Changes in melatonin receptor expression in a murine model of glaucoma.

Purpose: The objective of this study was to evaluate the changes in the melatoninergic receptors of DBA/2J and C57BL/6J mice with the development of glaucoma. DBA/2J mice are widely used to study the physiopathology of glaucoma due to the similarities of their eyes to human eyes and the resulting similarity in the development of their pathology. In addition, melatoninergic receptors are known for their control of intraocular pressure (IOP), reducing the production of aqueous humor; however, little is known about their relationship with the development of this pathology. Methods: mRNA expression of MT1, MT2, and GPR50 melatoninergic receptors was performed with quantitative real-time PCR. In addition, receptor expression was performed with immunohistochemical techniques on the ciliary processes. To further investigate the effect of melatonin and its analog 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) on IOP, animals were instilled with these compounds and the corresponding melatoninergic antagonists to assess their effect on IOP. Results: All melatoninergic receptor expression decayed with the development of the glaucomatous pathology in the DBA/2J mice, and was especially visible for the MT2 receptor. However, receptor expression was consistent in the C57BL/6J control mice across all ages investigated. Furthermore, IOP blockage was stronger with 4PPDOT (MT2 antagonist) only in the DBA/2J mice which suggests a correlation of this receptor with the development of the glaucomatous pathology in DBA/2J animals. Conclusions: Melatonin receptor expression decays with the development of the glaucomatous pathology. This implies that the physiologic hypotensive effect of endogenous melatonin reducing IOP is not possible. A solution for such changes in receptor expression is the exogenous application of melatonin or any of its analogs that permit the activation of the remaining melatonin receptors.

Neuronal P2X7 Receptor: Involvement in Neuronal Physiology and Pathology.

The proposed presence of P2X7 receptor (P2X7R) in neurons has been the source of some contention. Initial studies suggested an absence of P2X7R mRNA in neurons, and the apparent nonspecificity of the antibodies used to identify P2X7R raised further doubts. However, subsequent studies using new pharmacological and biomolecular tools provided conclusive evidence supporting the existence of functional P2X7Rs in neurons. The P2X7 receptor has since been shown to play a leading role in multiple aspects of neuronal physiology, including axonal elongation and branching and neurotransmitter release. P2X7R has also been implicated in neuronal pathologies, in which it may influence neuronal survival. Together, this body of research suggests that P2X7R may constitute an important therapeutic target for a variety of neurological disorders.

Transient P2X7 Receptor Antagonism Produces Lasting Reductions in Spontaneous Seizures and Gliosis in Experimental Temporal Lobe Epilepsy.

UNLABELLED: Neuroinflammation is thought to contribute to the pathogenesis and maintenance of temporal lobe epilepsy, but the underlying cell and molecular mechanisms are not fully understood. The P2X7 receptor is an ionotropic receptor predominantly expressed on the surface of microglia, although neuronal expression has also been reported. The receptor is activated by the release of ATP from intracellular sources that occurs during neurodegeneration, leading to microglial activation and inflammasome-mediated interleukin 1ß release that contributes to neuroinflammation. Using a reporter mouse in which green fluorescent protein is induced in response to the transcription of P2rx7, we show that expression of the receptor is selectively increased in CA1 pyramidal and dentate granule neurons, as well as in microglia in mice that developed epilepsy after intra-amygdala kainic acid-induced status epilepticus. P2X7 receptor levels were increased in hippocampal subfields in the mice and in resected hippocampus from patients with pharmacoresistant temporal lobe epilepsy. Cells transcribing P2rx7 in hippocampal slices from epileptic mice displayed enhanced agonist-evoked P2X7 receptor currents, and synaptosomes from these animals showed increased P2X7 receptor levels and altered calcium responses. A 5 d treatment of epileptic mice with systemic injections of the centrally available, potent, and specific P2X7 receptor antagonist JNJ-47965567 (30 mg/kg) significantly reduced spontaneous seizures during continuous video-EEG monitoring that persisted beyond the time of drug presence in the brain. Hippocampal sections from JNJ-47965567-treated animals obtained >5 d after treatment ceased displayed strongly reduced microgliosis and astrogliosis. The present study suggests that targeting the P2X7 receptor has anticonvulsant and possibly disease-modifying effects in experimental epilepsy. SIGNIFICANCE STATEMENT: Temporal lobe epilepsy is the most common and drug-resistant form of epilepsy in adults. Neuroinflammation is implicated as a pathomechanism, but the upstream mechanisms driving gliosis and how important this is for seizures remain unclear. In our study, we show that the ATP-gated P2X7 receptor is upregulated in experimental epilepsy and resected hippocampus from epilepsy patients. Targeting the receptor with a new centrally available antagonist, JNJ-47965567, suppressed epileptic seizures well beyond the time of treatment and reduced underlying gliosis in the hippocampus. The findings suggest a potential disease-modifying treatment for epilepsy based on targeting the P2X7 receptor.

