Synaptic activity protects against AD and FTD-like pathology via autophagic-lysosomal degradation.

Changes in synaptic excitability and reduced brain metabolism are among the earliest detectable alterations associated with the development of Alzheimer's disease (AD). Stimulation of synaptic activity has been shown to be protective in models of AD beta-amyloidosis. Remarkably, deep brain stimulation (DBS) provides beneficial effects in AD patients, and represents an important therapeutic approach against AD and other forms of dementia. While several studies have explored the effect of synaptic activation on beta-amyloid, little is known about Tau protein. In this study, we investigated the effect of synaptic stimulation on Tau pathology and synapses in in vivo and in vitro models of AD and frontotemporal dementia (FTD). We found that chronic DBS or chemically induced synaptic stimulation reduced accumulation of pathological forms of Tau and protected synapses, while chronic inhibition of synaptic activity worsened Tau pathology and caused detrimental effects on pre- and post-synaptic markers, suggesting that synapses are affected. Interestingly, degradation via the proteasomal system was not involved in the reduction of pathological Tau during stimulation. In contrast, chronic synaptic activation promoted clearance of Tau oligomers by autophagosomes and lysosomes. Chronic inhibition of synaptic activity resulted in opposite outcomes, with build-up of Tau oligomers in enlarged auto-lysosomes. Our data indicate that synaptic activity counteracts the negative effects of Tau in AD and FTD by acting on autophagy, providing a rationale for therapeutic use of DBS and synaptic stimulation in tauopathies.

Astrocytes and neurosteroids: metabolism of pregnenolone and dehydroepiandrosterone. Regulation by cell density.

The rat central nervous system (CNS) has previously been shown to synthesize pregnenolone (PREG) and convert it to progesterone (PROG) and 7 alpha-hydroxy-PREG (7 alpha-OH PREG). Astrocytes, which participate to the regulation of the CNS function, might be involved in the metabolism of neurosteroids. Purified type 1 astrocytes were obtained from fetal rat forebrain with the use of selective culture conditions and were identified by immunostaining with specific antibodies (GFAP+, A2B5-). They were plated at low, intermediate, or high densities (2.5-5 x 10(5), 1-2 x 10(6), or 4-8 x 10(6) cells/dish, respectively) and maintained for 21 d. They were then incubated with 14C-PREG and 14C-DHEA for 24 h and the steroids extracted from cells and media were analyzed. Most radioactive derivatives were released into incubation media. Two metabolic pathways were mainly observed. PREG and DHEA were oxidized to PROG and androstenedione (ADIONE), respectively, [3 beta-hydroxysteroid-dehydrogenase, delta 5-->4 3-ketosteroid-isomerase (3 beta-HSD) activity], and converted to 7 alpha-OH PREG and 7 alpha-OH DHEA, respectively (7 alpha-hydroxylase activity). After low density plating, the formation of PROG and ADIONE was approximately 10% of incubated radioactivity, tenfold larger than that of 7 alpha-hydroxylated metabolites. In contrast, after high density plating, low levels of PROG and ADIONE were formed, whereas the conversion to either 7 alpha-OH PREG or 7 alpha-OH DHEA was > or = 50%. The results expressed per cell indicated that the 3 beta-HSD activity was almost completely inhibited at high cell density, in contrast to the 7 alpha-hydroxylation which was maintained or increased. The pattern of steroid metabolism was related to cell density at the time of measurement and not to an early commitment of cells: when primary cultures were plated at high density (8 x 10(6) cells/dish), then subcultured after several dilutions (3-, 9-, or 27-fold), the 3 beta-HSD activity was recovered only at low density. Furthermore, when 5 x 10(5) cells were centrifuged and the resulting clusters were plated, 3 beta-HSD activity was decreased, whereas steroid 7 alpha-hydroxylation was enhanced. This implies that cell density per se, but neither cell number nor a diffusible factor(s) is involved in the regulation of steroid metabolism. We conclude that astrocytes in culture metabolize PREG and DHEA, and that the metabolic conversions and, therefore, the related enzymatic activities depend on cell-to-cell contacts.(ABSTRACT TRUNCATED AT 400 WORDS)

Progesterone synthesis and myelin formation by Schwann cells.

