JOURNAL OF NEUROCHEMISTRY | 2018 | 145 | 188–203 doi: 10.1111/jnc.14255 , *Molecular and Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, Michigan, USA †Department of Neurology, University of Michigan, Ann Arbor, Michigan, USA ‡Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, USA Abstract Using hES cell lines that inducibly express NEUROG2 as well Many neuropsychiatric disorders are thought to result from as GCaMP6f, we were able to characterize spontaneous subtle changes in neural circuit formation. We used human calcium activity in these neurons and show that calcium embryonic stem cells and induced pluripotent stem cells transients increase in the presence of FGF2. The FGF2- (hiPSCs) to model mature, post-mitotic excitatory neurons responsive genes were determined by RNA-Seq. FGF2- and examine effects of fibroblast growth factor 2 (FGF2). FGF2 regulated genes previously identified in non-neuronal cell types gene expression is known to be altered in brain regions of major were up-regulated (EGR1, ETV4, SPRY4, and DUSP6) as a depressive disorder (MDD) patients and FGF2 has anti- result of chronic FGF2 treatment of siNeurons. Novel neuron- depressive effects in animal models of depression. We gener- specific genes were also identified that may mediate FGF2- ated stable inducible neurons (siNeurons) conditionally dependent increases in synaptic efficacy including NRXN3, expressing human neurogenin-2 (NEUROG2) to generate a SYT2, and GALR1. Since several of these genes have been homogenous population of post-mitotic excitatory neurons and implicated in MDD previously, these results will provide the study the functional as well as the transcriptional effects of basis for more mechanistic studies of the role of FGF2 in MDD. FGF2. Upon induction of NEUROG2 with doxycycline, the vast Keywords: FGF2, glutamatergic, neurogenin-2, neuronal, majority of cells are post-mitotic, and the gene expression stem cells. profile recapitulates that of excitatory neurons within 6 days. J. Neurochem. (2018) 145, 188--203. Cover Image for this issue: doi: 10.1111/jnc.14175. Received August 4, 2017; revised manuscript received November 7, Major depressive disorder (MDD) is one of the most 2017; accepted November 10, 2017. common psychiatric disorders affecting about 28% of the Address correspondence and reprint requests to Dr Michael Uhler, Molecular and Behavioral Research Institute, University of Michigan, US population during their lifetime (Vandeleur et al. 2017). 109 Zina Pitcher Pl., Ann Arbor, MI 48109-2200, USA. E-mail: Because of the clinical and genetic heterogeneity of MDD, [email protected] its pathophysiology remains elusive (Tanti and Belzung Abbreviations used: ACC, anterior cingulate cortex; BDNF, brain- 2010). Previous research has demonstrated that alterations derived neurotrophic factor; DLPFC, dorsolateral prefrontal cortex; Dox, 0 in various neurotransmitters such as serotonin as well as doxycycline; EDTA, ethylenediaminetertaacetic acid; EdU, 5-ethynyl-2 - deoxyuridine; EGFP, enhanced green fluorescent protein; ESCs, embry- neurotrophic factors such as brain-derived neurotrophic onic stem cells; FGF, fibroblast growth factor; FGFR, fibroblast growth factors contribute to MDD (Duman and Aghajanian 2012). factor receptor; GDNF, glial cell line-derived neurotrophic factor; GO, More recently, reduced glutamatergic signaling has been gene ontology; GWAS, genome-wide association studies; iPSCs, linked to MDD (Eisch and Petrik 2012). Brain imaging of induced pluripotent stem cells; IRES, internal ribosome entry site; MDD patients suggests a volume reduction in hippocam- MAPK, mitogen-activated protein kinase; MDD, major depressive disorder; MEA, multielectrode array; PEI, polyethyleneimine; PLC/ pus, dorsolateral prefrontal cortex, and anterior cingulate PKA, phospholipase C/Protein kinase A; qRT-PCR, quantitative real- cortex (Sacher et al. 2012). Post-mortem analyses of these time PCR; RTK, receptor tyrosine kinase; STAT, signal transducer and same brain regions implicate several genes correlating with activator of transcription; TRE, tetracycline response elements. 