Neurosteroids are synthesized within the brain and act as endogenous anxiolytic, anticonvulsant, hypnotic, and sedative agents, actions that are principally mediated via their ability to potentiate phasic and tonic inhibitory neurotransmission mediated by γ-aminobutyric acid type A receptors (GABA A Rs). Although neurosteroids are accepted allosteric modulators of GABA A Rs, here we reveal they exert sustained effects on GABAergic inhibition by selectively enhancing the trafficking of GABA A Rs that mediate tonic inhibition. We demonstrate that neurosteroids potentiate the protein kinase C-dependent phosphorylation of S443 within α4 subunits, a component of GABA A R subtypes that mediate tonic inhibition in many brain regions. This process enhances insertion of α4 subunit-containing GABA A R subtypes into the membrane, resulting in a selective and sustained elevation in the efficacy of tonic inhibition. Therefore, the ability of neurosteroids to modulate the phosphorylation and membrane insertion of α4 subunit-containing GABA A Rs may underlie the profound effects these endogenous signaling molecules have on neuronal excitability and behavior.PKC | tonic current | receptor insertion | current rundown N eurosteroids are synthesized de novo in the brain from cholesterol, or steroid hormone precursors. Raising neurosteroid levels in the CNS causes anxiolysis, sedation/hypnosis, anticonvulsant action, and anesthesia and reduces depressivelike behaviors (1-3). Accordingly, dysregulation of neurosteroid signaling is associated with premenstrual dysphoric disorder, panic disorder, depression, schizophrenia, and bipolar disorder. Neurosteroids exert the majority of their actions via potentiating the activity of γ-aminobutyric acid receptors (GABA A Rs), which mediate the majority of fast synaptic inhibition in the adult brain. Accordingly, at low nanomolar concentrations they potentiate GABA-dependent currents, whereas at micromolar concentrations they directly activate GABA A Rs (4-8).GABA A Rs are Cl − -preferring pentameric ligand-gated ion channels that assemble from eight families of subunits: α(1-6), β(1-3), γ(1-3), δ, e, ө, π, and ρ(1-3) (9, 10). Receptor subtypes composed of α1-3βγ subunits largely mediate synaptic or phasic inhibition, whereas those constructed from α4-6β1-3, with or without γ/δ subunits, are principal determinants of tonic inhibition (11-13). Neurosteroids have been shown to bind GABA A Rs at an allosteric site distinct from that of GABA, benzodiazepines, or barbiturates (9, 14). Hosie et al. identified residues located within the transmembrane domain of GABA A R α and β subunits that are critical for the direct activation (α1-6; Threonine 236, β1-3; Tyrosine 284) and allosteric potentiation (α1-6 Asparagine 407, and α1-6 Glutamine 246) of neurosteroids (15-17). Accordingly, mutation of glutamine 241 (Q241) within the α1-6 subunits prevents allosteric potentiation of GABA A R composed of αβγ and αβδ subunits by neurosteroids (15,16).In addition to modulating channel gating, neurosteroids exert...

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Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABAARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABAARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2–1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development.

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The Tf-CPP liposomes showed efficient translocation of the anticancer drug across the brain endothelial barrier. In addition, an absolute and robust in vitro brain tumor model was successfully constructed to overcome the practical intricacies of developing a successful in vivo orthotopic brain tumor model.

