Regulation and function of Nurr1 in adult nigrostriatal dopamine neurons

描述（由申请人提供）：帕金森病是一种常见的神经系统疾病，其症状主要由黑质中多巴胺神经元的损失和纹状体中多巴胺的减少引起。转录因子Nurr 1是正常多巴胺神经元发育所必需的，如Nurr 1缺失纯合子小鼠中多巴胺神经元正常发育失败所证明的。Nurr 1似乎也在维持正常的多巴胺合成和多巴胺神经元存活中起作用，因为Nurr 1基因单一缺失的小鼠在这两个参数中均受损。此外，Nurr 1的表达已被证明波动的结果，在电活动的变化和治疗后，可以影响多巴胺自身受体的激活。这些因素对Nurr 1表达的相对贡献尚未确定。基于这些数据，我们假设增加神经元活性和抑制多巴胺自身受体将增加多巴胺神经元中Nurr 1的表达。此外，我们假设减弱Nurr 1表达将减弱Nurr 1、多巴胺合成和多巴胺神经元存活的靶基因。目前，对调节成年多巴胺神经元中Nurr 1表达的机制、Nurr 1的变化如何影响多巴胺合成或Nurr 1如何在多巴胺神经元存活中发挥作用知之甚少。本发明的具体目的是：1）通过比较神经元活性和多巴胺自身受体激活的变化的贡献，建立体内控制多巴胺神经元中Nurr 1表达的条件，所述神经元活性和多巴胺自身受体激活的变化是通过单独刺激或抑制活性或与通过反向微透析引入的多巴胺受体激动剂或拮抗剂组合来实现的，以及2）通过测量多巴胺神经元基因表达和多巴胺合成参数的变化以及在减弱Nurr 1表达的条件下多巴胺神经元存活的变化来鉴定Nurr 1控制多巴胺神经元功能的程度（反义寡核苷酸和Nurr 1缺失杂合小鼠的衰老）。通过使用激光捕获显微切割与基因表达技术的定量实时PCR和eXpress分析基于定量PCR的多重毛细管电泳相结合，这些实验将证明这些技术的实用性，研究转录因子在神经传递基因的调节中的作用，特别是多巴胺神经传递基因的调节Nurr 1。长期目标是更好地了解转录因子在多巴胺神经元的功能和存活中的作用，因为它们与帕金森病的发病机制有关。目前的建议将确定Nurr 1在多巴胺神经元中的表达是如何在体内调节的，以及Nurr 1如何发挥作用来调节多巴胺神经元的多巴胺合成和存活。这些数据将有助于确定靶向Nurr 1表达作为提高多巴胺合成和多巴胺神经元存活作为帕金森病治疗方法的潜力。由于帕金森病是由制造多巴胺的神经元的损失引起的;目前的治疗方法包括替代多巴胺的药物。然而，这些治疗并不能延缓多巴胺神经元的丧失或疾病的进展。通过了解特定基因如何控制多巴胺的产生并使多巴胺神经元免于死亡，可以开发针对这些基因表达的新治疗方法，既可以治疗帕金森病症状，又可以延缓这种疾病的进展。

DESCRIPTION (provided by applicant): Parkinson's disease is a common neurological disorder, the symptoms of which are caused primarily by a loss of dopamine neurons in the substantia nigra and reduced dopamine in the striatum. The transcription factor Nurr1 is necessary for normal dopamine neuron development as demonstrated by a failure of dopamine neurons to develop properly in Nurr1-null homozygous mice. Nurr1 also appears to function in maintaining normal dopamine synthesis and dopamine neuron survival as mice with a single deletion of the Nurr1 gene are impaired in both parameters. Additionally, the expression of Nurr1 has been shown to fluctuate as a result of changes in electrical activity and after treatments that can affect the activation of dopamine autoreceptors. The relative contribution of these factors on Nurr1 expression have not been determined. Based on this data, we hypothesize that increasing neuronal activity and inhibition of dopamine autoreceptors will increase Nurr1 expression in dopamine neurons. Furthermore, we hypothesize that attenuating Nurr1 expression will attenuate target genes of Nurr1, dopamine synthesis and dopamine neuron survival. Currently, little is known about the mechanism(s) that regulate Nurr1 expression in adult dopamine neurons, how changes in Nurr1 affect dopamine synthesis or how Nurr1 functions in dopamine neuron survival. The specific aims of the current proposal are 1) to establish the conditions that control Nurr1 expression in dopamine neurons in vivo by comparing the contributions of changes in neuronal activity and dopamine autoreceptor activation by stimulating or inhibiting activity alone or in combination with a dopamine receptor agonist or antagonist introduced via reverse microdialysis, and 2) to identify the extent by which Nurr1 controls dopamine neuron function by measuring changes in dopamine neuron gene expression and parameters of dopamine synthesis and changes in dopamine neuron survival in conditions of attenuated Nurr1 expression (antisense oligonucleotides and aging in Nurr1-null heterozygous mice). By using a combination of laser capture microdissection with gene expression technology of quantitative real-time PCR and eXpress profiling multiplex capillary electrophoresis based quantitative PCR; these experiments will demonstrate the utility of these techniques for investigating the role of transcription factors in the regulation of neurotransmission genes and specifically, the regulation of dopamine neurotransmission genes by Nurr1. The long term goal is to better understand the role of transcription factors in the function and survival of dopamine neurons as they relate to the pathogenesis of Parkinson's disease. The current proposal will ascertain how Nurr1 expression in dopamine neurons is regulated in vivo and how Nurr1 functions to regulate dopamine synthesis and survival of dopamine neurons. This data will aid in determining the potential for targeting Nurr1 expression as a means to elevate dopamine synthesis and dopamine neuron survival as a treatment of Parkinson's disease. PUBLIC HEALTH RELEVANCE Since Parkinson's disease is caused by a loss of neurons that make dopamine; the current treatments consist of drugs that replace dopamine. These treatments, however, do not delay the loss of dopamine neurons or the progression of the disease. By understanding how specific genes control the production of dopamine and keep dopamine neurons from dying, new treatments targeting the expression of these genes can be developed that both treat Parkinson's disease symptoms and/or delay the progression of this disease.