Physiological and Pathophysiological Roles of Transient Receptor Potential Channels in Microglia-Related CNS Inflammatory Diseases

Abstract

Central nervous system (CNS) inflammation is a potential therapeutic target for neurodegenerative diseases. In recent years, a number of studies have focused on the links between neurodegenerative diseases and CNS glial cells, especially microglia. Microglia are the main resident immune cells in the CNS and represent approximately 10–15% of all CNS cells. Microglia play an important role in maintaining brain homeostasis at rest by surveying the environment, and engulfing apoptotic cells and debris in the healthy brain. However, under certain pathological conditions, microglia can generate neurotoxic factors, such as pro-inflammatory cytokines and molecules like nitric oxide (NO), which lead to CNS inflammatory diseases. In this review, we discuss the evidence that regulation of microglial ion channels may modulate CNS inflammation and subsequent tissue damage in neurological disorders. In particular, we discuss the role of transient receptor potential (TRP) channels in microglia in both acute and chronic inflammatory conditions, and describe the physiological and pathophysiological roles of TRP channels in CNS inflammatory pathways. Additionally, we describe the benefits of stimulation/inhibition of TRP channels in animal models of microglia-related CNS inflammatory diseases.

1. INTRODUCTION

The brain has long been regarded as an immune-privileged organ, where inflammation can only occur after the breakdown of the blood–brain barrier and subsequent infiltration by peripheral immune cells.^1,2) However, immunocompetent cells residing in the central nervous system (CNS) express pattern recognition receptors (PRRs), such as toll-like receptors (TLRs) and nucleotide binding oligomerization domain (NOD)-like receptors, which mediate innate immunity.^3,4) PRRs recognize microbial molecular motifs, known as pathogen-associated molecular patterns (PAMPs), which are not generally found in normal brain tissue but which typically accumulate in infected tissues. Recent studies clearly indicate that, in the absence of CNS infection, endogenous PRR ligands—so-called damage associated molecular patterns (DAMPs), also known as alarmins—are released in damaged CNS tissue, resulting in activation of the innate immune system within the CNS.^3,4) These pro-inflammatory responses in the immunocompetent cells in the brain contribute to the progression of neurodegenerative diseases by sustained release of pro-inflammatory mediators, which leads to CNS inflammation.^5,6) Although the molecular mechanisms remain to be elucidated, the inflammatory response during a wide variety of chronic CNS diseases is likely controlled by microglia, the main type of immunocompetent cells residing in the CNS.^7,8) In various chronic disease states, microglia are primed by prior pathological changes in the CNS to respond more vigorously to subsequent inflammatory stimulation, leading to more deleterious consequences.^7,8) Conversely, microglia also engulf and degrade apoptotic cells in the injured brain, resulting in neuroprotection, tissue repair, and regeneration.^2,9) Microglia switch to an activated phenotype in response to multiple factors, including PAMPs and DAMPs^2,9); therefore, the mechanisms underlying microglial regulation of neuronal damage and dysfunction are the subject of much research.

Microglia express a wide variety of ion channels that are involved in their cellular inflammatory responses.¹⁰⁾ Some of these channels are of particular interest because of their potential as targets for treatment of neurological diseases.^10,11) In this review, we focus on transient receptor potential (TRP) channels, which are widely expressed in many different tissues and cell types. The TRP family currently comprises 28 mammalian cation channels, subdivided into six subfamilies according to their sequence homology: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPML (mucolipin) and TRPP (polycystic). Most TRP channels are nonselective Ca²⁺-permeable cation channels that serve as cellular sensors for mechanical, thermal, and chemical stimuli.^12,13) TRP channels are involved in cellular processes such as cytokine production, proliferation, and migration, all of which are important cellular activities in microglia.^13,14) In this review, we will discuss the preclinical evidence for a significant role of TRP channels in microglial activation, particularly in neurological diseases. Mechanisms by which microglia may affect neuronal activity will be highlighted.

2. PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL ROLES OF TRPC CHANNELS IN MICROGLIA

The TRPC subfamily comprises seven members, namely TRPC1–C7, all of which are Ca²⁺-permeable nonselective cation channels that are strongly expressed in neurons and glia in the brain.¹⁵⁾ TRPC members are classified into two groups on the basis of their sequence homology and functional similarity: the TRPC1/TRPC4/TRPC5 and TRPC3/TRPC6/TRPC7 groups.¹⁶⁾ TRPC3, TRPC6, and TRPC7 form the diacylglycerol (DAG)-sensitive group and are activated by DAG after stimulation of phospholipase C (PLC)-coupled receptors such as G_q/11-type G-protein-coupled receptors or receptor tyrosine kinases. In microglia, there are only a few studies on the functional expression of TRPC channels. Quantitative comparisons of mRNA expression have shown that TRPC1, TRPC3, and TRPC6 are highly expressed in cultured microglia.¹⁷⁾ In line with this finding, double-label immunofluorescence studies show that TRPC3 and TRPC6 are weakly localized in microglia in vivo.^18,19) Recently, Liu et al. reported that amyloid β-protein (Aβ) upregulates TRPC6 via nuclear factor-kappaB (NF-κB) and promotes the production of cyclooxygenase-2 (COX-2) in BV-2 microglia, which could be involved in microglial activation-induced hippocampus neuronal damage.²⁰⁾ Moreover, Mizoguchi et al. reported that brain-derived neurotrophic factor (BDNF) induces sustained elevation of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) through the upregulation of surface TRPC3 channels.²¹⁾ They also found that TRPC3 channels is important for the BDNF-induced suppression of nitric oxide (NO) production in activated microglia, which might be important for the regulation of inflammatory responses and involved in the treatment of neuropsychiatryic disorders including depression. Further investigation would be expected to reveal the physiological and pathophysiological roles of TRPC channels in microglia.

In additional to their physiological role, TRPC channels also play a pathophysiological role in microglia-related inflammatory diseases in the CNS. In this context, Munakata et al. demonstrated that, in the collagenase/autologous blood infusion mouse model of intracerebral hemorrhage (ICH) that is a subtype of hemorrhagic stroke with high morbidity and mortality, Pyr3, a selective TRPC3 inhibitor, can reduce the perihematomal accumulation of microglia and astrocytes and attenuate neurological deficits, neuronal injury, and brain edema, clearly suggesting that TRPC3 contributes to the outcomes after ICH.²²⁾ Since thrombin, a critical blood-derived factors that invades the brain tissue in ICH, has been shown to activate TRPC3 via protease-activated receptor-1 (PAR-1, a G-protein-coupled receptor that activated by thrombin proteolytically) in 1321N1 human astrocytoma cells²³⁾ and cultured rat primary astrocytes in vitro,²⁴⁾ thrombin in the hematoma may induce TRPC3-mediated astrocyte activation via PAR-1 and result in neuroinflammation and neurodegeneration after ICH. However, PAR-1 is important for ICH-induced microglia activation,²⁵⁾ implying TRPC3 in microglia might be a new therapeutic target for the prevention of secondary brain injury and neurological deficits after ICH.

