In a recent article published in Nature Communications, Zheng et al. report that heightened stimulation of A2A receptors (A2ARs) located in epithelial cells of the chrorioid plexus (ChP) is responsible for the development of hydrocephalus (Zheng et al., Nat Commun 17:83, 2025). They show evidence in hydrocephalus patients and mice models of hydrocephalus that the cerebrospinal fluid (CSF) concentration of adenosine is raised to about threefold of the control value. Under pathological conditions, CSF adenosine levels may be increased in response to infection, hemorrhage, traumatic insults, and tumors, all of which are known to be the initiators of hydrocephalus. In careful experiments, performed mostly in mice models, established by the injection of autologous blood (imitation of hemorrhage) or kaolin (imitation of obstruction) to the cerebral ventricles, the production of CSF, the level of adenosine, and the expression of A2ARs was increased and in consequence the ventricles enlarged. The signaling mechanisms leading to increased production of CSF were then identified by the genetic knockdown of proteins that were assumed to be involved in this process. The A2AR-SPAK (SPS1-related proline/alanine-rich kinase)-PI3K (phosphoinositide 3 kinase)-Akt (protein kinase B)-NF-κB (a nuclear factor)-ATP1A2 (a Na+,K+-ATPase) pathway was suggested to be instrumental in hydrocephalus development. The clinically important conclusion deriving from this observation was that A2AR blockade by istradefylline, a Food and Drug Administration-approved drug could be introduced into the therapy of human hydrocephalus.

Previous studies have suggested that running exerts anti-tumor effects on various cancers through multiple pathways. It has been reported that the P2X7 receptor (P2X7R) may display anti-tumor activity in cervical cancer cells, and running can regulate P2X7R expression and function in mice. However, the specific impact of running on cervical cancer and whether its underlying mechanism is associated with the regulation of P2X7R levels remains to be elucidated. In this study, mice bearing U14 cervical cancer tumors were used as a model to explore the association between running and P2X7R in cervical cancer. The results showed that running significantly inhibited tumor progression in mice, accompanied by an increase in P2X7R protein expression in tumor tissues. Additionally, treatment with the P2X7R antagonist JNJ-47965567 alone or in combination with running intervention promoted tumor growth and attenuated the anti-tumor effect of running. In contrast, the P2X7R agonist BzATP alone or combined with running intervention exerted anti-tumor effects and enhanced the anti-tumor effect of running. In conclusion, the present findings suggest only a pharmacological association between P2X7R and the anti-tumor effect of running against cervical cancer progression, given that genetic validation was not performed in this study. The potential involvement of immune mechanisms remains an inference and requires further experimental validation. As a possible therapeutic target, P2X7R provides supportive data for the treatment of cervical cancer and the clinical exploration of running as an adjuvant intervention.

P2X receptors (P2XR) are a family of seven cation channels gated by extracellular ATP (eATP). Activation of P2XRs results in diverse cellular responses, including cell signalling, proliferation, differentiation, and death-all critically important in multiple physiological and pathophysiological states. These receptors, therefore, represent therapeutic targets of considerable interest. However, P2XRs, while structurally related, exhibit highly divergent and context-dependent functions. Their spatiotemporal and functional complexity is evident by overlapping expression across multiple cell types that can shift dynamically during physiological processes or disease progression. Furthermore, P2XRs can assemble as homo- or hetero-trimers, with distinct functional properties. These factors complicate definitive identification of a given P2XR responsible for a specific pathophysiological effect. Receptor activity in vivo is transient because of receptor-specific mechanisms and follows eATP breakdown by ectonucleotidases. Any correlation of ATP release with receptor engagement, as assessed in vitro, often does not correspond with the in vivo dynamics. Translation from animal models to humans is complicated by the species-specific pharmacology of some P2XRs, confounded by many animal models in use not fully replicating human P2XR function and regulation in pathology. Furthermore, there are no clinical biomarkers to distinguish incomplete receptor blockade from lack of therapeutic effect. Thus, translation has been very limited. To identify and validate specific P2XR functionalities, future experimental designs should use approaches and assays that can reliably assess receptor involvement, while reducing methodologically flawed findings. We propose guidelines developed in consultation with the purinergic community for consistent and reliable research practices in P2XR studies.

