APOE4/4 is linked to harmful fat buildup in brain immune cells in Alzheimer’s disease

Frozen superior frontal gyrus and fusiform gyrus tissue blocks and pathology clinical reports were obtained from the Banner Sun Health Research Institute Brain and Body Donation Program in accordance with institutional review boards and policies at both Stanford School of Medicine and Banner Sun Health Research Institute. All samples obtained from Banner Sun Health Research Institute were stored at −80 °C until the time of processing. Isolation of nuclei from frozen brain tissue: 20–50 mg of flash-frozen human brain tissue isolated from the frontal cortex was thawed in 2 ml of ice-cold homogenization buffer (molecular biology grade water, 260 mM sucrose, 30 mM KCl, 10 mM MgCl, 20 mM tricine-KOH, 1 mM dithiothreitol, 500 μM spermidine, 150 μM spermine, 0.3% NP-40, protease inhibitor, RNAse inhibitor) for 5 min in a prechilled dounce homogenizer. Tissue was dounced with ‘A’ loose pestle for 10 strokes then dounced by 20 strokes with ‘B’ tight pestle. The resulting homogenate was passed through a 70 μm Flowmi strainer into a prechilled 1.5 ml tube. Nuclei were pelleted by centrifugation for 5 min at 4 °C at 350 relative centrifugal force in a fixed-angle centrifuge. All but 50 μl of supernatant (containing cytosolic RNA) was removed by pipetting and nuclei were resuspended in 1× homogenization buffer to a total volume of 400 μl and gently mixed by pipetting and transferred to a prechilled 2 ml protein-lobind tube. A total 400 μl of 50% iodixanol solution (OptiPrep Sigma catalogue no. D1556) was gently mixed by pipetting with 400 μl of resuspended nuclei to make a 25% iodixanol solution. A total 600 μl of a 30% iodixanol solution was gently layered underneath the 25% iodixanol solution containing isolated nuclei, without mixing iodixanol layers. Next, 600 μl of a 40% iodixanol solution was gently layered underneath the 30% iodixanol solution, without mixing iodixanol layers, resulting in a total volume of 2 ml. Samples were centrifuged in a prechilled swinging-bucket centrifuge for 20 min at 4 °C at 3,000 relative centrifugal force with the brake off. After centrifugation, the top 1 ml of the iodixanol layer (25% solution containing myelin and larger debris) was aspirated using a vacuum down to 1 ml total volume, containing the nuclei band. Nuclei were isolated from smaller cellular debris by removal by pipetting of the top 200 μl of the nuclei layer (at a volume between 800 μl and 1 ml in the 30%–40% iodixanol interface). A total 200 μl of isolated nuclei were diluted in 200 μl nuclei wash buffer (phosphate-buffered saline (PBS) with 0.1% BSA and 0.2 U μl⁻¹ of RNase inhibitor. The snRNA-seq libraries were prepared from nuclei using the Chromium Next GEM Single Cell 3′ v.3.1 according to the manufacturer’s protocol (10x Genomics). Nuclei were counted using with a TC20 Automated Cell Counter (Bio-Rad) and loaded into a 10× droplet generator at a concentration of 8,000 nuclei per sample. Thirteen polymerase chain reaction (PCR) cycles were applied to generate complementary DNA and 15 cycles for final library generation. The final snRNA-seq libraries were sequenced on a NovaSeq 6000.

Raw gene counts were obtained by aligning reads to the hg38 genome (refdata-gex-GRCh38-2020-A) using CellRanger software (v.4.0.0) (10x Genomics). To account for unspliced nuclear transcripts, reads mapping to pre-messenger RNA were also counted. All subsequent analysis was implemented in Python (v.3.9.12) based on the Scanpy⁴² (v.1.9.1) single-cell data analysis package, except where stated otherwise. Count data were first screened for doublets with the Scrublet (v.0.2.3) Python package⁴³. Once each cell was doublet scored, we applied a separate doublet score threshold per sample to discard doublets from the data. Thresholds were identified between 0.15 and 0.5 per sample on the basis of the sample-wise doublet score histograms (Extended Data Fig. 1a). We then applied standard filtering rules following the guideline of ref. ⁴⁴. We used the Scanpy (v.1.9.1) package to discard cells with (1) fewer than 500 genes or (2) less than total 1,000 reads or (3) more than 10% mitochondrial reads or (4) more than 10% ribosomal reads. Counts were then counts-per-million (c.p.m). scaled and log-normalized for downstream analysis.

We merged all data across the samples and used standard methods of Scanpy (v.1.9.1) to select the top 2,500 highly variable genes and calculate the top 20 principal components. The previous number of principal components were identified with the elbow method. We then used the Python implementation of BBKNN (v.1.5.1), a fast batch correction algorithm suitable for large datasets to integrate data across the samples⁴⁵. BBKNN calculates a batch-corrected neighbourhood graph from the imputed principal components. We set the individual sample IDs as batch labels to correct for potential sample-wise batch effects. We then used the batch-corrected neighbourhood graph to run Leiden clustering⁴⁶ and to calculate a global UMAP embedding with default parameters in Scanpy (v.1.9.1). We used these embeddings to annotate the cells. Note that BBKNN does not modify the count data in any ways but returns a neighbourhood graph which we use for the noted downstream analyses.

