Microglia-derived APOE2 improves remyelination even in the presence of endogenous APOE4

Abstract

Demyelination occurs with aging and is exacerbated in neurodegenerative diseases. During demyelination, microglia upregulate expression of APOE, the gene encoding for the brain's primary lipid transport protein apolipoprotein E (ApoE), which also mediates microglial engulfment and elimination of myelin debris. Compared to the E3 allele of APOE, the E2 allele decreases risk for Alzheimer's disease (AD), while the E4 allele increases AD risk and is associated with an increased severity and progression of multiple sclerosis. Previous work shows that mice expressing E2 exhibit improved microglial function and remyelination compared to mice expressing E4. However, whether microglial-derived APOE is responsible for driving these differences following demyelination, and if microglia-selective expression of E2 is sufficient to provide protection, is unknown. We sought to determine if microglia-specific replacement of the E4 allele with E2 can rescue myelin loss and promote remyelination, even in the presence of continued E4 expression by other central nervous system (CNS) cells. Using a novel APOE allelic "switch" model in which we can induce a replacement of E4 with E2 exclusively in microglia, we characterize the glial cell response and lipid profile of mice that underwent either lysophosphatidylcholine (LPC) or cuprizone (CPZ)-induced demyelination and subsequent remyelination. We found that although alterations to the brain lipid profile were subtle, microglial E2 replacement significantly improved remyelination, lessened microgliosis, and decreased astrocytic lipid droplet load following CPZ-remyelination. Our results indicate that microglia-specific E2 expression, in the presence of continued E4 expression, may provide protection against myelin loss via both cell-autonomous and non-autonomous immunometabolic mechanisms.

Keywords: Apolipoprotein E; Brain; Gliosis; Lipid metabolism; Microglia; Myelination.

Fig. 1

Microglia selectively and efficiently transition from E4 to E2. A, Graphic depicting the design of the tamoxifen inducible microglia specific APOE switch mice (APOE4s2^M) that undergo cell-specific replacement of E4 with E2. APOE4s2^flox/flox mice are crossed to microglia specific Tmem119-CreER^T2 mice to generate a model capable of an inducible, microglia specific APOE4 to APOE2 switch (4s2^M). B, Immunofluorescent staining of IBA1 + microglia and ai9 Cre-reporter + microglia in 4 different brain regions (cortex (CX), hippocampus (HIPP), corpus callosum (CC), and thalamus (THAL)) in a 4s2^M mouse that received tamoxifen. C, Quantification of the percent of cells positive for both tdTomato and IBA1 immunoreactivity in each region in 4s2^M (N = 3) compared to oil treated controls (N = 3); each dot is the average of 6 images per mouse; average manual quantification of 2 blinded individuals; red: vehicle (oil) treated 4s2^M mice, blue: 4s2^M mice injected with tamoxifen to induce allelic switching. D, Allelic discrimination plots depicting a shift from APOE4 to APOE2 mRNA expression in Cre-positive microglia sorted from other Cre-negative cells in the brain expressing APOE4 using an ai9 Cre-reporter. E, Schematic depicting the experimental design. 6-week old mice containing the APOE4s2 flox/flox with and without the Tmem119-CreER^T2 were injected with tamoxifen for 5 consecutive days to allow for allelic switching in Tmem119-CreER^T2 mice. At 8 weeks old, mice received either intracranial LPC injections or began the CPZ diet

Fig. 2

Microglial replacement with E2 decreases astrogliosis following LPC-induced demyelination. A, Graphic showing 2-point LPC intracranial injections targeting the CC in 4s2- (n = 7) and 4s2^M (n = 7) mice. Tamoxifen was administered at 6 weeks to induce allelic switching, LPC injections were performed at 8 weeks, and tissue was collected 10 days post-LPC injections. B-C, Representative images of dMBP staining in the whole brain (left hemi: contralateral, right hemi: ipsilateral) (B) and in the CC (C) of LPC injected 4s2- and 4s2^M mice. D, Percent area of dMBP + coverage in the CC of 4s2- and 4s2^M mice (D). E-F, Representative images of GFAP staining in the whole brain (E) and in the CC of LPC injected 4s2- and 4s2^M mice (F). G, Quantification of percent area coverage of GFAP in the CC of 4s2- and 4s2^M mice. H-I, Whole brain representative image of IBA1 staining in an LPC-injected mouse brain (H). IBA1 staining in the CC of 4s2- and 4s2^M mice (I). J, Quantification of percent area coverage of IBA1 in the CC of 4s2- and 4s2^M mice.) K-L, Whole brain representative image of CD68 staining in an LPC-injected mouse brain (K). CD68 staining in the CC of 4s2- and 4s2^M mice (L). M, Quantification of percent area coverage of CD68 in the CC of 4s2- and 4s2^M mice. D, G, J, M, Circles (male), squares (female). (2way ANOVA with Fisher’s LSD multiple comparisons; *p < 0.05; ***p < 0.0005; ****p < 0.0001). Anterior LPC-injection quantification can be found in Supplemental Fig. 2