Francesco Di Virgilio, one of the giants of purinergic signalling, died suddenly on September 22, 2024, which is an immense loss to so many friends and colleagues and especially to his beloved wife, Dorianna. Here, 1 year on, we pay tribute to him and his immense contribution to our field. Francesco graduated in medicine from the University of Padova, Italy, in 1979, with what became a lifelong interest in inflammation. He then held post-doctoral positions at University College London, Padova University, and Columbia University, New York, where he became acquainted with P2X7 receptors. He then returned to Padova as an Associate Professor of Molecular Pathology before moving to the University of Ferrara in 1992, where he set up a world-leading lab that studied the roles and underlying cellular mechanisms of the action of P2X7 receptors in inflammatory pathologies. Francesco published over 370 peer-reviewed articles, which have been cited > 35,000 times, giving him an H-index > 100. In addition, he filed several patents related to purinergic signalling. He also collaborated extensively, both within the University of Ferrara and worldwide, including in universities in the UK, Spain, Germany, the USA, and Brazil. Francesco was a man of great passion and intellect, who possessed scientific vision, intuition, and integrity, and he became the go-to world expert on P2X7 receptors and inflammation. We have lost a giant in the field and a dear friend, but he leaves behind an exceptional body of work, an outstanding legacy, and many friends who will miss him.

In a recent article published in Cell Research, Chen et al. reported that light flickering at 40 Hz effectively counteracts chronic pain in Complete Freund Adjuvant (CFA)–treated mice and in mice whose tibial and common peroneal nerves were ligated (spared nerve injury, SNI) [1]. These mice served as models for inflammatory and neuropathic pain, respectively. After establishing that 40 Hz light flickering exerted analgesia in both pain models, a systematic search started for the neuronal pathways involved and the conditions required for this effect. The combination of retrograde and anterograde tracing techniques indicated that retinal ganglion cells (RGCs) project monosynaptically to the central amygdala (CeA), and chemogenetic or optogenetic activation of this pathway simulates the effects of 40 Hz light stimulation. Genetic sensors for adenosine expressed in the CeA proved that such a light stimulation caused an increase in the local concentration of adenosine, via the promotion of the equilibrative adenosine transporter–mediated outflow of the nucleoside from CNS cells. The enriched adenosine levels apparently stimulated A2A receptors (Rs) as proved by the abolition of 40 Hz light flickering–induced analgesia by pharmacological blockade of A2ARs, or their genetic knockdown/knockout. The target neurons in the CeA were identified as belonging to the proenkephalin-containing type; their selective ablation abolished the effect of light stimulation. Finally, two capsaicin injections, 3 h apart, the second one either combined with saline or the protein synthesis inhibitor anisomycin, showed that anisomycin deleted chronic pain memory traces. Hence, 40 Hz light flickering may be a non-pharmacological manipulation for alleviating chronic pain in humans, without the cardiovascular and CNS side effects inherent to systemic adenosine application.

P2X receptors (P2XRs) are ATP-gated cation channels that play a pivotal role in chronic visceral pain (CVP). This review highlights the central contribution of the ATP-P2X3/4/7 axis in peripheral and central sensitization underlying CVP. Recent discoveries have identified the non-coding RNA miR-1306-3p as an endogenous nanomolar agonist of P2X3 receptors, coupling chronic stress to visceral pain via an epigenetic pathway. Moreover, persistent DNA methylation changes at the P2RX7 locus in spinal astrocytes create a "pain memory" that limits the durability of conventional antagonists. The first-generation P2X3/P2X7 antagonists (e.g., AF-219, AZD-9056, NC-2600) failed in clinical trials, primarily due to species-specific receptor pharmacology, the lack of ATP-based biomarkers for patient stratification, and irreversible central sensitization driven by epigenetic marks. To overcome these hurdles, we propose a precision-medicine framework that includes: (1) CRISPR-dCas9-based epigenome editing as a potential one-time "pain-memory eraser"; (2) patient stratification using sweat-ATP levels; and (3) human iPSC-derived neuron screening to improve translational predictability. This integrated approach holds promise not only for CVP related to irritable bowel syndrome (IBS), but also for other pain conditions.

Rationale: Cognitive impairment and depression are salient comorbidities of mesial temporal lobe epilepsy; it is still unclear whether this frequently drug resistant disease is a cause or consequence of hippocampal damage and its interplay with long-lasting seizure activity (status epilepticus; SE). Thus, a major therapeutic advance in this field is badly needed. Methods: We modeled enduring behavioral and electroencephalographic (EEG) seizures in mice by the intraperitoneal injection of kainic acid (KA), and measured the dynamics of the intracellular Ca2+ signals in the hippocampal CA1 area by fiber photometry. Learning and memory were controlled by the Morris Water-Maze and Novel Object Recognition tests on whole animals and by the induction of long-term potentiation in CA1 pyramidal neurons in brain slices. Depressive-like reactions were evaluated by the Tail Suspension, Forced Swim, and Sucrose Preference tests. Results: The intraperitoneal injection of the blood-brain permeable, highly selective, P2X7 and A2A receptor (R) antagonists, JNJ-47965567, and KW6002/SCH58261, respectively, counteracted the effects of KA-induced SE both on seizure activity and the increase of Ca2+ signals (as a measure of changes in the intracellular Ca2+ concentration) in neurons and astrocytes of the hippocampal CA1 area. In addition, these drugs also prevented the impairment of the hippocampus-dependent spatial and non-spatial learning abilities by KA-SE. The knockdown of P2X7Rs in CA1 astrocytes, but not neurons prevented the cognitive deterioration, suggesting that the release of astrocytic signaling molecules onto neighboring neurons might be the cause of this effect. In accordance with our observations, in hippocampal slices prepared from mice which underwent KA-SE, a selective sensitivity increases to the prototypic P2X7R agonist dibenzoyl-ATP (Bz-ATP) manifested in CA1 neurons. This sensitivity increase appeared to be due to a postsynaptic interference between P2X7Rs and the release of excitatory neurotransmitters during SE. In spite of a P2X7 and A2AR-mediated increase of Ca2+ signaling in the medial prefrontal cortex, no similar change was noted after KA-SE in depressive-like reactions or the open-field behavior. Conclusions: SE induced the release of ATP and adenosine from the hippocampus and in consequence decreased the cognitive abilities of mice. The pharmacological blockade of P2X7 and A2ARs prevented the SE-induced seizure activity and cognitive deterioration, but not depressive-like behavior.