Leiden clusters were annotated one-by-one on the basis of domain-specific expertise. We investigated each Leiden cluster separately on the basis of the expression of common cell-type markers (excitatory neurons, Slc17a7; inhibitory neurons, Gad1; oligodendrocytes, Mog; endothelial cells, Cldn5; astrocytes, Aqp4; microglia, Cd74) and annotated the clusters accordingly. Marker gene expression analysis was implemented in Python (v.3.9.12) and Scanpy (v.1.9.1).

We summed the raw counts per patient sample and hence derived ‘pseudobulk’ samples⁴⁷ from the single-cell counts. We then used Deseq2 (ref. ⁴⁸) to perform bulk data normalization and differential gene expression (DGE) in R (v.4.3). We followed the standard Deseq2 analysis steps and conducted sequencing depth based count normalization across all APOE4/4, APOE3/3 and control samples. Then, we performed DGE analysis at standard parameters. The resulting log-scaled fold changes were shrunken using the standard ‘apeglm’ approach. In every comparison, we discarded genes used for QC filtering (Rb* and Mt-*) from the DGE analysis as these may be biased by the quality-control process. P values were corrected with Benjamini–Hochberg procedure⁴⁹ (false discovery rate (FDR) = 0.05) per comparison.

To conduct unbiased single-cell level downstream analysis of the microglial cells which is not biased towards any of the patient samples, first we subsampled the microglia cluster with purification⁵⁰. We used the first 20 principal components calculated on the basis of the top 2,000 highly variable genes as input and selected 1,000 cells per individual group (control, AD-APOE3/3 and AD-APOE4/4).

We performed DGE analysis on the subsampled data with MAST⁵¹ in R (v.4.3) by using the Seurat package. We set gender, expired age, postmortem interval and the percentage of mitochondrial and ribosomal counts as covariates. We performed (1) pairwise comparisons (three in total) across the three individual groups (control, AD-APOE3/3 and AD-APOE4/4) and (2) ‘one-versus-rest’ comparisons between the three annotated microglial states (HOMEOSTATIC, DAM and LDAM). P values were corrected with Benjamini–Hochberg procedure⁴⁹ (FDR = 0.05) per comparison.

Subsampled microglia data were normalized as described previously^3,52. Briefly, starting from the raw counts, gender, expired age, postmortem interval and the percentage of mitochondrial/ribosomal counts were first regressed out with the regress_out function of the scanpy package (v.1.9.1). We then used the first 20 principal components based on the top 2,000 highly variable genes as input to repeat the Leiden clustering and UMAP visualization steps with default parameters in Scanpy (v.1.9.1) on this corrected microglia subcluster data. To characterize each subcluster, we calculated the mean expression level of marker genes (HOMEOSTATIC—P2RY12↗, P2RY13↗, CX3CR1, TMEM119; DAM—CD9, ITGAX, CLEC7A, CD63, SPP1, LPL, TREM2, APOE; LDAM—NAMPT, ACSL1, DPYD, CD163). We then calculated signature scores by averaging the minimum–maximum normalized expression values of these per cell. We used these signature scores to annotate the subclusters and also mapped them to the UMAP space and scanpy performed kernel density estimation to locate them in the UMAP space (the single-cell landscape).

Adjacent tissue processed for snRNA-seq was subjected to immunohistochemistry and immunofluorescence. Prefrontal cortex from each individual were cut with a razor blade and directly submerged in 4% paraformaldehyde at 4 °C for 24 h. They were then transferred to a 30% sucrose solution in 1× PBS and stored at 4 °C until the tissue sank in its vial. Tissues were then frozen in OCT compound, stored at −80 °C and sectioned on a cryostat (Leica CM3050S). For Oil Red O staining, slides with 10 μm sections of prefrontal cortex were removed from the −80 °C freezer and brought to room temperature for 5 min. The slides were first incubated in propylene glycol for 5 min and then Oil Red O solution from an Abcam Oil Red O stain kit (ab150678) for 2 h at room temperature. The sections were differentiated in 85% propylene glycol in distilled water for 1 min, incubated in haematoxylin for 1 min and rinsed with several changes of distilled water. The slides were sealed with a glass cover slide and ProLong Gold mounting media. Imaging was performed with a ZEISS Axioskop 2 Plus microscope. Oil Red O counts per image were quantified in ImageJ and we performed statistical analysis in Prism9 (Graphpad). For immunofluorescence, free-floating 50 μm sections were washed three times in PBST followed by blocking with 5% donkey serum in PBST for 1 h. Sections were incubated in PBS with 3% donkey serum and the following primary antibodies for 72 h at 4 °C: goat anti-Iba1 (1:500; Abcam, ab5076), rabbit anti-ACSL1 (1:100; Thermo Fisher PA5-78713) and mouse anti-β-amyloid (1:500; Cell Signaling Technologies, no. 15126). After primary antibody incubation, sections were washed three times in PBST and incubated in PBS and the following secondary antibodies for 2 h at room temperature: donkey anti-goat Alexa Fluor 488, donkey anti-rabbit Alexa Fluor 555 and donkey anti-mouse Alexa Fluor 647 (all 1:500; Invitrogen). Sections were incubated in DAPI (1:2,000; Thermo Fisher) for 10 min, then washed three times with PBST. Sections were mounted on microscope slides with ProLong Glass mounting media. Imaging was performed with a ZEISS LSM 900 confocal microscope ZEN 3.0 (Blue Edition) software.

