Microglial Hv1 Research Articles

Knockout of microglial Hv1 proton channel reduces neurotoxic A1 astrocytes and neuronal damage via the ROS/STAT3 pathway after spinal cord injury.

Spinal cord injury (SCI) causes severe functional deficits and neuronal damage, accompanied by intense glial activation. The voltage-gated proton channel Hv1, selectively expressed on microglia, is associated with SCI progression. However, the effect of Hv1 on the phenotypes and functions of reactive astrocytes after SCI remains unclear. Here, we combined Hv1 knockout (Hv1-/- ) mice and T10 spinal cord contusion to investigate the effects of microglial Hv1 on SCI pathophysiology and the phenotypes and functions of reactive astrocytes. After SCI, astrocytes proliferated and activated in the peri-injury area and exhibited an A1-dominant phenotype. Hv1 knockout reduced neurotoxic A1 astrocytes and shifted the dominant phenotype of reactive astrocytes from A1 to A2, enhancing synaptogenesis promotion, phagocytosis, and neurotrophy of astrocytes. Moreover, synaptic and axonal remodeling as well as motor recovery after SCI benefited from the improved astrocytic functions of Hv1 knockout. Furthermore, exogenous and endogenous reactive oxygen species (ROS) in astrocytes after SCI were reduced by Hv1 knockout. Our in vitro results showed that inhibition of ROS reduced the neurotoxic A1 phenotype in primary astrocytes via the STAT3 pathway. Similar to the effect of Hv1 knockout, the application of the ROS scavenger N-acetylcysteine reduced SCI-induced neurotoxic A1 astrocytes in vivo. Based on the in vivo and vitro results, we elucidated that microglial Hv1 knockout promotes synaptic and axonal remodeling in SCI mice by decreasing neurotoxic A1 astrocytes and increasing neuroprotective A2 astrocytes via the ROS/STAT3 pathway. Therefore, the Hv1 proton channel is a promising target for the treatment of SCI.

The Emerging Role of Microglial Hv1 as a Target for Immunomodulation in Myelin Repair.

In the central nervous system (CNS), the myelin sheath ensures efficient interconnection between neurons and contributes to the regulation of the proper function of neuronal networks. The maintenance of myelin and the well-organized subtle process of myelin plasticity requires cooperation among myelin-forming cells, glial cells, and neural networks. The process of cooperation is fragile, and the balance is highly susceptible to disruption by microenvironment influences. Reactive microglia play a critical and complicated role in the demyelination and remyelination process. Recent studies have shown that the voltage-gated proton channel Hv1 is selectively expressed in microglia in CNS, which regulates intracellular pH and is involved in the production of reactive oxygen species, underlying multifaceted roles in maintaining microglia function. This paper begins by examining the molecular mechanisms of demyelination and emphasizes the crucial role of the microenvironment in demyelination. It focuses specifically on the role of Hv1 in myelin repair and its therapeutic potential in CNS demyelinating diseases.

Microglial voltage-gated proton channel Hv1 in spinal cord injury.

After spinal cord injury, microglia as the first responders to the lesion display both beneficial and detrimental characteristics. Activated microglia phagocyte and eliminate cell debris, release cytokines to recruit peripheral immune cells to the injury site. Excessively activated microglia can aggravate the secondary damage by producing extravagant reactive oxygen species and pro-inflammatory cytokines. Recent studies demonstrated that the voltage-gated proton channel Hv1 is selectively expressed in microglia and regulates microglial activation upon injury. In mouse models of spinal cord injury, Hv1 deficiency ameliorates microglia activation, resulting in alleviated production of reactive oxygen species and pro-inflammatory cytokines. The reduced secondary damage subsequently decreases neuronal loss and correlates with improved locomotor recovery. This review provides a brief historical perspective of advances in investigating voltage-gated proton channel Hv1 and home in on microglial Hv1. We discuss recent studies on the roles of Hv1 activation in pathophysiological activities of microglia, such as production of NOX-dependent reactive oxygen species, microglia polarization, and tissue acidosis, particularly in the context of spinal cord injury. Further, we highlight the rationale for targeting Hv1 for the treatment of spinal cord injury and related disorders.

