-values: hE3-E3KI versus hEKO-E4KI (p = 0.0128) and hE4-E4KI versus hEKO-E4KI (p = 0.0213). (C) Representative images of pro-inflammatory microglial marker Cd68 staining within the hippocampus of chimeric mice. First column: Cd68 (green); second column: microglial marker Iba1 (magenta); third column: composite of Cd68 and Iba1 images. Scale bar, 100 μm. (D) Quantification of Cd68, as percent area of hippocampus (hE3-E3KI, n=5; hE4-E4KI, n=9; hEKO-E4KI, n=4). Identical adjusted p-values (q-values) displayed in this graph represent true data, with the following unadjusted p-values: hE3-E3KI versus hE4-E4KI (p = 0.0166) and hE4-E4KI versus hEKO-E4KI (p = 0.0226). For quantifications, each dot represents one mouse. All data are expressed as mean ± S.E.M. Differences between groups were determined by one-way ANOVA with Benjamini’s post hoc test for multiple comparisons. Adjusted p-values (q-values) are displayed.

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Update of

Microglia Depletion Reduces Human Neuronal APOE4-Driven Pathologies in a Chimeric Alzheimer's Disease Model.
Rao A, Chen N, Kim MJ, Blumenfeld J, Yip O, Hao Y, Liang Z, Nelson MR, Koutsodendris N, Grone B, Ding L, Yoon SY, Arriola P, Huang Y. Rao A, et al. bioRxiv [Preprint]. 2023 Nov 14:2023.11.10.566510. doi: 10.1101/2023.11.10.566510. bioRxiv. 2023. Update in: Cell Stem Cell. 2025 Jan 2;32(1):86-104.e7. doi: 10.1016/j.stem.2024.10.005. PMID: 38014339 Free PMC article. Updated. Preprint.

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Microglia depletion reduces human neuronal APOE4-related pathologies in a chimeric Alzheimer's disease model

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Microglia depletion reduces human neuronal APOE4-related pathologies in a chimeric Alzheimer's disease model

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