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.

The precise mechanism by which mutant huntingtin elicits its toxicity remains unknown. However, synaptic alterations and increased susceptibility to neuronal death are known contributors to Huntington's disease (HD) symptomatology. While decreased metabolism has long been associated with HD, recent findings have surprisingly demonstrated reduced neuronal apoptosis in Caenorhabditis elegans and Drosophila models of HD by drugs that diminish ATP production. Interestingly, extracellular ATP has been recently reported to elicit neuronal death through stimulation of P2X7 receptors. These are ATP-gated cation channels known to modulate neurotransmitter release from neuronal presynaptic terminals and to regulate cytokine production and release from microglia. We hypothesized that alteration in P2X7-mediated calcium permeability may contribute to HD synaptic dysfunction and increased neuronal apoptosis. Using mouse and cellular models of HD, we demonstrate increased P2X7-receptor level and altered P2X7-mediated calcium permeability in somata and terminals of HD neurons. Furthermore, cultured neurons expressing mutant huntingtin showed increased susceptibility to apoptosis triggered by P2X7-receptor stimulation. Finally, in vivo administration of the P2X7-antagonist Brilliant Blue-G (BBG) to HD mice prevented neuronal apoptosis and attenuated body weight loss and motor-coordination deficits. These in vivo data strongly suggest that altered P2X7-receptor level and function contribute to HD pathogenesis and highlight the therapeutic potential of P2X7 receptor antagonists.

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. Alzheimer disease (AD)3 is characterized by the loss of neurons and the presence of amyloid plaques and neurofibrillary tangles. The plaques are dense deposits of amyloid-␤ peptide and cellular material outside and around neurons, whereas the tangles are aggregates of the microtubule-associated protein tau, which has become hyperphosphorylated and accumulates inside the cells (1). In AD, tau pathology follows a reproducible pattern, in which hyperphosphorylated and aggregated tau first appears in the entorhinal cortex and hippocampus, and from there the disease spreads to the surrounding areas (2). During this process, neuronal loss occurs and tau protein may be found in the extracellular space in monomeric form or in aggregated form, assembled in extracellular ghost tangles. Indeed, an inverse correlation can be found between the number of extracellular tangles and the number of living neurons in the hippocampus (3-5). It has been also suggested that extracellular aggregated tau can promote the aggregation of intracellular tau (6). Moreover, it has been reported that extracellular monomeric tau is toxic for neurons, playing a role in the spreading of AD pathology (7-9). Monomeric tau-dependent toxicity occurs when extracellular tau binds and activates cell membrane receptors, identified as M1 and M3 muscarinic receptors (7).Sluggish disassembly of aggregated tau and slow degradation of its monomeric form in extracellular media provide this protein with a long stay outside the cell. In this location, hyperphosphorylated monomeric tau can be recognized as a substrate of several extracellular enzymes, some of which can remove the phosphates from the protein (10, 11). One of these enzymes is tissue-nonspecific alkaline phosphatas...

No abstract

Calcium is the most universal signal used by living organisms to convey information to many different cellular processes. In this review we present well-known and recently identified proteins that sense and decode the calcium signal and are key elements in the nucleus to regulate the activity of various transcriptional networks. When possible, the review also presents in vivo models in which the genes encoding these calcium sensors-transducers have been modified, to emphasize the critical role of these Ca(2+)-operated mechanisms in many physiological functions.

The Na+/Ca2+ exchangers NCX1, NCX2, and NCX3 are vital for the control of cellular Ca2+ homeostasis. Here, we show that a doublet of downstream regulatory element sites in the promoter of the NCX3 gene mediates transcriptional repression of NCX3 by the Ca2+-modulated transcriptional repressor downstream regulatory element antagonist modulator (DREAM). Overexpression of a DREAM EF-hand mutant insensitive to Ca2+ (EFmDREAM) in hippocampus and cerebellum of transgenic mice significantly reduced NCX3 mRNA and protein levels without modifying NCX1 and NCX2 expression. Cerebellar granules from EFmDREAM transgenic mice showed increased levels of cytosolic Ca2+ and were more vulnerable to increased Ca2+ influx after partial opening of voltage-gated plasma membrane Ca2+ channels induced by increasing K+ in the culture medium but survived better in the conditions of reduced Ca2+ influx prevailing in low extracellular K+. Overexpression of NCX3 in EFmDREAM transgenic granules using a lentiviral vector restored the normal survival response to high K+ observed in wild-type granules. Thus, the downregulation of the regulator of Ca2+ homeostasis NCX3 by Ca2+-regulated DREAM is a striking example of the autoregulatory property of the Ca2+ signal in neurons.

SummaryIn 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.

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 Ca 2+ ⁄ 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 Ca 2+ ⁄ 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.] i, intracellular free calcium concentration; BBG, brilliant blue G; BzATP, 2¢,3¢-O-(4-benzoyl)-benzoyl ATP; CaMKII, Ca 2+ ⁄ calmodulindependent kinase II; CNS, central nervous system; DiBucAMP, dibutyryl-cAMP; FAK, focal adhesion kinase; Fura-2 AM, fura-2 acetoxymethyl ester; GFP, green fluorescent protein; GSK3, glycogen synthase kinase 3; N2a, Neuro-2a; PFA, paraformaldehyde, PI3K, phosphatidylinositol 3-kinase; shRNA, small hairpin RNA; a,b-meATP, a,b-methylene-ATP.