Mass Spectrometric Studies of Neuropeptides in Feeding

描述（由申请人提供）：摄食行为对动物生存至关重要，也是能量稳态的基本方面。这一过程是由一个高度复杂的神经内分泌系统，其中涉及大量的神经肽和胺的调节。尽管对单个神经递质或肽能信号系统的研究已经进行了数十年，但神经调节的一般组织原理仍然知之甚少。这部分是由于缺乏在复杂微环境中测量和鉴定这些低丰度内源性信号分子的分析能力。我们的研究的长期目标是开发新的生物分析方法，以阐明复杂的身份和功能作用的神经肽的食物摄入，并扩大我们的基本理解的共传递和神经调节在分子水平上。在我们之前的赠款资助期间，已经取得了相当大的进展，导致发现了200多个新的神经肽，其中几个神经肽家族由一个简单的甲壳动物模型系统中的多达20-40个成员组成。这种令人惊叹的化学复杂性加上其最具特征的神经元回路提供了一个前所未有的机会来研究这些个体变体是否在调节食物摄入方面发挥独特作用的有趣问题。为了解决与肽的多样性和多样性的功能意义相关的重大生物学问题，我们提出了使用甲壳动物和哺乳动物神经系统的新的分析方法和能力的开发和应用。具体目标包括：（1）开发基于MALDI的质谱成像（MSI）技术，用于绘制已识别神经元和摄食回路中扩展肽家族和胺类神经递质的单个异构体的共定位模式。（2）建立一种基于纳米颗粒的亲和增强微透析体内采样技术，结合整体微尺度分离技术，用于MS检测和定量摄食后分泌的神经肽;（3）通过质谱和电生理技术的结合来确定神经肽异构体的功能后果。参与食物摄入的主要肽家族的个体变体将被研究其差异降解概况和对摄食回路的不同生理作用。拟议研究的结果将是开发用于在细胞和网络水平上探测神经化学的创新方法，以及更好地理解肽在调节食物摄入和其他生理功能方面的多样性。将这些新方法同时应用于甲壳类动物和哺乳动物的摄食神经系统，将加快我们开发摄食障碍新疗法的步伐。

DESCRIPTION (provided by applicant): Feeding behavior is critical for animal survival, and is also a fundamental aspect of energy homeostasis. This process is regulated by a highly complex neuroendocrine system which involves a multitude of neuropeptides and amines. Despite decades of work on individual neurotransmitter or peptidergic signaling systems, the general organizational principles underlying neuromodulation are still poorly understood. This is, in part, due to a lack of analytical capabilities to measure and identify these low abundance endogenous signaling molecules in a complex microenvironment. The long term goal of our research is to develop new bioanalytical methods to elucidate the complex identities and functional roles of neuropeptides in food intake and to expand our fundamental understanding of cotransmission and neuromodulation at the molecular level. During our previous grant funding period, considerable progress has been made leading to the discovery of more than 200 novel neuropeptides with several neuropeptide families consisting of as many as 20-40 members in a simple crustacean model system. This stunning chemical complexity coupled with its best characterized neuronal circuit offers an unprecedented opportunity to investigate the intriguing question whether these individual variants play distinct roles in regulation of food intake. To address significant biological questions related to functional significance of peptide multiplicity and diversity, we propose the development and application of new analytical methodologies and capabilities using both the crustacean and mammalian nervous systems. The specific aims of the proposal include: (1) To develop a MALDI-based mass spectral imaging (MSI) technique for mapping co-localization patterns of individual isoforms of extended peptide families and amine neurotransmitters in identified neurons and the feeding circuits. A set of novel multiplexed isobaric labeling reagents will be incorporated for quantitative assessment of neuropeptide expression upon feeding; (2) To develop a nanoparticle-based affinity-enhanced microdialysis in vivo sampling technique coupled with monolithic microscale separation for MS detection and quantitation of secreted neuropeptides in response to food intake; (3) To determine the functional consequences of neuropeptide isoforms via a combination of mass spectrometric and electrophysiological techniques. Individual variants of major peptide families involved in food intake will be investigated for their differential degradation profiles and distinct physiological actions on the feeding circuits. The outcome of the proposed research will be the development of innovative methodologies for probing neurochemistry at the cellular and network levels and an improved understanding of peptide multiplicity in regulation of food intake and other physiological functions. The parallel application of these new methods to both crustacean and mammalian nervous systems in feeding will accelerate our pace towards the development of new therapeutics for feeding disorders.