In vivo measurements of rates of protein synthesis with radiolabeled precursors are problematic because of uncertainties about the relative contribution of unlabeled amino acids derived from protein breakdown to the precursor pool for protein synthesis. The quantitative autoradiographic method for the determination of local rates of cerebral protein synthesis (lCPS) in vivo with a carboxyl-labeled, aliphatic, branched- chain amino acid as tracer takes into account recycling of the unlabeled species of the tracer amino acid. Rates of cerebral protein synthesis in rats have been shown to increase in regenerating nerve nuclei and decrease in most brain regions during postnatal brain development and with senescence. Cocaine treatment changes lCPS in selective brain regions; the direction of the changes depends on the history of treatment. Studies of normal sleep in monkeys indicate that slow wave sleep is linked with increased rates of lCPS in many brain regions. In hibernating ground squirrels, lCPS is profoundly and reversibly reduced throughout the brain. Light thiopental anesthesia in rats results in widespread but very small decreases in rates of cerebral protein synthesis. In general, changes in lCPS may mark brain regions undergoing long-term adjustments in response to a drug, a treatment, or a change in physiological state. We are studying cerebral protein synthesis in two genetic mouse models of mental retardation: 1) The Pahenu2 mouse model of phenylketonuria (PKU) and 2) The fragile X knockout mouse model of fragile X syndrome. In conjunction with these studies we are characterizing these animals with respect to physiology, behavior, and regional functional activity in brain. 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. Apart from Down's syndrome, fragile X syndrome is the most common inherited form of human mental retardation with an estimated incidence of 1 in 4,000 males. Clinical features include variable but generally severe mental retardation, autistic behavior, a typical facial appearance and enlarged testicles in adult males. The condition is transmitted as an X-linked trait. The name fragile X syndrome is derived from the expression of a folate-sensitive fragile site at Xq27.3. The molecular basis for this disease is a large expansion of a triplet repeat (CGG) in the 5'untranslated region of the fragile X gene (FMR1). The full mutation is characterized by a large repeat containing over 200 CGGs. As a result, the FMR1 promoter and the CGG repeat itself become methylated, leading to silencing of transcription and translation of the FMR1 gene for the fragile-X protein (RMRP). In normal brain both FMR1 mRNA and FMRP are enriched in nerve tissue. Although the function of FMRP is unknown, it has been shown to contain three RNA binding regions. FMRP binds approximately 4% of fetal human brain mRNAs. It seems clear that RNA binding is functionally important because mutations in the RNA-binding region are associated with severe mental retardation. The precise function of FMRP is still unknown, as are the causes of the abnormalities in fragile X syndrome. It is postulated that FMRP is involved in the transport of RNA and/or proteins from the nucleus to the cytoplasm. Since the absence of FMRP is not lethal, FMRP must be one of several such gene products that serve this function. The main clinical feature of the fragile X syndrome is mental retardation, so it seems likely that the function of FMRP may be important in brain development and possibly also in learning and memory. With the development of the fragile X knockout mouse by the Dutch-Belgian Fragile X Consortium it is now possible to study the consequences of the absence of FMRP during development and under controlled experimental conditions. The mouse model exhibits some subtle cognitive and behavioral deficits, hyperactivity, and enlarged testes. In both the mouse model and the human disease cerebral cortical spines appear to be immature, i.e., long, thin, and overabundant. We hypothesize that FMRP may function as a regulator of protein synthesis particularly during development and plasticity. We are measuring lCPS in adult, male hemizygous and wild type littermates to test for an effect of the lack of the FMR-1 mRNA or FMRP on regional brain protein synthesis.
