Molecular and neurochemical evidence for the ... - Wiley Online Library

Journal of Neurochemistry, 2005, 93, 1220–1230

doi:10.1111/j.1471-4159.2005.03113.x

Molecular and neurochemical evidence for the biosynthesis of dehydroepiandrosterone in the adult rat spinal cord Cherkaouia Kibaly, Christine Patte-Mensah and Ayikoe G. Mensah-Nyagan Laboratoire de Neurophysiologie Cellulaire et Inte´greé, Unite´ Mixte de Recherche 7519-Centre National de la Recherche Scientifique, Universite´ Louis Pasteur, Strasbourg, France

Abstract Various studies have indicated that exogenous dehydroepiandrosterone (DHEA) modulates several mechanisms in the CNS of rodents. As adult rodent glands do not secrete significant amounts of DHEA, its role as endogenous modulator of the CNS remains possible only if DHEA is produced by nerve cells. Therefore, the last decade has been marked by diverse unsuccessful investigations aiming to demonstrate the activity of cytochrome P450c17 (P450c17), the key DHEA-synthesizing enzyme, in adult rodent CNS. Here, we combined molecular, anatomical, cellular and neurochemical approaches to provide the first demonstration of the existence of P450c17 and bioactivity in adult rat spinal cord (SC). Realtime RT-PCR revealed P450c17 gene expression in all SC segments. Western blot analyses allowed identification of a

specific P450c17 protein in the SC and immunohistochemical studies localized P450c17 in neurones and glial cells. Pulsechase experiments combined with HPLC and radioactive steroid detection showed that SC slices converted [3H]pregnenolone into [3H]DHEA, a conversion markedly reduced by ketoconazole, a P450c17 inhibitor. Kinetics studies revealed accumulation of [3H]DHEA newly synthesized by SC slices in the incubation medium as its amount declined slowly. This first cellular mapping of an active P450c17 in adult rodent SC suggests that endogenous DHEA synthesized in spinal neural networks may control various spinally-mediated activities. Keywords: cytochrome P450c17, dehydroepiandrosterone, neurosteroid, real-time PCR, spinal cord, steroids and nervous system. J. Neurochem. (2005) 93, 1220–1230.

Dehydroepiandrosterone (DHEA) and its sulphate derivative (DHEAS) are the most abundant steroids secreted by the human adrenals (Bellino et al. 1995; Baulieu and Robel 1998). The biosynthesis of DHEA requires the catalytic actions of two different cytochromes; P450 side-chaincleavage converts cholesterol into pregnenolone (PREG) which is transformed successively into 17-hydroxy-PREG and DHEA by a single microsomal enzyme, cytochrome P450c17 (P450c17). Unlike in humans, plasma concentrations of DHEA/DHEAS are extremely low or undetectable in adult rodents (Vinson et al. 1978; Baulieu 1996). Concurrently, the P450c17 gene is expressed in human adrenals and gonads while, in rodents, the enzyme is present in gonads but not in adrenals (Chung et al. 1987; Di Blasio et al. 1987; Brentano et al. 1990; Durkee et al. 1992; Greco and Payne 1994). By using synthetic DHEA and the rodent brain as a model, pharmacological and behavioural studies suggested that DHEA may be a potent endogenous modulator of several neurobiological mechanisms and its decrease during ageing is correlated to various physiological deficits (Bellino

et al. 1995; Baulieu and Robel 1998; Baulieu et al. 1999). However, the validity of such a hypothesis remains speculative because the adult rodent endocrine glands do not secrete significant amounts of DHEA (Vinson et al. 1978; Baulieu 1996). Therefore, it appears that, in adult rodents,

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Received January 14, 2005; revised manuscript received January 25, 2005; accepted January 25, 2005. Address correspondence and reprint requests to Dr A. G. MensahNyagan, Laboratoire de Neurophysiologie Cellulaire et Inte´greé, Unite Mixte de Recherche 7519-Centre National de la Recherche Scientifique, Universite´ Louis Pasteur, 21 rue Rene´ Descartes, 67084 Strasbourg Cedex, France. E-mail: [email protected] Abbreviations used: DHEA, dehydroepiandrosterone; DHEAS, sulphate derivative of dehydroepiandrosterone; GalC, galactocerebroside; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; Gm, grey matter; [3H]DHEA, 1,2,6,7[3H](N)dehydroepiandrosterone; [3H]17OH-PROG, 17a-hydroxy 3 3 1,2,6,7-[ H]progesterone; [ H]PROG, 1,2,6,7-[3H](N)progesterone; MAP-2, microtubule-associated protein-2; NeuN, neuronal nuclei; P450c17, cytochrome P450c17; PB, phosphate buffer; PREG, pregnenolone; rt, real-time; SC, spinal cord; Wm, white matter.

