GLUTAMYLCYSTEUBE SYNTHETASE AND AGING

在各种老化组织和年龄- 相关疾病，并已被认为是负责 在老化组织中观察到的氧化损伤增加， 神经退行性疾病 直接和间接的证据都表明 降低γ-谷氨酰半胱氨酸合成酶（GCS）的活性， 谷胱甘肽从头合成的限速酶，是至少一种 导致衰老组织中GSH减少的因素。 但 这种GCS活性降低的潜在机制尚不清楚。 因此，本项目的具体目标是：1）确认 老年脑GCS活性和GSH含量均降低， 来自3个月、12个月和24个月大的小鼠的大脑的不同部分。 2)到 确定老化组织中GCS活性降低是否是由于 Western blotting显示蛋白质含量降低。 3)探讨 GCS表达下调的潜在机制 老年人的大脑 GCS蛋白的翻译和稳定性 年轻和年老小鼠的脑切片将使用短期的 [35S]蛋氨酸标记技术 GCS mRNA含量及 年轻人和老年人大脑中GCS基因的转录率将 分别通过北方印迹和核连续分析测量。 将通过注射年轻和年老的GCS mRNA来确定GCS mRNA的稳定性。 小鼠与转录抑制剂放线菌素D，然后测量半- 通过北方印迹分析来自脑切片的GCS mRNA的寿命。 此外，将处理来自年老和年幼动物的脑切片 用胰岛素加糖皮质激素看GCS是否下调 在老年脑中的表达是由于细胞对 荷尔蒙如果没有发现GCS蛋白质的量的差异， 年轻和年老动物的脑切片， 将按照目的4中所述检查GCS蛋白。 4)以确定 GCS蛋白的磷酸化和/或氧化 老年组织中GCS活性降低的原因。 长期目标 这个项目的目的是揭示衰老的潜在机制， 过程和年龄相关疾病，因此，提供线索， 对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.