Gene regulation in Parkinsons disease

这里提出的研究的长期目标是确定在多巴胺能神经元退化和死亡中发挥作用的基因。这些基因及其产物有可能成为治疗干预的靶点。为了实现这一目标，将使用一种名为SAGE(基因表达序列分析)的强大技术来全面分析单胺能神经元死亡的简便细胞培养模型-6-OHDA处理的大鼠PC12细胞中的基因调控。这种方法将被用来比较神经分化的PC12细胞在暴露于6-OHDA之前和之后的几次基因表达。对SAGE数据的分析将表明特定基因受到调控的程度，并将提供可用于鉴定和/或克隆的序列信息。接下来的一系列研究将确定这些受调控的基因是否存在于正常大鼠大脑中的多巴胺能神经元中，以及它们的表达是否在死亡过程中受到调控。这将通过使用原位定位和免疫细胞化学来检测正常大鼠和在正常发育过程中发生神经元死亡或因暴露于6-OHDA或靶点损伤而引起的黑质神经元中的转录和/或其产物。在SAGE PC12细胞筛选和大鼠脑黑质神经元中检测到的受调控基因随后将被检查是否存在帕金森氏病的存在和调控。这将通过免疫细胞化学定位进行，使用我们阿尔茨海默病和其他神经退行性疾病研究中心的神经病理学核心提供的材料。要检查的材料将包括帕金森病患者的大脑切片、没有神经退行性疾病的年龄匹配的对照组和其他神经退行性疾病患者的大脑。第四组研究将利用PC12细胞和交感神经元的细胞培养来检查调控基因在6-OHDA和其他方式引起的死亡中的作用。这将通过多种策略来实现，包括过度表达、下调和干扰功能。由于预计可能会检测到大量受调控的转录本，因此将使用一些标准来确定将在项目的每个步骤中研究的基因的优先顺序。

The long-term goal of the studies proposed here is to identify genes that play roles in the degeneration and death of dopaminergic neurons. Such genes and their products have the potential to serve as targets for therapeutic intervention. To accomplish this objective, a powerful technique designated SAGE (Serial Analysis of Gene Expression), will be used for comprehensive analysis of gene regulation in a convenient cell culture model of monoaminergic neuronal death, the 6-OHDA-treated rat PC12 cell. This approach will be used to compare the genes expressed in neuronally differentiated PC12 cells before and at several times after exposure to 6-OHDA. Analysis of the SAGE data will indicate the degree to which specific genes are regulated and will provide sequence information that can be used for their identification and/or cloning. A following set of studies will determine whether such regulated genes are present in dopaminergic neurons in the normal rat brain and whether their expression is regulated during death. This will be achieved by using in situ localization and immunocytochemistry to detect the transcripts and/or their products in neurons within the substantia nigra of normal rats and in rats in which death of such neurons occur during normal development or is evoked by exposure to 6-OHDA or by target injury. Regulated genes detected in the SAGE PC12 cell screen and also in rat brain substantia nigra neurons will then be examined for presence and regulation in Parkinson's Disease. This will be carried out by immunocytochemical localization using material available through the Neuropathology Core of our Center for Research on Alzheimer's Disease and other Neurodegenerative Disorders. Material to be examined will include sections from PD brains, age-matched controls without neurodegenerative disease and brains from patients with other neurodegenerative disorders. A fourth set of studies will exploit cell cultures of PC12 cells and sympathetic neurons to examine the roles of the regulated genes in death evoked by 6-OHDA and other means. This will be accomplished by a variety of strategies including over-expression, down-regulation and interference with function. Because it is anticipated that a large number of regulated transcripts may be detected, a number of criteria will be used to prioritize those genes that will be studied at each step of the project.