Deficiency of microglial Hv1 channel is associated with activation of autophagic pathway and ROS production in LPC-induced demyelination mouse model

BackgroundMultiple sclerosis (MS) is an immune-mediated demyelinated disease of the central nervous system. Activation of microglia is involved in the pathogenesis of myelin loss.ObjectiveThis study is focused on the role of Hv1 in regulating demyelination and microglial activation through reactive oxygen species (ROS) production after lysophosphatidylcholine (LPC)-mediated demyelination. We also explored autophagy in this process.MethodsA model of demyelination using two-point LPC injection into the corpus callosum was established. LFB staining, immunofluorescence, Western blot, and electron microscopy were used to study the severity of demyelination. Microglial phenotype and autophagy were detected by immunofluorescence and Western blot. Morris water maze was used to test spatial learning and memory ability.ResultsWe have identified that LPC-mediated myelin damage was reduced by Hv1 deficiency. Furthermore, we found that ROS and autophagy of microglia increased in the demyelination region, which was also inhibited by Hv1 knockout.ConclusionThese results suggested that microglial Hv1 deficiency ameliorates demyelination through inhibition of ROS-mediated autophagy and microglial phenotypic transformation.

Proton extrusion during oxidative burst in microglia exacerbates pathological acidosis following traumatic brain injury.

Acidosis is among the least studied secondary injury mechanisms associated with neurotrauma. Acute decreases in brain pH correlate with poor long‐term outcome in patients with traumatic brain injury (TBI), however, the temporal dynamics and underlying mechanisms are unclear. As key drivers of neuroinflammation, we hypothesized that microglia directly regulate acidosis after TBI, and thereby, worsen neurological outcomes. Using a controlled cortical impact model in adult male mice we demonstrate that intracellular pH in microglia and extracellular pH surrounding the lesion site are significantly reduced for weeks after injury. Microglia proliferation and production of reactive oxygen species (ROS) were also increased during the first week, mirroring the increase in extracellular ROS levels seen around the lesion site. Microglia depletion by a colony stimulating factor 1 receptor (CSF1R) inhibitor, PLX5622, markedly decreased extracellular acidosis, ROS production, and inflammation in the brain after injury. Mechanistically, we identified that the voltage‐gated proton channel Hv1 promotes oxidative burst activity and acid extrusion in microglia. Compared to wildtype controls, microglia lacking Hv1 showed reduced ability to generate ROS and extrude protons. Importantly, Hv1‐deficient mice exhibited reduced pathological acidosis and inflammation after TBI, leading to long‐term neuroprotection and functional recovery. Our data therefore establish the microglial Hv1 proton channel as an important link that integrates inflammation and acidosis within the injury microenvironment during head injury.

Deficiency of the microglial Hv1 proton channel attenuates neuronal pyroptosis and inhibits inflammatory reaction after spinal cord injury

BackgroundSpinal cord injury (SCI) causes neurological dysfunction with devastating consequences. SCI pathogenesis is accompanied by inflammasome activation and neuronal damage. But the spatial pattern and the time course of neuronal pyroptosis and apoptosis after SCI should be further elucidated. The microglial voltage-gated proton channel (Hv1) is implicated in reactive oxygen species (ROS)-induced neuronal damage following ischemic stroke. However, there is a lack of quantification on the neuronal pyroptosis and apoptosis associated with microglial Hv1 after SCI.MethodsWe analyzed spatial and temporal characteristics of neuronal pyroptosis and apoptosis following SCI and investigated the effects of Hv1 deficiency on neuronal pyroptosis and the nod-like receptor 3 (NLRP3) inflammasome pathway by using a mouse model of SCI. We tested the effects of Hv1-deficient microglia on ROS production in vivo and examined the relationship between ROS and neuronal pyroptosis in vitro.ResultsWe observed that apoptosis was detected closer to the injury core than pyroptosis. The incidence of neuronal apoptosis peaked on day 1 after SCI and occurred before pyroptosis. Hv1 deficiency reduced neuronal apoptosis and NLRP3-inflammasome-mediated pyroptosis, improved axonal regeneration, and reduced motor deficits. SCI led to elevated ROS levels, whereas Hv1 deficiency downregulated microglial ROS generation. In vitro, ROS upregulated neuronal pyroptosis and activated the NLRP3 inflammasome pathway, both of which were reversed by addition of a ROS scavenger. Our results suggested that microglial Hv1 regulated neuronal apoptosis and NLRP3-induced neuronal pyroptosis after SCI by mediating ROS production.ConclusionFollowing SCI, neuronal pyroptosis lasted longer and occurred farther away from the injury core compared with that of neuronal apoptosis. Microglial Hv1 deficiency downregulated microglial ROS generation and reduced apoptosis and NLRP3-induced neuronal pyroptosis. Our findings may provide novel insights into Hv1-associated mechanisms underlying neuronal damage after SCI.

