**3. Role of KCC3 in Stroke**

Here, we recall as stated earlier in Section 2 of this review that the stimulation/inhibition of NKCCs/KCCs pair via protein phosphorylation is through a reciprocal regulatory mechanism [67,72] (Figure 2). NKCC and KCC participations in cell volume regulations via RVI and RVD mechanisms, respectively, have also been earlier highlighted [19,53,65] (Figure 1).

It is only expected that in neuronal functions regulation, activation of KCC3 would play similar physiological roles to those that the inhibition of NKCC1 would. The WNK-Cab39 signaling increased expression of NKCC1 mRNA gene in brain tissues of rats subjected to ischemic stroke. It is proposed to have probable effects on the expression of other cotransporters such as KCC3 [106]. KCC3 expression in the brain requires NKCC1 expression for physiological regulation of cellular homeostasis in the CNS [72,86,87,113,114]. Hence, the roles of WNK-Cab39-KCC signalling in ischemic stroke should be further investigated [106]. In a mouse model study, Lucas et al. [114] demonstrated that alongside inhibited NKCC1, stimulation of KCC3 promoted decreased [Cl−]i in the sensory neuron of adult mice. This suggests their involvement in GABAergic/glycinergic transmission as adjudged by its influence on the hyperpolarization of GABAA equilibrium potential (EGABA-A) resulting in inhibitory GABAergic neurotransmission due to a decrease in [Cl−]i. Our recent functional kinomics study alluded that regulatory phosphorylation of KCC3 (Thr991/Thr1048 residues) is essential for cell volume homeostasis in the mammalian brain [86]. The notion that supports KCC3 physiological role in regulating [Cl−]i and consequent influence on GABA polarization state is fascinating and suggests possible relationship between neuronal excitability and cell volume homeostasis [86]. Moreover, this concept behind the physiological function of KCC3 is an indication that it might have a dual role in the regulation of both cell volume and [Cl−]i [66] which will be highly relevant in understanding its role in the etiology of stroke.

Furthermore, Byun and Delpire [115] reported that stimulation of KCC3 are involved in cell volume regulation (via RVD) in the nervous system, thereby emphasizing its role in the development and maintenance of myelin and peripheral nerves. The study further established that inhibition of KCC3 by knocking out its expression in mice caused anoxal and periaxonal swelling that ultimately led to neurodegeneration [115]. Another mousemodel study demonstrated that KCC3 gene knockout (KO) in parvalbumin neuron caused peripheral agenesis neuropathy associated with the agenesis of corpus callosum. Similarly, the post-mortem study by Auer and colleagues [116] suggested that neuropathological features observed in the central and peripheral nervous systems (CNS/PNS) could potentially link to genetic defects in axonal KCC3 of CNS/PNS. Indeed, sensory defects in KCC3 knockout (KCC3−/−) mice as well as its mutations in humans [63,66,117–119] confirm the fundamental role of the cotransporter in peripheral neurons (also see reviews [53,65,66]). Loss of function mutations function of KCC3 have contributed to the pathogenesis of motor and sensory peripheral neuropathy in adult animals and humans [114,115,120–122]. Manifestations of peripheral neuropathy or fluid-related axonopathy influence cell volume dysregulation [26,115] and may be involved in the pathogenesis of other neurological conditions such as stroke.

#### **4. Role of Regulatory WNK-SPAK/OSR1 Pathway in Stroke**

Certainly, the various cellular functional roles of CCCs in the biological system will be compromised without regulatory mechanisms in place. Thus, it is only principally reasonable that the cotransporters actively and continuously maintain their functional integrity through coordinated mechanisms of regulations [53,60,67,69]. Several reports owing to WNK-SPAK/OSR1 kinases as the most involved signaling pathway in the regulation of neuronal Cl− and cell volume homeostasis do exist [60,65,70,71,123] and these established roles of the WNK-SPAK/OSR1-CCC pathway have alluded to their connection with stroke [19,65,71].

