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Critical Re-Evaluation of Neuroglobin Expression Reveals Conserved Patterns Among Mammals

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Critical Re-Evaluation of Neuroglobin Expression Reveals Conserved Patterns Among Mammals Neuroscience 337 (2016) 339–354 CRITICAL RE-EVALUATION OF NEUROGLOBIN EXPRESSION REVEALS CONSERVED PATTERNS AMONG MAMMALS ANDREJ FABRIZIUS, a,b DANIEL ANDRE, a a a c TILMANN LAUFS, ANNE BICKER, STEFAN REUSS, Key words: mRNA expression, Hypothalamus, RNA-Seq. ELENA PORTO, a THORSTEN BURMESTER b AND THOMAS HANKELN a* INTRODUCTION a Institute of Molecular Genetics, Johannes Gutenberg-University Mainz, D-55099 Mainz, Germany Neuroglobin (Ngb) is an O2-binding respiratory protein b Institute of Zoology, Biocenter Grindel, University of Hamburg, (Burmester et al., 2000) predominantly expressed in the D-20146 Hamburg, Germany central and peripheral nervous system (CNS and PNS), c Department of Nuclear Medicine, University Medical Center, and is also found in some endocrine tissues (Reuss Johannes Gutenberg-University, Langenbeckstr. 1, D-55101 et al., 2002, 2016; Wystub et al., 2003; Laufs et al., Mainz, Germany 2004). Ngb is a monomer with about 150 amino acids and binds O2 with a similar affinity as myoglobin (Mb) Abstract—Neuroglobin (Ngb) is a respiratory protein that is (P50 1–2 Torr). It is a structurally highly conserved pro- almost exclusively expressed in the vertebrate nervous tein that occurs in almost all vertebrates except Agnatha system. Despite many years of research, the exact function (hagfish and lampreys) and Chondrichthyes (sharks and and even the expression sites of Ngb are still a matter of rays) (Schwarze et al., 2014; Opazo et al., 2015). Despite debate. However, to investigate hypotheses surrounding 15 years of research, the function of Ngb is still a matter of the potential roles of Ngb, a detailed knowledge of its debate, and a large variety of alternative and competing major and minor expression sites is indispensable. We hypotheses have been put forward (for reviews, see: have therefore evaluated Ngb expression by extensive Hankeln et al., 2005; Burmester and Hankeln, 2009, bioinformatic analysis using publicly available transcrip- 2014). Like other intracellular globins, Ngb may enhance tome data (RNA-Seq). During mammalian brain develop- ment, we observed low embryonic expression of Ngb O2 supply to metabolically highly active neurons mRNA and an increase after birth, arguing against a role (Burmester et al., 2000) or may be instrumental in the of Ngb in fetal hypoxia tolerance. In adult mouse brain, detoxification of reactive oxygen species (ROS) (Fordel we found highest Ngb mRNA levels in the hypothalamus, et al., 2006). Ngb may also be involved in NO metabolism where expression was up to 100-fold stronger than in cere- by detoxification of harmful NO under normoxic conditions bral cortex, cerebellum or hippocampus, as confirmed by (Brunori et al., 2005), or act as a nitrite reductase and qRT-PCR and Western blotting. High Ngb expression in generate NO under hypoxic conditions, thus preventing the hypothalamus was found conserved in humans and mitochondrial respiration in low-oxygen environment other mammals. Thus, Ngb mRNA is expressed at a basal (Tiso et al., 2011). Ngb may further interact with cyto- level in many mammalian brain regions, but shows distinc- chrome c, which is released from mitochondria upon an tive regional peaks. RNA-Seq analysis further revealed only low levels of Ngb mRNA in retina and testes and no apoptotic stimulus, thereby inhibiting the intrinsic apop- signal in standard tumor cell lines, thus raising questions totic pathway (Fago et al., 2006; Raychaudhuri et al., concerning previous studies and functional hypotheses. In 2010). It has also been proposed that Ngb interacts with conclusion, this broad-scale expression study may point the phosphatase and tensin homolog/protein kinase B to distinct Ngb functions for high- and low-expressing signaling pathway (PTEN/PI3K/AKT), implying a signaling cells and tissues and argues against a single, generic role role of Ngb in neuronal development (Li et al., 2014). of Ngb as an oxygen supplier or as an endogenous protec- Regardless of its true function(s), it has repeatedly been tant in all nerve cells. Ó 2016 Published by Elsevier Ltd on suggested that Ngb serves as a neuroprotective agent behalf of IBRO. (Sun et al., 2001; Khan et al., 2006; Greenberg et al., 2008; Raida et al., 2013). However, this hypothesis has also received criticism (Schmidt-Kastner et al., 2006; Kelsen et al., 2008; Raida et al., 2012; Di Pietro et al., 2014). Evidence has been reported that Ngb expression *Corresponding author. Address: Institute of Molecular Genetics, Johannes Gutenberg-University Mainz, J.