Stretch-activated ion channel TMEM63B associates with developmental and epileptic encephalopathies and progressive neurodegeneration.

By converting physical forces into electrical signals or triggering intracellular cascades, stretch-activated ion channels allow the cell to respond to osmotic and mechanical stress. Knowledge of the pathophysiological mechanisms underlying associations of stretch-activated ion channels with human disease is limited. Here, we describe 17 unrelated individuals with severe early-onset developmental and epileptic encephalopathy (DEE), intellectual disability, and severe motor and cortical visual impairment associated with progressive neurodegenerative brain changes carrying ten distinct heterozygous variants of TMEM63B, encoding for a highly conserved stretch-activated ion channel. The variants occurred de novo in 16/17 individuals for whom parental DNA was available and either missense, including the recurrent p.Val44Met in 7/17 individuals, or in-frame, all affecting conserved residues located in transmembrane regions of the protein. In 12 individuals, hematological abnormalities co-occurred, such as macrocytosis and hemolysis, requiring blood transfusions in some. We modeled six variants (p.Val44Met, p.Arg433His, p.Thr481Asn, p.Gly580Ser, p.Arg660Thr, and p.Phe697Leu), each affecting a distinct transmembrane domain of the channel, in transfected Neuro2a cells and demonstrated inward leak cation currents across the mutated channel even in isotonic conditions, while the response to hypo-osmotic challenge was impaired, as were the Ca2+ transients generated under hypo-osmotic stimulation. Ectopic expression of the p.Val44Met and p.Gly580Cys variants in Drosophila resulted in early death. TMEM63B-associated DEE represents a recognizable clinicopathological entity in which altered cation conductivity results in a severe neurological phenotype with progressive brain damage and early-onset epilepsy associated with hematological abnormalities in most individuals.

A2A Adenosine Receptor Antagonists: Are Triazolotriazine and Purine Scaffolds Interchangeable?

The A2A adenosine receptor (A2AAR) is one of the four subtypes activated by nucleoside adenosine, and the molecules able to selectively counteract its action are attractive tools for neurodegenerative disorders. In order to find novel A2AAR ligands, two series of compounds based on purine and triazolotriazine scaffolds were synthesized and tested at ARs. Compound 13 was also tested in an in vitro model of neuroinflammation. Some compounds were found to possess high affinity for A2AAR, and it was observed that compound 13 exerted anti-inflammatory properties in microglial cells. Molecular modeling studies results were in good agreement with the binding affinity data and underlined that triazolotriazine and purine scaffolds are interchangeable only when 5- and 2-positions of the triazolotriazine moiety (corresponding to the purine 2- and 8-positions) are substituted.

Ethanol neurotoxicity is mediated by changes in expression, surface localization and functional properties of glutamate AMPA receptors.

Modifications in the subunit composition of AMPA receptors (AMPARs) have been linked to the transition from physiological to pathological conditions in a number of contexts, including EtOH-induced neurotoxicity. Previous work from our laboratory showed that EtOH withdrawal causes CA1 pyramidal cell death in organotypic hippocampal slices and changes in the expression of AMPARs. Here, we investigated whether changes in expression and function of AMPARs may be causal for EtOH-induced neurotoxicity. To this aim, we examined the subunit composition, localization and function of AMPARs in hippocampal slices exposed to EtOH by using western blotting, surface expression assay, confocal microscopy and electrophysiology. We found that EtOH withdrawal specifically increases GluA1 protein signal in total homogenates, but not in the post-synaptic density-enriched fraction. This is suggestive of overall increase and redistribution of AMPARs to the extrasynaptic compartment. At functional level, AMPA-induced calcium influx was unexpectedly reduced, whereas AMPA-induced current was enhanced in CA1 pyramidal neurons following EtOH withdrawal, suggesting that increased AMPAR expression may lead to cell death because of elevated excitability, and not for a direct contribution on calcium influx. Finally, the neurotoxicity caused by EtOH withdrawal was attenuated by the non-selective AMPAR antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt as well as by the selective antagonist of GluA2-lacking AMPARs 1-naphthyl acetyl spermine. We conclude that EtOH neurotoxicity involves changes in expression, surface localization and functional properties of AMPARs, and propose GluA2-lacking AMPARs as amenable specific targets for the development of neuroprotective drugs in EtOH-withdrawal syndrome.

