The Steroid/Thyroid Hormone Receptor Superfamily

Nurr 1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。Nurr 1的表达似乎主要是脑特异性检测在胚胎阶段与峰值表达在出生时或之后不久。为了研究Nurr 1的生理作用，我们先前在Nurr 1基因中产生了无效突变的小鼠。Nurr 1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，缺乏神经递质多巴胺，多巴胺生物合成酶，转运蛋白和受体的多巴胺利用在中央多巴胺能区的新生幼崽。我们还表明，在Nurr 1的情况下，神经上皮细胞进行正常的腹侧化和迁移。这些多巴胺能神经元前体表达一般的神经元标记物，如神经元核标记物，并投射到纹状体。最近，我们还检测到另一个多巴胺能标记物，同源框基因Ptx 3，Ptx亚家族的成员的表达。此外，使用双重原位杂交，我们检测到胆囊收缩素的信息。这种脑肽共存于野生型脑中80%酪氨酸羟化酶阳性多巴胺能神经元中。此外，使用NIH图像程序，我们定量分析了Nurr 1基因敲除小鼠中脑腹侧的TUNEL阳性核团。Nurr 1基因敲除小鼠和野生型小鼠之间的凋亡细胞数量没有差异。因此，我们的研究结果表明，转录因子Nurr 1所需的中脑多巴胺能神经元的终端成熟，而多巴胺能前体的发生，他们的生存，并选择特定的目标神经支配在出生时没有Nurr 1基因功能的小鼠不受影响。为了研究Nurr 1在大脑其他区域中的作用，建立了原代神经元培养的程序。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。其他技术，如使用高压液相色谱法，免疫组织化学，原位杂交和原位细胞死亡检测使用TUNEL反应在原代脑培养物中的多巴胺的检测，以及设置。目前，这些技术被用来检查不同的基因表达和细胞存活的原代脑培养制备Nurr 1基因敲除幼崽相比，野生型对照。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。现有的原代脑培养物，目的是将Nurr 1基因引入中脑培养物，随后引入Nurr 1基因敲除小鼠的大脑，以进一步研究Nurr 1的功能。使用野生型和突变的Nurr 1序列产生重组腺病毒。病毒产生之后是绿色荧光蛋白。同时，我们正在使用PC 12细胞进一步研究Nurr 1的生理作用。初步结果表明，在PC 12细胞中，内源性Nurr 1表达可以由几种因素诱导。对于我们的体外研究，我们已经构建并将从HA-Nurr 1过表达细菌中纯化血凝素标记的Nurr 1（HA-Nurr 1）。纯化的HA-Nurr 1将用于使用足迹和迁移率变化实验来定义和表征Nurr 1的DNA靶标。任何Nurr 1辅助蛋白的身份，如RXR，将通过免疫沉淀和超移位研究进行研究。已经提出Nurr 1配体由中脑特异性星形胶质细胞分泌。我们将加入分级的条件神经胶质细胞培养基迁移率改变实验，试图确定配体。- 核受体; Nurr 1;多巴胺生物合成;中脑神经元

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