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Proline-Rich Transmembrane protein 2 (PRRT2) is a neuron-specific protein implicated in the control of neurotransmitter release and neural network stability. Accordingly, PRRT2 loss-of-function mutations associate with pleiotropic paroxysmal neurological disorders, including paroxysmal kinesigenic dyskinesia, episodic ataxia, benign familial infantile seizures, hemiplegic migraine. PRRT2 is a negative modulator of the membrane exposure and biophysical properties of Na+ channels NaV1.2/NaV1.6 predominantly expressed in brain glutamatergic neurons. NaV channels form complexes with β-subunits that facilitate the membrane targeting and the activation of the α-subunits. The opposite effects of PRRT2 and β-subunits on NaV channels raises the question of whether PRRT2 and β-subunits interact or compete for common binding sites on the α-subunit, generating Na+ channel complexes with distinct functional properties. Using a heterologous expression system, we have observed that β-subunits and PRRT2 do not interact with each other and act as independent non-competitive modulators of NaV1.2 channel trafficking and biophysical properties. PRRT2 antagonizes the β4-induced increase in expression and functional activation of the transient and persistent NaV1.2 currents, without affecting resurgent current. The data indicate that β4-subunit and PRRT2 form a push-pull system that finely tunes the membrane expression and function of NaV channels and the intrinsic neuronal excitability.

Neuroinflammation is involved in the pathogenesis of several neurologic disorders, including epilepsy. Both changes in the input/output functions of synaptic circuits and cell Ca2+ dysregulation participate in neuroinflammation, but their impact on neuron function in epilepsy is still poorly understood. Lipopolysaccharide (LPS), a toxic byproduct of bacterial lysis, has been extensively used to stimulate inflammatory responses both in vivo and in vitro. LPS stimulates Toll-like receptor 4, an important mediator of the brain innate immune response that contributes to neuroinflammation processes. Although we report that Toll-like receptor 4 is expressed in both excitatory and inhibitory mouse hippocampal neurons (both sexes), its chronic stimulation by LPS induces a selective increase in the excitatory synaptic strength, characterized by enhanced synchronous and asynchronous glutamate release mechanisms. This effect is accompanied by a change in short-term plasticity with decreased facilitation, decreased post-tetanic potentiation, and increased depression. Quantal analysis demonstrated that the effects of LPS on excitatory transmission are attributable to an increase in the probability of release associated with an overall increased expression of L-type voltage-gated Ca2+ channels that, at presynaptic terminals, abnormally contributes to evoked glutamate release. Overall, these changes contribute to the excitatory/inhibitory imbalance that scales up neuronal network activity under inflammatory conditions. These results provide new molecular clues for treating hyperexcitability of hippocampal circuits associated with neuroinflammation in epilepsy and other neurologic disorders. SIGNIFICANCE STATEMENT Neuroinflammation is thought to have a pathogenetic role in epilepsy, a disorder characterized by an imbalance between excitation/inhibition. Fine adjustment of network excitability and regulation of synaptic strength are both implicated in the homeostatic maintenance of physiological levels of neuronal activity. Here, we focused on the effects of chronic neuroinflammation induced by lipopolysaccharides on hippocampal glutamatergic and GABAergic synaptic transmission. Our results show that, on chronic stimulation with lipopolysaccharides, glutamatergic, but not GABAergic, neurons exhibit an enhanced synaptic strength and changes in short-term plasticity because of an increased glutamate release that results from an anomalous contribution of L-type Ca2+ channels to neurotransmitter release.

The repressor-element 1-silencing transcription/neuron-restrictive silencer factor (REST/NRSF) controls hundreds of neuron specific genes. We showed that REST/NRSF downregulates glutamatergic transmission in response to hyperactivity, thus contributing to neuronal homeostasis. However, whether GABAergic transmission is also implicated in the homeostatic action of REST/NRSF is unknown. Here, we show that hyperactivity-induced REST/NRSF activation triggers a homeostatic enhancement of GABAergic inhibition, with increased frequency of miniature inhibitory postsynaptic currents (IPSCs) and amplitude of evoked IPSCs. Notably, this effect was only observed at inhibitory-onto-excitatory neuron synapses, whose density increased at perisomatic sites, demonstrating a strict target-selectivity. These effects were occluded by TrkB receptor inhibition and resulted from a coordinated and sequential activation of the Npas4 and BDNF gene programs. The findings highlight the central role of REST/NRSF in the complex transcriptional responses aimed at preserving physiological levels of neuronal activity in front of the ever-changing environment.Impact StatementThis work elucidates the mechanisms by which the transcriptional regulator REST/NRSF selectively upregulates GABAergic transmission onto excitatory neurons in response to hyperactivity to rescue neuronal homeostasis.

Cultured hippocampal neurons represent the most widely used experimental substrate for in vitro electrophysiological studies. Nevertheless, in most cases, the nature of neuron under study is not identified as excitatory or inhibitory, or even worse, recorded neurons are considered as excitatory because of the paucity of GABAergic interneurons. Thus, the definition of reliable criteria able to guarantee an unequivocal identification of excitatory and inhibitory cultured hippocampal neurons is an unmet need. To reach this goal, we compared the electrophysiological properties and the localization and size of the axon initial segment (AIS) of cultured hippocampal neurons, taking advantage from GAD67-GFP knock-in mice, which expressing green fluorescent protein (GFP) in gamma-aminobutyric acid (GABA)-containing cells, allowed to unambiguously determine the precise nature of the neuron under study. Our results demonstrate that the passive electrophysiological properties, the localization and size of the AIS, and the shape and frequency of the action potential (AP) are not reliable to unequivocally identify neurons as excitatory or inhibitory. The only parameter, related to the shape of the single AP, showing minimal overlap between the sample-point distributions of the two neuronal subpopulations, was the AP half-width. However, the estimation of the AP failure ratio evoked by a short train of high-current steps applied at increasing frequency (40-140 Hz) resulted to be indisputably the safer and faster way to identify the excitatory or inhibitory nature of an unknown neuron. Our findings provide a precise framework for further electrophysiological investigations of in vitro hippocampal neurons.

