Molecular Psychiatry (2012) 17, 267–279 & 2012 Macmillan Publishers Limited All rights reserved 1359-4184/12 www.nature.com/mp ORIGINAL ARTICLE Transcriptional regulation of neurodevelopmental and metabolic pathways by NPAS3 L Sha1,7, L MacIntyre2,7, JA Machell1, MP Kelly3, DJ Porteous1, NJ Brandon3, WJ Muir4,{, DH Blackwood4, DG Watson2, SJ Clapcote5 and BS Pickard2,6 1Department of Medical Genetics, Institute for Genetics and Molecular Medicine, University of Edinburgh, Molecular Medicine Centre, Western General Hospital, Edinburgh, UK; 2Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK; 3Pfizer, Pfizer Global Research and Development, Neuroscience Research Unit, Groton, CT, USA; 4Division of Psychiatry, University of Edinburgh, Royal Edinburgh Hospital, Edinburgh, UK; 5Institute of Membrane and Systems Biology, University of Leeds, Leeds, UK and 6CeNsUS—Centre for Neuroscience, University of Strathclyde, Glasgow, UK The basic helix-loop-helix PAS (Per, Arnt, Sim) domain transcription factor gene NPAS3 is a replicated genetic risk factor for psychiatric disorders. A knockout (KO) mouse model exhibits behavioral and adult neurogenesis deficits consistent with human illness. To define the location and mechanism of NPAS3 etiopathology, we combined immunofluorescent, transcriptomic and metabonomic approaches. Intense Npas3 immunoreactivity was observed in the hippocampal subgranular zone—the site of adult neurogenesis—but was restricted to maturing, rather than proliferating, neuronal precursor cells. Microarray analysis of a HEK293 cell line over-expressing NPAS3 showed that transcriptional targets varied according to circadian rhythm context and C-terminal deletion. The most highly up-regulated NPAS3 target gene, VGF, encodes secretory peptides with established roles in neurogenesis, depression and schizophrenia. VGF was just one of many NPAS3 target genes also regulated by the SOX family of transcription factors, suggesting an overlap in neurodevelopmental function. The parallel repression of multiple glycolysis genes by NPAS3 reveals a second role in the regulation of glucose metabolism. Comparison of wild-type and Npas3 KO metabolite composition using high-resolution mass spectrometry confirmed these transcriptional findings. KO brain tissue contained significantly altered levels of NAD þ , glycolysis metabolites (such as dihydroxyacetone phosphate and fructose-1,6-bisphosphate), pentose phosphate pathway components and Kreb’s cycle intermediates (succinate and a-ketogluta- rate). The dual neurodevelopmental and metabolic aspects of NPAS3 activity described here increase our understanding of mental illness etiology, and may provide a mechanism for innate and medication-induced susceptibility to diabetes commonly reported in psychiatric patients. Molecular Psychiatry (2012) 17, 267–279; doi:10.1038/mp.2011.73; published online 28 June 2011 Keywords: schizophrenia; bipolar disorder; transcriptomics; metabolomics; glycolysis; SOX transcription factor Introduction A role for the NPAS3 gene in psychiatric illness risk was suggested through the study of a mother and Schizophrenia and bipolar disorder are common, daughter diagnosed with schizophrenia and mild lifelong psychiatric illnesses affecting mood, percep- learning disability who carried a chromosomal tion and cognition. A strong genetic contribution to abnormality disrupting the gene.1,2 Subsequent gene- these conditions is indicated by family and epide- specific and genome-wide case–control association miological studies. studies have linked single nucleotide polymorphisms at the NPAS3 locus with increased risk of schizo- phrenia,3,4 bipolar disorder3–5 and major depression.4 Correspondence: Dr BS Pickard, Strathclyde Institute of Phar- Three common NPAS3 exonic variants have also been macy and Biomedical Science, University of Strathclyde, Andrew recently associated with increased risk of schizo- Hamnett Wing, 161 Cathedral Street, Glasgow G4 0RE, UK. phrenia.6 Genetic variation at the NPAS3 locus has E-mail: [email protected] 7 also been linked with response to treatment with the Equal authorship status. 