Modeling the progression of SOD1-linked motor neuron disease

在过去的5年里，人们已经清楚，许多人类神经退行性疾病的蛋白质病理学 表现出朊病毒的特征，包括传播性，应变变化，以及从一个病毒传播的能力。 引入的焦点部位。肌萎缩侧索硬化症（ALS）突出的一个例子，其中的特点， 朊病毒样扩散是明显的，因为弱点沿着沿着解剖学上连接的通路扩散。家族性ALS 由超氧化物歧化酶1（SOD 1-ALS）突变引起，患者遗传了SOD 1的A4 V变体 虚弱传播迅速（发病后平均生存期<1.5年），而在遗传了 G37 R变异体虚弱传播缓慢（平均生存期约17年）。在该奖项的最初资助期间， 我们的实验室已经发现了证据，表明SOD 1-ALS的这种定义特征可以用朊病毒样蛋白来解释。 突变体SOD 1的特性。表达低水平ALS突变体SOD 1的转基因小鼠发育 在生命的后期，如果有。我们已经证明，在这些小鼠中注射脊髓灰质炎可以加速瘫痪， 从瘫痪的突变SOD 1转基因小鼠或从人类患者制备的脊髓匀浆。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引发疾病 这一过程非常类似于从一个肢体到另一个肢体的单侧虚弱扩散， 人类我们还成功地使用纯化的重组SOD 1在体外原纤化种子早发 在宿主小鼠中的麻痹，证明SOD 1能够像朊病毒一样起作用。因为我们可以传播疾病- 导致SOD 1构象幼稚SOD 1“宿主蛋白”，从下文中，我们将称为错误折叠的 与致病突变体SOD 1相关的构象作为ALS朊病毒。在我们成功的基础上， 初步工作，我们现在提出四个具体目标，旨在提高我们的理解， 朊病毒样扩散在SOD 1-ALS发病机制中的生物学作用在目标1中，我们试图调查是否 致病突变加密了错误折叠的SOD 1中独特的菌株样特征， 动物间传播研究中朊病毒样传播的速率。在目标2中，我们试图确定路线如何 传播途径和宿主受体鼠龄影响SOD 1-ALS朊病毒的传播。在我们的第三 第四个目的，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CNS中传播以及炎症是否 信令可以影响这种扩展。在目标3中，我们将使用新产生的loxp G85 R-SOD 1：YFP小鼠， 确定星形胶质细胞在繁殖SOD 1-ALS朊病毒中的作用。在目标4中，我们将使用腺- 相关载体表达促炎和抗炎细胞因子作为评估活化的 星形胶质细胞和小胶质细胞在SOD 1-ALS朊病毒的增殖和扩散中的作用。我们的终极目标是 确定SOD 1和外部非细胞自主的内在应变属性的贡献 过程的朊病毒样传播性质的疾病引起的SOD 1构象。

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.