We investigated the correlation between increased gene copy number of gamma aminobutyric acid type A (GABAA) receptor α5-containing subunits and electrophysiological and behavioral phenotypes in a mouse model of Dup15q syndrome (15q dup) and tested the hypothesis that selectively inhibiting the activity of GABAA-α5 receptors may have therapeutic effects. Dup15q syndrome is a rare neurodevelopmental disorder caused by copy number gains of the 15q11.2-q13.1 chromosomal region, which includes UBE3A and a cluster of three genes (GABRA5, GABRB3, and GABRG3) encoding GABAA receptor subunits, all of which are critical for neural development and function. Most affected children display hypotonia, motor delays, intellectual disability, and epilepsy, as well as a characteristic electroencephalography (EEG) beta-band phenotype. There is no disease-modifying therapy available. Autoradiography showed increased density of GABAA-α5 receptors in the brains of 15q dup mice, while electrophysiology revealed enhanced GABAergic transmission in hippocampal slices from these mice. A GABAA-α5 negative allosteric modulator, RO4938581, decreased inhibitory synaptic charge transfer in 15q dup hippocampal slices. The behavioral analyses confirmed inflexibility in learning and abnormal social behaviors in 15q dup mice, and both phenotypes were normalized following chronic treatment with RO4938581. EEG recordings showed increased beta-power in 15q dup mice – which resembled the spectral signature of subjects with Dup15q – and was partially normalized following RO4938581 treatment. Our results suggest that excessive expression and function of the GABAA-α5 receptor subtype plays a key role in the pathophysiology of Dup15q and GABAA-α5 NAMs may represent a potential precision medicine therapeutic option.

Background:Alterations in the GABAergic system contribute to the pathophysiology of neurodevelopmental disorders, including autism spectrum disorder (ASD) and Angelman syndrome (AS), particularly in cases involving large deletions in the 15q11-13 region. Positive modulation of GABAA-α5 receptors may provide a novel therapeutic approach without the typical side effects associated with non-selective GABAA positive allosteric modulators such as diazepam. Methods:Alogabat was assessed for binding and functional activity at GABAA-α5β3γ2 receptors in vitro and in electrophysiological studies using hippocampal slices. In vivo studies in rodents included receptor occupancy (RO) using a selective GABAA-α5 tracer (autoradiography), pharmacological MRI, and electroencephalography (EEG). Alogabat was evaluated for its effects on the repetitive behavior phenotype in BTBR and contactin-associated protein-like 2 (Cntnap2-/-) knockout mice, seizure models, cognitive performance in rats, and rotarod performance following combination treatment with diazepam. Results:Alogabat is a potent positive allosteric modulator of GABAA-α5 receptors, with binding and functional selectivity. Receptor occupancy studies provided direct proof of dose-dependent target engagement. Functional circuit modulation was demonstrated by dose-dependent regional perfusion changes in pharmacological MRI and changes in EEG theta- and beta-band power in rats. At >50% GABAA-α5 receptor occupancy, alogabat normalized elevated self-grooming behavior in both Cntnap2-/- and BTBR mice and exhibited antiepileptic activity in rats. Alogabat did not impair cognition in wildtype rats at GABAA-α5 receptor occupancy up to 75%, although impairment occurred at higher doses, probably due to increased activity at other receptor subtypes and/or saturation of α5 receptors. Alogabat did not worsen diazepam-induced impairment on the rotarod test. Conclusion:Alogabat showed beneficial effects in mouse models relevant to neurodevelopmental disorders and anti-seizure activity at doses that did not produce cognitive, sedative, or motoric side effects.

