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The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 3 1324-1331
Copyright © 2001 by The Endocrine Society

Original Studies

Characterization of the 5{alpha}-Reductase-3{alpha}-Hydroxysteroid Dehydrogenase Complex in the Human Brain1

Stephan Steckelbroeck, Mathias Watzka, Robert Reichelt, Volkmar H. J. Hans, Birgit Stoffel-Wagner, Dagmar D. Heidrich, Johannes Schramm, Frank Bidlingmaier and Dietrich Klingmüller

Departments of Clinical Biochemistry (S.S., M.W., R.R., B.S.-W., D.D.H., F.B., D.K.), Neuropathology (V.H.J.H.), and Neurosurgery (J.S.), University of Bonn, 53105 Bonn, Germany

Address all correspondence and requests for reprints to: Dr. Stephan Steckelbroeck, Institut für Klinische Biochemie, Universität Bonn, Sigmund-Freud-Strasse 25, D-53127 Bonn, Germany. E-mail: st_steckelbroeck{at}hotmail.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although androgen metabolism in the human brain was discovered almost 30 yr ago, conclusive studies on the enzymes involved are still lacking. We therefore investigated 5{alpha}-reductase and colocalized 3{alpha}-hydroxysteroid dehydrogenase (3{alpha}-HSD) activity in cerebral neocortex (CX) and subcortical white matter (SC) specimens neurosurgically removed from 44 patients suffering from epilepsy. We could demonstrate the presence of the 5{alpha}-reductase-3{alpha}-HSD complex in the biopsies of all patients under investigation. Inhibition experiments with specific inhibitors for 5{alpha}-reductase type 1 and type 2 revealed strong evidence for the exclusive activity of the type 1 isoform. We detected a significantly higher 5{alpha}-reductase activity in CX than in SC (P < 0.0001), but no sex-specific differences were observed. Furthermore, we found that, in contrast to liver, only 3{alpha}-HSD type 2 messenger RNA is expressed in the brain and that its expression is significantly higher in SC than in CX without sex-specific differences. The present study is the first to systematically characterize the 5{alpha}-reductase-3{alpha}-HSD complex in the human brain. The lack of sex-specific differences and also the colocalization of both enzymes at all life stages suggest a more general purpose of the complex, e.g. the synthesis of neuroactive steroids or the catabolism of neurotoxic steroids, rather than control of reproductive functions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANDROGENS HAVE IMPORTANT effects on the central nervous system. An extensive androgen metabolism including aromatization, 5{alpha}-reduction, and conversion via 3{alpha}-hydroxysteroid dehydrogenase (3{alpha}-HSD; EC 1.1.1.213, formerly EC 1.1.1.50) or 17ß-hydroxysteroid dehydrogenase (17ß-HSD; EC 1.1.62) activity occurs in several regions of the brain (1, 2, 3, 4, 5, 6). The steroid hormones synthesized by these enzymes influence the sexual differentiation of neural structures, the modulation of sexual behavior, and a variety of nonreproductive functions of the mature brain (7, 8, 9, 10). Specific intracellular receptors for androgens and estrogens were identified in several regions of the brain, indicating genomic actions of these steroid hormones on the brain (11, 12, 13). Recently, the existence of cognate membrane sex-steroid receptors for rapid nongenomic alterations of neuronal functions was also suggested (14, 15). Although androgen metabolism in the human brain via 5{alpha}-reductase (EC 1.3.99.5) and colocalized 3{alpha}-HSD was reported for the first time in the early 1970s (4), conclusive studies on the 5{alpha}-reductase-3{alpha}-HSD complex in the mature human brain are still lacking.

Besides its role in the androgen metabolism, 5{alpha}-reductase is thought to play an important role in the activation of neurosteroids via the 5{alpha}-reductase-3{alpha}-HSD complex (2, 10, 16, 17, 18). 5{alpha}-Reduced-3{alpha}-hydrogenated derivatives of progesterone and corticoids, but also androgens, are found to be potent mediators of the {gamma}-aminobutyric acid receptor-regulated chloride channel (19). Two isozymes catalyze the 5{alpha}-reduction of steroid substrates with 3-oxo-{Delta}4 structures (20, 21). The gonadal type 2 isoform is only transiently expressed in the late fetal and early postnatal rat brain, whereas the expression of the nongonadal type 1 isozyme was also detected in the adult rat brain (22). The conversion of 3-keto steroids into 3{alpha}-hydroxy compounds is catalyzed by 3{alpha}-HSD. Recently three highly homologous human 3{alpha}-HSD isoforms have been identified (23).

