Greene-5P01NS017771-220003. Homo sapiens

Parkinson's disease is a prevalent neurodegenerative disorder for which there is no cure. The cause of PD symptoms is loss of dopamine neurons in the midbrain, but it is not known why these neurons die. Pesticide exposure is epidemiologically associated with PD, and administration of the organic pesticide rotenone to rats recapitulates most of the behavioral, neurochemical, and neuropathological findings in PD, including specific death of dopamine neurons. We have developed an in vitro model of rotenone toxicity using a dopaminergic cell line (SK-N-MC neuroblastoma cells) that mimics many of the cellular changes seen with in vivo rotenone toxicity and with PD, such as alpha-synuclein aggregation and oxidative damage. We are currently using this simple model to explore mechanisms of dopaminergic neurodegeneration, with our ultimate goal being the discovery of novel mechanisms for dopaminergic neuroprotection in PD. We will examine gene expression profiles of cultured SK-N-MC cells at several time points during rotenone exposure to determine pathways involved in rotenone toxicity and dopaminergic degeneration. We will compare these profiles to baseline profiles of rodent dopaminergic neurons that we have already obtained, as well as to profiles of dopamine neurons from rotenone-treated rats that we will obtain in the near future. We will also compare these data to published results from SN neurons from human PD patients. This technique will not only help us to detect gene expression changes relevant to dopaminergic neurodegeneration in PD, but it will allow us to determine if the SK-N-MC system can be reliably used to screen for neuroprotective therapies for PD. We anticipate that SK-N-MC cells will show a relevant subset of the gene changes seen in dopamine neurons in vivo and that this will guide us in the sorts of mechanisms and drugs that can be screened in this system. Chronic exposure to low levels of rotenone causes changes in gene expression in SK-N-MC cells that sensitize the cells to toxic insults. We also hypothesize that there are several compensatory protective pathways that are stimulated by chronic rotenone, although these pathways are ultimately ineffective at preventing damage. We anticipate that gene expression profiling of rotenone-treated cells over time will suggest several novel strategies for neuroprotective intervention. SK-N-MC cells will be grown in three different media: media only, vehicle (EtOH), and rotenone (5 nM). All current experimental evidence in our lab indicates that vehicle-treated cells are indistinguishable from media-only cells. Rotenone-treated cellls have a stereotypical response in culture. At one week, the only noticed change is an increase in alph-synuclein aggregation. At two weeks, evidence of increased oxidative stress appears (increased protein carbonyls and lipid peroxidation). At four weeks, the cells are markedly sensitized to oxidative challenge with H2O2. Therefore, we will examine gene expression at baseline, and during 1, 2, and 4 weeks of rotenone treatment. Three experiments will be performed, each lasting 4 weeks. For each experiment, three separate dishes of vehicle-treated, and rotenone-treated cells will be harvested at 1, 2, and 4 weeks (18 independent samples). Untreated, media-only cells will be harvested after 1 week in vitro to serve as baseline cells. Total RNA will be isolated. An equal amount of RNA from one dish per experiment per group will be used to compose the final samples. Therefore, each independent sample will consist of RNA from 3 separate experiments. This will allow us to take advantage of a pooling strategy, yet not sacrifice technical and biological replication. 21 samples will be sent to the Consortium. Three will be from untreated cells. Nine will be vehicle-treated at 1, 2, and 4 weeks (3 each). Nine will be rotenone-treated at 1, 2 , and 4 weeks (3 each). Each sample will be labeled and hybridized to one Affymetrix Human Genome U133 Plus 2.0 Gene Chip. With assistance of the consortium, we will analyze the data using the Signifiance Analysis of Microarrays (SAM) program and self-organizing map algorithms. Keywords: time-course

帕金森病（Parkinson’s disease, PD）是一种尚无治愈方案的常见神经退行性疾病。其症状的核心成因是中脑多巴胺能神经元丢失，但目前尚不明确此类神经元死亡的具体机制。流行病学研究显示，农药暴露与帕金森病存在显著关联；向大鼠施用有机农药鱼藤酮（rotenone）可复现帕金森病绝大多数行为学、神经化学及神经病理学特征，包括多巴胺能神经元的特异性丢失。