Neuroprotection elicited by P2Y13 receptors against genotoxic stress by inducing DUSP2 expression and MAPK signaling recovery.

Nucleotides activating P2Y13 receptors display neuroprotective actions against different apoptotic stimuli in cerebellar granule neurons. In the present study, P2Y13 neuroprotection was analyzed in conditions of genotoxic stress. Exposure to cisplatin and UV radiation induced caspase-3-dependent apoptotic cell death, and p38 MAPK signaling de-regulation. Pre-treatment with P2Y13 nucleotide agonist, 2methyl-thio-ADP (2MeSADP), restored granule neuron survival and prevented p38 long-lasting activation induced by cytotoxic treatments. Microarray gene expression analysis in 2MeSADP-stimulated cells revealed over-representation of genes related to protein phosphatase activity. Among them, dual-specificity phosphatase-2, DUSP2, was validated as a transcriptional target for P2Y13 receptors by QPCR. This effect could explain 2MeSADP ability to dephosphorylate a DUSP2 substrate, p38, reestablishing the inactive form. In addition, cisplatin-induced p38 sustained activation correlated perfectly with progressive reduction in DUSP2 expression. In conclusion, P2Y13 receptors regulate DUSP2 expression and contribute to p38 signaling homeostasis and survival in granule neurons.

The vesicular nucleotide transporter (VNUT) is involved in the extracellular ATP effect on neuronal differentiation.

Before being released, nucleotides are stored in secretory vesicles through the vesicular nucleotide transporter (VNUT). Once released, extracellular ATP participates in neuronal differentiation processes. Thus, the expression of a functional VNUT could be an additional component of the purinergic system which regulates neuronal differentiation and axonal elongation. In vitro expression of VNUT decreases neuritogenesis in N2a cells differentiated by retinoic acid treatment, whereas silencing of VNUT expression increases the number and length of neurites in these cells. These results highlight the role of VNUT in the neuritogenic process because this transporter regulates the ATP content in neurosecretory vesicles.

α-Synuclein accumulates in huntingtin inclusions but forms independent filaments and its deficiency attenuates early phenotype in a mouse model of Huntington's disease.

Huntington's disease (HD) is the most common of nine inherited neurological disorders caused by expanded polyglutamine (polyQ) sequences which confer propensity to self-aggregate and toxicity to their corresponding mutant proteins. It has been postulated that polyQ expression compromises the folding capacity of the cell which might affect other misfolding-prone proteins. α-Synuclein (α-syn) is a small neural-specific protein with propensity to self-aggregate that forms Parkinson's disease (PD) Lewy bodies. Point mutations in α-syn that favor self-aggregation or α-syn gene duplications lead to familial PD, thus indicating that increased α-syn aggregation or levels are sufficient to induce neurodegeneration. Since polyQ inclusions in HD and other polyQ disorders are immunopositive for α-syn, we speculated that α-syn might be recruited as an additional mediator of polyQ toxicity. Here, we confirm in HD postmortem brains and in the R6/1 mouse model of HD the accumulation of α-syn in polyQ inclusions. By isolating the characteristic filaments formed by aggregation-prone proteins, we found that N-terminal mutant huntingtin (N-mutHtt) and α-syn form independent filamentous microaggregates in R6/1 mouse brain as well as in the inducible HD94 mouse model and that N-mutHtt expression increases the load of α-syn filaments. Accordingly, α-syn knockout results in a diminished number of N-mutHtt inclusions in transfected neurons and also in vivo in the brain of HD mice. Finally, α-syn knockout attenuates body weight loss and early motor phenotype of HD mice. This study therefore demonstrates that α-syn is a modifier of polyQ toxicity in vivo and raises the possibility that potential PD-related therapies aimed to counteract α-syn toxicity might help to slow HD.