7 { antipsychotic drug, iloperidone. Deceased. Received 24 October 2010; revised 6 May 2011; accepted 10 May NPAS3 encodes a member of the bHLH-PAS 2011; published online 28 June 2011 (basic helix-loop-helix—Per, Arnt, Sim) domain NPAS3 transcription factor targets L Sha et al 268 transcription factor family that form functional Prolong antifade reagent with DAPI (40,6-diamidino- heterodimers.8,9 Npas3 knockout (KO) mice display 2-phenylindole) nuclear stain (Invitrogen). neuroanatomical, memory and behavioral phenotypes typical of a model of human psychiatric disorders.10,11 NPAS3 and SOX expression constructs and standard/ They also exhibit a deficit in adult hippocampal circadian cell culture neurogenesis12—a potential cellular pathology of The full-length NPAS3 open reading frame (acc. schizophrenia and depression.13,14 Importantly, a novel NM_001164749) was cloned into a TET-inducible compound acting on mitochondria, P7C3, reverses the expression plasmid (pT-REx-DEST30; Invitrogen) neurogenesis, neuroanatomical, electrophysiological using a restriction digest, Gateway (Invitrogen) clon- and behavioral phenotypes of the Npas3 KO.15 ing linker ligation and the BP/LR reactions (Invitro- Here, we report results from parallel experimental gen). The truncated form, DNPAS3, was generated by approaches designed to identify the function of cleavage and removal of sequence between internal NPAS3 in health and mental illness. The hippocam- and multiple cloning site XhoI sites thus deleting pal neurogenesis deficit associated with Npas3 dele- the second PAS domain and the putative transactiva- tion suggested that NPAS3 might directly regulate tion domain. Plasmids were stably integrated into the developmental pathway associated with new HEK293 [T-REx-293] cells (Invitrogen), using Genet- neuron production and maturation and we describe icin and Blasticidin. HEK293 cells were maintained an immunofluorescence study to determine NPAS3’s in Dulbecco’s Modified Eagle Medium (DMEM) spatio-temporal contribution to this process. supplemented with 10% fetal bovine serum (‘stan- As a transcription factor, NPAS3 is particularly dard culture conditions’). amenable to a transcriptomics approach because SOX expression constructs were generously gifted by observed expression changes are likely to represent Chuanju Liu (SOX5/SOX6/SOX9) and Fabien Muri- direct activity rather than secondary or homeostatic sier/Friedrich Beerman (SOX10). SOX11 was cloned reactions. We employ an in vitro system; over- from human cDNA as described in Li et al. (submitted expression of full-length (FLNPAS3) and artificially for publication, Acta Neuropsychiatr). All SOX micro- truncated (DNPAS3) forms of NPAS3 in the human array experiments were carried out as transient embryonic kidney cell line, HEK293, followed by transfections of HEK293 cells using Optimem/Lipofec- microarray analysis not to model psychiatric illness tamine 2000/plasmid DNA (Invitrogen) incubation for but rather to efficiently identify target genes. We 6 h followed by 24 h in standard culture conditions. compare NPAS3 targets with those of a known For circadian induction, cells were maintained in family of neurodevelopmental regulators, the SOX DMEM alone for 36 h. At the zero hour time point, the family of transcription factors.16–18 We also describe cell medium was replaced with DMEM supplemented an immunofluorescence study to determine NPAS3’s with 50% horse serum plus tetracycline in order to spatio-temporal contribution to hippocampal neuro- induce circadian cycling/NPAS3 over-expression.21,22 genesis. The observation that the related NPAS2 At þ 2 h, cells were washed with DMEM and then protein acts as a functional equivalent of the CLOCK incubated with the same plus tetracycline for the circadian regulator in certain brain regions19 remaining period of the experiment. At either þ 12 or prompted us to examine NPAS3 targets in cells þ 24 h, cells were removed, washed and frozen. stimulated to commence synchronous circadian cy- cling. Finally, we assess the biological relevance of Illumina microarray analysis and data normalization the in vitro microarray data to the in vivo pathologies For baseline and circadian NPAS3 over-expression in the Npas3 KO mouse. This was achieved by high- experiments, parental HEK293 [T-REx-293] cells were resolution mass spectrometry-based metabonomic used as negative controls. Samples (n = 2) were comparison of wild-type and mutant brain tissue. assessed for FLNPAS3 over-expression, DNPAS3 Our findings indicate that NPAS3 contributes to over-expression and parental negative controls. In both neurodevelopmental transcription factor net- the SOX microarray studies, DNA-free Lipofecta- works and the regulation of brain glucose metabolism. mine2000 transfections (n = 4) were used as shared negative controls compared with transfections with each of the SOX expression constructs (n = 3). RNA Materials and methods extraction (RNeasy kit, Qiagen, Crawley, UK) and synthesized microarray probes (Illuminas TotalPrep Immunofluorescence RNA Amplification Kit, Ambion, Austin, TX, USA) Immunofluorescence of frozen brain sections was were quantified and quality checked using an Agilent carried out as previously described.20 Antibodies (Santa Clara, CA, USA) Bioanalyzer. An Illumina (San against Npas3, Gfap, Dcx and Nestin were all obtained Diego, CA, USA) Beadstation