Human APOE3-KI and APOE4-KI mice were cross-bred with mice overexpressing mutant human APP (J20 line) to generate J20/APOE4-KI and J20/APOE3-KI mice, as we reported previously¹⁵. All mouse lines were maintained on a C57Bl/6 J background. Sex- and age-matched wild-type mice were used as controls. Brains were collected from female J20/APOE4-KI and J20/APOE3-KI mice (n = 3 for each group) at 13 months of age. Brain sections were collected (30 μm) from paraformaldehyde-fixed right hemibrains on a sliding microtome fitted with a freezing stage as described previously. Free-floating 30 μm sections were washed three times in PBS followed by blocking with 10% donkey serum in PBS for 1 h. Sections were incubated in PBS with 5% donkey serum and Iba1 primary antibody (1:500; Wako 019-19741) for 72 h at 4 °C. After primary antibody incubation, sections were washed once in PBS and incubated in the following secondary antibody and LD dye for 2 h at room temperature: donkey anti-rabbit 555 (1:500; Invitrogen) and LipidSpot 488 (1:1000; Biotium). Sections were incubated in DAPI (1:2,000; Thermo Fisher) for 10 min, then washed three times with PBS. Sections were mounted on microscope slides with ProLong Glass mounting media. Imaging was performed with a ZEISS LSM 900 confocal microscope ZEN 3.0 (Blue Edition) software. All animal care and procedures complied with the Animal Welfare Act and were in accordance with institutional guidelines and approved by the institutional administrative panel of laboratory animal care at Stanford University.

Isogenic APOE4/4 and APOE3/3 iPS cells were generated as previously described¹⁶. iPS cells were maintained in StemFlex medium (Gibco, A3349401) and routinely passaged as clumps onto Matrigel(Corning, 40230)-coated plates. Differentiation into microglia was performed as previously described^17–19. The iPS cells were first differentiated into haematopoietic progenitor cells (HPCs) following the manufacturer’s instructions using the commercially available STEMdiff Hematopoietic Kit (Stemcell Technologies, 05310). HPCs in suspension were transferred to plates containing an adherent layer of confluent primary human astrocytes (Thermo Fisher, N7805100), in media containing Iscove’s Modified Dulbecco’s Medium (Thermo Fisher), fetal bovine serum (FBS) and penicillin–streptomycin and 20 ng ml⁻¹ of each of IL-3, GM-CSF and M-CSF (PeproTech) for 10 days. The iMG were harvested and transferred into homeostatic culture conditions adapted from ref. ¹⁹ (MGdM media) on Matrigel(Corning, 40230)-coated plates for 5–15 days before assay. For assays involving immunofluorescence for microscopy iMG were plated in homeostatic culture conditions (MGdM media) on fibronectin(StemCell, 07159)-coated plates to enhance cell adherence. All assays were performed under serum-free conditions (MGdM media).

iMicroglia or human macrophages were fixed in 4% paraformaldehyde for 10 min. Cells were washed with PBS followed by blocking with 5% donkey serum in PBS for 1 h. Cells were incubated in PBS with 3% donkey serum and the following primary antibodies overnight at 4 °C: rabbit anti-Perilipin 2 (1:200; Proteintech 15294-1-AP), rabbit anti-ACSL1 (1:100; Thermo Fisher PA5-78713), LC3B (1:300, Thermo Fisher Scientific, PA1-46286) and mouse anti-β-amyloid (1:500; Cell Signaling Technologies, no. 15126). After primary antibody incubation, cells were washed with PBS and incubated in PBS and the following secondary antibodies for 2 h at room temperature: donkey anti-rabbit Alexa Fluor 488, donkey anti-rabbit Alexa Fluor 555 and donkey anti-mouse Alexa Fluor 647 (all 1:500; Invitrogen). Sections were incubated in DAPI (1:2,000; Thermo Fisher) for 10 min, then washed three times with PBS. Cells grown and stained on coverslips were then mounted on glass microscope slides with ProLong Glass mounting media. Imaging was performed with a ZEISS LSM 900 confocal microscope ZEN 3.0 (Blue Edition) software. Additionally, cells were fixed in replicate wells of a 96-well plate and were imaged and quantified with Incucyte S3 analysis system (Essen Bioscience). For live cell microscopy, cells were untreated or treated with 5 μM Aβ fibrils or 10 μM GNE-317 (Selleckchem, S7798) for 24 h. After 24 h, media were changed and cells were incubated with LipidSpot 488 (biotium, 70065-T) in combination with pHrodo Red Zymosan Bioparticles (P35364↗, Thermo Fisher) or Lysotracker (L12492↗, Thermo Fisher), according to manufacturer’s instructions. For Aβ fibril formation, monomeric HFIP-treated Aβ protein (1–42) (Bachem catalogue no. 4090148) was formed into fibrils as previously described⁵³. Four phase-contrast, green and red fluorescent images per well were acquired every 1 h for 36 h using an Incucyte S3 analysis system (Essen Bioscience). For each time point, red and green fluorescence was normalized to the phase confluence per well.

Whole blood was obtained from Stanford Blood Center. Peripheral blood mononuclear cells isolation was performed by diluting sample in PBS (equivalent blood volume) and transferred on top of the Ficoll layer (GE Healthcare catalogue no. 17-5442-02). The tubes were then centrifuged at 400g, at room temperature, acceleration (slow) and brake (slow) for 30 min. After centrifugation, the upper layer was discarded and the peripheral blood mononuclear cells layer at the interphase was collected in a fresh 50 ml Falcon tube. The cells were washed twice with PBS and counted. Monocytes were then isolated using the Pan Monocyte Isolation Kit (Miltenyi Biotec catalogue no. 130-096-537) according to manufacturer’s instructions. Monocytes were cultured in X-VIVO 15 serum-free media (Lonza) and differentiated into macrophages with 20 ng ml⁻¹ of M-CSF (Peprotech).

