Dehydroepiandrosterone (3f3-hydroxy-5-androsten-17-one, I) sulfate (Ia) has been characterized in the anterior and the posterior parts of the brain of adult male rats. Its level (1.58 ± 0.14 and 4.89 ± 1.06 ng/g, mean ± SD, in anterior and posterior brain, respectively) largely exceeded that of I in brain (0.42 ± 0.10 and 0.12 ± 0.03 ng/g in anterior and posterior brain, respectively) and of Ia in plasma (0.26 ± 0.13 ng/ml). Brain Ia level did not seem to depend on adrenal secretion; it was unchanged after administration of corticotropin or dexamethasone for 3 days, and no meaningful change occurred in brain 15 days after adrenalectomy plus orchiectomy, compared with sham-operated controls. In contrast, stress conditions prevailing 2 days after adrenalectomy plus orchiectomy or after the corresponding sham operation resulted in a significantly increased concentration of Ia in the brain. Changes of Ia level in brain occurred irrespective of changes in corresponding plasma samples. It is proposed that Ia formation or accumulation (or both) in the rat brain depends on in situ mechanisms unrelated to the peripheral endocrine gland system. Dehydroepiandrosterone (3,3hydroxy-5-androsten-17-one, I) sulfate (Ia) is below detection limit in the plasma of most adult mammals (1); the exceptions are man and the highest nonhuman primates (1-3). It is a major secretory product ofhuman adrenals (4-7), and its concentration in adult plasma is larger than that of any other steroid. Although Ia is the main precursor of placental estrogens (8-10) and is occasionally converted into active androgens in peripheral tissues (11,12) was added to 2 ml of plasma or <1 g of tissue, and then 5 ml ofacetone/ethanol (1:1) was added. Tissues were homogenized in acetone/ethanol (1:1) with a Teflon/glass homogenizer and sonicated with a Branson Jl sonifier equipped with a minitip at a 100-W setting for 10 sec. The suspensions were kept at 390C overnight and centrifuged at 1000 x g for 10 min. The supernatant was saved, and the pellet was extracted with 4 ml of methanol/chloroform (1:1) with continuous shaking at room temperature for 30 min. The extract was centrifuged, and the two supernatants were combined and taken to dryness. The residue was dissolved in 4 ml of methanol/chloroform (1:1)/10 mM NaCl and deposited on a Sephadex LH-20 column (10 X 445 mm) equilibrated and developed in the same solvent system (15). The first 50 ml to run off contained unconjugated I, Ia was eluted in the next 75 ml. It was completely separated from free I and from I conjugated to fatty acids (16) 4704The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

The observation was made that dehydroepiandrosterone (DHEA), as the unconjugated steroid, and its sulfate ester (DHEAS) are present in the brain of adult male rats (1). This finding was unforeseen because the rodent steroidogenic glands, including the adrenals, do not secrete significant amounts of DHEA (2). It led to the discovery of a steroid biosynthetic machinery in the nervous system, in charge of producing neurosteroids.This term, neurosteroids, was proposed in 1981 (3). It applies to the steroids, the accumulation of which occurs in the nervous system independently, at least in part, of supply by the steroidogenic endocrine glands and which can be synthesized de novo in the nervous system from sterol precursors. The steroid precursors along their biosynthetic pathways can be formed in situ and assayed.This definition applies to DHEA. To demonstrate that brain DHEA is independent of peripheral steroidogenic glands, endocrine manipulations were conducted in rats. Injections for 3 days of long-acting preparations of corticotropin (␤1-24 ACTH), to stimulate adrenal steroidogenesis, or of dexamethasone to inhibit endogenous ACTH secretion, were not accompanied by clear-cut changes of brain DHEAS (1). Brain DHEAS was unchanged 1 day after castration, whereas testosterone completely disappeared from the brain. No obvious difference was observed when castrated adrenalectomized males were compared 15 days after operation with shamoperated controls (1, 4).It thus was logical to assume that DHEA synthesis evolves in two steps from cholesterol, as described for steroidogenic glands, principally catalyzed by two different cytochromeIndeed PREG was found in the brain, at concentrations about 1 order of magnitude larger than those of DHEA, as might be expected from a precursor-to-product relationship (5). The concentrations of PREG and of its sulfate ester PREGS were of the same order of magnitude, and about 10-fold larger than in plasma. They were found at least in part independent of the activity of steroidogenic glands.Solid evidence has been accrued for the biosynthesis of PREG in the nervous system (reviewed in ref. 6). Incubation of primary cultures of rat forebrain glial cells with a precursor to cholesterol led to the formation of cholesterol, PREG, progesterone, and 20␣-dihydro-PREG, and incubation of rat oligodendrocyte mitochondria with cholesterol yielded pregnenolone (7,8). There is only one cholesterol side-chain cleavage enzyme, named cytochrome P450 scc , with strong structural similarity between rodent, bovine, and human species. The presence of immunoreactive P450 scc protein in the rat and human brain has been established in the white matter and in primary cultures of the newborn rat forebrain glial cells (9).However, the abundance of P450 scc mRNA is exceedingly low and could be demonstrated only by reverse transcription-PCR (RT-PCR) (10-12).Major efforts were devoted to the elucidation of DHEA biosynthesis in the brain (13). However, incubations of [ 3 H]PREG (and sulfated or acetyl ester derivative...

