Using antisense oligonucleotides for the physiological modulation of the alternative splicing of NF1 exon 23a during PC12 neuronal differentiation

Meritxell Carrió,Conxi Lazaro,Bernat Gel,Ignacio Blanco,Eduard Serra,Helena Mazuelas,Elisabeth Castellanos,Josep Biayna,Ernest Terribas,Juana Fernández-Rodriguez,Gabrijela Dumbovic,Inma Rosas

doi:10.1038/s41598-021-83152-w

Abstract

Neurofibromatosis Type 1 (NF1) is a genetic condition affecting approximately 1:3500 persons worldwide. The NF1 gene codes for neurofibromin protein, a GTPase activating protein (GAP) and a negative regulator of RAS. The NF1 gene undergoes alternative splicing of exon 23a (E23a) that codes for 21 amino acids placed at the center of the GAP related domain (GRD). E23a-containing type II neurofibromin exhibits a weaker Ras-GAP activity compared to E23a-less type I isoform. Exon E23a has been related with the cognitive impairment present in NF1 individuals. We designed antisense Phosphorodiamidate Morpholino Oligomers (PMOs) to modulate E23a alternative splicing at physiological conditions of gene expression and tested their impact during PC12 cell line neuronal differentiation. Results show that any dynamic modification of the natural ratio between type I and type II isoforms disturbed neuronal differentiation, altering the proper formation of neurites and deregulating both the MAPK/ERK and cAMP/PKA signaling pathways. Our results suggest an opposite regulation of these pathways by neurofibromin and the possible existence of a feedback loop sensing neurofibromin-related signaling. The present work illustrates the utility of PMOs to study alternative splicing that could be applied to other alternatively spliced genes in vitro and in vivo.

Highlights

Neurofibromatosis Type 1 (NF1) is a genetic condition affecting approximately 1:3500 persons worldwide
Cell‐based systems to study the alternative splicing of exon 23a (E23a) of the NF1 gene
Results obtained by applying Phosphorodiamidate Morpholino Oligomers (PMOs) to the study of the alternative splicing of NF1 E23a highlight the importance of a precise quantitatively and time-dependent regulation of E23a splicing along the neuronal differentiation process

Summary

Introduction

Neurofibromatosis Type 1 (NF1) is a genetic condition affecting approximately 1:3500 persons worldwide. The NF1 gene codes for neurofibromin protein, a GTPase activating protein (GAP) and a negative regulator of RAS. The NF1 gene undergoes alternative splicing of exon 23a (E23a) that codes for 21 amino acids placed at the center of the GAP related domain (GRD). E23a-containing type II neurofibromin exhibits a weaker Ras-GAP activity compared to E23a-less type I isoform. We designed antisense Phosphorodiamidate Morpholino Oligomers (PMOs) to modulate E23a alternative splicing at physiological conditions of gene expression and tested their impact during PC12 cell line neuronal differentiation. Alternative splicing of exon 23a (E23a) of the NF1 gene generates two isoforms named type I and II. The increase in GAP activity due to the higher expression of neurofibromin type I isoform correlates with a down-regulation of Ras activity during PC12 neurite e longation[15], and in embryonic stem cell-derived n eurons[29]. NF1-dependent activation of cAMP/ PKA signaling has been demonstrated to be essential for mediating Drosophila learning and m emory[37]

Methods

Results

Conclusion