Seeded transmission of SOD1 misfolding

 描述(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)是一种非常不同的疾病，具有多种临床症状，可起源于上或下运动神经元，病程从一年到长达20年不等。疾病的持续时间在很大程度上取决于症状沿着神经轴传播的速度，直到参与呼吸的运动神经元受到影响。这种疾病似乎是如何传播的问题是ALS研究中尚未回答的主要问题之一。在过去的几年里，越来越多的证据表明，疾病传播的一种机制可能涉及到一种有毒的错误折叠蛋白沿着中枢神经系统的解剖连接路径在细胞之间进行类似Pron的传播。为了调查这一点，我们开始了研究，并获得了诱人的证据，证明我们可以传播ALS，并且不同的ALS突变体可能具有调节传播的类似菌株的属性。我们现在已经能够在一种表达突变的SOD1(G85R)与YFP融合的转基因小鼠中诱导出具有SOD1聚集性病理的瘫痪疾病。这种品系的小鼠的表达水平太低，本身不能诱发疾病。脊髓内注射G93A SOD1过表达的瘫痪小鼠的匀浆可在6-11个月内将运动神经元疾病传递给G85R-YFP小鼠，这些动物的脊髓匀浆连续传代给幼稚的G85R-YFP小鼠在不到3个月的时间内就会产生疾病。这种适应现象是普恩病毒病的典型表现。相比之下，来自瘫痪的G37R SOD1小鼠的匀浆未能在相同的受体小鼠中诱发疾病。这笔赠款寻求解决的问题是，不同的SOD1突变是否会产生产生菌株样特性的构象，这些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患者的疾病进展速度有关。为了确定与缓慢进展的ALS相关的突变是否代表了易传播的突变SOD1菌株，我们将测试在缓慢进展的SOD1连锁的FAL中发现的5种突变，以及在快速进展的疾病中发现的5种突变，并将这些突变与WT SOD1以及阴性对照进行比较。我们将检测瞬时转染突变SOD1和在无细胞系统中产生的重组hSOD1纤维作为接种物的细胞培养匀浆的可溶性和不溶性部分，并在出生后第P0天通过直接脊髓注射测试它们对G85R-YFP小鼠致病的可能性。总体而言，我们建议的研究可能会在该领域产生巨大影响，因为它们可能导致开发一种实验上易于传播的ALS模型，并有助于探索SOD1连锁FALS患者临床症状观察到的异质性的原因。

 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.