Acetylcholinergic Neurotransmission During Aging

 描述(由申请人提供)：我在未来五年的职业目标是获得R01资金，确保在特拉华州立大学的终身教职，并在利用果蝇阐明乙酰胆碱释放的分级变化对衰老期间生理和行为的作用方面发展一个利基研究。作为阿拉巴马州大学Janis O‘Donnell的研究生和加州大学洛杉矶分校的博士后研究员，我在果蝇神经遗传学和与人类疾病相关的神经元过程研究方面接受了广泛的培训。此外，与研究囊泡转运蛋白及其在神经传递中的作用的专家Krantz博士一起，我发展了自己在囊泡神经递质转运体功能方面的专业知识。我最近的项目涉及使用果蝇转运蛋白来测试基因与环境的相互作用，这可能有助于帕金森氏病(PD)的易感性。特别是，我确定囊泡单胺转运体(VMAT)的过度表达可以通过将胞质中的多巴胺隔离在作用部位之外来保护多巴胺神经元免受环境毒素的伤害。为了建立VMATs潜在的神经保护作用，我对能够增加其功能的小分子进行了筛选，并成功地确定了一类潜在的新的胺能药物。在我作为独立教员的职业生涯早期，我将继续研究果蝇囊泡神经递质转运体的神经遗传学，但将我的主要重点从VMAT转向果蝇囊泡乙酰胆碱转运体(Vacht)。在Krantz博士出色的指导下，我扩展了自己的技能，包括细胞培养、行为分析、生化分离和药理学。我现在正在利用这个背景，在我自己的实验室里作为PI来确定Vacht的生物功能。我已经制定了一个严格的培训计划，这将帮助我实现我的职业生涯过渡到研究衰老过程中的胆碱能神经传递。在K01大奖期间，我训练的一个重要方面就是学习电生理学。我接受过Diane O‘Dowd的基本培训，她是一名成年果蝇脑电生理学专家。我学会了如何准备果蝇的脑组织和鉴定果蝇的胆碱能神经元。此外，我还从加州大学洛杉矶分校的费利克斯·施韦泽那里收到了关于一般电生理学概念和技术的意见。在这两位导师的指导下，我能够建立修补神经元和记录动作电位的基础。我现在计划掌握这些基本技能，成为一名专家。Schweizer博士和O‘Dowd博士都表示愿意继续用他们的专业知识支持我。重要的是，特拉华州立大学的梅丽莎·哈林顿博士提出要加强我的培训，并将担任我亲身实践的初级生理学导师。她已经允许我完全使用她的钻井平台，在她的指导下，我将继续磨练我的技能。她还为当前的应用程序提供了大量的投入。我 最近，我在特拉华州立大学担任NIH/Cobre神经科学项目的助理教授，开始了自己的实验室。我收到了32万美元的创业套餐。这种支持为我提供了宝贵的资源/人员来实现我的目标(见培训计划)。我的目标是成为一名使用电生理学研究ACh释放差异如何影响突触功能的专家。我将把这套技术与我目前的专业知识结合起来，以确定胆碱能突触功能的变化如何与衰老过程中的行为下降相关。此外，我将很好地回答有关ACh释放、行为和衰老的基本问题，这些问题具有明显的翻译意义。为了研究这些问题，我将使用 果蝇Vacht中的一系列等位基因突变，是包装和运输ACh以释放突触的蛋白质。这些等位基因从轻微到严重不等，将使我能够确定乙酰胆碱释放的渐进性变化与乙酰胆碱介导的行为(如学习)进行性缺陷之间的关系。苍蝇的寿命很短，再加上这个系统独有的额外遗传工具，这将使我能够评估改变ACh释放对动物整个生命周期中的行为和突触生理的影响。此外，我还将测试这一假设，即通过过度表达Vacht而增加胆碱能张力将改善与年龄相关的认知功能缺陷。在未来的AIMS中，我将使用我在这里提出的模型系统作为一个平台来识别能够增加ACh释放的小分子。我相信，这些药物可以代表一种新的治疗策略，以改善与正常和病理性衰老的ACh神经传递减少相关的认知功能下降。

 DESCRIPTION (provided by applicant): My career objectives over the next five years are to obtain R01 funding, secure tenure at Delaware State University and develop a research niche in using Drosophila to elucidate the role of graded changes in acetycholine release on physiology and behavior during aging. As a graduate student with Janis O'Donnell (U. Alabama) and a post-doctoral fellow with David Krantz (UCLA), I received extensive training in Drosophila neurogenetics and on the study of neuronal processes relevant to human diseases. In addition, with Dr. Krantz, an expert in the study of vesicular transporters and their role in neurotransmission, I have developed my own expertise in the function of vesicular neurotransmitter transporters. My most recent projects have involved using Drosophila transporter to test gene-environment interactions that may contribute to the susceptibility to Parkinson's disease (PD). In particular, I determined that overexpression of the vesicular monoamine transporter (VMAT) can protect dopamine neurons from environmental toxins by sequestering cytosolic dopamine away from its site of action. To build on the potential neuroprotective role for VMATs, I conducted a screen for small molecules capable of increasing its function and I successfully identified a potentially new class of aminergic agents. I will continue to study the neurogenetics of Drosophila vesicular neurotransmitter transporters in the early stages of my career as an independent faculty, but change my primary focus from VMAT to the Drosophila vesicular acetylcholine transporter (VAChT). Under Dr. Krantz's excellent mentorship, I