Journal: Advanced Science

Article Title: Lilrb4a Suppression Reprograms Microglia to Mitigate APOE4‐Associated Amyloid Plaques and Cerebral Amyloid Angiopathy in Association With a PPAR‐Linked Pro‐Clearance State

doi: 10.1002/advs.202524167

Figure Lengend Snippet: LILRB4 shows stronger APOE4‐associated Co‐IP signals in the tested contexts and is upregulated in APOE4 and amyloid‐related settings. (A,B) Reciprocal Co‐IP of human APOE4 with Flag‐tagged human LILRB3 (A) or Flag‐tagged human LILRB4 (B) in HEK293T cells. Schematic representation of Co‐IP between LILRB3/LILRB4 and APOE4 protein. Immunoprecipitation with anti‐APOE or anti‐Flag antibodies, followed by immunoblotting for APOE4 and LILRB3/LILRB4. Input lysates are shown as positive controls, and IgG immunoprecipitation serves as a negative control; (C, D) Reciprocal Co‐IP of endogenous APOE and LILRB4 in microglia isolated from 12‐month‐old APOE4 (C) or APOE3 (D) mice 24 h after intraperitoneal LPS injection. Cell lysates were immunoprecipitated with anti‐APOE or anti‐LILRB4 antibodies and immunoblotted as indicated. Input lysates are shown as positive controls, and IgG immunoprecipitation serves as a negative control; (E) Representative Lilrb4a immunostaining in cortical sections from LPS‐treated APOE3 and APOE4 mice (10×, scale bar = 500 µm); (F) Quantification of Lilrb4a‐positive area in the cortex ( n = 4–5); (G) Schematic diagram of the experimental design; (H) Representative confocal images of X‐34 (blue), APOE (red), and IBA1 (green) staining in cortical sections from 5EL and 5ELKO mice; arrows indicate plaque‐associated microglia containing APOE signal (60× oil, scale bar = 50 µm); (I) Quantification of APOE‐positive area within IBA1‐positive microglia surrounding plaques (APOE/IBA1 area) ( n = 9–12); (J, K) Lilrb4a mRNA expression was analyzed by qPCR in cortical tissues from C57 and 5xFAD mice of different ages ( n = 5–8); Unless otherwise specified, experiments not shown as sex‐separated in the figures were performed using male mice only (Applicable to all figure legends). Data are presented as mean ± standard error of the mean (SEM). Each dot represents one mouse. Statistical significance was determined by unpaired two‐tailed Student's t test (F), one‐way ANOVA (J,K), or two‐way ANOVA (I), as appropriate. * p <0.05, ** p <0.01.

Article Snippet: For phagocytosis assays in sorted primary microglia from APOE3 and APOE4 mice, cells were treated with ASO‐L4a or control ASO (5 pmol per 1 × 10 4 cells) for 36 h, followed by an additional 24 h in medium containing either a PPAR‐γ agonist (MCE, HY‐13956, 20 μ m ) or a PPAR‐γ inhibitor (MCE, GW9662, 10 μ m ).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Negative Control, Isolation, Injection, Immunostaining, Staining, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: Lilrb4a Suppression Reprograms Microglia to Mitigate APOE4‐Associated Amyloid Plaques and Cerebral Amyloid Angiopathy in Association With a PPAR‐Linked Pro‐Clearance State

