Understanding the Role of ATP Release through Connexins Hemichannels during Neurulation.

Neurulation is a crucial process in the formation of the central nervous system (CNS), which begins with the folding and fusion of the neural plate, leading to the generation of the neural tube and subsequent development of the brain and spinal cord. Environmental and genetic factors that interfere with the neurulation process promote neural tube defects (NTDs). Connexins (Cxs) are transmembrane proteins that form gap junctions (GJs) and hemichannels (HCs) in vertebrates, allowing cell-cell (GJ) or paracrine (HCs) communication through the release of ATP, glutamate, and NAD+; regulating processes such as cell migration and synaptic transmission. Changes in the state of phosphorylation and/or the intracellular redox potential activate the opening of HCs in different cell types. Cxs such as Cx43 and Cx32 have been associated with proliferation and migration at different stages of CNS development. Here, using molecular and cellular biology techniques (permeability), we demonstrate the expression and functionality of HCs-Cxs, including Cx46 and Cx32, which are associated with the release of ATP during the neurulation process in Xenopus laevis. Furthermore, applications of FGF2 and/or changes in intracellular redox potentials (DTT), well known HCs-Cxs modulators, transiently regulated the ATP release in our model. Importantly, the blockade of HCs-Cxs by carbenoxolone (CBX) and enoxolone (ENX) reduced ATP release with a concomitant formation of NTDs. We propose two possible and highly conserved binding sites (N and E) in Cx46 that may mediate the pharmacological effect of CBX and ENX on the formation of NTDs. In summary, our results highlight the importance of ATP release mediated by HCs-Cxs during neurulation.

Roles of the Unsaturated Fatty Acid Docosahexaenoic Acid in the Central Nervous System: Molecular and Cellular Insights.

Fatty acids (FAs) are essential components of the central nervous system (CNS), where they exert multiple roles in health and disease. Among the FAs, docosahexaenoic acid (DHA) has been widely recognized as a key molecule for neuronal function and cell signaling. Despite its relevance, the molecular pathways underlying the beneficial effects of DHA on the cells of the CNS are still unclear. Here, we summarize and discuss the molecular mechanisms underlying the actions of DHA in neural cells with a special focus on processes of survival, morphological development, and synaptic maturation. In addition, we examine the evidence supporting a potential therapeutic role of DHA against CNS tumor diseases and tumorigenesis. The current results suggest that DHA exerts its actions on neural cells mainly through the modulation of signaling cascades involving the activation of diverse types of receptors. In addition, we found evidence connecting brain DHA and ω-3 PUFA levels with CNS diseases, such as depression, autism spectrum disorders, obesity, and neurodegenerative diseases. In the context of cancer, the existing data have shown that DHA exerts positive actions as a coadjuvant in antitumoral therapy. Although many questions in the field remain only partially resolved, we hope that future research may soon define specific pathways and receptor systems involved in the beneficial effects of DHA in cells of the CNS, opening new avenues for innovative therapeutic strategies for CNS diseases.

Modulation of glycine receptor single-channel conductance by intracellular phosphorylation.

Glycine receptors (GlyRs) are anion-permeable pentameric ligand-gated ion channels (pLGICs). The GlyR activation is critical for the control of key neurophysiological functions, such as motor coordination, respiratory control, muscle tone and pain processing. The relevance of the GlyR function is further highlighted by the presence of abnormal glycinergic inhibition in many pathophysiological states, such as hyperekplexia, epilepsy, autism and chronic pain. In this context, previous studies have shown that the functional inhibition of  GlyRs containing the α3 subunit is a pivotal mechanism of pain hypersensitivity. This pathway involves the activation of EP2 receptors and the subsequent PKA-dependent phosphorylation of α3GlyRs within the intracellular domain (ICD), which decrease the GlyR-associated currents and enhance neuronal excitability. Despite the importance of this mechanism of glycinergic dis-inhibition associated with dysfunctional α3GlyRs, our current understanding of the molecular events involved is limited. Here, we report that the activation of PKA signaling pathway decreases the unitary conductance of α3GlyRs. We show in addition that the substitution of the PKA-targeted serine with a negatively charged residue within the ICD of α3GlyRs and of chimeric receptors combining bacterial GLIC and α3GlyR was sufficient to generate receptors with reduced conductance. Thus, our findings reveal a potential biophysical mechanism of glycinergic dis-inhibition and suggest that post-translational modifications of the ICD, such as phosphorylation, may shape the conductance of other pLGICs.

17 Oxo Sparteine and Lupanine, Obtained from Cytisus scoparius, Exert a Neuroprotection against Soluble Oligomers of Amyloid-ß Toxicity by Nicotinic Acetylcholine Receptors.

