Enzymes Of Neurosteroid Biosynthesis In The Rodent Brain

Current View of Neurosteroid Biosynthesis

The definition stricto sensu of neurosteroids applies to PREG, DHEA, and their sulfate and fatty acid esters and to PROG and its 5a-reduced metabolites (5a-DH PROG, 3a,5a-TH PROG, and 3p,5a-TH PROG). It is not our purpose to provide a detailed description of the biosynthetic and metabolic enzymes, as they are accounted for extensively in other chapters of this book (refs. 19,20, and 53) We mainly wish to provide a critical appraisal of present knowledge. Our current view of neurosteroid biosynthesis and metabolism in the rat brain is summarized on Fig. 3.

Neurosteroids

Fig. 3. Neurosteroid biosynthesis and metabolism in the rat brain. Dotted arrows indicate metabolic conversions not yet formally demonstrated. Other metabolic reactions are not indicated such as the possible formation of 17p-hydroxylated steroids (e.g., A5-androstene-3p,17p-diol and testosterone), C19-steroids of the 5a series (e.g., androsterone and epiandrosterone), and phenolsteroids (e.g., estrogens).

Fig. 3. Neurosteroid biosynthesis and metabolism in the rat brain. Dotted arrows indicate metabolic conversions not yet formally demonstrated. Other metabolic reactions are not indicated such as the possible formation of 17p-hydroxylated steroids (e.g., A5-androstene-3p,17p-diol and testosterone), C19-steroids of the 5a series (e.g., androsterone and epiandrosterone), and phenolsteroids (e.g., estrogens).

Biosynthesis of Pregnenolone

Solid evidence has been provided for the biosynthesis of PREG in the nervous system. Incubation of primary cultures of newborn rat forebrain glial cells (a mixture of mature oligodendrocytes and astrocytes after 20 d of culture) with [3H]mevalonolactone, a precursor of cholesterol, led to the formation of cholesterol, PREG, PROG, and 2a-DH-PREG (45), and incubation of rat oligodendrocyte mitochondria with [3H] cholesterol yielded pregnenolone (54).

There is only one cholesterol side-chain cleavage enzyme, cytochrome P450scc with strong structural homology between rodent, bovine, and human species. The presence of immunoreactive P450scc protein in the rat brain has been established in the white matter and in primary cultures of newborn rat forebrain glial cells (45,55). However, the abundance of P45Oscc mRNA is exceedingly low and could be demonstrated only by reverse transcription polymerase chain reaction (RT-PCR) (56-58).

Biosynthesis of DHEA DHEA Biosynthesis in the Adult Rat Brain

Incubations of [3H]-PREG (and sulfate or acetyl esters) with brain slices, homogenates, or microsomes; with primary cultures of mixed glial cells, or with astrocytes and neurons of rat and mouse embryos never produced a radioactive metabolite with the chromatographic behavior of [3H]-DHEA (7). Moreover, all attempts to demonstrate the P450c17 antigen immunohistochemically in the rat brain with antibodies to the enzyme purified from pig testis and in the guinea pig brain with specific antibodies to the enzyme from guinea pig adrenal were unsuccessful (59). Accordingly, Mellon and Deschepper failed to detect the mRNA for P450c17 by RNAase protection assays and RT-PCR (56). Only a transient expression of the mRNA for this enzyme during embryonic life was reported (60), however a conflicting report indicated its presence was also in the adult rat brain (57). Thus, the pathway(s) by which DHEA biosynthesis occurs in the brain remain(s) controversial.

The Rat Retina

The biosynthesis of PREG also occurs in the rat retina where it is the most abundant steroid. Although suggested by Guarneri et al., the presence and activity of P450c17 in retina has not been conclusively demonstrated (50).

The Hydroperoxide Pathway

We also failed to demonstrate the direct conversion of radioactive cholesterol or sesterpene to DHEA (61). However, Prasad et al. have been able to generate PREG and DHEA from organic solvent extracts of rat brain by reactions with various reagents (62). They have suggested the intermediate formation of cholesterol 17,20-cycloperoxide or 17-hydroperoxide in an hypothetical biochemical pathway from cholesterol to DHEA. Their results are supported by a recent report of Cascio et al. (63). Previous reports had indicated that C6 rat glioma cells in culture biosynthesize both PREG and DHEA, despite the complete lack of expression of P450c17. Adding FeS04 to the culture medium increased the synthesis of both neurosteroids, even in the presence of specific inhibitors of P450scc and/or P450c17. These results were interpreted as caused by the fragmentation of in situ-formed tertiary hydroperoxides ("hydroperoxide pathway") (Fig. 3). Namely, the precursor of brain DHEA might be a steroid where both C-17 and C-20 are oxygenated. The enzyme(s) responsible for these conversions is (are) unknown.

