Modulating RBFOX1 Expression in Human Stem Cell-Derived Glutamatergic Neurons
Autism spectrum disorders (ASDs) are a group of neurodevelopmental conditions that currently afflict about 1 in every 68 children. The hunt is ongoing for the highly heterogeneous genetic causes of ASD. Both our group and others have identified potentially pathogenic copy number variants (CNVs) and point mutations in ASD individuals in the RNA binding protein, fox-1 homolog 1 (RBFOX1) gene (Griswold, et al, 2012). RBFOX1 is a neural splicing cofactor that regulates alternative splicing. Due to the high level of ASD genetic heterogeneity, investigations of ASD loci using genome wide association studies (GWAS) have identified relatively few consistent signals of interest. However, when we conducted a combined analysis of over 20,000 individuals from four independent family-based GWAS datasets, one of our strongest signals was in RBFOX1 (top SNP, rs74733079, discovery p=1.558 x 10-6, meta p=2.635 x 10-5). In addition to ASD, RBFOX1has been connected to bipolar disorder, epilepsy, and schizophrenia.
To better understand the impact of RBFOX1 on ASD, we are evaluating how RBFOX1 expression modulates neuronal function in iPSC-derived glutamatergic neuronal cells.
Given the neurodevelopmental nature of ASD, iPSC provide a unique opportunity to understand potential pathogenic mechanisms that may be at work during early phases of development. To that end, we have examined the impact that changes in RBFOX1 expression have on neuronal functionality in the developing neuronal cultures. The viability and functionality of the glutamatergic neurons in which RBFOX1 has either been overexpressed or silenced by RNA interference are assessed at multiple time points during in vitro neurogenesis. Overexpression experiments are performed with lentiviral transduction and genomic integration the wild type form of RBFOX1 variant 4 driven under a CMV promoter, as well as an ASD-specific nonsense mutation at R173X that we identified in a sporadic ASD family. Each condition is compared at a gross morphological level to determine if modulating RBFOX1 produces recognizable qualitative or quantitative phenotypes. Since RBFOX1 plays a key role in splicing and transcription, RNA-seq analysis of the iPSC-derived neurons under three conditions (RBFOX1 knockdown, overexpression and no treatment) will be used to complement the functional analysis to identify key networks regulated by RBFOX1.
Neuronal precursors cells (NPCs) from the control stem cell line were transduced with lentivirus on day 27 of the glutamatergic differentiation scheme. Cells overexpressing a RBFOX1-eGFP construct successfully glowed. RNA was extracted from all cells, purified, and reverse transcribed for quantification by qPCR. Following measurement of both RBFOX1 and the internal control GAPDH, the shRNA was determined to be successful at ~40-45% knockdown of endogenous RBFOX1 expression and the RBFOX1-eGFP vector was strongly (~35x) overexpressing the gene. The alteration of RBFOX1 levels through the RNAi-mediated silencing of RBFOX1 or the transgene overexpression showed alterations in transcriptional profiles, including differences in splice variant composition of the transcriptome. These changes are consistent with the known function of RBFOX1as a regulator of splicing.
Initial results suggest that RBFOX1 plays a central role in establishing key transcriptional networks in neural development.