Protein Synthesis And Long-Term Adaptive CNS Responses

神经系统的长期适应性变化建立在突触效能的持久改变上，需要从头合成蛋白质。在神经元中，蛋白质的生物合成对整个细胞（包括轴突、树突和突触末端）的生长和持续维持至关重要。蛋白质合成速率和特定蛋白质表达的调控对发育和突触发生、成熟、神经元可塑性、再生和对激素的反应等过程至关重要。为了能够定位这种长期变化，我们开发了定量放射自成像L-[1-14C]亮氨酸方法，用于测量体内脑蛋白合成（rCPS）的区域速率。该项目的目的是研究实验动物和人类神经系统的长期适应性反应。另一个目标是阐明蛋白质合成机制缺陷在长期适应性反应受损的疾病中的作用。本年度在以下两个方面取得进展：

Long-term adaptive changes in the nervous system are founded on lasting modifications in synaptic efficacy and require de novo protein synthesis. In neurons the biosynthesis of proteins is essential for growth and continued maintenance of the entire cell including axons, dendrites, and synaptic terminals. Regulation of the rate of protein synthesis and of the expression of specific proteins are crucial 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 the quantitative autoradiographic L-[1-14C]leucine method for the measurement of regional rates of cerebral protein synthesis (rCPS) in vivo. The objective of this project is to study long-term adaptive responses in the nervous system in both experimental animals and humans. A further objective is 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 two areas: 1) Modification of the L-[1-14C]leucine method for use in man with L-[1-11C]leucine and positron emission tomography (PET). The ability to measure rCPS quantitatively with PET will provide us with a new tool to investigate the human brain and its regional adaptive responses. A longstanding obstacle to quantitative measurement of rCPS with PET has been the confounding effect of recycling of tissue amino acids derived from protein breakdown into the precursor pool for protein synthesis. In animal studies we evaluate the effects of recycling in parallel terminal experiments. PET studies have been limited to measurement of incorporation rates of amino acids supplied by the circulation only. Without correction for recycling one cannot distinguish true changes in rCPS from apparent changes resulting from alterations in recycling of tissue amino acids. We have developed and validated a kinetic modeling approach to correct for the effect of recycling of tissue amino acids. We have completed a study in rhesus monkeys in which we demonstrated that, by use of the kinetic modeling approach, quantitatively accurate and reproducible measurement of rCPS is possible with L-[1-11C]leucine and PET. Manuscripts reporting these results are currently under review. 2) Studies of protein metabolism and neuroadaptation in experimental animals. Currently these studies are focused on genetic mouse models of mental retardation in an effort to try to understand underlying causes of the phenotype. In fragile X syndrome (FrX), an X-linked inherited form of mental retardation, methylation-induced transcriptional silencing of the fragile X mental retardation-1 (fmr1) gene leads to absence of the gene product, fragile X mental retardation protein (FMRP). Absence of FMRP in fmr1 knockout (KO) mice imparts many of the characteristics of the FrX phenotype. FMRP is an RNA-binding protein that has been shown to suppress translation of certain mRNAs in vitro. In brain FMRP is highly expressed in neuronal cytoplasm and is localized in dendrites and dendritic spines. The most striking neuropathological feature of FrX is the long, thin, and tortuous appearance of cortical dendritic spines, a similar morphology to that seen early in development. FMRP has been postulated to function as a suppressor of translation. Our in vivo studies of protein synthesis in fmr1 KO mice suggest that this indeed may be the case at least in selective brain regions. We also find that regional cerebral metabolic rates for glucose (rCMRglc) are elevated in male fmr1 KO mice and regions affected may correspond to behavioral abnormalities we have observed in these animals. We are also studying another genetic mouse model (pahenu2) of mental retardation that has a mutation in the gene for the enzyme phenylalanine hydroxylase. In many respects thee phenotype of animals with the mutation resembles human phenylketonuria (PKU). Phenylalanine hydroxylase activity is minimal in liver; concentrations of phenylalanine are 10-20 times normal in plasma; animals are hypopigmented and exhibit some subtle impairment in performance of several behavioral tests of cognitive function. We have studied the adult pahenu2 mouse and shown that brain size is reduced and rCPS is diminished throughout the brain. Whether this change in rCPS is a significant factor in the development of behavioral deficits or a consequence of the disease process remains to be determined. We have extended our studies of the PKU mouse to include analysis of behavioral abnormalities and regional functional activity as indicated by regional rates of cerebral glucose metabolism (rCMRglc). Our results show that the adult male pahenu2 mouse has regionally selective decreases in rCMRglc. Effects are noteworthy in regions of cerebral cortex involved in executive functions such as associative learning, working memory, and decision-making. In the hippocampus rCMRglc is not affected and performance of the pahenu2 mouse on a spatial memory task is normal. This is of interest because lesions of the hippocampus result in severe deficits in spatial memory. Some of these results were presented at the 33rd Annual Meeting of the Society for Neuroscience, 2003. Manuscripts reporting the results of these studies are currently being prepared for publication.