Neurexin 1 variants as risk factors for suicide death

Abstract

Suicide is a significant public health concern with complex etiology. Although the genetic component of suicide is well established, the scope of gene networks and biological mechanisms underlying suicide has yet to be defined. Previously, we reported genome-wide evidence that neurexin 1 (NRXN1), a key synapse organizing molecule, is associated with familial suicide risk. Here we present new evidence for two non-synonymous variants (rs78540316; P469S and rs199784139; H885Y) associated with increased familial risk of suicide death. We tested the impact of these variants on binding interactions with known partners and assessed functionality in a hemi-synapse formation assay. Although the formation of hemi-synapses was not altered with the P469S variant relative to wild-type, both variants increased binding to the postsynaptic binding partner, leucine-rich repeat transmembrane neuronal 2 (LRRTM2) in vitro. Our findings indicate that variants in NRXN1 and related synaptic genes warrant further study as risk factors for suicide death.

Fig. 1. Study design.

The table summarizes the number of familial and non-familial cases genotyped and analyzed in each study and serves as a reference for the study design. A total of 4382 cases were genotyped for both studies. The analysis strategy started with the prioritization of NRXN1, from a previous study of high-risk families (red arrow), led to the discovery of specific NRXN1 suicide variants (blue arrow) and finally resulted in the elucidation of their functional consequences (blue box). ¹Coon H, Darlington T, DiBlasi E, Callor W, Ferris E, Fraser A et al. Genome-wide significant regions in 43 Utah high-risk families implicate multiple genes involved in risk for completed suicide. Mol Psychiatry. 2020;25:3077-90.

Fig. 2. P469S variant shows increased binding to LRRTM2 in pull-down assay.

a Schematic of Nrxn1α protein lacking insert at splice site #4 (SS#4), including locations of the suicide-associated variants identified in this study (magenta). Laminin neurexin sex-hormone binding globulin domain (LNS); epidermal growth factor-like domain (EGF); transmembrane region (TM); O-glycosylation sequence (oGlyc); heparan sulfate (HS); PDZ bs.; PSD-95/Dlg/ZO-1 binding site. b In the left panel, ribbon diagram of variant P469S (magenta) in the αLNS2 domain, and binding of Nxph1 (green). In the right panel, stick model of variant H885Y, proximal to the Ca²⁺-binding site at the LNS4 domain. c Western blot of Nrxn1α and Nxph1 pull-downs. Note that neither variant alters binding. d Western blot of pull-down of α-DAG and β-DAG with the extracellular Fc-tagged domain of Nrxn1α shows undisturbed DAG binding with wild-type and variant Nrxn1α. e Quantification of 2d (n = 3); n.s. indicates not significant by one-way ANOVA. f Western blot pull-down shows Nrxn1α variants bind Nlgn1 and Nlgn2 at similar levels to WT Nrxn1α but the P469S variant binds YFP-LRRTM2 at increased levels to WT Nrxn1α. g Quantification of 2f (n = 3); One-way ANOVA using GraphPad Prism followed by pairwise posttests. *p < 0.05, n.s. indicates not significant.

Fig. 3. Suicide variants show increased binding to LRRTM2 in vitro.

a Representative confocal images of Fc cell surface binding assays. b-c Quantification of Fc-binding assays, normalized to control-Fc. b Quantification of experiments using 1.0 ug/mL concentration of Fc proteins. c Quantification of the same assay but using 0.1 and 0.5 ug/mL of P469S-Fc only. (****p < 0.0001, One-way ANOVA with Dunnett’s T3 multiple comparisons tests, *p = 0.02, multiple t tests; n = 39 (WT), n = 48 (P469S), n = 37 (H885Y) cells from three different cultures; ±SEM reported).

Fig. 4. P469S variant induces hemi-synapses similar to WT in vitro.

a Representative confocal images of the hemi-synapse assay. 293 cells were transfected with GFP (control) or co-transfected with full-length WT+GFP and variant+GFP constructs. MAP2 staining (white) indicates dendrites. For excitatory synapses, presynaptic vGLUT1 (blue) and postsynaptic PSD-95 (red) markers were used. For inhibitory synapses, presynaptic vGAT (blue) and postsynaptic gephyrin (red) markers were used. b-c Quantification of the excitatory and inhibitory hemi-synapse assays. b P469S variant induces excitatory synapses similar to WT(***p = 0.0004, **p = 0.0036, One-way ANOVA analysis with Dunnett’s T3 multiple comparisons tests, ns p > 0.05 for all other comparisons; n = 41 [GFP], n = 33 [WT], n = 29 [P469S] cells from three different cultures; ±SEM reported). c Nrxn1α does not induce inhibitory synapses and variants do not change its activity (n = 46 [GFP], n = 50 [WT], n = 27 [P469S] cells from three different cultures; ±SEM reported); no significant differences by one-way ANOVA.