Abnormal interneuron development in a new humanized mouse model of Arx dup24 which recapitulates the patients’ behavioural and fine motor alterations. Mus musculus

The Aristaless-related homeobox (ARX) gene is a transcription factor involved in the development of GABAergic and cholinergic neurons in the forebrain. ARX mutations have been associated with a wide spectrum of neurodevelopemental disorders in humans, among which the most frequent, the 24 bp duplication in the protein polyalanine tract 2 (c.428_451dup24), gives rise to intellectual disability (ID), fine motor defects with or without epilepsy. To understand the physiological and functional consequences of this mutation, we generated and characterized a humanized mouse model carrying the c.428_451dup24 duplication (Arxdup24/0). Arxdup24/0 males presented with hyperactivity, enhanced stereotypies and altered contextual fear memory. Specific fine motor skills were also affected in Arxdup24/0 males, with reaching and grasping abilities alteration and fine motor coordination and balance defect. Transcriptome analysis of Arxdup24/0 forebrains at E15.5 showed a down-regulation of genes specifically expressed in interneurons associated with an up-regulation of interneuron silenced genes, suggesting abnormal interneuron development. Accordingly, interneuron migration and development were altered particularly in the cortex and the striatum between stages E15.5, P0 and adult. We also revealed a perturbation of the inhibitory/excitatory balance in Arxdup24/0 basolateral amygdala. Altogether, we showed that the c.428_451dup24 mutation modifies Arx function with a direct consequence on interneuron development, leading to hyperactivity and defects in precise motor movement control and in associative memory. Finally, we presented a new review of the clinical features of 33 male patients with ARX c.428_451dup24 mutation and showed striking similarities between their clinical features and the mouse phenotype. Overall design: Total of 6 sequenced samples. RNA-seq (3 replicates) for ARX wt/0 and ARX dup24/0 in embryonic forebrain E15.5.

Aristaless相关同源框（ARX）基因是一类转录因子，参与前脑γ-氨基丁酸能（GABAergic）神经元与胆碱能（cholinergic）神经元的发育。ARX突变与人类多种神经发育障碍谱系相关，其中最为常见的是蛋白聚丙氨酸束2区域的24 bp重复突变（c.428_451dup24），该突变可引发智力障碍（ID）以及伴或不伴癫痫的精细运动缺陷。为阐明该突变的生理与功能效应，我们构建并鉴定了携带c.428_451dup24重复突变的人源化小鼠模型（Arxdup24/0）。Arxdup24/0雄性小鼠表现出多动症状、刻板行为增强以及情境恐惧记忆异常。该模型雄性小鼠的特定精细运动技能亦受影响，表现为抓取与抓握能力受损、精细运动协调与平衡功能缺陷。对胚胎发育第15.5天（E15.5）的Arxdup24/0小鼠前脑进行转录组分析显示，中间神经元特异性表达的基因出现下调，而中间神经元沉默的基因则出现上调，提示中间神经元发育存在异常。据此，在E15.5、出生后第0天（P0）至成年阶段，中间神经元的迁移与发育过程出现异常，尤其在皮层与纹状体中表现显著。我们还发现Arxdup24/0小鼠的基底外侧杏仁核（basolateral amygdala）中抑制/兴奋平衡被打破。综上，我们证实c.428_451dup24突变改变了Arx的功能，直接对中间神经元发育造成影响，进而导致多动症状、精细运动控制缺陷以及关联记忆障碍。最后，我们回顾了33例携带ARX c.428_451dup24突变的男性患者的临床特征，并发现其临床表型与该小鼠模型的表型具有显著相似性。整体实验设计：共设置6个测序样本，针对E15.5天胚胎前脑的野生型（wt/0）与c.428_451dup24突变型（dup24/0）样本，每组各3个生物学重复，开展RNA测序（RNA-seq）。