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.

抽象的肌萎缩侧索硬化症（ALS）和额颞叶变性（FTLD）是两种致命疾病目前尚无治疗方法可以预防、减缓或阻止神经元死亡的神经退行性疾病 ALS 和 FTLD 在临床上是不同的，但在死后脑病理学和遗传上表现出重叠。因素：受影响的中枢神经系统 (CNS) 中 TDP-43 的核清除和细胞质积聚 98% 的 ALS 和 50% 的 FTLD 患者可观察到该区域，而最初症状可导致诊断。无论是 ALS 还是 FTLD，高达 50% 的 ALS 患者最终会出现 FTLD 症状，其中约 15% 的患者会出现 FTLD 症状最终接受两种诊断（FTLD 伴有运动神经元疾病，FTLD/MND 基因突变）。编码 TDP-43 (TARDBP) 会导致罕见的 ALS 病例，而在患者中观察到 TDP-43 病理学携带更常见的突变，例如病理性 C9orf72 六核苷酸重复扩增 (C9orf72+)——迄今为止发现的 ALS 和 FTLD 最常见的遗传原因是 TDP-43。然而，尽管如此，它仍然是 ALS 和 FTLD 发病机制中的关键和趋同因素。运动神经元选择性脆弱的原因、TDP-43 错误定位的机制、以及核 TDP-43 排除和异常液-液相分离对神经健康的影响潜在的细胞质分层仍然未知。为了解决这一挑战，在目标 1 中，我们系统地进行了研究。以单细胞分辨率分析 ALS 和 FTLD/MND 患者的转录和表观基因组改变在目标 2 中，我们整合生成的数据集来研究之间的联系。我们将 ALS 和 FTLD/MND 的遗传、表观基因组、转录和细胞特征联系起来。利用现有的临床信息，阐明每个基因和生物学途径，并预测新的在目标 3 中，我们通过评估这些靶标的分子和细胞效应来验证它们。使用高通量定向扰动对神经活力和 TDP-43 功能/聚集的影响我们在实验中研究了这些扰动的细胞自主和非细胞自主效应。在目标 4 中，我们进行了神经病理学分析。 TDP-43修饰剂在ALS和FTLD/MND死后组织中的作用，并努力挽救体内病理学和所得数据集、分析和硬脑膜衍生的 iPSC 将提供小鼠模型中的表型。了解 ALS 和 FTLD/MND 中 TDP-43 病理机制的宝贵资源，并且可能揭示了能够通过基因操作减轻 TDP-43 病理的假定治疗靶点。