Progesterone is shown here to be produced from pregnenolone by Schwann cells in peripheral nerves. After cryolesion of the sciatic nerve in male mice, axons regenerate and become myelinated. Blocking either the local synthesis or the receptor-mediated action of progesterone impaired remyelination. Administration of progesterone or its precursor, pregnenolone, to the lesion site increased the extent of myelin sheath formation. Myelination of axons was also increased when progesterone was added to cultures of rat dorsal root ganglia. These observations indicate a role for locally produced progesterone in myelination, demonstrate that progesterone is not simply a sex steroid, and suggest a new therapeutic approach to promote myelin repair.

DHEA decreases HIF-1alpha accumulation under hypoxia in human pulmonary artery cells: potential role in the treatment of pulmonary arterial hypertension.

Previous work showed that dehydroepiandrosterone (DHEA) prevents and reverses chronic hypoxic pulmonary artery hypertension in rat via targeting smooth muscle cells. In our study, DHEA was tested on human pulmonary arterial smooth muscle cells (HPASMC) to identify its mechanism of action under hypoxia in vitro. We show that DHEA decreased HIF-1alpha accumulation under both "chemical hypoxia" with treatment by the iron chelator deferroxamin and gas hypoxia (1% O2). The mRNA levels of HIF-1alpha were unchanged whether or not DHEA was applied under chemical and gas hypoxia, as compared to controls in normoxia, suggesting a post-transcriptional effect of the steroid. Protein levels of prolyl hydroxylases responsible for HIF-1alpha degradation were not modified by DHEA treatment. In addition, a synthetic derivative of DHEA, 3beta-methyl-Delta5-androsten-17-one (which cannot be metabolized), was as active as DHEA on HIF-1alpha accumulation, as well as testosterone and 17beta-estradiol (E2). In HPASMC cultures under normoxia and both types of hypoxia, DHEA gave rise to Delta5-androstene-3beta,17beta-diol (ADIOL) and DHEA-sulfate (DHEA-S). Neither testosterone, nor E2 were found. In addition, ADIOL, DHEA-S, 7alpha-hydroxy-DHEA and Delta4-androstene-3,17-dione were ineffective on HIF-1alpha accumulation. The effect of DHEA per se reducing HIF-1alpha accumulation may be relevant to reduced hypoxia effects in pulmonary arterial hypertension.

Steroid hormones and neurosteroids in normal and pathological aging of the nervous system.

Without medical progress, dementing diseases such as Alzheimer's disease will become one of the main causes of disability. Preventing or delaying them has thus become a real challenge for biomedical research. Steroids offer interesting therapeutical opportunities for promoting successful aging because of their pleiotropic effects in the nervous system: they regulate main neurotransmitter systems, promote the viability of neurons, play an important role in myelination and influence cognitive processes, in particular learning and memory. Preclinical research has provided evidence that the normally aging nervous system maintains some capacity for regeneration and that age-dependent changes in the nervous system and cognitive dysfunctions can be reversed to some extent by the administration of steroids. The aging nervous system also remains sensitive to the neuroprotective effects of steroids. In contrast to the large number of studies documenting beneficial effects of steroids on the nervous system in young and aged animals, the results from hormone replacement studies in the elderly are so far not conclusive. There is also little information concerning changes of steroid levels in the aging human brain. As steroids present in nervous tissues originate from the endocrine glands (steroid hormones) and from local synthesis (neurosteroids), changes in blood levels of steroids with age do not necessarily reflect changes in their brain levels. There is indeed strong evidence that neurosteroids are also synthesized in human brain and peripheral nerves. The development of a very sensitive and precise method for the analysis of steroids by gas chromatography/mass spectrometry (GC/MS) offers new possibilities for the study of neurosteroids. The concentrations of a range of neurosteroids have recently been measured in various brain regions of aged Alzheimer's disease patients and aged non-demented controls by GC/MS, providing reference values. In Alzheimer's patients, there was a general trend toward lower levels of neurosteroids in different brain regions, and neurosteroid levels were negatively correlated with two biochemical markers of Alzheimer's disease, the phosphorylated tau protein and the beta-amyloid peptides. The metabolism of dehydroepiandrosterone has also been analyzed for the first time in the aging brain from Alzheimer patients and non-demented controls. The conversion of dehydroepiandrosterone to Delta5-androstene-3beta,17beta-diol and to 7alpha-OH-dehydroepiandrosterone occurred in frontal cortex, hippocampus, amygdala, cerebellum and striatum of both Alzheimer's patients and controls. The formation of these metabolites within distinct brain regions negatively correlated with the density of beta-amyloid deposits.