2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 1220–1230

DHEA formation in the spinal cord 1221

DHEA could be a potent endogenous modulator of the CNS activity only if this steroid is synthesized within the nervous tissue. Baulieu and co-workers have shown the accumulation of DHEA in the adult rat brain independently of steroidogenic glands, an observation which led to the neurosteroid discovery (Corpećhot et al. 1981; Baulieu et al. 1999). However, all attempts by these authors to demonstrate the presence and activity of P450c17 in the rat brain were unsuccessful and the biosynthetic pathway of DHEA in the CNS has become an important source of controversy over the past two decades (Baulieu and Robel 1998; Baulieu et al. 1999). RNAse protection assays coupled with PCR revealed P450c17 expression only in foetal but not adult rodent brain (Mellon and Deschepper 1993; Compagnone et al. 1995). Conflicting results reported the isolation of mRNA encoding P450c17 from homogenates of young adult rat cerebellum and brainstem (Stro¨mstedt and Waterman 1995; Kohchi et al. 1998). None of these molecular studies has investigated whether P450c17 mRNA detected in the foetal or young adult rat brain corresponded to an active form of the enzyme. Owing to the difficulty of demonstrating P450c17 activity in adult rat brain, a hypothetical biochemical pathway was suggested to explain DHEA synthesis from cholesterol without P450c17 involvement (Prasad et al. 1994; Cascio et al. 1998). A recent study has shown P450c17 activity in adult rat hippocampal neurones (Hojo et al. 2004). However, as this work was focused only on hippocampal slices, the anatomical localization, cellular distribution and occurrence of P450c17 activity in various important structures of the adult rodent CNS remain unknown. We and others have recently observed that the adult rat spinal cord (SC), which controls many neurophysiological mechanisms including somatosensory transmission, motor activities and neurovegetative functions, is an active biosynthetic centre of various neurosteroids such as PREG, progesterone and allopregnanolone (Coirini et al. 2002; Patte-Mensah et al. 2003, 2004a,b). To clarify the controversy about DHEA biosynthesis in the CNS, we decided to combine molecular, anatomical, cellular and neurochemical approaches to investigate the presence and activity of P450c17 in the SC of adult rats.

Materials and methods Animals Adult male Sprague-Dawley rats weighing 350–450 g were used in this study. Animal care and manipulations were performed according to the European Community Council Directives (86/609/EC) and under the supervision of authorized investigators. The animals were obtained from a commercial source (Harlan, Le Malcourlet, France) and housed under standard laboratory conditions in a 12-h light/dark cycle with food and water ad libitum. To avoid variations

due to circadian rhythms, tissues were always sampled between 09:30 and 11:30 h. Antisera The rabbit anti-porcine P450c17 was a generous gift from Dr Dale Buchanan Hales (University of Illinois, Chicago, IL, USA). The mouse anti-neuronal nuclei (NeuN) monoclonal antibody, monoclonal anti-galactocerebroside (GalC), monoclonal anti-microtubuleassociated protein-2 (MAP-2) and goat anti-rabbit/FITC antibodies were purchased from Chemicon (Temecula, CA, USA). Mouse monoclonal anti-glial fibrillary acidic protein (GFAP) was from Sigma (St Louis, MO, USA). Alexa-488-conjugated donkey antimouse and Alexa-555-conjugated goat anti-rabbit were supplied by Interchim Molecular Probes (Eugene, OR, USA). Chemicals and reagents Synthetic steroids, including allopregnanolone, DHEA, PREG and progesterone, were purchased from Steraloids (Newport, RI, USA). Dichloromethane, hexane, isopropanol and propylene glycol were obtained from Merck (Darmstadt, Germany). Ketoconazole was supplied by Sigma. Dulbecco’s modified Eagle’s medium was purchased from Invitrogen Corporation (Paisley, UK). Tritiated steroids, such as 9,11,12-[3H](N)allopregnanolone, 1,2,6,7[3H](N)DHEA ([3H]DHEA) and 1,2,6,7-[3H](N)progesterone ([3H]PROG), were obtained from PerkinElmer (Boston, MA, USA). 17a-Hydroxy 1,2,6,7-[3H]progesterone ([3H]17OH-PROG) was purchased from Amersham (Piscataway, NJ, USA). RT and real-time PCR The TRIzol reagent (Invitrogen Corporation) was used according to the manufacturer’s instructions for the extraction of total RNA from the SC and testis. The quality of RNA was electrophoretically determined by ethidium bromide-stained agarose gels and by an optical density (OD) absorption ratio (OD260 nm : OD280 nm) > 1.7. RT of 20 lg total RNA was performed at 37C for 1 h and the reaction system for first-strand cDNA was as follows: 160 U Moloney murine leukaemia virus (Amersham Biosciences Europe, Freiburg, Germany), 0.16 lg random hexamer [pd(N)6 random primer; Pharmacia, Milan, Italy] and 2 mM dNTP mixture (Fermentas Inc., Hanover, MD, USA) in a final volume of 40 lL. Real-time (rt) PCRs were performed using a LightCycler (Roche Diagnostics GmbH, Mannheim, Germany). The specific primer sequences for P450c17 were: forward, 5¢-GACCAAGGGAAAGGCGT-3¢ (nucleotides 351–368) and reverse, 5¢-GCATCCACGATACCCTC-3¢ (nucleotides 636–653) (Sakaue et al. 2002). The primers for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were: forward, 5¢-ACCACAGTCCATGCCATCAC-3¢ (nucleotides 3069–3088) and reverse, 5¢-TCCACCACCCTGTTGCTGTA-3¢ (nucleotides 3624–3605) (Clontech Inc., Palo Alto, CA, USA). PCR was performed in a total volume of 20 lL including 0.5 lg cDNA, 0.5 lM specific primers, 2 lL LightCycler-DNA Master SYBR Green I kit (Roche Diagnostics GmbH) containing Taq polymerase, dNTP, MgCl2 and SYBR Green I dye. A negative control without cDNA template (called water) was run simultaneously with every assay. Standard curves were obtained by using serial dilutions of testis cDNA. The amplification programme consisted of one denaturing step of 8 min at 95C followed by different amplification cycles of 5 s at 95C, 5 s at