Presence of diadenosine polyphosphates in microdialysis samples from rat cerebellum in vivo: effect of mild hyperammonemia on their receptors.

Diadenosine triphosphate (Ap(3)A), diadenosine tetraphosphate (Ap(4)A), and diadenosine pentaphosphate (Ap(5)A) have been identified in microdialysis samples from the cerebellum of conscious freely moving rats, under basal conditions, by means of a high-performance liquid chromatography method. The occurrence of Ap(3)A in the cerebellar microdyalisates is noteworthy, as the presence of this compound in the interstitial medium in neural tissues has not been previously described. The concentrations measured for the diadenosine polyphosphates in the cerebellar dialysate were (in nanomolar) 10.5 ± 2.9, 5.4 ± 1.2, and 5.8 ± 1.3 for Ap(3)A, Ap(4)A, and Ap(5)A, respectively. These concentrations are in the range that allows the activation of the presynaptic dinucleotide receptor in nerve terminals. However, a possible interaction of these dinucleotides with other purinergic receptors cannot be ruled out, as rat cerebellum expresses a variety of P2X or P2Y receptors susceptible to be activated by diadenosine polyphosphates, such as the P2X1-4, P2Y(1), P2Y(2), P2Y(4), and P2Y(12) receptors, as demonstrated by quantitative real-time PCR. Also, the ecto-nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP3, able to hydrolyze the diadenosine polyphosphates and terminate their extracellular actions, are expressed in the rat cerebellum. All these evidences contribute to reinforce the role of diadenosine polyphosphates as signaling molecules in the central nervous system. Finally, we have analyzed the possible differences in the concentration of diadenosine polyphosphates in the cerebellar extracellular medium and changes in the expression levels of their receptors and hydrolyzing enzymes in an animal model of moderate hyperammonemia.

The specificity protein factor Sp1 mediates transcriptional regulation of P2X7 receptors in the nervous system.

P2X7 receptors are involved not only in physiological functions but also in pathological brain processes. Although an increasing number of findings indicate that altered receptor expression has a causative role in neurodegenerative diseases and cancer, little is known about how expression of P2rx7 gene is controlled. Here we reported the first molecular and functional evidence that Specificity protein 1 (Sp1) transcription factor plays a pivotal role in the transcriptional regulation of P2X7 receptor. We delimited a minimal region in the murine P2rx7 promoter containing four SP1 sites, two of them being highly conserved in mammals. The functionality of these SP1 sites was confirmed by site-directed mutagenesis and Sp1 overexpression/down-regulation in neuroblastoma cells. Inhibition of Sp1-mediated transcriptional activation by mithramycin A reduced endogenous P2X7 receptor levels in primary cultures of cortical neurons and astrocytes. Using P2rx7-EGFP transgenic mice that express enhanced green fluorescent protein under the control of P2rx7 promoter, we found a high correlation between reporter expression and Sp1 levels in the brain, demonstrating that Sp1 is a key element in the transcriptional regulation of P2X7 receptor in the nervous system. Finally, we found that Sp1 mediates P2X7 receptor up-regulation in neuroblastoma cells cultured in the absence of serum, a condition that enhances chromatin accessibility and facilitates the exposure of SP1 binding sites.

Seizure suppression and neuroprotection by targeting the purinergic P2X7 receptor during status epilepticus in mice.