Sulfotransferases and Sulfatases

In the rat brain, the concentrations of PREG S and DHEA S (in mol/g) are significantly higher than those in plasma (in mol/mL) and are maintained for several weeks after adrenalectomy and castration.

These observations, together with the blood-brain barrier's low permeability to steroid sulfates (64) argue for the changes in brain PREG S and DHEA S levels being independent of direct uptake from the circulation. This would mean that they are either synthesized in situ or stored in some other form, possibly analogous to the labile sulfolipid derivative(s), for which indirect evidence has been obtained (65).

Sulfotransferases

Where estrogen sulfotransferase activity in mammalian brain is ascertained, the search for A5-3^-hydrosteroid sulfotransferase activity has been disappointing (66). Low hydroxysteroid sulfotransferase activity was detected in all regions of the rat brain, with the highest one in the hypothalamus (67). The cytosolic enzyme has different properties from those of hepatic isozymes, with a pH optimum of 6.5 and a high Km of 2.8 mM for DHEA. The enzyme was equally active with PREG as the substrate. The specific activity (per mg cytosolic protein) in the brain was approximately 300-fold lower than in the liver and was higher in females than in males. Relatively high activities were found in the fetal brain and these declined at birth. There was a major peak in activity in pubertal female brain and a less important one in males. The low brain hydroxysteroid sulfotransferase activity explains the lack of information in the literature, and it is not known whether the enzyme cloned from rat liver is the one expressed in brain (68). There is still the question as to whether such low activity is adequate to explain the origin of DHEA S and PREG S in the brain. With a number of assumptions, it was calculated that it would take the rat brain over 5 d to synthesize 6 ng DHEA S/g (67). These data are compatible with the lack of increase of DHEA S in the brain after subcutaneous injection of DHEA in oil solution (12).

Sulfatase

Steroid sulfatase (STS) activity in the rat brain is associated with the nuclear and cytosolic fractions (69). Purification of the murine enzyme allowed to measure the protein by enzyme-linked immunosorbent assay (ELISA) in the brain (70). Rat and mouse STS have been cloned (70,71). Expression of STS was investigated in mouse brain during embryogenesis (E) (72). On days E16.5-E18.5, an in situ hybridization signal was found in the thalamus, hippoccampus, cerebellum, and spinal chord, and expression in these regions persisted 9 d after birth. Expression was also observed in the peripheral nervous system.

STS activity may be involved in the regulation of steroid sulfate concentrations in the brain, although no direct measurement has been reported. Inhibition of sulfatase activity by selective sulfatase inhibitors might increase DHEA S (and PREG S) concentrations in brain and might be involved in the slow effect of selective STS inhibitors on memory performance, as they were shown to increase plasma DHEA S and counteract scopo-lamine-induced amnesia as measured by a passive avoidance test (73).

Acyltransferase

Because DHEA and PREG were also characterized in the rat brain as unpolar complexes converted to the respective unconjugated steroids by saponification, which is known to split fatty acid esters (39), the characterization of a A5-3^-hydroxysteroid acyltransferase activity in rat brain microsomes was undertaken (74).

Endogenous fatty acids in the microsomal fraction served for the esterification of steroids. The enzyme system had a pH optimum of 4.5 with [3H]DHEA as the substrate. The apparent Km was 92 ± 31 (iM and the Vmax was 18.6 ± 3.4 nmol/h per mg protein (mean ± standard error of the mean). The highest activity in 1-to 3-wk-old rats may be related to the development of the brain, particularly to myelin formation which occurs at that stage.

The inhibition constants of PREG and testosterone for the sulfonation of [3H]DHEA were 123 and 64 ^M, respectively, and results were compatible with a competitive type of inhibition. The main endogenous fatty acids coupled to DHEA and PREG were palmitate, oleate, linoleate, stearate, and myristate.

