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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 in neural cells of the 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 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 ). 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 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 with a immunohistochemistry and in situ hybridization methods, CBG has molecular mass of ~55kDa (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.

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In the current study, we examined CBG expression in various 4l of first strand buffer (5ϫ), 1l (0.1moll–1) dithiothreitol (DTT), brain regions of wild-type and CBG-deficient mice using 1l RNaseOUT Inhibitor (Invitrogen) and 1l 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 55min 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 10min. 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 2l cDNA, 5l of the sequence-specific primers mix (2moll–1), –1 MATERIALS AND METHODS 10l PCR buffer mix 2ϫ (GoTaq Flexi Buffer 2ϫ, 3mmoll MgCl2 Animals 0.8mmoll–1 dNTPs), 0.1l GoTaq Hotstart Polymerase (Invitrogen), The generation of Cbg–/– mice has been previously described made up to 20l 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- Table1. 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 (5min), followed by 40 cycles at 94°C selecting Cbg–/– and Cbg+/+ by specific genotyping from tail (30s), including annealing (58°C for 1min) and extension (72°C biopsies. for 1.5min). The final extension was performed at 72°C for 10min. 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 12h:12h light:dark cycle (lights on at 07:00h) 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 (Table1) 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 (N5) and Cbg+/+ mice (N5) collected from mice killed between Biosystems) and migrated on a ABI 3130xl 16-capillary sequencer 19:00h and 20:00h (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’ (1.2mm; 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). Total RNA isolated from the hypothalamus, pituitary, 260nm. RNA was considered of high quality at a 260/280nm ratio prefrontal cortex, amygdala and hippocampus from Cbg–/– and in the range 1.8–2.0. Total RNA was reverse transcribed with Cbg+/+ mice was reverse transcribed into cDNA as described above. Superscript III (Invitrogen) in 13l of reaction mixture containing Then, 5l of cDNA (diluted 1:20) was amplified using 10l of 2g of RNA, 1l (50ngml–1) of random hexamers, 1l Mesagreen qPCR master mix (Eurogentec, Seraing, Belgium) and (10mmoll–1) of desoxynucleoside triphosphates (dNTPs) and 5l of primer mix at 300nmoll–1 into a total volume of 20l onto diethylpyrocarbonate (DEPC) water to volume. This mixture was an ABI7500 thermocycler (Applied Biosystems). The PCR program heated in a water bath at 65°C for 5min and then cooled on ice. consisted of 40 cycles, each at 95°C (15s) and 60°C (1min). The

Table 1. List of oligonucleotide sets used in this study Sequence Amplicon Position Application Fwd 5Ј-AGCAGACGACCTGGTCAACC-3Ј 718bp 29-746 RT-PCR Rev 5Ј-CCTGACTGGACCATCATGGG-3Ј Fwd 5Ј-CTCAAAGGAATATGGAAACTTC-3Ј 689bp 640-1328 RT-PCR Rev 5Ј-GGGGAGACCCTGAAGAAGAC-3Ј Fwd 5Ј-ACCAAAACAATGTCGCTCGC-3Ј 633bp 46-678 Nested PCR Rev 5Ј-ATTTTCTGGGCTGAAGGGAAG-3Ј (sequencing) Fwd 5Ј-CTCAAAGGAATATGGAAACTTC-3Ј 621bp 640-1260 Nested PCR Rev 5Ј-CGGGACAAGCTTTTAGGCTG-3Ј (sequencing) Fwd 5Ј-AGCAGACGACCTGGTCAACC-3Ј 50bp 29-78 q-PCR Rev 5Ј-ACAGGTATACAGGGCAAGCG-3Ј

