NRF2/ARE PATHWAY AS A THERAPEUTIC TARGET FOR AMYOTROPHIC LATERAL SCLEROSIS

描述(申请人提供)：氧化损伤、神经炎症和线粒体功能障碍的病理激活与肌萎缩侧索硬化症(ALS)的发病机制有关。这些病理生理方面受到Nrf2/ARE(核因子-E2相关因子2/抗氧化反应元件)信号的严格调控，Nrf2/ARE信号的受损会触发有害的信号事件，导致ALS运动神经元死亡。因此，Nrf2/ARE通路被认为是非常重要的通路，在ALS等神经退行性疾病的神经治疗中具有广阔的前景。Nrf2已被认为是协调诱导Are驱动的神经保护基因“电池”的关键调控因素，例如抗氧化和抗炎蛋白。Nrf2系统受胞浆蛋白Keap1(Kelch样ECH相关蛋白1)的调节，该蛋白与Nrf2结合并阻止其移位到细胞核。KEAP 1中一个关键的半胱氨酸的氧化允许Nrf2移位到细胞核中，在那里它激活了大量编码II期解毒酶的基因的转录。我们合成的三萜类化合物经过结构修饰，在中枢神经系统实现了更高的生物利用度，是Nrf2/ARE途径的有效激活剂。ALS小鼠口服这些Nrf2/ARE激活剂后，Nrf2从胞浆移位到胞核，参与抗氧化反应和线粒体生物发生的基因表达上调，而促炎基因表达下调。在G93A SOD1小鼠的初步实验中，这些三萜类化合物的治疗导致体重减轻，运动能力改善，最重要的是显著提高了存活率。我们推测Nrf2/ARE信号通路在ALS中受损，合成的三萜类化合物在预防运动神经元死亡方面具有很大的潜力。为了检验我们的假设，本文提出了两个具体目标。在目标1中，我们将检查三萜类药物CDDO(2-氰基-3，12-二氧代-3，12-二氧代-1，9-二烯-28-酸)乙酰胺和CDDO-三氟乙胺在ALS小鼠模型中的疗效。这将使我们能够验证和确定在预防小鼠ALS样疾病方面最有效的三萜类化合物。在目标2中，我们将确定Nrf2下游的靶点是否受到最有效的三萜类药物的调节，以努力在ALS小鼠模型中确定准确的神经保护作用机制。通过激活Nrf2/ARE信号通路来揭示这些合成的三萜类化合物所调控的神经保护机制，将使我们能够将这些化合物开发成潜在的治疗药物，以延缓ALS运动神经元的死亡。公共卫生相关性：这项研究建议研究Nrf2/ARE通路在肌萎缩侧索硬化症(ALS)中的作用。长期目标是了解Nrf2/ARE基因程序在ALS中的作用，并确定合成的三萜类化合物激活ALS小鼠模型运动神经元中神经保护性Nrf2/ARE信号通路的神经保护效果。这项研究将使我们能够表征Nrf2/ARE途径，并确定最佳的合成三萜类化合物，作为开发有效的ALS治疗策略的潜在候选药物。

DESCRIPTION (provided by applicant): Pathological activation of oxidative damage, neuroinflammation, and mitochondrial dysfunction are implicated in pathogenesis of Amyotrophic lateral sclerosis (ALS). These pathophysiolgical aspects are tightly regulated by Nrf2/ARE (NF-E2 related factor 2/antioxidant response element) signaling, the impairment of which triggers detrimental signaling events leading to motor neuron death in ALS. Due to this, the Nrf2/ARE pathway is considered important and has great promise to be explored for neurotherapeutics in neurodegenerative diseases such as ALS. The Nrf2 has been identified as a key regulatory factor in the coordinated induction of ARE driven "battery" of neuroprotective genes; e.g. antioxidant and anti-inflammatory proteins. The Nrf2 system is regulated by the cytosolic protein Keap1 (Kelch like ECH associated protein 1) that binds to Nrf2 and prevents its translocation to the nucleus. Oxidation of a critical cysteine in Keap 1 allows Nrf2 to translocate into the nucleus, where it activates transcription of a large number of genes encoding phase II detoxification enzymes. Our synthetic triterpenoids, are structurally modified to achieve increased bioavailability in central nervous system and are potent activators of Nrf2/ARE pathway. Oral administration of these Nrf2/ARE activators in ALS mice resulted in translocation of Nrf2 from cytoplasm to nucleus and upregulated expression of genes involved in antioxidant response and mitochondrial biogenesis while pro-inflammatory genes were downregulated in ALS mice. In pilot experiments in G93A SOD1 mice, treatment with these triterpenoids resulted in decrease in weight loss, improvement in motor performance and most importantly a significant increase in survival. We hypothesize that Nrf2/ARE signaling pathway is impaired in ALS and synthetic triterpenoids possess great potential as therapeutic candidates in preventing motor neuron death. Two specific aims are proposed to test our hypothesis. In aim 1, we will examine the efficacy of triterpenoid drugs CDDO (2-cyano-3, 12- dioxooleana-1,9-dien-28-oic acid) ethylamide, and CDDO-trifluoroethylamide in blocking motor deficits, neuronal loss, oxidative damage and inflammation in a mouse model of ALS when triterpenoids are administered during symptomatic and presymptomatic stages. This will enable us to validate and identify the most potent triterpenoid in preventing ALS-like disease in mice. In aim 2, we will determine the targets downstream of the Nrf2/ARE modulated by the most potent triterpenoid drug in an effort to identify the precise neuroprotective mechanism of action in a mouse model of ALS. Unraveling the neuroprotective mechanisms modulated by these synthetic triterpenoids through activation of Nrf2/ARE signaling pathway will enable us to develop these compounds into potential therapeutic drugs to retard the death of motor neurons in ALS. PUBLIC HEALTH RELEVANCE: This study proposes to examine the role of Nrf2/ARE pathway in Amyotrophic Lateral Sclerosis (ALS). The long-term goal is to understand the role of Nrf2/ARE genetic program in ALS and to determine the neuroprotective efficacy of synthetic triterpenoids that activate the neuroprotective Nrf2/ARE signaling pathway in motor neurons in the mouse model of ALS. The study will enable us to characterize Nrf2/ARE pathway and to determine the best synthetic triterpenoid as a potential drug candidate for the development of an effective therapeutic strategy for ALS.