SORTING AND TRANSPORT OF SPECIFIC NEURONAL GLYCOPROTEINS

特定神经元糖蛋白的分选和运输

• 批准号: 6484429
• 负责人: RICHARD T AMBRON
• 金额: $ 3.27万
• 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/6484429
• 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（来自申请人的摘要）神经损伤长期触发 需要改变蛋白质合成并可能导致的改变 在恢复功能中。 但是，再生通常会失败， 导致感觉缺陷，慢性疼痛和麻痹。 努力 如果我们知道，促进增长并最大程度地减少感觉缺陷将得到促进 信号的身份通知细胞体的轴突已是 受伤以及这些信号如何调节转录程序 负责成功再生。 使用神经系统 申请人发现阳性伤害的aplysia加利福尼亚州 在轴突损伤部位激活的信号逆转到 细胞核。 当将包含这些信号的轴突注入 无损伤的神经元，它诱导了相同的生长和过度兴奋性 这些细胞的轴突受伤时会出现。 类似 哺乳动物神经元的轴突切开术后过度兴奋性发生，被认为 负责慢性疼痛。 确定负责的信号 这些变化，他们分析了轴质，发现它富含2 激酶，ERK和SAPK。 大多数ERK在磷酸化（活动）中 形式，申请人假设激活发生时发生 病变位点的钙激活磷酸激酶C。它们将操纵 使用离子团的钙水平查看是否受到PKC的影响。 如何ERK 逆行运输尚不清楚。 他们会注入重组ERK 直接进入轴突以监视其运输，并将使用特定 轴突的抗体和亚细胞分级，以查看它是否发生在 与细胞器的关联。 一旦ERK到达核 磷酸化转录因子C/EBP。 这可能会增加 C/EBP对DNA的亲和力，更改转录或调节其进入 核。 每种可能性将使用重组野生型评估 并突变C/EBP。 有趣的是，ERK也被神经激活 炎症，这也引起过度刺激性。 这表明这一点 过度兴奋性是由于ERK作用于C/EBP。 他们会尝试 通过显微注射抗体和 寡核苷酸以及使用突变的ERK和C/EBP。 相比之下， 逆转运输的SAPK具有组成性活跃，尽管它 受伤后的活动增加，并且可能与 C-Jun。 研究人员有抗体和重组蛋白 调查这种可能性，并将使用与那些类似的策略 为ERK雇用。