This work on human subjects was conducted under the protocol 06-M-0214, NCT00362843. During the 2018 funding period, we addressed the following: 1) rCPS measured with the L-1-C-11leucine PET method in participants with FXS and healthy volunteers studied either under dexmedetomidine sedation or in the awake state, 2) Effects of treatment with an inhibitor of phosphodiesterase-4D (PDE-4D) on behavior and rCPS in Fmr1 KO mice, 3) rCPS in Tsc2+/- mice, 4) Effects of rapamycin treatment on Fmr1 KO mice, 5) Sleep abnormalities in Fmr1 KO and Tsc2+/- mice. 1) Measurement of rCPS in human subjects with fragile X syndrome. We used the L-1-C-11leucine PET method to measure rCPS. As most fragile X participants were not able to undergo the PET study awake, we used dexmedetomidine for sedation during the imaging studies. We first studied the effects of dexmedetomidine sedation on rCPS in ten healthy controls, and we found no significant effects. Next, we studied nine fragile X participants under sedation and compared results with sedated healthy controls. Mean rates of protein synthesis were similar in both groups. Our concern was that sedatives could have a differential effect in FXS subjects, so we focused our efforts on trying to study these subjects without the use of sedatives. We modified our PET protocol to enable some fragile X participants to undergo the study awake. We compared rates of cerebral protein synthesis in six fragile X participants in the awake state with results in ten healthy controls studied with the same procedure. Contrary to our hypothesis, results indicate that rates of cerebral protein synthesis in the awake fragile X participants are decreased throughout the brain by 15-33% compared to healthy controls. Our results indicate that the absence of fragile X mental retardation protein may trigger an imbalance of pathways involved in the regulation of translation resulting in significantly decreased rates of protein synthesis. A manuscript reporting these results is in preparation. 2) 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 (Univ. Penn.) 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 (BPN-14770). We chronically treated Fmr1 KO mice with the PDE-4D NAM and measured its effects on behavior and rCPS. We are currently analyzing results of these studies. 3) Effects of rapamycin treatment on Fmr1 KO mice. The mammalian target of rapamycin pathway (mTORC1) is reported to be upregulated in Fmr1 KO mice and is thought to be important for the pathogenesis of this disorder. We treated Fmr1 KO mice chronically with an mTORC1 inhibitor, rapamycin, to determine if rapamycin treatment could reverse behavioral phenotypes. We found that pS6 was upregulated in Fmr1 KO mice and normalized by rapamycin treatment, but, except for an anxiogenic effect, rapamycin did not reverse any of the behavioral phenotypes examined. Moreover, rapamycin treatment had an adverse effect on sleep and social behavior in both control and Fmr1 KO mice. These results suggest that targeting the mTOR pathway in FXS is not a good treatment strategy and that other pathways should be considered. The results of these studies were published this year. 4) rCPS in Tsc2+/- mice. 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 measured the effects of Tsc2 haploinsufficiency (Tsc2+/-) in three month old male mice on rCPS by means of the in vivo L-1-14Cleucine method. This quantitative autoradiographic method includes an estimate of the integrated specific activity of the tracer amino acid in brain tissue. The estimate accounts for recycling of unlabeled amino acids from tissue protein breakdown by means of a factor () that was determined in control and Tsc2+/- mice. The value of was higher in Tsc2+/- mice, indicating that a greater fraction of leucine in the tissue precursor pool for protein synthesis is derived from the plasma compared to controls, consistent with reduced rates of protein degradation. We determined rCPS in freely-moving, awake male Tsc2+/- and control mice, and we used the genotype-specific values of in the calculation of rCPS. Unexpectedly, we found that rCPS were significantly decreased in 16 of the 17 brain regions analyzed in Tsc2+/- mice compared to controls. Our results indicate a complex role of mTORC1 in the regulation of cerebral protein synthesis that has not been previously recognized. The results of these studies were published this year. 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 two and six months of age sleep less than WT mice in the light phase. In younger animals (at P21), we found no difference between Fmr1 KO and WT mice in sleep times. Sleep deficits were not restored by treatment with a GABA-B agonist, R-baclofen. Ongoing studies will address the ability of other drugs to improve sleep and improve behavioral phenotypes. Future studies will also address sleep in other single gene neurodevelopmental disorders.
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