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S.01.01 Molecular studies of enzymes involved in steroid biosynthesis in the central nervous system
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Symposia S.01 Neurosteroids ~
Molecular studies of enzymes involved in steroid biosynthesis in the central nervous system
N.A. Compagnone ~, S.H. Mellon. Universi~_ of California San Francisco, Dept. OB&GYN, Box 0556, SF CA 94143, USA, I lNSERM U33, 80 av du general Leclerc 94276 Le kremlin Bic~tre, France Persistence of steroids, such as pregnenonlone, pregnenolone-sulfate, allopregnanolone, dehydroepiandrosterone (DHEA) and DHEA-sulfate in the nervous system of adrenalectomized and gonadectomized animals has lead to the concept that steroids could be synthesized de novo in the central (CNS) or peripheral nervous systems (PNS). These steroids have been given the name neurosteroids. Neurosteroids are capable of neuromodulation at both GABAA and NMDA receptors in the brain. Synthesis of neurosteroids in the CNS and 1,NS thus suggest that contrary to classically held notions of steroid synthesis and action, neurosteroids do not need to be transported to their target, but are able to act locally, and mediate their actions through neurotransmitter membrane receptors rather than through intracellular steroid hormone receptors. Hence, neurosteroids do not have "endocrine", but rather have paracrine and/or autocrine action in the nervous system. Since the initial observation of de novo steroidogenesis in the brain, several research laboratories, including ours, have been involved in determining whether steroidogenic enzymes necessary for steroidogenesis in endocrine glands were also necessary for neurosteroidogenesis. Identification of mRNAs specific for each steroidogenic cytochrome P450 in the nervous system was difficult, suggesting very low levels of expression of all these mRNAs in the nervous system. We and others have described the expression of mRNA specific for P450 scc, P450cl 1/3, 3/3HSD, 5or reductase and 3c~ hydroxysteroid dehydrogenase, enzymes necessary for the local synthesis of pregnenolone, progesterone, allopregnanolone and corticosterone. However, although DHEA was one of the first steroids identified in the brain of adrenalectomized and gonadectomized animals, the expression of P450c17 (17ct hydroxylase, c17-20 lyase), crucial for local synthesis of DHEA, was not detected in the adult brain. We detected P450c17 in the nervous system of embryonic rodents in a developmentally- and regionally-regulated pattern. Our developmental studies demonstrated the expression of both P450 scc and P450c17 in the developing brain, and showed that P450eI7 was transiently expressed in the neocortex at a time corresponding to its cytoarchitectural organization. We showed that DHEA was able to promote axonal growth in embryonic neocortical cultures, while its expression in vivo paralleled the expression of other axotonines involved in the growth and guidance of thalamo-cortical axons. Furthermore, DHEA increased calcium entry into neocortical cells through activation of NMDA receptors. DHEA-sulfate, however, did not elicit any changes in intracellular calcium, and specifically promoted dendritic growth. Hence, addition and removal of sulfate moieties may participate in the regulation of DHEA/S actions within the neocortex. Regulation of enzymes with these opposing activities in a developmentally- and regionally-specific pattern could thus allow local modulation of neurotransmitter action, and/or modulation of morphological differentiation of neocortical neurons. Preliminary data on the expression of the steroid sulfatase support such an hypothesis.
•
Neurosteroids and the peripheral benzodiazepine receptor
V. Papadopoulos, R.C. Brown, C. Cascio. Department of Cell Biology, Georgetown University Medical Center, Washington, D.C. 20007, USA The specific interactions of steroids with binding sites in the brain together with the rapid effects of various steroids on neuronal function has prompted the investigation of the steroidogenic potential of central
nervous system structures. The pioneering work of Baulieu et al (1) demonstrated that glial cells can convert cholesterol to pregnenolone and give origin to steroid metabolites, potential modulators of neuronal function. It was then shown that a glioma cell line, oligodendrocytes, and Schwann cells all have the ability to metabolize cholesterol to pregnenolone, the first step in steroid biosynthesis. In order to understand the mechanisms of neurosteroid synthesis we looked at mechanisms well established in peripheral steroidogenic tissues. Trophic hormone regulation of steroid synthesis cart be thought as being either "'acute", occurring within minutes and resulting in the rapid synthesis of steroids, or "chronic", occurring over a long period of time and resulti~g in continued steroid production. In the ctuonic regulation, peptide hormones and cAMP act by inducing the expression of steroidogenic enzymes. In acute regulation, the rate of steroid fonxnation depends on the rate of cholesterol transport from intracellalar stores to the inner mitochondrial membrane and loading of the cytochrome P450 side chain cleavage (17'450 scc; the enzyme which metabolizes cholesterol to pregnenolone) with cholesterol. In previous in vitro studies, we demonstrated that a key element in the acute regulation of cholesterol transport is the mitochondrial peripheral-type benzodiazepine receptor (PBR), and that the presence of the polypeptide diazepam binding inhibitor (DBI) was vital for steroidogenesis (3). We also showed that DBI, the endogenous PBR ligand, stimulates cholesterol transport and promotes loading of cholesterol to P450 scc. Based on these data and the obse,rvations that: (i) the mitochondrial PBR binding and topography is regulated by hormones, (ii) the 18 kDa PBR protein is associated with mitochondrial membrane contact site proteins, (iii) the 18 kDa PBR protein is a channel for cholesterol, as