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57C and 12 s at 72C. Amplifications were followed by melting curve analysis to ensure the specificity of PCR products and the absence of non-specific products. Based on analyses and calculation integrating adequate standard curves, levels of transcripts encoding P450c17 and GAPDH were determined in samples from gonads and SC using the LightCycler Software available in the rt-PCR system. The concentration of P450c17 mRNA in each sample was calculated after normalization of rt-PCR P450c17 product to GAPDH. For each sample, the rt-PCR experiment was repeated four times. The results are expressed as mean ± SEM.

3% bovine serum albumin. They were then incubated for 15 h at 4C with the polyclonal anti-P450c17 diluted at 1 : 10 000 in phosphate-buffered saline with 0.1% Tween 20 containing 3% bovine serum albumin. Membranes were then incubated for 2 h at 25C with a goat anti-rabbit immunoglobulin antibody conjugated to horseradish peroxidase diluted at 1 : 4000 in phosphate-buffered saline with 0.1% Tween 20 with 3% bovine serum albumin. Immunoreacting bands were detected using the enhanced chemiluminescence visualization system (ECL; Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Immunofluorescence procedure Animals were deeply anaesthetized with 25% urethane (0.5 mL/ 100 g, i.p.) and perfused transcardially with 100 mL of 0.1 M phosphate buffer (PB, pH 7.4). The perfusion was carried out with 450 mL of fixative solution (4% formaldehyde and 0.2% picric acid in PB). The SC was rapidly dissected and post-fixed in the same fixative solution for 24 h at 4C. The tissues were embedded in 5% agar after the post-fixation period, cut in transverse 20-lm-thick sections with a vibratome (VT 1000 S; Leica, Wetzlar, Germany) and collected in PB. The sections were incubated for 48 h at 4C in the P450c17 antiserum diluted at 1 : 200 in PB containing 0.3% triton X 100 and 0.5% bovine serum albumin. The procedure was continued by rinsing the sections three times in PB (10 min/rinse) and transferring them for 2 h into goat anti-rabbit/FITC antibody diluted at 1 : 100 in PB containing 0.3% triton X 100. Finally, the sections were rinsed three times in PB and mounted in vectashield (Vector Laboratories, Burlingame, CA, USA). The preparations were examined under a fluorescence DMR microscope equipped with a digital camera and a pentium IV PC (Leica Microsystems, Wetzlan, Germany) or under a multichannel confocal laser-scanning microscope (LSM510; Zeiss, Go¨ttingen, Germany). The specificity of the immunoreaction was controlled by: (i) substitution of P450c17 antiserum with PB; (ii) replacement of P450c17 antiserum by non-immune rabbit serum and (iii) preincubation of P450c17 antiserum with microsomal proteins from the SC. For cell type identification, double-labelling experiments were conducted by incubating the sections with a mixture containing P450c17 antiserum (1 : 200) and the monoclonal antibodies against GalC (1 : 200), GFAP (1 : 200), NeuN (1 : 1000) or MAP-2 (1 : 20 000), respectively. In these conditions, P450c17 immunoreactivity was revealed with Alexa-555-conjugated goat anti-rabbit and GalC-, GFAP-, NeuN- or MAP-2-like immunostaining was revealed with Alexa-488-conjugated donkey anti-mouse. The preparations were examined under the confocal laser-scanning microscope using specific channels for the detection of Alexa-488 (green) and Alexa-555 (red).