Paroxysmal dyskinesias are a group of hyperkinetic movement disorders characterized by episodes of dystonia, chorea, athetosis, and ballism that appear isolated or in combination without loss of consciousness. We can differentiate various forms of paroxysmal dyskinesia based on genotypic and phenotypic features: Paroxysmal kinesigenic dyskinesia, paroxysmal non-kinesigenic dyskinesia, and paroxysmal exercise-induced dyskinesia. However, all disorders display common traits, such as the paroxysmal nature and the presence of triggering factors. The episodic nature of these diseases suggests that they result from neuronal network instability and from a general state of hyperexcitability. This augmented excitability is also responsible for the epileptic phenotype that is often associated with paroxysmal dyskinesia.

Mutations in PRoline Rich Transmembrane protein 2 (PRRT2) cause pleiotropic syndromes including benign infantile epilepsy, paroxysmal kinesigenic dyskinesia, episodic ataxia, that share the paroxysmal character of the clinical manifestations. PRRT2 is a neuronal protein that plays multiple roles in the regulation of neuronal development, excitability, and neurotransmitter release. To better understand the physiopathology of these clinical phenotypes, we investigated PRRT2 interactome in mouse brain by a pulldown-based proteomic approach and identified α1 and α3 Na+/K+ ATPase (NKA) pumps as major PRRT2-binding proteins. We confirmed PRRT2 and NKA interaction by biochemical approaches and showed their colocalization at neuronal plasma membrane. The acute or constitutive inactivation of PRRT2 had a functional impact on NKA. While PRRT2-deficiency did not modify NKA expression and surface exposure, it caused an increased clustering of α3-NKA on the plasma membrane. Electrophysiological recordings showed that PRRT2-deficiency in primary neurons impaired NKA function during neuronal stimulation without affecting pump activity under resting conditions. Both phenotypes were fully normalized by re-expression of PRRT2 in PRRT2-deficient neurons. In addition, the NKA-dependent afterhyperpolarization that follows high-frequency firing was also reduced in PRRT2-silenced neurons. Taken together, these results demonstrate that PRRT2 is a physiological modulator of NKA function and suggest that an impaired NKA activity contributes to the hyperexcitability phenotype caused by PRRT2 deficiency.

Summary Loss-of-function mutations in proline-rich transmembrane protein-2 (PRRT2) cause paroxysmal disorders associated with defective Ca2+ dependence of glutamatergic transmission. We find that either acute or constitutive PRRT2 deletion induces a significant decrease in the amplitude of evoked excitatory postsynaptic currents (eEPSCs) that is insensitive to extracellular Ca2+ and associated with a reduced contribution of P/Q-type Ca2+ channels to the EPSC amplitude. This synaptic phenotype parallels a decrease in somatic P/Q-type Ca2+ currents due to a decreased membrane targeting of the channel with unchanged total expression levels. Co-immunoprecipitation, pull-down assays, and proteomics reveal a specific and direct interaction of PRRT2 with P/Q-type Ca2+ channels. At presynaptic terminals lacking PRRT2, P/Q-type Ca2+ channels reduce their clustering at the active zone, with a corresponding decrease in the P/Q-dependent presynaptic Ca2+ signal. The data highlight the central role of PRRT2 in ensuring the physiological Ca2+ sensitivity of the release machinery at glutamatergic synapses., Graphical abstract, Highlights • PRRT2 deficiency reduces the contribution of P/Q-type Ca2+ channels to the EPSC • PRRT2 deficiency decreases P/Q-type currents and membrane targeting of the channels • PRRT2 directly interacts with P/Q-type Ca2+ channels • PRRT2 deficiency reduces P/Q-channel clustering and Ca2+ signals in nerve terminals, PRRT2 deficiency causes paroxysmal disorders associated with defective Ca2+ dependence of glutamatergic transmission. Ferrante et al. now find that, in the absence of PRRT2, the membrane targeting of P/Q-type Ca2+ channels is reduced, and the channels fail to concentrate at the nanodomain where the machinery for synchronous release is assembled.

Transient receptor potential vanilloid 1 (TRPV1) is a polymodal cation channel gated by a large array of chemical and physical stimuli and distributed across different brain regions on neuronal and glial cells. Preclinical studies indicate that TRPV1 might be a target for the treatment of anxiety, depression and addictive disorders. The aim of this narrative review is to focus on studies examining the effects of TRPV1 antagonism on neuroinflammation, neuroprotection and epigenetic regulation. Results suggest that TRPV1 modulation leads to pro- or anti-inflammatory effects depending on the cytokine environment and that the TRPV1 antagonism can switch the microglia towards an anti-inflammatory phenotype. Moreover, TRPV1 inhibitors have neuroprotective properties through the regulation of calcium levels. Finally, TRPV1 antagonism exerts regulatory effects on genes involved in synaptic and cognitive functions through histone deacetylase 2 inhibition. These findings highlight different mechanisms that may underlie the efficacy of TRPV1 antagonists in animal models of severe psychiatric disorders.