Cells from the mouse microglial BV2 cell line were originally obtained from Banca Biologica e Cell Factory, IRCCS Azienda Ospedaliera Universitaria San Martino, Italy. Cells were maintained in DMEM (Life Technologies) supplemented with 5% FBS and antibiotics (penicillin 100 U ml⁻¹, streptomycin 100 U ml⁻¹ (Gibco), 10 mM glutamax (Gibco) under standard culture conditions (95% relative humidity with 5% CO₂ at 37 °C). Adherent cells were split using 1× TrypLE (Gibco). Experiments with deuterated lipids were conducted by replacing glucose with 10 mM d-glucose (U-13C6, Cambridge isotope laboratories CLM-1396-PK) for 24 h before treatment with 5 μM Aβ fibrils or no treatment.

Primary rat microglia were cultured as previously described⁵⁴. P10-20 rats were transcardially perfused with ice-cold PBS and, immediately after perfusion, brains were rapidly dissected and placed into ice-cold PBS. Brain material was minced and transferred to an ice-cold dounce homogenizer (Wheaton) with ice-cold PBS containing 200 μl of 0.4% DNaseI per 50 ml of PBS. Tissue chunks were subjected to three successive rounds of three to ten gentle strokes of the homogenizer piston and centrifuged. Percoll PLUS was added during centrifugation to separate myelin from cells. The cell suspension volume was adjusted to 33.4 ml with PBS and 10 ml of 100% isotonic Percoll (9 ml of Percoll PLUS (GE Healthcare), 1 ml of 10× PBS without Ca and Mg, 9 μl of 1 M CaCl₂, 5 μl of 1 M MgCl₂) was added and thoroughly mixed (23% isotonic Percoll final). Suspensions were centrifuged (15 min, 500g, 4 °C) and the supernatant and top layer of myelin were discarded. Rat CD11b/c (Microglia) MicroBeads (Miltenyi, catalogue no. 130-105-643) were used to enrich rat microglia using MACS columns according to manufacturer’s instructions. Serum-free rat microglia basal microglial growth medium was sterile-filtered and stored at 4 °C for up to 1 month and was comprised of: DMEM/F12 containing 100 U ml⁻¹ of penicillin, 100 μg ml⁻¹ of streptomycin, 2 mM glutamine, 5 μg ml⁻¹ of N-acetyl cysteine, 5 μg ml⁻¹ of insulin, 100 μg ml⁻¹ of apo-transferrin and 100 ng ml⁻¹ of sodium selenite, ovine wool cholesterol (1.5 μg ml⁻¹, Avanti Polar Lipids), heparan sulfate (1 μg ml⁻¹, Galen Laboratory Supplies). The final medium was comprised of basal media containing human TGFβ2 (2 ng ml⁻¹, Peprotech), mouse IL-34 (100 ng ml⁻¹, R&D Systems). Cells were plated on 24-well plates coated with poly-d-lysine (Gibco). Cells were grown in a humidified incubator held at 37 °C and 10% CO₂. The 50% medium changes were performed every 2–3 days. For live cell microscopy, cells were untreated or treated with 5 μM Aβ fibrils (as described above) pre-incubated with an Aβ antibody (Cell Signaling Technologies, no. 15126) conjugated to an Alexa Fluor 555 secondary (Invitorogen), along with LipidSpot 488 (Biotium, 70065-T) and imaged for 24 h. All animal care and procedures complied with the Animal Welfare Act and were in accordance with institutional guidelines and approved by the institutional administrative panel of laboratory animal care at Stanford University.

The cell cultures, adherent to circular cover glasses, were positioned on secondary no. 1.5H cover glasses (Thorlabs) with a thin layer of PBS. To circumvent interference with lipid CARS signal generation, neither mounting media, antifade agents nor surfactants were used. The edges of the cover glass were sealed with VALAP, a homogeneous blend of equal parts petroleum jelly, lanolin and paraffin, which served to prevent sample desiccation.

