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

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.