expanded my repertoire of skills to include cell culture, behavioral analyses, biochemical fractionation and pharmacology. I am now exploiting this background to determine the biological function for VAChT in my own lab as a PI. I have developed a rigorous training plan that will help me to achieve my career transition into the study of cholinergic neurotransmission during aging. One important aspect of my training during the K01 award will be to learn electrophysiology. I have received basic training from Diane O'Dowd, a specialist in adult Drosophila brain electrophysiology. I learned how to prepare fly brain tissue and identify Drosophila cholinergic neurons. In addition, I received input on general electrophysiological concepts and techniques from Felix Schweizer at UCLA. Under these two mentors, I was able to establish the basics of patching onto a neuron and recording action potentials. I now plan to master these foundational skills and become an expert. Both Drs. Schweizer and O'Dowd have offered to continue to support me with their expertise. Importantly, Dr. Melissa Harrington at Delaware State has offered to enhance my training and will serve as my hands-on primary physiology mentor. She has given me full access to her rig and under her mentorship I will continue to hone my skills. She has also provided substantial input into the current application. I recently started my own lab as an Assistant Professor in the NIH/COBRE Neuroscience program at Delaware State. I received a start-up package of $320,000. This support has afforded me valuable resources/personnel to achieve my goals (see Training Plan). My goal is to become an expert in using electrophysiology to investigate how differences in ACh release effect synaptic function. I will couple this skill set with my current expertise to determine how te changes in the function of cholinergic synapses may correlate with behavioral decline during aging. Moreover, I will be well-positioned to address fundamental questions about ACh release, behavior and aging that have clear translational significance. To study these issues, I will use an allelic series of point mutants in the Drosophila VAChT, the protein that packages and transports ACh for synaptic release. These alleles which range from mild to severe, and will allow me to determine the relationship between graded changes in acetylcholine release and progressive deficits in acetylcholine-mediated behaviors such as learning. The short life span of the fly coupled with additional genetic tools unique to this system will allow me to assess the impact of altered ACh release on behavior and synaptic physiology throughout the lifespan of the animal. In addition, I will test the hypothesis that an increase in cholinergic tone through overexpression of VAChT will improve age-related deficits in cognitive functions. In future aims, I will use the model system I propose here as a platform to identify small molecules capable of increasing ACh release. I believe that these agents could represent a new therapeutic strategy to improve the cognitive decline associated with decreased ACh neurotransmission in both normal and pathological aging.