To date, only limited information is available on 5{alpha}-reductase and colocalized 3{alpha}-HSD activities in the human brain (24, 25), but we recently determined the exclusive expression of the 5{alpha}-reductase type 1 isoform messenger RNA (mRNA) in the postnatal human temporal lobe (26). In the present study, we investigated the 5{alpha}-reductase and the colocalized 3{alpha}-HSD activities in microsomal preparations of macroscopically and microscopically inconspicuous surgical brain biopsies from patients suffering from epilepsy. We used androstenedione as the substrate because, in human fetal brain preparations, the formation of 5{alpha}-reductase products was highest with androstenedione, when compared with testosterone, progesterone, and 17{alpha}-hydroxy-progesterone, respectively (25). Moreover, human 5{alpha}-reductase type 1 shows a clear preference for androstenedione over testosterone as substrate (20).

To elucidate the isoform patterns of 5{alpha}-reductase and to verify our recent RT-PCR experiments, we investigated the inhibitory effects of MK386 [a specific inhibitor of the 5{alpha}-reductase type 1 isoform (27)] and of finasteride [a specific inhibitor of the 5{alpha}-reductase type 2 isoform (28)] on brain tissue 5{alpha}-reductase activity and compared it with that on the prostate isoform type 2. A further important aim of our study was to determine possible sex-, age-, and tissue-specific differences of 5{alpha}-reductase activity in the human cerebral neocortex (CX) and subcortical white matter (SC).

To further extend the knowledge of the 5{alpha}-reductase-3{alpha}-HSD complex in the human brain, we investigated the mRNA expression of the three known 3{alpha}-HSD isoforms in the human temporal lobe. This was conducted to determine the predominant isoform and to detect possible sex- or tissue-specific differences.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Sources of tissues

Human brain tissue was obtained neurosurgically from 44 patients suffering from focal epilepsy. The specimens used for the present study derived in 42 cases from the temporal lobe (19 female patients, 4–47 yr old; 23 male patients, 1–67 yr old); whereas, in 2 cases, the epileptogenic focus was located occipital (male patient, 8 yr old) and frontal (female patient, 11 yr old), respectively. Tissue situated around the presumable epileptic focus was not taken for our study. We used only specimens that appeared macroscopically and microscopically inconspicuous. Immunohistochemistry, using an antibody against the astrocyte-marker GFAP (glial fibrillary acidic protein), revealed fibrillary gliosis in SC and some reactive astrocytes in CX, as well as in the subpial zone; this is a common finding in samples from epileptic patients (not shown). Extensive neuronal cell loss was not evident in any specimen. Tissue was excluded from the study if it was histologically suspicious for tumor or inflammation.

Macroscopically normal human liver tissue was obtained from biopsies carried out to test for liver diseases in a transplantation program, and macroscopically normal human prostate tissue was obtained from a patient with bladder cancer undergoing cystectomy and prostatectomy. The tissues were transferred to liquid nitrogen immediately after removal and were stored at -80 C.

The study was approved by the local ethics committee, and informed consent was received from all tissue donors or their parents.

Steroids and chemicals

[1ß,2ß-3H]androstenedione (42 Ci/mmol) was obtained from NEN Life Science Products Co. (Dreieich, Germany) and was purified by thin-layer chromatography (TLC) before use. Unlabeled steroids, EDTA, Folin & Ciocalteu’s phenol reagent, TRIZMA ({alpha},{alpha},{alpha}-Tris-(hydroxymethyl)-methylamin), TRIZMA-HCl, citric acid, and sodium potassium tartrate were purchased from Sigma Chemical Company (Deisenhofen, Germany). 17ß-(N-tert-butylcarbamoyl)-4-aza-5{alpha}-androst-1-ene-3-one (finasteride) and 4,7-dimethyl-4-azacholestan-3-one (MK386) were kindly provided by Merck & Co., Inc. (Rahway, NJ). Pyridine nucleotides, PCR buffer, Taq polymerase, and deoxynucleotide triphosphates were obtained from Roche Molecular Biochemicals (Mannheim, Germany). Trizol LS reagent and Superscript II were purchased from Life Technologies, Inc. (Karlsruhe, Germany). Primers for PCR were obtained from Life Technologies, Inc. and PE Applied Biosystems (Weiterstadt, Germany). The liquid scintillation cocktail, Ultima Gold, was supplied by Packard-Instrument, B.V., Chemical Operations (Groningen, Netherlands). All other chemicals and all solvents were purchased from Merck KGaA (Darmstadt, Germany). Solvents were distilled before use, and all reagents were at the highest grade commercially available.