Time-dependent induction of anxiogenic-like effects after central infusion of urocortin or corticotropin-releasing factor in the rat.

RATIONALE: Corticotropin-releasing factor (CRF) and urocortin (Ucn) belong to the CRF-related family, share a high degree of structural homology and bind to CRF receptors. However, compared with CRF, Ucn was shown to display either weaker or similar anxiogenic-like effects in vivo. OBJECTIVE: To compare the anxiogenic-like responses of rats injected intracerebroventricularly (ICV) with different doses of either rat/human CRF (r/hCRF) or rat Ucn (rUcn) at different intervals after injection. METHODS: Rats were tested on three validated paradigms of emotional behavior [i.e. elevated plus-maze (EPM), defensive withdrawal (DW) and conflict test (CT)] 5 and 30 min after treatment. RESULTS: In the EPM test only r/hCRF, but not rUcn, produced anxiogenic-like effects at the dose of 1.0 microg, when the peptides were injected 5 min before testing. At 30 min after injection, both peptides caused a significant reduction of open arms exploration, rUcn being effective at 0.01 microg. In the DW test both peptides were equally potent in decreasing the exploratory behavior and increasing the time spent in the chamber at the dose of 1.0 microg when tested 30 min after injection. In the CT both rUcn (0.25-1.0 microg) and r/hCRF (0.75-1.0 microg) decreased significantly the responding in the punished component. However, rUcn reduced food responding also in the unpunished component possibly due to its powerful anorectic activity. CONCLUSIONS: Comparison of anxiogenic-like activities of r/hCRF and rUcn at doses up to 1.0 microg revealed striking differential effects that depended on the time of testing after ICV peptide injection, and on the paradigm of anxiety used. These results suggest that the onset of r/hCRF and rUcn actions related to behavioral responses to anxiety is likely to depend on brain peptide-specific mechanisms including binding properties to CRF-receptors, differential distribution to specific functional brain sites and the distribution and effectiveness of binding-protein interactions.

Neurosteroids: biosynthesis, metabolism and function of pregnenolone and dehydroepiandrosterone in the brain.

