REGULATION OF CA2+-RELATED MRNA/PROTEIN EXPRESSION BY AGING AND HORMONES

衰老和激素对 CA2 相关 mRNA/蛋白质表达的调节

• 批准号: 6299337
• 负责人: KUEY-CHU CHEN
• 金额: $ 20.95万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-15 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6299337
• 关键词: Alzheimer's disease; aging; antisense nucleic acid; autoradiography; calcium channel; calmodulin; confocal scanning microscopy; electrophysiology; gene expression; glucocorticoids; hippocampus; homeostasis; hormone regulation /control mechanism; immunocytochemistry; in situ hybridization; laboratory rat; messenger RNA; neural degeneration; neurons; oligonucleotides; peptidylprolyl isomerase; polymerase chain reaction; protein structure function; tissue /cell culture; western blottings

This project has the primary overall goal of seeking changes in calcium regulatory mRNA and protein expression in hippocampus that are common to both aging and glucocorticoid (GC) activation. Those studies test the hypothesis that changes in Ca/2+ regulation common to aging and GC activation are important in neuronal vulnerability. In the prior period we developed extensive ribonuclease protection assay (RPA) approaches to examine effects of aging on hippocampal mRNA expression of 11 calcium- related genes (e.g., the alpha/1D subunit, alpha/1C subunit and beta subunit of the L-type calcium channel, plasma membrane calcium membrane calcium ATPase (PMCA) isoforms 1 and 2, calmodulin II, calcineurin cyclophilin D, glucocorticoid receptor (GR), mineralocorticoid receptor (MR) and as a control, glyceraldehyde phosphate dehydrogenase, (GAPDH)). The equivalent proteins were measured in aging for most of these genes. Surprisingly few changes common to aging and GC activation were found. However, both aging and GCs increased 3 genes: cycophilin A, calmodulin II, and the alpha/1D subunit of the L-type calcium channel. In situ hybridization showed that A/1D changes in aging were most pronounced in field CA1. Protein measurements (westerns) did not show clear effects of aging, but required more tissue and are mess sensitive than RPAs. The need to homogenize tissues for both RPAs and Westerns appears to be a major obscuring factor. In the next period, therefore, it is focused these studies at the single cell and/or regional level, and to introduce direct collections with functional measure (electrophysiology, calcium imaging, collaboration with Project 3) to pursue these leads and more accurately test the main hypotheses. To achieve this improved accuracy and localization, the next phase will utilize single-cell RT-PCR method methods in neurons from which electrophysiological recordings are obtained, and extensive in situ hybridization in aged or adrenalectomized rats versus controls. Proteins assays will be enhanced by performing immunoautoradiography (IAR) and regional dissections prior to western blotting analyses. These studies will more accurately be able to test the test that increased expression of the mRNA for the L-type calcium channel, for calmodulin or for cyclophilin induced by aging or hormonal modulation alters calcium channel activity and/or calcium homeostasis and modifies neuronal vulnerability.

该项目的主要总体目标是寻求钙的变化 海马中的调节mRNA和蛋白质表达是常见的 衰老和糖皮质激素（GC）激活。这些研究测试了 假设CA/2+调节的变化是衰老和GC的常见 激活在神经元脆弱性中很重要。前期 我们开发了广泛的核糖核酸酶保护测定法（RPA）的方法 检查衰老对11钙的海马mRNA表达的影响 相关基因（例如，alpha/1d亚基，alpha/1c亚基和beta L型钙通道的亚基，质膜钙膜 钙ATPase（PMCA）同工型1和2，钙调蛋白II，钙调蛋白 环磷脂D，糖皮质激素受体（GR），矿物皮质受体受体 （MR）和作为对照，磷酸甘油醛脱氢酶（GAPDH））。 对于大多数这些基因，在衰老中测量了等效蛋白。 令人惊讶的是，很少发现衰老和GC激活的变化。 但是，衰老和GC都增加了3个基因：环粒蛋白A，钙调蛋白 II和L型钙通道的α/1D亚基。原位 杂交表明，衰老的A/1D变化最为明显 场CA1。蛋白质测量（西方）没有显示出明确的影响 衰老，但需要比RPA的组织更敏感。这 需要将RPA和西方人的组织均匀化似乎是 主要的模糊因素。 因此，在下一个时期，将这些研究集中在单一时期 单元和/或区域级别，并引入直接收藏 功能度量（电生理学，钙成像，协作 使用项目3）追求这些潜在客户并更准确地测试主要 假设。为了提高准确性和本地化，下一个 阶段将利用来自来自的神经元中的单细胞RT-PCR方法 获得了哪些电生理记录，并广泛 老年或肾上腺切除大鼠与对照组的原位杂交。 蛋白质测定将通过执行免疫功能摄影来增强 （IAR）和区域解剖之前进行蛋白质印迹分析。这些 研究将更准确地测试增加的测试 mRNA的表达L型钙通道，钙调蛋白或 对于通过衰老或激素调节诱导的环糖素 通道活动和/或钙稳态，并修饰神经元 脆弱性。