Kv 1 . 3 In Microglia : Neuroinflammatory Determinant and Promising Pharmaceutical Target

Microglial cells are responsible for brain immunosurveillance. These cells maintain brain homeostasis, phagocytose cellular debris, present antigens to T-lymphocytes and secrete cytokines and chemokines. Upon activation, microglia can polarize into two different phenotypes in particular environments: the classical M1 proinflammatory state and the alternative M2 anti-inflammatory state. Chronically activated M1 microglial cells are involved in neuroinflammation in various brain diseases. In multiple sclerosis, Alzheimer’s disease, Parkinson’s disease and neuropathic pain, brain inflammation is involved in initiation and pathological progression, which is partially mediated by activated microglia. Molecular studies revealed that expression of the voltage-gated potassium channel Kv1.3 is a M1 phenotypic feature. Indeed, an increase in Kv1.3 expression and function was detected in chronically activated microglial cells in patients. Therefore, recent works evaluated Kv1.3targeted therapies for neuroimmune diseases and showed encouraging results. Inhibition of Kv1.3 activity led to amelioration of some pathology-related features in brain diseases. This review summarizes the latest findings concerning microglial function in neuroinflammatory processes, focusing on the involvement of Kv1.3 and highlighting this channel as a promising therapeutic target.


Microglia in Neuroinflammatory Diseases
Microglial cells are the main innate immune effector cells of the brain.Constantly scrutinizing the environment, these cells detect and react to injury and insults (immunosurveillance). Upon activation, microglia phagocytoses cellular debris, present antigens to T-cells and produce the cytokines and chemokines governing an intricate pluricellular response 1 .Microglia also plays key roles in homeostasis by regulating cell death and neurogenesis engulfing synaptic material 2 .Likewise, synaptic maturation links to microglial function 3 .The acute inflammatory response is advantageous and necessary for resolving a transient pathophysiological situation.However, chronic inflammation can lead to functional alteration and tissue damage.Nervous system inflammation (called neuroinflammation) can be particularly deleterious, contributing to pathogenesis in the peripheral and central nervous systems 2 .Indeed, low-level sustained neuroinflammation accompanied by a redox imbalance might cause and drive the disease progression 4 .
Alzheimer's disease (AD) is the most prevalent neurodegenerative chronic disease.The main pathological features of this disorder include extracellular senile plaques formed by amyloid-β peptide (Aβ) and intracellular aggregates of the microtubule-associated and phosphorylated protein tau 2 .In addition to these neuronal events, AD progression and even initiation also involves the immune system 5 .Amyloid precursor protein (APP) activates microglia 6 .Indeed, AD brains exhibited activated microglia, which endocytoses pathogenic Aβ peptides while producing survival-promoting signals.
However, prolonged inflammation generates synaptotoxic cytokines, chemokines and reactive oxygen/nitrogen species 7 .Further studies in the human brain showed Aβ plaques surrounded by IL-1β-positive microglia 8 .Moreover, impairment of microglial activation in a murine AD model exerted protective effects on hippocampal neurogenesis and improved cognitive defects 9 .
Parkinson's disease (PD) is characterized by the progressive death of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of Lewy bodies enriched in α-synuclein protein.Similar to the situation in AD, neuroinflammation participates in the exacerbation of the illness 2 .Dopamine neurons are highly sensitive to inflammatory injury, suggesting a role for microglia in PD progression 5 .Evidence indicates that pathological α-synuclein facilitates the progression of PD via the activation of the transcription factor NF-κB, which further increases the expression of proinflammatory cytokines, such as TNF-α and IL-1β 10 .Moreover, mutations in leucine-rich repeat kinase 2 compromise the ability of microglial cells to internalize and degrade α-synuclein, thus triggering neuroinflammation 11 .
During the progression of multiple sclerosis (MS), the involvement of microglia seems minor, although these cells are recruited to MS plaques.In this context, mast cells play major roles during the neuroinflammatory MS progression 2 .However, activation of microglia and astrocytes is a characteristic of amyotrophic lateral sclerosis (ALS) 12 .The crosstalk between mast cells and microglia further activates both cell types.Thus, microglia becomes more sensitive to damage-associated molecular patterns (i.e., high mobility group box 1), which are elevated in the spinal cord of ALS patients 13 .The association between mast cells and neuromuscular junction (NMJ) denervation indicates that after pathogenic events, mast cells infiltrate and degranulate at NMJs, which correlates with progressive NMJ degradation 2 .
The main cause of disability in the developed world is stroke, and 8 of 10 cases are due to cerebral ischemia 2 .The stroke-associated pathology suggests activation of microglia,

