Trans-Cellular Activation of Transcription to Analyze Dopaminergic Axon Reorganiz

跨细胞转录激活分析多巴胺能轴突重组

基本信息

批准号：
8352193
负责人：
Josh Leitch Bonkowsky
金额：
$ 223.61万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-30 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8352193
关键词：
Animal Model Automobile Driving Axon Behavior Binding Binding Sites Biological Process Caenorhabditis elegans Cell Nucleus Cells Cleaved cell Clinical Complex Dopamine Enhancers Genetic Genetic Transcription Homologous Gene Imagery Injury Label Ligand Binding Ligands Lobular Neoplasia Maps Mental disorders Methods Molecular Nematoda Nervous system structure Neurobiology Neurons Neurosciences Organism Reagent Receptor Cell Reporting Solutions Specificity Synapses System Therapeutic Transcription Coactivator Transcriptional Activation Transgenes Work Yeasts Zebrafish Proteins abstracting base cell type cross reactivity design dopaminergic neuron in vivo innovation nervous system disorder neural circuit notch protein novel novel strategies prevent public health relevance receptor research study tissue fixing transgene expression

项目摘要

DESCRIPTION (Provided by the applicant) Abstract: Specific visualization and manipulation of neural circuitry has remained a vexing problem in neurobiology. Classical methods rely upon analysis in fixed tissue, preventing characterization of function or behavior. Newer methods allow genetic targeting to specific neuron types and even identify single neurons, but synaptic partners and functional circuits are not accessible by these current methods. A more general related issue is how to induce expression of a transgene in a vertebrate system when two cells make contact. A solution to these issues could have wide applicability, both for experimental studies, as well as for potentially a variety of therapeutic options. My project, Trans-Cellular Activation of Transcriptin to Analyze Dopaminergic Axon Reorganization, describes a novel strategy to analyze vertebrate circuit construction and function. It is the first genetic method for visualizing and driving expression in two cells that make contact, and offers the potential to identify and manipulate neuronal circuits in a vertebrate organism. I designed TCAT (trans-cellular activation of transcription) based on components from the conserved receptor/ligand pair of Notch/Delta. Upon ligand binding to receptor, the intracellular domain of Notch is cleaved and translocates to the nucleus. But by replacing the intracellular domain of Notch with the yeast transcriptional activator Gal4, I can specifically drive expression of transgenes at the Gal4-binding site UAS. TCAT uses the homologs LAG-2 (Delta) and LIN-12 (Notch) from the nematode C. elegans to prevent cross-reactivity with the endogenous zebrafish proteins. Specificity of labeling is provided by restricting expression of LAG-2 or LIN-12 to specific cell types. Expression from the UAS occurs when Gal4 is targeted to the nucleus, and this only occurs in the presence of ligand-receptor (cell-cell) LAG-2 to LIN-12 binding. Critically, this method is more than a means for labeling cells, but allows any inducible form of manipulation to be driven. There has been no other comparable method reported that activates transcription when two cells come into contact. Thus, TCAT will have applicability not only for use in the nervous system, but also in the study of other biological processes. In this proposal I introduce and explain TCAT, and show proof-of-principle that TCAT works in vivo, including when it is expressed by cell-type-specific enhancers. I describe how I will use it in mapping functional neural circuitry, by designing reagents to target TCAT to synapses. Finally, I outline how I will use TCAT to characterize dopamine circuit reorganization following injury, and the relevance of this for both basic and clinical neuroscience. TCAT is a significant technical innovation for mapping and manipulating circuits, but its real power is the ability it provides to create a full and functional understandig of the vertebrate CNS connectome. Public Health Relevance: The project develops a novel method to analyze and understand complex nervous systems, based on genetic approaches in the vertebrate model organism D. rerio. We apply this method to study the molecular mechanisms by which dopamine neurons alter their connections after injury, a clinically important cause of neurological and psychiatric diseases.

描述（申请人提供）摘要：神经回路的特定可视化和操纵在神经生物学中仍然是一个烦人的问题。经典方法依赖于固定组织中的分析，从而阻止了功能或行为的表征。较新的方法允许遗传靶向特定的神经元类型，甚至可以识别单个神经元，但是这些当前方法无法访问突触伙伴和功能电路。一个更一般的相关问题是，当两个细胞接触时，如何在脊椎动物系统中诱导转基因的表达。解决这些问题的解决方案可能具有广泛的适用性，包括实验研究，以及潜在的各种治疗选择。我的项目是转录蛋白的跨细胞激活以分析多巴胺能轴突重组，描述了一种分析脊椎动物电路构建和功能的新型策略。这是在两个建立接触的细胞中可视化和驱动表达的遗传方法，并提供了识别和操纵脊椎动物中神经元电路的潜力。我基于来自保守的受体/配体对Notch/Delta的成分设计TCAT（转录的透射激活）。配体与受体结合时，将Notch的细胞内结构域切割并易位到细胞核。但是，通过用酵母转录激活剂gal4代替Notch的细胞内结构域，我可以在GAL4结合位点UAS上特异性地驱动转基因的表达。 TCAT使用线虫C.秀丽隐杆线虫的同源物滞后-2（Delta）和Lin-12（Notch）来防止与内源性斑马鱼蛋白发生交叉反应。标记的特异性是通过将滞后-2或LIN-12的表达限制为特定细胞类型来提供的。当GAL4靶向核时，来自UAS的表达发生，并且仅在配体受体（细胞细胞）滞后-2与LIN-12结合的情况下发生。至关重要的是，此方法不仅仅是标记细胞的一种手段，而是允许驱动任何可诱导形式的操纵形式。没有其他可比较的方法报道当两个细胞接触时激活转录。因此，TCAT不仅适用于神经系统，而且还适用于其他生物学过程。在此提案中，我介绍并解释了TCAT，并显示了原理证明TCAT在体内工作，包括当细胞类型特异性增强剂表达时。我描述了如何通过将试剂靶向TCAT来映射功能神经电路来映射功能神经回路。最后，我概述了如何使用TCAT来表征受伤后多巴胺回路的重组，以及与基本和临床神经科学的相关性。 TCAT是用于映射和操纵电路的重要技术创新，但其真正的力量是它提供了对脊椎动物CNS Connectome创建完整且功能强大的理解的能力。公共卫生相关性：基于脊椎动物模型有机体D. Rerio的遗传方法，该项目开发了一种新颖的方法来分析和理解复杂的神经系统。我们应用这种方法来研究多巴胺神经元在受伤后改变其联系的分子机制，这是神经系统和精神病的临床重要原因。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Chiari I Malformation Causing Developmental Regression in a 4 Month Old.

Chiari I 畸形导致 4 个月大的婴儿发育退化。

DOI：
10.1177/2333794x14560819
发表时间：
2014
期刊：
Global pediatric health
影响因子：
2.2
作者：
Doll,ElizabethS;Bonkowsky,JoshuaL;Brown,LauraL;deHavenon,AdamH;Brockmeyer,DouglasL;Glasgow,TiffanyS;Morita,DeniseC
通讯作者：
Morita,DeniseC

Seizure Action Plans Do Not Reduce Health Care Utilization in Pediatric Epilepsy Patients.

癫痫行动计划不会减少小儿癫痫患者的医疗保健利用率。