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The brain: A vulnerable target
ExperimentalGerontology,Vol. 32, Nos. 4/5, pp. 355-362, 1997 Copyright© 1997 ElsevierScienceInc. Printed in the USA.All rights reserved 0531-5565/97 $17.00 + .00 ELSEVIER
PII S0531-5565(96)00172-6
THE
BRAIN:
A VULNERABLE
TARGET
L. MARTINI and R.C. MELCANG1 Department of Endocrinology, University of Milano, Via G. Balzaretti 9, Milano 20133, Italy
AbstractmThis article addresses the description of several endocrine-related functions of the brain. It has emerged that steroids and growth factors may influence brain functions, and that brain cells may metabolize sex steroids. The crosstalk between different types of brain cells (neurons, astrocytes, the LHRH (luteinizing hormone-releasing hormone) producing GT1 cell line, etc.) has been described. Of relevance is that brain enzymes may convert sex steroids into compounds able to bind the GABAa receptor, creating a link between brain steroids and one neurotransmitter system. The data presented also provide the first demonstration that glial-neuronal interactions may intervene, in conjunction with neuronal-neuronal communications, in the control of the secretion of hypothalamic hormones. The detailed discussion of all these mechanisms has provided a long list of possible targets during the aging process. Fortunately, the demonstration that stem cells may be rescued in the "adult" CNS by the proper manipulations with growth factors opens new hope and directions for future interventions. © 1997 Elsevier Science Inc. Key Words: aging, brain, steroids, growth factors, neurons, astrocytes, the LHRH-producing GT1 cell line, GABAa receptor, glial-neuronal interactions, hypothalamic hormones
INTRODUCTION IT WOULD be difficult to count and describe the billions of physiological functions that are organized, supervised, directed, and controlled by the brain. Likewise, it seems impossible to count the millions of different biological and biochemical mechanisms present and simultaneously operating in each brain cell. It is obvious that each of these functions and each of these mechanisms may be subjected to deterioration via genetic and/or pathological reasons. One peculiar type of deterioration is that induced by the process of "physiological" aging. At the CNS level, like elsewhere, this process is multifactorial, and involves both intrinsic as well as extrinsic factors (vascular, nutritional, etc.). It is well known that hormones are involved in the control of several brain functions. It is extremely important to realize that endocrine factors start modulating many brain functions from the very beginning of life, i.e., already during early fetal development. It is known, for instance,
Correspondence to: L. Martini. E-mail: [email protected] 355
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that estrogens influence neuronal sprouting and the number of dendritic spines (Frankfurt et al., 1990), as well as the development and remodeling of synapses, in particular brain areas (Olmos et al., 1987; Naftolin et al., 1990). Testosterone is involved in the control of the sexual differentiation of specific centers present in the brain, and those that control sexual behavior, gonadotropin, and prolactin secretion (for references, see Martini, 1982). But it is also crucial to understand that, in addition to these "organizational" properties, hormones may also exert "activational" effects, which are operating throughout the life span of an animal, and which result in the expression of normal behavioral patterns, in a correct, chronobiologically adjusted secretion of several pituitary hormones. Finally, it is sad to say that hormones may also facilitate, and sometime precipitate brain cell degeneration. The most studied example of the intervention of peripheral hormones in inducing neuronal degeneration and death is provided by the effects of corticoids on the hippocampus; these effects become particularly evident when corticoid blood levels are enhanced by stress and by aging, which in turn creates a dangerous vicious cycle (Sapolsky, 1996). The present article will concentrate on the description of the results obtained in the authors' laboratories, which were aimed at elucidating endocrinologicaily modulated physiological mechanisms crucial for the normal function of several brain activities. It is obvious that each one of the physiological steps discussed may be seen as a possible target for pathological or age-induced alterations. HORMONES AND SEXUAL DIFFERENTIATION The sexual differentiation of the brain is a multifactorial event, in which both genetic and hormonal factors are involved. The best examples of hormonally induced sexual dimorphism are provided by the mechanisms controlling sexual behavior, gonadotropin, growth hormone, and prolactin secretion. For reasons of space, only the effects hormones exert on the sexual dimorphism of prolactin (PRL) secretion will be analyzed. It is important to emphasize that the majority of the studies in this field have been performed in rodent species, but that recent data show that the results obtained may also be applicable to primates and humans. The fact that prolactin secretion is subject to different mechanisms of control in mate and female rodents is supported by the following observations. In the normal male rat PRL is secreted in a tonic acyclic pattern, whereas in normal females, PRL release is cyclic, with periodic surges. The stimulation of the hypothalamic medial preoptic area increases PRL secretion in male rats, but inhibits the proestrous surge of PRL in female rats. On the contrary, lesions of the medial preoptic area, or the stimulation of the hypothalamic dorsoventromedial nucleus, bring about nocturnal PRL surges in ovariectomized females, but are ineffective in castrated males. Moreover, sexual differences have been reported in the brain concentrations and turnover of dopamine and serotonin, two of the neurotransmitters involved in the control of PRL secretion (for references, see Limonta et al., 1989). Additional evidence supporting the view that there are major sexual differences in the neural control of PRL secretion has been derived from studies that have used the opioids and their antagonists. Naloxone, a typical opioid antagonist, has been repeatedly found to decrease serum PRL levels in adult male rats, but not in adult females (Limonta et al., 1989). The sex differences in the mechanisms controlling PRL secretion have been ascribed to a different pattern of organization of the brain in the two sexes. It is presently recognized that, in the male rat, the neuroendocrine brain develops towards a male pattern of control of PRL