Neuroin ammation Pathogen removal
In ammation attenuation Homeostasis restablishment   -γ).M1-microglia releases cytokines and chemokines such as IL-1β and IL-6.These molecules mediate in neuroinflammation and pathogen removal.On the other hand, IL-4 and IL-10 enhance the M2 anti-inflammatory state (bottom, right), elevating the Kir2.1 expression.M2-microglia releases cytokines and chemokines such as Arg-1 and IGF-1.They mediate in inflammation attenuation and homeostasis reestablishment.The dichotomic phenotype is, nowadays, considered as a continuum for in vivo experiments.
release of cytokines/chemokines and infiltration of neutrophils/macrophages 14 .Interestingly, microglia, together with astrocytes and oligodendrocytes are pivotal for conditioning the microenvironment after traumatic brain injury (TBI) 15,16 .Moreover, a long-term investigation in humans showed chronically activated microglia years after the traumatic event 17 .Similarly, studies in rodents have shown upregulated proinflammatory markers, chronically activated microglia, injury extension and myelin damage 18 .Therefore, the therapeutic inhibition of microglial activation has been proposed as beneficial 2 .
Treatments attenuating peripheral neuropathic pain (NP) mainly focus on reducing pain transduction 19 .However, the origin and maintenance of this pathological condition depend mainly on astrocytes, microglia and Schwann cells, together with systemic immune cells 20 .Activated spinal microglia produces IL-1β to modulate neuronal activity via either membrane receptors or proinflammatory signals.Indeed, the expression of P2X and P2Y purinergic receptors in dorsal horn microglia is linked to NP development 21 .In fact, the inhibition of both receptors alleviates NP symptoms 22 .A recent study implicates microglia and monocytes in the switch from acute to chronic pain 23 .Therefore, targeting microglia may be beneficial in future NP therapies.Similarly, research on depression mainly focuses on neuronal pathophysiology.Nevertheless, evidence links depression to the impairment of normal microglial structure and function 24 .Both high activation and a lack of microglial senescence are associated with depression-related impairment of neuroplasticity and neurogenesis 2 .

Kv1.3 Pivotal Role in Altered Microglial Function
Within the limited repertoire of potassium channels in leukocytes, the voltage-gated potassium channel Kv1.3 is crucial during the immune system response.Kv1.3 participates in physiological processes and in the onset of several pathophysiological dysfunctions in immune system cells 25 .Kv1.3, together with the calcium-dependent potassium channel KCa3.1, sustains the ionic driving force necessary for the T-cell activation.Moreover, proliferation and activation of macrophages is linked to an upregulation of Kv1.3.On the other hand, lupus erythematosus, psoriasis, type I diabetes and rheumatoid arthritis are associated with impaired Kv1.3 activity.In addition to autoimmune diseases, cancer, metabolic disorders and chronic inflammatory pathologies are linked to this channel 26 .
During the last twenty years, evidence about the Kv1.3 function in microglia reveal a strong association with neuropathology.Microglial activation leads to two different functional phenotypes: M1 (classic/proinflammatory) and M2 (alternative/neuroprotective) 27  support a continuum of the activation status between the M1 and M2 phenotypes 28 .Some authors propose abandoning this oversimplification for a stimulus-based terminology.Similarly to the closely related macrophages 29 , Kv1.3 was functionally characterized in microglial cells and was strongly related to their proliferation 30 .The role of Kv1.3 in microglial activation was initially controversial and dependent on either the culture conditions or the mode of stimulation 31 .Some authors described that both classical M1 and an alternative M2 microglial subtype (M2a) exhibit an increase in Kv1.3 expression.Additionally, the M2a phenotype shows increases in anti-inflammatory genes and ROS production 32 .However, other lines of evidence indicate that Kv1.3 activity, together with KCa3.1, is confined exclusive to the M1 phenotype.On the other hand, cells in the M2 state have larger inward-rectifier potassium channel Kir2.1 currents than unstimulated microglial cells 31,33 .Concomitantly, Kv1.3 and KCa3.1 blockers impair the production of proinflammatory cytokines, such as IL-1β and TNF-α (Fig. 1).Moreover, activated microglia can kill neurons in a Kv1.3-dependent manner 34 .
Microglia is a central player in AD-associated neuroinflammation, and Kv1.3 regulates microglial function.Resting microglia also express Kv1.5, but upon activation, Kv1.3 expression increases at the plasma membrane 30 .Indeed, the exposure of rat microglial cells to Aβ increases Kv1.3 currents and leads to Kv1.3-dependent microglial priming 35 .Concomitantly, studies of the human brain pointed out that microglial cells from AD patients present increased expression of Kv1.3 36 .Apart from microglial alterations, AD patients have Kv1.3 channels that are insensitive to glutamate-dependent modulation in T-cells.This condition results in a 4-fold increase in the Kv1.3 resting channel activity 37 .Indeed, AD treatment with N-methyl-D-aspartate receptor (NMDAR) antagonists, such as memantine, decreases the Kv1.3 T-cell conductivity 38 .In healthy donors, memantine also inhibits CD3 antibodyinduced effects, impairs alloantigen-induced proliferation and suppresses the chemokine-induced migration of peripheral blood T-cells.CD4+ T-cells from AD patients receiving therapeutic doses of memantine exhibited a longlasting reduced proliferative response to alloantigens 39 .Thus, inhibition of Kv1.3 in AD might exert a beneficial bipartite effect on T-cells and microglia.Actually, PAP-1, a Kv1.3 inhibitor, reduces neuroinflammation, decreases amyloid load, enhances hippocampal neuronal plasticity and improves behavioral deficits in a murine AD model.Concomitantly, PAP-1 stimulates an uptake by microglia 40 .

