Chronic administration of XBD173 ameliorates cognitive deficits and neuropathology via 18 kDa translocator protein (TSPO) in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the accumulation of ß-amyloid peptide (Aß). It affects cognition and leads to memory impairment. The mitochondrial translocator protein (TSPO) plays an essential role in maintaining mitochondrial homeostasis and has been implicated in several neuronal disorders or neuronal injuries. Ligands targeting the mitochondrial translocator protein (18 kDa), promote neurosteroidogenesis and may be neuroprotective. To study whether the TSPO ligand XBD173 may exert early neuroprotective effects in AD pathology we investigated the impact of XBD173 on amyloid toxicity and neuroplasticity in mouse models of AD. We show that XBD173 (emapunil), via neurosteroid-mediated signaling and delta subunit-containing GABAA receptors, prevents the neurotoxic effect of Aß on long-term potentiation (CA1-LTP) in the hippocampus and prevents the loss of spines. Chronic but not acute administration of XBD173 ameliorates spatial learning deficits in transgenic AD mice with arctic mutation (ArcAß). The heterozygous TSPO-knockout crossed with the transgenic arctic mutation model of AD mice (het TSPOKO X ArcAß) treated with XBD173 does not show this improvement in spatial learning suggesting TSPO is needed for procognitive effects of XBD173. The neuroprotective profile of XBD173 in AD pathology is further supported by a reduction in plaques and soluble Aß levels in the cortex, increased synthesis of neurosteroids, rescued spine density, reduction of complement protein C1q deposits, and reduced astrocytic phagocytosis of functional synapses both in the hippocampus and cortex. Our findings suggest that XBD173 may exert therapeutic effects via TSPO in a mouse model of AD.

Disease stage-dependent changes in brain levels and neuroprotective effects of neuroactive steroids in Parkinson's disease.

Neuroactive steroids are known neuroprotective agents and neurotransmitter regulators. We previously found that expression of the enzymes synthesizing 5α-dihydroprogesterone (5α-DHP), allopregnanolone (ALLO), and dehydroepiandrosterone sulfate (DHEAS) were reduced in the substantia nigra (SN) of Parkinson's Disease (PD) brain. Here, concentrations of a comprehensive panel of steroids were measured in human post-mortem brains of PD patients and controls. Gas chromatography-mass spectrometry (GC/MS) was used to measure steroid levels in SN (involved in early symptoms) and prefrontal cortex (PFC) (involved later in the disease) of five control (CTR) and nine PD donors, divided into two groups: PD4 (PD-Braak stages 1-4) and PD6 (PD-Braak stages 5-6). In SN, ALLO was increased in PD4 compared to CTR and 5α-DHP and ALLO levels were diminished in PD6 compared to PD4. The ALLO metabolite 3α5α20α-hexahydroprogesterone (3α5α20α-HHP) was higher in PD4 compared to CTR. In PFC, 3α5α20α-HHP was higher in PD4 compared to both CTR and PD6. The effects of 5α-DHP, ALLO and DHEAS were tested on human post-mortem brain slices of patients and controls in culture. RNA expression of genes involved in neuroprotection, neuroinflammation and neurotransmission was analysed after 5 days of incubation with each steroid. In PD6 slices, both 5α-DHP and ALLO induced an increase of the glutamate reuptake effector GLAST1, while 5α-DHP also increased gene expression of the neuroprotective TGFB. In CTR slices, ALLO caused reduced expression of IGF1 and GLS, while DHEAS reduced the expression of p75 and the anti-apoptotic molecule APAF1. Together these data suggest that a potentially protective upregulation of ALLO occurs at early stages of PD, followed by a downregulation of progesterone metabolites at later stages that may exacerbate the pathological changes, especially in SN. Neuroprotective effects of neurosteroids are thus dependent on the neuropathological stage of the disease.

The Comprehensive Steroidome in Complete TSPO/PBR Knockout Mice under Basal Conditions.