Microglial Hv1 exacerbates secondary damage after spinal cord injury in mice

The pathological process of spinal cord injury (SCI) is complex, particularly during secondary damage that triggers a multiphasic glial reaction consisting of both detrimental and beneficial effects. Deletion of a novel voltage-gated proton channel (Hv1) functionally expressed in microglia has been shown to confer neuroprotection during ischemic stroke. Here, we hypothesized that microglial Hv1 may also participate in the process of SCI through modulating glial responses. To test this hypothesis, we employed an SCI model in Hv1-knockout (Hv1−/−) and wild type (WT) mice and assessed resulting microglial polarization, accumulation of pro-inflammatory cytokines, astrocytic activation, oligodendrocytic apoptosis, lesion sizes, and demyelinated areas. Compared with post-SCI results in WT mice, post-SCI Hv1−/− mice exhibited an M2-dominant microglial polarization, decreased accumulation of microglia, and reduced production of pro-inflammatory factors such as tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β). Additionally, Hv1−/− mice had significantly attenuated reactive astrogliosis and reduced expression of chondroitin sulphate proteoglycans (CSPGs) after SCI. Furthermore, Hv1 deficiency reduced SCI-induced oligodendrocytic apoptosis, demyelinated areas, and cavity formation. Collectively, our results provide the first evidence suggesting that microglial Hv1 may be a multi-mechanism therapeutic target for the treatment of SCI.

Microglial Hv1 proton channels promote white matter injuries after chronic hypoperfusion in mice.

Microglia are critical in damage/repair processes during ischemic white matter injury (WMI). Voltage-gated proton channel (Hv1) is expressed in microglia and contributes to nicotinamide adenine dinucleotide phosphate oxidase complex-dependent production of reactive oxygen species (ROS). Recent findings have shown that Hv1 is involved in regulating luminal pH of M1-polarized microglial phagosomes and inhibits endocytosis in microglia. We previously reported that Hv1 facilitated production of ROS and pro-inflammatory cytokines in microglia and enhanced damage to oligodendrocyte progenitor cells from oxygen and glucose deprivation. To investigate the role of Hv1 in hypoperfusion-induced WMI, we employed mice that were genetically devoid of Hv1 (Hv1-/- ), as well as a model of subcortical vascular dementia via bilateral common carotid artery stenosis. Integrity of myelin was assessed using immunofluorescent staining and transmission electron microscopy, while cognitive impairment was assessed using an eight-arm radial maze test. Hv1 deficiency was found to attenuate bilateral common carotid artery stenosis-induced disruption of white matter integrity and impairment of working memory. Immunofluorescent staining and western blotting were used to assay changes in oligodendrocytes, OPCs, and microglial polarization. Compared with that in wild-type (WT) mice, Hv1-/- mice exhibited reduced ROS generation, decreased pro-inflammatory cytokines production, and an M2-dominant rather than M1-dominant microglial polarization. Furthermore, Hv1-/- mice exhibited enhanced OPC proliferation and differentiation into oligodendrocytes. Results of mouse-derived microglia-OPC co-cultures suggested that PI3K/Akt signaling was involved in Hv1-deficiency-induced M2-type microglial polarization and concomitant OPC differentiation. These results suggest that microglial Hv1 is a promising therapeutic target for reducing ischemic WMI and cognitive impairment.

