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

描述（由申请人提供）：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。这些病理生理学方面受到Nrf 2/ARE（NF-E2相关因子2/抗氧化反应元件）信号传导的严格调节，其损伤触发导致ALS中运动神经元死亡的有害信号传导事件。因此，Nrf 2/ARE通路被认为是重要的，并且在神经退行性疾病如ALS的神经治疗中具有很大的潜力。Nrf 2已被鉴定为协调诱导ARE驱动的神经保护基因（例如抗氧化剂和抗炎蛋白）“电池”的关键调节因子。Nrf 2系统由细胞溶质蛋白Keap 1（Kelch样ECH相关蛋白1）调节，该蛋白结合Nrf 2并阻止其易位至细胞核。Keap 1中关键半胱氨酸的氧化允许Nrf 2易位到细胞核中，在那里它激活大量编码II期解毒酶的基因的转录。我们的合成三萜类化合物在结构上进行了修饰，以提高中枢神经系统的生物利用度，并且是Nrf 2/ARE途径的有效激活剂。在ALS小鼠中口服这些Nrf 2/ARE激活剂导致Nrf 2从细胞质易位到细胞核，并上调参与抗氧化反应和线粒体生物合成的基因的表达，而促炎基因在ALS小鼠中下调。在G93 A SOD 1小鼠的初步实验中，用这些三萜类化合物治疗导致体重减轻减少，运动性能改善，最重要的是存活率显著增加。我们推测Nrf 2/ARE信号通路在ALS中受损，并且合成的三萜类化合物具有作为预防运动神经元死亡的治疗候选物的巨大潜力。提出了两个具体的目标来检验我们的假设。在目的1中，我们将检查三萜类药物CDDO（2-氰基-3，12-二氧代齐墩果-1，9-二烯-28-酸）乙酰胺和CDDO-三氟乙酰胺在ALS小鼠模型中阻断运动缺陷、神经元损失、氧化损伤和炎症的功效，其中在症状和症状前阶段期间施用三萜类。这将使我们能够验证和鉴定预防小鼠ALS样疾病的最有效的三萜类化合物。在目标2中，我们将确定由最有效的三萜类药物调节的Nrf 2/ARE下游的靶点，以确定ALS小鼠模型中精确的神经保护作用机制。揭示这些合成三萜类化合物通过激活Nrf 2/ARE信号通路调节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：本研究旨在研究Nrf 2/ARE通路在肌萎缩侧索硬化症（ALS）中的作用。长期目标是了解Nrf 2/ARE遗传程序在ALS中的作用，并确定合成三萜类化合物在ALS小鼠模型中激活运动神经元中的神经保护性Nrf 2/ARE信号通路的神经保护功效。该研究将使我们能够表征Nrf 2/ARE途径，并确定最佳合成三萜类化合物作为开发ALS有效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.