GLUTAMYLCYSTEUBE SYNTHETASE AND AGING

在不同年龄的组织中发现谷胱甘肽含量降低，而在年龄- 相关疾病，并被建议对 老年组织和年龄相关组织中观察到的氧化损伤增加 神经退行性疾病。直接和间接证据都表明 降低γ-谷氨酰半胱氨酸合成酶(GCS)的活性， GSH从头合成中的限速酶，至少是 导致衰老组织中GSH降低的因素。然而， GCS活性下降的机制尚不清楚。 因此，该项目的具体目标是：1)确认 老年脑内GCS活性和GSH含量均降低 3月龄、12月龄和24月龄小鼠的不同脑组织切片。2)至 确定衰老组织中GCS活性降低是否是由于 Western blotting显示蛋白质含量减少。3)探索 GCS表达下调的可能机制 在衰老的大脑中。日本血吸虫GCS蛋白的翻译及其稳定性 幼年和老年小鼠的脑切片将使用短期 [35S]蛋氨酸标记技术。GCS信使核糖核酸含量和 年轻人和老年人大脑中GCS基因的转录速率将是 分别通过Northern印迹分析和核连续分析进行测量。 GCS mRNAs的稳定性将通过注射年轻人和老年人来确定 小鼠注射转录抑制剂放线菌素D，然后测量一半 Northern blotting分析脑切片GCS mRNAs的寿命。 此外，来自老年和幼年动物的脑切片将被处理 用胰岛素对糖皮质激素是否下调GCS进行观察 衰老大脑中的表达是由于细胞对 荷尔蒙。如果没有发现GCS蛋白的量在 幼年和老年动物的脑切片，结构修改 GCS蛋白将按照目标4.4)中的描述进行检测，以确定 是否与GCS蛋白的磷酸化和/或氧化有关 对于衰老组织中GCS活性的降低。长期目标 这个项目的目的是揭示衰老背后的机制 过程和年龄相关的疾病，因此，为 年龄相关疾病的潜在治疗方法。

Decreased GSH content has been found in various aged tissues and in age- related diseases, and has been suggested to be responsible for the increased oxidative damage observed in aged tissues and age-related neurodegenerative diseases. Both direct and indirect evidence indicates that decreased activity of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo GSH synthesis, is at least one of the factors that contribute to decreased GSH in aged tissues. However, the mechanism underlying such decreased GCS activity is not clear. Therefore, the specific aims for this project are: 1) To confirm that both GCS activity and GSH content decrease in aged brain by comparing different sections of brains from 3, 12, and 24 month-old mice. 2) To determine whether the decreased GCS activity in aged tissue is due to decreased protein amount by Western blotting. 3) To explore the potential mechanism responsible for down-regulation of GCS expression in aged brain. The translation and the stability of GCS proteins from brain sections of young and aged mice will be measured using short-term [35S] methionine labeling technique. GCS mRNA contents and the transcription rates of GCS genes in young and aged brains will be measured by Northern blotting and nuclear run-on analyses, respectively. Stability of GCS mRNAs will be determined by injecting young and aged mice with transcription inhibitor actinomycin D and then measuring half- life of GCS mRNAs from the brain sections by Northern blotting analysis. Furthermore, brain slices from aged and young animals will be treated with insulin of glucocorticoid to see whether the down-regulation of GCS expression in aged brain is due to a decreased response of the cells to hormones. If no difference in the amount of GCS protein is found between brain sections from young and aged animals, structural modification of GCS protein will be examined as described in aim 4. 4) To determine whether phosphorylation and/or oxidation of GCS proteins is responsible for the decreased GCS activity in aged tissues. The long-term objective of this project is to uncover the mechanisms underlying the aging process and age-related diseases and therefore, to provide clues for potential therapeutic approaches to the age-related diseases.