Abstract Background There is compelling evidence that dysfunction of the GABAergic system, the main inhibitory neurotransmitter system in the brain, contributes to the pathophysiology of neurodevelopmental disorders including autism spectrum disorder and deletion Angelman syndrome (1, 2, 3, 4). Genetic studies highlight the critical role of GABAA receptor gene dosage and function within the 15q11-13 chromosomal region in the etiology of these conditions (5, 6, 7, 8). This region encompasses several genes including UBE3A and GABRB3, GABRA5, GABRG3 encoding the β3, α5, and g3 GABAA receptor subunits, respectively. These subunits together with the g2 subunit co-assemble to form the GABAA- α5 receptor subtype (9). Therefore, positive modulation of GABAA-α5 receptors may provide a novel therapeutic approach to restore deficient GABAergic signaling without the typical side effects of non- selective GABAA positive allosteric modulators (PAM) i.e., diazepam. Aims & Objectives Assess the in vitro and in vivo pharmacological profile of alogabat, a novel small molecule, selective GABAA-α5 receptor positive allosteric modulator. Provide preclinical proof of concept data to support clinical studies in ASD and Angelman syndrome. Methods Alogabat was evaluated for selective binding and functional activity in vitro at GABAA-α5β3g2 receptors, including electrophysiological studies in hippocampal slices. In vivo studies included receptor occupancy (RO) using a selective GABAA-α5 tracer (autoradiography), pharmacological magnetic resonance imaging (phMRI) and EEG in rodents. Alogabat was assessed on the repetitive behavior phenotype in BTBR and contactin-associated protein-like 2 knockout (Cntnap2-/-) mice, on seizure models and cognitive performance in rats, and on rotarod performance following a combination treatment with diazepam. Results Alogabat is a potent PAM of the GABAA-α5 receptor with binding and functional selectivity. RO studies provided direct proof of dose-dependent target engagement. Functional circuit modulation was demonstrated by dose-dependent regional perfusion changes in phMRI and change in EEG theta and beta band power. Alogabat at >50% RO at GABAA-α5 normalized elevated self-grooming in both Cntnap2-/- and BTBR mice and exhibited antiepileptic activity in rats. Alogabat did not impair cognition in rats at RO up to 88%, although impairment occurred at higher doses probably due to decreased α-subunit selectivity. Alogabat did not worsen diazepam-induced rotarod impairment. Conclusions Alogabat showed beneficial effects in mouse models relevant for NDD, as well as anti- seizure activity, at doses without cognitive, sedative, and motoric side effects. In addition, translational biomarkers were identified to guide clinical studies: PET, phMRI, EEG. References 1. Braat S, Kooy RF. The GABAA receptor as a therapeutic target for neurodevelopmental disorders. Neuron 2015;86:1119-30. 2. Ali Rodriguez R, Joya C, Hines RM. Ribs of inhibitory synaptic dysfunction in the umbrella of neurodevelopmental disorders. Front Mol Neurosci. 2018;11:132. 3. Tang X, Jaenisch R, Sur M. The role of GABAergic signalling in neurodevelopmental disorders. Nature Reviews Neuroscience. 2021 (5):290-307. 4. Zhao H, Mao X, Zhu C, et al. GABAergic system dysfunction in autism spectrum disorders. Front Cell Dev Biol. 2022; 9:781327. 5. Warrier V, Baron-Cohen S, Chakrabarti B. Genetic variation in GABRB3 is associated with Asperger syndrome and multiple endophenotypes relevant to autism. Mol Autism 2013;4:48. 6. Zurek AA, Kemp SW, Aga Z, et al. a5GABAA receptor deficiency causes autism-like behaviors. Ann Clin Transl Neurol 2016;3:392-8. 7. Hogart A, Wu D, LaSalle JM, et al. The comorbidity of autism with the genomic disorders of chromosome 15q11.2-q13. Neurobiol Dis 2010;38:181-91. 8. Frohlich J, Miller MT, Bird LM, et al. Electrophysiological phenotype in Angelman syndrome differs between genotypes. Biol Psychiatry 2019; 85(9):752-9. 9. Sur C, Fresu L, Howell O, et al. Autoradiographic localization of alpha5 subunit-containing GABAA receptors in rat brain. Brain Res 1999; 822:265-70.

BACKGROUND:Impairments in behavioral pattern separation (BPS)-the ability to distinguish between similar contexts or experiences-contribute to memory interference and overgeneralization seen in many neuropsychiatric conditions, including depression, anxiety, PTSD, dementia, and age-related cognitive decline. While BPS relies on the dentate gyrus and is sensitive to changes in adult hippocampal neurogenesis (AHN), its significance as a pharmacological target has not been tested. METHODS:In this study, we applied a human neural stem cell high-throughput screening cascade to identify compounds that increase human neurogenesis. One compound with a favorable profile, RO6871135, was then tested in BPS in mice. RESULTS:Chronic treatment with RO6871135, 7.5 mg/kg increased AHN and improved BPS in a fear discrimination task in both young and aged mice. RO6871135 treatment also lowered innate anxiety-like behavior, which was more apparent in mice exposed to chronic corticosterone. Ablation of AHN by hippocampal irradiation supported a neurogenesis-dependent mechanism for RO6871135-induced improvements in BPS. To identify possible mechanisms of action, in vitro and in vivo kinase inhibition and chemical proteomics assays were performed. These tests indicated that RO6871135 inhibited CDK8, CDK11, CaMK2a, CaMK2b, MAP2K6, and GSK3b. An analog compound also demonstrated high affinity for CDK8, CaMK2a, and GSK3b. CONCLUSIONS:These studies demonstrate a method for empirical identification and preclinical testing of novel neurogenic compounds that can improve BPS, and points to possible novel mechanisms that can be interrogated for the development of new therapies to improve specific endophenotypes such as impaired BPS.