TRPV1, a member of the transient receptor potential (TRP) family, is a nonselective calcium permeable ion channel gated by physical and chemical stimuli. In the skin, TRPV1 plays an important role in neurogenic inflammation, pain and pruritus associated to many dermatological diseases. Consequently, TRPV1 modulators could represent pharmacological tools to respond to important patient needs that still represent an unmet medical demand. Previously, we reported the design of capsaicinoid-based molecules that undergo dermal deactivation (soft drugs), thus preventing their long-term dermal accumulation. Here, we investigated the pharmacological properties of the lead antagonist, 2-((4-hydroxy-2-iodo-5-methoxybenzyl) amino)-2-oxoethyl dodecanoate (AG1529), on heterologously expressed human TRPV1 (hTRPV1), on nociceptor excitability and on an in vivo model of acute pruritus. We report that AG1529 competitively blocked capsaicin-evoked activation of hTRPV1 with micromolar potency, moderately affected pH-induced gating, and did not alter voltage- and heat-mediated responses. AG1529 displays modest receptor selectivity as it mildly blocked recombinant hTRPA1 and hTRPM8 channels. In primary cultures of rat dorsal root ganglion (DRG) neurons, AG1529 potently reduced capsaicin-evoked neuronal firing. AG1529 exhibited lower potency on pH-evoked TRPV1 firing, and TRPA1-elicited nociceptor excitability. Furthermore, AG1529 abolished histaminergic and inflammation mediated TRPV1 sensitization in primary cultures of DRG neurons. Noteworthy, dermal wiping of AG1529, either in an acetone-based formulation or in an anhydrous ointment, dose-dependently attenuated acute histaminergic itch in a rodent model. This cutaneous anti-pruritic effect was devoid of the normal nocifensive action evoked by the burning sensation of capsaicin. Taken together, these preclinical results unveil the mode of action of AG1529 on TRPV1 channels and substantiate the tenet that this capsaicinoid-based soft drug is a promising candidate for drug development as a topical anti-pruritic and anti-inflammatory medication.

Loss-of-function mutations in proline-rich transmembrane protein 2 (PRRT2) cause paroxysmal disorders associated with defective Ca2+ dependence of glutamatergic transmission. We found that either acute or constitutive PRRT2 deletion induced a significant decrease in the amplitude of evoked excitatory postsynaptic currents (eEPSCs) that was insensitive to extracellular Ca2+ and associated with a reduced contribution of P/Q-type Ca2+ channels to the EPSC amplitude. This synaptic phenotype was paralleled by a decrease of somatic P/Q-type Ca2+ currents due to a decreased membrane targeting of the channel with unchanged total expression levels. Co-immunoprecipitation assays and proteomic screens revealed an interaction between PRRT2 and P/Q-type Ca2+ channels. At presynaptic terminals lacking PRRT2, P/Q-type Ca2+ channels reduce their clustering at the active zone, with a corresponding decrease of the P/Q-dependent presynaptic Ca2+ signal. The data highlight the central role of PRRT2 in assuring the physiological Ca2+ sensitivity of the release machinery at glutamatergic synapses.

Neuroinflammation is involved in the pathogenesis of several neurologic disorders, including epilepsy. Both changes in the input/output functions of synaptic circuits and cell Ca(2+) dysregulation participate in neuroinflammation, but their impact on neuron function in epilepsy is still poorly understood. Lipopolysaccharide (LPS), a toxic byproduct of bacterial lysis, has been extensively used to stimulate inflammatory responses both in vivo and in vitro. LPS stimulates Toll-like receptor 4, an important mediator of the brain innate immune response that contributes to neuroinflammation processes. Although we report that Toll-like receptor 4 is expressed in both excitatory and inhibitory mouse hippocampal neurons (both sexes), its chronic stimulation by LPS induces a selective increase in the excitatory synaptic strength, characterized by enhanced synchronous and asynchronous glutamate release mechanisms. This effect is accompanied by a change in short-term plasticity with decreased facilitation, decreased post-tetanic potentiation, and increased depression. Quantal analysis demonstrated that the effects of LPS on excitatory transmission are attributable to an increase in the probability of release associated with an overall increased expression of L-type voltage-gated Ca(2+) channels that, at presynaptic terminals, abnormally contributes to evoked glutamate release. Overall, these changes contribute to the excitatory/inhibitory imbalance that scales up neuronal network activity under inflammatory conditions. These results provide new molecular clues for treating hyperexcitability of hippocampal circuits associated with neuroinflammation in epilepsy and other neurologic disorders. SIGNIFICANCE STATEMENT Neuroinflammation is thought to have a pathogenetic role in epilepsy, a disorder characterized by an imbalance between excitation/inhibition. Fine adjustment of network excitability and regulation of synaptic strength are both implicated in the homeostatic maintenance of physiological levels of neuronal activity. Here, we focused on the effects of chronic neuroinflammation induced by lipopolysaccharides on hippocampal glutamatergic and GABAergic synaptic transmission. Our results show that, on chronic stimulation with lipopolysaccharides, glutamatergic, but not GABAergic, neurons exhibit an enhanced synaptic strength and changes in short-term plasticity because of an increased glutamate release that results from an anomalous contribution of L-type Ca(2+) channels to neurotransmitter release.

Mutations in PRoline-Rich Transmembrane protein 2 (PRRT2) underlie a group of paroxysmal disorders including epilepsy, kinesigenic dyskinesia and migraine. Most of the mutations lead to impaired PRRT2 expression and/or function, emphasizing the pathogenic role of the PRRT2 deficiency. In this work, we investigated the phenotype of primary hippocampal neurons obtained from mouse embryos in which the PRRT2 gene was constitutively inactivated. Although PRRT2 is expressed by both excitatory and inhibitory neurons, its deletion decreases the number of excitatory synapses without significantly affecting the number of inhibitory synapses or the nerve terminal ultrastructure. Analysis of synaptic function in primary PRRT2 knockout excitatory neurons by live imaging and electrophysiology showed slowdown of the kinetics of exocytosis, weakened spontaneous and evoked synaptic transmission and markedly increased facilitation. Inhibitory neurons showed strengthening of basal synaptic transmission, accompanied by faster depression. At the network level these complex synaptic effects resulted in a state of heightened spontaneous and evoked activity that was associated with increased excitability of excitatory neurons in both PRRT2 knockout primary cultures and acute hippocampal slices. The data indicate the existence of network instability/hyperexcitability as the possible basis of the paroxysmal phenotypes associated with PRRT2 mutations.

The striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific phosphatase involved in synaptic transmission. The current hypothesis on STEP function holds that it opposes synaptic strengthening by dephosphorylating and inactivating key neuronal proteins involved in synaptic plasticity and intracellular signaling, such as the MAP kinases ERK1/2 and p38, as well as the tyrosine kinase Fyn. Although STEP has a predominant role at the post-synaptic level, it is also expressed in nerve terminals. To better investigate its physiological role at the presynaptic level, we functionally investigated brain synaptosomes and autaptic hippocampal neurons from STEP knockout (KO) mice. Synaptosomes purified from mutant mice were characterized by an increased basal and evoked glutamate release compared with wild-type animals. Under resting conditions, STEP KO synaptosomes displayed increased cytosolic Ca2+ levels accompanied by an enhanced basal activity of Ca2+/calmodulin-dependent protein kinase type II (CaMKII) and hyperphosphorylation of synapsin I at CaMKII sites. Moreover, STEP KO hippocampal neurons exhibit an increase of excitatory synaptic strength attributable to an increased size of the readily releasable pool of synaptic vesicles. These results provide new evidence that STEP plays an important role at nerve terminals in the regulation of Ca2+ homeostasis and neurotransmitter release.

PRoline-Rich Transmembrane protein-2 (PRRT2) is a recently described neuron-specific type-2 integral membrane protein with a large cytosolic N-terminal domain that distributes in presynaptic and axonal domains where it interacts with several presynaptic proteins and voltage-gated Na+ channels. Several PRRT2 mutations are the main cause of a wide and heterogeneous spectrum of paroxysmal disorders with a loss-of-function pathomechanism. The highest expression levels of PRRT2 in brain occurs in cerebellar granule cells (GCs) and cerebellar dysfunctions participate in the dyskinetic phenotype of PRRT2 knockout (KO) mice. We have investigated the effects of PRRT2 deficiency on the intrinsic excitability of GCs and the input-output relationships at the mossy fiber-GC synapses. We show that PRRT2 KO primary GCs display increased expression of Na+ channels, increased amplitude of Na+ currents and increased length of the axon initial segment, leading to an overall enhancement of intrinsic excitability. In acute PRRT2 KO cerebellar slices, GCs were more prone to action potential discharge in response to mossy fiber activation and exhibited an enhancement of transient and persistent Na+ currents, in the absence of changes at the mossy fiber-GC synapses. The results support a key role of PRRT2 expressed in GCs in the physiological regulation of the excitatory input to the cerebellum and are consistent with a major role of a cerebellar dysfunction in the pathogenesis of the PRRT2-linked paroxysmal pathologies.

// Pierluigi Valente 1, * , Pasqualina Farisello 1, 2, * , Flavia Valtorta 3 , Pietro Baldelli 1, 2 and Fabio Benfenati 1, 2 1 Department of Experimental Medicine, Section of Physiology, University of Genoa, 16132 Genova, Italy 2 Center for Synaptic Neuroscience and Technology, Istituto Italiano di Tecnologia, 16132 Genova, Italy 3 S. Raffaele Scientific Institute and Vita-Salute University, 20132 Milano, Italy * These authors have contributed equally to this work Correspondence to: Fabio Benfenati, email: fabio.benfenati@iit.it Keywords: epilepsy, excitatory transmission, GABA receptors, facilitation, synaptic depression Received: June 22, 2017 Accepted: September 03, 2017 Published: September 30, 2017 ABSTRACT Synapsins are a family of synaptic vesicle phosphoproteins regulating synaptic transmission and plasticity. SYN1/2 genes are major epilepsy susceptibility genes in humans. Consistently, synapsin I/II/III triple knockout (TKO) mice are epileptic and exhibit severe impairments in phasic and tonic GABAergic inhibition that precede the appearance of the epileptic phenotype. These changes are associated with an increased strength of excitatory transmission that has never been mechanistically investigated. Here, we observed that an identical effect in excitatory transmission could be induced in wild-type (WT) Schaffer collateral-CA1 pyramidal cell synapses by blockade of GABA B receptors (GABA B Rs). The same treatment was virtually ineffective in TKO slices, suggesting that the increased strength of the excitatory transmission results from an impairment of GABA B presynaptic inhibition. Exogenous stimulation of GABA B Rs in excitatory autaptic neurons, where GABA spillover is negligible, demonstrated that GABA B Rs were effective in inhibiting excitatory transmission in both WT and TKO neurons. These results demonstrate that the decreased GABA release and spillover, previously observed in TKO hippocampal slices, removes the tonic brake of presynaptic GABA B Rs on glutamate transmission, making the excitation/inhibition imbalance stronger.

The expression and function of TRPV1 are influenced by its interaction with cellular proteins. Here, we identify Whirlin, a cytoskeletal PDZ-scaffold protein implicated in hearing, vision and mechanosensory transduction, as an interacting partner of TRPV1. Whirlin associates with TRPV1 in cell lines and in primary cultures of rat nociceptors. Whirlin is expressed in 55% of mouse sensory C-fibers, including peptidergic and non-peptidergic nociceptors, and co-localizes with TRPV1 in 70% of them. Heterologous expression of Whirlin increased TRPV1 protein expression and trafficking to the plasma membrane, and promoted receptor clustering. Silencing Whirlin expression with siRNA or blocking protein translation resulted in a concomitant degradation of TRPV1 that could be prevented by inhibiting the proteasome. The degradation kinetics of TRPV1 upon arresting protein translation mirrored that of Whirlin in cells co-expressing both proteins, suggesting a parallel degradation mechanism. Noteworthy, Whirlin expression significantly reduced TRPV1 degradation induced by prolonged exposure to capsaicin. Thus, our findings indicate that Whirlin and TRPV1 are associated in a subset of nociceptors and that TRPV1 protein stability is increased through the interaction with the cytoskeletal scaffold protein. Our results suggest that the Whirlin-TRPV1 complex may represent a novel molecular target and its pharmacological disruption might be a therapeutic strategy for the treatment of peripheral TRPV1-mediated disorders. (C) 2015 Elsevier B.V. All rights reserved.