Measurement of cytochrome P450c17 activity For each experiment, 220 mg of SC (cervical, thoracic, lumbar or sacral segment) slices were pre-incubated for 15 min in 2 mL 0.9% NaCl at 37C. The SC slices were incubated at 37C for 3 h in 1.5 mL of Dulbecco’s modified Eagle’s medium (pH 7.4) containing 100 nM [3H]PREG supplemented with 1% propylene glycol. The incubation was in a water-saturated atmosphere (95% air, 5% CO2) which made it possible to maintain the pH at 7.4. At the end of the incubation period, the reaction was stopped by adding 500 lL of ice-cold Dulbecco’s modified Eagle’s medium and transferring the tubes into a cold water-bath (0C). Newly synthesized neurosteroids released by the SC slices were extracted from the incubation medium. The SC slices were homogenized with a glass potterelvehjem homogenizer in 2 mL of Dulbecco’s modified Eagle’s medium (pH 7.4) for the extraction of neurosteroids remaining in the spinal tissue. Newly synthesized neurosteroids were extracted, either from the incubation medium or from the slice homogenates, three times with 2 mL of dichloromethane and the organic phase was evaporated on ice under a stream of nitrogen. The dry extracts were redissolved in 2 mL of hexane and pre-purified on Sep-Pak C18 cartridges (Waters Associates, Milford, MA, USA). Steroids were eluted with a solution of 60% isopropanol and 40% hexane. The solvent was evaporated in an RC-10-10 Speed Vac Concentrator and the dry extracts were kept at )20C until HPLC analysis. To determine the recovery of newly synthesized steroids during the extraction procedure, SC homogenates were incubated for 10 min with 105 cpm of [3H]PREG and extracted as described above. The extraction efficiency was 89 ± 7%.

Western immunoblot analysis Microsomal and total proteins (50 lg) from the SC and testis were separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis for 5 h at 60 mA in electrode buffer (15 mM Tris, 130 mM glycine, pH 8.3, 0.1% sodium dodecyl sulphate). Separated proteins were transferred onto polyvinylidene difluoride membranes (Immobilon P; Millipore Corp., Bedford, MA, USA) at 40 V for 15 h in transfer buffer. Membranes were blocked for 2 h at 25C in phosphate-buffered saline with 0.1% Tween 20 supplemented with

HPLC-Flo/One characterization of steroids The newly synthesized steroids extracted from the incubation medium or tissue homogenates already purified on Sep-Pak cartridges were characterized using a previously validated method which combines HPLC analysis and flow scintillation detection (Mensah-Nyagan et al. 1994, 1996a,b, 2001a,b). Briefly, the prepurified extracts were analysed by reversed-phase HPLC on a liquid chromatograph (322 pump, UV/VIS 156 detector, Unipoint system; Gilson, Middleton, WI, USA) equipped with a 4.6 · 250-mm SymetryShield C18 column (Waters Associates) equilibrated with 100% hexane. The radioactive steroids were eluted at a flow rate of 0.5 mL/min using a gradient of isopropanol (0–60% over 65 min) including five isocratic steps at 0% (0–10 min), 1% (30–35 min), 2% (40–45 min), 30% (50–55 min) and 60% (60–65 min). The tritiated steroids eluted from the HPLC column were directly quantified with a flow scintillation analyser (Radiomatic Flo/One-Beta A 500; Packard Instruments, Meriden, CT, USA) equipped with a pentium IV PC for measurement of the percentage of total radioactivity



contained in each peak. Synthetic steroids used as reference standards were chromatographed under the same conditions as tissue extracts and their elution positions were determined by UV absorption using a UV/VIS 156 detector (Gilson). To optimize the characterization of newly synthesized neurosteroids, synthetic tritiated well-known neurosteroids, including 9,11,12-[3H](N)allopregnanolone, [3H]DHEA, [3H]PREG and [3H]PROG (for reviews see Baulieu et al. 1999; Mensah-Nyagan et al. 1999, 2001b; Mellon and Griffin 2002), were also used as reference standards, chromatographed under the same conditions as tissue extracts and identified by their elution times with the Flo/One computer system. Quantification of steroid biosynthesis and statistical analysis The amount of radioactive steroids formed by the conversion of [3H]PREG was expressed as a percentage of the total radioactivity contained in all peaks resolved by the HPLC-Flo/One system, including [3H]PREG itself. Each value is the mean of four independent experiments. Statistical analysis was performed using Student’s t-test.