Pregnenolone (P) and dehydroepiandrosterone (D) accumulate in the brain as unconjugated steroids and their sulfate (S) and fatty acid (L) esters. The microsomal acyl-transferase activity is highest in immature (1-3 weeks old) male rats. The immunocytochemical and biochemical evidence for P biosynthesis by differentiated oligodendrocytes is reviewed. The importance of P synthesis for its brain accumulation is assessed by the intracysternal injection of the inhibitor aminoglutethimide. Primary glial cell cultures convert P to 20-OH-P, PL, progesterone, 5 alpha-pregnane-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnane-20-one (Polone). Astroglial cell cultures also produce these metabolites, whereas neurons from 17-day mouse embryos only form 20-OH-P. P and D are converted to the corresponding 7 alpha-hydroxylated metabolites by a very active P-450 enzyme from rat brain microsomes. Several functions of neurosteroids are documented. P decreases in olfactory bulb of intact male rats exposed to the scent of estrous females. D inhibits the aggressive behavior of castrated male mice towards lactating female intruders. The D analog 3 beta-methyl-androst-5-en-17-one, which cannot be metabolized into sex steroids and is not demonstrably androgenic or estrogenic is at least as efficient as D. Both compounds elicit a marked decrease of PS in rat brain. The Cl- conductance of gamma-aminobutyric (GABAA) receptor is stimulated by GABA agonists, an effect which is enhanced by Polone and antagonized by PS. Thus, P metabolites in brain as well as steroids of extraencephalic sources may be involved physiologically in GABAA receptor function. The neurosteroids accumulated in brain may be precursors of sex steroid hormones and progesterone receptors have been localized in glial cells. P and D do not bind to any known intracellular receptor. A heat stable P binding protein has been found in brain cytosol with distinct ligand specificity. A binding component specific for steroids sulfates, including Polone S, DS and PS, in the order of decreasing affinity is localized in adult rat brain synaptosomal membranes. Its relationship to the GABAA receptor is under current investigation.

Neuropeptide Y blocks anxiogenic-like behavioral action of corticotropin-releasing factor in an operant conflict test and elevated plus maze.

Central administration of neuropeptide Y (NPY) produces anxiolytic-like behavioral effects in rat models of anxiety. Because previous evidence has suggested a relationship between NPY and corticotropin-releasing factor (CRF) in the brain, we have focused on the interaction of these neuropeptide systems in emotional responsiveness to stressful stimuli. Intracerebroventricular administration of CRF produced a marked response suppression in an operant incremental shock conflict paradigm. NPY [(1 microg, intracerebroventricularly (i.c.v.)] significantly antagonized the response-suppressing effects of CRF (0.75 microg, i.c.v.) on punished responding in the conflict test at doses that produced little or no behavioral effect when administered alone. Central administration of the CRF antagonist [D-Phe(12), Nle(21,38),C(alpha) MeLeu(37)]CRF (D-Phe CRF(12-41)) alone did not alter punished or unpunished responding in the conflict test. However, pretreatment with the CRF antagonist before a subthreshold dose of NPY (1 microg, i.c.v.) produced a significant potentiation of the release of punished responding relative to NPY alone and untreated controls. NPY also antagonized the "anxiogenic-like" behavioral effects of CRF in the elevated plus maze. These findings support the hypothesis that NPY and CRF may reciprocally modulate an animal's behavioral response to stressful stimuli.

The amygdala mediates the anxiolytic-like effect of the neurosteroid allopregnanolone in rat.

The neurosteroid allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one) possesses clear anxiolytic-like effects. Other neurosteroids namely pregnenolone sulfate (PREG-S) and dehydroepiandrosterone sulfate (DHEA-S) influence anxiety-related behavior differently. In the present study, the implication of the amygdala, a key structure in mechanisms of fear and anxiety, was investigated as a potential neural substrate for the effects of neurosteroids on anxiety-like behavior in rat. Animals implanted with bilateral cannulae aimed at the central nucleus of the amygdala (CeA) and infused with neurosteroids, were tested in two animal models of anxiety. Allopregnanolone (8 microg/side) produced a significant increase in responding suppressed by punishment in the conflict test. In the elevated plus maze, allopregnanolone (8 microg/side) induced a significant increase in the time spent and the number of entries in open arms compared with the vehicle-infused controls. No significant changes in punished and unpunished responding of the conflict test were observed with PREG-S (0.001-8 microg/side) and DHEA-S (2-8 microg/side) administered into the CeA or into the lateral ventricle (1-20 microg). The results reveal the lack of activity of PREG-S and DHEA-S in the operant conflict test, but suggest that the central nucleus of the amygdala is a key region involved in the mechanisms underlying the anxiolytic-like action of allopregnanolone.