doi: 10.1002/advs.202524167

Figure Lengend Snippet: Exploratory bulk RNA‐seq nominates a PPAR‐related signature, and PPAR‑γ activation phenocopies clearance‐associated outcomes. (A) Volcano plot of bulk RNA‐seq data from thalamic tissue of 5EL and 5ELKO mice; colored points indicate genes meeting an exploratory threshold of nominal p <0.05 and absolute fold change >1.2; (B) KEGG pathway enrichment analysis of transcriptomic alterations in 5ELKO versus 5EL mice; (C) qPCR validation of selected genes related to the PPAR signaling pathway, including Apoa1 , Fabp7 , Pck1 , Plin2 , Apoa2 , Pparg , Cyp2e1 in thalamus from 5EL and 5ELKO mice ( n = 4–5); (D) Representative RNAscope images of Pparg mRNA in brain sections from 5EL and 5ELKO mice, combined with X‐34 and IBA1 co‐staining (left) and Imaris‐based reconstruction (right). White arrows indicate PPAR‐γ‐positive signals within microglia (60× oil, scale bar = 20 µm); (E) Quantification of Pparg RNAscope signal volume within plaque‐associated microglia in 5EL and 5ELKO mice; (F) Representative Incucyte images of pHrodo‐labeled Aβ uptake in BV2 cells treated with vehicle or a PPAR‐γ agonist at 0 h and 1.5 h; (G) Quantification of the phagocytosis rate in BV2 cells under the indicated agonist or inhibitor treatments, expressed as the percentage of pHrodo‐positive area relative to total cell area ( n = 3); (H) qPCR analysis of Lilrb4a and Pparg expression in BV2 cells treated with vehicle or a PPAR‐γ agonist ( n = 3); (I) Schematic diagram of the FAM‐labeled Aβ phagocytosis and degradation assays in BV2 cells; (J) Representative flow cytometry histograms showing intracellular Alexa‐488 fluorescence in BV2 cells after 1.5 h phagocytosis and after 3 h loading followed by 24 h degradation under the indicated PPAR‐γ agonist or inhibitor conditions; (K) Quantification of mean intracellular Alexa‐488 fluorescence in BV2 cells after 1.5 h phagocytosis (left) and after 24 h degradation (right) ( n = 3); (L) Representative flow cytometry histograms showing intracellular Alexa‐488 fluorescence in sorted primary microglia from LPS‐treated APOE4 mice under the indicated ASO and PPAR‐γ inhibitor conditions. Left, phagocytosis assay, in which cells were incubated with FAM‐Aβ and analyzed after 1.5 h to assess uptake. Right, degradation assay, in which cells were first allowed to internalize FAM‐Aβ for 3 h, then switched to substrate‐free medium, and analyzed 24 h later to assess residual intracellular signal; (M) Quantification of mean intracellular Alexa‐488 fluorescence in APOE4 microglia in the phagocytosis (left) and degradation (right) assays ( n = 3); (N,O) Corresponding representative histograms and quantification for sorted primary microglia from LPS‐treated APOE3 mice under identical assay conditions and treatment paradigms, with phagocytosis measured at 1.5 h and degradation measured 24 h after 3 h loading with FAM‐Aβ ( n = 3); (P) Representative immunofluorescence images of WT primary neuron–microglia co‐cultures treated with vehicle‐ or PPAR‐γ agonist‐treated microglia, showing DAPI (blue), MAP2 (green), and IBA1 (red) (10×, scale bar = 200 µm); (Q) Quantification of neuronal survival in co‐culture, measured as MAP2‐positive area ( n = 8–11). Data are presented as mean ± SEM. Statistical significance was determined by unpaired two‐tailed Student's t test (C, E, H, and Q) or one‐way ANOVA (G, K, M, and O), as appropriate. ns, not significant. * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: For phagocytosis assays in sorted primary microglia from APOE3 and APOE4 mice, cells were treated with ASO‐L4a or control ASO (5 pmol per 1 × 10 4 cells) for 36 h, followed by an additional 24 h in medium containing either a PPAR‐γ agonist (MCE, HY‐13956, 20 μ m ) or a PPAR‐γ inhibitor (MCE, GW9662, 10 μ m ).

Techniques: RNA Sequencing, Activation Assay, Biomarker Discovery, RNAscope, Staining, Labeling, Expressing, Flow Cytometry, Fluorescence, Phagocytosis Assay, Incubation, Degradation Assay, Immunofluorescence, Co-Culture Assay, Two Tailed Test

Journal: Frontiers in Aging Neuroscience

Article Title: APOE4 accelerates menopause-associated brain metabolic shift and disrupts bioenergetic adaptation

doi: 10.3389/fnagi.2026.1796680

Figure Lengend Snippet: Pathway enrichment analysis comparing chronological and endocrinological groups within each APOE genotype. Top enriched metabolic pathways (adjusted p < 0.1) are shown for each pairwise comparison between chronological and endocrinological groups within (A) APOE3/3, (B) APOE3/4, and (C) APOE4/4 groups. For each panel, comparisons are presented from top to bottom as follows: 9M-Reg vs. 6M-Reg, 9M-Irreg vs. 9M-Reg, 15M-Irreg vs. 9M-Irreg, and 15M-Acyc vs. 15M-Irreg. Amino acid pathway included the amino acids shown in . Long-chain fatty acids (FA) pathway included long-chain saturated, monounsaturated, and polyunsaturated fatty acids shown in . Acylcarnitine pathway included medium-chain, long-chain saturated, monounsaturated, and polyunsaturated acylcarnitines as shown in .

