SORTING AND TRANSPORT OF SPECIFIC NEURONAL GLYCOPROTEINS

特定神经元糖蛋白的分选和运输

• 批准号: 6393370
• 负责人: RICHARD T AMBRON
• 金额: $ 30.93万
• 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 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6393370
• 关键词: Aplysia; Cephalopoda; action potentials; axon reaction; axoplasm; cell nucleus; dynein ATPase; enzyme activity; glycoproteins; 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

DESCRIPTION (from applicant's abstract) Nerve injury triggers long-term alterations that require changes in protein synthesis and which may result in the restoration of function. Often, however, regeneration fails, resulting in sensory deficits, chronic pain, and paralysis. Efforts to promote growth and minimize sensory defects would be facilitated if we knew the identity of the signals that inform the cell soma that its axon has been injured and how these signals regulated the transcriptional programs that are responsible for successful regeneration. Using the nervous system of Aplysia californica as a model the applicants found that positive injury signals activated at the site of axon injury are retrogradely transported to the cell nucleus. When axoplasm containing these signals is injected into non-injured neurons, it induces the same growth and hyperexcitability that appears when the axons of these cells are injured. A similar hyperexcitability occurs after axotomy in mammalian neurons and is thought to be responsible for chronic pain. To identify the signals responsible for these changes, they analyzed the axoplasm and found it to be enriched in 2 kinases, ERK and SAPK. Most of the ERK is in the phosphorylated (active) form and the applicants hypothesize that activation occurs when an influx of calcium at the lesion site activates phosphokinase C. They will manipulate calcium levels using an ionophore to see whether PKC is affected. How ERK is retrogradely transported is not known. They will inject recombinant ERK directly into the axon to monitor its transport and will use specific antibodies and subcellular fractionation of axoplasm to see if it occurs in association with an organelle. Once ERK reaches the nucleus it phosphorylates the transcription factor C/EBP. This could increase the affinity of C/EBP for DNA, alter transcription, or regulate its entry into the nucleus. Each possibility will be assessed using recombinant wild type and mutated C/EBP. Interestingly ERK is also activated by nerve inflammation, which also induces hyperexcitability. This suggests that hyperexcitability is due to ERK acting on C/EBP. They will attempt to interfere with this process by microinjecting antibodies and oligonucleotides and by using mutated ERK and C/EBP. In contrast, retrogradely transported SAPK is constitutively active, although its activity increases after injury, and it may be involved in growth through c-Jun. The investigators have antibodies and recombinant proteins to investigate this possibility and will use strategies similar to those employed for ERK.

描述(摘自申请者的摘要)神经损伤会触发长期 需要改变蛋白质合成的改变，并可能导致 在恢复功能方面。然而，再生往往会失败， 导致感觉障碍、慢性疼痛和瘫痪。努力实现 如果我们知道，促进生长和最大限度地减少感官缺陷将会更容易 通知细胞胞体轴突的信号标识 以及这些信号是如何调节转录程序的 对成功再生负有责任。利用人类的神经系统 作为海兔模型的申请者发现，积极的伤害 在轴突损伤处激活的信号被逆行传输到 细胞核。当含有这些信号的轴浆被注射到 无损伤的神经元，它诱导的生长和过度兴奋与 当这些细胞的轴突受到损伤时就会出现。一种类似的 哺乳动物神经元在轴突切断后发生过度兴奋性，并被认为 对慢性疼痛负责。以确定负责的信号 这些变化，他们分析了轴浆，并发现它在2 激酶、ERK和SAPK。大部分ERK处于磷酸化(激活)状态 表格和申请人假设激活发生在涌入 病变部位的钙激活了磷酸激酶C，他们会操纵 钙水平使用离子载体来观察PKC是否受到影响。如何使用ERK 是不是被逆行运输的还不得而知。他们将注射重组ERK 直接进入轴突来监控其运输，并将使用特定的 抗体和轴浆亚细胞分级以了解它是否发生在 与细胞器联系在一起。一旦ERK到达细胞核，它就会 使转录因子C/EBP磷酸化。这可能会增加 C/EBP与DNA的亲和力、改变转录或调节其进入 原子核。每种可能性都将使用重组野生型进行评估 C/EBP基因突变。有趣的是，ERK也被神经激活 炎症，这也会导致过度兴奋。这表明 过度兴奋是由于ERK作用于C/EBP所致。他们将尝试 通过微量注射抗体和 通过使用突变的ERK和C/EBP。相比之下， 倒退运输的SAPK在本质上是活跃的，尽管它的 受伤后活动量增加，可能通过以下途径参与生长 C-Jun.研究人员拥有抗体和重组蛋白来 调查这种可能性，并将使用类似于 受雇于ERK。