Cell Model of Parkinson's Disease and Screening for Therapeutic Compounds

描述（由申请人提供）：基因细胞模型将加强神经保护策略的发展，以防止帕金森病的神经元细胞死亡。筛选这种小分子细胞模型可以确定药物开发的主要候选物。培养中突变蛋白的表达可用于模拟常染色体显性神经退行性疾病。我们建立了一种在PC12细胞中表达突变亨廷顿蛋白的诱导细胞模型。通过测试FDA批准的1040种NINDS化合物，我们已经证明该模型可以用于中通量筛选。该屏幕的命中率约为0.4 - 0.5%。我们现在建议开发一个类似的帕金森病遗传细胞模型，使用诱导性表达突变α -突触核蛋白。我们有初步的数据表明，在分化的PC12细胞中，过度表达E46K突变体α -突触核蛋白本身会导致神经元细胞死亡。我们将测试当前的候选神经保护剂，并对NINDS化合物集合进行筛选，以证明中等通量筛选的实用性。在Specific Aim 1中，我们将描述几种诱导表达E46K α -突触核蛋白的PC12细胞系，以获得低背景表达、高诱导表达和一致毒性的细胞系。我们将优化条件并获得可靠的筛选方法。在Specific Aim 2中，我们将测试目前被认为是PD神经保护治疗候选者的化合物，如辅酶Q10、caspase抑制剂z-VAD等，以确定它们在我们的模型中是否具有神经保护活性。在Specific Aim 3中，我们将对fda批准的1040种NINDS化合物和相关化合物进行中等通量筛选，以抑制毒性。所有阳性结果将被确认，并建立剂量-反应曲线以确定近似的抑制效力。经过验证的阳性命中可以在未来的PD动物模型中进行测试。帕金森病的细胞模型及治疗化合物的筛选

DESCRIPTION (provided by applicant): The development of neuroprotective strategies to prevent neuronal cell death in Parkinson's disease would be enhanced by genetic cell models. Screening of such a cell model with small molecules could identify lead candidates for drug development. Expression of mutant proteins in culture can be used to model autosomal dominant neurodegenerative diseases. We have developed an inducible cell model expression of mutant huntingtin in PC12 cells. We have shown that this model can be used for medium throughput screens by testing the NINDS compound collection of 1040 FDA approved compounds. The hit rate in this screen was approximately 0.4 - 0.5%. We now propose to develop a similar genetic cell model for Parkinson's disease, using inducible expression of mutant alpha-synuclein. We have preliminary data indicating that over expression E46K mutant alpha-synuclein itself causes neuronal cell death in differentiated PC12 cells. We will test current candidate neuroprotective agents, and perform a screen of the NINDS compound collection to demonstrate the utility for moderate throughput screens. In Specific Aim 1, we will characterize several PC12 cell lines inducibly expressing E46K alpha-synuclein, in order to obtain lines with low background expression, high inducible expression, and consistent toxicity. We will optimize conditions and get a reliable assay for screening. In Specific Aim 2, we will test compounds currently considered candidates for neuroprotective therapy for PD, such as Co-enzyme Q10, caspase inhibitor z-VAD and others to determine whether they have neuroprotective activity in our model. In Specific Aim 3, we will carry out a medium throughput screen of the 1040 NINDS compound collection of FDA-approved and related compounds, for inhibition of toxicity. All positives will be confirmed, and dose- response curve will be established to determine approximate inhibitory potencies. Validated positive hits could then be taken to testing in PD animal models in future.Cell Model of Parkinson's Disease and Screening for Therapeutic Compounds Project Narrative: Parkinson's disease (PD) is the second most common neurodegenerative disease. There is currently no cure or effective neuroprotective treatment. Our long-term goal is to develop effective neuroprotective agents in PD models in order to prepare for clinical therapeutic trials in PD. Our proposed research has potential to find compounds which are neuroprotective and further be taken to preclinical and clinical trials of PD.