Type I interferon response-related microglial Mef2c deregulation at the onset of Alzheimer's pathology in 5×FAD mice

Abstract

Alzheimer's disease (AD) is a chronic neurodegenerative disorder with multifactorial etiology. The role of microglia in the pathogenesis of AD has been increasingly recognized in recent years; however, the detailed mechanisms shaping microglial phenotypes in AD-relevant pathological settings remain largely unresolved. Myocyte-specific enhancer factor 2C (Mef2C) is a transcription factor with versatile functions. Recent studies have attributed aging-related microglial changes to type I interferon (IFN-I)-associated Mef2C deregulation. In view of the close relationship between brain aging and AD, it is of great interest to determine microglial Mef2C changes in AD-related conditions. In this study, we have found that suppressed Mef2C nuclear translocation was an early and prominent microglial phenotype in a mouse model of brain amyloidosis (5×FAD mice), which exacerbated with age. Echoing the early Mef2C deregulation and its association with microglial activation, transcriptional data showed elicited IFN-I response in microglia from young 5×FAD mice. Amyloid beta 42 (Aβ42) in its oligomeric forms promoted Mef2C deregulation in microglia on acute organotypic brain slices with augmented microglial activation and synapse elimination via microglial phagocytosis. Importantly, these oligomeric Aβ42-mediated microglial changes were substantially attenuated by blocking IFN-I signaling. The simplest interpretation of the results is that Mef2C, concurring with activated IFN-I signaling, constitutes early microglial changes in AD-related conditions. In addition to the potential contribution of Mef2C deregulation to the development of microglial phenotypes in AD, Mef2C suppression in microglia may serve as a potential mechanistic pathway linking brain aging and AD.

Keywords: Alzheimer's disease; Amyloid beta; Microglia; Myocyte-specific enhancer factor 2C; Type I interferon.

Fig. 1.

Decreased microglial Mef2C nuclear translocation in 5×FAD mice brain. A. Measurement of total Mef2C expression in microglia from 3–4–month– and 9–10–month–old nonTg and 5×FAD mice. Two-way ANOVA followed by Bonferroni post hoc analysis. 3–4 months: nonTg, n = 6; 5×FAD, n = 5. 9–10 months: nonTg, n = 6; 5×FAD, n = 6. B&C. Analysis of nuclear (B) and cytoplasmic (C) expression of Mef2C in microglia from 3–4–month– and 9–10–month–old nonTg and 5×FAD mice. Two-way ANOVA followed by Bonferroni post hoc analysis. 3–4 months: nonTg, n = 6; 5×FAD, n = 5. 9–10 months: nonTg, n = 6; 5×FAD, n = 6. D. Calculation of nuclear/cytoplasmic ratio of Mef2C expression in microglia from 3–4–month– and 9–10–month–old nonTg and 5×FAD mice. Two-way ANOVA followed by Bonferroni post hoc analysis. 3–4 months: nonTg, n = 6; 5×FAD, n = 5. 9–10 months: nonTg, n = 6; 5×FAD, n = 6. E. Representative confocal microscopy images of quantification in (A-D). Iba1 was used to visualize the microglia. Nuclei were stained with DAPI. Scale bar = 10 μm.

Fig. 2.

Early activation of IFN-I signaling in microglia from 5×FAD mice. A. Volcano plot showing differences in gene expression in purified microglia from 3–4–month–old 5×FAD mice with respect to nonTg littermates. Unpaired two-way Student’s t-test. Significance is plotted against fold change. Up- or down- regulated genes are highlighted in red and green, respectively, with adjusted significance of P < 0.05. n = 3 per group. B. Top 20 significantly enriched Gene Ontology (GO) terms from genes differentially expressed in purified microglia from 3–4–month–old 5×FAD mice compared to nonTg littermates. The significance of the observed GO enrichment was estimated by the P value plotted on a log10 scale. n = 3 per group. C. Heat map showing clustering of upregulated and downregulated interferon stimulated genes (ISGs) expressed in purified microglia from 3–4–month–old nonTg and 5×FAD mice. The major upregulated and downregulated genes were listed. D&E. Venn diagram showing upregulated (D) and downregulated (E) interferon stimulated genes (ISGs) induced by Type I and/or Type II interferons in purified microglia from 3–4–month–old 5×FAD mice compared to nonTg littermates.

Fig. 3.

Involvement of IFN-I signaling in microglial activation in soluble Aβ-rich milieus. A, B & C. Effect of Aβ (1 μM, 2 h) treatment on microglia convex hull area (A), number of nodes (B), and total processes length (C) from acute organotypic brain slices in the presence or absence of a 20 min pretreatment of α-IFNAR (10 μg/ml). The brain slices were prepared from 3–month–old nonTg mice. Two-way ANOVA followed by Bonferroni post hoc analysis. n = 18–24 microglia. D. Representative confocal microscopy images of quantification in (A-C). Iba1 was used to visualize microglia. Nuclei were stained with DAPI. Scale bar = 10 μm. E. Effect of Aβ (1 μM, 2 h) treatment on microglial engulfment of synapses from acute organotypic brain slices in the presence or absence of a 20 min pretreatment of α-IFNAR (10 μg/ml). The brain slices were prepared from 3–month–old nonTg mice. Two-way ANOVA followed by Bonferroni post hoc analysis. n = 27–34 microglia. F. Representative confocal microscopy images of microglial engulfment of synapses. Iba1 (red) and PSD95 (green) were used to visualize microglia and synapses respectively. The overlapped staining of microglia and PSD95 indicates synapses engulfment. Scale bar = 10 μm.

Fig. 4.

Reduced microglia Mef2C nuclear translocation in soluble Aβ-rich milieus is IFN-I dependent. A, B & C. Effect of Aβ (1 μM, 2 h) treatment on nuclear Mef2C (A), cytoplasmic Mef2C (B), and nuclear/cytoplasmic ratio of Mef2C (C) in microglia of acute organotypic brain slices in the presence or absence of a 20 min pretreatment of α-IFNAR (10 μg/ml). The brain slices were prepared from 3–month–old nonTg mice. Two-way ANOVA followed by Bonferroni post hoc analysis. n = 18–24 microglia. D. Effect of Aβ (1 μM, 2 h) treatment on total Mef2C expression in microglia of acute organotypic brain slices in the presence or absence of a 20 min pretreatment of α-IFNAR (10 μg/ml). The brain slices were prepared from 3–month–old nonTg mice. Two-way ANOVA followed by Bonferroni post hoc analysis. n = 18–24 microglia. E. Representative confocal microscopy images of quantification in (A-D). Iba1 was used to visualize the microglia. Nuclei were stained with DAPI. Scale bar = 10 μm.