Intracellular lipids in iPS cell-derived cells were visualized and quantified using coherent anti-Stokes Raman scattering (CARS) microscopy. An inverted microscope (Nikon, Ti2-E equipped with a C2 confocal scanning head and a Nikon CFI Apochromat TIRF 100XC oil immersion objective) was used for this purpose. The C2 scanner was retrofitted with a slidable mirror (Optique Peter), allowing for a convenient switch between fluorescence excitation using the laser diodes (at wavelengths 405, 488, 561 and 647 nm) and CARS excitation. In CARS imaging mode, carbon–hydrogen (C–H) vibrations were coherently driven by temporally and spatially overlapping two near-infrared laser beams, generated by a picosecond-pulsed laser system (APE America, picoEmerald S with 2 ps pulse length, 80 MHz repetition rate and 10 cm⁻¹ bandwidth) consisting of a 1,031 nm mode-locked ytterbium fibre laser and an optical parametric oscillator (OPO) tunable between 700 and 960 nm (pumped by the second harmonic of the 1,031 nm laser). The OPO wavelength was set to 797 nm to drive the symmetric stretching vibration of CH₂ at 2,850 cm⁻¹. The quadratic dependence of the CARS signal on the number density of the probed C–H vibrational group rendered sharp contrast for the lipid-dense regions without requiring external labels or disruptive sample preparations. The CARS signal generated by simultaneously scanning the two excitation beams over the sample was detected pixel-by-pixel with a photomultiplier tube (Hamamatsu, R6357) in the forward direction with optical filters which minimized background signals (Semrock; two FF01-640-20 and one FF01-750/SP). The excitation powers at the sample position were 18 mW for the pump (OPO) beam and 15 mW for the Stokes (1,031 nm) beam. For the iMG-like cells, 5–16 image stacks were acquired for each APOE genotype and Aβ treatment condition. Each stack comprised 19 slices at a resolution of 1,024 × 1,024 pixels (77.14 × 77.14 μm²) per image with a dwell time of 10.8 μs per pixel. Slices were spaced 0.4 μm apart, yielding a total imaging depth of 7.2 μm. Cell-specific immunohistochemistry facilitated the identification of the microglia (IBA1) with confocal fluorescence, which was collected in the same positions as the nonlinear imaging with a dwell time of 5.3 μs per pixel. The microglia were stained with Alexa Fluor 488. For selected APOE/treatment combinations, a CARS spectrum was acquired in the C–H stretching region for one field of view by varying the OPO wavelength between 785.5 and 801 nm, with 0.5 nm intervals per acquisition, amounting to a total of 32 image stacks making up the spectrum. All images were analysed using the Fiji distribution package of ImageJ. The CARS stacks underwent east shadows correction, Gaussian blur filtering (σ = 1) and background subtraction (30 pixel rolling ball radius). Lipid particles were identified by thresholding using the FindFoci method, with a search parameter for half peak value of 0.1, peak parameter for minimum peak height relative to background of 0 and a minimum peak size of 200 pixels. Cells were identified by thresholding with the Triangle method. Intracellular lipid features were quantified using the ‘3D Objects Counter’ command. To generate spectral line plots, the spectra from all individual intracellular lipid features in each cell were normalized and averaged.

APOE4/4 iMG were treated with 5 μM Aβ fibrils stained with LipidSpot 488 as described above then top 10% highest and 10% lowest LipidSpot fluorescent cells were seeded into 96-well plates at 5,000 cells per well by FACS (Sony, MA900) in 100 μl of MGdM media for 24 h. Media supernatant was collected and secreted signalling proteins were measured in culture supernatants by the Human Immune Monitoring Center at Stanford University using a Human 48-plex Luminex Procarta Immuno-assay (Thermo Fisher).

Approximately 100,000 iMG were pelleted at 300g and 4 °C. Next, pellets were gently resuspended in ice-cold 50 μl of lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% IGEPAL CA-630) and spun down at 500g for 10 min and 4 °C. The supernatants were discarded and pellets gently resuspended in 50 μl of transposition reaction mix (25 μl of tagment DNA buffer (Nextera, Illumina), 2.5 μl of tagment DNA enzyme (Nextera, Illumina), 22.5 μl of nuclease free water) and incubated at 37 °C for 30 min. Tagmented DNA was purified using MinElute PCR purification kit (Qiagen) and size selected for 70–500 base pairs (bp) using AmpureXP beads (Beckman Coulter). Libraries were constructed and amplified using 1.25 μM Nextera index primers and NEBNext High-Fidelity 2× PCR Master Mix (New England BioLabs). A quantitative PCR was run to determine the optimal number of cycles. Libraries were gel size selected for 165–300 bp fragments and single-end sequenced for 100 cycles (PE100) on an Illumina NovaSeq 6000.

Bowtie2 with default parameters was used to map ATAC-seq. HOMER was used to convert aligned reads into ‘tag directories’ for further analysis⁵⁵. ATAC-seq experiments were performed in replicate and peaks were called with parameters -L 0 -C 0 -fdr 0.9 -minDist 200 -size 200. Irreproducible discovery rate (IDR) was used to test for reproducibility between replicates. IDR peaks were merged using HOMER mergePeaks and annotated with HOMER annotatePeaks.pl with a size parameter of 250. Differential enhancer peaks (±3 kilobases from transcription start site) were identified using DESeq2 with FC > 1 and adjusted P < 0.05. HOMER motif analysis (findMotifsGenome.pl) including known default motifs and de novo motifs was used to identify motifs enriched in enhancer peak regions over background. The background sequences were from random GC-matched genome sequences. The UCSC genome browser was used to visualize ATAC-seq data.

For Aβ-treated conditions, iMG were exposed to 5 μM Aβ fibrils for 24 h or DMSO before RNA isolation. For LD-high versus LD-low conditions, iMG were exposed to 5 μM Aβ fibrils for 24 h, stained with LipidSpot 488 as described above and top 10% highest and 10% lowest LipidSpot fluorescent cells were separated by FACS (Sony, MA900). iMicroglia RNA was isolated from the cell pellets using an RNeasy Plus Micro kit (Qiagen, 74034). The mRNA was transcribed into full-length cDNA using a SMART-Seq v.4 Ultra Low Input RNA kit (Clontech) according to the manufacturer’s instructions. Full-length cDNA (150 pg) was processed using a Nextera XT DNA library preparation kit (Illumina) according to the manufacturer’s protocol. Library quality was verified using the Agilent 2100 Bioanalyzer and the Agilent High Sensitivity DNA kit. Sequencing was carried out using an Illumina NexSeq 550, paired-end, 2× 100 bp depth sequencer. Reads were mapped to the human hg38 reference genome using STAR (v.2.5.1b). Raw read counts were generated with STAR using the GeneCounts function. Differential expression in RNA-seq was analysed using the R package DESeq2 (ref. ⁴⁸).

