Role of Single Cell mRNA Variation in Systems Associated Electrically Excitable C

描述（由申请人提供）：本U01的目标是表征和理解人类可兴奋细胞表达转录组的可变性。人体内有两种主要的可兴奋细胞：神经元细胞和肌肉细胞，包括心肌细胞。许多人类中枢神经系统疾病是由神经元电反应性的调节引起的，而心律失常是心脏相关死亡的主要原因。然而，在单个细胞的水平上，在功能、反应和功能障碍方面存在相当大的异质性。在这里，我们提出的初步数据显示大规模单细胞变异性是很难解释为简单的分子噪声。我们假设在转录组状态和细胞表型之间存在多对一的关系。在这种关系中，RNA的功能分子比例由细胞系统的化学计量限制决定，这低于转录组状态。由于广泛的多基因组合支持特定的表型，转录组状态的变化并不一定导致表型的变化，这可能解释了表型对不同条件（如治疗分子的应用）的反应中的细胞异质性。为了验证这一假设，我们建议研究整体单细胞变异的程度

DESCRIPTION (provided by applicant): The goal of this U01 is to characterize and understand the variability in the expressed transcriptome of human excitable cells. There are two predominant types of excitable cell in the human body, neurons and muscle cells, including cardiac cells. Many human CNS diseases result from modulation of the electrical responsiveness of neurons while cardiac arrhythmias account for most of heart associated deaths. However, at the level of individual cells there is considerable heterogeneity in function, response, and dysfunction. Here, we present preliminary data showing large-scale single cell variability that is difficult to explain as simple molecular noise. We hypothesize that there is a many-to-one relationship between transcriptome states and a cell's phenotype. In this relationship the functional molecular ratios of the RNA are determined by the cell systems' stoichiometric constraints, which underdetermine the transcriptome state. Because a broad set of multi-genic combinations support a particular phenotype, changes in the transcriptome state do not necessarily lead to changes in the phenotype potentially explaining cellular heterogeneity in phenotype response to variant conditions such as the application of therapeutic molecules. To test this hypothesis we propose to investigate the extent of single cell variation for the whole transcriptome for excitable cells that are in their natural environment using a novel mRNA capture methodology (TIVA-tag), and on a subset of the transcriptome, the mRNAs encoding the therapeutically important and manipulable G protein-coupled receptor (GPRC) pathways. The use of functional genomics techniques developed in the Eberwine and Kim labs (TIPeR) will permit an assessment of the biological role of multigenic transcriptome variation. These studies are truly interdisciplinary involving the collaboration of two clinicians (Drs. Grady, Neurosurgeon and Kuhn, Cardiologist), two genomicists (Drs. Eberwine and Kim) one of whom is a computational scientist (Dr. Kim), a neuro/cardio- pharmacologist (Dr. Bartfai) and a biophotonics expert (Dr. Sul).! PUBLIC HEALTH RELEVANCE: The goal of this proposal is to generate a compendium of single cells sequencing data from live human excitable cells that are in contact with endogenous neighboring cells. These sequencing data will be analyzed for variability in gene expression and the biological function of this variability assessed using a novel in vivo functional genomics methodology.