Mechanisms of Prion Aggregation

Pron病是一种无情的进行性神经退行性疾病，通常在6个月内死亡 神经系统症状的出现。病理特征包括广泛的细胞外Pron聚集体， 海绵状变性、突触和神经元丢失，以及严重的星形胶质细胞增多症和小胶质细胞增多症。结构性的 Prion蛋白(PrP)的决定因素和驱动聚集的内源性辅助因子，支配Prion 集合体，以及影响集合体通过中枢神经系统扩散尚不清楚。这样做的一个主要目标是 应用是确定内源性辅助因子--硫酸乙酰肝素(HS)何时以及如何促进纤维聚集 在实质和血管中使用，并减缓PrP通过间质液体的清除 活体模型系统。在此之前，我们已经使用了一系列基于细胞的Pron转换方法 与结构生物学家合作进行检测和新产生的转基因和敲入小鼠模型 定义PrP自组装的基础机制和PrP转化的物种障碍。我们发现 利用PrP上N-连接的葡聚糖减少海绵状变性，阻碍斑块的敲入小鼠模型 形成，并排斥HS结合。此外，我们还发现，形成空斑的普恩病毒由质量较差的 糖基化的、无GPI锚定的PrP与高度硫酸盐化的HS结合，强调了PrP后的关键作用。 翻译修饰在驱动聚集体构象和疾病表型中的作用。我们还发现， 减少HS链长可减少实质斑块的形成，延长存活期。最后，我们确定了 PrP序列中控制跨物种Prion转换的高度淀粉样变性片段，如 谷氨酰胺和天冬酰胺残基在PrP中的位置提高或降低了PrP的蛋白传递屏障。在这 更新，我们的目标是确定PrP-HS相互作用，促进PrP-HS聚集组装并加速 疾病。我们长期观察到PrP的结构特征与宿主一起 糖胺多聚糖，推动高效的朊病毒转化。首先，我们将从基因上操纵神经元、星形胶质细胞、 和内皮HS链，并利用小鼠模型确定对Pron细胞靶点和存活率的影响。 其次，我们将定义内源性HS如何通过间质液体调节PrP清除，使用条件 HS小鼠模型和放射性标记PrP。第三，我们将测试反义寡核苷酸(ASOS)的效果 以HS生物合成酶或PRNP mRNA为靶点在PrP斑块形成的早期和中期 Pron病模型。我们预计这些机制研究将(I)定义内生辅助因素HS， 加速和修饰PrP疾病，以及(Ii)确定减少PrP与这一潜力的相互作用 治疗靶点阻止了普恩病毒的传播。

Prion diseases are relentlessly progressive neurodegenerative disorders with death often within six months of the onset of neurologic symptoms. Pathologic features include widespread extracellular prion aggregates, spongiform degeneration, synaptic and neuronal loss, and severe astrogliosis and microgliosis. The structural determinants of the prion protein (PrP) and endogenous co-factors that drive aggregation, govern prion assembly, and impact aggregate spread through the central nervous system are unclear. A major goal of this application is to define when and how the endogenous co-factor, heparan sulfate (HS), promotes fibril assembly in the parenchyma and blood vessels and slows PrP clearance through the interstitial fluid using in vitro and in vivo model systems. We have previously pursued a range of approaches using cell-based prion conversion assays and newly generated transgenic and knock-in mouse models in collaboration with structural biologists to define the mechanisms that underlie PrP self-assembly and species barriers to prion conversion. We discovered using knock-in mouse models that N-linked glycans on PrP reduce spongiform degeneration, hinder plaque formation, and repel HS binding. Further, we found that plaque-forming prions were composed of poorly glycosylated, GPI-anchorless PrP bound to highly sulfated HS, underscoring the pivotal role of PrP post- translational modifications in driving the aggregate conformation and disease phenotype. We also found that reducing HS chain length decreases parenchymal plaque formation and prolongs survival. Finally, we identified highly amyloidogenic segments in the PrP sequence that control cross species prion conversion, as the number and location of glutamine and asparagine residues in PrP raise or lower the prion transmission barrier. In this renewal, we aim to determine the PrP-HS interactions that promote prion aggregate assembly and accelerate disease. We build on our long-standing observation that structural features of PrP, together with host glycosaminoglycans, drive efficient prion conversion. First, we will genetically manipulate neuronal, astrocytic, and endothelial HS chains and determine the impact on prion cell targets and survival using mouse models. Second, we will define how endogenous HS regulates PrP clearance through the interstitial fluid using conditional HS mouse models and radiolabeled PrP. Third, we will test the efficacy of antisense oligonucleotides (ASOs) targeting HS biosynthetic enzymes or Prnp mRNA in the early and mid-stages of prion plaque development in a prion disease model. We expect these mechanistic studies will (i) define how an endogenous co-factor, HS, accelerates and modifies prion disease, and (ii) determine whether reducing PrP interactions with this potential therapeutic target blocks prion spread.