SORTING AND TRANSPORT OF SPECIFIC NEURONAL GLYCOPROTEINS

最近发现的途径，轴浆蛋白在Aaplasia 神经元通过轴突逆行运输到细胞体， 然后进入细胞核，在轴突和神经元之间提供了一种独特的连接， 细胞内的蛋白质合成机制。 我们 假设这条途径在这一过程中提供了一个重要的环节， 轴突和末梢经历结构变化的神经可塑性 对受伤或环境线索的反应 长期目标是 项目是探索逆行的机制和功能， 运输/核进口途径。 我们现在的重点是伤病， 有证据表明，使用这一途径的蛋白质之一是损伤 信号了 可同时使用逆行运输和核进口 过程是通过包含信号的碱性氨基酸的短序列 肽（sp）。 一种抗sp的抗体可以识别两种细胞中的几种蛋白质。 脊髓神经元的轴浆和细胞核。 其中最丰富的， sp 83是一种糖蛋白，含有单个O-连接的N-乙酰葡糖胺 部分。 Sp 83在周围神经结扎后积聚， 被组成性地运送到细胞核。 另一种蛋白质Sp 97， 轴突破碎后逆行运输，表现为损伤 信号了 这两种蛋白质都以可溶性和膜结合的形式存在， 轴突 我们将使用亲和色谱法纯化这两种蛋白质， 并将获得部分序列以生成 多克隆抗体。 抗体用于确定 sp 83和sp 97在神经系统中的分布及其作为特异性探针 的功能。 为了研究转运/输入，将纯化的蛋白质 直接注射到体外生长的轴突中， 通过共聚焦显微镜监测细胞中的脂肪。 我们还将测试 假设它是蛋白质的膜相关形式， 运输，从可溶性到膜相关的过渡是 由磷酸化调节。 此外，SP- 蛋白质在囊泡表面逆行运输， 通过EM免疫细胞化学检查，动力蛋白的可能作用， 逆行马达 轴质中的两种蛋白质（SPR）识别sp和 可以将SP-蛋白偶联到运输和输入机构。 这 通过纯化SPRs并将其注入轴突来测试想法， 观察它们是否进入细胞核。 感觉神经元缺失 在受伤后经历其电特性的变化。 我们将看到 如果将sp 97注射到未受伤的感觉神经元中可以引起这些变化。 有趣的是，巨大的神经元R2对损伤有不同的反应， 然而. SP 97将被注射到这个细胞中，以测试这种独特的想法， 神经元具有独特的损伤信号。 类似的一系列实验是 进行，看看是否sp 83调节轴突生长在体外和体内。 如果 我们是正确的，神经元中的转运/输入途径传递了 向调节生长和可塑性的细胞核发送信号， 深刻地影响了我们对周边事件如何影响 神经元表型。 此外，损伤信号的识别将 对神经后的治疗干预有重要意义， 损伤

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.