The protocol involved in this project is 06-M-0214, NCT00362843. During the 2016 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 treatment with metformin on rCPS and behavior in Fmr1 KO mice, 3) The effects of treatment with an inhibitor of phosphodiesterase-4D (PDE-4D) on behavior and rCPS 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 propofol 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 altered in patients with FXS compared with age-matched controls. These studies are ongoing. 2) 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 on rCPS and behavior in Fmr1 KO mice. Metformin is a biguanide drug used in the treatment of type 2 diabetes. Mice are treated chronically as adults and tested on tests of memory and rCPS are measured. 3) Treatment of Fmr1 KO mice with an inhibitor of PDE-4D. Work from Berry-Kravis (Rush College of Medicine) indicates that cAMP is reduced in peripheral cells from patients with FXS and that in neuroblastoma cells cAMP production is regulated by FMRP. Moreover, work from the laboratory of T. Jongens shows that inhibition of PDE-4 ameliorates memory deficits in the dfmr1 fly and restores mGluR-dependent LTD to WT levels in the Fmr1 KO mouse. In collaboration with Tetra Discovery Partners, we are testing the efficacy of a negative allosteric modulator (NAM) of PDE-4D that they have developed (BPN-14770). We are chronically treating Fmr1 KO mice with the PDE-4D NAM and measuring its effects on behavior 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. To date, our in vivo measurements of rCPS in freely-moving awake, adult, male Tsc2+/- mice indicate that rCPS is decreased in selective brain regions. Current experiments are addressing the effects of the mutation in TSC2 on recycling of leucine derived from protein degradation in the tissue; this is a critical factor in the equation for rCPS. 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 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. 6) We also sought to determine the effects of chronic sleep-restriction during development on subsequent adult behavior 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 minutes 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 with sleep ad libitum, 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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