Hv1 proton channel facilitates production of ROS and pro-inflammatory cytokines in microglia and enhances oligodendrocyte progenitor cells damage from oxygen-glucose deprivation in vitro

The contribution of microglial activation to oligodendrocyte precursor cell (OPC) damage in the brain is considered to be a principal pathophysiological feature of periventricular leukomalacia (PVL). Nicotinamide adenine dinucleotide phosphate oxidase (NOX)-dependent reactive oxygen species (ROS) produced in microglia has been shown to be significantly toxic to OPCs. The voltage-gated proton channel Hv1 is selectively expressed in microglia and is essential for NOX-dependent ROS production in the central nervous system. This study aimed to investigate the effects of microglial Hv1 deficiency on the protection of OPCs from oxygen-glucose deprivation (OGD)-induced injury in vitro. In the present study, the levels of OGD-induced ROS and pro-inflammatory cytokine production were dramatically lower in Hv1-deficient microglia (Hv1−/−) than in wild-type (WT) microglia. Following OGD, OPCs co-cultured with WT microglia had increased apoptosis and decreased proliferation and maturation, while those co-cultured with Hv1−/− microglia had attenuated apoptosis and greater proliferation and differentiation. Furthermore, the attenuated damage and enhanced regeneration of OPCs were associated with decreases in extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase phosphorylation. These results indicate that the protective effects of Hv1 deficiency on OPCs are due to the suppression of ROS and pro-inflammatory cytokine production in microglia. We thus suggest that the microglial proton channel Hv1 may be a potential therapeutic target in PVL.

Deficiency in the voltage‐gated proton channel Hv1 increases M2 polarization of microglia and attenuates brain damage from photothrombotic ischemic stroke

Microglia become activated during cerebral ischemia and exert pro-inflammatory or anti-inflammatory role dependent of microglial polarization. NADPH oxidase (NOX)-dependent reactive oxygen species (ROS) production in microglia plays an important role in neuronal damage after ischemic stroke. Recently, NOX and ROS are consistently reported to participate in the microglial activation and polarization; NOX2 inhibition or suppression of ROS production are shown to shift the microglial polarization from M1 toward M2 state after stroke. The voltage-gated proton channel, Hv1, is selectively expressed in microglia and is required for NOX-dependent ROS generation in the brain. However, the effect of Hv1 proton channel on microglial M1/M2 polarization state after cerebral ischemia remains unknown. In this study, we investigated the role of microglial Hv1 proton channel in modulating microglial M1/M2 polarization during the pathogenesis of ischemic cerebral injury using a mouse model of photothrombosis. Following photothrombotic ischemic stroke, wild-type mice presented obvious brain infarct, neuronal damage, and impaired motor coordination. However, mice lacking Hv1 (Hv1(-/-)) were partially protected from brain damage and motor deficits compared to wild-type mice. These rescued phenotypes in Hv1(-/-) mice in ischemic stroke is accompanied by reduced ROS production, shifted the microglial polarization from M1 to M2 state. Hv1 deficiency was also found to shift the M1/M2 polarization in primary cultured microglia. Our study suggests that the microglial Hv1 proton channel is a unique target for modulation of microglial M1/M2 polarization in the pathogenesis of ischemic stroke. The voltage-gated proton channel, Hv1, is selectively expressed in microglia and is required for NOX-dependent generation of reactive oxygen species (ROS) in the brain. ROS participate in microglial activation and polarization. However, the effect of Hv1 on microglial M1/M2 polarization state after cerebral ischemia remains unknown. Hv1 deficiency was found to shift the microglial polarization from M1 to M2 state in ischemic stroke accompanied by reduced ROS production. Our study suggests that the microglial Hv1 proton channel is a unique target for modulation of microglial M1/M2 polarization in the pathogenesis of ischemic stroke.

Microglial Hv1 proton channel promotes cuprizone-induced demyelination through oxidative damage.