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secretion because it is exposed, during the nenatal period, to the presence of testosterone (T) secreted by the testes. It has also been reported that it is possible to masculinize the feminine neuroendocrine brain by administering T to the neonatal female rat, and conversely, to demasculinize the neuroendocrine male brain by orchidectomizing the male rat immediately after birth. It is important to recall here that this type of "organizational" effects of testosterone is linked to the aromatization of the steroid to yield estrone and/or estradiol (for references, see Martini, 1982). The experiments that will be summarize here have been designed: (1) in order to analyze in more detail the sexual differences in the responses of PRL to naloxone; and (2) to try to understand the mechanisms underlying these differences. To this purpose, the hormonal responses to naloxone have been tested acutely at different ages (days 16, 26, and 60 of life) in the following four groups of animals: (1) normal male rats, (2) normal female rats, (3) neonatally androgenized female rats (treated with 1.25 mg of T), and (4) neonatally demasculinized male rats (castrated at three days of life). These experiments are based on the hypothesis that the presence or absence of androgens in the perinatal period might influence the development of the central opioid systems (pathways and/or receptors) controlling PRL release. The results obtained have shown that the acute injection of naloxone significantly decreases serum levels of PRL at all ages considered in normal male rats and in androgenized females. On the contrary, the opioid antagonist was always ineffective in normal females and in neonatally castrated males. These data suggest then that, in the rat, a sexual difference exists in the opiatergic control of PRL secretion; apparently, the central opioid systems that regulate PRL secretion develop towards a male pattern because of the presence of androgens in the neonatal period. It is clear that the present results underline two important aspects of the control of PRL secretion: first of all, they confirm that the dimorphic mode of secretion of PRL is linked to an effect on the brain of neonatal androgens; secondly, they clearly prove that this effect is linked to an action modifying the activity of one of the most important neurotransmitter systems operating in the brain. METABOLISM OF SEX STEROIDS IN THE BRAIN As previously mentioned, T may exert some of its "organizational" effects via the process of aromatization, which occurs in some specialized areas of the brain like the limbic system and the hypothalamus. Another very important pathway for the metabolism of T has been shown to be present in the central nervous system (CNS) of all animal species studied so far. Following this pathway, T may be first reduced to 5o~-androstane-1713-ol-3-one (dihydrotestosterone, DHT), and subsequently metabolized to 5ot-androstane-3ot, 1713-diol (3ot-diol) through the action of the enzymes 5ot-reductase (5a-R) and 3ot-hydroxysteroid dehydrogenase (3ot-HSD). Previously, only a few data were available on the localization of these two enzymes in the various cellular components of the CNS (neurons, oligodendrocytes, astrocytes, etc.). The cellular distribution of the 5et-R has been analyzed in this laboratory by using either freshly isolated cell preparations (neurons, astrocytes, and oligodendrocytes) obtained from the brain of adult male rats, or brain cell cultures derived from the fetal (neurons) or newborn (mixed glia) rat brain. For the sake of brevity, only the data on cell cultures will be reported (Melcangi et al., 1990, 1993). The activities of the 5ot-R and of the 3ot-HSD have been evaluated in primary cultures of neurons, of oligodendrocytes, and of type 1 and type 2 astrocytes, obtained from the fetal or neonatal rat brain. All the cultures were used on the fifth day. The formation of DHT
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and 3(x-diol were evaluated incubating respectively the different cultures with (14C)-T or (14C)-DHT as substrates. The results obtained indicate that the formation of DHT from T takes place preferentially in neurons; however, also type 2 astrocytes and oligodendrocytes possess a measurable 5e~-R activity, while type 1 astrocytes show a much lower enzymatic activity. A completely different localization was observed for the 3et-HSD, because the formation of 3e~-diol from DHT appears to be take place preferentially, if not exclusively, in type 1 astrocytes. The compartmentalization of two strictly correlated enzymes (5e~-R and 3e~-HSD) in separate CNS cell populations suggests the simultaneous participation of neurons and glial ceils in the 5ot-reductive metabolism of T and possibly of other hormonal steroids. It is interesting that, in parallel studies, it has been tbund that the aromatase, the enzyme that transforms T into estrogens, is present only in neurons (Negri-Cesi et al., 1992). It is known that, in different androgen-depending structures and/or tissues, the 5e~-R is not specific for T but may also utilize progesterone (P) as the substrate. The hypothesis on whether this may occur also in the brain has been consequently verified. It was found that, in the rat CNS, P can be converted into a series of 5co-reduced metabolites. Through the action of the 5e~-R, P is first metabolized into 5tx-pregnan-3,20-dione (dihydroprogesterone, DHP); this steroid can be further converted to 5e~-pregnan-3e~-ol-20-one (THP) by a 3e~-HSD (for references, see Celotti et al., 1992). On the basis of the data discussed in the preceding paragraphs, it was decided to repeat an experiment similar to the preceding one, using P rather than T as the substrate. The same types of brain cell cultures were used. To analyze the 5ot-R activity of the different cell cultures the amounts of DHP formed from labeled P were quantified; to analyze the 3c~-HSD activity of the same cell cultures the formation of THP from labeled DHP was evaluated. The data obtained have indicated that, similarly to what happens when T is used as the substrate, the 5oL-R that metabolizes P shows a significantly higher activity in neurons than in glial cells; however, also type 1 and type 2 astrocytes as well as oligodendrocytes possess some ability to 5tx-reduce P. On the contrary, the 3et-HSD, the enzyme that converts DHP into THP appears to be mainly present in type 1 astrocytes; much lower levels of this enzyme are present in neurons and in type 2 astrocytes. At variance with the previous results obtained using androgens as precursors, oligodendrocytes have been shown to possess a considerable 3(x-HSD activity, even if this is statistically lower than that present in type 1 astrocytes (Melcangi et al., 1994a). There are two facts that emerge from these data and that must be underlined. First of all, in brain cells like in any other structure possessing the 5c~-R, P appears to be a preferential substrate tbr this enzyme. Second, it is important to emphasize that the brain is able to form THP, and that the formation of this metabolite occurs through the ping-pong game between neurons and type l astrocytes. While P and DHP act through their binding to the P receptor, THP has been shown to interact with the GABAa receptor, and consequently, to posses sedative properties. Consequently, these findings open a new pathway in the interpretation of the mode of action of sex steroids in the brain. It is important to note at this point that two 5o~-R isozymes have been recently cloned (type 1 and type 2). They have different functional and biochemical properties, such as the pH optima for their reactions, the affinity for the various substrates (T, P, androstenedione, corticosterone, etc.), which is generally higher in the case of the type 2 isoform, etc. (for references, see Poletti et al., 1996). It is, however, important to point out that for both 5oL-R isoforms P represents the preferential substrate. There are no clear-cut data on whether both isoenzymes are simultaneously expressed in the brain. Work is presently in progress in this laboratory to clarify this issue.
