Mechanisms of Prion Spread and Neuronal Toxicity

突触功能障碍和神经炎性营养不良是朊病毒的突出病理特征， 阿尔茨海默病影响的大脑。还经常观察到泛素化蛋白质内含物， 提供了蛋白质抑制途径受损的有力证据。细胞膜泛素化 蛋白质和清除通过ESCRT途径（内体分选复合物所需的 运输）对于维持突触稳态至关重要。在这里，我们将深入调查 ESCRT通路对朊病毒疾病中突触稳态破坏的贡献在朊病毒感染的小鼠中，我们发现ESCRT-0（一种Hrs和STAM 1蛋白复合物）显著减少。 以及突触体中泛素化蛋白的富集。显著地，消耗神经元Hrs 在朊病毒感染的小鼠中，缩短了存活时间并加速了突触的退化， 生物化学和结构上。此外，在一项对朊病毒感染者的纵向研究中， 海马，我们发现突触活性反应基因Arc/Arg3.1上调， 磷酸化CaMKII和磷酸化AMPA受体的慢性升高，提示 突触功能的增强和改变始于疾病早期。我们的长期目标是 破解朊病毒和β淀粉样蛋白寡聚体如何破坏与细胞朊病毒相关的信号通路 蛋白质，诱导蛋白质抑制功能障碍和突触变性。利用电生理学， 未感染和朊病毒感染的培养细胞的相关光电子显微镜和蛋白质组学 神经元，我们将首先确定Hrs表达如何影响突触，评估活动，突触前和突触后蛋白，结构和信号。然后我们将测试朊病毒 构象异构体影响ESCRT途径和神经元信号传导在突触。 最后，我们将研究谷氨酸受体活性对朊病毒扩散的贡献， 神经变性我们将直接测试这些基因操纵模型中的发现 与人类朊病毒感染的大脑相比。这些研究是第一次测试神经元活动 影响朊病毒传播、突触变性和疾病进展， 预计将提供关键的见解去调节突触信号，驱动神经元损失， 从而揭示了新的治疗靶点。

Synaptic dysfunction and neuritic dystrophy are prominent pathologic features of the prion- and Alzheimer’s disease-affected brain. Ubiquitinated protein inclusions are also commonly observed, providing strong evidence of impaired proteostatic pathways. Ubiquitination of cell membrane proteins and clearance through the ESCRT pathway (endosomal sorting complex required for transport) is critical to maintaining synaptic homeostasis. Here we will deeply investigate the ESCRT pathway contributions to disrupted synaptic homeostasis in prion disease. In prion-infected mice, we have found markedly reduced ESCRT-0 (an Hrs and STAM1 protein complex) and an enrichment of ubiquitinated proteins in synaptosomes. Strikingly, depleting neuronal Hrs in prion-infected mice shortened survival time and accelerated the degeneration of synapses, biochemically and structurally. Additionally, in a longitudinal study of the prion-infected hippocampus, we found an upregulation in the synaptic activity response gene, Arc/Arg3.1, and a chronic elevation in phosphorylated CaMKII and phosphorylated AMPA receptors, suggestive of enhanced and altered synapse function beginning in early disease. Our long-term goal is to decipher how prion and amyloid-β oligomers disrupt signaling pathways linked to the cellular prion protein, inducing proteostatic dysfunction and synaptic degeneration. Using electrophysiology, correlative light-electron microscopy, and proteomics on uninfected and prion-infected cultured neurons, we will first determine how Hrs expression impacts synapses, assessing activity, pre-and post-synaptic proteins, structure, and signaling. We will then test how distinct prion conformers impact the ESCRT pathway and neuronal signaling at glutamatergic synapses. Finally, we will investigate the contribution of glutamate receptor activity to prion spread and neurodegeneration. We will directly test how the findings in these genetically manipulated models compare to human prion-affected brain. These studies are the first to test how neuronal activity impacts prion dissemination, synaptic degeneration, and disease progression, and outcomes are expected to provide key insights into the deregulated synaptic signaling that drives neuron loss, thus revealing new therapeutic targets.