Roles of glial ion transporters in brain diseases

Abstract

Glial ion transporters are important in regulation of ionic homeostasis, cell volume, and cellular signal transduction under physiological conditions of the central nervous system (CNS). In response to acute or chronic brain injuries, these ion transporters can be activated and differentially regulate glial functions, which has subsequent impact on brain injury or tissue repair and functional recovery. In this review, we summarized the current knowledge about major glial ion transporters, including Na⁺/H⁺ exchangers (NHE), Na⁺/Ca²⁺ exchangers (NCX), Na⁺–K⁺–Cl⁻ cotransporters (NKCC), and Na⁺–HCO₃⁻ cotransporters (NBC). In acute neurological diseases, such as ischemic stroke and traumatic brain injury (TBI), these ion transporters are rapidly activated and play significant roles in regulation of the intra- and extracellular pH, Na⁺, K⁺, and Ca²⁺ homeostasis, synaptic plasticity, and myelin formation. However, overstimulation of these ion transporters can contribute to glial apoptosis, demyelination, inflammation, and excitotoxicity. In chronic brain diseases, such as glioma, Alzheimer’s disease (AD), Parkinson’s disease (PD), and multiple sclerosis (MS), glial ion transporters are involved in the glioma Warburg effect, glial activation, neuroinflammation, and neuronal damages. These findings suggest that glial ion transporters are involved in tissue structural and functional restoration, or brain injury and neurological disease development and progression. A better understanding of these ion transporters in acute and chronic neurological diseases will provide insights for their potential as therapeutic targets.

1. |. GLIAL ION TRANSPORT IN ACUTE NEUROLOGICAL DISEASES

1.1 |. Glial cells and glial ion transporters

Increasing evidence suggests that glial cells, including astrocytes, microglia, and oligodendrocytes, are not only neuron supporters, but also master regulators that control various aspects of brain functions (Zuchero & Barres, 2015). Astrocytes regulate normal brain functions through blood flow and vasculature support (Mishra, Pal, Gupta, & Carmel, 2017), blood-brain barrier (BBB) formation and function (Erdo, Denes, & de Lange, 2017), neurotrophic factor secretion (Jha et al., 2018), and formation of the tripartite synapses and neural circuit remodeling (Chai et al., 2017; Kim, Nabekura, & Koizumi, 2017). Microglia are the resident phagocytes in the central nervous system (CNS) and function as the first line of defense against injury (Ma, Wang, Wang, & Yang, 2017). Under physiological conditions, microglial cells extend their branching processes in a ramified morphology and constantly scan their surrounding environment for surveillance (Wolf, Boddeke, & Kettenmann, 2017). The microglial processes can also make direct contact with neuronal synapses (Wake, Moorhouse, Jinno, Kohsaka, & Nabekura, 2009) and plays an important role in synapse elimination and pruning, synapse formation, and neuronal network synchronization in both developing and adult brains (Akiyoshi et al., 2018; Miyamoto et al., 2016; Reshef et al., 2017). Oligodendrocyte precursor cells (OPCs) migrate within the brain parenchyma and differentiate into mature oligodendrocytes, which can extend their cell membrane protrusions to form myelinating sheaths around the neuronal axons for fast saltatory nerve conduction between nodes of Ranvier (Domingues et al., 2018). Myelination by oligodendrocytes is especially enriched in the white matter, and is regulated by neuronal activity (Gibson et al., 2014). Maintenance of oligodendrocytes and myelination homeostasis is vital for normal neurological functions, such as cognitive, perceptual, and sensorimotor functions (Wang, Liu, et al., 2016). Collectively, glial cells are required for physiological brain functions by providing structural and material support, as well as interacting with axons and synapses for signal transduction and neuronal network regulation.

Increasingly accumulated research shows that several major glial ion transporters, including Na⁺/H⁺ exchangers (NHE), Na⁺/Ca²⁺ exchangers (NCX), Na⁺–K⁺–Cl⁻ cotransporters (NKCC), and Na⁺–HCO₃⁻ cotransporters (NBC), play important roles in regulation of glial functions. In acute neurological diseases, such as ischemic stroke and traumatic brain injury (TBI), these ion transporters are rapidly activated to regulate the intra- and extracellular pH, Na⁺, K⁺, and Ca²⁺ homeostasis, synaptic plasticity, and myelin formation. However, pathological overstimulation of these ion transporters can contribute to glial apoptosis, demyelination, inflammation, and excitotoxicity (Figure 1). In this section, we will summarize the roles of these glial ion transporters in the functional changes of astrocytes, microglia, and oligodendrocytes. These findings reveal the potential of targeting these ion transporter proteins for therapeutic treatment for acute neurological diseases.

FIGURE 1.

Roles of glial ion transporters in ischemic brain damage. Ischemic stroke results in an immediate marked reductions of intracellular pH (pH_i) as a consequence of oxygen depletion, which necessitates a switch from aerobic metabolism to anaerobic glycolysis, leading to generation of intracellular lactic acid as well as protons. Several ion transporters/exchangers in astrocytes, microglia, and/or oligodendrocytes are activated to prevent the pH_i dysregulation. In astrocytes, Na⁺–HCO₃⁻ cotransporter (NBC) transports HCO₃⁻ to alkalinize pH_i. Stimulation of Na⁺/H⁺ exchanger isoform-1 (NHE1) extrudes H⁺ in exchange of Na⁺ influx, which leads to astrocytic swelling and disruption of astrocytic end-feet function at the blood-brain barrier (BBB), and contributes to inflammation by secretion of various cytokines. The increased Na⁺ overload triggers the reversal operation of Na⁺/Ca²⁺ exchanger (NCX_rev) and causes intracellular overload of Ca²⁺, and reduces glutamate uptake via the Na⁺-dependent excitatory amino acid transporter (EAAT). Astrocytic Na⁺–K⁺–Cl⁻ cotransporter (NKCC1) activation also contributes to astrocyte swelling and cascade events as described above. In microglia, NHE1 maintains the optimal pH_i for NADPH oxidase (NOX2) activation, together with NCX_rev activation, promoting the release of reactive oxygen species (ROS) and contributes to oxidative injury. In oligodendrocytes, NKCC1 is activated via the upstream WNK-SPAK/OSR1 pathway, and triggers NCX_rev function and enhances excitotoxicity and apoptosis. Taken together, NHE1, NCX, NKCC1, and NBC play important roles in regulation of astrocytic, microglial, and oligodendrocytic functions in response to ischemic stroke

1.1.1 |. Ion transporters in reactive astrocytes after ischemic stroke

Ischemic stroke results from occlusions in major cerebral arteries, most commonly the middle cerebral artery, and leads to irreversible neuronal as well as glial cell death in the ischemic core due to lack of of glucose and oxygen (Puig, Brenna, & Magnus, 2018). Astrocytes, microglia, and oligodendrocytes in the brain tissue surrounding the ischemic core (the penumbra) alter their functions in response to the ischemic insult for restricting injury and restoring brain functions or accelerating cell death and ischemic infarct (Liu & Chopp, 2016; Ma et al., 2017; Shindo et al., 2016).

Within minutes after ischemic stroke, astrocytes in the penumbra are rapidly activated by cytokines produced by injured neurons in the ischemic lesion (Liu & Chopp, 2016; Pekny et al., 2016). Within a few days after the ischemic insult, these reactive astrocytes exhibit stimulated proliferation and elongated processes to form a glial scar, a physical wall surrounding the ischemic lesion, which functions in isolating the ischemic core and preventing the diffusion of detrimental factors into the nonlesioned remote area (Liu & Chopp, 2016; Pekny et al., 2016). The reactive astrocytes can be beneficial by secreting restorative growth factors such as brain derived neurotrophic factor (BDNF; Miyamoto et al., 2015), transforming growth factor (TGF)-β (Cekanaviciute et al., 2014), and ciliary neurotrophic factor (CNTF) for poststroke tissue repair (Kang, Keasey, Cai, & Hagg, 2012). However, the astrocytic glial scar can also secrete chondroitin sulfate proteoglycans (CSPG) that inhibits axonal regeneration and hinders the functional recovery during the late phase after ischemic stroke (Liu & Chopp, 2016). A transcriptome analysis of the reactive astrocytes isolated from the ischemic brains showed that the signal transducer and activator of transcription-3 (STAT3) pathway was activated and contributed to the increased secretion of detrimental molecules, such as galectin-3, lipocalin-2, and osteopontin (Rakers et al., 2019). Selective deletion of the Stat3 gene in astrocytes reduced ischemic infarction and improved motor functions in a mouse model of ischemic stroke (transient middle cerebral artery occlusion, tMCAO; Rakers et al., 2019).

These studies illustrate that reactive astrocytes have both detrimental and restorative functions, and transformation of astrocytes toward the protective phenotype is important for the resolution of ischemic brain injury. Multiple ion transporters have been reported to mediate the switch between beneficial or detrimental phenotypes of astrocytes.

Astrocytic NHE1 in pH_i, homeostasis, BBB breakdown, ER stress, and inflammation

Among nine members of the NHE family (encoded by SLC9A1-SLC9A9 genes), NHE1 protein is the most abundantly expressed isoform in the CNS (Orlowski & Grinstein, 2004). NHE1 is ubiquitously expressed in all cell types in the brain and regulates pH_i and cell volume homeostasis by mediating H⁺ extrusion in exchange of Na⁺ influx at a 1:1 ratio (Shi, Kim, Caldwell, & Sun, 2013).

The steady-state pH_i in mouse cortical astrocytes are reported at 7.0–7.2 in CO₂/HCO₃⁻ buffered saline when both NBC and NHE1 are functioning (Chesler, 2005; Theparambil & Deitmer, 2015). In the absence of HCO₃⁻, NHE1 functions as the major regulator for astrocytic pH_i and maintains their resting pH_i at about 6.86 (Kintner et al., 2004). Knockout of Nhe1 gene acidified the astrocytic pH_i to 6.53 (Kintner et al., 2004). Under hypoxic conditions, in part mediated by the activation of extracellular signal regulated kinase (ERK) signaling pathway (Kintner, Look, Shull, & Sun, 2005), NHE1 in astrocytes was rapidly activated for extrusion of the excess cytosolic H⁺ and pH_i recovery, which was abolished by either genetic knockout or pharmacological inhibition of NHE1 in astrocytes (Kintner et al., 2004; Wang, Li, et al., 2016).