188 © 2017 International Society for Neurochemistry, J. Neurochem. (2018) 145, 188--203 FGF2 regulation of excitatory neurons 189 fi MDD but the most signi cantly dysregulated genes in Materials and methods MDD patients belong to the fibroblast growth factor (FGF) family (Turner et al. 2016). Microarray hybridization Cell culture and transfection of human embryonic stem cells studies of the dorsolateral prefrontal cortex region of H9 embryonic stem cells (ESCs) were obtained from WiCell Research MDD patients show that the ligand FGF9 is up-regulated Resources (WiCell, Madison, WI, USA; RRID: CVCL_9773); the relative to controls while the ligand FGF2 and the FGF control induced pluripotent stem cells (iPSC) line was derived from fi receptors (FGFR2 and FGFR3) are down-regulated (Evans commercially available human foreskin broblasts (MTI Global Systems, Gaithersburg, Maryland, USA; Cat. No. GSC-3002) and et al. 2004). reprogrammed using episomal plasmids as described (Okita et al. Animal studies using various models of depression in 2011). Both ESCs and iPSCs were maintained in feeder-free rodents have shown anti-depressant effects of FGF2 (Jarosik conditions using Matrigel substrate (Corning; Corning, NY, USA et al. 2011). Rats undergoing social defeat stress showed Cat. No. CB40234B), mTeSR8 medium (E8-Stem Cell Technologies, decreased hippocampal FGF2 expression and administration Cambridge, MA, USA; Cat. No. 05940), and passaged every 4– of FGF2 reversed the behavioral correlates of depression 5 days with EDTA as described (Beers et al. 2012). The H9 ESC line (Elsayed et al. 2012). Furthermore, treatment of adult rats is not a commonly misidentified cell line (iclac.org/databases/cross- with anti-depressants resulted in increased FGF2 expression contaminations). All human cell culture experiments were approved in cerebral cortex and hippocampus (Mallei et al. 2002; by the University of Michigan Human Pluripotent Stem Cell Research Bachis et al. 2008). FGF2 knockout mice displayed Oversight Committee and the University of Michigan Institutional increased anxiety and the phenotype was completely rescued Review Board. upon treatment with FGF2 in adulthood (Salmaso et al. 2016). It has also been established that FGF2 treatment of rat Generation of siNeuron hES and hiPSC lines using Tol2 recombinase – cortical neurons enhances of glutamatergic signaling (Li Stem cells were plated onto Matrigel-coated plates at a density of 1 5 9 104 cells per well in the presence of 1 lM Y-26732 (Tocris et al. 2002). These reports together implicate a role for FGF2 Bio, Minneapolis, MN, USA; Cat. No. 1254) in E8 media. The regulation of fully developed neurons in mediating anti- following day, the media was changed to E8 alone, and cells were depressant effect. transfected with a total of 2.5 lg of Tol2-plasmids (Balciunas et al. FGF2 is the most abundant ligand in the developing brain 2006). Plasmids to be chromosomally integrated contained the Tol2 (Vaccarino et al. 1999; Ford-Perriss et al. 2001) and the arms and were derived from the pMiniTol2 parental plasmid human genome contains 22 genes encoding FGF ligands as (Balciunas et al. 2006). These plasmids included the pMT-US2- well as four genes encoding receptors, most of which are TetOn-internal ribosome entry site-enhanced green fluorescent expressed in the developing nervous system (Guillemot and protein (EGFP) (20% of transfected DNA), pMT-tetracycline Zimmer 2011). All four FGFR genes encode single trans- response elements-hNeurog2 (20% of transfected DNA), and – membrane protein with extracellular immunoglobulin pMT-US2-puro (5 10%) which were generated from previously domain and intracellular tyrosine kinase domains. The described plasmids lacking the minitol integration arms (Huang et al. 2010, 2015). In addition to the integrating plasmids, the binding of an FGF ligand to an FGFR causes dimerization transfected DNA contained 40–50% of the pUS2-Tol2 plasmid resulting in autophosphorylation and activation of four major which expresses the Tol2 recombinase. pUS2-Tol2 was generated intracellular pathways: Ras/MAP kinase, phospholipase C/ by excising the Tol2 coding region from pCMV-Tol2 (Balciunas protein kinase A, signal transducer and activator of tran- et al. 2006) and inserting it into the multiple cloning site of the scription, and/or PI3 kinase signaling pathway. Once pUS2 plasmid containing the human ubiquitin C promoter. activated, these pathways phosphorylate transcription factors Following transfection of 2.5 lg of mixed plasmid DNA using to alter the expression of FGF-dependent genes (Ornitz and Mirus LT1 (Thermo Fisher, Waltham, MA, USA; Cat. No. MIR2300), Itoh 2015). the cells were fed with E8 media after 24 h. At 72 h after transfection, In order to better understand the specific role of FGF2 in cells were treated with 0.8 lg/mL puromycin in E8 media for 24–48 h human neuronal function, we investigated the global tran- followed by growth in E8 media. Approximately 1 week later, scriptional effects of FGF2 in post-mitotic human gluta- colonies of approximately 500 cells were picked manually into matergic neurons and the signaling mechanisms that control individual wells of a 24-well plate. The clones were expanded and replicate cultures were tested for differentiation in the presence of dox. these transcriptional changes. We generated doxycycline (dox)-inducible stable human