The protocol involved in this project is 06-M-0214, NCT00362843. During the 2013 funding period, we addressed the following: Based on results of our studies in the Fmr1 KO mouse, we proposed that changes in rCPS are a core phenotype of FXS and that in human subjects we would find an increased rCPS in selective regions of the brain, e.g., hippocampus, thalamus and parietal cortex. We measured rCPS in 15 male subjects (18-24 y) with the full fragile X mutation and 12 age-matched healthy volunteers. We excluded subjects on any psychotropic medication and subjects with a history of seizures. We determined rCPS in whole brain and 10 regions. Because of their impairments it was necessary to study FXS subjects under deep sedation. For comparisons all subjects underwent PET scans while sedated with propofol. Subjects also underwent MRI studies for placement of regions of interest (ROIs). Presence of a full mutation allele was confirmed in the FXS subjects by analysis of the CGG repeat length;all had greater than 200 CGG-repeats. In control subjects repeat lengths ranged from 20 to 32 with a mean of 27. Control subjects had full scale IQ scores above average (mean, 130). FXS subjects scores were in the cognitively impaired range (mean, 53). Of the 15 FXS subjects, two met criteria for an anxiety disorder and four for ADHD (combined type). All of the FXS subjects met at least one of 12 DSM IV criteria for the diagnosis of autism and six subjects met the diagnostic criterion for autism. Each healthy volunteer was studied twice with the 11Cleucine PET method, once awake and once under deep propofol-sedation. We compared rCPS in the two states with paired t-tests, and we found no difference in whole brain or in any of the regions examined. Contrary to our hypothesis, FXS subjects under propofol-sedation had statistically significantly reduced rCPS in whole brain, cerebellum, frontal and parietal cortex compared with sedated controls. We considered the possibility that propofol could have a disparate effect on rCPS in FXS subjects thereby masking a baseline elevation in rCPS, and we examined this possibility in the Fmr1 KO mouse model. We studied four groups of mice: wild type (WT) and Fmr1 KO mice, vehicle- and propofol-injected. We confirm in the vehicle-treated mice our previous finding that rCPS was increased in the absence of FMRP in selective brain regions. Effects of propofol sedation on rCPS differed dramatically between WT and KO mice. In WT mice, propofol resulted in decreased rCPS in two of the regions and an increase in CA1 of dorsal hippocampus. In KO mice, propofol treatment decreased rCPS by 14-26% (average decrease of 19%) in all regions examined except CA1. Comparison of propofol-sedated WT and propofol-sedated KO mice indicates that rCPS was lower in the KO mice in somatosensory cortex by about 10%. We think that the increased rCPS seen in the awake KO mouse model (Qin et al, 2005) is a core phenotype of FXS and is likely also pathogenic in the human disease. Our observation that treatment with a drug that alters neuronal and possibly synaptic activity also alters rCPS in KO mice has implications for the development of therapeutic strategies for FXS. A manuscript reporting these results is published in the J. Cerebr. Blood Flow &Metab. (Qin et al., 2013). There are numerous clinical trials of proposed treatments for FXS currently underway in the U.S. and abroad. Drugs recently being tested include mGluR antagonists and GABA agonists. Ongoing work in collaboration with Seaside Therapeutics addresses the involvement of the GABA-B receptor in FXS. We are measuring the effects of acute r-baclofen treatment in three month old mice on behavior in the open field, behavior in a test of social interaction, and on rCPS. Our preliminary results indicate that vehicle-injected Fmr1-KO mice exhibit hyperactivity and lower anxiety-like behavior in the open field compared to WT as we and others have seen before, but our preliminary data indicate that r-baclofen (1.5 mg/kg, i.p. administered 30 min prior to the test) has little or no effect on activity or anxiety-like behavior in either genotype. Social interaction behavior in the Fmr1 KOs, however, appears to be profoundly affected by the r-baclofen treatment. We use the three-chambered test of social interaction and confirm our previous finding in vehicle-injected mice that there is clear phenotype in Fmr1 KO mice on the social novelty phase of the test. Following r-baclofen treatment, Fmr1 KO mice have interactions with the novel mouse that appear to be very similar to WT indicating that activation of GABA-B receptors appears to promote normalized social interaction in the Fmr1 KO mice. Also underway are determinations of the effects of acute r-baclofen treatment on rCPS in these mice. 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 hypothesize that the phenotype in the KI mice is due to the profound reduction in FMRP rather than the excess message. We are extending 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 find that FMRP levels are 50% of WT and Fmr1 mRNA levels are increased 2-4 fold over WT. The behavioral phenotype is also milder than the Usdin model (Van Dam et al, 2005). In collaboration with R. Willemsen (Erasmus University) we are repeating our measurements of rCPS in the Erasmus premutation model to ascertain whether an effect on rCPS can be detected in the presence of a less profound decrease in FMRP. Our preliminary results indicate that rCPS is not significantly affected in this KI mouse model suggesting that reductions greater than 50% in FMRP concentration are needed to affect rCPS.

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Qin, Mei; Schmidt, Kathleen C; Zametkin, Alan J et al. (2013) Altered cerebral protein synthesis in fragile X syndrome: studies in human subjects and knockout mice. J Cereb Blood Flow Metab 33:499-507
Lokanga, Rachel Adihe; Entezam, Ali; Kumari, Daman et al. (2013) Somatic expansion in mouse and human carriers of fragile X premutation alleles. Hum Mutat 34:157-66
Liu, Zhong-Hua; Chuang, De-Maw; Smith, Carolyn Beebe (2011) Lithium ameliorates phenotypic deficits in a mouse model of fragile X syndrome. Int J Neuropsychopharmacol 14:618-30
Tomasi, Giampaolo; Bertoldo, Alessandra; Bishu, Shrinivas et al. (2009) Voxel-based estimation of kinetic model parameters of the L-[1-(11)C]leucine PET method for determination of regional rates of cerebral protein synthesis: validation and comparison with region-of-interest-based methods. J Cereb Blood Flow Metab 29:1317-31
Bishu, Shrinivas; Schmidt, Kathleen C; Burlin, Thomas V et al. (2009) Propofol anesthesia does not alter regional rates of cerebral protein synthesis measured with L-[1-(11)C]leucine and PET in healthy male subjects. J Cereb Blood Flow Metab 29:1035-47
Liu, Zhong-Hua; Smith, Carolyn Beebe (2009) Dissociation of social and nonsocial anxiety in a mouse model of fragile X syndrome. Neurosci Lett 454:62-6
Smith, Carolyn B; Schmidt, Kathleen C; Bishu, Shrinivas et al. (2008) Use of acute hyperphenylalaninemia in rhesus monkeys to examine sensitivity and stability of the L-[1-11C]leucine method for measurement of regional rates of cerebral protein synthesis with PET. J Cereb Blood Flow Metab 28:1388-98
Bishu, Shrinivas; Schmidt, Kathleen C; Burlin, Thomas et al. (2008) Regional rates of cerebral protein synthesis measured with L-[1-11C]leucine and PET in conscious, young adult men: normal values, variability, and reproducibility. J Cereb Blood Flow Metab 28:1502-13
Qin, Mei; Smith, Carolyn Beebe (2008) Unaltered hormonal response to stress in a mouse model of fragile X syndrome. Psychoneuroendocrinology 33:883-9