MOLECULAR DETERMINANTS OF HUMAN PRION DISEASES

我提议的调查的目的是确定 在遗传性朊病毒疾病中朊病毒通过其从头产生， 发展成为一名物理学家和科学家。 我计划研究分子 朊病毒病的遗传学研究。变性梯度 凝胶电泳（DGGE）将用于调查患者的突变 产生遗传性神经系统疾病的PrP基因， 可能改变CNS表型表达的多态性 退化 将评估DGGE检测到的所有遗传变异 通过DNA测序。我计划确定人类是否携带 PrP基因产生感染性朊病毒， 脑脊液或其他体液在神经系统疾病发作前 功能障碍 转基因（Tg）小鼠允许人类传播 (Hu)朊病毒将用于我的研究临床和 小鼠神经系统疾病的神经病理学特征 携带携带P102 L突变的小鼠（Mo）PrP转基因， 计划好了 我将确定这些动物是否会产生传染性朊病毒 在血液、脑脊髓液或其他体液中可以检测到 在神经功能障碍发作之前。 调查可能的 治疗方法，我将尝试防止人类CJD的传播 通过共表达HuPrPc和朊病毒的C末端片段， MoPrPpc。 PrPc的C末端被认为与蛋白X结合， 推测的分子伴侣参与PrPc转化为 PrPSc。 过氧化氢对HuPrPpc与蛋白X结合的竞争性抑制 MoPrPc C-末端片段的表达可以预防疾病。 在 此外，还建议将散发性和遗传性朊病毒疾病模型化， 通过用人朊病毒感染一些培养的人神经元细胞， 用含有P102 L或 E200 K突变。 如果形成HuPrPSc并产生Hu朊病毒感染性， 在这些细胞中，我将试图通过外源性的逆转感染， 肽给药。 我的研究将检查表型和 朊病毒病的基因型多样性，并研究 人、转基因动物和有丝分裂后的人神经元中的朊病毒， 携带PrP基因突变靶向蛋白X用于基因治疗 朊病毒疾病可能提供一种独特的治疗方法， 神经退行性疾病

The objectives of my proposed investigations are to define the mechanism by which prions are generated de-novo in the inherited prion diseases and to develop as a physician-scientist. I plan to study the molecular genetics of prion disease using several approaches. Denaturing gradient gel electrophoresis (DGGE) will be used to survey patients for mutations of the PrP gene that produce inherited neurological diseases, and for polymorphisms which may alter the phenotypic expression of CNS degeneration. All genetic alterations detected by DGGE will be assessed by DNA sequencing. I plan to determine if humans carrying a mutation of the PrP gene produce infectious prions that are detectable in their blood, cerebrospinal fluid or other body fluids prior to the onset of neurologic dysfunction. Transgenic (Tg) mice permissive for transmission of human (Hu) prions will be used in my studies. A comparison of the clinical and neuropathologic features of the neurological disease that occurs in mice harboring the mouse (Mo) PrP transgene which carries the P102L mutation is planned. I shall determine if these animals produce infectious prions that are detectable in the blood, cerebrospinal fluid or other body fluids prior to the onset of neurologic dysfunction. To investigate a possible therapeutic approach, I shall attempt to prevent transmission of human CJD prions to Tg mice by co-expressing HuPrPc and a C-terminal fragment of MoPrPpc. The C-terminus of PrPc is thought to bind to protein X, a putative molecular chaperone involved in the conversion of PrPc into PrPSc. Competitive inhibition of HuPrPpc binding to protein X by over expression of a MoPrPc C-terminal fragment, may prevent disease. In addition, propose to model sporadic and inherited prion disease in cultured human neuronal cells by infecting some with human prions and transfecting others with the HuPrP gene containing either the P102L or E200K mutation. If HuPrPSc is formed and Hu prion infectivity is produced in these cells, I shall attempt to reverse the infection by exogenous peptide administration. My studies will examine the phenotypic and genotypic diversity of prion disease, and investigate the biogenesis of prions in humans, transgenic animals, and post mitotic human neurons which carry a mutation of the PrP gene. Targeting protein X for gene therapy in prion diseases may offer a unique approach to the treatment of neurodegenerative disorders.