Transient Receptor Potential Vanilloid 1 (TRPV1) channels are non-selective cationic polymodal receptors gated by several different chemical and physical stimuli. TRPV1 receptors are distributed in several brain areas and interact with important neurotransmitter systems linked to mental disorders, such as endocannabinoid and opioid systems. The increasing number of results obtained in this field has recently attracted growing attention to these receptors as potential targets for the treatment of different psychiatric conditions. To review the available results on this topic, we searched on PubMed, Embase and Science Direct databases up to May 2020 using the following search string: "TRPV1", thus including a total of 48 studies. The results, still limited to preclinical studies, suggest that TRPV1 antagonism could represent a potential mechanism for the treatment of depression and anxiety, as well as for opioids, methamphetamine and cocaine addiction. Few available results consider schizophrenia-like behaviours, suggesting an intriguing role of TRPV1 receptors in the neurobiology of major psychoses. Single studies report the effectiveness of TRPV1 antagonists in animal models of obsessive-compulsive disorder and fibromyalgia. Future preclinical and clinical studies are required to shed further light on the feasibility of the use of TRPV1 modulators in psychopharmacology.

See Lerche (doi:10.1093/brain/awy073) for a scientific commentary on this article.Proline-rich transmembrane protein 2 (PRRT2) is the causative gene for a heterogeneous group of familial paroxysmal neurological disorders that include seizures with onset in the first year of life (benign familial infantile seizures), paroxysmal kinesigenic dyskinesia or a combination of both. Most of the PRRT2 mutations are loss-of-function leading to haploinsufficiency and 80% of the patients carry the same frameshift mutation (c.649dupC; p.Arg217Profs*8), which leads to a premature stop codon. To model the disease and dissect the physiological role of PRRT2, we studied the phenotype of neurons differentiated from induced pluripotent stem cells from previously described heterozygous and homozygous siblings carrying the c.649dupC mutation. Single-cell patch-clamp experiments on induced pluripotent stem cell-derived neurons from homozygous patients showed increased Na+ currents that were fully rescued by expression of wild-type PRRT2. Closely similar electrophysiological features were observed in primary neurons obtained from the recently characterized PRRT2 knockout mouse. This phenotype was associated with an increased length of the axon initial segment and with markedly augmented spontaneous and evoked firing and bursting activities evaluated, at the network level, by multi-electrode array electrophysiology. Using HEK-293 cells stably expressing Nav channel subtypes, we demonstrated that the expression of PRRT2 decreases the membrane exposure and Na+ current of Nav1.2/Nav1.6, but not Nav1.1, channels. Moreover, PRRT2 directly interacted with Nav1.2/Nav1.6 channels and induced a negative shift in the voltage-dependence of inactivation and a slow-down in the recovery from inactivation. In addition, by co-immunoprecipitation assays, we showed that the PRRT2-Nav interaction also occurs in brain tissue. The study demonstrates that the lack of PRRT2 leads to a hyperactivity of voltage-dependent Na+ channels in homozygous PRRT2 knockout human and mouse neurons and that, in addition to the reported synaptic functions, PRRT2 is an important negative modulator of Nav1.2 and Nav1.6 channels. Given the predominant paroxysmal character of PRRT2-linked diseases, the disturbance in cellular excitability by lack of negative modulation of Na+ channels appears as the key pathogenetic mechanism.

The functional consequence of γ-aminobutyric acid (GABA) release at mossy fiber terminals is still a debated topic. Here, we provide multiple evidence of GABA release in cultured autaptic hippocampal granule cells. In ∼50% of the excitatory autaptic neurons, GABA, VGAT, or GAD67 colocalized with vesicular glutamate transporter 1-positive puncta, where both GABAB and GABAA receptors (Rs) were present. Patch-clamp recordings showed a clear enhancement of autaptic excitatory postsynaptic currents in response to the application of the GABABR antagonist CGP58845 only in neurons positive to the selective granule cell marker Prox1, and expressing low levels of GAD67. Indeed, GCP non-responsive excitatory autaptic neurons were both Prox1- and GAD67-negative. Although the amount of released GABA was not sufficient to activate functional postsynaptic GABAARs, it effectively activated presynaptic GABABRs that maintain a tonic "brake" on the probability of release and on the size of the readily releasable pool and contributed to resting potential hyperpolarization possibly through extrasynaptic GABAAR activation. The autocrine inhibition exerted by GABABRs on glutamate release enhanced both paired-pulse facilitation and post-tetanic potentiation. Such GABABR-mediated changes in short-term plasticity confer to immature granule cells the capability to modulate their filtering properties in an activity-dependent fashion, with remarkable consequences on the dynamic behavior of neural circuits.

Synapsins are a family of synaptic vesicle phosphoproteins regulating synaptic transmission and plasticity. SYN1/2 genes are major epilepsy susceptibility genes in humans. Consistently, synapsin I/II/III triple knockout (TKO) mice are epileptic and exhibit severe impairments in phasic and tonic GABAergic inhibition that precede the appearance of the epileptic phenotype. These changes are associated with an increased strength of excitatory transmission that has never been mechanistically investigated. Here, we observed that an identical effect in excitatory transmission could be induced in wild-type (WT) Schaffer collateral-CA1 pyramidal cell synapses by blockade of GABAB receptors (GABABRs). The same treatment was virtually ineffective in TKO slices, suggesting that the increased strength of the excitatory transmission results from an impairment of GABAB presynaptic inhibition. Exogenous stimulation of GABABRs in excitatory autaptic neurons, where GABA spillover is negligible, demonstrated that GABABRs were effective in inhibiting excitatory transmission in both WT and TKO neurons. These results demonstrate that the decreased GABA release and spillover, previously observed in TKO hippocampal slices, removes the tonic brake of presynaptic GABABRs on glutamate transmission, making the excitation/inhibition imbalance stronger.