Buffers

Homogenization buffer consisted of 10 mmol/L TRIZMA-HCl (pH 7.4) and 1 mmol/L EDTA. Assay buffer consisted of 160 mmol/L TRIZMA-citrate and 10 mmol/L MgCl2 at the indicated pH value. PCR buffer consisted of 10 mmol/L Tris-HCl (pH 8.3), 40 mmol/L potassium chloride, 1.5 mmol/L MgCl2, 200 µmol/L of each deoxynucleotide triphosphate, and 0.5 U Taq polymerase.

Preparation of tissues

All steps of tissue preparation were carried out at 4 C. Immediately after neurosurgical removal, the tissue specimens were macroscopically separated into CX and SC, transferred into liquid nitrogen, and stored at -80 C until further processing. To ensure optimal protein concentrations in the assays, 150–200 mg wet brain tissue and 50 mg cut-up wet prostate tissue, respectively, were homogenized in 1 mL ice-cold homogenization buffer using a motor-driven Teflon-glass homogenizer (Potter S, B. Braun, Melsungen, Germany) with 2 x 10 strokes at 1000 rpm, followed by a ultrasonication [3 times, for 10 sec each time, at 50 W (Labsonic 2000, B. Braun)]. The final homogenates were centrifuged at 4000 x g for 15 min to provide a nuclear pellet and a postnuclear supernatant. The resultant cell-free supernatant was employed as microsomal enzyme source. Protein concentrations were measured according to the procedure of Lowry et al. (29).

Measurement of 5{alpha}-reductase activity

Determination of 5{alpha}-reductase in vitro activity by TLC analysis was performed as described previously (5). Briefly, in a final vol of 200 µL, the reaction mixture contained either 50 µL of the microsomal tissue preparation or 50 µL homogenization buffer for control incubations and 100 µL assay buffer, at the indicated pH value, containing dissolved [1ß,2ß-3H]androstenedione (plus unlabeled androstenedione for substrate concentrations of more than 0.1 µmol/L). The reactions were started by the addition of 50 µL homogenization buffer containing NADPH as cofactor (3 mmol/L final concentration). All incubations were performed in duplicate, for 1 h, with constant shaking, at 37 C. Reactions were terminated, and organic compounds were extracted by the addition of 1 mL ice-cold chloroform/methanol (2:1, vol/vol). An aliquot of each organic phase was evaporated to dryness and dissolved in 50 µL chloroform containing nonradioactive reference steroids: 5{alpha}-androstane-3,17-dione (androstanedione), androst-4-ene-3,17-dione (androstenedione), 5{alpha}-androstane-17ß-ol-3-one (dihydrotestosterone), 5{alpha}-androstane-3{alpha}-ol-17-one (androsterone), androst-4-ene-17ß-ol-3-one (testosterone), and 5{alpha}-androstane-3{alpha},17ß-diol (3{alpha}-androstanediol). Estrogens were not added because the microsomal human CX and SC aromatase cytochrome P450 (EC 1.14.14.1) activity amounts to just 1/1000th of the 5{alpha}-reductase activity and, therefore, is not detectable by TLC analysis (6). Consequently, it does not interfere with the determination of brain tissue 5{alpha}-reductase activity by TLC analysis.

Dissolved incubation extracts were separated by TLC using a TLC sheet with plastic back precoated with a 0.25-mm layer of silica gel (Polygram Sil G, Macherey & Nagel, Düren, Germany). Dichloromethane/acetone (92.5:7.5, vol/vol) was used as the mobile phase. Within each lane, the zones corresponding to the stained reference steroids were cut out and transferred into counting vials containing 15 mL liquid scintillation cocktail. Radioactivity was counted, as automatically quench-corrected decay per minute, with a Wallac, Inc. (Turku, Finland) 1409 liquid scintillation counter.

The relative amount of each corresponding radioactive steroid was calculated, in percentage, with the total radioactivity recovered from a single TLC lane set as 100%. Blank values were subtracted from tissue metabolism rates. Enzyme activities were expressed as femtokatal per mg protein (fkat/mgprotein). 5{alpha}-Reductase activity was assessed by quantifying the formation of androstanedione and the formation of androsterone subsequently formed from androstanedione via colocalized 3{alpha}-HSD activity. 17ß-HSD activity was assessed by quantifying the formation of testosterone.

Digital autoradiography analysis

For digital autoradiography analysis, after incubation, substrate and metabolites were extracted as described above. Total corresponding organic phases were combined, transferred into a 10-mL glass tube, and dried under a stream of nitrogen. Dried extracts were dissolved in 100 µL chloroform containing the above mentioned nonradioactive reference steroids. Each reconstituted extract was applied to a 20 x 20-cm TLC glass plate precoated with a 0.25-mm layer of silica gel 60 F254 (Merck KGaA, Darmstadt, Germany). For the separation of the substances, dichloromethane/acetone (92.5:7.5, vol/vol) was used as the mobile phase. The radiodetection system consisted of an automatic TLC-linear analyzer LB 285 equipped with a one-dimensional position multiisotope head detector of high resolution, LB 2821-HR (EG & G Berthold, Wildbad, Germany). The TLC-linear analyzer measures linear radiodistributions in the Y direction. Two-dimensional distributions have to be scanned in the X direction, i.e. approximately 100 sections of a 20 x 20-cm TLC plate have to be measured. Two-dimensional radiodistribution was reconstructed from these sections by the computer program CHROMA 2D (EG & G Berthold).