Prolonged seizures [status epilepticus (SE)] constitute a neurological emergency that can permanently damage the brain. SE results from a failure of the normal mechanisms to terminate seizures; in particular, γ-amino butyric acid-mediated inhibition, and benzodiazepine anticonvulsants are often incompletely effective. ATP acts as a fast neurotransmitter via ionotropic ligand-gated P2X receptors. Here we report that SE induced by intra-amygdala kainic acid in mice selectively increased hippocampal levels of P2X7 receptors relative to other P2X receptors. Using transgenic P2X7 reporter mice expressing enhanced green fluorescent protein, we identify dentate granule neurons as the major cell population transcribing the P2X7 receptor after SE. Pretreatment of mice with an intracerebroventricular microinjection of 1.75 nmol A438079, a P2X7 receptor antagonist, reduced seizure duration by 58% and reduced seizure-induced neuronal death by 61%. Injection of brilliant blue G (1 pmol), another selective antagonist, reduced seizure duration by 48% and was also neuroprotective. A438079 was seizure-suppressive when injected shortly after induction of SE, and coinjection of A438079 with lorazepam 60 min after triggering SE, when electrographic seizure-responsiveness to lorazepam had decreased, also terminated SE. Our results suggest that P2X7 receptor antagonists may be a promising class of drug for seizure abrogation and neuroprotection in SE.

Tissue-nonspecific alkaline phosphatase promotes the neurotoxicity effect of extracellular tau.

There is solid evidence indicating that hyperphosphorylated tau protein, the main component of intracellular neurofibrillary tangles present in the brain of Alzheimer disease patients, plays a key role in progression of this disease. However, it has been recently reported that extracellular unmodified tau protein may also induce a neurotoxic effect on hippocampal neurons by activation of M1 and M3 muscarinic receptors. In the present work we show an essential component that links both effects, which is tissue-nonspecific alkaline phosphatase (TNAP). This enzyme is abundant in the central nervous system and is mainly required to keep control of extracellular levels of phosphorylated compounds. TNAP dephosphorylates the hyperphosphorylated tau protein once it is released upon neuronal death. Only the dephosphorylated tau protein behaves as an agonist of muscarinic M1 and M3 receptors, provoking a robust and sustained intracellular calcium increase finally triggering neuronal death. Interestingly, activation of muscarinic receptors by dephosphorylated tau increases the expression of TNAP in SH-SY5Y neuroblastoma cells. An increase in TNAP activity together with increases in protein and transcript levels were detected in Alzheimer disease patients when they were compared with healthy controls.

Update of P2Y receptor pharmacology: IUPHAR Review 27.

Eight G protein-coupled P2Y receptor subtypes respond to extracellular adenine and uracil mononucleotides and dinucleotides. P2Y receptors belong to the δ group of rhodopsin-like GPCRs and contain two structurally distinct subfamilies: P2Y1 , P2Y2 , P2Y4 , P2Y6 , and P2Y11 (principally Gq protein-coupled P2Y1 -like) and P2Y12-14 (principally Gi protein-coupled P2Y12 -like) receptors. Brain P2Y receptors occur in neurons, glial cells, and vasculature. Endothelial P2Y1 , P2Y2 , P2Y4 , and P2Y6 receptors induce vasodilation, while smooth muscle P2Y2 , P2Y4 , and P2Y6 receptor activation leads to vasoconstriction. Pancreatic P2Y1 and P2Y6 receptors stimulate while P2Y13 receptors inhibits insulin secretion. Antagonists of P2Y12 receptors, and potentially P2Y1 receptors, are anti-thrombotic agents, and a P2Y2 /P2Y4 receptor agonist treats dry eye syndrome in Asia. P2Y receptor agonists are generally pro-inflammatory, and antagonists may eventually treat inflammatory conditions. This article reviews recent developments in P2Y receptor pharmacology (using synthetic agonists and antagonists), structure and biophysical properties (using X-ray crystallography, mutagenesis and modelling), physiological and pathophysiological roles, and present and potentially future therapeutic targeting.

P2X7 and P2Y13 purinergic receptors mediate intracellular calcium responses to BzATP in rat cerebellar astrocytes.

