Differential contribution of THIK-1 K+ channels and P2X7 receptors to ATP-mediated neuroinflammation by human microglia.

Neuroinflammation is highly influenced by microglia, particularly through activation of the NLRP3 inflammasome and subsequent release of IL-1ß. Extracellular ATP is a strong activator of NLRP3 by inducing K+ efflux as a key signaling event, suggesting that K+-permeable ion channels could have high therapeutic potential. In microglia, these include ATP-gated THIK-1 K+ channels and P2X7 receptors, but their interactions and potential therapeutic role in the human brain are unknown. Using a novel specific inhibitor of THIK-1 in combination with patch-clamp electrophysiology in slices of human neocortex, we found that THIK-1 generated the main tonic K+ conductance in microglia that sets the resting membrane potential. Extracellular ATP stimulated K+ efflux in a concentration-dependent manner only via P2X7 and metabotropic potentiation of THIK-1. We further demonstrated that activation of P2X7 was mandatory for ATP-evoked IL-1ß release, which was strongly suppressed by blocking THIK-1. Surprisingly, THIK-1 contributed only marginally to the total K+ conductance in the presence of ATP, which was dominated by P2X7. This suggests a previously unknown, K+-independent mechanism of THIK-1 for NLRP3 activation. Nuclear sequencing revealed almost selective expression of THIK-1 in human brain microglia, while P2X7 had a much broader expression. Thus, inhibition of THIK-1 could be an effective and, in contrast to P2X7, microglia-specific therapeutic strategy to contain neuroinflammation.

Pannexin 1: a novel regulator of acute hypoxic pulmonary vasoconstriction.

AIMS: Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to alveolar hypoxia that diverts blood flow from poorly ventilated to better aerated lung areas to optimize ventilation-perfusion matching. Yet, the exact sensory and signalling mechanisms by which hypoxia triggers pulmonary vasoconstriction remain incompletely understood. Recently, ATP release via pannexin 1 (Panx1) and subsequent signalling via purinergic P2Y receptors has been identified as regulator of vasoconstriction in systemic arterioles. Here, we probed for the role of Panx1-mediated ATP release in HPV and chronic hypoxic pulmonary hypertension (PH). METHODS AND RESULTS: Pharmacological inhibition of Panx1 by probenecid, spironolactone, the Panx1 specific inhibitory peptide (10Panx1), and genetic deletion of Panx1 specifically in smooth muscle attenuated HPV in isolated perfused mouse lungs. In pulmonary artery smooth muscle cells (PASMCs), both spironolactone and 10Panx1 attenuated the increase in intracellular Ca2+ concentration ([Ca2+]i) in response to hypoxia. Yet, genetic deletion of Panx1 in either endothelial or smooth muscle cells did not prevent the development of PH in mice. Unexpectedly, ATP release in response to hypoxia was not detectable in PASMC, and inhibition of purinergic receptors or ATP degradation by ATPase failed to attenuate HPV. Rather, transient receptor potential vanilloid 4 (TRPV4) antagonism and Panx1 inhibition inhibited the hypoxia-induced [Ca2+]i increase in PASMC in an additive manner, suggesting that Panx1 regulates [Ca2+]i independently of the ATP-P2Y-TRPV4 pathway. In line with this notion, Panx1 overexpression increased the [Ca2+]i response to hypoxia in HeLa cells. CONCLUSION: In the present study, we identify Panx1 as novel regulator of HPV. Yet, the role of Panx1 in HPV was not attributable to ATP release and downstream signalling via P2Y receptors or TRPV4 activation, but relates to a role of Panx1 as direct or indirect modulator of the PASMC Ca2+ response to hypoxia. Panx1 did not affect the development of chronic hypoxic PH.

P2Y13 receptors regulate microglial morphology, surveillance, and resting levels of interleukin 1ß release.

