Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease of the central nervous system. The manifestation of MS is related to steroid changes during the menstrual cycle and pregnancy. As data focusing on the effect of anti-MS drug treatment on steroidome are scarce, we evaluated steroidomic changes (79 steroids) in 61 female MS patients of reproductive age 39 (29, 47) years (median with quartiles) after treatment with anti-MS drugs on the GC-MS/MS platform and immunoassays (cortisol and estradiol). The changes were assessed using steroid levels and steroid molar ratios (SMRs) that may reflect the activities of steroidogenic enzymes (SMRs). A repeated measures ANOVA, followed by multiple comparisons and OPLS models, were used for statistical analyses. The anti-MS treatment decreased steroid levels in the follicular phase. Anti-CD20 monoclonal antibodies (mAb), such as ofatumumab and ocrelizumab; inhibitors of the sphingosine-1-phosphate receptor (S1PRI); and IFNβ-1a decreased circulating 17-hydroxy-pregnanes and shifted the CYP17A1 functioning from the hydroxylase- toward the lyase step. Decreased conjugated/unconjugated steroid ratios were found after treatment with anti-MS drugs, especially for glatiramer acetate and anti-CD20 mAb. In the luteal phase, IFN-β1a treatment increased steroidogenesis; both IFN-β1a and ocrelizumab increased AKR1D1, and S1PRI increased SRD5A functioning. Anti-CD20 mAb reduced the functioning of enzymes catalyzing the synthesis of immunomodulatory 7α/β and 16α-hydroxy-androgens, which may affect the severity of MS. The above findings may be important concerning the alterations in bioactive steroids, such as cortisol; active androgens and estrogens; and neuroactive, neuroprotective, and immunomodulatory steroids in terms of optimization of anti-MS treatment.

• Keywords
• GC-MS/MS, anti-MS drugs, multiple sclerosis, multivariate statistics, steroidomics,
• MeSH
• Adult MeSH
• Glatiramer Acetate therapeutic use   MeSH
• Middle Aged MeSH
• Humans MeSH
• Menstrual Cycle drug effects   MeSH
• Multiple Sclerosis * drug therapy   metabolism   blood   MeSH
• Steroids * metabolism   blood   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Glatiramer Acetate MeSH
• Steroids * MeSH

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) mainly afflicting young women. Various steroids can influence the onset and development of the disease or, on the contrary, mitigate its course; however, a systematic review of steroidomic changes in MS patients is lacking. Based on the gas chromatography tandem mass spectrometry (GC-MS/MS) platform and, in the case of estradiol, also using immunoassay, this study performed a comprehensive steroidomic analysis in 25 female MS patients aged 39(32, 49) years compared to 15 female age-matched controls aged 38(31, 46) years. A significant trend towards higher ratios of conjugated steroids to their unconjugated counterparts was found in patients, which is of particular interest in terms of the balance between excitatory and inhibitory steroid modulators of ionotropic receptors. Patients showed altered metabolic pathway to cortisol with decreased conversion of pregnenolone to 17-hydroxypregnenolone and 17-hydroxypregnenolone to 17-hydroxyprogesterone and increased conversion of 17-hydroxypregnenolone to dehydroepiandrosterone (DHEA), resulting in lower levels of 17-hydroxyprogesterone, as well as indications of impaired conversion of 11-deoxy-steroids to 11β-hydroxy-steroids but reduced conversion of cortisol to cortisone. Due to over-activation of hypothalamic-pituitary-adrenal axis (HPAA), however, cortisol and cortisone levels were higher in patients with indications of depleted cortisol synthesizing enzymes. Patients showed lower conversion of DHEA to androstenedione, androstenedione to testosterone, androstenedione to estradiol in the major pathway, and testosterone to estradiol in the minor pathway for estradiol synthesis at increased conversion of androstenedione to testosterone. They also showed lower conversion of immunoprotective Δ5 androstanes to their more potent 7α/β-hydroxy metabolites and had lower circulating allopregnanolone and higher ratio 3β-hydroxy-steroids to their neuroprotective 3α-hydroxy-counterparts.