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GLIA-NEURON INTERACTIONS Metabolism of sex steroids in the brain
For many years glial cells have been considered to represent only a mechanical support for neurons and other elements of the CNS. The existence of important functional relationships between specific glia components (oligodendrocytes, astrocytes, etc.) on one side, and neurons on the other have become apparent only recently. The interactions of glial cells with neurons have been shown to play an important role in neuronal migration, in neurite outgrowth, and in axonal guidance during ontogenetic development. Moreover, it has been recently demonstrated that glial cells are able to synthesize, and possibly release, an array of bioactive principles, like neurotransmitters, growth factors, interleukins, prostaglandins, excitatory amino acids, etc. It is probable that several of these principles may exert definite and specific influences on neuronal activity (for references, see Melcangi et al., 1994b, 1996a). The glia-neuronal interactions are not a one-way phenomenon, because several data indicate that neurons may interfere with the proliferation and the maturation of glial elements. For example, neuronal activity has been shown to upregulate the expression of the glial fibrillary acidic protein (GFAP), an astroglia intermediate filament protein that is considered a specific marker of astrocytes. Moreover, other parameters of astrocytic function, like the enzyme glutamine synthetase, the formation of gangliosides, the assembly of voltage-sensitive calcium channels are activated only in the presence of neurons (for references, see Melcangi et al., 1994b, 1996a). As previously mentioned, in the CNS, the metabolism of androgens and of other hormonal steroids (e.g., P) is a very active process, which occurs, even if with different yields and with different end products, practically in all brain cells (see above). The peculiar compartmentalization of the two enzymes (5~x-R and 3et-HSD), which characterize the 5et-reductive metabolism of T and P which we have just described, has prompted us to investigate whether humoral mechanisms of communication might exist between neurons and type 1 astrocytes, and whether these might be relevant for the coordination of the metabolism of androgens and other hormonal steroids in the brain. To examine the reciprocal interactions between astrocytes and neurons, the following two approaches have been used: (a) a coculture system has been established that allows the transfer of secretory products of the cocultured cells, even if these remain physically separated; (b) the addition of neuron-conditioned medium (CM) to cultures of astrocytes, and vice versa of astrocyte CM to cultures of neurons. T metabolism was studied in neurons and type 1 astrocytes either cocultured with the other type of cells, or exposed to the "other" CM. In both types of experiments, the activity of the two enzymes was studied using labeled T (to quantitate the formation of DHT, 5a-R activity), or labeled DHT (to quantitate the formation of 3a-diol, 3ot-HSD activity) as precursors. The results indicate that both the 5o~-reductase and the 3(x-HSD activities are stimulated in type 1 astrocytes by the coculture with neurons and by the addition of neuron CM. However, neither the coculture with type 1 astrocytes nor the addition of astrocyte CM alters the metabolism of T or DHT in neurons (Melcangi et al., 1994b). Control of gonadotropin secretion
The secretion of pituitary gonadotropins is controlled by the hypothalamic hormone LHRH, which is manufactured in a small number of highly specialized neurons. LHRH release is
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regulated by several types of feedback signals (long, short, ultrashort), as well as by multiple nervous influences (for references, see Melcangi et al., 1995). Until recently, it has been believed that these might be transferred to the LHRH-producing neurons only via inputs of neuronal origin, expressed through the liberation, at synaptic or axo-axonal junctions, of classical neurotransmitters (acetylcholine, catecholamines, serotonin, etc.) or of several neuropeptides (opioids, galanin, neuropeptide Y, etc.) (for references, see Melcangi et al., 1995). As mentioned in the preceding paragraph, in the last few years several reports have indicated that glial cells may play an important role in controlling neuronal morphology and function. With regard to the control of gonadotropin secretion, it has been recently reported that feedback signals that typically influence LHRH release may modify the glia surrounding the LHRH neurons. For instance, Witkin et al. (1991) have observed that ovariectomy increases the degree of glial ensheathment (i.e., the apposition of glial processes to perikaria membranes) around the LHRH-producing neurons in the hypothalamus of the female rhesus monkeys, but not in other brain regions; these authors (Witkin et al., 1991) have also shown that this phenomenon is partially reversed by ovarian steroid replacement. These data have been interpreted as suggesting that alterations in the circulating levels of gonadal steroids may produce changes in the glial component located in the immediate environment of the LHRH-producing neurons. However, one may also infer that these morphological alterations represent the expression of metabolic changes occurring within the glial cells, and aimed at modifying the activity of the LHRHproducing neurons, and consequently, their ability to secrete LHRH. This hypothesis has been tested. Advantage has been taken of the existence of a cell line (GTI) derived from a hypothalamic LHRH-producing tumor, induced in a female transgenic mouse by genetically targeted tumorigenesis (Mellon et al., 1990). The GTI cells appear to be highly differentiated neurons, which express the LHRH gene at a high degree (Mellon et al., 1990). These cells present a basal, pulsatile release of LHRH, which can be influenced by a series of neurotransmitters and neuromodulators physiologically involved in the control of LHRH release (norepinephrine, dopamine, GABA, activin-A, endothelin-3, high concentration of K +, prostaglandins, etc.) (for references, see Melcangi et al., 1995). In order to evaluate the possible effects of glial cell products on LHRH secretion from GT1-1, cells two different approaches have been used: (a) GTI-1 cells were coincubated with purified cultures of type I astrocytes; (b) GTI-I cells were exposed to the conditioned medium (CM, untreated or submitted to different experimental procedures) in which type 1 astrocytes were grown for 24 h. In both sets of experiments LHRH was measured by RIA in the incubation media of GTI-1 cells at different time intervals. The data show that coculture of GT1 cells with type I astrocytes and their exposure to previously frozen or heated (but not fresh) type 1 astrocyte CM significantly increase the release of LHRH from the GTI-1 cells. The stimulatory effect on LHRH release appears to be specific for type 1 astrocytes (either cortical or hypothalamic), because the CM of oligodendrocytes does not possess stimulating activities. The fact that fresh CM proved to be inactive, while frozen or heated CM proved effective suggested that the factor(s) involved might be "activated" by the process of freezing or heating. Because it is known that TGF[3 may be liberated from its supporting proteins by freezing or heating, the effects of TGF[3 and of a TGF[3 neutralizing antibody were analyzed. It was found that the stimulatory effect exerted by the frozen astrocyte CM is completely abolished by the antibody, while direct treatment with TGF[3 proved able to significantly increase LHRH release from GTI-I cells. In conclusion, the present experiments demonstrate that type 1 astrocytes may secrete in the medium some factor(s) (most probably TGFI3) able to stimulate the release of LHRH from the
