Unexpected expression of ␣- and ␤-globin in mesencephalic dopaminergic neurons and glial cells Marta Biagiolia,b,1, Milena Pintoa,1, Daniela Cessellic, Marta Zaninelloa, Dejan Lazarevica,b,d, Paola Roncagliaa, Roberto Simonea,b, Christina Vlachoulia, Charles Plessye, Nicolas Bertine, Antonio Beltramic, Kazuto Kobayashif, Vittorio Gallog, Claudio Santoroh, Isidro Ferreri, Stefano Rivellaj, Carlo Alberto Beltramic, Piero Carnincie, Elio Raviolak, and Stefano Gustincicha,b,2 aSector of Neurobiology, International School for Advanced Studies, bThe Giovanni Armenise–Harvard Foundation Laboratory, and dConsorzio per il Centro di Biomedicina Molecolare, AREA Science Park, Basovizza, 34012 Trieste, Italy; cCentro Interdipartimentale Medicina Rigenerativa, University of Udine, 33100 Udine, Italy; eRIKEN Omics Science Center, Yokohama Institute 1-7-22 Suehiro-cho Tsurumi-ku Yokohama, Kanagawa 230-0045, Japan; fDepartment of Molecular Genetics, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan; gCenter for Neuroscience Research, Children’s National Medical Center, 111 Michigan Avenue NW, Washington, DC 20010; hDepartment of Medical Sciences, University of Eastern Piedmont, 28100 Novara, Italy; iInstitute of Neuropathology, Institut d’Investigacio´Biome`dica de Bellvitge–University Hospital Bellvitge, University of Barcelona, 08907 Llobregat, Spain; jDepartment of Pediatric Hematology–Oncology, Weill Medical College of Cornell University, 515 East 71st Street, New York, NY 10021; and kDepartment of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115 Edited by Emilio Bizzi, Massachusetts Institute of Technology, Cambridge, MA, and approved July 6, 2009 (received for review December 26, 2008) The mesencephalic dopaminergic (mDA) cell system is composed A crucial requirement for metabolically active aerobic cells is of two major groups of projecting cells in the substantia nigra a steady supply of oxygen. To this purpose, hemoglobin (Hb)-like (SN) (A9 neurons) and the ventral tegmental area (VTA) (A10 molecules occur widely in organisms ranging from bacteria to cells). A9 neurons form the nigrostriatal pathway and are human (6). Vertebrate Hb is the oxygen- and carbon dioxide- involved in regulating voluntary movements and postural re- carrying protein in cells of erythroid lineage and is responsible flexes. Their selective degeneration leads to Parkinson’s disease. for oxygen delivery to the respiring tissues of the body. Addi- Here, we report that gene expression analysis of A9 dopami- tional vertebrate heme-containing proteins with structural ho- nergic neurons (DA) identifies transcripts for ␣- and ␤-chains of mology to globin chains include cytoglobin, mostly described in hemoglobin (Hb). Globin immunoreactivity decorates the ma- connective tissues (7), and neuroglobin, broadly expressed in the jority of A9 DA, a subpopulation of cortical and hippocampal brain (8). astrocytes and mature oligodendrocytes. This pattern of expres- Surprisingly, Hb chains have been recently detected in noner- sion was confirmed in different mouse strains and in rat and ythroid cells including macrophages, alveolar cells, eye’s lens, and human. We show that Hb is expressed in the SN of human mesangial cells of the kidney (9–12). postmortem brain. By microarray analysis of dopaminergic cell By a combination of different gene expression platforms with lines overexpressing ␣- and ␤-globin chains, changes in genes laser capture microdissection (LCM), we have identified the involved in O2 homeostasis and oxidative phopshorylation were transcripts of Hb ␣, adult chain 1 (Hba-a1), and Hb ␤, adult chain observed, linking Hb expression to mitochondrial function. Our 1 (Hbb-b1) in A9 neurons. Interestingly, Hb immunoreactivity data suggest that the most famed oxygen-carrying globin is not (Hb-IR) decorated the large majority of A9 cells, whereas it exclusively restricted to the blood, but it may play a role in the stained only Ͻ5% of A10 neurons. Furthermore, we detected Hb normal physiology of the brain and neurodegenerative diseases. expression in almost all oligodendrocytes and cortical and hippocampal astrocytes and proved that this pattern of expres- astrocytes ͉ hemoglobin ͉ oligodendrocytes ͉ oxidative phosphorylation ͉ sion was conserved in mammals. Importantly, A9 DA neurons Parkinson from human postmortem brain showed Hb expression. By gene expression analysis of mouse dopaminergic cell lines ␣ ␤ opaminergic neurons (DA) are an anatomically and func- stably transfected with - and -chains, we observed changes in Dtionally heterogeneous group of cells involved in a wide genes involved in O2 homeostasis and oxidative phosphorylation, range of neuronal network activities and behavior. Among them, suggesting a