ABSENCE OF FMRP MODULATES BRAIN DEVELOPMENT IN A SEX- SPECIFIC MANNER
Authors: Kikkawa *Takako , Ebrahimiazar Sara , Osumi Noriko 
Published: February 2025

Abstract

Background
Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene, leading to the absence of Fragile X mental retardation protein (FMRP), an RNA-binding protein. There is sex-specificity in the symptoms of FXS patients, such as social interaction defects in female patients (Reiss et al., 1988). Sex-specific behaviors are also observed in Fmr1-knockout (KO) mice, such as more repetitive behavior in females (Nolan et al., 2017). The expression of FMRP starts from the embryonic stage and localizes to the neural stem/progenitor cells and neurons, contributing to neurogenesis (Saffary and Xie, 2011). Molecular dysregulations of FMRP during brain development may cause brain malformation. However, it is still unclear whether the sex-biased phenotypes of Fmr1-KO mice are related to the regulatory functions of FMRP during brain development.

Aims & Objectives
Our purpose is to investigate sex differences in the molecular basis of FXS by exploring the transcriptome status of mouse brain in the presence and absence of FMRP. We aim to reveal the sex differences in the expression, localization, and function of potential FMRP targets during brain development.

Methods
To comprehensively analyze the transcriptomic features with regards to FMRP, we performed RNA sequencing on the telencephalic samples from WT and Fmr1-KO male and female mice, as well as the Fmr1-heterozygous females at the embryonic day 14.5 (E14.5), when neurogenesis is at its peak. Among the differentially expressed genes (DEGs) in the sex comparison of WT or Fmr1-KO condition, we focused on the Fmr1-KO specific DEGs based on Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) categories. We focused on DEGs with higher expression levels and fold change&gt;1.2, identifying sex- biased target genes in the absence of FMRP. To confirm the mRNA expression of our targets, we performed quantitative real-time PCR using E14.5 cortical samples of all the genotypes.

Results
DEG number analysis in WT or Fmr1-KO condition revealed a higher number of sex-biased DEGs in the Fmr1-KO condition (p&lt;0.05; 929 genes) compared to WT (p&lt;0.05; 519 genes) with only 4.1% of them being shared between these conditions. Consistently, GO analysis and IPA exhibited highly specific enriched categories for sex-biased DEGs. The Fmr1-KO condition consists of 486 male- and 443 female- biased DEG, and these are enriched in specific GO categories including neuron-related and stem cell proliferation-related categories in males and females, respectively. Additionally, we detected sex- specific alterations in the mRNA levels several targets.

Discussion & Conclusion
These data show an increase in sex-biased DEG numbers in the absence of FMRP rather than that of WT. The GO analysis and IPA of Fmr1-KO specific sex-biased DEGs further suggest a notable transcriptome shift during brain development in each sex. We assume that FMRP can differentially affect the expression of our potential targets at the level of mRNA localization and stability in the cortex with a sex-specific manner, leading to their sex-biased functions during brain development.