ARHGEF39, a Gene Implicated in Developmental Language Disorder, Activates RHOA and Is Involved in Cell De-Adhesion and Neural Progenitor Cell Proliferation

Abstract

ARHGEF39 was previously implicated in developmental language disorder (DLD) via a functional polymorphism that can disrupt post-transcriptional regulation by microRNAs. ARHGEF39 is part of the family of Rho guanine nucleotide exchange factors (RhoGEFs) that activate small Rho GTPases to regulate a wide variety of cellular processes. However, little is known about the function of ARHGEF39, or how its function might contribute to neurodevelopment or related disorders. Here, we explore the molecular function of ARHGEF39 and show that it activates the Rho GTPase RHOA and that high ARHGEF39 expression in cell cultures leads to an increase of detached cells. To explore its role in neurodevelopment, we analyse published single cell RNA-sequencing data and demonstrate that ARHGEF39 is a marker gene for proliferating neural progenitor cells and that it is co-expressed with genes involved in cell division. This suggests a role for ARHGEF39 in neurogenesis in the developing brain. The co-expression of ARHGEF39 with other RHOA-regulating genes supports RHOA as substrate of ARHGEF39 in neural cells, and the involvement of RHOA in neuropsychiatric disorders highlights a potential link between ARHGEF39 and neurodevelopment and disorder. Understanding the GTPase substrate, co-expression network, and processes downstream of ARHGEF39 provide new avenues for exploring the mechanisms by which altered expression levels of ARHGEF39 may contribute to neurodevelopment and associated disorders.

Keywords: ARHGEF39; RHOA; Rho GTPases; cell adhesion; cell division; neural progenitor cells (NPCs); scRNA-seq.

FIGURE 1

(A) Diagram of FRET-based Rho GTPase activity biosensor. If the Rho GTPase is an inactive GDP-bound state only a cyan fluorescent protein (mTFP) is excited. When the Rho GTPase is in an activated GTP-bound state, it is able to bind a Rho GTPase binding domain (RBD) in the biosensor. This induces a conformational change that enables a cyan fluorescent protein (mTFP) to excite a yellow fluorescent protein (Venus) through FRET. Activity is measured as the ratio between emissions at 528 nm and 492 nm. (B) Normalised ratio of emissions at 528 and 492 nm from Rho GTPase biosensors. Multiple ratios of biosensor vs. ARHGEF39 co-transfection were used: 1:0 (no ARHGEF39 control), 1:1, 1:3, and 1:5. Mean and standard error are indicated in red. p-values are determined with a Tukey HSD test after ANOVA. ***Indicates p < 0.001, **p < 0.01.

FIGURE 2

(A) Representative images of cells after transfection with an ARHGEF39 overexpressing vector (pcDNA3.1-ARHGEF39) or empty vector control (pcDNA3.1-empty). Scale bar indicates 500 um. (B) Counts of cells in suspension and attached cells 48 h after transfection with pcDNA3.1-ARHGEF39 and pcDNA3.1-empty. Concentrations for cells in suspension and attached cells were measured in 100 ul and 1 ml resuspension volumes, respectively (see Section “Materials and Methods”). Mean and standard error are indicated in red. (C) Viability of cells in suspension and attached cells 48 h after transfection with pcDNA3.1-ARHGEF39 and pcDNA3.1-empty. p-values are determined with two-sided t-test. ***Indicates p < 0.001, and **p < 0.01.

FIGURE 3

Differential gene expression analysis between ARHGEF39-positive and ARHGEF39-negative cells in ARHGEF39 expressing cell clusters. (A) Overlap between lists of DEGs. Purple curves link identical genes between lists. Genes that hit multiple lists are coloured in dark orange, and genes unique to a list are shown in light orange. Counts of DEGs are between brackets. For Loo_RG2 and Polioudakis_PgG2M, the number of cluster marker genes is added in oblique type. (B) Heatmap of top 20 enriched terms across lists of DEGs, coloured by p-values. Grey indicates a lack of significance. Dendrograms indicate similarity between terms (rows) and lists of DEGs (columns). Terms that are directly related to cell cycle processes are marked with a dark blue bar, other terms are marked with a light blue bar.