Results

Expression of cytochrome P450c17 gene in the spinal cord Figure 1 shows representative amplification curves for the P450c17 gene, GAPDH used as a housekeeping gene and the negative control. These curves were obtained by the fluorescence reporter SYBR Green I dye whose emission is directly proportional to the quantity of amplicons produced during the PCR. Through the denaturing step (see Materials and methods), the dye could not be activated and was free in the solution. At the annealing temperature (57C), the first newly synthesized double-strand DNA stimulated the dye whose emission was recorded in real time during the polymerization as shown in Fig. 1. Therefore, the continuous fluorescence monitoring of the copy numbers within an ample dynamic range provided a highly sensitive detection of specific template signals for P450c17 (Fig. 1a) and GAPDH (Fig. 1b) in the SC and testis, a classical steroidogenic tissue. Amplification reactions were followed by melting-curve analysis to ensure the specificity of PCR products. This analysis, based on melting temperatures, was performed for each PCR product in the same closed capillary used for amplification and thus the risk of contamination could be avoided enabling easy differentiation of specific fragments from non-specific products (Figs 2a and b). The ranges of P450c17 mRNA concentrations detected in the nervous tissue were 102-fold lower than that measured in testis (Figs 1a and b). Occurrence of cytochrome P450c17 protein in the spinal cord The expression of P450c17 protein (54 kDa) in the SC and testis was assessed by western blot analysis (Fig. 3). By

using the antiserum against P450c17, we detected a 54-kDa protein in total homogenates and microsomal fractions from the SC and testis. The specific band disappeared when the P450c17 antibody was pre-incubated with microsomal proteins (data not shown). Cellular distribution of cytochrome P450c17 in the spinal cord Immunoreactivity for P450c17 was detected in the white matter (Wm) and grey matter (Gm) of the SC from the cervical to sacral segments (Figs 4a–d). Pre-incubation of the P450c17 antibody with microsomal proteins extracted from the spinal tissue resulted in a complete disappearance of the immunostaining in the SC (Figs 4c–f). Similar observations were made when the P450c17 antiserum was replaced by PB or non-immune rabbit serum during the immunohistochemical procedure (data not shown). In the Gm, numerous cell bodies expressing the enzyme were localized in the ventral horn (Figs 4a–d) and superficial layers, laminae I–II of the dorsal horn (Figs 5a and b). Most of the P450c17-positive cells detected in the Wm were characterized by thick and linear processes (Fig. 5c). Double-labelling experiments were performed using P450c17 antiserum and monoclonal antibodies against NeuN, MAP-2, GFAP or GalC (Fig. 6). It appeared that

Fig. 1 Real-time (rt)-PCR amplification of (a) cytochrome P450c17 (P450c17) and (b) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes. rt-PCR obtained in the testis and spinal cord (SC).



65% of the P450c17-positive cell bodies detected in the dorsal horn also expressed immunoreactivity for NeuN (Figs 6a–c). A high magnification made it possible to observe that the P450c17-immunoreactive material, which was present in the cytoplasm of the majority of labelled cells, was also expressed in the nucleoplasm of certain positive neuronal perykarya (Fig. 6d). Several P450c17immunoreactive fibres located in the Gm also exhibited immunostaining for MAP-2 (Fig. 6e). These P450c17/ MAP2-positive fibres were connected to various cell bodies containing P450c17 immunoreactivity (Fig. 6f). The majority of P450c17-positive fibres localized in the Wm coexpressed immunoreactivity for GFAP (Fig. 6g) but not for MAP-2 (Fig. 6e). The percentage of colocalization between GFAP and P450c17 in perikarya of the Gm was less than 1% (Fig. 6h) while 34% of P450c17-positive cell bodies also contained the GalC-immunoreactive material (Fig. 6i).

Fig. 3 Western immunoblot analysis of cytochrome P450c17 in rat spinal cord (SC) and testis (Te). From left to right, total and microsomal proteins (50 lg) were prepared from SC. Microsomal and total proteins (50 lg) from rat testis were used as positive controls. The estimated molecular size is shown on the left.

Cytochrome P450c17 activity in the spinal cord A 3-h incubation of SC slices with [3H]PREG yielded the formation of several radioactive metabolites (Fig. 7).

Fig. 4 Cytochrome P450c17 (P450c17)-immunoreactive cells and fibres in the spinal cord (SC). (a–d) Transverse sections through the SC showing the distribution of P450c17 immunoreactivity in the white matter (Wm) and grey matter (Gm). An intense immunostaining was found in the ventral horn and the superficial layers of the dorsal horn in cervical (a), thoracic (b), lumbar (c) and sacral (d) regions. (e and f) Adjacent sections of (c and d) incubated with P450c17 antiserum preabsorbed with purified microsomal proteins. Scale bars, 200 lm.

