We have shown that Hv1 is expressed and functions as the voltage–gated proton–selective current in mouse and cultured human brain microglia. We found that microglial Hv1 is required for NOX–dependent ROS generation both in vitro
and in vivo
. Hv1, by rapidly exporting positive charge (H+
), is required for NOX to effectively transport electrons and thus generate ROS. Experimentally induced ischemia or metabolic challenge elicits ROS production from microglia and other inflammatory cells. ROS are not entirely detrimental, as they are also cerebral vasodilators and mediators of tissue repair and remodeling following ischemic injury37
. Nevertheless, neuronal lipids, membrane receptors, intracellular kinases and phosphatases, as well as pro–apoptotic transcription factors1
are all targets of ROS that damage brain function (Suppl. Fig. 13
Our ROS imaging experiments demonstrate that mouse microglial Hv1 is activated and indeed required for the majority of NOX–dependent ROS production by microglia in situ. The Hv1 channel is ideally suited for this function since it is activated only upon depolarization. The amount of depolarization needed is inversely proportional to the pH gradient. Under most physiologically relevant conditions this means that upon cell acidification, less depolarization from rest is required to allow protons to exit the cell, exactly the conditions under which NADPH oxidase is active. Without Hv1, NOX activation will cause significant depolarization and intracellular acidification that will eventually inhibit NOX activation.
One of the many changes defining microglia activation in response to ischemia is translocation of NOX cytosolic subunits to the plasma membrane where it becomes active32, 34
. Here, we have shown that microglial Hv1 contributes significantly to NOX ROS production, supporting NOX–mediated neuronal cell death after stroke. First, PMA– or OGD–induced ROS production was significantly reduced in Hv1−/−
microglia compared with wt
microglia. Second, ROS production in brain microglia in situ
after stroke was reduced in Hv1−/−
mice. Third, reduced neuronal death and brain damage was observed in Hv1−/−
mice compared to wt
mice after stroke. Fourth, OGD–induced neuronal death in microglia (Hv1−/−
)–neuron co–cultures was less than that in wt
co–cultures. Fifth, rescue by ROS scavengers was less in Hv1−/−
compared to wt mice. Finally, chimeric mice with wt
microglia, but Hv1−/−
circulating blood cells (Hv1−/−>wt
), showed significantly more brain damage than mice with Hv1−/−
microglia and wt
blood cells (wt> Hv1−/−
), establishing that 24h after stroke, resident microglia are responsible for most of the ROS–mediated brain damage.
All cell types in the brain contain NADPH oxidases and mice deficient in NOX2 or NOX4 have better stoke outcomes5, 6
. Astrocytes comprise a significant proportion of cells in the brain and express NOX1, 2 and 338
, but we did not detect Hv1 currents in mouse astrocytes. We assessed brain tissue damage at 1d after MCAO, but large numbers of circulating blood cells enter the brain at later times after stroke39, 40
. Therefore, we do not exclude the contribution of Hv1 in late stages of stroke. A caveat to the interpretation of bone marrow chimera experiments is the potential complication of irradiation41
. Nonetheless, it is unlikely that microglia are the only sources of ROS–mediated brain damage since ROS are released from all metabolically stressed mitochondria. Moreover, microglia may exert important protective effects by producing IL–10 and TGF–β, as well as growth factors in the post–ischemic brain42
. Therefore, the dual protective/destructive effects of microglia should be considered when targeting microglia for stroke treatment.
We found no voltage–gated proton current in hippocampal or cortical neurons in acute brain slices. Moreover, hippocampal basal synaptic transmission, plasticity or NMDA receptor function did not differ between wt
mice. NMDA induced similar degree of cell death in wt
neurons. Thus, glutamate neurotoxicity is unlikely to underlie Hv1’s contribution to brain damage. Finally, since Hv1 function is important in peripheral blood cells13,14
, late effects after stroke requires future study.
Currently, therapies for ischemic stroke are limited43
. Recent studies using NOX inhibitors show conflicting results, perhaps due to poor NOX selectivity43
. We suggest that Hv1 channels may be more tractable targets for prevention of brain injury during ischemia. Notably, since Hv1 currents below detection levels in neurons, Hv1 inhibitors should not limit neuronal NOX activity. Our results may also be relevant for other ischemic disorders and ROS–related neurodegeneration.