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 Ca-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 HO 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. 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 Ca-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.

Microglia are immune cells in the central nervous system (CNS) and essential for homeostasis that are important for both neuroprotection and neurotoxicity, and are activated in a variety of CNS diseases. Microglia aggravate cognitive impairment induced by chronic cerebral hypoperfusion, but their precise roles under these conditions remain unknown. Here, we used PLX3397, a colony-stimulating factor 1 receptor inhibitor, to deplete microglia in mice with chronic cerebral hypoperfusion induced by bilateral common carotid artery stenosis (BCAS). Cognitive impairment induced 28 days after BCAS was significantly improved in mice fed a diet containing PLX3397. In PLX3397-fed mice, microglia were depleted and white matter injury induced by BCAS was suppressed. In addition, the expression of proinflammatory cytokines, interleukin 6 and tumor necrosis factor alpha, was suppressed in PLX3397-fed mice. Taken together, these findings suggest that microglia play destructive roles in the development of cognitive impairment and white matter injury induced by chronic cerebral hypoperfusion. Thus, microglia represent a potential therapeutic target for chronic cerebral hypoperfusion-related diseases.

Chronic inflammatory bladder disorders, such as interstitial cystitis/bladder pain syndrome, are associated with poor quality of life. The exact pathological processes remain unclear, but accumulating evidence suggests that reactive oxidative species (ROS) are involved in urinary bladder disorders. Transient receptor potential ankyrin 1 (TRPA1), the most sensitive TRP channel to ROS, was shown to be responsible for urinary bladder abnormalities and hyperalgesia in an acute cystitis model. However, the roles of TRPA1 in chronic inflammatory bladder are not fully understood. We previously established a novel mouse cystitis model induced by intravesical injection of hydrogen peroxide (H2O2), resulting in long-lasting frequent urination, bladder inflammation, pain-related behavior, and histopathological changes. In the present study, we investigated the pathophysiological role of TRPA1 in the H2O2-induced long-lasting cystitis mouse model. Under anesthesia, 1.5% H2O2 solution was introduced transurethrally into the bladder of female wild-type (WT) and TRPA1-knockout mice and maintained for 30 min. This increased the number of voids in WT mice at 1 and 7 days after injection, but reduced the number in TRPA1-knockout mice at 1 day but not 7 days after injection. Spontaneous locomotor activities (increase in freezing time and decrease in distance moved) were reduced at 3 h after injection in WT mice, whereas the spontaneous visceral pain-related behaviors were attenuated in TRPA1-knockout mice. Furthermore, upregulation of c-fos mRNA in the spinal cord at 1 day after injection was observed in WT but not TRPA1-knockout mice. However, there was no difference in histopathological changes in the urinary bladder, such as edematous thickening in the submucosa, between WT and TRPA1-knockout mice at 1 or 7 days after injection. Finally, Trpa1 mRNA levels in the L5-S1 dorsal root ganglion were not altered, but levels in the urinary bladder were drastically increased at 1 and 7 days after injection. Taken together, these results suggest that TRPA1 contributes to acute bladder hyperactivity such as frequent urination and bladder pain, but does not appear to play a major role in the pathological processes of long-lasting cystitis.

Aging causes various functional changes, including cognitive impairment and inflammatory responses in the brain. Transient receptor potential melastatin 2 (TRPM2), a Ca 2+-permeable channel expressed abundantly in immune cells, exacerbates inflammatory responses. Previously, we reported that TRPM2 on resident microglia plays a critical role in exacerbating inflammation, white matter injury, and cognitive impairment during chronic cerebral hypoperfusion; however, the physiological or pathophysiological role of TRPM2 during age-associated inflammatory responses remains unclear. Therefore, we examined the effects of TRPM2 deletion in young (2-3 months) and older (12-24 months) mice. Compared with young wild-type (WT) mice, middle-aged (12-16 months) WT mice showed working and cognitive memory dysfunction and aged (20-24 months) WT mice exhibited impaired spatial memory. However, these characteristics were not seen in TRPM2 knockout (TRPM2-KO) mice. Consistent with the finding of cognitive impairment, aged WT mice exhibited white matter injury and hippocampal damage and an increase in the number of Iba1-positive cells and amounts of pro-inflammatory cytokines in the brain; these characteristics were not seen in TRPM2-KO mice. These findings suggest that TRPM2 plays a critical role in exacerbating inflammatory responses and cognitive dysfunction during aging.