Previous work has established the presence of functional P2X(7) subunits in rat cerebellar astrocytes, which after stimulation with 3'-O-(4-benzoyl)benzoyl ATP (BzATP) evoked morphological changes that were not reproduced by any other nucleotide. To further characterize the receptor(s) and signaling mechanisms involved in the action of BzATP, we have employed fura-2 microfluorometry and the patch-clamp technique. BzATP elicited intracellular calcium responses that typically exhibited two components: the first one was transient and metabotropic in nature--sensitive to phospholipase C inhibition and pertussis toxin treatment, whereas the second one was sustained and depended on the presence of extracellular calcium. The ionotropic nature of this latter component was corroborated by measurements of Mn(2+) entry and macroscopic non-selective cation currents evoked by either BzATP (100 muM) or ATP (1 mM). The two components of the calcium response to BzATP differed in their pharmacological sensitivity. The metabotropic component was partially sensitive to pyridoxalphosphate-5'-phosphate-6-azo-(-2-chloro-5-nitrophenyl)-2,4-disulfonate, a selective antagonist of P2Y(13) receptors, while the ionotropic component was modulated by external magnesium and markedly reduced by brilliant blue G and 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine (A438079), thus implying the involvement of P2X(7) purinergic receptors. It is concluded that P2Y(13) and P2X(7) purinergic receptors are functionally expressed in rat cerebellar astrocytes and mediate the increase in intracellular calcium elicited by BzATP in these cells.

MicroRNA-22 Controls Aberrant Neurogenesis and Changes in Neuronal Morphology After Status Epilepticus.

Prolonged seizures (status epilepticus, SE) may drive hippocampal dysfunction and epileptogenesis, at least partly, through an elevation in neurogenesis, dysregulation of migration and aberrant dendritic arborization of newly-formed neurons. MicroRNA-22 was recently found to protect against the development of epileptic foci, but the mechanisms remain incompletely understood. Here, we investigated the contribution of microRNA-22 to SE-induced aberrant adult neurogenesis. SE was induced by intraamygdala microinjection of kainic acid (KA) to model unilateral hippocampal neuropathology in mice. MicroRNA-22 expression was suppressed using specific oligonucleotide inhibitors (antagomir-22) and newly-formed neurons were visualized using the thymidine analog iodo-deoxyuridine (IdU) and a green fluorescent protein (GFP)-expressing retrovirus to visualize the dendritic tree and synaptic spines. Using this approach, we quantified differences in the rate of neurogenesis and migration, the structure of the apical dendritic tree and density and morphology of dendritic spines in newly-formed neurons.SE resulted in an increased rate of hippocampal neurogenesis, including within the undamaged contralateral dentate gyrus (DG). Newly-formed neurons underwent aberrant migration, both within the granule cell layer and into ectopic sites. Inhibition of microRNA-22 exacerbated these changes. The dendritic diameter and the density and average volume of dendritic spines were unaffected by SE, but these parameters were all elevated in mice in which microRNA-22 was suppressed. MicroRNA-22 inhibition also reduced the length and complexity of the dendritic tree, independently of SE. These data indicate that microRNA-22 is an important regulator of morphogenesis of newly-formed neurons in adults and plays a role in supressing aberrant neurogenesis associated with SE.

Nucleotides in neuroregeneration and neuroprotection.

Brain injury generates the release of a multitude of factors including extracellular nucleotides, which exhibit bi-functional properties and contribute to both detrimental actions in the acute phase and also protective and reparative actions in the later recovery phase to allow neuroregeneration. A promising strategy toward restoration of neuronal function is based on activation of endogenous adult neural stem/progenitor cells. The implication of purinergic signaling in stem cell biology, including regulation of proliferation, differentiation, and cell death has become evident in the last decade. In this regard, current strategies of acute transplantation of ependymal stem/progenitor cells after spinal cord injury restore altered expression of P2X4 and P2X7 receptors and improve functional locomotor recovery. The expression of both receptors is transcriptionally regulated by Sp1 factor, which plays a key role in the startup of the transcription machinery to induce regeneration-associated genes expression. Finally, general signaling pathways triggered by nucleotide receptors in neuronal populations converge on several intracellular kinases, such as PI3K/Akt, GSK3 and ERK1,2, as well as the Nrf-2/heme oxigenase-1 axis, which specifically link them to neuroprotection. In this regard, regulation of dual specificity protein phosphatases can become novel mechanism of actions for nucleotide receptors that associate them to cell homeostasis regulation. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.

Characterization of a functional P2X(7)-like receptor in cerebellar granule neurons from P2X(7) knockout mice.

