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

BACKGROUND: Neurosteroids are investigated as effective antidotes for the poisoning induced by tetramethylenedisulfotetramine (TMDT) as well as treatments for epileptic spasms during infancy. Both these conditions are quite resistant to pharmacotherapy; thus, a search for new treatments is warranted. METHODS: In this study, we determined the efficacy of two novel neurosteroids, pregnanolone glutamate (PAG) and pregnanolone pyroglutamate (PPG), and tested these drugs in doses of 1-10 mg/kg (ip) against the TMDT syndrome and in our rodent model of infantile spasms. RESULTS: Only PPG in doses 5 and 10 mg/kg suppressed the severity of the TMDT syndrome and TMDT-induced lethality, while the 1 mg/kg dose was without an effect. Interestingly, the 1 mg/kg dose of PPG in combination with 1 mg/kg of diazepam was also effective against TMDT poisoning. Neither PAG nor PPG were effective against experimental spasms in the N-methyl-D-aspartate (NMDA)-triggered model of infantile spasms. CONCLUSIONS: While evidence suggests that PAG can act through multiple actions which include allosteric inhibition of NMDA-induced and glycine receptor-evoked currents as well as augmentation of ɣ-aminobutyric acid subtype A (GABAA) receptor-induced currents, the agent appears to neither have the appropriate mechanistic signature for activity in the infantile spasm model, nor the adequate potency, relative to PPG, for ameliorating the TMDT syndrome. The full mechanisms of action of PPG, which may become a potent TMDT antidote either alone or in combination with diazepam are yet unknown and thus require further investigation.

• Klíčová slova
• Antidote, Neurosteroids, Neurotoxicity, Severe seizures, Tetramethylenedisulfotetramine,
• MeSH
• diazepam farmakologie   MeSH
• hlodavci MeSH
• křeče u dětí * chemicky indukované   farmakoterapie   MeSH
• kyselina glutamová MeSH
• kyselina pyrrolidonkarboxylová MeSH
• N-methylaspartát toxicita   terapeutické užití   MeSH
• neurosteroidy * MeSH
• neurotoxické syndromy * MeSH
• pregnanolon škodlivé účinky   MeSH
• spasmus MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• diazepam MeSH
• kyselina glutamová MeSH
• kyselina pyrrolidonkarboxylová MeSH
• N-methylaspartát MeSH
• neurosteroidy * MeSH
• pregnanolon MeSH
• tetramethylenedisulfotetramine MeSH Prohlížeč

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

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.

• Klíčová slova
• Munc13-1, glutamate, neurosteroid, phorbol ester, pregnanolone, presynaptic,
• MeSH
• HEK293 buňky MeSH
• krysa rodu Rattus MeSH
• kyselina glutamová MeSH
• lidé MeSH
• neurosteroidy * MeSH
• pregnanolon * farmakologie   MeSH
• sírany MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyselina glutamová MeSH
• neurosteroidy * MeSH
• pregnanolon * MeSH
• sírany MeSH

Evidence from clinical and preclinical studies implicates dysfunction of N-methyl-D-aspartate receptors (NMDARs) in schizophrenia progression and symptoms. We investigated the antipsychotic effect of two neuroactive steroids in an animal model of schizophrenia induced by systemic application of MK-801. The neuroactive steroids differ in their mechanism of action at NMDARs. MS-249 is positive, while PA-Glu is a negative allosteric NMDAR modulator. We hypothesized that the positive NMDA receptor modulator would attenuate deficits caused by MK-801 co-application more effectively than PA-Glu. The rats were tested in a battery of tests assessing spontaneous locomotion, anxiety and cognition. Contrary to our expectations, PA-Glu exhibited a superior antipsychotic effect to MS-249. The performance of MS-249-treated rats in cognitive tests differed depending on the level of stress the rats were exposed to during test sessions. In particular, with the increasing severity of stress exposure, the performance of animals worsened. Our results demonstrate that enhancement of NMDAR function may result in unspecific behavioral responses. Positive NMDAR modulation can influence other neurobiological processes besides memory formation, such as anxiety and response to stress.

• Klíčová slova
• MK-801, anxiety, cognition, neurosteroids, schizophrenia, stress,
• MeSH
• antipsychotika farmakologie   MeSH
• bicyklické sloučeniny heterocyklické metabolismus   MeSH
• chování zvířat účinky léků   MeSH
• dizocilpinmaleát farmakologie   MeSH
• HEK293 buňky MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• potkani Long-Evans MeSH
• potkani Wistar MeSH
• pregnenolon metabolismus   farmakologie   MeSH
• receptory N-methyl-D-aspartátu antagonisté a inhibitory   metabolismus   MeSH
• schizofrenie farmakoterapie   metabolismus   MeSH
• steroidy farmakologie   MeSH
• test vyvýšeného křížového bludiště MeSH
• úleková reakce účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antipsychotika MeSH
• bicyklické sloučeniny heterocyklické MeSH
• dizocilpinmaleát MeSH
• pregnenolon MeSH
• pregnenolone sulfate MeSH Prohlížeč
• receptory N-methyl-D-aspartátu MeSH
• steroidy MeSH
• tert-butylbicyclophosphorothionate MeSH Prohlížeč

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

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.

• Klíčová slova
• NMDA receptor, amide, neurosteroid, plasma stability, structure-activity relationship,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• NMDA receptor, memantine, neuroprotection, neurosteroid, pregnanolone sulfate, synaptic transmission,
• MeSH
• excitační postsynaptické potenciály účinky léků   MeSH
• HEK293 buňky MeSH
• hipokampus metabolismus   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• metoda terčíkového zámku MeSH
• nervový přenos účinky léků   MeSH
• neuroprotektivní látky farmakologie   MeSH
• pohybová aktivita účinky léků   MeSH
• potkani Wistar MeSH
• pregnanolon chemie   farmakologie   MeSH
• pregnany chemie   farmakologie   MeSH
• receptory N-methyl-D-aspartátu antagonisté a inhibitory   účinky léků   genetika   MeSH
• učení vyhýbat se účinky léků   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• N-methyl D-aspartate receptor subtype 2A MeSH Prohlížeč
• neuroprotektivní látky MeSH
• NR2B NMDA receptor MeSH Prohlížeč
• pregnanolon MeSH
• pregnany MeSH
• receptory N-methyl-D-aspartátu MeSH

The active place avoidance task is a dry-arena task used to assess spatial navigation and memory in rodents. In this task, a subject is put on a rotating circular arena and avoids an invisible sector that is stable in relation to the room. Rotation of the arena means that the subject's avoidance must be active, otherwise the subject will be moved in the to-be-avoided sector by the rotation of the arena and a slight electric shock will be administered. The present experiment explored the effect of variable arena rotation speed on the ability to avoid the to-be-avoided sector. Subjects in a group with variable arena rotation speed learned to avoid the sector with the same speed and attained the same avoidance ability as rats in a group with a stable arena rotation speed. Only a slight difference in preferred position within the room was found between the two groups. No difference was found between the two groups in the dark phase, where subjects could not use orientation cues in the room. Only one rat was able to learn the avoidance of the to-be-avoided sector in this phase. The results of the experiment suggest that idiothetic orientation and interval timing are not crucial for learning avoidance of the to-be-avoided sector. However, idiothetic orientation might be sufficient for avoiding the sector in the dark.

• Klíčová slova
• Inertial idiothetic navigation, Interval timing, Rats, Spatial navigation, Substratal idiothetic navigation,
• 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