Direct measurements of neurosteroid binding to specific sites on GABAA receptors.

BACKGROUND AND PURPOSE: Neurosteroids are allosteric modulators of GABAA currents, acting through several functional binding sites although their affinity and specificity for each site are unknown. The goal of this study was to measure steady-state binding affinities of various neurosteroids for specific sites on the GABAA receptor. EXPERIMENTAL APPROACH: Two methods were developed to measure neurosteroid binding affinity: (1) quenching of specific tryptophan residues in neurosteroid binding sites by the neurosteroid 17-methylketone group, and (2) FRET between MQ290 (an intrinsically fluorescent neurosteroid) and tryptophan residues in the binding sites. The assays were developed using ELIC-α1GABAAR, a chimeric receptor containing transmembrane domains of the α1-GABAA receptor. Tryptophan mutagenesis was used to identify specific interactions. KEY RESULTS: Allopregnanolone (3α-OH neurosteroid) was shown to bind at intersubunit and intrasubunit sites with equal affinity, whereas epi-allopregnanolone (3ß-OH neurosteroid) binds at the intrasubunit site. MQ290 formed a strong FRET pair with W246, acting as a site-specific probe for the intersubunit site. The affinity and site-specificity of several neurosteroid agonists and inverse agonists was measured using the MQ290 binding assay. The FRET assay distinguishes between competitive and allosteric inhibition of MQ290 binding and demonstrated an allosteric interaction between the two neurosteroid binding sites. CONCLUSIONS AND IMPLICATIONS: The affinity and specificity of neurosteroid binding to two sites in the ELIC-α1GABAAR were directly measured and an allosteric interaction between the sites was revealed. Adaptation of the MQ290 FRET assay to a plate-reader format will enable screening for high affinity agonists and antagonists for neurosteroid binding sites.

Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum.

Protein homeostasis (proteostasis) deficiency is an important contributing factor to neurological and metabolic diseases. However, how the proteostasis network orchestrates the folding and assembly of multi-subunit membrane proteins is poorly understood. Previous proteomics studies identified Hsp47 (Gene: SERPINH1), a heat shock protein in the endoplasmic reticulum lumen, as the most enriched interacting chaperone for gamma-aminobutyric acid type A (GABAA) receptors. Here, we show that Hsp47 enhances the functional surface expression of GABAA receptors in rat neurons and human HEK293T cells. Furthermore, molecular mechanism study demonstrates that Hsp47 acts after BiP (Gene: HSPA5) and preferentially binds the folded conformation of GABAA receptors without inducing the unfolded protein response in HEK293T cells. Therefore, Hsp47 promotes the subunit-subunit interaction, the receptor assembly process, and the anterograde trafficking of GABAA receptors. Overexpressing Hsp47 is sufficient to correct the surface expression and function of epilepsy-associated GABAA receptor variants in HEK293T cells. Hsp47 also promotes the surface trafficking of other Cys-loop receptors, including nicotinic acetylcholine receptors and serotonin type 3 receptors in HEK293T cells. Therefore, in addition to its known function as a collagen chaperone, this work establishes that Hsp47 plays a critical and general role in the maturation of multi-subunit Cys-loop neuroreceptors.

Decreased extrasynaptic δ-GABAA receptors in PNN-associated parvalbumin interneurons correlates with anxiety in APP and tau mouse models of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is associated with gradual memory loss and anxiety which affects ~75% of AD patients. This study investigated whether AD-associated anxiety correlated with modulation of extrasynaptic δ-subunit-containing GABAA receptors (δ-GABAARs) in experimental mouse models of AD. EXPERIMENTAL APPROACH: We combined behavioural experimental paradigms to measure cognition performance, and anxiety with neuroanatomy and molecular biology, using familial knock-in (KI) mouse models of AD that harbour ß-amyloid (Aß) precursor protein App (AppNL-F) with or without humanized microtubule-associated protein tau (MAPT), age-matched to wild-type control mice at three different age windows. RESULTS: AppNL-F KI and AppNL-F/MAPT AD models showed a similar magnitude of cognitive decline and elevated magnitude of anxiety correlated with neuroinflammatory hallmarks, including triggering receptor expressed on myeloid cells 2 (TREM2), reactive astrocytes and activated microglia consistent with accumulation of Aß, tau and down-regulation of Wnt/ß-catenin signalling compared to aged-matched WT controls. In both the CA1 region of the hippocampus and dentate gyrus, there was an age-dependent decline in the expression of δ-GABAARs selectively expressed in parvalbumin (PV)-expressing interneurons, encapsulated by perineuronal nets (PNNs) in the AD mouse models compared to WT mice. In vivo positive allosteric modulation of the δ-GABAARs, using a δ-selective-compound DS2, decreased the level of anxiety in the AD mouse models, which was correlated with reduced hallmarks of neuroinflammation, and 'normalisation' of the expression of δ-GABAARs. CONCLUSIONS: Our data show that the δ-GABAARs could potentially be targeted for alleviating symptoms of anxiety, which would greatly improve the quality of life of AD individuals.

