Mechanisms of Prion Aggregation

朊病毒疾病是一种无情的进行性神经退行性疾病，通常在发病后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朊病毒聚集， 海绵状变性、突触和神经元损失以及严重的星形胶质细胞增生和小胶质细胞增生。结构性 朊病毒蛋白（PrP）的决定因素和驱动聚集的内源性辅因子，支配朊病毒 组装，并通过中枢神经系统传播的影响聚集体尚不清楚。一个主要目标是 应用是定义内源性辅因子硫酸乙酰肝素（HS）何时以及如何促进纤维组装 在实质和血管中，并减缓PrP通过组织液的清除， 体内模型系统。我们以前已经采用了一系列基于细胞的朊病毒转化方法 分析和新产生的转基因和敲入小鼠模型与结构生物学家合作， 定义PrP自我组装和朊病毒转化的物种障碍的机制。我们发现 使用敲入小鼠模型，PrP上的N-连接聚糖减少海绵状变性，阻碍斑块， 形成，并排斥HS结合。此外，我们发现，噬斑形成朊病毒是由不良的 糖基化，GPI无锚PrP结合高度硫酸化的HS，强调PrP后的关键作用， 在驱动聚集体构象和疾病表型中的翻译修饰。我们还发现 降低HS链长度减少实质斑块形成和动脉存活。最后，我们确定了 PrP序列中控制跨物种朊病毒转化的高度淀粉样蛋白生成片段， PrP中谷氨酰胺和天冬酰胺残基的位置提高或降低朊病毒传递屏障。在这 更新，我们的目标是确定PrP-HS相互作用，促进朊病毒聚集体组装和加速 疾病我们建立在我们长期观察的基础上，即PrP的结构特征，以及宿主 糖胺聚糖，驱动朊病毒的高效转化。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，并使用小鼠模型确定对朊病毒细胞靶和存活的影响。 第二，我们将使用条件性免疫荧光技术来确定内源性HS如何调节PrP通过间质液的清除。 HS小鼠模型和放射性标记的PrP。第三，我们将测试反义寡核苷酸（ASO）的功效。 在朊病毒空斑发育的早期和中期阶段靶向HS生物合成酶或Prnp mRNA， 朊病毒病模型我们期望这些机制研究将（i）定义内源性辅因子HS， 加速和修改朊病毒疾病，和（ii）确定是否减少PrP与这种潜力的相互作用 治疗靶点阻断朊病毒扩散。

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.