SPATIAL CELL BIOLOGY OF RETINAL CIRCUIT DEVELOPMENT

描述(申请人提供)：神经元通过其树突上的突触连接接收信息。树突跨越很长的距离，并错综复杂地分支以联系适当的突触伙伴。树突的大小和复杂模式构成了独特的细胞生物学挑战。因此，细胞器需要远离胞体运输，以维持树突结构和支持突触功能。线粒体--细胞的动力源--遍布神经元树突。除了产生三磷酸腺苷外，线粒体还能吸收钙离子并参与神经元信号传递。线粒体对神经元特别重要的事实是，虽然所有细胞都含有线粒体，但影响线粒体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)，即眼睛的输出神经元，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。这项提案使用多学科方法来解决完整视网膜的RGC中的这些问题。基因策略的组合将被用来同时标记RGC树突、突触和线粒体。利用静态高分辨率成像和时间推移显微镜，分析了这些结构的分布、发展和动态相互作用。接下来，突触活动在发育过程中引导线粒体的运动并在局部控制其功能的假设将在转基因小鼠身上进行测试，在转基因小鼠中，RGCs的突触输入在体内被修改。双分子传感器已被开发和测试，以动态监测线粒体在其自然环境中使用光学方法的功能。最后，将使用基因技术来测试线粒体对树突和突触发育和功能的特定贡献，以选择性地干扰线粒体的定位或使其对激光消融敏感。这些操作对RGC发育的影响将使用实时成像进行评估，其对视觉功能的影响将使用膜片钳电生理学进行评估。总之，拟议的实验不仅将促进我们对视网膜电路发育和功能背后的基本细胞生物学的理解，而且还将为深入了解越来越多的神经系统疾病的机制提供洞察力，这些神经系统疾病涉及眼睛和大脑，由线粒体基因突变(例如显性视神经萎缩)和/或与线粒体功能障碍(例如帕金森氏症和阿尔茨海默病)有关。

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).