Antiseizure properties of fenamate NSAIDs determined in mature human stem-cell derived neuroglial circuits.

Repeated and uncontrolled seizures in epilepsy result in brain cell loss and neural inflammation. Current anticonvulsants primarily target ion channels and receptors implicated in seizure activity. Identification of neurotherapeutics that can inhibit epileptiform activity and reduce inflammation in the brain may offer significant benefits in the long-term management of epilepsy. Fenamates are unique because they are both non-steroidal anti-inflammatory drugs (NSAIDs) and highly subunit selective modulators of GABAA receptors. In the current study we have investigated the hypothesis that fenamates have antiseizure properties using mature human stem cell-derived neuro-glia cell cultures, maintained in long-term culture, and previously shown to be sensitive to first, second and third generation antiepileptics. Mefenamic acid, flufenamic acid, meclofenamic acid, niflumic acid, and tolfenamic acid (each tested at 10-100 µM) attenuated 4-aminopyridine (4-AP, 100 µM) evoked epileptiform activity in a dose-dependent fashion. These actions were as effective diazepam (3-30 µM) and up to 200 times more potent than phenobarbital (300-1,000 µM). The low (micromolar) concentrations of fenamates that inhibited 4-AP evoked epileptiform activity correspond to those reported to potentiate GABAA receptor function. In contrast, the fenamates had no effect on neural spike amplitudes, indicating that their antiseizure actions did not result from inhibition of sodium-channels. The antiseizure actions of fenamates were also not replicated by either of the two non-fenamate NSAIDs, ibuprofen (10-100 µM) or indomethacin (10-100 µM), indicating that inhibition of cyclooxygenases is not the mechanism through which fenamates have anticonvulsant properties. This study therefore shows for the first time, using functionally mature human stem cell-derived neuroglial circuits, that fenamate NSAIDs have powerful antiseizure actions independent of, and in addition to their well-established anti-inflammatory properties, suggesting these drugs may provide a novel insight and new approach to the treatment of epilepsy in the future.

Advances in Therapeutics to Alleviate Cognitive Decline and Neuropsychiatric Symptoms of Alzheimer's Disease.

Dementia exists as a 'progressive clinical syndrome of deteriorating mental function significant enough to interfere with activities of daily living', with the most prevalent type of dementia being Alzheimer's disease (AD), accounting for about 80% of diagnosed cases. AD is associated with an increased risk of comorbidity with other clinical conditions such as hypertension, diabetes, and neuropsychiatric symptoms (NPS) including, agitation, anxiety, and depression as well as increased mortality in late life. For example, up to 70% of patients diagnosed with AD are affected by anxiety. As aging is the major risk factor for AD, this represents a huge global burden in ageing populations. Over the last 10 years, significant efforts have been made to recognize the complexity of AD and understand the aetiology and pathophysiology of the disease as well as biomarkers for early detection. Yet, earlier treatment options, including acetylcholinesterase inhibitors and glutamate receptor regulators, have been limited as they work by targeting the symptoms, with only the more recent FDA-approved drugs being designed to target amyloid-ß protein with the aim of slowing down the progression of the disease. However, these drugs may only help temporarily, cannot stop or reverse the disease, and do not act by reducing NPS associated with AD. The first-line treatment options for the management of NPS are selective serotonin reuptake inhibitors/selective noradrenaline reuptake inhibitors (SSRIs/SNRIs) targeting the monoaminergic system; however, they are not rational drug choices for the management of anxiety disorders since the GABAergic system has a prominent role in their development. Considering the overall treatment failures and side effects of currently available medication, there is an unmet clinical need for rationally designed therapies for anxiety disorders associated with AD. In this review, we summarize the current status of the therapy of AD and aim to highlight novel angles for future drug therapy in our ongoing efforts to alleviate the cognitive deficits and NPS associated with this devastating disease.