On the other hand, NHE1-mediated Na⁺ influx led to astrocytic swelling and was associated with reduced astrocyte viability (Wang, Li, et al., 2016). Both genetic knockout and pharmacological inhibition of astrocytic NHE1 significantly reduced Na⁺_i overload (Kintner et al., 2004), decreased astrocytic aquaporin (AQP)-4 expression, and increased resistance of astrocytes to swelling in response to oxygen glucose deprivation (OGD; Kintner et al., 2004; Rutkowsky, Wallace, Wise, & O’Donnell, 2011). Selective deletion of Nhe1 gene in astrocytes also reduced brain edema, along with alleviated BBB breakdown, swelling of endothelial cells, and structural interruption of tight junctions, altogether leading to an increased regional cerebral blood flow in mice after tMCAO (Begum et al., 2018). These astrocytic Nhe1-null mice also exhibited reduced astrogliosis and infarct volume, as well as improved neurological functions after tMCAO (Begum et al., 2018). In addition, the elevated Na⁺_i led to activation of the reverse mode of the NCX (NCX_rev) in astrocytes, which subsequently caused the overload of Ca²⁺ stores in the endoplasmic reticulum (ER) and the mitochondria, contributing to ER stress, mitochondrial dysfunction, and astrocytic apoptosis after OGD (Bondarenko, Svichar, & Chesler, 2005; Cengiz et al., 2014; Kintner et al., 2005). The NHE1-mediated Na⁺_i increase also triggered reversal of the Na⁺-dependent excitatory amino acid transporter (EAAT), which mediated glutamate release from astrocytes, leading to excitotoxicity after OGD (Cengiz et al., 2014). Moreover, astrocytic NHE1 is involved in proinflammatory cytokine release after ischemia, such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and matrix metallopeptidase (MMP)-9 (Begum et al., 2018; Cengiz et al., 2014). Pharmacological inhibition or genetic knockout of astrocytic NHE1 protein significantly reduced these cytokine levels after ischemia (Begum et al., 2018; Cengiz et al., 2014).

Therefore, upon ischemic stroke, the overstimulated astrocytic NHE1 activity plays an important role in maintaining the astrocytic pH_i homeostasis, but also mediates astrocytic end-feet swelling, BBB breakdown, and inflammation. Inhibition of astrocytic NHE1 activity reduces brain edema and inflammation for ischemic brain tissue repair.

Astrocytic NCX in synaptic plasticity, excitotoxicity, ER stress, and astrocyte apoptosis

NCX mediates three Na⁺ ion influx and one Ca²⁺ extrusion across the cell membrane under physiological conditions, whose stoichiometric and electrogenic characteristics regulate Na⁺- and Ca²⁺-dependent signaling, as well as membrane potentials in the brain (Khananshvili, 2013). Depending on the intracellular and extracellular Na⁺ and Ca²⁺ levels, NCX can operate either in forward mode, extruding Ca²⁺ in exchange of Na⁺ influx, or in reverse mode, coupling Na⁺ efflux to the Ca²⁺ influx (Khananshvili, 2013).

Astrocytes express all three isoforms of NCX (NCX1-3; Parpura, Sekler, & Fern, 2016; Rose & Verkhratsky, 2016), with NCX1 transcript being the most predominant (Sakaue et al., 2000). Glutamatergic neuronal activity caused drastic elevations of Na⁺_i through the astrocytic NCX in neocortical but not hippocampal astrocytes (Ziemens, Oschmann, Gerkau, & Rose, 2019), which promoted astrocytic metabolism (Chatton, Magistretti, & Barros, 2016), and drove the Ca²⁺-mediated signaling (Ziemens et al., 2019). Astrocytic NCX in reverse mode (NCX_rev) is the major contributor for maintaining Na⁺ and Ca²⁺ homeostasis in astrocytes both at resting states and with injury stimulus (Reyes, Verkhratsky, & Parpura, 2012). Specific blockade of NCX_rev activity with KB-R7943 showed a significant decrease in the astrocytic cytosol Ca²⁺ concentration, while blockade of NCX with benzamil did not show such effect (Reyes et al., 2012).

Astrocytic NCX activation could play multiple roles in healthy and ischemic brains. On one hand, the astrocytic NCX is located on the plasma membrane directly opposing the ER (Blaustein, Juhaszova, Golovina, Church, & Stanley, 2002), indicating that NCX-mediated Ca²⁺ flux closely relates to ER functions in astrocytes. In fact, in cultured astrocytes, NCX_rev activity can be induced by increased local Na⁺_i concentration, either mediated by Na⁺ pump inhibition or elevated NKCC1 activity, and led to cytosolic Ca²⁺_i elevation and subsequent augmentation of the adjacent ER Ca²⁺ stores, which contributed to ER stress in ischemic conditions (Blaustein et al., 2002; Golovina, Song, James, Lingrel, & Blaustein, 2003; Kintner et al., 2007; Lenart, Kintner, Shull, & Sun, 2004). On the other hand, the NCX_rev-mediated Ca²⁺ influx can also be stored at the proximal processes of astrocytes called “branchlets” (Semyanov, 2019), where the release of the Ca²⁺ store is triggered by synaptic activity near the peri-synaptic distal end processes of astrocytes (“leaflets”) through inositol-3-phosphate (IP₃)-dependent mechanisms, and in turn spreads the Ca²⁺-mediated events and regulated neuronal network and synaptic plasticity (Minelli et al., 2007; Semyanov, 2019). In addition, the mitochondrial subtype of NCX in astrocytes, primarily the Na⁺, Li⁺/Ca²⁺ exchanger (NCLX), which exchanges Na⁺ or Li⁺ for Ca²⁺ (Sharma, Roy, Sekler, & O’Halloran, 2017), can mediate Ca²⁺ release in post-tetanic potentiation, a form of short-term synaptic plasticity (Castaldo et al., 2009; Gobbi et al., 2007). We speculate that changes of NCX activity in different brain regions/astrocytic cellular locations could be involved in learning, memory, and information processing functions in the brain. These regulations are also Na⁺-dependent, and thus are possible to occur in the ischemic brains with prolonged Na⁺_i elevation (Brazhe, Verisokin, Verveyko, & Postnov, 2018; Gerkau, Rakers, Durry, Petzold, & Rose, 2018; Verkhratsky, Trebak, Perocchi, Khananshvili, & Sekler, 2018).

The involvement of astrocytic NCX in synaptic transmission and plasticity is also through glutamate release/uptake from the astrocytes, as blockade of NCX_rev by KB-R7943 in cultured astrocytes significantly reduced the glutamate release induced by mild polarization (Reyes et al., 2012). The NCX-mediated Ca²⁺ mobilization (increase) led to the internalization of the glutamate transporter GLT-1 in astrocytes, which reduced glutamate reuptake upon ischemic stroke injury (Ibanez, Bartolome-Martin, Piniella, Gimenez, & Zafra, 2019).

In addition, the NCX_rev-mediated Ca²⁺ influx is responsible for the nitric oxide (NO)-induced cytotoxicity in cultured astrocytes via the ERK, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) pathways (Kitao et al., 2010; Takuma, Ago, & Matsuda, 2013). Pharmacological inhibition of NCX_rev attenuated the Ca²⁺-induced production of reactive oxygen species (ROS), DNA ladder formation, and nuclear condensation, increasing cell viability and reducing apoptosis in the cultured astrocytes (Matsuda et al., 2001). NCX activity contributed to ischemic brain damage, as pharmacological inhibition of NCX significantly reduced infarct volume and neuronal damage in tMCAO models (Matsuda et al., 2001; Shenoda, 2015).

Taken together, considering the multiple functions of astrocytic NCX in regulation of synapse and intracellular Ca²⁺ signaling, additional studies are needed to investigate their impacts on brain functions under both healthy and pathological conditions.

Astrocytic NKCC1 in cell volume regulation, excitotoxicity, and ER stress

Na⁺–K⁺–2Cl⁻ cotransporters (NKCCs) belong to the SLC12 superfamily of cation-chloride cotransporters (CCCs) and exist in two isoforms, NKCC1 and NKCC2 (Ben-Ari, 2017). NKCC1 is the predominant isoform in the brain, while NKCC2 is mostly expressed in the thick ascending limb of the loop of Henle in the kidney (Zeuthen & MacAulay, 2012). NKCC1 couples Na⁺, K⁺, and Cl⁻ import at a 1:1:2 ratio (Ben-Ari, 2017), and regulates these ion concentrations along with cell volume homeostasis by cotransporting 600 molecules of water influx per turnover (Zeuthen & MacAulay, 2012). NKCC1 counteracts the K⁺–Cl⁻ cotransporter (KCC), which is another member of the CCCs that mediates K⁺ and Cl⁻ export along with water efflux (Zhang et al., 2018). Upon ischemic stroke, both NKCC1 and KCCs are phosphorylated via the upstream with-no-lysine (K) kinase (WNK)-Ste20/SPS1-related proline-alanine-rich protein kinase (SPAK)/oxidative stress responsive kinase (OSR1) signaling pathway, leading to NKCC1 activation and KCC inhibition (Baudel, Poole, & Darlison, 2017; Zhang et al., 2018). The WNK-calcium binding protein (Cab39; Bhuiyan et al., 2017) or the MAPK (p38, ERK, JNK) pathways (Yu et al., 2018) have also been reported to be associated with NKCC1 activation after ischemia. The human subacute ischemic stroke brain tissues display increased expression of NKCC1 protein (Bhuiyan et al., 2017). NKCC1 protein activation contributed to ischemic brain damages, because genetic deletion of NKCC1 or its upstream regulator WNK3 exhibited reduced infarction, edema, and white matter damage in mouse tMCAO models (Begum et al., 2015; Chen, Luo, Kintner, Shull, & Sun, 2005). Pharmacological inhibition of NKCC1 with its potent inhibitor bumetanide or a novel inhibitor STS66 also reduced ischemic infarction and white matter disruption, and improved neurological functions in rodent models of ischemic stroke (Huang et al., 2019; Xu et al., 2017; Yu et al., 2018), especially comorbid with hypertension (Bhuiyan et al., 2017).