Opposing effects of different steroid sulfates on GABAA receptor-mediated chloride uptake.

Steroids with the 3alpha-hydroxy-5alpha- or 5beta-reduced configurations of the A ring interact with the gamma-aminobutyric acid (GABA) type A receptor chloride channel complex and potentiate the stimulation of Cl- uptake by GABA agonists. Conversely, the sulfate esters of 3beta-hydroxy-5-ene neurosteroids pregnenolone and dehydroepiandrosterone behave as inhibitory modulators. In the present work, steroid sulfates were tested for their ability to modulate muscimol-induced chloride ion uptake into cortical synaptoneurosomes. 3alpha-Hydroxy-5alpha-pregnan-20-one sulfate and several other 3alpha-hydroxy-steroid sulfates potentiated, whereas 3beta-hydroxy-steroid sulfates inhibited muscimol effect. It is concluded that GABA-agonistic or antagonistic properties of steroid sulfates depend on the alpha or beta orientation of the sulfate moiety linked to the A ring.

Structural and functional neuropathology in transgenic mice with CNS expression of IFN-alpha.

Cytokines belonging to the type I interferon (e.g. interferon-alpha) family are important in the host response to infection and may have complex and broad ranging actions in the central nervous system (CNS) that may be beneficial or harmful. To better understand the impact of the CNS expression of the type I interferons (IFN), transgenic mice were developed that produce IFN-alpha(1) chronically from astrocytes. In two independent transgenic lines with moderate and low levels of astrocyte IFN-alpha mRNA expression respectively, a spectrum of transgene dose- and age-dependent structural and functional neurological alterations are induced. Structural changes include neurodegeneration with loss of cholinergic neurons, gliosis, angiopathy with mononuclear cell cuffing, progressive calcification affecting basal ganglia and cerebellum and the up-regulation of a number of IFN-alpha-regulated genes. At a functional level, in vivo and in vitro electrophysiological studies revealed impaired neuronal function and disturbed synaptic plasticity with pronounced hippocampal hyperexcitability. Severe behavioral alterations were also evident in higher expressor GFAP-IFNalpha mice which developed fatal seizures around 13 weeks of age precluding their further behavioral assessment. Modest impairments in discrimination learning were measured in lower expressor GFAP-IFNalpha mice at various ages (7-42 weeks). The behavioral and electrophysiological findings suggest regional changes in hippocampal excitability which may be linked to abnormal calcium metabolism and loss of cholinergic neurons in the GIFN mice. Thus, these transgenic mice provide a novel animal model in which to further evaluate the mechanisms that underlie the diverse actions of type I interferons in the intact CNS and to link specific structural changes with functional impairments.

The anxiolytic-like effects of the neurosteroid allopregnanolone: interactions with GABA(A) receptors.

The neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone) was administered systemically to rats which were tested in the Geller-Seifter conflict paradigm, an established animal model of anxiety. Allopregnanolone was found to produce significant anxiolytic-like effects at a dose of 8 mg/kg. When three ligands that function at different sites on the gamma-aminobutyric acid/benzodiazepine receptor-chloride ionophore complex (GABA(A) receptors) were examined in conjunction with allopregnanolone, the anti-conflict effects of allopregnanolone were effectively reversed only by the benzodiazepine receptor inverse agonist RO15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5-alpha]-[1,4]benzodiazepine-3-carboxylate). Since this inverse agonist has been reported to inhibit the GABA(A)-activated chloride flux in neuronal membranes, it is likely that the stimulation of the chloride channel in GABA(A) receptors is an important component of the effects of allopregnanolone. In contrast, the benzodiazepine receptor antagonist flumazenil (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5-alpha]-[1,4]benzodiazepine-3-carboxylate) did not block the anxiolytic-like actions of allopregnanolone, indicating that allopregnanolone does not bind at the benzodiazepine site directly. Isopropylbicyclophosphate, which binds at the picrotoxinin site on the GABA(A) receptors and blocks the behavioral actions of ethanol, also dose-dependently reversed the anti-conflict effect of this neurosteroid. The results suggest that allopregnanolone may be working either at a site specific for the benzodiazepine receptor inverse agonist RO15-4513 or at the picrotoxinin site to produce its potent anxiolytic-like behavioral effects.

