Gene regulation in Parkinsons disease

本文提出的研究的长期目标是确定在多巴胺能神经元变性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，一个强大的技术SAGE（基因表达的系列分析），将用于综合分析基因调控的一个方便的细胞培养模型的单胺能神经元死亡，6-OHDA处理的大鼠PC 12细胞。 该方法将用于比较暴露于6-OHDA之前和之后几次在神经元分化的PC 12细胞中表达的基因。 SAGE数据的分析将指示特定基因被调节的程度，并将提供可用于其鉴定和/或克隆的序列信息。 接下来的一系列研究将确定这些受调节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节。 这将通过使用原位定位和免疫细胞化学来检测正常大鼠黑质内的神经元中的转录物和/或其产物以及在正常发育期间发生此类神经元死亡或通过暴露于6-OHDA或通过靶损伤诱发的大鼠中的转录物和/或其产物来实现。 在SAGE PC 12细胞筛选中以及在大鼠脑黑质神经元中检测到的受调节基因随后将被检查在帕金森病中的存在和调节。 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神经病理学核心提供的材料。 待检查的材料将包括PD脑切片、无神经退行性疾病的年龄匹配对照和其他神经退行性疾病患者的脑切片。 第四组研究将利用PC 12细胞和交感神经元的细胞培养物来检查受调节的基因在由6-OHDA和其他手段诱发的死亡中的作用。 这将通过多种策略来实现，包括过表达、下调和干扰功能。 由于预计可能会检测到大量的受调控转录物，因此将使用一些标准来优先考虑将在项目的每个步骤中研究的那些基因。

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.