Role of Local Protein Synthesis in CNS Axon Regeneration

局部蛋白质合成在中枢神经系统轴突再生中的作用

• 批准号: 9903455
• 负责人: MICHAEL EDGAR SELZER
• 金额: $ 34.67万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9903455
• 关键词: ATF2 gene; Actins; Address; Antisense Oligonucleotides; Appearance; Aspirate substance; Axon; Axotomy; Brain; Cell Survival; Cells; Cytoplasm; Cytoskeletal Proteins; Development; Elements; F-Actin; Failure; Filopodia; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Growth Cones; Heterogeneity; Image; Immunohistochemistry; In Vitro; Injury; JUN gene; Lampreys; Lasers; MAPK8 gene; Messenger RNA; Mitogen-Activated Protein Kinase Inhibitor; Mitogen-Activated Protein Kinases; Molecular; Molecular Weight; Motor; Natural regeneration; Nerve Fibers; Nerve Regeneration; Nervous system structure; Neuraxis; Neurofilament-L; Neurons; Nuclear Translocation; Paralysed; Pathway interactions; Perikaryon; Peripheral nerve injury; Phosphorylation; Play; Polyribosomes; Protein Biosynthesis; Protein Synthesis Inhibitors; Proteins; Ribosomes; Risk; Role; Sampling; Signal Pathway; Signal Transduction; Signaling Protein; Site; Specificity; Spinal Cord; Spinal Cord transection injury; Spinal cord injury; Structural Protein; Structure; Surgeon; Testing; Time; Transcript; Translating; Translations; Trees; Western Blotting; Work; axon growth; axon injury; axon regeneration; axoplasm; base; cell growth; cell injury; cell motility; functional restoration; in vivo; inhibitor/antagonist; laser capture microdissection; multiphoton microscopy; neurofilament; neuron development; neuronal cell body; overexpression; p38 Mitogen Activated Protein Kinase; peripheral nerve regeneration; polyadenylated messenger RNA; postnatal development; response; response to injury; retrograde transport; targeted treatment; tissue culture; tissue injury; transcriptome sequencing

Project Summary We will use the unique advantages of the lamprey nervous system to determine whether after spinal cord injury (SCI), local protein synthesis in the axon tip plays a role in the mechanism of axon regeneration. It has long been assumed that axon regeneration depends on protein synthesis in the cell body, the proteins then being transported to the growing axon tips. However, based mainly on observations in tissue culture and injured peripheral nerve, where ribosomes and mRNAs have been detected in axons, a role has been proposed for local protein synthesis in axon regeneration. Locally synthesized proteins might be used to form new axon constituents, or be transported retrogradely to the cell body to signal cell survival and growth-associated gene expression. Until now, evidence for local protein synthesis in the central nervous system (CNS) has been limited primarily to growth cones in early stages of neuronal development in vivo and in vitro. In mature mammalian CNS, where axons do not regenerate, the capacity for local translation is maintained primarily in the dendritic tree; the axons have minimal capacity to synthesize proteins. However, work in lamprey spinal cord, where axons do regenerate, suggests that mature CNS axons regenerate without growth cones, i.e., their tips lack filopodia, have relatively little F-actin, are packed with neurofilaments (NFs), and elongate much more slowly than developing axons. This raises the question whether local protein synthesis plays a role in regeneration of mature CNS axons, even though they do not have growth cones. We previously showed that after SCI in lamprey, mRNA and ribosomes accumulate in the axon tips, and that actively growing tips have more mRNA than static or retracting tips. Moreover, the mRNAs include abundant neurofilament transcripts, but much less actin transcripts, the opposite of findings in mammalian axons growing in vitro or in injured peripheral nerve. Now we will use RNAseq to analyze laser microdissected cytoplasm from large, identified reticulospinal neurons, and micro-aspirated axoplasm from their injured axon tips, to identify mRNAs associated with axon regeneration. We will determine how the mRNAs are regulated in the cell body and get to the tip by determining a) the role of the MAP kinase pathway in signaling axotomy and triggering regeneration, and b) the time course of mRNA appearance along the axon and in the growing tip. Then we will determine whether we can enhance regeneration by locally overexpressing some proteins with micro-injected mRNAs, and whether we can inhibit regeneration by a) inhibiting local protein synthesis with locally applied protein synthesis inhibitors, and b) locally applying morpholino antisense oligonucleotides. This study will help us determine where and how to target therapies to enhance axon regeneration and restore function after SCI.

项目摘要 我们将利用七鳃鳗神经系统的独特优势来确定脊髓损伤后 (SCI)轴突尖端的局部蛋白质合成在轴突再生机制中起作用。长期 假设轴突再生取决于细胞体中的蛋白质合成，然后蛋白质被 运输到生长的轴突尖端。然而，主要基于对组织培养和损伤的观察， 外周神经，其中在轴突中检测到核糖体和mRNA，已经提出了 轴突再生中的局部蛋白质合成。局部合成的蛋白质可能被用来形成新的轴突 成分，或被逆行转运到细胞体，以信号细胞存活和生长相关基因 表情到目前为止，中枢神经系统（CNS）中局部蛋白质合成的证据已经被证实。 主要限于体内和体外神经元发育早期阶段的生长锥。在成熟 在哺乳动物中枢神经系统中，轴突不能再生，局部翻译的能力主要在 树突树;轴突合成蛋白质的能力最小。然而，在七鳃鳗脊椎的工作 索，轴突再生的地方，表明成熟的CNS轴突再生没有生长锥，即，他们的 尖端缺乏丝状伪足，具有相对较少的F-肌动蛋白，充满了神经丝（NF），并且伸长得更多 比发育中的轴突要慢这就提出了一个问题，即局部蛋白质合成是否在 成熟CNS轴突的再生，即使它们没有生长锥。我们之前已经证明， 七鳃鳗脊髓损伤后，mRNA和核糖体在轴突尖端积累，活跃生长的尖端 更多的mRNA比静态或缩回尖端。此外，mRNA包括丰富的神经丝转录物， 但肌动蛋白转录物的数量要少得多，这与体外生长的哺乳动物轴突或损伤的 外周神经现在，我们将使用RNAseq来分析来自大的、鉴定的、 网状脊髓神经元和从其受损轴突尖端微抽吸的轴浆，以鉴定mRNA 与轴突再生有关我们将确定mRNA在细胞体中是如何调节的， a）MAP激酶途径在发信号传导轴突切断和触发再生中的作用， 和B）mRNA沿沿着轴突和在生长尖端出现的时间过程。然后我们将确定 我们是否可以通过微注射mRNA局部过表达某些蛋白质来增强再生， 以及我们是否可以通过a）用局部施用的蛋白质抑制局部蛋白质合成来抑制再生 合成抑制剂，和B）局部应用吗啉代反义寡核苷酸。这项研究将帮助我们 确定在哪里以及如何靶向治疗以增强SCI后的轴突再生和恢复功能。