Molecular basis of prion protein-induced neurodegeneration

项目总结 Pron病是一种罕见的、总是致命的神经退行性疾病，其病理特征是 常见于阿尔茨海默病，包括细胞外蛋白聚集体、突触丢失和 神经性营养不良。在Prion和阿尔茨海默病模型中，神经细胞Prion的耗竭 蛋白质(PrPC)显著改善突触损伤和临床疾病 在改变的信号转导通路中暗示神经元PrPC的表达 可能是突触毒性和内溶酶体功能障碍的基础。我们已经设计了第一个 具有PRNP点突变的敲入小鼠模型，该模型发展为显著的和 严重海绵状脑病、神经性营养不良和突触后受体改变 在没有普恩聚集体的情况下，发生磷酸化。从这些细胞培养出的皮质神经元 转基因小鼠对谷氨酸和树突状静脉曲张的敏感性增加， 暗示有兴奋性毒性。因此，这种PrP敲入模式提供了一个独特的机会 阐明突触中关键的PrPC相互作用和改变的信号转导途径 并确定将PrPC与突触丢失和 内溶酶体调节失调。我们的长期目标是了解PrPC如何触发 可能导致蛋白稳定和突触毒性受损的神经信号异常 疾病。利用培养的原代神经元和小鼠，我们将首先确定突变体是如何 PrPC相互作用影响突触前、后神经元蛋白水平和谷氨酸 受体功能。然后我们将确定突变的PrPC是如何失调内溶酶体的 和蛋白抑制活性。最后，我们使用高度敏感和定量的蛋白质组学来 通过串联确定大脑中PrP相互作用组和磷酸蛋白质组网络的改变 质量标签质谱分析。对于所有目标，我们将直接测试来自 表达PrPC的突变型大脑与感染病毒的鼠脑和人脑进行了比较。这些 首次有研究针对脑内神经细胞内溶酶体和突触通路。 表达突变PrPC的敲入小鼠模型和结果有望提供关键 PrPC在突触维持和刺激信号通路中的作用 突触丢失，从而揭示了Pron病的新治疗靶点。

PROJECT SUMMARY Prion diseases are rare, invariably fatal neurodegenerative disorders with pathologic features in common with Alzheimer’s disease, including extracellular protein aggregates, synaptic loss, and neuritic dystrophy. In prion and Alzheimer’s disease models, depletion of neuronal cellular prion protein (PrPC) ameliorates synaptic impairment and clinical disease, strongly implicating neuronal PrPC expression in the altered signal transduction cascades that may underlie synaptotoxicity and endolysosomal dysfunction. We have engineered the first knock-in mouse model with a point mutation in Prnp that develops a striking and severe spongiform encephalopathy, neuritic dystrophy, and altered post-synaptic receptor phosphorylation, in the absence of prion aggregates. Cultured cortical neurons from these knock-in mice show an increased sensitivity to glutamate and dendritic varicosities, suggestive of excitotoxicity. Thus, this PrP knock-in model provides a unique opportunity to elucidate key PrPC interactions and altered signal transduction pathways at the synapse and to determine the molecular mechanisms that link PrPC to synaptic loss and endolysosomal dysregulation. Our long-term goal is to understand how PrPC triggers aberrant neuronal signaling that may drive impaired proteostasis and synaptotoxicity in prion disease. Using cultured primary neurons and mice, we will first determine how the mutant PrPC interactions impact pre- and post-synaptic neuronal protein levels and glutamate receptor function. We will then identify how mutant PrPC dysregulates endolysosomal and proteostatic activity. Finally, we use highly sensitive and quantitative proteomics to define the PrP interactome and phosphoproteome network alterations in the brain by tandem mass tag mass spectrometry analysis. For all aims, we will directly test how the findings from the mutant PrPC-expressing brain compare to prion-infected mouse and human brain. These studies are the first to target the neuronal endolysosomal and synaptic pathways in a knock-in mouse model expressing mutant PrPC, and outcomes are expected to provide key insights into the role of PrPC in synapse maintenance and the signaling pathways inciting synaptic loss, thus revealing new therapeutic targets for prion disease.