The Journal of Neuroscience, December 15, 2002, 22(24):10613–10620 An Essential Component in Steroid Synthesis, the Steroidogenic Acute Regulatory Protein, Is Expressed in Discrete Regions of the Brain Steven R. King,1,2,3 Pulak R. Manna,4 Tomohiro Ishii,6 Peter J. Syapin,5 Stephen D. Ginsberg,7 Kevin Wilson,4 Lance P. Walsh,4 Keith L. Parker,6 Douglas M. Stocco,4 Roy G. Smith,2,3 and Dolores J. Lamb1,3 1Scott Department of Urology, 2Huffington Center on Aging, and 3Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, Departments of 4Cell Biology and Biochemistry and 5Pharmacology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, 6Departments of Internal Medicine and Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, and 7Center for Dementia Research, Nathan Kline Institute, New York University School of Medicine, Orangeburg, New York 10962 Recent data implicate locally produced steroids, termed neuro- sistent with a role in de novo neurosteroidogenesis, StAR co- steroids, as regulators of neuronal function. Adrenal and go- localizes with the cholesterol side-chain cleavage enzyme nadal steroidogenesis is controlled by changes in the steroido- P450scc in both mouse and human brains. These data support genic acute regulatory protein (StAR); however, little is known a role for StAR in the production of neurosteroids and identify about the regulation of neurosteroid production. We now dem- potential sites of active de novo steroid synthesis in the brain. onstrate unequivocally that StAR mRNA and protein are ex- Key words: neurosteroid; steroidogenic acute regulatory pro- pressed within glia and neurons in discrete regions of the tein; P450scc ; steroidogenesis; cholesterol side-chain cleavage mouse brain, and that glial StAR expression is inducible. Con- enzyme; StAR The rate-limiting step in steroid biosynthesis is regulated by the Genazzani et al., 1995; Frye et al., 1996; Calogero et al., 1998; steroidogenic acute regulatory protein (StAR) (Stocco and Clark, Akwa et al., 2001). Deficits in neurosteroid production may con- 1996; Stocco, 2001). StAR mediates the transfer of cholesterol tribute to a variety of disorders, including dementia, epilepsy, from the outer to the inner mitochondrial membrane, where premenstrual syndrome, and postpartum depression (Vallee et cholesterol is then converted by cytochrome P450scc to preg- al., 1997; Smith et al., 1998; Beyenburg et al., 1999). Conse- nenolone, the precursor for all steroids. The critical role of StAR quently, it is critical that we understand the mechanism of neu- is demonstrated in humans and mice by the observation that rosteroid biosynthesis and regulation. mutations in StAR result in congenital lipoid adrenal hyperplasia In response to trophic stimulation, StAR is rapidly synthesized (Lin et al., 1995; Bose et al., 1996; Caron et al., 1997). Patients as a 37 kDa preprotein. Before or during import into the mito- carrying this mutation cannot synthesize sufficient levels of adre- chondria and processing to 30 kDa forms, StAR stimulates inter- nal and gonadal steroids and, if untreated, die shortly after birth. membrane cholesterol transfer (Krueger and Orme-Johnson, The essential function of StAR in steroidogenesis is established 1983; Epstein and Orme-Johnson, 1991; Stocco and Sodeman, for all tissues that synthesize steroid, except for the human 1991; Clark et al., 1994; King et al., 1995). Direct evidence in placenta and the brain. support of this function was illustrated by stimulation of steroi- The brain synthesizes neurosteroids de novo, especially within dogenesis in COS1 cells after transient coexpression of StAR and glia (Corpechot et al., 1981; Koenig et al., 1995; Zwain and Yen, P450 (Sugawara et al., 1995a). Thus, given adequate availability 1999; Compagnone and Mellon, 2000; Tsutsui et al., 2000), but scc of reducing equivalents for the enzymatic reaction, the sole re- the relative roles of locally produced neuroactive steroids and quirements for the production of steroid in a cell are the presence those converted from circulating precursors remain to be defined. of P450 and continual de novo synthesis of StAR. Unlike Alterations in levels of locally produced neurosteroids in the scc hypothalamus, hippocampus, and other regions may serve as P450scc, StAR activity is acutely regulated, and thus, StAR serves crucial paracrine modulators of essential brain functions, includ- as a useful protein marker for ongoing steroidogenesis. ing sexual drive, learning, and memory (Majewska et al., 1986; Expression of P450scc in the CNS has been established previ- ously (Le Goascogne et al., 1987), proving that the brain has the Received June 28, 2002; revised Sept. 5, 2002; accepted Sept. 30, 2002. capability to synthesize the steroid. However, a lack of informa- This research was supported by grants from the Gilson Longenbaugh Foundation tion on StAR localization has hampered efforts to characterize and Alzheimer’s Association Grant NIRG-00-2250 (S.D.G.); by Texas Advanced