Ponomareva, O.; Seabrook, L. T.; Maya-Martinez, M.; Klengel, C.; Balakundi, V.; Kini, S.; Catt, E.; Zion, J.; Shah, R.; Martinez, P.; Hernandez, E.; Beatty, Z.; Millet, M. S.; Flanagan-Burt, Q.; Lussier, A.; Nievergelt, C. M.; Maihofer, A. X.; Koenen, K.; PTSD Working Group of Psychiatric Genomics Consortium, ; PsychENCODE PTSD BrainOmics Project, ; Kleinman, J. E.; Suh, J.; Carlezon, W. A.; Daskalakis, N. P.; Ressler, K. J.

Post-traumatic Stress Disorder (PTSD) is a debilitating psychiatric condition caused by severe trauma exposure and characterized by ongoing dysregulation of fear processing, hyperarousal, and amygdala activation, but with limited effective treatments. Recent large-scale genome-wide association studies (GWAS) of PTSD have identified the transcription factor FOXP2 as a highly-significant, top putative risk gene. Both fine-mapping of the PTSD GWAS with amygdala-specific expression quantitative trait loci (eQTL) data, and transcriptome-wide association analyses, show that altered expression of FOXP2 is associated with increased PTSD risk. In vertebrates, FOXP2 mRNA is most densely expressed in the intercalated cells (ITCs) of the amygdala. ITC neurons receive excitatory input from external regulatory and sensory brain regions, as well as the basolateral amygdala, and send inhibitory projections to the central amygdala, which regulates downstream fear responses. While ITCs are critical for conditioned fear acquisition and extinction, the role of the FOXP2 gene in modulating fear-related behaviors remains unknown. Here, we used complementary bioinformatic, molecular, circuit, behavioral, and electrophysiological approaches to characterize the function of mouse (Foxp2) and human (FOXP2) orthologs in amygdala-mediated fear learning. To assess Foxp2 function in vivo, we first used shRNA-mediated knockdown (KD) of Foxp2 in ITC neurons of adult mice. Targeted Foxp2 KD robustly and significantly reduced freezing (threat/fear expression) during and after auditory fear conditioning. Whole-cell recordings from individual ITC neurons revealed that Foxp2 KD increased their intrinsic membrane excitability and action potential frequency, consistent with hypothesized enhanced inhibitory output to the central amygdala and thus reduced fear expression. This hyperexcitability was associated with reduced potassium channel conductance. Bulk RNA sequencing (RNA-seq) of mouse amygdala after ITC Foxp2 KD confirmed decreased potassium channel transcription and revealed broader Foxp2-dependent regulation of multiple genes implicated in fear learning, including Wnt, Crh (which encodes corticotropin-releasing hormone), and neurokinin signaling pathways. Consistent with these findings, bulk RNA-seq of medial amygdala postmortem tissue from humans with PTSD versus neurotypical controls showed decreased potassium channel transcription in samples with low FOXP2 expression. Downstream transcriptional changes following Foxp2 KD in the mouse amygdala also showed marked enrichment of genes identified in PTSD risk loci from the largest PTSD GWAS to date. Specifically, downregulated genes were enriched for mouse orthologs of Tier 1 PTSD GWAS risk genes. This enrichment appears to reflect subcortical Foxp2 signaling within the amygdala, driven predominantly by decreased expression of genes lacking promoter-anchored chromatin loops. This finding suggests that Foxp2 may directly bind regulatory elements of multiple top PTSD risk genes, acting as a key regulatory node for fear-related pathways in the amygdala. Collectively, our findings establish FOXP2 as a central transcriptional regulator of fear-related gene networks in the amygdala and potential regulatory hub for PTSD genetic risk.