Epigenetic mechanisms relevant to the pathogenesis of ALS

ALS发病机制相关的表观遗传机制

• 批准号: 8043487
• 负责人: Neil W. Kowall
• 金额: --
• 依托单位: VA BOSTON HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8043487
• 关键词: Affect; Age-Months; Amino Acids; Amyotrophic Lateral Sclerosis; Antibodies; Antioxidants; Arginine; Behavior; Behavioral; Biological Assay; Cell Death; Cell Line; Cells; Cessation of life; Clinical; Data; Development; Disease; Disease Progression; Embryo; Environment; Enzymes; Epigenetic Process; Exposure to; Familial Amyotrophic Lateral Sclerosis; Foundations; Free Radical Scavenging; Future; Gene Expression; Generations; Genes; Genetic; Genetic Transcription; Gliosis; Gulf War; Histone H3; Histones; Human; Hydrogen Peroxide; In Vitro; Injury; Intervention; Link; Luciferases; Measures; Mediating; Methods; Methylation; Motor; Motor Neurons; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Neurotoxins; Oxidative Stress; Paralysed; Parnate; Pathogenesis; Pathway interactions; Persian Gulf; Pharmaceutical Preparations; Phenotype; Polyamines; Process; Production; Reporting; Research; Resistance; Risk; Role; Spermidine; Spinal Cord; Staining method; Stains; System; Techniques; Testing; Tetanus Helper Peptide; Therapeutic; Therapeutic Effect; Time; Transgenic Mice; Transgenic Organisms; Translations; Tranylcypromine; Veterans; Western Blotting; base; design; effective therapy; enzyme activity; follow-up; inhibitor/antagonist; knock-down; motor neuron degeneration; mouse model; mutant; nervous system disorder; neuroblastoma cell; neuron loss; neuropathology; neurotoxicity; novel; polyamine oxidase; research study; small hairpin RNA; superoxide dismutase 1

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.

描述（由申请人提供）： 肌萎缩性侧索硬化症（ALS）是一种致命疾病，其特征是由于运动神经元变性引起的进行性瘫痪。已经测试了许多疗法，但没有治愈或有效的治疗方法。超氧化物歧化酶1（SOD1）的突变是家族性ALS的原因，并且被广泛认为氧化损伤可能是导致疾病发病机理的主要因素。尽管如此，尚未确定运动​​神经元死亡的原因尚不清楚，突变体SOD1在ALS中产生氧化应激中的作用尚未确定。我们最近报道，氨基酸L-精氨酸在突变SOD1（G93A）转基因ALS小鼠中减缓疾病进展。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多胺是LSD1潜在底物的神经保护多胺，这是一种调节蛋白甲基化的关键酶，与FAD依赖性的多胺氧化酶具有相当多的同源性。我们发现，LSD1表达抑制了瞬时转染的SY5Y神经母细胞瘤细胞中的转录，这表明LSD1表达可以调节神经元中的转录活性。我们还发现，LSD1酶活性增加了G93A小鼠的脊髓，并降低了LSD1的二甲基化组蛋白H3 Lys4（DMH3K4）的水平。相反，未经LSD1脱甲基化的三甲基化组蛋白H3 Lys4（TMH3K4）的水平不变。因此，我们检查了LSD1表达与多胺精子的神经保护作用之间的关系。我们发现，精子治疗减少了G93A小鼠的神经元丧失和神经胶质病，并将LSD1活性恢复到正常水平。为了直接测试LSD1在神经元中氧化损伤的发病机理中的作用，我们处理了用过氧化氢转染的NSC-34运动神经元细胞，并发现LSD1表达降低与氧化损伤的抗性相关，从而进一步支撑了LSD1表达和神经化学的氧化性损伤之间的可能联系，从而进一步支撑了氧化性损伤。基于这些观察结果，我们假设增加的LSD1表达可能通过改变组蛋白和/或非固定蛋白蛋白甲基化而导致ALS运动神经元变性。因此，降低LSD1表达的干预措施可以保护神经元免受G93A小鼠的变性。在当前的建议中，我们计划研究G93A小鼠中LSD1表达，蛋白甲基化和神经退行性的关系。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。该研究的具体目的是：1）确定运动神经元中LSD1诱导与体外氧化应激介导的细胞死亡之间的关系； 2）确定LSD1在具有G93A SOD1突变的运动神经元存活中的作用； 3）研究LSD1调节剂对运动神经元变性，神经病理学，临床进展和G93A小鼠存活的治疗作用。将使用各种补充方法来实现我们的目标。我们将通过瞬时在NSC-34细胞和培养的原发性运动神经元中使用SHRNA击倒LSD1表达以及在NSC-34细胞中稳定转染的NSC-34细胞中的LSD1介导的神经毒性的重要性，并用TET-TET-TET-TET-TET-诱导的LSD1或SHRNA表达系统稳定转染以替代LSD1 trransels LSD1。我们将使用实时PCR，Western印迹，免疫荧光染色以及使用荧光素酶测定法和使用特定抗体对组蛋白H3K4的甲基化评估转录活性，以测量LSD1基因表达和活性水平。我们将确定精子定和跨环肽，LSD1抑制剂对G93A小鼠中LSD1活性和DMH3K4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G93A小鼠神经病理学，行为和存活的影响。我们希望我们的研究将为这种致命状况的更好治疗奠定基础，这种致命状况不成比例地影响已部署到波斯湾地区的退伍军人。 公共卫生相关性： 肌萎缩性侧索硬化症（ALS或Lou Gehrig病）是一种不可治疗的致命性神经系统疾病，其特征是运动系统的变性。与不在海湾任职的退伍军人相比，海湾战争退伍军人的风险可能会增加。尚不清楚这种增加风险的原因，但被认为是由于环境中暴露于神经毒素的原因。我们提出的研究旨在更好地了解ALS的特征良好的小鼠模型中运动神经元变性的原因。该转基因小鼠是通过添加异常的人类基因来产生的，该基因将ALS引起小鼠胚胎。这些小鼠在大约4个月大的时候就会出现渐进的弱点并过早死亡。我们预计，我们对运动神经元退化如何为这种不可逆转的渐进性和致命状况的更好治疗奠定基础。对这种研究的迫切需求是由成千上万的未来退伍军人可能会增加这种毁灭性疾病的风险的前景。