THE JOURNAL OF EXPERIMENTAL BIOLOGY CBG expression and structure in mouse brain 247 primers amplified a 50bp fragment of mouse Cbg (Table1). RESULTS Specificity of the PCR reaction was validated by a melting-curve Expression of CBG immunoreactivity in the mouse brain analysis and evaluation of PCR efficiency using five dilution points Slight to moderate specific CBG immunoreactivity was observed of the calibrator sample. mRNA levels of target were as a granulated reaction product in the perinuclear cytoplasm of normalized using a 18S RNA expression for each sample (cDNA neurons in the different brain regions of Cbg+/+ mice diluted 1:2000). 18S proved to be the best internal control in our (Fig.1A–G,L,N) and was absent in Cbg–/– (Fig.1J,K,M represents experiments, compared to cyclophilin and 2 microglobulin genes. cerebral cortex, paraventricular and supraoptic hypothalamic nuclei, Relative quantification of normalized mRNA levels was calculated respectively). In the cerebellum of Cbg+/+ mice, CBG using SDS2.1 software (Applied Biosystems). immunoreactivity was observed in large neurons within the granule cell layer (Fig.1A, arrows), and many Purkinje cells and their Tissue preparation and immunohistochemistry processes were also stained for CBG (Fig.1A, arrowhead). Some For morphological investigations, intact Cbg–/– (N3) and Cbg+/+ CBG-stained cells were distributed throughout the amygdala mice (N3) were anesthetized with a rapid isoflurane exposure complex and the hypothalamic periventricular region (Fig.1B, (Aerrane; Baxter SA, Maurepas, France) and sacrificed by arrowheads). In the hippocampus, CBG immunoreactivity was transcardiac perfusion with 4% paraformaldehyde in isotonic observed in the cytoplasm of granule cells (Fig.1C, arrowhead) and phosphate-buffered saline solution (PBS, pH 7.4). These mice were of some interneurons (Fig.1C, arrow). Various regions of the sacrificed between 19:00h and 20:00h (at the onset of the dark cerebral cortex contained CBG-positive perykariya, some of which phase). After fixation, brains, pituitaries and liver (used as a positive had a pyramidal cell-like morphology (Fig.1D). CBG-positive cells control) were removed and post-fixed in the same fixative at 4°C. were also found in brain areas known to be associated with stress Samples were embedded in 6% agarose and sectioned on vibratome response and regulation of the HPA axis: throughout the (VT1000E; Leica Instruments, Nußloch, Germany). Brains were cut hypothalamus, CBG immunoreactivity was observed in cell bodies coronally, pituitaries and liver horizontally (30–50m thick) and (Fig.1E, arrowheads; Fig.1L) as well as their processes within the collected in PBS until immunostaining was performed. paraventricular nucleus (Fig.1E, arrow). Intense CBG The following commercially available antibodies were used at immunoreactivity was observed in some endocrine cells of the optimal dilutions: anti-Serpin A6 (R&D Systems, Minneapolis, MN, anterior pituitary lobe (Fig.1F, arrowheads). Numerous USA; polyclonal, 1:100), anti-neuronal nuclear antigen (NeuN, magnocellular CBG-immunoreactive cells were also present in the Chemicon, Merck Millipore, Billerica, MA, USA; mouse monoclonal, supraoptic nucleus (Fig.1G, arrowheads; Fig.1N). CBG clone A60, 1:100), anti-cyclic nucleotide phosphodiesterase (CNPase, immunoreactivity was found in axon terminals within the median Sigma-Aldrich, St Louis, MO, USA; mouse monoclonal, clone 11- eminence and in Herring bodies of the posterior pituitary lobe. 5B, 1:1000), anti-glial fibrillary acidic protein (GFAP, Merck Compared to the liver (Fig.1H), the level of CBG staining