The Steroid/Thyroid Hormone Receptor Superfamily

类固醇/甲状腺激素受体超家族

• 批准号: 6227925
• 负责人: Vera M Nikodem
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6227925
• 关键词: Adenoviridae; DNA footprinting; cholecystokinin; developmental genetics; developmental neurobiology; dopamine; gel mobility shift assay; gene expression; gene therapy; genetic techniques; genetic transduction; genetically modified animals; homeobox genes; in situ hybridization; laboratory mouse; mesencephalon; organ culture; stainings; steroid hormone receptor; transcription factor

Nurr1 is a transcription factor that structurally resembles members of the superfamily of nuclear hormone receptors. Nurr1 expression appears to be predominantly brain specific detected in embryonic stages with the peak expression at birth or soon thereafter. To investigate the physiological role of Nurr1, we previously generated mice with a null mutation in the Nurr1 gene. Nurr1-null mice appear to develop normally but die within 12 hours of birth. Subsequent analysis revealed the absence of neurotransmitter dopamine, dopamine biosynthetizing enzymes, transporters and receptors for dopamine utilization in the central dopaminergic area of newborn pups. We have also shown that in the absence of Nurr1, the neuroepithelial cells undergo normal ventralization and migration. These dopaminergic neuron precursors express general neuronal markers such as a neuronal nuclear marker and project to the striatum. Recently, we have also detected the expression of another dopaminergic marker, homeobox gene Ptx3, a member of the Ptx subfamily. Furthermore, using double in situ hybridization we detected the message for cholecystokinin. This brain peptide coexists in 80% tyrosine hydroxylase positive dopaminergic neurons in the wild type brain. In addition, using the NIH image program we quantitatively analyzed the TUNEL positive nuclei in the ventral midbrain of Nurr1 null mice. No difference was detected in the number of apoptotic cells between Nurr1-null mice and wild type mice. Hence, our results show that the transcription factor Nurr1 is required for terminal maturation of mesencephalic dopaminergic neurons, while genesis of dopaminergic precursors, their survival, and selection of specific target innervation are not affected at the time of birth in mice lacking Nurr1 gene function. In order to investigate the role of Nurr1 in other areas of the brain, a procedure for primary neuronal cultures was established. The system includes midbrain cultures with dopaminergic neurons, olfactory bulb and hippocampal neuronal cultures. Other techniques such as detection of dopamine using high pressure liquid chromatography, immunohistochemistry, in situ hybridization, and in situ cell death detection using the TUNEL reaction in primary brain cultures were setup as well. Currently, these techniques are being utilized to examine the differential expression of various genes and cell survival in primary brain cultures prepared from Nurr1 knockout pups compared to wild-type controls. Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. Having primary brain cultures on hand, the aim is to introduce the Nurr1 gene into midbrain cultures and subsequently into the brain of Nurr1 knockout mice to further study the Nurr1 function. Nurr1 sequences, wild type and mutated, were used to generate recombinant adenoviruses. Viral production was followed with green fluorescent protein. In parallel, we are further investigating the physiological role of Nurr1, using PC12 cells. Preliminary results have shown that in PC12 cells, endogenous Nurr1 expression can be induced by several factors. For our in vitro studies we have constructed and will purify hemagluttinin tagged Nurr1 (HA-Nurr1) from HA-Nurr1 overexpressing bacteria. Purified HA-Nurr1 will be used to define and characterize the DNA target of Nurr1 using footprint and mobility shift experiments. The identity of any Nurr1 accessory proteins, such as RXR, will be investigated by immunoprecipitation and supershift studies. It has been proposed that a Nurr1 ligand is secreted by midbrain specific astrocytes. We will add fractionated conditioned glial cell media to mobility shift experiments attempting to identify the ligand. - nuclear receptors; Nurr1; dopamine biosynthesis: midbrain neurons

Nurr1是一种转录因子，在结构上类似于核激素受体超家族的成员。Nurr1的表达似乎主要是在胚胎阶段检测到的大脑特异性，在出生时或之后不久表达达到高峰。为了研究Nurr1的生理作用，我们先前培育了Nurr1基因零突变的小鼠。Nurr1基因缺失的小鼠似乎发育正常，但在出生后12小时内死亡。随后的分析显示，新生幼崽的中央多巴胺能区缺乏神经递质多巴胺、多巴胺生物合成酶、多巴胺利用的转运体和受体。我们还表明，在没有Nurr1的情况下，神经上皮细胞经历正常的腔化和迁移。这些多巴胺能神经元前体表达一般的神经元标记，如神经元核标记，并投射到纹状体。最近，我们还检测到了另一个多巴胺能标记物，PTX亚家族成员同源盒基因Ptx3的表达。此外，通过双重原位杂交，我们检测到了缩胆囊素的信息。这种脑肽共存于野生型大脑中80%的酪氨酸羟基酶阳性的多巴胺能神经元中。此外，我们还利用NIH图像分析程序对Nurr1基因缺失小鼠腹侧中脑的TUNEL阳性核进行了定量分析。Nurr1基因缺失小鼠与野生型小鼠之间的凋亡细胞数无明显差异。因此，我们的结果表明，转录因子Nurr1是中脑多巴胺能神经元最终成熟所必需的，而在出生时缺乏Nurr1基因功能的小鼠，其多巴胺能前体的发生、存活和特定靶神经的选择不受影响。为了研究Nurr1在大脑其他区域的作用，建立了一种原代神经元培养程序。该系统包括含有多巴胺能神经元的中脑培养物、嗅球和海马神经元培养物。还建立了其他技术，如使用高压液相色谱检测多巴胺，免疫组织化学，原位杂交，以及在原代脑培养中使用TUNEL反应原位检测细胞死亡。目前，这些技术正被用于检测从Nurr1基因敲除幼鼠制备的原代脑培养中各种基因的差异表达和细胞存活情况，并与野生型对照进行比较。重组腺病毒为基因表达研究和治疗应用提供了一个通用的系统。有了原代脑培养，目的是将Nurr1基因引入中脑培养，随后进入Nurr1基因敲除小鼠的大脑，以进一步研究Nurr1的功能。用野生型和突变型Nurr1序列构建重组腺病毒。病毒产生后加入绿色荧光蛋白。同时，我们正在利用PC12细胞进一步研究Nurr1的生理作用。初步结果表明，在PC12细胞中，内源性Nurr1的表达可以被多种因素诱导。在我们的体外研究中，我们已经构建并将从高表达HA-Nurr1的细菌中纯化血凝素标记的Nurr1(HA-Nurr1)。纯化的HA-Nurr1将用于通过足迹和迁移率移动实验来定义和表征Nurr1的DNA靶标。任何Nurr1辅助蛋白，如RXR，都将通过免疫沉淀和超移位研究进行鉴定。有人认为Nurr1配体是由中脑特异的星形胶质细胞分泌的。我们将在流动性改变实验中加入分级条件神经胶质细胞培养液，试图确定配体。-核受体；Nurr1；多巴胺生物合成：中脑神经元