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Background and Purpose: GABAA receptors are regulated by numerous classes of allosteric modulators. However, regulation of receptor macroscopic desensitisation remains largely unexplored and may offer new therapeutic opportunities. Here, we report the emerging potential for modulating desensitisation with analogues of the endogenous inhibitory neurosteroid, pregnenolone sulfate. Experimental Approach: New pregnenolone sulfate analogues were synthesised incorporating various heterocyclic substitutions located at the C‐21 position on ring D. The pharmacological profiles of these compounds were assessed using electrophysiology and recombinant GABAA receptors together with mutagenesis, molecular dynamics simulations, structural modelling and kinetic simulations. Key Results: All seven analogues retained a negative allosteric modulatory capability whilst exhibiting diverse potencies. Interestingly, we observed differential effects on GABA current decay by compounds incorporating either a six‐ (compound 5) or five‐membered heterocyclic ring (compound 6) on C‐21, which was independent of their potencies as inhibitors. We propose that differences in molecular charges, and the targeted binding of analogues to specific states of the GABAA receptor, are the most likely cause of the distinctive functional profiles. Conclusions and Implications: Our findings reveal that heterocyclic addition to inhibitory neurosteroids not only affected their potency and macroscopic efficacy but also affected innate receptor mechanisms that underlie desensitisation. Acute modulation of macroscopic desensitisation will determine the degree and duration of GABA inhibition, which are vital for the integration of neural circuit activity. Discovery of this form of modulation could present an opportunity for next‐generation GABAA receptor drug design and development. [ABSTRACT FROM AUTHOR]

Abstract Background Migraine is a common brain disorder that predominantly affects women. Migraine pain seems mediated by the activation of mechanosensitive channels in meningeal afferents. Given the role of transient receptor potential melastatin 3 (TRPM3) channels in mechanical activation, as well as hormonal regulation, these channels may play a role in the sex difference in migraine. Therefore, we investigated whether nociceptive firing induced by TRPM3 channel agonists in meningeal afferents was different between male and female mice. In addition, we assessed the relative contribution of mechanosensitive TRPM3 channels and that of mechanosensitive Piezo1 channels and transient receptor potential vanilloid 1 (TRPV1) channels to nociceptive firing relevant to migraine in both sexes. Methods Ten- to 13-week-old male and female wildtype (WT) C57BL/6 J mice were used. Nociceptive spikes were recorded directly from nerve terminals in the meninges in the hemiskull preparations. Results Selective agonists of TRPM3 channels profoundly activated peripheral trigeminal nerve fibres in mouse meninges. A sex difference was observed for nociceptive firing induced by either PregS or CIM0216, both agonists of TRPM3 channels, with the induced firing being particularly prominent for female mice. Application of Yoda1, an agonist of Piezo1 channels, or capsaicin activating TRPV1 channels, although also leading to increased nociceptive firing of meningeal fibres, did not reveal a sex difference. Cluster analyses of spike activities indicated a massive and long-lasting activation of TRPM3 channels with preferential induction of large-amplitude spikes in female mice. Additional spectral analysis revealed ​a dominant contribution of spiking activity in the α- and β-ranges following TRPM3 agonists in female mice. Conclusions Together, we revealed a specific mechanosensitive profile of nociceptive firing in females and suggest TRPM3 channels as a potential novel candidate for the generation of migraine pain, with particular relevance to females.

Abstract: The transient receptor potential ion channel TRPM3 is highly prevalent on nociceptive dorsal root ganglion (DRG) neurons, but its functions in neuronal plasticity of chronic pain remain obscure. In an animal model of nonspecific low back pain (LBP), latent spinal sensitization known as nociceptive priming is induced by nerve growth factor (NGF) injection. Here, we address the TRPM3-associated molecular basis of NGF-induced latent spinal sensitization at presynaptic level by studying TRPM3-mediated calcium transients in DRG neurons. By investigating TRPM3-expressing HEK cells, we further show the dynamic mitochondrial activity downstream of TRPM3 activation. NGF enhances TRPM3 function, attenuates TRPM3 tachyphylaxis, and slows intracellular calcium clearance; TRPM3 activation triggers more mitochondrial calcium loading than depolarization does, causing a steady-state mitochondrial calcium elevation and a delayed recovery of cytosolic calcium; mitochondrial calcium buffering accounts for approximately 40% of calcium influx subsequent to TRPM3 activation. TRPM3 activation provokes an outbreak of pulsatile superoxide production (mitoflash) that comes in the form of a surge in frequency being tunable. We suggest that mitoflash pulsations downstream of TRPM3 activation might be an early signaling event initiating pain sensitization. Tuning of mitoflash activity would be a novel bottom-up therapeutic strategy for chronic pain conditions such as LBP and beyond. [ABSTRACT FROM AUTHOR]