GABA system as the cause and effect in early development.

GABA is the primary inhibitory neurotransmitter in the adult brain and through its actions on GABAARs, it protects against excitotoxicity and seizure activity, ensures temporal fidelity of neurotransmission, and regulates concerted rhythmic activity of neuronal populations. In the developing brain, the development of GABAergic neurons precedes that of glutamatergic neurons and the GABA system serves as a guide and framework for the development of other brain systems. Despite this early start, the maturation of the GABA system also continues well into the early postnatal period. In this review, we organize evidence around two scenarios based on the essential and protracted nature of GABA system development: 1) disruptions in the development of the GABA system can lead to large scale disruptions in other developmental processes (i.e., GABA as the cause), 2) protracted maturation of this system makes it vulnerable to the effects of developmental insults (i.e., GABA as the effect). While ample evidence supports the importance of GABA/GABAAR system in both scenarios, large gaps in existing knowledge prevent strong mechanistic conclusions.

TRPM2 and CaMKII Signaling Drives Excessive GABAergic Synaptic Inhibition Following Ischemia.

Excitotoxicity and the concurrent loss of inhibition are well-defined mechanisms driving acute elevation in excitatory/inhibitory (E/I) balance and neuronal cell death following an ischemic insult to the brain. Despite the high prevalence of long-term disability in survivors of global cerebral ischemia (GCI) as a consequence of cardiac arrest, it remains unclear whether E/I imbalance persists beyond the acute phase and negatively affects functional recovery. We previously demonstrated sustained impairment of long-term potentiation (LTP) in hippocampal CA1 neurons correlating with deficits in learning and memory tasks in a murine model of cardiac arrest/cardiopulmonary resuscitation (CA/CPR). Here, we use CA/CPR and an in vitro ischemia model to elucidate mechanisms by which E/I imbalance contributes to ongoing hippocampal dysfunction in male mice. We reveal increased postsynaptic GABAA receptor (GABAAR) clustering and function in the CA1 region of the hippocampus that reduces the E/I ratio. Importantly, reduced GABAAR clustering observed in the first 24 h rebounds to an elevation of GABAergic clustering by 3 d postischemia. This increase in GABAergic inhibition required activation of the Ca2+-permeable ion channel transient receptor potential melastatin-2 (TRPM2), previously implicated in persistent LTP and memory deficits following CA/CPR. Furthermore, we find Ca2+-signaling, likely downstream of TRPM2 activation, upregulates Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, thereby driving the elevation of postsynaptic inhibitory function. Thus, we propose a novel mechanism by which inhibitory synaptic strength is upregulated in the context of ischemia and identify TRPM2 and CaMKII as potential pharmacological targets to restore perturbed synaptic plasticity and ameliorate cognitive function.

γ-Aminobutyric acid transporter and GABAA receptor mechanisms in Slc6a1+/A288V and Slc6a1+/S295L mice associated with developmental and epileptic encephalopathies.