link between Hb and mitochondrial activity. mesencephalic DA (mDA) are the major source of dopamine in These results open a scenario for a role for Hb in brain physiology the brain. They present two main groups of projecting cells: the and PD pathogenesis. A9 neurons of the substantia nigra (SN) and the A10 cells of the Results ventral tegmental area (VTA). ␣ ␤ A9 neurons form the nigrostriatal pathway and are involved Identification of - and -Globin Transcripts by Expression Analysis of in regulating voluntary movements and postural reflexes. A9 DA Neurons. To study the cellular physiology of A9 DA Their selective degeneration leads to Parkinson’s disease neurons, we determined their gene expression profiles with two (PD), and the loss of DA synapses in the striatum is believed to be primary cause for the disruption of the ability to control Author contributions: M.B., M.P., E.R., and S.G. designed research; M.B., M.P., D.C., M.Z., movements (1). A10 cells constitute the mesocorticolimbic D.L., R.S., C.V., and C.P. performed research; A.B., K.K., V.G., C.S., I.F., S.R., C.A.B., and P.C. pathway, playing a fundamental role in reward and attention. contributed new reagents/analytic tools; M.B., M.P., D.C., D.L., P.R., R.S., C.V., C.P., N.B., S.R., Their abnormal function has been linked to schizophrenia, C.A.B., P.C., and S.G. analyzed data; and M.B. and S.G. wrote the paper. attention deficit, and addiction, whereas they are relatively The authors declare no conflict of interest. spared in PD (2). This article is a PNAS Direct Submission. The description of the repertory of genes of mDA neurons Freely available online through the PNAS open access option. may provide crucial information on their physiology and on the Data deposition: The data reported in this paper have been deposited in the Gene mechanisms of cell-type specific dysfunction. Interestingly, in Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE16192). previous gene expression profiling experiments, mDA cell 1M.B. and M.P. contributed equally to this work. groups presented a limited number of differentially expressed 2To whom correspondence should be addressed. E-mail: [email protected]. genes with A9-enriched transcripts mainly related to energy This article contains supporting information online at www.pnas.org/cgi/content/full/ metabolism and mitochondrial function (3–5). 0813216106/DCSupplemental. 15454–15459 ͉ PNAS ͉ September 8, 2009 ͉ vol. 106 ͉ no. 36 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813216106 Downloaded by guest on September 27, 2021 A B A9 C TH probe merge zoom qPCR analysis of globin transcripts expression in A9 AS and A10 neurons ** Ct) 2.5 5‘-------------------------------------------------------------------------------------3’ ∆∆ alpha chain (Hba-a1) NM_008218 TSS S 2 alpha-globin Hba-a1 TPM count A9 31.849 1.5 1 AS 0.5 0 Normalized fold expression ( Normalized fold 3‘---------------------------------------------------------------------5’ Hba-a1 Hbb-b1 Alas Spna TH beta chain (Hbb-b1) NM_008220 TSS S beta-globin Hbb-b1 TPM count A9 18.888 Fig. 1. Expression of ␣- and ␤-globin transcripts in A9 and A10 neurons. (A) NanoCAGE tracks visualization concerning Hbb-a1 (Chr.11) and Hbb-b1 (Chr.7) gene loci. A Genome Browser view is presented. A9 library is indicated, and the tags per million (TPM) for each gene are reported. The structure of Hbb-a1 and Hbb-b1 transcripts is depicted at the bottom. Transcriptional start sites (TSS) are indicated at the 5Ј untranscribed region of each transcript. (B) In situ hybridization of ␣- and ␤-globin transcripts on A9 DA neurons: ventral midbrain slices were processed with antisense (AS) and sense (S) probes for the two globins transcripts. DA neurons were visualized by immunohistochemistry using an anti-TH antibody (green). The overlay (merge) shows colocalization of the transcripts of ␣- and ␤-globin in the cytoplasm of A9 neurons. Probes synthesis from sense transcripts were used as negative controls. The zoom offers magnifications of the area in the boxes of the overlay images. (Scale bars: 20 ␮m.) (C) qPCR starting from 500 LCM-isolated neurons from A9 (black column) and A10 (grey column) regions of the ventral midbrain of TH-GFP mice. TH, ␣-globin, and ␤-globin transcripts were amplified and the absence of blood contamination was evaluated by using primers for Alas and Spna. ␣- and ␤-transcripts are more expressed in A9 neurons (Ϸ2-fold), four biological replicas, P Ͻ 0.05. independent techniques: cDNA microarrays and a nanoscale We studied the precise distribution of ␣- and ␤-globin version of the cap analysis of gene expression (nanoCAGE). To transcripts in the mouse midbrain by in situ hybridization. this purpose we took advantage of transgenic