• Keywords
• GC-MS/MS, differential diagnostics, multiple sclerosis, multivariate statistics, steroidomics,
• MeSH
• Adult MeSH
• Hydrocortisone metabolism   blood   MeSH
• Middle Aged MeSH
• Humans MeSH
• Gas Chromatography-Mass Spectrometry MeSH
• Multiple Sclerosis * metabolism   blood   MeSH
• Steroids metabolism   MeSH
• Case-Control Studies MeSH
• Tandem Mass Spectrometry MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Hydrocortisone MeSH
• Steroids MeSH

Pregnanolone glutamate (PA-G) is a neuroactive steroid that has been previously demonstrated to be a potent neuroprotective compound in several biological models in vivo. Our in vitro experiments identified PA-G as an inhibitor of N-methyl-D-aspartate receptors and a potentiator of γ-aminobutyric acid receptors (GABAARs). In this study, we addressed the hypothesis that combined GABAAR potentiation and NMDAR antagonism could afford a potent anticonvulsant effect. Our results demonstrated the strong age-related anticonvulsive effect of PA-G in a model of pentylenetetrazol-induced seizures. PA-G significantly decreased seizure severity in 12-day-old animals, but only after the highest dose in 25-day-old animals. Interestingly, the anticonvulsant effect of PA-G differed both qualitatively and quantitatively from that of zuranolone, an investigational neurosteroid acting as a potent positive allosteric modulator of GABAARs. Next, we identified 17-hydroxy-pregnanolone (17-OH-PA) as a major metabolite of PA-G in 12-day-old animals. Finally, the administration of PA-G demonstrated direct modulation of unexpected neurosteroid levels, namely pregnenolone and dehydroepiandrosterone sulfate. These results suggest that compound PA-G might be a pro-drug of 17-OH-PA, a neurosteroid with a promising neuroprotective effect with an unknown mechanism of action that may represent an attractive target for studying perinatal neural diseases.

• Keywords
• GABA, NMDA, anticonvulsant, metabolomics, neurosteroids, zuranolone,
• Publication type
• Journal Article MeSH

BACKGROUND AND PURPOSE: Neurosteroids influence neuronal function and have multiple promising clinical applications. Direct modulation of postsynaptic neurotransmitter receptors by neurosteroids is well characterized, but presynaptic effects remain poorly understood. Here, we report presynaptic glutamate release potentiation by neurosteroids pregnanolone and pregnanolone sulfate and compare their mechanisms of action to phorbol 12,13-dibutyrate (PDBu), a mimic of the second messenger DAG. EXPERIMENTAL APPROACH: We use whole-cell patch-clamp electrophysiology and pharmacology in rat hippocampal microisland cultures and total internal reflection fluorescence (TIRF) microscopy in HEK293 cells expressing GFP-tagged vesicle priming protein Munc13-1, to explore the mechanisms of neurosteroid presynaptic modulation. KEY RESULTS: Pregnanolone sulfate and pregnanolone potentiate glutamate release downstream of presynaptic Ca2+ influx, resembling the action of a phorbol ester PDBu. PDBu partially occludes the effect of pregnanolone, but not of pregnanolone sulfate. Calphostin C, an inhibitor that disrupts DAG binding to its targets, reduces the effect PDBu and pregnanolone, but not of pregnanolone sulfate, suggesting that pregnanolone might interact with a well-known DAG/phorbol ester target Munc13-1. However, TIRF microscopy experiments found no evidence of pregnanolone-induced membrane translocation of GFP-tagged Munc13-1, suggesting that pregnanolone may regulate Munc13-1 indirectly or interact with other DAG targets. CONCLUSION AND IMPLICATIONS: We describe a novel presynaptic effect of neurosteroids pregnanolone and pregnanolone sulfate to potentiate glutamate release downstream of presynaptic Ca2+ influx. The mechanism of action of pregnanolone, but not of pregnanolone sulfate, partly overlaps with that of PDBu. Presynaptic effects of neurosteroids may contribute to their therapeutic potential in the treatment of disorders of the glutamate system.

• Keywords
• Munc13-1, glutamate, neurosteroid, phorbol ester, pregnanolone, presynaptic,
• MeSH
• HEK293 Cells MeSH
• Rats MeSH
• Glutamic Acid MeSH
• Humans MeSH
• Neurosteroids * MeSH
• Pregnanolone * pharmacology   MeSH
• Sulfates MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glutamic Acid MeSH
• Neurosteroids * MeSH
• Pregnanolone * MeSH
• Sulfates MeSH