Cardiac and neuronal HCN channelopathies.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed as four different isoforms (HCN1-4) in the heart and in the central and peripheral nervous systems. In the voltage range of activation, HCN channels carry an inward current mediated by Na+ and K+, termed If in the heart and Ih in neurons. Altered function of HCN channels, mainly HCN4, is associated with sinus node dysfunction and other arrhythmias such as atrial fibrillation, ventricular tachycardia, and atrioventricular block. In recent years, several data have also shown that dysfunctional HCN channels, in particular HCN1, but also HCN2 and HCN4, can play a pathogenic role in epilepsy; these include experimental data from animal models, and data collected over genetic mutations of the channels identified and characterized in epileptic patients. In the central nervous system, alteration of the Ih current could predispose to the development of neurodegenerative diseases such as Parkinson's disease; since HCN channels are widely expressed in the peripheral nervous system, their dysfunctional behavior could also be associated with the pathogenesis of neuropathic pain. Given the fundamental role played by the HCN channels in the regulation of the discharge activity of cardiac and neuronal cells, the modulation of their function for therapeutic purposes is under study since it could be useful in various pathological conditions. Here we review the present knowledge of the HCN-related channelopathies in cardiac and neurological diseases, including clinical, genetic, therapeutic, and physiopathological aspects.

The Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels: from Biophysics to Pharmacology of a Unique Family of Ion Channels.

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels are important members of the voltage-gated pore loop channels family. They show unique features: they open at hyperpolarizing potential, carry a mixed Na/K current, and are regulated by cyclic nucleotides. Four different isoforms have been cloned (HCN1-4) that can assemble to form homo- or heterotetramers, characterized by different biophysical properties. These proteins are widely distributed throughout the body and involved in different physiologic processes, the most important being the generation of spontaneous electrical activity in the heart and the regulation of synaptic transmission in the brain. Their role in heart rate, neuronal pacemaking, dendritic integration, learning and memory, and visual and pain perceptions has been extensively studied; these channels have been found also in some peripheral tissues, where their functions still need to be fully elucidated. Genetic defects and altered expression of HCN channels are linked to several pathologies, which makes these proteins attractive targets for translational research; at the moment only one drug (ivabradine), which specifically blocks the hyperpolarization-activated current, is clinically available. This review discusses current knowledge about HCN channels, starting from their biophysical properties, origin, and developmental features, to (patho)physiologic role in different tissues and pharmacological modulation, ending with their present and future relevance as drug targets.

NLRP3 inflammasome as a target of berberine in experimental murine liver injury: interference with P2X7 signalling.

Berberine (BRB) is commonly used in herbal medicine, but its mechanisms of action are poorly understood. In the present study, we tested BRB in steatohepatitis induced by a methionine- and choline-deficient (MCD) diet, in acute acetaminophen intoxication and in cultured murine macrophages. BRB markedly improved parameters of liver injury and necroinflammation induced by the MCD diet, although increased mortality was observed by mechanisms independent of bacterial infections or plasma levels of BRB. The MCD diet induced up-regulation of all components of the NLRP3 (NACHT, LRR and PYD domain-containing protein 3) inflammasome, and increased hepatic levels of mature IL-1ß (interleukin 1ß). All of these parameters were significantly reduced in mice treated with BRB. In mice administered an acetaminophen overdose, a model dependent on inflammasome activation, BRB reduced mortality and ALT (alanine aminotransferase) elevation, and limited the expression of inflammasome components. In vitro, LPS (lipopolysaccharide)-induced activation of NLRP3 inflammasome in RAW264.7 murine macrophages was markedly decreased by pre-incubation with BRB. BRB significantly limited the activation of the purinergic receptor P2X7, involved in the late phases of inflammasome activation. Upon P2X7 knockdown, the ability of BRB to block LPS-induced secretion of IL-1ß was lost. These data indicate that administration of BRB ameliorates inflammation and injury in two unrelated murine models of liver damage. We demonstrate for the first time that BRB interferes with activation of the NLRP3 inflammasome pathway in vivo and in vitro, through a mechanism based on interference with activation of P2X7, a purinergic receptor involved in inflammasome activation.