The presence of ionotropic P2X(7) receptor has been studied in mice brain from wild type and P2X(7) receptor knockout animals. Western blot and immunocytochemical assays show the presence of a protein containing the P2X(7) immunogenic epitopes in the brain of knockout model. Reverse transcriptase polymerase chain reaction experiments demonstrate the absence of the disrupted sequence, but other sequences of P2X(7) specific mRNA expression have been detected. Functional calcium imaging experiments in cultured granule neurons from P2X(7) knockout cerebella show the existence of a functional P2X(7)-like receptor that keeps some of the properties of the genuine receptor.

Presence of diverse functional P2X receptors in rat cerebellar synaptic terminals.

Studies in individual synaptic terminals have demonstrated the presence of diverse functional P2X receptors in rat cerebellum. No immunolabelling for P2X1, P2X4, P2X5 and P2X6, and scarce presence of P2X2 were found at the cerebellar synaptic terminals. P2X3 immunolabelling was present in 28% of isolated synaptosomes. At these synaptic terminals, nucleotides as ATP or alpha,beta-meATP induced Ca2+ transients in the presence of extracellular Ca2+, showing homologous and heterologous receptor desensitization in 60% of cases. Ip5I 10 nM did not block responses to alpha,beta-meATP, but inhibition occurred when antagonist concentrations were equal or higher than 100 nM. These data agree with the presence of abundant P2X3 homomeric receptors. P2X7 immunolabelling was present in 60% of terminals and P2X7 receptor hallmarks in Ca2+ responses have been found. BzATP was more potent than ATP and responses were potentiated when assayed in Mg2+-free medium. EC50 values were, respectively, 39.4+/-0.4 and 0.3+/-0.1 microM for ATP in the presence or absence of Mg2+. Maximal values of synaptosomal calcium transients, in the presence or absence of Mg2+, were, respectively, 91.6+/-11.9 and 132.9+/-12.9 nM for ATP; and 104.3+/-9.4 and 169.7+/-17.1 nM for BzATP. In addition, Zn2+ inhibited ATP responses in the absence of Mg2+ and the P2X7 specific antagonist Brilliant Blue G completely blocked these responses in one half of synaptosomes. This study reports the presence of functional P2X3 and P2X7 receptors at synaptic sites, which provides complexity and regulatory possibilities to the cerebellar neurotransmission.

PI3K/Akt signaling pathway triggers P2X7 receptor expression as a pro-survival factor of neuroblastoma cells under limiting growth conditions.

The expression of purinergic P2X7 receptor (P2X7R) in neuroblastoma cells is associated to accelerated growth rate, angiogenesis, metastasis and poor prognosis. Noticeably, P2X7R allows the survival of neuroblastoma cells under restrictive conditions, including serum and glucose deprivation. Previously we identified specificity protein 1 (Sp1) as the main factor involved in the transcriptional regulation of P2rx7 gene, reporting that serum withdrawal triggers the expression of P2X7R in Neuro-2a (N2a) neuroblastoma cell line. Here we demonstrate that PI3K/Akt pathway is crucial for the upregulation of P2X7R expression in serum-deprived neuroblastoma cells, circumstance that facilitates cell proliferation in the absence of trophic support. The effect exerted by PI3K/Akt is independent of both mTOR and GSK3, but requires the activation of EGF receptor (EGFR). Nuclear levels of Sp1 are strongly reduced by inhibition of PI3K/Akt pathway, and blockade of Sp1-dependent transcription with mithramycin A prevents upregulation of P2rx7 gene expression following serum withdrawal. Furthermore, atypical PKCζ plays a key role in the regulation of P2X7R expression by preventing phosphorylation and, consequently, activation of Akt. Altogether, these data indicate that activation of EGFR enhanced the expression of P2X7R in neuroblastoma cells lacking trophic support, being PI3K/Akt/PKCζ signaling pathway and Sp1 mediating this pro-survival outcome.

Role of P2X7 and P2Y2 receptors on α-secretase-dependent APP processing: Control of amyloid plaques formation "in vivo" by P2X7 receptor.