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hypothalamic LHRH-producing GTI-1 cells. The data provide the first clear-cut demonstration that glial cells may directly intervene in the control of LHRH release, integrating the old concept that only neuronal influences may participate in such a phenomenon (for references, see Melcangi et al., 1995). In most recent experiments it has been shown that astrocyte CM and TGF~3 may modify also LHRH gene expression in GTI-1 cells (Galbiati et al., 1996). RESCUE OF PLURIPOTENTIAL STEM CELLS It has been shown that in a variety of animal species neurons and glial cells may arise from common multipotent precursors that are present in the embryonic and neonatal CNS (for references, see Melcangi et al., 1996b). Recently, one of these multipotential cells has been isolated from the mouse striatum; in the absence of an adhesive substrate, these may be induced to proliferate by epidermal growth factor (EGF), producing floating clones (neurospheres). Cells within these neurospheres retain undifferentiated features, and do not express either neuronal or glial markers for the first 10-14 days in vitro (DIV). When the cultures are plated on an adhesive substrate, in the absence of EGF, massive cell differentiation is triggered. It must be emphasized, however, that upon removal of EGF, neurons develop in serum-free conditions, while serum is required for the full differentiation of EGF-generated precursors into glial cells (Melcangi et al., 1996b). As discussed in the preceding portions of this article, fully differentiated CNS cells (neurons and glial cells) are able to convert T and P into the corresponding 5or-reduced metabolites via the 5ct-R-3ot-HSD pathways. The present work has been devoted to analyze whether the enzymes 5ct-R and 3ot-HSD are also present in the multipotential striatum stern cells obtained from the fetal mouse; it has also been investigated whether their differentiation into glial cells (astrocytes and oligodendrocytes) and neurons brings about modifications of the activity of these two enzymes. To this purpose, striatal EGF-responsive stem cells isolated from the mouse brain at the 14th day of embryonic life have been cultured and passaged four times, at weekly intervals, in the presence of EGF. Starting from this point (28 DIV), the cells have been plated on an adhesive substrate, and cultured either in the continuous presence of EGF (that allows proliferation, but maintains the stem cells in an undifferentiated state), or in the absence of EGF but in the presence of fetal bovine serum (FBS) for additional 14 DIV to induce cell differentiation. The relative proportions of the various cell subtypes present were evaluated at different time intervals (i.e., at 35, 38, and 42 DIV); at the same time intervals, the conversion of T and P (5ct-R activity) and of their respective 5et-reduced derivatives DHT and DHP (3ct-HSD activity) were measured both in the differentiated cultures (experimental), and in undifferentiated cells (controls). The differentiation is completed by 35 DIV, when the total cell count comprises: 86 _ 2.0% of astrocytes, 6 _ 0.7% of neurons, 1.6 +- 0.5% of oligodendrocytes, and 6.4 +_ 0.5% of undifferentiated cells. No percent changes in cell population composition are observed thereafter (38 and 45 DIV). The undifferentiated cells (controls) possess a much higher ability to form DHP than DHT (about l0 times). On the contrary, the conversions of DHP and DHT into THP and 3ot-diol are very similar. In the population of differentiating cells, the 5t~-R converting P becomes higher at 38 DIV, and remains similarly elevated at 42 DIV (four times). On the other hand, the conversion of T into DHT remains at the levels of controls up to 42 DIV when an increase is observed. The 3ct-HSD converting DHP but not that converting DHT is increased at 38 and 42 days. These results underline the fact that undifferentiated CNS cells possess steroid metabolizing enzymes for androgens and progestagens that are strongly influenced by the
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cellular differentiation/maturation process. It also appears from the data that during the differentiation process the cells acquire the ability to convert P much earlier than that of converting T. There may be different explanations for this finding (Melcangi et al., 1996b). The most attractive one suggests the existence of a third 5~-R isozyme (see above) more specific for P. This concept emerges also from other recent data of this laboratory, which have shown that the CM of brain tumor cells (C6 glioma and 1321Nl astrocytoma) is able to inhibit the conversion of P into DHP but not that of T into DHT in type 1 astrocytes (R.C. Melcangi, I. Cavarretta, V. Magnaghi, M. Ballabio, L. Martini, and M. Motta, unpublished data). REFERENCES CELOTTI, F., MELCANGI R.C., and MARTINI, L. The 5e~-reductase in the brain: Molecular aspects and relation to brain function. In: Frontiers in Neuroendocrinology, Martini, L. and Ganong, W.F. (Editors), pp. 163-215, vol. 13, Raven Press, New York, 1992. FRANKFURT, M , GOULD, E., WOOLEY, C.S., and MCEWEN, B.S. Gonadal steroids modify dendritic spine density in ventromedial bypothalamic neurons: A Golgy study in the adult rat. Neuroendocrinology 51, 530-535, 1990, GALBIATI, M., ZANISI, M., MESSI, E.. CAVARRETTA, I., MARTINI, L., and MELCANGI, R.C. Transforming growth factor-[?, and astrocytic conditioned medium influence luteinizing hormone-releasing hormone gene expression in the hypothalamic cell line GTI. Endocrinology 137, 5605-5609, 1996. LIMONTA, P., DONDI, D., MAGGI, R., MARTINI, L., and PIVA, F. Neonatal organization of the brain opioid systems controlling prolactin and luteinizing hormone secretion. Endocrinology 124, 681-686, 1989. MARTINI, L. The 5e~-reduction of testosterone in the neuroendocrine structures. Biochemical and physiological implications. Endocr. Rev. 3, 1-25, 1982. MELCANGI, R.C., CELOTT1, F., BALLABIO, M., CASTANO. P.. MASSARELLI, R., POLETTI, A., and MARTINI, L. 5~-Reductase activity in isolated and cultured neuronal and glial cells of the rat. Brain Res. 516, 229-236, 1990. MELCANGI, R.C., CELOTTI, F., CASTANO, P., and MARTINI, L. Differential localization of the 5~-reductase and the 3~-hydroxysteroid dehydrogenase in neuronal and glial cultures. Endocrinology 132, 1252-1259, 1993. MELCANGI, R.C., CELOTTI, F., and MARTINI, L. Progesterone 5e~-reduction in neuronal and in different types of glial cell cultures: Type 1 and 2 astrocytes and oligodendrocytes. Brain Res. 639, 202-206, 1994a. MELCANGI, R.C., CELOTTI, F., and MARTINI, L. Neurons influence the metabolism of testosterone in cultured astrocytes via humoral signals. Endocrine 2, 709-713, 1994b. MELCANGI, R.C., GALBIATI, M., MESSI, E., PIVA, F., MARTINI, L., and MOTTA, M. Type 1 astrocytes influence luteinizing hormone-releasing hormone release from the hypothalamic cell line GTI-I: Is transforming growth factor-J3 the principle involved? Endocrinology 136, 679-686, 1995. MELCANGI, R.C., RIVA, M.A., FUMAGALLI, F., MAGNAGHI, V., RACAGNI, G_ and MARTINI, L. Effect of progesterone, testosterone and their 5e~-reduced metabolites on GFAP gene expression in type 1 astrocytes. Brain Res. 711, 10-15, 1996a. MELCANGI, R.C., FROELICHSTHAL, P., MARTINI, L., and VESCOVL A.L. Steroid metabolizing enzymes in pluripotential progenitor central nervous system cells: Effect of differentiation and maturation. Neuroscience 72, 467-475, 1996b. MELLON, P.L.. WINDLE, J.J., GOLDSMITH, P.C., PADULA, C.A., ROBERTS, J.L., and WEINER, R.I. Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 5, 1-10, 1990. NAFTOLIN, F., GARCIA-SEGURA, L.M., KEEFE, D., LERANTH. C., MACLUSKY, N.J., and BRAWER, J.R. Estrogen effects on the synaptology and neuronal membranes of the rat hypothalamic arcuate nucleus. Biol. Reprod. 42, 21-28, 1990. NEGRI-CESI, P., MELCANGI, R.C., CELOTTI, F., and MARTINI, L. Aromatase activity in cultured brain cells: Difference between neurons and glia. Brain Res. 589, 327-332, 1992. OLMOS, G., AGUILERA, P., TRANQUE, P., NAFTOLIN, F., and GARCIA-SEGURA, L.M. Estrogen-induced synaptic remodelling in adult rat brain is accompanied by the reorganization of neuronal membranes. Brain Res. 425, 57-64, 1987. POLETTI, A., CELOTTI, F., MOTTA, M., and MARTIN1, L. Characterisation of rat 5e~-reductases type 1 and type 2 expressed in yeast Saceharomyces cerevisiae. ,L Biochem. 314, 1047-1052, 1996. SAPOLSKY, R.M. Why stress is bad for your brain. Science 273, 749-750, 1996. WITKIN, J.W., FERIN, M., POPILSKIS, S.J., and SILVERMAN, A.J. Effects of gonadal steroids on the ultrastructure of GnRH neurons in the reshus monkey: Synaptic input and glial apposition. Endocrinology 129, 1083-1092, 1991.
