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Mechanisms of prion strain dynamics

朊病毒应变动力学机制

基本信息

批准号：
10188653
负责人：
Jason C Bartz
金额：
$ 37.64万
依托单位：
CREIGHTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-30 至 2023-06-30
项目状态：
已结题

项目摘要

Prion diseases are inevitably fatal neurodegenerative zoonotic disorders of animals, including humans, with no known treatment or cure. Prions are comprised largely, if not entirely, of PrPSc, a misfolded form of the normal non-infectious prion protein PrPC. Prion strains are operationally defined by differences in neuropathology that breed true under controlled conditions that are encoded by strain-specific conformations of PrPSc. The long- term goal of this work is to understand the dynamics of prion strains. The objective of this application is to determine if prion strains exist as a mixture of substrains or as a homogeneous population. We hypothesize that prions strains are a dynamic mixture of prion substrains (i.e. quasispecies). This would fundamentally impact our understanding of the mechanism of interspecies transmission, adaptation of prions in response to anti-prion drugs, the ecology of prion transmission in natural settings and may be relevant to other protein misfolding diseases that share prion strain-like features (e.g. Alzheimer’s). Since the structure of PrPSc is poorly defined and technologies do not exist to measure the structure of an individual protein in a mixture, we will test the hypothesis based on the predicted properties of quasispecies that are experimentally feasible. First we will determine the effect of repeated limiting dilutions on prion fitness. Repeated bottleneck passage of a quasispecies leads to a reduction in fitness (i.e. Muller’s ratchet) since, on average, the fitness of an individual infectious unit is lower compared to the fitness of the overall population. We anticipate that repeated transmission of prions at limiting dilutions in PMCA or in cell culture will result in a reduction in the fitness of the prion agent compared to agent passaged at high titer. Second we will establish if biologically cloned prion strains contain substrains. Quasispecies hypothesis predicts that any given prion strain is comprised of a dynamic population of substrains. We will test this by amplifying PrPSc in vitro or passaging in cells under conditions that favor the selection of a minor substrain. In a second series of experiments, we will select for subpopulations of PrPSc that have common shared biochemical features to seed PMCA reactions or prion susceptible RK13 cells. Serial repeated rounds of selection followed by transmission in animals will determine if biologically cloned prion strains contain additional strains and if biochemical selective pressure results in the emergence of strains with the selected properties. The results of these experiments will directly test the hypothesis that prions are quasispecies.

朊病毒疾病是动物（包括人类）不可避免的致命性神经退行性人畜共患病，已知的治疗或治愈。朊病毒如果不是全部，也是大部分由PrPSc组成，PrPSc是正常朊病毒的错误折叠形式。非感染性朊病毒蛋白PrPC。朊病毒株在操作上由神经病理学的差异来定义，在由PrPSc的菌株特异性构象编码的受控条件下繁殖。很长的- 这项工作的长期目标是了解朊病毒株的动力学。本申请的目的是确定朊病毒株是作为亚株的混合物存在还是作为同质群体存在。我们假设朊病毒株是朊病毒亚株（即准种）的动态混合物。这将从根本上影响我们对种间传播机制的理解，朊病毒的适应性，抗朊病毒药物，朊病毒在自然环境中传播的生态学，并可能与其他蛋白质具有朊病毒株样特征的错误折叠疾病（如阿尔茨海默病）。由于PrPSc的结构是由于定义不清，而且不存在测量混合物中单个蛋白质结构的技术，我们将根据实验上可行的准物种的预测特性来检验假设。第一我们将确定重复有限稀释对朊病毒适应性的影响。重复的瓶颈通道，准种导致适应性降低（即穆勒棘轮），因为平均而言，个体的适应性感染单位的适应度低于总体群体的适应度。我们预计，朊病毒在PMCA或细胞培养物中的有限稀释度下的传播将导致朊病毒因子与以高滴度传代的因子相比。其次，我们将确定生物克隆朊病毒菌株含有亚菌株。准种假说预测，任何给定的朊病毒株是由一个亚株的动态种群。我们将通过体外扩增PrPSc或在细胞中传代来测试这一点。有利于选择次要亚株的条件。在第二系列实验中，我们将选择具有共同的生物化学特征的PrPSc亚群，用于接种PMCA反应或朊病毒易感RK 13细胞。一系列重复的选择，然后在动物中传播将决定如果生物克隆的朊病毒菌株含有额外的菌株，如果生物化学选择压力导致具有所选特性的菌株的出现。这些实验的结果将直接检验朊病毒是准种的假说