Astrocytic expression of NKCC1 was detected in cortex, corpus callosum, hippocampus, cerebellum, and optic nerves in rats (MacVicar, Feighan, Brown, & Ransom, 2002; Yan, Dempsey, & Sun, 2001). NKCC1 in astrocytes maintains Na⁺ homeostasis along with Na⁺/K⁺-ATPase and NBC (Rose & Karus, 2013; Rose & Verkhratsky, 2016). Astrocytic NKCC1 activity is the main cell volume regulatory mechanism to revert the osmotic stress-mediated astrocytic shrinkage during regulatory volume increase (RVI; Deng et al., 2016; Zhang et al., 2018). During ischemia, the astrocytic NKCC1 activation is dependent on the hypertonicity induced by Na⁺/K⁺-ATPase activity (Hertz et al., 2013; Hertz & Chen, 2016) via the β1-adrenergic (Hertz, Song, Xu, Peng, & Gibbs, 2015; Song et al., 2014; Song, Xu, Hertz, & Peng, 2015) or ERK1/2 MAPK signaling pathways (Cai et al., 2011; Yuen et al., 2014). Overstimulation of astrocytic NKCC1 activity contributed to brain edema formation under hypotonic osmotic stress or after ischemic stroke (Deng et al., 2016; Zhang et al., 2018). Both pharmacological inhibition and genetic deficiency of NKCC1 reduced astrocytic swelling, cerebral edema, and BBB breakdown during the reperfusion phase after ischemic stroke (Chen & Sun, 2005; Hertz et al., 2014; Kahle et al., 2009). Moreover, NKCC1-mediated elevation in Na⁺_i can subsequently trigger NCX_rev activity in astrocytes, which led to ER Ca²⁺ overload and mitochondrial dysfunction, contributing to the ischemic brain damage (Kintner et al., 2007; Lenart et al., 2004). Pharmacological inhibition or genetic knockout of NKCC1 abolished the increase in Na⁺_i (Chen et al., 2005; Lenart et al., 2004), as well as the Ca²⁺_i accumulation in astrocytes (Kintner et al., 2007) in OGD models.

The role of astrocytic NKCC1 activity in K⁺ homeostasis remains incompletely defined. First, it has been reported that knockout of NKCC1 in astrocytes completely abolished the K⁺ influx under hyperosmotic solution in response to high extracellular K⁺ stress (Chen & Sun, 2005; Su, Kintner, Flagella, Shull, & Sun, 2002; Su, Kintner, & Sun, 2002). The K⁺ uptake via NKCC1 requires glycogenolysis (Hertz et al., 2013; Xu et al., 2013). The astrocytic glycogenolysis mediates glutamate synthesis from astrocytes (Hertz et al., 2013), while the astrocytic NKCC1 is an important contributor for glutamate release, as genetic knockout of NKCC1 in astrocytes reduced 30% of glutamate release under hyperosmotic solution with high extracellular K⁺ (Chen & Sun, 2005; Su, Kintner, Flagella, et al., 2002; Su, Kintner, & Sun, 2002), collectively contributing to learning and memory functions (Hertz et al., 2013; Hertz & Chen, 2016; Xu et al., 2013). The astrocytic NKCC1-mediated glutamate release activated non-NMDA glutamate receptors on oligodendrocytes in the white matter, which resulted in oligodendrocyte death in a rat optic nerve model (Wilke, Thomas, Allcock, & Fern, 2004). On the other hand, however, recent studies showed that inhibition of NKCC1 failed to affect the rate of K⁺ removal from the extracellular space in rat hippocampal slices under normoxic conditions (Larsen et al., 2014). Inhibition of either Na⁺/K⁺-ATPase or Kir4.1 reduced the clearance of K⁺_o transients in the hippocampal slices in response to increased extracellular K⁺, indicating that Na⁺/K⁺-ATPase and Kir4.1, but not NKCC1, played vital roles in extracellular K⁺ clearance (Larsen et al., 2014). The contribution of each astrocytic K⁺ regulatory mechanisms (NKCC1, Na⁺/K⁺-ATPase, and Kir4.1) in brain extracellular K⁺ clearance under ischemic pathological conditions warrants further study.

In addition to Na⁺, K⁺, and glutamate, NKCC1 also maintains Cl⁻ homeostasis, as the Cl⁻ influx was completely abolished in the NKCC1-null astrocytes (Chen & Sun, 2005; Su, Kintner, Flagella, et al., 2002; Su, Kintner, & Sun, 2002). The activation of glutamate N-methyl-d-aspartic acid (NMDA) receptors is dependent on the NKCC1-mediated Cl⁻ accumulation (Beck, Lenart, Kintner, & Sun, 2003). γ-aminobutyric acid (GABA) exerts its action via synaptic and extra-synaptic GABA_A receptors, mediating phasic and tonic inhibition, respectively (Cellot & Cherubini, 2013). The Cl⁻ influx regulated by NKCC1 suppressed the inhibitory GABAergic responses and resulted in neuron depolarization, which mediated postsynaptic firing and contributed to excitotoxicity following ischemic stroke (Baudel et al., 2017; Ben-Ari, 2017; Schulte, Wierenga, & Bruining, 2018). Inhibition of NKCC1 using bumetanide promoted the inhibitory GABAergic transmission and eliminated seizures in a genetically induced mouse astrogliosis model (Robel et al., 2015). The astrocytic regulation of NKCC1 on GABAergic synaptic transmission was mediated by phospho-activation of the WNK-SPAK/OSR1 signaling pathways (Wilson & Mongin, 2019; Zhang et al., 2018).

In summary, astrocytic NKCC1 activity is the main cell volume regulatory mechanism mediated by conducting Na⁺, K⁺, and Cl⁻ influx. Overstimulation of NKCC1 activity affects astrocyte metabolism and GABAergic transmission regulation, which present NKCC1 as a therapeutic target for ischemic stroke, as well as epilepsy.

Astrocytic NBC in pH_i, homeostasis, cell volume regulation, membrane potential, and energy metabolism

NBC mediates two or three HCO3⁻ cotransport per Na⁺, thus eletrogenic, and maintains the astrocytic membrane potential at −70 to −80 mV (Rose & Verkhratsky, 2016). Among the three isoforms of NBC (NBC1-3), NBC1 is the most widely expressed in astrocytes throughout the CNS, including cerebral cortex, hippocampus, and cerebellum (Annunziato, Boscia, & Pignataro, 2013; Schmitt et al., 2000), and can operate in either inward or outward modes for regulations of Na⁺ signaling, pH_i homeostasis, as well as glial membrane potential in the CNS (Boscia et al., 2016; Rose & Verkhratsky, 2016). Rose and Verkhratsky (2016) reported that the inward mode of NBC activation mediated influx of Na⁺ into astrocytes and contributed to setting the baseline [Na⁺]_i under physiological conditions (Figure 1).

In addition, because NBC is electrogenic, it is activated by astrocytic membrane depolarization induced by neuronal activity and the subsequent K⁺ influx, which resulted in intracellular accumulation of K⁺, Na⁺, and HCO₃⁻, creating an osmotic gradient which drives water influx, the neuronal activity-dependent astrocytic swelling, and extracellular space shrinkage without affecting the K⁺ transients (Florence, Baillie, & Mulligan, 2012; Larsen & MacAulay, 2017; Ostby et al., 2009). The depolarization-induced alkalization of astrocytes also resulted in a stimulation of glycolysis through NBC activation, as the glycolytic response to increased K⁺ was absent when NBC was blocked by pharmacological inhibition or genetic knockout of NBC (Ruminot et al., 2011). NBC activation stimulated soluble adenylyl cyclase (sAC) and intracellular cyclic adenosine monophosphate (cAMP) levels, which enhanced glycolysis and the release of lactate into the extracellular space (Choi et al., 2012), whereas lactate can be transported through the monocarboxylate transporters and in turn stimulate NBC activity (Becker, Broer, & Deitmer, 2004; Choi et al., 2012), forming a positive feedback loop for metabolic communication between astrocytes and neurons (Becker et al., 2004; Choi et al., 2012). The NBC-mediated alkalization in astrocytes was also highly correlated with astrocytic membrane potential changes during seizure-like events in hippocampal astrocytes, confirming the role of the electrogenic NBC in tissue excitability through regulation of astrocytic membrane potential (Raimondo et al., 2016). This view has been further supported by a finding showing that elevated NBC immunoreactivity was detected in gerbil hippocampus on seizure onset, but not in the seizure-resistant gerbil hippocampus (Kang et al., 2002).

Oppositely, hyperpolarization induced by prompt reduction of the extracellular K⁺ concentration concurrently led to intracellular acidosis, which was dependent on extracellular Ca²⁺, as removal of Ca²⁺_o led to an instant pH_i recovery (increase) in mouse neural stem cell-derived astrocytes (Nordstrom, Andersson, & Akerman, 2019). This pH_i recovery was mediated by the inward mode of NBC activation, since pharmacological inhibition of NBC prevented such pH_i increase in the hyperpolarized astrocytes upon Ca²⁺_o removal (Nordstrom et al., 2019). In addition, with the presence of high extracellular K⁺, the NBC-mediated pH_i increase can override the Ca²⁺-mediated acidosis even in prehyperpolarized and acidified astrocytes (Nordstrom et al., 2019), further indicating the importance of NBC in astrocytic pH_i regulation. The NBC-mediated regulation on pH_i is essential for astrocytic development (Giffard et al., 2000), and has been confirmed in NBC knockout astrocytes (Theparambil, Ruminot, Schneider, Shull, & Deitmer, 2014). Upon ischemic stroke, NBC protein expression was upregulated in the ischemic penumbra in a rat pMCAO model (Jung, Choi, & Kwon, 2007), and in astrocytes in the CA1 region, but not in CA3 region of the hippocampus (Sohn et al., 2011), which played a vital role in astrocytic survival by buffering the ischemic stroke-induced intracellular acidosis (Yao et al., 2016). Silencing NBC expression rendered astrocytes more vulnerable to acidic injury (Giffard et al., 2000), and pharmacological inhibition of NBC by a generic NBC blocker S0859 exacerbated the ischemia-induced cell death in human stem cell-derived astrocytes (Yao et al., 2016).

In conclusion, NBC is important for maintaining pH_i homeostasis, cell volume, membrane potential, and energy metabolism in astrocytes. While the upregulated NBC activity plays a positive role in astrocytic survival by alleviating the intracellular acidosis after ischemic stroke, increased NBC activity also exerts an effect on membrane potential, contributing to tissue excitability and seizure-like events.

1.1.2 |. Ion transporters in activated microglia after ischemic stroke

Upon brain injury, microglia are rapidly activated, migrate toward the lesion site and trigger proinflammatory responses by secreting cytokines, such as IL-1β, IL-6, and TNF-α (Bar & Barak, 2019; Liu et al., 2010). Alternatively, activated microglia transform their activity and contribute to tissue repair and remodeling by secretion of anti-inflammatory cytokines and growth factors, as well as phagocytic activity (Bar & Barak, 2019; Parkhurst et al., 2013; Song et al., 2018). Increased phagocytic activity of microglia is essential for clearance of tissue debris after brain injury, favoring oligodendrogenesis and remyelination (Bar & Barak, 2019; Olah et al., 2012; Rinaldi et al., 2016). Microglia also recognize and eliminate injured synapses in a phagocytosis-dependent manner, which subsequently promote synaptic remodeling and plasticity in ischemic brains (Bar & Barak, 2019; Wake et al., 2009). Increasing evidence suggests that microglial ion transporters play important roles in regulation of microglial function after ischemic stroke, which is further discussed in the following sections.

