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Fucosylation, especially core fucosylation of N-glycans catalyzed by α1-6 fucosyltransferase (fucosyltransferase 8 or FUT8), plays an important role in regulating the peripheral immune system and inflammation. However, its role in microglial activation is poorly understood. Here we used human induced pluripotent stem cells-derived microglia (hiMG) as a model to study the role of FUT8-catalyzed core fucosylation in amyloid-β oligomer (AβO)-induced microglial activation, in view of its significant relevance to the pathogenesis of Alzheimer's disease (AD). HiMG responded to AβO and lipopolysaccharides (LPS) with a pattern of pro-inflammatory activation as well as enhanced core fucosylation and FUT8 expression within 24 h. Furthermore, we found increased FUT8 expression in both human AD brains and microglia isolated from 5xFAD mice, a model of AD-like cerebral amyloidosis. Inhibition of fucosylation in AβO-stimulated hiMG reduced the induction of pro-inflammatory cytokines, suppressed the activation of p38MAPK, and rectified phagocytic deficits. Specific inhibition of FUT8 by siRNA-mediated knockdown also reduced AβO-induced pro-inflammatory cytokines. We further showed that p53 binds to the two consensus binding sites in the Fut8 promoter, and that p53 knockdown abolished FUT8 overexpression in AβO-activated hiMG. Taken together, our evidence supports that FUT8-catalyzed core fucosylation is a signaling pathway required for AβO-induced microglia activation and that FUT8 is a component of the p53 signaling cascade regulating microglial behavior. Because microglia are a key driver of AD pathogenesis, our results suggest that microglial FUT8 could be an anti-inflammatory therapeutic target for AD.

Microglia-mediated inflammation exerts adverse effects in ischemic stroke and in neurodegenerative disorders such as Alzheimer's disease (AD). Expression of the voltage-gated potassium channel Kv1.3 is required for microglia activation. Both genetic deletion and pharmacological inhibition of Kv1.3 are effective in reducing microglia activation and the associated inflammatory responses, as well as in improving neurological outcomes in animal models of AD and ischemic stroke. Here we sought to elucidate the molecular mechanisms underlying the therapeutic effects of Kv1.3 inhibition, which remain incompletely understood. Using a combination of whole-cell voltage-clamp electrophysiology and quantitative PCR (qPCR), we first characterized a stimulus-dependent differential expression pattern for Kv1.3 and P2X4, a major ATP-gated cationic channel, both in vitro and in vivo. We then demonstrated by whole-cell current-clamp experiments that Kv1.3 channels contribute not only to setting the resting membrane potential but also play an important role in counteracting excessive membrane potential changes evoked by depolarizing current injections. Similarly, the presence of Kv1.3 channels renders microglia more resistant to depolarization produced by ATP-mediated P2X4 receptor activation. Inhibiting Kv1.3 channels with ShK-223 completely nullified the ability of Kv1.3 to normalize membrane potential changes, resulting in excessive depolarization and reduced calcium transients through P2X4 receptors. Our report thus links Kv1.3 function to P2X4 receptor-mediated signaling as one of the underlying mechanisms by which Kv1.3 blockade reduces microglia-mediated inflammation. While we could confirm previously reported differences between males and females in microglial P2X4 expression, microglial Kv1.3 expression exhibited no gender differences in vitro or in vivo. MAIN POINTS: The voltage-gated K+ channel Kv1.3 regulates microglial membrane potential. Inhibition of Kv1.3 depolarizes microglia and reduces calcium entry mediated by P2X4 receptors by dissipating the electrochemical driving force for calcium.

Microglia show a rich repertoire of activation patterns regulated by a complex ensemble of surface ion channels, receptors, and transporters. We and others have investigated whether microglia vary their K+ channel expression as a means to achieve functional diversity. However, most of the prior studies were conducted using in vitro models such as BV2 cells, primary microglia, or brain slices in culture, which may not accurately reflect microglia physiology in adult individuals. Here we employed an in vivo mouse model of selective innate immune activation by intracerebroventricular injection of lipopolysaccharides (ICV-LPS) to determine the role of the voltage-gated Kv1.3 channel in LPS-induced M1-like microglial activation. Using microglia acutely isolated from adult brains, we detected Kv1.3 and Kir2.1 currents, and found that ICV-LPS increased the current density and RNA expression of Kv1.3 but did not affect those of Kir2.1. Genetic knockout of Kv1.3 abolished LPS-induced microglial activation exemplified by Iba-1 immunoreactivity and expression of pro-inflammatory mediators such as IL-1β, TNF-α, IL-6, and iNOS. Moreover, Kv1.3 knockout mitigated the LPS-induced impairment of hippocampal long-term potentiation (hLTP), suggesting that Kv1.3 activity regulates pro-inflammatory microglial neurotoxicity. Pharmacological intervention using PAP-1, a small molecule that selectively blocks homotetrameric Kv1.3 channels, achieved anti-inflammatory and hLTP-recovery effects similar to Kv1.3 knockout. We conclude that Kv1.3 is required for microglial M1-like pro-inflammatory activation in vivo. A significant implication of our in vivo data is that Kv1.3 blockers could be therapeutic candidates for neurological diseases where microglia-mediated neurotoxicity is implicated in the pathogenesis.

Microglia are highly plastic cells that can assume different phenotypes in response to microenvironmental signals. Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) promote differentiation into classically activated M1-like microglia, which produce high levels of pro-inflammatory cytokines and nitric oxide and are thought to contribute to neurological damage in ischemic stroke and Alzheimer's disease. IL-4 in contrast induces a phenotype associated with anti-inflammatory effects and tissue repair. We here investigated whether these microglia subsets vary in their K+ channel expression by differentiating neonatal mouse microglia into M(LPS) and M(IL-4) microglia and studying their K+ channel expression by whole-cell patch-clamp, quantitative PCR and immunohistochemistry. We identified three major types of K+ channels based on their biophysical and pharmacological fingerprints: a use-dependent, outwardly rectifying current sensitive to the KV 1.3 blockers PAP-1 and ShK-186, an inwardly rectifying Ba2+ -sensitive Kir 2.1 current, and a Ca2+ -activated, TRAM-34-sensitive KCa 3.1 current. Both KV 1.3 and KCa 3.1 blockers inhibited pro-inflammatory cytokine production and iNOS and COX2 expression demonstrating that KV 1.3 and KCa 3.1 play important roles in microglia activation. Following differentiation with LPS or a combination of LPS and IFN-γ microglia exhibited high KV 1.3 current densities (∼50 pA/pF at 40 mV) and virtually no KCa 3.1 and Kir currents, while microglia differentiated with IL-4 exhibited large Kir 2.1 currents (∼ 10 pA/pF at -120 mV). KCa 3.1 currents were generally low but moderately increased following stimulation with IFN-γ or ATP (∼10 pS/pF). This differential K+ channel expression pattern suggests that KV 1.3 and KCa 3.1 inhibitors could be used to inhibit detrimental neuroinflammatory microglia functions. GLIA 2016;65:106-121.