Homeostatic plasticity is a regulatory feedback response in which either synaptic strength or intrinsic excitability can be adjusted up or down to offset sustained changes in neuronal activity. Although a growing number of evidences constantly provide new insights into these two apparently distinct homeostatic processes, a unified molecular model remains unknown. We recently demonstrated that REST is a transcriptional repressor critical for the downscaling of intrinsic excitability in cultured hippocampal neurons subjected to prolonged elevation of electrical activity. Here, we report that, in the same experimental system, REST also participates in synaptic homeostasis by reducing the strength of excitatory synapses by specifically acting at the presynaptic level. Indeed, chronic hyperactivity triggers a REST-dependent decrease of the size of synaptic vesicle pools through the transcriptional and translational repression of specific presynaptic REST target genes. Together with our previous report, the data identify REST as a fundamental molecular player for neuronal homeostasis able to downscale simultaneously both intrinsic excitability and presynaptic efficiency in response to elevated neuronal activity. This experimental evidence adds new insights to the complex activity-dependent transcriptional regulation of the homeostatic plasticity processes mediated by REST.

The thermosensory transient receptor potential vanilloid 1 channel (TRPV1) is a polymodal receptor activated by physical and chemical stimuli. TRPV1 activity is drastically potentiated by proinflammatory agents released upon tissue damage. Given the pivotal role of TRPV1 in human pain, there is pressing need for improved TRPV1 antagonists, the development of which will require identification of new pharmacophore scaffolds. Uncompetitive antagonists acting as open-channel blockers might serve as activity-dependent blockers that preferentially modulate the activity of overactive channels, thus displaying fewer side effects than their competitive counterparts. Herein we report the design, synthesis, biological evaluation, and SAR analysis of a family of triazine-based compounds acting as TRPV1 uncompetitive antagonists. We identified the triazine 8aA as a potent, pure antagonist that inhibits TRPV1 channel activity with nanomolar efficacy and strong voltage dependency. It represents a new class of activity-dependent TRPV1 antagonists and may serve as the basis for lead optimization in the development of new analgesics., This work was supported by grants from Spanish Ministry of Science and Innovation (Grant SAF2008-00048 to A.M., Grant BFU2009-08346 to A.F.-M., CONSOLIDER-INGENIO 2010 (Grant CSD2008-00005) to A.F.-M., J.M.G.-R., and A.M.), from Fundació La Marató de TV3 (to A.F.-M. and A.M.), and from PROMETEO/2010/046 from the GVA to A.F.-M.

L1 is a trans-membrane glycoprotein subserving neuron-neuron adhesion via homophilic and heterophilic interactions. Although experimental evidences have implicated L1 in axonal outgrowth, fasciculation and pathfinding, its contribution to voltage-gated sodium channels (NaChs) function and membrane excitability has remained unknown. Here, we show that firing rate, single cell spiking frequency and Na+ current density are all reduced in hippocampal excitatory neurons from L1-deficient mice both in culture and in slices, due to an overall reduced membrane expression of NaChs. Remarkably, normal firing activity was restored when L1 was reintroduced into L1-deficient excitatory neurons, indicating that abnormal firing patterns are not related to developmental abnormalities, but are a direct consequence of L1 deletion. Moreover, L1-deficiency leads to impairment of action potential (AP) initiation, most likely due to the loss of the interaction of L1 with Ankyrin G that produces the delocalization of NaChs at the at the axonal initial segment. We conclude that L1 contributes to functional expression and localization of NaChs to the neuronal plasma membrane, ensuring correct initiation of AP and normal firing activity.

Voltage-gated ion channels are modular proteins designed by the structural linkage of a voltage sensor and a pore domain. The functional coupling of these two protein modules is a subject of intense research. A major focus has been directed to decipher the role of the S4-S5 linker and the C-end of the inner pore helix in channel gating. However, the contribution of the cytosolic N terminus of S5 remains elusive. To address this issue, we used a chimeric subunit that linked the voltage sensor of the Shaker channel to the prokaryotic KcsA pore domain (denoted as Shaker-KcsA). This chimera preserved the Shaker sequences at both the N terminus of S5 and the C-end of S6. Chimeric Shaker-KcsA subunits did not form functional homomeric channels but were synthesized, folded, and trafficked to the cell surface, as evidenced by their co-assembly with Shaker wild type subunits. Sequential substitution of Shaker amino acids at the C-end of S6 and the N terminus of S5 by the corresponding KcsA created voltage-sensitive channels with voltage-dependent properties that asymptotically approached those of the wild type Shaker channel. Noteworthy, substitution of the region encompassing Phe(401)-Phe(404) at the N-end of Shaker S5 by KcsA residues resulted in a significant gain in voltage sensitivity of the chimeras. Furthermore, analysis of channel function at high [K(+)](o) revealed that the Phe(401)-Phe(404) region is an important molecular determinant for competent coupling of voltage sensing and pore opening. Taken together, these findings indicate that complete replacement of Shaker S5 and S6 by KcsA M1 and M2 is required for voltage-dependent gating of the prokaryotic channel. In addition, our results imply that the region encompassing Phe(401)-Phe(404) in Shaker is involved in protein-protein interactions with the voltage sensor, and signal to the Phe(401) in the S5 segment as a key molecular determinant to pair the voltage sensor and the pore domain.

Incubation of ECV304 cells with 1 micro M staurosporine (STS) causes apoptotic cell death. In the present study, we investigate whether a significant apoptotic volume decrease (AVD) was apparent during the very early times (1 h) of the apoptotic process. Our data suggest that upregulation of Cl(-) (and possibly K(+)) channels by STS may be a very early primary event required for the subsequent onset of AVD, which results in apoptosis.

