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 1×Hank 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 μm 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.