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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（由申请人提供）翻译后摘要：具体的可视化和操纵的神经回路仍然是一个令人烦恼的问题，在神经生物学。经典方法依赖于固定组织中的分析，从而阻止功能或行为的表征。较新的方法允许遗传靶向特定的神经元类型，甚至识别单个神经元，但突触伴侣和功能回路无法通过这些现有方法获得。更普遍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。这些问题的解决方案可能具有广泛的适用性，无论是实验研究，以及潜在的各种治疗方案。我的项目，跨细胞转录激活分析多巴胺轴突重组，描述了一种新的策略来分析脊椎动物电路的建设和功能。这是第一个可视化和驱动两个接触细胞表达的遗传方法，并提供了识别和操纵脊椎动物生物体神经元回路的潜力。我基于Notch/Delta的保守受体/配体对的组分设计了TCAT（trans-cellular activation of transcription）。在配体与受体结合后，Notch的细胞内结构域被切割并易位至细胞核。但通过用酵母转录激活因子Gal 4替换Notch的胞内结构域，我可以特异性地驱动Gal 4结合位点UAS处的转基因表达。TCAT使用来自线虫C的同源物LAG-2（Delta）和LIN-12（Notch）。elegans以防止与内源性斑马鱼蛋白的交叉反应。通过将LAG-2或LIN-12的表达限制于特定细胞类型来提供标记的特异性。当Gal 4靶向细胞核时，UAS发生表达，并且这仅在配体-受体（细胞-细胞）LAG-2与LIN-12结合的情况下发生。重要的是，这种方法不仅仅是标记细胞的手段，而是允许驱动任何诱导形式的操纵。还没有其他类似的方法报道，当两个细胞接触时激活转录。因此，TCAT不仅适用于神经系统，而且适用于其他生物过程的研究。在这个提案中，我介绍和解释TCAT，并显示TCAT在体内工作的原理证明，包括当它由细胞类型特异性增强子表达时。我描述了我将如何使用它在映射功能神经电路，通过设计试剂靶向TCAT突触。最后，我概述了我将如何使用TCAT来表征损伤后多巴胺回路重组，以及这对基础和临床神经科学的相关性。TCAT是映射和操纵电路的重要技术创新，但其真实的力量是它提供创建脊椎动物CNS连接体的完整和功能性理解的能力。公共卫生相关性：该项目开发了一种新的方法来分析和理解复杂的神经系统，基于脊椎动物模式生物D。雷里奥。我们应用这种方法来研究损伤后多巴胺神经元改变其连接的分子机制，这是神经和精神疾病的临床重要原因。

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

Microfluidic-aided genotyping of zebrafish in the first 48 h with 100% viability.