within Millipore; polyclonal, 1:2000), anti-ionized calcium binding the brain was low. Immunohistochemistry for CBG in the liver adaptor molecule 1 (Iba1, Biocare Medical, Concord, CA, USA; rabbit revealed strong staining in numerous hepatocytes throughout the polyclonal, 1:600). Prior to the immunofluorescence staining, blocking liver parenchyma (Fig.1H, arrowheads) as well as in the close of nonspecific binding sites was performed for 1.5h at room vicinity of sinusoids (Fig.1H, arrow) in Cbg+/+ mice. No specific temperature using PBS (pH 7.4), containing 1% Triton X-100 (Roth, staining was observed in the liver of Cbg–/– animals (Fig.1I). Karlsruhe, Germany) and 10% normal donkey serum (Sigma- In order to analyze cellular localization of CBG within the brain, Aldrich). For double staining, the mixture of primary antibodies, double immunofluorescence labeling with well-known neuronal and diluted in PBS containing 0.1% Aurion BSA-c (Aurion Immuno Gold glial markers was performed. CBG was expressed in neurons, as Reagents & Accessories, Wageningen, The Netherlands) was used. demonstrated by double staining with NeuN in amygdala Incubation with the primary antibody was carried out for 3days at (Fig.2A–AЉ), but was also observed in other populations of brain 4°C (Irintchev et al., 2005; Hoffmann et al., 2009). After washing in cells. CBG immunoreactivity was also found in a few glial fibrillary PBS, the mixture of appropriate secondary antibody conjugated with acidic protein (GFAP)-positive cells in the periventricular Alexa Fluor® 488 Dye and Alexa Fluor® 568 Dye (Invitrogen), diluted hypothalamic region (Fig.2B–BЉ). Staining for CBG was also 1:300 in PBS containing 0.1% Aurion BSA-c, was applied for 2.5h observed in oligodendrocyte cell bodies and processes, as at room temperature. Finally, after subsequent washes in PBS, demonstrated by double labeling with 2Ј,3Ј-cyclic nucleotide 3Ј- sections were affixed onto microscopic slides and mounted with an phosphodiesterase (CNPase) in cerebellum (Fig.2C–CЉ) and cerebral anti-fading medium (Fluoromount G; Southern Biotechnology cortex (Fig.2D–DЉ). Double immunohistochemistry with ionized Associates, Biozol, Eching, Germany). For immunohistochemical calcium binding adaptor molecule 1 (Iba1) showed that microglial controls, primary antibodies were omitted and were devoid of specific cells were devoid of specific CBG signal in any of the regions studied staining. (Fig.2E–EЉ). Immunostained sections were examined under a confocal laser- scanning microscope TCS SP5 (Leica, Mannheim, Germany). Qualitative analysis of Cbg mRNA in brain regions Digital images were analyzed using Adobe Photoshop (Adobe To answer the question concerning intrinsic expression of Cbg in Systems, v. 8.0.1). Anatomical identification of labeled structures the brain, RT-PCR was applied to amplify the full-length Cbg cDNA was based on the cytoarchitectonic descriptions of the mouse brain in Cbg+/+ and Cbg–/– mice using two sets of overlapping primers (Franklin and Paxinos, 2007). (Fig.3, Table1), which produced a 718bp 5Ј cDNA fragment and a 689bp 3Ј cDNA fragment. Statistical analysis As illustrated in Fig.4, specific bands of the expected size were Values were expressed as means ± s.e.m. Statistics were performed seen in the pituitary, prefrontal cortex, hypothalamus, hippocampus using the non-parametric Mann–Whitney U-test. The level of and liver, while in the amygdala this band was very weak or absent. significance was set at P<0.05. On graphs, * indicates P<0.05 and No amplification of the 689bp and 718bp bands was detected in ** indicates P<0.01. Cbg–/– mice (data not shown).