Differential contribution of Ih to the integration of excitatory synaptic inputs in substantia nigra pars compacta and ventral tegmental area dopaminergic neurons.

The selective vulnerability of substantia nigra pars compacta (SNc) dopaminergic (DA) neurons is an enigmatic trait of Parkinson's disease (PD), especially if compared to the remarkable resistance of closely related DA neurons in the neighboring ventral tegmental area (VTA). Overall evidence indicates that specific electrophysiological, metabolic and molecular factors underlie SNc vulnerability, although many pieces of the puzzle are still missing. In this respect, we recently demonstrated that 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of the parkinsonizing toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), alters the electrophysiological properties of SNc DA neurons in vitro by inhibiting the hyperpolarization-activated current (Ih). Here, we present an electrophysiological investigation of the functional role of Ih in the integration of synaptic inputs in identified SNc and VTA DA neurons, comparatively, in acute midbrain slices from TH-GFP mice. We show that pharmacological suppression of Ih increases the amplitude and decay time of excitatory postsynaptic potentials, leading to temporal summation of multiple excitatory potentials at somatic level. Importantly, these effects are quantitatively more evident in SNc DA neurons. We conclude that Ih regulates the responsiveness to excitatory synaptic transmission in SNc and VTA DA neurons differentially. Finally, we present the hypothesis that Ih loss of function may be linked to PD trigger mechanisms, such as mitochondrial failure and ATP depletion, and act in concert with SNc-specific synaptic connectivity to promote selective vulnerability.

Pharmacological blockage of NOP receptors decreases ventral tegmental area dopamine neuronal activity through GABAB receptor-mediated mechanism.

The Nociceptin/Orphanin FQ (N/OFQ) peptide and its receptor NOP are highly expressed within several regions of the mesolimbic system, including the ventral tegmental area (VTA). Evidence indicates that the N/OFQ-NOP receptor system is involved in reward processing and historically it has been proposed that activation of NOP receptors attenuates the motivation for substances of abuse. However, recent findings demonstrated that drug self-administration and relapse to drug-seeking are also attenuated after administration of NOP receptor antagonists. Here, to shed light on the mechanisms through which NOP receptor blockers modulate these processes, we utilized ex vivo patch-clamp recordings to investigate the effect of the selective NOP receptor antagonist LY2817412 on VTA dopaminergic (DA) function in male rats. Results showed that, similar to the endogenous NOP receptor agonist N/OFQ, LY2817412 reduced the spontaneous basal firing discharge of VTA DA neurons. Consistently, we found that NOP receptors are expressed both in VTA DA and GABA cells and that LY2817412 slice perfusion increased GABA release onto VTA DA cells. Finally, in the attempt to dissect the role of postsynaptic and presynaptic NOP receptors, we tested the effect of N/OFQ and LY2817412 in the presence of GABA receptors blockers. Results showed that the effect of LY2817412 was abolished following pretreatment with GABABR, but not GABAAR, blockers. Conversely, inhibition of DA neuronal activity by N/OFQ was unaffected by blockade of GABA receptors. Altogether, these results suggest that both NOP receptor agonists and antagonists can decrease VTA DA neuronal activity, but through distinct mechanisms of action. The effect of NOP receptor antagonists occurs through a GABABR-mediated mechanism while NOP receptor agonists seem to act via a direct effect on VTA DA neurons.

