A rare variant in GPR156 associated with depression in a Mennonite pedigree causes habenula hyperactivity and stress sensitivity in mice

Abstract

Major depressive disorder (MDD) is a leading cause of disability worldwide. Risk for MDD is heritable, and the genetic structure of founder populations enables investigation of rare susceptibility alleles with large effect. In an extended Old Order Mennonite family cohort, we identified a rare missense variant in GPR156 (c.1599G>T, p.Glu533Asp) associated with a two-fold increase in the relative risk of MDD. GPR156 is an orphan G protein-coupled receptor localized in the medial habenula, a region implicated in mood regulation. Insertion of a human sequence containing c.1599G>T into the murine Gpr156 locus induced medial habenula hyperactivity and abnormal stress-related behaviors. This work reveals a human variant that is associated with depression, implicates GPR156 as a target for mood regulation, and introduces informative murine models for investigating the pathophysiology and treatment of affective disorders.

Keywords: GPR156; genetics; major depressive disorder; medial habenula; orphan G protein-coupled receptor.

Fig. 1.

Old Order Mennonite pedigree harboring GPR156 c.1599G>T (p.Glu533Asp) allele. Participants with MDD and bipolar disorder type 1 (BP1) are shown with red and yellow shading, respectively. The proband is indicated with a vertical arrow. 0/1 denotes a GPR156 c.1599G>T heterozygous genotype.

Fig. 2.

Association of GPR156 c.1599G>T with affective illness. (A) In the informative sibships, 48% of offspring heterozygous for the variant had a major affective disorder diagnosis, whereas 24% of offspring homozygous for the reference allele had major affective disorder diagnosis; RR = 2.03, 95% CI [0.94, 4.84], P = 0.089 (Fisher exact test, 2- tailed). (B) The age of onset (Left axis), number of depressive symptoms (Right axis), and frequency of episodes (Right axis) did not differ between individuals with MDD who harbored the GPR156 c.1599G>T variant (red circles, n = 12) and affected individuals who did not (gray circles, n = 7).

Fig. 3.

Expression of Gpr156 in the medial habenula. (A) Replacement of the endogenous mouse Gpr156 locus with a LacZ reporter cassette shows the pattern of Gpr156 expression in adult mouse brain. A sagittal brain section reveals the highest expression in the habenula as indicated by the intensely dark staining. Areas of lower but significant expression include the olfactory bulb, fasciculus retroflexus, interpeduncular nucleus (IPN), colliculus, and dorsal brainstem nuclei. (B) In situ mRNA hybridization (RNAscope) of wildtype mouse brain shows abundant Gpr156 mRNA (red) in the medial habenula; no such expression was detected in Gpr156^−/−animals (SI Appendix, Fig. S3). (Scale bar, 400 µm.) (C) Higher magnification image of medial habenula demonstrates high level of Gpr156 expression. (Scale bar, 100 µm.) (D) Within the medial habenula, Gpr156 mRNA (green, perinuclear) localizes with Vglut2 expressing neurons, designated by td-tomato (red, cytoplasmic) expression. (Scale bar, 10 µm.) (E) The lateral (LHb) and medial (MHb) habenula receive diverse excitatory inputs (green lines), inhibitory inputs (red lines), and combined excitatory and inhibitory inputs (purple lines) from limbic and basal ganglia structures of the forebrain (, , –50). They project via the rostromedial (RM) tegmental and interpeduncular nuclei to brainstem nuclei with broad dopaminergic and serotonergic projections. These circuits are believed to play a critical role in mediating adaptive (and maladaptive) responses to rewarding or aversive stimuli. Black dotted line represents unknown neurotransmitter. Abbreviations. LHb: Lateral habenula. MHb: Medial habenula. RM: Rostromedial. N.: Nucleus.

Fig. 4.

The medial habenula is hyperactive in Gpr156 mutant mouse brains. (A) Schematic of cell-attached electrophysiological recordings from the medial habenula. (B) Representative traces from Gpr156 wildtype (Gpr156^+/+) and Gpr156 knockout (Gpr156^−/−) brain slices. (C) Increased spontaneous firing rate in the medial habenula of Gpr156−/− mice (14.7 Hz in versus 7.3 Hz, P < 0.0001, t test; n = 11 cells from 3 Gpr156^+/+ animals and 11 cells from 2 Gpr156^−/− animals. (D) Representative traces from brain slices of humanized Gpr156 control mice (Gpr156^hum/hum) and humanized Gpr156 c.1599G>T mutants (Gpr156^E533D/E533D). (E) Mean spontaneous firing rate in the medial habenula of Gpr156^E533D/E533D mutants was 12.8 Hz versus 7.1 Hz in humanized Gpr156^hum/hum controls (P = 0.0120, t test; n = 9 cells from 2 Gpr156^hum/hum animals and 12 cells from 2 Gpr156^E533D/E533D animals). MHb: Medial habenula. LHb: Lateral habenula. DG: Dentate gyrus. Thal: Thalamus.

Fig. 5.

Gpr156^−/− and Gpr156^E533D/E533D mice exhibit markedly aberrant behavior in the FST that can be rescued with antidepressant medications. (A) On the 6 min FST, Gpr156^+/+ and Gpr156^hum/hum mice swam vigorously around the reservoir with muzzle above water searching for an escape. In contrast, most Gpr156^−/− and Gpr156 ^E533D/E533D mice began to swim in an erratic and disorganized manner within 4 min, diving repeatedly beneath the water’s surface until they required rescue. (B) Only 14% and 12.5% of Gpr156^−/− and Gpr156^E533D/E533D mice, respectively, completed the 6 min FST. In contrast, 98% of Gpr156^+/+ and Gpr156^hum/hum mice completed the test unassisted. (C) Gpr156^hum/hum and Gpr156^E533D/E533D mice received intraperitoneal (i.p.) fluoxetine (10 mg/kg•day) 6 d weekly for 9 wk and were subjected to the FST at weeks 3, 6, and 9 of treatment. After 9 wk of fluoxetine therapy, 53% of Gpr156^E533D/E533D completed the 6 min FST as compared to 9% of the untreated group. The probability of completing the FST for 6 min improved significantly in Gpr156^E533D/E533D mice treated with either fluoxetine (panel D) or imipramine (panel E) (logrank and Gehan–Breslow–Wilcoxon tests, P < 0.0001).