Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability.

Tzu-Ting Chiou,Kirsten Harvey,Stefan A Haas,Angel L De Blas,Alexandra Schumann-Gillett,Megan L O’Mara,Philip Long,Robert J Harvey,Vera M Kalscheuer,Venkateswarlu Kanamarlapudi

doi:10.3389/fnmol.2019.00060

Abstract

The recruitment of inhibitory GABAA receptors to neuronal synapses requires a complex interplay between receptors, neuroligins, the scaffolding protein gephyrin and the GDP-GTP exchange factor collybistin (CB). Collybistin is regulated by protein-protein interactions at the N-terminal SH3 domain, which can bind neuroligins 2/4 and the GABAAR α2 subunit. Collybistin also harbors a RhoGEF domain which mediates interactions with gephyrin and catalyzes GDP-GTP exchange on Cdc42. Lastly, collybistin has a pleckstrin homology (PH) domain, which binds phosphoinositides, such as phosphatidylinositol 3-phosphate (PI3P/PtdIns3P) and phosphatidylinositol 4-monophosphate (PI4P/PtdIns4P). PI3P located in early/sorting endosomes has recently been shown to regulate the postsynaptic clustering of gephyrin and GABAA receptors and consequently the strength of inhibitory synapses in cultured hippocampal neurons. This process is disrupted by mutations in the collybistin gene (ARHGEF9), which cause X-linked intellectual disability (XLID) by a variety of mechanisms converging on disrupted gephyrin and GABAA receptor clustering at central synapses. Here we report a novel missense mutation (chrX:62875607C>T, p.R356Q) in ARHGEF9 that affects one of the two paired arginine residues in the PH domain that were predicted to be vital for binding phosphoinositides. Functional assays revealed that recombinant collybistin CB3SH3-R356Q was deficient in PI3P binding and was not able to translocate EGFP-gephyrin to submembrane microaggregates in an in vitro clustering assay. Expression of the PI3P-binding mutants CB3SH3-R356Q and CB3SH3-R356N/R357N in cultured hippocampal neurones revealed that the mutant proteins did not accumulate at inhibitory synapses, but instead resulted in a clear decrease in the overall number of synaptic gephyrin clusters compared to controls. Molecular dynamics simulations suggest that the p.R356Q substitution influences PI3P binding by altering the range of structural conformations adopted by collybistin. Taken together, these results suggest that the p.R356Q mutation in ARHGEF9 is the underlying cause of XLID in the probands, disrupting gephyrin clustering at inhibitory GABAergic synapses via loss of collybistin PH domain phosphoinositide binding.

Highlights

The Dbl-family guanine nucleotide exchange factor collybistin was initially identified as an interactor of the inhibitory postsynaptic clustering protein gephyrin (Kins et al, 2000)
We report the identification of a novel pathogenic missense variant in ARHGEF9 using next-generation sequencing and variant filtering in a family with mild NS-X-linked intellectual disability (XLID), which was recently included in a case series (Alber et al, 2017)
X-chromosome exome resequencing of individual II:8 followed by bioinformatics analysis and filtering against public datasets revealed four novel missense changes: ARHGEF9, chrX:62875607C>T, p.R356Q (Figures 1B–D), consensus score 5.13, predicted as probably damaging (PolyPhen-2; Adzhubei et al, 2013) and damaging (SIFT; Kumar et al, 2009) with a CADD score (Kircher et al, 2014) of 26; CCDC22, chrX: 49105321A>G, p.A492G with a conservation score of 5.56 and predicted as benign (Polyphen-2) and tolerated (SIFT) and CADD of 18; FAM3A, chrX:153736880T>C, p.E37G with a conservation score 2.67 and predicted as benign and tolerated and CADD of 15; GLUD2, chrX:120181572T>C, p.S12P with a low conservation score (0.06) and predicted as benign and damaging with a CADD score of 11

Summary

Introduction

The Dbl-family guanine nucleotide exchange factor collybistin was initially identified as an interactor of the inhibitory postsynaptic clustering protein gephyrin (Kins et al, 2000). Since collybistin variants containing SH3 domains predominate in the brain and spinal cord (Harvey et al, 2004; Soykan et al, 2014) this triggered a search for neuronal regulatory proteins that could bind to the SH3 domain and trigger collybistin activity This resulted in the identification of neuroligin-2 (NL2), neuroligin (NL4) and the GABAA receptor α2 subunit as collybistin SH3 domain interactors (Poulopoulos et al, 2009; Saiepour et al, 2010; Hoon et al, 2011). Disruption of collybistin-GABAA receptor α2 subunit interactions in mice leads to loss of a subset of inhibitory synapses, spontaneous seizures and early mortality, with surviving animals showing anxiety-like behavior (Hines et al, 2018)

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