SYNAPTIC ORGANIZATION OF THE OPTIC TECTUM

视顶盖的突触组织

• 批准号: 3255677
• 负责人: JOHN A FREEMAN
• 金额: $ 33.5万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-03-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3255677
• 关键词: autoradiography; calcium flux; casein kinase; electron microscopy; gene expression; guanine nucleotide binding protein; in situ hybridization; laboratory rabbit; laboratory rat; membrane channels; nervous system regeneration; neurogenesis; neuronal transport; nuclear runoff assay; optic nerve; phosphorylation; posttranscriptional RNA processing; protein kinase C; protein structure function; retinal ganglion; site directed mutagenesis; tissue /cell culture; transcription factor; video recording system; voltage /patch clamp

The overall objective of the proposed research is to identify the cellular functions and mechanisms of regulation of GAP43, the most prominent of a recently discovered class of growth associated proteins whose synthesis is selectively enhanced during periods of axonal growth and regeneration in retinal ganglion cells and other CNS neurons. We have obtained preliminary evidence suggesting that GAP43 expression is dually regulated by transcriptional as well as by post-transcriptional mechanisms, and postulate that retinal ganglion cells constitutively express the gene for GAP43, but fail to upregulate GAP43 levels following injury due to inhibitory factors, associated with contact with non-neuronal cells, conveyed by retrograde transport from the nerve terminal to the nucleus. We propose experiments designed to provide definitive information about the cellular function of this protein, to identify molecules that control its expression, and to identify the mechanisms by which its expression is regulated post-transcriptionally. Specific residues associated with known functional domains of GAP43 will be modified by oligonucleotide-directed site-specific mutagenesis. These include sequences associated with membrane attachment, phosphorylation by protein kinase C (pKC) and by casein kinase II (CKII), and GTP-G-O protein binding. Resulting functional effects will be measured in growth cones of rat retinal ganglion cells, including transport targeting, motility (using quantitative time-lapse video microscopy), intracellular calcium levels (using fura-2 imaging), and calcium channel conductance (using patch clamp analysis). Retrograde regulatory mechanisms will be investigated using blockers of axonal transport and quantitative 2D gel autoradiography. Experiments are designed to determine whether retrogradely transported molecules endocytosed from or modified by contact with mature oligodendrocytes in the optic nerve regulate the expression of GAP43, and if so, to isolate and test the regulatory molecules. The regulation of GAP43 message will be analyzed using quantitative RNA hybridization analysis and nuclear run-on assays to examine GAP43 message quantitatively. This will determine the extent to which GAP43 mRNA is regulated post-transcriptionally by alterations of message stability and translation efficiency during development and following optic nerve injury. Understanding the functions and molecular genetic control of this protein is likely to provide important insights more generally into the mechanisms of axonal growth, and in particular, might provide information critical to attempts to promote optic nerve regeneration and repair in the mammalian CNS. This information is of paramount importance in understanding the development of the visual system, and its response to injury.

拟议研究的总体目标是确定细胞 GAP 43的功能和调节机制，最突出的一个 最近发现的一类生长相关蛋白，其合成是 在轴突生长和再生期间选择性增强， 视网膜神经节细胞和其他CNS神经元。我们已经获得了初步的 有证据表明，GAP 43的表达受到以下因素的双重调节： 转录以及转录后机制，和 假设视网膜神经节细胞组成型表达基因， GAP 43，但在损伤后不能上调GAP 43水平， 与非神经元细胞接触相关的抑制因子， 通过从神经末梢到核的逆行运输传递。 我们提出的实验旨在提供明确的信息， 这种蛋白质的细胞功能，以确定控制 它的表达，并确定其表达的机制， 转录后调控。与已知的 GAP 43的功能结构域将通过寡核苷酸定向修饰 定点诱变。这些序列包括与 膜附着，蛋白激酶C（pKC）和 酪蛋白激酶II（CKII）和GTP-G-O蛋白结合。结果泛函 将在大鼠视网膜神经节细胞的生长锥中测量效果， 包括转运靶向、运动性（使用定量时间推移 视频显微术），细胞内钙水平（使用Fura-2成像）， 和钙通道电导（使用膜片钳分析）。逆行 调节机制将使用轴突的阻断剂进行研究。 运输和定量2D凝胶放射自显影。实验 旨在确定逆行转运的分子 从成熟的少突胶质细胞内吞或通过与成熟的少突胶质细胞接触而修饰， 视神经调节GAP 43的表达，如果是这样， 并测试调节分子。GAP 43报文的规定将 使用定量RNA杂交分析和细胞核运行分析 定量检测GAP 43信息。这将决定 GAP 43 mRNA在转录后受到调节的程度 改变消息稳定性和翻译效率， 发展和视神经损伤后。了解功能 这种蛋白质的分子遗传控制可能会提供 更普遍地对轴突生长机制的重要见解， 特别是，可能会提供关键信息， 促进哺乳动物中枢神经系统视神经再生和修复。这 信息对于理解发展至关重要 视觉系统及其对损伤的反应。