Microglial NHE1 in pH_i, homeostasis and proinflammatory microglial activation

NHE1 maintains the basal pH_i of microglia at about 7.19 in resting conditions, and pharmacological inhibition of NHE1 with its potent inhibitor HOE642 acidified the basal pH_i to 6.82 (Liu et al., 2010). In response to lipopolysaccharides (LPS) or hypoxia stimulation, NHE1 protein is activated for excess H⁺ extrusion and maintains an alkaline optimal pH_i at 7.2 during the microglial respiratory burst and superoxide production by NADPH oxidase (NOX; Lam et al., 2013;Liu et al., 2010 ; Shi, Kim, et al., 2013). This pH_i homeostasis is important for microglial chemotaxis and migration (Shi et al., 2013). Pharmacological inhibition of NHE1 during this process failed to buffer the pH_i changes, and led to sustained intracellular acidification and cessation of the NOX activity, and abolished microglial proinflammatory polarization (Liu et al., 2010; Shi, Chanana, Watters, Ferrazzano, & Sun, 2011). Selective deletion of the Nhe1 gene in microglia also prevented microglial proinflammatory activation as well as their proliferation in the ischemic brains (Song et al., 2018).

On the other hand, it is reported that increased expression of Na⁺ channels such as the voltage-gated Na⁺ channel (Na_v; Pappalardo, Black, & Waxman, 2016) and the acid-sensing ion channels (ASICs; Yu et al., 2015), as well as the Na⁺ transporters/exchangers such as NHE1 (Liu et al., 2010) collectively contributed to the increased Na⁺ loading in microglia under pathological ischemic or inflammatory conditions. Activation of NHE1 caused cell swelling through increased Na⁺ influx, while inhibition of NHE1 caused microglial shrinkage (Nishioka et al., 2016). Importantly, increased Na⁺ loading activates NCX_rev (Liu et al., 2010; Shi, Yuan, et al., 2013) and led to a two-fold increase of the Ca²⁺ level in microglial cells (Liu et al., 2010), which could subsequently trigger the Ca²⁺-dependent signaling pathways, such as the Ca²⁺-calcium/calmodulin-dependent protein kinase (CaMKII)/transforming growth factor β-activated kinase (TAK1)-nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) signaling, and contributed to increased inflammation (Huang et al., 2015). Pharmacological inhibition or genetic knockout of NHE1 in microglia reduced proinflammatory cytokines secretion, such as IL-1β, IL-6, and TNF-α (Liu et al., 2010; Shi et al., 2011; Song et al., 2018), as well as reduced CD86 protein expression (Song et al., 2018). Selective deletion of Nhe1 in microglia switched them to anti-inflammatory phenotypes with increased TGF-β and IL-10 secretion, along with elevated Ym1 protein expression after ischemic stroke (Song et al., 2018). Recent findings also revealed that NHE1 activation suppressed microglial phagocytic activity and contributed to sustained white matter demyelination and neurological dysfunction, possibly through reduced myelin debris clearance after ischemic stroke (Song et al., 2018).

Taken together, these findings collectively suggest that NHE1 plays an important role in maintaining the microglial pH_i homeostasis, and is required for proinflammatory microglial activation and oxidative injury after ischemic stroke. Blocking NHE1 activity promotes transformation of activated microglia to restorative phenotypes for ischemic brain tissue repair.

Microglial NCX in proinflammatory microglial activation and microglial apoptosis

Microglia express all three isoforms of NCX, while NCX1 is the most abundant isoform (Newell, Stanley, & Schlichter, 2007; Parpura et al., 2016). NCX1 plays a major role in regulation of Ca²⁺ in microglia, as silencing NCX1 with small interfering RNA (siRNA) completely abolished the Ca²⁺ increase in cultured microglia after OGD (Boscia et al., 2009, 2013). NCX1_rev activation can be induced by inflammatory factors such as interferon (IFN)-γ in cultured microglia, which was mediated by upregulation of protein kinase C (PKC), tyrosine kinase, and MAPK/ERK (Nagano, Kawasaki, Baba, Takemura, & Matsuda, 2004). The NCX1_rev-mediated Ca²⁺ influx is important for microglial activation, migration, and phagocytosis after LPS stimulation in early postnatal mice (Sunkaria, Bhardwaj, Halder, Yadav, & Sandhir, 2016). However, the NCX1-dependent microglial migration is restricted to the bradykinin-induced chemotaxis, but not ATP-induced migration, as proven in the NCX1 knockout mouse model (Ifuku et al., 2007; Noda, Ifuku, Mori, & Verkhratsky, 2013). NCX plays a critical role in microglial phagocytic activity through the Ca²⁺-dependent activation of the purinergic receptors (P2Y2, P2Y6, and P2Y12) during postnatal development (Sunkaria et al., 2016), therefore might also be important in microglial phagocytosis of tissue debris after ischemic stroke. Indeed, upon ischemic stroke, NCX1_rev activity was significantly enhanced in the round phagocytic microglia in the infarct core at 3–7 days after pMCAO, which was accompanied by downregulation of NCX2 and NCX3 protein expressions (Boscia et al., 2009, 2013). In addition, the microglia-mediated respiratory burst was suppressed by pharmacological inhibition of NCX1_rev activity (Newell et al., 2007). This is at least partially through Na⁺-dependent mechanism and the activation of NHE1, since the reduced Ca²⁺ level with NCX inhibitor was similar to that with the NHE1 inhibitor in a microglial OGD model (Liu et al., 2010; Shi, Kim, et al., 2013). NCX can also couple with NHE1 activation in inflammatory responses, and contributes to the production of inducible nitric oxide synthase (iNOS) through the NHE1-NCX-CaMKN/TAK1-NFκB signaling in cultured microglia (Huang et al., 2015). Moreover, the NCX-mediated Ca²⁺ elevation concurrently led to ER stress and apoptosis in cultured microglia induced by NO, while pharmacological inhibition of NCX protected microglia from these events (Matsuda, Nagano, Takemura, & Baba, 2006; Nagano et al., 2005; Takuma et al., 2013). Thus, NCX is important for mediating Ca²⁺ signaling in microglia, which contributes to microglial proinflammatory activation and microglial apoptosis during ischemic stroke. Inhibition of NCX activity suppresses the microglia-mediated respiratory burst for tissue repair after ischemic stroke.

1.1.3 |. Ion transporters in oligodendrocytes after ischemic stroke

Due to limited collateral blood flow in the white matter, oligodendrocytes are the most vulnerable type of glial cells for ischemic damage (Wang, Liu, et al., 2016). Oligodendrocyte cell death can result from excessive release of glutamate (Doyle et al., 2018), increased oxidative stress (Takase et al., 2018), and elevated neuroinflammation (Choi et al., 2016), altogether leading to sustained loss of myelin sheath and prolonged neurological function deficits after ischemic stroke. Brain injury triggers immediate proliferation and migration of the OPCs, which can differentiate into the mature myelin-secreting oligodendrocytes (Hughes, Kang, Fukaya, & Bergles, 2013). However, concurrent activation of several signaling pathways (such as Nogo receptor-1) in OPCs prevents their maturation (Sozmen et al., 2016). Therefore, a better understanding of the regulatory mechanisms for white matter repair after ischemic stroke is important.

NHE1 and NBC have been reported to regulate pH_i in cultured oligodendrocytes under homeostatic conditions (Ro & Carson, 2004). The NHE1-mediated H⁺ extrusion is the predominant alkalinizing mechanism in the perikaryon and proximal processes of oligodendrocytes, while the HCO₃⁻ efflux mediated by the outward mode of NBC activity functions as the predominant acidifying mechanism in the distal dendrites of oligodendrocytes (Ro & Carson, 2004). However, no studies on oligodendrocytic NHE1 or NBC under ischemic stroke conditions were reported. Oligodendrocytic NCX and NKCC1 have been shown to play important roles in oligodendrocyte damages and genesis after ischemic stroke.

NCX in oligodendrocytic survival, maturation, myelin formation

All three isoforms of NCX are expressed in oligodendrocytes (Parpura et al., 2016; Quednau, Nicoll, & Philipson, 1997), and can mediate myelin synthesis by modulating Ca²⁺ signaling in OPCs (Friess et al., 2016). Among the three isoforms, NCX1 and NCX3, but not NCX2, are involved in the OPC differentiation into mature oligodendrocytes during development, with NCX1 downregulated and NCX3 upregulated during this process (Boscia et al., 2012, 2013).

Oligodendrocytic NCX3 activation is beneficial and promotes myelination. In response to OGD, only NCX3 mRNA level in cultured rat OPCs was significantly decreased, but not NCX1 or NCX2 (Cai, Ma, Tian, Li, & Li, 2018). Pharmacological inhibition of NCX3 led to elevated lactate dehydrogenase (LDH) release in these OPCs, which correlated with increased OPC apoptosis, mitochondrial damage, and the activation of ERK1/2 and poly [ADP-ribose] polymerase (PARP1) pathways (Cai et al., 2018). Knockdown of NCX3 using siRNA in OPC cultures inhibited the expression of myelin proteins 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNPase) and myelin basic protein (MBP), while overexpression of NCX3 promoted these myelin protein expressions (Boscia et al., 2012, 2013). Genetic knockout of NCX3 increased NG2⁺ OPC counts in the spinal cord in mice, but concurrently exhibited reduced size of the spinal cord as well as hypomyelination with decreased MBP and axonal neurofilament (NF200) protein expression (Boscia et al., 2012, 2013). These findings indicate that loss of NCX3 protein arrests OPC differentiation and myelination, but not OPC proliferation. The NCX3-mediated modulation on myelin synthesis is through Ca²⁺ signaling, and thus can be induced by increased Na⁺_i level which subsequently triggers the NCX_rev activity, such as through the inhibition of the α2 isoform of Na⁺–K⁺–ATPase (Hammann, Bassetti, White, Luhmann, & Kirischuk, 2018). Increased resting Ca²⁺_i level stimulated MBP synthesis in cultured OPCs, while blockade of NCX_rev reduced resting Ca²⁺_i level and led to less MBP synthesis in acute slices of the corpus callosum from mice at P20-30 but not P10 (Friess et al., 2016). In addition, a recent study also demonstrated that NCX3 played an important role in the glutamate-mediated remyelination in a cuprizone-induced demyelination model in mice (de Rosa et al., 2019). This could relate to the study that optic nerve myelin responded to axonal action potentials with a rise in Ca²⁺ levels, which was mediated by glutamatergic NMDA receptors, and played a role in modulating oligodendrocyte myelination in an activity-dependent manner (Micu et al., 2016).

