Long-term Pannexin 1 Ablation Produces an Imbalance Between Small Rho-GTPases Activity and Actin Polymerization Leading to Structural and Functional Modifications in Hippocampal Neurons

Enhanced activity and overexpression of Pannexin 1 (PANX1) channels contribute to neuronal pathologies, such as epilepsy and Alzheimer’s disease (AD). In the hippocampus, the PANX1 channel ablation alters glutamatergic neurotransmission, synaptic plasticity, and memory flexibility. Nevertheless, PANX1-knockout (PANX1-KO) mice still preserve the ability to learn, suggesting that compensatory mechanisms work to stabilize neuronal activity. Here, we show that the absence of PANX1 in the adult brain promotes a series of structural and functional modifications in PANX1-KO CA1 hippocampal synapses, preserving spontaneous activity. Adult CA1 neurons of PANX1-KO mice exhibit enhanced excitability, a more complex dendritic branching, enhanced spine maturation, and multiple synaptic contacts compared to the WT condition. These modifications seem to rely on the actin-cytoskeleton dynamics as an increase in actin polymerization and an imbalance between Rac1 and RhoA GTPase activity is observed in the absence of PANX1. Our findings highlight a novel interaction between PANX1, actin, and small Rho GTPases, which appear to be relevant for synapse stability.

Inherent P2X7 Receptors Regulate Macrophage Functions during Inflammatory Diseases

Macrophages are mononuclear phagocytes which derive either from blood-borne monocytes or reside as resident macrophages in peripheral (Kupffer cells of the liver, marginal zone macrophages of the spleen, alveolar macrophages of the lung) and central tissue (microglia). They occur as M1 (pro-inflammatory; classic) or M2 (anti-inflammatory; alternatively activated) phenotypes. Macrophages possess P2X7 receptors (Rs) which respond to high concentrations of extracellular ATP under pathological conditions by allowing the non-selective fluxes of cations (Na+, Ca2+, K+). Activation of P2X7Rs by still higher concentrations of ATP, especially after repetitive agonist application, leads to the opening of membrane pores permeable to ~900 Da molecules. For this effect an interaction of the P2X7R with a range of other membrane channels (e.g., P2X4R, transient receptor potential A1 [TRPA1], pannexin-1 hemichannel, ANO6 chloride channel) is required. Macrophage-localized P2X7Rs have to be co-activated with the lipopolysaccharide-sensitive toll-like receptor 4 (TLR4) in order to induce the formation of the inflammasome 3 (NLRP3), which then activates the pro-interleukin-1β (pro-IL-1β)-degrading caspase-1 to lead to IL-1β release. Moreover, inflammatory diseases (e.g., rheumatoid arthritis, Crohn’s disease, sepsis, etc.) are generated downstream of the P2X7R-induced upregulation of intracellular second messengers (e.g., phospholipase A2, p38 mitogen-activated kinase, and rho G proteins). In conclusion, P2X7Rs at macrophages appear to be important targets to preserve immune homeostasis with possible therapeutic consequences.

A dual voltage clamp technique to study gap junction hemichannels in astrocytes cultured from neonatal rodent spinal cords

Astrocytes express surface channels involved in purinergic signaling, and among these channels, pannexin-1 (Px1) and connexin-43 (Cx43) hemichannels (HCs) mediate ATP release that acts directly, or through its derivatives, on neurons and glia via purinergic receptors. Although HCs are functional, i.e., open and close, under physiological and pathological conditions, single channel conductance of Px1 HCs is not well defined. Here, we developed a dual voltage clamp technique in HeLa cells overexpressing human Px1-YFP, and then applied this system to rodent spinal astrocytes. Single channels were recorded in cell attached patches and evoked with ramp cycles of 2 s duration and -/+ 80-100 mV amplitude or rectangular pulses through another pipette in whole cell clamp. Conductance of Px1 HC openings recorded during ramp stimuli ranged 25-110 pS. Based on their single channel conductances, Px1 HCs could be distinguished from Cx43 HCs and P2X7 receptors (P2X7Rs) in spinal astrocytes during dual voltage clamp experiments. Furthermore, we found that single channel activity of Cx43 HCs and P2X7Rs was increased, and that of Px1 HCs was decreased, in spinal astrocytes treated for 7h with FGF-1, a growth factor implicated in neurodevelopment, repair and inflammation.