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Vascular cognitive impairment (VCI) refers to cognitive alterations caused by vascular disease, which is associated with various types of dementia. Because chronic cerebral hypoperfusion (CCH) induces VCI, we used bilateral common carotid artery stenosis (BCAS) mice as a CCH-induced VCI model. Transient receptor potential ankyrin 1 (TRPA1), the most redox-sensitive TRP channel, is functionally expressed in the brain. Here, we investigated the pathophysiological role of TRPA1 in CCH-induced VCI. During early-stage CCH, cognitive impairment and white matter injury were induced by BCAS in TRPA1-knockout but not wild-type mice. TRPA1 stimulation with cinnamaldehyde ameliorated BCAS-induced outcomes. RNA sequencing analysis revealed that BCAS increased leukemia inhibitory factor (LIF) in astrocytes. Moreover, hydrogen peroxide–treated TRPA1-stimulated primary astrocyte cultures expressed LIF, and culture medium derived from these cells promoted oligodendrocyte precursor cell myelination. Overall, TRPA1 in astrocytes prevents CCH-induced VCI through LIF production. Therefore, TRPA1 stimulation may be a promising therapeutic approach for VCI.

Vascular cognitive impairment (VCI) is a syndrome defined as cognitive decline caused by vascular disease, which is associated with Alzheimer's disease and vascular dementia. Since chronic cerebral hypoperfusion (CCH) is commonly present in various types of dementia and induces VCI, we used bilateral common carotid artery stenosis (BCAS) mice as a CCH-induced VCI model. Transient receptor potential ankyrin 1 (TRPA1), the most redox-sensitive TRP channel, is functionally expressed in the brain and seems to function as a polymodal sensor in vascular disease. To clarify the involvement of TRPA1 in CCH-induced VCI, we used genetically engineered mice: TRPA1-knockout (TRPA1-KO) and cell-specific conditional TRPA1-KO mice. We showed that TRPA1 deficiency exacerbated BCASinduced cognitive impairment and white matter injury during early-stage CCH. TRPA1 stimulation with cinnamaldehyde ameliorated BCAS-induced outcomes. We also revealed that BCAS increased a cytokine in astrocytes. Moreover, TRPA1-stimulated primary astrocyte cultures expressed the cytokine, and culture medium derived from these TRPA1-stimulated cells promoted oligodendrocyte precursor cell myelination. Overall, TRPA1 stimulation in astrocytes plays a protective role in CCH-induced VCI through the cytokine production.

Astrocytes are glial cells that serve homeostatic functions in the central nervous system (CNS). Recent research, however, suggests that under pathological conditions, astrocytes are stimulated by various factors and actively participate in CNS inflammation. In the present study, we found that astrocytes upregulate various inflammatory factors including prostaglandin E 2 (PGE 2 ) by co-stimulation with tumor necrosis factor-alpha (TNFα) and interleukin-1alpha (IL1α). These TNFα/IL1α-stimulated astrocytes also showed increased Ca 2+ release from the endoplasmic reticulum (ER) and increased expression of Orai2, a member of the store-operated calcium channel (SOCC) family. To reveal the role of Orai2, we used astrocytes in which Orai2 was knocked-down (KD) or knocked-out (KO). The expression of the prostaglandin E synthase Ptges and the production of PGE 2 were higher in Orai2-KD astrocytes than in WT astrocytes when stimulated with TNFα and IL1α. Orai2-KO astrocytes also showed increased expression of Ptges and increased PGE 2 production. The expression of Ptgs2, another PGE 2 synthetic enzyme, was also upregulated in Orai2-KO astrocytes. Moreover, Orai2-KO astrocytes showed increased store-operated calcium entry (SOCE) and increased Orai1 expression. These results suggest that Orai2 is upregulated in TNFα/IL1α-stimulated astrocytes and reduces PGE 2 production to some extent, modulating CNS inflammation. Our findings may aid in understanding how astrocytes are associated with inflammatory responses, and the identification of new targets that modulate astrocytic reactivity.