Natalia Lozovaya

GRIN2A mutations in acquired epileptic aphasia and related childhood focal epilepsies and encephalopathies with speech and language dysfunction

Epileptic encephalopathies are severe brain disorders with the epileptic component contributing to the worsening of cognitive and behavioral manifestations. Acquired epileptic aphasia (Landau-Kleffner syndrome, LKS) and continuous spike and waves during slow-wave sleep syndrome (CSWSS) represent rare and closely related childhood focal epileptic encephalopathies of unknown etiology. They show electroclinical overlap with rolandic epilepsy (the most frequent childhood focal epilepsy) and can be viewed as different clinical expressions of a single pathological entity situated at the crossroads of epileptic, speech, language, cognitive and behavioral disorders. Here we demonstrate that about 20% of cases of LKS, CSWSS and electroclinically atypical rolandic epilepsy often associated with speech impairment can have a genetic origin sustained by de novo or inherited mutations in the GRIN2A gene (encoding the N-methyl-D-aspartate (NMDA) glutamate receptor α2 subunit, GluN2A). The identification of GRIN2A as a major gene for these epileptic encephalopathies provides crucial insights into the underlying pathophysiology.

Glycine receptors in CNS neurons as a target for nonretrograde action of cannabinoids

At many central synapses, endocannabinoids released by postsynaptic cells act retrogradely on presynaptic G-protein-coupled cannabinoid receptors to inhibit neurotransmitter release. Here, we demonstrate that cannabinoids may directly affect the functioning of inhibitory glycine receptor (GlyR) channels. In isolated hippocampal pyramidal and Purkinje cerebellar neurons, endogenous cannabinoids anandamide and 2-arachidonylglycerol, applied at physiological concentrations, inhibited the amplitude and altered the kinetics of rise time, desensitization, and deactivation of the glycine-activated current (IGly) in a concentration-dependent manner. These effects of cannabinoids were observed in the presence of cannabinoid CB1/CB3, vanilloid receptor 1 antagonists, and the G-protein inhibitor GDPβS, suggesting a direct action of cannabinoids on GlyRs. The effect of cannabinoids on IGly desensitization was strongly voltage dependent. We also demonstrate that, in the presence of a GABAA receptor antagonist, GlyRs may contribute to the generation of seizure-like activity induced by short bursts (seven stimuli) of high-frequency stimulation of inputs to hippocampal CA1 region, because this activity was diminished by selective GlyR antagonists (strychnine and ginkgolides B and J). The GlyR-mediated rhythmic activity was also reduced by cannabinoids (anandamide) in the presence of a CB1 receptor antagonist. These results suggest that the direct inhibition of GlyRs by endocannabinoids can modulate the hippocampal network activity.

Selective suppression of excessive GluN2C expression rescues early epilepsy in a tuberous sclerosis murine model

Tuberous sclerosis complex (TSC), caused by dominant mutations in either TSC1 or TSC2 tumour suppressor genes is characterized by the presence of brain malformations, the cortical tubers that are thought to contribute to the generation of pharmacoresistant epilepsy. Here we report that tuberless heterozygote Tsc1+/− mice show functional upregulation of cortical GluN2C-containing N-methyl-D-aspartate receptors (NMDARs) in an mTOR-dependent manner and exhibit recurrent, unprovoked seizures during early postnatal life (<P19). Seizures are generated intracortically in the granular layer of the neocortex. Slow kinetics of aberrant GluN2C-mediated currents in spiny stellate cells promotes excessive temporal integration of persistent NMDAR-mediated recurrent excitation and seizure generation. Accordingly, specific GluN2C/D antagonists block seizures in Tsc1+/− mice in vivo and in vitro. Likewise, GluN2C expression is upregulated in TSC human surgical resections, and a GluN2C/D antagonist reduces paroxysmal hyperexcitability. Thus, GluN2C receptor constitutes a promising molecular target to treat epilepsy in TSC patients.