On the contrary, unlike NCX3, increased NCX1 activity in axons exerted detrimental effects on axonal injury and myelination through increased Ca²⁺_i overload and elevated Na⁺ influx, as ~73% of the injured axons expressing β-amyloid precursor protein (β-APP) co-expressed NCX1 and the voltage-gated Na⁺ channel Na_v1.6, while only ~4% coexpression was found in the β-APP negative axons in the spinal cord from a demyelination model of EAE in mice (Craner, Hains, Lo, Black, & Waxman, 2004). Moreover, oligodendrocytic NCX activity is also induced by NKCC1-mediated Na⁺ influx, and the NCX-mediated Ca²⁺_i increase is involved in the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced excitotoxicity for mitochondrial damage and oligodendrocyte cell death in cultured oligodendrocytes (Chen et al., 2007). Whether the NCX1 isoform plays a role in this damaging process remains unknown.

Therefore, different isoforms of NCX play extinct roles in regulation of white matter injury and repair, with NCX3 promotes oligodendrocytic survival, maturation, and myelin formation, while NCX1 induces demyelination and excitotoxicity. Inhibition of NCX1 and enhancement of NCX3 may be beneficial for ischemic white matter repair.

NKCC1 in oligodendrocytic survival, proliferation, maturation, and myelin formation

NKCC1 is present in oligodendrocytes (Chen & Sun, 2005), and regulates the formation of OPCs at the early stage of neurogenesis at E14.5, as genetic ablation of NKCC1 displayed a significantly reduced number of Olig2⁺ oligodendrocytes at this embryonic stage (Magalhaes & Rivera, 2016). The postnatal expression of oligodendrocytic NKCC1 started to increase from P3-7 (Jantzie et al., 2015; Wang, Yan, Kintner, Lytle, & Sun, 2003), and this was in parallel with the increase of MBP expression from P6 to P21 (Wang et al., 2003), indicating its role in myelination during development. Selective deletion of Nkcc1 gene from the PDGFRα⁺ OPCs showed increased proliferation of NG2⁺Ki67⁺ OPC counts, but suppressed differentiation of OPCs with reduced CC1⁺ mature oligodendrocyte counts and decreased MBP expression in the cerebellum white matter of neonatal mice (Zonouzi et al., 2015). These effects resembled the results from administration of GABA_A receptor antagonists in neonatal wild-type mice (Zonouzi et al., 2015), suggesting that the NKCC1-mediated regulation on neonatal OPC maturation and white matter myelination could relate to the activation of GABA_A receptors. Indeed, NKCC1 activity was stimulated by the Cl⁻ efflux and cell shrinkage mediated by the activation of GABA_A receptors in oligodendrocytes (Wang et al., 2003). The upregulated NKCC1 activity in turn maintained a high Cl⁻_i to allow for the GABAergic trophic effects, which played an important role in oligodendrocytic survival following the withdrawal of growth factors (Wang et al., 2003). Inhibition of NKCC1 with bumetanide completely abolished the GABA_A receptor-mediated trophic effects and resulted in higher oligodendrocyte mortality (Wang et al., 2003).

However, activation of NKCC1 concurrently mediates Na⁺ influx, which triggers Ca²⁺ overload through NCX_rev and non-NMDA type glutamate receptors in oligodendrocytes, eliciting compromised mitochondrial functions, enhanced glutamergic excitotoxicity induced by AMPA, and increased oligodendrocyte death (Chen et al., 2007; Wilke et al., 2004). During ischemia, elevated oligodendrocytic NKCC1 activity increased the vulnerability to ischemic white matter injury (Jantzie et al., 2015). Blockade of NKCC1 activity using bumetanide reverted the ischemic stroke-induced MBP loss and white matter damage in neonatal rats (Jantzie et al., 2015). NKCC1 inhibition also promoted OPC proliferation through increased DNA synthesis and cell cycle progression, with elevated expressions of cyclin D1, CDK4, and cyclin E in an OGD model (Fu et al., 2015). Similarly, bumetanide induced OPC proliferation in a bilateral common carotid artery stenosis model in adult mice, along with improved white matter integrity and working memory functions (Yu et al., 2018). The detrimental role of NKCC1 on oligodendrocytes and white matter damages after ischemic stroke was through the upstream regulation of WNK3-SPAK/OSR1 signaling pathway, since genetic knockout of WNK3 decreased the phospho-activation and overall protein expression of NKCC1, along with reduced axonal demyelination and improved neurological functions after ischemic stroke (Begum et al., 2015). Genetic knockout of WNK3 or knockdown of SPAK/OSR1 using siRNA also illustrated an increased tolerance of oligodendrocytes after OGD (Begum et al., 2015). However, regarding relationship between WNK-SAPK/OSR1-NKCC1 complex and ERK signaling pathway, inconsistent results have been reported: bumetanide reduced p-ERK and elevated p-p38 MAPK expressions in the OPCs in response to OGD (Fu et al., 2015), but elevated p-ERK and decreased p-JNK and p-p38 MAPK signaling were detected in the poststroke corpus callosum after bumetanide treatment (Yu et al., 2018).

Hence, NKCC1 is essential for oligodendrocytic survival, maturation, and myelin formation during development. However, upon ischemic stroke, the overstimulated NKCC1 plays harmful roles in stimulating excitotoxicity, which increases oligodendrocyte apoptosis, as well as suppressing OPC proliferation and causing white matter demyelination, which contributes to cognitive dysfunctions. The regulative mechanisms of the WNK3-SPAK/OSR1-NKCC1 signaling pathway in oligodendrogenesis during development and postinjury remyelination are not well defined and require more study. This will shed light on their potentials as new therapeutic targets.

1.2 |. Glial ion transporters in TBI

TBI directly causes primary injury in neurons, and differential polarization of astrocytes (Burda, Bernstein, & Sofroniew, 2016), microglia (Jassam, Izzy, Whalen, McGavern, & El Khoury, 2017), and oligodendrocyte (Takase et al., 2018) can either sustain the damage through a cascade of events, causing secondary injury, or accelerate tissue repair, leading to lesion resolving. Increasing evidence suggest that glial ion transporters play important roles in the regulation of these glial functions for tissue damage or repair after TBI.

1.2.1 |. Ion transporters in reactive astrocytes for astrogliosis, brain edema, astrocyte apoptosis, and synaptic transmission after TBI

Astrocytes are pivotal responders to TBI and can form reactive astrogliosis in areas of injured, but surviving and functioning neural tissue (Burda et al., 2016). Similar to that in the ischemic stroke, the reactive astrocytes can form an astroglial scar that surrounds and restricts the spread of inflammatory response from the lesion core to the remote nonlesioned area (Burda et al., 2016). The secretion of restorative factors from the astroglial scar, such as BDNF, TGF-β, and CNTF, are important for synaptogenesis and axonal outgrowth after TBI or spinal cord injury (SCI; Bei et al., 2016; Chen et al., 2017; Diniz, Matias, Siqueira, Stipursky, & Gomes, 2018), while its concurrent secretion of CSPG is detrimental for neurite outgrowth in a mouse TBI model (Galindo et al., 2018).

Astrocytic NCX_rev activity is involved in astrocyte migration and proliferation, which contributes to astrogliosis in a mechanical injury model using cultured primary astrocytes (Pappalardo, Samad, Black, & Waxman, 2014). This is dependent on the increased Na⁺_i mediated by Na_v1.5, as knockdown of Na_v1.5 mRNA attenuated the NCX-mediated Ca²⁺ rise as well as astrogliosis (Pappalardo et al., 2014). However, brain astrocytes exhibit a significant regional heterogeneity that hippocampal astrocytes are different from neocortical astrocytes in multiple ways (Ziemens et al., 2019). In response to brain injury, hippocampal astrocytes expressed reduced NCX mRNA and protein expression, which was opposite to that in the neocortical astrocytes (Hwang et al., 2006; Matsuda & Baba, 1998; Zhao, Gorin, Berman, & Lyeth, 2008).

Astrocytic NKCC1 activity contributes to brain edema following TBI. In an in vitro fluid percussion (FPI) injury model, the cultured astrocytes showed extensive swelling with an upregulation of AQP4 expression in the plasma membrane, which was dependent on the astrocytic NKCC1 activation (Rao, Reddy, Curtis, & Norenberg, 2011) via the MAPKs and oxidative/nitrosative stress pathways (Jayakumar et al., 2011). The astrocytic swelling was also triggered by glutamate and proinflammatory cytokines, such as TNF-α and IL-1β, released from microglia after TBI (Jayakumar et al., 2018). Pharmacological inhibition of NKCC1 with bumetanide or silencing NKCC1 with siRNA significantly reduced the astrocytic swelling (Jayakumar et al., 2011). In addition, elevated expression of NKCC1 was detected along with increased astrocyte apoptosis in a stretch injury model using cultured astrocytes, with increased Bax level and cleaved caspase-3 activity, enhanced proinflammatory astrocytic responses of elevated IL-1β, IL-6, and TNF-α, and the upregulation of the NF-κB pathway (Zhang, Cui, Cui, Wang, & Zhong, 2017). Inhibition of NF-κB significantly reduced the astrocytic NKCC1 activation, while inhibition of NKCC1 using bumetanide exhibited significantly decreased cleaved caspase-3 activity (Zhang et al., 2017). On the other hand, astrocytic NKCC1 can be beneficial and plays an important role in K⁺ uptake after TBI, which reverses the neuronal inactivity, enhances synaptic transmission and neuronal excitability, and facilitates learning and memory functions (Hertz & Chen, 2016; Sajja, Hlavac, & VandeVord, 2016).