PII S0531-5565(96)00172-6
THE
BRAIN:
A VULNERABLE
TARGET
L. MARTINI and R.C. MELCANG1 Department of Endocrinology, University of Milano, Via G. Balzaretti 9, Milano 20133, Italy
AbstractmThis article addresses the description of several endocrine-related functions of the brain. It has emerged that steroids and growth factors may influence brain functions, and that brain cells may metabolize sex steroids. The crosstalk between different types of brain cells (neurons, astrocytes, the LHRH (luteinizing hormone-releasing hormone) producing GT1 cell line, etc.) has been described. Of relevance is that brain enzymes may convert sex steroids into compounds able to bind the GABAa receptor, creating a link between brain steroids and one neurotransmitter system. The data presented also provide the first demonstration that glial-neuronal interactions may intervene, in conjunction with neuronal-neuronal communications, in the control of the secretion of hypothalamic hormones. The detailed discussion of all these mechanisms has provided a long list of possible targets during the aging process. Fortunately, the demonstration that stem cells may be rescued in the "adult" CNS by the proper manipulations with growth factors opens new hope and directions for future interventions. © 1997 Elsevier Science Inc. Key Words: aging, brain, steroids, growth factors, neurons, astrocytes, the LHRH-producing GT1 cell line, GABAa receptor, glial-neuronal interactions, hypothalamic hormones
INTRODUCTION IT WOULD be difficult to count and describe the billions of physiological functions that are organized, supervised, directed, and controlled by the brain. Likewise, it seems impossible to count the millions of different biological and biochemical mechanisms present and simultaneously operating in each brain cell. It is obvious that each of these functions and each of these mechanisms may be subjected to deterioration via genetic and/or pathological reasons. One peculiar type of deterioration is that induced by the process of "physiological" aging. At the CNS level, like elsewhere, this process is multifactorial, and involves both intrinsic as well as extrinsic factors (vascular, nutritional, etc.). It is well known that hormones are involved in the control of several brain functions. It is extremely important to realize that endocrine factors start modulating many brain functions from the very beginning of life, i.e., already during early fetal development. It is known, for instance,
Correspondence to: L. Martini. E-mail: [email protected] 355
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that estrogens influence neuronal sprouting and the number of dendritic spines (Frankfurt et al., 1990), as well as the development and remodeling of synapses, in particular brain areas (Olmos et al., 1987; Naftolin et al., 1990). Testosterone is involved in the control of the sexual differentiation of specific centers present in the brain, and those that control sexual behavior, gonadotropin, and prolactin secretion (for references, see Martini, 1982). But it is also crucial to understand that, in addition to these "organizational" properties, hormones may also exert "activational" effects, which are operating throughout the life span of an animal, and which result in the expression of normal behavioral patterns, in a correct, chronobiologically adjusted secretion of several pituitary hormones. Finally, it is sad to say that hormones may also facilitate, and sometime precipitate brain cell degeneration. The most studied example of the intervention of peripheral hormones in inducing neuronal degeneration and death is provided by the effects of corticoids on the hippocampus; these effects become particularly evident when corticoid blood levels are enhanced by stress and by aging, which in turn creates a dangerous vicious cycle (Sapolsky, 1996). The present article will concentrate on the description of the results obtained in the authors' laboratories, which were aimed at elucidating endocrinologicaily modulated physiological mechanisms crucial for the normal function of several brain activities. It is obvious that each one of the physiological steps discussed may be seen as a possible target for pathological or age-induced alterations. HORMONES AND SEXUAL DIFFERENTIATION The sexual differentiation of the brain is a multifactorial event, in which both genetic and hormonal factors are involved. The best examples of hormonally induced sexual dimorphism are provided by the mechanisms controlling sexual behavior, gonadotropin, growth hormone, and prolactin secretion. For reasons of space, only the effects hormones exert on the sexual dimorphism of prolactin (PRL) secretion will be analyzed. It is important to emphasize that the majority of the studies in this field have been performed in rodent species, but that recent data show that the results obtained may also be applicable to primates and humans. The fact that prolactin secretion is subject to different mechanisms of control in mate and female rodents is supported by the following observations. In the normal male rat PRL is secreted in a tonic acyclic pattern, whereas in normal females, PRL release is cyclic, with periodic surges. The stimulation of the hypothalamic medial preoptic area increases PRL secretion in male rats, but inhibits the proestrous surge of PRL in female rats. On the contrary, lesions of the medial preoptic area, or the stimulation of the hypothalamic dorsoventromedial nucleus, bring about nocturnal PRL surges in ovariectomized females, but are ineffective in castrated males. Moreover, sexual differences have been reported in the brain concentrations and turnover of dopamine and serotonin, two of the neurotransmitters involved in the control of PRL secretion (for references, see Limonta et al., 1989). Additional evidence supporting the view that there are major sexual differences in the neural control of PRL secretion has been derived from studies that have used the opioids and their antagonists. Naloxone, a typical opioid antagonist, has been repeatedly found to decrease serum PRL levels in adult male rats, but not in adult females (Limonta et al., 1989). The sex differences in the mechanisms controlling PRL secretion have been ascribed to a different pattern of organization of the brain in the two sexes. It is presently recognized that, in the male rat, the neuroendocrine brain develops towards a male pattern of control of PRL