Residues I696 and W697 are crucial to coupling between the TRPV1 ligand- and voltage-sensing domains and the channel pore., The transient receptor potential vanilloid receptor subtype I (TRPV1) channel acts as a polymodal sensory receptor gated by chemical and physical stimuli. Like other TRP channels, TRPV1 contains in its C terminus a short, conserved domain called the TRP box, which is necessary for channel gating. Substitution of two TRP box residues—I696 and W697—with Ala markedly affects TRPV1’s response to all activating stimuli, which indicates that these two residues play a crucial role in channel gating. We systematically replaced I696 and W697 with 18 native l-amino acids (excluding cysteine) and evaluated the effect on voltage- and capsaicin-dependent gating. Mutation of I696 decreased channel activation by either voltage or capsaicin; furthermore, gating was only observed with substitution of hydrophobic amino acids. Substitution of W697 with any of the 18 amino acids abolished gating in response to depolarization alone, shifting the threshold to unreachable voltages, but not capsaicin-mediated gating. Moreover, vanilloid-activated responses of W697X mutants showed voltage-dependent gating along with a strong voltage-independent component. Analysis of the data using an allosteric model of activation indicates that mutation of I696 and W697 primarily affects the allosteric coupling constants of the ligand and voltage sensors to the channel pore. Together, our findings substantiate the notion that inter- and/or intrasubunit interactions at the level of the TRP box are critical for efficient coupling of stimulus sensing and gate opening. Perturbation of these interactions markedly reduces the efficacy and potency of the activating stimuli. Furthermore, our results identify these interactions as potential sites for pharmacological intervention.

The capsaicin receptor TRPV1 (Transient Receptor Potential, Vanilloid family member 1), the founding member of the heat-sensitive TRP ("thermo-TRP") channel family, plays a pivotal role in pain transduction. There is mounting evidence that TRPV1 regulation is complex and is manifest at many levels, from gene expression through post-translational modification and formation of receptor heteromers to subcellular compartmentalization and association with regulatory proteins. These mechanisms are believed to be involved both in disease-related changes in TRPV1 expression, and the long-lasting refractory state, referred to as "desensitization", that follows TRPV1 agonist treatment. The signaling cascades that regulate TRPV1 and related thermo-TRP channels are only beginning to be understood. Here we review our current knowledge in this rapidly changing field. We propose that the complex regulation of TRPV1 may be exploited for therapeutic purposes, with the ultimate goal being the development of novel, innovative agents that target TRPV1 in diseased, but not healthy, tissues. Such compounds are expected to be devoid of the side-effects (e.g. hyperthermia and impaired noxious heat sensation) that plague the clinical use of existing TRPV1 antagonists.

Ana Gomis, [et al.]. 13 p., 7 figures, 1 table and references, The transient receptor potential vanilloid 1 (TRPV1) channel is a thermosensory receptor implicated in diverse physiological and pathological processes. The TRP domain, a highly conserved region in the C terminus adjacent to the internal channel gate, is critical for subunit tetramerization and channel gating. Here, we show that cell-penetrating, membrane-anchored peptides patterned after this protein domain are moderate and selective TRPV1 antagonists both in vitro and in vivo, blocking receptor activity in intact rat primary sensory neurons and their peripheral axons with mean decline time of 30 min. The most potent lipopeptide, TRP-p5, blocked all modes of TRPV1 gating with micromolar efficacy (IC 50100 μM). TRP-p5 did not affect the capsaicin sensitivity of the vanilloid receptor. Our data suggest that TRP-p5 interferes with protein-protein interactions at the level of the TRP domain that are essential for the "conformational" change that leads to gate opening. Therefore, these palmitoylated peptides, which we termed TRPducins, are noncompetitive, voltage-independent, sequence-specific TRPV1 blockers. Our findings indicate that TRPducin-like peptides may embody a novel molecular strategy that can be exploited to generate a selective pharmacological arsenal for the TRP superfamily of ion channels., This work was supported by Spain Ministry of Science and Innovation grants BFU2009-08346 to A.F.-M., BFU2008-0062 to J.M.G.R., SAF2007-63193 to R.P.-C., BFU2009- 07835 to A.G., BFU2007-61855 to F.V., and BFU2008-04425 to C.B.; Consolider-Ingenio 2010 CSD2008-00005 to A.F.-M., J.M.G.-R., and R.P.-C., and CSD2007-00002 to A.G., F.V., and C.B.; la Generalitat Valenciana Prometeo/2010/046 to A.F.-M., F.V., and C.B.; and Intramural (Consejo Superior de Investigaciones Cientificas) Ref. 2009-20I098 to A.G.

Item does not contain fulltext Transient receptor potential vanilloid (TRPV1) transduces noxious chemical and physical stimuli in high-threshold nociceptors. The pivotal role of TRPV1 in the physiopathology of pain transduction has thrust the identification and characterization of interacting partners that modulate its cellular function. Here, we report that TRPV1 associates with gamma-amino butyric acid A-type (GABA(A)) receptor associated protein (GABARAP) in HEK293 cells and in neurons from dorsal root ganglia coexpressing both proteins. At variance with controls, GABARAP augmented TRPV1 expression in cotransfected cells and stimulated surface receptor clustering. Functionally, GABARAP expression attenuated voltage and capsaicin sensitivity of TRPV1 in the presence of extracellular calcium. Furthermore, the presence of the anchor protein GABARAP notably lengthened the kinetics of vanilloid-induced tachyphylaxia. Notably, the presence of GABARAP selectively increased the interaction of tubulin with the C-terminal domain of TRPV1. Disruption of tubulin cytoskeleton with nocodazole reduced capsaicin-evoked currents in cells expressing TRPV1 and GABARAP, without affecting the kinetics of vanilloid-induced desensitization. Taken together, these findings indicate that GABARAP is an important component of the TRPV1 signaling complex that contributes to increase the channel expression, to traffic and cluster it on the plasma membrane, and to modulate its functional activity at the level of channel gating and desensitization. 01 juni 2010

