Seeded transmission of SOD1 misfolding

 描述（由申请人提供）：铜锌超氧化物歧化酶1（SOD 1）相关家族性肌萎缩性侧索硬化症（fALS）是一种表型极其异质性的疾病，具有多种临床症状，可起源于上或下运动神经元，疾病持续时间范围广泛，从短至1年到长达20年。疾病的持续时间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。疾病如何传播的问题是ALS研究中尚未解答的主要问题之一。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一种有毒的错误折叠蛋白质沿着沿着解剖学上连接的CNS通路从一个细胞到另一个细胞的朊病毒样传播。为了调查这一点，我们启动了研究，并获得了诱人的证据，我们可以传播ALS，不同的ALS突变体可能具有调节传播的“应变”属性。我们现在已经能够在表达与YFP融合的突变体SOD 1（G85 R）的转基因小鼠品系中诱导具有聚集性SOD 1病理的麻痹性疾病。这种小鼠的表达水平太低，无法自行诱发疾病。从瘫痪的G93 A SOD 1过表达小鼠的脊髓内注射匀浆在6-11个月内将运动神经元疾病传递给G85 R-YFP小鼠，并且将这些动物的脊髓匀浆连续传代给幼稚的G85 R-YFP小鼠在不到3个月内产生疾病。这种适应性的表现是朊病毒病的典型特征。相比之下，施用来自瘫痪的G37 R SOD 1小鼠的匀浆未能在相同的受体小鼠中诱导疾病。这项研究旨在解决的问题是，不同的SOD 1突变是否会产生产生菌株样性质的构象，这些性质表现为不同的传播运动神经元疾病的能力，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突变型SOD 1的传播性差的菌株，我们将测试在缓慢进展的SOD 1相关的fALS中发现的5种突变体和在快速进展的疾病中发现的5种突变体，并将这些突变体与WT SOD 1以及阴性对照进行比较。我们将测试来自用突变SOD 1瞬时转染的细胞的细胞培养物匀浆的可溶性和不溶性部分以及在无细胞系统中产生的重组hSOD 1原纤维作为我们的接种物，并在出生后第0天通过在G85 R-YFP小鼠中直接脊髓注射来测试它们引起疾病的潜力。总的来说，我们提出的研究可能会在该领域产生巨大的影响，因为它们可能导致开发一种实验简便的传播性ALS模型，并有助于探索在SOD 1相关的fALS患者中观察到的临床症状异质性的原因。

 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.