Alzheimer's disease (AD) is a neurodegenerative pathology, which is characterized by progressive and irreversible cognitive impairment. Most of the neuronal perturbations described in AD can be associated with soluble amyloid- ß oligomers (SO-Aß). There is a large amount of evidence demonstrating the neuroprotective effect of Nicotine neurotransmission in AD, mainly through nicotinic acetylcholine receptor (nAChR) activation and antiapoptotic PI3K/Akt/Bcl-2 pathway signaling. Using HPLC and GC/MS, we isolated and characterized two alkaloids obtained from C. scoparius, Lupanine (Lup), and 17- oxo-sparteine (17- ox), and examined their neuroprotective properties in a cellular model of SO-Aß toxicity. Our results showed that Lup and 17- ox (both at 0.03µM) prevented SO-Aß-induced toxicity in PC12 cells (Lup: 64±7%; 17- ox: 57±6%). Similar results were seen in hippocampal neurons where these alkaloids prevented SO-Aß neurotoxicity (Lup: 57±2%; 17- ox: 52±3%) and increased the frequency of spontaneous calcium transients (Lup: 60±4%; 17- Ox: 40±3%), suggesting an enhancing effect on neural network activity and synaptic activity potentiation. All of the neuroprotective effects elicited by both alkaloids were completely blocked by α-bungarotoxin. Additionally, we observed that the presence of both Lup and 17- ox increased Akt phosphorylation levels (52±4% and 35±7%, respectively) in cells treated with SO-Aß (3 h). Taken together, our results suggest that the activation of nAChR by Lup and 17- ox induces neuroprotection in different cellular models, and appears to be an interesting target for the development of new pharmacological tools and strategies against AD.

P2X receptor overexpression induced by soluble oligomers of amyloid beta peptide potentiates synaptic failure and neuronal dyshomeostasis in cellular models of Alzheimer's disease.

The most common cause of dementia is Alzheimer's disease. The etiology of the disease is unknown, although considerable evidence suggests a critical role for the soluble oligomers of amyloid beta peptide (Aß). Because Aß increases the expression of purinergic receptors (P2XRs) in vitro and in vivo, we studied the functional correlation between long-term exposure to Aß and the ability of P2XRs to modulate network synaptic tone. We used electrophysiological recordings and Ca2+ microfluorimetry to assess the effects of chronic exposure (24 h) to Aß oligomers (0.5 µM) together with known inhibitors of P2XRs, such as PPADS and apyrase on synaptic function. Changes in the expression of P2XR were quantified using RT-qPCR. We observed changes in the expression of P2X1R, P2X7R and an increase in P2X2R; and also in protein levels in PC12 cells (143%) and hippocampal neurons (120%) with Aß. In parallel, the reduction on the frequency and amplitude of mEPSCs (72% and 35%, respectively) were prevented by P2XR inhibition using a low PPADS concentration. Additionally, the current amplitude and intracellular Ca2+ signals evoked by extracellular ATP were increased (70% and 75%, respectively), suggesting an over activation of purinergic neurotransmission in cells pre-treated with Aß. Taken together, our findings suggest that Aß disrupts the main components of synaptic transmission at both pre- and post-synaptic sites, and induces changes in the expression of key P2XRs, especially P2X2R; changing the neuromodulator function of the purinergic tone that could involve the P2X2R as a key factor for cytotoxic mechanisms. These results identify novel targets for the treatment of dementia and other diseases characterized by increased purinergic transmission.

Reversal of Ethanol-induced Intoxication by a Novel Modulator of Gßγ Protein Potentiation of the Glycine Receptor.

The acute intoxicating effects of ethanol in the central nervous system result from the modulation of several molecular targets. It is widely accepted that ethanol enhances the activity of the glycine receptor (GlyR), thus enhancing inhibitory neurotransmission, leading to motor effects, sedation, and respiratory depression. We previously reported that small peptides interfered with the binding of Gßγ to the GlyR and consequently inhibited the ethanol-induced potentiation of the receptor. Now, using virtual screening, we identified a subset of small molecules capable of interacting with the binding site of Gßγ. One of these compounds, M554, inhibited the ethanol potentiation of the GlyR in both evoked currents and synaptic transmission in vitro When this compound was tested in vivo in mice treated with ethanol (1-3.5 g/kg), it was found to induce a faster recovery of motor incoordination in rotarod experiments and a shorter sedative effect in loss of righting reflex assays. This study describes a novel molecule that might be relevant for the design of useful therapeutic compounds in the treatment of acute alcohol intoxication.

The basic property of Lys385 is important for potentiation of the human α1 glycine receptor by ethanol.