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Fig.1. Analysis of CBG immunoreactivity in the mouse brain and liver. Representative single confocal images throughout the cerebellum (A; arrows, large neurons within the granule cell layer; arrowheads, Purkinje cells), periventricular hypothalamic region (B; arrowheads), hippocampus (C; arrow, interneuron; arrowhead, granule cell), cerebral cortex (D; arrowheads, pyramidal-like cells), paraventricular nucleus (E; arrowheads, cell bodies; arrow, cell processes), anterior pituitary lobe (F; arrowheads) and supraoptic hypothalamic nucleus (G; arrowheads), demonstrating slight to moderate immunofluorescence for CBG in wild-type mice. Strong specific CBG expression found in the liver of wild-type mice (H; arrowheads, cells throughout the liver parenchyma; arrow, cells in the close vicinity of sinusoids) was not observed in the liver (I) and brain (J) of Cbg knockout mice. Representative images at a lower power magnification throughout the paraventricular (K,L) and the supraoptic hypothalamic nuclei (M,N) demonstrate specific CBG immunoreactivity in wild-type mice (L,N), which is absent in Cbg–/– mice (K,M). Abbreviations: III, the third ventricle; ox, optic chiasma. Scale bars, 50m.

To identify the structure of Cbg cDNA in the brain, a sequencing compared to its expression in the liver; about 200 times less analysis of the full-length cDNA obtained from hypothalamic and expressed in the pituitary and hippocampus when compared to the liver mRNA of a wild-type mice was performed. We first applied liver. However, in every tissue examined, the level of Cbg mRNA nested PCR, using primers within the first PCR products in order was significantly higher in wild-type when compared with Cbg–/– to increase the amount of Cbg cDNA (Fig.3, Table1). Sequencing mice (P<0.05 for each tissue). Moreover, the analysis of the melting of both strands of a 633bp corresponding to the 5Ј half and of a curve of PCR products showed a single specific product in each 621bp fragment corresponding to the 3Ј half of Cbg cDNA revealed tissue of Cbg+/+ mice that was absent in Cbg–/– animals. a 100% homology with the liver Cbg cDNA. DISCUSSION Quantitative expression of Cbg mRNA in brain regions The expression of CBG in the brain has been reported before by In order to estimate the relative level of Cbg mRNA expression in various authors in (de Kloet et al., 1984; Perrot-Applanat et al., the various brain regions and in the liver, primers amplifying a short 1984; Möpert et al., 2006; Jirikowski et al., 2007) and humans fragment (Table1) were used for quantitative real-time PCR (Fig.5). (Sivukhina et al., 2006). Our previous findings using As expected based on the immunohistochemical study, relative immunohistochemistry, in situ hybridization, western blot (Möpert mRNA expression of Cbg in the brain was significantly lower when et al., 2006) and RT-PCR (Jirikowski et al., 2007) suggested

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Fig.2. Analysis of the cellular localization of CBG immunoreactivity in the mouse brain. Representative single confocal images throughout the amygdala (A), periventricular hypothalamic region (B), cerebellum (C) and cerebral cortex (D,E) of CBG wild-type mice. Double fluorescence immunostainings revealed partial co- localization of CBG with NeuN (A–AЉ), GFAP (B–BЉ) and CNPase (C–CЉ,D–DЉ). No coexistence of CBG with the microglial marker Iba1 was observed (E–EЉ). Double-labeled cells are indicated with arrowheads. Scale bars, 50m (A–C,E); 25m (D).

