Targeted investigation of tissue specific oxidative stress in the etiology of ALS

描述(由申请人提供)： 本研究的目的是确定脂质过氧化应激对肌萎缩侧索硬化症(ALS)发病机制中单个组织的影响。许多研究表明，氧化应激在肌萎缩侧索硬化症中起着重要的作用；然而，在肌萎缩侧索硬化症中重要的氧化损伤的具体类型和靶细胞尚未阐明。这些信息可能有助于为这种疾病设计急需的有效治疗干预措施。在这个方案中，我们将检验这样的假设，即过氧化脂质的增加在ALS的启动和/或进展中起着关键作用，并且参与疾病的细胞类型可能受到不同的影响。为此，我们将使用我们实验室开发的小鼠模型来操纵脂质过氧化氢的水平，这是一种携带磷脂氢谷胱甘肽过氧化物酶(Gpx4flf1小鼠)的等位基因的条件性基因敲除小鼠。Gpx4在膜保护中起着重要作用，它通过清除脂质过氧化氢来保护细胞膜。我们将通过将Gpx4flf1与携带G93AGur1 ALS突变SOD1转基因的小鼠杂交，来测量特定组织/细胞类型中脂质过氧化增加对疾病发生和发展的影响。通过在Gpx4flf1条件性基因敲除ALS突变小鼠的特定组织(如运动神经元、骨骼肌、小胶质细胞和星形胶质细胞)靶向表达Cre重组酶，我们将产生关键抗氧化酶Gpx4的组织特异性缺失，并增加目标组织/细胞类型中脂质氢过氧化诱导的氧化应激。我们将测量野生型对照小鼠、G93A1Gur转基因小鼠、Gpx4flfl小鼠x组织特异性Cre小鼠和G93A1Gur Gpx4flfl x组织特异性Cre小鼠的疾病发病(体重减轻和神经功能评分)、进展(轮状突起、运动神经元数量、肌肉神经支配丧失)和寿命。我们还将在疾病症状前、发病和结束阶段检测脊髓和腓肠肌中脂质(F2-异前列腺素，神经前列腺素)、DNA(Oxo8dG)和蛋白质(羰基，HNE加合物)的氧化损伤，以确定氧化应激增加对这些组织中疾病发生和发展的影响。我们提出的研究将增加我们对氧化应激增加的影响的理解，特别是在可能参与神经退行性疾病ALS的发生和发展的特定组织/细胞中。这种测量特定类型氧化损伤的组织特异性影响的新方法具有巨大的潜力，可以产生信息，不仅有助于设计有效的ALS治疗方法，而且还有助于其他涉及运动神经元退化的疾病的治疗。

DESCRIPTION (provided by applicant): The purpose of this study is to determine the impact of lipid peroxide induced oxidative stress on individual tissues involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). Many studies have suggested an important role for oxidative stress in ALS; however, the specific types of oxidative damage and the target cells that are important in ALS have not been elucidated. This information could be instrumental in helping to design much needed effective therapeutic interventions for this disease. In this proposal, we will test the hypothesis that an increase in lipid peroxides plays a critical role in the initiation and/or progression of ALS and that the cell types involved in the disease may be differentially affected. To do this, we will use a mouse model developed in our laboratory to manipulate the levels of lipid hydroperoxides, a conditional knockout mouse carrying floxed alleles for phospholipid hydroperoxide glutathione peroxidase (Gpx4flfl mice). Gpx4 plays a critical role in protecting membranes by removal of lipid hydroperoxides. We will measure the effect of increased lipid peroxides in specific tissues/cell types on disease initiation and progression by crossing the Gpx4flfl to mice carrying the G93AGur1 ALS mutant SOD1 transgene. Through targeted expression of Cre recombinase to specific tissues (e.g., motor neurons, skeletal muscle, microglia and astrocytes) in the Gpx4flfl conditional knockout ALS mutant mice, we will generate tissue-specific deletion of Gpx4, a key antioxidant enzyme, and increase lipid hydroperoxide induced oxidative stress in the targeted tissues/cell types. We will measure disease onset (loss of body weight and neurologic score), progression (rotarod, motor neuron numbers, loss of muscle innervation) and lifespan in wild type control mice, G93A1Gur transgenic mice, Gpx4flfl mice x tissue specific Cre mice and G93A1Gur Gpx4flfl x tissue specific Cre mice. We will also measure oxidative damage to lipid (F2-isoprostanes, neuroprostanes), DNA (oxo8dG) and protein (carbonyls, HNE adducts) in spinal cord and gastrocnemius muscle at pre-symptomatic, onset and end stages of the disease to determine the impact of increased oxidative stress on disease initiation and progression in these tissues. The studies we have proposed will increase our understanding of the effect of increased oxidative stress in general, and lipid peroxides in particular, in specific tissues/cells potentially involved in the initiation and progression of the neurodegenerative disease ALS. This novel approach to measure tissue specific effects of a specific type of oxidative damage has significant potential to generate information that will facilitate the design of effective treatments not only for ALS, but also for and other diseases involving motor neuron degeneration.