Retrograde Tracers with Suicide Enzyme?Prodrug to Study Specific Neural Circuits

描述（由申请人提供）：追踪技术长期以来一直用于分析特定的神经通路，但并未揭示其功能。病变技术也一直被用于分析神经通路的功能，但不是很有选择性。目前的损伤方法使用兴奋毒性，电解或机械损伤，以及有毒基因产物的转基因表达。在这里，我们建议开发一种利用逆行转运自杀酶或重组腺病毒和局部注射前药的系统，该系统将根据神经元细胞体的位置和轴突的投射来实现神经元的特异性消融。如果成功，这项创新技术将显著提高我们损伤神经系统中特定通路并表征其功能的能力。第一种方法采用了一种逆行示踪剂霍乱毒素B亚基（CTB）的偶联物，该偶联物与硝化还原酶（NTR）、嘌呤核苷磷酸化酶（PNP）或辣根过氧化物酶（HRP）等自杀酶相连。第二种方法使用含有编码自杀酶基因的复制缺陷腺病毒作为一种有效工具，通过逆行运输将这种酶运送到神经元中。结合物，或立体定向注射到特定区域的病毒载体，被局部轴突末端吸收，并通过轴突逆行运输到支配该大脑区域的神经元的远端细胞体。注射后2-5天，将酶的无毒底物，即所谓的“前药”注射到大脑的一个或多个选定区域。这种底物随后在逆行标记的细胞体内被自杀酶转化为剧毒产物。因此，毒素只诱导那些含有自杀酶的神经元退化。拟议项目的具体目标是：1。制备CTB-HRP、CTB-NTR或CTB-PNP的偶联物，使其能有效地将自杀酶从轴突末端转运到其细胞体中，并构建含有自杀酶cdna的重组腺病毒载体，通过逆行转运有效地进入神经元。2. 在体外测定偶联自杀酶的活性，并研究“示踪-自杀酶-前药”系统在大鼠体内产生的选择性和神经毒性。我们创新的“示踪-自杀酶-前药”方法应该产生选择性病变，只破坏那些含有逆行转运偶联物或腺病毒结构的神经元，同时保留大脑中的其他神经元。我们相信，我们的用户友好的完整工具包将是高度重要的神经科学界。我们的最终目标是开发新的研究工具包，以促进神经回路的研究和发现神经和金属健康领域的新型药物靶点。公共卫生相关性：病变技术已被用于研究大脑中神经回路的结构和功能。但目前的方法选择性较差，耗时较长。我们建议开发一种新的工具包，使研究人员能够快速分析特定神经解剖通路或特定神经回路的结构和功能。这一创新研究工具包将促进神经和金属健康领域的研究和药物发现。

DESCRIPTION (provided by applicant): Tracing techniques have long been used to analyze specific neural pathways but do not reveal their function. Lesion techniques have also long been employed to analyze the function of neural pathways but are not very selective. Current lesion methods use excitotoxic, electrolytic or mechanical lesions, and transgenic expression of a toxic gene product. Here we propose to develop a system utilizing retrogradely transported suicide enzyme or recombinant adenovirus and locally injected prodrug that will enable the specific ablation of neurons based upon the location of their cell bodies and the projection of their axons. If successful, this innovative technique would significantly advance our ability to lesion particular pathways in the nervous system and characterize their function. The first approach employs a conjugate of a retrograde tracer cholera toxin B subunit (CTB) linked to a suicide enzyme such as nitroreductase (NTR), purine nucleoside phosphorylase (PNP), or horseradish peroxidase (HRP). The second approach uses a replication-defective adenovirus containing genes encoding the suicide enzyme as an efficient tool to deliver this enzyme into neurons via retrograde transport. The conjugate, or the viral vector stereotaxically injected into a specific region of interest, is taken up by local axon terminals, and retrogradely transported through the axons to the distant cell bodies of neurons that innervate this brain area. Following 2-5 days after injection, the nontoxic substrate of the enzyme, so called 'prodrug' is injected into one or more selected areas in the brain. This substrate is then converted into a highly toxic product by the suicide enzyme within the retrogradely labeled cell body. Consequently, the toxin induces degeneration of only those neurons that contain the suicide enzymes. The Specific Aims of the proposed project are to: 1. Make conjugates of CTB-HRP, CTB-NTR or CTB-PNP that can effectively transport the suicide enzymes from axonal terminals into their cell bodies and construct recombinant adenoviral vectors containing the suicide enzyme cDNAs which can move efficiently by retrograde transport into neurons. 2. Determine the conjugated suicide enzyme activity in vitro and investigate the selectivity and neurotoxicity produced by the 'tracer-suicide enzyme-prodrug' systems in rats in vivo Our innovative 'tracer-suicide enzyme-prodrug' approach should produce selective lesions, destroying only those neurons that contain retrogradely transported conjugates or adenoviral construct, while sparing other neurons in the brain. We believe that our user-friendly complete toolkits will be of high importance to the neuroscience community. Our ultimate goal is to develop novel research toolkits to facilitate studies of neural circuits and discovery of novel drug targets in the neurological and metal health areas. PUBLIC HEALTH RELEVANCE: Lesion techniques have been used to study structure and function of neural circuits in the brain. But the current methods are less selective and time-consuming. We propose to develop a novel toolkit that allows investigators to rapidly analyze the structure and function of a particular neuroanatomical pathway or a specific neural circuit. This innovative research toolkit will facilitate research and drug discovery in the neurological and metal health areas.