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.

• Klíčová slova
• GC-MS/MS, anti-MS drugs, multiple sclerosis, multivariate statistics, steroidomics,
• MeSH
• dospělí MeSH
• glatiramer acetát terapeutické užití   MeSH
• lidé středního věku MeSH
• lidé MeSH
• menstruační cyklus účinky léků   MeSH
• roztroušená skleróza * farmakoterapie   metabolismus   krev   MeSH
• steroidy * metabolismus   krev   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glatiramer acetát MeSH
• steroidy * MeSH

BACKGROUND: Causative genetic variants cannot yet be found for many disorders with a clear heritable component, including chronic fatigue disorders like myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). These conditions may involve genes in difficult-to-align genomic regions that are refractory to short read approaches. Structural variants in these regions can be particularly hard to detect or define with short reads, yet may account for a significant number of cases. Long read sequencing can overcome these difficulties but so far little data is available regarding the specific analytical challenges inherent in such regions, which need to be taken into account to ensure that variants are correctly identified. Research into chronic fatigue disorders faces the additional challenge that the heterogeneous patient populations likely encompass multiple aetiologies with overlapping symptoms, rather than a single disease entity, such that each individual abnormality may lack statistical significance within a larger sample. Better delineation of patient subgroups is needed to target research and treatment. METHODS: We use nanopore sequencing in a case of unexplained severe fatigue to identify and fully characterise a large inversion in a highly homologous region spanning the AKR1C gene locus, which was indicated but could not be resolved by short-read sequencing. We then use GC-MS/MS serum steroid analysis to investigate the functional consequences. RESULTS: Several commonly used bioinformatics tools are confounded by the homology but a combined approach including visual inspection allows the variant to be accurately resolved. The DNA inversion appears to increase the expression of AKR1C2 while limiting AKR1C1 activity, resulting in a relative increase of inhibitory GABAergic neurosteroids and impaired progesterone metabolism which could suppress neuronal activity and interfere with cellular function in a wide range of tissues. CONCLUSIONS: This study provides an example of how long read sequencing can improve diagnostic yield in research and clinical care, and highlights some of the analytical challenges presented by regions containing tandem arrays of genes. It also proposes a novel gene associated with a novel disease aetiology that may be an underlying cause of complex chronic fatigue. It reveals biomarkers that could now be assessed in a larger cohort, potentially identifying a subset of patients who might respond to treatments suggested by the aetiology.

• Klíčová slova
• AKR1C1, AKR1C2, Allopregnanolone, Fatigue, Long read sequencing, ME/CFS diagnosis, Neurosteroids, Structural variants,
• MeSH
• biologické markery MeSH
• hydroxysteroiddehydrogenasy MeSH
• lidé MeSH
• syndrom chronické únavy * MeSH
• tandemová hmotnostní spektrometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AKR1C2 protein, human MeSH Prohlížeč
• biologické markery MeSH
• hydroxysteroiddehydrogenasy MeSH

Multiple molecular targets have been identified to mediate membrane-delimited and nongenomic effects of natural and synthetic steroids, but the influence of steroid metabolism on neuroactive steroid signaling is not well understood. To begin to address this question, we set out to identify major metabolites of a neuroprotective synthetic steroid 20-oxo-5β-pregnan-3α-yl l-glutamyl 1-ester (pregnanolone glutamate, PAG) and characterize their effects on GABAA and NMDA receptors (GABARs, NMDARs) and their influence on zebrafish behavior. Gas chromatography-mass spectrometry was used to assess concentrations of PAG and its metabolites in the hippocampal tissue of juvenile rats following intraperitoneal PAG injection. PAG is metabolized in the peripheral organs and nervous tissue to 20-oxo-17α-hydroxy-5β-pregnan-3α-yl l-glutamyl 1-ester (17-hydroxypregnanolone glutamate, 17-OH-PAG), 3α-hydroxy-5β-pregnan-20-one (pregnanolone, PA), and 3α,17α-dihydroxy-5β-pregnan-20-one (17-hydroxypregnanolone, 17-OH-PA). Patch-clamp electrophysiology experiments in cultured hippocampal neurons demonstrate that PA and 17-OH-PA are potent positive modulators of GABARs, while PAG and 17-OH-PA have a moderate inhibitory effect at NMDARs. PAG, 17-OH-PA, and PA diminished the locomotor activity of zebrafish larvae in a dose-dependent manner. Our results show that PAG and its metabolites are potent modulators of neurotransmitter receptors with behavioral consequences and indicate that neurosteroid-based ligands may have therapeutic potential.