In mice, adult hippocampal neurogenesis is elevated by interventions that improve affect and cognition. Here, we test whether chronic treatment with a novel neurogenic compound, RO6871135, can alter behaviors relevant to cognitive, anxiety, mood, and trauma-related disorders. We also test which behavioral effects are neurogenesis-dependent by ablating neurogenesis with irradiation.

The K‐Cl cotransporter KCC2 is essential in the development of the “GABA switch” that produces a change in neuronal responses to GABA signaling from excitatory to inhibitory early in brain development, and alterations in this progression have previously been hypothesized to play a causal role in autism spectrum disorder (ASD). We investigated the KCC2b (Slc12a5) heterozygous knockout mouse using a battery of rodent behavioral tests relevant to core and comorbid ASD symptoms. Compared to wild‐type littermates, KCC2+/− mice were normal in standard measures of locomotor activity, grooming and digging behaviors, and social, vocalization, and anxiety‐like behaviors. However, KCC2+/− mice exhibited increased social dominance behaviors and increased amplitude of spontaneous postsynaptic currents in the medial prefrontal cortex (PFC) that were previously implicated in governing social hierarchy and dominance behaviors. Treatment of wild‐type mouse brain slices with the KCC2 inhibitor VU0240511 increased the amplitude and frequency of excitatory postsynaptic currents, partially recapitulating the phenotype of KCC2+/− mice. These findings indicate that the activity of KCC2 plays a role in social dominance, in parallel with effects on PFC signaling, further suggesting that KCC2 function has some relevance to social behavior but without the breadth of impact on autism‐like behavior suggested by previous studies. Further testing could assess whether KCC2 alters other circuits and whether additional factors such as environmental insults may precipitate autism‐related behavioral phenotypes. Autism Research 2019, 12: 732–743. © 2019 International Society for Autism Research, Wiley Periodicals, Inc.

Study Objectives:Although recent innovations have enabled modification of the rat genome, it is unclear whether enhanced utility of rodents as human disease models will result. We compared electroencephalogram (EEG) and behavioral phenotypes of rats and mice with homozygous deletion of Cntnap2, a gene associated with cortical dysplasia-focal epilepsy (CDFE) and autism spectrum disorders (ASD).Methods:Male contactin-associated protein-like 2 (Cntnap2) knockout (KO) and wild-type (WT) rats and male Cntnap2 KO and WT mice were implanted with telemeters to record EEG, electromyogram, body temperature, and locomotor activity. Animals were subjected to a test battery for ASD-related behaviors, followed by 24-hr EEG recordings that were analyzed for sleep-wake parameters and subjected to spectral analysis.Results:Cntnap2 KO rats exhibited severe motor seizures, hyperactivity, and increased consolidation of wakefulness and REM sleep. By contrast, Cntnap2 KO mice demonstrated absence seizure-like events, hypoactivity, and wake fragmentation. Although seizures observed in Cntnap2 KO rats were more similar to those in CDFE patients than in KO mice, neither model fully recapitulated the full spectrum of disease symptoms. However, KOs in both species had reduced spectral power in the alpha (9-12 Hz) range during wake, suggesting a conserved EEG biomarker.Conclusions:Deletion of Cntnap2 impacts similar behaviors and EEG measures in rats and mice, but with profound differences in nature and phenotypic severity. These observations highlight the importance of cross-species comparisons to understand conserved gene functions and the limitations of single- species models to provide translational insights relevant to human diseases.