3. PHYSIOLOGICAL ROLES OF TRPM CHANNELS IN GLIA

The TRPM subfamily comprises eight members, namely TRPM1–M8, some of which are sensitive to oxidative stress.²⁶⁾ TRPM2 was the first TRP channel to be identified as redox-sensitive.²⁷⁾ It is strongly expressed in the brain and immune cells and its role in the CNS has been studied extensively. TRPM2 forms a Ca²⁺-permeable cation channel that is activated by oxidative stress mediated by reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂), through the production of nicotinamide adenine dinucleotide and its metabolites, such as ADP-ribose (ADPR) and cyclic ADPR.^27,28) Studies indicate that TRPM2 mediates H₂O₂-induced Ca²⁺ influx, which modulates physiological and pathological cellular functions. TRPM2 is expressed in both neurons and glia, and oxidative-stress-induced TRPM2 activation is implicated in neuronal diseases. Several studies have focused on the physiological and pathophysiological roles of TRPM2 in microglia because of the myeloid–monocytic lineage of microglia. TRPM2 functions as a Ca²⁺-permeable channel in mouse monocytes.²⁹⁾ Kraft et al. reported that TRPM2 is functionally expressed at high levels in cultured rat microglia.³⁰⁾ Using cultured microglia derived from wild-type and TRPM2 knockout mice, Haraguchi et al. showed that TRPM2 is involved in NO production.³¹⁾ Subsequently, Miyake et al. examined the intracellular signaling mechanisms underlying these phenomena and demonstrated that combined application of lipopolysaccharide (LPS) and interferon-γ can stimulate TRPM2-mediated extracellular Ca²⁺ influx in cultured microglia.³²⁾ They also showed that activation of TRPM2 results in proline-rich tyrosine kinase 2 (Pyk2)-mediated activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase (JNK) signaling, leading to the increased NO production in microglia. The sulfonylurea receptor 1-TRPM4 channel regulates NO synthase 2 transcription in TLR4-activated microglia.³³⁾ TRPM7 is functionally expressed in cultured rat microglia,³⁴⁾ and is essential for the enhanced ability of microglia to migrate and invade during anti-inflammatory states.³⁵⁾

4. PATHOPHYSIOLOGICAL ROLES OF TRPM CHANNELS IN MICROGLIA

4.1. Microglial/Macrophagic TRPM2 Channels in Neuropathic Pain

TRPM2 is thought to be involved in a range of pathological pain states, including neuropathic pain.^31,36,37) Evidence suggests that peripheral and spinal neuroinflammation mediated by the interaction between nociceptive neurons and immune/glial cells plays a pivotal role in neuropathic pain.³⁸⁾ After peripheral nerve injury, pro-nociceptive inflammatory mediators, such as pro-inflammatory cytokines, chemokines, and excess ROS produced by peripheral tissues and the spinal cord, can lead to peripheral and central sensitization of nociceptive neurons. In this context, Haraguchi et al., demonstrate that TRPM2 expressed in macrophages and microglia plays a critical role in neuropathic pain. TRPM2 is responsible for chemokine (C-X-C motif) ligand 2 (CXCL2) and NO production in macrophages and microglia, which aggravate peripheral and spinal pro-nociceptive inflammatory responses, respectively, in inflammatory and neuropathic pain models.^31,32) Furthermore, TRPM2 may play a role in the infiltration of peripheral immune cells into the spinal cord after peripheral nerve injury.³⁶⁾ Consequently, TRPM2 is involved in a wide range of pathological pain states induced by peripheral and spinal neuroinflammation—such as inflammatory pain, osteoarthritic pain, neuropathic pain induced by peripheral nerve injury, chemotherapy-induced peripheral neuropathy, and painful diabetic neuropathy—rather than in physiological nociceptive pain.³⁷⁾

4.2. TRPM2 Channels in Cerebrovascular Disease

TRPM2 is thought to be involved in stroke, especially in ischemic cerebral infarction, which is the leading cause of death and permanent disability in adults worldwide. TRPM2 is thus a potential therapeutic target for stroke. The injury mechanisms following ischemic stroke are multifaceted. Oxidative stress induced by cerebral ischemia–reperfusion injury is considered to be the main event leading to neuronal death. Several lines of evidence indicate that TRPM2 mediates ROS-induced neuronal death. TRPM2 acts as a redox-sensitive Ca²⁺-permeable channel and thus has a pivotal role in H₂O₂-induced neuronal death in primary cultured neurons.³⁹⁾ TRPM2 is activated by intracellular ADPR that is overproduced in response to oxidative stress and ROS, such as H₂O₂.²⁷⁾ In line with these findings, several studies indicate that TRPM2 mediates ischemic brain damage. For example, Alim et al. used wild-type and TRPM2 knockout mice to show that the genetic ablation of TRPM2 causes a shift in the expression ratio of the GluN2A/GluN2B subunits of the N-methyl-D-aspartate receptor, which may selectively upregulate survival pathways and result in neuroprotection from cerebral-ischemia-induced neuronal cell death in vivo.⁴⁰⁾ Shimizu et al. reported an extended therapeutic window for treatment with a novel peptide inhibitor of TRPM2 channels after focal cerebral ischemia, demonstrating that TRPM2 is a promising candidate for treatment of acute cerebral ischemic infarction.⁴¹⁾ However, recent studies have focused extensively on the pathophysiological role of TRPM2 in microglia/macrophages. Application of lysophosphatidylcholine induces intracellular Ca²⁺ influx and increases phosphorylation of p38 mitogen-activated protein kinase (MAPK) via TRPM2, which in turn activates microglia.⁴²⁾ In addition, TRPM2-mediated Ca²⁺ influx induces the production of pro-inflammatory cytokines/chemokines in monocytes.²⁹⁾ TRPM2 is also involved in chemokine CXCL2 and NO production in cultured macrophages and microglia.^31,32) In line with these findings, TRPM2 regulates the migratory capacity of neutrophils and macrophages in response to ischemic brain injury, thereby secondarily perpetuating brain injury after the ischemic event.⁴³⁾ Taken together, these results suggest that TRPM2 in microglia/macrophages could mainly contribute to the development of cerebral ischemic injury.