Chemogenetic activation or inhibition of histaminergic neurons bidirectionally modulates recognition memory formation and retrieval in male and female mice.

Several lines of evidence demonstrate that the brain histaminergic system is fundamental for cognitive processes and the expression of memories. Here, we investigated the effect of acute silencing or activation of histaminergic neurons in the hypothalamic tuberomamillary nucleus (TMNHA neurons) in vivo in both sexes in an attempt to provide direct and causal evidence of the necessary role of these neurons in recognition memory formation and retrieval. To this end, we compared the performance of mice in two non-aversive and non-rewarded memory tests, the social and object recognition memory tasks, which are known to recruit different brain circuitries. To directly establish the impact of inactivation or activation of TMNHA neurons, we examined the effect of specific chemogenetic manipulations during the formation (acquisition/consolidation) or retrieval of recognition memories. We consistently found that acute chemogenetic silencing of TMNHA neurons disrupts the formation or retrieval of both social and object recognition memory in males and females. Conversely, acute chemogenetic activation of TMNHA neurons during training or retrieval extended social memory in both sexes and object memory in a sex-specific fashion. These results suggest that the formation or retrieval of recognition memory requires the tonic activity of histaminergic neurons and strengthen the concept that boosting the brain histaminergic system can promote the retrieval of apparently lost memories.

Ethanol-induced AMPA alterations are mediated by mGLU5 receptors through miRNA upregulation in hippocampal slices.

Prenatal alcohol exposure (PAE) affects neuronal networks and brain development causing a range of physical, cognitive and behavioural disorders in newborns that persist into adulthood. The array of consequences associated with PAE can be grouped under the umbrella-term 'fetal alcohol spectrum disorders' (FASD). Unfortunately, there is no cure for FASD as the molecular mechanisms underlying this pathology are still unknown. We have recently demonstrated that chronic EtOH exposure, followed by withdrawal, induces a significant decrease in AMPA receptor (AMPAR) expression and function in developing hippocampus in vitro. Here, we explored the EtOH-dependent pathways leading to hippocampal AMPAR suppression. Organotypic hippocampal slices (2 days in cultures) were exposed to EtOH (150 mM) for 7 days followed by 24 h EtOH withdrawal. Then, the slices were analysed by means of RT-PCR for miRNA content, western blotting for AMPA and NMDA related-synaptic proteins expression in postsynaptic compartment and electrophysiology to record electrical properties from CA1 pyramidal neurons. We observed that EtOH induces a significant downregulation of postsynaptic AMPA and NMDA subunits and relative scaffolding protein expression and, accordingly, a decrease of AMPA-mediated neurotransmission. Simultaneously, we found that chronic EtOH induced-upregulation of miRNA 137 and 501-3p and decreased AMPA-mediated neurotransmission are prevented by application of the selective mGlu5 antagonist MPEP during EtOH withdrawal. Our data indicate mGlu5 via miRNA137 and 501-3p expression as key factors in the regulation of AMPAergic neurotransmission that may contribute, at least in part, to the pathogenesis of FASD.

Induction of vertebrate regeneration by a transient sodium current.

Amphibians such as frogs can restore lost organs during development, including the lens and tail. To design biomedical therapies for organ repair, it is necessary to develop a detailed understanding of natural regeneration. Recently, ion transport has been implicated as a functional regulator of regeneration. Whereas voltage-gated sodium channels play a well known and important role in propagating action potentials in excitable cells, we have identified a novel role in regeneration for the ion transport function mediated by the voltage-gated sodium channel, Na(V)1.2. A local, early increase in intracellular sodium is required for initiating regeneration following Xenopus laevis tail amputation, and molecular and pharmacological inhibition of sodium transport causes regenerative failure. Na(V)1.2 is absent under nonregenerative conditions, but misexpression of human Na(V)1.5 can rescue regeneration during these states. Remarkably, pharmacological induction of a transient sodium current is capable of restoring regeneration even after the formation of a nonregenerative wound epithelium, confirming that it is the regulation of sodium transport that is critical for regeneration. Our studies reveal a previously undetected competency window in which cells retain their intrinsic regenerative program, identify a novel endogenous role for Na(V) in regeneration, and show that modulation of sodium transport represents an exciting new approach to organ repair.