Fig. 2 Specificity of cytochrome P450c17 (P450c17) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR products. Meltingcurve analysis of P450c17 (a) and GAPDH (b) PCR products showing the very high degree of specificity of amplified double-strand DNA. Performance of melting analysis for each PCR product in the same closed capillary used for amplification meant that the risk of contamination could be avoided and enabled easy differentiation of specific fragments from non-specific products. SC, spinal cord.

Reversed-phase HPLC analysis coupled to a flow scintillation detection showed that one of the newly synthesized tritiated metabolites had the same retention time as synthetic DHEA and [3H]DHEA obtained from a commercial source. In addition, the SC slices were also capable of converting [3H]PREG into [3H]PROG and [3H]17OHPROG. The amounts of newly synthesized neurosteroids detected in the incubation medium were higher than that



remaining in the tissue at the end of the incubation period (Figs 7a and b). Time-course studies were performed to investigate the kinetics of [3H]PREG conversion into [3H]DHEA by SC slices. A significant level of newly synthesized [3H]DHEA was measured 30 min after the beginning of the incubation. This level reached a maximum within 3 h and slowly declined during the next 9 h (Fig. 8a). After 12 h of incubation, the relative amount of newly synthesized [3H]DHEA was 0.16 ± 0.012% compared with the maximal level obtained at 3 h which was 0.20 ± 0.02%. The selective inhibitor of P450c17 activity, ketoconazole (50 lM), significantly reduced the conversion of [3H]PREG into [3H]DHEA in SC slices ()58%; ***p < 0.001) (Fig. 8b). Discussion

Fig. 5 Cytochrome P450c17 (P450c17)-immunoreactive cells and fibres in the grey matter and white matter (Wm) of the spinal cord (SC). (a and b) Confocal laser-scanning microscope photomicrographs showing P450c17-positive cell bodies in laminae I and II of the dorsal horn (DH) in cervical (a) and lumbar (b) regions of the SC. (c) Transverse section of the lumbar SC showing P450c17-positive cells and their thick and linear processes in the Wm. Scale bars, 200 lm.

Within nanomolar and micromolar concentration ranges, synthetic DHEA and DHEAS control various mechanisms in the CNS of rodents (for reviews see Baulieu and Robel 1998; Mellon and Griffin 2002). DHEA and DHEAS induce prominent increases in the numbers of neurones and astrocytes with extensions of the processes of both cell types (Roberts et al. 1987). In particular, DHEA promotes axonal growth and morphological indices of synaptic contacts whereas DHEAS stimulates dendritic growth and branching in cultured embryonic neuronal cells (Compagnone and Mellon 1998). In addition, DHEA has been reported to increase neuronal excitability when directly applied to septal-pre-optic neurones (Carette and Poulain 1984). Several studies have also indicated that DHEA and DHEAS modulate NMDA receptors which are involved in numerous activities of the nervous system (Baulieu and Robel 1998; Mellon and Griffin 2002). As the adult rodent steroidogenic glands do not secrete significant amounts of DHEA and DHEAS, the only possible explanation for all of these neuroactive actions being exerted by DHEA and DHEAS in vivo remains the local formation of DHEA and DHEAS in the nervous system. Therefore, the last two decades have been marked by several attempts to demonstrate the presence and activity of P450c17, the key DHEA-synthesizing enzyme in the CNS of adult rodents (Mellon and Deschepper 1993; Compagnone et al. 1995; Stro¨mstedt and Waterman 1995; Kohchi et al. 1998). This enzyme is crucial for life in rodents as the deletion of the mouse P450c17 gene causes early embryonic lethality (Bair and Mellon 2004). The present report provides the first anatomical and cellular localization of P450c17 in the adult rat SC and shows the occurrence of P450c17 activity leading to DHEA formation in spinal neural tissue. The observations were possible by a coherent combination of various molecular, cellular and neurochemical techniques.



Fig. 6 Dual-channel confocal laser-scanning microscope photomicrographs of spinal cord transverse sections comparing the distribution of cytochrome P450c17 (P450c17) immunoreactivity with that of neuronal nuclei (NeuN) (a–d), microtubule-associated protein-2 (MAP-2) (e and f), glial fibrillary acidic protein (GFAP) (g and h) or galactocerebroside (GalC) (i). (a) Photomicrograph of the dorsal horn (DH) superficial layers labelled with the monoclonal anti-NeuN revealed with Alexa-488-conjugated donkey anti-mouse. (b) Photomicrograph of the same section as in (a) labelled with P450c17 antiserum revealed with Alexa-555-conjugated goat anti-rabbit. (c) Combination of the two images in (a and b) showing the colocalization of NeuN and P450c17 in neuronal cell bodies in the DH (arrows). (d) High-magnification photomicrograph showing a cell body in the DH expressing both NeuN and P450c17 immunostaining. In the cytoplasm where the intensity of NeuN immunoreactivity was

