Identification of a Potential Entry-Fusion Complex Based on Sequence Homology of African Swine Fever and Vaccinia Virus.

African swine fever virus (ASFV) belongs to the family of Asfarviridae, part of the group of nucleocytoplasmic large DNA viruses (NCLDV). Little is known about the internalization of ASFV in the host cell and the fusion membrane events that take place at early stages of the infection. Poxviruses, also members of the NCLDV and represented by vaccinia virus (VACV), are large, enveloped, double-stranded DNA viruses. Poxviruses were considered unique in having an elaborate entry-fusion complex (EFC) composed of 11 highly conserved proteins integrated into the membrane of mature virions. Recent advances in methodological techniques have again revealed several connections between VACV EFC proteins. In this study, we explored the possibility of an analogous ASFV EFC by identifying ten candidate proteins exhibiting structural similarities with VACV EFC proteins. This could reveal key functions of these ASFV proteins, drawing attention to shared features between the two virus families, suggesting the potential existence of an ASFV entry-fusion complex.

Kidins220 sets the threshold for survival of neural stem cells and progenitors to sustain adult neurogenesis.

In the adult mammalian brain, neural stem cells (NSCs) located in highly restricted niches sustain the generation of new neurons that integrate into existing circuits. A reduction in adult neurogenesis is linked to ageing and neurodegeneration, whereas dysregulation of proliferation and survival of NSCs have been hypothesized to be at the origin of glioma. Thus, unravelling the molecular underpinnings of the regulated activation that NSCs must undergo to proliferate and generate new progeny is of considerable relevance. Current research has identified cues promoting or restraining NSCs activation. Yet, whether NSCs depend on external signals to survive or if intrinsic factors establish a threshold for sustaining their viability remains elusive, even if this knowledge could involve potential for devising novel therapeutic strategies. Kidins220 (Kinase D-interacting substrate of 220 kDa) is an essential effector of crucial pathways for neuronal survival and differentiation. It is dramatically altered in cancer and in neurological and neurodegenerative disorders, emerging as a regulatory molecule with important functions in human disease. Herein, we discover severe neurogenic deficits and hippocampal-based spatial memory defects accompanied by increased neuroblast death and high loss of newly formed neurons in Kidins220 deficient mice. Mechanistically, we demonstrate that Kidins220-dependent activation of AKT in response to EGF restraints GSK3 activity preventing NSCs apoptosis. We also show that NSCs with Kidins220 can survive with lower concentrations of EGF than the ones lacking this molecule. Hence, Kidins220 levels set a molecular threshold for survival in response to mitogens, allowing adult NSCs growth and expansion. Our study identifies Kidins220 as a key player for sensing the availability of growth factors to sustain adult neurogenesis, uncovering a molecular link that may help paving the way towards neurorepair.

Elucidation of the Cellular Interactome of African Swine Fever Virus Fusion Proteins and Identification of Potential Therapeutic Targets.

African swine fever virus (ASFV) encodes more than 150 proteins, most of them of unknown function. We used a high-throughput proteomic analysis to elucidate the interactome of four ASFV proteins, which potentially mediate a critical step of the infection cycle, the fusion and endosomal exit of the virions. Using affinity purification and mass spectrometry, we were able to identify potential interacting partners for those ASFV proteins P34, E199L, MGF360-15R and E248R. Representative molecular pathways for these proteins were intracellular and Golgi vesicle transport, endoplasmic reticulum organization, lipid biosynthesis, and cholesterol metabolism. Rab geranyl geranylation emerged as a significant hit, and also Rab proteins, which are crucial regulators of the endocytic pathway and interactors of both p34 and E199L. Rab proteins co-ordinate a tight regulation of the endocytic pathway that is necessary for ASFV infection. Moreover, several interactors were proteins involved in the molecular exchange at ER membrane contacts. These ASFV fusion proteins shared interacting partners, suggesting potential common functions. Membrane trafficking and lipid metabolism were important categories, as we found significant interactions with several enzymes of the lipid metabolism. These targets were confirmed using specific inhibitors with antiviral effect in cell lines and macrophages.

New insights into the role of endosomal proteins for African swine fever virus infection.

