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The m6A-mediated epi-transcriptomic dysregulation drives synaptic dysfunction in fragile X syndrome
Lu Lu1,2, Avijite Kumer Sarkar1,2, Lan Dao1,2
1Center for Stem Cell and Organoid Medicine (CuSTOM), Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, 45229, USA.
Abstract:
Fragile X syndrome (FXS), the leading genetic cause of intellectual disability, arises from FMR1 gene silencing and the subsequent loss of the RNA-binding protein FMRP. N6-methyladenosine (m6A) is a prevalent mRNA modification essential for post-transcriptional regulation. FMRP binds and regulates the stability of m6A-containing transcripts. However, how FMRP deficiency impacts transcriptome-wide m6A modifications in FXS remains unknown. To address this, we generated cortical neurons from induced pluripotent stem cells (iPSCs) derived from healthy individuals and FXS patients. Electrophysiology recordings revealed synaptic and neuronal network defects in FXS iPSC-derived neurons. Transcriptome-wide analysis revealed striking m6A hypermethylation predominantly affecting synapse-associated transcripts. Mechanistically, we demonstrated that FMRP deficiency drives the aberrant translational upregulation of core m6A writers, a causal relationship definitively validated using CGG-corrected isogenic control lines. Targeted genetic knockdown of the m6A writer METTL3 successfully rescued synaptic phenotypes in FXS neurons, whereas its overexpression in control neurons phenocopied these synaptic defects, confirming the causal role of m6A dysregulation in FXS pathology. Notably, pharmacological intervention with the METTL3 inhibitor STM-2457 normalized methylation on synapse-associated transcripts and restored synaptic transmission in FXS neurons. Together, our findings uncover an FMRP-dependent epitranscriptomic mechanism contributing to FXS pathogenesis and suggest a promising avenue for m6A-targeted therapies.
Insights
Fragile X syndrome (FXS) involves FMRP loss, impacting RNA methylation. We found FXS neurons show increased m6A on synapse genes due to METTL3 upregulation, which STM-2457 treatment reversed, suggesting new therapies.
Area of Science:
- Neuroscience
- Genetics
- Molecular Biology
Background:
- Fragile X syndrome (FXS) is a leading genetic cause of intellectual disability, linked to FMR1 gene silencing and FMRP protein loss.
- N6-methyladenosine (m6A) is a key mRNA modification regulating gene expression, and FMRP influences m6A-modified transcripts.
- The impact of FMRP deficiency on transcriptome-wide m6A patterns in FXS was previously unknown.
Purpose of the Study:
- To investigate the role of m6A modifications in FXS pathogenesis.
- To explore the relationship between FMRP deficiency and m6A dysregulation in neuronal function.
- To identify potential therapeutic targets for FXS based on epitranscriptomic mechanisms.
Main Methods:
- Generated induced pluripotent stem cell (iPSC)-derived cortical neurons from FXS patients and healthy controls.
- Performed electrophysiology recordings to assess neuronal and synaptic function.
- Conducted transcriptome-wide m6A analysis and validated molecular mechanisms using genetic manipulation and pharmacological interventions.
Main Results:
- FXS neurons exhibited synaptic and network defects.
- Transcriptome-wide m6A analysis revealed hypermethylation in synapse-associated transcripts in FXS neurons.
- FMRP deficiency caused METTL3 upregulation, driving m6A changes and synaptic defects, which were rescued by METTL3 inhibition or STM-2457 treatment.
Conclusions:
- FMRP deficiency leads to aberrant m6A epitranscriptomic modifications, contributing to FXS pathology.
- METTL3-mediated m6A dysregulation plays a causal role in FXS-related synaptic dysfunction.
- Targeting m6A pathways, such as with METTL3 inhibitors, offers a promising therapeutic strategy for FXS.
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