Role of Local Protein Synthesis in CNS Axon Regeneration

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着重要作用。它已经有很长时间了 一直认为轴突的再生依赖于细胞体中蛋白质的合成，然后蛋白质被 被运送到不断生长的轴突尖端。然而，主要基于组织培养中的观察和损伤 周围神经，在轴突中检测到核糖体和mRNAs，已经提出了在 轴突再生中的局部蛋白质合成。局部合成的蛋白质可能用于形成新的轴突 成分，或被逆行运输到细胞体，以信号细胞生存和生长相关基因 表情。到目前为止，有证据表明中枢神经系统(CNS)中存在局部蛋白质合成 主要局限于体内和体外神经元发育的早期阶段的生长锥体。成熟期 哺乳动物中枢神经系统，轴突不再生，局部翻译的能力主要维持在 树状突起；轴突合成蛋白质的能力最小。然而，在七鳃鳗脊椎动物中工作 轴突确实再生的索表明，成熟的中枢神经系统轴突在没有生长锥体的情况下再生，即它们的 顶端缺乏丝足，相对较少的F-肌动蛋白，充满了神经细丝(Nf)，并且延长得更多 比发育中的轴突要慢。这就提出了这样一个问题：局部蛋白质合成是否在 成熟的中枢神经系统轴突再生，即使它们没有生长锥体。我们之前已经证明了 在七鳃鳗脊髓损伤后，mRNA和核糖体在轴突尖端积累，并活跃地生长在轴突尖端 比静止或回缩的尖端更多的mRNA。此外，mRNAs含有丰富的神经丝转录本， 但肌动蛋白转录本要少得多，这与在体外生长或损伤的哺乳动物轴突的研究结果相反 周围神经。现在我们将使用RNAseq来分析激光显微切割的细胞质 网状脊髓神经元及其损伤轴突顶端的微量抽吸轴浆，以识别mRNAs 与轴突再生有关。我们将确定mRNAs在细胞体中是如何调节的，并获得 通过确定a)MAP激酶通路在信号转导轴突切断和触发再生中的作用， B)mRNA沿轴突和生长端出现的时间进程。然后我们将确定 我们能否通过显微注射的mRNAs局部过度表达一些蛋白质来促进再生， 以及我们是否可以通过a)用局部应用的蛋白质抑制局部蛋白质合成来抑制再生 合成抑制剂，以及b)局部应用吗啉反义寡核苷酸。这项研究将对我们有所帮助 确定在哪里以及如何进行靶向治疗以增强脊髓损伤后轴突再生和恢复功能。

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.