SORTING AND TRANSPORT OF SPECIFIC NEURONAL GLYCOPROTEINS

特定神经元糖蛋白的分选和运输

• 批准号: 2379609
• 负责人: RICHARD T AMBRON
• 金额: $ 29.16万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-04-01 至 1998-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2379609
• 关键词: Aplysia; Cephalopoda; action potentials; affinity chromatography; axon reaction; axoplasm; cell nucleus; confocal scanning microscopy; dynein ATPase; glycoproteins; immunoelectron microscopy; nerve /myelin protein; nervous system regeneration; neural degeneration; neuronal transport; phosphorylation; protein sequence; protein signal sequence; protein transport; receptor; receptor coupling; squid; tissue /cell culture; vesicle /vacuole; western blottings

The recently discovered pathway whereby axoplasmic proteins in Aplysia neurons are transported retrogradely through the axon to the cell body and then into the nucleus provides a unique connection between the axonal periphery ad the protein synthesizing machinery in the cell body. We hypothesize that this pathway provides a vital link in the process of neuronal plasticity in which axons and terminals undergo structural changes in response to injury or environmental clues. The long-term goals of this project are to explore the mechanism and functions of the retrograde transport/nuclear import pathway. Our immediate focus is on injury and we have evidence that one of the proteins that uses this pathway is an injury signal. Access to both the retrograde transport and nuclear import processes is via a short sequence of basic amino acids comprising a signal peptide (sp). An antibody to the sp recognizes several proteins in both the axoplasm and nucleus of Aplysia neurons. The most abundant of these, sp83, is a glycoprotein that contains single O-linked N-Acetylglucosamine moieties. Sp83 accumulates behind a ligation on peripheral nerves and may be constituitively transported to the nucleus. Another protein, Sp97, is retrogradely transported after axon crush and behaves like an injury signal. Both proteins exist in soluble and membrane-associated forms in the axon. We will use affinity chromatography to purify both proteins from the Aplysia nervous system and will obtain a partial sequence to generate polyclonal antibodies. The antibodies are used to determine the distribution of sp83 and sp97 in the nervous system and as specific probes of t heir function. To study transport/import, the purified proteins are injected directly into axons of Aplysia neurons growing in vitro and their fat in the cell is monitored by confocal microscopy. We will also test the hypotheses that it is the membrane-associated form of the protein that is transported and that the transition from soluble to membrane-associated is regulated by phosphorylation. In addition, the possibility that the sp- proteins are retrogradely transported on the surface of vesicles will be examined by EM immunocytochemistry, as will the possible role of dynein as the retrograde motor. Two proteins (SPRs) in axoplasm recognize the sp and may couple sp-proteins to both the transport and import machinery. This idea is tested by purifying the SPRs and injecting them into the axon and cell body to see if they enter the nucleus. Aplysia sensory neurons undergo changes in their electrical properties after injury. We will see if injecting sp97 into noninjured sensory neurons can elicit these changes. Interestingly, the giant neuron R2 has a different response to injury, however. Sp97 will be injected into this cell to test the idea that unique neurons have unique injury signals. A similar series of experiments are carried out to see if sp83 regulates axon growth in vitro and in vivo. If we are correct, that the transport/import pathway in neurons conveys signals to the nucleus that regulate growth and plasticity, it would have profound affects on our understanding of how peripheral events influence the neuronal phenotype. Moreover, identification of an injury signal would have important implications for therapeutic intervention after nerve injury.

最近发现的途径，轴质蛋白在垂体中 神经元通过轴突逆转到细胞体，然后 然后进入核提供轴突之间的独特联系 外围广告细胞体中的蛋白质合成机械。 我们 假设该途径在过程中提供了至关重要的联系 轴突和末端发生结构变化的神经元可塑性 响应伤害或环境线索。 这个长期目标 项目将探索逆行的机制和功能 运输/核进口途径。 我们的直接重点是伤害，我们 有证据表明使用该途径的一种蛋白质是伤害 信号。 进入逆行运输和核进口 过程是通过一系列构成信号的碱性氨基酸的短序列 肽（SP）。 SP的抗体识别两者中的几种蛋白质 Aplysia神经元的轴突和核。 其中最丰富的 SP83是一种糖蛋白，含有单个O连接的N-乙酰葡萄糖胺 部分。 SP83积聚在周围神经的连接后面，可能 被组成式运输到核。 另一种蛋白质Sp97是 轴突压碎后逆行运输，表现得像受伤 信号。 两种蛋白质都以可溶性和膜相关形式存在 轴突。 我们将使用亲和力色谱法纯化两种蛋白质 Aplysia神经系统，将获得部分序列以生成 多克隆抗体。 抗体用于确定 SP83和SP97在神经系统中的分布以及特定的探针的分布 T Heir功能。 为了研究运输/进口，纯化的蛋白质是 直接注射到体外生长的腹腔神经元的轴突及其 通过共聚焦显微镜监测细胞中的脂肪。 我们还将测试 假设是蛋白质的膜相关形式 运输，从可溶性到膜相关的过渡是 受磷酸化调节。 另外，SP-的可能性 蛋白质在囊泡表面逆行转运 通过EM免疫细胞化学检查，Dynein可能作用 逆行电动机。 轴突中的两个蛋白质（SPR）识别SP和 可以将SP蛋白授予运输和进口机械。 这 通过净化SPR并将其注入轴突来测试想法，然后 细胞体看它们是否进入核。 Aplysia感觉神经元 受伤后其电性能发生变化。 我们会看到 如果将SP97注射到未造成的感觉神经元中可以引起这些变化。 有趣的是，巨型神经元R2对伤害有不同的反应， 然而。 SP97将被注入该单元格，以测试独特的想法 神经元具有独特的伤害信号。 类似的一系列实验是 进行以查看SP83是否在体外和体内调节轴突生长。 如果 我们是正确的，神经元中的运输/进口途径传达 向调节生长和可塑性的细胞核的信号，它将具有 深刻影响我们对外围事件如何影响的理解 神经元表型。 此外，识别受伤信号将 对神经后的治疗干预具有重要意义 受伤。