Microglia sense their environment using an array of membrane receptors. While P2Y12 receptors are known to play a key role in targeting directed motility of microglial processes to sites of damage where ATP/ADP is released, little is known about the role of P2Y13 , which transcriptome data suggest is the second most expressed neurotransmitter receptor in microglia. We show that, in patch-clamp recordings in acute brain slices from mice lacking P2Y13 receptors, the THIK-1 K+ current density evoked by ADP activating P2Y12 receptors was increased by ~50%. This increase suggested that the P2Y12 -dependent chemotaxis response should be potentiated; however, the time needed for P2Y12 -mediated convergence of microglial processes onto an ADP-filled pipette or to a laser ablation was longer in the P2Y13 KO. Anatomical analysis showed that the density of microglia was unchanged, but that they were less ramified with a shorter process length in the P2Y13 KO. Thus, chemotactic processes had to grow further and so arrived later at the target, and brain surveillance was reduced by ~30% in the knock-out. Blocking P2Y12 receptors in brain slices from P2Y13 KO mice did not affect surveillance, demonstrating that tonic activation of these high-affinity receptors is not needed for surveillance. Strikingly, baseline interleukin-1ß release was increased fivefold while release evoked by LPS and ATP was not affected in the P2Y13 KO, and microglia in intact P2Y13 KO brains were not detectably activated. Thus, P2Y13 receptors play a role different from that of their close relative P2Y12 in regulating microglial morphology and function.

Amyloid ß oligomers constrict human capillaries in Alzheimer's disease via signaling to pericytes.

Cerebral blood flow is reduced early in the onset of Alzheimer's disease (AD). Because most of the vascular resistance within the brain is in capillaries, this could reflect dysfunction of contractile pericytes on capillary walls. We used live and rapidly fixed biopsied human tissue to establish disease relevance, and rodent experiments to define mechanism. We found that in humans with cognitive decline, amyloid ß (Aß) constricts brain capillaries at pericyte locations. This was caused by Aß generating reactive oxygen species, which evoked the release of endothelin-1 (ET) that activated pericyte ETA receptors. Capillary, but not arteriole, constriction also occurred in vivo in a mouse model of AD. Thus, inhibiting the capillary constriction caused by Aß could potentially reduce energy lack and neurodegeneration in AD.

Effects of the ecto-ATPase apyrase on microglial ramification and surveillance reflect cell depolarization, not ATP depletion.

Microglia, the brain's innate immune cells, have highly motile processes which constantly survey the brain to detect infection, remove dying cells, and prune synapses during brain development. ATP released by tissue damage is known to attract microglial processes, but it is controversial whether an ambient level of ATP is needed to promote constant microglial surveillance in the normal brain. Applying the ATPase apyrase, an enzyme which hydrolyzes ATP and ADP, reduces microglial process ramification and surveillance, suggesting that ambient ATP/ADP maintains microglial surveillance. However, attempting to raise the level of ATP/ADP by blocking the endogenous ecto-ATPase (termed NTPDase1/CD39), which also hydrolyzes ATP/ADP, does not affect the cells' ramification or surveillance, nor their membrane currents, which respond to even small rises of extracellular [ATP] or [ADP] with the activation of K+ channels. This indicates a lack of detectable ambient ATP/ADP and ecto-ATPase activity, contradicting the results with apyrase. We resolve this contradiction by demonstrating that contamination of commercially available apyrase by a high K+ concentration reduces ramification and surveillance by depolarizing microglia. Exposure to the same K+ concentration (without apyrase added) reduced ramification and surveillance as with apyrase. Dialysis of apyrase to remove K+ retained its ATP-hydrolyzing activity but abolished the microglial depolarization and decrease of ramification produced by the undialyzed enzyme. Thus, applying apyrase affects microglia by an action independent of ATP, and no ambient purinergic signaling is required to maintain microglial ramification and surveillance. These results also have implications for hundreds of prior studies that employed apyrase to hydrolyze ATP/ADP.

Microglial Ramification, Surveillance, and Interleukin-1ß Release Are Regulated by the Two-Pore Domain K+ Channel THIK-1.