Strategically designed amphiphilic invertible polymers (AIPs) are capable of (i) self-assembling into invertible micellar assemblies (IMAs) in response to changes in polarity of environment, polymer concentration, and structure, (ii) accommodating (solubilizing) substances that are otherwise insoluble in water, and (iii) inverting their molecular conformation in response to changes in the polarity of the local environment. The unique ability of AIPs to invert the molecular conformation depending on the polarity of the environment can be a decisive factor in establishing the novel stimuli-responsive mechanism of solubilized drug release that is induced just in response to a change in the polarity of the environment. The IMA capability to solubilize lipophilic drugs and deliver and release the cargo molecules by conformational inversion of polymer macromolecules in response to a change of the polarity of the environment was demonstrated by loading IMA with a phytochemical drug, curcumin. It was demonstrated that four sets of micellar vehicles based on different AIPs were capable of delivering the curcumin from water to an organic medium (1-octanol) by means of unique mechanism: AIP conformational inversion in response to changing polarity from polar to nonpolar. The IMAs are shown to be nontoxic against human cells up to a concentration of 10 mg/L. On the other hand, the curcumin-loaded IMAs are cytotoxic to breast carcinoma cells at this concentration, which confirms the potential of IMA-based vehicles in controlled delivery of poorly water-soluble drug candidates and release by means of this novel stimuli-responsive mechanism.

The accumulation of ␥-aminobutyric acid receptors (GABA A Rs) at the appropriate postsynaptic sites is critical for determining the efficacy of fast inhibitory neurotransmission. Although we know that the majority of synaptic GABA A R subtypes are assembled from ␣1-3, ␤, and ␥2 subunits, our understanding of how neurons facilitate their targeting to and stabilization at inhibitory synapses is rudimentary. To address these issues, we have created knock-in mice in which the pH-sensitive green fluorescent protein (GFP) and the Myc epitope were introduced to the extracellular domain of the mature receptor ␣2 subunit (pH␣2). Using immunoaffinity purification and mass spectroscopy, we identified a stable complex of 174 proteins that were associated with pH␣2, including other GABA A R subunits, and previously identified receptor-associated proteins such as gephyrin and collybistin. 149 of these proteins were novel GABA A R binding partners and included G-protein-coupled receptors and ion channel subunits, proteins that regulate trafficking and degradation, regulators of protein phosphorylation, GTPases, and a number of proteins that regulate their activity. Notably, members of the postsynaptic density family of proteins that are critical components of excitatory synapses were not associated with GABA A Rs. Crucially, we demonstrated for a subset of these novel proteins (including cullin1, ephexin, potassium channel tetramerization domain containing protein 12, mitofusin2, metabotropic glutamate receptor 5, p21-activated kinase 7, and Ras-related protein 5A) bind directly to the intracellular domains of GABA A Rs, validating our proteomic analysis. Thus, our experiments illustrate the complexity of the GABA A R proteome and enhance our understanding of the mechanisms neurons use to construct inhibitory synapses.

Zuranolone (SAGE-217) is a novel, synthetic, clinical stage neuroactive steroid GABA A receptor positive allosteric modulator designed with the pharmacokinetic properties to support oral daily dosing. In vitro , zuranolone enhanced GABA A receptor current at nine unique human recombinant receptor subtypes, including representative receptors for both synaptic (γ subunit-containing) and extrasynaptic (δ subunit-containing) configurations. At a representative synaptic subunit configuration, α 1 β 2 γ 2 , zuranolone potentiated GABA currents synergistically with the benzodiazepine diazepam, consistent with the non-competitive activity and distinct binding sites of the two classes of compounds at synaptic receptors. In a brain slice preparation, zuranolone produced a sustained increase in GABA currents consistent with metabotropic trafficking of GABA A receptors to the cell surface. In vivo , zuranolone exhibited potent activity, indicating its ability to modulate GABA A receptors in the central nervous system after oral dosing by protecting against chemo-convulsant seizures in a mouse model and enhancing electroencephalogram β-frequency power in rats. Together, these data establish zuranolone as a potent and efficacious neuroactive steroid GABA A receptor positive allosteric modulator with drug-like properties and CNS exposure in preclinical models. Recent clinical data support the therapeutic promise of neuroactive steroid GABA A receptor positive modulators for treating mood disorders; brexanolone is the first therapeutic approved specifically for the treatment of postpartum depression. Zuranolone is currently under clinical investigation for the treatment of major depressive episodes in major depressive disorder, postpartum depression, and bipolar depression.