Gene expression regulatory circuitry and microRNAs in midbrain dopamine neurons

描述（由申请人提供）：许多发育和成人脑部疾病与中脑多巴胺神经元（mDNs）有关，包括帕金森病（PD）、精神分裂症、自闭症、运动障碍和药物成瘾。因此，调控这些细胞发育和功能的基本机制是非常重要的。在之前的研究中，我们和其他人已经研究了多巴胺神经元发育、功能和生存的基本调控过程，例如多巴胺生物合成途径中关键酶的表达调控。然而，从这些研究中可以清楚地看出，所有这些过程都具有高度的复杂性。例如，在简化的ES细胞培养模型中，至少有2个转录因子Nurr1和Pitx3协同作用控制发育晚期mDN标记物的表达。最近，在初步数据和发表的手稿中，我的实验室发现了在mDN发育和功能背景下，microRNAs （miRNAs）转录后调控的额外复杂性层的证据。mirna是进化上保守的，具有18-25个核苷酸的非蛋白质编码转录物，在发育过程中对基因表达的转录后调控起重要作用。具体来说，我们确定了microRNA miR-133b，它富含mdn，并在Pitx3的调节反馈回路中发挥作用。在这里，我们建议更广泛地定义miRNA在mdn中调控基因表达的复杂程度，并确定这些形式的调控在体内的功能。最终，这种形式的调节可能在mdn相关疾病中发挥作用，并且进一步操纵这些机制为治疗提供了潜在的途径。我们希望检验两个假设：1。miRNAs通过与mDN转录因子的反馈回路和直接调控关键的mDN靶点，在mDN调控中发挥作用。2. 这种调节网络在体内的mdn中发挥着重要的功能作用。许多发育和成人脑部疾病与中脑多巴胺能神经元（mDNs）有关，包括帕金森病（PD）、精神分裂症、自闭症、运动障碍和药物成瘾。因此，调控这些细胞发育和功能的基本机制是非常重要的。在这里，我们提出揭示决定中脑多巴胺神经元发育和功能的复杂分子调控信号。我们专注于microrna的作用，这是一种短RNA分子，调节关键多巴胺能神经元基因的表达。我们最初使用简化的模型系统，包括胚胎干细胞衍生的多巴胺神经元和原代神经元培养，这允许进行详细的分子分析。最终，我们将这些研究扩展到确认调控分子电路在完整行为的啮齿动物中的作用。

DESCRIPTION (provided by applicant): A number of developmental and adult brain disorders are associated with midbrain dopamine neurons (mDNs), including Parkinson's disease (PD), schizophrenia, autism, dyskinesias, and drug addiction. Thus, the fundamental mechanisms that regulate the development and function of these cells are of great import. In prior studies, we and others have investigated basic regulatory processes, such as regulation of the expression of key enzymes in the dopamine biosynthetic pathway in the context of dopamine neuron development, function, and survival. However, it is clear from these studies that there is a high level of complexity governing all of these processes. For instance, at least 2 transcription factors, Nurr1 and Pitx3, function synergistically to govern expression of late developmental mDN markers in simplified ES cell-based culture models. More recently, in preliminary data and a published manuscript, my lab has found evidence of an added layer of complexity involving post-transcriptional regulation by microRNAs (miRNAs) in the context of mDN development and function. miRNAs are evolutionarily conserved, 18-25 nucleotide non-protein coding transcripts that play an important function in post-transcriptional regulation of gene expression during development. Specifically, we identified microRNA, miR-133b that is enriched in mDNs and functions within a regulatory feedback circuit with Pitx3. Here we propose to more broadly define the level of complexity of gene expression regulation by miRNA in mDNs, and to determine the function of these forms of regulation in vivo. Ultimately, such forms of regulation are likely to play a role in mDN-associated diseases, and furthermore manipulations of these mechanisms offer potential avenues for therapies. We wish to test two hypotheses: 1. miRNAs function in the regulation of mDNs, both within feedback circuits with mDN transcription factors and by the direct regulation of key mDN targets. 2. Such regulatory networks play functionally important roles in mDNs in vivo. PUBLIC HEALTH RELEVANCE A number of developmental and adult brain disorders are associated with midbrain dopaminergic neurons (mDNs), including Parkinson's disease (PD), schizophrenia, autism, dyskinesias, and drug addiction. Thus, the fundamental mechanisms that regulate the development and function of these cells are of great import. Here we propose to unravel the complex molecular regulatory signals that determine the development and function of midbrain dopamine neurons. We focus on the role of microRNAs, which are short RNA molecules that regulate the expression of key dopaminergic neuron genes. We initially use simplified model systems, including embryonic stem cell derived dopamine neurons and primary neuron cultures, which allow for a detailed molecular analysis. Ultimately, we extend these studies to confirming the role of regulatory molecular circuits in the intact behaving rodent.