RT-PCR-based 3{alpha}-HSD isozyme identification

Total liver and brain RNA were extracted from 50 mg tissue using the Trizol reagent as described previously (26). For the RT of 2 µg total RNA, we pooled 4 respective brain RNA samples (either SC or CX of both 4 women and 4 men). RT was performed at 42 C for 60 min by using 100 U Superscript II ribonuclease-free reverse transcriptase. For the analysis of the gene expression of the three known 3{alpha}-HSD isoforms, we used oligonucleotide primers that have been published previously (30, 31). PCR was conducted using 50 ng of the resulting complementary DNA in a final vol of 20 µL PCR buffer containing 4 pmol of the respective primer. After initial denaturation at 95 C for 3 min, 35 cycles of PCR amplification were routinely performed under the following conditions: 35 sec at 94 C, 45 sec at 55 C, and 90 sec at 72 C. After a final extension step of 5 min at 72 C, PCR products were resolved on 2% agarose gel containing ethidium bromide and then visualized under ultraviolet light using the Gel Doc 1000 System (Bio-Rad Laboratories, Inc. Heidelberg, Germany).

mRNA quantification

Quantification of mRNA was carried out according to a competitive RT-PCR protocol as described previously (32). We used competitive RNA standards with small deletions, and we investigated the mRNA expression of the 3{alpha}-HSD type 2 isozyme and GAPDH as the housekeeping gene. PCR products, labeled with fluorescent dyes, were separated on 6% denaturing acrylamide gel and analyzed. Peak areas were calculated with the Genescan Software (Version 1.2.1; PE Applied Biosystems). The ratio of the native PCR product to the standard PCR product was used for differential determination of gene expression. Initial differences in the amount of total RNA, which was subjected to RT, were corrected by calculating the ratios of native GAPDH PCR products to standard GAPDH PCR products.

Data analysis

The statistical differences between 5{alpha}-reductase activity in CX and in SC were calculated using the Wilcoxon rank test. To determine sex or age differences, a Mann-Whitney U test was performed. Results for statistical analysis were calculated as mean ± SD. P < 0.05 was considered to reflect statistical significance.

Kinetic analyses were performed with a computer-assisted nonlinear curve-fitting method using the Michaelis-Menten metabolism model (FigP 2.7, Biosoft, Cambridge, UK).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dose-response analyses of inhibition of 5{alpha}-reductase activity by finasteride and MK386

To identify the isoform responsible for the 5{alpha}-reductase reaction in the human brain, we investigated the dose-responsive inhibition of the enzyme activity by the 4-aza-steroids finasteride and MK386. Dose-response analyses of inhibition of androstenedione metabolism (0.1 µmol/L) by increasing inhibitor concentrations (0.0001–100 µmol/L vs. incubations without inhibitor) were performed either at pH 7.5 using a microsomal temporal lobe tissue (equal amounts of CX and SC from a 26-yr-old man) or at pH 5.5 using a microsomal prostate tissue preparation (58-yr-old man). Both finasteride and MK386 demonstrated dose-responsive inhibitory activity on brain as well as on prostate 5{alpha}-reductase activity (Fig. 1Go). We found inhibitor concentrations resulting in 50% inhibition (IC50 values) to be either 2.0 nmol/L for MK386 and 142.8 nmol/L for finasteride with the brain tissue preparation or 998.7 nmol/L for MK386 and 1.6 nmol/L for finasteride with prostate tissue preparation.



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  		Figure 1. Dose-response curves for the inhibition of human brain (A) and prostate tissue (B) 5{alpha}-reductase in vitro activity by finasteride ({circ}) and MK386 (•). Dose-response analyses were carried out either at pH 7.5 using a microsomal temporal lobe tissue preparation (26-yr-old man; equal amounts of CX and SC) or at pH 5.5 using a microsomal prostate tissue preparation (58-yr-old man). Inhibition of androstenedione metabolism (0.1 µmol/L) was studied at increasing inhibitor concentrations vs. control incubations without inhibitor.