African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. Then, the virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. Similar to other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. While the endosomal maturation entails luminal acidification, the decrease in pH acts on the multilayer structure of the virion dissolving the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Viral fusion is then necessary for the egress of incoming virions from endosomes into the cytoplasm, however this remains an intriguing and yet essential process for infection, specifically for the egress of viral nucleic acid into the cytoplasm for replication. ASFV proteins E248R and E199L, located at the exposed inner viral membrane, might be implicated in the fusion step. An interaction between these viral proteins and cellular endosomal proteins such as the Niemann-Pick C type 1 (NPC1) and lysosomal membrane proteins (Lamp-1 and -2) was shown. Furthermore, the silencing of these proteins impaired ASFV infection. It was also observed that NPC1 knock-out cells using CRISPR jeopardized ASFV infection and that the progression and endosomal exit of viral cores was arrested within endosomes at viral entry. These results suggest that the interactions of ASFV proteins with some endosomal proteins might be important for the membrane fusion step. In addition to this, reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by fewer and smaller viral factories. Our findings pave the way to understanding the role of proteins of the endosomal membrane in ASFV infection.

African Swine Fever Virus Ubiquitin-Conjugating Enzyme Is an Immunomodulator Targeting NF-κB Activation.

African swine fever virus (ASFV) is an acute and persistent swine virus with a high economic burden that encodes multiple genes to evade host immune response. In this work, we have revealed that early viral protein UBCv1, the only known conjugating enzyme encoded by a virus, modulates innate immune and inflammatory signaling. Transient overexpression of UBCv1 impaired activation of NF-κB and AP-1 transcription factors induced by several agonists of these pathways. In contrast, activation of IRF3 and ISRE signaling upon stimulation with TRIFΔRIP, cGAS/STING or RIG-I-CARD remained unaltered. Experiments aimed at mapping UBCv1 inhibitory activity indicated that this viral protein acts upstream or at the level step of IKKß. In agreement with this, UBCv1 was able to block p65 nuclear translocation upon cytokine stimulation, a key event in NF-ĸB signaling. Additionally, A549 stably transduced for UBCv1 showed a significant decrease in the levels of NF-ĸB dependent genes. Interestingly, despite the well-defined capacity of UBCv1 to conjugate ubiquitin chains, a mutant disabled for ubiquitylation activity retained similar immunomodulatory activity as the wild-type enzyme, suggesting that the two functions are segregated. Altogether these data suggest that ASFV UBCv1 manipulates the innate immune response targeting the NF-κB and AP-1 pathways and opens new questions about the multifunctionality of this enzyme.

Kidins220 deficiency causes ventriculomegaly via SNX27-retromer-dependent AQP4 degradation.

Several psychiatric, neurologic and neurodegenerative disorders present increased brain ventricles volume, being hydrocephalus the disease with the major manifestation of ventriculomegaly caused by the accumulation of high amounts of cerebrospinal fluid (CSF). The molecules and pathomechanisms underlying cerebral ventricular enlargement are widely unknown. Kinase D interacting substrate of 220 kDa (KIDINS220) gene has been recently associated with schizophrenia and with a novel syndrome characterized by spastic paraplegia, intellectual disability, nystagmus and obesity (SINO syndrome), diseases frequently occurring with ventriculomegaly. Here we show that Kidins220, a transmembrane protein effector of various key neuronal signalling pathways, is a critical regulator of CSF homeostasis. We observe that both KIDINS220 and the water channel aquaporin-4 (AQP4) are markedly downregulated at the ventricular ependymal lining of idiopathic normal pressure hydrocephalus (iNPH) patients. We also find that Kidins220 deficient mice develop ventriculomegaly accompanied by water dyshomeostasis and loss of AQP4 in the brain ventricular ependymal layer and astrocytes. Kidins220 is a known cargo of the SNX27-retromer, a complex that redirects endocytosed plasma membrane proteins (cargos) back to the cell surface, thus avoiding their targeting to lysosomes for degradation. Mechanistically, we show that AQP4 is a novel cargo of the SNX27-retromer and that Kidins220 deficiency promotes a striking and unexpected downregulation of the SNX27-retromer that results in AQP4 lysosomal degradation. Accordingly, SNX27 silencing decreases AQP4 levels in wild-type astrocytes whereas SNX27 overexpression restores AQP4 content in Kidins220 deficient astrocytes. Together our data suggest that the KIDINS220-SNX27-retromer-AQP4 pathway is involved in human ventriculomegaly and open novel therapeutic perspectives.

African Swine Fever Virus Ubiquitin-Conjugating Enzyme Interacts With Host Translation Machinery to Regulate the Host Protein Synthesis.