We have previously characterized the molecular mechanisms for variants in γ-aminobutyric acid transporter 1-encoding solute carrier family 6-member 1 (SLC6A1) in vitro and concluded that a partial or complete loss of γ-aminobutyric acid uptake due to impaired protein trafficking is the primary aetiology. Impairment of γ-aminobutyric acid transporter 1 function could cause compensatory changes in the expression of γ-aminobutyric acid receptors, which, in turn, modify disease pathophysiology and phenotype. Here we used different approaches including radioactive 3H γ-aminobutyric acid uptake in cells and synaptosomes, immunohistochemistry and confocal microscopy as well as brain slice surface protein biotinylation to characterize Slc6a1+/A288V and Slc6a1+/S295L mice, representative of a partial or a complete loss of function of SLC6A1 mutations, respectively. We employed the γ-aminobutyric acid transporter 1-specific inhibitor [3H]tiagabine binding and GABAA receptor subunit-specific radioligand binding to profile the γ-aminobutyric acid transporter 1 and GABAA receptor expression in major brain regions such as cortex, cerebellum, hippocampus and thalamus. We also determined the total and surface expression of γ-aminobutyric acid transporter 1, γ-aminobutyric acid transporter 3 and expression of GABAA receptor in the major brain regions in the knockin mice. We found that γ-aminobutyric acid transporter 1 protein was markedly reduced in cortex, hippocampus, thalamus and cerebellum in both mutant mouse lines. Consistent with the findings of reduced γ-aminobutyric acid uptake for both γ-aminobutyric acid transporter 1(A288V) and γ-aminobutyric acid transporter 1(S295L), both the total and the γ-aminobutyric acid transporter 1-mediated 3H γ-aminobutyric acid reuptake was reduced. We found that γ-aminobutyric acid transporter 3 is only abundantly expressed in the thalamus and there was no compensatory increase of γ-aminobutyric acid transporter 3 in either of the mutant mouse lines. γ-Aminobutyric acid transporter 1 was reduced in both somatic regions and nonsomatic regions in both mouse models, in which a ring-like structure was identified only in the Slc6a1+/A288V mouse, suggesting more γ-aminobutyric acid transporter 1 retention inside endoplasmic reticulum in the Slc6a1+/A288V mouse. The [3H]tiagabine binding was similar in both mouse models despite the difference in γ-aminobutyric acid uptake function and γ-aminobutyric acid transporter 1 protein expression for both mutations. There were no differences in GABAA receptor subtype expression, except for a small increase in the expression of α5 subunits of GABAA receptor in the hippocampus of Slc6a1S295L homozygous mice, suggesting a potential interaction between the expression of this GABAA receptor subtype and the mutant γ-aminobutyric acid transporter 1. The study provides the first comprehensive characterization of the SLC6A1 mutations in vivo in two representative mouse models. Because both γ-aminobutyric acid transporter 1 and GABAA receptors are targets for anti-seizure medications, the findings from this study can help guide tailored treatment options based on the expression and function of γ-aminobutyric acid transporter 1 and GABAA receptor in SLC6A1 mutation-mediated neurodevelopmental and epileptic encephalopathies.

Effect of GABAa-receptors on neuronal discharge and ion activity in focal seizures.

Focal seizures are a type of epileptic event that has plagued the medical community for a long time, and the existing drug treatment is mainly based on the modulation of ${GABA}_a$-receptors to affect GABAergic signaling to achieve the therapeutic purpose. The majority of research currently focuses on the impact of ${GABA}_a$-receptors on neuronal firing, failing to analyze the molecular and ionic mechanisms involved. Specifically, the research on deeper-level mechanisms on how ${GABA}_a$-receptors affect neuronal firing by altering ion activity has not been addressed. This research aimed to study the effects of different ${GABA}_a$-receptor structures on ion activity in focal seizures model by adjusting parameters of the ${GABA}_a$-receptors: the rise time constant (${tau}_1$) and decay time constant (${tau}_2$). The research indicates that as the values of ${tau}_1$ and ${tau}_2$ of the ${GABA}_a$-receptor change, the ion concentration will vary based on the change of the ${GABA}_a$-receptor potential. To a certain extent, the duration of epileptic activity will also be affected to a certain extent. In conclusion, the alteration of ${GABA}_a$-receptor structure will affect the inhibitory effect of interneurons on pyramidal neurons, and different parameters of the ${GABA}_a$-receptor will directly impact the therapeutic effect.

Reducing the harms of alcohol: nutritional interventions and functional alcohol alternatives.