Article Snippet: Humanized APOE4/4 targeted replacement (APOE4/4) homozygous mice were obtained from Jackson Laboratory (#027894).

Techniques: Comparison

Journal: Frontiers in Aging Neuroscience

Article Title: APOE4 accelerates menopause-associated brain metabolic shift and disrupts bioenergetic adaptation

doi: 10.3389/fnagi.2026.1796680

Figure Lengend Snippet: Pathway enrichment analysis comparing APOE genotypes within each chronological and endocrinological group. Top enriched metabolic pathways (adjusted p < 0.1) are shown for each pairwise comparison between APOE genotypes within each chronological and endocrinological group [ (A) 6M-Reg; (B) 9M-Reg; (C) 9M-Irreg; (D) 15M-Irreg; (E) 15M-Acyc]. For each panel, comparisons are presented from left to right as follows: APOE3/4 vs. APOE3/3, APOE4/4 vs. APOE3/3 and APOE4/4 vs. APOE3/4. Amino acid pathway included the amino acids shown in . Long-chain fatty acids (FA) pathway included long-chain saturated, monounsaturated, and polyunsaturated fatty acids shown in . Acylcarnitine pathway included medium-chain, long-chain saturated, monounsaturated, and polyunsaturated acylcarnitines as shown in .

Article Snippet: Humanized APOE4/4 targeted replacement (APOE4/4) homozygous mice were obtained from Jackson Laboratory (#027894).

Techniques: Comparison

Journal: Frontiers in Aging Neuroscience

Article Title: APOE4 accelerates menopause-associated brain metabolic shift and disrupts bioenergetic adaptation

doi: 10.3389/fnagi.2026.1796680

Figure Lengend Snippet: Glucose and lipid metabolism. (A) Heatmap of glucose and glycolytic intermediate levels. Diphosphates included fructose 1,6-diphosphate, glucose 1,6-diphosphate, and myo-inositol diphosphates. (B) Heatmap of long-chain fatty acid levels. (C) Heatmap of acylcarnitine levels. Metabolite name*: Indicated a compound that had not been confirmed based on a standard, but Metabolon was confident in its identity. (D) Volcano plots of triacylglycerols (TAGs) comparing the following groups: 9M-Irreg APOE4/4 vs. APOE3/3, 9M-Irreg APOE4/4 vs. APOE3/4, 15M-Irreg APOE4/4 vs. APOE3/3, and 15M-Irreg APOE4/4 vs. APOE3/4.

Article Snippet: Humanized APOE4/4 targeted replacement (APOE4/4) homozygous mice were obtained from Jackson Laboratory (#027894).

Techniques:

Journal: Frontiers in Aging Neuroscience

Article Title: APOE4 accelerates menopause-associated brain metabolic shift and disrupts bioenergetic adaptation

doi: 10.3389/fnagi.2026.1796680

Figure Lengend Snippet: Brain lipidomic profile. (A) Brain lipid composition. (B) Heatmap of lipid concentrations. (C) Volcano plots of ceramides comparing the following groups: 9M-Irreg APOE3/4 vs. APOE3/3 and 9M-Irreg APOE4/4 vs. APOE3/3. (D) Volcano plots of phosphatidylcholines comparing the following groups: 15M-Acyc APOE3/4 vs. APOE3/3 and 15M-Acyc APOE4/4 vs. APOE3/3.

Article Snippet: Humanized APOE4/4 targeted replacement (APOE4/4) homozygous mice were obtained from Jackson Laboratory (#027894).

