One of the fundamental properties of nervous tissue is its capacity to undergo long-term adaptive responses. In neurons the biosynthesis of proteins essential for growth and continued maintenance of the entire cell including axons, dendrites, and synaptic terminals is clearly one of the important biochemical processes underlying adaptive changes. Regulation of the rate of protein synthesis and of the expression of specific proteins are essential to the processes of development and synaptogenesis, maturation, neuronal plasticity, regeneration, and responses to hormones. In order to be able to localize such long-term changes we have developed a quantitative autoradiographic method for the measurement of regional rates of cerebral protein synthesis in vivo. The objective of this project is to study long-term adaptive responses in the nervous system and to elaborate the role of deficiencies in protein synthetic mechanisms in diseases in which long-term adaptive responses are impaired. In the current year work progressed in the following three areas: 1) Studies of cerebral protein synthesis in a genetic mouse model of fragile X syndrome have been completed. In humans failure to express the fragile X mental retardation protein (FMRP) gives rise to fragile X syndrome, the most common form of inherited mental retardation. A fragile X knockout (fmr1 KO) mouse has been described that has some of the characteristics of patients with fragile X syndrome including immature dendritic spines and subtle behavioral deficits. In our behavioral studies fmr1 KO mice exhibited hyperactivity and a higher rate of entrance into the center of an open field compared with controls suggesting decreased levels of anxiety. Our finding of impaired performance of fmr1 KO mice on a passive avoidance task is suggestive of a deficit in learning and memory. In an effort to understand what brain regions are involved in the behavioral abnormalities we applied the [14C]deoxyglucose method for the determination of cerebral metabolic rates for glucose (CMRglc). We measured CMRglc in 38 regions in adult, male fmr1 KO and wild type littermates. We found CMRglc was higher in all 38 regions in fmr1 KO mice, and in 26 of the regions differences were statistically significant. Differences in CMRglc ranged from 12% to 46%, and the greatest differences occurred in regions of the limbic system and primary sensory and posterior parietal cortical areas. Regions most affected are consistent with the behavioral deficiencies and the regions in which FMRP expression is highest. Higher CMRglc in fragile X mice may be a function of abnormalities found in dendritic spines. These results are published in PNAS. We have completed parallel studies in female fmr1 KO mice to examine the sex difference in disease severity and the effect of heterozygocity. Our results indicate that the female fmr1 KO also exhibits hyperactivity and a deficit in performance on the passive avoidance test, but CMRglc is unchanged from wild type except possibly in the dorsal raphe. Females homozygous or heterozygous for the fmr1 KO exhibited hyperactivity, but only the homozygous animals had the deficit on the passive avoidance test and the effect on CMRglc in dorsal raphe. These results have been presented at the 34th Annual Meeting of the American Society for Neurochemistry in May 2003. A manuscript reporting these findings is in preparation. We have also completed studies of cerebral protein synthesis in adult, male fmr1 KO mice. FMRP is an RNA-binding protein and is postulated to be a negative regulator of cerebral protein synthesis. To our knowledge our studies are the first to demonstrate that the absence of FMRP is accompanied by increased rates of cerebral protein synthesis (lCPS) in vivo. Differences in lCPS ranged from 6 to 40% with the greatest differences occurring in specific hypothalamic nuclei. Effects in the hypothalamus may be related to abnormalities in reproductive system structure and function found in the human disease. A manuscript reporting these results is in preparation. 2) Studies of hibernation in the Arctic ground squirrel are ongoing in collaboration with colleagues in Alaska and NINDS. The primary objective is to investigate central control of metabolic suppression during hibernation. These animals hibernating at minus15 degrees C have oxygen consumption that is 10 times that of an animal hibernating at 0 degrees C. We are also investigating metabolic and protein changes in these animals during arousal from hibernation; within 2 h of arousal hippocampal dendrites exhibit remarkable changes in morphology. We are interested in the regulation of these changes in state. 3) The confounding effect of recycling of tissue amino acids into the precursor pool for protein synthesis has been an obstacle in adaptation of in vivo methods for determination of regional rates of cerebral protein synthesis (CPS) for use with PET. We used a kinetic modeling approach and validated our fitted results with biochemical measurements in the same animals. In the adaptation of the L-[1-14C]leucine method for PET we used L-[1-11C]leucine as the tracer and estimated blood volume and kinetic rate constants from time activity curves and measured blood data. From the rate constants we calculated lambda, the fraction of the precursor pool for protein synthesis derived from arterial plasma; the remainder comes from recycling. We studied two adult, male, rhesus monkeys. Each monkey was subjected to four [1-11C]leucine PET studies while under isoflurane anaesthesia at intervals of at least one month. By means of kinetic modeling, the regional value of lambda was estimated for whole brain, cerebellum, cortex, basal ganglia, and white matter. Following the PET studies we determined the value of lambda biochemically in a terminal experiment on each animal. We found very good agreement among results of the four PET studies for each animal and between the means for the two animals. Mean rates of CPS calculated with estimated ls range from 1.8 to 2.8 nmol/g/min in corpus callosum and occipital cortex, respectively. The average CPS for whole brain was 2.5 0.2 nmol/g/min. This value compares well with the mean whole brain CPS of 2.3 0.4 nmol/g/min of seven awake, adult monkeys studied previously with the autoradiographic method. Our results indicate that adaptation of the leucine method for CPS for use with PET is feasible. We show that in studies of monkeys it is possible to evaluate lambda and to measure reproducibly actual rates of protein synthesis with a kinetic modeling approach. We have validated the approach by evaluating lambda in the same animals with a biochemical technique. The results of the modeling and biochemical approaches are in good agreement with each other. These results have been presented at the XXIst Meeting of the International Society on Cerebral Blood flow, Metabolism, and Function in July 2003. A manuscript reporting these results is in preparation.