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N-Methyl-D-aspartate (NMDA, 200 pzM) evokes the release of [3H]norepinephrine ([3H]NE) from preloaded hippocampal slices. This effect is potentiated by dehydroepiandrosterone sulfate (DHEA S), whereas it is inhibited by pregnenolone sulfate (PREG S) and the highaffinity r inverse agonist 1,3-di(2-tolyl)guanidine, at concentrations of .100 nM. Neither 3a-hydroxy-5a-pregnan-20-one nor its sulfate ester modified NMDA-evoked [3H] NE overflow. Eighty-four female Sprague-Dawley rats (180-225 g), purchased from Iffa Credo, were kept at 21°C on a 12 hr:12 hr light/dark cycle with free access to water and Purina chow. At least 4 weeks prior to the release experiments, rats were anesthetized under ether and bilateral ovariectomy was carried out by lateral access. In a subgroup of 16 rats, anesthetized with chloral hydrate (400 mg/kg, i.p.) 3-11 days prior to the experiments, PTX (1 ,g/2 ,ul of physiological saline) was injected (using a 10-uAl Hamilton syringe) bilaterally into the dorsal hippocampus at A: 4.5, L: 4, and D: 4, according to the atlas of Paxinos and Watson (25) as described (3). Twelve control rats received an equal volume of the vehicle. When appropriate, the rats were killed by decapitation and their brains were rapidly dissected. Coronal slices (0.4-mm thick) of the hippocampus were prepared with a McIlwain tissue chopper. The slices were incubated in Krebs' solution containing[3H]NE (0.1 ,uM) and bubbled with a mixture of 95% 02/5% CO2 at 37°C for 30 min. The composition of the Krebs' solution (in mM) was NaCl 118, KCl 4.7, CaCl2 1.3, MgCl2 1.2, NaH2PO4 1, NaHCO3 25, glucose 11.1, Na2EDTA 0.04, and ascorbic acid 0.06. At the end of the incubation period, each glass chamber received two slices that were superfused continuously at a rate of 0.5 ml/min with oxygenated Mg2+-free Krebs' solution at 37°C for 68 min. As indicated in Results, one steroid and/or one of the cr ligands DTG, haloperidol, BD-1063, or spiperone were added in Mg2+-free Krebs' solution throughout the superfusion period. The prototypic a-ligand DTG was chosen since it acts on both a-i and o'2 receptors (3, 26). The universal o-antagonist haloperidol also binds to dopaminergic, serotoninergic, adrenergic, and cholinergic sites Abbreviations: 3a,5a-THP, 3a-hydroxy-Sa-pregnan-20-one; CNS, central nervous system; DHEA, dehydroepiandrosterone; DTG, 1,3-di(2-tolyl)guanidine; Gi/o protein, guanine nucleotide-binding pro-

