Plasticity in the Aging Olfactory System

描述（由申请人提供）：定义中枢神经系统中产生广泛神经元表型的调控机制是发育领域的一个关键挑战，对神经疾病和神经变性的病因学具有重要意义。神经元表型是由特定基因的表达决定的，但调节这些基因转录的机制在很大程度上是不明确的。为了解决我们对具有双多巴胺和GABA神经递质表型的嗅球（OB）小球旁（JG）神经元的这一关键知识空白，本提案将建立调节酪氨酸羟化酶（Th）和谷氨酸脱羧酶1 （Gad1）基因共表达的分子机制。TH和GAD1酶分别是多巴胺和GABA神经递质的生物合成所必需的。本研究将验证一种假设，即通过新的上游增强子区域募集部分重叠的转录因子组合集，可以激活OB中Th和Gad1的转录。本研究将使用系统发育和蛋白质组学相结合的方法，这是一种建立Th和Gad1表达调控机制的新方法。由于关键的顺式调控元件通常表现出进化保守性，因此将通过系统发育方法分析几种哺乳动物物种的Th和Gad1的上游区域。体外和体内方法将证明保守的基因组元件是调节小鼠OB表达所必需的。质谱法和蛋白质组学方法将鉴定与保守元件结合的转录因子。功能研究将表明，这些蛋白在小鼠OB中调节Th或Gad1的转录。Specific Aim 1将表明，与保守的Th上游区域结合的转录因子蛋白协同增强Th近端启动子介导的基因表达。初步研究已经确定hnRNP-A1是由Th近端启动子募集的一种新的调节因子，验证了系统发育和蛋白质组学相结合的方法用于鉴定新的调节因子及其结合序列。特异性Aim 2将鉴定与Gad1上游新保守区域结合的转录因子蛋白，并证明这些蛋白是OB中Gad1表达所必需的。这些发现将表明一些Gad1表达所必需的蛋白也是OB中Th表达所必需的，从而为Th和Gad1在OB JG神经元亚群中共表达提供机制基础。这一建议将大大促进对OB - DA神经元分化和神经元表型多样性产生的机制理解。

DESCRIPTION (provided by applicant): Defining the regulatory mechanisms that generate the wide array of neuronal phenotypes in the central nervous system is a critical challenge for the field of development and has important implications for the etiology of neurological disease and neurodegeneration. Neuronal phenotypes are defined by the expression of specific genes, but the mechanisms that regulate transcription of these genes are largely undefined. To address this critical gap in our knowledge for olfactory bulb (OB) juxtaglomerular (JG) neurons that have a dual dopamine and GABA neurotransmitter phenotype, this proposal will establish the molecular mechanisms regulating the co-expression of tyrosine hydroxylase (Th) and glutamic acid decarboxylase 1 (Gad1) genes. The TH and GAD1 enzymes are necessary for the biosynthesis of dopamine and GABA neurotransmitters, respectively. This proposal will test the hypothesis that recruitment of partially overlapping combinatorial sets of transcription factors by novel upstream enhancer regions activate Th and Gad1 transcription in the OB. This proposal will use a combined phylogenetic and proteomic methodology that is a novel approach for establishing the regulatory mechanisms for Th and Gad1 expression. Since critical cis- regulatory elements typically show evolutionary conservation, upstream regions of both Th and Gad1 from several mammalian species will be analyzed by phylogenetic methods. In vitro and in vivo methods will demonstrate that conserved genomic elements are necessary to regulate expression in the mouse OB. Mass spectrometry and proteomic methods will identify the transcription factors that bind to the conserved elements, and functional studies will show that these proteins regulate either Th or Gad1 transcription in the mouse OB. Specific Aim 1 will show that transcription factor proteins bound to conserved Th upstream regions synergistically enhance gene expression mediated by the Th proximal promoter. Preliminary studies have identified hnRNP-A1 as a novel regulatory factor recruited by the Th proximal promoter, validating the combined phylogenetic and proteomic methodology for identifying novel regulatory factors and their binding sequences. Specific Aim 2 will identify the transcription factor proteins bound to a novel and conserved Gad1 upstream region, and demonstrate that these proteins are necessary for Gad1 expression in the OB. These findings will show that some proteins necessary for Gad1 expression are also necessary for Th expression in the OB, and thus, provide a mechanistic basis for the co-expression of Th and Gad1 in a subset of OB JG neurons. This proposal will significantly advance the mechanistic understanding of both OB DA neuronal differentiation and the generation of neuronal phenotypic diversity.