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secretion because it is exposed, during the nenatal period, to the presence of testosterone (T) secreted by the testes. It has also been reported that it is possible to masculinize the feminine neuroendocrine brain by administering T to the neonatal female rat, and conversely, to demasculinize the neuroendocrine male brain by orchidectomizing the male rat immediately after birth. It is important to recall here that this type of "organizational" effects of testosterone is linked to the aromatization of the steroid to yield estrone and/or estradiol (for references, see Martini, 1982). The experiments that will be summarize here have been designed: (1) in order to analyze in more detail the sexual differences in the responses of PRL to naloxone; and (2) to try to understand the mechanisms underlying these differences. To this purpose, the hormonal responses to naloxone have been tested acutely at different ages (days 16, 26, and 60 of life) in the following four groups of animals: (1) normal male rats, (2) normal female rats, (3) neonatally androgenized female rats (treated with 1.25 mg of T), and (4) neonatally demasculinized male rats (castrated at three days of life). These experiments are based on the hypothesis that the presence or absence of androgens in the perinatal period might influence the development of the central opioid systems (pathways and/or receptors) controlling PRL release. The results obtained have shown that the acute injection of naloxone significantly decreases serum levels of PRL at all ages considered in normal male rats and in androgenized females. On the contrary, the opioid antagonist was always ineffective in normal females and in neonatally castrated males. These data suggest then that, in the rat, a sexual difference exists in the opiatergic control of PRL secretion; apparently, the central opioid systems that regulate PRL secretion develop towards a male pattern because of the presence of androgens in the neonatal period. It is clear that the present results underline two important aspects of the control of PRL secretion: first of all, they confirm that the dimorphic mode of secretion of PRL is linked to an effect on the brain of neonatal androgens; secondly, they clearly prove that this effect is linked to an action modifying the activity of one of the most important neurotransmitter systems operating in the brain. METABOLISM OF SEX STEROIDS IN THE BRAIN As previously mentioned, T may exert some of its "organizational" effects via the process of aromatization, which occurs in some specialized areas of the brain like the limbic system and the hypothalamus. Another very important pathway for the metabolism of T has been shown to be present in the central nervous system (CNS) of all animal species studied so far. Following this pathway, T may be first reduced to 5o~-androstane-1713-ol-3-one (dihydrotestosterone, DHT), and subsequently metabolized to 5ot-androstane-3ot, 1713-diol (3ot-diol) through the action of the enzymes 5ot-reductase (5a-R) and 3ot-hydroxysteroid dehydrogenase (3ot-HSD). Previously, only a few data were available on the localization of these two enzymes in the various cellular components of the CNS (neurons, oligodendrocytes, astrocytes, etc.). The cellular distribution of the 5et-R has been analyzed in this laboratory by using either freshly isolated cell preparations (neurons, astrocytes, and oligodendrocytes) obtained from the brain of adult male rats, or brain cell cultures derived from the fetal (neurons) or newborn (mixed glia) rat brain. For the sake of brevity, only the data on cell cultures will be reported (Melcangi et al., 1990, 1993). The activities of the 5ot-R and of the 3ot-HSD have been evaluated in primary cultures of neurons, of oligodendrocytes, and of type 1 and type 2 astrocytes, obtained from the fetal or neonatal rat brain. All the cultures were used on the fifth day. The formation of DHT
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and 3(x-diol were evaluated incubating respectively the different cultures with (14C)-T or (14C)-DHT as substrates. The results obtained indicate that the formation of DHT from T takes place preferentially in neurons; however, also type 2 astrocytes and oligodendrocytes possess a measurable 5e~-R activity, while type 1 astrocytes show a much lower enzymatic activity. A completely different localization was observed for the 3et-HSD, because the formation of 3e~-diol from DHT appears to be take place preferentially, if not exclusively, in type 1 astrocytes. The compartmentalization of two strictly correlated enzymes (5e~-R and 3e~-HSD) in separate CNS cell populations suggests the simultaneous participation of neurons and glial ceils in the 5ot-reductive metabolism of T and possibly of other hormonal steroids. It is interesting that, in parallel studies, it has been tbund that the aromatase, the enzyme that transforms T into estrogens, is present only in neurons (Negri-Cesi et al., 1992). It is known that, in different androgen-depending structures and/or tissues, the 5e~-R is not specific for T but may also utilize progesterone (P) as the substrate. The hypothesis on whether this may occur also in the brain has been consequently verified. It was found that, in the rat CNS, P can be converted into a series of 5co-reduced metabolites. Through the action of the 5e~-R, P is first metabolized into 5tx-pregnan-3,20-dione (dihydroprogesterone, DHP); this steroid can be further converted to 5e~-pregnan-3e~-ol-20-one (THP) by a 3e~-HSD (for references, see Celotti et al., 1992). On the basis of the data discussed in the preceding paragraphs, it was decided to repeat an experiment similar to the preceding one, using P rather than T as the substrate. The same types of brain cell cultures were used. To analyze the 5ot-R activity of the different cell cultures the amounts of DHP formed from labeled P were quantified; to analyze the 3c~-HSD activity of the same cell cultures the formation of THP from labeled DHP was evaluated. The data obtained have indicated that, similarly to what happens when T is used as the substrate, the 5oL-R that metabolizes P shows a significantly higher activity in neurons than in glial cells; however, also type 1 and type 2 astrocytes as well as oligodendrocytes possess some ability to 5tx-reduce P. On the contrary, the 3et-HSD, the enzyme that converts DHP into THP appears to be mainly present in type 1 astrocytes; much lower levels of this enzyme are present in neurons and in type 2 astrocytes. At variance with the previous results obtained using androgens as precursors, oligodendrocytes have been shown to possess a considerable 3(x-HSD activity, even if this is statistically lower than that present in type 1 astrocytes (Melcangi et al., 1994a). There are two facts that emerge from these data and that must be underlined. First of all, in brain cells like in any other structure possessing the 5c~-R, P appears to be a preferential substrate tbr this enzyme. Second, it is important to emphasize that the brain is able to form THP, and that the formation of this metabolite occurs through the ping-pong game between neurons and type l astrocytes. While P and DHP act through their binding to the P receptor, THP has been shown to interact with the GABAa receptor, and consequently, to posses sedative properties. Consequently, these findings open a new pathway in the interpretation of the mode of action of sex steroids in the brain. It is important to note at this point that two 5o~-R isozymes have been recently cloned (type 1 and type 2). They have different functional and biochemical properties, such as the pH optima for their reactions, the affinity for the various substrates (T, P, androstenedione, corticosterone, etc.), which is generally higher in the case of the type 2 isoform, etc. (for references, see Poletti et al., 1996). It is, however, important to point out that for both 5oL-R isoforms P represents the preferential substrate. There are no clear-cut data on whether both isoenzymes are simultaneously expressed in the brain. Work is presently in progress in this laboratory to clarify this issue.
