MOLECULAR BASES OF NEURONAL CONNECTIVITY

为了使受损的通路在损伤后再生，神经细胞 必须开始表达重建基因所需的基因产物 它们的轴突；这反过来又受到细胞外环境的影响 由非神经元支持细胞提供。低等脊椎动物能够 损伤后再生视神经，视力完全恢复 这一现象可能使我们能够理解分子和细胞 脊椎动物中枢的生长和可塑性的基础变化 神经系统。我们将利用生物化学和基因克隆方法来 确定神经元和非神经元分子对 金鱼视神经的再生。目标1将确定部分 参与最佳基因表达的分子级联 鉴定了生长相关蛋白GAP-43。我们的预赛 有证据表明，GAP-43的表达在很大程度上被控制在 转录后水平，并涉及特定的结合 蛋白质与信使RNA的3‘未翻译端的序列 可能起到保护其免受核酸酶降解的作用。我们将隔离 这些蛋白质并鉴定它们与之结合的核苷酸序列。 目标2将分离并测序一种不太典型的 视神经通路的生长相关蛋白GAP-24。我们会比较一下 调节其表达的机制与目标1至 对内部发生的一连串事件有更广泛的了解 神经元在再生轴突时。目标3专注于一种蛋白质 由神经的非神经性鞘细胞分泌，可能 作为其他两个部分研究的分子变化的触发器。 我们将结合经典的蛋白质纯化方法，克隆 抗体技术部分氨基酸测序，cDNA文库 筛选、识别生长因子的抗体和探针，以及 几种生物测定系统来分离和测序这种营养因子。 总而言之，这些研究代表了一种理解 视觉再生的细胞和分子生物学基础 系统，最终可能被应用于诱导视觉的知识 人类的康复。

In order for damaged pathways to regenerate after injury, nerve cells must begin to express gene products required for the reconstruction of their axons; this in turn is influenced by the extracellular milieu provided by non-neuronal support cells. Lower vertebrates are able to regenerate their optic nerve after injury and recover vision fully, a phenomenon that may enable us to understand the molecular and cellular changes that underlie growth and plasticity in the vertebrate central nervous system. We will utilize biochemistry and gene cloning methods to identify neuronal and non-neuronal molecules that are critical for the regeneration of the goldfish optic nerve. Aim 1 will identify portions of the molecular cascade involved in the expression of the best characterized growth-associated protein, GAP-43. Our preliminary evidence indicates that the expression of GAP-43 is controlled largely at the post-transcriptional level, and involves the binding of specific proteins to sequences in the 3'untranslated end of the messenger RNA that may serve to protect it from degradation by nucleases. We will isolate these proteins and identify the nucleotide sequences to which they bind. Aim 2 will isolate and sequence a less well-characterized growth-associated protein of the optic pathway, GAP-24. We will compare the mechanisms that regulate its expression with those found in Aim 1 to gain a broader understanding of the cascade of events occurring within the neuron as it regenerates its axon. Aim 3 focuses on a protein secreted from the non-neuronal sheath cells of the nerve that may serve as a trigger for the molecular changes studied in the other two sections. We will combine classical protein purification methods, monoclonal antibody technology partial amino acid sequencing, cDNA library screening, antibodies and probes to identified growth factors, and several bioassay systems to isolate and sequence this trophic factor. Together, these studies represent an integrated approach to understanding the cellular and molecular biology underlying regeneration in the visual system, knowledge that may eventually be applied for inducing visual recovery in man.