The Sulfated Steroids Pregnenolone Sulfate and Dehydroepiandrosterone Sulfate Inhibit the α1ß3γ2L GABAA Receptor by Stabilizing a Novel Nonconducting State.

The GABAA receptor is inhibited by the endogenous sulfated steroids pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS). It has been proposed in previous work that these steroids act by enhancing desensitization of the receptor. Here, we have investigated the modulatory effects of the steroids on the human α1ß3γ2L GABAA receptor. Using electrophysiology and quantitative model-based data analysis, we show that exposure to the steroid promotes occupancy of a nonconducting state that retains high affinity to the transmitter but whose properties differ from those of the classic, transmitter-induced desensitized state. From the analysis of the inhibitory actions of two combined steroids, we infer that PS and DHEAS act through shared or overlapping binding sites. SIGNIFICANCE STATEMENT: Previous work has proposed that sulfated neurosteroids inhibit the GABAA receptor by enhancing the rate of entry into the desensitized state. This study shows that the inhibitory steroids pregnenolone sulfate and dehydroepiandrosterone sulfate act through a common interaction site by stabilizing a distinct nonconducting state.

Neuroactive Type-A γ-Aminobutyric Acid Receptor Allosteric Modulator Steroids from the Hypobranchial Gland of Marine Mollusk, Conus geographus.

In a program to identify pain treatments with low addiction potential, we isolated five steroids, conosteroids A-E (1-5), from the hypobranchial gland of the mollusk Conus geographus. Compounds 1-5 were active in a mouse dorsal root ganglion (DRG) assay that suggested that they might be analgesic. A synthetic analogue 6 was used for a detailed pharmacological study. Compound 6 significantly increased the pain threshold in mice in the hot-plate test at 2 and 50 mg/kg. Compound 6 at 500 nM antagonizes type-A γ-aminobutyric acid receptors (GABAARs). In a patch-clamp experiment, out of the six subunit combinations tested, 6 exhibited subtype selectivity, most strongly antagonizing α1ß1γ2 and α4ß3γ2 receptors (IC50 1.5 and 1.0 µM, respectively). Although the structures of 1-6 differ from those of known neuroactive steroids, they are cell-type-selective modulators of GABAARs, expanding the known chemical space of neuroactive steroids.

De novo Neurosteroidogenesis in Human Microglia: Involvement of the 18 kDa Translocator Protein.

Neuroactive steroids are potent modulators of microglial functions and are capable of counteracting their excessive reactivity. This action has mainly been ascribed to neuroactive steroids released from other sources, as microglia have been defined unable to produce neurosteroids de novo. Unexpectedly, immortalized murine microglia recently exhibited this de novo biosynthesis; herein, de novo neurosteroidogenesis was characterized in immortalized human microglia. The results demonstrated that C20 and HMC3 microglial cells constitutively express members of the neurosteroidogenesis multiprotein machinery-in particular, the transduceosome members StAR and TSPO, and the enzyme CYP11A1. Moreover, both cell lines produce pregnenolone and transcriptionally express the enzymes involved in neurosteroidogenesis. The high TSPO expression levels observed in microglia prompted us to assess its role in de novo neurosteroidogenesis. TSPO siRNA and TSPO synthetic ligand treatments were used to reduce and prompt TSPO function, respectively. The TSPO expression downregulation compromised the de novo neurosteroidogenesis and led to an increase in StAR expression, probably as a compensatory mechanism. The pharmacological TSPO stimulation the de novo neurosteroidogenesis improved in turn the neurosteroid-mediated release of Brain-Derived Neurotrophic Factor. In conclusion, these results demonstrated that de novo neurosteroidogenesis occurs in human microglia, unravelling a new mechanism potentially useful for future therapeutic purposes.

Physiopathological Role of Neuroactive Steroids in the Peripheral Nervous System.