Cells were grown on aclar and then fixed in Karnovsky’s fixative: 2% glutaraldehyde (EMS catalogue no. 16000) and 4% paraformaldehyde (EMS catalogue no. 15700) in 0.1 M sodium cacodylate (EMS catalogue no. 12300) pH 7.4 for 1 h, chilled and sent to Stanford Cell Sciences Imaging Facility on ice. They were then postfixed in cold 1% osmium tetroxide (EMS catalogue no. 19100) in water and allowed to warm for 2 h in a hood, washed 3× with ultrafiltered water, then bloc stained 2 h in 1% uranyl acetate at room temperature. Samples were then dehydrated in a series of ethanol washes for 10 min each at room temperature beginning at 30%, 50%, 70%, 95%, changed to 100% 2X, then propylene oxide for 10 min. Samples are infiltrated with EMbed-812 resin (EMS catalogue no. 14120) mixed 1:1 and 2:1 with propylene oxide for 2 h each. The samples are then placed into EMbed-812 for 2 h opened then placed into flat moulds with labels and fresh resin and placed into 65 °C oven overnight. Sections were taken around 90 nm, picked up on formvar/carbon-coated Cu grids, stained for 40 s in 3.5% uranyl acetate in 50% acetone followed by staining in Sato’s Lead for 2 min. These were observed in the JEOL JEM-1400 120 kV and photos were taken using a Gatan Orius 2k × 4k digital camera.

U937 cells were acquired from the American Type Culture Collection (CRL-1593.2). Cells were maintained in suspension culture using spinner flasks for library propagation and tissue culture plates for single-gene knockout lines, all in sterile-filtered U937 growth medium (RPMI-1640 supplemented with 2 mM glutamine, 100 U ml⁻¹ of penicillin, 100 mg ml⁻¹ of streptomycin and 10% heat-inactivated FBS. Cells were cultured in a humidified 37 °C incubator set at 5% CO₂. A ten-sgRNA-per-gene CRISPR–Cas9 deletion library (Human CRISPR knockout library was a gift from M. Bassik (Addgene no. 101926-101934)) was infected into Cas9-expressing U937 cells as described⁵⁶. Briefly about 300 million U937 cells stably expressing SFFV-Cas9-BFP were infected with the ten-guide-per-gene genome-wide sgRNA library at a multiplicity of infection less than one. Infected cells underwent puromycin selection (1 μg ml⁻¹) for 5 d, after which puromycin was removed and cells were resuspended in normal growth medium without puromycin. After selection, sgRNA infection was measured as more than 90% of cells as indicated by measuring mCherry-positive cells with flow cytometry. For the LD screen, the 10% FBS in the media was replaced with 10% aged (more than 80 years of age) human plasma pooled from several donors, dialysed and delipidated to better represent the aged circulating monocyte environment. U937s were then stained with BODIPY 493/503 (1:2,000 from 1 mg ml⁻¹ of stock solution in DMSO; Thermo Fisher) and top 10% highest and 10% lowest BODIPY fluorescent cells were separated by FACS (Sony, MA900). Screens were performed in duplicate. At the end of each screen genomic DNA (gDNA) was extracted for all screen populations separately according to the protocol included with QIAGEN Blood Maxi Kit. Using known universal sequences present in the lentivirally incorporated DNA, sgRNA sequences were amplified and prepared for sequencing by two sequential PCR reactions as described¹⁶. Products were sequenced using an Illumina Nextseq to monitor library composition (30–40 million reads per library). Trimmed sequences were aligned to libraries using Bowtie, with zero mismatches tolerated and all alignments from multimapped reads included. Guide composition and comparisons across bound and unbound fractions were analysed using casTLE⁵⁷ v.1.0. Enrichment of individual sgRNAs was then calculated as a median-normalized log ratio of the fraction of counts, as described⁵⁷. For each gene, a maximum likelihood estimator was used to identify the most likely effect size and associated log-likelihood ratio (confidence score) by comparing the distribution of gene-targeting guides to a background of non-targeting and safe-targeting guides.