Techniques:

Journal: Frontiers in Aging Neuroscience

Article Title: Accelerated midlife endocrine and bioenergetic brain aging in APOE4 females

doi: 10.3389/fnagi.2025.1632877

Figure Lengend Snippet: Accelerated endocrine aging in APOE4 females. (A) Percentage of APOE3 and APOE4 females by cycling status (total n = 122, 17 APOE3 6M, 20 APOE3 9M, 21 APOE3 15M, 15 APOE4 6M, 22 APOE4 9M, and 27 APOE4 15M). Plasma levels of estradiol ( B , n = 3–11/group) and progesterone ( C , n = 3–7/group) in all groups. (D) Uterine weights of the animals ( n = 5–13/group). Data were presented as Mean ± SEM with individual data plotted. Statistically significant differences were determined using two-way ANOVA, followed by Tukey′s post hoc test. * p < 0.05, ** p < 0.01. CEA, chronological and endocrinological aging.

Article Snippet: Humanized APOE4 targeted replacement (APOE4) homozygous mice were obtained from Jackson Laboratory (#027894).

Techniques: Clinical Proteomics

Journal: Frontiers in Aging Neuroscience

Article Title: Accelerated midlife endocrine and bioenergetic brain aging in APOE4 females

doi: 10.3389/fnagi.2025.1632877

Figure Lengend Snippet: Shift in peripheral metabolism in APOE4 PAM females. Body weight (A) , adipose composition (B) and muscle composition (C) of APOE3 and APOE4 females with different age and endocrine status ( n = 5–17/group). Fasting levels of glucose (D) , triglyceride (E) and ketone bodies (F) in all groups ( n = 3–14/group). Data were presented as Mean ± SEM with individual data plotted. Statistically significant differences were determined using two-way ANOVA, followed by Tukey′s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. CEA, chronological and endocrinological aging.

Article Snippet: Humanized APOE4 targeted replacement (APOE4) homozygous mice were obtained from Jackson Laboratory (#027894).

Techniques:

Journal: Frontiers in Aging Neuroscience

Article Title: Accelerated midlife endocrine and bioenergetic brain aging in APOE4 females

doi: 10.3389/fnagi.2025.1632877

Figure Lengend Snippet: Deficits in upstream regulators required for brain metabolic reprogramming in APOE4 PAM brains. (A) Volcano plots showing differentially expressed genes involved in estrogen receptor, AMPK and CREB signaling pathways in the 9M-Irreg APOE4 group compared to the APOE3 group, with the corresponding gene list provided in . (B) Volcano plots showing differentially expressed genes involved in estrogen receptor, AMPK and CREB signaling pathways in the 15M-Irreg APOE4 group compared to the APOE3 group, with the corresponding gene list provided in . Hippocampal expression levels of Ppargc1a (C) and Nrf1 (D) of APOE3 and APOE4 mice ( n = 3–7). (E) Cortical levels of PGC-1α and TFAM of 15M-Irreg and 15M-Acyc APOE3 and APOE4 mice ( n = 3–5). Pathway-level hippocampal gene expression of glycolysis (F) and TCA cycle (G) of 15M-Irreg and 15M-Acyc APOE3 and APOE4 mice ( n = 3–7). Data were presented as Mean ± SEM with individual data plotted. (C,D) Statistically significant differences were determined using two-way ANOVA, followed by Tukey′s post hoc test. ( E–G) Statistically significant differences were determined using two-way ANOVA with Holm-Sidak multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001. CEA: chronological and endocrinological aging.

Article Snippet: Humanized APOE4 targeted replacement (APOE4) homozygous mice were obtained from Jackson Laboratory (#027894).

Techniques: Protein-Protein interactions, Expressing, Gene Expression

Journal: Frontiers in Aging Neuroscience

Article Title: Accelerated midlife endocrine and bioenergetic brain aging in APOE4 females

doi: 10.3389/fnagi.2025.1632877

Figure Lengend Snippet: APOE4 perimenopausal brain exhibited greater mitochondrial deficits. (A) Mitochondrial DNA copy number in all groups ( n = 3–9). Statistically significant differences were determined using two-way ANOVA, followed by Tukey′s post hoc test. Cortical complex I (B) and IV (C) activities and complex protein levels (D) of 6M-Reg APOE3 and APOE4 females ( n = 3–5/group). Cortical complex I (E) and IV (F) activities and complex protein levels (G) of 9M-Reg and 9M-Irreg APOE3 and APOE4 females ( n = 4–5/group). At 15 months, cortical complex I (H) and IV (I) activities ( n = 4–7/group) and complex protein levels ( J , n = 3–5/group) were significantly reduced in APOE4 15M-Acyc groups. Data were presented as Mean ± SEM with individual data plotted. ( E – J) Statistically significant differences were determined using two-way ANOVA with Holm-Sidak multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. CEA: chronological and endocrinological aging.