4.3. Microglial TRPM2 Channels in Cognitive Impairment in CNS Diseases

Recently, TRPM2 channels in resident microglia were shown to play a critical role in the pathogenesis of chronic cerebral hypoperfusion through the aggravation of inflammatory responses.⁴⁴⁾ Chronic cerebral hypoperfusion manifests in a wide range of CNS diseases, including neurodegenerative disorders and mental disorders that are accompanied by cognitive impairment; however, the underlying mechanisms remain unclear. In this study, chronic cerebral hypoperfusion can be modeled in mice by bilateral common carotid artery stenosis (BCAS), which results in a decline in cognitive function and excessive release of pro-inflammatory cytokines such as tumor necrosis factor α (TNFα) and interleukin 6 (IL6), all of which were suppressed in TRPM2 knockout mice. TRPM2 knockout mice also had significantly decreased accumulation of microglia in the corpus callosum, although there was no difference in the number of glial fibrillary acidic protein (GFAP)-positive cells between genotypes. Moreover, BCAS mice treated daily with the anti-inflammatory antibiotic minocycline showed significant improvements in their cognitive function.⁴⁴⁾ The schematic in Fig. 1 summarizes the role of TRPM2 in pathology caused by chronic cerebral hypoperfusion. Taken together, these findings suggest a new hypothesis, in which TRPM2-mediated activation of microglia specifically contributes to the pathology through the aggravation of inflammatory responses.

Fig. 1. Schematic of the Pathophysiological Role of TRPM2 during Chronic Cerebral Hypoperfusion

ROS are produced locally to the chronic cerebral hypoperfusion, resulting in the specific activation of microglial TRPM2 channels. Excessive release of pro-inflammatory cytokines such as TNFα, IL1β, and IL6, presumably from microglia, can induce CNS inflammation and white matter injury, ultimately leading to cognitive impairment (see ref. 44).

In this context, Ostapchenko et al. clearly demonstrate that genetic elimination of TRPM2 normalizes deficits in synaptic markers in aged mice, improves age-dependent spatial memory deficits and reduces microglial activation of hippocampus in mice designed to model Alzheimer’s disease.⁴⁵⁾ More recently, Alawieyah Syed Mortadza et al. report that exposure to 10–300 nM Aβ42 induces concentration-dependent microglial activation and generation of TNFα that are ablated by genetically deleting of TRPM2 or pharmacologically inhibiting TRPM2, revealing a critical role of TRPM2 in Aβ42-induced microglial activation.⁴⁶⁾ These reports shed light on our understanding of the mechanisms of chronic cerebral hypoperfusion-related inflammation, and suggest a novel therapeutic target for the treatment of cognitive impairment in CNS diseases.

5. PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL ROLES OF TRPV CHANNELS IN MICROGLIA

The TRPV subfamily is expressed in glial cells, especially astrocytes and microglia. Microglia express several TRPV isoforms, particularly TRPV1, TRPV2, and TRPV4. Activation of TRPV1 triggers Ca²⁺ signaling-dependent cell death in microglia⁴⁷⁾ and contributes to reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase-mediated production of ROS in microglia.⁴⁸⁾ Miyake et al. demonstrated that activation of TRPV1 triggers an increase in intramitochondrial Ca²⁺ concentration and following depolarization of mitochondria, which results in mitochondrial ROS production, MAPK activation, and enhancement of chemotactic activity in microglia⁴⁹⁾ (see Fig. 2). Marrone et al. clearly demonstrated that stimulation of microglial TRPV1 controls cortical microglia activation per se and indirectly enhances glutamatergic transmission in neurons by promoting extracellular microglial microvesicles shedding.⁵⁰⁾ In this context, in an experimental model of Parkinson’s disease, the TRPV1 selective agonist capsaicin inhibits microglia-mediated ROS production, leading to the resultant blockade of the death of dopaminergic neurons.⁵¹⁾ Moreover, TRPV1 partially regulates cytokine production induced by elevated hydrostatic pressure in retinal microglia.⁵²⁾ Further investigations are warranted to clarify the pathophysiological roles of microglial TRPV1 in CNS diseases.

Fig. 2. Schematic of the Physiological Role of TRPV1 in Microglial Migration

TRPV1 is functionally expressed in mitochondria and its activation by capsaicin or endovanilloids triggers an increase in intramitochondrial Ca²⁺ concentration, depolarization of mitochondria, resultant mtROS production, JNK/p38 activation and enhancement of chemotactic activity in microglia (see ref. 49).

With respect to TRPV2 and TRPV4, activation of TRPV2 (and also TRPV1) by cannabidiol results in enhanced microglial phagocytosis.⁵³⁾ In addition, Konno et al. demonstrates that stimulation of TRPV4 in cultured rat microglia suppresses LPS-induced TNFα release and galectin-3 upregulation, and also suppresses augmentation of voltage-dependent K⁺ currents, suggesting that depolarization in response to opening of the TRPV4 channel attenuates the driving force for extracellular Ca²⁺ and suppresses microglial activation⁵⁴⁾ (see Fig. 3).

Fig. 3. Schematic of the Role of TRPV4 in Microglial Activation

TRPV4 is functionally expressed in mitochondria and opening of TRPV4 channels, not by blocking of TRPV4, induces membrane depolarization mainly by increasing Na⁺ influx. The LPS-induced morphological changes and increases in TNFα release, galectin-3 expression and K⁺ current amplitude are all attenuated by TRPV4-mediated depolarization of microglia, leading to the attenuation of Ca²⁺ influx and activation of microglia are finally suppressed (see ref. 54).

6. CONCLUSION

Recent studies emphasize the significance of TRP channels and microglial activation in CNS inflammation and the pathologies of various neurological diseases. In this review, we summarized the evidence for the potential molecular mechanisms underlying stimulation or inhibition of TRP channels, and the pathways leading from TRP activation to the modulation of microglia and resultant CNS inflammation. Notably, microglial TRPV1 and TRPM2 play essential roles in various pathological CNS conditions, such as Parkinson’s disease, neuropathic pain, Alzheimer’s disease and chronic cerebral hypoperfusion, and, as such, are promising therapeutic targets for these diseases. Of note, suppression of microglial activation and subsequent downstream signaling can be achieved not only through the loss or inhibition of TRP channel function, but also through TRP stimulation (for example, stimulation of microglial TRPV1 and TRPV4). This suggests that the different types of TRP channel affect microglial activation independently. Although the precise mechanisms underlying TRP channel function remain to be verified in vivo, our hope is that TRP channel activators and/or inhibitors will be developed as therapeutic tools to treat neurological diseases in which CNS inflammation is a factor.

Acknowledgments

This work was supported by Ministry of Education, Culture, Sports, Science and Technology (MEXT)/JSPS KAKENHI Grant Numbers 17K19486 (to H.S.), 24390016 (to S.K.), and also supported by the Takeda Science Foundation and the Novartis Foundation (to H.S.).

Conflict of Interest

The authors declare no conflict of interest.

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