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

描述(申请人提供)：追踪技术长期以来一直被用来分析特定的神经通路，但并没有揭示它们的功能。长期以来，损伤技术也被用于分析神经通路的功能，但选择性不高。目前的损伤方法使用兴奋性损伤、电解性损伤或机械性损伤，以及转基因表达有毒基因产物。在这里，我们建议开发一种系统，利用逆行运输的自杀酶或重组腺病毒和局部注射的前药，可以根据神经元胞体的位置和轴突的投射来特异性地消融神经元。如果成功，这项创新技术将极大地提高我们损伤神经系统中特定路径并表征其功能的能力。第一种方法使用逆行示踪霍乱毒素B亚单位(CTB)与自杀酶(如硝基还原酶(NTR)、嘌呤核苷磷酸化酶(PNP)或辣根过氧化物酶(HRP))连接的结合物。第二种方法使用含有自杀酶编码基因的复制缺陷型腺病毒作为一种有效的工具，通过逆行运输将这种酶运送到神经元中。这种结合物或病毒载体通过立体定向注射到特定的感兴趣区域，被局部轴突终末摄取，并通过轴突逆行运输到支配这个大脑区域的神经元的远端细胞体。注射后2-5天，这种酶的无毒底物，即所谓的“前体药物”，被注射到大脑中一个或多个选定的区域。然后，这种底物被逆行标记的细胞体内的自杀酶转化为剧毒产品。因此，这种毒素只会导致含有自杀酶的神经元退化。本研究的具体目的是：1.将CTB-HRP、CTB-NTR或CTB-PNP与能有效地将自杀酶从轴突末端输送到细胞体的结合物进行偶联，构建能逆行进入神经元的自杀酶基因重组腺病毒载体。2.在体外测定结合自杀酶的活性，并在大鼠体内研究“示踪剂-自杀酶-前药物”系统产生的选择性和神经毒性。我们创新的“示踪剂-自杀酶-前药物”方法应该会产生选择性损伤，只破坏那些含有逆行转运的偶联物或腺病毒结构的神经元，而不破坏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对神经科学界具有高度的重要性。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，并在神经学和金属健康领域发现新的药物靶点。公共卫生相关性：损伤技术已被用于研究大脑中神经回路的结构和功能。但目前的方法没有那么有选择性，也不那么耗时。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路径或特定神经回路的结构和功能。这一创新的研究工具包将促进神经学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.