245 The Journal of Experimental Biology 216, 245-252 © 2013. Published by The Company of Biologists Ltd doi:10.1242/jeb.076893 RESEARCH ARTICLE Intrinsic expression of transcortin in neural cells of the mouse brain: a histochemical and molecular study Elena Sivukhina1, Jean-Christophe Helbling2,3, Amandine M. Minni2,3, H. Hendrik Schäfer1, Véronique Pallet2,3, Gustav F. Jirikowski1 and Marie-Pierre Moisan2,3,* 1Institute of Anatomy II, Friedrich-Schiller University, Jena University Hospital, Jena, Germany, 2INRA, Nutrition and Integrative Neurobiology, UMR 1286, 33076 Bordeaux, France and 3Université de Bordeaux, Nutrition and Integrative Neurobiology, UMR 1286, 33076 Bordeaux, France *Address for correspondence ([email protected]) SUMMARY Corticosteroid binding globulin (CBG, transcortin) has been shown to be expressed in the brain of rat and human species. In this study, we examined the CBG brain expression and cDNA structure in mice, comparing wild-type (Cbg+/+) and Cbg knockout mice (Cbg–/–, obtained by genetic disruption of the SerpinA6 alias Cbg gene). We used double immunofluorescence labeling with specific neuronal and glial markers to analyze the cellular localization of CBG in various regions of the mouse brain. In wild-type (Cbg+/+) mice, we found CBG immunoreactivity in neuronal perikarya of the magnocellular hypothalamic nuclei, amygdala, hippocampus, cerebral cortex, cerebellum and pituitary. A portion of glial cells (astrocytes, oligodendrocytes) contained CBG immunoreactivity, including some of the ependymal cells and choroid plexus cells. No CBG immunoreactivity was detected in Cbg–/– brain tissues. Using RT-PCR, we showed that the full-length Cbg mRNA is present in those regions, indicating an intrinsic expression of the steroid-binding globulin. Furthermore, sequencing analysis showed that Cbg cDNA obtained from the mouse hypothalamus was homologous to Cbg cDNA obtained from the liver. Finally, we have evaluated the relative levels of CBG expression in various brain regions and in the liver by quantitative PCR. We found that brain levels of Cbg mRNA are low compared with the liver but significantly higher than in CBG-deficient mice. Although derived from the same gene as liver CBG, brain CBG protein may play a specific or complementary role that requires the production and analysis of brain-specific Cbg knockout models. Key words: corticosteroid-binding globulin, transcortin, glucocorticoid, brain, glial cell. Received 2 July 2012; Accepted 11 September 2012 INTRODUCTION hypothalamic neurons as well as in neurons of the rat hypothalamo- Most systemic glucocorticoids (GCs) are bound to corticosteroid- hypophyseal system, partially co-localized with the two important binding globulin (CBG), also referred to as transcortin, a member peptides of stress response – vasopressin and oxytocin (Sivukhina of the SERPIN family of proteins (for recent reviews, see Moisan, et al., 2006; Möpert et al., 2006; Jirikowski et al., 2007). With 2010; Henley and Lightman, 2011). CBG is a glycoprotein with a immunohistochemistry and in situ hybridization methods, CBG has molecular mass of ~55kDa (Westphal, 1986; Westphal, 1971; also been observed in a portion of the periventricular neurons, the Hammond, 1990). CBG-bound corticosteroid transport into the brain ependymal cells lining the third ventricle and in the choroid plexus has been shown to be insignificant (Pardridge and Mietus, 1979), (Möpert et al., 2006; Jirikowski et al., 2007). However, detailed presumably because the size of the glycated CBG prevents it from morphological analysis of CBG distribution in the different crossing the blood–brain barrier. In addition to being a buffer for populations of brain cells has not been performed, and the brain bioavailability of adrenal steroids, plasma CBG acts as a reservoir Cbg structure has not been reported. of GC hormones and allows GC delivery to target tissues (reviewed Recently, we successfully developed a mouse model of full CBG by Rosner, 1991; Hammond, 1995), particularly in sites of deficiency by specific deletion of the gene encoding CBG, i.e. Cbg, inflammation, where CBG–GC complexes are cleaved by elastase, also called SerpinA6 gene (Richard et al., 2010). These knockout thereby releasing GCs and creating high concentrations of GCs at mice display the features of the HPA axis regulation observed in the target cell. Finally, according to some authors, CBG may be very rare CBG-deficient patients (for review, see Gagliardi et al., directly involved in membrane effects of GCs, with the existence 2010; Henley and Lightman, 2011), i.e. very low total circulating of a CBG receptor (Orchinik et al., 1997). In a recent study, we levels of GC hormones but normal free active GC levels in basal showed the endogenous expression of CBG in a human astrocytoma conditions. After stress exposure, CBG-deficient mice (Cbg–/–) cell line (Pusch et al., 2009). In this culture system, CBG secretion demonstrated insufficient free GC levels, leading to insufficient GC could be rapidly induced after corticosterone stimulation in a dose- signaling and