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（PMNDs），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宿主蛋白的毒性引起的。现有的治疗方法旨在改善症状，但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疾病修饰治疗发展的主要障碍。为了研究这些机制，在过去的4年里，我们开发了一个朊病毒蛋白（PrP）诱导的神经元死亡模型。该模型基于单体错误折叠形式的重组PrP （TPrP）的高神经元特异性毒性；它再现了朊病毒疾病小鼠模型中神经元死亡的形态学和分子特征。我们现在开始使用这个模型来研究错误折叠的PrP诱导神经元死亡的机制。我们观察到TPrP进入神经元细胞，而不是成纤维细胞，并通过adp核糖基化依赖途径诱导NAD+的深度消耗。NAD+的消耗最终导致神经元死亡，因为NAD+的补充导致tprp诱导的损伤后细胞的完全恢复。这项重点研究计划的目的是确定tprp诱导的毒性途径中导致NAD+耗竭和神经元死亡的关键效应物。我们的工作假设是，对执行TPrP诱导死亡途径至关重要的细胞因子的敲除将防止神经元死亡，因此这些关键因素可以通过在我们的TPrP神经毒性试验中实施基因组siRNA筛选来识别。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，从而解决神经保护治疗干预领域进展的一个关键障碍。因为其他PMNDs，如朊病毒疾病，是由于宿主蛋白错误折叠形式的毒性造成的，鉴于越来越多的证据表明共同的机制特征，这项研究的结果可能对蛋白质错误折叠疾病的治疗产生广泛的影响。后续项目将致力于利用已识别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。

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.!