-J. Becher-Weg 30a, levels are positively correlated with oxidative metabolism D-55099 Mainz, Germany. (Bentmann et al., 2005; Mitz et al., 2009) and hypoxia tol- E-mail address: [email protected] (T. Hankeln). erance of a species (Roesner et al., 2008; Nayak et al., Abbreviations: NGB/Ngb, human/mouse neuroglobin gene and mRNA; 2009; Avivi et al., 2010; Schneuer et al., 2012). NGB/Ngb, human/mouse neuroglobin protein; qRT-PCR, quantitative real-time reverse-transcriptase PCR; RNA-Seq, RNA sequencing; Detailed knowledge of Ngb expression is an essential RPKM, reads per kilobase exon model per million mapped reads. prerequisite for the understanding of Ngb function(s) http://dx.doi.org/10.1016/j.neuroscience.2016.07.042 0306-4522/Ó 2016 Published by Elsevier Ltd on behalf of IBRO. 339 340 A. Fabrizius et al. / Neuroscience 337 (2016) 339–354 (Hankeln et al., 2004; Burmester and Hankeln, 2014). Western blotting However, there is no consensus concerning the levels Tissues were homogenized in heated RIPA lysis buffer and patterns of Ngb expression in the mammalian CNS (10 mM Tris, 1 mM CaCl , 150 mM NaCl, 10 mM NaF, (cf., e.g., Mammen et al., 2002; Hankeln et al., 2004; 2 25 mM b-Glycerophosphate, 0.5% NP-40, 0.5% Hundahl et al., 2010). The distribution and relative expres- Deoxycholic acid, 0.1% SDS, pH 7.4). Total protein sion levels vary between different studies, probably extracts were cleared by centrifugation and protein depending on the methods applied to evaluate protein concentrations were determined by a Bradford assay and/or mRNA expression. In fact, many techniques for (Bradford, 1976). Extracts were supplemented with an detecting protein or mRNA levels are potentially error- equal volume of 2Â Laemmli buffer and heat denatured prone, since they rely on indirect staining and hybridiza- at 95 °C for 5 min before they were loaded onto a 12%- tion techniques, potentially causing misleading interpreta- SDS polyacrylamide gel. Proteins were transferred onto tions. We therefore decided to evaluate (and re-assess) nitrocellulose membranes using the Mini Trans Blot Sys- the published, diverging results of Ngb expression pat- tem (Bio-Rad, Hercules, California, USA). The mem- terns and levels via a hybridization-free approach. We branes were incubated overnight with anti-Ngb serum made use of the vast and steadily growing amount of tran- diluted 1:150 in 5% non-fat dry milk in PBS-T (80 mM Na - scriptome sequencing (RNA-Seq) datasets, which pro- 2 HPO , 20 mM NaH PO , 100 mM NaCl, 0.2% Tween 20, vide gene-specific mRNA expression information via 4 2 4 pH 7.5) at 4 °C. For Western blot analysis of the develop- digital read-counting and mapping of cDNA sequences, ing mouse brain, we used anti-Ngb1, a polyclonal rabbit obtained by Illumina next-generation sequencing. More antibody raised against a peptide that corresponds to than 380 RNA-Seq data sets were analyzed bioinformat- amino acids 55–70 of human NGB (tested as described ically to determine the expression of Ngb in the CNS of in Schmidt et al., 2003, 2005; Wystub et al., 2003). Wes- mouse and man, other land-living mammals and, for com- tern analysis of mouse adult brain regions was performed parison, the zebrafish. In addition, we studied Ngb using a polyclonal rabbit anti-Ngb serum raised against a expression during embryonic development of the mouse combination of peptides that correspond to amino acids brain, and in human cancer cell lines. Selected transcrip- 55–70 and 138–151 of human NGB. The latter was suc- tome sequencing results were additionally confirmed by cessfully tested for specific Western blot binding to Ngb qRT-PCR and Western blotting. We observed a con- using tissues from our Ngb knockout mouse model (to served pattern of regionalized Ngb mRNA expression in be described elsewhere). As loading control, an anti-b- adult mammalian brains, with low expression levels in actin antibody raised against the C11-peptide (Sigma– many regions and consistent expression peaks in the Aldrich, #A2066) was used. After incubation with a sec- hypothalamus. Ngb transcription in embryonic brain ondary goat anti-rabbit antibody coupled with development and in the retina was very low. Altogether, horseradish-peroxidase (HRP) (Biozym, Hamburg, Ger- the data suggest different functions of Ngb, depending many) for 1 h at RT, detection was carried out using on its strength of expression, and argue against a single enhanced chemiluminescence ECL reagent. Signals generic role in oxygen supply or neuroprotection. were collected by exposure to an X-ray film (Kodak, Bio- Max). Quantification of Western blot signals was per- formed with ImageJ (Schneider et al., 2012)by EXPERIMENTAL PROCEDURES measuring the area under curve (AUC) or calculating the number of pixels for each specific signal and the load- Animals ing control. Ngb signals were normalized to either the Animals were handled according to the Directive 2010/63/ used loading controls or the unspecific band produced EU EEC for animal experiments. To quantify Ngb by the anti-Ngb serum. expression in developing mouse brain by qRT-PCR and Western blotting, total RNA and proteins were extracted Quantitative real-time RT-PCR from BALB/c embryos and adult mice. Mouse embryos were collected at days 10, 15 and 19 of pregnancy Total RNA was isolated from mouse tissues that had been (E10, E15, E19) and shortly after birth (D1).
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