The Function And Mechanism Of Eftud2 In Purkinje Neuron Development And Microglia Activation

Elongation factor Tu GTP-binding domain inclusion 2（Eftud2）plays an important role in the RNA splicing process as an essential component of the RNA spliceosome U5 sn RNPs.In zebrafish mutants,Eftud2 was shown to regulate neural progenitor fate.On the other hand,specific deletion of Eftud2 in mouse neural crest cells resulted in defects in mouse craniofacial development.However,the function of Eftud2 in cerebellar Purkinje cells remains unknown.In this study,by constructing a Purkinje cell-specific knockout mouse model of Eftud2(L7-Cre;Eftud2^F/F c KO),we revealed the function of Eftud2 in Purkinje cells.The study found that specific knockout of Eftud2 in mouse Purkinje cells did not affect mouse survival,but exhibited phenotypic changes such as ataxia,impaired balance,and social deficits.Histomorphological analysis revealed that Purkinje cells from L7-Cre;Eftud2^F/F c KO mice first showed impaired synaptic connections and then progressively died.We also further dissected the death process of Purkinje cells.The study found that the process of Purkinje cell death was accompanied by the loss of dendritic spines and the reduction of dendritic complexity.Interestingly,these changes tended to occur first in dendrites close to the soma and gradually spread to dendrites farther from the soma.By constructing L7-Cre;Eftud2^F/F c KO mice,we demonstrated that Eftud2 is essential for Purkinje cell survival.Therefore,we next conducted an in-depth study around the phenotype and possible mechanism of Purkinje cell death.Previous work has shown that deletion of Eftud2 in zebrafish leads to P53-dependent apoptosis in neural progenitor cells.In this study,we also found an apoptotic phenotype in Eftud2-deficient Purkinje cells.And at the same time as apoptosis,autophagy is activated in Purkinje cells.We subsequently inhibited apoptosis and autophagy,respectively,by pharmacological inhibitors,confirming that the activation of autophagy may be a self-protective mechanism in response to cell death.Interestingly,in addition to the morphological changes in apoptosis observed in Eftud2-deficient Purkinje cells,we also found more and smaller mitochondria.Obviously.This mitochondrial alteration was not caused by apoptosis,but was consistent with the morphological features of ferroptosis,another mode of cell death.Subsequently,we demonstrated that Purkinje cells also undergo lipid peroxidation in the absence of Eftud2.On the other hand,we found that the ferroptosis inhibitor Ferrostain-1 could significantly rescue Purkinje cell death and dendrite loss.The results showed that Eftud2-deficient Purkinje cells underwent ferroptosis.Finally,the molecular mechanism by which Eftud2 deletion leads to ferroptosis in Purkinje cells was further explored by combining with transcriptome sequencing.We found that,upon Eftud2 deletion,a large number of splicing events were altered in Purkinje cells,mainly exon skipping and intron retention.In particular,we found significant transcriptional changes in the ferroptosis-related genes Scd1 and Sat1.Therefore,we speculate that Eftud2 may be involved in the occurrence of ferroptosis by regulating the alternative splicing of Scd1 and/or Sat1,but the specific molecular mechanism remains to be further studied.Microglia are innate immune cells that play an important role in the immune response of the central nervous system.Preliminary findings suggest that Eftud2 is involved in innate immune regulation and plays an important role in limiting viral infection.However,its role in microglia,key immune regulatory cells in the central nervous system,is unknown.In the present study,we first found that Eftud2 was consistently upregulated in lipopolysaccharide（LPS）-induced inflammatory responses.Next,we constructed microglia-specific Eftud2 knockout mice(CX3CR1-Cre ERT2;Eftud2^F/F c KO)to reveal the function of Eftud2 in microglia.We found that deletion of Eftud2 resulted in a marked increase in the number of microglia in various brain regions（hippocampus,striatum,prefrontal cortex,cerebellum）.Further analysis revealed abnormal proliferation of Eftud2-deficient microglia.To clarify the function of Eftud2in microglia,we also analyzed the morphological changes of microglia in different brain regions after Eftud2 deletion.The results showed that Eftud2-deficient microglia exhibited an amoeba-like activation morphology,that is,shorter branches,fewer branch and terminal points,and enlarged soma.It is currently believed that the activation of microglia is mainly divided into two types,namely pro-inflammatory subtype and anti-inflammatory subtype.Both our in vivo and in vitro results demonstrate that Eftud2deletion leads to activation of an anti-inflammatory phenotype in microglial cells.Furthermore,we also found that Eftud2-mediated activation of the anti-inflammatory phenotype of microglia may depend on NF-κB signaling pathway activation.Thus,our findings suggest that Eftud2 also plays a key role in regulating brain microglia homeostasis.In conclusion,this study revealed that Eftud2,as an important regulator of neural development,plays an important regulatory function in the development and survival of cerebellar Purkinje cells,and its role may be exerted by inhibiting ferroptosis.Because the death of Purkinje cells is closely related to diseases such as spinocerebellar ataxia.Therefore,our work is not only of great significance for in-depth understanding of the molecular mechanisms regulating Purkinje cell survival,but also is expected to provide new targets for targeted therapy of Purkinje cell-related diseases.On the other hand,this study also revealed that Eftud2,as an innate immune regulator,also plays an important role in the regulation of the pro-/anti-inflammatory phenotype of central microglia.Given that the role of microglia in the development of central nervous system diseases such as spinal cord injury,stroke,Alzheimer’s disease has been widely reported.Therefore,our work also provides new experimental evidence for further revealing and understanding the activation mechanism of microglia in central nervous cells and the mechanism of action in the development of major brain diseases.