Subcellular Compartmentilization Of Neuronal Gene Expres

神经元基因表达的亚细胞区室化

• 批准号: 6824201
• 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/6824201
• 关键词: RNA binding protein; autoradiography; axon; bioenergetics; electronic spectra; gene deletion mutation; gene expression; in situ hybridization; intracellular transport; messenger RNA; motor neurons; nerve endings; nervous system regeneration; neural plasticity; neurogenetics; neuronal transport; polymerase chain reaction; polysomes; protein binding; protein biosynthesis; protein transport; 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.

轴突和神经末端是神经元的独特亚细胞结构，在神经连通性的发展和维持中起着至关重要的作用。神经科学中的中心原则之一是，这些远端神经元区室的蛋白质成分在神经细胞体中合成，然后转运到其最终功能部位。因此，神经元的这些高度专业的远端结构域的结构和功能完全取决于缓慢的顺行轴向传输。与这个观点相反，我的实验室工作着重于以下假设：神经元中的微型群中发生从头蛋白质合成，以包括轴突和突触前神经末端。我们的研究采用鱿鱼巨型轴突，该轴突充当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，轴突包含大约100-200个不同mRNA的异质种群。这些mRNA是能够在无细胞翻译系统中合成蛋白质的全长基因转录本。我们已经克隆并表征了几种编码B-肌动蛋白，B-微管蛋白，光谱，动力素，MAP I，神经丝蛋白和烯醇酶的轴突mRNA。此外，我们已经确定了几个mRNA，这些mRNA代码为新型蛋白质。这些mRNA物种的轴突定位通过原位杂交组织化学明确证明了，并且这些序列在多层体组中的存在由{{请阐明RT代表什么；除非我们的副本编辑者坚持首次参考的聚合酶链反应：} 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含有生物活性的多核糖体。与这项工作同时，我们证明了蛋白质合成发生在鱿鱼视网膜感受器神经元的较大突触前末端。使用无细胞的翻译分析，高分辨率放射自显影和电子光谱成像获得了这一发现。我们最近的结果表明，这些突触前末端中蛋白质合成水平受钙离子的影响，因此可以受末端本身的活性调节。基于该无脊椎动物模型系统收集的信息，我们假设细胞瘤，轴突传输系统的分子电机的关键要素以及参与能量代谢的蛋白质在神经元的远端结构和功能域中局部合成。在成熟的神经元中，局部蛋白质合成系统可以显着促进轴突结构的维持和重塑，以及神经末端的动态特性。该系统可能在大型不对称运动和感觉神经元中尤其重要，在大型的不对称电动机和感觉神经元中，轴突和末端场与细胞体的去除远。目前，我和我的同事正在使用差异mRNA显示方法来识别轴突mRNA种群的新成分，我们开始探索与轴突mRNA细胞内运输有关的机制。这些后者的研究将涉及mRNA-蛋白结合测定法，以及将荧光标记的mRNA标记为分离的鱿鱼巨型轴突制剂的缺失突变分析和显微注射。我们希望这些研究能够增强我们对在神经元发展，再生和可塑性中起关键作用的分子机制的理解。