Epigenetic regulation of GABA catabolism in iPSC-derived neurons: The molecular links between FGF21 and histone methylation.

OBJECTIVE: Fibroblast growth factor 21 (FGF21) analogs have been tested as potential therapeutics for substance use disorders. Prior research suggests that FGF21 administration might affect alcohol consumption and reward behaviors. Our recent report showed that plasma FGF21 levels were positively correlated with alcohol use in patients with alcohol use disorder (AUD). FGF21 has a short half-life (0.5-2 h) and crosses the blood-brain barrier. Therefore, we set out to identify molecular mechanisms for both the naïve form of FGF21 and a long-acting FGF21 molecule (PF-05231023) in induced pluripotent stem cell (iPSC)-derived forebrain neurons. METHODS: We performed RNA-seq in iPSC-derived forebrain neurons treated with naïve FGF21 or PF-05231023 at physiologically relevant concentrations. We obtained plasma levels of FGF21 and GABA from our previous AUD clinical trial (n = 442). We performed ELISA for FGF21 in both iPSC-derived forebrain neurons and forebrain organoids. We determined protein interactions using co-immunoprecipitation. Finally, we applied ChIP assays to confirm the occupancy of REST, EZH2 and H3K27me3 by FGF21 using iPSC-derived forebrain neurons with and without drug exposure. RESULTS: We identified 4701 and 1956 differentially expressed genes in response to naïve FGF21 or PF-05231023, respectively (FDR < 0.05). Notably, 974 differentially expressed genes overlapped between treatment with naïve FGF21 and PF-05231023. REST was the most important upstream regulator of differentially expressed genes. The GABAergic synapse pathway was the most significant pathway identified using the overlapping genes. We also observed a significant positive correlation between plasma FGF21 and GABA concentrations in AUD patients. In parallel, FGF21 and PF-05231023 significantly induced GABA levels in iPSC-derived neurons. Finally, functional genomics studies showed a drug-dependent occupancy of REST, EZH2, and H3K27me3 in the promoter regions of genes involved in GABA catabolism which resulted in transcriptional repression. CONCLUSIONS: Our results highlight a significant role in the epigenetic regulation of genes involved in GABA catabolism related to FGF21 action. (The ClinicalTrials.gov Identifier: NCT00662571).