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）摘要：神经回路的具体可视化和操作仍然是神经生物学中一个棘手的问题。经典方法依赖于固定组织的分析，无法表征功能或行为。较新的方法允许对特定神经元类型进行遗传靶向，甚至识别单个神经元，但这些当前方法无法访问突触伙伴和功能电路。一个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，既适用于实验研究，也适用于各种潜在的治疗选择。我的项目“转录蛋白跨细胞激活分析多巴胺能轴突重组”描述了一种分析脊椎动物回路结构和功能的新策略。它是第一个用于可视化和驱动接触的两个细胞中表达的遗传方法，并提供了识别和操纵脊椎动物生物体中神经元回路的潜力。我根据 Notch/Delta 保守受体/配体对的成分设计了 TCAT（跨细胞转录激活）。当配体与受体结合时，Notch 的胞内结构域被切割并转移到细胞核。但通过用酵母转录激活剂 Gal4 替换 Notch 的胞内结构域，我可以特异性驱动 Gal4 结合位点 UAS 处转基因的表达。 TCAT 使用来自线虫秀丽隐杆线虫的同源物 LAG-2 (Delta) 和 LIN-12 (Notch) 来防止与内源斑马鱼蛋白发生交叉反应。通过将 LAG-2 或 LIN-12 的表达限制在特定细胞类型来提供标记的特异性。当 Gal4 靶向细胞核时，UAS 就会表达，并且只有在配体-受体（细胞-细胞）LAG-2 与 LIN-12 结合的情况下才会发生。重要的是，这种方法不仅仅是一种标记细胞的方法，而且允许驱动任何可诱导形式的操作。目前还没有其他类似的方法可以在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。在本提案中，我介绍并解释了 TCAT，并展示了 TCAT 在体内发挥作用的原理证明，包括当它由细胞类型特异性增强子表达时。我将描述如何通过设计将 TCAT 靶向突触的试剂来使用它来绘制功能神经回路图。最后，我概述了如何使用 TCAT 来表征损伤后的多巴胺回路重组，以及这与基础和临床神经科学的相关性。 TCAT 是绘制和操纵电路的一项重大技术创新，但其真正的力量在于它提供了对脊椎动物中枢神经系统连接组进行全面和功能性理解的能力。公共卫生相关性：该项目基于脊椎动物模型生物斑马鱼的遗传方法，开发了一种分析和理解复杂神经系统的新方法。我们应用这种方法来研究多巴胺神经元损伤后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

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。