There is a growing body of evidence that the WNK-SPAK/OSR1-CCC pathway is involved in pathogenesis of stroke [53,65,70,106,124]. The established roles of the WNK-SPAK-CCC pathway on GABA signaling and cell volume homeostasis are linked to several neurological diseases such as cerebral stroke [53,60,125]. WNK and SPAK/OSR1 kinases are copiously expressed in the CNS [75]. After an ischemic stroke, both NKCC1 and KCCs are phosphorylated via the WNK-SPAK/OSR1 signaling pathway, leading to activation and inhibition of NKCC1 and KCCs, respectively [22,65]. However, inactivating the WNK- SPAK-CCC cascade through concurrent inhibition of NKCC-mediated ionic influx and stimulation of the KCC-mediated ion efflux has been shown to reduce cellular swelling in ischemic stroke brains [53,65]. The regulatory role of WNK-SPAK-CCC in cellular ionic homeostasis have also been shown to contribute to post-ischemic stroke infarction and cerebral edema [66]. Thus, inactivation of the WNK-SPAK-CCC cascade would trigger the simultaneous inhibition of NKCC mediated ionic import and stimulation of KCC mediated ionic export to eradicate cellular osmotic imbalance [53,65]. It has also been reported that estradiol increases NKCC1 phosphorylation consequently promoting GABA-mediated depolarization [126]. This occurs through stimulation of SPAK and OSR1 that is transcription dependent [127]. Studies using focal ischemia rat model have shown that estradiol treatment promotes neurogenesis in the subventricular zone of the brain, probably by increased expression of HIF-1α and VEGF [128]. WNK phosphorylate SPAK/OSR1, which in turn, phosphorylate NKCC1 and KCC3 at key regulatory sites [129]. Previous reports have shown that SPAK has a CCT domain to interact with NKCC1 and the KCCs [11,89,91,130]. However, the understanding of their physiological functions in normal and ischemic brains are still elusive [92].

Indeed, WNK isoforms are selectively expressed in the CNS [131] and WNK3 is mostly expressed in the brain [132]. This particular WNK isoform exerts its action on NKCCs and KCCs reciprocally [53,113]. Thus, the reciprocal actions of WNK3 on NKCC1 and the KCCs along with its concurrent expression with cotransporters in GABAergic neurotransmission that undergo dynamic changes in [Cl−]i, suggest its involvement in regulation of neuronal CCCs [53,133,134]. In fact, Kahle et al. [113] provided a compendium of data that suggested WNK3 as a dynamic regulator of NKCC1 and KCCs physiological activities. Simultaneous expression of WNK3 and NKCC1 in neurons may lead to enhanced phosphorylation of regulatory sites in NKCC1 and a consequent increase in the activity of NKCC1 [32,135]. The target of protein phosphatase 1 (which recognizes the consensus motif: RVNFXD) is a highly conserved RVNFVD sequence that is located in the amino-terminus of NKCC1. The RVNF binding motif overlaps with the SPAK binding motif (RFRV). A slight mutation of this sequence will cause NKCC1 activity to increase [136]. Interestingly, phenotypes of NKCC1 inhibition and KCC activation due to inactive WNK3 signaling pathway are reversed by potential protein phosphatase 1 inhibitors such as calyculin A and cyclosporine A [133,137]. According to Melo et al. [138], WNK3 inhibits the activity of KCC3 by promoting the phosphorylation of Thr<sup>991</sup> and Thr<sup>1048</sup> as well as Ser96, a third phospho-site involved in KCC3 regulation (also see Figure 2). Double (KCC3-T991A/T1048A) or triple (KCC3-S96A/T991A/T1048A) alanine mutations of KCC3, activated the cotransporter, which further increased hypotonicity. Thus, the study suggested that the phosphorylation of WNK3 signaling pathway was disabled, subsequently activating KCC3 by cell swelling [138].