NMDARs are ligand-gated ion channels that cause an influx of Na+ and Ca2+ into postsynaptic neurons. The resulting intracellular Ca2+ transient triggers synaptic plasticity. When prolonged, it may induce excitotoxicity, but it may also activate negative feedback to control the activity of NMDARs. Here, we report that a transient rise in intracellular Ca2+ (Ca2+ challenge) increases the sensitivity of NMDARs but not AMPARs/kainate receptors to the endogenous inhibitory neurosteroid 20-oxo-5β-pregnan-3α-yl 3-sulfate and to its synthetic analogs, such as 20-oxo-5β-pregnan-3α-yl 3-hemipimelate (PAhPim). In cultured hippocampal neurons, 30 μm PAhPim had virtually no effect on NMDAR responses; however, following the Ca2+ challenge, it inhibited the responses by 62%; similarly, the Ca2+ challenge induced a 3.7-fold decrease in the steroid IC50 on recombinant GluN1/GluN2B receptors. The increase in the NMDAR sensitivity to PAhPim was dependent on three cysteines (C849, C854, and C871) located in the carboxy-terminal domain of the GluN2B subunit, previously identified to be palmitoylated (Hayashi et al., 2009). Our experiments suggested that the Ca2+ challenge induced receptor depalmitoylation, and single-channel analysis revealed that this was accompanied by a 55% reduction in the probability of channel opening. Results of in silico modeling indicate that receptor palmitoylation promotes anchoring of the GluN2B subunit carboxy-terminal domain to the plasma membrane and facilitates channel opening. Depalmitoylation-induced changes in the NMDAR pharmacology explain the neuroprotective effect of PAhPim on NMDA-induced excitotoxicity. We propose that palmitoylation-dependent changes in the NMDAR sensitivity to steroids serve as an acute endogenous mechanism that controls NMDAR activity.SIGNIFICANCE STATEMENT There is considerable interest in negative allosteric modulators of NMDARs that could compensate for receptor overactivation by glutamate or de novo gain-of-function mutations in neurodevelopmental disorders. By a combination of electrophysiological, pharmacological, and computational techniques we describe a novel feedback mechanism regulating NMDAR activity. We find that a transient rise in intracellular Ca2+ increases NMDAR sensitivity to inhibitory neurosteroids in a process dependent on GluN2B subunit depalmitoylation. These results improve our understanding of the molecular mechanisms of steroid action at the NMDAR and indeed of the basic properties of this important glutamate-gated ion channel and may aid in the development of therapeutics for treating neurologic and psychiatric diseases related to overactivation of NMDARs without affecting normal physiological functions.

• Keywords
• NMDAR, carboxy-terminal domain, molecular dynamics simulation, neurosteroid, palmitoylation, single-channel recording,
• MeSH
• HEK293 Cells MeSH
• Hippocampus physiology   MeSH
• Rats MeSH
• Humans MeSH
• Lipoylation drug effects   physiology   MeSH
• Neuroprotection physiology   MeSH
• Rats, Wistar MeSH
• Pregnanes metabolism   pharmacology   MeSH
• Receptors, N-Methyl-D-Aspartate metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 20-oxo-5beta-pregnan-3alpha-yl sulfate MeSH Browser
• NR2B NMDA receptor MeSH Browser
• Pregnanes MeSH
• Receptors, N-Methyl-D-Aspartate MeSH

N-methyl-D-aspartate receptor (NMDAR) hypofunction has been implicated in several neurodevelopmental disorders. NMDAR function can be augmented by positive allosteric modulators, including endogenous compounds, such as cholesterol and neurosteroid pregnenolone sulfate (PES). Here we report that PES accesses the receptor via the membrane, and its binding site is different from that of cholesterol. Alanine mutagenesis has identified residues that disrupt the steroid potentiating effect at the rat GluN1 (G638; I642) and GluN2B (W559; M562; Y823; M824) subunit. Molecular dynamics simulation indicates that, in the absence of PES, the GluN2B M1 helix residue W559 interacts with the M4 helix residue M824. In the presence of PES, the M1 and M4 helices of agonist-activated receptor rearrange, forming a tighter interaction with the GluN1 M3 helix residues G638 and I642. This stabilizes the open-state position of the GluN1 M3 helices. Together, our data identify a likely binding site for the NMDAR-positive allosteric modulator PES and describe a novel molecular mechanism by which NMDAR activity can be augmented.SIGNIFICANCE STATEMENT There is considerable interest in drugs that enhance NMDAR function and could compensate for receptor hypofunction associated with certain neuropsychiatric disorders. Positive allosteric modulators of NMDARs include an endogenous neurosteroid pregnenolone sulfate (PES), but the binding site of PES on the NMDAR and the molecular mechanism of potentiation are unknown. We use patch-clamp electrophysiology in combination with mutagenesis and in silico modeling to describe the interaction of PES with the NMDAR. Our data indicate that PES binds to the transmembrane domain of the receptor at a discrete group of residues at the GluN2B membrane helices M1 and M4 and the GluN1 helix M3, and that PES potentiates NMDAR function by stabilizing the open-state position of the GluN1 M3 helices.