The health risks and harm associated with regular alcohol consumption are well documented. In a recent WHO statement published in The Lancet Public Health alcohol consumption has been estimated to contribute worldwide to 3 million deaths in 2016 while also being responsible for 5·1% of the global burden of disease and injury. The total elimination of alcohol consumption, which has been long imbedded in human culture and society, is not practical and prohibition policies have proved historically ineffective. However, valuable strategies to reduce alcohol harms are already available and improved alternative approaches are currently being developed. Here, we will review and discuss recent advances on two main types of approaches, that is nutritional interventions and functional alcohol alternatives.

Membranes and Synaptosomes Used to Investigate Synaptic GABAergic Currents in Epileptic Patients.

Among the most prevalent neurological disorders, epilepsy affects about 1% of the population worldwide. We previously found, using human epileptic tissues, that GABAergic neurotransmission impairment is a key mechanism that drives the pathological phenomena that ultimately lead to generation and recurrence of seizures. Using both a "microtransplantation technique" and synaptosomes preparations from drug-resistant temporal lobe epilepsies (TLEs), we used the technique of two-electrode voltage clamp to record GABA-evoked currents, focusing selectively on the synaptic "fast inhibition" mediated by low-affinity GABAA receptors. Here, we report that the use-dependent GABA current desensitization (i.e., GABA rundown, which is evoked by applying to the cells consecutive pulses of GABA, at high concentration), which is a distinguishing mark of TLE, is mainly dependent on a dysfunction that affects synaptic GABAA receptors. In addition, using the same approaches, we recorded a depolarized GABA reversal potential in synaptosomes samples from the human epileptic subicula of TLE patients. These results, which confirm previous experiments using total membranes, suggest an altered chloride homeostasis in the synaptic area. Finally, the lack of a Zn2+ block of GABA-evoked currents using the synaptosomes supports the enrichment of "synaptic fast inhibitory" GABAA receptors in this preparation. Altogether, our findings suggest a pathophysiological role of low-affinity GABAA receptors at the synapse, especially during the fast and repetitive GABA release underlying recurrent seizures.

The gephyrin scaffold modulates cortical layer 2/3 pyramidal neuron responsiveness to single whisker stimulation.

Gephyrin is the main scaffolding protein at inhibitory postsynaptic sites, and its clusters are the signaling hubs where several molecular pathways converge. Post-translational modifications (PTMs) of gephyrin alter GABAA receptor clustering at the synapse, but it is unclear how this affects neuronal activity at the circuit level. We assessed the contribution of gephyrin PTMs to microcircuit activity in the mouse barrel cortex by slice electrophysiology and in vivo two-photon calcium imaging of layer 2/3 (L2/3) pyramidal cells during single-whisker stimulation. Our results suggest that, depending on the type of gephyrin PTM, the neuronal activities of L2/3 pyramidal neurons can be differentially modulated, leading to changes in the size of the neuronal population responding to the single-whisker stimulation. Furthermore, we show that gephyrin PTMs have their preference for selecting synaptic GABAA receptor subunits. Our results identify an important role of gephyrin and GABAergic postsynaptic sites for cortical microcircuit function during sensory stimulation.

Phenols and GABAA receptors: from structure and molecular mechanisms action to neuropsychiatric sequelae.

γ-Aminobutyric acid type A receptors (GABAARs) are members of the pentameric ligand-gated ion channel (pLGIC) family, which are widespread throughout the invertebrate and vertebrate central nervous system. GABAARs are engaged in short-term changes of the neuronal concentrations of chloride (Cl-) and bicarbonate (HCO3 -) ions by their passive permeability through the ion channel pore. GABAARs are regulated by various structurally diverse phenolic substances ranging from simple phenols to complex polyphenols. The wide chemical and structural variability of phenols suggest similar and different binding sites on GABAARs, allowing them to manifest themselves as activators, inhibitors, or allosteric ligands of GABAAR function. Interest in phenols is associated with their great potential for GABAAR modulation, but also with their subsequent negative or positive role in neurological and psychiatric disorders. This review focuses on the GABAergic deficit hypotheses during neurological and psychiatric disorders induced by various phenols. We summarize the structure-activity relationship of general phenol groups concerning their differential roles in the manifestation of neuropsychiatric symptoms. We describe and analyze the role of GABAAR subunits in manifesting various neuropathologies and the molecular mechanisms underlying their modulation by phenols. Finally, we discuss how phenol drugs can modulate GABAAR activity via desensitization and resensitization. We also demonstrate a novel pharmacological approach to treat neuropsychiatric disorders via regulation of receptor phosphorylation/dephosphorylation.

