Selective Activation in Specific Neuronal Populations Summary: Molecules targeted towards cell surface markers have been used for years to identify specific cell types. It has been demonstrated over the course of the past decade that biologically-active molecules, when attached to these cell surface-binding molecules, can be delivered in a specific manner, utilizing the tendency of a bound receptor to be internalized. Frequently, delivery of biologically-active molecules has resulted in cell death or inhibition. It is proposed in this project to direct this technology toward specific neuronal populations with the intention of activating these cells temporarily, thereby increasing neurotransmission. The proof of concept will include synthesis of a conjugate of an antibody to the mouse low-affinity neurotrophin receptor (mu p75) and the enzymatic A1 fragment of cholera toxin (CTA1), and to examine the effects on neuronal cells that express the p75 receptor. CTA1 is an ADP-ribosylating enzyme that causes temporary elevation of cAMP levels, which in neuronal cells should lead to increased neurotransmission for a period of three days, peaking at one day post-treatment. The effect would be limited to the cells expressing the p75 receptor, as CTA1 contains no binding domain for independent entry into cells. The conjugate is allowed entry only through binding to, and internalization of, the p75 receptor. The project requires the cloning and expression of CTA1, optimization of an assay to test CTA1 activity, synthesis of a mu p75-CTA1 conjugate, and optimization of an assay to test for elevated cAMP levels in target cell populations. The success of this project would revolutionize the already established targeted conjugate technology. The ability for researchers to study the effects of an activated or amplified neuronal system, rather than the results of a neuronal deficit through transgenics or immunotoxins, would allow for greater understanding of the neuronal function and physiology. Additionally, the return to basal levels after only a three-day period, makes this new research tool valuable in its temporary and quickly repeatable nature, and also as a control for the experiment. Therapeutic applications for the treatment of neuropsychiatric and other maladies could result from this technology, and while they are not the focus of this proposal, certainly the application of proven ATS research tools could significantly enhance the possibility of success in those areas as well. Selective Activation in Specific Neuronal Populations PUBLIC HEALTH RELEVANCE This proposal seeks to create a new line of research tools to advance understanding of neuronal systems. Specifically targeting cholera toxin A (CTA) to neurons will temporarily activate neurotransmission and allow the study of the effects. This information will provide vital clues for drug design in neuronal- based diseases.
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