Optical methods in the study of protein-protein interactions.

Förster (or Fluorescence) resonance energy transfer (FRET) is a physical process in which energy is transferred nonradiatively from an excited fluorophore, serving as a donor, to another chromophore (acceptor). Among the techniques related to fluorescence microscopy, FRET is unique in providing signals sensitive to intra- and intermolecular distances in the 1-10 nm range. Because of its potency, FRET is increasingly used to visualize and quantify the dynamics of protein-protein interaction in living cells, with high spatio-temporal resolution. Here we describe the physical bases of FRET, detailing the principal methods applied: (1) measurement of signal intensity and (2) analysis of fluorescence lifetime (FLIM). Although several technical complications must be carefully considered, both methods can be applied fruitfully to specific fields. For example, FRET based on intensity detection is more suitable to follow biological phenomena at a finely tuned spatial and temporal scale. Furthermore, a specific fluorescence signal occurring close to the plasma membrane (< or = 100 nm) can be obtained using a total internal reflection fluorescence (TIRF) microscopy system. When performing FRET experiments, care must be also taken to the method chosen for labeling interacting proteins. Two principal tools can be applied: (1) fluorophore tagged antibodies; (2) recombinant fluorescent fusion proteins. The latter method essentially takes advantage of the discovery and use of spontaneously fluorescent proteins, like the green fluorescent protein (GFP). Until now, FRET has been widely used to analyze the structural characteristics of several proteins, including integrins and ion channels. More recently, this method has been applied to clarify the interaction dynamics of these classes of membrane proteins with cytosolic signaling proteins. We report two examples in which the interaction dynamics between integrins and ion channels have been studied with FRET methods. Using fluorescent antibodies and applying FRET-FLIM, the direct interaction of beta1 integrin with the receptor for Epidermal Growth Factor (EGF-R) has been proved in living endothelial cells. A different approach, based on TIRFM measurement of the FRET intensity of fluorescently labeled recombinant proteins, suggests that a direct interaction also occurs between integrins and the ether-a-go-go-related-gene 1 (hERG1) K+ channel.

Convergence of integrins and EGF receptor signaling via PI3K/Akt/FoxO pathway in early gene Egr-1 expression.

The early gene early growth response (Egr-1), a broadly expressed member of the zing-finger family of transcription factors, is induced in many cell types by a variety of growth and differentiation stimuli, including epidermal growth factor (EGF). Here we demonstrate that Egr-1 expression is mainly regulated by integrin-mediated adhesion. Integrin-dependent adhesion plays a dual role in Egr-1 regulation, either being sufficient "per se" to induce Egr-1, or required for EGF-dependent expression of Egr-1, which occurs only in adherent cells and not in cells in suspension. To dissect the molecular basis of integrin-dependent Egr-1 regulation, we show by FLIM-based FRET that in living cells beta1-integrin associates with the EGF receptor (EGFR) and that EGF further increases the extent complex formation. Interestingly, Egr-1 induction depends on integrin-dependent PI3K/Akt activation, as indicated by the decrease in Egr-1 levels in presence of the pharmacological inhibitor LY294002, the kinase-defective Akt mutant and Akt1/2 shRNAs. Indeed, upon adhesion activated Akt translocates into the nucleus and phosphorylates FoxO1, a Forkhead transcription factors. Consistently, FoxO1silencing results in Egr-1-increased levels, indicating that FoxO1 behaves as a negative regulator of Egr-1 expression. These data demonstrate that integrin/EGFR cross-talk is required for expression of Egr-1 through a novel regulatory cascade involving the activation of the PI3K/Akt/Forkhead pathway.