low, P450c17-positive elements appeared in red. The P450c17-immunoreactive material was also found in the nucleoplasm where it colocalized with NeuN and appeared in yellow. (e) Dual-channel image of a lumbar section showing the colocalization of MAP-2 and P450c17 immunoreactivities in the DH superficial layers. (f) Highmagnification photomicrograph of the DH showing MAP-2-positive fibres connected to P450c17-immunoreactive cell bodies (arrows). (g) Dual-channel image of a lumbar section showing the colocalization of GFAP and P450c17 immunoreactivities in the white matter (Wm). (h) High-magnification photomicrograph showing the absence of colocalization between P450c17- and GFAP-like immunostaining in the DH superficial layers. (i) High-magnification photomicrograph of the DH showing perikarya simultaneously expressing GalC and P450c17 immunostaining. Scale bars (a–c, f, h and i), 50 lm; (d) 10 lm; (e and g) 100 lm. Gm, grey matter.

Cytochrome P450c17 gene expression and biological activity in the spinal cord By using rt-PCR after RT we have detected significant amounts of specific mRNA encoding P450c17 in all segments of adult rat SC. In particular, we confirmed the specificity of PCR products by performing analyses based on melting temperature in the same closed capillary used for amplification, an approach which made it possible to avoid the risk of contamination and enabled easy differentiation of specific fragments from non-specific products (Morrison et al. 1998; Patte-Mensah et al. 2004b). The normalization of P450c17

product to GAPDH revealed that concentrations of P450c17 mRNA present in the adult rat SC were sufficient to justify a substantial expression of the enzymatic protein in spinal tissue. We have taken advantage of the availability of a specific antiserum to assess, by western blot, the occurrence of P450c17 protein in the SC and testis. The P450c17 antiserum, which allowed the detection of a specific protein in total homogenates and microsomal fractions from the SC and testis, has also been used successfully in previous studies to localize P450c17 in Leydig cells (Hales et al. 1987; Hales 1992).



Fig. 7 Characterization of neurosteroids released in the incubation medium by spinal cord slices (a) and remaining in the spinal tissue (b) after a 3-h incubation with [3H]pregnenolone (PREG). Analyses were performed using a hexane/isopropanol gradient and a reversedHPLC system coupled to a flow scintillation detector. The ordinate indicates the radioactivity measured in the HPLC eluent. The dashed line represents the gradient of secondary solvent (% isopropanol). The arrows indicate elution positions of standard steroids. ALLOPREG, allopregnanolone; DHEA, dehydroepiandrosterone; PROG, progesterone; 17OH-PROG, 17-hydroxyprogesterone.

The anti-P450c17 also made it possible to determine the anatomical and cellular distribution of P450c17-like immunoreactivity in the Wm and Gm of the SC. Double-labelling experiments with specific markers for neurones, astrocytes and oligodendrocytes (Raff et al. 1978; Kennedy 1982; Matus 1990; Mullen et al. 1992) revealed that P450c17 immunostaining was expressed in both neurones and glial cells throughout the adult rat SC. However, in the Wm, P450c17 was mainly localized in astrocytes while the enzyme was detected in neurones and oligodendrocytes in the Gm. In particular, an important population of neurones of the dorsal horn expressed P450c17 immunoreactivity, suggesting that the enzyme may be involved in the modulation of sensory activity (Willis et al. 1995; Millan 1999, 2002). Numerous neurones of the ventral horn also contained P450c17, indicating a possible correlation of the enzyme

Fig. 8 (a) Kinetics of the conversion of [3H]pregnenolone (PREG) into [3H]dehydroepiandrosterone (DHEA) by spinal cord (SC) slices. The values were obtained from experiments similar to that presented in Fig. 7. Each value was calculated as the relative amount of [3H]DHEA compared with the total amount of [3H]-labelled compounds resolved by HPLC-Flo/One characterization (·100). Each value is the mean ± SEM of four independent experiments. (b) Effect of the cytochrome P450c17 inhibitor ketoconazole (50 lM) on the conversion of [3H]PREG into [3H]DHEA by SC slices after a 3-h incubation period. The values were obtained from experiments similar to that presented in Fig. 7. Each value was calculated as the relative amount of [3H]PREG compared with the total [3H]-labelled compounds resolved by HPLC-Flo/One characterization (·100). Each value is the mean ± SEM of four independent experiments. ***p < 0.001.

