Studies On Protein Synthesis And Long-Term Adaptive Responses in the CNS

为了将rCPS作为FXS临床试验的结果衡量标准，我们实现了以下具体目标：

To establish rCPS as an outcome measure for clinical trials in FXS, we addressed the following specific aims: 1) Determine the effects of FXS in human subjects on rCPS. 2) Test the effects of treatments for FXS on rCPS in a mouse model of FXS. 3) Establish the effect of the fragile X premutation on rCPS in a mouse model. 1) We have implemented in human subjects the L-1-11Cleucine PET method for measurement of rCPS and have completed two studies in healthy young men. The first was a study of the reproducibility and variability of the measurement of rCPS in human subjects. We determined rCPS in 18 regions and whole brain in ten healthy males (21-24 years). Subjects underwent two 90 min dynamic PET studies in the conscious state and at least two weeks apart. rCPS varied regionally ranging from 0.97 +/- 0.70 (mean +/- SD) nmol/g/min in corona radiata to 2.25 +/- 0.20 in occipital cortex. Values of rCPS were in good agreement between the two PET studies in an individual. Mean differences in rCPS between studies ranged from 9% in cortical regions to 15% in white matter. Intersubject variability in rCPS was on average 6% in cortical areas, 9% in subcortical regions, and 12% in white matter. Our data indicate that in human subjects low variance and highly reproducible measures of rCPS can be made with the L-1- 11Cleucine PET method. The second study completed this year was a study of the effects of propofol anesthesia on rCPS. This study was undertaken in anticipation of our proposed studies of subjects with FXS. High quality PET data acquisition requires that patient motion in the scanner be minimized. However, many patients with FXS and other neurodevelopmental disorders may not tolerate long periods of motionlessness while awake. It may therefore be necessary to study such subjects under sedation or anesthesia. This presents the potential problem that anesthesia itself may alter rCPS and may mask effects of the disease or the potential treatment under evaluation. We studied a group of ten healthy males (20-24 years). Each subject was studied twice: once awake and once under propofol anesthesia. Across 18 regions and whole brain, mean differences in rCPS between studies ranged from -5 to 5% and were within the variability of rCPS in awake studies (coefficient of variation range: 7-14%). Intersubject variances and patterns of regional variation were also similar under both conditions. In awake subjects, rCPS varied regionally from 1.00 +/- 0.10 to 2.52 +/- 0.34 nmol/g/min in corona radiata and occipital cortex, respectively; and values were in excellent agreement with our first study on conscious young men. Our data indicate that the values, variances, and patterns of regional variation in rCPS measured by the L-1-11Cleucine PET method are not significantly altered by anesthesia with propofol. Results of these two investigations indicate that measurements of rCPS with the L-1-11Cleucine PET method are reproducible and of sufficiently low variability that they can be used to study group differences between age-matched controls and subjects with FXS. The lack of an effect of propofol anesthesia on rCPS further indicates that it will be possible to study subjects with FXS while they are anesthetized with propofol without affecting rCPS. This finding increases the utility of rCPS as an outcome measure in clinical trials in developmental disorders. 2) Our studies of the effects of potential treatments for FXS were carried out in the fragile X knockout (FXKO) mouse model. We have initiated studies of two potential treatments. The first is treatment with a metabotropic glutamate receptor subtype 5 (mGluR5) antagonist, 2-methyl-6-phenylethynyl-pyridine hydrochloride (MPEP) to test the mGluR theory of FXS. According to the mGluR theory of FXS mGluR-stimulated protein synthesis is normally constrained by FMRP; in FXS, in the absence of FMRP, protein synthesis is unchecked and the process set in motion by activation of the receptor does not return to baseline. Blockade of mGluRs by MPEP would therefore be expected to constrain rCPS in the FXKO mice and modulate some of the symptoms of FXS in human subjects. We studied four groups of 5-7 mice: WT and FXKO mice treated with either vehicle or MPEP (20 mg/kg) 2 minutes before injection of L-1-14Cleucine. We found that in FXKO mice, MPEP treatment reversed the increased rCPS in the hippocampus. These findings support the notion that measurement of rCPS in subjects with FXS may provide us with a tool with which to monitor efficacy of proposed treatments. The second treatment under study is lithium carbonate (Li) (0.24% added to normal mouse chow at weaning). We tested for effects on one morphological and four behavioral phenotypes in FXKO mice: immature dendritic spines, hyperactivity, reduced general anxiety, impaired social interactions, and deficits on a fear learning/memory test. Chronic Li treatment normalized spine morphology, partially reversed hyperactivity, alleviated effects on general anxiety, and ameliorated effects on social interactions and fear learning/memory. Studies of the effects of Li on rCPS in FXKO mice are underway. These results in mice suggest that chronic lithium treatment may have therapeutic value in FXS. 3) A knockin (FXKI) mouse model of the fragile X premutation developed by Dr. K Usdin (Laboratory of Molecular & Cellular Biology, NIDDK) has many of the attributes found in human subjects, i.e., intranuclear inclusions, increased levels of fmr1 mRNA, and a loss of Purkinje cells. Dr Usdin has also demonstrated that in some regions of the brain and, despite the increases in fmr1 mRNA, FMRP concentrations are reduced. We have undertaken a study of these mice to characterize the phenotype and to determine if the regional reductions in FMRP can be linked to changes in brain function and to effects on rCPS. Our results indicate that FXKI mice do exhibit abnormalities in behavior and morphology. Young adult male FXKI mice are hyperactive, have reduced generalized anxiety and impaired social interactions. They have deficits in fear-motivated learning/memory test, but appear to behave normally on a test of motor function. We also found that dendritic spines were elongated and increased in density in some regions of the brain. Studies of the effects of the premutation on rCPS in FXKI mice are underway. Studies described address the central question that symptoms of FXS are caused by the lack of regulation of translation that occurs in the absence of FMRP. Our focus is on FXS because the process of translation regulation is likely at the heart of the malady in this single gene disease. It is also likely that dysregulation of protein metabolism underlies other neurodevelopmental disorders.