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UPTAKE, TRANSPORT, AND SPREAD OF PRIONS

朊病毒的摄取、运输和传播

基本信息

批准号：
7894842
负责人：
DAVID A HARRIS
金额：
$ 40.63万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-15 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7894842
关键词：
Animals Antibodies Applications Grants Area Astrocytes Axon Axonal Transport Biological Assay Blood Bovine Spongiform Encephalopathy Brain Cattle Cells Cellular biology Chimeric Proteins Chronic Wasting Disease Creutzfeldt-Jakob Syndrome Deer Dendrites Deposition Detection Etiology Exposure to Fluorescence Microscopy Food Safety Genetic Glycolipids Human Image Imagery Infection Infectious Agent Kuru Label Life Ligand Binding Light Lymphoid Mediating Membrane Glycoproteins Methods Microscopic Molecular Monitor Morphologic artifacts Motor Movement Mus Mutation Nerve Nerve Endings Neuraxis Neurodegenerative Disorders Neurons New Guinea Nucleic Acids Oral Organ Organelles Pathway interactions Peripheral Peripheral Nerves Pharmaceutical Preparations PrP PrP gene PrPSc Proteins Prion Diseases Prions Process Protein Isoforms Proteins Public Health Purkinje Cells Research Role Route Scrapie Sheep Site Slice Somatotropin Source Spinal Cord Staining method Stains Testing Therapeutic Time Tissues Transgenic Mice Travel Tribes Variant cell type design enhanced green fluorescent protein extracellular granule cell improved insight interest intestinal epithelium novel prevent promoter prophylactic receptor research study sciatic nerve selective expression trafficking transmission process uptake wild-type PrP

项目摘要

Prion diseases are fatal neurodegenerative disorders of humans and animals. They result from conversion of PrPC, a normal membrane glycoprotein into PrPSc, a conformationally altered isoform that is infectious in the absence of nucleic acid. Most exogenously acquired prion diseases arise by exposure to the infectious agent outside of the central nervous system (CNS). For this reason, a major focus of research in the field has been to understand how prions gain access to the CNS from the periphery, and how they spread within the spinalcord and brain. To facilitate studies of prion trafficking, we propose to utilize PrP molecules fused to enhanced green fluorescent protein (EGFP). Such PrP-EGFP fusion proteins make it possible to analyze the cellular trafficking of PrPSc in living cells in real time, and to avoid the artifacts associated with conventional immunocytochemical detection of PrPSc. We previously created Tg(PrP-EGFP) mice in which EGFP was inserted adjacent to the glycolipid attachment site of PrP. This form of PrP-EGFP serves a highly specific ligand that binds to and labels intracellular and extracellular deposits of PrPSc in prion-infected animals. However, this particular fusion protein is not itself converted into PrPSc-EGFP, and so is subject to several experimental limitations. In this two-year project, we propose to design improved PrP-EFGP constructs that can be converted efficiently into PrPSc-EGFP. We will then use these fluorescent proteins to visualize the transport of PrPSc along the axons of living neurons to determine whether this movement is an intra-axonal, motor-driven process, or occurs via a "domino mechanism" on the axolemma. We expect that the proposed experiments will provide important insights into how prions spread along nerves,and suggest how this process can be manipulated as a therapeutic or prophylactic strategy.

朊病毒病是人类和动物的致命性神经退行性疾病。它们是由PrPC（一种正常的膜糖蛋白）转化为PrPSc（一种构象改变的同种型，没有核酸。大多数外源性获得性朊病毒疾病是由于暴露于中枢神经系统（CNS）以外的感染因子而引起的。由于这个原因，该领域研究的一个主要焦点是了解朊病毒如何从外周进入中枢神经系统，以及它们如何在脊髓和大脑中传播。为了促进朊病毒运输的研究，我们建议利用PrP分子融合增强型绿色荧光蛋白（EGFP）。这种PrP-EGFP融合蛋白使得能够真实的分析活细胞中PrPSc的细胞运输，并避免与常规免疫细胞化学相关的伪影 PrPSc的检测。我们先前创建了Tg（PrP-EGFP）小鼠，其中EGFP插入邻近PrP的糖脂附着位点。这种形式的PrP-EGFP作为高度特异性的配体，结合并标记朊病毒感染动物中PrPSc的细胞内和细胞外沉积物。然而，这种特定的融合蛋白本身不会转化为PrPSc-EGFP，因此受到几个实验限制。在这个为期两年的项目中，我们建议设计改进的PrP-EFGP结构，可以有效地转化为PrPSc-EGFP。然后，我们将使用这些荧光蛋白来观察PrPSc沿着活神经元的轴突，以确定这种运动是否是轴突内的，马达驱动的过程，或通过轴膜上的“多米诺机制”发生。我们期望所提出的实验将提供重要的见解朊病毒如何沿沿着神经传播，并建议如何操纵这一过程作为一种治疗或预防策略。