E2f1 transcription factor is required for neurodevelopment through regulation of conserved miR-33 and RhoGEF trio

The E2f1 transcription factor exhibits diverse and complex functions beyond its canonical roles in cell cycle regulation, DNA damage response, and apoptosis. Aberrant expression and activity of E2f1 in neurons have been observed in neurodegenerative disorders such as Parkinson’s disease (PD) and Alzheimer’s disease (AD). However, it remains unclear whether the E2f1 is required for neurodevelopment. Functions of the E2f1 in neurodevelopment were explored using mutant flies and over-expression flies. The CNS axons in embryos and synaptic growth at larval neuromuscular junctions were assessed by morphological and immunofluorescence analysis. Reverse transcription-quantitative PCR (RT-qPCR) was performed to quantify gene expression of miR-33, hmiR-33, e2f1, he2f1, and dp. Western blot analysis was performed to assess Trio protein level. Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) was employed to detect direct regulation of miR-33 by the E2f1. The dual-luciferase reporter assay was utilized to investigate the post-transcriptional regulation of trio by the miR-33. Grid crossing and peristalsis contraction assays were performed to analyze the locomotor behavior. Flies with dysfunctional E2f1, reduced expression or overexpression of E2f1 in neurons exhibit disrupted axons and abnormal synaptic growth at neuromuscular junctions. The miR-33, an important regulator of lipid and glucose metabolisms with uncharacterized roles in neurodevelopment, is directly regulated by the E2f1 and works downstream of the E2f1 to regulate neurodevelopment. The RhoGEF Trio, a central regulator of neurodevelopment and synaptic plasticity, acts as a target of the miR-33 and the downstream effector of the E2f1-miR-33 axis to modulate neurodevelopment. Notably, flies with dysfunctional e2f1 or loss of miR-33 display aberrant locomotor behaviors. Furthermore, the E2f1-miR-33-Trio signaling and its functions in neurons are conserved in Drosophila and human. This study uncovers a novel role of the E2f1 transcription factor in the central nervous system, identifies upstream regulators of the RhoGEF Trio, and reveals an evolutionarily conserved E2f1-miR-33-Trio signaling cascade critical for neurodevelopment.