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GLIA-NEURON INTERACTIONS Metabolism of sex steroids in the brain
For many years glial cells have been considered to represent only a mechanical support for neurons and other elements of the CNS. The existence of important functional relationships between specific glia components (oligodendrocytes, astrocytes, etc.) on one side, and neurons on the other have become apparent only recently. The interactions of glial cells with neurons have been shown to play an important role in neuronal migration, in neurite outgrowth, and in axonal guidance during ontogenetic development. Moreover, it has been recently demonstrated that glial cells are able to synthesize, and possibly release, an array of bioactive principles, like neurotransmitters, growth factors, interleukins, prostaglandins, excitatory amino acids, etc. It is probable that several of these principles may exert definite and specific influences on neuronal activity (for references, see Melcangi et al., 1994b, 1996a). The glia-neuronal interactions are not a one-way phenomenon, because several data indicate that neurons may interfere with the proliferation and the maturation of glial elements. For example, neuronal activity has been shown to upregulate the expression of the glial fibrillary acidic protein (GFAP), an astroglia intermediate filament protein that is considered a specific marker of astrocytes. Moreover, other parameters of astrocytic function, like the enzyme glutamine synthetase, the formation of gangliosides, the assembly of voltage-sensitive calcium channels are activated only in the presence of neurons (for references, see Melcangi et al., 1994b, 1996a). As previously mentioned, in the CNS, the metabolism of androgens and of other hormonal steroids (e.g., P) is a very active process, which occurs, even if with different yields and with different end products, practically in all brain cells (see above). The peculiar compartmentalization of the two enzymes (5~x-R and 3et-HSD), which characterize the 5et-reductive metabolism of T and P which we have just described, has prompted us to investigate whether humoral mechanisms of communication might exist between neurons and type 1 astrocytes, and whether these might be relevant for the coordination of the metabolism of androgens and other hormonal steroids in the brain. To examine the reciprocal interactions between astrocytes and neurons, the following two approaches have been used: (a) a coculture system has been established that allows the transfer of secretory products of the cocultured cells, even if these remain physically separated; (b) the addition of neuron-conditioned medium (CM) to cultures of astrocytes, and vice versa of astrocyte CM to cultures of neurons. T metabolism was studied in neurons and type 1 astrocytes either cocultured with the other type of cells, or exposed to the "other" CM. In both types of experiments, the activity of the two enzymes was studied using labeled T (to quantitate the formation of DHT, 5a-R activity), or labeled DHT (to quantitate the formation of 3a-diol, 3ot-HSD activity) as precursors. The results indicate that both the 5o~-reductase and the 3(x-HSD activities are stimulated in type 1 astrocytes by the coculture with neurons and by the addition of neuron CM. However, neither the coculture with type 1 astrocytes nor the addition of astrocyte CM alters the metabolism of T or DHT in neurons (Melcangi et al., 1994b). Control of gonadotropin secretion
The secretion of pituitary gonadotropins is controlled by the hypothalamic hormone LHRH, which is manufactured in a small number of highly specialized neurons. LHRH release is
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regulated by several types of feedback signals (long, short, ultrashort), as well as by multiple nervous influences (for references, see Melcangi et al., 1995). Until recently, it has been believed that these might be transferred to the LHRH-producing neurons only via inputs of neuronal origin, expressed through the liberation, at synaptic or axo-axonal junctions, of classical neurotransmitters (acetylcholine, catecholamines, serotonin, etc.) or of several neuropeptides (opioids, galanin, neuropeptide Y, etc.) (for references, see Melcangi et al., 1995). As mentioned in the preceding paragraph, in the last few years several reports have indicated that glial cells may play an important role in controlling neuronal morphology and function. With regard to the control of gonadotropin secretion, it has been recently reported that feedback signals that typically influence LHRH release may modify the glia surrounding the LHRH neurons. For instance, Witkin et al. (1991) have observed that ovariectomy increases the degree of glial ensheathment (i.e., the apposition of glial processes to perikaria membranes) around the LHRH-producing neurons in the hypothalamus of the female rhesus monkeys, but not in other brain regions; these authors (Witkin et al., 1991) have also shown that this phenomenon is partially reversed by ovarian steroid replacement. These data have been interpreted as suggesting that alterations in the circulating levels of gonadal steroids may produce changes in the glial component located in the immediate environment of the LHRH-producing neurons. However, one may also infer that these morphological alterations represent the expression of metabolic changes occurring within the glial cells, and aimed at modifying the activity of the LHRHproducing neurons, and consequently, their ability to secrete LHRH. This hypothesis has been tested. Advantage has been taken of the existence of a cell line (GTI) derived from a hypothalamic LHRH-producing tumor, induced in a female transgenic mouse by genetically targeted tumorigenesis (Mellon et al., 1990). The GTI cells appear to be highly differentiated neurons, which express the LHRH gene at a high degree (Mellon et al., 1990). These cells present a basal, pulsatile release of LHRH, which can be influenced by a series of neurotransmitters and neuromodulators physiologically involved in the control of LHRH release (norepinephrine, dopamine, GABA, activin-A, endothelin-3, high concentration of K +, prostaglandins, etc.) (for references, see Melcangi et al., 1995). In order to evaluate the possible effects of glial cell products on LHRH secretion from GT1-1, cells two different approaches have been used: (a) GTI-1 cells were coincubated with purified cultures of type I astrocytes; (b) GTI-I cells were exposed to the conditioned medium (CM, untreated or submitted to different experimental procedures) in which type 1 astrocytes were grown for 24 h. In both sets of experiments LHRH was measured by RIA in the incubation media of GTI-1 cells at different time intervals. The data show that coculture of GT1 cells with type I astrocytes and their exposure to previously frozen or heated (but not fresh) type 1 astrocyte CM significantly increase the release of LHRH from the GTI-1 cells. The stimulatory effect on LHRH release appears to be specific for type 1 astrocytes (either cortical or hypothalamic), because the CM of oligodendrocytes does not possess stimulating activities. The fact that fresh CM proved to be inactive, while frozen or heated CM proved effective suggested that the factor(s) involved might be "activated" by the process of freezing or heating. Because it is known that TGF[3 may be liberated from its supporting proteins by freezing or heating, the effects of TGF[3 and of a TGF[3 neutralizing antibody were analyzed. It was found that the stimulatory effect exerted by the frozen astrocyte CM is completely abolished by the antibody, while direct treatment with TGF[3 proved able to significantly increase LHRH release from GTI-I cells. In conclusion, the present experiments demonstrate that type 1 astrocytes may secrete in the medium some factor(s) (most probably TGFI3) able to stimulate the release of LHRH from the