• Keywords
• glutamate receptors, neurosteroids, patch clamp, structure,
• MeSH
• Alanine genetics   MeSH
• Cell Membrane drug effects   MeSH
• Cholesterol metabolism   MeSH
• Electrophysiological Phenomena MeSH
• HEK293 Cells MeSH
• Protein Conformation MeSH
• Rats MeSH
• Humans MeSH
• Patch-Clamp Techniques MeSH
• Pregnenolone pharmacology   MeSH
• Receptors, N-Methyl-D-Aspartate drug effects   MeSH
• Molecular Dynamics Simulation MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Alanine MeSH
• Cholesterol MeSH
• NMDA receptor A1 MeSH Browser
• NR2B NMDA receptor MeSH Browser
• Pregnenolone MeSH
• pregnenolone sulfate MeSH Browser
• Receptors, N-Methyl-D-Aspartate MeSH

Spa treatment can effectively reestablish mood balance in patients with psychiatric disorders. In light of the adrenal gland's role as a crossroad of psychosomatic medicine, this study evaluated changes in 88 circulating steroids and their relationships with a consolidation of somatic, psychosomatic and psychiatric components from a modified N-5 neurotic questionnaire in 46 postmenopausal 50+ women with anxiety-depressive complaints. The patients underwent a standardized one-month intervention therapy with physical activity and an optimized daily regimen in a spa in the Czech Republic. All participants were on medication with selective serotonin reuptake inhibitors. An increase of adrenal steroidogenesis after intervention indicated a reinstatement of the hypothalamic-pituitary-adrenal axis. The increases of many of these steroids were likely beneficial to patients, including immunoprotective adrenal androgens and their metabolites, neuroactive steroids that stimulate mental activity but protect from excitotoxicity, steroids that suppress pain perception and fear, steroids that consolidate insulin secretion, and steroids that improve xenobiotic clearance. The positive associations between the initial values of neurotic symptoms and their declines after the intervention, as well as between initial adrenal activity and the decline of neurotic symptoms, indicate that neurotic impairment may be alleviated by such therapy provided that the initial adrenal activity is not seriously disrupted.

• Keywords
• adrenal, mood balance, postmenopausal females, spa treatment, steroid metabolome,
• MeSH
• Affect * MeSH
• Exercise * MeSH
• Middle Aged MeSH
• Humans MeSH
• Adrenal Glands metabolism   MeSH
• Postmenopause * MeSH
• Projective Techniques MeSH
• Psychotherapy * MeSH
• Aged MeSH
• Steroids biosynthesis   MeSH
• Symptom Assessment MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Steroids MeSH

Herein, we report the synthesis, structure-activity relationship study, and biological evaluation of neurosteroid inhibitors of N-methyl-D-aspartate receptors (NMDARs) receptors that employ an amide structural motif, relative to pregnanolone glutamate (PAG) - a compound with neuroprotective properties. All compounds were found to be more potent NMDAR inhibitors (IC50 values varying from 1.4 to 21.7 μM) than PAG (IC50 = 51.7 μM). Selected compound 6 was evaluated for its NMDAR subtype selectivity and its ability to inhibit AMPAR/GABAR responses. Compound 6 inhibits the NMDARs (8.3 receptors (8.3 ± 2.1 μM) more strongly than it does at the GABAR and AMPARs (17.0 receptors (17.0 ± 0.2 μM and 276.4 ± 178.7 μM, respectively). In addition, compound 6 (10 μM) decreases the frequency of action potentials recorded in cultured hippocampal neurons. Next, compounds 3, 5-7, 9, and 10 were not associated with mitotoxicity, hepatotoxicity nor ROS induction. Lastly, we were able to show that all compounds have improved rat and human plasma stability over PAG.

