Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by intellectual disabilities and a plethora of neuropsychiatric symptoms. FXS is the leading monogenic cause of autism spectrum disorder (ASD), which is defined clinically by repetitive and/or restrictive patterns of behavior and social communication deficits. Epilepsy and anxiety are also common in FXS and ASD. Serotonergic neurons directly innervate and modulate the activity of neurobiological circuits altered in both disorders, providing a rationale for investigating serotonin receptors (5-HTRs) as targets for FXS and ASD drug discovery. Previously we unveiled an orally active aminotetralin, (S)-5-(2'-fluorophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (FPT), that exhibits partial agonist activity at 5-HT1ARs, 5-HT2CRs, and 5-HT7Rs and that reduces repetitive behaviors and increases social approach behavior in wild-type mice. Here we report that in an Fmr1 knockout mouse model of FXS and ASD, FPT is prophylactic for audiogenic seizures. No FPT-treated mice displayed audiogenic seizures, compared to 73% of vehicle-treated mice. FPT also exhibits anxiolytic-like effects in several assays and increases social interactions in both Fmr1 knockout and wild-type mice. Furthermore, FPT increases c-Fos expression in the basolateral amygdala, which is a preclinical effect produced by anxiolytic medications. Receptor pharmacology assays show that FPT binds competitively and possesses rapid association and dissociation kinetics at 5-HT1ARs and 5-HT7Rs, yet has slow association and rapid dissociation kinetics at 5-HT2CRs. Finally, we reassessed and report FPT’s affinity and function at 5-HT1ARs, 5-HT2CRs, and 5-HT7Rs. Collectively, these observations provide mounting support for further development of FPT as a pharmacotherapy for common neuropsychiatric symptoms in FXS and ASD.

No abstract

No abstract

There are no approved medicines for fragile X syndrome (FXS), a monogenic, neurodevelopmental disorder. Electroencephalogram (EEG) studies show alterations in restingstate cortical EEG spectra, such as increased gamma-band power, in patients with FXS that are also observed in Fmr1 knockout models of FXS, offering putative biomarkers for drug discovery. Genes encoding serotonin receptors (5-HTRs), including 5-HT 1A , 5-HT 1B , and 5-HT 1D Rs, are differentially expressed in FXS, providing a rationale for investigating them as pharmacotherapeutic targets. Previously we reported pharmacological activity and preclinical neurotherapeutic effects in Fmr1 knockout mice of an orally active 2-aminotetralin, (S)-5-(2′-fluorophenyl)-N,Ndimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (FPT). FPT is a potent (low nM), high-efficacy partial agonist at 5-HT 1A Rs and a potent, low-efficacy partial agonist at 5-HT 7 Rs. Here we report new observations that FPT also has potent and efficacious agonist activity at human 5-HT 1B and 5-HT 1D Rs. FPT's K i values at 5-HT 1B and 5-HT 1D Rs were <5 nM, but it had nil activity (>10 μM K i ) at 5-HT 1F Rs. We tested the effects of FPT (5.6 mg/kg, subcutaneous) on EEG recorded above the somatosensory and auditory cortices in freely moving, adult Fmr1 knockout and control mice. Consistent with previous reports, we observed significantly increased relative gamma power in untreated or vehicle-treated male and female Fmr1 knockout mice from recordings above the left somatosensory cortex (LSSC). In addition, we observed sex effects on EEG power. FPT did not eliminate the genotype difference in relative gamma power from the LSSC. FPT, however, robustly decreased relative alpha power in the LSSC and auditory cortex, with more pronounced effects in Fmr1 KO mice. Similarly, FPT decreased relative alpha power in the right SSC but only in Fmr1 knockout mice. FPT also increased relative delta power, with more pronounced effects in Fmr1 KO mice and caused small but significant increases in relative beta power. Distinct impacts of FPT on cortical EEG were like effects caused by certain FDA-approved psychotropic medications (including baclofen, allopregnanolone, and clozapine). These results advance the understanding of FPT's pharmacological and neurophysiological effects.

Fragile X syndrome (FXS)—caused by FMR1 gene silencing—is a severe neurodevelopmental disorder characterized by intellectual disabilities that are often comorbid with seizures, sensory hypersensitivities, anxiety, social deficits, and repetitive behaviors. Neuronal hyperexcitability is an overarching neurophysiological characteristic of FXS that may underlie FXS symptoms. About 33% of Fmr1 KO mice from our colony exhibit spontaneous seizures, a newly observed phenotype that more closely parallels seizures in FXS, compared to the audiogenic seizure phenotype in Fmr1 KO mice. In addition, we and others show that Fmr1 KO mice, like individuals with FXS, have cortical EEG gamma‐band power alterations, and at the single‐cell level, have hyperexcitable pyramidal neurons in multiple brain regions. We are using a combinatorial approach—from behavioral to EEG to single‐cell experiments—to advance FXS drug discovery. Based on our observations of altered brain expression and in vivo function of serotonin 1A receptors (5‐HT1ARs) in Fmr1 KO mice and correction of the audiogenic seizure phenotype by our novel 2‐aminotetralin‐type 5‐HT1R modulator, FPT, we are testing the hypothesis that selectively activating 5‐HT1ARs prevents seizures and corrects neurophysiological abnormalities. We evaluated the efficacy of FPT (5.6 mg/kg), a potent and efficacious 5‐HT1AR agonist, to correct EEG abnormalities in Fmr1 KO mice. We also tested the antiepileptic effects of the selective 5‐HT1AR agonist, NLX‐112 (0.25‐2.5 mg/kg), and are currently testing the effects of FPT and NLX‐112 on CA1 pyramidal neuron hyperexcitability in Fmr1 KO mice. In parallel experiments, we are evaluating the pharmacology of FPT and NLX‐112 at each of the 5‐HT G protein‐coupled receptors. Recordings from above the left somatosensory cortex showed a significantly elevated high gamma (65‐100 Hz) power ratio in Fmr1 KO mice relative to control mice at baseline (n=16, P=0.0357) and after vehicle injection (n=16, P=0.0066), a genotype difference that FPT eliminated (n=15‐16, P=0.6279). Comparisons between baseline and first injection conditions also revealed an increased delta power in Fmr1 KO mice relative to controls. Separately, NLX‐112 prevented audiogenic seizures in Fmr1 KO mice (n=10‐12, P≤0.0002), and preliminary data suggest NLX‐112 and FPT modulate CA1 pyramidal neuron activity. For example, FPT (10 µM) showed a reversible reduction of firing frequency of hippocampal CA1 neurons in Fmr1 KO mice (n=8, P<0.05). Forthcoming experiments will include evaluating the effects of NLX‐112 on cortical EEG activity in Fmr1 KO and control mice and the effects of chronic NLX‐112 and FPT on spontaneous seizures in Fmr1 KO mice. Tests of the selective 5‐HT1AR antagonist, WAY100635, will be conducted to examine a 5‐HT1AR mechanism underlying positive outcomes of NLX‐112 and FPT. At present, our convergent data suggest that 5‐HT1AR activation may ameliorate neuronal hyperexcitability, at multiple levels of analysis, in Fmr1 KO mice. Potent and selective 5‐HT1AR agonists might ...