1.2.2 |. Ion transporters in activated microglia for microglial migration, inflammation, and hypersensitivity after TBI

Microglia are rapidly activated and migrate toward the lesion site in response to CNS injury, such as TBI (Bar & Barak, 2019). Microglial migration can be induced by bradykinin, mainly through the B1 bradykinin receptor, as B1 receptor antagonist markedly inhibited the microglial motility in cultured microglia, and genetic knockout of B1 receptor suppressed microglial chemotaxis in response to bradykinin (Ifuku et al., 2007). This bradykinin-induced microglial migration is dependent on Ca²⁺ influx via the NCX_rev activity, since both pharmacological blockade and genetic knockout of NCX abolished the bradykinin-induced microglial migration (Ifuku et al., 2007). NCX also plays a role in microglial migration and phagocytosis induced by the active form of thyroid hormone T3 (3, 3′, 5-triiodothyronine) in a stab wound model in mice (Mori et al., 2015). However, the subsequently activated channels are different between these NCX-mediated mechanisms, as the bradykinin-triggered NCX activation resulted in the intermediate-conductance (IK) type of Ca²⁺-dependent K⁺ channel activation, while the T3-induced NCX activation led to the apaminsensitive small-conductance (SK) type of activation in microglia (Ifuku et al., 2007; Mori et al., 2015). The chemoattracted microglia can exert inflammatory responses in the lesion site, where overexpression of NKCC1 and downregulation of KCCs are needed, for microglial secretions of cytotoxic cytokines and neurotrophic factors, such as TNF-α and BDNF, as well as MMPs and ROS, altogether contributing to hypersensitivity and neuropathic pain in rodent SCI or peripheral nerve injury models (Lopez-Alvarez, Modol, Navarro, & Cobianchi, 2015; Schomberg, Ahmed, Miranpuri, Olson, & Resnick, 2012). In addition, NKCC1 also plays a role in increasing the seizure susceptibility induced by TBI using the controlled cortical impact (CCI) procedures in awake un-anesthetized mice (Wang et al., 2017). Microglial secretion of TGF-β can upregulate NKCC1 expressions in the cortex and hippocampus, and leads to increased latency and duration of seizure after TBI, but it remains unclear about the specific role of microglial NKCC1 during this process (Wang et al., 2017). Recent findings also suggest that microglia-derived but not astrocyte-derived cytokines (such as TNF-α and IL-1β) upregulated NKCC1 activity in cultured neurons and promoted neuronal swelling in a severe FPI model of TBI, while no swelling in microglia themselves was observed (Jayakumar et al., 2018). Exposure of microglia to the conditioned media from traumatized neurons or astrocytes also had no effects on the microglial cell volume (Jayakumar et al., 2018).

In conclusion, a few recent studies reveal the roles of NCX and NKCC1 in astrogliosis, astrocyte apoptosis, brain edema formation, synaptic transmission, as well as microglial migration, inflammation, and hypersensitivity after TBI. However, extensive studies on glial ion transporters in the pathogenesis and progression of TBI are lacking. Further investigations are needed for a better understanding of the roles of these ion transporters in reactive astrocytes, microglia-mediated inflammation and phagocytosis for tissue repair, and oligodendrocyte-mediated remyelination after TBI.

2 |. GLIAL ION TRANSPORT IN CHRONIC NEUROLOGICAL DISEASES

2.1 |. Glial ion transporters in glioma tumors

Glioma is the malignant tumor that starts in the glial cells of the brain or the spine. Based on the specific cell types, glioma includes malignancy of astrocytes (astrocytoma and glioblastoma), oligodendrocytes (oligodendroglioma), and ependymal cells (ependymoma; Ahmed, Gull, Khuroo, Aqil, & Sultana, 2017). The World Health Organization (WHO) classifies glioma into four grades (I, II, III, and IV). Grade I and II are low-grade gliomas (LGGs), whereas Grade III and IV are high-grade gliomas (HGGs; Claus et al., 2015; Delgado-Lopez, Corrales-Garcia, Martino, Lastra-Aras, & Duenas-Polo, 2017). Standard of treatment in both LGGs and HGGs includes maximal safe surgical resection followed by radiation and chemotherapy (Oberheim Bush & Chang, 2016; Weller, 2011). Patients with LGGs have an indolent course with longer-term survival (~7 years) in comparison with HGGs. Moreover, the majority of LGGs patients (over 80%) present seizures, and 35% continue to have spontaneous seizure recurrence, which is termed as tumor-associated epilepsy (Campbell et al., 2015; Schiff, 2017). Thus, LGGs are considered malignant because of their invasive growth, resistance to therapy, and high risk of transforming into HGGs (Kohanbash et al., 2017).

Glioblastoma (GBM) belongs to Grade IV and is characterized as the most aggressive, primary malignant tumor (Louis et al., 2016). Under the standard care, GBM patients have a mean 5-year survival rate of less than 10% (Hide, Shibahara, & Kumabe, 2019). The difficulty in effectively treating GBM patients is largely due to the heterogeneous nature of these tumors and to the complex network of interactions with the tumor microenvironment (TME; Oliveira et al., 2017). The GBM microenvironment consists of a complex network of a variety of glioma-associated stromal cell types within the brain, including microglia, macrophages, astrocytes, and oligodendrocytes (Oliveira et al., 2017; van der Vos et al., 2016). Presence of tumor-associated stromal cells in the microenvironment of glioma promotes tumor progression, driven by paracrine signals and secreting trophic factors (Oliveira et al., 2017), and they consequently increase glioma resistance to current therapies (Brandao, Simon, Critchley, & Giamas, 2018; O’Brien, Howarth, & Sibson, 2013). Among various cells of the TME, glial cells, including astrocytes and microglia/macrophages (TAMs), are the most common cellular entities that interact with GBM (Matias et al., 2018). The TAM population is usually the dominant glioma-infiltrating immune cells, occupied around 30% of glioma mass (Miller et al., 2014). Astrocytes, which comprise 40–50% of the cells in TME, become reactive, resulting in a layer of reactive astrocytes surrounding the tumor (Guan, Hasan, Maniar, Jia, & Sun, 2018; Placone, Quinones-Hinojosa, & Searson, 2016). Therefore, better understanding of the TME is important for developing effective therapies for GBM patients.

2.1.1 |. Ion transporters in glioma proliferation and migration

New studies show that ion transporter proteins play a role in glioma pathophysiology. They are involved in cell proliferation, migration and invasion (Guan et al., 2018). Recent bioinformatics analysis of the Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA) datasets revealed that SLC9A1 mRNA expression (encoding for NHE1 protein) is significantly higher in gliomas and is associated with glioma malignancy. Worsened survival probabilities were also correlated with the elevated SLC9A1 mRNA levels in glioma (Guan et al., 2018). The underlying mechanisms of elevated NHE1 protein in glioma progression may include promoting angiogenesis, and extracellular matrix remodeling (Guan, Luo, Begum, et al., 2018). NHE1 protein in gliomas maintains alkaline pH_i of 7.3–7.5 by extruding H⁺ in exchange of Na⁺ influx (Boedtkjer, Bunch, & Pedersen, 2012), which is a driving force for glycolytic metabolism (Stock & Pedersen, 2017; Figure 2). Cancer cells rely on oxidative glycolysis by increasing glucose uptake and lactate production even in the presence of oxygen and fully functioning mitochondria, known as Warburg effect (Guan, Hasan, Begum, et al., 2018). Upregulation of NHE1 protein expression in glioma also plays a vital role in glioma migration, survival and their resistance to TMZ-mediated apoptosis via activation of ERK signaling pathways (Cong et al., 2014; Figure 2). Moreover, ezrin-radixinmoesin (ERM) proteins play an important role in glioma migration and invasion and interact with NHE1 protein (Figure 2). NHE1 is partially colocalized with ezrin and increased colocalization of NHE1 and ezrin was detected in glioma cells which was abolished in cells treated with NHE1 inhibitor HOE642 (Cong et al., 2014). Furthermore, direct interactions between NHE1 and ERM can recruit phosphoinositide 3-kinase and Akt to regulate cell survival (Cong et al., 2014). In addition, glioma cells migrate through narrow and tortuous extracellular spaces posing significant demands on cell volume changes, which can also be mediated by Cl⁻ channels and transporters (Ernest & Sontheimer, 2007; Habela, Ernest, Swindall, & Sontheimer, 2009). The Cl⁻ channels, including ClC-2, ClC-3, ClC-5, ClC-6, and ClC-7, carry Cl⁻ influx or efflux along with obligated water across the cell membrane and are involved in glioma cell volume dynamics and glioma cell migration (Ernest, Weaver, Van Duyn, & Sontheimer, 2005). Among these Cl⁻ channels and transporters, NKCC1 protein was also detected in human gliomas and correlated with the tumor malignancy (Schiapparelli et al., 2017) and plays a fundamental role in regulation of intracellular volume by transport of Na⁺, K⁺, and Cl⁻ ions across the plasma membrane into glioma cells. Expression of NKCC1 protein localized to the leading edge of glioma cells which contributes to tumor invasion through interacting with ERM cytoskeleton proteins (Lin, Liu, Ling, & Xu, 2016; Zhu et al., 2014; Figure 2). NKCC1 protein also promotes epithelial mesenchymal transformation-like process via facilitating the binding of Rac1 and RhoA to GTP during glioma cell invasion (Ma et al., 2019). Ilkhanizadeh et al. (2018) recently found that anti-secretory factor (AF) inhibited cell volume regulation of GBM cells, an effect that was phenocopied in vitro by the NKCC1 inhibitor bumetanide (BMT) through reducing interstitial fluid pressure (IFP) and increasing drug uptake (Ilkhanizadeh et al., 2018). In short, these new findings indicate that ion transporters play an important role in tumorigenesis and emerge as new therapeutic targets for glioma.

FIGURE 2.

Schematic illustration of ion transporters in glioma-TAM interaction. NHE1 is expressed by both glioma cells and tumor-associated microglia/macrophages (TAMs). NHE1 functions to maintain an alkaline pH_i and drives the Warburg effect for glycolysis and glioma proliferation. Moreover, NHE1 and NKCC1 localize to the leading edge of glioma cells and promote glioma migration by interacting with ezrin-radixin-moesin (ERM) cytoskeleton proteins, together with the Cl⁻ channels (ClCs), K⁺ channels (e.g., Kv channels), and water channels (e.g., AQP4) in conducting K⁺ and Cl⁻ along with obligated water efflux across the cell membrane. Soluble factors secreted by glioma cells can stimulate microglial NHE1 activation and induce release of various cytokines (TGF-β, IL-10, and Arg-1). NHE1 activation also leads to upregulation of ERK pathways, which triggers pro-survival signal transduction in glioma, and creates an acidic extracellular environment, which enhances activation of membrane Type 1 metalloprotease (MT1-MMP) and MMP9 for matrix degradation and tumor invasion. Taken together, ion transporters have multiple functions in promoting glioma progression

2.1.2 |. Ion transporters in TAMs for glioma proliferation and invasion

Microglia are considered to share macrophages characteristic of acquiring different phenotypes, like M1/M2 polarization (Matias et al., 2018). TAMs can display polarized M1-like phenotypes that contribute to both innate and adaptive anti-tumor immunity by presenting antigen to adaptive immune cells, producing proinflammatory cytokines and phagocytosing tumor cells (Wei, Gabrusiewicz, & Heimberger, 2013). Whereas in M2 state, TAMs become tumor supportive and secrete chemokines and immunosuppressive cytokines, such as IL-10, TGF-β, and arginase-1 (Arg-1). Tumor-supportive TAMs play a prominent role in angiogenesis, activating matrix-metalloproteases (MMPs) and remodeling extracellular matrix, therefore, promote tumor growth (Coniglio & Segall, 2013; Li & Graeber, 2012).

