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 four areas: 1)Studies of cerebral protein synthesis were completed on a genetic mouse model of phenylketonuria (PKU) to determine if a partial inhibition of protein synthesis in the central nervous system contributes to the mental retardation that accompanies untreated PKU. The primary cause of mental retardation in PKU is unknown but it is clearly linked to persistent hyperphenylalaninemia during the developmental period. It has been shown that high arterial plasma phenylalanine concentrations competitively inhibit transport of other essential neutral amino acids across the blood brain barrier via the L-amino acid carrier, and it is hypothesized that reduced concentrations of essential amino acids in brain may limit rates of protein synthesis resulting in abnormal brain development. Prior to the recent establishment of a genetic-based mouse model (pahenu2) all attempts to test this hypothesis in experimental animals have used some form of administration of phenylalanine to achieve hyperphenylalaninemia. Results of some of these studies suggest that hyperphenylalaninemia particularly in developing animals affects protein synthesis, but direct measurements of rates of protein synthesis have yielded conflicting results probably due to methodological difficulties in the estimation of the specific activity of the amino acid precursor pool in the tissue. Local rates of in vivo cerebral protein synthesis (lCPS) were determined with the quantitative autoradiographic L-[1-14C]leucine method in the adult pahenu2 mouse. Arterial plasma concentrations of phenylalanine were elevated in both homozygous (HMZ) and heterozygous (HTZ) mutants by 21 times and 38%, respectively compared with the background strain (BTBR). In the total acid-soluble pool in brain, concentrations of phenylalanine were higher and other neutral amino acids lower in HMZ mice compared with either HTZ or BTBR mice indicating a partial saturation of the L-amino acid carrier at the blood brain barrier by the elevated plasma phenylalanine concentrations. In the HMZ mice there were on average 20% reductions in lCPSleu throughout the brain compared with the other two groups, but no reductions in brain concentrations of tRNA-bound neutral amino acids. In a series of steady state experiments the contribution of leucine from the arterial plasma to the tRNA-bound pool in brain was statistically significantly reduced in HMZ mice compared with the other groups, indicating that a greater fraction of leucine in the precursor pool for protein synthesis is derived from protein degradation. Our results in the mouse model suggest that in untreated phenylketonuria in adults, the partial saturation of the L-amino acid transporter at the BBB may not underlie a reduction in lCPSleu. The results of these studies have been published in PNAS. Our current work on this model includes studies of protein synthesis in developing animals and cerebral metabolic mapping studies in adults. 2)Metabolic mapping studies of 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 ?in press? in PNAS. Our current work is aimed at measuring CMRglc in female fmr1 KO mice and female littermates with a single allele for FMRP as a model of fragile X carriers. In addition, we are studying the effect of the mutation on lCPSleu because FMRP is an RNA-binding protein and is postulated to be involved in cerebral protein synthesis. 3)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 ?15 C have oxygen consumption that is 10 times that of an animal hibernating at 0 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. 4)Work is progressing on the adaptation of the quantitative autoradiographic [14C]leucine method for measurement of rates of cerebral protein synthesis for use in man with [11C]leucine and PET. We are using a compartmental modeling approach to the estimation of the parameters of the model. Studies are underway in which experimental animals undergo PET scans following a pulse iv injection of [11C]leucine. We obtain best-fitting estimates of the parameters from the time activity curves of the 11C in brain and fractions of blood. Terminal biochemical and autoradiographic experiments in the animals will test the validity of these estimates.