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hypothalamic LHRH-producing GTI-1 cells. The data provide the first clear-cut demonstration that glial cells may directly intervene in the control of LHRH release, integrating the old concept that only neuronal influences may participate in such a phenomenon (for references, see Melcangi et al., 1995). In most recent experiments it has been shown that astrocyte CM and TGF~3 may modify also LHRH gene expression in GTI-1 cells (Galbiati et al., 1996). RESCUE OF PLURIPOTENTIAL STEM CELLS It has been shown that in a variety of animal species neurons and glial cells may arise from common multipotent precursors that are present in the embryonic and neonatal CNS (for references, see Melcangi et al., 1996b). Recently, one of these multipotential cells has been isolated from the mouse striatum; in the absence of an adhesive substrate, these may be induced to proliferate by epidermal growth factor (EGF), producing floating clones (neurospheres). Cells within these neurospheres retain undifferentiated features, and do not express either neuronal or glial markers for the first 10-14 days in vitro (DIV). When the cultures are plated on an adhesive substrate, in the absence of EGF, massive cell differentiation is triggered. It must be emphasized, however, that upon removal of EGF, neurons develop in serum-free conditions, while serum is required for the full differentiation of EGF-generated precursors into glial cells (Melcangi et al., 1996b). As discussed in the preceding portions of this article, fully differentiated CNS cells (neurons and glial cells) are able to convert T and P into the corresponding 5or-reduced metabolites via the 5ct-R-3ot-HSD pathways. The present work has been devoted to analyze whether the enzymes 5ct-R and 3ot-HSD are also present in the multipotential striatum stern cells obtained from the fetal mouse; it has also been investigated whether their differentiation into glial cells (astrocytes and oligodendrocytes) and neurons brings about modifications of the activity of these two enzymes. To this purpose, striatal EGF-responsive stem cells isolated from the mouse brain at the 14th day of embryonic life have been cultured and passaged four times, at weekly intervals, in the presence of EGF. Starting from this point (28 DIV), the cells have been plated on an adhesive substrate, and cultured either in the continuous presence of EGF (that allows proliferation, but maintains the stem cells in an undifferentiated state), or in the absence of EGF but in the presence of fetal bovine serum (FBS) for additional 14 DIV to induce cell differentiation. The relative proportions of the various cell subtypes present were evaluated at different time intervals (i.e., at 35, 38, and 42 DIV); at the same time intervals, the conversion of T and P (5ct-R activity) and of their respective 5et-reduced derivatives DHT and DHP (3ct-HSD activity) were measured both in the differentiated cultures (experimental), and in undifferentiated cells (controls). The differentiation is completed by 35 DIV, when the total cell count comprises: 86 _ 2.0% of astrocytes, 6 _ 0.7% of neurons, 1.6 +- 0.5% of oligodendrocytes, and 6.4 +_ 0.5% of undifferentiated cells. No percent changes in cell population composition are observed thereafter (38 and 45 DIV). The undifferentiated cells (controls) possess a much higher ability to form DHP than DHT (about l0 times). On the contrary, the conversions of DHP and DHT into THP and 3ot-diol are very similar. In the population of differentiating cells, the 5t~-R converting P becomes higher at 38 DIV, and remains similarly elevated at 42 DIV (four times). On the other hand, the conversion of T into DHT remains at the levels of controls up to 42 DIV when an increase is observed. The 3ct-HSD converting DHP but not that converting DHT is increased at 38 and 42 days. These results underline the fact that undifferentiated CNS cells possess steroid metabolizing enzymes for androgens and progestagens that are strongly influenced by the
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cellular differentiation/maturation process. It also appears from the data that during the differentiation process the cells acquire the ability to convert P much earlier than that of converting T. There may be different explanations for this finding (Melcangi et al., 1996b). The most attractive one suggests the existence of a third 5~-R isozyme (see above) more specific for P. This concept emerges also from other recent data of this laboratory, which have shown that the CM of brain tumor cells (C6 glioma and 1321Nl astrocytoma) is able to inhibit the conversion of P into DHP but not that of T into DHT in type 1 astrocytes (R.C. Melcangi, I. Cavarretta, V. Magnaghi, M. Ballabio, L. Martini, and M. Motta, unpublished data). REFERENCES CELOTTI, F., MELCANGI R.C., and MARTINI, L. The 5e~-reductase in the brain: Molecular aspects and relation to brain function. In: Frontiers in Neuroendocrinology, Martini, L. and Ganong, W.F. (Editors), pp. 163-215, vol. 13, Raven Press, New York, 1992. FRANKFURT, M , GOULD, E., WOOLEY, C.S., and MCEWEN, B.S. Gonadal steroids modify dendritic spine density in ventromedial bypothalamic neurons: A Golgy study in the adult rat. Neuroendocrinology 51, 530-535, 1990, GALBIATI, M., ZANISI, M., MESSI, E.. CAVARRETTA, I., MARTINI, L., and MELCANGI, R.C. Transforming growth factor-[?, and astrocytic conditioned medium influence luteinizing hormone-releasing hormone gene expression in the hypothalamic cell line GTI. Endocrinology 137, 5605-5609, 1996. LIMONTA, P., DONDI, D., MAGGI, R., MARTINI, L., and PIVA, F. Neonatal organization of the brain opioid systems controlling prolactin and luteinizing hormone secretion. Endocrinology 124, 681-686, 1989. MARTINI, L. The 5e~-reduction of testosterone in the neuroendocrine structures. Biochemical and physiological implications. Endocr. Rev. 3, 1-25, 1982. MELCANGI, R.C., CELOTT1, F., BALLABIO, M., CASTANO. P.. MASSARELLI, R., POLETTI, A., and MARTINI, L. 5~-Reductase activity in isolated and cultured neuronal and glial cells of the rat. Brain Res. 516, 229-236, 1990. MELCANGI, R.C., CELOTTI, F., CASTANO, P., and MARTINI, L. Differential localization of the 5~-reductase and the 3~-hydroxysteroid dehydrogenase in neuronal and glial cultures. Endocrinology 132, 1252-1259, 1993. MELCANGI, R.C., CELOTTI, F., and MARTINI, L. Progesterone 5e~-reduction in neuronal and in different types of glial cell cultures: Type 1 and 2 astrocytes and oligodendrocytes. Brain Res. 639, 202-206, 1994a. MELCANGI, R.C., CELOTTI, F., and MARTINI, L. Neurons influence the metabolism of testosterone in cultured astrocytes via humoral signals. Endocrine 2, 709-713, 1994b. MELCANGI, R.C., GALBIATI, M., MESSI, E., PIVA, F., MARTINI, L., and MOTTA, M. Type 1 astrocytes influence luteinizing hormone-releasing hormone release from the hypothalamic cell line GTI-I: Is transforming growth factor-J3 the principle involved? Endocrinology 136, 679-686, 1995. MELCANGI, R.C., RIVA, M.A., FUMAGALLI, F., MAGNAGHI, V., RACAGNI, G_ and MARTINI, L. Effect of progesterone, testosterone and their 5e~-reduced metabolites on GFAP gene expression in type 1 astrocytes. Brain Res. 711, 10-15, 1996a. MELCANGI, R.C., FROELICHSTHAL, P., MARTINI, L., and VESCOVL A.L. Steroid metabolizing enzymes in pluripotential progenitor central nervous system cells: Effect of differentiation and maturation. Neuroscience 72, 467-475, 1996b. MELLON, P.L.. WINDLE, J.J., GOLDSMITH, P.C., PADULA, C.A., ROBERTS, J.L., and WEINER, R.I. Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 5, 1-10, 1990. NAFTOLIN, F., GARCIA-SEGURA, L.M., KEEFE, D., LERANTH. C., MACLUSKY, N.J., and BRAWER, J.R. Estrogen effects on the synaptology and neuronal membranes of the rat hypothalamic arcuate nucleus. Biol. Reprod. 42, 21-28, 1990. NEGRI-CESI, P., MELCANGI, R.C., CELOTTI, F., and MARTINI, L. Aromatase activity in cultured brain cells: Difference between neurons and glia. Brain Res. 589, 327-332, 1992. OLMOS, G., AGUILERA, P., TRANQUE, P., NAFTOLIN, F., and GARCIA-SEGURA, L.M. Estrogen-induced synaptic remodelling in adult rat brain is accompanied by the reorganization of neuronal membranes. Brain Res. 425, 57-64, 1987. POLETTI, A., CELOTTI, F., MOTTA, M., and MARTIN1, L. Characterisation of rat 5e~-reductases type 1 and type 2 expressed in yeast Saceharomyces cerevisiae. ,L Biochem. 314, 1047-1052, 1996. SAPOLSKY, R.M. Why stress is bad for your brain. Science 273, 749-750, 1996. WITKIN, J.W., FERIN, M., POPILSKIS, S.J., and SILVERMAN, A.J. Effects of gonadal steroids on the ultrastructure of GnRH neurons in the reshus monkey: Synaptic input and glial apposition. Endocrinology 129, 1083-1092, 1991.