Increased mRNA expression of SLC9A1 gene encoding NHE1 protein is related to TAM accumulation (Guan, Luo, Begum, et al., 2018). TAMs exhibited increased NHE1 expression and positively correlated with Iba1 level in glioma xenografts and human GBM tissue microarray (Zhu et al., 2016). NHE1 activity is involved in upregulation of iNOS, Arg-1, and microglial cytokine IL-6, IL-10, and TGF-β expression, indicating an important role of NHE1 in glioma-mediated microglia activation (Zhu et al., 2016). Interestingly, increased transcriptional expression and release of TGF-β1 by glioma-stimulated microglia can be abolished when NHE1 activity was attenuated either by inhibition with its inhibitor HOE642 or by siRNA knockdown, indicating that microglial NHE1 protein may promote glioma cell proliferation through the production and release of TGF-β (Zhu et al., 2016). In addition, MMPs are involved in the expansion of malignant gliomas by facilitating their penetration of anatomical barriers and migration within the neuropil (Valastyan & Weinberg, 2011). Zhu et al. (2016) observed that GC22 glioma xenograft tissues exhibited abundant expression of membrane Type 1 metalloprotease (MT1-MMP), MMP2, and MMP9, whereas MT1-MMP appears more abundant in Iba1⁺ cells (Zhu et al., 2016). When applied glioma-conditioned media to microglia resulted in increased expression of MT1-MMP and MMP9 proteins, but not MMP2. Interestingly, this increased MT1-MMP and MMP9 expression were abolished after exposing microglia to NHE1 inhibitor HOE642, indicating that NHE1 activity is required for these changes (Zhu et al., 2016; Figure 2).

2.1.3 |. Ion transporters of TAAs in tumor-associated epilepsy and peritumoral edema formation

The roles of TAMs on brain tumor progression have been actively studied, while astrocytes, which comprise nearly half of the total healthy brain cells (Brandao et al., 2018), have received less attention (Placone et al., 2016). As glioma development involves healthy tissue invasion and destruction, astrocytes undergo a number of molecular, cellular, and functional changes to form tumor-associated reactive astrocytes (TAAs; Brandao et al., 2018). These reactive astrocytes initially aim to fight the progression of the tumor and repair the brain tissue. Some signaling mechanisms, such as NF-κB signaling, may then stimulate astrocytes into TAAs that subsequently support tumor growth (Brandao et al., 2018). TAAs have been shown to be involved in tumor cell proliferation (Bonavia et al., 2003), invasion (Rath, Fair, Jamal, Camphausen, & Tofilon, 2013), apoptosis evasion (Oliveira et al., 2017), immunoprotection (Brandao et al., 2018) and chemoprotection (Chen et al., 2015) by releasing different cytokines. For instance, TAAs-released IL-6 appears to be at the crossroads of multiple TAAs’ effects on GBM development, including promoting proliferation, invasion, and also modulating immune response to tumor progression (Brandao et al., 2018).

The interactions between glioma cells and TAAs in TME are facilitated via ion channels and ion transporters (Guan, Hasan, Maniar, et al., 2018). Among the ions, K⁺ homeostasis in astrocytes and/or neurons plays an important role in tumor-associated epilepsy. In normal conditions, astrocytes show a very negative resting membrane potential (between −80 and −90 mV) and prefer K⁺ uptake, caused by inwardly rectifying currents (Guan, Hasan, Maniar, et al., 2018), and that astrocytes are essential in maintaining the cerebral ionic homeostasis by clearance of excess K⁺ from the extracellular space (Schousboe, Sarup, Bak, Waagepetersen, & Larsson, 2004). The initial clearance of extracellular K⁺ following neuronal excitation and increase in the [K⁺]_o is mediated by astrocytic uptake, because elevated [K⁺]_o activates astrocytic but not neuronal Na⁺, K⁺-ATPases (Hertz & Chen, 2016). Subsequently, astrocytic K⁺ is re-released via Kir4.1 channel after distribution in the astrocytic functional syncytium via gap junctions. This K⁺ uptake by astrocytes serves a crucial purpose to prevent sustained rise in [K⁺]_o in neural tissues, which would cause neurons to remain depolarized and unable to fire action potentials for signaling (Guan, Hasan, Maniar, et al., 2018). However, glioma cells have a more positive resting membrane potential than astrocytes, between −20 and −40 mV (Guan, Hasan, Maniar, et al., 2018; Molenaar, 2011), which leads to accumulation of K⁺_o and may explain the prevalence of epileptic seizures seen in glioma patients (Guan, Hasan, Maniar, et al., 2018). Moreover, the less negative resting potential in glioma cells impaired the ability of Na⁺ gradient across the plasma membrane. Therefore, decreased Na⁺-dependent glutamate transporters causing a higher glutamate presence extracellularly lead to epilepsy in glioma patients (Campbell et al., 2015; Molenaar, 2011). Furthermore, the increased [K⁺]_o leading to abnormal K⁺ uptake through Na–K–ATPases or stimulated Na–K–Cl cotransporters produces cell swelling and depolarization of astrocytes (Coulter & Steinhauser, 2015). The dysfunction of astrocytes in epilepsy revealed alterations in expression, subcellular localization, and function of astrocytic K⁺ channels and gap junctions, resulting in impaired K⁺ buffering and disturbance of delivery of energetic substrates to neurons (Steinhauser, Seifert, & Bedner, 2012).

In addition, high [K⁺]_o appears to play an important role in tumor cell volume and could be involved in peritumoral edema. Peritumoral brain edema is defined as an increase in brain water content around the tumor (Blystad et al., 2017). Peritumoral brain edema is a leading cause of morbidity and mortality in patients with brain tumors. There are two types of brain edema: breakdown of the structural integrity of the BBB and the subsequent increase in interstitial fluid (vasogenic brain edema); and intracellular ionic dysregulation-mediated cell swelling (cytotoxic edema; Murayi & Chittiboina, 2016). The cytotoxic brain edema was believed to be mainly caused by swelling of astrocytes, but also includes neuronal swell (Zhang et al., 2019). High [K⁺]_o induces swelling in astrocytes, leading to cytotoxic edema and cell death in the brain, such as after stroke and TBI (Murakami & Kurachi, 2016). The underlying mechanism involves in enhanced activity of Na, K-ATPases, and NKCC1 and results in the absorption of excess extracellular K⁺ and water into astrocytes, whereas, the efflux of K⁺ through K–Cl cotransporters (KCCs) and K_ir channels was suppressed following the elevation of [K⁺]_o (Murakami & Kurachi, 2016). Astrocytic swelling has been studied in nontumor brain edema, however, its contribution to peri-tumoral brain edema and the underlying mechanisms remain unknown, which should be further investigated.

2.2 |. Glial ion transporters in neurodegenerative diseases

The degeneration of the CNS is characterized by chronic, progressive loss of the structure and functions of neurons, leading to neurological function deficits (Chen, Zhang, & Huang, 2016). Various neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS), involve lesions in different brain regions and display different etiology. Cumulative data suggest common cellular and molecular mechanisms (Volkman & Offen, 2017). A growing number of evidences suggest that glial cells play an important role in neural survival, nerve repair and regeneration, synaptic transmission and immune inflammation, and associated with progression of neurodegenerative diseases (Xu, Wang, Song, Jiang, & Xie, 2016). Neuroinflammatory processes, mainly mediated by reactive astrocytes and microglia, are observed in almost all neurodegenerative diseases (Ettle, Schlachetzki, & Winkler, 2016). In addition, the involvement of astrocytes in the neuropathology of these diseases is likely a consequence of both the loss of normal homeostatic functions and gain of toxic functions (Phatnani & Maniatis, 2015). Furthermore, oligodendrocytes and their widely distributed progenitor cells profoundly influence and control processes known to be frequently dysregulated in neurodegenerative diseases, including ionic homeostasis and nerve impulse conduction (Ettle et al., 2016). Studies of glial ion transporters relating to these neurodegenerative diseases will be discussed in the following sections.

2.2.1 |. Astrocytic ion transporters are involved in AD pathology

AD, the most prevalent cause of dementia, is pathologically characterized by extracellular amyloid β (Aβ) plaques and intraneuronal deposits of neurofibrillary tangles (NFTs; Wang & Colonna, 2019). However, several investigations suggest that inflammation may be the key neuropathological event leading to neurodegeneration in AD (Fakhoury, 2018). Astrocytes in AD become reactive as a result of deposition of Aβ, which has detrimental consequences, including decreased glutamate uptake due to decreased expression of glutamate transporters, altered energy metabolism, altered ion homeostasis (K⁺ and Ca²⁺), increased tonic GABA-mediated inhibition, and increased release of cytokines and inflammatory mediators (Assefa, Gebre, & Altaye, 2018). It was recently shown that exposing cultured astrocytes to Aβ^25-35 causes a significant increase in intracellular Na⁺ levels, which is associated with reduced Na⁺/K⁺-ATPase protein containing the α2 subunit expression and activity (Boscia et al., 2016). Moreover, elevated NHE1 activity was detected in cultured astrocytes in response to HIV-linked gp-120 and cytokines, likely resulting in increased pH_i and enhanced release of glutamate via reversal of Na⁺-dependent glutamate transporters in the development of AIDS-mediated dementia (Boscia et al., 2016). In addition, it has been hypothesized that disturbance in Ca²⁺ homeostatic cascades may progressively affect cellular homeostasis, synaptic transmission, and ultimately cell survival (Verkhratsky, Rodriguez-Arellano, Parpura, & Zorec, 2017). Generation of astrocytic Ca²⁺ signals is mainly due to Ca²⁺ release from the ER, which is mediated by a plethora of ionotropic glutamatergic, purinergic and cholinergic receptors, transient receptor potential (TRP) channels, and NCX_rev (Verkhratsky et al., 2017). Alberdi et al. (2013) found that exposure Aβ (Aβ 1–42) oligomers to cultured astrocytes led to a cytoplasmic Ca²⁺ increase and triggered increased expression of GFAP, as well as oxidative and ER stress, as indicated by eIF2α phosphorylation and overexpression of chaperone GRP78 (Alberdi et al., 2013). These effects were decreased by inhibitors of phospholipase C and inhibitors of ryanodine receptors and InsP₃ receptors, respectively (Alberdi et al., 2013; Lim et al., 2013). These results provide evidence that Aβ disrupts ER Ca²⁺ homeostasis and induces ER stress that leads to astrogliosis, which may be relevant to AD pathophysiology (Alberdi et al., 2013). Taken together, astrocytic ion homeostasis is important in AD progression and future study is needed to further examine roles of ion transporters in reactive astrocytes in AD brains.