shown by molecular modeling simulations and hz vitro reconstitution experiments, (iv) targeted disruption of the 1,BR gene in steroidogerdc cells dramatically reduces the steroidogenic ability of the cells, and (v) in vivo reduction of adrenal PBR expression results in reduced circulating glucocorticoid levels, we concluded that PBR is an indispensable element of the steroidogenic machinery. Using the C6-2B glioma cell line, we demonstrated the presence of both regulatory mechanisms - cAMP and mitochondrial PBR - in the control of neurosteroid synthesis. ]First, with chronic treatment of C6-2B cells with cAMP, we showed that neurosteroid synthesis increased. Second, we demonstrated that these cells express high levels of PBR, and that 1'BR drug ligands stimulated the formation of pregnenolone by glial cells and isolated mitochondria, by accelerating the rate of cholesterol transport to the 1,450scc resulting in increased pregnenolone formation. Howeve;, discrepancies exist between levels of the enzymatic activity, the amount of immunoreactive protein, and mRNA of the P450 scc present in brain. In addition, despite the high levels of DHEA (the first neurosteroid described) found in brain, no one has demonstrated the presen~-e of the 17c~-hydroxylase cytochrome P450 activity in brain (P450c 17). Thus, it seems that brain steroid synthesis may not fit the well defined scheme of adrenal, gonadal, and placental sternidogenesis, and that new pathways should be explored. Understanding the mechanism of DHEA formation is paramount to all further speculation and hypotheses about DHEA and its role in aging and brain function. The levels of DHEA and its sulfated form in brain are distinct from the peripheral steroid levels, and its function as neuroactive steroid at the GABAA and NMDA receptor level has been well established. We showed that C6-2B rat glioma tumor cells express the mRNA, protein, and activity for P450scc but not for P450c17, although both pregnenolone and DHEA are synthesized. In addition, we demonstrated th~.t human glioma ceils in culture are also able to synthesize neurosteroids, in a similar manner to C6-2B cells, thas validating the use of this model system. We then investigated whether DHEA could be formed from alternate precursors in C6-2B cells. Addition of the reducing agent FeSO4 to the cells increased DHEA synthesis 10-fold in a dosedependent manner without affecting progesterone biosynthesis. The 1"450 scc inhibitor aminoglutethimide and the P450cl 7 inhibb:or SU-10603 did
Symposia S.01 Neurosteroids ~
Molecular studies of enzymes involved in steroid biosynthesis in the central nervous system
N.A. Compagnone ~, S.H. Mellon. Universi~_ of California San Francisco, Dept. OB&GYN, Box 0556, SF CA 94143, USA, I lNSERM U33, 80 av du general Leclerc 94276 Le kremlin Bic~tre, France Persistence of steroids, such as pregnenonlone, pregnenolone-sulfate, allopregnanolone, dehydroepiandrosterone (DHEA) and DHEA-sulfate in the nervous system of adrenalectomized and gonadectomized animals has lead to the concept that steroids could be synthesized de novo in the central (CNS) or peripheral nervous systems (PNS). These steroids have been given the name neurosteroids. Neurosteroids are capable of neuromodulation at both GABAA and NMDA receptors in the brain. Synthesis of neurosteroids in the CNS and 1,NS thus suggest that contrary to classically held notions of steroid synthesis and action, neurosteroids do not need to be transported to their target, but are able to act locally, and mediate their actions through neurotransmitter membrane receptors rather than through intracellular steroid hormone receptors. Hence, neurosteroids do not have "endocrine", but rather have paracrine and/or autocrine action in the nervous system. Since the initial observation of de novo steroidogenesis in the brain, several research laboratories, including ours, have been involved in determining whether steroidogenic enzymes necessary for steroidogenesis in endocrine glands were also necessary for neurosteroidogenesis. Identification of mRNAs specific for each steroidogenic cytochrome P450 in the nervous system was difficult, suggesting very low levels of expression of all these mRNAs in the nervous system. We and others have described the expression of mRNA specific for P450 scc, P450cl 1/3, 3/3HSD, 5or reductase and 3c~ hydroxysteroid dehydrogenase, enzymes necessary for the local synthesis of pregnenolone, progesterone, allopregnanolone and corticosterone. However, although DHEA was one of the first steroids identified in the brain of adrenalectomized and gonadectomized animals, the expression of P450c17 (17ct hydroxylase, c17-20 lyase), crucial for local synthesis of DHEA, was not detected in the adult brain. We detected P450c17 in the nervous system of embryonic rodents in a developmentally- and regionally-regulated pattern. Our developmental studies demonstrated the expression of both P450 scc and P450c17 in the developing brain, and showed that P450eI7 was transiently expressed in the neocortex at a time corresponding to its cytoarchitectural organization. We showed that DHEA was able to promote axonal growth in embryonic neocortical cultures, while its expression in vivo paralleled the expression of other axotonines involved in the growth and guidance of thalamo-cortical axons. Furthermore, DHEA increased calcium entry into neocortical cells through activation of NMDA receptors. DHEA-sulfate, however, did not elicit any changes in intracellular calcium, and specifically promoted dendritic growth. Hence, addition and removal of sulfate moieties may participate in the regulation of DHEA/S actions within the neocortex. Regulation of enzymes with these opposing activities in a developmentally- and regionally-specific pattern could thus allow local modulation of neurotransmitter action, and/or modulation of morphological differentiation of neocortical neurons. Preliminary data on the expression of the steroid sulfatase support such an hypothesis.