African Swine Fever virus (ASFV) causes one of the most relevant emerging diseases affecting swine, now extended through three continents. The virus has a large coding capacity to deploy an arsenal of molecules antagonizing the host functions. In the present work, we have studied the only known E2 viral-conjugating enzyme, UBCv1 that is encoded by the I215L gene of ASFV. UBCv1 was expressed as an early expression protein that accumulates throughout the course of infection. This versatile protein, bound several types of polyubiquitin chains and its catalytic domain was required for enzymatic activity. High throughput mass spectrometry analysis in combination with a screening of an alveolar macrophage library was used to identify and characterize novel UBCv1-host interactors. The analysis revealed interaction with the 40S ribosomal protein RPS23, the cap-dependent translation machinery initiation factor eIF4E, and the E3 ubiquitin ligase Cullin 4B. Our data show that during ASFV infection, UBCv1 was able to bind to eIF4E, independent from the cap-dependent complex. Our results provide novel insights into the function of the viral UBCv1 in hijacking cellular components that impact the mTORC signaling pathway, the regulation of the host translation machinery, and the cellular protein expression during the ASFV lifecycle.

Differential regulation of Kidins220 isoforms in Huntington's disease.

Huntington's disease (HD) is an inherited progressive neurodegenerative disease characterized by brain atrophy particularly in the striatum that produces motor impairment, and cognitive and psychiatric disturbances. Multiple pathogenic mechanisms have been proposed including dysfunctions in neurotrophic support and calpain-overactivation, among others. Kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is an essential mediator of neurotrophin signaling. In adult brain, Kidins220 presents two main isoforms that differ in their carboxy-terminal length and critical protein-protein interaction domains. These variants are generated through alternative terminal exon splicing of the conventional exon 32 (Kidins220-C32) and the recently identified exon 33 (Kidins220-C33). The lack of domains encoded by exon 32 involved in key neuronal functions, including those controlling neurotrophin pathways, pointed to Kidins220-C33 as a form detrimental for neurons. However, the functional role of Kidins220-C33 in neurodegeneration or other pathologies, including HD, has not been explored. In the present work, we discover an unexpected selective downregulation of Kidins220-C33, in the striatum of HD patients, as well as in the R6/1 HD mouse model starting at early symptomatic stages. These changes are C33-specific as Kidins220-C32 variant remains unchanged. We also find the early decrease in Kidins220-C33 levels takes place in neurons, suggesting an unanticipated neuroprotective role for this isoform. Finally, using ex vivo assays and primary neurons, we demonstrate that Kidins220-C33 is downregulated by mechanisms that depend on the activation of the protease calpain. Altogether, these results strongly suggest that calpain-mediated Kidins220-C33 proteolysis modulates onset and/or progression of HD.

Lipid Exchange Factors at Membrane Contact Sites in African Swine Fever Virus Infection.

African swine fever (ASF) is a hemorrhagic fever of wild and domestic pigs with a high rate of mortality. Originally endemic in Africa, this disease is currently disseminating in Europe and China, causing a large socioeconomic impact. ASF is caused by a DNA virus, African swine fever virus (ASFV). There is no vaccine available against ASFV, limiting the options for disease control. ASFV reorganizes intracellular membranes to generate viral factories (VFs) in order to amplify its genome. However, little is known about the process involved in the formation of these viral replication organelles. Membrane contact sites (MCSs) allow nonvesicular lipids and ion exchange between organelles. Lipid exchange to form VFs apparently requires a number of proteins at MCSs, such as the oxysterol-binding protein (OSBP), the acyl-coenzyme A binding domain containing 3 (ACBD3) and the phosphatidylinositol-phosphate-4-kinase III beta (PI4Kß). Itraconazole (ITZ) is an antifungal agent that targets sterol-transport molecules such as OSBP and OSBP-related protein 4 (ORP4). 25-Hydroxycholesterol (25-HC) inhibits lipid transport by high affinity binding OSBP. In this work, we analyzed the antiviral function of ITZ and 25-HC against ASFV in Vero cell cultures using the cell-adapted Ba71V isolate. ITZ and 25-HC decreased significantly ASFV replication. Our study revealed OSBP distribution in cytoplasmic membranes in uninfected Vero cells and to the periphery of VFs in infected cells. In addition, we showed that OSBP and OSBP-related proteins, PI4Kß and ACBD3 were recruited to VFs in the context ASFV infection.

Author Correction: Excitotoxic inactivation of constitutive oxidative stress detoxification pathway in neurons can be rescued by PKD1.

