Compartmentilization of Neuronal Gene Expression

神经元基因表达的区室化

• 批准号: 6111234
• 负责人: BARRY B. KAPLAN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6111234
• 关键词: RNA binding protein; autoradiography; axon; bioenergetics; electronic spectra; gene deletion mutation; gene expression; in situ hybridization; intracellular transport; messenger RNA; nerve endings; nervous system regeneration; neural plasticity; neuronal transport; polymerase chain reaction; polysomes; protein biosynthesis; protein transport; retina; squid; synapses

Axons and nerve terminals are unique subcellular structures of the neuron that play a critical role in the development and maintenance of neural connectivity. One of the central tenets in neuroscience is that the protein constituents of these distal neuronal compartments are synthesized in the nerve cell body and subsequently transported to their ultimate sites of function. Hence, the structure and function of these highly specialized distal domains of the neuron are totally dependent on slow anterograde axoplasmic transport. In contrast to this viewpoint, work in my laboratory focuses on the hypothesis that de novo protein synthesis occurs within microcompartments in the neuron to include the axon and presynaptic nerve terminal. Our studies employ the squid giant axon, which serves as a model invertebrate motor neuron system. Using this model, my colleagues and I have shown that the axon contains a heterogeneous population of approximately 100-200 different mRNAs. These mRNAs are full-length gene transcripts capable of synthesizing protein in a cell-free translation system. We have cloned and characterized several axonal mRNAs that encode B-actin, B-tubulin, spectrin, kinesin, MAP I, neurofilament protein, and enolase. In addition, we have identified several mRNAs that code for novel proteins. The axonal localization of these mRNA species was definitively demonstrated by in situ hybridization histochemistry, and the presence of these sequences in the polysome fraction was established by {please spell out what RT stands for; we'll let pcr stand unless our copy editor insists on polymerase chain reaction on first reference:} RT-PCR methodology. Using biochemical labeling experiments and electron spectroscopic phosphate imaging, we were also able to show that the giant axon contained biologically active polyribosomes. Concurrent with this work, we have demonstrated that protein synthesis occurs in the large presynaptic terminals of squid retinal photoreceptor neurons. This finding was obtained using cell-free translation analysis, high-resolution autoradiography, and electron spectroscopic imaging. Our most recent results suggest that the level of protein synthesis in these presynaptic terminals is effected by calcium ions and, hence, could be regulated by the activity of the terminal itself. Based upon the information gleaned from this invertebrate model system, we have postulated that key elements of the cytomatrix, molecular motors of the axon transport systems, and proteins involved in energy metabolism are locally synthesized in the distal structural and functional domains of the neuron. In the mature neuron, a local system of protein synthesis could contribute significantly to the maintenance and remodeling of axonal architecture, as well as the dynamic properties of the nerve terminal. This system might prove especially important in large asymmetric motor and sensory neurons, where the axon and terminal fields are far removed from the cell body. Currently, my colleagues and I are using differential mRNA display methodology to identify novel constituents of the axonal mRNA population, and we are beginning to explore the mechanisms involved in intracellular trafficking of axonal mRNAs. These latter studies will involve mRNA-protein binding assays, as well as deletion mutation analysis and microinjection of fluorescently labeled mRNAs into isolated squid giant axon preparations. We hope that these investigations will augment our understanding of the molecular mechanisms that play a key role in neuronal development, regeneration, and plasticity.

轴突和神经末端是独特的亚细胞 神经元的结构在发展中起关键作用 和神经连通性的维护。中央宗旨之一 神经科学是这些远端神经元的蛋白质成分 隔室在神经细胞体中合成，并 随后运输到其最终功能部位。因此， 这些高度专业的远端结构域的结构和功能 神经元完全取决于缓慢的顺行轴突 运输。与这个观点相反，在我的实验室工作 侧重于从头蛋白质合成的假设 在神经元中的微室内，包括轴突和 突触前神经末端。我们的研究采用鱿鱼巨人 Axon，作为模型无脊椎动物运动神经元系统。 使用此模型，我和我的同事表明了轴突 包含大约100-200的异质种群 不同的mRNA。这些mRNA是全长基因转录本 能够在无细胞翻译系统中合成蛋白质。我们 已经克隆并表征了几个编码的轴突mRNA B-肌动蛋白，B-微管蛋白，光谱，驱动蛋白，MAP I，神经丝蛋白， 和烯醇酶。此外，我们已经确定了几个mRNA 新颖蛋白质的代码。这些mRNA的轴突定位 原位杂交明确地证明了物种 组织化学以及这些序列在 多层分数是由{请阐明什么RT建立的 代表；除非我们的副本编辑坚持要求，否则我们会让PCR站立 首次参考时聚合酶链反应：} rt-pcr 方法论。使用生化标记实验和电子 光谱磷酸盐成像，我们还能够证明 巨型轴突包含具有生物活性的多核糖体。 与这项工作同时，我们已经证明了蛋白质 合成发生在鱿鱼视网膜的较大突触前端子中 光感受器神经元。该发现是使用无细胞的 翻译分析，高分辨率自显影和电子 光谱成像。我们最近的结果表明 在这些突触前末端的蛋白质合成水平受到影响 通过钙离子，因此可以通过 终端本身。根据从中收集的信息 无脊椎动物模型系统，我们假设 Cytomatrix，轴突传输系统的分子电动机， 和涉及能量代谢的蛋白质是局部合成的 在神经元的远端结构和功能结构域中。在 成熟的神经元，局部蛋白质合成系统可能有助于 显着进行轴突的维护和重塑 建筑以及神经的动态特性 终端。该系统在整个中可能尤其重要 轴突和轴突的不对称运动和感觉神经元 末端场远离细胞体。目前，我的 同事和我正在使用差异mRNA显示方法 确定轴突mRNA种群的新成分，以及 我们开始探索涉及的机制 轴突mRNA的细胞内贩运。这些后一个研究将 涉及mRNA-蛋白结合测定以及缺失突变 分析和显微注射荧光标记的mRNA 孤立的鱿鱼巨型轴突制剂。我们希望这些 调查将增强我们对分子的理解 在神经元发展中起关键作用的机制， 再生和可塑性。