Research Program Grant 010674-0013 (P.J.S.); by National Institutes of Health steroid-producing cells and investigate the regulation of neuros- (NIH) Grants DK54028 (K.L.P.), NS43939 (S.D.G.), and HD17481 (D.M.S.); by the teroidogenesis. Although StAR mRNA was identified recently in Robert A. Welch Foundation (D.M.S.); and by NIH Grant T32-DK07763 and the Lalor Foundation (S.R.K.). We thank J. C. Hutson, Y. O. Lukyanenko, J. Koss- the brain (Furukawa et al., 1998; Wehrenberg et al., 2001), the Dertien, C. Y. Cutler, J. T. Le, and D. Alberts for their assistance. protein was not detected. Despite an initial report suggesting Correspondence should be addressed to Dr. Dolores J. Lamb, Scott Department expression in the hippocampus (Kimoto et al., 2001), controversy of Urology, Room N730, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030-3498. E-mail: [email protected]. has been raised with issues of specificity of the antisera used. Copyright © 2002 Society for Neuroscience 0270-6474/02/2210613-08$15.00/0 Therefore, we set out to establish unambiguously whether or not 10614 J. Neurosci., December 15, 2002, 22(24):10613–10620 King et al. • StAR Expression in the Brain StAR is expressed in the brain and to identify potential sites of de computer-assisted image analysis system (BioImage, Ann Arbor, MI). novo neurosteroidogenesis. For RT-PCR, bands of interest were normalized to L19 ribosomal protein mRNA levels and compared with those of unstimulated cells. MATERIALS AND METHODS Statistical analyses for these results are detailed in the figure legends. Preparation of brain tissue. We perfused, fixed, and dissected brains Materials. Media were obtained from Invitrogen (Grand Island, NY). We from terminally anesthetized male and female adult C57BL/6 or used primary antisera against amino acids 88–98 of StAR (previously Ϫ Ϫ Ϫ Ϫ StAR / mice of different ages. Adult animals were perfused transcar- used to verify the loss of StAR in the StAR / mouse) (Clark et al., dially with PBS, followed by phosphate-buffered 4% paraformaldehyde, 1994; Caron et al., 1997), an N-terminally truncated form of StAR (N-62; pH 7.4, in phosphate buffer. Brains were removed and placed into PBS. kindly provided by W. L. Miller, University of San Francisco, San Brains were then fixed in 4% p-formaldehyde for 2 hr before incubation Francisco, CA) (Bose et al., 1999), recombinant StAR (a gift from D. B. on consecutive nights with 12% and then 30% sucrose solutions in Hales, University of Illinois, Chicago, IL) (Hales et al., 2000), and glial phosphate buffer, pH 7.4, at 4°C. The brains were mounted in Tissue Tek fibrillary acidic protein (GFAP; Sigma, St. Louis, MO), as well as amino optimal cutting temperature compound (Miles Inc., Elkhart, IN) on a acids 421–441 and 509–526 of P450scc and monoclonal antisera against chuck and, after equilibration to Ϫ20°C in a cryostat, 40-␮m-thick coro- neuronal-specific nuclear protein (NeuN) and GFAP (Chemicon, Te- nal, sagittal, and horizontal sections were cut and stored at Ϫ20°Cina mecula, CA). cryoprotectant solution (30% glycerol and 30% ethylene glycol in 0.2ϫ Preparation of astrocyte cultures and measurement of steroid production. phosphate buffer). Astrocyte cultures were prepared from the mesencephalon of 15- to Neutral buffered formalin (10%) fixed nondemented human brain 16-d-old Sprague-Dawley rat embryos as detailed previously (Syapin et tissues with no pathological evidence of neurodegenerative disease were al., 2001) and dissected with the aid of a stereomicroscope. The cells were obtained from the Harvard Brain Tissue Resource Center (McLean maintained in standard glia medium (HEPES-buffered F12/DMEM plus Hospital, Belmont, MA). Forty micrometer sections were cut using a 10% FBS) to eliminate neuronal contaminants for 9–12 d, until stable in vibratome and treated with methanol and hydrogen peroxide to quench appearance. We further purified and expanded cultures twice by replat- endoperoxidase activity before antibody incubation. ing (1:2 splits) to obtain confluent, quiescent, tertiary cultures in 2% FBS Immunohistochemistry and immunofluorescence. Selected sections were glia medium. Cultures devoid of oligodendrocytes and microglia were washed several times in TBS and then left for 1 hr in blocking solution used 2–14 d later. Mouse MA-10 Leydig tumor cells (kindly provided by (0.01% Triton X-100, 2% serum, and 0.1 M Tris/HCl, pH 7.4). Samples M. Ascoli, University of Iowa, Iowa City, IA) were maintained in culture were incubated overnight at 4°C with primary antibody appropriately in Waymouth’s medium with 15% heat-inactivated horse serum (Clark et diluted in a solution of 0.005% Triton X-100, 1% serum, and 0.1 M Tris. al., 1994). Rat C6 glioma cells from the American Type Culture Collec- Immunoperoxidase labeling was then performed using the Vectastain tion (Manassas, VA) were grown in high glucose-containing DMEM with Elite ABC kit (Vector Laboratories, Burlingame, CA) according to the 5% FBS (Syapin, 1995).