Neuroactive steroids are a group of steroid molecules that are involved in the regulation of functions of the nervous system. The nervous system is not only the site of their action, but their biosynthesis can also occur there. Neuroactive steroid levels depend not only on the physiological state of an individual (person's sex, age, diurnal variation, etc.), but they are also affected by various pathological processes in the nervous system (some neurological and psychiatric diseases or injuries), and new knowledge can be gained by monitoring these processes. The aim of our research was to develop and validate a comprehensive method for the simultaneous determination of selected steroids with neuroactive effects in human serum. The developed method enables high throughput and a sensitive quantitative analysis of nine neuroactive steroid substances (pregnenolone, progesterone, 5α-dihydroprogesterone, allopregnanolone, testosterone, 5α-dihydrotestosterone, androstenedione, dehydroepiandrosterone, and epiandrosterone) in 150 μL of human serum by ultrahigh-performance liquid chromatography with tandem mass spectrometry. The correlation coefficients above 0.999 indicated that the developed analytical procedure was linear in the range of 0.90 nmol/L to 28.46 μmol/L in human serum. The accuracy and precision of the method for all analytes ranged from 83 to 118% and from 0.9 to 14.1%, respectively. This described method could contribute to a deeper understanding of the pathophysiology of various diseases. Similarly, it can also be helpful in the search for new biomarkers and diagnostic options or therapeutic approaches.

• Klíčová slova
• neuroactive steroids, neurosteroids, serum, ultrahigh-performance liquid chromatography−tandem mass spectrometry,
• MeSH
• lidé MeSH
• neurosteroidy krev   MeSH
• reprodukovatelnost výsledků MeSH
• steroidy krev   analýza   MeSH
• tandemová hmotnostní spektrometrie * metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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.

• Klíčová slova
• glutamate receptors, neurosteroids, patch clamp, structure,
• MeSH
• alanin genetika   MeSH
• buněčná membrána účinky léků   MeSH
• cholesterol metabolismus   MeSH
• elektrofyziologické jevy MeSH
• HEK293 buňky MeSH
• konformace proteinů MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• metoda terčíkového zámku MeSH
• pregnenolon farmakologie   MeSH
• receptory N-methyl-D-aspartátu účinky léků   MeSH
• simulace molekulární dynamiky MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alanin MeSH
• cholesterol MeSH
• NMDA receptor A1 MeSH Prohlížeč
• NR2B NMDA receptor MeSH Prohlížeč
• pregnenolon MeSH
• pregnenolone sulfate MeSH Prohlížeč
• receptory N-methyl-D-aspartátu MeSH

Background: Neuroactive steroids (NAS) affect neurotransmitter systems and cognition; thus, they play role in etiopathogenesis of psychiatric disorders. Aims: The primary aim was to examine cognition and effects of NAS on cognitive functioning in first-episode psychosis patients and in their healthy siblings. The secondary aims were to verify whether cognitive deficit is an endophenotype of psychosis and whether higher NAS levels represent a high-risk factor for psychosis. Methods: Studied participants were 1) patients with first episode of psychosis, 2) healthy siblings of the patients, and 3) matching healthy controls. Study procedures included administration of a battery of neuropsychological tests assessing six cognitive domains and examination of NAS plasma levels [cortisol (CORT), 11-deoxycorticosterone (DOC), testosterone (TEST), dehydroepiandrostendione (DHEA), dihydrotestosterone (DHT), and progesterone (PROG)]. Results: A total of 67 subjects were analyzed (16 patients, 22 siblings, and 29 controls). Significant group differences were found in most of the cognitive domains; the patients had the lowest scores. The Kruskal-Wallis test revealed significant group differences in CORT levels (p < 0.01), TEST (p < 0.01), and DHT (p < 0.001); no difference was found in PROG, DHEA, and DOC. All cognitive domains, except for attention, were affected by the NAS levels. CORT levels of patients correlated with speed of processing (r = 0.55) and working memory (r = 0.52), while PROG levels correlated with abstraction (r = -0.63). In siblings, there was a negative correlation between TEST levels and verbal memory (r = -0.51) and PROG with attention (r = -0.47). Conclusions: Our results verified that individual domains of cognitive deficit (abstraction and verbal memory) can be considered as an endophenotype of psychosis. Higher levels of cortisol and testosterone in siblings are consistent with high-risk states for psychosis. Multiple interactions between NAS and cognitive functioning, particularly memory functions, were observed. Study limitations (small sample size and administration of antipsychotic medication) did not allow us to establish unequivocally NAS as an endophenotype.

• Klíčová slova
• cognition, endophenotype, neuroactive steroids, psychosis, siblings,
• Publikační typ
• časopisecké články 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
• aminokyselinové motivy MeSH
• lidé MeSH
• mutace * MeSH
• pregnanolon * chemie   metabolismus   MeSH
• receptory N-methyl-D-aspartátu * antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• vestibulární aparát * chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• pregnanolon * MeSH
• receptory N-methyl-D-aspartátu * MeSH