iMG CRISPR–Cas9 screens were performed using modified sgRNA lentiviral infection with Cas9 protein electroporation (SLICE) approach⁵⁸. The human CRISPR knockout library was a gift from M. Bassik (Addgene no. 101927). In brief, about 40 million APOE4/4 iPS cells were lentiviral infected with the ten-guide-per-gene sgRNA sublibraries at a multiplicity of infection less than one. Infected cells underwent puromycin selection (0.8 μg ml⁻¹) for 2 d after which point puromycin was removed and cells were resuspended in normal growth media without puromycin. iPS cells were differentiated into iMG as described above. At day 15 of iMG–human astrocyte coculture, iMG cells were pelleted and resuspended in Lonza electroporation buffer P3 (Lonza, V4XP-3032) at 5 million cells per 100 ml. Cas9 protein (MacroLab, 40 mM stock) was added to the cell suspension at a 1:10 v/v ratio. Cells were electroporated at 5 million cells per 100 ml of cells per cuvette using a Lonza 4-D nucleofector with pulsecode DP-148 (Lonza, VVPA-1002). Cells were cocultured for five more days with human astrocyte before transferring cells to homeostatic culture conditions (MGdM media) as described above. In duplicate culture conditions, cells were treated with 5 μM Aβ fibrils for 24 h, stained with BODIPY 493/503 (1:2,000 from 1 mg ml⁻¹ of stock solution in DMSO; Thermo Fisher) and top 10% highest and 10% lowest BODIPY fluorescent cells were separated by FACS (Sony, MA900). The gDNA was extracted for all populations separately using a QIAGEN Blood Midi Kit, sgRNA sequences were amplified by PCR using common flanking primers and indices and adaptors were attached to amplicons in a second PCR. Deep sequencing of sgRNA sequences on an Illumina Nextseq550 was used to monitor library composition. Guide composition was analysed and compared to the plasmid library and between conditions using casTLE⁵⁷. The enrichment of individual guides was calculated as the log ratio between LD-high and LD-low populations and gene-level effects were calculated from ten guides targeting each gene. P values were then calculated by permutating the targeting guides as previously described⁵⁷.

Human induced pluripotent stem (hiPS) cells were derived into neurons as previously described¹⁶, with slight modifications to increase yield. The hiPS cells were dissociated with Accutase followed by quenching with warm (37 °C) N2B27 medium. N2B27 medium consisted of 1:1 DMEM/F12 (11330032, Thermo Fisher) and Neurobasal Media (21103049, Thermo Fisher), 1% N2 Supplement (21103049, Thermo Fisher), 1% B27 (17504044, Thermo Fisher), 1% MEM non-essential amino acids (11140050, Thermo Fisher), 1% Glutamax (35050061, Thermo Fisher) and 0.5% penicillin–streptomycin (15140122, Thermo Fisher). Dissociated hiPS cells were then pelleted and resuspended in embryoid body media (10 µM SB431542 (1614, Tocris) and 0.25 µM LDN (04-0074, Stemgent) in N2B27) with 10 µM ROCK inhibitor (1254, Tocris), followed by growth in suspension in a T-75 flask (12-565-349, Fisher Scientific). For the first 3 h, the flasks were shaken manually once per hour. On days 2, 4 and 6, the media were replaced with fresh embryoid body medium without ROCK inhibitor. On day 8, spheres were plated as neural progenitors onto a 10 cm dish precoated with growth factor-reduced (GFR) Matrigel (CB-40230A, Fisher Scientific). Neural progenitors were allowed to form neuronal rosettes and sustained in N2B27 media alone for days 8–15. During this period, half of the media were replaced every 48–72 h, depending on confluency and media consumption. On day 16, the neuronal rosettes were lifted using STEMdiff Neural Rosette Selection Reagent (05832, StemCell Tech) and plated onto three wells of a six-well plate precoated with GFR Matrigel in N2B27 with 100 ng ml⁻¹ of FGFb (100-18B, Peprotech) and 100 ng ml⁻¹ of EGF (AF-100-15, Peptrotech). This N2B27 medium with FGFb and EGF was replaced daily. On day 20, the neural progenitors were passaged by dissociating with Accutase, quenching with N2B27 and resuspending in STEMdiff Neural Progenitor Medium (05833, StemCell Tech) at 1.2 × 10⁶ cells per 2 ml for one well of a six-well plate, precoated with GFR Matrigel. For days 21–27, neural progenitor cells were fed with fresh Neural Progenitor Medium every day. On day 28, the neural progenitor cells were dissociated with Accutase, N2B27 media was added to bring the volume of cell suspension to 40 ml and cells were filtered through a 40 µm cell strainer (08-771-1, Fisher). Cells were then collected by centrifugation and resuspended in complete neuronal medium. Neuronal media consisted of 10 ng ml⁻¹ of BDNF (450-02, Peprotech) and 10 ng ml⁻¹ of GDNF (450-10, Peprotech) in N2B27 with 10 nM DAPT (2634, Tocris). Next, the cells were counted and plated at a concentration of 2 × 10⁵ cells per well onto 12 mm coated glass coverslips (354087, Corning) in a 24-well plate. Coverslips were coated with poly-l-lysine (P4707, Sigma-Aldrich) and mouse-Laminin (23017015, Gibco) before plating. Then 50% of culture medium was replaced on maturing neurons every 3–4 days. DAPT was removed after the first week. Experiments were performed on neuronal cultures that had been differentiated for 4 weeks.

LDAM-high and LDAM-low conditioned media from 1 million APOE4/4, APOE3/3 and APOE-KO iMG were prepared by exposing iMG to 5 μM fAβ for 24 h, washed 3× with PBS, stained with LipidSpot 488 as described above then top 10% highest and 10% lowest LipidSpot fluorescent cells were sorted by FACS (Sony, MA900) and grown in N2B27 medium (as described above) supplemented with 100 ng ml⁻¹ of IL-34, 10 ng ml⁻¹ of CSF1 and 10 ng ml⁻¹ of TGFβ for 12 h. Conditioned media were collected by centrifuging media supernatant at 2,000g for 10 min to remove cell debris. The 10% of the total volume from the iMG-conditioned media was added to fresh N2B27 with 10 ng ml⁻¹ of BDNF (450-02, Peprotech) and 10 ng ml⁻¹ of GDNF (450-10, Peprotech) before treating neurons. To treat neurons with the various LDAM media, N2B27 neuronal media were completely removed from the cells and replaced with the iMG-conditioned media containing GDNF and BDNF. After 48 h, the neurons were fixed for immunocytochemistry.