NOP Receptor Agonist Ro 64-6198 Decreases Escalation of Cocaine Self-Administration in Rats Genetically Selected for Alcohol Preference.

Cocaine dependence is a psychiatric condition for which effective medications are still lacking. Published data indicate that an increase in nociceptin/orphanin FQ (N/OFQ) transmission by NOP receptor activation attenuates cocaine-induced place conditioning and the locomotor sensitization effects of cocaine. This suggests that the activation of the N/OFQ receptor (NOP) may attenuate the motivation for psychostimulants. To further explore this possibility, we investigated the effect of the potent and selective NOP receptor agonist Ro 64-6198 on cocaine intake under 1 h short access (ShA) and 6 h long access (LgA) operant self-administration conditions in rats. We used Marchigian Sardinian alcohol-preferring (msP) rats and Wistar control rats. msP rats were used because we recently found that this rat line, originally selected for excessive alcohol drinking and preference, exhibits a greater propensity to escalate cocaine self-administration following LgA training. msP rats are also characterized by innate overexpression of the N/OFQ-NOP system compared with Wistar rats. Wistar and msP rats both exhibited an increase in cocaine self-administration under LgA conditions, with a higher trend toward escalation in msP rats. In Wistar rats, the intraperitoneal administration of Ro 64-6198 (0. 1 and 3 mg/kg) significantly decreased ShA cocaine self-administration. In Wistar rats that underwent LgA cocaine self-administration training, Ro 64-6198 induced no significant effect either during the first hour of self-administration or after the entire 6 h session. In msP rats, Ro 64-6198 significantly reduced cocaine self-administration both under ShA conditions and in the first hour of the LgA session. At the end of the 6 h session, the effect of Ro 64-6198 was no longer observed in msP rats. The highest dose of Ro 64-6198 (3 mg/kg) did not affect saccharin self-administration in msP rats but reduced saccharin self-administration in Wistar rats. Altogether, these data suggest that NOP receptor activation attenuates cocaine self-administration, and this effect tends to be more pronounced in a rat line with innately higher NOP receptor expression and that more robustly escalates cocaine intake.

Building better strategies to develop new medications in Alcohol Use Disorder: Learning from past success and failure to shape a brighter future.

Alcohol Use Disorder (AUD) is a chronic disease that develops over the years. The complexity of the neurobiological processes contributing to the emergence of AUD and the neuroadaptive changes occurring during disease progression make it difficult to improve treatments. On the other hand, this complexity offers researchers the possibility to explore new targets. Over years of intense research several molecules were tested in AUD; in most cases, despite promising preclinical data, the clinical efficacy appeared insufficient to justify futher development. A prototypical example is that of corticotropin releasing factor type 1 receptor (CRF1R) antagonists that showed significant effectiveness in animal models of AUD but were largely ineffective in humans. The present article attempts to analyze the most recent venues in the development of new medications in AUD with a focus on the most promising drug targets under current exploration. Moreover, we delineate the importance of using a more integrated translational framework approach to correlate preclinical findings and early clinical data to enhance the probability to validate biological targets of interest.

Reward-Related Behavioral, Neurochemical and Electrophysiological Changes in a Rat Model of Autism Based on Prenatal Exposure to Valproic Acid.