Why Can Modulation of α6-Containing GABAA Receptors Reduce the Symptoms of Multiple Neuropsychiatric Disorders?

α6-containing GABAA receptors (α6GABAARs) are strongly expressed in cerebellar granule cells, where they mediate a correctly timed and precise coordination of all muscle groups that execute behavior and protect the brain from information overflow. Recently, it was demonstrated that positive modulators with a high selectivity for α6GABAARs (α6-modulators) can reduce the symptoms of multiple neuropsychiatric disorders in respective animal models to an extent comparable with established clinical therapeutics. Here, these incredible findings are discussed and explained. So far, the beneficial actions of α6-modulators and their lack of side effects have only been demonstrated in animal models of the respective disorders. Preclinical studies have demonstrated their suitability for further drug development. Future human studies have to investigate their safety and possible side effects, and to clarify to which extent individual symptoms of the respective disorders can be reduced by α6-modulators in patients during acute and chronic dosing. Due to their broad therapeutic potential, α6-modulators might become a valuable new treatment option for multiple neuropsychiatric disorders.

Vascular effects of midazolam, flumazenil, and a novel imidazobenzodiazepine MP-III-058 on isolated rat aorta.

Hypotensive influences of benzodiazepines and other GABAA receptor ligands, recognized in clinical practice, seem to stem from the existence of "vascular" GABAA receptors in peripheral blood vessels, besides any mechanisms in the central and peripheral nervous systems. We aimed to further elucidate the vasodilatatory effects of ligands acting through GABAA receptors. Using immunohistochemistry, the rat aortic smooth muscle layer was found to express GABAA γ2 and α1-5 subunit proteins. To confirm the role of "vascular" GABAA receptors, we investigated the vascular effects of standard benzodiazepines, midazolam, and flumazenil, as well as the novel compound MP-III-058. Using two-electrode voltage clamp electrophysiology and radioligand binding assays, MP-III-058 was found to have modest binding but substantial functional selectivity for α5ß3γ2 over other αxß3γ2 GABAA receptors. Tissue bath assays revealed comparable vasodilatory effects of MP-III-058 and midazolam, both of which at 100 µmol/L concentrations had efficacy similar to prazosin. Flumazenil exhibited weak vasoactivity per se, but significantly prevented the relaxant effects of midazolam and MP-III-058. These studies indicate the existence of functional GABAA receptors in the rat aorta, where ligands exert vasodilatory effects by positive modulation of the benzodiazepine binding site, suggesting the potential for further quest for leads with optimized pharmacokinetic properties as prospective adjuvant vasodilators.

4-Phenylbutyrate promoted wild-type γ-aminobutyric acid type A receptor trafficking, reduced endoplasmic reticulum stress, and mitigated seizures in Gabrg2+/Q390X mice associated with Dravet syndrome.

OBJECTIVE: γ-Aminobutyric acid type A (GABAA ) receptor subunit gene mutations are major causes of various epilepsy syndromes, including severe kinds such as Dravet syndrome. Although the GABAA receptor is a major target for antiseizure medications, treating GABAA receptor mutations with receptor channel modulators is ineffective. Here, we determined the effect of a novel treatment with 4-phenylbutyrate (PBA) in Gabrg2+/Q390X knockin mice associated with Dravet syndrome. METHODS: We used biochemistry in conjunction with differential tagging of the wild-type and the mutant alleles, live brain slice surface biotinylation, microsome isolation, patch-clamp whole-cell recordings, and video-monitoring synchronized electroencephalographic (EEG) recordings in Gabrg2+/Q390X mice to determine the effect of PBA in vitro with recombinant GABAA receptors and in vivo with knockin mice. RESULTS: We found that PBA reduced the mutant γ2(Q390X) subunit protein aggregates, enhanced the wild-type GABAA receptor subunits' trafficking, and increased the membrane expression of the wild-type receptors. PBA increased the current amplitude of GABA-evoked current in human embryonic kidney 293T cells and the neurons bearing the γ2(Q390X) subunit protein. PBA also proved to reduce endoplasmic reticulum (ER) stress caused by the mutant γ2(Q390X) subunit protein, as well as mitigating seizures and EEG abnormalities in the Gabrg2+/Q390X mice. SIGNIFICANCE: This research has unveiled a promising and innovative approach for treating epilepsy linked to GABAA receptor mutations through an unconventional antiseizure mechanism. Rather than directly modulating the affected mutant channel, PBA facilitates the folding and transportation of wild-type receptor subunits to the cell membrane and synapse. Combining these findings with our previous study, which demonstrated PBA's efficacy in restoring GABA transporter 1 (encoded by SLC6A1) function, we propose that PBA holds significant potential for a wide range of genetic epilepsies. Its ability to target shared molecular pathways involving mutant protein ER retention and impaired protein membrane trafficking suggests broad application in treating such conditions.

