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基础上，需要从头合成蛋白质。在神经元中，蛋白质的生物合成对于整个细胞的生长和持续维持是必不可少的，包括轴突、树突和突触终末。蛋白质合成速率和特定蛋白质表达的调节对发育和突触形成、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，我们发展了定量放射自显影L-[1-14C]亮氨酸方法，用于测量活体脑蛋白质合成的区域速率(RCP)。这个项目的目标是研究神经系统在实验动物和人类中的长期适应性反应。另一个目标是阐述蛋白质合成机制缺陷在长期适应反应受损的疾病中的作用。本年度在以下两个方面取得了进展： 1)用L-[1-11C]亮氨酸和正电子发射断层扫描对人用L-[1-14C]亮氨酸法进行了改进。用PET定量测量RCP的能力将为我们提供一种新的工具来研究人脑及其局部适应性反应。用PET定量测定RCP的一个长期障碍是蛋白质分解产生的组织氨基酸循环进入蛋白质合成前体池的混杂效应。在动物实验中，我们在平行的终端实验中评估循环的效果。PET研究仅限于测量由循环供应的氨基酸的掺入率。如果不对循环进行修正，就不能区分RCP的真实变化和组织氨基酸循环变化引起的明显变化。我们开发并验证了一种动力学建模方法，以校正组织氨基酸循环的影响。我们已经在恒河猴身上完成了一项研究，在这项研究中，我们证明了使用动力学建模方法，可以用L-[1-11C]亮氨酸和正电子发射计算机断层扫描定量准确和重复性地测量RCP。报告这些结果的手稿目前正在审查中。 2)实验动物蛋白质代谢和神经适应的研究。目前，这些研究主要集中在精神发育迟滞的小鼠遗传模型上，试图了解表型的潜在原因。脆性X综合征(FRX)是一种X连锁的遗传性精神发育迟滞，甲基化导致脆性X智力低下-1(Fmr1)基因转录沉默，导致基因产物脆性X智力低下蛋白(FMRP)的缺失。在fmr1基因敲除(KO)小鼠中，FMRP的缺失赋予了FRX表型的许多特征。FMRP是一种RNA结合蛋白，已被证明在体外可以抑制某些mRNAs的翻译。在脑内，FMRP高表达于神经元胞浆，定位于树突和树突棘。FRX最显著的神经病理特征是皮质树突棘细长而曲折的外观，与发育早期所见的形态相似。FMRP被认为是翻译的抑制者。我们对fmr1KO小鼠体内蛋白质合成的研究表明，至少在选择性脑区可能确实是这样。我们还发现，在雄性fmr1KO小鼠中，局部脑葡萄糖代谢率(RCMRglc)升高，受影响的区域可能与我们在这些动物中观察到的行为异常相对应。 我们还在研究另一种精神发育迟缓的遗传性小鼠模型(Pahenu2)，这种模型的苯丙氨酸羟基酶基因发生了突变。在许多方面，该突变动物的表型类似于人类苯丙酮尿症(PKU)。苯丙氨酸羟基酶在肝脏中的活性很低；苯丙氨酸在血浆中的浓度是正常的10-20倍；动物色素减退，在几项认知功能的行为测试中表现出一些轻微的损害。我们对成年Pahenu2小鼠进行了研究，结果表明，大脑体积缩小，整个大脑的RCPS都减少了。RCP的这种变化是行为缺陷发展的一个重要因素，还是疾病过程的结果仍有待确定。我们已经扩展了我们对PKU小鼠的研究，包括行为异常和局部功能活动的分析，如局部大脑葡萄糖代谢速率(RCMRglc)所指示的。我们的结果表明，成年雄性Pahenu2小鼠rCMRglc具有区域选择性降低。值得注意的是，在大脑皮层参与执行功能的区域，如联想学习、工作记忆和决策。在海马区，rCMRglc不受影响，pahenu2小鼠在空间记忆任务中的表现正常。这很有趣，因为海马体的损伤会导致严重的空间记忆缺陷。其中一些成果已在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稿，以便出版。

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.