Ethanol alters the function of several members of the Cys-loop ligand-gated ion channel superfamily. Recent studies have shown that the sensitivity of the α1 glycine receptor (GlyR) to ethanol can be affected by the state of G protein activation mediated by the interaction of Gßγ with intracellular amino acids in the GlyR. Here, we evaluated the physicochemical property of Lys385 that contributes to ethanol modulation by using mutagenesis, patch-clamp, and biochemical techniques. A conserved substitution (K385R) did not affect either the apparent glycine EC50 (40 ± 1 versus 41 ± 0.5 µM) or the ethanol-induced potentiation (53 ± 5 versus 46 ± 5%) of the human α1 GlyR. On the other hand, replacement of this residue with glutamic acid (K385E), an acidic amino acid, reduced the potentiation of the GlyR to 10 ± 1%. Furthermore, mutations with a hydrophobic leucine (K385L), a hydrogen bond donor glutamine (K385Q), or a neutral residue (K385A) also reduced ethanol modulation. Finally, substitution by a large and hydrophobic residue (K385F) and deletion of 385 (Lys385_) reduced ethanol modulation to 10 ± 4 and 17 ± 0.4%, respectively. Experiments using dynamic cysteine substitution with a methanethiosulfonate reagent and homology modeling indicate that the basic property and the position of Lys385, probably because of its interaction with Gßγ, is critical for ethanol potentiation of the receptor.

Activated G protein α s subunits increase the ethanol sensitivity of human glycine receptors.

It is well known that ethanol modulates the function of the Cys loop ligand-gated ion channels, which include the inhibitory glycine receptors (GlyRs). Previous studies have consistently shown that transmembrane and extracellular sites are essential for ethanol actions in GlyRs. In addition, recent evidence has shown that the ethanol modulation of GlyRs is also affected by G protein activation through Gßγ subunits. However, more specific roles of G protein α subunits on ethanol actions are unknown. Here, we show that the allosteric effect of ethanol on the human α(1) GlyR is selectively enhanced by the expression of Gα(s) Q-L. For example, constitutively active Gα(s), but not Gα(q) or Gα(i), was able to displace the alcohol sensitivity of GlyRs toward low millimolar concentrations (17 ± 4 versus 48 ± 5% at 100 mM). Experiments under conditions that increased cAMP and protein kinase A (PKA)-mediated signaling, on the contrary, did not produce the same enhancement in sensitivity, suggesting that the Gα(s) Q-L effect was not dependent on cAMP/PKA-dependent signaling. On the other hand, the effect of Gα(s) Q-L was blocked by a Gßγ scavenger (9 ± 3% of control). Furthermore, two mutant receptors previously shown to have impaired interactions with Gßγ were not affected by Gα(s) Q-L, suggesting that Gßγ is needed for enhancing ethanol sensitivity. These results support the conclusion that activated Gα(s) can facilitate the Gßγ interaction with GlyRs in presence of ethanol, independent of increases in cAMP signaling. Thus, these data indicate that the activated form of Gα(s) is able to positively influence the effect of ethanol on a type of inhibitory receptor important for motor control, pain, and respiration.

Regulation of GABAergic synapse formation and plasticity by GSK3beta-dependent phosphorylation of gephyrin.

Postsynaptic scaffolding proteins ensure efficient neurotransmission by anchoring receptors and signaling molecules in synapse-specific subcellular domains. In turn, posttranslational modifications of scaffolding proteins contribute to synaptic plasticity by remodeling the postsynaptic apparatus. Though these mechanisms are operant in glutamatergic synapses, little is known about regulation of GABAergic synapses, which mediate inhibitory transmission in the CNS. Here, we focused on gephyrin, the main scaffolding protein of GABAergic synapses. We identify a unique phosphorylation site in gephyrin, Ser270, targeted by glycogen synthase kinase 3ß (GSK3ß) to modulate GABAergic transmission. Abolishing Ser270 phosphorylation increased the density of gephyrin clusters and the frequency of miniature GABAergic postsynaptic currents in cultured hippocampal neurons. Enhanced, phosphorylation-dependent gephyrin clustering was also induced in vitro and in vivo with lithium chloride. Lithium is a GSK3ß inhibitor used therapeutically as mood-stabilizing drug, which underscores the relevance of this posttranslational modification for synaptic plasticity. Conversely, we show that gephyrin availability for postsynaptic clustering is limited by Ca(2+)-dependent gephyrin cleavage by the cysteine protease calpain-1. Together, these findings identify gephyrin as synaptogenic molecule regulating GABAergic synaptic plasticity, likely contributing to the therapeutic action of lithium.

Modulation of glycine-activated ion channel function by G-protein betagamma subunits.

Glycine receptors (GlyRs), together with GABA(A) and nicotinic acetylcholine (ACh) receptors, form part of the ligand-activated ion channel superfamily and regulate the excitability of the mammalian brain stem and spinal cord. Here we report that the ability of the neurotransmitter glycine to gate recombinant and native ionotropic GlyRs is modulated by the G protein betagamma dimer (Gbetagamma). We found that the amplitude of the glycine-activated Cl- current was enhanced after application of purified Gbetagamma or after activation of a G protein-coupled receptor. Overexpression of three distinct G protein alpha subunits (Galpha), as well as the Gbetagamma scavenger peptide ct-GRK2, significantly blunted the effect of G protein activation. Single-channel recordings from isolated membrane patches showed that Gbetagamma increased the GlyR open probability (nP(o)). Our results indicate that this interaction of Gbetagamma with GlyRs regulates both motor and sensory functions in the central nervous system.