Some neurosteroids have been shown to display beneficial effects on neuroprotection in rodents. To investigate the physiopathological significance of neurosteroids in Alzheimer's disease (AD), we compared the concentrations of pregnenolone, pregnenolone sulfate (PREGS), dehydroepiandrosterone, dehydroepiandrosterone sulfate (DHEAS), progesterone, and allopregnanolone, measured by gas chromatography-mass spectrometry, in individual brain regions of AD patients and aged nondemented controls, including hippocampus, amygdala, frontal cortex, striatum, hypothalamus, and cerebellum. A general trend toward decreased levels of all steroids was observed in all AD patients' brain regions compared with controls: PREGS and DHEAS were significantly lower in the striatum and cerebellum, and DHEAS was also significantly reduced in the hypothalamus. A significant negative correlation was found between the levels of cortical beta-amyloid peptides and those of PREGS in the striatum and cerebellum and between the levels of phosphorylated tau proteins and DHEAS in the hypothalamus. This study provides reference values for steroid concentrations determined by gas chromatography-mass spectrometry in various regions of the aged human brain. High levels of key proteins implicated in the formation of plaques and neurofibrillary tangles were correlated with decreased brain levels of PREGS and DHEAS, suggesting a possible neuroprotective role of these neurosteroids in AD.

Determining the functional relationship between Tau phosphorylation and aggregation has proven a challenge owing to the multiple potential phosphorylation sites and their clustering in the Tau sequence. We use here in vitro kinase assays combined with NMR spectroscopy as an analytical tool to generate well-characterized phosphorylated Tau samples and show that the combined phosphorylation at the Ser202/Thr205/Ser208 sites, together with absence of phosphorylation at the Ser262 site, yields a Tau sample that readily forms fibers, as observed by thioflavin T fluorescence and electron microscopy. On the basis of conformational analysis of synthetic phosphorylated peptides, we show that aggregation of the samples correlates with destabilization of the turn-like structure defined by phosphorylation of Ser202/Thr205.

Letter to the Editor this criteria correspond to known functional groups of A Unified Nomenclature System for receptors. This procedure yields six subfamilies. All the the Nuclear Receptor Superfamily unusual receptors that contain only one of the two conserved domains (C or E) were grouped into a separate subfamily (subfamily 0) irrespective of their evolutionary Nuclear hormone receptors (NRs) are important tranorigin. Within subfamilies, groups of receptors are descriptional regulators involved in widely diverse physiofined as the most internal branches with bootstrap vallogical functions such as control of embryonic developues above 90%. In this nomenclature system, the numment, cell differentiation, and homeostasis (Gronemeyer ber of a given receptor inside a group does not carry and Laudet, 1995; Mangelsdorf et al., 1995). In addition, any specific information. In many cases these groups these molecules are extremely important in medical recontain arthropod and vertebrate members. The various search since a large number of them are implicated in homologs of the same gene in invertebrates (e.g., Drodiseases such as cancer, diabetes, or hormone resissophila and Caenorhabditis) have the same name. It is tance syndromes. Some of the NRs act as ligand-inducible transcription factors, while a large number of them have no defined ligand and are hence described as "orphan" receptors (Enmark and Gustafsson, 1996). Over the last decade, workers in the field have described more than 300 sequences of NRs using an increasingly complex and baroque nomenclature. The existence of several names for the same gene is an acute problem for the orphan receptors, which often cannot be described by their function, particularly at the moment of their discovery. As discussed during the Seventh International CBT Symposium on "Nuclear Orphan Receptors" in Huddinge, Sweden (September 9-12, 1995), this plethora of names has become more and more confusing and now constitutes a barrier for understanding of newly acquired knowledge to researchers outside as well as within the field. For that reason, four of us (V. L., J. A., J.-A. G., and W. W.) agreed to form a committee for the nomenclature of NRs. It is the purpose of this paper to recommend names for the subfamilies and groups of receptors based on a phylogenetic tree connecting all known NR sequences. This system, based on the evolution of the two well-conserved domains of NRs (the DNA-binding C domain and the ligand-binding E domain), offers a practical and significant framework to which subsequent genes can be easily added. The resulting nomenclature has now been endorsed by over 40 scientists 1 many of whom contributed to defining the nomenclature and to preparing this letter. This nomenclature has been discussed with the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR). A subcommittee of NC-IUPHAR entitled "Nuclear Receptors" will be set up to further clarify receptor nomenclature to integrate structure and function.A summa...

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