NADPH oxidase (NOX)-dependent reactive oxygen species (ROS) production in inflammatory cells including microglia plays an important role in demyelination and free radical-mediated tissue injury in multiple sclerosis (MS). However, the mechanism underlying microglial ROS production and demyelination remains largely unknown. The voltage-gated proton channel, Hv1, is selectively expressed in microglia and is required for NOX-dependent ROS generation in the brain. In the present study, we sought to determine the role of microglial Hv1 proton channels in a mouse model of cuprizone-induced demyelination, a model for MS. Following cuprizone exposure, wild-type mice presented obvious demyelination, decreased myelin basic protein expression, loss of mature oligodendrocytes, and impaired motor coordination in comparison to mice on a normal chow diet. However, mice lacking Hv1 (Hv1(-/-) ) are partially protected from demyelination and motor deficits compared with those in wild-type mice. These rescued phenotypes in Hv1(-/-) mice in cuprizone-induced demyelination is accompanied by reduced ROS production, ameliorated microglial activation, increased oligodendrocyte progenitor cell (NG2) proliferation, and increased number of mature oligodendrocytes. These results demonstrate that the Hv1 proton channel is required for cuprizone-induced microglial oxidative damage and subsequent demyelination. Our study suggests that the microglial Hv1 proton channel is a unique target for controlling NOX-dependent ROS production in the pathogenesis of MS.

Abstract T P229: Deletion of Voltage Gated Proton Channel in Microglial Cells is Neurovascular Protective After Ischemic Brain Injury

Despite the failure of antioxidant treatments in clinical trials, the undoubted role of reactive oxygen species (ROS) in neurovascular damage after ischemic stroke calls for a more targeted approach. ROS production by microglia, the primary resident immune cells in the brain, is a key event of this process in ischemic stroke. Voltage gated proton channel, Hv1, is localized primarily to microglia and sustains NADPH oxidase activity. Deletion of Hv1 is neuroprotective after permanent middle cerebral artery occlusion (MCAO). We hypothesized that Hv1-mediated microglial ROS generation is also critical for vascular integrity and contributes to reperfusion injury after transient ischemic stroke. The wildtype (WT) and Hv1 knockout (KO) rats (n=4) were subjected to permanent or 3/24 h transient MCAO. The neurological deficiency, infarct, hemorrhagic transformation, and edema ratio were assessed. We found that in both permanent and transient MCAO model, KO rats develop smaller infarct, less vascular injury, edema, and hemorrhagic transformation, resulting in better short-term functional outcome. These results suggest that deletion of microglial Hv1 channel is vasculoprotective after ischemia/reperfusion and the underlying mechanisms need to be further studied.

Microglial Voltage-Gated Proton Channel Hv1 in Ischemic Stroke

Microglia, resident immune cells in the brain, contribute both to the damage and resolution of ischemic stroke. However, the mechanisms of microglia's detrimental or beneficial role in the disease are poorly understood. The voltage-gated proton channel, Hv1, rapidly removes protons from depolarized cytoplasm, and is highly expressed in the immune system. In the brain, Hv1 is selectively and functionally expressed in microglia but not neurons. Although the physiological function of microglial Hv1 is still not clear, Hv1 is one of major ion channels expressed in resting microglia. Under pathological conditions, microglial Hv1 is required for NADPH oxidase (NOX)-dependent generation of reactive oxygen species (ROS) by providing charge compensation for exported electrons and relieving intracellular acidosis. In a mouse model of cerebral middle artery occlusion, Hv1 knockout mice are protected from ischemic damage, showing reduced NOX-dependent ROS production, microglial activation and neuronal cell death. Therefore, microglial Hv1 aids in NOX-dependent ROS generation, which subsequently induces neuronal cell death and a significant fraction of brain damage after ischemic stroke. These studies illuminate a critical role of microglial Hv1 in ischemic brain injury, providing a rationale for Hv1 as a potential therapeutic target for the treatment of ischemic stroke. The current understanding of Hv1 in ischemic injury through NOX-dependent ROS production may serve as a common model to reveal the deleterious role of microglia in neurological diseases other than ischemic stroke, such as multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, and neuropathic pain.