Certainly, the upstream WNK3-SPAK/OSR1 pathway regulation of NKCC1 activity coupled with inhibition of KCC3 is implicated in the pathology of ischemic stroke [86]. Previous studies have demonstrated that WNK3 KO mice exhibited a reduction in infarct volume and axonal demyelination coupled with diminished cerebral edema and improved neurological behaviors following cerebral stroke when compared to WT mice with significantly activated WNK3 [32,86,92]. However, it is important to note that WT mice showed better survival and functional outcomes after a brain edema in comparison to mouse models lacking aquaporin-4 (AQP4) [139], a water transport system that allows for bidirectional water flux. As such, further research will need to elucidate the distinction between the role of AQP4 and WNK3 in cerebral edema and stroke. Thus, Begum and colleagues [32] observed stimulation of WNK3 and SPAK kinases in cortical neurons and primary oligodendrocytes cultured from the brain of mice subjected to transient MCA stroke [32]. They further established that cerebral ischemia facilitates hyperphosphorylation of the WNK3-SPAK/OSR1 catalytic T-loop and of NKCC1 stimulatory sites (Thr203/Thr207/Thr212); thus, increased expression of NKCC1 in the brain cells [32]. However, transgenic KO of WNK3, abridged ischemia-mediated SPAK/OSR1-NKCC1

phosphorylation and displayed reduced cerebral edema, axonal demyelination, and infarct volume, as well as improved post-stroke neurological recovery when compared to WT mice [32]. Briefly, the data presented by Begum et al. [32] identify the role of WNK3- SPAK/OSR1-NKCC1 signaling pathway in ischemic neuroglial injury and suggested that obstruction of this pathway could reduce NKCC1 expression in the brain and avert poststroke neuronal cell death following [32]. Similarly, Zhao et al. [92] demonstrated that KO of the WNK3-SPAK kinase complex in mice instigates decreased expression of NKCC1 and subsequently ameliorated cerebral infarction and edema after MCA stroke. Generally, deletion of the WNK3-SPAK kinase complex significantly produced less cytotoxic edema, less demyelination, and improved post-ischemic stroke neurological outcomes in the transgenic mice [92]. However, it is worth noting that the mechanism(s) of regulations employed by WNK3-SPAK/OSR1-NKCC1 signaling pathway in oligodendrogenesis is still elusive and requires further studies [26]. In addition, we recently demonstrated that WNK3 KO mice exhibit reduced endothelial and perivascular cytotoxic edema of astrocytes following post-ischemic stroke [86]. We further alluded that WNK3-SPAK inhibition confer neuroprotection on mammalian brain through concurrent stimulation of KCC3 activity at Thr991 and Thr1048 residues and inhibition of NKCC1 activity at Thr203, Thr207, and Thr212 residues [86].

In a recent rat model study, Bhuiyan et al. [106] demonstrated that WNK-Cab39- NKCC1 signaling pathway is implicated in ischemia. Furthermore, they suggested that activated WNK-Cab39 pathway increased NKCC1 activity in brain tissues of spontaneous hypertensive rats following subacute ischemic stroke [106]. A more recent similar report by Huang et al. [124] demonstrated that ischemic stroke with hypertension comorbidity further stimulates the WNK-SPAK/OSR1-NKCC1 signaling pathway, which contributes to deteriorated neurological functions/behavior [124]. In fact, the established role of WNK-SPAK/OSR1 signaling pathway in stimulating NKCC1 and inhibiting KCC3, which contribute to the pathogenesis of stroke, are the reasons for our recent pharmacological studies [70,71].

### **5. Current Pharmacological Treatments for Stroke**

We underscored in the earliest section of this review that stroke is one of the major threats to global health. Over the years, stroke had been a chief contributor to mortality and disabled lives across the globe; there are projections that the impact of this disease on global health may be worse in the near future. Presently, there are only few pharmacological strategies available to reduce the health and socio-economic burden triggered by this disease. Thus, there is an urgent need to tackle this disease. In this regard, research on the role of CCCs in the pathogenesis of stroke to inform future drug development is needed. Accordingly, this section of the review highlights current pharmacological approaches in the management of stroke with particular focus on molecular compounds that potentially inhibit SPAK/OSR1 pathway and NKCC1 and stimulate KCC3.
