Selective Activation in Specific Neuronal Populations

概述：针对细胞表面标记物的分子多年来一直用于识别特定的细胞类型。在过去的十年中已经证明，当生物活性分子附着在这些细胞表面结合分子上时，可以利用结合受体被内化的趋势以特定的方式传递。通常，生物活性分子的递送会导致细胞死亡或抑制。在这个项目中，建议将这项技术用于特定的神经元群体，目的是暂时激活这些细胞，从而增加神经传递。概念验证将包括合成针对小鼠低亲和力神经营养因子受体（mu p75）和霍乱毒素酶促A1片段（CTA1）的抗体偶联物，并检查其对表达p75受体的神经元细胞的影响。CTA1是一种adp核糖基化酶，可导致cAMP水平暂时升高，在神经细胞中，cAMP水平升高可导致神经传递增加3天，并在治疗后1天达到峰值。这种作用将仅限于表达p75受体的细胞，因为CTA1不包含独立进入细胞的结合域。缀合物只能通过与p75受体的结合和内化而进入。该项目需要克隆和表达CTA1，优化检测CTA1活性的方法，合成mu p75-CTA1偶联物，并优化检测目标细胞群中cAMP水平升高的方法。该项目的成功将彻底改变已经建立的靶向共轭技术。研究人员有能力研究激活或扩增的神经元系统的影响，而不是通过转基因或免疫毒素研究神经元缺陷的结果，这将有助于更好地了解神经元的功能和生理学。此外，仅在三天之后就恢复到基础水平，这使得这个新的研究工具在其临时和快速可重复的性质中具有价值，并且也可以作为实验的控制。这项技术可以用于治疗神经精神疾病和其他疾病，虽然它们不是本提案的重点，但肯定的是，经过验证的ATS研究工具的应用也可以显著提高这些领域成功的可能性。特定神经元群体的选择性激活与公共卫生相关本提案旨在创建一条新的研究工具线，以促进对神经元系统的理解。特别针对霍乱毒素A （CTA）的神经元将暂时激活神经传递，并允许研究的影响。这些信息将为神经性疾病的药物设计提供重要线索。

DESCRIPTION (provided by applicant): 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.