Screen for determinants of synaptic specificity in outer retina.

筛选外视网膜突触特异性的决定因素。

• 批准号: 8869733
• 负责人: JOSHUA R SANES
• 金额: $ 25.35万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8869733
• 关键词: Address; Amacrine Cells; Antibodies; Attenuated; Axon; Biological Models; Blindness; Brain; Caenorhabditis elegans; Candidate Disease Gene; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Cone; Confocal Microscopy; Data; Dendrites; Drosophila melanogaster; Electroporation; Gene Expression; Gene Expression Profile; Gene Transfer; Genes; Genetic Screening; Genome; Germ Lines; Interneurons; Intervention; Label; Mammals; Mediating; Methods; Modification; Mus; Neurons; Pattern; Phenotype; Photoreceptors; Process; Reagent; Replacement Therapy; Retina; Retinal Cone; Retinal Ganglion Cells; Role; Sorting - Cell Movement; Specificity; Synapses; Time; Transgenic Organisms; Vertebrate Photoreceptors; Vision; Visual system structure; Zebrafish; base; differential expression; gain of function; horizontal cell; in vivo; insight; loss of function; molecular marker; mutant; outer plexiform layer; public health relevance; relating to nervous system; research study; retinal rods; selective expression; small hairpin RNA; success; synaptogenesis; transcriptome sequencing; transcriptomics; visual information

 DESCRIPTION (provided by applicant): Neural processing of visual information begins at the first synapses of the retina, which are made by rod and cone photoreceptors with horizontal and bipolar cells (HCs, BCs) in a thin synaptic layer called the outer plexiform layer (OPL). These interneurons, along with amacrine cells, pass the information to retinal ganglion cells, which send it to the brain. Connectivity in the OPL is specific in at least three ways: rods and cones synapse almost entirely on rod BCs and cone BCs, respectively (cellular specificity); they synapse with axons and dendrites of HCs, respectively (subcellular specificity); and their synapses are confined to outer and inner strata of the OPL, respectively (laminar specificity). To date, few molecules have been found that mediate any of these aspects of synaptic recognition. The objective of this proposal is to identify such molecules. Our approach is to screen candidates in vivo in mice. Few such screens have been performed in any mammal, but the large size and accessibility of OPL synapses, along with recent technical advances in gene transfer and genome modification, now make it possible to analyze dozens of genes in a manageable period. To prepare for this screen, we have: (a) characterized molecular markers that label all synaptic partners in the OPL; (b) analyzed their expression during the period of synapse formation; (c) optimized gene transfer methods by electroporation in vivo; (d) shown that these methods can be used to effectively attenuate gene expression in rods and cones using shRNA and Cas9/CRISPRs for loss of function studies, and to ectopically express genes for gain-of-function studies; and (e) purified developing rods and cones by FACS sorting and used RNA-Seq to obtain transcriptome information from them. We will now use transcriptomic data to select ~50 genes that encode transmembrane or secreted molecules differentially expressed by developing rods and cones. We will attenuate their expression in developing retina, then use multi-label confocal microscopy to seek altered synaptic patterns in the OPL. Finally, for the most promising of these genes, we will conduct expression analysis as well as additional loss- and gain-of-function studies to elucidate their roles in synapse formation. In addition to initiating a deep analysis of synaptogenesis and synaptic selectivity at this clinicall important synapse, our results will be useful in two ways. First, they will provide reagents and insights for studies of less accessible synapses elsewhere in the brain. Second, they may guide optimization of methods to restore vision by photoreceptor replacement. Replacement methods have shown recent promise, but may fail if the new photoreceptors fail to make appropriate synapses. Molecules we identify could be useful in enhancing the efficacy of this strategy.

 描述（由申请人提供）：视觉信息的神经处理开始于视网膜的第一个突触，这些突触由视杆细胞和视锥细胞感光细胞组成，在称为外丛状层（OPL）的薄突触层中具有水平和双极细胞（HC、BC）。这些中间神经元与无长突细胞一起将信息传递给视网膜神经节细胞，视网膜神经节细胞将信息发送到大脑。 OPL 中的连接性至少在三个方面具有特异性：杆状突触和锥状突触几乎完全分别位于杆状 BC 和锥状 BC 上（细胞特异性）；它们分别与 HC 的轴突和树突发生突触（亚细胞特异性）；它们的突触分别局限于 OPL 的外层和内层（层流特异性）。迄今为止，很少有分子能够介导突触识别的这些方面。该提案的目的是识别此类分子。我们的方法是在小鼠体内筛选候选者。很少在任何哺乳动物中进行过这样的筛选，但 OPL 突触的大尺寸和可访问性，以及基因转移和基因组修饰方面的最新技术进步，现在使得在可管理的时间内分析数十个基因成为可能。为了准备这个筛选，我们有：（a）特征分子标记，标记 OPL 中的所有突触伙伴； (b) 分析它们在突触形成期间的表达； (c) 通过体内电穿孔优化基因转移方法； (d) 表明这些方法可用于使用shRNA和Cas9/CRISPR有效减弱视杆细胞和视锥细胞中的基因表达以进行功能丧失研究，并异位表达基因以进行功能获得研究； (e) 通过 FACS 分选纯化发育中的视杆细胞和视锥细胞，并使用 RNA-Seq 从中获取转录组信息。现在，我们将使用转录组数据来选择约 50 个基因，这些基因编码通过发育中的视杆细胞和视锥细胞差异表达的跨膜或分泌分子。我们将减弱它们在发育中的视网膜中的表达，然后使用多标记共聚焦显微镜来寻找 OPL 中改变的突触模式。最后，对于这些基因中最有希望的，我们将进行表达分析以及额外的功能丧失和获得研究，以阐明它们在突触形成中的作用。除了对这个临床上重要的突触的突触发生和突触选择性进行深入分析之外，我们的结果还将在两个方面发挥作用。首先，他们将为研究大脑其他地方不易接近的突触提供试剂和见解。其次，它们可以指导优化通过光感受器替代来恢复视力的方法。替代方法最近已显示出希望，但如果新的光感受器无法形成适当的突触，则可能会失败。我们发现的分子可能有助于增强该策略的功效。