N -Methyl-d-aspartate (NMDA) Receptor NR2 Subunit Selectivity of a Series of Novel Piperazine-2,3-dicarboxylate Derivatives: Preferential Blockade of Extrasynaptic NMDA Receptors in the Rat Hippocampal CA3-CA1 Synapse

N-Methyl-d-aspartate (NMDA) receptor antagonists that are highly selective for specific NMDA receptor 2 (NR2) subunits have several potential therapeutic applications; however, to date, only NR2B-selective antagonists have been described. Whereas most glutamate binding site antagonists display a common pattern of NR2 selectivity, NR2A > NR2B > NR2C > NR2D (high to low affinity), (2S*,3R*)-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA) has a low selectivity for NR2C- and NR2D-containing NMDA receptors. A series of PPDA derivatives were synthesized and then tested at recombinant NMDA receptors expressed in Xenopus laevis oocytes. In addition, the optical isomers of PPDA were resolved; the (−) isomer displayed a 50- to 80-fold greater potency than the (+) isomer. Replacement of the phenanthrene moiety of PPDA with naphthalene or anthracene did not improve selectivity. However, phenylazobenzoyl (UBP125) or phenylethynylbenzoyl (UBP128) substitution significantly improved selectivity for NR2B-, NR2C-, and NR2D-containing receptors over NR2A-containing NMDA receptors. Phenanthrene attachment at the 3 position [(2R*,3S*)-1-(phenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP141); (2R*,3S*)-1-(9-bromophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP145); (2R*,3S*)-1-(9-chlorophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP160); and (2R*,3S*)-1-(9-iodophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP161)] displayed improved NR2D selectivity. UBP141 and its 9-brominated homolog (UBP145) both display a 7- to 10- fold selectivity for NR2D-containing receptors over NR2B- or NR2A-containing receptors. Schild analysis indicates that these two compounds are competitive glutamate binding site antagonists. Consistent with a physiological role for NR2D-containing receptors in the hippocampus, UBP141 (5 μM) displayed greater selectivity than PPDA for inhibiting the slow-decaying component of the NMDA receptor-mediated CA3-CA1 synaptic response in rat hippocampal slices. UBP125, UBP128, UBP141, and UBP145 may be useful tools for determining the function of NMDA receptor subtypes.

Tubacin prevents neuronal migration defects and epileptic activity caused by rat Srpx2 silencing in utero

Altered development of the human cerebral cortex can cause severe malformations with often intractable focal epileptic seizures and may participate in common pathologies, notably epilepsy. This raises important conceptual and therapeutic issues. Two missense mutations in the sushi repeat-containing protein SRPX2 had been previously identified in epileptic disorders with or without structural developmental alteration of the speech cortex. In the present study, we aimed to decipher the precise developmental role of SRPX2, to have a better knowledge on the consequences of its mutations, and to start addressing therapeutic issues through the design of an appropriate animal model. Using an in utero Srpx2 silencing approach, we show that SRPX2 influences neuronal migration in the developing rat cerebral cortex. Wild-type, but not the mutant human SRPX2 proteins, rescued the neuronal migration phenotype caused by Srpx2 silencing in utero, and increased alpha-tubulin acetylation. Following in utero Srpx2 silencing, spontaneous epileptiform activity was recorded post-natally. The neuronal migration defects and the post-natal epileptic consequences were prevented early in embryos by maternal administration of tubulin deacetylase inhibitor tubacin. Hence epileptiform manifestations of developmental origin could be prevented in utero, using a transient and drug-based therapeutic protocol.

Intracellular Na+ inhibits voltage‐dependent N‐type Ca2+ channels by a G protein βγ subunit‐dependent mechanism

N‐type  voltage‐dependent  Ca2+ channels (N‐VDCCs) play important roles in neurotransmitter release and certain postsynaptic phenomena. These channels are modulated by a number of intracellular factors, notably by Gβγ subunits of G proteins, which inhibit N‐VDCCs in a voltage‐dependent (VD) manner. Here we show that an increase in intracellular Na+ concentration inhibits N‐VDCCs  in hippocampal pyramidal neurones and in Xenopus oocytes. In acutely dissociated hippocampal neurones, Ba2+ current via N‐VDCCs was inhibited by Na+ influx caused by the activation of NMDA receptor channels. In Xenopus oocytes expressing N‐VDCCs, Ba2+ currents were inhibited by Na+ influx and enhanced by depletion of Na+, after incubation in a Na+‐free extracellular solution. The Na+‐induced inhibition was accompanied by the development of  VD facilitation, a hallmark of a Gβγ‐dependent process. Na+‐induced regulation of N‐VDCCs is Gβγ dependent, as suggested by the blocking of Na+ effects by Gβγ scavengers and by excess Gβγ, and may be mediated by the Na+‐induced dissociation of Gαβγ heterotrimers. N‐VDCCs may be novel effectors of Na+ion, regulated by the Na+ concentration via Gβγ.