It may be envisioned that the persistent accumulation of PREG and DHEA observed in the rat brain after combined orchiectomy and adrenalectomy occurs at the expenses of PREG L and DHEA L stores. In fact, the concentrations of lipoidal derivatives did not decrease significantly in the brain of operated rats, thus excluding the possibility that they serve as storage molecules (62).

3^-Hydroxysteroid Dehydrogenase A5 ^ 4 Isomerase (3^-HSD)

The rat brain can convert [3H]PREG into [3H]progesterone (75) and [3H]DHEA into [3H]androstenedione (38). Converting PREG to PROG was demonstrated in cultured rodent glia (45), neurons (76), and astrocytes (77). Four different isoforms (I-IV) of rat 3^-HSD have been thus far characterized (78). An in situ hybridization study, using an oligonucleotide probe common to the four known isoforms, demonstrated 3^-HSD mRNA in neurons of several brain regions (79) and in rat sensory neurons and Schwann cells (80). Using selective RNA probes, in situ hybridization indicated that 3^-HSD isoforms I, II, and IV are expressed throughout the brain at a low level and mainly in white matter (58).

Cell culture experiments showed that 3^-HSD activity may be regulated by cell density. Purified type 1 astrocytes were obtained from fetal rat forebrain, plated at low, intermediate or high density and maintained for 21 d. They were then incubated with [14C]DHEA or [3H] PREG for 24 h and the radioactive metabolites formed (androstene-dione or PROG, respectively) were analyzed (81). It appeared that the 3^-HSD activity observed at low cell density was almost completely inhibited at high density. Another example of regulation 3^-HSD activity by cellular interaction has been observed in the peripheral nervous system. Schwann cells can synthesize PROG from [3H]PREG, but only in response to a diffusible factor produced by dorsal root ganglia sensory neurons (80). In accordance with this finding, neurons induced a 20-fold increase of 3^-HSD mRNA levels in Schwann cells. The dependence of 3^-HSD expression on a neuronal signal could also be demonstrated in vivo: its RNA was easily detected by RT-PCR in the intact rat sciatic nerve but was down-regulated to undetectable levels 3 d after cryolesion when axons have degenerated. By 6 d when Schwann cells made new contact with the regenerating axons, 3^-HSD mRNA was again present. After cutting and ligat-ing the nerve fibers, thus preventing their regeneration, 3^-HSD mRNA was not reexpressed (F. Robert, R. Guennoun, F. Desarnaud, A. D. Thi, U. Sueter, E.-E. Baulieu, and M. Schumacher, unpublished results).

DHEA and DHEA S did not increase in the brain of male rats treated for 7 d with trilostane, an inhibitor of 3^-HSD, thus suggesting that the DHEA ^ androstenedione conversion is very low in the CNS in vivo (82). Conversely, PREG and PREG S were increased, mostly as a result of increased adrenocortical secretion.

7a-Hydroxylase (CYP 7b)

DHEA and PREG are converted by rat brain microsomes into polar metabolites, identified as the respective 7a-hydroxylated (7a-OH) derivatives by gas chromatography mass spectrometry (GC-MS) of deuterated substrates (83). Under optimal conditions, the Km values for DHEA and PREG are 13.8 and 4.41 ^M and the Vmax values are 322 and 38.8 pmol/min/mg of microsomal protein, respectively. Formation of 7a-hydroxylated metabolites is low in prepubertal rats and increases fivefold in adults. Activity might decrease with aging (84). The biochemical properties of the brain microsomal enzyme were reminiscent of those of 5a-androstane-3p, 17^-diol hydroxylase (85), and of 27-hydroxy cholesterol 7a-hydroxylase (86) measured in the rat brain. Indeed, a novel cytochrome P450 was cloned from rat hippocampus (87), and later identified as the neurosteroid 7a-hydroxylase and designated as CYP 7b, distinct from the previously cloned liver cholesterol 7a-hydroxylase (88). Extracts from Hela cells infected with a recombinant virus were very active on DHEA and PREG and less active on 25-hydroxycholesterol, 17^-estradiol, and 5a-androstane-3p,17^-diol, with low to undectable activity toward steroids devoid of a 3^-hydroxyl. 7a-Hydroxylation might serve as a control mechanism of neuroactive neurosteroids in brain (83,89). Moreover 7a-OH DHEA and 7a-OH PREG might have activities of their own. Stimulation of immune responses has been reported (90,91).