The 18 kDa translocator protein (TSPO/PBR) is a multifunctional evolutionary highly conserved outer mitochondrial membrane protein. Decades of research has reported an obligatory role of TSPO/PBR in both mitochondrial cholesterol transport and, thus, steroid production. However, the strict dependency of steroidogenesis on TSPO/PBR has remained controversial. The aim of this study was to provide insight into the steroid profile in complete C57BL/6-Tspotm1GuWu(GuwiyangWurra)-knockout male mice (TSPO-KO) under basal conditions. The steroidome in the brain, adrenal glands, testes and plasma was measured by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). We found that steroids present in wild-type (WT) mice were also detected in TSPO-KO mice, including pregnenolone (PREG), progestogens, mineralo-glucocorticosteroids and androgens. The concentrations of PREG and most metabolites were similar between genotypes, except a significant decrease in the levels of the 5α-reduced metabolites of progesterone (PROG) in adrenal glands and plasma and of the 5α-reduced metabolites of corticosterone (B) in plasma in TSPO-KO compared to WT animals, suggesting other regulatory functions for the TSPO/PBR. The expression levels of the voltage-dependent anion-selective channel (VDAC-1), CYP11A1 and 5α-reductase were not significantly different between both groups. Thus, the complete deletion of the tspo gene in male mice does not impair de novo steroidogenesis in vivo.

Aging Is Associated With Lower Neuroactive Steroids and Worsened Outcomes Following Cerebral Ischemia in Male Mice.

Ischemic stroke is a leading cause of disability and death, and aging is the main nonmodifiable risk factor. Following ischemia, neuroactive steroids have been shown to play a key role in cerebroprotection. Thus, brain steroid concentrations at the time of injury as well as their regulation after stroke are key factors to consider. Here, we investigated the effects of age and cerebral ischemia on steroid levels, behavioral outcomes, and neuronal degeneration in 3- and 18-month-old C57BL/6JRj male mice. Ischemia was induced by middle cerebral artery occlusion for 1 hour followed by reperfusion (MCAO/R) and analyses were performed at 6 hours after MCAO. Extended steroid profiles established by gas chromatography coupled with tandem mass spectrometry revealed that (1) brain and plasma concentrations of the main 5α-reduced metabolites of progesterone, 11-deoxycorticosterone, and corticosterone were lower in old than in young mice; (2) after MCAO/R, brain concentrations of progesterone, 5α-dihydroprogesterone, and corticosterone increased in young mice; and (3) after MCAO/R, brain concentrations of 5α-reduced metabolites of progesterone, 3α5α-tetrahydrodeoxycorticosterone, and 3ß5α-tetrahydrodeoxycorticosterone were lower in old than in young mice. After ischemia, old mice showed increased sensori-motor deficits and more degenerating neurons in the striatum than young mice. Altogether, these findings strongly suggest that the decreased capacity of old mice to metabolize steroids toward the 5α-reduction pathway comparatively to young mice may contribute to the worsening of their stroke outcomes.

Bisphenol S Alters the Steroidome in the Preovulatory Follicle, Oviduct Fluid and Plasma in Ewes With Contrasted Metabolic Status.

Bisphenol A (BPA), a plasticizer and endocrine disruptor, has been substituted by bisphenol S (BPS), a structural analogue that had already shown adverse effects on granulosa cell steroidogenesis. The objective of this study was to assess the effect of chronic exposure to BPS, a possible endocrine disruptor, on steroid hormones in the ovary, oviduct and plasma using the ewe as a model. Given the interaction between steroidogenesis and the metabolic status, the BPS effect was tested according to two diet groups. Eighty adult ewes were allotted to restricted (R) and well-fed (WF) groups, that were further subdivided into two subgroups. Ewes were exposed to 50 µg BPS/kg/day in their diet (R50 and WF50 groups) or were unexposed controls (R0 and WF0 groups). After at least 3 months of BPS exposure, preovulatory follicular fluid, oviduct fluid and plasma were collected and steroid hormones were analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). A deleterious effect of restricted diet on the volume of oviduct fluid and numbers of pre-ovulatory follicles was observed. Exposure to BPS impaired estradiol concentrations in both follicular and oviduct fluids of well-fed ewes and progesterone, estradiol and estrone concentrations in plasma of restricted ewes. In addition, a significant interaction between metabolic status and BPS exposure was observed for seven steroids, including estradiol. In conclusion, BPS acts in ewes as an endocrine disruptor with differential actions according to metabolic status.