Neurons were washed with 1× DPBS (14080055, Gibco) and fixed in 4% paraformaldehyde for 15 min. The neurons were then washed three times for 5 min with 1× DPBS (14080055, Gibco) containing 0.1% Tween. Next, the neurons were permeabilized and blocked with a 1 h wash in 1× DPBS (14080055, Gibco) containing 10% Normal Donkey Serum (017000121, Jackson Immuno) and 0.5% Triton-X. Cells were then stained with primary antibodies overnight at 4 °C targeting the following proteins: MAP2 (PA1-10005, Thermo Fisher Scientific, 1:5,000), AT8 (MN1020, Thermo Fisher Scientific, 1:500) and caspase-3 (9661, Cell Signaling Technology, 1:500). The secondary antibodies were IgG conjugated to Alexa Fluor 488 (donkey anti-mouse, A-21202, Life Technologies Corporation, 1:1,000), Alexa Fluor 594 (donkey anti-rabbit, A-21207, Life Technologies Corporation, 1:1,000) and Alexa Fluor 647 (donkey anti-chicken, 703-605-155, Jackson Immuno, 1:500). Coverslips were mounted to microscope slides with VECTASHIELD Prolong Gold with DAPI (H-1200-10, Vector Labs). Images were taken with a FV3000 confocal laser scanning microscope (Olympus) at ×20 or ×40. Image analysis to quantify AT8 or caspase-3 positivity in hiPS cell-derived neuron stains was performed using custom macros written in the open-source Fiji (ImageJ) software. For all image analyses, a standard threshold value was chosen and automatically applied to each channel of each image before measurement. For quantification of AT8 immunofluorescence, the total area of AT8 immunofluorescence was normalized to the total area of MAP2 immunofluorescence. For quantification of caspase-3 immunofluorescence, the total count of caspase-3 positivity was normalized to the total count of DAPI positivity.

Newborn mouse pups on the first day after birth were humanely killed through decapitation and their cortices were carefully collected using microdissection techniques. The freshly obtained cortices were then rinsed with a dissection medium (Thermo Fisher Scientific, 14170161) before being subjected to tissue dissociation using scissors, 0.25% trypsin and pipette trituration. Cell strainers with a pore size of 70 µm were used to remove any remaining tissue fragments from the digested cortices. The dissociated neurons were then plated onto 24-well plates with coverslips coated with poly-l-lysine (Newcomer Supply, 1339 A) using minimum essential medium (Thermo Fisher Scientific, 21010046) supplemented with 10% inactivated fetal calf serum (Thermo Fisher Scientific, 10438026), 2 mM glutamine and penicillin and streptomycin (Thermo Fisher Scientific, SV30010). After 24 h of initial seeding, a complete medium change was performed using neurobasal medium (Thermo Fisher Scientific, 21103049) supplemented with B27 (Invitrogen, 17504044) and 2 mM GlutaMax (Thermo Fisher Scientific, 35050061). The primary neuronal cells were maintained at a temperature of 37 °C with a 5% CO₂ environment. A detailed protocol is described in ref. ⁵⁹.

Dried lipids were reconstituted by a buffer consisting of 50 μl of ACN:IPA:water in a ratio of 13:6:1 (v/v/v). The mixture was then vigorously mixed using a vortex for a duration of 10 min at 4 °C. Afterward, the samples were centrifuged at maximum speed for 10 min at 4 °C. A volume of 45 μl of the supernatant was then carefully transferred into glass insert vials for further analysis using liquid chromatography–mass spectrometry.

Lipid profiling was conducted using an ID-X tribrid mass spectrometer equipped with a heated electrospray ionization probe. C18-based lipid separation was performed using an Ascentis Express C18 column coupled with a guard column. The mobile phases consisted of ammonium formate and formic acid dissolved in water, acetonitrile and 2-propanol. The mass spectrometer parameters, including temperatures, resolutions, voltages and gas settings, were optimized for lipid analysis. HCD fragmentation and data-dependent tandem mass spectrometry were used for comprehensive lipid identification. LipidSearch and Compound Discoverer software were used for unbiased differential analysis and lipid annotation. Metabolite abundance was accurately quantified using TraceFinder (Thermo Fisher Scientific). Mass tolerance of 5 ppm was applied for the extraction of ion chromatograms, ensuring accurate measurement of lipid concentrations. The full detail of the method can be found in ref. ⁶⁰.

Further information on research design is available in thelinked to this article. Nature Portfolio Reporting Summary

Reporting SummarySupplementary Table 1Metadata for brain tissue used in single-nucleus RNA-seq.Supplementary Table 2DEG and KEGG enrichment analyses of microglia from single-nucleus RNA-seq of brain tissue.Supplementary Table 3Genome-wide CRISPR-KO screen analysis for LD levels.Supplementary Table 4iMG bulk RNA-seq DEG analyses.Supplementary Table 5ATAC-seq analysis comparing LD⁺ and LD⁻ iMGs.Supplementary Table 6Drug target, kinase, phosphatase CRISPR-KO screen analysis comparing LD⁺ and LD⁻ iMGs.Supplementary Table 7Lipidomics analysis comparing neurons ± MG LD conditioned media.