Study Objectives Neuroligin-3 (NLGN3) is one of the many genes associated with autism spectrum disorder (ASD). Sleep dysfunction is highly prevalent in ASD, but has not been rigorously examined in ASD models. Here, we evaluated sleep/wake physiology and behavioral phenotypes of rats with genetic ablation of Nlgn3. Methods Male Nlgn3 knockout (KO) and wild-type (WT) rats were assessed using a test battery for ASD-related behaviors and also implanted with telemeters to record the electroencephalogram (EEG), electromyogram, body temperature, and locomotor activity. 24-h EEG recordings were analyzed for sleep/wake states and spectral composition. Results Nlgn3 KO rats were hyperactive, exhibited excessive chewing behavior, and had impaired prepulse inhibition to an auditory startle stimulus. KO rats also spent less time in non-rapid eye movement (NREM) sleep, more time in rapid eye movement (REM) sleep, exhibited elevated theta power (4-9 Hz) during wakefulness and REM, and elevated delta power (0.5-4 Hz) during NREM. Beta (12-30 Hz) power and gamma (30-50 Hz) power were suppressed across all vigilance states. Conclusions The sleep disruptions in Nlgn3 KO rats are consistent with observations of sleep disturbances in ASD patients. The EEG provides objective measures of brain function to complement rodent behavioral analyses and therefore may be a useful tool to study ASD.

Autism spectrum disorder comprises several neurodevelopmental conditions presenting symptoms in social communication and restricted, repetitive behaviors. A major roadblock for drug development for autism is the lack of robust behavioral signatures predictive of clinical efficacy. To address this issue, we further characterized, in a uniform and rigorous way, mouse models of autism that are of interest because of their construct validity and wide availability to the scientific community. We implemented a broad behavioral battery that included but was not restricted to core autism domains, with the goal of identifying robust, reliable phenotypes amenable for further testing. Here we describe comprehensive findings from two known mouse models of autism, obtained at different developmental stages, using a systematic behavioral test battery combining standard tests as well as novel, quantitative, computer-vision based systems. The first mouse model recapitulates a deletion in human chromosome 16p11.2, found in 1% of individuals with autism. The second mouse model harbors homozygous null mutations in Cntnap2, associated with autism and Pitt-Hopkins-like syndrome. Consistent with previous results, 16p11.2 heterozygous null mice, also known as Del(7Slx1b-Sept1)4Aam weighed less than wild type littermates displayed hyperactivity and no social deficits. Cntnap2 homozygous null mice were also hyperactive, froze less during testing, showed a mild gait phenotype and deficits in the three-chamber social preference test, although less robust than previously published. In the open field test with exposure to urine of an estrous female, however, the Cntnap2 null mice showed reduced vocalizations. In addition, Cntnap2 null mice performed slightly better in a cognitive procedural learning test. Although finding and replicating robust behavioral phenotypes in animal models is a challenging task, such functional readouts remain important in the development of therapeutics and we anticipate both our positive and negative findings will be utilized as a resource for the broader scientific community.

INTRODUCTION:Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental condition characterized by core differences and impairments in social behavioral functioning. There are no approved medications for improving social cognition and behavior in ASD, and the underlying mechanisms needed to discover safer, more effective medications are unclear.DISCUSSION:In this review, we diagram the basic neurocircuitry governing social behaviors in order to provide a neurobiological framework for the origins of the core social behavioral symptoms of ASD. In addition, we discuss recent technological innovations in research tools that provide unprecedented observation of cellular morphology and activity deep within the intact brain and permit the precise control of discrete brain regions and specific cell types at distinct developmental stages.CONCLUSIONS:The use of new technologies to reveal the neural circuits underlying social behavioral impairments associated with ASD is advancing our understanding of the brain changes underlying ASD and enabling the discovery of novel and effective therapeutic interventions.

PURPOSE: Autism spectrum disorder (ASD) is a neuropsychiatric disorder characterised by stereotyped behaviours and impairments in communication and social interactions. Despite its neurodevelopmental origin and high incidence (>1 in 100 children), the aetiology and pathology of ASD are still largely unknown. Substantial heterogeneity of clinical manifestations of ASD has been reported as a major obstacle in further uncovering disease aetiology and specific biomarkers and therefore animal models that mimic specific facets of the disease, i.e. endophenotypes, have been developed. These animal models of ASD have been assessed mainly by behavioural testing and/or ex vivo structural MRI. Here we leveraged neurofunctional and neurochemical appraisals with the goal of bridging the gap between genetic/molecular findings and ASD-related behavioural phenotypes. A potentially translational approach was taken with in vivo fMRI and MRS that were carried out in five distinct mouse models of idiopathic ASD ranging from inbred strains and environmental challenges to specific gene mutations and copy number variants.