本研究团队已利用多巴胺能细胞系SK-N-MC神经母细胞瘤细胞（SK-N-MC neuroblastoma cells）构建了鱼藤酮毒性的体外模型，该模型可复现体内鱼藤酮毒性及帕金森病中观察到的多种细胞变化，如α-突触核蛋白（alpha-synuclein）聚集与氧化损伤。目前本团队正利用该简便模型探索多巴胺能神经元退行性变的分子机制，最终目标是发掘帕金森病中多巴胺能神经元神经保护的全新机制。

本研究将检测鱼藤酮处理过程中多个时间点的培养SK-N-MC细胞的基因表达谱，以明确参与鱼藤酮毒性及多巴胺能神经元退行性变的信号通路。我们将把这些基因表达谱与本团队已获取的大鼠多巴胺能神经元基线表达谱，以及未来将获取的鱼藤酮处理大鼠的多巴胺能神经元表达谱进行比对；此外还将把这些数据与已发表的人类帕金森病患者黑质（substantia nigra, SN）神经元的研究结果进行对比。

该技术不仅可帮助我们检测与帕金森病多巴胺能神经元退行性变相关的基因表达变化，还能让我们明确SK-N-MC细胞体系是否可可靠地用于筛选帕金森病的神经保护疗法。我们预计SK-N-MC细胞将呈现体内多巴胺能神经元中观察到的相关部分基因表达变化，这将为我们确定可在该体系中筛选的机制与药物提供指导。长期低剂量鱼藤酮暴露可改变SK-N-MC细胞的基因表达谱，使细胞对毒性损伤更加敏感。我们同时提出假说：慢性鱼藤酮暴露会激活多种代偿性保护通路，但这些通路最终无法有效阻止细胞损伤。我们预计，对鱼藤酮处理细胞的动态基因表达谱分析将提出多种全新的神经保护干预策略。

SK-N-MC细胞将在三种不同培养基中培养：仅基础培养基、溶媒（乙醇，EtOH）以及含5 nM鱼藤酮的培养基。本实验室当前所有实验证据均表明，溶媒处理组细胞与基础培养基组细胞无显著差异。鱼藤酮处理组细胞在培养体系中呈现典型的反应模式：培养1周时，仅能观察到α-突触核蛋白聚集水平升高；培养2周时，可观察到氧化应激增强的证据（蛋白质羰基化水平升高与脂质过氧化加剧）；培养4周时，细胞对过氧化氢（H₂O₂）介导的氧化攻击敏感性显著升高。因此，我们将在基线以及鱼藤酮处理1、2、4周时检测基因表达水平。

本研究将开展3次独立实验，每次实验周期为4周。每次实验中，我们将在1、2、4周时分别收集3个独立培养皿的溶媒处理组与鱼藤酮处理组细胞，共计18份独立样本。未处理的基础培养基组细胞将在体外培养1周后收集，作为基线对照样本。将提取总RNA，每组每次实验中各1个培养皿的等量RNA混合后制备最终样本。因此，每份独立样本将来自3次独立实验的RNA混合产物。该策略可在兼顾技术重复与生物学重复的前提下，利用混合样本的优势。

共计21份样本将提交至联合研究团队：其中3份为未处理组细胞样本，9份为溶媒处理组样本（分别在1、2、4周时收集，每组3份），另有9份为鱼藤酮处理组样本（同样在1、2、4周时收集，每组3份）。每份样本将进行标记，并与Affymetrix Human Genome U133 Plus 2.0基因芯片进行杂交。在联合团队的协助下，我们将使用微阵列显著性分析（Significance Analysis of Microarrays, SAM）程序与自组织映射（self-organizing map）算法对数据进行分析。

关键词：时间进程（time-course）