Involvement of P2X7 receptors in chronic pain disorders

Chronic pain is caused by cellular damage with an obligatory inflammatory component. In response to noxious stimuli, high levels of ATP leave according to their concentration gradient, the intracellular space through discontinuities generated in the plasma membrane or diffusion through pannexin-1 hemichannels, and activate P2X7Rs localized at peripheral and central immune cells. Because of the involvement of P2X7Rs in immune functions and especially the initiation of macrophage/microglial and astrocytic secretion of cytokines, chemokines, prostaglandins, proteases, reactive oxygen, and nitrogen species as well as the excitotoxic glutamate/ATP, this receptor type has a key role in chronic pain processes. Microglia are equipped with a battery of pattern recognition receptors that detect pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) from bacterial infections or danger associated molecular patterns (DAMPs) such as ATP. The co-stimulation of these receptors leads to the activation of the NLRP3 inflammasome and interleukin-1β (IL-1β) release. In the present review, we invite you to a journey through inflammatory and neuropathic pain, primary headache, and regulation of morphine analgesic tolerance, in the pathophysiology of which P2X7Rs are centrally involved. P2X7R bearing microglia and astrocyte-like cells playing eminent roles in chronic pain will be also discussed.

On the molecular mechanism of excitation–transcription coupling in skeletal muscle

An important question in neuromuscular biology is how skeletal muscle cells decipher the stimulation pattern coming from motoneurons to define their phenotype-activating transcriptional changes in a process named excitation–transcription coupling. We have shown in adult muscle fibers that 20 Hz electrical stimulation (ES) activates a signaling cascade that starts with Cav1.1 activation, ATP release trough pannexin-1 channel, activation of purinergic receptors, and IP3-dependent Ca2+ signals inducing transcriptional changes related to muscle plasticity from fast to slow phenotype. Extracellular addition of 30 µM ATP mimics transcriptional changes induced by ES at 20 Hz. ATP release occurs in two peaks, the first around 15 s after ES and a second around 300 s after ES. In the present work, we used apyrase to hydrolyze ATP 60 s after ES, maintaining the first peak and eliminating the second peak. In this condition, transcriptional changes were abolished, indicating that the second peak is the one crucial to activate transcription. Additionally, we observed a small depolarization of fibers after ES. The addition of 30 to 100 µM external ATP also induced depolarization of muscle fibers. This depolarization was unable to activate contraction but was able to induce transcriptional changes induced by 20 Hz ES. These changes were completely inhibited by the IP3R blocker xestospongin B, suggesting that IP3-dependent events are triggered at these membrane depolarization values. Moreover, transcriptional changes induced by addition of 30 µM extracellular ATP was blocked by incubation of fibers with 25 µM Nifedipine. These results suggest that the second ATP peak observed after 20 Hz ES is responsible for transcriptional activation by inducing small depolarizations of fiber membranes that are also sensed by Cav1.1. Finally, we show evidence that downstream of purinergic receptors, PKC is activated, likely causing phosphorylation of ClC-1 chloride channels, possibly responsible for depolarization after 20 Hz.

Ion Channel Dysfunction and Neuroinflammation in Migraine and Depression

Migraine and major depression are debilitating disorders with high lifetime prevalence rates. Interestingly these disorders are highly comorbid and show significant heritability, suggesting shared pathophysiological mechanisms. Non-homeostatic function of ion channels and neuroinflammation may be common mechanisms underlying both disorders: The excitation-inhibition balance of microcircuits and their modulation by monoaminergic systems, which depend on the expression and function of membrane located K+, Na+, and Ca+2 channels, have been reported to be disturbed in both depression and migraine. Ion channels and energy supply to synapses not only change excitability of neurons but can also mediate the induction and maintenance of inflammatory signaling implicated in the pathophysiology of both disorders. In this respect, Pannexin-1 and P2X7 large-pore ion channel receptors can induce inflammasome formation that triggers release of pro-inflammatory mediators from the cell. Here, the role of ion channels involved in the regulation of excitation-inhibition balance, synaptic energy homeostasis as well as inflammatory signaling in migraine and depression will be reviewed.

Long-term Pannexin 1 ablation promotes structural and functional modifications in hippocampal neurons through the regulation of actin cytoskeleton and Rho GTPases activity.

Enhanced activity and overexpression of Pannexin 1 (PANX1) channels contribute to neuronal pathologies, such as epilepsy and Alzheimers disease (AD). In the hippocampus, the PANX1 channels ablation alters glutamatergic neurotransmission, synaptic plasticity, and memory flexibility. Nevertheless, PANX1-knockout (KO) mice still preserve the ability to learn, suggesting that compensatory mechanisms work to stabilize neuronal activity. Here, we show that the absence of PANX1 in the adult brain promotes a series of structural and functional modifications in KO hippocampal synapses, preserving spontaneous activity. Adult CA1 neurons of KO mice exhibit enhanced excitability, complex dendritic branching, spine maturation, and multiple synaptic contacts compared to the WT condition. These modifications seem to rely on the actin-cytoskeleton dynamics as an increase in actin polymerization and an imbalance between Rac1 and RhoA GTPase activity is observed in the absence of PANX1. Our findings highlight a novel interaction between PANX1, actin, and small Rho GTPases that appear to be relevant for synapse maintenance as a long-term compensatory mechanism for PANX1 deficiency.