FAAH genetic variation enhances fronto-amygdala function in mouse and human

Abstract

Cross-species studies enable rapid translational discovery and produce the broadest impact when both mechanism and phenotype are consistent across organisms. We developed a knock-in mouse that biologically recapitulates a common human mutation in the gene for fatty acid amide hydrolase (FAAH) (C385A; rs324420), the primary catabolic enzyme for the endocannabinoid anandamide. This common polymorphism impacts the expression and activity of FAAH, thereby increasing anandamide levels. Here, we show that the genetic knock-in mouse and human variant allele carriers exhibit parallel alterations in biochemisty, neurocircuitry and behaviour. Specifically, there is reduced FAAH expression associated with the variant allele that selectively enhances fronto-amygdala connectivity and fear extinction learning, and decreases anxiety-like behaviours. These results suggest a gain of function in fear regulation and may indicate for whom and for what anxiety symptoms FAAH inhibitors or exposure-based therapies will be most efficacious, bridging an important translational gap between the mouse and human.

Figure 1. Generation and validation of FAAH C385A knock-in mice

(a) A portion of coding region in the FAAH gene is replaced with C385A SNP. (b) The region encompassing the SNP has high homology between human and mouse FAAH genes. (c) FAAH protein levels in knock-in mice (FAAH^C/A; FAAH^A/A) and wild-type (FAAH^C/C) littermates from 3 independent western blot analyses. Brain homogenates from FAAH^−/− mice, and lysates from heterologous 293 cells overexpressing FAAH, were used as controls. (d) FAAH hydrolytic activity, and (e) anandamide (AEA) content in FAAH knock-in mouse forebrain homogenates (n = 4 per group). (EA; ethanolamine) Means ± SEM presented. *P < 0.05, **P < 0.01

Figure 2. Functional and structural connectivity between ventromedial prefrontal cortex (vmPFC) and amygdala in humans and mice with FAAH C385A

(a) fMRI functional connectivity compared between subgenual vmPFC (x, y, z = 0, 40, −3) and bilateral amygdala in A-allele carriers (n = 17) relative to C homozygotes (n = 18). (b) Anterograde tracer (AAV2-eGFP; eGFP), targeted to IL, labeled afferents in BLA in FAAH^A/A mice (n = 4) and controls (FAAH^C/C; n = 4). Drawing illustrates anatomical boundaries. (c) Retrograde tracer (fluorogold; FG), targeted to IL, labeled BLA cell bodies in FAAH^A/A mice (n = 4) and controls (FAAH^C/C; n = 4). (Scale bars: 100µm) Means ± SEM presented. *P < 0.05, ***P < 0.001

Figure 3. Enhanced cued fear extinction in humans and mice with FAAH C385A

(a) Extinction, indexed by differential skin conductance response (SCR) [(CS+) – (CS−)], in 18 A-allele carriers and 22 C homozygotes. Trials were binned into early (average of the first 5 trials) and late (average of the following 6 trials). (b) Fear extinction, time spent freezing to cue, was tested in wild type (FAAH^C/C; n = 21), heterozygous (FAAH^C/A; n = 20) and homozygous knock-in mice (FAAH^A/A; n = 20). Extinction trials were binned into early (average of extinction day 1) and late trials (average of extinction day 4). Means ± SEM presented. *P < 0.05, #P < 0.05 heterozygous knock-in mice vs wild-type controls, **P < 0.01 homozygous knock-in mice vs wild-type controls

Figure 4. Decreased anxiety in humans and mice with FAAH C385A

(a) Trait anxiety levels in A-allele carriers (n = 57) and C homozygotes (n = 80). Anxiety-related behavior in FAAH C385A mice in (b) elevated plus maze (FAAH^C/C, n = 17; FAAH^C/A, n = 11; FAAH^A/A, n = 12) and (c) novelty induced hypophagia (NIH) (FAAH^C/C, n = 16; FAAH^C/A, n = 21; FAAH^A/A, n = 10). (d) Photomicrographs of NIH-induced c-Fos (red) labeling in the BLA (parvalbumin (green)) in FAAH C385A mice. Density of c-Fos immunoreactive (IR) cells in BLA, n = 5 per group. (Scale bars: 50µm upper row, 100µm lower row) Means ± SEM presented. *P < 0.05, **P < 0.01, ****P < 0.0001