The Immediate Early Gene Egr3 Is Required for Hippocampal Induction of Bdnf by Electroconvulsive Stimulation.

Kimberly T Meyers,Diano F Marrone,Amelia L Gallitano,Milad Charbel,Ketan K Marballi,Briana Renda,Samuel J Brunwasser

doi:10.3389/fnbeh.2018.00092

Abstract

Early growth response 3 (Egr3) is an immediate early gene (IEG) that is regulated downstream of a cascade of genes associated with risk for psychiatric disorders, and dysfunction of Egr3 itself has been implicated in schizophrenia, bipolar disorder, and depression. As an activity-dependent transcription factor, EGR3 is poised to regulate the neuronal expression of target genes in response to environmental events. In the current study, we sought to identify a downstream target of EGR3 with the goal of further elucidating genes in this biological pathway relevant for psychiatric illness risk. We used electroconvulsive stimulation (ECS) to induce high-level expression of IEGs in the brain, and conducted expression microarray to identify genes differentially regulated in the hippocampus of Egr3-deficient (-/-) mice compared to their wildtype (WT) littermates. Our results replicated previous work showing that ECS induces high-level expression of the brain-derived neurotrophic factor (Bdnf) in the hippocampus of WT mice. However, we found that this induction is absent in Egr3-/- mice. Quantitative real-time PCR (qRT-PCR) validated the microarray results (performed in males) and replicated the findings in two separate cohorts of female mice. Follow-up studies of activity-dependent Bdnf exons demonstrated that ECS-induced expression of both exons IV and VI requires Egr3. In situ hybridization demonstrated high-level cellular expression of Bdnf in the hippocampal dentate gyrus following ECS in WT, but not Egr3-/-, mice. Bdnf promoter analysis revealed eight putative EGR3 binding sites in the Bdnf promoter, suggesting a mechanism through which EGR3 may directly regulate Bdnf gene expression. These findings do not appear to result from a defect in the development of hippocampal neurons in Egr3-/- mice, as cell counts in tissue sections stained with anti-NeuN antibodies, a neuron-specific marker, did not differ between Egr3-/- and WT mice. In addition, Sholl analysis and counts of dendritic spines in golgi-stained hippocampal sections revealed no difference in dendritic morphology or synaptic spine density in Egr3-/-, compared to WT, mice. These findings indicate that Egr3 is required for ECS-induced expression of Bdnf in the hippocampus and suggest that Bdnf may be a downstream gene in our previously identified biologically pathway for psychiatric illness susceptibility.

Highlights

The risk to develop neuropsychiatric illnesses is determined by both genetic and environmental factors
brain-derived neurotrophic factor (Bdnf) was of particular interest to us, as it has been shown to contribute to the therapeutic effects of antidepressant treatments, including electroconvulsive seizure (ECS), in rodent models (Altar et al, 2003; Adachi et al, 2008; Inta et al, 2013)
Results of the expression microarray demonstrated that, in WT mice, ECS produced a 2.5-fold increase in Bdnf mRNA levels, measured 1 h following seizure, compared to baseline unstimulated controls. This induction is dependent upon early growth response 3 (Egr3), as Egr3−/− mice did not show a change in Bdnf mRNA expression following seizure (Figure 1A)

Summary

Introduction

The risk to develop neuropsychiatric illnesses is determined by both genetic and environmental factors. We have hypothesized that immediate early gene (IEG) transcription factors are poised to modulate this dual contribution, as they are rapidly activated in the brain in response to environmental stimuli and, in turn, influence numerous neurobiological processes that are dysfunctional in the brains of patients with mental illness (Gallitano-Mendel et al, 2007, 2008; Huentelman et al, 2015). EGR3 was not identified within one of the 108 loci found to be associated with schizophrenia risk in the Psychiatric Genomics Consortium genome-wide association study (GWAS), numerous genes encoding proteins that regulate or are regulated by EGR3 do map to these schizophrenia-associated loci (Schizophrenia Working Group of the Psychiatric Genomics Consortium, 2014; Marballi and Gallitano, 2018)

Methods

Results

Conclusion