Fig. 3

Microglial replacement of E4 with E2 drives increased remyelination. A, Timeline of cuprizone-mediated demyelination and remyelination following microglial-specific allelic switching induced at 6 weeks. At 8 weeks, mice received CPZ diet (normal chow for controls) for 5 weeks before tissue collection (Demyelination/DM) or for 5 weeks before returning to Chow diet for 1 additional week before tissue collection (Remyelination/RM). B, Normalized weekly body weight (normalized to Control) for DM and RM mice of each genotype throughout the duration of the experiment. C-F, Fluorescent staining using FluoroMyelin (C) and dMBP (E) in the CC of 4s2- and 4s2M mice from Control, DM, and RM paradigms. Quantification of the percent area coverage of FluoroMyelin (D) and dMBP (F) in the CC of 4s2- and 4s2M mice in each treatment group. Circles (male), squares (female). A significant Genotype x Treatment interaction effect was observed (p < 0.0091) for FluoroMyelin quantification. (*p < 0.05; ***p < 0.0005; ****p < 0.0001)

Fig. 4

Microglial E2 replacement decreases microgliosis following a short remyelination period. A-H, Immunofluorescent staining of IBA1 (A), CD68 (C), and GFAP (G) in the hippocampus and CC of 4s2- and 4s2^M mice in each treatment group. Quantification of percent area coverage of IBA1 (B), CD68 (D), CD68 + IBA1+ (E), and GFAP (H), in the CC of 4s2- and 4s2^M mice in each treatment group. Regional quantification can be found in Supplemental Fig. 4. Log2-transformed Trem2 fold change in the brains of 4s2- and 4s2^M mice from each treatment group (F). Circles (male), squares (female). (2way ANOVA with Tukey’s multiple comparisons; *p < 0.05; ***p < 0.0005; ****p < 0.0001)

Fig. 5

Microglia expression of E2 subtly alters oligodendrocyte lineage marker expression. A-B, Representative images of immunofluorescent staining of Olig2, PDGFRα, and CC1 from the CC of 4s2- and 4s2^M following Control, DM, RM. C-E, Quantification of percent area coverage of Olig2 (C), PDGFRα (D), and CC1 (E) in the CC of 4s2- and 4s2^M mice. Circles (male), squares (female). (2way ANOVA with Tukey’s multiple comparisons; *p < 0.05; ***p < 0.0005; ****p < 0.0001)

Fig. 6

Microglial E2 replacement has a modest effect on the E4 brain lipidome following de/remyelination. A, PCA plot including all identified lipids depicting separation between Control and DM/RM treatment groups. B, Heatmap displaying lipid abundance profiles grouped into six clusters corresponding to the experimental conditions (2 genotypes × 3 treatments). Clustering was based on predefined experimental grouping rather than unsupervised data-driven analysis. Colors indicate relative (log₂-scaled) lipid abundances. C-E, Volcano plots for lipid species differentially expressed between 4s2- and 4s2^M following Control (C), DM (D), and RM (E) treatments. (2way ANOVA with FDR-corrected multiple comparisons (Benjamini-Hochberg); *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001)

Fig. 7

Microglial APOE genotype modulates ApoE protein expression and lipid droplet distribution. A-B, Representative images from the CC of immunofluorescent staining of IBA1 and Plin2 (A) and GFAP and Plin2 (B). C-E, Quantification of percent area coverage of Plin2 (C), Plin2 + IBA1+ (D), and Plin2 + GFAP+ (E) within the CC of 4s2- and 4s2^M mice. F, ApoE immunofluorescent staining in the CC of 4s2- and 4s2^M mice. G, Quantification of percent area coverage of ApoE in the CC of 4s2- and 4s2^M mice following de/remyelination. H-I, Representative images from the CC of immunofluorescent staining of IBA1 and ApoE (H), and GFAP and ApoE (I). J-K, Quantification of percent area coverage of microglial ApoE (APOE + IBA1+) (J) and astrocytic ApoE (APOE + GFAP+) (K) within the CC of 4s2- and 4s2^M mice. Circles (male), squares (female). A significant Genotype x Treatment interaction (p < 0.0276) was observed for APOE + IBA1 + quantification. (2way ANOVA with Tukey’s multiple comparisons; *p < 0.05; ***p < 0.0005; ****p < 0.0001)