Microglial Calhm2 regulates neuroinflammation and contributes to Alzheimer's disease pathology

Abstract

Alzheimer's disease (AD) is the most common neurodegenerative disease in the world. Neuronal calcium dysfunction and microglial-mediated neuroinflammation are closely associated with the development of AD. However, it remains unknown whether calcium dysfunction contributes to microglial activation and, in turn, AD pathology in vivo. In this study, we demonstrated that the expression of calcium homeostasis modulator family protein 2 (Calhm2) is increased in an AD mouse model. In 5×FAD mice carrying five familial AD gene mutations, both conventional knockout of Calhm2 and conditional microglial knockout of Calhm2 significantly reduced amyloid β deposition, neuroinflammation, and cognitive impairments. Mechanistically, knockout of Calhm2 inhibited microglial proinflammatory activity but increased phagocytic activity, leading to restoration of the balance between inflammation and phagocytosis. In addition, knockout of Calhm2 reduced acute LPS-induced neuroinflammation. These results highlight an important role for Calhm2 in microglial activation and provide a potential therapeutic target for diseases related to microglia-mediated neuroinflammation.

Fig. 1. Calhm2 levels are increased in patients with AD and in AD mice.

(A and B) Transcriptional up-regulation of Calhm2 in hippocampal (Hip) tissue of patients with AD in the GSE48350 database (control, n = 19; AD patient, n = 19) and GSE5281 database (control, n = 13; AD patient, n = 10). (C) Transcriptional up-regulation of Calhm2 in the hippocampus of 6-month-old 5×FAD mice (WT mice, n = 9; AD mice, n = 9). (D and E) Coexpression and quantification of Calhm2 mRNA [in situ hybridization (ISH)] and Cx3cr1 mRNA (ISH), Gfap mRNA (ISH), and NeuN mRNA (ISH) in the hippocampus of 6-month-old WT mice. (F) RNA ISH of Calhm2 expression and its colocalization with Iba1 (immunostaining)– and GFAP (immunostaining)–positive cells in 6-month-old WT and 5×FAD mice. (G) The quantification of Calhm2 levels in microglia and astrocytes from 6-month-old WT and 5×FAD mice. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 2. Calhm2 knockout restores cognitive functions, decreases Aβ deposition, and decreases glial numbers in 5×FAD mice.

(A) Schematic for the generation of Calhm2^−/−:5×FAD mice, behavioral tests, and pathological analysis. KO, knockout. (B) Transcriptional levels of Calhm2 in the hippocampus of 6-month-old WT (n = 7), Calhm2^−/− (n = 6), 5×FAD (n = 6), and Calhm2^−/−:5×FAD mice (n = 10). (C) MWM analysis as latency (s) to target in the invisible platform trainings. (D to G) MWM analysis as the latency (s), target quarter preference (%), target cross number, and mean speed (cm/s) in the invisible platform tests in 5-month-old WT (n = 10 mice), Calhm2^−/− (n = 10 mice), 5×FAD (n = 10 mice), and Calhm2^−/−:5×FAD mice (n = 10 mice). (H) Representative images of the track plots in the MWM tests. (I and J) Immunohistochemistry (IHC) and statistical analysis of Aβ plaques per pixel area in the prefrontal cortex (PFC) (n = 13 to 18 slices from three mice per group), CA1 (n = 6 to 11 slices from three mice per group), and dentate gyrus (DG) (n = 11 to 13 slices from three mice per group) of 5×FAD and Calhm2^−/−:5×FAD mice. (K to N) ELISA method for detecting soluble [radioimmunoprecipitation assay (RIPA) fraction] and insoluble (SDS fraction) contents of Aβ_1–40 and Aβ_1–42 of 5×FAD and Calhm2^−/−:5×FAD mice (n = 8 per group). (O and P) IHC and statistical analysis of Iba1-positive cells (microglia) per pixel area in 6-month-old WT, Calhm2^−/− mice, 5×FAD, and Calhm2^−/−:5×FAD mice. (Q and R) IHC and statistical analysis of GFAP-positive cells (astrocytes) per pixel area in 6-month-old WT mice, Calhm2^−/− mice, 5×FAD mice, and Calhm2^−/−:5×FAD mice (n = 8 to 13 slices from three mice per group). Scale bar, 50 μm. One-way analysis of variance (ANOVA) for multiple groups in (C) to (G) were based on mouse number. Statistical analysis of (I), (O), (P), (Q), and (R) were dependent on multiple sampling. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 3. Microglial Calhm2 knockout restores cognitive function and decreases amyloid beta deposition in 5×FAD mice.

(A) Schematic for the generation of Calhm2^{flox/flox:Cx3cr1-CreER}:5×FAD mice, intragastric tamoxifen administration, behavioral testing, and pathological analysis. P0, postnatal day 0. (B) Transcriptional levels of Calhm2 in isolated microglia of 5-month-old Calhm2^flox/flox and Calhm2^{flox/flox:Cx3cr1-CreER} mice (n = 3 mice). (C) MWM analysis as latency (s) to target in the invisible platform trainings. (D to G) MWM analysis as the latency (s), target quarter preference (%), target cross number, and mean speed (centimeters per second) in the invisible platform tests in 5-month-old Calhm2^flox/flox (n = 11 mice), Calhm2^{flox/flox:Cx3cr1-CreER} (n = 11 mice), Calhm2^flox/flox:5×FAD (n = 11 mice), and Calhm2^{flox/flox:Cx3cr1-CreER}:5×FAD (n = 11 mice) mice. (H) Representative images of track plots in the MWM tests. cKO, conditional knockout. (I) Open-field test and locomotor activity (distance traveled) over three consecutive days. (J and K) IHC and immunofluorescent staining and statistical analysis of Aβ plaques per pixel area in the PFC, CA1, and DG of Calhm2^flox/flox:5×FAD and Calhm2^{flox/flox:Cx3cr1-CreER}:5×FAD mice (n = 7 to 11 slices from three mice per group). (L and M) Statistical analysis of thioflavin S staining (compact Aβ plaque) of Aβ plaques in the PFC of Calhm2^flox/flox:5×FAD and Calhm2^{flox/flox:Cx3cr1-CreER}:5×FAD mice (n = 8 slices from three mice per group). (N to Q) ELISA analysis of soluble (RIPA fraction) and insoluble (SDS fraction) contents of Aβ_1–40 and Aβ_1–42 of Calhm2^flox/flox:5×FAD and Calhm2^{flox/flox:Cx3cr1-CreER}:5×FAD mice (n = 8 per group). One-way ANOVA for multiple groups in (C) to (G) and (I) were based on mouse number. Statistical analysis of (J) to (M) were dependent on multiple sampling. NS, no significance; *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 4. Microglial Calhm2 knockout increases microglial phagocytosis activation.

(A to C) IHC and statistical analysis of Iba1- and GFAP-positive cells in PFC and CA1 of 6-month-old Calhm2^flox/flox:5×FAD and Calhm2^{flox/flox:Cx3cr1-CreER}:5×FAD mice (n = 7 to 11 slices from three mice per group). (D and E) Immunoblotting and statistical analysis of Iba1 and GFAP levels of 6-month-old Calhm2^flox/flox, Calhm2^{flox/flox:Cx3cr1-CreER}, Calhm2^flox/flox:5×FAD, and Calhm2^{flox/flox:Cx3cr1-CreER}:5×FAD mice (n = 3 per group). (F and G) Thioflavin S staining, immunofluorescent staining of Iba1 and Aβ, and statistical analysis of microglia number per Aβ plaque of 6-month-old Calhm2^flox/flox:5×FAD and Calhm2^{flox/flox:Cx3cr1-CreER}:5×FAD mice (n > 50 from three mice per group). (H and I) Immunofluorescent staining of Iba1, CD68, and Aβ and statistical analysis of CD68 density of 6-month-old Calhm2^flox/flox:5×FAD and Calhm2^{flox/flox:Cx3cr1-CreER}:5×FAD mice (n > 25 from three mice per group). (J and K) Immunofluorescent staining of Iba1, TREM2, and Aβ and statistical analysis of TREM2 density of 6-month-old Calhm2^flox/flox:5×FAD and Calhm2^{flox/flox:Cx3cr1-CreER}:5×FAD mice (n > 50 from three mice per group). (L) Immunoblotting TREM2 levels in primary microglia of WT and Calhm2^−/− with and without Aβ (1 μg/ml, 3 hours) treatment. (M) Immunoblotting of Aβ levels of WT and Calhm2^−/− in the supernatant (Sup) and pellet (Pel) of primary microglia. Statistical analysis of (B), (C), and (F) to (K) were dependent on multiple sampling. *P < 0.05 and ***P < 0.001. SE, short exposure; LE, long exposure.

Fig. 5. Microglial transcriptome changes reveal a protective role for Calhm2 knockout in 5×FAD mice.

(A) Schematic for microglia isolation, RNA extraction, RNA-seq, and bioinformatic analysis. (B) Heatmap shows four groups of expressed genes that differentially expressed between 6-month-old Calhm2^flox/flox mice and Calhm2^flox/flox:5×FAD mice (log₂ fold change > 0.5, adjusted false discovery rate < 0.05). (C and D) GSEA of the ROS pathway and IL-6/JAK/Stat3 signaling of 6-month-old Calhm2^{flox/flox:Cx3cr1-CreER}:5×FAD versus Calhm2^flox/flox:5×FAD. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (E) Heatmap of microglial markers, complement, NF-κB signaling, cytokines, chemokines, and cytokines receptors in microglia from these four groups of mice. (F) qPCR assay to test the expression levels of indicated genes in isolated microglial cells from these four groups mice (n = 6 per group). (G) Schematic for intraperitoneal LPS injections in 2-month-old WT and Calhm2^−/− mice. (H to J) Transcriptional levels of IL-1β, TNF-α, and IL-6 in 2-month-old WT and Calhm2^−/− mice after intraperitoneal LPS injections (n = 3 mice per group). (K) Immunofluorescence staining of Iba1-positive cells within the hippocampus of WT and Calhm2^−/− mice. (L to O) Skeletonized analysis: Representative images and statistical analysis of branch numbers, total branch length (micrometers), average branch length (micrometers), and soma area (square micrometers) of Iba1-positive cells within the hippocampus of WT and Calhm2^−/− mice (n > 20 from three mice per group). Statistical analysis of (L) to (O) were dependent on multiple sampling. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 6. Calhm2 regulates microglial calcium influx and inflammatory activation.

(A and B) The fluorescence intensity and statistical analysis of fluorescence change (ΔF/F₀) in WT and Calhm2 knockout microglia cells stimulated with 1 mM ATP in the detecting buffer with 2 mM CaCl₂. (C) Immunoblotting of phosphorylated CaMKII (p-CaMKII) and total CaMKII levels in LPS-primed WT and Calhm2 knockout primary microglia in response to the stimulations induced by ATP and A438079. (D and E) Representative images and statistical analysis of peak and steady current density (pA/pF) of WT and Calhm2 knockout primary microglia in response to ATP stimulation. The peak current consisted of the response of both P2X4 and P2X7 receptors. Meanwhile, the steady current was the response of only P2X7 receptor. (F) Immunoblotting of P2X7 levels in biotin-bound membrane protein. (G) Immunoblotting analysis of IL-1β levels in the supernatants of LPS primed WT and Calhm2 knockout primary microglia in response to the stimulations with ATP and A438079. (H) Transcriptional levels of IL-1β and TNF-α of WT and Calhm2 knockout primary microglia after LPS stimulation and A438079 treatment. (I and J) Immunoblotting and quantification of P2X7 levels in 6-month-old WT, Calhm2^−/−, 5×FAD, and Calhm2^−/−:5×FAD mice. One-way ANOVA for multiple groups was based on mouse number. (K) Immunoblotting analysis of cleaved caspase-1 in hippocampal tissue of 6-month-old WT, Calhm2^−/−, 5×FAD, and Calhm2^−/−:5×FAD mice. (L) Immunoblotting analysis of cleaved caspase-1 in hippocampal tissue of 6-month-old Calhm2^flox/flox, Calhm2^{flox/flox:Cx3cr1-CreER}, Calhm2^flox/flox:5×FAD, and Calhm2^{flox/flox:Cx3cr1-CreER}:5×FAD mice. (M) The working model of microglial Calhm2 in AD mouse model. *P < 0.05 and **P < 0.01.