[Neurosteroids: behavioral aspects and physiological implications]. / Neurostéroïdes: aspects comportementaux et implications physiologiques.

The term "neurosteroids" applies to those steroids that are both formed in the nervous system from sterol precursors, and accumulate in the nervous system, at least in part, independently of peripheral steroidogenic glands secretion. Neurosteroids that are active on the central nervous system include, mainly, pregnenolone (PREG), dehydroepiandrosterone (DHEA) and their sulfate esters (PREG-S and DHEA-S), as well as the reduced metabolite of progesterone, 3 alpha,5 alpha-TH PROG also called allopregnanolone. These neuroactive neurosteroids alter neuronal excitability by modulating the activity of several neurotransmitter receptors and thus can influence behavior. PREG-S decreases the sleeping time in rats anesthetized with a barbiturate, which is consistent with its antagonist action on the GABAA receptor (GABAA-R). Allopregnanolone is anxiolytic in rats tested in a conflict paradigm, through an interaction at a site specific for the benzodiazepine (BZ) receptor inverse agonist RO15-4513 and/or at the picrotoxinin site on GABAA-R. The contribution of the amygdala, a key region involved in the control of anxiety, is also demonstrated for the anxiolytic action of allopregnanolone. An anti-agressive effect of DHEA can be observed in castrated male mice who become agressive in the presence of lactating females. This inhibition of agressiveness by DHEA is associated to a selective decrease in the brain of PREG-S, which may, in turn, trigger an increase of endogenous GABAergic tone. Finally, cognitive performances of aged rats tested in the Morris water maze and the Y-maze can be correlated with individual concentrations of PREG-S in the hippocampus, i.e. poor performance in both tasks with low levels of PREG-S. Remarkably, the memory deficits are significantly improved, albeit transiently, by an intra-hippocampal injection of PREG-S in impaired aged rats. Promnesiant PREG-S may then reinforce some neurotransmitter systems that can decline with age. This brief review provides evidence of the pharmacology and physiological correlates of neurosteroids involved in behavioral phenomena. However, neurobiological mechanisms of behavioral effects of neurosteroids await further investigation.

7 Alpha-hydroxylation of 27-hydroxycholesterol in rat brain microsomes.

27-hydroxycholesterol is shown to be 7 alpha-hydroxylated by microsomal preparations of rat brain. The apparent Km was about 2 microM and Vmax about 15 pmol/min x mg protein. The reaction might modulate biological functions of 27-hydroxycholesterol, such as its suppressive effect on the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and supports the possibility of bile acid formation in the brain.

Neurosteroid metabolism. 7 alpha-Hydroxylation of dehydroepiandrosterone and pregnenolone by rat brain microsomes.

Two 'neurosteroids', dehydroepiandrosterone (DHEA) and pregnenolone (PREG), are converted by rat brain microsomes into polar metabolites, identified as the respective 7 alpha-hydroxylated (7 alpha-OH) derivatives by the 'twin ion' technique of g.l.c.-m.s. with deuterated substrates. The enzymic reaction requires NADPH and is stimulated 2-4-fold by EDTA. Under optimal conditions (pH 7.4, 0.5 mM-NADPH, 1 mM-EDTA), the Km values for DHEA and PREG are 13.8 and 4.4 microM respectively, and the Vmax. values are 322 and 38.8 pmol/min per mg of microsomal protein respectively. Trace amounts of putative 7 beta-OH derivatives of DHEA and PREG are detected. Oestradiol, at a pharmacological concentration of 5 microM, inhibits DHEA and PREG 7 alpha-hydroxylation. Formation of 7 alpha-hydroxylated metabolites is low in prepubertal rats and increases 5-fold in adults. Derivatives of PREG and DHEA, such as PREG sulphate, DHEA sulphate, progesterone and 3 alpha-hydroxy-5 alpha-pregnan-20-one, are known to be neuroactive. Therefore the quantitatively important metabolism to 7 alpha-OH compounds may contribute to the control of neurosteroid activity in brain.