The original version of this Article contained an error in the spelling of the author Álvaro Sebastián-Serrano, which was incorrectly given as Álvaro Sebastián Serrano. This has now been corrected in both the PDF and HTML versions of the Article.

Excitotoxic inactivation of constitutive oxidative stress detoxification pathway in neurons can be rescued by PKD1.

Excitotoxicity, a critical process in neurodegeneration, induces oxidative stress and neuronal death through mechanisms largely unknown. Since oxidative stress activates protein kinase D1 (PKD1) in tumor cells, we investigated the effect of excitotoxicity on neuronal PKD1 activity. Unexpectedly, we find that excitotoxicity provokes an early inactivation of PKD1 through a dephosphorylation-dependent mechanism mediated by protein phosphatase-1 (PP1) and dual specificity phosphatase-1 (DUSP1). This step turns off the IKK/NF-κB/SOD2 antioxidant pathway. Neuronal PKD1 inactivation by pharmacological inhibition or lentiviral silencing in vitro, or by genetic inactivation in neurons in vivo, strongly enhances excitotoxic neuronal death. In contrast, expression of an active dephosphorylation-resistant PKD1 mutant potentiates the IKK/NF-κB/SOD2 oxidative stress detoxification pathway and confers neuroprotection from in vitro and in vivo excitotoxicity. Our results indicate that PKD1 inactivation underlies excitotoxicity-induced neuronal death and suggest that PKD1 inactivation may be critical for the accumulation of oxidation-induced neuronal damage during aging and in neurodegenerative disorders.

Cannabinoid Receptors Modulate Neuronal Morphology and AnkyrinG Density at the Axon Initial Segment.

Neuronal polarization underlies the ability of neurons to integrate and transmit information. This process begins early in development with axon outgrowth, followed by dendritic growth and subsequent maturation. In between these two steps, the axon initial segment (AIS), a subcellular domain crucial for generating action potentials (APs) and maintaining the morphological and functional polarization, starts to develop. However, the cellular/molecular mechanisms and receptors involved in AIS initial development and maturation are mostly unknown. In this study, we have focused on the role of the type-1 cannabinoid receptor (CB1R), a highly abundant G-protein coupled receptor (GPCR) in the nervous system largely involved in different phases of neuronal development and differentiation. Although CB1R activity modulation has been related to changes in axons or dendrites, its possible role as a modulator of AIS development has not been yet explored. Here we analyzed the potential role of CB1R on neuronal morphology and AIS development using pharmacological and RNA interference approaches in cultured hippocampal neurons. CB1R inhibition, at a very early developmental stage, has no effect on axonal growth, yet CB1R activation can promote it. By contrast, subsequent dendritic growth is impaired by CB1R inhibition, which also reduces ankyrinG density at the AIS. Moreover, our data show a significant correlation between early dendritic growth and ankyrinG density. However, CB1R inhibition in later developmental stages after dendrites are formed only reduces ankyrinG accumulation at the AIS. In conclusion, our data suggest that neuronal CB1R basal activity plays a role in initial development of dendrites and indirectly in AIS proteins accumulation. Based on the lack of CB1R expression at the AIS, we hypothesize that CB1R mediated modulation of dendritic arbor size during early development indirectly determines the accumulation of ankyrinG and AIS development. Further studies will be necessary to determine which CB1R-dependent mechanisms can coordinate these two domains, and what may be the impact of these early developmental changes once neurons mature and are embedded in a functional brain network.

ATP-P2X7 Receptor Modulates Axon Initial Segment Composition and Function in Physiological Conditions and Brain Injury.

Axon properties, including action potential initiation and modulation, depend on both AIS integrity and the regulation of ion channel expression in the AIS. Alteration of the axon initial segment (AIS) has been implicated in neurodegenerative, psychiatric, and brain trauma diseases, thus identification of the physiological mechanisms that regulate the AIS is required to understand and circumvent AIS alterations in pathological conditions. Here, we show that the purinergic P2X7 receptor and its agonist, adenosine triphosphate (ATP), modulate both structural proteins and ion channel density at the AIS in cultured neurons and brain slices. In cultured hippocampal neurons, an increment of extracellular ATP concentration or P2X7-green fluorescent protein (GFP) expression reduced the density of ankyrin G and voltage-gated sodium channels at the AIS. This effect is mediated by P2X7-regulated calcium influx and calpain activation, and impaired by P2X7 inhibition with Brilliant Blue G (BBG), or P2X7 suppression. Electrophysiological studies in brain slices showed that P2X7-GFP transfection decreased both sodium current amplitude and intrinsic neuronal excitability, while P2X7 inhibition had the opposite effect. Finally, inhibition of P2X7 with BBG prevented AIS disruption after ischemia/reperfusion in rats. In conclusion, our study demonstrates an involvement of P2X7 receptors in the regulation of AIS mediated neuronal excitability in physiological and pathological conditions.

