SORTING AND TRANSPORT OF SPECIFIC NEURONAL GLYCOPROTEINS

海兔新发现的轴浆蛋白途径 神经元通过轴突逆行运输到细胞体， 然后进入核团提供了轴突之间独特的联系 外周和细胞体中的蛋白质合成机器。我们 假设这条途径提供了一个至关重要的环节 轴突和终末经历结构变化的神经元可塑性 作为对伤害或环境线索的反应。这样做的长期目标是 本项目旨在探索逆行的机制和功能 运输/核进口途径。我们现在的关注点是伤病，我们 有证据表明使用这种途径的蛋白质之一是一种损伤 信号。获得逆行运输和核进口的机会 过程是通过一短序列的碱性氨基酸组成的信号 多肽(SP)。抗SP的抗体可识别这两种蛋白中的几种。 海兔神经元的轴浆和胞核。其中最丰富的是， Sp83是一种含有单一O-连接N-乙酰氨基葡萄糖的糖蛋白 半个。Sp83在周围神经结扎后积聚，可能 在宪法上被输送到原子核。另一种蛋白质SP97是 轴突挤压后逆行运输，表现为受伤 信号。这两种蛋白质都以可溶性和膜结合的形式存在于 轴突。我们将使用亲和层析来纯化这两种蛋白质 并将获得海兔神经系统的部分序列生成 多克隆抗体。抗体被用来确定 SP83和SP97在神经系统中的分布及其作为特异性探针 继承人的职能。为了研究运输/进口，纯化的蛋白质是 直接注射入体外生长的海兔神经元轴突 细胞中的脂肪通过共聚焦显微镜进行监测。我们还将测试 假设它是膜相关形式的蛋白质，即 从可溶到膜结合的转变是 受磷酸化调节。此外，SP-的可能性- 蛋白质在囊泡表面的逆行运输会被 通过EM免疫细胞化学检查，以及动力蛋白作为 逆行马达。轴浆中的两种蛋白质(SPR)识别SP和 可以将SP蛋白偶联到运输和进口机械上。这 通过提纯SPR并将它们注入轴突和 细胞体观察它们是否进入细胞核。海兔感觉神经元 在受伤后会发生电学性质的变化。我们将拭目以待 如果将SP97注射到未损伤的感觉神经元可以引起这些变化。 有趣的是，巨型神经元R2对损伤有不同的反应， 然而。SP97将被注入到这个细胞中，以测试这种独特的想法 神经元有独特的损伤信号。类似的一系列实验是 以了解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/输入途径传递 调节生长和可塑性的核信号，它会有 深刻影响我们对外围事件如何影响的理解 神经细胞的表型。此外，识别伤害信号将 对神经后遗症的治疗干预有重要意义 受伤。

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.