Modeling the progression of SOD1-linked motor neuron disease

在过去的5年里，许多人类神经退行性疾病的蛋白质病理学已经变得清晰起来。 展示了普恩病毒的特征，包括传递性、菌株变异和从 介绍的重点地点。肌萎缩侧索硬化症(ALS)是一个突出的例子，它的特征是 当虚弱沿着解剖学上连接的路径扩散时，Pron样的扩散是明显的。家族性肌萎缩侧索硬化 由超氧化物歧化酶1(SOD1-ALS)突变引起，遗传SOD1的A4V变体的患者 虚弱迅速蔓延(发病后平均生存期为1.5年)，而在遗传的患者中 G37R变异弱扩散缓慢(平均生存期17年)。在本奖项最初的资助期内， 我们实验室发现的证据表明，SOD1-ALS的这一决定性特征可以用类普里子来解释 突变体SOD1的特性。表达低水平ALS突变SOD1的转基因小鼠出现 晚年的疾病，如果有的话。我们已经证明，通过注射脊髓可以加速这些小鼠的瘫痪 从瘫痪的突变SOD1转基因小鼠或从人类患者制备的脐带匀浆。我们有 还表明，我们可以将这些匀浆注射到脆弱小鼠的坐骨神经中，从而引发一种疾病 一种类似于虚弱从一条肢体向另一条肢体单侧扩散的过程 人类。我们还成功地使用纯化的重组SOD1在体外成纤维细胞来播种早期发病 在宿主小鼠中出现瘫痪，证明SOD1能够像普恩病毒一样发挥作用。因为我们可以传播疾病- 导致SOD1的构象为幼稚的SOD1“宿主蛋白”，此后我们将称为错误折叠 与致病突变体SOD1相关联的构象，作为肌萎缩侧索硬化症的Prion。以我们的成功为基础 在最初的工作中，我们现在提出四个具体目标，旨在提高我们对 朊病毒样扩散在SOD1-ALS发病机制中的生物学作用在目标1中，我们试图调查 致病突变加密了错误折叠的SOD1中独特的毒株样特征，影响了 动物间传播研究中类病毒传播的速率。在目标2中，我们试图确定路线如何 宿主小鼠的传播和年龄影响SOD1-ALS蛋白的繁殖。在我们的第三个 第四个目标，使用我们的新模型系统，我们将把注意力转向确定外在的 决定错误折叠的蛋白质构象如何在中枢神经系统扩散以及是否具有炎症性的因素 信号可能会影响这种传播。在目标3中，我们将使用新产生的loxP G85R-SOD1：YFP小鼠 确定星形胶质细胞在繁殖SOD1-ALS普恩病毒中的作用。在目标4中，我们将使用腺- 联合载体表达促炎和抗炎细胞因子作为评估活化作用的手段 星形胶质细胞和小胶质细胞在SOD-ALS蛋白的繁殖和扩散中的作用。我们的首要目标是 确定SOD1和外源性非细胞自主的内在应变样属性的贡献 致病SOD1型构象的类病毒传播特性的过程。

In the past 5 years, it has become clear that the protein pathology of many human neurodegenerative diseases exhibits characteristics of prions, including transmissibility, strain variation, and the ability to spread from a focal site of introduction. Amyotrophic lateral sclerosis (ALS) stands out as an example where the hallmarks of prion-like spreading is evident as weakness spreads along anatomically connected pathways. In familial ALS caused by mutations in superoxide dismutase 1 (SOD1-ALS), patients inheriting the A4V variant of SOD1 weakness spreads rapidly (average survival <1.5 years after the onset), whereas in patients inheriting the G37R variant weakness spreads slowly (average survival ~17 years). In the initial funding period of this award, our laboratory has uncovered evidence that this defining feature of SOD1-ALS may be explained by prion-like characteristics of mutant SOD1. Transgenic mice that express low levels of ALS mutant SOD1 develop disease late in life if at all. We have shown that paralysis can be accelerated in these mice by injecting spinal cord homogenates prepared from paralyzed mutant SOD1 transgenic mice or from human patients. We have also shown that we can inject these homogenates into the sciatic nerve of vulnerable mice to initiate a disease process that closely mimics the unilateral spread of weakness from one limb to another limb that is seen in humans. We have also successfully used purified recombinant SOD1 fibrilized in vitro to seed early onset paralysis in host mice, proving that SOD1 is capable of acting like a prion. Because we can propagate disease- causing conformations of SOD1 to naïve SOD1 “host proteins”, from hereafter we will refer to the misfolded conformation associated with disease-causing mutant SOD1 as an ALS prion. Building on the success of our initial work, we now propose four Specific Aims that are designed to improve our understanding of the biological role of prion-like spread in the pathogenesis of SOD1-ALS. In Aim 1, we seek to investigate whether the disease-causing mutations encrypt unique strain-like characteristics in misfolded SOD1 that influences the rate of prion-like spread in animal to animal transmission studies. In Aim 2, we seek to determine how the route of transmission and age of the host recipient mouse influence the propagation of SOD1-ALS prions. In our third and fourth Aims, using our novel model system we will turn our attention towards determining the extrinsic factors that determine how misfolded protein conformations may spread in the CNS and whether inflammatory signaling may influence such spread. In Aim 3, we will use newly generated loxp G85R-SOD1:YFP mice to determine the contribution of astrocytes in propagating SOD1-ALS prions. In Aim 4, we will use adeno- associated vectors to express pro- and anti-inflammatory cytokines as a means to assess the role of activated astrocytes and microglia in the propagation and spreading of SOD1-ALS prions. Our over-arching goal is to determine the contribution of intrinsic strain-like attributes in SOD1 and extrinsic non-cell autonomous processes to the prion-like propagation properties of disease-causing SOD1 conformations.