Transient receptor potential vanilloid receptor subtype 1 (TRPV1) is an ionotropic receptor activated by temperature and chemical stimuli. The C-terminal region that is adjacent to the channel gate, recognized as the TRP domain, is a molecular determinant of receptor assembly. However, the role of this intracellular domain in channel function remains elusive. Here, we show that replacement of the TRP domain of TRPV1 with the cognate region of TRPV channels (TRPV2–TRPV6) did not affect receptor assembly and trafficking to the cell surface, although those receptors containing the TRP domain of the distantly related TRPV5 and TRPV6 did not display ion channel activity. Notably, functional chimeras exhibited an impaired sensitivity to the activating stimuli, consistent with a significant contribution of this protein domain to channel function. At variance with TRPV1, voltage-dependent gating of chimeric channels could not be detected in the absence of capsaicin and/or heat. Biophysical analysis of functional chimeras revealed that the TRP domain appears to act as a molecular determinant of the activation energy of channel gating. Together, these findings uncover a role of the TRP domain in intersubunit interactions near the channel gate that contribute to the coupling of stimulus sensing to channel opening., This work was supported by Spanish Ministry of Education and Science Grants SAF2003-0509 and SAF2006-2580 (A.F.-M.) and BFU2005-03986 (A.G.), the Fundación Ramón Areces (A.F.-M.), and Generalitat Valenciana Grants GV-ACOMP06/202 (A.F.-M.) and GV05/076 (A.G.).

A pathophysiological increase in free arachidonic acid (AA) is thought to regulate the channel-mediated astrocytic swelling occurring in several brain injuries. We report that in cultured rat type-1 cortical astrocytes, exposure to 10 microM AA activates an open rectifier K(+) channel, which exhibits many similarities with TREK/TRAAK members of the two-pore-domain K(+) channel family KCNK. Patch-clamp experiments showed that the current developed with a long latency and was preceded by a depression of the previously described outward rectifier K(+) conductance. Pharmacologic studies indicate that the K(+) open rectifier was differentially sensitive to classic K(+)-channel blockers (quinine, quinidine, tetraethylammonium, and barium) and was inhibited potently by gadolinium ions. The activation of this K(+) current occurred independently of the AA metabolism as pharmacologic inhibition of the lipoxygenase, cyclooxygenase, and cytochrome P450 epoxygenase signaling cascades did not alter the AA effect. Moreover, neither the neutralization of the NADPH-oxidase pathway nor scavenging intracellular free radicals modified the AA response. Finally, the AA-induced K(+) current was unaffected by protein kinase C inhibitors. The activation mechanism of the K(+) open rectifier was through an extracellular interaction of AA with the plasma membrane. RT-PCR analysis revealed that the AA-induced K(+) conductance was mediated likely by TREK-2 channels. Collectively, the results demonstrate that in cultured cortical astrocytes, pathological levels of AA directly activate an open rectifier K(+) channel, which may play a role in the control of K(+) homeostasis under pathophysiological conditions.

IFSCC Magazine, 11 (2008) (4) 311–315 This paper was presented as a keynote lecture at the IFSCC Congress 2008, Barcelona, Spain. Cutaneous neurogenic inflammation is emerging as an underlying mechanism for several skin conditions. The intimate cross-talk between the cutaneous immune system and the peripheral nervous system is fundamental for skin biology. However, an imbalance or dysfunction results in the onset of an inflammatory state that is reinforced by the synergic and complementary action of both systems. Cumulative evidence indicates that the thermoreceptor TRPV1 is a key player of neurogenic inflammation. This receptor is activated by both physical and chemical stimuli, and its activity is potentiated by pro-inflammatory mediators. An increase in TRPV1 activity results in an increment of neuronal excitability that leads to the release of proalgesic agents that stimulate the immune system. Therefore, the TRPV1 receptor is being considered as a cosmeceutical target, and agents that reduce its activity will be useful cosmeceuticals. Keywords: Algogens, epidermis, immune system, nociceptor, sensitization

A feasibility study on superconducting toroids for the Empact Detector of SSC has been carried out by Ansaldo (1) under contract with Grumman. In particular problems connected with the cable stability and quench evolution compared with the radiation length thickness are investigated. Industrial analysis of superconducting coils, cable design, cryogenic cooling system, manufacturing and assembly problems is carried out.

Coffee, one of the most excessively used beverages worldwide, commences the risk of gastroesophageal reflux (GER), which may lead to gastric ulcers and increase the risk of gastric cancer. Many attempts have been made by the coffee industry to diminish the irritating effect on mucosa by means of altering the extraction methods concerning gerbic acids and the roasting processes. This paper describes the effect of differently produced coffees involving two brands of Darhoven and two brands of other coffee roasters. The aim of this study was to prove the results of gastric potential measurements we found in literature by using human AGS gastric epithelial cells (human adenocarcinoma). All four coffee extracts tested differentially affected the membrane resting potential of AGS cells Coffees no. I and no. 2 depolarized the cells, presumably by increasing the cation entry into the cytosol. In marked contrast, coffee no. 4 hyperpolarizes the cells, possibly by H + extrusion and/or Cl - influx, suggesting that this coffee might increase acidity in the stomach, which might negatively affect the stomach, especially in people with gastroesophageal reflux symptoms. Overall, our data suggest that different roasting methods of coffees affect the membrane potentials of AGS stomach cells, resulting in increased influx of H + possibly resulting in decreased stomach acidity and thus reducing GER. These results are in good accordance with clinical pharmacological results from potential difference measurements in healthy volunteers we found in the literature.