 
Inhibitory effects of finasteride and MK386 on pH-dependent 5{alpha}-reductase activity

To clearly identify the isozyme responsible for 5{alpha}-reduction of androstenedione in the human brain, we also investigated how finasteride and MK386 inhibited the pH-dependent metabolism of 0.1 µmol/L androstenedione. 5{alpha}-Reductase activity was determined within a pH range from 4.5–9.5 using microsomal temporal lobe tissue preparations (equal amounts of CX and SC) from a 36-yr-old woman and a 30-yr-old man, either with 0.1 µmol/L finasteride or with 0.1 µmol/L MK386. Incubations without inhibitor were conducted as controls.

5{alpha}-Reductase activity in both preparations exhibited a broad pH optimum between 6.5 and 8.5, centered at pH 8.0 (Fig. 2Go). Incubations with MK386 revealed a potent inhibition of 5{alpha}-reductase activity within the whole pH range, whereas the inhibition was much lower with finasteride, especially within the acidic range.



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  		Figure 2. Inhibitory effects of finasteride ({circ}) and MK386 (•) on pH-dependent microsomal 5{alpha}-reductase activity were investigated using temporal lobe tissue preparations (equal amounts of CX and SC) of a 36-yr-old woman (A) and a 30-yr-old man (B). Inhibition of androstenedione metabolism (0.1 µmol/L) was determined over a pH range from 4.5–9.5 with 3 mM NADPH and with either 0.1 µmol/L of finasteride, 0.1 µmol/L of MK386, or without inhibitor as control ({blacksquare}).

 
All our experiments showed that, in addition to the 5{alpha}-reduction of androstenedione into androstanedione, a high amount of the 5{alpha}-reductase metabolite was subsequently converted into androsterone via colocalized 3{alpha}-HSD activity (Fig. 3Go). Furthermore, Fig. 3Go demonstrates that, in human brain tissue preparations, testosterone is formed from androstenedione via colocalized 17ß-HSD. This we observed in all our experiments as well. At pH 8.0, for example, 0.67 (0.51) fkat/mgprotein of androstanedione and 0.37 (0.38) fkat/mgprotein of androsterone were formed in the tissue preparation from the woman (man). In this case, 0.32 (0.35) fkat/mgprotein of testosterone was formed in the tissue preparation from the woman (man). The incubation with finasteride resulted in a minor reduction of the formation of androstanedione (46.7% and 44.3%, respectively) and of androsterone (46.0% and 41.2%, respectively), but the application of MK386 almost completely inhibited the formation of both metabolites. In contrast to that, the formation of testosterone was not impaired by the use of any of the inhibitors.



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  		Figure 3. Digital autoradiogram of TLC analysis of androstenedione metabolites. The autoradiogram shows the radiosignals of substrate and metabolites derived from incubations with microsomal temporal lobe tissue preparations (equal amounts of CX and SC) of a 36-yr-old woman and a 30-yr-old man, as well as from tissue-less control incubations [marked with (C)]. 5{alpha}-Reductase in vitro activity was determined at pH 8.0, using 3 mM NADPH as the cofactor and 0.1 µmol/L 3H-labeled androstenedione as the substrate, to investigate the inhibitory effects of 0.1 µmol/L of the 5{alpha}-reductase type 2 inhibitor finasteride (F) or the 5{alpha}-reductase type 1 inhibitor MK386 (MK) in relation to incubations without an inhibitor (—). The plotted rims are assigned to: androstanedione (5{alpha}-A), androstenedione ({Delta}4), dihydrotestosterone (DHT), androsterone (A), testosterone (T), and 3{alpha}-androstanediol (DIOL). They were obtained by staining nonlabeled reference steroids added to the incubation extracts. The origins of TLC are marked OR. Acquisition parameters: gain, 4; voltage, 1380 V; stepwidth, 2 mm; slit-width, 2 mm; step-numbers, 100; counting time, 200 min.

 
Tissue- and sex-specific microsomal 5{alpha}-reductase activity

To determine possible tissue- and sex-specific differences, we investigated microsomal 5{alpha}-reductase activity in both CX and SC specimens from 13 female (4–47 yr old) and 16 male (1–67 yr old) patients using 2 µM androstenedione as the substrate. Data analyses yielded neither a statistical difference between the age groups of 1–11 and 18–67 yr, in CX as well as in SC, nor were significant sex-specific differences observed. Therefore, we combined all data for males and females across age groups to investigate differences between CX and SC specimens. Statistical analysis revealed a highly significant difference between 5{alpha}-reductase activity in SC [5.3 ± 2.2 fkat/mgprotein (mean ± SD)] and in CX (7.8 ± 1.9 fkat/mgprotein) as shown in Fig. 4AGo (P < 0.0001).