Control of Inhibition by the Direct Action of Cannabinoids on GABAA Receptors

Cannabinoids are known to regulate inhibitory synaptic transmission via activation of presynaptic G protein-coupled cannabinoid CB1 receptors (CB1Rs). Additionally, recent studies suggest that cannabinoids can also directly interact with recombinant GABAA receptors (GABAARs), potentiating currents activated by micromolar concentrations of γ-aminobutyric acid (GABA). However, the impact of this direct interaction on GABAergic inhibition in central nervous system is unknown. Here we report that currents mediated by recombinant GABAARs activated by high (synaptic) concentrations of GABA as well as GABAergic inhibitory postsynaptic currents (IPSCs) at neocortical fast spiking (FS) interneuron to pyramidal neuron synapses are suppressed by exogenous and endogenous cannabinoids in a CB1R-independent manner. This IPSC suppression may account for disruption of inhibitory control of pyramidal neurons by FS interneurons. At FS interneuron to pyramidal neuron synapses, endocannabinoids induce synaptic low-pass filtering of GABAAR-mediated currents evoked by high-frequency stimulation. The CB1R-independent suppression of inhibition is synapse specific. It does not occur in CB1R containing hippocampal cholecystokinin-positive interneuron to pyramidal neuron synapses. Furthermore, in contrast to synaptic receptors, the activity of extrasynaptic GABAARs in neocortical pyramidal neurons is enhanced by cannabinoids in a CB1R-independent manner. Thus, cannabinoids directly interact differentially with synaptic and extrasynaptic GABAARs, providing a potent novel context-dependent mechanism for regulation of inhibition.

Diadenosine Polyphosphate Analog Controls Postsynaptic Excitation in CA3-CA1 Synapses via a Nitric Oxide-Dependent Mechanism

Previously, we have described the modulatory effect of diadenosine polyphosphates Ap4A and Ap5A on synaptic transmission in the rat hippocampal slices mediated by presynaptic receptors (Klishin et al., 1994). In contrast, we now describe how nonhydrolyzable Ap4A analog diadenosine-5′,5′′′-P1,P4-[β,β′-methylene]tetraphosphate (AppCH2ppA) at low micromolar concentrations exerts strong nondesensitizing inhibition of orthodromically evoked field potentials (OFPs) without affecting the amplitude of excitatory postsynaptic currents and antidromically evoked field potentials, as recorded in hippocampal CA1 zone. The effects of AppCH2ppA on OFPs are eliminated by a P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) but not mimicked by purinoceptor agonists α,β-methylene-ATP and adenosine 5′-O-(3-thio)-triphosphate, indicating that a P2-like receptor is involved but not one belonging to the conventional P2X/P2Y receptor classes. Diadenosine polyphosphate receptor (P4) antagonist Ip4I (diinosine tetraphosphate) was unable to modulate AppCH2ppA effects. Thus, the PPADS-sensitive P2-like receptor for AppCH2ppA seems to control selectively dendritic excitation of the CA1 neurons. The specific nitric oxide (NO)-scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide is shown to significantly attenuate AppCH2ppA-mediated inhibitory effects, indicating that NO is involved in the cascade of events initiated by AppCH2ppA. Further downstream mediation by adenosine A1 receptors is also demonstrated. Hence, AppCH2ppA-mediated effects involve PPADS-sensitive P2-like receptor activation leading to the production of NO that stimulates intracellular synthesis of adenosine, causing in turn postsynaptic A1 receptor activation and subsequent postsynaptic CA1 dendritic inhibition. Such spatially selective postsynaptic dendritic inhibition may influence dendritic electrogenesis in pyramidal neurons and consequently mediate control of neuronal network activity.