• Klíčová slova
• glutamate, negative allosteric modulator, steroid, thigmotaxis, zebrafish,
• MeSH
• dánio pruhované MeSH
• estery MeSH
• GABA MeSH
• krysa rodu Rattus MeSH
• kyselina glutamová MeSH
• pregnanolon * farmakologie   chemie   MeSH
• receptory GABA-A MeSH
• receptory N-methyl-D-aspartátu * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• estery MeSH
• GABA MeSH
• kyselina glutamová MeSH
• pregnanolon * MeSH
• receptory GABA-A MeSH
• receptory N-methyl-D-aspartátu * MeSH

Steroid sulfation and desulfation participates in the regulation of steroid bioactivity, metabolism and transport. The authors focused on sulfation and desulfation balance in three neurodegenerative diseases: Alzheimer´s disease (AD), Parkinson´s disease (PD), and multiple sclerosis (MS). Circulating steroid conjugates dominate their unconjugated counterparts, but unconjugated steroids outweigh their conjugated counterparts in the brain. Apart from the neurosteroid synthesis in the central nervous system (CNS), most brain steroids cross the blood-brain barrier (BBB) from the periphery and then may be further metabolized. Therefore, steroid levels in the periphery partly reflect the situation in the brain. The CNS steroids subsequently influence the neuronal excitability and have neuroprotective, neuroexcitatory, antidepressant and memory enhancing effects. They also exert anti-inflammatory and immunoprotective actions. Like the unconjugated steroids, the sulfated ones modulate various ligand-gated ion channels. Conjugation by sulfotransferases increases steroid water solubility and facilitates steroid transport. Steroid sulfates, having greater half-lives than their unconjugated counterparts, also serve as a steroid stock pool. Sulfotransferases are ubiquitous enzymes providing massive steroid sulfation in adrenal zona reticularis and zona fasciculata.. Steroid sulfatase hydrolyzing the steroid conjugates is exceedingly expressed in placenta but is ubiquitous in low amounts including brain capillaries of BBB which can rapidly hydrolyze the steroid sulfates coming across the BBB from the periphery. Lower dehydroepiandrosterone sulfate (DHEAS) plasma levels and reduced sulfotransferase activity are considered as risk factors in AD patients. The shifted balance towards unconjugated steroids can participate in the pathophysiology of PD and anti-inflammatory effects of DHEAS may counteract the MS.

• Klíčová slova
• Alzheimer’s disease, Parkinson's disease, brain, multiple sclerosis, neuroactive steroids, neurosteroids, steroid sulfatase, steroid sulfotransferases,
• Publikační typ
• časopisecké články MeSH
• přehledy 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.

• Klíčová slova
• GABA, NMDA, anticonvulsant, metabolomics, neurosteroids, zuranolone,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• adrenal, mood balance, postmenopausal females, spa treatment, steroid metabolome,
• MeSH
• afekt * MeSH
• cvičení * MeSH
• lidé středního věku MeSH
• lidé MeSH
• nadledviny metabolismus   MeSH
• postmenopauza * MeSH
• projektivní techniky MeSH
• psychoterapie * MeSH
• senioři MeSH
• steroidy biosyntéza   MeSH
• určení symptomu MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• steroidy MeSH