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  		Figure 4. 5{alpha}-reductase in vitro activity (A) in the human brain was investigated at pH 7.4, using 3 mmol/L NADPH as the cofactor and 2 µmol/L androstenedione as the substrate, in relation to tissue-, sex- and age-specific differences. Enzyme activity was studied in microsomal tissue preparations of both CX (solid symbols) and SC specimens (open symbols) of 13 female [4–47 yr old (circles)] and 16 male patients [1–67 yr old (squares)]. B shows the amount of androstanedione being subsequently converted into androsterone (via colocalized 3{alpha}-HSD activity).

 
In all tissue preparations under investigation, both the 5{alpha}-reduction of androstenedione into androstanedione and the subsequent conversion of the 5{alpha}-reductase metabolite into androsterone were observed (Fig. 4BGo). In SC, on an average, 23.9 ± 4.0% (mean ± SD) of the androstanedione formed was subsequently converted into androsterone; whereas in CX, it was 20.2 ± 4.5%. No attempt was made to estimate significant differences concerning 3{alpha}-HSD activity, because the activity of this enzyme was not determined directly.

mRNA expression of 3{alpha}-HSD isozymes in the human temporal lobe

To discover the expression of the human 3{alpha}-HSDs types 1 to 3 in the human temporal lobe, we conducted a PCR-based identification using four pooled RNA samples of the respective brain tissue samples and two human liver RNA samples. As shown in Fig. 5Go, in contrast to liver, only the type 2 isoform was found to be expressed in the human temporal lobe. 3{alpha}-HSDs type 2 mRNA was detected in both CX and SC specimens of women and men.



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  		Figure 5. RT-PCR based identification of the expression of the 3{alpha}-HSD types 1, 2, and 3 in human brain and liver tissue. We pooled total RNA samples isolated from CX (lane 1) and SC (lane 2) of 4 men and from CX (lane 3) and SC (lane 4) of 4 women. Also, total RNA isolated from liver tissue of a 20-yr-old woman (lane 5) and a 38-yr-old woman (lane 6) was used. Oligonucleotide primers used in the RT-PCR experiments were specific for the genes of 3{alpha}-HSD type 1 (top), 3{alpha}-HSD type 2 (middle), and 3{alpha}-HSD type 3 (bottom), respectively. Lane 7 is an H2O no-template negative control. A 50-bp ladder (M) as DNA size marker is given on the left and on the right.

 
To validate our mRNA quantification protocol, we determined the expression of the 3{alpha}-HSD type 2 mRNA in brain tissue and compared it with that in liver. Employment of competitive RT-PCR requires knowledge of the amount of the RNA standard, which yields a signal of approximately equal density when coamplified with a defined amount of total RNA. Using 250 ng total RNA, we found the equivalent titration point for 3{alpha}-HSD type 2 to be 5 pg standard RNA in temporal lobe tissue and 50 pg standard RNA in liver tissue; whereas for GAPDH, it was 250 pg standard RNA in both tissues. 3{alpha}-HSD type 2 mRNA concentration was investigated in both the CX and SC of six female (20–46 yr old) and six male (26–48 yr old) patients. Data analysis did not reveal sex-specific differences (female patients: 2.5 ± 0.9 arbitrary units (aU) in CX, mean ± SD; 6.6 ± 2.7 aU in SC; male patients: 3.5 ± 2.1 aU in CX, 5.1 ± 2.5 aU in SC). Therefore, statistical analyses of differences between mRNA expression in SC and CX were calculated from the data of all patients, regardless of their sex. We determined a significantly higher 3{alpha}-HSD type 2 mRNA concentration in SC (5.8 ± 2.6 aU) than in CX (3.0 ± 1.7 aU; P < 0.05).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
An extensive sex steroid metabolism occurs in several regions of the brain (1, 2, 3, 4). The metabolism of androgens in the human central nervous system includes 3{alpha}-HSD, 17ß-HSD, aromatase cytochrome P450, and 5{alpha}-reductase activity (5, 6, 23, 24, 26, 30, 32, 33, 34). Especially in the postnatal and mature human brain, the 5{alpha}-reductase-3{alpha}-HSD complex has, to date, not been studied in detail. Two isozymes of 5{alpha}-reductase (type 1 and 2) with differential tissue distribution and biochemical properties have been identified (20, 21). Previously, we determined that only the type 1 mRNA of both 5{alpha}-reductase isoforms is expressed in the human temporal lobe (26). In the present study, we were able to provide strong biochemical evidence for the predominant, if not exclusive, activity of the 5{alpha}-reductase type 1 isozyme in the human brain by determining the inhibitor sensitivity of the in vitro reaction ( Figs. 1–3GoGoGo). We could demonstrate the presence of 5{alpha}-reductase and colocalized 3{alpha}-HSD in the brain tissue biopsies of all patients under investigation ( Figs. 1–4GoGoGoGo). Three highly homologous human 3{alpha}-HSD isoforms catalyze the reduction of 3-keto steroids (23). We ascertained that, in contrast to liver, only the 3{alpha}-HSD type 2 is expressed in the human temporal lobe (Fig. 5Go). Furthermore, we determined, in all our experiments, the formation of testosterone and therefore confirmed our previous results concerning testosterone formation in human brain tissue most likely due to 17ß-HSD isozyme type 3 expression (5, 33).

