The protocol involved in this project is 06-M-0214, NCT00362843. Based on pathophysiological studies in animal models of FXS a number of compounds, including mGluR5 antagonists and GABA agonists, are being tested in human subjects. Preliminary preclinical and clinical studies indicated that treatment with the GABA-B agonist, r-baclofen, was promising. We have completed a study addressing the involvement of the GABA-B receptor in the Fmr1 KO mouse. Strikingly, we found that a single acute treatment with r-baclofen (1.5 mg/kg, i.p) 30 min prior to initiation of in vivo measurement of rCPS reversed the increased rCPS in Fmr1 KO mice with little effect on WT mice. Our behavioral results indicated that vehicle-injected Fmr1-KO mice exhibit hyperactivity and lower anxiety-like behavior in the open field and abnormal social interaction in the three-chambered test compared to WT as we and others have seen before. Administration of r-baclofen (1.5 mg/kg, i.p) 30 min prior to the test had little or no effect on activity or anxiety-like behavior in either genotype. Abnormal social interaction behavior in the Fmr1 KOs, however, was profoundly affected by the r-baclofen treatment. We have also observed effects of acute r-baclofen on components of signaling pathways known to be altered in Fmr1 KO mice. Increased p-mTOR and p-Akt in frontal cortex were both reversed by r-baclofen treatment. Our study suggests that the GABA-B pathway remains a promising therapeutic target for FXS. These results were presented at the Gordon Research Conference, Fragile X and Autism-Related Disorders, June 2014. Sleep disturbances represent one of the most prevalent concurrent disorders in patients with autism. Between 50%-80% of patients have difficulty initiating sleep, difficulty staying asleep, and overall decreased time sleeping. Sleep has critical functions in brain development and synaptic plasticity, and studies have shown a correlation between the severities of sleep abnormalities and autistic behavioral problems. Despite the prevalence of sleep disturbances in patients with autism, little is known about the role that sleep abnormalities may have in autism. We used home cage monitoring to investigate total sleep times in wild type (WT) and Fmr1 KO mice, and we found that Fmr1 KO mice spend significantly less time sleeping than WT mice. Effects were seen in animals at Postnatal Day 21 in the light phase only and at Postnatal Day 30 time in both the light and dark phases. These preliminary results, presented at the Gordon Research Conference, Fragile X and Autism-Related Disorders, June 2014, demonstrate that sleep abnormalities are present in Fmr1 KO mice. Current studies address the ability of drugs that can facilitate sleep in control animals to effect reversal of the sleep phenotype in Fmr1 KO mice. In collaboration with Karen Usdin (NIDDK) we tested whether primary fibroblasts derived from patients with FXS have disease-specific molecular phenotypes observed in the brains of Fmr1 KO mice. Primary human fibroblasts are readily accessible, adherent and untransformed cells that might serve to identify novel therapeutic targets for FXS as well as identify markers to test the efficacy of targeted therapies prior to clinical trials. Such markers may provide quantifiable measures that could be used in conjunction with the more subjective clinical criteria that are currently used for assessing outcomes of therapeutic drug trials. Our results in a small sample of subjects indicate that rates of protein synthesis are significantly elevated in these cells and levels of p-mTOR, p-ERK , and p-S6K1 are elevated. Treatment with small molecules that inhibit S6K1 or phosphoinositide 3-kinase (P13K) catalytic subunit p110-beta lowered rates of protein synthesis in both control and patient fibroblasts. Our data demonstrate that fibroblasts from FXS patients may be a useful in vitro model to test the efficacy and toxicity of potential therapeutics prior to clinical trials, as well as for drug screening and designing personalized treatment approaches. A manuscript reporting these results is under review. In our previous study of the premutation knockin (KI) mouse model developed by K. Usdin (NIDDK) we found a phenotypic profile very similar to Fmr1 KO mice including increased rCPS (Qin et al., 2011). We also found that Fmr1 mRNA levels were increased 2-6-fold and FMRP concentrations in 12 regions of the brain were reduced to about 10-20% of WT. We hypothesized that the phenotype in the KI mice is due to the profound reduction in FMRP rather than the excess Fmr1 message. We have extended these studies to another model of the premutation developed at Erasmus University (Bontekoe et al, 2001). In the model developed by K. Usdin's lab, a CGG repeat sequence was generated by serial ligation and inserted in the Fmr1 gene. In the Erasmus model the endogenous CGG repeat tract was replaced with a cloned human premutation CGG allele. In the Erasmus model, we found that FMRP levels were 50% of WT and Fmr1 mRNA levels were increased 2-4 fold over WT. The behavioral phenotype was also milder than that of the Usdin model (Van Dam et al, 2005). Our results of studies of rCPS in the Erasmus model show that rCPS is similar to WT throughout the brain. These results suggest that there may be a threshold level of FMRP necessary for maintenance of normal rates of protein synthesis in the nervous system. As animals age and FMRP levels in brain decline further symptoms may unfold. A manuscript reporting these results is in press. 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 many brain regions, including hippocampus (-17%), cortex (-16.5%), thalamus (-16%), and hypothalamus (-20%). Our results suggest a possible novel role/ regulation of protein synthesis in the brain.
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