Abstract Background Neurosteroids affect the balance between neuroexcitation and neuroinhibition but have been little studied in migraine. We compared the serum levels of pregnenolone sulfate, pregnanolone and estradiol in women with menstrually-related migraine and controls and analysed if a correlation existed between the levels of the three hormones and history of migraine and age. Methods Thirty women (mean age ± SD: 33.5 ± 7.1) with menstrually-related migraine (MM group) and 30 aged- matched controls (mean age ± SD: 30.9 ± 7.9) participated in the exploratory study. Pregnenolone sulfate and pregnanolone serum levels were analysed by liquid chromatography-tandem mass spectrometry, while estradiol levels by enzyme-linked immunosorbent assay. Results Serum levels of pregnenolone sulfate and pregnanolone were significantly lower in the MM group than in controls (pregnenolone sulfate: P = 0.0328; pregnanolone: P = 0.0271, Student’s t-test), while estradiol levels were similar. In MM group, pregnenolone sulfate serum levels were negatively correlated with history of migraine (R2 = 0.1369; P = 0.0482) and age (R2 = 0.2826, P = 0.0025) while pregnenolone sulfate levels were not age-related in the control group (R2 = 0.04436, P = 0.4337, linear regression analysis). Conclusion Low levels of both pregnanolone, a positive allosteric modulator of the GABAA receptor, and pregnenolone sulfate, a positive allosteric modulator of the NMDA receptor, involved in memory and learning, could contribute either to headache pain or the cognitive dysfunctions reported in migraine patients. Overall, our results agree with the hypothesis that migraine is a disorder associated with a loss of neurohormonal integrity, thus supporting the therapeutic potential of restoring low neurosteroid levels in migraine treatment.

Background: Migraine is a common brain disorder that predominantly affects women. Migraine pain seems mediated by the activation of mechanosensitive channels in meningeal afferents. Given the role of transient receptor potential melastatin 3 (TRPM3) channels in mechanical activation, as well as hormonal regulation, these channels may play a role in the sex difference in migraine. Therefore, we investigated whether nociceptive firing induced by TRPM3 channel agonists in meningeal afferents was different between male and female mice. In addition, we assessed the relative contribution of mechanosensitive TRPM3 channels and that of mechanosensitive Piezo1 channels and transient receptor potential vanilloid 1 (TRPV1) channels to nociceptive firing relevant to migraine in both sexes. Methods: Ten- to 13-week-old male and female wildtype (WT) C57BL/6 J mice were used. Nociceptive spikes were recorded directly from nerve terminals in the meninges in the hemiskull preparations. Results: Selective agonists of TRPM3 channels profoundly activated peripheral trigeminal nerve fibres in mouse meninges. A sex difference was observed for nociceptive firing induced by either PregS or CIM0216, both agonists of TRPM3 channels, with the induced firing being particularly prominent for female mice. Application of Yoda1, an agonist of Piezo1 channels, or capsaicin activating TRPV1 channels, although also leading to increased nociceptive firing of meningeal fibres, did not reveal a sex difference. Cluster analyses of spike activities indicated a massive and long-lasting activation of TRPM3 channels with preferential induction of large-amplitude spikes in female mice. Additional spectral analysis revealed ​a dominant contribution of spiking activity in the α- and β-ranges following TRPM3 agonists in female mice. Conclusions: Together, we revealed a specific mechanosensitive profile of nociceptive firing in females and suggest TRPM3 channels as a potential novel candidate for the generation of migraine pain, with particular relevance to females. [ABSTRACT FROM AUTHOR]

Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated ion channels that play a crucial role in excitatory synaptic transmission in the central nervous system. Each subunit contributes with three helical transmembrane segments (M1, M3, and M4) and a pore loop (M2) to form the channel pore. Recent studies suggest that the architecture of all eukaryotic iGluRs derives from a common prokaryotic ancestral receptor that lacks M4 and consists only of transmembrane segments M1 and M3. Although significant contribution has emerged in the last years, the role of this additionally evolved transmembrane segment in iGluR assembly and function remains unclear. Here, we have investigated how deletions and mutations of M4 in members of the NMDA receptor (NMDAR) subfamily, the conventional heteromeric GluN1/GluN2 and glycine-gated GluN1/GluN3 NMDARs, affect expression and function in Xenopus oocytes. We show that deletion of M4 in the GluN1, GluN2A, or GluN3A subunit, despite retained receptor assembly and cell surface expression, results in nonfunctional membrane receptors. Coexpression of the corresponding M4 as an isolated peptide in M4-deleted receptors rescued receptor function of GluN1/GluN2A NMDARs without altering the apparent affinity of glutamate or glycine. Electrophysiological analyses of agonist-induced receptor function and its modulation by the neurosteroid pregnenolone sulfate (PS) at mutations of the GluN1-M4/GluN2/3-transmembrane interfaces indicate a crucial role of position M813 in M4 of GluN1 for functional coupling to the core receptor and the negative modulatory effects of PS. Substitution of residues and insertion of interhelical disulfide bridges confirmed interhelical interactions of positions in M4 of GluN1 with residues of transmembrane segments of neighboring subunits. Our results show that although M4s in NMDARs are not important for receptor assembly and surface expression, the residues at the subunit interface are substantially involved in M4 recognition of the core receptor and regulation of PS efficacy. Because mutations in the M4 of GluN1 specifically resulted in loss of PS-induced inhibition of GluN1/GluN2A and GluN1/GluN3A NMDAR currents, our results point to distinct roles of M4s in NMDAR modulation and highlight the importance of the evolutionarily newly evolved M4 for selective in vivo modulation of glutamate- and glycine-activated NMDARs by steroids. [ABSTRACT FROM AUTHOR]

Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated ion channels that play a crucial role in excitatory synaptic transmission in the central nervous system. Each subunit contributes with three helical transmembrane segments (M1, M3, and M4) and a pore loop (M2) to form the channel pore. Recent studies suggest that the architecture of all eukaryotic iGluRs derives from a common prokaryotic ancestral receptor that lacks M4 and consists only of transmembrane segments M1 and M3. Although significant contribution has emerged in the last years, the role of this additionally evolved transmembrane segment in iGluR assembly and function remains unclear. Here, we have investigated how deletions and mutations of M4 in members of the NMDA receptor (NMDAR) subfamily, the conventional heteromeric GluN1/GluN2 and glycine-gated GluN1/GluN3 NMDARs, affect expression and function in Xenopus oocytes. We show that deletion of M4 in the GluN1, GluN2A, or GluN3A subunit, despite retained receptor assembly and cell surface expression, results in nonfunctional membrane receptors. Coexpression of the corresponding M4 as an isolated peptide in M4-deleted receptors rescued receptor function of GluN1/GluN2A NMDARs without altering the apparent affinity of glutamate or glycine. Electrophysiological analyses of agonist-induced receptor function and its modulation by the neurosteroid pregnenolone sulfate (PS) at mutations of the GluN1-M4/GluN2/3-transmembrane interfaces indicate a crucial role of position M813 in M4 of GluN1 for functional coupling to the core receptor and the negative modulatory effects of PS. Substitution of residues and insertion of interhelical disulfide bridges confirmed interhelical interactions of positions in M4 of GluN1 with residues of transmembrane segments of neighboring subunits. Our results show that although M4s in NMDARs are not important for receptor assembly and surface expression, the residues at the subunit interface are substantially involved in M4 recognition of the core receptor and regulation of PS efficacy. Because mutations in the M4 of GluN1 specifically resulted in loss of PS-induced inhibition of GluN1/GluN2A and GluN1/GluN3A NMDAR currents, our results point to distinct roles of M4s in NMDAR modulation and highlight the importance of the evolutionarily newly evolved M4 for selective in vivo modulation of glutamate- and glycine-activated NMDARs by steroids.

