SPATIAL CELL BIOLOGY OF RETINAL CIRCUIT DEVELOPMENT

描述（申请人提供）：神经元通过树突上的突触连接接收信息。树突跨越很长的距离和复杂的分支来接触合适的突触伴侣。树突的大小和复杂的模式构成了独特的细胞生物学挑战。因此，细胞器需要远离索马运输以维持树突结构和支持突触功能。线粒体--细胞的发电厂--遍布神经元树突。除了产生ATP外，线粒体还吸收Ca 2+并参与神经元信号传导。线粒体对神经元的特殊重要性由以下事实强调：虽然所有细胞都含有线粒体，但影响其运输和功能的突变特别表现为神经系统疾病。特别是，这些疾病经常影响视网膜神经节细胞（RGC），眼睛的输出神经元，并导致视力丧失。尽管它们很重要，但我们对树突状线粒体如何在神经元中移动，它们如何靶向树突内的特定区域，以及它们的局部功能如何塑造和支持神经发育和功能知之甚少。该提案使用多学科方法来解决完整视网膜中RGC中的这些问题。将使用遗传策略的组合来同时标记RGC树突、突触和线粒体。使用静态高分辨率成像和延时显微镜，这些结构的分布，发展和动态相互作用进行分析。接下来，将在转基因小鼠中测试突触活性在发育期间引导线粒体的运动并局部控制其功能的假设，其中在转基因小鼠中，对RGC的突触输入被体内修饰。双分子传感器已被开发和测试，以监测线粒体功能动态在其自然环境中使用光学方法。最后，将使用遗传技术来测试线粒体对树突和突触发育和功能的具体贡献，以选择性地干扰线粒体定位或使其对激光消融敏感。将使用实时成像评估这些操作对RGC发育的影响，并使用膜片钳电生理学评价其对视觉功能的影响。总之，拟议的实验不仅将促进我们对视网膜回路发育和功能的基本细胞生物学的理解，而且还提供了对越来越多的神经系统疾病（涉及眼睛和大脑）机制的深入了解，这些疾病是由线粒体基因突变引起的。在一些实施方案中，所述疾病与线粒体功能障碍（例如显性视神经萎缩）和/或与线粒体功能障碍（例如帕金森病和阿尔茨海默病）相关。

DESCRIPTION (provided by applicant): Neurons receive information through synaptic connections on their dendrites. Dendrites span long distances and branch intricately to contact the appropriate synaptic partners. The size and complex patterns of dendrites pose unique cell biological challenges. Thus, cellular organelles need to traffic far from the soma to maintain dendritic structure and support synaptic function. Mitochondria - the cell's power plants - are found throughout neuronal dendrites. In addition to producing ATP, mitochondria take up Ca2+ and participate in neuronal signaling. The special importance of mitochondria to neurons is highlighted by the fact, that, while all cells contain mitochondria, mutations that affect their trafficking and function manifest specifically as diseases of the nervous system. In particular, these diseases frequently affect retinal ganglion cells (RGCs), the output neurons of the eye, and cause vision loss. Despite their importance, we know little about how dendritic mitochondria move through neurons, how they target specific regions within dendrites, and how their local function shapes and supports neural development and function. This proposal uses a multidisciplinary approach to address these questions in RGCs in the intact retina. A combination of genetic strategies will be used to simultaneously label RGC dendrites, synapses and mitochondria. Using static high resolution imaging and time-lapse microscopy, the distribution, development and dynamic interaction of these structures will be analyzed. Next, the hypothesis that synaptic activity guides the movements of mitochondria during development and locally controls their function will be tested in transgenic mice in which synaptic input to RGCs is modified in vivo. Bimolecular sensors have been developed and tested to monitor mitochondrial function dynamically in their natural environment using optical approaches. Finally, the specific contribution of mitochondria to dendritic and synaptic development and function will be tested using genetic techniques to selectively interfere with mitochondrial localization or sensitize them to laser-ablation. The consequences of these manipulations for RGC development will be assessed using live imaging and their impact on visual function will be evaluated using patch-clamp electrophysiology. Together, the proposed experiments will not only advance our understanding of the fundamental cell biology underlying retinal circuit development and function, but also provide insight into the mechanisms of a growing number of nervous system disorders - involving eye and brain - that are caused by mutations in mitochondrial genes (e.g. dominant optic atrophy) and/or associated with mitochondrial dysfunction (e.g. Parkinson's and Alzheimer's disease).