Behavioural and functional evidence revealing the role of RBFOX1 variation in multiple psychiatric disorders and traits

Abstract

Common variation in the gene encoding the neuron-specific RNA splicing factor RNA Binding Fox-1 Homolog 1 (RBFOX1) has been identified as a risk factor for several psychiatric conditions, and rare genetic variants have been found causal for autism spectrum disorder (ASD). Here, we explored the genetic landscape of RBFOX1 more deeply, integrating evidence from existing and new human studies as well as studies in Rbfox1 knockout mice. Mining existing data from large-scale studies of human common genetic variants, we confirmed gene-based and genome-wide association of RBFOX1 with risk tolerance, major depressive disorder and schizophrenia. Data on six mental disorders revealed copy number losses and gains to be more frequent in ASD cases than in controls. Consistently, RBFOX1 expression appeared decreased in post-mortem frontal and temporal cortices of individuals with ASD and prefrontal cortex of individuals with schizophrenia. Brain-functional MRI studies demonstrated that carriers of a common RBFOX1 variant, rs6500744, displayed increased neural reactivity to emotional stimuli, reduced prefrontal processing during cognitive control, and enhanced fear expression after fear conditioning, going along with increased avoidance behaviour. Investigating Rbfox1 neuron-specific knockout mice allowed us to further specify the role of this gene in behaviour. The model was characterised by pronounced hyperactivity, stereotyped behaviour, impairments in fear acquisition and extinction, reduced social interest, and lack of aggression; it provides excellent construct and face validity as an animal model of ASD. In conclusion, convergent translational evidence shows that common variants in RBFOX1 are associated with a broad spectrum of psychiatric traits and disorders, while rare genetic variation seems to expose to early-onset neurodevelopmental psychiatric disorders with and without developmental delay like ASD, in particular. Studying the pleiotropic nature of RBFOX1 can profoundly enhance our understanding of mental disorder vulnerability.

Fig. 1. Genetic risk variants in RBFOX1 in different psychiatric conditions and traits.

A Common single-nucleotide variants in RBFOX1 showed a gene-based association with most disorders and traits tested; B Copy number variants (CNVs) identified in ASD and SCZ patients. Top panel, copy number gains identified in ASD and SCZ patients. Bottom panel, CN losses identified in ASD and SCZ patients. Each bar represents a CNV. ADHD attention-deficit/hyperactivity disorder, AGG aggression, ANO anorexia, ANX anxiety, ASD autism spectrum disorder, BIP bipolar disorder, MDD major depressive disorder, OCD obsessive-compulsive disorder, RT risk tolerance behaviour, SCZ schizophrenia, TS Tourette’s syndrome, CD cross-disorder meta-analysis. p-val p-value.

Fig. 2. Effects of the rs6500744 RBFOX1 genotype on brain responses during implicit emotion processing and executive functioning in healthy adults, and on fear learning in patients with panic disorder and agoraphobia.

A left panel: Schematic overview of the face matching task. Participants had to select either one of the two faces or forms shown at the bottom of the screen that was identical to the target stimulus shown at the top of the screen. A right panel: C-allele carrier (C/C and C/T) showed increased brain responses in the dorsal anterior cingulate cortex (dACC) compared to those with the T/T genotype during matching faces vs. forms (faces > forms; MNI coordinate: x = 15, y = 23, z = 27, peak-voxel family-wise error-corrected [FWE] P = 0.010, T = 3.9 within bilateral ACC). B left panel: Schematic overview of the Flanker/Go-NoGo task. Participants had to respond to the direction of the arrow shown in the centre (red box for illustration purposes only) B right panel: C-allele carriers (C/C and C/T) showed reduced brain responses in the left dorsolateral prefrontal cortex (L dlPFC) compared to those homozygous for the T allele during executive functioning (contrast: [incongruent & nogo] > [congruent & neutral]; MNI coordinates: x = −54, y = 32, z = 21, peak-voxel pFWE-corrected=0.039, T = 4.55, across the whole-brain). Brain maps were thresholded at p < 0.001 uncorrected for display purposes. Error bars indicate ± 1 standard error. C left panel: Schematic overview of the fear conditioning and extinction task. During the acquisition phase, 50% of CS+ was paired pseudo-randomly with the US and 50% were not. Only those trials in which no US was delivered were analysed during acquisition to avoid overlap with neuron activation directly related to the presentation of the US. C right panel: Using ROI analysis within the ACC, homozygote risk allele carriers (C/C) compared to T/T homozygotes revealed increased activation in the dACC for CS+ after fear acquisition (CS+ in the late acquisition> CS+ in the late familiarization; cluster size = 61; peak-voxel family-wise error-corrected [FWE] P = 0.014, T = 3.87), and activation reduction for CS+ after fear extinction (CS+ in the late acquisition >CS+ in the late extinction; cluster size = 11; peak-voxel family-wise error-corrected [FWE] P = 0.018, T = 3.86). Brain maps were thresholded at p < 0.001 uncorrected for display purposes. Error bars indicate ± 1 standard error.

Fig. 3. Effects of neuron-specific Rbfox1 deletion on behavioural measures in male mice.

A open field test: Rbfox1-KO mice displayed hyperactivity and reduced time in the centre in the open field test (CTRL: n = 21; HET: n = 8; KO: n = 8; **p < 0.01; *** p < 0.001 vs CTRL; ^##p < 0.01; ^###p < 0.001 vs HET; one-way ANOVA, Bonferroni test); B open field test and novel object exploration in 8-month-old mice: KO mice spent longer investigating a novel object placed into the open field (CTRL: n = 8; KO: n = 4; **p < 0.01 vs CTRL, Mann-Whitney test); C light-dark box test: KO mice again were hyperactive and spent more time in the dark zone (CTRL: n = 21; HET: n = 8; KO: n = 8; ***p < 0.001 vs CTRL; two-way ANOVA (genotype x zone), Bonferroni test); D pre-pulse inhibition test: KO mice had markedly reduced startle amplitude without changes in the sensorimotor gating (CTRL: n = 21; HET: n = 8; KO: n = 8; **p < 0.01; ***p < 0.001 vs CTRL; ^#p < 0.05 vs HET; repeated measures ANOVA, Bonferroni test); E auditory fear conditioning and extinction: fear acquisition and extinction was impaired in the KO mice, and HET mice displayed impaired fear retention (CTRL: n = 21; HET: n = 8; KO: n = 8; *p < 0.05; **p < 0.01; ***p < 0.001 vs CTRL; repeated measures ANOVA, Bonferroni test); F touchscreen visual pairwise discrimination task: acquisition of the task was similar in CTRL and KO (CTRL: n = 5; KO: n = 4; repeated measures ANOVA); G spontaneous alternations in the Y-maze: the number of spontaneous alternations was not changed in KO (Kruskall-Wallis test) although the distance travelled during the test was significantly higher than CTRL (n = 8–16 per group; ***p < 0.001 vs CTRL; ^##p < 0.01 vs HET; one-way ANOVA, Bonferroni test); H social interaction: KO spent significantly less time investigating unfamiliar stimulus mice (CTRL: n = 7; KO: n = 4; *p < 0.05; **p < 0.01; ***p < 0.001 vs CTRL; Welch’s t-tests); I escalated aggression paradigm: while aggressive behaviour increased during repeated sessions in CTRL, KO remained non-aggressive throughout testing (CTRL: n = 6; KO: n = 5; repeated measures ANOVA). Data is presented as means ± S.E.M.