Microglia exhibit two modes of motility: they constantly extend and retract their processes to survey the brain, but they also send out targeted processes to envelop sites of tissue damage. We now show that these motility modes differ mechanistically. We identify the two-pore domain channel THIK-1 as the main K+ channel expressed in microglia in situ. THIK-1 is tonically active, and its activity is potentiated by P2Y12 receptors. Inhibiting THIK-1 function pharmacologically or by gene knockout depolarizes microglia, which decreases microglial ramification and thus reduces surveillance, whereas blocking P2Y12 receptors does not affect membrane potential, ramification, or surveillance. In contrast, process outgrowth to damaged tissue requires P2Y12 receptor activation but is unaffected by blocking THIK-1. Block of THIK-1 function also inhibits release of the pro-inflammatory cytokine interleukin-1ß from activated microglia, consistent with K+ loss being needed for inflammasome assembly. Thus, microglial immune surveillance and cytokine release require THIK-1 channel activity.

The role of pannexin hemichannels in the anoxic depolarization of hippocampal pyramidal cells.

Neuronal gap junctional hemichannels, composed of pannexin-1 subunits, have been suggested to play a crucial role in epilepsy and brain ischaemia. After a few minutes of anoxia or ischaemia, neurons in brain slices show a rapid depolarization to ∼-20 mV, called the anoxic depolarization. Glutamate receptor blockers can prevent the anoxic depolarization, suggesting that it is produced by a cation influx through glutamate-gated channels. However, in isolated hippocampal pyramidal cells, simulated ischaemia evokes a large inward current and an increase in permeability to large molecules, mediated by the opening of pannexin-1 hemichannels. N-methyl-d-aspartate is also reported to open these hemichannels, suggesting that the activation of N-methyl-d-aspartate receptors, which occurs when glutamate is released in ischaemia, might cause the anoxic depolarization by evoking a secondary ion flux through pannexin-1 hemichannels. We tested the contribution of pannexin hemichannels to the anoxic depolarization in CA1 pyramidal cells in the more physiological environment of hippocampal slices. Three independent inhibitors of hemichannels-carbenoxolone, lanthanum and mefloquine-had no significant effect on the current generating the anoxic depolarization, while a cocktail of glutamate and gamma-aminobutyric acid class A receptor blockers abolished it. We conclude that pannexin hemichannels do not generate the large inward current that underlies the anoxic depolarization. Glutamate receptor channels remain the main candidate for generating the large inward current that produces the anoxic depolarization.

Progesterone potentiates calcium release through IP3 receptors by an Akt-mediated mechanism in hippocampal neurons.

Progesterone (P4) is a steroid hormone that plays multiple roles in the central nervous system (CNS) including promoting neuroprotection. However, the precise mechanisms involved in its neuroprotective effects are still unknown. Given that the regulation of the intracellular calcium (Ca(2+)) concentration is critical for cell survival, we determined if inositol 1, 4, 5-trisphosphate receptors (IP(3)Rs) are relevant targets of P4. Using primary hippocampal neurons, we tested the hypothesis that P4 controls the gain of IP3R-mediated intracellular Ca(2+) signaling in neurons and characterized the subcellular distribution and phosphorylation of potential signaling intermediates involved in P4s actions. Our results reveal that P4 treatment altered the intensity and distribution of IP3R immunoreactivity and induced the nuclear translocation of phosphorylated Akt. Further, P4 potentiated IP(3)R-mediated intracellular Ca(2+) responses. These results suggest a potential involvement of P4 in particular and of steroid hormone signaling pathways in general in the control of intracellular Ca(2+) signaling and its related functions.

Progesterone potentiates IP(3)-mediated calcium signaling through Akt/PKB.

The activity of cells critically depends on the control of their cytosolic free calcium ion (Ca(2+)) concentration. The objective of the present study was to identify mechanisms of action underlying the control of the gain of intracellular Ca(2+) release by circulating gonadal steroid hormones. Acute stimulation of isolated neurons with progesterone led to IP(3)R-mediated Ca(2+) transients that depend on the activation of the PI3 kinase/Akt/PKB signaling pathway. These results were confirmed at the molecular level and phosphorylation of IP(3)R type 1 by Akt/PKB was identified as the mechanism of action. Hence, it is likely that circulating gonadal steroid hormones control neuronal activity including phosporylation status through receptor- and kinase-mediated signaling. With a direct control of the gain of the Ca(2+) second messenger system as a signaling gatekeeper for neuronal activity the present study identifies a novel pathway for interaction of the endocrine and central nervous system.