Mass Spectrometric Studies of Neuropeptides in Feeding

描述(申请人提供)：摄食行为是动物生存的关键，也是能量平衡的一个基本方面。这一过程受到高度复杂的神经内分泌系统的调控，该系统涉及多种神经肽和胺。尽管几十年来对单个神经递质或肽能信号系统的研究，但神经调节背后的一般组织原则仍然知之甚少。这在一定程度上是由于缺乏分析能力，无法在复杂的微环境中测量和识别这些低丰度的内源信号分子。我们研究的长期目标是开发新的生物分析方法来阐明神经肽在食物摄入中的复杂身份和功能作用，并在分子水平上扩大我们对共传递和神经调节的基本理解。在我们之前的资助期间，已经取得了相当大的进展，导致在一个简单的甲壳类动物模型系统中发现了200多个新的神经肽，其中有几个神经肽家族，由多达20-40个成员组成。这种令人震惊的化学复杂性，加上其最具特征的神经回路，为研究这些单个变异是否在调节食物摄入量中发挥不同作用这一有趣的问题提供了一个前所未有的机会。为了解决与多肽多样性和多样性的功能意义相关的重大生物学问题，我们建议开发和应用新的分析方法和能力，使用甲壳类和哺乳动物的神经系统。该提案的具体目标包括：(1)开发一种基于MALDI的质谱成像(MSI)技术，用于在已识别的神经元和馈入回路中定位扩展肽家族的各个亚型和胺类神经递质的共定位模式。将纳入一套新型的多重等压标记试剂，用于定量评估进食时神经肽的表达；(2)开发基于纳米颗粒的亲和力增强的体内微透析采样技术，结合整体微尺度分离，用于MS检测和定量响应食物摄取的分泌神经肽；(3)通过结合质谱学和电生理技术，确定神经肽亚型的功能后果。涉及食物摄取的主要多肽家族的个别变体将被研究其不同的降解谱和对喂养回路的不同生理作用。拟议研究的结果将是在细胞和网络水平上探索神经化学的创新方法的发展，以及更好地理解多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摄食中的甲壳类和哺乳动物神经系统，将加快我们开发治疗摄食障碍的新疗法的步伐。

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.