The protocol involved in this project is 06-M-0214, NCT00362843. During the 2015 funding period, we addressed the following: 1) rCPS measured with the L-1-C-11leucine PET method in patients with FXS and healthy volunteers sedated with dexmedetomidine, 2) The effects of propofol on a test of aversive memory in Fmr1 KO mice mediated by the endocannabinoid system, 3) The effects of treatment with metformin on rCPS and behavior in Fmr1 KO mice, 4) rCPS in Tsc2+/- mice, 5) Sleep abnormalities in Fmr1 KO and Tsc2+/- mice, 6) The effects of sleep restriction in WT mice on behavioral phenotype in adulthood. 1) Fragile X syndrome. We continue our studies of rCPS in patients with FXS. We are measuring rCPS with the L-1-C-11leucine PET method in patients and healthy volunteers while sedated with dexmedetomidine. Dexmedetomidine is an alpha-2-adrenergic receptor agonist. With this strategy we expect to avoid the selective effects that propfol had on rCPS in patients because dexmedetomidine acts via a completely different mechanism. When possible we are also studying patients awake with a modified protocol. Our hypothesis is that rCPS is increased in patients with FXS compared with age-matched controls. These studies are ongoing. 2) Endocannabinoid system in Fmr1 KO mice. We further investigated the genotype-selective effects of propofol treatment. We used a behavioral test as an end-point, a test of aversive memory. In our hands, deficiency on the passive avoidance test of aversive memory is a robust behavioral phenotype in Fmr1 KO mice. We assessed the effects of propofol on the passive avoidance test in WT and Fmr1 KO mice, and we found that propofol treatment 30 minutes after the training improved performance of Fmr1 KO, but not WT mice. Additionally we explored the mechanism by which propofol effects this genotype-specific change in the hope that it might aid in the discovery of new therapeutics. Propofol acts via a wide range of sites, including positive modulation of GABA-A receptors and inhibition of fatty acid amide hydrolase (FAAH), an enzyme that catalyzes the metabolism of the endogenous cannabinoid, anandamide. We focused our study on the latter site of action because it has been implicated in an effect of propofol on the enhancement of memory consolidation in rats. Moreover, endocannabinoid systems have been reported to be altered in Fmr1 KO mice. The effect of propofol was blocked by prior administration of the cannabinoid receptor-1 antagonist, AM-251. To see if inhibition of FAAH could have therapeutic relevance in FXS, we tested the effects of a specific inhibitor of FAAH (URB-597) on passive avoidance performance and on tests of other behavioral phenotypes in Fmr1 KO mice. Treatment with URB-597 improved performance on passive avoidance in Fmr1 KO mice but had no effect on WT. Results of other behavior tests indicated that URB-597 normalized anxiety-related behavior in the elevated plus maze, but did not improve social behavior in Fmr1 KO mice. Our results indicate that the endocannabinoid system is involved in FXS and suggest that the endocannabinoid system is a promising target for treatment of FXS. A manuscript reporting these results was published in Behavioral Brain Research. 3) Metformin treatment of Fmr1 KO mice. T. Jongens (University of Pennsylvania) reported recently that elevated insulin signaling is involved in the expression of behavioral phenotypes in the Drosophila model of FXS (dfmr1). We are following up this finding by testing the efficacy of metformin, a biguanide drug used in the treatment of type 2 diabetes, on rCPS and behavior in Fmr1 KO mice. Our initial studies are designed to determine the optimal dose. Mice are treated chronically as adults and tested on the novel object recognition test and rCPS. 4) Another syndromic form of autism under study in the SNPM is tuberous sclerosis complex (TSC). TSC is an autosomal dominant neurogenetic disorder manifested by a high incidence of seizures, intellectual disability, and autism. TSC is caused by mutations in either TSC1 or TSC2, which encode for proteins that form a complex and interact with a small GTP-binding protein, RHEB, to inhibit mTORC1. mTORC1 is a central regulator of ribosomal biogenesis and translation initiation, and loss of TSC1/2 function results in increased activity of mTORC1. We hypothesized that haploinsufficiency of TSC2 (Tsc2+/-) in mice would lead to increased rCPS. Our in vivo measurements of rCPS in freely-moving awake, adult, male Tsc2+/- mice indicate that rCPS is statistically significant decreased in selective brain regions. Acute treatment with rapamycin, an inhibitor of mTOR, reversed the decreased rCPS in the Tsc2+/- mice, but had no effect on WT. Our results suggest a possible novel role/ regulation of protein synthesis in the brain. 5) Sleep and neurodevelopmental disorders. Sleep abnormalities are one of the most prevalent concurrent disorders in patients diagnosed with neurodevelopmental syndromes. In these patients, the severity of behavioral abnormalities and the severity of sleep abnormalities are correlated. Given the importance of sleep in developmental plasticity, we sought to examine sleep behavior in two animal models of single gene neurodevelopmental disorders, FXS and TSC. We also sought to determine the effects of chronic sleep-restriction during development on subsequent adult behavior in WT mice. a. Studies in transgenic mice. We used home cage monitoring to investigate total sleep times during the light and dark phases. We found that Fmr1 KO mice at one month of age spend less time sleeping than WT mice in both the light and dark phases. In Tsc+/- mice at two months of age we found that total sleep time was lower than WT particularly in the dark phase. Future studies will address the ability of drugs that can facilitate sleep in control animals to effect reversal of the reduced sleep duration and to improve behavioral phenotypes. b. Studies in WT mice. We sleep-restricted developing WT mice from P5-P42 for three hours per day by means of gentle handling and compared behavioral outputs to controls who were handled ten min daily. We assayed activity in the open field, social behavior, repetitive behavior, and anxiety immediately following sleep restriction and after four weeks recovery. At six weeks of age, immediately following chronic sleep-restriction, mice were less active in an open field arena. Sociability was increased, but repetitive behaviors were unchanged in both males and females. After a 4-week period of recovery, some behavioral abnormalities persisted and some became apparent. Sleep-restricted mice had decreased activity in the beginning of an open field test. Female mice continued to have increased sociability and, in addition, increased preference for social novelty. We saw a trend toward decreased sociability in male mice. Repetitive behavior was decreased in sleep-restricted female mice and increased in males. Measures of anxiety were not affected in the sleep-restricted mice. These results indicate that chronic sleep restriction during development can lead to long-lasting behavioral changes that are modulated by sex. Our study may have implications for a role of sleep disorders in childhood on the unfolding of neurodevelopmental disorders.
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