Ferroptosis Triggered by Spliceosomal GTPase EFTUD2 Deficiency Leads to Purkinje Cell Degeneration

Neurology Biochemistry Genetics Basic Medical Sciences Life Sciences Medicine Cell Biology
spliceosome EFTUD2 ferroptosis Purkinje cells neurodegeneration cerebellar development

Depletion of EFTUD2 Triggers Ferroptosis-Induced Degeneration of Cerebellar Purkinje Cells

Background and Motivation

The cerebellum plays a crucial role in motor coordination and higher cognitive functions, with the health of cerebellar Purkinje cells (PCs) being essential for maintaining cerebellar function. Gene regulation based on alternative splicing (AS) is key in the development of the nervous system, particularly in maintaining PC survival. Studies have shown that abnormalities in spliceosomes and RNA-binding proteins (RBPs) can lead to a range of neurodevelopmental and degenerative diseases, including the rapid degeneration of PCs. This study focuses on the EFTUD2 gene, a vital GTPase in the spliceosome, indispensable during RNA splicing. Previous research has shown that mutations in EFTUD2 can lead to a syndrome called Mandibulofacial Dysostosis with Microcephaly (MFDM), which causes cerebellar dysplasia and motor disorders. However, the exact molecular mechanisms by which EFTUD2 depletion leads to these symptoms remain unclear.

This research was conducted by Guochao Yang and his team, from institutions such as the Beijing Institute of Basic Medical Sciences and Shandong University, and published in the journal “Neuron.” The study aims to clarify the role of EFTUD2 in cerebellar PC growth and survival and how it maintains PC function by inhibiting ferroptosis. The research team explored the mechanisms of cell damage caused by EFTUD2 deficiency using a PC-specific EFTUD2 knockout mouse model (EFTUD2 cKO) and verified whether inhibiting ferroptosis could reverse the degenerative symptoms caused by PC deficiency.

Research Methods

Experimental Procedures and Design

This study included several key experimental steps designed to investigate EFTUD2’s function from cellular to systemic levels.

  1. Construction of the PC-Specific EFTUD2 Knockout Mouse Model: The research team crossed EFTUD2f/f mice with L7-Cre mice to obtain PC-specific EFTUD2 knockout mice (EFTUD2 cKO). In these mice, EFTUD2 was specifically knocked out to observe its impact on PC structure and function.

  2. Morphological and Functional Assessment: Through immunofluorescence and tissue staining techniques, researchers examined the number and morphological changes of PCs in EFTUD2 cKO mice. The experiments showed a significant reduction in cerebellar volume and PC number during development in mice lacking EFTUD2, accompanied by motor coordination impairments. Additionally, the research team conducted behavioral experiments like the accelerated rotarod and balance beam tests to evaluate changes in motor coordination and social behavior in mice.

  3. Gene Expression and Molecular Mechanism Analysis: The study utilized RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) to analyze the impact of EFTUD2 knockout on gene expression in cerebellar PCs. Differential gene expression and enrichment analysis revealed significant changes in genes related to lipid metabolism, oxidative stress, and ferroptosis in EFTUD2 cKO mice.

  4. Detection of Ferroptosis Signaling Pathways: Observations of intracellular reactive oxygen species (ROS) levels and mitochondrial morphology showed significant increases in ROS levels in PC of mice lacking EFTUD2, with reduced and ruptured mitochondrial volumes, which are typical features of ferroptosis.

  5. Ferroptosis Inhibition Experiment: To further validate the role of ferroptosis in EFTUD2 deficiency-induced PC death, the research team treated the mice with ferroptosis inhibitors, ferrostatin-1 (Fer-1), and vitamin E (Vitamin E). The results showed that these inhibitors effectively alleviated PC degeneration and improved motor impairments in mice.

