Gene expression regulatory circuitry and microRNAs in midbrain dopamine neurons

描述(申请人提供)：许多发育和成人大脑障碍与中脑多巴胺神经元(MDN)有关，包括帕金森氏病(PD)、精神分裂症、自闭症、运动障碍和药物成瘾。因此，调节这些细胞的发育和功能的基本机制是非常重要的。在以前的研究中，我们和其他人已经研究了基本的调控过程，例如在多巴胺神经元发育、功能和生存的背景下，对多巴胺生物合成途径中关键酶的表达的调控。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，在简化的基于ES细胞的培养模型中，至少有两种转录因子，Nurr1和Pitx3协同作用，控制后期发育的MDN标记的表达。最近，在初步数据和一份发表的手稿中，我的实验室发现了证据表明，在MDN的发育和功能背景下，涉及microRNAs(MiRNAs)转录后调控的复杂性增加了一层。MiRNAs是进化上保守的18-25个核苷酸的非蛋白质编码转录本，在发育过程中对基因表达的转录后调控起着重要作用。具体地说，我们确定了microRNA，miR-133b，它富含mDN，并与Pitx3一起在调节反馈电路中发挥作用。在这里，我们建议更广泛地定义mDns中miRNA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，这种形式的调控很可能在MDN相关疾病中发挥作用，此外，对这些机制的操纵为治疗提供了潜在的途径。我们希望验证两个假设：1.miRNAs在mDN的调节中发挥作用，既包括在与MDN转录因子的反馈电路中，也包括通过直接调节关键的MDN靶标。2.在体内，这种调控网络在mDNS中发挥着重要的功能。公共卫生相关性许多发育和成人大脑疾病与中脑多巴胺能神经元(MDN)有关，包括帕金森氏病(PD)、精神分裂症、自闭症、运动障碍和药物成瘾。因此，调节这些细胞的发育和功能的基本机制是非常重要的。在这里，我们建议解开复杂的分子调控信号，这些信号决定了中脑多巴胺神经元的发育和功能。我们专注于microRNAs的作用，microRNAs是一种短RNA分子，调节关键的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，包括胚胎干细胞来源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回路在完整的行为啮齿动物中的作用。

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.