Integration of postmortem amygdala expression profiling, GWAS, and functional cell culture assays: neuroticism-associated synaptic vesicle glycoprotein 2A (SV2A) gene is regulated by miR-133a and miR-218

Magdalena Jurkiewicz,Dirk Moser,Lei Yu,Turhan Canli,Antonius Koller,Emily I Chen,David A Bennett

doi:10.1038/s41398-020-00966-4

Abstract

Recent genome-wide studies have begun to identify gene variants, expression profiles, and regulators associated with neuroticism, anxiety disorders, and depression. We conducted a set of experimental cell culture studies of gene regulation by micro RNAs (miRNAs), based on genome-wide transcriptome, proteome, and miRNA expression data from twenty postmortem samples of lateral amygdala from donors with known neuroticism scores. Using Ingenuity Pathway Analysis and TargetScan, we identified a list of mRNA–protein–miRNA sets whose expression patterns were consistent with miRNA-based translational repression, as a function of trait anxiety. Here, we focused on one gene from that list, which is of particular translational significance in Psychiatry: synaptic vesicle glycoprotein 2A (SV2A) is the binding site of the anticonvulsant drug levetiracetam ((S)-α-Ethyl-2-oxo-1-pyrrolidineacetamide), which has shown promise in anxiety disorder treatments. We confirmed that SV2A is associated with neuroticism or anxiety using an original GWAS of a community cohort (N = 1,706), and cross-referencing a published GWAS of multiple cohorts (Ns ranging from 340,569 to 390,278). Postmortem amygdala expression profiling implicated three putative regulatory miRNAs to target SV2A: miR-133a, miR-138, and miR-218. Moving from association to experimental causal testing in cell culture, we used a luciferase assay to demonstrate that miR-133a and miR-218, but not miR-138, significantly decreased relative luciferase activity from the SV2A dual-luciferase construct. In human neuroblastoma cells, transfection with miR-133a and miR-218 reduced both endogenous SV2A mRNA and protein levels, confirming miRNA targeting of the SV2A gene. This study illustrates the utility of combining postmortem gene expression data with GWAS to guide experimental cell culture assays examining gene regulatory mechanisms that may contribute to complex human traits. Identifying specific molecular mechanisms of gene regulation may be useful for future clinical applications in anxiety disorders or other forms of psychopathology.

Highlights

Neuroticism is a heritable personality trait[1,2,3,4] that shares genetic overlap with anxiety and depression[5,6,7,8,9,10].Large-scale genome-wide association studies (GWAS)have identified single nucleotide variants (SNPs) associated with neuroticism, anxiety disorders, and depression[10,11,12]
Since we focused on translational repression, we only included gene sets where a given protein and target miRNA showed an inverse expression relationship, and the protein-coding mRNA showed no expression change as a function of trait anxiety status
16 mRNA–protein–miRNA sets whose expression patterns were consistent with translational repression as a function of trait anxiety (Supplementary Table S2)

Summary

Introduction

Have identified single nucleotide variants (SNPs) associated with neuroticism, anxiety disorders, and depression[10,11,12]. Genome-wide expression profiling studies of Jurkiewicz et al Translational Psychiatry (2020)10:297 human brain have begun to identify genes that are differentially expressed as a function of major depressive disorder[13] or that affect brain structures[14]. Recent work has begun to identify epigenetic regulators of gene expression as potentially useful biomarkers and therapeutic agents in depression[15,16,17,18,19]. One gene regulatory mechanism of interest with potential therapeutic applications involves microRNAs (miRNAs), which are short (20–23 nucleotides in length), single-stranded, endogenous RNAs that interact with mRNA to regulate gene expression through mechanisms that include mRNA transcript degradation and downregulation of translation of mRNA-to-protein through translational repression[20,21]. Each miRNA can regulate hundreds of genes and affects multiple cellular processes relevant to health and disease including psychiatric and neurological disorders[19,22,23]

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Results

Conclusion