Pregnane steroidogenesis is altered by HIV-1 Tat and morphine: Physiological allopregnanolone is protective against neurotoxic and psychomotor effects.

Pregnane steroids, particularly allopregnanolone (AlloP), are neuroprotective in response to central insult. While unexplored in vivo, AlloP may confer protection against the neurological dysfunction associated with human immunodeficiency virus type 1 (HIV-1). The HIV-1 regulatory protein, trans-activator of transcription (Tat), is neurotoxic and its expression in mice increases anxiety-like behavior; an effect that can be ameliorated by progesterone, but not when 5α-reduction is blocked. Given that Tat's neurotoxic effects involve mitochondrial dysfunction and can be worsened with opioid exposure, we hypothesized that Tat and/or combined morphine would perturb steroidogenesis in mice, promoting neuronal death, and that exogenous AlloP would rescue these effects. Like other models of neural injury, conditionally inducing HIV-1 Tat in transgenic mice significantly increased the central synthesis of pregnenolone and progesterone's 5α-reduced metabolites, including AlloP, while decreasing central deoxycorticosterone (independent of changes in plasma). Morphine significantly increased brain and plasma concentrations of several steroids (including progesterone, deoxycorticosterone, corticosterone, and their metabolites) likely via activation of the hypothalamic-pituitary-adrenal stress axis. Tat, but not morphine, caused glucocorticoid resistance in primary splenocytes. In neurons, Tat depolarized mitochondrial membrane potential and increased cell death. Physiological concentrations of AlloP (0.1, 1, or 10 nM) reversed these effects. High-concentration AlloP (100 nM) was neurotoxic in combination with morphine. Tat induction in transgenic mice potentiated the psychomotor effects of acute morphine, while exogenous AlloP (1.0 mg/kg, but not 0.5 mg/kg) was ameliorative. Data demonstrate that steroidogenesis is altered by HIV-1 Tat or morphine and that physiological AlloP attenuates resulting neurotoxic and psychomotor effects.

Dose-dependent and long-term cerebroprotective effects of intranasal delivery of progesterone after ischemic stroke in male mice.

Intranasal administration is emerging as a very promising route to deliver therapeutics to the brain. We have recently shown that the intranasal delivery of progesterone at 8 mg/kg is neuroprotective after stroke in male mice. To explore the translational potential of intranasal progesterone treatment, we performed a dose-response study and analyzed outcomes at 48 h after middle cerebral artery occlusion (MCAO). The effects on functional outcomes at long-term were examined by using the optimal dose. In the first experiment, male C57BL/6JRj mice were treated with progesterone at 8, 16 or 24 mg/kg, or with placebo at 1, 6 and 24 h post-MCAO. Our results show that the dose of 8 mg/kg was optimal in counteracting the early histopathological impairments as well as in improving functional recovery. Steroid profiling in plasma showed that the dose of 8 mg/kg is the one that leads to sustained high levels of progesterone and its neuroactive metabolites. In the second experiment, the dose of 8 mg/kg was used and analyzes were performed at 2, 7 and 21 days post-MCAO. Progesterone increased survival, glycemia and body weight. Furthermore, progesterone decreased neurological deficits and improved performances of mice on the rotarod and pole as early as 2 days and up to 21 days post-MCAO. These findings show that intranasal administration of progesterone has a significant translational potential as a cerebroprotective treatment after stroke that can be effective to reduce mortality, to limit tissue and cell damage at the acute phase; and to confer a long-term functional recovery.

Steroid profiles in quail brain and serum: Sex and regional differences and effects of castration with steroid replacement.

