Genetics of Prion Susceptibility in vitro

体外朊病毒敏感性遗传学

• 批准号: 8085701
• 负责人: George A. Carlson
• 金额: $ 140.17万
• 依托单位: MC LAUGHLIN RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8085701
• 关键词: Genetics; Prion; Susceptibility; vitro

Prion diseases are neurodegenerative disorders of humans and animals caused by misfolding of prion protein (PrP). Diseases caused by prions can be transmitted to experimental animals whether their origin is genetic, infectious, or sporadic. The transmission of prion disease to humans by ingestion of bovine spongiform encephalopathy (BSE) prions has been documented. The spread of chronic wasting disease (CWD) through herds of deer and elk in North America is a serious concern and the susceptibility of humans to CWD prions is unknown. All prion diseases involve changes in the conformation of PrP from its benign cellular isoform, PrPc, to a disease-specific isoform, PrPSc. PrPSc is sufficient to transmit disease and its conformation enciphers prion strain properties. Appreciation of the biological significance of PrP came from incubation time studies in normal and transgenic mice. However, genes in addition to the prion protein gene (Prnp) also are important in prion replication and disease susceptibility but their identification has proven exceptionally difficult due to the length and expense of incubation time studies in mice. Similarly, synthetic prions created from recombinant PrP produced in E. coli cause disease in transgenic (Tg) mice but incubation times often exceed 500 days. Only a few cell lines were available that could be infected with prions, offering limited genetic diversity and sensitivity to only one or two prion strains. CNS stem cells can be isolated from adult or fetal brains of normal or Tg mice and grown in culture. Aggregates of these cells, called neurospheres, can be infected with prions and offer an exciting new technology for prion research. Efficiency of infection, spread from cell to cell, and rate of prion replication can be discriminated in neurosphere cultures and conformation dependent epitopes allow identification of individual cells with intracellular PrPSc. The four senior investigators on this Program Project application will apply this novel in vitro model to develop rapid and sensitive bioassays for prions, including those of humans and ungulates. The influence of conformational stability of infectious prions on the rate of prion propagation can be addressed more efficiently in culture than in mice. The ability to isolate neurosphere lines from any strain or Tg line offers the opportunity for genetic analysis of prion disease in culture and a system to screen candidates for the genes within chromosomal regions containing prion incubation time modifier loci. Neurosphere lines also offer a refinement over mice for the systems approach to identify gene networks perturbed by prion replication and pathogenic processes. CNS stem cells can be directed to differentiate to neurons and glia. Effects of infection on differentiation and effects of infection on differentiated cells will be evaluated in neurospheres and in "matured brain spheres", emphasizing the Notch-Hes pathway.

朊病毒疾病是由朊病毒蛋白（PrP）错误折叠引起的人类和动物神经退行性疾病。由朊病毒引起的疾病可以传播给实验动物，无论其来源是遗传性的、传染性的还是散发性的。已有文献记载，人类摄入牛海绵状脑病 (BSE) 朊病毒可将朊病毒病传播给人类。慢性消耗性疾病 (CWD) 通过北美鹿群和麋鹿的传播是一个严重的问题，而人类对 CWD 朊病毒的易感性尚不清楚。所有朊病毒疾病都涉及 PrP 构象的变化，从良性细胞亚型 PrPc 转变为疾病特异性亚型 PrPSc。 PrPSc 足以传播疾病，其构象加密了朊病毒株的特性。对 PrP 生物学意义的认识来自 正常和转基因小鼠的孵育时间研究。然而，除朊病毒蛋白基因 (Prnp) 之外的基因在朊病毒复制和疾病易感性中也很重要，但由于小鼠孵化时间研究的长度和费用，它们的鉴定已被证明异常困难。同样，由大肠杆菌产生的重组 PrP 产生的合成朊病毒会导致转基因 (Tg) 小鼠患病，但孵化时间通常超过 500 天。只有少数细胞系可以被感染 朊病毒，仅对一种或两种朊病毒株提供有限的遗传多样性和敏感性。 CNS 干细胞可以从正常或 Tg 小鼠的成年或胎儿大脑中分离出来并在培养物中生长。这些细胞的聚集体，称为神经球，可以被朊病毒感染，并为朊病毒研究提供了令人兴奋的新技术。可以在神经球培养物中区分感染效率、从细胞到细胞的传播以及朊病毒的复制速率，并且构象依赖性表位允许识别单个细胞 细胞内 PrPSc。该计划项目申请的四名高级研究人员将应用这种新颖的体外模型来开发快速、灵敏的朊病毒生物测定方法，包括人类和有蹄类动物的朊病毒。 与小鼠相比，在培养物中可以更有效地解决传染性朊病毒构象稳定性对朊病毒繁殖速率的影响。从任何菌株或 Tg 系中分离神经球系的能力为培养物中朊病毒疾病的遗传分析提供了机会，并为筛选包含朊病毒孵育时间修饰基因座的染色体区域内的候选基因提供了系统。 神经球系还对小鼠的系统方法进行了改进，以识别受朊病毒复制和致病过程扰动的基因网络。中枢神经系统干细胞可以定向分化为神经元和神经胶质细胞。将在神经球和“成熟脑球”中评估感染对分化的影响以及感染对分化细胞的影响，重点是Notch-Hes途径。

项目成果

Systems biology and the discovery of diagnostic biomarkers.

系统生物学和诊断生物标志物的发现。

• DOI: 10.3233/dma-2010-0697
• 发表时间: 2010
• 期刊: Disease markers
• 作者: Wang K;Lee I;Carlson G;Hood L;Galas D
• 通讯作者: Galas D

A systems approach to prion disease.

• DOI: 10.1038/msb.2009.10
• 发表时间: 2009
• 期刊: MOLECULAR SYSTEMS BIOLOGY
• 影响因子: 9.9
• 作者: Hwang, Daehee;Lee, Inyoul Y.;Yoo, Hyuntae;Gehlenborg, Nils;Cho, Ji-Hoon;Petritis, Brianne;Baxter, David;Pitstick, Rose;Young, Rebecca;Spicer, Doug;Price, Nathan D.;Hohmann, John G.;DeArmond, Stephen J.;Carlson, George A.;Hood, Leroy E.
• 通讯作者: Hood, Leroy E.

Levels of the Mahogunin Ring Finger 1 E3 ubiquitin ligase do not influence prion disease.

• DOI: 10.1371/journal.pone.0055575
• 发表时间: 2013
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Silvius D;Pitstick R;Ahn M;Meishery D;Oehler A;Barsh GS;DeArmond SJ;Carlson GA;Gunn TM
• 通讯作者: Gunn TM

P4 medicine: how systems medicine will transform the healthcare sector and society.

• DOI: 10.2217/pme.13.57
• 发表时间: 2013
• 期刊: Personalized medicine
• 影响因子: 2.3
• 作者: Flores M;Glusman G;Brogaard K;Price ND;Hood L
• 通讯作者: Hood L

Down-regulation of Shadoo in prion infections traces a pre-clinical event inversely related to PrP(Sc) accumulation.

• DOI: 10.1371/journal.ppat.1002391
• 发表时间: 2011-11
• 期刊: PLoS pathogens
• 影响因子: 6.7
• 作者: Westaway D;Genovesi S;Daude N;Brown R;Lau A;Lee I;Mays CE;Coomaraswamy J;Canine B;Pitstick R;Herbst A;Yang J;Ko KW;Schmitt-Ulms G;Dearmond SJ;McKenzie D;Hood L;Carlson GA
• 通讯作者: Carlson GA