Metabolism of 27-, 25- and 24-hydroxycholesterol in rat glial cells and neurons.

The metabolism of 27-, 25- and 24-hydroxycholesterol in cultures of rat astrocytes, Schwann cells and neurons was studied. 27- and 25-Hydroxycholesterol, but not 24-hydroxycholesterol, underwent 7 alpha-hydroxylation with subsequent oxidation to 7 alpha-hydroxy-3-oxo-delta 4 steroids in all three cell types. When cells were incubated for 24 h with 0.28 nmol of 27-hydroxycholesterol in 10 ml of medium, the rates of conversion into 7 alpha-hydroxylated metabolites were 0.21, 0.12 and 0.02 nmol/24 h per 10(6) cells in the media of astrocytes, Schwann cells and neurons respectively. The corresponding values for 25-hydroxycholesterol were 0.26, 0.16 and 0.04. A minor fraction of 27-hydroxycholesterol and its 7 alpha-hydroxylated metabolites was oxidized to 3 beta-hydroxy-5-cholestenoic acid. 3 beta, 7 alpha-dihydroxy-5-cholestenoic acid and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid. In addition to the two hydroxycholesterols, other 3 beta-hydroxy-delta 4 steroids, dehydro-epiandrosterone, pregnenolone, 3 beta-hydroxy-5-cholestenoic acid and 3 beta-hydroxy-5-cholenoic acid underwent 7 alpha-hydroxylation. Competitive experiments did not distinguish between the presence of one or several 7 alpha-hydroxylases. In astrocyte incubations, 27-hydroxycholesterol also underwent 25-hydroxylation, and 12% of its metabolites carried a 25-hydroxy group. 25-Hydroxylation of added 24-hydroxycholesterol was also observed in the astrocyte incubations, as was the formation of 7 alpha, 25-dihydroxy-4-cholesten-3-one, 25-hydroxycholesterol and 7 alpha, 25-dihydroxycholesterol from endogenous precursor(s). Our study indicates that side-chain oxygenated cholesterol can undergo metabolic transformations that may be of importance for cholesterol homoeostasis in the brain.

The synthetic enantiomer of pregnenolone sulfate is very active on memory in rats and mice, even more so than its physiological neurosteroid counterpart: distinct mechanisms?

The demonstration that the neurosteroid pregnenolone sulfate (PREGS) is active on memory function at both the physiological and pharmacological levels led to us examining in detail the effects of the steroid on spatial working memory by using a two-trial recognition task in a Y-maze, a paradigm based on the natural drive in rodents to explore a novel environment. Dose-response studies in young male adult Sprague-Dawley rats and Swiss mice, after the postacquisition intracerebroventricular injection of steroid, showed an U-inverted curve for memory performance and indicated a greater responsiveness in rats compared with mice. Remarkably, the synthetic (-) enantiomer of PREGS not only also displayed promnesiant activity, but its potency was 10 times higher than that of the natural steroid. Intracerebroventricular coadministration experiments with DL-2-amino-5-phosphonovaleric acid, a competitive selective antagonist of the N-methyl-D-aspartate receptor, abolished the memory-enhancing effect of PREGS, but not that of the PREGS enantiomer, evoking enantiomeric selectivity at the N-methyl-d-aspartate receptor and/or different mechanisms for the promnestic function of the two enantiomers.