Ellsworth et al. (27) demonstrated that MK386 is a specific inhibitor of human 5{alpha}-reductase type 1, with an IC50 value of approximately 20 nmol/L for the native type 1 isoform, while an IC50 of approximately 3100 nmol/L for the native type 2 isoform was observed. According to Harris et al. (28), finasteride is a potent inhibitor of human 5{alpha}-reductase type 2 (with an IC50 value of approximately 5 nmol/L) and a poor inhibitor of the type 1 isoform (with an IC50 value of approximately 500 nmol/L). Therefore, our dose-response analyses with finasteride and MK386 as inhibitors and 0.1 µmol/L androstenedione as substrate suggest the predominant, if not exclusive, activity of the 5{alpha}-reductase type 1 isozyme in the mature human brain. MK386 is a strong inhibitor of human brain tissue 5{alpha}-reductase activity, with an IC50 value of 2.0 nmol/L, whereas finasteride turned out to be a poor inhibitor of the reaction, with an IC50 value of 142.8 nmol/L (Fig. 1AGo). In contrast to that, we observed an IC50 value of 998.7 nmol/L with MK386 and of 1.6 nmol/L with finasteride for the prostate 5{alpha}-reductase type 2 (Fig. 1BGo). Moreover, our dose-response analyses did not show biphasic inhibition curves; but experiments by others, with equal amounts of both human 5{alpha}-reductase isoforms, did (35).

The pH profiles of 5{alpha}-reductase in vitro activity in the brain tissue of both sexes and the potent inhibition of the pH-dependent reaction by MK386 (but not by finasteride), which we observed in our studies, further substantiate an at-least predominant activity of the 5{alpha}-reductase type 1 isozyme in the human brain (Fig. 2Go). According to Andersson et al. (21), the 5{alpha}-reductase type 1 isoform expressed in Hek-293 cells shows a broad pH optimum, between pH 6.0 and pH 8.5, whereas the type 2 isoform has a sharp pH optimum at pH 5.0. In our experiments, 5{alpha}-reductase activity in the human temporal lobe of both sexes exhibited a broad pH optimum, between pH 6.5 and pH 8.5, centered at pH 8.0. The additional presence of 5{alpha}-reductase type 2 would be characterized by a second distinct peak of activity approximately at pH 5 (35) and a potent finasteride inhibition at least within the acidic pH range. In our studies, however, we observed no second peak, and the inhibition of the pH-dependent 5{alpha}-reductase activity in brain tissue was considerably less potent with finasteride than with MK386, especially within the acidic range (Fig. 2Go). These findings suggest that, in both sexes, microsomal 5{alpha}-reduction of androstenedione must be attributed to the 5{alpha}-reductase type 1 isozyme; and this confirms our previous report of exclusive mRNA expression of the 5{alpha}-reductase type 1 isoform in the human temporal lobe (26).

In addition to this, to the authors’ knowledge, the present study is the first to investigate conclusively differences of human brain tissue 5{alpha}-reductase activity in relation to sex or age. Investigation of 5{alpha}-reductase activity in CX and SC specimens with 2 µM androstenedione as substrate revealed significantly higher enzyme activities (P < 0.0001) in CX than in SC (Fig. 4AGo), which is in good agreement with results obtained in experiments with brain tissue from bull, pig, hamster, monkey, and one woman (36) but is obviously in contradiction to the findings in rat and mouse (36, 37). Consequently, the present experiments confirm the unique difference between muridae and other vertebrate species studied so far. 5{alpha}-Reductase activity in CX and in SC did not differ between both sexes; this is consistent with previous studies in which no significant sex-specific differences regarding 5{alpha}-reductase activity were found in neural tissue of nonhuman primates (38) and of rodents (39, 40). We also discovered no differences between the age groups of 1–11 and 18–67 yr in CX and in SC tissue preparations.

Our findings concerning tissue-specific differences and age-related enzyme activities are in contradiction with our previous RT-PCR experiments, where we determined a significantly higher mRNA expression of the 5{alpha}-reductase type 1 in CX of adults than in that of children and where we did not find a significant specific difference between CX and SC specimens (26). Our enzyme activity data concerning possible sex differences are, however, consistent with those of our previous RT-PCR experiments, where also no sex-specific differences in the mRNA expression of the 5{alpha}-reductase type 1 in CX and in SC specimens were detected. These inconsistent findings may indicate the regulation of 5{alpha}-reductase at the posttranscriptional or posttranslational level. Therefore, we strongly recommend that, not only should the mRNA expression of an enzyme be investigated, but its activity should also always be measured.

