Studies On Protein Synthesis And Amino Acid Compartmenta

在体内用放射性标记的前体测量蛋白质合成速率是有问题的，因为不确定来自蛋白质分解的未标记氨基酸对蛋白质合成前体池的相对贡献。用羧基标记的脂肪族支链氨基酸作为示踪剂，定量放射自显影法测定体内脑蛋白合成（lCPS）的局部速率，该方法考虑了示踪氨基酸中未标记物种的再循环。大鼠的大脑蛋白质合成率在神经核再生过程中增加，而在出生后的大脑发育和衰老过程中，大多数大脑区域的蛋白质合成率下降。可卡因治疗改变了选择性脑区的lCPS；变化的方向取决于治疗的历史。对猴子正常睡眠的研究表明，慢波睡眠与大脑许多区域中lCPS的增加有关。在冬眠的地松鼠中，lCPS在整个大脑中都被深刻地、可逆地减少了。小剂量硫喷妥钠麻醉会导致大鼠大脑蛋白质合成率的广泛但非常小的下降。一般来说，lCPS的变化可能标志着大脑区域对药物、治疗或生理状态变化的长期调整。我们正在研究两种智力低下小鼠遗传模型的脑蛋白合成：1)苯丙酮尿症（PKU） Pahenu2小鼠模型和2)脆性X基因敲除小鼠模型脆性X综合征。结合这些研究，我们在生理、行为和大脑区域功能活动方面对这些动物进行了表征。PKU智力迟钝的主要原因尚不清楚，但它显然与发育期间持续的高苯丙氨酸血症有关。研究表明，高浓度的动脉血浆苯丙氨酸竞争性地抑制了其他必需中性氨基酸通过l -氨基酸载体穿过血脑屏障的运输，并且假设大脑中必需氨基酸浓度的降低可能会限制蛋白质合成的速度，从而导致大脑发育异常。在最近建立的基于基因的小鼠模型（Pahenu2）之前，所有在实验动物中测试这一假设的尝试都使用了某种形式的苯丙氨酸管理来实现高苯丙氨酸血症。其中一些研究结果表明，高苯丙氨酸血症，特别是在发育中的动物中，影响蛋白质合成，但蛋白质合成速率的直接测量产生了相互矛盾的结果，这可能是由于在估计组织中氨基酸前体库的特定活性方面的方法困难。采用定量放射自显影L-[1-14C]亮氨酸法测定成年Pahenu2小鼠体内脑蛋白合成（lCPS）的局部率。纯合子（HMZ）和杂合子（HTZ）突变体的动脉血浆苯丙氨酸浓度分别比背景菌株（BTBR）高21倍和38%。在脑总酸溶池中，与HTZ或BTBR小鼠相比，HMZ小鼠苯丙氨酸浓度较高，其他中性氨基酸浓度较低，表明血浆苯丙氨酸浓度升高导致血脑屏障处l -氨基酸载体部分饱和。在HMZ小鼠中，与其他两组相比，整个大脑的lCPSleu平均减少了20%，但大脑中trna结合的中性氨基酸浓度没有减少。在一系列稳态实验中，与其他组相比，HMZ小鼠动脉血浆中亮氨酸对脑内trna结合池的贡献在统计学上显著减少，这表明蛋白质合成前体池中亮氨酸的更大一部分来自蛋白质降解。我们在小鼠模型中的结果表明，在未经治疗的成人苯丙酮尿中，血脑屏障上l -氨基酸转运体的部分饱和可能不是lCPSleu减少的基础。除了唐氏综合症，脆性X染色体综合症是人类智力迟钝最常见的遗传形式，估计每4000名男性中就有1人患病。临床特征包括可变但通常严重的智力迟钝，自闭症行为，典型的面部外观和成年男性的睾丸肿大。这种情况以x连锁性状的形式传播。脆性X综合征的名称来源于Xq27.3叶酸敏感脆性位点的表达。这种疾病的分子基础是脆弱的X基因（FMR1） 5'非翻译区域的三联体重复序列（CGG）的大量扩增。完整突变的特征是包含超过200个cgg的大重复序列。结果，FMR1启动子和CGG重复本身被甲基化，导致FMR1基因转录和翻译脆性x蛋白（RMRP）的沉默。在正常脑中，神经组织中FMR1 mRNA和FMRP均富集。虽然FMRP的功能尚不清楚，但它已被证明包含三个RNA结合区。FMRP结合了大约4%的胎儿人脑mrna。很明显，RNA结合在功能上很重要，因为RNA结合区域的突变与严重的智力迟钝有关。FMRP的确切功能尚不清楚，脆性X综合征异常的原因也不清楚。据推测，FMRP参与RNA和/或蛋白质从细胞核到细胞质的运输。由于缺乏FMRP并不致命，所以FMRP一定是具有这种功能的几个基因产物之一。脆性X综合征的主要临床特征是智力迟钝，因此FMRP的功能可能在大脑发育中很重要，也可能在学习和记忆中很重要。随着荷兰-比利时脆性X联盟对脆性X基因敲除小鼠的开发，现在有可能在开发过程中和受控实验条件下研究缺乏FMRP的后果。小鼠模型表现出一些微妙的认知和行为缺陷，多动和睾丸肿大。在小鼠模型和人类疾病中，大脑皮质棘都表现为不成熟，即长、薄和过多。我们假设FMRP可能作为蛋白质合成的调节器，特别是在发育和可塑性期间。我们正在测量成年、雄性半合子和野生型窝鼠的lCPS，以测试缺乏FMR-1 mRNA或FMRP对区域脑蛋白合成的影响。

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.