Both systemic and local production contribute to the concentration of steroids measured in the brain. This idea was originally based on rodent studies and was later extended to other species, including humans and birds. In quail, a widely used model in behavioural neuroendocrinology, it was demonstrated that all enzymes needed to produce sex steroids from cholesterol are expressed and active in the brain, although the actual concentrations of steroids produced were never investigated. We carried out a steroid profiling in multiple brain regions and serum of sexually mature male and female quail by gas chromatography coupled with mass spectrometry. The concentrations of some steroids (eg, corticosterone, progesterone and testosterone) were in equilibrium between the brain and periphery, whereas other steroids (eg, pregnenolone (PREG), 5α/ß-dihydroprogesterone and oestrogens) were more concentrated in the brain. In the brain regions investigated, PREG sulphate, progesterone and oestrogen concentrations were higher in the hypothalamus-preoptic area. Progesterone and its metabolites were more concentrated in the female than the male brain, whereas testosterone, its metabolites and dehydroepiandrosterone were more concentrated in males, suggesting that sex steroids present in quail brain mainly depend on their specific steroidogenic pathways in the ovaries and testes. However, the results of castration experiments suggested that sex steroids could also be produced in the brain independently of the peripheral source. Treatment with testosterone or oestradiol restored the concentrations of most androgens or oestrogens, respectively, although penetration of oestradiol in the brain appeared to be more limited. These studies illustrate the complex interaction between local brain synthesis and the supply from the periphery for the steroids present in the brain that are either directly active or represent the substrate of centrally located enzymes.

Abnormal steroidogenesis and aromatase activity in preeclampsia.

INTRODUCTION: Estrogens and progesterone play critical roles in angiogenesis and vasodilation. Moreover, placental aromatase deficiency is detected in women with preeclampsia (PE) at delivery. We hypothesized that abnormal steroidogenesis occurs much earlier than typical PE diagnosis. Thus, we investigated whether the circulating steroid profile was already disturbed at 24-29 weeks of gestation in women with subsequent PE, and compared the profile with that of women with "placental" small gestational age (SGA) without PE. METHODS: We selected nulliparous women (n = 90) from the MOMA trial, including women with PE (n = 25), SGA (n = 25), and controls (NP; n = 40), for plasma steroid profiling by gas chromatography/mass spectrometry and to measure placental growth factor and soluble fms-like tyrosine kinase-1. Placental aromatase expression was evaluated in a new set of women. RESULTS: Compared with that of controls, the women with PE had a significantly lower estrone/androstenedione ratio, and exhibited a decreasing trend for estradiol and estrone levels. Lower estriol levels were observed in the SGA group compared to the NP group. Compared with that of controls, the women with PE and SGA had significantly higher levels of 20α-dihydroprogesterone (20α-DHP) and 20α-DHP/progesterone ratios. Pregnenolone sulfate levels were lower in the PE group than in the NP and SGA groups. Decreased expression of aromatase was observed in the PE group compared to the control group. DISCUSSION: Preeclampsia appears to be characterized by specific steroidogenesis dysregulation long before PE diagnosis, highlighting potential new biomarkers of PE.

Long-lasting masculinizing effects of postnatal androgens on myelin governed by the brain androgen receptor.

The oligodendrocyte density is greater and myelin sheaths are thicker in the adult male mouse brain when compared with females. Here, we show that these sex differences emerge during the first 10 postnatal days, precisely at a stage when a late wave of oligodendrocyte progenitor cells arises and starts differentiating. Androgen levels, analyzed by gas chromatography/tandem-mass spectrometry, were higher in males than in females during this period. Treating male pups with flutamide, an androgen receptor (AR) antagonist, or female pups with 5α-dihydrotestosterone (5α-DHT), revealed the importance of postnatal androgens in masculinizing myelin and their persistent effect into adulthood. A key role of the brain AR in establishing the sexual phenotype of myelin was demonstrated by its conditional deletion. Our results uncover a new persistent effect of postnatal AR signaling, with implications for neurodevelopmental disorders and sex differences in multiple sclerosis.

A Role of Endogenous Progesterone in Stroke Cerebroprotection Revealed by the Neural-Specific Deletion of Its Intracellular Receptors.

