Potential mechanisms of the antidepressant effects of FGF2: Gene expression changes in postmitotic human excitatory neurons

Abstract

Many neuropsychiatric disorders are hypothesized to be due to subtle changes in neural circuit formation during development. Here we utilized human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to model mature, postmitotic excitatory neurons to examine the molecular effects of FGF2. In postmortem studies of major depressive disorder (MDD) patients FGF2 gene expression has been demonstrated to be altered in brain regions known to be dysfunctional from fMRI imaging studies. In addition, FGF2 has been shown to have anti‐depressive affects in animal models of depression. Recently it has been demonstrated that viral transduction of neurogenin2, a neurogenic bHLH transcription factor into hiPSCs yields populations of post‐mitotic neurons homogeneously expressing glutamatergic markers.In our studies, we have used a Tol2 recombinase system for gene transfer that allows generation of stable clonal hESC and hIPSC lines. These lines can be reproducibly differentiated into neurons by treatment with doxycycline (dox) to generate a homogenous population of stably induced glutamatergic neurons (siNeurons). We have used these siNeurons to study the gene expression changes involved in the transition from stem cell to neuron and to study the effects of FGF2 on neuronal gene expression. Approximately 95% of the cells are post‐mitotic after 6 days of induction with dox. Using RNA‐Seq over the time course of 11 days of dox treatment, we identified many key neuronal genes that were upregulated and several pluripotency genes that were downregulated. The RNA‐Seq findings were confirmed using qPCR and key protein expression changes were verified using western blotting. The morphological changes over the time course were also confirmed using immunostaining experiments for neuronal marker proteins. Using stem cell lines that inducibly express neurogenin2 as well as GCamp6f, we were able to characterize spontaneous calcium activity in these neurons and showed that 60% cells are able to spontaneously fire after 6 days of differentiation.In further studies, FGF2‐responsive genes were determined by RNA‐Seq after two independent siNeuron lines were treated in presence or absence of 20nM FGF2. Over one hundred genes were identified to be upregulated as a result of chronic FGF2 treatment. Many of these gene expression changes were confirmed using qRT‐PCR and western blotting. To determine the downstream signaling pathway that mediates FGF2 transcriptional regulation, we are using pharmacological inhibitors of the four major pathways activated by FGF2 binding to FGFRs (RAS‐MAP kinase, PI3‐kinase, STAT, and PLCγ‐PKA). Further investigation of FGF2 regulation of the excitatory neuron network activity by calcium imaging and its impact of maturation of neurons and synapses are underway in our lab. We believe that these dox‐inducible siNeuron stem cell lines will serve as a tractable model to understand the cellular events and the transcriptional changes that ultimately lead to formation of functional human neuronal circuitry.Support or Funding InformationThis work was funded by the Pritzker Neuropsychiatric Disorders Research Consortium, which is supported by the Pritzker Neuropsychiatric Disorders Research Fund L.L.C. A shared intellectual property agreement exists between this philanthropic fund and the University of Michigan, Stanford University, the Weill Medical College of Cornell University, the University of California at Irvine, and the Hudson Alpha Institute for Biotechnology to encourage the development of appropriate findings for research and clinical applications.