Epigenetic mechanisms relevant to the pathogenesis of ALS

描述（由申请人提供）： 肌萎缩性侧索硬化症（ALS）是一种以运动神经元变性导致的进行性瘫痪为特征的致死性疾病。许多疗法已经过测试，但没有治愈或有效的治疗方法。超氧化物歧化酶-1（SOD 1）的突变是家族性ALS的原因之一，并且广泛认为氧化损伤可能是疾病发病机制的主要贡献者。尽管如此，运动神经元死亡的原因是未知的，突变体SOD 1在ALS中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-精氨酸减缓突变型SOD 1（G93 A）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，但一种可能的机制可能涉及神经保护性多胺的产生，这些多胺是LSD 1的潜在底物，LSD 1是调节蛋白质甲基化的关键酶，与FAD依赖性多胺氧化酶具有相当大的同源性。我们发现LSD 1表达抑制瞬时转染的SH-SY 5 Y神经母细胞瘤细胞的转录，表明LSD 1表达可以调节神经元的转录活性。我们还发现LSD 1酶活性在G93 A小鼠的脊髓中增加，并且LSD 1的底物二甲基化组蛋白H3 Lys 4（DMH 3 K4）的水平降低。相比之下，未被LSD 1去甲基化的三甲基化组蛋白H3 Lys 4（TMH 3 K4）的水平没有变化。因此，我们研究了LSD 1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G93 A小鼠的神经元丢失和神经胶质增生，并将LSD 1活性恢复到正常水平。为了直接测试LSD 1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染有LSD 1 shRNA的NSC-34运动神经元细胞，发现LSD 1表达减少与对氧化损伤的抗性相关，进一步支持G93 A小鼠中氧化损伤、LSD 1表达和神经变性之间的可能联系。基于这些观察结果，我们假设LSD 1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中运动神经元变性。因此，减少LSD 1表达的干预措施可能会保护G93 A小鼠的神经元免于变性。在目前的建议中，我们计划在G93 A小鼠中研究LSD 1表达、蛋白质甲基化和神经变性之间的关系。我们还将测试LSD 1抑制剂对G93 A小鼠的行为和神经病理表型的影响。本研究的具体目的是：1）确定LSD 1在运动神经元中的诱导与体外氧化应激介导的细胞死亡之间的关系：2）确定LSD 1在携带G93 A SOD 1突变的运动神经元存活中的作用;和3）研究LSD 1调节剂对运动神经元变性、神经病理学、临床进展和G93 A小鼠的存活率。将使用各种补充方法来实现我们的目标。我们将确定LSD 1在氧化损伤和G93 A介导的神经毒性中的重要性，通过在NSC-34细胞和培养的原代运动神经元中用shRNA瞬时敲低LSD 1表达，以及在用泰特诱导的LSD 1或shRNA表达系统稳定转染以抑制LSD 1翻译的NSC-34细胞中瞬时敲低LSD 1表达。我们将使用实时PCR、Western印迹、免疫荧光染色和使用荧光素酶测定评估转录活性以及使用特异性抗体评估组蛋白H3 K4的甲基化来测量LSD 1基因表达和活性水平。我们将确定亚精胺和反苯环丙胺（一种LSD 1抑制剂）对G93 A小鼠LSD 1活性和DMH 3 K4水平以及抗氧化基因表达的影响。此外，我们将研究这些药物对G93 A小鼠的神经病理学、行为学和存活率的影响。我们希望我们的研究将为开发更好的治疗方法奠定基础，这种致命的疾病对部署到波斯湾地区的退伍军人造成了不成比例的影响。 公共卫生相关性： 肌萎缩侧索硬化症（ALS或Lou Gehrig病）是一种无法治愈的致命性进行性神经系统疾病，其特征在于运动系统的变性。海湾战争退伍军人可能有ALS的风险增加相比，谁没有在海湾服役的退伍军人。这种风险增加的原因尚不清楚，但据认为是由于暴露于环境中的神经毒素。我们提出的研究旨在更好地了解ALS小鼠模型中运动神经元变性的原因。这种转基因小鼠是通过将导致ALS的异常人类基因添加到小鼠胚胎中而产生的。这些小鼠发展为进行性虚弱，并在约4个月大时过早死亡。我们希望我们对运动神经元如何退化的研究将为开发更好的治疗这种不可逆的、进行性的和致命的疾病奠定基础。前景表明，未来数十万退伍军人患这种毁灭性疾病的风险可能会增加，这突出了对此类研究的迫切需要。

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. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS or Lou Gehrig disease) is an untreatable fatal progressive neurological disease characterized by degeneration of the motor system. Gulf War veterans may have an increased risk of ALS compared to veterans who did not serve in the Gulf. The reasons for this increased risk are not known but it is thought to be due to exposure to a neurotoxin in the environment. The studies we propose are designed to better understand the causes of motor neuron degeneration in a well-characterized mouse model of ALS. This transgenic mouse is produced by adding the abnormal human gene that causes ALS to the mouse embryo. These mice develop progressive weakness and die prematurely at about 4 months of age. We expect that our studies on how motor neurons degenerate will lay the foundation for the development of better treatments for this irreversible progressive and fatal condition. The urgent need for such studies is highlighted by the prospect that hundreds of thousands of future veterans may be at increased risk for this devastating disease.