Treatment with progesterone protects the male and female brain against damage after middle cerebral artery occlusion (MCAO). However, in both sexes, the brain contains significant amounts of endogenous progesterone. It is not known whether endogenously produced progesterone enhances the resistance of the brain to ischemic insult. Here, we used steroid profiling by gas chromatography-tandem mass spectrometry (GC-MS/MS) for exploring adaptive and sex-specific changes in brain levels of progesterone and its metabolites after MCAO. We show that, in the male mouse brain, progesterone is mainly metabolized via 5α-reduction leading to 5α-dihydroprogesterone (5α-DHP), also a progesterone receptor (PR) agonist ligand in neural cells, then to 3α,5α-tetrahydroprogesterone (3α,5α-THP). In the female mouse brain, levels of 5α-DHP and 3α,5α-THP are lower and levels of 20α-DHP are higher than in males. After MCAO, levels of progesterone and 5α-DHP are upregulated rapidly to pregnancy-like levels in the male but not in the female brain. To assess whether endogenous progesterone and 5α-DHP contribute to the resistance of neural cells to ischemic damage, we inactivated PR selectively in the CNS. Deletion of PR in the brain reduced its resistance to MCAO, resulting in increased infarct volumes and neurological deficits in both sexes. Importantly, endogenous PR ligands continue to protect the brain of aging mice. These results uncover the unexpected importance of endogenous progesterone and its metabolites in cerebroprotection. They also reveal that the female reproductive hormone progesterone is an endogenous cerebroprotective neurosteroid in both sexes.SIGNIFICANCE STATEMENT The brain responds to injury with protective signaling and has a remarkable capacity to protect itself. We show here that, in response to ischemic stroke, levels of progesterone and its neuroactive metabolite 5α-dihydroprogesterone are upregulated rapidly in the male mouse brain but not in the female brain. An important role of endogenous progesterone in cerebroprotection was demonstrated by the conditional inactivation of its receptor in neural cells. These results show the importance of endogenous progesterone, its metabolites, and neural progesterone receptors in acute cerebroprotection after stroke. This new concept could be exploited therapeutically by taking into account the progesterone status of patients and by supplementing and reinforcing endogenous progesterone signaling for attaining its full cerebroprotective potential.

Differential effects of the 18-kDa translocator protein (TSPO) ligand etifoxine on steroidogenesis in rat brain, plasma and steroidogenic glands: Pharmacodynamic studies.

Etifoxine is indicated in humans for treating anxiety. In rodents, besides its anxiolytic-like properties, it has recently shown neuroprotective and neuroregenerative activities. It acts by enhancing GABAA receptor function and by stimulating acute steroid biosynthesis via the activation of the 18-kDa translocator protein. However, the regulatory action of etifoxine on steroid production is not well characterized. In this work, we performed dose-response, acute and chronic time-course experiments on the effects of intraperitoneal injections of etifoxine on steroid levels in adult male rat brain and plasma analyzed by gas chromatography-mass spectrometry. Concentrations of pregnenolone, progesterone and its 5α-reduced metabolites were significantly increased in both tissues in response to 25 and 50mg/kg of etifoxine, as compared with vehicle controls, and reached maximal values at 0.5-1h post-injection. Daily injections of etifoxine (50mg/kg, 15days) kept them increased at day 15. Comparisons between steroidogenic tissues revealed that 1h after 50mg/kg of etifoxine treatment, levels of pregnenolone, progesterone and corticosterone were highest in adrenal glands and markedly increased together with their reduced metabolites. They were also increased by etifoxine in brain and plasma, but not in testis except for corticosterone and its metabolites. In contrast, testosterone level was significantly decreased in testis while with its 5α-reduced metabolites, it was unchanged in brain. Results demonstrate that the modulation of steroid concentrations by etifoxine is dependent on the type of steroid and on the steroidogenic organ. They further suggest that adrenal steroids upregulated by etifoxine make an important contribution to the steroids present in brain. This work provides a precise and complete view of steroids regulated by etifoxine that could be useful in therapeutic research.

Steroid Profiling in Male Wobbler Mouse, a Model of Amyotrophic Lateral Sclerosis.