Subthalamic Lesion or Levodopa Treatment Rescues Giant GABAergic Currents of PINK1-Deficient Striatum

Cellular electrophysiological signatures of Parkinsons disease described in the pharmacological 6-hydroxydopamine (6-OHDA) animal models of Parkinsons disease include spontaneous repetitive giant GABAergic currents in a subpopulation of striatal medium spiny neurons (MSNs), and spontaneous rhythmic bursts of spikes generated by subthalamic nucleus (STN) neurons. We investigated whether similar signatures are present in Pink1−/− mice, a genetic rodent model of the PARK6 variant of Parkinsons disease. Although 9- to 24-month-old Pink1−/− mice show reduced striatal dopamine content and release, and impaired spontaneous locomotion, the relevance of this model to Parkinsons disease has been questioned because mesencephalic dopaminergic neurons do not degenerate during the mouse lifespan. We show that 75% of the MSNs of 5- to 7-month-old Pink1−/− mice exhibit giant GABAergic currents, occurring either singly or in bursts (at 40 Hz), rather than the low-frequency (2 Hz), low-amplitude, tonic GABAergic drive common to wild-type MSNs of the same age. STN neurons from 5- to 7-month-old Pink1−/− mice spontaneously generated bursts of spikes instead of the control tonic drive. Chronic kainic acid lesion of the STN or chronic levodopa treatment reliably suppressed the giant GABAergic currents of MSNs after 1 month and replaced them with the control tonic activity. The similarity between the in vitro resting states of Pink1 MSNs and those of fully dopamine (DA)-depleted MSNs of 6-OHDA-treated mice, together with the beneficial effect of levodopa treatment, strongly suggest that dysfunction of mesencephalic dopaminergic neurons in Pink1−/− mice is more severe than expected. The beneficial effect of the STN lesion also suggests that pathological STN activity strongly influences striatal networks in Pink1−/− mice.

Stable, synthetic analogs of diadenosine tetraphosphate inhibit rat and human P2X3 receptors and inflammatory pain

Background A growing body of evidence suggests that ATP-gated P2X3 receptors (P2X3Rs) are implicated in chronic pain. We address the possibility that stable, synthetic analogs of diadenosine tetraphosphate (Ap4A) might induce antinociceptive effects by inhibiting P2X3Rs in peripheral sensory neurons. Results The effects of two stable, synthetic Ap4A analogs (AppNHppA and AppCH2ppA) are studied firstly in vitro on HEK293 cells expressing recombinant rat P2XRs (P2X2Rs, P2X3Rs, P2X4Rs, and P2X7Rs) and then using native rat brain cells (cultured trigeminal, nodose, or dorsal root ganglion neurons). Thereafter, the action of these stable, synthetic Ap4A analogs on inflammatory pain and thermal hyperalgesia is studied through the measurement of antinociceptive effects in formalin and Hargreaves plantar tests in rats in vivo. In vitro inhibition of rat P2X3Rs (not P2X2Rs, P2X4Rs nor P2X7Rs) is shown to take place mediated by high-affinity desensitization (at low concentrations; IC50 values 100–250 nM) giving way to only weak partial agonism at much higher concentrations (EC50 values ≥ 10 µM). Similar inhibitory activity is observed with human recombinant P2X3Rs. The inhibitory effects of AppNHppA on nodose, dorsal root, and trigeminal neuron whole cell currents suggest that stable, synthetic Ap4A analogs inhibit homomeric P2X3Rs in preference to heteromeric P2X2/3Rs. Both Ap4A analogs mediate clear inhibition of pain responses in both in vivo inflammation models. Conclusions Stable, synthetic Ap4A analogs (AppNHppA and AppCH2ppA) being weak partial agonist provoke potent high-affinity desensitization-mediated inhibition of homomeric P2X3Rs at low concentrations. Therefore, both analogs demonstrate clear potential as potent analgesic agents for use in the management of chronic pain associated with heightened P2X3R activation.