Amyloid precursor protein (APP) is expressed in a large variety of neural and non-neural cells. The balance between non-pathogenic and pathologic forms of APP processing, mediated by α-secretase and ß-secretase respectively, remains a crucial step to understand ß-amyloid, Aß42 peptide, formation and aggregation that are at the origin of the senile plaques in the brain, a characteristic hallmark of Alzheimer's disease (AD). In Neuro-2a, a neuroblastoma cell line that constitutively expresses APP, activation of the P2X7 receptor leads to reduction of α-secretase activity, the opposite effect being obtained by P2Y2 receptor activation. The in vivo approach was made possible by the use of J20 mice, a transgenic mouse model of familial Alzheimer's disease (FAD) expressing human APP mutant protein. This animal exhibits prominent amyloid plaques by six months of age. In vivo inhibition of the P2X7 receptor induced a significant decrease in the number and size of hippocampal amyloid plaques. This reduction is mediated by an increase in the proteolytic processing of APP through α-secretase activity, which correlates with an increase in the phosphorylated form of GSK-3, a less active form of this enzyme. The in vivo findings corroborate the therapeutic potential of P2X7 antagonists in the treatment of FAD.

Neuroprotection Mediated by P2Y13 Nucleotide Receptors in Neurons.

ADP-specific P2Y13 receptor constitutes one of the most recently identified nucleotide receptor and the understanding of their physiological role is currently under investigation. Cerebellar astrocytes and granule neurons provide excellent models to study P2Y13 expression and function since the first identification of ADP-evoked calcium responses not attributable to the related P2Y1 receptor was performed in these cell populations. In this regard, all responses induced by ADP analogues in astrocytes resulted to be Gi-coupled activities mediated by P2Y13 instead of P2Y1 receptors. Similarly, both glycogen synthase kinase-3 (GSK3) and ERK1/2 signaling triggered by 2MeSADP in cerebellar granule neurons were also dependent on Gi-coupled receptors, and mediated by PI3K activity. In granule neurons, P2Y13 receptor was specifically coupled to the main neuronal survival PI3K/Akt-cascade targeting GSK3 phosphorylation. GSK3 inhibition led to nuclear translocation of transcriptional targets, including ß-catenin and Nrf2. The activation of the Nrf2/heme oxygenase-1 (HO-1) axis was responsible for the prosurvival effect against oxidative stress. In addition, P2Y13-mediated ERK1/2 signaling in granule neurons also triggered activation of transcription factors, such as CREB, which underlined the antiapoptotic action against glutamate-induced excitotoxicity. Finally, a novel signaling mechanism has been recently described for a P2Y13 receptor in granule neurons that involved the expression of a dual protein phosphatase, DUSP2. This activity contributed to regulate MAPK activation after genotoxic stress. In conclusion, P2Y13 receptors harbored in cerebellar astrocytes and granule neurons exhibit specific signaling properties that link them to specialized functions at the level of neuroprotection and trophic activity in both cerebellar cell populations.

microRNA targeting of the P2X7 purinoceptor opposes a contralateral epileptogenic focus in the hippocampus.

The ATP-gated ionotropic P2X7 receptor (P2X7R) modulates glial activation, cytokine production and neurotransmitter release following brain injury. Levels of the P2X7R are increased in experimental and human epilepsy but the mechanisms controlling P2X7R expression remain poorly understood. Here we investigated P2X7R responses after focal-onset status epilepticus in mice, comparing changes in the damaged, ipsilateral hippocampus to the spared, contralateral hippocampus. P2X7R-gated inward currents were suppressed in the contralateral hippocampus and P2rx7 mRNA was selectively uploaded into the RNA-induced silencing complex (RISC), suggesting microRNA targeting. Analysis of RISC-loaded microRNAs using a high-throughput platform, as well as functional assays, suggested the P2X7R is a target of microRNA-22. Inhibition of microRNA-22 increased P2X7R expression and cytokine levels in the contralateral hippocampus after status epilepticus and resulted in more frequent spontaneous seizures in mice. The major pro-inflammatory and hyperexcitability effects of microRNA-22 silencing were prevented in P2rx7(-/-) mice or by treatment with a specific P2X7R antagonist. Finally, in vivo injection of microRNA-22 mimics transiently suppressed spontaneous seizures in mice. The present study supports a role for post-transcriptional regulation of the P2X7R and suggests therapeutic targeting of microRNA-22 may prevent inflammation and development of a secondary epileptogenic focus in the brain.