Seeded transmission of SOD1 misfolding

SOD1 错误折叠的种子传播

基本信息

批准号：
9060410
负责人：
DAVID R BORCHELT
金额：
$ 18.75万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-05-01 至 2018-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9060410
关键词：
Address Affect Age Age-Months Amino Acids Amyotrophic Lateral Sclerosis Animals Appearance Biological Models Cell Culture Techniques Cell-Free System Cells Clinical Cultured Cells Cuprozinc Superoxide Dismutase Data Development Disease Disease Progression Escherichia coli Exhibits Familial Amyotrophic Lateral Sclerosis Follow-Up Studies Genes Grant Health Heterogeneity Indium Lead Life Link Mediating Modeling Molecular Conformation Monitor Motor Motor Neuron Disease Motor Neurons Mouse Strains Mus Mutagenesis Mutation Nervous system structure Neuraxis Paralysed Pathology Pathway interactions Patients Preparation Prion Diseases Prions Property Proteins Recombinant Proteins Recombinants Respiration Serial Passage Source Speed Spinal Spinal Cord Spinal Injections Staging Symptoms System Testing Tissues Transgenic Mice Transgenic Organisms Variant Work aged base humane endpoint illness length in vivo mouse model mutant overexpression postnatal prion-like protein misfolding superoxide dismutase 1 transmission process

项目摘要

DESCRIPTION (provided by applicant): Cu-Zn superoxide dismutase 1 (SOD1)-linked familial amyotrophic lateral sclerosis (fALS) is an extremely heterogeneous disease phenotypically with diverse clinical symptoms that can originate in upper or lower motor neurons and with a wide range of disease durations, from as short as a year to as long as 20 years. The duration of disease is largely a function of the speed with which symptoms spread along the neuraxis until motor neurons involved in respiration become affected. The question of how the disease seems to spread is one of the major unanswered questions in the study of ALS. Over the past few years, there has been increasing evidence that one mechanism by which the disease spreads may involve a prion-like propagation of a toxic misfolded protein from cell to cell along anatomically connected pathways of the CNS. To investigate this, we initiated studies and obtained tantalizing evidence that we can transmit ALS and that different ALS mutants may have "strain-like" attributes that modulate transmission. We have now been able to induce paralytic disease with aggregate SOD1 pathology in a strain of transgenic mice that express mutant SOD1 (G85R) fused to YFP. This strain of mice expresses at levels too low to induce disease on their own. Intraspinal injection of homogenates from a paralyzed G93A SOD1-overexpressing mouse transmits motor neuron disease to G85R-YFP mice in 6-11 months, and serial passage of spinal cord homogenates of these animals to naïve G85R-YFP mice produces disease in less than 3 months. This appearance of adaptation is typical of prion disease. In contrast, administration of homogenate from paralyzed G37R SOD1 mice failed to induce disease in the same recipient mice. The question this grant seeks to resolve is whether different SOD1 mutations produce conformations that produce strain-like properties that manifest as differing abilities to transmit motor neuron disease and if these abilities relate to rates of disese progression in patients. To establish whether mutations associated with slowly progressing ALS represent poorly transmissible strains of mutant SOD1, we will test 5 mutants that are found in slowly progressing SOD1-linked fALS and 5 found in rapidly progressing disease and compare these to WT SOD1 as well as negative controls. We will test both soluble and insoluble fractions of cell culture homogenates from cells transiently transfected with mutant SOD1 and recombinant hSOD1 fibrils created in a cell-free system as our inoculum, and test their potential to cause disease by direct spinal injection in G85R-YFP mice at postnatal day P0. Overall, our proposed studies could have an enormous impact in the field as they could lead to the development of an experimentally facile model of transmitted ALS and facilitate exploration of the causes for the heterogeneity of clinical symptoms observed in SOD1-linked fALS patients.

描述（由申请人提供）：铜锌超氧化物歧化酶 1 (SOD1) 相关的家族性肌萎缩侧索硬化症 (fALS) 是一种表型极其异质的疾病，具有多种临床症状，可起源于上运动神经元或下运动神经元，并且具有多种症状疾病持续时间，短至一年，长至 20 年。疾病持续时间很大程度上取决于症状沿神经轴传播的速度。参与呼吸的运动神经元受到影响，这种疾病如何传播是 ALS 研究中尚未解决的主要问题之一，越来越多的证据表明该疾病可能通过一种机制传播。涉及有毒错误折叠蛋白沿着中枢神经系统解剖学上相连的通路从一个细胞到另一个细胞的类似朊病毒的传播，为了研究这一点，我们启动了研究并获得了诱人的证据，证明我们可以传播 ALS，并且不同的 ALS 突变体可能具有这种能力。我们现在已经能够在表达与 YFP 融合的突变型 SOD1 (G85R) 的转基因小鼠品系中诱导出具有聚合 SOD1 病理学的麻痹性疾病。脊髓内注射来自瘫痪 G93A SOD1 过表达小鼠的匀浆，可将运动神经元疾病传播给 6-11 中的 G85R-YFP 小鼠。几个月内，将这些动物的脊髓匀浆连续传代给幼稚的 G85R-YFP 小鼠，会在不到 3 个月的时间内产生疾病，这种适应现象是朊病毒疾病的典型表现，相比之下，给予瘫痪的 G37R SOD1 小鼠匀浆则未能诱发疾病。这项资助试图解决的问题是，不同的 SOD1 突变是否会产生类似品系的特性，表现为传播运动神经元疾病的不同能力，以及这些能力是否与速率相关。为了确定与缓慢进展的 ALS 相关的突变是否代表了 SOD1 突变体的传播性较差，我们将测试在缓慢进展的 SOD1 相关 fALS 中发现的 5 个突变体和在快速进展的疾病中发现的 5 个突变体，并将它们与 WT 进行比较。 SOD1 以及阴性对照我们将测试来自瞬时转染突变 SOD1 和重组 hSOD1 原纤维的细胞的可溶性和不溶性部分。总体而言，我们提出的研究可能会在该领域产生巨大影响，因为它们可能会导致疾病。开发一种实验简便的传播性 ALS 模型，并有助于探索 SOD1 相关 fALS 患者中观察到的临床症状异质性的原因。