Peripheral neuropathy (PN) refers to many conditions involving damage to the peripheral nervous system (PNS). Usually, PN causes weakness, numbness and pain and is the result of traumatic injuries, infections, metabolic problems, inherited causes, or exposure to chemicals. Despite the high prevalence of PN, available treatments are still unsatisfactory. Neuroactive steroids (i.e., steroid hormones synthesized by peripheral glands as well as steroids directly synthesized in the nervous system) represent important physiological regulators of PNS functionality. Data obtained so far and here discussed, indeed show that in several experimental models of PN the levels of neuroactive steroids are affected by the pathology and that treatment with these molecules is able to exert protective effects on several PN features, including neuropathic pain. Of note, the observations that neuroactive steroid levels are sexually dimorphic not only in physiological status but also in PN, associated with the finding that PN show sex dimorphic manifestations, may suggest the possibility of a sex specific therapy based on neuroactive steroids.

Site-specific effects of neurosteroids on GABAA receptor activation and desensitization.

This study examines how site-specific binding to three identified neurosteroid-binding sites in the α1ß3 GABAA receptor (GABAAR) contributes to neurosteroid allosteric modulation. We found that the potentiating neurosteroid, allopregnanolone, but not its inhibitory 3ß-epimer epi-allopregnanolone, binds to the canonical ß3(+)-α1(-) intersubunit site that mediates receptor activation by neurosteroids. In contrast, both allopregnanolone and epi-allopregnanolone bind to intrasubunit sites in the ß3 subunit, promoting receptor desensitization and the α1 subunit promoting effects that vary between neurosteroids. Two neurosteroid analogues with diazirine moieties replacing the 3-hydroxyl (KK148 and KK150) bind to all three sites, but do not potentiate GABAAR currents. KK148 is a desensitizing agent, whereas KK150 is devoid of allosteric activity. These compounds provide potential chemical scaffolds for neurosteroid antagonists. Collectively, these data show that differential occupancy and efficacy at three discrete neurosteroid-binding sites determine whether a neurosteroid has potentiating, inhibitory, or competitive antagonist activity on GABAARs.

Analysis of Modulation of the ρ1 GABAA Receptor by Combinations of Inhibitory and Potentiating Neurosteroids Reveals Shared and Distinct Binding Sites.

The ρ1 GABAA receptor is prominently expressed in the retina and is present at lower levels in several brain regions and other tissues. Although the ρ1 receptor is insensitive to many anesthetic drugs that modulate the heteromeric GABAA receptor, it maintains a rich and multifaceted steroid pharmacology. The receptor is negatively modulated by 5ß-reduced steroids, sulfated or carboxylated steroids, and ß-estradiol, whereas many 5α-reduced steroids potentiate the receptor. In this study, we analyzed modulation of the human ρ1 GABAA receptor by several neurosteroids, individually and in combination, in the framework of the coagonist concerted transition model. Experiments involving coapplication of two or more steroids revealed that the receptor contains at least three classes of distinct, nonoverlapping sites for steroids, one each for the inhibitory steroids pregnanolone (3α5ßP), 3α5ßP sulfate, and ß-estradiol. The site for 3α5ßP can accommodate the potentiating steroid 5αTHDOC. The findings are discussed with respect to receptor modulation by combinations of endogenous neurosteroids. SIGNIFICANCE STATEMENT: The study describes modulation of the ρ1 GABAA receptor by neurosteroids. The coagonist concerted transition model was used to determine overlap of binding sites for several inhibitory and potentiating steroids.

Psychoactive properties of BNN27, a novel neurosteroid derivate, in male and female rats.