Neuronal and glial purinergic receptors functions in neuron development and brain disease.

Brain development requires the interaction of complex signaling pathways, involving different cell types and molecules. For a long time, most attention has focused on neurons in a neuronocentric conceptualization of central nervous system development, these cells fulfilling an intrinsic program that establishes the brain's morphology and function. By contrast, glia have mainly been studied as support cells, offering guidance or as the cells that react to brain injury. However, new evidence is appearing that demonstrates a more fundamental role of glial cells in the control of different aspects of neuronal development and function, events in which the influence of neurons is at best weak. Moreover, it is becoming clear that the function and organization of the nervous system depends heavily on reciprocal neuron-glia interactions. During development, neurons are often generated far from their final destination and while intrinsic mechanisms are responsible for neuronal migration and growth, they need support and regulatory influences from glial cells in order to migrate correctly. Similarly, the axons emitted by neurons often have to reach faraway targets and in this sense, glia help define the way that axons grow. Moreover, oligodendrocytes and Schwann cells ultimately envelop axons, contributing to the generation of nodes of Ranvier. Finally, recent publications show that astrocytes contribute to the modulation of synaptic transmission. In this sense, purinergic receptors are expressed widely by glial cells and neurons, and recent evidence points to multiple roles of purines and purinergic receptors in neuronal development and function, from neurogenesis to axon growth and functional axonal maturation, as well as in pathological conditions in the brain. This review will focus on the role of glial and neuronal secreted purines, and on the purinergic receptors, fundamentally in the control of neuronal development and function, as well as in diseases of the nervous system.

GSK3 and ß-catenin determines functional expression of sodium channels at the axon initial segment.

Neuronal action potentials are generated through voltage-gated sodium channels, which are tethered by ankyrinG at the membrane of the axon initial segment (AIS). Despite the importance of the AIS in the control of neuronal excitability, the cellular and molecular mechanisms regulating sodium channel expression at the AIS remain elusive. Our results show that GSK3α/ß and ß-catenin phosphorylated by GSK3 (S33/37/T41) are localized at the AIS and are new components of this essential neuronal domain. Pharmacological inhibition of GSK3 or ß-catenin knockdown with shRNAs decreased the levels of phosphorylated-ß-catenin, ankyrinG, and voltage-gated sodium channels at the AIS, both "in vitro" and "in vivo", therefore diminishing neuronal excitability as evaluated via sodium current amplitude and action potential number. Thus, our results suggest a mechanism for the modulation of neuronal excitability through the control of sodium channel density by GSK3 and ß-catenin at the AIS.

Adenylate cyclase 5 coordinates the action of ADP, P2Y1, P2Y13 and ATP-gated P2X7 receptors on axonal elongation.

In adult brains, ionotropic or metabotropic purinergic receptors are widely expressed in neurons and glial cells. They play an essential role in inflammation and neurotransmission in response to purines secreted to the extracellular medium. Recent studies have demonstrated a role for purinergic receptors in proliferation and differentiation of neural stem cells although little is known about their role in regulating the initial neuronal development and axon elongation. The objective of our study was to investigate the role of some different types of purinergic receptors, P2Y1, P2Y13 and P2X7, which are activated by ADP or ATP. To study the role and crosstalk of P2Y1, P2Y13 and P2X7 purinergic receptors in axonal elongation, we treated neurons with specific agonists and antagonists, and we nucleofected neurons with expression or shRNA plasmids. ADP and P2Y1-GFP expression improved axonal elongation; conversely, P2Y13 and ATP-gated P2X7 receptors halted axonal elongation. Signaling through each of these receptor types was coordinated by adenylate cyclase 5. In neurons nucleofected with a cAMP FRET biosensor (ICUE3), addition of ADP or Blue Brilliant G, a P2X7 antagonist, increased cAMP levels in the distal region of the axon. Adenylate cyclase 5 inhibition or suppression impaired these cAMP increments. In conclusion, our results demonstrate a crosstalk between two metabotropic and one ionotropic purinergic receptor that regulates cAMP levels through adenylate cyclase 5 and modulates axonal elongation triggered by neurotropic factors and the PI3K-Akt-GSK3 pathway.