2.2.2 |. Astrocytic ion transporters are involved in dopaminergic neuron degeneration in PD

PD, the common progressive neurodegenerative movement disorder, is pathologically characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc; Booth, Hirst, & Wade-Martins, 2017) and intraneuronal aggregates of α-synuclein in neuronal cell bodies (Lewy body) and axons (Lewy neurites; Lees, Hardy, & Revesz, 2009). Although the cellular mechanisms of dopaminergic loss in PD is not completely understood, several abnormalities including inflammation, mitochondrial dysfunction, iron accumulation, and oxidative stress are likely involved in the cascade of events (Aquilano, Baldelli, Rotilio, & Ciriolo, 2008; Lee, Kim, & Lee, 2017). It has been shown that astrocytes accumulate nonfibrillized α-synuclein in their cytoplasm during the initiation and progression of PD (Halliday & Stevens, 2011). Accumulated α-synuclein in astrocytes have been shown to produce proinflammatory cytokines and chemokines which lead to microglia activation and the dysregulation of other astrocyte functions, such as glutamate uptake and BBB integrity (Booth et al., 2017), and further cause loss of dopaminergic and motor neurons (Gu et al., 2010; Halliday & Stevens, 2011; Phatnani & Maniatis, 2015).

Oxidative stress is increasingly recognized as a central event contributing to the degeneration of dopaminergic neurons in the pathogenesis of PD (Guo, Zhao, Li, Li, & Liu, 2018). Moreover, the role of NO in the pathogenesis and progression of neurodegenerative illnesses such as PD and AD has become prominent over the years (Hannibal, 2016). Much of the oxidative damage of proteins due to the overproduction of NO by nitric oxide synthases (NOS) and its subsequent reactivity with ROS(Hannibal, 2016). High level of NOS was observed in the nigrostriatal region and basal ganglia in postmortem brains with PD (Aquilano et al., 2008). Astrocytic NCX plays an important role in the regulation of intracellular Ca²⁺ concentration and can be stimulated by NO in astrocytes (Ago et al., 2011). SEA0400, the specific NCX inhibitor, protects against MPTP-induced neurotoxicity by inhibiting NCX-mediated Ca²⁺ influx and blocking ERK phosphorylation and lipid peroxidation (Ago et al., 2011). Additionally, NCX1 mRNA is detected in the rat midbrain, where dopaminergic cell bodies are localized (Canitano et al., 2002), and in the rat striatum, where the terminal projections of dopaminergic nigrostriatal neurons are found (Papa et al., 2003). Moreover, NCX in mitochondria also plays a role in PD. PTEN-induced kinase 1 (PINK1) is a mitochondrial kinase that regulates Ca²⁺ efflux from the mitochondria via the mitochondrial NCX (Gandhi et al., 2009). The cause of mitochondrial Ca²⁺ accumulation in PINK1 deficiency is a direct impairment of Ca²⁺ efflux from the mitochondria secondary to dysfunction of the NCX (Gandhi et al., 2009). Ca²⁺ overload stimulates ROS production via NADPH oxidase, which inhibits the glucose transporter, resulting in reduced substrate delivery and impaired respiration (Gandhi et al., 2009). Taken together, NCX is an important protein in PD but the underlying mechanism of astrocytic NCX in reactive astrocyte dysfunction and its contribution to PD pathology need to be further studied.

2.2.3 |. Microglial ion transporters in inflammation and neurodegeneration in PD

In PD, neuroinflammation and microglia activation are considered neuropathological hallmarks (Joers, Tansey, Mulas, & Carta, 2017). Activated microglia were reported to cluster around dead or dystrophic, pigmented dopamine neurons in the PD substantia nigra and elevated cytokines and other inflammatory mediators were subsequently found both in the nigra and striatum of PD patient autopsy (Rogers, Mastroeni, Leonard, Joyce, & Grover, 2007). Microglia is recognized as a double-edged sword, depending on the activation state of microglia and showing both neurotoxic and neuroprotective effects (Okolie et al., 2016). In the early stage of the PD, microglia can be activated directly or indirectly by a range of misfolded proteins or pathogens, such as α-synuclein, and produce a variety of proinflammatory mediators such as cytokines, chemokines, which contribute to engulf infectious organisms or invading pathogens, and clearing toxic proteins and cell debris from the injury site by phagocytosis (Okolie et al., 2016). However, prolonged or excessive activation of microglia may result in pathological forms of inflammation that contribute to the progression of neurodegeneration, causing the loss of dopaminergic neurons (Cao, Standaert, & Harms, 2012; Ramirez et al., 2017). Among the three different NCX isoforms (NCX1, NCX2, and NCX3), NCX1 is the most highly expressed isoform in microglia (Boscia et al., 2009). Upregulation of microglial NCX1 plays a crucial role in regulation of microglia activation (Boscia et al., 2009). Sirabella et al. (2018) found that high levels of NCX1 protein expression are detected in IBA-1-positive microglial cells in the striatum of A53T mice (an animal model mimicking a familial form of PD). Whereas decreased NCX3 protein was observed in tyrosine hydroxylase (TH)-positive neurons in SNc of A53T mice, a phenomenon that was accompanied by an increase in mitochondrial Ca²⁺ concentrations and neuronal death (Sirabella et al., 2018). These new findings demonstrated that changes in NCXs expression and activity in the midbrain and striatum of A53T mice contribute to the loss of dopaminergic neurons and also suggested that an imbalance of the mechanisms involved in the regulation of cytosolic and mitochondrial Ca²⁺ homeostasis might be associated with the degeneration of dopaminergic neurons in the presence of a mutated form of α-synuclein A53T, thus revealing new potential players in the pathophysiology of PD (Sirabella et al., 2018). Limited information is available on the underlying mechanism of NCXs in astrocytes and microglia involved in PD pathology by regulating Ca²⁺ homeostasis. Future studies are needed to investigate glial ion transporters in specific processes of the pathological development of PD.

2.2.4 |. Glial ion transporters in axonal damage in MS

MS is a chronic demyelinating disease of the CNS, characterized by immune-mediated inflammatory and degenerative changes (Lemus, Warrington, & Rodriguez, 2018). This axonal demyelination and neurodegeneration play an important role in accumulating nonremitting neurologic deficits in chronic lesions, showing a variety of clinical presentations and unclear pathogenesis (Lemus et al., 2018; Ludwin, Rao, Moore, & Antel, 2017; Plemel, Liu, & Yong, 2017). Although the mechanisms involved in MS are incompletely understood, myelin membranes and oligodendrocytes are destroyed through chronic inflammation in the CNS (Dulamea, 2017). In general, a multifactorial interplay of genetic and environmental factors leads to a chronic activation of the immune cells and cerebral tissue injury in MS (Dulamea, 2017). NCX3, identified as a myelin membrane component, regulates [Na⁺]_i and [Ca²⁺]_i in oligodendrocytes during physiological and demyelinating conditions (Boscia et al., 2016). Ca²⁺ signaling mediated by the NCX3 (forward mode) is involved in oligodendrocyte maturation and myelin formation. Knockdown of NCX3 isoform in OPCs prevented expressions of two major myelinating oligodendrocyte markers 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) and myelin basic protein (MBP), while overexpression of NCX3 isoform triggered an upregulation of CNPase and MBP expression (Boscia et al., 2012). Furthermore, NCX3 knockout mice showed a reduced size of spinal cord with marked hypo-myelination, a reduced diameter of axons and a dramatic decrease in OPC and premyelinating cells (Boscia et al., 2012; Casamassa et al., 2016). Moreover, during the chronic phase of an EAE model, elevated NCX3 protein expression was detected and coexpressed with the marker NG2 and CNPase while knockout of NCX3 accelerated the early onset of symptoms (Casamassa et al., 2016). In addition, astrocytes function as CNS immune cells and release inflammatory molecules in the pathogenesis of MS (Guerrero-Garcia, 2017). Astrocytes exert active, dual, and paradoxical roles during disease development in MS (Williams, Piaton, & Lubetzki, 2007). They could contribute to MS through the following mechanisms: (a) as part of the innate immune system, (b) as a source of cytotoxic factors, (c) inhibiting remyelination and axonal regeneration by forming a glial scar, and (d) contributing to axonal mitochondrial dysfunction (Correale & Farez, 2015). Chronic MS plaques contain demyelinated axons that are often surrounded by a dense meshwork of astrocytic processes (Holley, Gveric, Newcombe, Cuzner, & Gutowski, 2003). These astrocytic processes exhibited immunofluorescence staining signals of Na_v1.2 and NCX (no specific isomer is determined). Astrocytic NCX can contribute to the regulation of Ca²⁺_i levels via forward Ca²⁺ extrusion or reverse Ca²⁺ influx operation. Black, Newcombe, Trapp, and Waxman (2007) examined the expression of Na_v1.2 and NCX in chronic MS plaques within the spinal cord. They found Na_v1.2 was not detected along demyelinated axons in chronic lesions but was expressed by scar and reactive astrocytes within the plaque (Black et al., 2007). Moreover, NCX protein was clearly present within the scar astrocytes surrounding the demyelinated axons (Black et al., 2007). Although the functional significance of NCX and Nav1.2 in these astrocytes is not clear at this time, the presence of NCX in these cells implies that these molecules may be involved in Ca²⁺ flux into or out of glial cells within chronic lesions. Future studies are needed to understand function of astrocytic NCX and other ion transporters in peri-plaque scars and demyelination in MS.

3. |. CONCLUSION

The significance of glial cells (astrocytes, microglia, and oligodendrocytes) in neurological diseases has now been widely recognized. Several major ion transporters (NCX, NHE1, and NKCC1, and so on) are well documented to play critical and distinct roles in regulation of glial functions under physiological and pathophysiological conditions. During acute brain injuries (such as ischemic stroke and TBI), these ion transporters are rapidly activated in glial cells and affect excitotoxicity, ion homeostasis, inflammation, and myelination in experimental models. However, in comparison to ischemic stroke research, studies of these glial ion transporters in post-TBI brains and their impact on tissue repair and chronic functional deficits are lacking. Some newly emerged studies suggest that ion transporters (NHE1 and NKCC1) play important roles in activation and transformation of TAMs and TAAs in developing protumoral microenvironment of glioma. Glial NCX (in astrocytes and microglia) is shown to regulate Ca²⁺ signaling and glutamate uptake in experimental AD, MS, and PD models. Considering the significant roles of glial cells in both acute and chronic neurological diseases, our knowledge of glial ion transporters in transformation of glial functions and in disease pathogenesis is limited, which warrants further exploration, especially for chronic neurodegenerative diseases.