Astrocytes play a vital role in neuroinflammatory processes within the central nervous system (CNS). Increasing evidence suggests that purinergic G protein-coupled receptor P2Y14 is associated with neuroinflammation. However, the expression, distribution, and function of P2Y14 in astrocytes remain unclear. This study aimed to investigate the expression of P2Y14 receptors in astrocytes and their regulatory role in astrocytic inflammatory responses. P2Y14 expression was confirmed in primary astrocytes isolated from the mouse cerebral cortex using immunofluorescence techniques. Western blot and RT-qPCR analyses showed that lipopolysaccharide (LPS) stimulation significantly increased P2Y14 expression in astrocytes. The antagonist of P2Y14 receptor, 4-(piperidin-4-yl)-phenyl)-7-(4-(trifluoromethyl)-phenyl2-naphthoic acid (PPTN) reduced glial fibrillary acidic protein (GFAP) expression, while the agonist of P2Y14 receptor, uridine diphosphate glucose (UDPG) promoted it in astrocytes. Mechanistically, PPTN inhibited the expression of signal transducer and activator of transcription 1 (STAT1) and its phosphorylated form (p-STAT1), while UDPG enhanced the activation of STAT1/p-STAT1. Treatment with the P2Y14 antagonist PPTN resulted in significant inhibition of interleukin (IL)-6, IL-18, and tumor necrosis factor-alpha (TNF-α) release. By contrast, stimulation of the P2Y14 receptor with its agonist UDPG induced a substantial increase in the levels of these inflammatory mediators. These results were further validated in the cell line of mouse astrocyte C8-D1A using pharmacological approaches and lentiviral transduction to modulate P2Y14 expression. This study confirmed that astrocytes express functional P2Y14 receptors and demonstrated their key role in regulating astrocyte activation and inflammatory responses. This regulation may be achieved through the UDPG/P2Y14-STAT1 signaling axis, which provides a new molecular basis for understanding the regulatory mechanisms of astrocytic inflammatory responses.

Astrocyte atrophy is the main histopathological hallmark of major depressive disorder (MDD) in humans and in animal models of depression. Here we demonstrated that manipulating Ezrin expression specifically in astrocytes significantly increases the resilience of mice to chronic unpredictable mild stress (CUMS). Overexpression of Ezrin in astrocytes from the medial prefrontal cortex (mPFC) rescued depressive-like behaviours induced by CUMS, whereas down-regulation of Ezrin in astrocytes from the mPFC increased mouse susceptibility to CUMS and promoted depressive-like behaviours. These behavioural changes correlated with astrocytic morphology. Astrocytes from the mPFC of mice sensitive to CUMS demonstrated significant atrophy; similar atrophy was found in astrocytes from animals with down-regulated Ezrin expression. On the contrary, morphology of astrocytes remained unchanged in animals resistant to CUMS and in animals with astrocytic overexpression of Ezrin. Morphological changes also correlated with Ezrin immunoreactivity, which was low in mice with depressive-like behaviours and high in mice resistant to stress. We conclude that Ezrin-dependent morphological remodelling of astrocytes defines the sensitivity of mice to stress; high Ezrin expression renders them stress resilient, whereas low Ezrin expression promotes depressive-like behaviour in response to chronic stress.