Efficacy and safety of Dimdazenil in adults with insomnia disorder: results from a multicenter, randomized, double-blind, placebo-controlled phase III trials.

STUDY OBJECTIVES: To evaluate the efficacy and safety of Dimdazenil, a novel partial positive allosteric modulator for GABAA receptor in adults with insomnia disorder. METHODS: This was a 2-week, multicenter, randomized, double-blind, placebo-controlled, parallel-group phase III study of Dimdazenil. The primary efficacy outcome was total sleep time (TST) analyzed by polysomnography (PSG) on day 13/14. Latency to persistent sleep (LPS), sleep efficiency (SE), and wake after sleep onset (WASO) were analyzed in the same way by polysomnography (PSG). The other secondary outcomes including the average subjective sleep latency (sSL), subjective TST (sTST), subjective SE (sSE), subjective WASO (sWASO), and subjective number of awakenings (sNAW) were analyzed from sleep diary data, and the insomnia severity index (ISI) was also assessed. Treatment-emergent adverse events (TEAEs) were monitored throughout the study. RESULTS: A total of 546 participants with insomnia (age ≥18 years) were randomized (2:1), received treatment with an oral dose of Dimdazenil (2.5 mg) or placebo, and analyzed. Compared to baseline and placebo, Dimdazenil demonstrated significant improvements in PSG measures, increased TST (71.09, 31.68 minutes, respectively; both p < 0.001), increased SE (13.26%, 5.55%, respectively; both < 0.001), reduced WASO (49.67, 20.16 minutes, respectively; both p < 0.001), and reduced LPS (21.65 minutes, p < 0.001; 6.46 minutes, p = 0.023). Compared to placebo, Dimdazenil also improved key self-reported measures of sTST (18.33 minutes, p < 0.001), sWASO (14.60 minutes, p < 0.001), sSL (4.23 minutes, p < 0.001), sSE (2.97%, p < 0.001), and sNAW (0.29, p < 0.001). Participants treated with Dimdazenil reported a significant improvement in ISI. Dimdazenil was well tolerated. The majority of TEAEs were mild or moderate. There were no clinically relevant treatment-related serious AEs and no deaths. CONCLUSIONS: Dimdazenil of 2.5 mg provided significant benefit on sleep maintenance and sleep onset in individuals with insomnia disorder versus placebo, with a favorable safety profile and was well tolerated. CLINICAL TRIAL INFORMATION: A multicenter, randomized, double-blind phase III clinical study evaluating the efficacy and safety of EVT201 capsules compared to placebo in patients with insomnia disorders (http://www.chinadrugtrials.org), with the number of CTR20201068.

Efficacy and safety of Dimdazenil in the adult insomnia patients: a phase II randomized, multicenter, double-blind, placebo-controlled, and parallel-group study.