RATIONALE: Νeurosteroids, like dehydroepiandrosterone (DHEA), play an important role in neurodegeneration and neural protection, but they are metabolized in androgens, estrogens, or other active metabolites. A newly developed synthetic DHEA analog, BNN27 ((20R)-3ß,21-dihydroxy-17R,20-epoxy-5-pregnene), exerts neurotrophic and neuroprotective actions without estrogenic or androgenic effects. OBJECTIVES: This study aimed to investigate potential anxiolytic or antidepressant properties of BNN27. METHODS: Male and female adult Wistar rats were treated with BNN27 (10, 30, or 90 mg/kg, i.p.) and subjected to behavioral tests measuring locomotion, exploration, and "depressive-like" behavior (open field, light/dark box, hole-board, and forced swim tests). The hippocampus and prefrontal cortex were collected for glutamate and GABA measurements, and trunk blood was collected for gonadal hormone analysis. RESULTS: Acute high-dose BNN27 reduced locomotion and exploratory behavior in both sexes. Intermediate acute doses (30 mg/kg) of BNN27 reduced exploration and testosterone levels only in males, and enhanced progesterone levels in both sexes. Notably, with the present design, BNN27 had neither anxiolytic nor antidepressant effects and did not affect estrogen levels. Interestingly, acute administration of a low BNN27 dose (10 mg/kg) increased glutamate turnover, GABA, and glutamine levels in the hippocampus. The same dose also enhanced glutamate levels in the prefrontal cortex of males only. Sex differences were apparent in the basal levels of behavioral, hormonal, and neurochemical parameters, as expected. CONCLUSIONS: BNN27 affects locomotion, progesterone, and testosterone levels, as well as the glutamatergic and GABAergic systems of the hippocampus and prefrontal cortex in a sex-dependent way.

Mitochondrial dysfunction and Alzheimer's disease: prospects for therapeutic intervention.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease and has become a major socioeconomic issue in many developed countries. Currently available therapeutic agents for AD provide only symptomatic treatments, mainly because the complete mechanism of the AD pathogenesis is still unclear. Although several different hypotheses have been proposed, mitochondrial dysfunction has gathered interest because of its profound effect on brain bioenergetics and neuronal survival in the pathophysiology of AD. Various therapeutic agents targeting the mitochondrial pathways associated with AD have been developed over the past decade. Although most of these agents are still early in the clinical development process, they are used to restore mitochondrial function, which provides an alternative therapeutic strategy that is likely to slow the progression of the disease. In this mini review, we will survey the AD-related mitochondrial pathways and their small-molecule modulators that have therapeutic potential. We will focus on recently reported examples, and also overview the current challenges and future perspectives of ongoing research. [BMB Reports 2020; 53(1): 47-55].

The effect of finasteride and dutasteride on the synthesis of neurosteroids by glioblastoma cells.

Glioblastoma (GBM) is the most aggressive local brain tumor and effective treatments are lacking. Many studies have proposed an important participation of steroid hormones in the development of gliomas. Evidence was provided by statistics analysis where the incidence in adult population is 50% higher in men than in women. Female patients have a better prognosis for survival compared to male patients with GBM. Also, the expression of receptors to estrogen, progesterone and androgens in glioma cell lines and tumor biopsies, and glucocorticoid receptors in GBM cell lines had been reported. Here we have investigated the effect of the pharmacological inhibition of 5-α reductases on the capacity of GBM derived cell lines C6 (rat) and U87 (human) to synthesize neurosteroids. As the knowledge of the pathways used to synthesize neurosteroids by GBM derived cells was incomplete, we have investigated the synthesis of these steroids by C6 and U87 cells using tritiated precursors and thin layer chromatography (TLC). Increasing concentrations of finasteride and dutasteride were added to U87 culture media that was collected after 24 and 48 h. The results of the study showed that C6 cells incubated with 3H-cholesterol yielded dihydroandrosterone, hydroxytestosterone, androstenediol, androstenedione and estriol, while U87 cells also synthesized progesterone, and androstanedione. Incubation with 3H-androstenedione or 3H-testosterone mainly yielded dihydrotestosterone, androsterone, dihydroandrosterone, hydroxytestosterone, and estradiol in both lines. To note, we showed here for the first time that U87 cells synthesize corticosteroids. Addition of finasteride or dutasteride to U87 cells reduced androgen and estrogen synthesis. Dutasteride also decreased the synthesis of dihydrocorticosterone and allotetrahydrodesoxycorticosterone while deoxycorticosterone was accumulated. In summary, both GBM cell lines synthesize numerous neurosteroids, including 5-α reductase products and 3α-HSD pathways that were inhibited by finasteride and dutasteride. These inhibitors may be considered as tools to control neurosteroid synthesis of potential relevance for GBM survival.