The Wobbler mouse is an animal model for human motoneuron diseases, especially amyotrophic lateral sclerosis (ALS), used in the investigation of both pathology and therapeutic treatment. ALS is a fatal neurodegenerative disease, characterized by the selective and progressive death of motoneurons, leading to progressive paralysis. Previous limited studies have reported steroidal hormone dysregulation in Wobbler mouse and in ALS patients, suggesting endocrine dysfunctions which may be involved in the pathogenesis of the disease. In this study, we established a steroid profiling in brain, spinal cord, plasma, adrenal glands, and testes in 2-month-old male Wobbler mice and their littermates by gas chromatography coupled to mass spectrometry. Our results show in Wobbler mice the following: 1) a marked up-regulation of corticosterone levels in adrenal glands, plasma, spinal cord regions (cervical, thoracic, lumbar) and brain; 2) a strong decrease in T levels in the testis, plasma, spinal cord, and brain; and 3) increased levels of progesterone and especially of its reduced metabolites 5α-dihydroprogesterone, allopregnanolone, and 20α-dihydroprogesterone in the brain, spinal cord, and adrenal glands. Furthermore, Wobbler mice showed a hypothalamic-pituitary-gonadal hypoactivity. Interestingly, plasma concentrations of corticosterone and T correlate well with their respective levels in cervical spinal cord in both control and Wobbler mice. T down-regulation is probably the consequence of adrenal hyperactivity, and the up-regulation of progesterone and its reduced metabolites may correspond to an endogenous protective mechanism in response to motoneuron degeneration. Our findings suggest that increased levels of corticosterone and decreased levels of T in plasma could be a signature of motoneuron degeneration.

Steroid hormones in bovine oviductal fluid during the estrous cycle.

Ovarian steroid hormones are major regulators of the physiology of the oviduct and reproductive events occurring within the oviduct. To establish a whole steroid profiling of the bovine oviductal fluid (OF) during the estrous cycle, contralateral and ipsilateral (to the corpus luteum or preovulatory follicle) oviducts were classified into four stages of the estrous cycle (n = 18-27 cows per stage): postovulatory (Post-ov), mid-luteal (Mid-lut), late luteal (Late-lut), and preovulatory on the basis of the ovarian morphology and intrafollicular steroid concentrations. Steroids were extracted from pools of 150 to 200 µL OF (three to 10 cows per pool; three to four pools per "stage × side" group), purified, fractioned by high-performance liquid chromatography, and analyzed by gas chromatography coupled with tandem mass spectrometry. The concentrations of progesterone (P4) in ipsilateral OF increased from Post-ov (56.9 ± 13.4 ng/mL) to Mid-lut (120.3 ± 34.3 ng/mL), then decreased from Late-lut (76.7 ± 1.8 ng/mL) to Pre-ov (6.3 ± 1.7 ng/mL), and were four to 16 times higher than in contralateral OF. Most P4 metabolites followed similar patterns of variation. Concentrations of 17beta-estradiol (E2) were significantly higher at Pre-ov (290.5 ± 63.2 pg/mL) compared with all other stages (<118.3 pg/mL), with no difference regarding the side of ovulation. Concentrations of androstenedione displayed a pattern similar to that of E2, whereas other androgens, estrone, and corticoids did not vary between stages or sides. In conclusion, a highly concentrated and fluctuating hormonal environment was evidenced in the bovine OF. These results could be useful to improve media for IVF, embryo development, and culture of oviductal cells.

Intranasal delivery of progesterone after transient ischemic stroke decreases mortality and provides neuroprotection.