Transient receptor potential melastatin 3 channel (TRPM3) is a calcium-permeable nonselective cation channel that plays an important role in modulating glucose homeostasis in the pancreatic beta cells. However, how TRPM3 is regulated under physiological and pathological conditions is poorly understood. In this study, we found that both intracellular and extracellular protons block TRPM3 through its binding sites in the pore region. We demonstrated that external protons block TRPM3 with an inhibitory pH50 of 5.5. whereas internal protons inhibit TRPM3 with an inhibitory pH50 of 6.9. We identified three titratable residues, D1059, D1062, and D1073, at the vestibule of the channel pore that contributes to pH sensitivity. The mutation of D1073Q reduced TRPM3 current by low external pH 5.5 from 62 ± 3% in wildtype to 25 ± 6.0% in D1073Q mutant. These results indicate that D1073 is essential for pH sensitivity. In addition, we found that a single mutation of D1059 or D1062 enhanced pH sensitivity. In summary, our findings identify molecular determinants respionsible for the pH regulation of TRPM3. The inhibition of TRPM3 by protons may indicate an endogenous mechanism governing TRPM3 gating and its physiological/pathological functions.

Already for centuries, humankind is driven to understand the physiological and pathological mechanisms that occur in our brains. Today, we know that ion channels play an essential role in the regulation of neural processes and control many functions of the central nervous system. Ion channels present a diverse group of membrane-spanning proteins that allow ions to penetrate the insulating cell membrane upon opening of their channel pores. This regulated ion permeation results in different electrical and chemical signals that are necessary to maintain physiological excitatory and inhibitory processes in the brain. Therefore, it is no surprise that disturbances in the functions of cerebral ion channels can result in a plethora of neurological disorders, which present a tremendous health care burden for our current society. The identification of ion channel-related brain disorders also fuel the research into the roles of ion channel proteins in various brain states. In the last decade, mounting evidence has been collected that indicates a pivotal role for transient receptor potential (TRP) ion channels in the development and various physiological functions of the central nervous system. For instance, TRP channels modulate neurite growth, synaptic plasticity and integration, and are required for neuronal survival. Moreover, TRP channels are involved in numerous neurological disorders. TRPM3 belongs to the melastatin subfamily of TRP channels and represents a non-selective cation channel that can be activated by several different stimuli, including the neurosteroid pregnenolone sulfate, osmotic pressures and heat. The channel is best known as a peripheral nociceptive ion channel that participates in heat sensation. However, recent research identifies TRPM3 as an emerging new player in the brain. In this review, we summarize the available data regarding the roles of TRPM3 in the brain, and correlate these data with the neuropathological processes in which this ion channel may be involved.

Background: Neurosteroids affect the balance between neuroexcitation and neuroinhibition but have been little studied in migraine. We compared the serum levels of pregnenolone sulfate, pregnanolone and estradiol in women with menstrually-related migraine and controls and analysed if a correlation existed between the levels of the three hormones and history of migraine and age. Methods: Thirty women (mean age ± SD: 33.5 ± 7.1) with menstrually-related migraine (MM group) and 30 aged- matched controls (mean age ± SD: 30.9 ± 7.9) participated in the exploratory study. Pregnenolone sulfate and pregnanolone serum levels were analysed by liquid chromatography-tandem mass spectrometry, while estradiol levels by enzyme-linked immunosorbent assay. Results: Serum levels of pregnenolone sulfate and pregnanolone were significantly lower in the MM group than in controls (pregnenolone sulfate: P = 0.0328; pregnanolone: P = 0.0271, Student's t-test), while estradiol levels were similar. In MM group, pregnenolone sulfate serum levels were negatively correlated with history of migraine (R2 = 0.1369; P = 0.0482) and age (R2 = 0.2826, P = 0.0025) while pregnenolone sulfate levels were not age-related in the control group (R2 = 0.04436, P = 0.4337, linear regression analysis). Conclusion: Low levels of both pregnanolone, a positive allosteric modulator of the GABAA receptor, and pregnenolone sulfate, a positive allosteric modulator of the NMDA receptor, involved in memory and learning, could contribute either to headache pain or the cognitive dysfunctions reported in migraine patients. Overall, our results agree with the hypothesis that migraine is a disorder associated with a loss of neurohormonal integrity, thus supporting the therapeutic potential of restoring low neurosteroid levels in migraine treatment. [ABSTRACT FROM AUTHOR]