STUDY OBJECTIVES: To evaluate the efficacy and safety of Dimdazenil, a positive allosteric modulator with selectivity for α1, α5 subunit-containing GABAA receptors, on sleep variables in patients with insomnia disorder. METHODS: In this randomized, double-blind, placebo-controlled trial, adults (18-65 years) with insomnia disorder were randomized (1:1:1:1 to receive daily oral placebo, Dimdazenil (1.5, 2.5, or 5 mg) for 14 days. The primary efficacy outcome was the total sleep time (TST) on day 1/2 and day 13/14, measured by polysomnography. The secondary outcome measures included (1) latency to persistent sleep (LPS), sleep efficiency (SE), wake after sleep onset (WASO) and number of awakenings (NAW) on days 1/2 and day 13/14, and (2) the average subjective sleep latency (sSL), total sleep time (sTST), wake after sleep onset (sWASO) and number of awakenings (sNAW) recorded in sleep diary and sleep questionnaire, and the evaluation of insomnia severity index. Rebound insomnia, withdrawal, and treatment-emergent adverse events were also assessed. RESULTS: Of 569 patients screened, 288 (76.4% female) were randomized and received one dose. For the primary outcomes, TST was significantly improved in the Dimdazenil 1.5, 2.5, and 5 mg group compared with the placebo group at day 1/2, and significantly improved in the Dimdazenil 2.5 and 5 mg groups compared with the placebo group at day 13/14. The Least Squares Means (standard errors) and 95% Confidence Intervals for the three active doses compared to placebo are 25.5 (8.31), (9.16, 41.89) for the 1.5 mg dose; 17.4 (8.19), (1.29, 33.55) for the 2.5 mg dose; 22.8 (8.15), (6.72, 38.80) for the 5 mg dose on day 1/2. Corresponding data on day 13/14 are 7.6 (8.07), (-8.24, 23.53) and 19.3 (8.06), (3.43, 35.17) and 18.2 (7.95), (2.49, 33.80). LPS was significantly reduced in the Dimdazenil 5 mg group compared with the placebo group on day 1/2. SE was significantly improved in the Dimdazenil 1.5 and 5 mg group compared with the placebo group at day 1/2. In the subjective sleep parameters, sSL on average was significantly lower in the Dimdazenil 1.5, 2.5, and 5 mg groups compared with the placebo group. sTST on average was significantly higher in the Dimdazenil 1.5, 2.5, and 5 mg groups compared with the placebo group. The most common TEAEs were dizziness, vertigo, and weakness with no clinically relevant treatment-related serious adverse events. CONCLUSIONS: Dimdazenil of 1.5, 2.5, and 5 mg improved certain objective and subjective sleep outcomes in people with insomnia disorder, with a favorable safety profile. These findings suggested that Dimdazenil may represent a promising new treatment for insomnia disorder, a prevalent condition with limited effective and safe treatments available. CLINICAL TRIAL INFORMATION: A multicenter, randomized, double-blind, multidose, placebo parallel controlled phase II clinical study of EVT201 in the treatment of insomnia disorders (http://www.chinadrugtrials.org), with the number of CTR20150664.

Disinhibition does not play a role in endomorphin-2-induced changes in inspiratory motoneuron output produced by in vitro neonatal rat preparations.

Low level activation of mu-opioid receptors (MORs) in neonatal rat brainstem-spinal cord preparations increases inspiratory burst amplitude recorded on cervical spinal roots. We tested whether: (1) MOR activation with an endogenous ligand, such as endomorphin-2, increases inspiratory burst amplitude, (2) disinhibition of GABAergic or glycinergic inhibitory synaptic transmission is involved, and (3) inflammation alters endomorphin-2 effects. Using neonatal rat (P0-P3) brainstem-spinal cord preparations, bath-applied endomorphin-2 (10-200 nM) increased inspiratory burst amplitude and decreased burst frequency. Blockade of GABAA receptors (picrotoxin), glycine receptors (strychnine), or both (picrotoxin and strychnine) did not abolish endomorphin-2-induced effects. In preparations isolated from neonatal rats injected 3 h previously with lipopolysaccharide (LPS, 0.1 mg/kg), endomorphin-2 continued to decrease burst frequency but abolished the burst amplitude increase. Collectively, these data indicate that disinhibition of inhibitory synaptic transmission is unlikely to play a role in endomorphin-2-induced changes in inspiratory motor output, and that different mechanisms underlie the endomorphin-2-induced increases in inspiratory burst amplitude and decreases in burst frequency.