A progesterone metabolite stimulates the release of gonadotropin-releasing hormone from GT1-1 hypothalamic neurons via the gamma-aminobutyric acid type A receptor.

The reduced progesterone metabolite tetrahydroprogesterone (3 alpha-hydroxy-5 alpha-pregnan-20-one; 3 alpha,5 alpha-THP) is a positive modulator of the gamma-aminobutyric acid type A (GABAA) receptor. Experiments performed in vitro with hypothalamic fragments have previously shown that GABA could modulate the release of gonadotropin-releasing hormone (GnRH). Using GT1-1 immortalized GnRH neurons, we investigated the role of GABAA receptor ligands, including 3 alpha,5 alpha-THP, on the release of GnRH. We first characterized the GABAA receptors expressed by these neurons. [3H]Muscimol, but not [3H]flunitrazepam, bound with high affinity to GT1-1 cell membranes (Kd = 10.9 +/- 0.3 nM; Bmax = 979 +/- 12 fmol/mg of protein), and [3H]muscimol binding was enhanced by 3 alpha,5 alpha-THP. mRNAs encoding the alpha 1 and beta 3 subunits of the GABAA receptor were detected by the reverse transcriptase polymerase chain reaction. In agreement with binding data, the benzodiazepine-binding gamma subunit mRNA was absent. GnRH release studies showed a dose-related stimulating action of muscimol. 3 alpha,5 alpha-THP not only modulated muscimol-induced secretion but also stimulated GnRH release when administered alone. Bicuculline and picrotoxin blocked the effects of 3 alpha,5 alpha-THP and muscimol. Finally, we observed that GT1-1 neurons convert progesterone to 3 alpha,5 alpha-THP. We propose that progesterone may increase the release of GnRH by a membrane mechanism, via its reduced metabolite 3 alpha,5 alpha-THP acting at the GABAA receptor.

Progesterone as a neurosteroid: actions within the nervous system.

1. Some progesterone is synthesized within both the central and the peripheral nervous systems, where it regulates neurotransmission and important glial functions, such as the formation of myelin. Progesterone can thus be designated a "neurosteroid." 2. Steroids act not only on the brain, but also on peripheral nerves, which offer many advantages to study the biological significance of locally produced neurosteroids: their remarkable plasticity and regenerative capacity and their relatively simple structure. 3. By using the regenerating mouse sciatic nerve as a model, we have shown that progesterone synthesized by rat Schwann cells promotes the formation of new myelin sheaths. Progesterone also increases the number of myelinated axons when added at a low concentration to cocultures of Schwann cells and sensory neurons. 4. These findings show a function on myelination for locally produced progesterone and suggest a new pharmacological approach of myelin repair.

Transgenic models to study the actions of cytokines in the central nervous system.

To better understand the actions of cytokines in the mammalian central nervous system (CNS), we have developed transgenic mice in which the expression of various cytokines including interleukin (IL)-3, IL-6, IL-12, interferon-alpha or tumor necrosis factor-alpha was targeted to astrocytes under the transcriptional control of the glial fibrillary acidic protein (GFAP) promoter. Transgenic lines displaying low astrocyte expression of the respective cytokine were developed and characterized. The findings indicate that expression of these different cytokines in the intact CNS produces divergent inflammatory responses which are associated with the development of wide-ranging and progressive molecular, cellular and functional CNS impairments. These transgenic mice provide a powerful tool which we are now exploiting further to define novel mechanisms that might underlie the individual cytokine-driven neuroinflammatory responses. To date the results clearly show there are distinct model-associated patterns of cerebral expression of key molecules involved in the inflammatory response including the cellular adhesion molecules, chemokines, major histocompatibility complex molecules and the matrix metalloproteinases. In conclusion, these GFAP-cytokine transgenic mice highlight the potent and diverse array of actions mediated by cytokines when expressed in the CNS and provide a valuable resource to further our knowledge of the mechanisms by which cytokines exert their effects.