In vivo P2X7 inhibition reduces amyloid plaques in Alzheimer's disease through GSK3ß and secretases.

ß-Amyloid (Aß) peptide production from amyloid precursor protein (APP) is essential in the formation of the ß-amyloid plaques characteristic of Alzheimer's disease. However, the extracellular signals that maintain the balance between nonpathogenic and pathologic forms of APP processing, mediated by α-secretase and ß-secretase respectively, remain poorly understood. In the present work, we describe regulation of the processing of APP via the adenosine triphosphate (ATP) receptor P2X7R. In 2 different cellular lines, the inhibition of either native or overexpressed P2X7R increased α-secretase activity through inhibition of glycogen synthase kinase 3 (GSK-3). In vivo inhibition of the P2X7R in J20 mice, transgenic for mutant human APP, induced a significant decrease in the number of hippocampal amyloid plaques. This reduction correlated with a decrease in glycogen synthase kinase 3 activity in J20 mice, increasing the proteolytic processing of APP through an increase in α-secretase activity. The in vivo findings presented here demonstrate for the first time the therapeutic potential of P2X7R antagonism in the treatment of familiar Alzheimer's disease (FAD).

Ca2+/calmodulin-dependent kinase II signalling cascade mediates P2X7 receptor-dependent inhibition of neuritogenesis in neuroblastoma cells.

ATP, via purinergic P2X receptors, acts as a neurotransmitter and modulator in both the central and peripheral nervous systems, and is also involved in many biological processes, including cell proliferation, differentiation and apoptosis. Previously, we have reported that P2X7 receptor inhibition promotes axonal growth and branching in cultured hippocampal neurons. In this article, we demonstrate that the P2X7 receptor negatively regulates neurite formation in mouse Neuro-2a neuroblastoma cells through a Ca2+/calmodulin-dependent kinase II-related mechanism. Using both molecular and immunocytochemical techniques, we characterized the presence of endogenous P2X1, P2X3, P2X4 and P2X7 subunits in these cells. Of these, the P2X7 receptor was the only functional receptor, as its activation induced intracellular calcium increments similar to those observed in primary neuronal cultures, exhibiting pharmacological properties characteristic of homomeric P2X7 receptors. Patch-clamp experiments were also conducted to fully demonstrate that ionotropic P2X7 receptors mediate nonselective cation currents in this cell line. Pharmacological inhibition of the P2X7 receptor and its knockdown by small hairpin RNA interference resulted in increased neuritogenesis in cells cultured in low serum-containing medium, whereas P2X7 overexpression significantly reduced the formation of neurites. Interestingly, P2X7 receptor inhibition also modified the phosphorylation state of focal adhesion kinase, Akt and glycogen synthase kinase 3, protein kinases that participate in the Ca2+/calmodulin-dependent kinase II signalling cascade and that have been related to neuronal differentiation and axonal growth. Taken together, our results provide the first mechanistic insight into P2X7 receptor-triggered signalling pathways that regulate neurite formation in neuroblastoma cells.

Inhibition of the ATP-gated P2X7 receptor promotes axonal growth and branching in cultured hippocampal neurons.

During the establishment of neural circuits, the axons of neurons grow towards their target regions in response to both positive and negative stimuli. Because recent reports show that Ca2+ transients in growth cones negatively regulate axonal growth, we studied how ionotropic ATP receptors (P2X) might participate in this process. Our results show that exposing cultured hippocampal neurons to ATP induces Ca2+ transients in the distal domain of the axon and the concomitant inhibition of axonal growth. This effect is mediated by the P2X7 receptor, which is present in the growth cone of the axon. Pharmacological inhibition of P2X7 or its silencing by shRNA interference induces longer and more-branched axons, coupled with morphological changes to the growth cone. Our data suggest that these morphological changes are induced by a signalling cascade in which CaMKII and FAK activity activates PI3-kinase and modifies the activity of its downstream targets. Thus, in the absence or inactivation of P2X7 receptor, axons grow more rapidly and form more branches in cultured hippocampal neurons, indicative that ATP exerts a negative influence on axonal growth. These data suggest that P2X7 antagonists have therapeutic potential to promote axonal regeneration.