• Keywords
• NMDA receptor, amide, neurosteroid, plasma stability, structure-activity relationship,
• Publication type
• Journal Article MeSH

UNLABELLED: Postsynaptic N-methyl-d-aspartate receptors (NMDARs) phasically activated by presynaptically released glutamate are critical for synaptic transmission and plasticity. However, under pathological conditions, excessive activation of NMDARs by tonically increased ambient glutamate contributes to excitotoxicity associated with various acute and chronic neurological disorders. Here, using heterologously expressed GluN1/GluN2A and GluN1/GluN2B receptors and rat autaptic hippocampal microisland cultures, we show that pregnanolone sulfate inhibits NMDAR currents induced by a prolonged glutamate application with a higher potency than the NMDAR component of EPSCs. For synthetic pregnanolone derivatives substituted with a carboxylic acid moiety at the end of an aliphatic chain of varying length and attached to the steroid skeleton at C3, the difference in potency between tonic and phasic inhibition increased with the length of the residue. The steroid with the longest substituent, pregnanolone hemipimelate, had no effect on phasically activated receptors while inhibiting tonically activated receptors. In behavioral tests, pregnanolone hemipimelate showed neuroprotective activity without psychomimetic symptoms. These results provide insight into the influence of steroids on neuronal function and stress their potential use in the development of novel therapeutics with neuroprotective action. SIGNIFICANCE STATEMENT: Synaptic activation of N-methyl-d-aspartate receptors (NMDARs) plays a key role in synaptic plasticity, but excessive tonic NMDAR activation mediates excitotoxicity associated with many neurological disorders. Therefore, there is much interest in pharmacological agents capable of selectively blocking tonically activated NMDARs while leaving synaptically activated NMDARs intact. Here, we show that an endogenous neurosteroid pregnanolone sulfate is more potent at inhibiting tonically than synaptically activated NMDARs. Further, we report that a novel synthetic analog of pregnanolone sulfate, pregnanolone hemipimelate, inhibits tonic NMDAR currents without inhibiting the NMDAR component of the EPSC and shows neuroprotective activity in vivo without inducing psychomimetic side effects. These results suggest steroids may have a clinical advantage over other known classes of NMDAR inhibitors.

• Keywords
• NMDA receptor, memantine, neuroprotection, neurosteroid, pregnanolone sulfate, synaptic transmission,
• MeSH
• Excitatory Postsynaptic Potentials drug effects   MeSH
• HEK293 Cells MeSH
• Hippocampus metabolism   MeSH
• Rats MeSH
• Humans MeSH
• Patch-Clamp Techniques MeSH
• Synaptic Transmission drug effects   MeSH
• Neuroprotective Agents pharmacology   MeSH
• Motor Activity drug effects   MeSH
• Rats, Wistar MeSH
• Pregnanolone chemistry   pharmacology   MeSH
• Pregnanes chemistry   pharmacology   MeSH
• Receptors, N-Methyl-D-Aspartate antagonists & inhibitors   drug effects   genetics   MeSH
• Avoidance Learning drug effects   MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• N-methyl D-aspartate receptor subtype 2A MeSH Browser
• Neuroprotective Agents MeSH
• NR2B NMDA receptor MeSH Browser
• Pregnanolone MeSH
• Pregnanes MeSH
• Receptors, N-Methyl-D-Aspartate MeSH

N-methyl-D-aspartate receptors (NMDARs) mediate synaptic plasticity, and their dysfunction is implicated in multiple brain disorders. NMDARs can be allosterically modulated by numerous compounds, including endogenous neurosteroid pregnanolone sulfate. Here, we identify the molecular basis of the use-dependent and voltage-independent inhibitory effect of neurosteroids on NMDAR responses. The site of action is located at the extracellular vestibule of the receptor's ion channel pore and is accessible after receptor activation. Mutations in the extracellular vestibule in the SYTANLAAF motif disrupt the inhibitory effect of negatively charged steroids. In contrast, positively charged steroids inhibit mutated NMDAR responses in a voltage-dependent manner. These results, in combination with molecular modeling, characterize structure details of the open configuration of the NMDAR channel. Our results provide a unique opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with dysfunction of the glutamate system.

• MeSH
• Amino Acid Motifs MeSH
• Humans MeSH
• Mutation * MeSH
• Pregnanolone * chemistry   metabolism   MeSH
• Receptors, N-Methyl-D-Aspartate * antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• Vestibule, Labyrinth * chemistry   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Pregnanolone * MeSH
• Receptors, N-Methyl-D-Aspartate * MeSH