inappropriate behavioral responses such as despair- and time-dependent manner whereas dexamethasone, a potent GC like behaviors (Richard et al., 2010). The availability of these Cbg agonist that is not bound by CBG, was ineffective (Pusch et al., knockout mice provides a unique opportunity to evaluate the 2009). Previously, we described CBG in human magnocellular importance of CBG within the brain. THE JOURNAL OF EXPERIMENTAL BIOLOGY 246 The Journal of Experimental Biology 216 (2) In the current study, we examined CBG expression in various 4l of first strand buffer (5ϫ), 1l (0.1moll–1) dithiothreitol (DTT), brain regions of wild-type and CBG-deficient mice using 1l RNaseOUT Inhibitor (Invitrogen) and 1l reverse transcriptase immunohistochemistry and reverse-transcription polymerase chain (Superscript III Reverse Transcriptase; Invitrogen) were added. The reaction (RT-PCR). Double immunostaining for neuronal and glial mixture was incubated for 55min at 50°C in a water bath and, finally, markers followed by confocal laser scanning microscopy were used the reaction was stopped by heating the mixture at 75°C for 10min. for further characterization of CBG-positive cells. RT-PCR of the full-length cDNA was then used to confirm the local synthesis of Molecular analysis by RT-PCR CBG in the brain and to identify its cDNA structure. Finally, real- cDNA was obtained after the reverse transcription of total RNA time quantitative PCR was employed to evaluate the relative isolated from the different brain regions, pituitaries and liver of expression of Cbg mRNA in various brain regions and liver. Cbg–/– and Cbg+/+ mice as described above. The PCR consisted of 2l cDNA, 5l of the sequence-specific primers mix (2moll–1), –1 MATERIALS AND METHODS 10l PCR buffer mix 2ϫ (GoTaq Flexi Buffer 2ϫ, 3mmoll MgCl2 Animals 0.8mmoll–1 dNTPs), 0.1l GoTaq Hotstart Polymerase (Invitrogen), The generation of Cbg–/– mice has been previously described made up to 20l with PCR water. (Richard et al., 2010). These mice bred and developed normally The oligonucleotides used for specific amplification are listed in and showed no gross anatomical abnormalities. Three to 6-month- Table1. PCR was carried out using a Thermal Cycler 2720 (Applied old Cbg–/– mice and their wild-type littermates (later referred to as Biosystems, Villebon-sur-Yvette, France). PCR was programmed Cbg+/+) were obtained by breeding Cbg+/– males and females and to conduct one cycle at 94°C (5min), followed by 40 cycles at 94°C selecting Cbg–/– and Cbg+/+ by specific genotyping from tail (30s), including annealing (58°C for 1min) and extension (72°C biopsies. for 1.5min). The final extension was performed at 72°C for 10min. All mice of the study were males and had a C57BL/6J genetic PCR products were separated by electrophoresis in 2% agarose gel background exceeding 98%. Mice were kept in an animal room in a Tris–Borate–EDTA buffer stained with ethidium bromide. The (23°C) with a 12h:12h light:dark cycle (lights on at 07:00h) and cDNA bands were visualized under ultraviolet light. with ad libitum access to chow and water. All experiments were For the sequencing analysis, in order to obtain sufficient cDNA conducted in strict compliance with current European Conventions material, nested PCR using the Platinum® Taq DNA Polymerase and approved by the Institutional Committee (protocol validated on High Fidelity (Invitrogen) and specific primers sets (Table1) was 25 February 2011) as well by the Regional veterinary services performed under the same PCR conditions, using as template the (agreement no. A33-063-920). same reverse-transcription products as above. PCR products obtained by nested PCR for hypothalamus and liver were first gel- RNA extraction and cDNA synthesis purified on a spin-column using a QIAquick PCR Purification Kit For detection of Cbg mRNA, total RNA was extracted from brains, (Qiagen, Courtaboeuf, France). The sequencing reactions were then pituitaries and portions of liver (used as a positive control) of Cbg–/– performed using the BigDye Terminator v. 3.1 kit (Applied (N5) and Cbg+/+ mice (N5) collected from mice killed between Biosystems) and migrated on a ABI 3130xl 16-capillary sequencer 19:00h and 20:00h (at the onset of the dark phase), frozen (Applied Biosystems). immediately on dry ice and stored at –80°C. Dissection of brain Primer sequences were designed using Primer Express software regions (cortex, amygdala, hippocampus and hypothalamus) was (Applied Biosystems). performed bilaterally from the whole frozen brains using a ‘Micro Punch’ (1.2mm; Harris, Saint-Quentin Fallavier, France) in a RNA- Analysis of Cbg gene expression by quantitative RT-PCR free environment at –20°C. Total RNA was isolated with TRIzol® In order to establish a comparative quantification of Cbg mRNA in reagent (Invitrogen, Cergy Pontoise, France) according to the the mouse brain, we applied real-time quantitative PCR from manufacturer’s protocol, and RNA quality was evaluated by reverse-transcribed total RNA as described previously (Richard et spectrophotometry (Thermo Scientific, Wilmington, DE, USA) at al., 2010).