All our experiments showed that a high amount of the 5{alpha}-reductase metabolite androstanedione is subsequently converted into androsterone. PCR-identification experiments revealed that, in contrast to liver, only the 3{alpha}-HSD type 2 isoform, which is also designated as 17ß-HSD type 5 (41), is expressed in the human temporal lobe (Fig. 5Go). In contrast to its 3{alpha}-HSD activity, its 17ß-HSD activity is highly labile and greatly reduced by homogenization (42). For that reason, it is unlikely that 3{alpha}-HSD type 2 is responsible for the catalyzation of the determined testosterone formation but that it is possibly the candidate for the 3-keto steroid reduction. 5{alpha}-Reductase activity is significantly higher in CX than in SC (Fig. 4aGo), but the amount of androsterone subsequently formed from the original 5{alpha}-reductase metabolite is almost equal in both tissues (Fig. 4bGo), so that a higher 3{alpha}-HSD activity in SC than in CX has to be assumed. These findings are in good agreement with the significantly higher mRNA expression of the 3{alpha}-HSD type 2 isozyme in SC than in CX. Interestingly, 3{alpha}-HSD type 2 is thought to eliminate active androgens from the prostate (23), which might be inconsistent with an anabolic function of the enzyme in the brain. Penning et al. (23) are presently demonstrating that, apart from 3{alpha}-HSD type 2 being expressed, 20{alpha}-HSD (EC 1.1.1.149) is also expressed to a larger extend in the human brain. Moreover, they showed that all human 3{alpha}-HSD isoforms and the human 20{alpha}-HSD act as 3-, 17-, and 20-ketosteroid reductases as well as 3-, 17-, and 20-hydroxysteroid oxidases. Thus, the meaning of the differential expression of the single isoforms is less established than ever.

The ubiquitous presence of 5{alpha}-reductase activity in the mature human CX and SC and the lack of any sex- and age-specific differences suggest that it has more general effects, e.g. the synthesis of neurosteroids, rather than the control of the reproductive function and the sexual behavior. We always observed a high amount of androstanedione being subsequently converted into androsterone (Figs. 3Go and 4BGo). Therefore, our findings prove that 5{alpha}-reductase and 3{alpha}-HSD activity are colocalized in the investigated human neural tissues and support the assumption that the nongonadal isoform of 5{alpha}-reductase may play an important role in the synthesis of neurosteroids via the 5{alpha}-reductase-3{alpha}-HSD pathway (2, 10, 16, 17, 18).

This is in accordance with a previous study in adult rats, which demonstrated that the anesthetic effects of progesterone, but not of its 5{alpha}-reduced/3{alpha}-hydroxylated metabolite 3{alpha}-hydroxy-5{alpha}-pregnan-20-one (allopregnanolone), is impaired by the pretreatment with a 5{alpha}-reductase inhibitor (43). Interestingly, in humans (just as in the mature rat brain), only the 5{alpha}-reductase type 1 isozyme is expressed in the postnatal brain (22, 26). Because the potencies of 5{alpha}-reduced/3{alpha}-hydroxylated neuroactive steroids in biochemical and electrophysiological assays correlate with their sedative, anti-seizure, anxiolytic, and neuroprotective effects, the specific activities of these neurosteroids may become useful in enlarging the therapeutic approaches to functional alterations of the nervous system (16, 17, 18, 44).

Finally, an important neuroprotective role of the brain tissue 5{alpha}-reductase-3{alpha}-HSD complex may also be proposed (45). Because 5{alpha}-reductase type 1 and 3{alpha}-HSD activity are present at all life stages, they might possibly be involved in the catabolism of neurotoxic steroids (e.g. glucocorticoids) via hydroxylation, after glucuronidation or sulfatation and the final elimination.


   Acknowledgments
 
We are grateful to Merck & Co., Inc., who kindly provided us with finasteride and MK386. We thank Dr. P. Albers (University of Bonn, Department of Urology) for supply of prostate tissue and Dr. M. Wolff (University of Bonn, Department of Surgery) for supply of liver tissue. In addition, we thank Prof. Dr. H. U. Schweikert for the seminal contribution made to this project.


   Footnotes
 
1 Supported by a grant from the Deutsche Forschungsgemeinschaft (Kl 524/5-1). Back

Received March 27, 2000.

Revised October 9, 2000.

Accepted November 26, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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