scRNA-seq and MAS-seq in Eftud2-deficient Purkinje cells

Spliceosomal GTPase EFTUD2 (Elongation factor Tu GTP binding domain containing 2) is a causative gene for mandibulofacial dysostosis with microcephaly (MFDM) in humans, a complex syndrome with multiple neurological symptoms including cerebellar hypoplasia and motor dysfunction. How EFTUD2 deficiency contributes to these neurological symptoms remains elusive. In this study, we demonstrate that specific ablation of Eftud2 in cerebellar Purkinje cells (PCs) in mice results in severe ferroptosis, PC degeneration, dyskinesia and cerebellar atrophy, which are similar to phenotypes observed in MFDM patients. Mechanistically, Eftud2 promotes the expression of Scd1 and Gch1, upregulates monounsaturated fatty acids-phospholipids (MUFA-PLs), and enhances systematic antioxidant activity, thereby suppressing ferroptotic cell death of PCs. Importantly, inhibiting ferroptosis efficiently rescues PC loss and motor deficits in Eftud2 cKO mice. Our data reveals a novel role of Eftud2 in maintaining the survival of PCs, and that inhibiting ferroptosis via pharmacological or genetic means may be a promising therapeutic strategy for EFTUD2 deficiency-induced MFDM and related disorders. The entire cerebellum is separated from the entire brain with tweezers and the meninges are removed. The sample was then cut in 1Hank equilibrium salt solution (HBSS) and digested in 0.25% trypsin solution (Thermo Fisher Scientific) for 15-20 minutes. The dissociated cells were filtered through a 22 m cell filter (Millipore), centrifuged at 300 g for 3 min, and then suspended in a solution containing 0.04% BSA (Sigma) at a concentration of 2000-3000 cells /ml (without calcium and magnesium). Trypan Blue staining was used to detect cell health status and cell number. The dissociated cells in each sample were segmented into nanoscale gel bead emulsions (GEMs) at limited dilution to reduce the multiplication rate. Single-cell GEMs are then incubated with reverse transcription reagents, including several primers for cell and transcript barcoding in cDNA synthesis, followed by cDNA amplification and library construction. All procedures follow the guidelines provided by 10x Genomics, Inc.剪接体GTP酶EFTUD2（Elongation factor Tu GTP binding domain containing 2，含延伸因子Tu GTP结合结构域2）是人类小头畸形伴下颌面骨发育不全（mandibulofacial dysostosis with microcephaly，MFDM）的致病基因，该综合征为伴随多种神经症状的复杂疾病，包括小脑发育不全与运动功能障碍。目前EFTUD2缺失如何诱发此类神经症状的机制尚未阐明。本研究证实，小鼠小脑浦肯野细胞（Purkinje cells，PCs）中特异性敲除Eftud2，会引发严重铁死亡、浦肯野细胞变性、运动障碍与小脑萎缩，其表型与MFDM患者的临床表现高度相似。机制层面研究显示，Eftud2可促进Scd1与Gch1的表达，上调单不饱和脂肪酸-磷脂（monounsaturated fatty acids-phospholipids，MUFA-PLs）水平，并增强系统性抗氧化活性，从而抑制浦肯野细胞的铁死亡性细胞死亡。值得注意的是，抑制铁死亡可有效挽救Eftud2条件性敲除（cKO）小鼠中的浦肯野细胞丢失与运动功能缺陷。本研究数据揭示了Eftud2在维持浦肯野细胞存活中的全新功能，且通过药理学或遗传学手段抑制铁死亡，或可成为治疗EFTUD2缺失诱导的MFDM及相关疾病的潜在治疗策略。 实验流程如下：首先用镊子将整个小脑与全脑分离，去除脑膜。随后将样本置于1×汉克平衡盐溶液（Hank equilibrium salt solution，HBSS）中切割，并在0.25%胰蛋白酶溶液（Thermo Fisher Scientific）中消化15-20分钟。将解离后的细胞通过22μm细胞过滤器（Millipore）过滤，以300g离心3分钟，随后重悬于含0.04%牛血清白蛋白（Sigma，BSA）的无钙镁溶液中，调整细胞浓度至2000-3000个/毫升。采用台盼蓝染色检测细胞活力与细胞数量。将每份样本中的解离细胞以有限稀释法分配至纳米级凝胶微滴（GEMs）中，以降低细胞增殖速率。随后将单细胞GEMs与逆转录试剂共同孵育，其中包含若干用于cDNA合成中细胞与转录本条形码标记的引物，后续进行cDNA扩增与文库构建。所有实验流程均遵循10x Genomics公司提供的操作指南。