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

摘要 肌萎缩侧索硬化症（ALS）和额颞叶变性（FTLD）是两种致命的 神经退行性疾病，目前没有预防、减缓或阻止神经元死亡的治疗方法， 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显示出重叠。 因素：TDP-43在受累中枢神经系统（CNS）中的核清除和细胞质蓄积 在98%的ALS患者和50%的FTLD患者中观察到区域。虽然最初的症状导致诊断为 无论是ALS还是FTLD，高达50%的ALS患者最终会出现FTLD症状，约15%的患者 最终接受两种诊断（FTLD伴运动神经元疾病，FTLD/MND）。的基因突变 编码TDP-43（TARDBP）的基因导致罕见的ALS病例，而在患者中观察到TDP-43病理学 携带更普遍的突变，如病理性C9 orf 72六核苷酸重复扩增 （C9 orf 72+）-迄今为止确定的ALS和FTLD的最常见遗传原因。因此，TDP-43似乎 在ALS和FTLD的发病机制中是一个关键的和会聚的因素。尽管如此， 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排斥和异常液-液相分离对神经元健康的影响 潜在的细胞质分层仍然未知。为了应对这一挑战，在目标1中，我们系统地 以单细胞分辨率描绘ALS和FTLD/MND患者的转录和表观基因组改变 使用死后的中枢神经系统样本在目标2中，我们整合了得到的数据集，以研究 ALS和FTLD/MND的遗传、表观基因组、转录和细胞特征。我们将这些链接 利用现有的临床信息，阐明每种疾病中改变的基因和生物学途径，并预测新的 治疗目标在目标3中，我们通过评估这些靶点的分子和细胞效应， 使用高通量定向扰动对神经元活力和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主效应， 人硬脑膜成纤维细胞衍生的iPSC神经元和星形胶质细胞。在目标4中，我们进行神经病理学分析， TDP-43修饰剂在ALS和FTLD/MND死后组织中的作用，并奋进挽救体内病理学， 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将提供一个新的研究。 了解TDP-43在ALS和FTLD/MND中的病理机制的宝贵资源， 揭示了能够通过遗传操作减轻TDP-43病理的推定治疗靶点。

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.