Preclinical characterization of zuranolone (SAGE-217), a selective neuroactive steroid GABAA receptor positive allosteric modulator

Zuranolone (SAGE-217) is a novel, synthetic, clinical stage neuroactive steroid GABAA receptor positive allosteric modulator designed with the pharmacokinetic properties to support oral daily dosing. In vitro, zuranolone enhanced GABAA 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, α1β2γ2, 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 GABAA receptors to the cell surface. In vivo, zuranolone exhibited potent activity, indicating its ability to modulate GABAA 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 GABAA receptor positive allosteric modulator with drug-like properties and CNS exposure in preclinical models. Recent clinical data support the therapeutic promise of neuroactive steroid GABAA 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

Examination of the role of PTEN in ionotropic glutamate receptor expression

The phosphatase Pten negatively regulates PI3K/Akt/mTOR signaling, a pathway critical for cell growth and protein synthesis. Germline PTEN mutations are implicated in seizure and autism, suggesting that alterations in PTEN affect neuronal function and development. Several brain-specific conditional Pten knockout (KO) mice exhibit enlarged brains, neuronal hypertrophy and increased seizure susceptibility, which may be indicative of altered glutamate receptor function. mTOR inhibition can suppress seizure activity observed in these Pten mutant models, revealing the importance of mTOR signaling in Pten-dependent phenotypes. To better understand how Pten may regulate neuronal excitability, ionotropic glutamate receptor expression was examined in NEX-Pten homozygous KO mice, which lack Pten in nearly all forebrain excitatory neurons. Biochemical analyses revealed alterations in select NMDA and AMPA receptor subunit protein levels in the forebrains of newly born NEX-Pten KO mice, suggesting developmental loss of Pten can affect synaptic proteins important for neurotransmission. Similarly, initial analysis of CaMKIIα-Pten KO mice indicated postnatal loss of Pten in excitatory neurons may also alter NMDA receptor subunits in the cortex, but not the hippocampus, underscoring the importance of Pten for proper synaptic protein expression. To further characterize the effects of Pten deletion on glutamate receptor subunit expression, dissociated cortical neuronal cultures were used to evaluate how chronic Pten deficiency alters glutamate receptors over time. NMDA receptor abnormalities were modest and transient, indicating that alterations in glutamate receptor subunits may normalize due to homeostatic mechanisms. Further, pharmacological inhibition of PI3K reduced select NMDA receptor subunits in dissociated cortical cultures. Together, these data suggest that in vivo activation of PI3K through loss of Pten leads to selective increases in NMDA receptor subunits in cortical neurons, but not hippocampal neurons, since no alterations were detected in this region. Additional studies with rapamycin and second generation mTOR inhibitors are required to determine how mTOR function contributes to the glutamate receptor phenotype. The NEX-Pten model demonstrates that Pten may be crucial in controlling neuronal excitability at the synapse. Dysregulation of these functions may underlie some of the phenotypes associated with PTEN mutations in the human population.Ph. D.Includes bibliographical referencesby Tatiana Maria Kazdoba-Leac

Behavioral phenotypes of genetic mouse models of autism

More than a hundred de novo single gene mutations and copy-number variants have been implicated in autism, each occurring in a small subset of cases. Mutant mouse models with syntenic mutations offer research tools to gain an understanding of the role of each gene in modulating biological and behavioral phenotypes relevant to autism. Knockout, knockin and transgenic mice incorporating risk gene mutations detected in autism spectrum disorder and comorbid neurodevelopmental disorders are now widely available. At present, autism spectrum disorder is diagnosed solely by behavioral criteria. We developed a constellation of mouse behavioral assays designed to maximize face validity to the types of social deficits and repetitive behaviors that are central to an autism diagnosis. Mouse behavioral assays for associated symptoms of autism, which include cognitive inflexibility, anxiety, hyperactivity, and unusual reactivity to sensory stimuli, are frequently included in the phenotypic analyses. Over the past 10 years, we and many other laboratories around the world have employed these and additional behavioral tests to phenotype a large number of mutant mouse models of autism. In this review, we highlight mouse models with mutations in genes that have been identified as risk genes for autism, which work through synaptic mechanisms and through the mTOR signaling pathway. Robust, replicated autism-relevant behavioral outcomes in a genetic mouse model lend credence to a causal role for specific gene contributions and downstream biological mechanisms in the etiology of autism

Evaluation of the neuroactive steroid ganaxolone on social and repetitive behaviors in the BTBR mouse model of autism

RationaleAbnormalities in excitatory/inhibitory neurotransmission are hypothesized to contribute to autism spectrum disorder (ASD) etiology. BTBR T (+) Itpr3 (tf) /J (BTBR), an inbred mouse strain, displays social deficits and repetitive self-grooming, offering face validity to ASD diagnostic symptoms. Reduced GABAergic neurotransmission in BTBR suggests that GABAA receptor positive allosteric modulators (PAMs) could improve ASD-relevant BTBR phenotypes. The neuroactive steroid ganaxolone acts as a PAM, displaying anticonvulsant properties in rodent epilepsy models and an anxiolytic-like profile in the elevated plus-maze.ObjectivesWe evaluated ganaxolone in BTBR and C57BL/6J mice in standardized assays for sociability and repetitive behaviors. Open field and anxiety-related behaviors were tested as internal controls and for comparison with the existing neuroactive steroid literature.ResultsGanaxolone improved aspects of social approach and reciprocal social interactions in BTBR, with no effect on repetitive self-grooming, and no detrimental effects in C57BL/6J. Ganaxolone increased overall exploratory activity in BTBR and C57BL/6J in the open field, social approach, and elevated plus-maze, introducing a confound for the interpretation of social improvements. Allopregnanolone and diazepam similarly increased total entries in the elevated plus-maze, indicating that behavioral activation may be a general property of GABAA receptor PAMs in these strains.ConclusionsGanaxolone shows promise for improving sociability. In addition, ganaxolone, as well as other GABAA receptor PAMs, enhanced overall BTBR activity. The translational implications of specific sociability improvements and nonspecific behavioral activation by ganaxolone in the BTBR model remain to be determined. Future studies to explore whether PAMs provide a novel profile with unique benefits for ASD treatment will be worthwhile