Development and Maintenance of the Human Photoreceptor-Bipolar Cell Synapse.

项目摘要 视觉系统的第一个突触--在光感受器和双极细胞之间--对视觉处理至关重要 但在光感受器功能障碍的情况下显示异常的特异性和维持。功能 恢复，例如基因或细胞为基础的治疗，将取决于更好地了解改变的 人类视网膜疾病中的突触连接。人类视网膜类器官系统允许详细的研究 视网膜组织中突触发育和维持的可扩展性和实验可访问的方式。 拟议的实验将使用人类视网膜类器官来解决视杆双极细胞（RBC） 树突最初与视杆细胞和视锥细胞形成非特异性接触，然后在生长过程中变得越来越特异。 视网膜疾病中的突触混杂代表了这些非特异性的 早期视网膜发育中的相互作用。这一假设将在三个具体目标中得到检验。目标1确定 光感受器双极细胞突触发生的长期发育阶段，并具体检查 视杆细胞-红细胞突触特异性是否通过修剪不适当的接触来实现。目标2调查了 使用CRISPR工程化荧光报告基因在肝类器官中RBC突触发生的短期动力学 线，以确定是否探索树突行为促进突触特异性。目标3审查了 突触前神经元Cav1.4介导的谷氨酸释放在突触形成、维持和特异性中的作用 钙通道与X连锁先天性静止性夜盲症有关。拟议的工作将提供 有机会在理解人类视网膜组织中的突触布线方面取得重大进展， 建立这个系统来识别维持或恢复突触连接的分子靶点。这 应用构成了我的职业发展奖（K 08）作为一个学术儿科视网膜的基础 具有很高积极性和机构支持的专家，以开展独立的实验室研究 小儿视网膜疾病的研究项目在追求这些目标的过程中，我将接受著名科学家的指导， 儿童医院洛杉矶和南加州大学，因为我获得了必要的技能，在人类 视网膜类器官技术，视网膜和突触神经科学，活细胞成像，以实现独立 R 01融资最终，我的工作将首次探索人类视网膜突触连接， 开发并建立一个研究项目，专注于恢复人类疾病中的这些联系， 状态

Project Summary The first synapse of the visual system—between photoreceptors and bipolar cells—is critical to visual processing but displays abnormal specificity and maintenance in the setting of photoreceptor dysfunction. Functional restoration, for example with gene or cell-based therapies, will depend upon a greater understanding of altered synaptic connectivity in human retinal disease. The human retinal organoid system permits the detailed study of synaptic development and maintenance in retinal tissue in a scalable and experimentally accessible fashion. The proposed experiments will use human retinal organoids to address the hypothesis that rod bipolar cell (RBC) dendrites initially form non-specific contacts with rods and cones, which then become increasingly specific during development, and that synaptic promiscuity in retinal diseases represents a recapitulation of these non-specific interactions in early retinal development. This hypothesis will be tested in three Specific Aims. Aim 1 determines the long-term developmental stages of photoreceptor-bipolar cell synaptogenesis and specifically examines whether rod-RBC synaptic specificity is achieved by pruning inappropriate contacts. Aim 2 investigates the short-term dynamics of RBC synaptogenesis in live organoids using a CRISPR-engineered fluorescent reporter line to ascertain whether exploratory dendrite behavior facilitates synaptic specificity. Aim 3 examines the role of glutamate release in synapse formation, maintenance, and specificity as mediated by Cav1.4, the presynaptic calcium channel implicated in X-linked congenital stationary night blindness. The proposed work will provide opportunities to make significant advances in understanding synaptic wiring in human retinal tissue, thus establishing this system to identify molecular targets that maintain or restore synaptic connectivity. This application constitutes the foundation of my career development award (K08) as an academic pediatric retina specialist with very high motivation and institutional support to develop an independent laboratory research program in pediatric retinal disease. In pursuit of these aims, I will be mentored by renowned scientists at Children's Hospital Los Angeles and the University of Southern California, as I gain the necessary skills in human retinal organoid techniques, retinal and synaptic neuroscience, and live cell imaging to achieve independence through R01 funding. Ultimately, my work will explore for the first time human retinal synaptic connectivity during development and establish a research program focused on restoring these connections in the human disease state.