Transcriptome analyses of the nervous system of Familial Dysautonomia-model mice identify novel cellular pathways dependent on Ikbkap/Elp1 (CNS)

Familial Dysautonomia (FD; OMIM #223900) is both a developmental and progressive autosomal recessive neurodegenerative disorder that results from a nervous-system reduction in the IKAP/ELP1 protein due to a mutation in a splice acceptor site of the IKBKAP/ELP1 gene. The function of this gene in the nervous system is unresolved. To obviate the embryonic lethality of mice completely null for Ikbkap, we generated conditional knock out (CKO) mouse models for FD that recapitulate hallmarks of the human disease. To derive insight into potential intracellular functions for Ikbkap, we conducted a genome-wide transcriptome analysis of both the peripheral and central nervous systems from Ikbkap CKO mice, and identify over 100 shared misregulated genes that reveal roles for IKAP in several metabolic and signaling pathways in addition to synaptic transmission. Importantly, our data are the first to demonstrate that in the absence of IKAP, neurons undergo intracellular stress that is marked by transcriptional elevations in ATF5, p53, and several CREB target genes, as well as an increase in reactive oxygen species. These data will aid in the identification of common upstream and downstream targets for therapeutics for preventing the progressive demise of neurons in FD and potentially other neuropathies. Examining the major genetic pathways that require IKAP are hampered by that Ikbkap null mice are embryonic lethal (E10), but can circumvented by genome-wide transcriptome analysis in the brains in adult Tuba1a-Cre;IkbkapLoxP/LoxP mice, which live on average 6 months. These lines were obtained by crossing mice homozygous for a floxed allele of Ikbkap (exon 4) mice to mice that are heterozygous for this floxed allele, and a Tuba1a-Cre transgene. For this study, brain tissue (cerebellum and brainstem) was removed from 2-5 month old adult Tuba1a-cre;IkbkapLoxP/LoxP mice and stored at -80 until RNA was isolated. Four wildtype and four conditional knock-out animals were analyzed for this experiment.家族性自主神经功能障碍（Familial Dysautonomia，FD；在线人类孟德尔遗传数据库（Online Mendelian Inheritance in Man，OMIM）编号#223900）是一种兼具发育性与进展性的常染色体隐性遗传神经退行性疾病，其致病机制为IKBKAP/ELP1基因的剪接接受位点发生突变，导致神经系统中IKAP/ELP1蛋白表达水平降低。该基因在神经系统内的具体功能尚未阐明。为解决完全敲除Ikbkap的小鼠会出现胚胎致死的问题，我们构建了可重现人类FD疾病特征的条件性敲除（conditional knock out，CKO）小鼠模型。为深入探究Ikbkap潜在的细胞内功能，我们对Ikbkap CKO小鼠的外周与中枢神经系统开展了全基因组转录组分析，鉴定出超过100个共同失调的基因，揭示了IKAP除参与突触传递外，还在多条代谢与信号通路中发挥重要作用。尤为重要的是，本研究数据首次证实，在IKAP缺失的神经元中会出现细胞内应激反应，具体表现为ATF5、p53及多个CREB靶基因的转录水平上调，同时活性氧水平升高。上述研究数据将有助于识别可用于干预FD及其他潜在神经病变中神经元进行性死亡的共同上下游治疗靶点。此前，由于Ikbkap全敲除小鼠会在胚胎期（E10）出现致死，阻碍了对依赖IKAP的主要遗传通路的研究，但该限制可通过对成年Tuba1a-Cre;IkbkapLoxP/LoxP小鼠的脑组织开展全基因组转录组分析得以规避，该类小鼠的平均存活时长可达6个月。该模型小鼠通过将Ikbkap第4外显子floxed等位基因纯合小鼠与携带该floxed等位基因杂合子及Tuba1a-Cre转基因的小鼠杂交获得。本实验选取2-5月龄的成年Tuba1a-cre;IkbkapLoxP/LoxP小鼠，提取其脑组织（小脑与脑干）并于-80℃保存直至RNA提取完成。本实验共分析了4只野生型小鼠与4只条件性敲除小鼠。