•
Neurosteroids and the peripheral benzodiazepine receptor
V. Papadopoulos, R.C. Brown, C. Cascio. Department of Cell Biology, Georgetown University Medical Center, Washington, D.C. 20007, USA The specific interactions of steroids with binding sites in the brain together with the rapid effects of various steroids on neuronal function has prompted the investigation of the steroidogenic potential of central
nervous system structures. The pioneering work of Baulieu et al (1) demonstrated that glial cells can convert cholesterol to pregnenolone and give origin to steroid metabolites, potential modulators of neuronal function. It was then shown that a glioma cell line, oligodendrocytes, and Schwann cells all have the ability to metabolize cholesterol to pregnenolone, the first step in steroid biosynthesis. In order to understand the mechanisms of neurosteroid synthesis we looked at mechanisms well established in peripheral steroidogenic tissues. Trophic hormone regulation of steroid synthesis cart be thought as being either "'acute", occurring within minutes and resulting in the rapid synthesis of steroids, or "chronic", occurring over a long period of time and resulti~g in continued steroid production. In the ctuonic regulation, peptide hormones and cAMP act by inducing the expression of steroidogenic enzymes. In acute regulation, the rate of steroid fonxnation depends on the rate of cholesterol transport from intracellalar stores to the inner mitochondrial membrane and loading of the cytochrome P450 side chain cleavage (17'450 scc; the enzyme which metabolizes cholesterol to pregnenolone) with cholesterol. In previous in vitro studies, we demonstrated that a key element in the acute regulation of cholesterol transport is the mitochondrial peripheral-type benzodiazepine receptor (PBR), and that the presence of the polypeptide diazepam binding inhibitor (DBI) was vital for steroidogenesis (3). We also showed that DBI, the endogenous PBR ligand, stimulates cholesterol transport and promotes loading of cholesterol to P450 scc. Based on these data and the obse,rvations that: (i) the mitochondrial PBR binding and topography is regulated by hormones, (ii) the 18 kDa PBR protein is associated with mitochondrial membrane contact site proteins, (iii) the 18 kDa PBR protein is a channel for cholesterol, as shown by molecular modeling simulations and hz vitro reconstitution experiments, (iv) targeted disruption of the 1,BR gene in steroidogerdc cells dramatically reduces the steroidogenic ability of the cells, and (v) in vivo reduction of adrenal PBR expression results in reduced circulating glucocorticoid levels, we concluded that PBR is an indispensable element of the steroidogenic machinery. Using the C6-2B glioma cell line, we demonstrated the presence of both regulatory mechanisms - cAMP and mitochondrial PBR - in the control of neurosteroid synthesis. ]First, with chronic treatment of C6-2B cells with cAMP, we showed that neurosteroid synthesis increased. Second, we demonstrated that these cells express high levels of PBR, and that 1'BR drug ligands stimulated the formation of pregnenolone by glial cells and isolated mitochondria, by accelerating the rate of cholesterol transport to the 1,450scc resulting in increased pregnenolone formation. Howeve;, discrepancies exist between levels of the enzymatic activity, the amount of immunoreactive protein, and mRNA of the P450 scc present in brain. In addition, despite the high levels of DHEA (the first neurosteroid described) found in brain, no one has demonstrated the presen~-e of the 17c~-hydroxylase cytochrome P450 activity in brain (P450c 17). Thus, it seems that brain steroid synthesis may not fit the well defined scheme of adrenal, gonadal, and placental sternidogenesis, and that new pathways should be explored. Understanding the mechanism of DHEA formation is paramount to all further speculation and hypotheses about DHEA and its role in aging and brain function. The levels of DHEA and its sulfated form in brain are distinct from the peripheral steroid levels, and its function as neuroactive steroid at the GABAA and NMDA receptor level has been well established. We showed that C6-2B rat glioma tumor cells express the mRNA, protein, and activity for P450scc but not for P450c17, although both pregnenolone and DHEA are synthesized. In addition, we demonstrated th~.t human glioma ceils in culture are also able to synthesize neurosteroids, in a similar manner to C6-2B cells, thas validating the use of this model system. We then investigated whether DHEA could be formed from alternate precursors in C6-2B cells. Addition of the reducing agent FeSO4 to the cells increased DHEA synthesis 10-fold in a dosedependent manner without affecting progesterone biosynthesis. The 1"450 scc inhibitor aminoglutethimide and the P450cl 7 inhibb:or SU-10603 did