coexpression of the steroid-binding globulin with the classical colocalized in secretory vesicles (Herbert et al., 2006). We thus neuroendocrine peptides within the rat hypothalamus as well as conclude that there may be a similar situation for CBG in mice. intrinsic synthesis of Cbg mRNA in various brain regions. Here, Our colocalization studies with neuronal marker protein (NeuN) we extend the data obtained previously by the use of a mouse model showed that many neuronal systems other than the hypothalamic in which expression of the Cbg gene is totally abolished in each neuroendocrine nuclei contained CBG-immunostaining. Such tissue of the animal. We present data refining CBG localization within brain regions; we show that the same gene encodes brain ATG AAA and liver CBG and we confirm that Cbg mRNA is produced within cerebral regions and thus not transported from blood. These data 621 bp suggest a specific role for CBG protein in brain. 633 bp 718 bp First, we located CBG in the hypothalamic magnocellular 689 bp perikarya, in axonal varicosities in the periventricular nucleus, in the internal zone of the median eminence, and in Herring bodies of 0 500 1000 1500 the posterior pituitary lobe, which shows that magnocelluar CBG in mouse is subject to axonal transport and terminal release in the Fig.3. Schematic illustration of the Cbg cDNA fragments obtained after RT- respective neurohemal organs. In a former immunoelectron PCR. The thin lines represent the 718bp and 689bp fragments amplified in the liver and the various brain regions. The thick lines represent the 633bp microscopical study, we observed a similar morphology for sex and 631bp fragments obtained after nested PCR for the sequencing hormone-binding globulin (SHBG) and oxytocin in the rat analysis. ATG is the translation start codon, and AAA is the translation stop hypothalamo-neurohypophyseal system, where they were codon within Cbg cDNA.

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A 5Ј end amplification B 3Ј end amplification Fig.4. Expression of full-length Cbg mRNA in the mouse brain by RT-PCR. Specific Cbg RT-PCR product of 718bp (A) and 689bp (B) observed in the pituitary, prefrontal cortex, hypothalamus, amygdala, hippocampus

O O +/+ 2 2 and liver in wild-type mice (Cbg ). Liver Pituitary Cortex Hypothalamus Amygdala Hippocampus Pituitary Amygdala Hippocampus H Liver Pituitary Cortex Hypothalamus Ladder Cortex Hypothalamus Amygdala Hippocampus H Cortex Hypothalamus Ladder Examples of gels are presented here. 718 bp 800 Specific amplification of 718bp or 689bp 800 600 bands did not appear in the amygdala of 600 689 bp 400 400 200 each animal or appeared only as a very 200 faint band. neurons occurred in functionally quite diverse cerebral regions in the brain contains CBG. Immunostaining for GFAP and CBG including the amygdala, the prefrontal cortex and the hippocampus, was found in many of the ependymal cells, lining the third implicated in an interconnected way in the central stress response ventricle, in tanycytes, in epithelial cells of the choroid plexus (McEwen, 2007; Joëls and Baram, 2009). Interestingly, phenotypic and in small fractions of fibrillary and protoplasmic astrocytes. analysis of Cbg–/– mice demonstrated that these mice show brain- All these cell types are known to be derived from astroglia, and specific deficit in situations of stress (Richard et al., 2010). In therefore contain GFAP. Astrocytes are known as supportive cells particular, despair-like behavior and altered memory response that interact with neurons and are able to transfer ions to other during stress has been demonstrated in these mice. These brain- astrocytes acting as a sufficient ‘buffer’ for the glutamate. GCs specific alterations have been linked to the lack of plasma CBG stimulate an increase of glutamate, with initial reversible although a possible contribution of brain CBG cannot be excluded remodeling and eventual cell death (Campbell and Macqueen, (Minni et al., 2012). Thus, the specific involvement of brain CBG 2004). This could indicate that CBG may be important as a buffer compared to plasma CBG has yet to be defined. The production in this cell fraction if local CBG levels were high enough for and analysis of brain specific knockout for the Cbg gene would be such a function. The low levels of Cbg mRNA found in this study very useful in this respect. do not favor this hypothesis but these levels might be upregulated Finally, positive staining was found in the granule and Purkinje in stress conditions. A portion of the oligodendrocytes also cells of the mouse cerebellum. Little is known about the effects contained CBG, as shown by double immunostaining with of steroids on cerebellar structures; however, data show that CNPase. The complex role of GCs in the maturation of postnatal exposure to clinically routine doses of hydrocortisone oligodendrocytes and myelinogenesis has been demonstrated or dexamethasone was associated with impaired cerebellar growth (Preston and McMorris, 1984; Raschke et al., 2008) and could (Tam et al., 2011). In another study, data showed that GCs be explained by documented expression of GC receptors in increase cerebellar cell death and induce apoptosis in immature oligodendrocyte progenitor cells (Bohn et al., 1991). Currently, granule neurons via a non-genomic mechanism (Aden et al., high doses of GCs are widely used in the treatment of multiple 2008). sclerosis, a well-described disease characterized by While our earlier results showed the presence of CBG in several oligodendrocyte degeneration. However, the potentially negative regions of the brain, we have not provided a detailed analysis of influence of GCs on remyelination has also been demonstrated cellular distribution of CBG throughout the brain. Double (Clarner et al., 2011), and patients with multiple sclerosis show immunostaining for CBG and for glial markers GFAP and decreased GC receptor affinity and sensitivity when compared to CNPase showed that a considerable portion of non-neuronal cells controls (Ysrraelit et al., 2008). Altogether, it might be a further indication for a possible dysregulation of the intracellular CBG expression. Microglia cells were, in all cases, CBG negative in +/+ 10 ** Cbg animals. Given the fact that microglia cells are resident Cbg+/+ macrophages in the brain and thus form the immunoreactive cell 8 ** Cbg–/– fraction, their lack of CBG underlines their specific role. In a 0.10 former study, we investigated the CBG expression and stimulus- dependent liberation of CBG in a human glioblastoma cell line 0.08 (Pusch et al., 2009). Here, we found clear evidence for the 0.06 ** expression of the steroid-binding globulin in glia-like non- neuronal cells. The functional properties of the different glial cell 0.04 types are diverse. While astrocytes and tanycytes are involved in blood–brain barrier functions and may somehow take up CBG

