Dysregulation of Protein Synthesis in Fragile X Syndrome and Other Developmental Disorders

National Institute Of Mental Health

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Abstract

The work on humans was conducted under the protocol 06-M-0214, NCT00362843. During the 2019 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 in the awake state, 2) protein synthesis rates in hippocampal slices from WT and Fmr1 KO mice, 3) protein synthesis rates in mouse models of other neurodevelopmental disorders, 4) mTORC1 activity as a central regulator of brain protein synthesis in some neurodevelopmental disorders, 5) sleep and neurodevelopmental disorders. 1) Measurement of rCPS in humans with fragile X syndrome. We used the L-1-C-11leucine PET method to measure rCPS. We have focused our efforts on trying to study individuals with fragile X syndrome without the use of sedatives. We use our modified PET protocol to enable some fragile X participants to undergo the study awake. Our preliminary 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. We continue to recruit participants to this study. 2) Protein synthesis in hippocampal slices from Fmr1 KO mice. Synaptic plasticity, protein synthesis and cellular growth pathways have been studied extensively in hippocampal slices from Fmr1 KO mice. Enhanced metabotropic glutamate receptor 5 (mGluR5)-dependent long-term depression (LTD), increased rates of protein synthesis, and effects on signaling molecules have been reported. These phenotypes were found under conditions commonly used in electrophysiological studies in which slices were incubated in a balanced salt solution with the addition of compounds to support energy metabolism, but without amino acids. Amino acid starvation is a condition that has widespread, powerful effects on activation and translation of proteins involved in regulating protein synthesis. We asked if this unphysiological condition could have effects on reported Fmr1 KO hippocampal slice phenotypes. We performed hippocampal slice experiments in the presence and absence of amino acids. We measured rates of incorporation of a radiolabeled amino acid into protein to determine protein synthesis rates. By means of Western blots, we assessed relative levels of total and phosphorylated forms of proteins involved in signaling pathways regulating translation. We measured evoked field potentials in area CA1 to assess the strength of the long-term depression response to mGluR activation. In the absence of amino acids, we replicate many of the reported findings in Fmr1 KO hippocampal slices, but in the more physiological condition of inclusion of amino acids in the medium, we did not find evidence of enhanced mGluR5-dependent LTD. Activation of mGluR5 increased protein synthesis in both WT and Fmr1 KO. Moreover, mGluR5-activation increased eIF2alpha phosphorylation and decreased phosphorylation of p70S6k in slices from Fmr1 KO. We propose that the eIF2alpha response is a cellular attempt to compensate for the lack of regulation of translation by FMRP. The results of our studies are in press in the Journal of Neurochemistry. 3) The genetic bases of several other neurodevelopmental disorders suggest that defects in translational control may be a core phenotype. For example, tuberous sclerosis complex (TSC) is caused by heterozygous mutations in either the TSC1 or TSC2 gene, both of which encode proteins that are negative regulators of mTOR activity. The mTOR pathway is one of the nodes regulating protein synthesis. Disruption of this control is thought to underlie some of the phenotypes observed in TSC. We have measured rCPS in a mouse model of TSC, TSC2+/-, and found that rCPS are decreased throughout the brain. We continue studies of rCPS in mouse models of other neurodevelopmental disorders such as Shank3 KO mice. We are also measuring rCPS in a conditional knockout mouse model of Tsc2. These studies are ongoing in the laboratory. 4) It has been reported that the mTOR pathway is overactivated in Fmr1 KO mice. We are investigating the consequences of increased mTORC1 activation in Fmr1 KO mice. 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. Studies of rCPS in rapamycin-treated Fmr1 KO mice are ongoing. We also have crossed Fmr1 KO mice with Tsc+/- mice to try to reproduce previously reported finding that such a cross cancels out abnormalities in protein synthesis measured in vitro in hippocampal slices. We are measuring rCPS in vivo and behavioral phenotypes (including sleep, social behavior, activity, and learning and memory) in these mice. 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 continue studies on the role of sleep in neurodevelopmental disorders. We are collecting sleep data on numerous mouse models of neurodevelopmental disorders including Shank3 KO, Oxytocin receptor KO, and Tsc2+/- mice. Additionally, we are treating Fmr1 KO mice with hypnotics to determine the effects on sleep and other behavioral phenotypes.