Genome-wide screening for effectors of toxic prion protein-induced neuronal death

描述（由申请人提供）：朊病毒疾病是致命的蛋白质错误折叠神经退行性疾病（PMND），如阿尔茨海默病、帕金森病、亨廷顿病或肌萎缩侧索硬化症。这些疾病是由于错误折叠的宿主蛋白质的毒性。可用的治疗针对症状的改善，并且随着疾病的进展而变得无效。缺乏关于神经退行性变的确切机制的信息是发展疾病修饰治疗的主要障碍。为了研究这些机制，在过去的4年里，我们建立了朊蛋白（PrP）诱导的神经元死亡模型。该模型是基于高，神经元特异性，毒性的单体错误折叠形式的重组PrP（TPrP），它再现的形态和分子特征的神经元死亡发生在小鼠模型朊病毒疾病。我们现在开始使用这个模型来研究错误折叠的PrP诱导的神经元死亡的机制。我们观察到TPrP进入神经元细胞，但不是成纤维细胞，并通过ADP-核糖基化依赖性途径诱导深刻的NAD+耗竭。NAD+耗竭最终导致神经元死亡，因为NAD+补充导致TPrP诱导的损伤后细胞完全恢复。这项重点研究提案的目的是确定TPrP诱导的毒性途径中导致NAD+耗尽和神经元死亡的关键效应物。我们的工作假设是，敲除对TPrP诱导的死亡途径的执行至关重要的细胞因子将防止神经元死亡，因此可以通过在我们的TPrP神经毒性试验中实施基因组SiRNA筛选来识别这些关键参与者。拟议的研究将确定朊病毒蛋白诱导的神经变性的新治疗靶点，从而解决神经保护性治疗干预领域进展的关键障碍。因为其他PMND，如朊病毒疾病，是由于宿主蛋白质的错误折叠形式的毒性，并且鉴于越来越多的证据表明共同的机制特征，拟议研究的结果可能对蛋白质错误折叠疾病的治疗产生广泛影响。后续项目将致力于使用已鉴定的细胞因子来破译整个神经元死亡途径，并在朊病毒疾病和可能的其他蛋白质错误折叠神经退行性疾病中开发新的神经保护治疗策略。 公共卫生相关性：朊病毒疾病是致命的蛋白质错误折叠的神经退行性疾病，如阿尔茨海默病、帕金森病、亨廷顿病或肌萎缩性侧索硬化症。缺乏对神经退行性变机制的了解是疾病改善治疗发展的主要障碍。使用我们最近开发的测定法，我们建议进行全基因组siRNA筛选以鉴定执行错误折叠朊病毒蛋白诱导的神经元死亡所必需的细胞因子。

DESCRIPTION (provided by applicant): Prion diseases are fatal protein misfolding neurodegenerative diseases (PMNDs) like Alzheimer, Parkinson, Huntington's Diseases or Amyotrophic Lateral Sclerosis. These diseases are due to the toxicity of a misfolded host protein. Available treatments are directed towards amelioration of symptoms and become ineffective as disease progresses. The lack of information concerning the exact mechanisms of neurodegeneration is a major hurdle to the development of disease- modifying treatments. With the aim to study these mechanisms, during the last 4 years, we developed a model for prion protein (PrP)-induced neuronal death. This model is based on the high, neuron-specific, toxicity of a monomeric misfolded form of recombinant PrP (TPrP); it reproduces morphological and molecular features of neuronal death occurring in mouse models of prion disease. We now set out to use this model to study the mechanism of neuronal death induced by misfolded PrP. We observed that TPrP enters neuronal cells, but not fibroblasts, and induces a profound NAD+ depletion via an ADP-ribosylation-dependent pathway. NAD+ depletion is ultimately responsible for neuronal death as NAD+ replenishment leads to full cellular recovery after TPrP-induced injury. The objective of this focused research proposal is to identify key effectors in the TPrP-induced toxicity pathway leading to NAD+ depletion and neuronal death. Our working hypothesis is that the knock-down of cellular factors critical to the execution of TPrP-induced death pathway will prevent neuronal death, and therefore these key players can be identified by implementing a genomic siRNA screen in our TPrP neurotoxicity assay. The proposed research will identify new therapeutic targets for prion protein-induced neurodegeneration thereby addressing a critical barrier to progress in the field of neuroprotective therapeutic interventions Because other PMNDs, like prion diseases, are due to the toxicity of a misfolded form of a host protein, and in view of the growing evidence for common mechanistic features, the results of the proposed research are likely to have a broad impact for the treatment of protein misfolding diseases. Subsequent projects will aim at using identified cellular factors to decipher the entire neuronal death pathway and developing new neuroprotective therapeutic strategies in prion diseases and possibly other protein misfolding neurodegenerative diseases.! PUBLIC HEALTH RELEVANCE: Prion diseases are fatal protein misfolding neurodegenerative diseases like Alzheimer, Parkinson, Huntington's diseases or Amyotrophic Lateral Sclerosis. The lack of knowledge about mechanisms of neurodegeneration is a major hurdle to the development of disease-modifying treatments. Using an assay that we have recently developed, we propose to perform a genome-wide siRNA screen to identify cellular factors necessary to execute misfolded prion protein -induced neuronal death.