Major Findings

  1. EFTUD2 Deficiency Triggers PC Ferroptosis and Leads to Cerebellar Dysplasia: In the cerebella of EFTUD2 cKO mice, there was a significant reduction in PC number, accompanied by cerebellar atrophy. This phenomenon resembles the symptoms in MFDM patients, highlighting EFTUD2’s key role in maintaining PC structure and function.

  2. EFTUD2 Inhibits Ferroptosis Through Lipid Metabolism Regulation: The study found that EFTUD2 deficiency led to the downregulation of ferroptosis-resistance genes SCD1 (stearyl-CoA desaturase 1) and GCH1 (GTP cyclohydrolase 1), indicating its role in inhibiting ferroptosis through the regulation of monounsaturated fatty acids (MUFAs) phospholipids and antioxidant activity.

  3. Inhibition of Ferroptosis Through a p53-Independent Pathway: The research showed that the ferroptosis triggered by EFTUD2 deficiency occurs via a p53-independent pathway. Although p53 typically plays a crucial role in apoptosis and ferroptosis regulation, the study revealed that EFTUD2 suppresses ferroptosis through the downregulation of SCD1 and GCH1 rather than relying on the p53 signaling pathway.

  4. EFTUD2 Knockout Causes Aberrant Splicing of ATF4, Further Affecting Anti-Ferroptosis Genes: RNA-seq and long-read RNA sequencing indicated that EFTUD2 deficiency leads to aberrant splicing of the ATF4 (Activating Transcription Factor 4) gene, impairing its function. Since ATF4 directly regulates the expression of SCD1 and GCH1, such splicing errors further reduce the levels of anti-ferroptosis gene expression.

  5. Ferroptosis Inhibition Effectively Alleviates PC Degeneration: In EFTUD2 cKO mice treated with ferroptosis inhibitors, PC survival rate significantly increased, and motor behavior improved. Furthermore, double knockout of EFTUD2 and ACSL4 (long-chain acyl-CoA synthetase family member 4) in mice showed notable alleviation of degeneration, further confirming the role of ferroptosis in EFTUD2 deficiency-induced PC degeneration.

Research Significance

This study reveals the essential function of EFTUD2 in the survival of cerebellar PCs and highlights that EFTUD2 maintains PC structure and function by inhibiting ferroptosis, providing a new perspective for potential treatment strategies for MFDM and other neurodegenerative diseases. By regulating the expression of SCD1 and GCH1, EFTUD2 reduces intracellular lipid peroxidation and ROS levels, thereby halting ferroptosis through a novel non-p53 dependent pathway. The findings suggest that the application of ferroptosis inhibitors could be a promising therapeutic approach for cerebellar degenerative diseases caused by EFTUD2 deficiency. Furthermore, the study indicates that detecting and intervening in cerebellar developmental abnormalities early in MFDM patients has potential clinical value.

Research Highlights

  1. Discovery of EFTUD2’s Inhibitory Mechanism on PC Ferroptosis: Using a PC-specific EFTUD2 knockout model, the research is the first to reveal the molecular mechanism of EFTUD2’s inhibition of ferroptosis through a non-p53 dependent pathway.

  2. Proposal of Ferroptosis Inhibitors as Potential Treatment for EFTUD2 Deficiency-Related Diseases: Ferroptosis inhibitors like Fer-1 and vitamin E showed positive effects in mouse models, offering a novel therapeutic approach for diseases related to EFTUD2 deficiency.

  3. Exploration of Ferroptosis’s Importance in Nervous System Development: This study directly links ferroptosis to cerebellar development and motor coordination dysfunction, expanding ferroptosis’s application scope in neurodevelopmental and degenerative disease research.

Conclusion

The study elucidates the PC ferroptosis mechanism induced by EFTUD2 deficiency and presents the therapeutic potential of ferroptosis inhibition. By investigating the relationship between the spliceosomal GTPase EFTUD2 and the ferroptosis pathway, the study further unveils the significance of RNA splicing in neuronal survival and neurodevelopment.