Progesterone is a potential neuroprotective agent for cerebral stroke. One of the STAIR's recommendations is to test different routes of delivery of therapeutic agents. Here, we investigated the neuroprotective efficacy of intranasal delivery of progesterone in oleogel. Male mice were subjected to transient middle cerebral occlusion (MCAO) for 1 h. Mice received intranasal or intraperitoneal administrations of progesterone (8 mg/kg) at 1, 6, and 24 h post-MCAO. Plasma and brain levels of steroids were measured by gas chromatography-mass spectrometry 2 and 24 h after the last administration of progesterone. Behavioral and histopathological analyzes were performed at 48 h post-MCAO. For blood-brain barrier (BBB) permeability analysis, mice received one intranasal administration of progesterone or placebo at reperfusion and Evans Blue and sodium fluorescein extravasations were assessed at 4 h post-MCAO. Two hours after its nasal administration, progesterone reached elevated levels in brain and plasma and was bioconverted to its 5α-reduced metabolites and to 20α-dihydroprogesterone. However, brain levels of progesterone and its metabolites were about half those measured after intraperitoneal injections, whereas levels of 11-deoxycorticosterone and corticosterone were 5-times lower. In contrast, after 24 h, higher levels of progesterone were measured in brain and plasma after intranasal than after intraperitoneal delivery. Intranasal progesterone decreased the mortality rate, improved motor functions, reduced infarct, attenuated neuronal loss, and decreased the early BBB disruption. This study demonstrates a good bioavailability, a prolonged absorption and a good neuroprotective efficacy of intranasal delivery of progesterone, thus potentially offering an efficient, safe, non-stressful and very easy mode of administration in stroke patients.

Effect of Sex Differences on Brain Mitochondrial Function and Its Suppression by Ovariectomy and in Aged Mice.

Sex steroids regulate brain function in both normal and pathological states. Mitochondria are an essential target of steroids, as demonstrated by the experimental administration of 17ß-estradiol or progesterone (PROG) to ovariectomized female rodents, but the influence of endogenous sex steroids remains understudied. To address this issue, mitochondrial oxidative stress, the oxidative phosphorylation system, and brain steroid levels were analyzed under 3 different experimental sets of endocrine conditions. The first set was designed to study steroid-mediated sex differences in young male and female mice, intact and after gonadectomy. The second set concerned young female mice at 3 time points of the estrous cycle in order to analyze the influence of transient variations in steroid levels. The third set involved the evaluation of the effects of a permanent decrease in gonadal steroids in aged male and female mice. Our results show that young adult females have lower oxidative stress and a higher reduced nicotinamide adenine dinucleotide (NADH)-linked respiration rate, which is related to a higher pyruvate dehydrogenase complex activity as compared with young adult males. This sex difference did not depend on phases of the estrous cycle, was suppressed by ovariectomy but not by orchidectomy, and no longer existed in aged mice. Concomitant analysis of brain steroids showed that pregnenolone and PROG brain levels were higher in females during the reproductive period than in males and decreased with aging in females. These findings suggest that the major male/female differences in brain pregnenolone and PROG levels may contribute to the sex differences observed in brain mitochondrial function.

Progesterone and nestorone promote myelin regeneration in chronic demyelinating lesions of corpus callosum and cerebral cortex.

Multiple Sclerosis affects mainly women and consists in intermittent or chronic damages to the myelin sheaths, focal inflammation, and axonal degeneration. Current therapies are limited to immunomodulators and antiinflammatory drugs, but there is no efficient treatment for stimulating the endogenous capacity of myelin repair. Progesterone and synthetic progestins have been shown in animal models of demyelination to attenuate myelin loss, reduce clinical symptoms severity, modulate inflammatory responses and partially reverse the age-dependent decline in remyelination. Moreover, progesterone has been demonstrated to promote myelin formation in organotypic cultures of cerebellar slices. In the present study, we show that progesterone and the synthetic 19-nor-progesterone derivative Nestorone® promote the repair of severe chronic demyelinating lesions induced by feeding cuprizone to female mice for up to 12 weeks. Progesterone and Nestorone increase the density of NG2(+) oligodendrocyte progenitor cells and CA II(+) mature oligodendrocytes and enhance the formation of myelin basic protein (MBP)- and proteolipid protein (PLP)-immunoreactive myelin. However, while demyelination in response to cuprizone was less marked in corpus callosum than in cerebral cortex, remyelination appeared earlier in the former. The remyelinating effect of progesterone was progesterone receptor (PR)-dependent, as it was absent in PR-knockout mice. Progesterone and Nestorone also decreased (but did not suppress) neuroinflammatory responses, specifically astrocyte and microglial cell activation. Therefore, some progestogens are promising therapeutic candidates for promoting the regeneration of myelin.

Naturally occurring steroids in Xenopus oocyte during meiotic maturation. Unexpected presence and role of steroid sulfates.