Pregnenolone-7^-hydroxylating activity has been assigned to another enzyme, cytochrome P450-lA1 (92).

20a-Hydroxysteroid Oxidoreductase

Pregnenolone and progesterone can be converted to the respective 20a-dihydro-derivatives by cultures of rat brain glial cells and neurons (20). The yields vary according to experimental conditions. For example, after the release of aminoglutethimide blockade, which inhibits P450scc activity and thus produces the accumulation of [3H]choles-terol in cultured glial cells, [3H]Pregn-5-ene-3p, 20a-diol (20a-dihydro-PREG) was the major steroid released in culture medium (93).

20a-Hydroxysteroid oxidoreductase has been mainly investigated in rat corpus luteum, where it serves to regulate the concentration of PROG. The ovarian enzyme was cloned, sequenced, and shown to belong to the NADP aldo-keto reductase family (94). Its expression and distribution in brain have not yet been reported.

5a-Reductase and 3a-Hydroxysteroid Oxido Reductase (3a-HOR)

These enzymes are responsible for the conversion of PROG and deoxycorticosterone into their neuroactive metabolites 3a,5a-TH PROG and 3a,5a-TH DOC, respectively. They have been the subject of extensive reviews (5,95,96) and are described in two other chapters of this book (19,20). Although 5a-reductase is found both in neurons and glial cells, the reductive form of 3a-HOR is expressed mainly in glial cells (23). Therefore, the neuroactive 3a,5a-TH PROG seems provided to neurons by surrounding astrocytes in a paracrine mode of action.

PHYSIOLOGICAL CORRELATES OF NEUROSTEROIDS Pregnenolone Sulfate and Progesterone

Although it was already known that there are steroids active on the nervous system (neuroactive), the discovery of neurosteroids gave a big boost to pharmacological studies of neuroactive steroids since the early 1980s, and a new physiological role for steroids in the nervous system was envisioned.

The pharmacology of neurosteroids is the subject of several chapters in this book, dealing with their effects on GABAareceptors (24,97), glutamate receptors (98), Sigma receptors (99), or calcium channels (100).

However, there are still little functional correlates of neurosteroids suggesting their physiological implication in the functioning of the nervous system. Here we review three such examples. Two of them deal with the implication of PREG S in a particular type of aggressive behavior and in memory performance, the third one relates to the role of progesterone in peripheral nerve regeneration.

Pregnenolone Sulfate and the Aggressive Behavior of Mice Against Lactating Female Intruders

This peculiar model of aggressiveness has been discovered and characterized by Marc Haug (101). This behavior is triggered by a pheromonal signal emitted in the urine of lactating mice (102). It is influenced by the genotype of the mice (103) and by their sex: females are more aggressive than males (103). Moreover, castration of males triggers a marked increase of aggressiveness, suggesting an inhibitory role of male gonadal hormones (104). Indeed, treatment of castrated males with testosterone or estradiol counteracts the effect of castration.

DHEA Inhibits the Aggressiveness of Castrated Male Mice

DHEA also inhibits the aggressiveness of castrated male mice (105). This effect was mimicked neither by DHEAS nor by its estrogenic metabolite androst-5-ene-3p, 17^-diol. It is well known that DHEA can be converted in vivo into active androgens and/or estrogens (106). However, the conversion of the injected amounts of DHEA (280 nmol daily in oil vehicle for 2 wk) to testosterone in the brain was extremely small (105): the concentration of testosterone in intact mice was 2.8 ± 1.2 ng/g (mean ± standard deviation), in castrated mice it was 0.04 ± 0.02 ng/g, and in DHEA treated castrated mice it was 0.16 ± 0.06 ng/g. To completely eliminate the possibility of an androgenic action of DHEA, the effect of its analog 3^-methyl-androst-5-en-17-one (CH3-DHEA) was investigated. This molecule cannot be metabolized into sex steroids and is not demonstrably estrogenic or androgenic (12). Nevertheless, it inhibited the aggressive behavior of castrated mice dose relatedly, at least as efficiently as DHEA itself (107).

DHEA and CH -DHEA Decrease the Concentration of PREG S

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