activity with motor function (Haines et al. 1997). Surprisingly, the P450c17-immunoreactive material was detected in the nucleoplasm of certain nerve cell bodies even though the enzyme is well known to be a microsomal protein in classical steroidogenic tissues. It is possible that this apparent nuclear labelling corresponds to a localization of P450c17 in the endoplasmic reticulum associated with the nuclear mem-



brane, although our observations were made with a confocal microscope on single or several superimposed optical sections. Nevertheless, it is noteworthy that previous studies have also mentioned the nuclear localization of steroidogenic enzymes, such as 17b-hydroxysteroid dehydrogenase and 5a-reductase isozymes, in nuclear and cytoplasmic compartments of human and rat prostatic cells (Abalain et al. 1989; Aumuller et al. 1996; Bonkhoff et al. 1996; Span et al. 1996). To demonstrate that P450c17-like immunoreactivity detected in the SC corresponds to an active form of the enzyme, pulse-chase experiments were performed to study the conversion of [3H]PREG into radioactive metabolites by SC slices. Our results showed that [3H]DHEA was synthesized de novo from [3H]PREG in SC slices and the newly produced steroid was characterized by the HPLC/Flo-One method previously validated (Mensah-Nyagan et al. 1994, 1996a,b; Patte-Mensah et al. 2003, 2004b). In addition, among the newly synthesized radioactive steroids from [3H]PREG, two corresponded to [3H]PROG and [3H]17OHPROG, indicating that the precursor ([3H]PREG) was converted by 3b-hydroxysteroid dehydrogenase into [3H]PROG which was, in turn, transformed into [3H]17OH-PROG by P450c17. In agreement with this observation, 3b-hydroxysteroid dehydrogenase gene expression and activity have been shown in adult rat SC (Coirini et al. 2002). Furthermore, the conversion of [3H]PREG into [3H]DHEA or [3H]17OH-PROG was significantly reduced when the pulse-chase experiments were performed in the presence of ketoconazole, a selective inhibitor of P450c17 (Kuhn-Velten and Lessmann 1992; Swart et al. 1993). Moreover, kinetics studies showed that the absolute amount of [3H]DHEA synthesized from [3H]PREG reached a maximum within 3 h but declined slowly as only a 20% decrease was observed during the next 9 h. This result suggests that endogenous DHEA produced in the SC may be accumulated and reach sufficient or required concentrations to induce various neuroactive actions. The 20% decrease may correspond to the percentage of DHEA converted into DHEAS or into endogenous estradiol as observed in hippocampal neurones (Hojo et al. 2004). DHEA can also be reobtained from DHEAS by the activity of steroid sulphatase, the presence of which has been shown in the human and rodent nervous system (Compagnone et al. 1997; Steckelbroeck et al. 2004). Physiological implications It has been demonstrated that DHEA promotes recovery of motor behaviour after contusive SC injury in adult rodents (Fiore et al. 2004). This observation was made by treating the injured SC with synthetic DHEA. Therefore, it is possible that local production of DHEA in the SC may be an endogenous mechanism activated in the spinal neural tissue to cope with aggressive or traumatic situations. In support of

this suggestion, our results revealed that, in the Wm, P450c17 immunoreactivity was mainly expressed in astrocytes, a cell type strongly involved in reactive gliosis characterizing the spinal neural tissue in traumatic states (for review see Profyris et al. 2004). It has also been reported that DHEA, PREG and sex steroids may affect brain repair by down-regulating gliotic tissue (Garcia-Estrada et al. 1999). Moreover, implication of DHEA was shown in the regulation of the astroglial reaction to denervation of olfactory glomeruli (Hoyk et al. 2004). Collectively, these data indicate that the endogenous DHEA synthesized within the nervous system may be important in the control of neural plasticity. Various pharmacological studies have also suggested the involvement of synthetic DHEA/DHEAS in the modulation of sensory processes. A pro-nociceptive effect of DHEAS has been described in mice using the peripheral flexor response test (Ueda et al. 2001; Uchida et al. 2003). Electrophysiological data also revealed that DHEA potentiates native ionotropic ATP receptors containing the P2X2 subunit in rat sensory neurones (De Roo et al. 2003). A competitive inhibition of the capsaicin receptor-mediated current by DHEA has recently been shown on dorsal root sensory neurones (Chen et al. 2004). Altogether, these studies suggest a potentially pivotal role for endogenous DHEA in the control of nociceptive transmission. This hypothesis is strongly supported by the localization of P450c17 in numerous neurones of the dorsal horn, an important structure involved in nociception (Millan 1999, 2002), and also by the local synthesis of DHEA in spinal neuronal networks described herein. In conclusion, this study provides the first demonstration of the presence and activity of P450c17 in the SC of adult rodents and suggests that endogenous DHEA produced in spinal neural tissue may participate in the regulation of neuroprotective mechanisms and sensorimotor activities. Acknowledgements This work was supported by grants from Conseil Re´gional d’Alsace, Centre National de la Recherche Scientifique (CNRS, France) and Universite´ Louis Pasteur (Strasbourg, France). CK was the recipient of a fellowship from the Ministe`re de l’Education Nationale et de la Recherche.

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