In the ovary, oocytes are surrounded by follicle cells and arrested in prophase of meiosis I. Although steroidogenic activity of follicle cells is involved in oogenesis regulation, clear qualitative and quantitative data about the steroid content of follicles are missing. We measured steroid levels of Xenopus oocytes and follicles by gas chromatography-mass spectrometry. We show that dehydroepiandrosterone sulfate is the main steroid present in oocytes. Lower levels of free steroids are also detected, e.g., androgens, whereas progesterone is almost undetectable. We propose that sulfatation is a protective mechanism against local variations of active steroids that could be deleterious for follicle-enclosed oocytes. Steroid levels were measured after LH stimulation, responsible for the release by follicle cells of a steroid signal triggering oocyte meiosis resumption. Oocyte levels of androgens rise slowly during meiosis re-entry whereas progesterone increases abruptly to micromolar concentration, therefore representing the main physiological mediator of meiosis resumption in Xenopus oocyte.

Age-related Purkinje cell death is steroid dependent: RORα haplo-insufficiency impairs plasma and cerebellar steroids and Purkinje cell survival.

A major problem of ageing is progressive impairment of neuronal function and ultimately cell death. Since sex steroids are neuroprotective, their decrease with age may underlie age-related neuronal degeneration. To test this, we examined Purkinje cell numbers, plasma sex steroids and cerebellar neurosteroid concentrations during normal ageing (wild-type mice, WT), in our model of precocious ageing (Rora(+/sg), heterozygous staggerer mice in which expression of the neuroprotective factor RORα is disrupted) and after long-term hormone insufficiency (WT post-gonadectomy). During normal ageing (WT), circulating sex steroids declined prior to or in parallel with Purkinje cell loss, which began at 18 months of age. Although Purkinje cell death was advanced in WT long-term steroid deficiency, this premature neuronal loss did not begin until 9 months, indicating that vulnerability to sex steroid deficiency is a phenomenon of ageing Purkinje neurons. In precocious ageing (Rora(+/sg)), circulating sex steroids decreased prematurely, in conjunction with marked Purkinje cell death from 9 months. Although Rora(+/sg) Purkinje cells are vulnerable through their RORα haplo-insufficiency, it is only as they age (after 9 months) that sex steroid failure becomes critical. Finally, cerebellar neurosteroids did not decrease with age in either genotype or gender; but were profoundly reduced by 3 months in male Rora(+/sg) cerebella, which may contribute to the fragility of their Purkinje neurons. These data suggest that ageing Purkinje cells are maintained by circulating sex steroids, rather than local neurosteroids, and that in Rora(+/sg) their age-related death is advanced by premature sex steroid loss induced by RORα haplo-insufficiency.

Steroid profiling in preeclamptic women: evidence for aromatase deficiency.

OBJECTIVE: Experimental data have revealed the critical role played by 2-methoxy-estradiol, a metabolite of 17ß-estradiol, in the pathophysiology of preeclampsia. We used gas chromatography/mass spectrometry to measure a whole panel of hormonal steroids in the plasma from women during the third trimester of their pregnancy. STUDY DESIGN: The population study consists of 24 pregnant patients with different outcomes: normal, or complicated by isolated preeclampsia or by severe preeclampsia with Hemolysis Enzyme Liver Low Platelets (HELLP) syndrome. RESULTS: 17ß-estradiol was reduced by 50% in isolated preeclampsia, and by 70% in severe preeclampsia with HELLP syndrome (normal: 8.54 ± 0.9 ng/mL; isolated preeclampsia: 4.65 ± 1.0 ng/mL; severe preeclampsia with HELLP syndrome: 2.64 ± 0.4 ng/mL), as is estrone. Downstream, 2-methoxy-estradiol was decreased only in severe preeclampsia with HELLP syndrome. The concentrations of estrone and 17ß-estradiol precursors were comparable between groups, suggesting that placental aromatase is deficient in preeclampsia. CONCLUSION: The gradual decrease of estrogen levels with increasing severity of preeclampsia suggests an impairment of placental steroidogenesis.