Prenatal exposure to the antiepileptic drug valproic acid (VPA) induces autism spectrum disorder (ASD) in humans and autistic-like behaviors in rodents, which makes it a good model to study the neural underpinnings of ASD. Rats prenatally exposed to VPA show profound deficits in the social domain. The altered social behavior displayed by VPA-exposed rats may be due to either a deficit in social reward processing or to a more general inability to properly understand and respond to social signals. To address this issue, we performed behavioral, electrophysiological and neurochemical experiments and tested the involvement of the brain reward system in the social dysfunctions displayed by rats prenatally exposed to VPA (500 mg/kg). We found that, compared to control animals, VPA-exposed rats showed reduced play responsiveness together with impaired sociability in the three-chamber test and altered social discrimination abilities. In addition, VPA-exposed rats showed altered expression of dopamine receptors together with inherent hyperexcitability of medium spiny neurons (MSNs) in the nucleus accumbens (NAc). However, when tested for socially-induced conditioned place preference, locomotor response to amphetamine and sucrose preference, control and VPA-exposed rats performed similarly, indicating normal responses to social, drug and food rewards. On the basis of the results obtained, we hypothesize that social dysfunctions displayed by VPA-exposed rats are more likely caused by alterations in cognitive aspects of the social interaction, such as the interpretation and reciprocation of social stimuli and/or the ability to adjust the social behavior of the individual to the changing circumstances in the social and physical environment, rather than to inability to enjoy the pleasurable aspects of the social interaction. The observed neurochemical and electrophysiological alterations in the NAc may contribute to the inability of VPA-exposed rats to process and respond to social cues, or, alternatively, represent a compensatory mechanism towards VPA-induced neurodevelopmental insults.

Anticonvulsant and Neuroprotective Effects of the Thyroid Hormone Metabolite 3-Iodothyroacetic Acid.

BACKGROUND: 3-Iodothyroacetic acid (TA1) is among the thyroid hormone (T3) metabolites that can acutely modify behavior in mice. This study aimed to investigate whether TA1 is also able to reduce neuron hyper-excitability and protect from excitotoxic damage. METHODS: CD1 male mice were treated intraperitoneally with saline solution or TA1 (4, 7, 11, or 33 µg/kg) before receiving 90 mg/kg pentylenetrazole subcutaneously. The following parameters were measured: latency to first seizure onset, number of mice experiencing seizures, hippocampal levels of c-fos, and PI3K/AKT activation levels. Organotypic hippocampal slices were exposed to vehicle or to 5 µM kainic acid (KA) in the absence or presence of 0.01-10 µM TA1. In another set of experiments, slices were exposed to vehicle or 5 µM KA in the absence or presence of 10 µM T3, 3,5,3'-triiodothyroacetic acid (TRIAC), T1AM, thyronamine (T0AM), or thyroacetic acid (TA0). Neuronal cell death was measured fluorimetically. The ability of TA1 and T3, TRIAC, T1AM, T0A, and TA0 to activate the PI3K/AKT cascade was evaluated by Western blot. The effect of TA1 on KA-induced currents in CA3 neurons was evaluated by patch clamp recordings on acute hippocampal slices. RESULTS: TA1 (7 and 11 µg/kg) significantly reduced the number of mice showing convulsions and increased their latency of onset, restored pentylenetrazole-induced reduction of hippocampal c-fos levels, activated the PI3K/AKT, and reduced GSK-3ß activity. In rat organotypic hippocampal slices, TA1 reduced KA-induced cell death by activating the PI3K/AKT cascade and increasing GSK-3ß phosphorylation levels. Protection against KA toxicity was also exerted by T3 and other T3 metabolites studied. TA1 did not interact at KA receptors. Both the anticonvulsant and neuroprotective effects of TA1 were abolished by pretreating mice or organotypic hippocampal slices with pyrilamine, an histamine type 1 receptor antagonist (10 mg/kg or 1 µM, respectively). CONCLUSIONS: TA1 exerts anticonvulsant activity and is neuroprotective in vivo and in vitro. These findings extend the current knowledge on the pharmacological profile of TA1 and indicate possible novel clinical use for this T3 metabolite.

3-Iodothyroacetic acid (TA1), a by-product of thyroid hormone metabolism, reduces the hypnotic effect of ethanol without interacting at GABA-A receptors.

