Defining the altered FUS-PARP-1-DNA Ligase III axis and its implications to nuclear and mitochondrial genome damage response in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD)

定义改变的 FUS-PARP-1-DNA 连接酶 III 轴及其对肌萎缩侧索硬化症 (ALS) 和额颞叶痴呆 (FTD) 中核和线粒体基因组损伤反应的影响

• 批准号: 9980670
• 负责人: Muralidhar L Hegde
• 金额: $ 201.88万
• 依托单位: METHODIST HOSPITAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9980670
• 关键词: ALS patients; Affect; Amyotrophic Lateral Sclerosis; Architecture; Astrocytes; Basic Science; CRISPR/Cas technology; Cells; Cessation of life; Clinical; Communication; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Ligases; DNA Ligation; DNA Repair; DNA Single Strand Break; DNA ligase III; DNA strand break; DNA-Binding Proteins; Data; Defect; Disease Progression; Double Strand Break Repair; Down-Regulation; Energy Metabolism; Etiology; Event; Familial Amyotrophic Lateral Sclerosis; Follow-Up Studies; Frontotemporal Dementia; Genes; Genome; Genome Stability; Genomic Instability; Goals; Hospitals; Human; In Vitro; Individual; Intervention; Knock-in; Lead; Ligase; Ligation; Light; Link; Maintenance; Mediating; Methodist Church; Mitochondria; Molecular; Motor Neurons; Muscular Atrophy; Mutation; NADH; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nuclear; Oxidative Stress; Oxides; Pathologic; Pathology; Pathway interactions; Patients; Physiological; Point Mutation; Proteins; Publications; RNA; Reactive Oxygen Species; Recipe; Research; Research Institute; Resources; Role; Signal Transduction; Site; Spinal Cord; Stress; Symptoms; Techniques; Testing; Tissues; Toxic effect; Transgenic Organisms; Translational Research; Untranslated RNA; XRCC1 gene; autosomal dominant mutation; brain tissue; effective therapy; follow-up; frontal lobe; frontotemporal lobar dementia-amyotrophic lateral sclerosis; genome integrity; induced pluripotent stem cell; innovation; insight; loss of function; member; mitochondrial dysfunction; mitochondrial genome; mouse model; mutant; mutation correction; nerve stem cell; neuron loss; neuroprotection; neurotoxicity; novel; oxidative damage; recruit; repaired; response; sarcoma; senescence; stress granule; targeted treatment; therapeutic target; transcriptome sequencing; translational impact

Genome damage and defective repair are etiologically linked to Fused in Sarcoma (FUS)-associated amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the underlying mechanisms remain enigmatic, which is a roadblock for exploiting genome repair-targeted therapies for ALS/FTD. Our recent publication (Wang et al, Nature Communications, 2018) identified defects in DNA nick ligation and oxidative damage repair in a subset of ALS patients, caused by mutations in the RNA/DNA-binding protein FUS. In healthy neurons, FUS protects the genome by facilitating PARP1-dependent recruitment of XRCC1/DNA Ligase IIIα (LigIII) to oxidized genome sites and activates LigIII via direct interaction. We discovered that FUS toxicity caused significantly decreased recruitment of XRCC1/LigIII to DNA strand breaks. DNA ligation defects in ALS patient-derived iPSC lines carrying FUS mutations and in subsequently generated motor neurons were rescued by CRISPR/Cas9-mediated mutation correction. Moreover, our follow-up studies showed substantially reduced auto and total PARylation activity of PARP-1 both in vitro and in cell, after loss of FUS or mutant expression, which in addition to regulating LigIII/XRCC1 recruitment at damage sites, could impact neuronal energy metabolism by uncoupling NAD+/NADH levels and stress granule dynamics in motor neurons. Collectively these events may provide a recipe for neurodegeneration. These findings that uncovered a new pathway of defective DNA ligation and PARP-1 functions in FUS-linked ALS-FTD, raised three key questions that need to be investigated to understand their implications in neuronal death and to develop a comprehensive strategy of PARylation and LigIII targeted interventions for ameliorating FUS-associated ALS-FTD. These questions are: (1) How does FUS affect PARP-1's PARylation activity and what is its impact on genome maintenance and energy metabolism? (2) What is the effect of FUS-mediated LigIII inhibition on the mitochondrial genome and its functions? This is important as LigIII is the only DNA ligase for both replication and repair in mitochondria, and both FUS and PARP-1 localize in mitochondria. (3) What is the effect of FUS pathology on microhomology-mediated alternative end-joining (MMEJ) pathway of DNA double strand break repair, which involves LigIII, XRCC1 and PARP-1?. This project, will utilize human patient-derived iPSC lines harboring FUS mutations, their isogenic controls with mutation correction by CRISPR/Cas9 knock-in strategy, a transgenic FUS-∆NLS mouse model and human ALS, FTD patient spinal cord/brain tissue, to test our novel hypothesis that FUS pathology-mediated DNA ligation defects via reduced PARylation inhibits oxidative genome damage repair and promotes neurodegeneration. We will further show that rescuing Ligase and PARP functions are promising avenues for neuroprotection. Our studies investigating the previously unexplored link between altered FUS-PARP-1-LigIII axis and ALS-FTD are both technically and conceptually innovative, have important immediate and long term goals and will strongly impact translational ALS-FTD research.

