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功效的持久修饰上，并且需要从头蛋白质合成。在神经元中，蛋白质的生物合成对于包括轴突、树突和突触末梢在内的整个细胞的生长和持续维持是必不可少的。蛋白质合成速率和特定蛋白质表达的调节对于发育和突触发生、成熟、神经元可塑性、再生和对激素的反应过程至关重要。为了能够定位这种长期变化，我们开发了定量放射自显影L-[1- 14 C]亮氨酸方法，用于测量体内脑蛋白质合成（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。另一个目标是阐述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。本年度的工作在以下两个领域取得了进展： 1)L-[1- 14 C]亮氨酸方法的改良，用于L-[1- 11 C]亮氨酸和正电子发射断层扫描（PET）。PET定量测量rCPS的能力将为我们研究人脑及其区域适应性反应提供一种新的工具。用PET定量测量rCPS的一个长期存在的障碍是来自蛋白质分解的组织氨基酸再循环到蛋白质合成的前体库中的混杂效应。在动物研究中，我们在平行终端实验中评估回收的影响。PET研究仅限于测量由循环提供的氨基酸的掺入率。如果不对再循环进行校正，就无法区分rCPS的真实变化与组织氨基酸再循环改变引起的表观变化。我们已经开发并验证了一种动力学建模方法，以纠正组织氨基酸再循环的影响。我们已经在恒河猴中完成了一项研究，其中我们证明，通过使用动力学建模方法，可以使用L-[1- 11 C]亮氨酸和PET定量准确和可重复地测量rCPS。目前正在审查报告这些结果的患者。 2)实验动物蛋白质代谢与神经适应性研究。目前，这些研究集中在智力迟钝的遗传小鼠模型上，试图了解表型的根本原因。在脆性X综合征（FrX）中，一种X连锁遗传形式的智力低下，脆性X智力低下-1（fmr 1）基因的甲基化诱导的转录沉默导致基因产物脆性X智力低下蛋白（FMRP）的缺失。在fmr 1敲除（KO）小鼠中FMRP的缺失赋予FrX表型的许多特征。FMRP是一种RNA结合蛋白，已显示其在体外抑制某些mRNA的翻译。在脑中，FMRP在神经元细胞质中高度表达，并且定位于树突和树突棘。FrX最显著的神经病理学特征是皮质树突棘的长、薄和曲折的外观，与发育早期所见的形态相似。FMRP被认为是翻译的抑制因子。我们对fmr 1基因敲除小鼠体内蛋白质合成的研究表明，至少在选择性脑区确实可能是这种情况。我们还发现，区域脑葡萄糖代谢率（rCMRglc）升高，在雄性fmr 1基因敲除小鼠和受影响的地区可能对应于我们在这些动物中观察到的行为异常。 我们还在研究另一种智力迟钝的遗传小鼠模型（pahenu 2），它的苯丙氨酸羟化酶基因发生了突变。在许多方面，具有突变的动物的表型类似于人类苯丙酮尿症（PKU）。肝脏中苯丙氨酸羟化酶活性极低;血浆中苯丙氨酸浓度为正常值的10-20倍;动物色素减退，并在认知功能的几项行为测试中表现出一些细微的损害。我们已经研究了成年pahenu 2小鼠，并表明大脑尺寸减小，整个大脑的rCPS减少。rCPS的这种变化是否是行为缺陷发展的重要因素或疾病过程的后果仍有待确定。我们已经扩展了我们的PKU小鼠的研究，包括行为异常和区域功能活动的分析，如区域脑葡萄糖代谢率（rCMRglc）所示。我们的研究结果表明，成年雄性pahenu 2小鼠具有区域选择性降低rCMRglc。在涉及执行功能（如联想学习、工作记忆和决策）的大脑皮层区域，效果值得注意。在海马中，rCMRglc不受影响，并且pahenu 2小鼠在空间记忆任务上的表现是正常的。这是令人感兴趣的，因为海马体的损伤导致空间记忆的严重缺陷。其中一些结果在2003年第33届神经科学学会年会上发表。目前正在编写报告这些研究结果的手册供出版。

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.