Major depressive disorder (MDD) is a mental illness characterized primarily by persistent low mood and anhedonia. Traditional monoamine antidepressants have not achieved entirely satisfactory clinical efficacy. Recent studies have revealed that extracellular adenosine 5'-triphosphate (ATP), adenosine, and their P2/P1 receptor (R)-mediated purinergic signaling play a significant role in the pathogenesis of depression. This review first describes what is known regarding the mechanisms of ATP release in glial cells and neurons, particularly the role of reduced ATP release by astrocytes in decreasing synaptic plasticity and dopaminergic signaling, leading to depressive-like behavior. Subsequently, it summarizes the distinct roles of the purinergic ionotropic P2XRs (especially P2X7) and metabotropic P2YRs in regulating neuroinflammation and synaptic plasticity. It then reviews the role of adenosine metabolic imbalance and altered function of adenosine A1 and A2ARs in further promoting pathological processes in depression. Finally, based on the aforementioned mechanisms, intervention strategies targeting different purinergic pathways are briefly outlined, including P2X7R antagonists, adenosine receptor modulators and S-adenosylmethionine (SAMe), including outstanding challenges for their translation towards a clinical application.

In a recent paper published in Nature Communications, Fu et al. provided evidence that microglial CD39 is essen-tial for the generation of adenosine from the (co)transmit-ter adenosine 5 '-triphosphate (ATP), which then activates vasodilatory A2A receptors (A2ARs) at the cerebral vascu-lature to increase cerebral blood flow (CBF) [1]. CBF is, among other processes, regulated by adenosine via A2ARs to satisfy the brain's energy demand for glucose/nutrient and oxygen supply during phases of altered neuronal activity. Diverse pharmacological and genetic approaches aimed at ablating microglial populations, identified the microglial enzyme, CD39, as the key target of this purinergic regula-tory mechanism. The following experimental approaches were used: (a) PLX3397 reversibly depleted microglia; (b) the crossing of CXCR1-CreER and CAG-26R-TDA mice resulted in ablation of microglia in their offspring, due to damage via the expression of diphtheria A-toxin; and (c) fms-intronic regulatory element (FIRE)-deleted mice showed selective microglial depletion, but no change in the number of perivascular macrophages. All of these manipula-tions caused the blunting of whisker stimulation (stimulus for the release of ATP/adenosine)-induced increases of CBF in the barrel cortex. The genetic deletion of CD39 and its pharmacological blockade by ARL67156 both blocked the effect of whisker stimulation. In conclusion, CD39 appears to be the key enzyme in degrading neuronal ATP to adeno-sine and thereby exerts a feedback control imposed on the neurovascular tone regulating the delivery of nutrients and oxygen to the brain

A recent study published by Zhang et al.1 in Nature suggested that the endogenous ceramide receptors cysteinyl leukotriene receptor 2(CYSLTR2)and the pyrimidinergic receptor P2Y6(P2Y6R)are potential novel targets for the treatment of athero-sclerosis and related cardiovascular conditions beyond main-stream cholesterol control strategies.This new finding paves the way for another milestone in the emerging innovative search for novel atherosclerosis therapies.

In a recent article published in Neuron, Li et al. (Neuron 112(22):3734-3749.e5, 2024) accomplished a major scientific advance by reporting that ATP-sensitive P2X3 receptor-channels (P2X3Rs) in the paraventricular hypothalamus (PVH) specifically regulate visceral pain without affecting somatic pain. On the other hand, vasoactive intestinal polypeptide-sensing receptors (VIPR2) selectively process somatic pain without altering visceral pain. Function-dependent laser capture microdissection sequencing (fLCM-Seq) and immunohistochemistry demonstrated that P2X3Rs and VIPR2 have different transcriptional profiles and belong to the colorectal distension (CRD) and von Frey filament (VFF)-stimulated subgroups of PVH neurons, respectively. An anterograde tracing strategy, in which green fluorescent protein (GFP) was selectively expressed in CRD-labeled or VFF-labeled PVH neurons, showed that PVHP2X3R+ neuronal projections terminated exclusively at the ventral part of the lateral septal nucleus (LSV) while the PVHVIPR2+ neuronal projections terminated at the caudal part of the zona incerta (ZIC). The PVHP2X3R+ circuit selectively responded to visceral pain while remaining unresponsive to somatic pain. By contrast, the PVHVIPR2+ circuit selectively responded to somatic pain, while it did not react to visceral pain. Knockdown of P2X3R expression in PVH neurons enhanced the visceral pain threshold without affecting somatic nociception, and the reverse findings were true for the knockdown of the VIPR2 expressing PVH neurons. All these results provide possible new strategies based on central-targeted therapies for the future treatment of visceral and somatic pain, respectively.

Dénes and his co-workers recently published a paper in Nature Neuroscience,documenting that neurological abnormalities in COVID are based on microglial dysfunction in the brain.1 In case of acute respiratory syndrome of COVID infection,the central nervous system symptomatology significantly contribute to the severity of this disease.The authors used an autopsy platform allowing morphological and biochemical/molecular biological investigations in postmortem mirror blocks prepared from the brain and peripheral organs of 13 COVID and 23 non-COVID-infected patients.