3-iodothyroacetic acid (TA1) is among the by-products of thyroid hormone metabolism suspected to mediate the non-genomic effects of the hormone (T3). We aim to investigate whether TA1 systemically administered to mice stimulated mice wakefulness, an effect already described for T3 and for another T3 metabolite (i.e. 3-iodothryonamine; T1AM), and whether TA1 interacted at GABA-A receptors (GABA-AR). Mice were pre-treated with either saline (vehicle) or TA1 (1.32, 4 and 11 µg/kg) and, after 10 min, they received ethanol (3.5 g/kg, i.p.). In another set of experiments, TA1 was administered 5 min after ethanol. The latency of sleep onset and the time of sleep duration were recorded. Voltage-clamp experiments to evaluate the effect of 1 µM TA1 on bicuculline-sensitive currents in acute rat hippocampal slice neurons and binding experiments evaluating the capacity of 1, 10, 100 µM TA1 to displace [3H]flumazenil from mice brain membranes were also performed. 4 µg/kg TA1 increases the latency of onset and at 1.32 and 4 µg/kg it reduces the duration of ethanol-induced sleep only if administered before ethanol. TA1 does not functionally interact at GABA-AR. Overall these results indicate a further similarity between the pharmacological profile of TA1 and that of T1AM.

Dexpramipexole improves bioenergetics and outcome in experimental stroke.

BACKGROUND AND PURPOSE: Dexpramipexole, a drug recently tested in patients with amyotrophic lateral sclerosis (ALS,) is able to bind F1Fo ATP synthase and increase mitochondrial ATP production. Here, we have investigated its effects on experimental ischaemic brain injury. EXPERIMENTAL APPROACH: The effects of dexpramipexole on bioenergetics, Ca2+ fluxes, electrophysiological functions and death were evaluated in primary neural cultures and hippocampal slices exposed to oxygen-glucose deprivation (OGD). Effects on infarct volumes and neurological functions were also evaluated in mice following proximal or distal middle cerebral artery occlusion (MCAo). Distribution of dexpramipexole within the ischaemic brain was evaluated by means of mass spectrometry imaging. KEY RESULTS: Dexpramipexole increased mitochondrial ATP production in cultured neurons or glia and reduces energy failure, prevents intracellular Ca2+ overload and affords cytoprotection when cultures are exposed to OGD. This compound also counteracted ATP depletion, mitochondrial swelling, anoxic depolarization, loss of synaptic activity and neuronal death in hippocampal slices subjected to OGD. Post-ischaemic treatment with dexpramipexole, at doses consistent with those already used in ALS patients, reduced brain infarct size and ameliorated neuroscore in mice subjected to transient or permanent MCAo. Notably, the concentrations of dexpramipexole reached within the ischaemic penumbra equalled those found neuroprotective in vitro. CONCLUSION AND IMPLICATIONS: Dexpramipexole, a compound able to increase mitochondrial F1Fo ATP-synthase activity reduced ischaemic brain injury. These findings, together with the excellent brain penetration and favourable safety profile in humans, make dexpramipexole a drug with realistic translational potential for the treatment of stroke. LINKED ARTICLES: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

In vivo multiphoton nanosurgery on cortical neurons.

Two-photon microscopy has been used to perform high spatial resolution imaging of spine plasticity in the intact neocortex of living mice. Multiphoton absorption has also been used as a tool for the selective disruption of cellular structures in living cells and simple organisms. In this work, we exploit the spatial localization of multiphoton excitation to perform selective lesions on the neuronal processes of cortical neurons in living mice expressing fluorescent proteins. Neurons are irradiated with a focused, controlled dose of femtosecond laser energy delivered through cranial optical windows. The morphological consequences are then characterized with time lapse 3-D two-photon imaging over a period of minutes to days after the procedure. This methodology is applied to dissect single dendrites with submicrometric precision without causing any visible collateral damage to the surrounding neuronal structures. The spatial precision of this method is demonstrated by ablating individual dendritic spines, while sparing the adjacent spines and the structural integrity of the dendrite. The combination of multiphoton nanosurgery and in vivo imaging in mammals represents a promising tool for neurobiology and neuropharmacology research.