Identification of TDP-43 modifiers through single-cell transcriptional and epigenomic dissection of ALS and FTLD-MND

通过 ALS 和 FTLD-MND 的单细胞转录和表观基因组解剖鉴定 TDP-43 修饰物

基本信息

批准号：
10494102
负责人：
Veronique Belzil
金额：
$ 166.23万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-29 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10494102
关键词：
ALS patients Address Affect Amyotrophic Lateral Sclerosis Astrocytes Autopsy Bayesian Method Biological Biological Models Brain Pathology C9ORF72 CRISPR screen Candidate Disease Gene Cell model Cells Characteristics Clinical Coculture Techniques Computational Technique DNA Data Data Set Deposition Development Diagnosis Disease Dissection Distal Dura Mater Enhancers Exclusion Exhibits Family Fibroblasts Frequencies Frontotemporal Lobar Degenerations Gene Expression Profile Gene-Modified Genes Genetic Genetic Risk Genetic Transcription Health Human Individual Induced pluripotent stem cell derived neurons Lead Light Link Liquid substance Maps Mediating Mediation Molecular Motor Motor Cortex Motor Neuron Disease Motor Neurons Mutation Nerve Degeneration Neuraxis Neurodegenerative Disorders Neurons Nuclear Nucleic Acid Regulatory Sequences Pathogenesis Pathologic Pathology Pathway interactions Patients Phase Phase Transition Phenotype Physiological Population Prefrontal Cortex Protein Isoforms RNA RNA-Binding Proteins Recording of previous events Regulatory Element Research Resolution Resources Sampling Single Nucleotide Polymorphism Symptoms Therapeutic Therapeutic Intervention Thoracic spinal cord structure Tissues Transcription Alteration Validation Variant cell type clinical phenotype differential expression epigenomics experimental study familial amyotrophic lateral sclerosis frontotemporal lobar dementia-amyotrophic lateral sclerosis genetic information genetic manipulation genome wide association study genome-wide genomic locus in vivo in vivo Model induced pluripotent stem cell insight mouse model neuron loss neuropathology neurotoxicity new therapeutic target novel pre-clinical predictive signature prevent protein TDP-43 risk variant single-cell RNA sequencing sporadic amyotrophic lateral sclerosis therapeutic target trait transcriptome

项目摘要

Abstract Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two fatal neurodegenerative conditions with no current treatment to prevent, decelerate or stop neuronal death in patients. ALS and FTLD are clinically distinct but show an overlap in postmortem brain pathology and genetic factors: nuclear clearance and cytoplasmic accumulation of TDP-43 in affected central nervous system (CNS) regions is observed in 98% of ALS and 50% of FTLD patients. While initial symptoms lead to the diagnosis of either ALS or FTLD, up to 50% of ALS patients eventually develop symptoms of FTLD, with ~15% of patients ultimately receiving both diagnoses (FTLD with motor neuron disease, FTLD/MND). Mutations in the gene encoding TDP-43 (TARDBP) lead to rare cases of ALS, while TDP-43 pathology is observed in patients carrying more prevalent mutations, such as a pathological C9orf72 hexanucleotide repeat expansion (C9orf72+)—the most common genetic cause of ALS and FTLD identified thus far. TDP-43 therefore appears to be a pivotal and convergent factor in the pathogenesis of both ALS and FTLD. Despite this, however, the reasons for selective vulnerability of motor neurons, the mechanisms responsible for TDP-43 mislocalization, and the impact on neuronal health of nuclear TDP-43 exclusion and aberrant liquid-liquid phase separation underlying cytoplasmic demixing remain unknown. To address this challenge, in Aim 1, we systematically profile the transcriptional and epigenomic alterations of ALS and FTLD/MND patients at single-cell resolution using post-mortem CNS samples. In Aim 2, we integrate the resulting datasets to study the link between genetic, epigenomic, transcriptional, and cellular signatures of ALS and FTLD/MND. We associate these links with available clinical information, elucidate the genes and biological pathways altered in each, and predict new therapeutic targets. In Aim 3, we validate the molecular and cellular effects of these targets by assessing their impact on neuronal viability and TDP-43 functions/aggregation using high-throughput directed perturbation experiments. We study both cell-autonomous and non-cell-autonomous effects of these perturbations in human dura fibroblast-derived iPSC neurons and astroglia. In Aim 4, we perform neuropathological analyses of TDP-43 modifiers in ALS and FTLD/MND postmortem tissues, and endeavor to rescue in vivo pathology and phenotypes in a mouse model. The resulting datasets, analyses, and dura-derived iPSCs will provide an invaluable resource to understand the mechanisms of TDP-43 pathology in ALS and FTLD/MND, and may reveal putative therapeutic targets able to mitigate TDP-43 pathology through genetic manipulation.

抽象的肌萎缩性侧索硬化症（ALS）和额颞叶变性（FTLD）是两个致命的神经退行性疾病没有当前治疗以预防，减速或阻止神经元死亡患者。 ALS和FTLD在临床上是不同的，但在死后脑病理学和遗传学上表现出重叠因素：TDP-43在受影响的中枢神经系统（CNS）中的核清除率和细胞质积累在98％的ALS和50％的FTLD患者中观察到区域。虽然初始症状导致诊断 ALS或FTLD，多达50％的ALS患者最终会出现FTLD症状，约有15％的患者最终接受两个诊断（患有运动神经元疾病的FTLD，FTLD/MND）。基因突变编码TDP-43（TARDBP）导致ALS的罕见病例，而患者观察到TDP-43病理携带更普遍的突变，例如病理C9ORF72六核苷酸重复扩展（C9orf72+） - 到目前为止确定的ALS和FTLD的最常见遗传原因。因此出现TDP-43 在ALS和FTLD的发病机理中成为关键和收敛因子。尽管如此，但是运动神经元选择性脆弱性的原因，导致TDP-43错误定位的机制，以及对核TDP-43排除和异常液态液相的神经元健康的影响潜在的细胞质分解仍然未知。为了应对这一挑战，在AIM 1中，我们系统地介绍了单细胞分辨率的ALS和FTLD/MND患者的转录和表观基因组改变使用后CNS样品。在AIM 2中，我们集成了结果数据集，以研究 ALS和FTLD/MND的遗传，表观基因组，转录和细胞特征。我们关联这些链接有了可用的临床信息，阐明每种基因和生物学途径都发生了变化，并预测了新的治疗靶标。在AIM 3中，我们通过评估它们的分子和细胞效应来验证它们的分子和细胞效应使用高通量的扰动对神经元的生存能力和TDP-43功能/聚集的影响实验。我们研究了这些扰动的细胞自主和非细胞自治作用人硬脑膜成纤维细胞衍生的IPSC神经元和星形胶质细胞。在AIM 4中，我们进行神经病理学分析 ALS和FTLD/MND验尸组织中的TDP-43修饰符，并努力营救体内病理学和小鼠模型中的表型。由此产生的数据集，分析和硬脑膜衍生的IPSC将提供了解ALS和FTLD/MND中TDP-43病理机制的宝贵资源，并且可能揭示假定的治疗靶标可以通过遗传操纵来减轻TDP-43病理。