Although the precise mechanisms determining the neurotoxic or neuroprotective activation phenotypes in microglia remain poorly characterized, metabolic changes in these cells appear critical for these processes. As cellular metabolism can be tightly regulated by changes in intracellular pH, we tested whether pharmacological targeting of the microglial voltage-gated proton channel 1 (Hv1), an important regulator of intracellular pH, is critical for activated microglial reprogramming. Using a mouse microglial cell line and mouse primary microglia cultures, either alone, or co-cultured with rat cerebrocortical neurons, we characterized in detail the microglial activation profile in the absence and presence of Hv1 inhibition. We observed that activated microglia neurotoxicity was mainly attributable to the release of tumor necrosis factor alpha, reactive oxygen species, and zinc. Strikingly, pharmacological inhibition of Hv1 largely abrogated inflammatory neurotoxicity not only by reducing the production of cytotoxic mediators but also by promoting neurotrophic molecule production and restraining excessive phagocytic activity. Importantly, the Hv1-sensitive change from a pro-inflammatory to a neuroprotective phenotype was associated with metabolic reprogramming, particularly via a boost in NADH availability and a reduction in lactate.Most critically, Hv1 antagonism not only reduced inflammatory neurotoxicity but also promoted microglia-dependent neuroprotection against a separate excitotoxic injury.Our results strongly suggest that Hv1 blockers may provide an important therapeutic tool against a wide range of inflammatory neurodegenerative disorders.

Background The precise mechanisms determining the neurodestructive or neuroprotective activation phenotypes in microglia remain unknown. Emerging evidence suggest, however, that metabolic changes are important for microglial phenotype determination. Metabolism, in turn, can be tightly regulated by changes in intracellular pH. The aim of this work was to establish whether pharmacological targeting of the microglial voltage gated proton channel 1 (Hv1), an important regulator of intracellular microglial pH, is critical for reducing inflammatory neurotoxicity while maintaining the neuroprotective components of activation, as well as to describe the mechanism behind this process. Methods We used lipopolysaccharide/gamma interferon (LPS/IFNγ) to activate a microglial cell line and primary microglia isolated from mouse and established the activation profile in the absence and presence of pharmacological Hv1 inhibition. Additionally, we utilized co-cultures of microglia and cortical rat neurons to assess the role of Hv1 in inflammatory neurotoxicity and the modulation of excitotoxic injury using a range of neuronal viability assays. Results Activation with LPS/IFNγ induced widespread production of proinflammatory mediators, as well as reactive species and phagocytic activity. The ensuing neurotoxicity was mainly attributable to the release of tumor necrosis factor alpha (TNFα), reactive oxygen species and zinc. Strikingly, pharmacological inhibition of Hv1 largely abrogated inflammatory neurotoxicity not only by reducing the production of cytotoxic mediators, but also by promoting neurotrophic molecule production and restraining phagocytic activity. Furthermore, we demonstrate that this Hv1-mediated change from a pro-inflammatory to a neuroprotective phenotype is associated with metabolic microglial reprogramming. Finally, we show that Hv1 antagonism not only reduces inflammatory neurotoxicity, but also promotes neuroprotection against excitotoxic injury. Conclusions We present a comprehensive characterization of the molecular components that are modulated by Hv1 activity during microglial activation in both a cell line and in primary microglial cells. Moreover, we show that Hv1 antagonism retains the beneficial aspects of microglial activation, not only by reducing inflammatory neurotoxicity, but also by providing neuroprotection in an in vitro model of excitotoxicity. Our results suggest that Hv1 blockers are likely to provide an important therapeutic tool against a wide range of inflammatory neurodegenerative disorders.