mRNA relative quantification mRNA * 0.02 * from serum, ependymal cells line the ventricles and may accumulate CBG from the cerebrospinal fluid CSF (Schwarz and 0 Pohl, 1992; Predine et al., 1984; Orchinik et al., 1997) in addition to possible intrinsic biosynthesis. Liver Pituitary Cortext Previous RT-PCR of a Cbg cDNA fragment suggested the HippocampusHypothalamus presence of Cbg mRNA in the respective brain regions, indicating intrinsic expression of CBG within the brain (Jirikowski et al., 2007). Fig.5. Quantitative analysis of Cbg mRNA by real-time PCR. mRNA expression is shown as relative expression. **P<0.01; *P<0.05 by Although CBG has been shown to be expressed in several organs Mann–Whitney U-test. Cbg+/+, N4 in pituitary, hypothalamus and cortex, (Miska et al., 2004; Misao et al., 1994; Misao et al., 1999; Scrocchi N5 in liver and hippocampus. Cbg–/–, N5 in each cerebral region. et al., 1993; del Mar Grasa et al., 2001; de Kloet and McEwen,

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Invest. 64, Real-time PCR experiments and sequencing analysis were performed at the 145-154. Genotyping and Sequencing facility of Bordeaux. We express our thanks to Perrot-Applanat, M., Racadot, O. and Milgrom, E. (1984). Specific localization of Sabine Hitschke (University of Jena, Germany) for her excellent technical plasma corticosteroid-binding globulin immunoreactivity in pituitary corticotrophs. assistance, to Dr Hartmut Oehring (University of Jena, Germany) for helpful Endocrinology 115, 559-569. suggestions, to Dr Valery Grinevich (MPI for Medical Research, Heidelberg, Predine, J., Brailly, S., Delaporte, P. and Milgrom, E. (1984). Protein binding of Germany) for his critical discussion and helpful comments and to Martine Schuler cortisol in human cerebrospinal fluid. J. Clin. Endocrinol. Metab. 58, 6-11. for revising the writing of the manuscript. Preston, S. L. and McMorris, F. A. (1984). Adrenalectomy of rats results in hypomyelination of the central nervous system. J. 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