Article Snippet: Humanized APOE4 targeted replacement (APOE4) homozygous mice were obtained from Jackson Laboratory (#027894).

Techniques:

Journal: Frontiers in Aging Neuroscience

Article Title: Accelerated midlife endocrine and bioenergetic brain aging in APOE4 females

doi: 10.3389/fnagi.2025.1632877

Figure Lengend Snippet: Increased microglial activation in APOE4 PAM brains. (A) Aif1 RNA levels in all groups ( n = 3–7). (B) Protein levels of IBA1 and GFAP of 15M-Irreg and 15M-Acyc APOE3 and APOE4 groups ( n = 4–5/group). The actin control for IBA1 is the same as the actin control used for TFAM in . (C) Anti-IBA1 staining on brain sections from 15M-Irreg and 15M-Acyc APOE3 and APOE4 groups ( n = 3–5, Bregma –2.2 mm). Cortical levels of IL-10 (D) , IL-1β (E) and IL-6 (F) in all groups ( n = 3–11). (G) IL1B was predicted to be activated in APOE4 15M-Irreg group compared to APOE3 counterparts. (H) IPA canonical pathways indicated activation of multiple cytokine, mTOR and CXCR4 signaling pathways in APOE4 15M-Acyc group compared to APOE3 counterparts. Data were presented as Mean ± SEM with individual data plotted. ( A, D–F) Statistically significant differences were determined using two-way ANOVA, followed by Tukey′s post hoc test. (B,C) Statistically significant differences were determined using two-way ANOVA with Holm-Sidak multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001. CEA: chronological and endocrinological aging.

Article Snippet: Humanized APOE4 targeted replacement (APOE4) homozygous mice were obtained from Jackson Laboratory (#027894).

Techniques: Activation Assay, Control, Staining, Protein-Protein interactions

Journal: Frontiers in Aging Neuroscience

Article Title: Accelerated midlife endocrine and bioenergetic brain aging in APOE4 females

doi: 10.3389/fnagi.2025.1632877

Figure Lengend Snippet: Increased demyelination in APOE4 PAM brains. (A) Myelin metabolic and generation related gene expression in all groups (z-score). (B) CNPase and MBP protein levels in APOE3 and APOE4 9M groups ( n = 4–5). (C) CNPase and MBP protein levels in APOE3 and APOE4 15M groups ( n = 3–5). (D) Anti-MBP staining on brain sections from 15M-Irreg and 15M-Acyc APOE3 and APOE4 females ( n = 3–5, Bregma –2.2 mm). (E) Corpus callosum thickness in APOE3 and APOE4 15M groups ( n = 3–5, Bregma 0.2 mm). Data were presented as Mean ± SEM with individual data plotted. Statistically significant differences were determined using two-way ANOVA with Holm-Sidak multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Humanized APOE4 targeted replacement (APOE4) homozygous mice were obtained from Jackson Laboratory (#027894).

Techniques: Gene Expression, Staining

Journal: Frontiers in Aging Neuroscience

Article Title: Accelerated midlife endocrine and bioenergetic brain aging in APOE4 females

doi: 10.3389/fnagi.2025.1632877

Figure Lengend Snippet: APOE4 is associated with early menopause in women. (A) Study design. (B) APOE4 is associated with early menopause age. (C) Participant characteristics. (D) Odds ratio of AD risk for APOE4 carriers and non-carriers with early or normal menopausal age. ** p < 0.01, *** p < 0.001.

Article Snippet: Humanized APOE4 targeted replacement (APOE4) homozygous mice were obtained from Jackson Laboratory (#027894).

Techniques: