TRPM2 Channel Aggravates CNS Inflammation and Cognitive Impairment via Activation of Microglia in Chronic Cerebral Hypoperfusion

Abstract

Chronic cerebral hypoperfusion is a characteristic seen in widespread CNS diseases, including neurodegenerative and mental disorders, and is commonly accompanied by cognitive impairment. Recently, several studies demonstrated that chronic cerebral hypoperfusion can induce the excessive inflammatory responses that precede neuronal dysfunction; however, the precise mechanism of cognitive impairment due to chronic cerebral hypoperfusion remains unknown. Transient receptor potential melastatin 2 (TRPM2) is a Ca²⁺-permeable channel that is abundantly expressed in immune cells and is involved in aggravation of inflammatory responses. Therefore, we investigated the pathophysiological role of TRPM2 in a mouse chronic cerebral hypoperfusion model with bilateral common carotid artery stenosis (BCAS). When male mice were subjected to BCAS, cognitive dysfunction and white matter injury at day 28 were significantly improved in TRPM2 knock-out (TRPM2-KO) mice compared with wild-type (WT) mice, whereas hippocampal damage was not observed. There were no differences in blood-brain barrier breakdown and H₂O₂ production between the two genotypes at 14 and 28 d after BCAS. Cytokine production was significantly suppressed in BCAS-operated TRPM2-KO mice compared with WT mice at day 28. In addition, the number of Iba1-positive cells gradually decreased from day 14. Moreover, daily treatment with minocycline significantly improved cognitive perturbation. Surgical techniques using bone marrow chimeric mice revealed that activated Iba1-positive cells in white matter could be brain-resident microglia, not peripheral macrophages. Together, these findings suggest that microglia contribute to the aggravation of cognitive impairment by chronic cerebral hypoperfusion, and that TRPM2 may be a potential target for chronic cerebral hypoperfusion-related disorders.SIGNIFICANCE STATEMENT Chronic cerebral hypoperfusion is manifested in a wide variety of CNS diseases, including neurodegenerative and mental disorders that are accompanied by cognitive impairment; however, the underlying mechanisms require clarification. Here, we used a chronic cerebral hypoperfusion mouse model to investigate whether TRPM2, a Ca²⁺-permeable cation channel highly expressed in immune cells, plays a destructive role in the development of chronic cerebral hypoperfusion-induced cognitive impairment, and propose a new hypothesis in which TRPM2-mediated activation of microglia, not macrophages, specifically contributes to the pathology through the aggravation of inflammatory responses. These findings shed light on the understanding of the mechanisms of chronic cerebral hypoperfusion-related inflammation, and are expected to provide a novel therapeutic molecule for cognitive impairment in CNS diseases.

Keywords: TRPM2; cerebral hypoperfusion; cognitive impairment; cytokine; microglia; white matter injury.

Figure 1.

BCAS-induced cognitive decline at day 28 was not observed in TRPM2-KO mice. A–D, Alternation behavior at day 14 (A) and day 28 (B) and the number of arm entries at day 14 (C) and day 28 (D) after BCAS in the Y-maze test. E, F, Exploratory preference to the novel object in the novel object recognition test session at days 14 (E) and 28 (F) after BCAS. *p < 0.05 and ***p < 0.001 vs WT sham; ^#p < 0.05 and ^###p < 0.001 vs WT BCAS. Values are mean ± SEM. A, C, n = 12–14; B, D, n = 14–25; E, n = 10–13; F, n = 11–13.

Figure 2.

BCAS-induced white matter injury at day 28 was not observed in TRPM2-KO mice. A–C, Representative images of white matter in the corpus callosum by fluoromyelin staining (A) and the relative myelin density in WT (B) and TRPM2-KO mice (C). D–F, Representative images of immunostaining with GSTpi antibody in the corpus callosum (D) and the number of positive cells counted in WT (E) and TRPM2-KO mice (F). A, D, Bottom, Magnified images from the location marked by the boxed area of the above panels. *p < 0.05 vs WT sham. Values are mean ± SEM. B, C, n = 8–12; E, F, n = 6–9.

Figure 3.

Neuronal death was not observed in either WT or TRPM2-KO mice at day 28 after BCAS. A–D, Representative images of NeuN-positive neurons in CA1 (A), CA3 (B), DG (C), and cerebral cortex (D) at 28 d after BCAS. E–H, The number of NeuN-positive cells measured in each area. Values are mean ± SEM. n = 3–6.

Figure 4.

No differences were observed in BBB permeability and ROS production in the corpus callosum between WT and TRPM2-KO mice at days 14 and 28 after BCAS. A, B, Representative images (A) and relative leakage (B) evaluated by IgG staining. C, The level of measured H₂O₂ content. **p < 0.01 vs WT sham. Values are mean ± SEM. B, n = 4–8; C, n = 5–6. n.s., Not significant.

Figure 5.

TRPM2 mRNA upregulation and proinflammatory cytokine production were detected in the corpus callosum at days 14 and 28 after BCAS. A, The mRNA expression level of TRPM2 by real-time PCR. B–D, The protein levels of IL1β (B), TNFα (C), and IL6 (D) measured by ELISA. *p < 0.05, **p < 0.01, and ***p < 0.001 vs WT sham; ^#p < 0.05 and ^###p < 0.001 vs WT BCAS. Values are mean ± SEM. A, n = 10–13; B–D, n = 9–14. n.s., Not significant.

Figure 6.

The number of Iba1-positive cells in the corpus callosum and microglia-related genes were decreased in TRPM2-KO mice at days 14 and 28, with the pharmacological inhibition ameliorating the cognitive outcomes in BCAS-operated mice at day 28. A, B, Representative images (A) and the number (B) of GFAP-positive cells in the corpus callosum. C, D, Representative images (C) and the number (D) of Iba1-positive cells in the corpus callosum. A, C, Bottom, Magnified images from the location marked by the boxed area of the above panels. *p < 0.05 and **p < 0.01 vs sham groups; ^#p < 0.05 vs WT BCAS. E–K, The mRNA expression levels of CX3CR1 (E), CX3CL1 (F), MHCII (H2-Aa, G), CD68 (H), TLR4 (I), CD14 (J), and TREM2 (K) by real-time PCR. **p < 0.01 vs WT sham; ^#p < 0.05 and ^##p < 0.01 vs WT BCAS. L–N, Representative images (L) and the number (M) of Iba1-positive cells in the corpus callosum, and exploratory preference to the novel object in the test session (N), at day 28 after daily treatment with minocycline or vehicle. L, Bottom, Magnified images from the location marked by the boxed area of the above panels. *p < 0.05 and ***p < 0.001 vs vehicle sham; ^##p < 0.01, and ^###p < 0.001 vs vehicle BCAS. Values are mean ± SEM. B, D, n = 5–12; E–K, n = 6–16; M, n = 4–5; N, n = 12–13.

Figure 7.

The numbers of GFAP- and Iba1-positive cells were increased but there was a slight change of cytokines in the hippocampus of WT mice at 28 d after BCAS. A, B, E, F, I, J, Representative images (A, E, I) and the number (B, F, J) of GFAP-positive cells in CA1 (A, B), CA3 (E, F), and DG (I, J). C, D, G, H, K, L, Representative images (C, G, K) and the number (D, H, L) of Iba1-positive cells in CA1 (C, D), CA3 (G, H), and DG (K, L). *p < 0.05 and **p < 0.01 vs WT sham; ^#p < 0.05 vs WT BCAS. M–R, The mRNA expression level of TRPM2 (M), CX3CR1 (N), CX3CL1 (O), IL1β (P), TNFα (Q), and IL6 (R) by real-time PCR. **p < 0.01 vs WT sham; ^#p < 0.05 vs WT BCAS. S, The level of measured H₂O₂ content. Values are mean ± SEM. B, D, F, H, J, L, n = 4–6; M–R, n = 5–13; S, n = 5–6.

Figure 8.

In chimeric mice, Iba1-positive cells activated 28 d after BCAS were microglia, not macrophages. A–C, Representative images (A) and the number of GFP-positive cells (B) and Iba1-positive cells (C) in the corpus callosum of sham-operated chimeric mice. D–F, Representative images (D) and the number of GFP-positive cells (E) and Iba1-positive cells (F) in the corpus callosum of BCAS-operated chimeric mice. D, Bottom, Magnified images from the location marked by the boxed area of the above panels. G, H, Representative images (G) and the number of GSTpi-positive cells (H) in the corpus callosum. I, J, Representative image (I) and the number of GFP and CD3 double-positive cells (J) in the corpus callosum. K, Alternation behavior in the Y-maze test at day 28 after BCAS. L, Exploratory preferences to the novel object in the novel object recognition test session at day 28 after BCAS. *p < 0.05 and **p < 0.01 vs WT^WT-BM. Values are mean ± SEM. B, C, n = 3; E, F, H, J–L, n = 9–15.

Figure 9.

Schematics for the role of TRPM2 under chronic cerebral hypoperfusion.