MS exhibits a tight link to T-cell malfunction. Evidence indicates that Kv1.3 blockers ameliorate MS symptoms via
T-cell modulation 26 .Moreover, Kv1.3 expression increases in activated microglia, macrophages and reactive astrocytes at the peak of experimental autoimmune encephalomyelitis (EAE), a murine model of MS 41 .Indeed, the anti-Kv1.3vaccination of EAE animals reduces pathogenic T-cells and increases protective T subsets, as well as decreases the infiltration of microglia/macrophages.After vaccine administration, microglial cells shift towards the M2 antiinflammatory phenotype in the central nervous system 42 .The involvement of Kv1.3 has been further evaluated in other neuroinflammatory diseases.After status epilepticus, Kv1.3 expression increases in activated microglia 43 .Moreover, human microglia exhibits increased Kv1.3, KCa3.1 and Kir2.1 currents in brain stroke patients 44 .Therapeutically, the Kv1.3-inhibition diminishes the brain damage following a traumatic brain injury 45 .Therefore, a direct connection between microglia-mediated neuroinflammation and Kv1.3 expression is noticeable.
Due to its wide expression and prominent role during immune response, Kv1.3 blockers have been developed and tested as a potential treatment for diverse diseases.Studies in mice suggest Kv1.3 blockers as a possible therapy for obesity and insulin resistance.Moreover, Kv1.3 inhibition with Margatoxin reduces proliferative capacity of cancer cells.In EAE, the Kv1.3 channel blockage by ShK-22 ameliorated the clinical course of both moderate and severe EAE.Finally, two different treatments are being developed for treating psoriasis patients.Subcutaneous administration of dalatazatide (ShK-186) is demonstrated to improve the psoriatic skin lesions.Currently, a clinical trial is ongoing for studying the cutaneous effect of the Kv1.3 blocker PAP-1 26 .
In this pathophysiological scenario, the modulation of Kv1.3 is crucial.Elevated Kv1.3 activity in the M1 microglial phenotype is linked to neuroinflammatory processes 30,33 .Kv1.3 is effectively targeted to the plasma membrane, thus controlling the membrane potential, which is crucial for cell activation.Therefore, the determinants responsible for Kv1.3 cell surface targeting are worth studying 46 .In this context, we have identified KCNE4, an endogenous β subunit in leukocytes that reduces Kv1.3 membrane abundance.KCNE4, initially named MinK-related peptide 3, is an auxiliary subunit that promiscuously regulates a wide variety of voltage-gated channels mostly functioning as a channel inhibitor 47 .KCNE4 mutations have been linked to several human immune system diseases, such as allergic rhinitis and acute lymphoblastic leukemia 48,49 .KCNE4, which is tightly regulated 50 , is highly expressed in mononuclear phagocytes, such as dendritic cells and macrophages, modulating Kv1.3.Thus, KCNE4, which fine-tunes Kv1.3 function, is an important regulator of leukocyte physiology.KCNE4 impairs Kv1.3 trafficking to the cell surface via two synergistic mechanisms (Fig. 2) 47 .Therefore, the pharmacological targeting of KCNE4 during neuroinflammation could decrease the harmful effects of the Kv1.3-dependent activation of leukocytes.
Furthermore, limiting the effect to KCNE4-expressing cells might decrease treatment side-effects.

Concluding Remarks
Evidence demonstrates that the voltage-dependent Kv1.3 plays important roles in the cell physiology.Because this channel participates during the immune system response, a precise regulation of Kv1.3 activity and expression would fine-tune the magnitude of either the proor anti-inflammatory events.Microglia, being sentinel cells in the brain, functions as a first barrier against neuronal inflammation.Therefore, a deeper investigation of the Kv1.3 modulation by either a pharmacological targeting or by controlling the regulatory subunit expression in microglial cells is highly warranted to generate new treatment strategies 50 .Thereby, the control of Kv1.3 expression is a promising objective for improved treatment of neuroinflammatory diseases.