Epigenetic mechanisms relevant to the pathogenesis of ALS

描述(由申请人提供)： 肌萎缩侧索硬化症(ALS)是一种以运动神经元变性引起的进行性瘫痪为特征的致命性疾病。已经测试了许多治疗方法，但没有治愈或有效的治疗方法。超氧化物歧化酶-1(SOD1)基因突变是家族性肌萎缩侧索硬化症的病因之一，人们普遍认为氧化损伤可能是疾病发病的主要因素。尽管如此，运动神经元死亡的原因尚不清楚，突变的SOD1在肌萎缩侧索硬化症氧化应激产生中的作用尚未确定。我们最近报道了氨基酸L-精氨酸延缓了突变型SOD1G93A转基因ALS小鼠的疾病进展。这种神经保护作用的基础尚不清楚，但一个可能的机制可能涉及神经保护性多胺的产生，这些多胺是LSD1的潜在底物，LSD1是调节蛋白质甲基化的关键酶，与FAD依赖的多胺氧化酶有相当大的同源性。我们发现LSD1的表达抑制了瞬时转染的SH-SY5Y神经母细胞瘤细胞的转录，提示LSD1的表达可以调节神经元的转录活性。我们还发现，LSD1酶的活性在G93A小鼠的脊髓中增加，而作为LSD1底物的二甲基化组蛋白H3Lys4(DMH3K4)的水平降低。相反，未被LSD1去甲基化的三甲基化组蛋白H3Lys4(TMH3K4)的水平没有变化。因此，我们研究了LSD1的表达与多胺、亚精胺的神经保护作用之间的关系。我们发现，亚精胺治疗减少了G93A小鼠的神经元丢失和胶质细胞增生，并将LSD1活性恢复到正常水平。为了直接测试LSD1在神经元氧化损伤发病机制中的作用，我们用过氧化氢处理转LSD1 shRNA的NSC-34运动神经元细胞，发现LSD1表达降低与G93A小鼠对氧化损伤的抵抗有关，进一步支持了氧化损伤、LSD1表达与神经变性之间的可能联系。基于这些观察，我们推测LSD1表达增加可能通过改变组蛋白和/或非组蛋白甲基化而导致ALS运动神经元变性。因此，减少LSD1表达的干预措施可能会保护G93A小鼠的神经元免受退化。在目前的方案中，我们计划研究G93A小鼠LSD1表达、蛋白质甲基化和神经退行性变之间的关系。我们还将测试LSD1抑制剂对G93A小鼠行为和神经病理表型的影响。本研究的具体目的是：1)确定体外氧化应激诱导运动神经元LSD1表达与细胞死亡的关系；2)确定LSD1在携带G93A SOD1突变的运动神经元存活中的作用；3)探讨LSD1调节剂对G93A小鼠运动神经元变性、神经病理、临床进展和存活的治疗作用。我们将使用多种互补的方法来实现我们的目标。我们将通过在NSC-34细胞和培养的原代运动神经元中用shRNA瞬时下调LSD1的表达，以及在稳定转染Tet诱导的LSD1或shRNA表达系统以抑制LSD1翻译的NSC-34细胞中，确定LSD1在氧化损伤和G93A介导的神经毒性中的重要性。我们将使用Real-Time PCR、Western blotting、免疫荧光染色以及使用荧光素酶实验评估转录活性和使用特定抗体评估组蛋白H3K4的甲基化来测量LSD1基因的表达和活性水平。我们将确定亚精胺和三羟环丙胺(一种LSD1抑制剂)对G93A小鼠LSD1活性、DMH3K4水平和抗氧化基因表达的影响。此外，我们还将研究这些药物对G93A小鼠神经病理、行为和存活的影响。我们希望，我们的研究将为开发更好的治疗这种致命疾病奠定基础，这种致命疾病对已被部署到波斯湾地区的退伍军人造成不成比例的影响。

DESCRIPTION (provided by applicant): Amyotrophic Lateral Sclerosis (ALS) is a fatal disease characterized by progressive paralysis due to motor neuron degeneration. Many therapies have been tested but no cure or effective therapy is available. Mutations of superoxide dismutase-1 (SOD1) are a cause of familial ALS and it is widely held that oxidative injury is likely to be a major contributor to disease pathogenesis. Despite this, the cause of motor neuron death is unknown and the role of mutant SOD1 in the generation of oxidative stress in ALS has not been established. We recently reported that the amino acid L-arginine slows disease progression in mutant SOD1 (G93A) transgenic ALS mice. The basis for this neuroprotective effect is not known but one possible mechanism may involve the production of neuroprotective polyamines that are potential substrates for LSD1, a key enzyme regulating protein methylation that shares considerable homology with FAD-dependent polyamine oxidases. We found that LSD1 expression suppresses transcription in transiently transfected SH-SY5Y neuroblastoma cells suggesting that LSD1 expression can regulate transcriptional activity in neurons. We also found that LSD1 enzyme activity is increased the spinal cords of G93A mice and that levels of dimethylated histone H3 Lys4 (DMH3K4), a substrate of LSD1, were reduced. In contrast, levels of trimethylated histone H3 Lys4 (TMH3K4), which is not demethylated by LSD1, were unchanged. Because of this, we examined the relationship between the expression of LSD1 and the neuroprotective effects of the polyamine, spermidine. We found that spermidine treatment reduced neuronal loss and gliosis in G93A mice and restored LSD1 activity to normal levels. To directly test the role of LSD1 in the pathogenesis of oxidative injury in neurons, we treated NSC-34 motor neuronal cells transfected with LSD1 shRNA with hydrogen peroxide and found that reduced LSD1 expression was associated with resistance to oxidative injury further supporting a possible link between oxidative injury, LSD1 expression and neurodegeneration in G93A mice. Based on these observations we hypothesize that increased LSD1 expression may contribute to motor neuron degeneration in ALS by altering histone and/or non-histone protein methylation. Interventions that reduce LSD1 expression may therefore protect neurons from degeneration in G93A mice. In the current proposal we plan to study the relationship between LSD1 expression, protein methylation and neurodegeneration in the G93A mice. We will also test the effects of LSD1 inhibitors on the behavioral and neuropathological phenotype of G93A mice. The specific aims of the study are: 1) To determine the relationship between LSD1 induction in motor neurons and cell death mediated by oxidative stress in vitro; 2) To determine the role of LSD1 in the survival of motor neurons harboring the G93A SOD1 mutation; and 3) To investigate the therapeutic effect of LSD1 modulators on motor neuron degeneration, neuropathology, clinical progression, and the survival of G93A mice. A variety of complementary methods will be used to accomplish our aims. We will determine the importance of LSD1 in oxidative injury and G93A-mediated neurotoxicity by transiently knocking down LSD1 expression with shRNA in NSC-34 cells and in cultured primary motor neurons and in NSC-34 cells stably transfected with either Tet- inducible LSD1 or a shRNA expression system to suppress LSD1 translation. We will measure LSD1 gene expression and activity levels using Real-Time PCR, Western blotting, immunofluorescent staining and by assessing transcriptional activity using a luciferase assay and the methylation of histone H3K4 using specific antibodies. We will determine the effect of spermidine and tranylcypromine, an LSD1 inhibitor on LSD1 activity and DMH3K4 levels and anti-oxidant gene expression in G93A mice. In addition, we will investigate the effects of these drugs on neuropathology, behavior and survival of G93A mice. We hope that our studies will lay the foundation for the development of better treatments for this fatal condition that disproportionately affects veterans who have been deployed to the Persian Gulf region.