Plasticity in the Aging Olfactory System

描述（由申请人提供）：定义在中枢神经系统中产生广泛神经元表型的调节机制是开发领域的关键挑战，对神经系统疾病和神经退行性变的病因学具有重要意义。神经元表型由特定基因的表达来定义，但调节这些基因转录的机制在很大程度上是不确定的。为了解决这个关键的差距，在我们的知识嗅球（OB）神经球（JG）神经元，具有双重多巴胺和GABA神经递质表型，这个建议将建立调节酪氨酸羟化酶（Th）和谷氨酸脱羧酶1（Gad 1）基因的共表达的分子机制。TH和GAD 1酶分别是多巴胺和GABA神经递质生物合成所必需的。这个建议将测试的假设，招募部分重叠的组合集的转录因子的新的上游增强子区域激活Th和Gad 1转录的OB。该提案将使用相结合的系统发育和蛋白质组学的方法，这是一种新的方法建立Th和Gad 1表达的调控机制。由于关键的顺式调控元件通常表现出进化保守性，Th和Gad 1的上游区域从几个哺乳动物物种将通过系统发育方法进行分析。在体外和体内的方法将证明，保守的基因组元件是必要的，以调节在小鼠OB的表达。质谱和蛋白质组学方法将确定结合保守元件的转录因子，并且功能研究将表明这些蛋白质调节小鼠OB中Th或Gad 1的转录。具体目标1将表明，转录因子蛋白结合保守的Th上游区域协同增强Th近端启动子介导的基因表达。初步研究已经确定hnRNP-A1作为一种新的调节因子，由Th近端启动子招募，验证了系统发育和蛋白质组学相结合的方法，用于确定新的调节因子及其结合序列。具体目标2将确定一个新的和保守的Gad 1上游区域结合的转录因子蛋白，并证明这些蛋白质是Gad 1在OB中表达所必需的。这些研究结果将表明，Gad 1表达所必需的一些蛋白质也是必要的Th在OB的表达，因此，提供了一个机制的基础上，Th和Gad 1在OB JG神经元的一个子集的共表达。这一建议将显着推进OB DA神经元分化和神经元表型多样性的产生机制的理解。 公共卫生相关性：本研究旨在阐明调节中枢神经系统神经元表型多样性的分子机制。这些基础知识可以为神经系统和神经退行性疾病的分子基础提供重要的见解，并影响治疗这些综合征的新治疗策略的发展。

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. PUBLIC HEALTH RELEVANCE: This research proposal is designed to elucidate the molecular mechanisms that regulate the diversity of neuron phenotypes in the central nervous system. This fundamental knowledge can provide critical insight into the molecular basis of neurological and neurodegenerative diseases as well as impact the development of novel therapeutic strategies to treat these syndromes.