Neuroactive Steroid N-Methyl-d-aspartate Receptor Positive Allosteric Modulators: Synthesis, SAR, and Pharmacological Activity.

Neuroactive steroids (NASs) play a pivotal role in maintaining homeostasis is the CNS. We have discovered that one NAS in particular, 24(S)-hydroxycholesterol (24(S)-HC), is a positive allosteric modulator (PAM) of NMDA receptors. Using 24(S)-HC as a chemical starting point, we have identified other NASs that have good in vitro potency and efficacy. Herein, we describe the structure activity relationship and pharmacokinetic optimization of this series that ultimately led to SGE-301 (42). We demonstrate that SGE-301 enhances long-term potentiation (LTP) in rat hippocampal slices and, in a dose-dependent manner, improves cognition in a rat social recognition study.

Mapping the neurosteroid binding sites on glycine receptors.

Glycine is a major inhibitory neurotransmitter in the CNS, where it modulates both sensory and motor transduction throughout its binding to glycine receptors (GlyRs), pentameric chloride channels that share structural and functional properties with type A γ-aminobutyric acid receptors (GABAAR). A large number of structurally diverse organic compounds have been identified as GlyR and GABAAR allosteric modulators, making these receptors attractive pharmacological targets. Taking into account the recent resolved crystal structures of GABAAR/neurosteroid complexes, and due to the high sequence identity between the GABAAR and GlyR transmembrane domains, in this work we applied molecular modeling methods to explore the neurosteroid binding to GlyR. Our results indicated that neurosteroid binding sites of GABAARs are also conserved in the GlyRs. Furthermore, docking and molecular dynamics simulations predicted that neurosteroids are stably recognized at these sites, providing precise information on the molecular basis of the neurosteroid binding mode to GlyR. The comparison of how allopregnanolone and pregnanolone 3-OH moieties are recognized by the GlyR binding pocket revealed significant differences that may be associated to opposite effects of these isomers on the GlyR response.

The molecular determinants of neurosteroid binding in the GABA(A) receptor.

Neurosteroids positively modulate GABA-A receptor (GABAAR) channel activity by binding to a transmembrane domain intersubunit site. Understanding the interactions in this site that determine neurosteroid binding and its effect is essential for the design of neurosteroid-based therapeutics. Using photo-affinity labeling and an ELIC-α1GABAAR chimera, we investigated the impact of mutations (Q242L, Q242W and W246L) within the intersubunit site on neurosteroid binding. These mutations, which abolish the thermal stabilizing effect of allopregnanolone on the chimera, reduce neither photolabeling within the intersubunit site nor competitive prevention of labeling by allopregnanolone. Instead, these mutations change the orientation of neurosteroid photolabeling. Molecular docking of allopregnanolone in WT and Q242W receptors confirms that the mutation favors re-orientation of allopregnanolone within the binding pocket. Collectively, the data indicate that mutations at Gln242 or Trp246 that eliminate neurosteroid effects do not eliminate neurosteroid binding within the intersubunit site, but significantly alter the preferred orientation of the neurosteroid within the site. The interactions formed by Gln242 and Trp246 within this pocket play a vital role in determining the orientation of the neurosteroid that may be necessary for its functional effect.

In Search of GABAA Receptor's Neurosteroid Binding Sites.

Neurosteroids (NS) are the main modulators of γ-aminobutyric acid type A receptors (GABAARs), which are the ligand-gated channels target of the major inhibitory neurotransmitter in vertebrates. As a consequence of their ability to modify inhibitory functions in the brain, NS have high physiological and clinical relevance. Accumulated evidence has strongly suggested that NS binding sites were located in the GABAAR transmembrane domain; however the specific localization of these sites has remained an enigma for decades. Fortunately, recent resolution of GABAARs crystal structures, together with computational strategies applied to investigate the NS binding, has paved the way to rationalizing the molecular basis of NS modulation. This work reviews from a historical perspective the road followed for establishing the GABAAR/NS binding mode, from their initial molecular modeling to the latest findings. Furthermore, a comparative analysis describing the NS binding is provided, plus a preliminary analysis of putative NS sites in other assemblies.