基因组损伤和修复缺陷在病因学上与融合肉瘤（FUS）相关 肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）。然而，潜在的机制 仍然是个谜，这是开发ALS/FTD基因组修复靶向疗法的障碍。我们最近 出版物（Wang et al，Nature Communications，2018）鉴定了DNA切口连接和氧化损伤中的缺陷。 ALS患者亚群中的损伤修复，由RNA/DNA结合蛋白FUS突变引起。健康 FUS通过促进PARP 1依赖的XRCC 1/DNA连接酶IIIα的募集来保护基因组 （LigIII）与氧化的基因组位点结合并通过直接相互作用激活LigIII。我们发现FUS毒性导致 显著降低了XRCC 1/LigIII对DNA链断裂的募集。在携带FUS突变的ALS患者来源的iPSC系和随后产生的运动神经元中的DNA连接缺陷通过以下方法得到拯救： CRISPR/Cas9介导的突变校正。此外，我们的后续研究显示， 以及在FUS或突变体表达丧失后，PARP-1在体外和细胞中的总PAR化活性， 除了调节损伤部位的LigIII/XRCC 1募集外，还可以通过以下方式影响神经元能量代谢： 解偶联NAD+/NADH水平和运动神经元中的应激颗粒动力学。总的来说，这些事件可能 提供了一个神经退化的处方。这些发现揭示了一种新的缺陷DNA连接途径， 和PARP-1功能，提出了三个需要研究的关键问题， 了解它们在神经元死亡中的意义，并制定一个全面的PAR化策略， 用于改善FUS相关ALS-FTD的LigIII靶向干预。这些问题是：（1）FUS如何 影响PARP-1的PAR化活性，它对基因组维持和能量代谢有什么影响？（二） FUS介导的LigIII抑制对线粒体基因组及其功能有何影响？这是 重要的是LigIII是线粒体中复制和修复的唯一DNA连接酶，FUS和 PARP-1定位于线粒体。(3)FUS病理学对微同源介导的替代性 DNA双链断裂修复的末端连接（MMEJ）途径，涉及LigIII、XRCC 1和PARP-1？。 该项目将利用携带FUS突变的人类患者来源的iPSC系，其具有FUS突变的同基因对照， 通过CRISPR/Cas9敲入策略的突变校正，转基因FUS-NLS小鼠模型和人ALS， FTD患者脊髓/脑组织，以检验我们的新假设，即FUS病理介导的DNA连接 通过减少PAR化的缺陷抑制氧化性基因组损伤修复，并促进 神经变性我们将进一步表明，拯救连接酶和PARP功能是有前途的途径 神经保护我们的研究调查了改变的FUS-PARP-1-LigIII轴和ALS-FTD之间先前未探索的联系，在技术和概念上都是创新的，具有重要的直接和长期意义。 长期目标，并将强烈影响翻译ALS-FTD研究。

项目成果

Mitochondria-Targeted Oligomeric α-Synuclein Induces TOM40 Degradation and Mitochondrial Dysfunction in Parkinson's Disease and Parkinsonism-Dementia of Guam.

线粒体靶向寡聚 α-突触核蛋白在关岛帕金森病和帕金森痴呆症中诱导 TOM40 降解和线粒体功能障碍。

• DOI: 10.21203/rs.3.rs-3970470/v1
• 发表时间: 2024
• 期刊: Research square
• 作者: Hegde,Muralidhar;Vasquez,Velmarini;Kodavati,Manohar;Mitra,Joy;Vendula,Indira;Hamilton,Dale;Garruto,Ralph;Rao,KS
• 通讯作者: Rao,KS