GENE EXPRESSION IN AXOTOMIZED RETINAL GANGLION CELLS

轴切视网膜神经节细胞中的基因表达

基本信息

批准号：
2710767
负责人：
Leonard A Levin
金额：
$ 8.92万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-08-01 至 1999-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2710767
关键词：
RNase protection assay apoptosis denervation developmental genetics electron microscopy fusion gene gene expression genetic library immunocytochemistry laboratory rat neurogenetics neuronal transport northern blottings retinal ganglion

项目摘要

Death of retinal ganglion cells is the final common pathway underlying virtually all diseases of the optic nerve, including anterior ischemic optic neuropathy, optic neuritis, and compressive optic neuropathy. Despite a wealth of studies examining the direct effect of hypoxia, excitotoxins, and other injuries on retinal ganglion cells, the effect on these cells of axonal injury, which underlies most optic neuropathies, is far less clearly understood. Attempts to promote regeneration of ganglion cell axons after axotomy presupposes an intact ganglion cell soma; only by preventing ganglion cell death can it be expected that regenerative strategies will succeed. Therefore the overall goal of this proposal is to use recently developed techniques to better understand the subcellular events underlying death of retinal ganglion cells due to axonal injury. The working hypothesis is that axotomy of retinal ganglion cells causes expression of genes that turn on a program for cell death, and that these genes and their products can be characterized. The long-term goal is to modulate these genes or their products and therefore allow rescue of retinal ganglion cells when the optic nerve is injured. The first specific aim is to identify genes differentially expressed in axotomized retinal ganglion cells. A subtracted copy DNA (cDNA) library will be made from messenger RNA (mRNA) extracted from axotomized and control rat retinas. Candidate cDNAs will be screened with Northern blotting or ribonuclease protection assay using RNA probes. The second specific aim is to study the tissue, species, and developmental expression of the identified genes. Gene sequences will be used to look for homologies to other genes associated with cell death. The third specific aim is to express the protein products of the genes and determine their localization and possible function. The health-relatedness of this project is to diseases affecting the anterior and retrobulbar optic nerve. While these disorders directly damage axons contained within the optic nerve, the irreversible loss of vision reflects the death of ganglion cells contained within the retina. Understanding the subcellular nature of the ganglion cell response to axonal injury will allow development of therapies for these diseases. The opportunity to receive formal training in molecular biology and carry out this research program will also further the applicant's goal of becoming an independent investigator.

视网膜神经节细胞的死亡是最终的公共途径几乎所有视神经的疾病，包括前缺血性视神经病变，视神经炎和压缩视神经病变。尽管大量研究了缺氧的直接影响，但兴奋毒素和视网膜神经节细胞上的其他损伤，对这些轴突损伤细胞是大多数视神经病变的基础，是不清楚地理解。试图促进神经节再生轴切开后的细胞轴突以完整的神经节细胞体为前提。仅由可以预期再生神经节细胞死亡策略将成功。因此，该提议的总体目标是使用最近开发的技术来更好地了解亚细胞由于轴突损伤导致的视网膜神经节细胞死亡的事件。工作假设是视网膜神经节细胞的轴切开术会导致表达打开细胞死亡程序的基因，这些基因及其产品可以被表征。长期目标是调节这些基因或其产品，因此允许营救视网膜神经节细胞受伤时。第一个具体目的是识别以差异表达的基因轴向视网膜神经节细胞。减去拷贝DNA（cDNA）库将由从轴测质中提取的使者RNA（mRNA）制成控制大鼠视网膜。候选cDNA将与北部筛选使用RNA探针的印迹或核糖核酸酶保护测定法。第二个具体目的是研究组织，物种和发育表达确定的基因。基因序列将用于寻找与细胞死亡相关的其他基因的同源物。第三个特定目的是表达基因的蛋白质产物并确定其本地化和可能的功能。该项目与健康相关的是影响影响的疾病前后侧面视神经。而这些疾病直接损伤轴突包含的视神经内，不可逆转的损失视力反映了视网膜中包含的神经节细胞的死亡。了解神经节细胞反应的亚细胞性质轴突损伤将允许开发这些疾病的疗法。这有机会接受分子生物学的正式培训并进行该研究计划还将进一步进一步成为申请人的目标独立研究者。