Molecular basis of prion protein-induced neurodegeneration

朊病毒病是一种罕见的，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，包括细胞外蛋白质聚集，突触丢失， 神经炎性营养不良在朊病毒和阿尔茨海默病模型中， 蛋白（PrPC）改善突触损伤和临床疾病，强烈暗示 神经元PrPC表达在改变的信号转导级联中可能是 突触毒性和内溶酶体功能障碍。我们设计了第一只基因敲入的老鼠 一个Prnp点突变的模型，该模型发展出一种引人注目的严重海绵状 脑病、神经炎性营养不良和突触后受体磷酸化改变， 没有朊病毒聚集体。来自这些基因敲入小鼠的培养皮层神经元显示出 对谷氨酸和树突静脉曲张的敏感性增加，提示兴奋性毒性。因此，在本发明中， 这种PrP基因敲入模型提供了一个独特的机会来阐明关键的PrPC相互作用， 改变了突触的信号转导途径，并确定了 PrPC与突触丢失和内溶酶体失调的联系机制。我们的长期 目的是了解PrPC如何触发异常的神经元信号传导， 朊病毒疾病中的蛋白质稳态和突触毒性。使用培养的原代神经元和小鼠，我们 将首先确定突变的PrPC相互作用如何影响突触前和突触后神经元 蛋白质水平和谷氨酸受体功能。然后我们将确定突变的PrPC 内溶酶体和蛋白抑制活性失调。最后，我们使用高度敏感和 定量蛋白质组学，以确定PrP相互作用组和磷酸化蛋白质组网络的改变 在大脑中的串联质谱分析。为了所有的目的，我们将直接测试 表达突变PrPC的大脑与朊病毒感染的小鼠相比， 人脑这些研究是第一个针对神经元内溶酶体和突触 在表达突变型PrPC的敲入小鼠模型中， 为PrPC在突触维持和信号通路中的作用提供关键见解 从而揭示朊病毒疾病的新治疗靶点。

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.