Albuminuria is not an effective marker for early diagnosis of diabetic renal complication with several subjects progressing to chronic kidney disease without any albuminuria. A biomarker that can predict early changes of the diabetic kidney will be useful in effective management of type 2 diabetes. Mass spectrometry based metabolomics approach offers tremendous promise for the identification of novel metabolite biomarkers. A case-control approach was carried out to identify renal biomarkers among Asian Indian subjects in a hospital setting. A total of 29 subjects were included in the study that included groups of diabetic controls, diabetic subjects with eGFR >90 ml/min/1.72 m2 and diabetic subjects with eGFR between 60 and 89 ml/min/1.72 m2 and eGFR between 15 and 30 ml/min/1.72 m2. We employed an un-targeted mass spectrometry method for the identification of plasma metabolites. A total of 1414 and 975 metabolites were identified in the positive and negative ion mode respectively. 19 metabolites were up regulated and 18 metabolites were down regulated in CKD2 and CKD4 groups when compared to control. Correlation analysis of the differential metabolites revealed Pregnenolone sulfate, creatinine and ganglioside GA1 to be negatively correlated and hexyl glucoside, all-trans-carophyll yellow and PG to be positively correlated with eGFR. We have identified Pregnenolone sulfate, GA1, PG and all-trans-Carophyll yellow as biomarkers for early identification of diabetic nephropathy. These markers could aid in better management of diabetic nephropathy that may result delaying the progression of the disease. • Pregnenolone sulfate is identified as a novel biomarker for diabetic renal disease. • The level of this marker is correlated with glomerular filtration rate. • Plasma levels of these biomarkers are increased in early stages of CKD. [ABSTRACT FROM AUTHOR]

Memory dysfunction is a symptomatic feature of many neurologic and neuropsychiatric disorders; however, the basic underlying mechanisms of memory and altered states of circuitry function associated with disorders of memory remain a vast unexplored territory. The initial discovery of endogenous neurosteroids triggered a quest to elucidate their role as neuromodulators in normal and diseased brain function. In this review, based on the perspective of our own research, the advances leading to the discovery of positive and negative neurosteroid allosteric modulators of GABA type-A (GABAA), NMDA, and non-NMDA type glutamate receptors are brought together in a historical and conceptual framework. We extend the analysis toward a state-of-the art view of how neurosteroid modulation of neural circuitry function may affect memory and memory deficits. By aggregating the results from multiple laboratories using both animal models for disease and human clinical research on neuropsychiatric and age-related neurodegenerative disorders, elements of a circuitry level view begins to emerge. Lastly, the effects of both endogenously active and exogenously administered neurosteroids on neural networks across the life span of women and men point to a possible underlying pharmacological connectome by which these neuromodulators might act to modulate memory across diverse altered states of mind. [ABSTRACT FROM AUTHOR]

The Transient Receptor Potential Melastatin 3 (TRPM3) is a Ca2+-permeable non-selective cation channel activated by the neurosteroid pregnenolone sulfate (PS). This compound was previously shown to contract mouse aorta by activating TRPM3 in vascular smooth muscle cells (VSMC), and proposed as therapeutic modulator of vascular functions. However, PS effects and the role of TRPM3 in resistance arteries remain unknown. Thus, we aimed at determining the localization and physiological role of TRPM3 in mouse mesenteric arteries. Real-time qPCR experiments, anatomical localization using immunofluorescence microscopy and patch-clamp recordings in isolated VSMC showed that TRPM3 expression in mesenteric arteries is restricted to perivascular nerves. Pressure myography experiments in wild type (WT) mouse arteries showed that PS vasodilates with a concentration-dependence that was best fit by two Hill components (effective concentrations, EC 50 , of 14 and 100 μM). The low EC 50 component was absent in preparations from Trpm3 knockout (KO) mice and in WT arteries in the presence of the CGRP receptor antagonist BIBN 4096. TRPM3-dependent vasodilation was partially inhibited by a cocktail of K+ channel blockers, and not mediated by β-adrenergic signaling. We conclude that, contrary to what was found in aorta, PS dilates mesenteric arteries, partly via an activation of TRPM3 that triggers CGRP release from perivascular nerve endings and a subsequent activation of K+ channels in VSMC. We propose that TRPM3 is implicated in the regulation of the tone of resistance arteries and that its activation by yet unidentified endogenous damage-associated molecules lead to protective vasodilation responses in mesenteric arteries. Unlabelled Image • TRPM3 expression is restricted to sensory nerve endings in mesenteric arteries. • TRPM3 activation dilates mesenteric arteries via CGRP release from nerve endings. • Pregnenolone sulfate is a specific TRPM3 aganist at concentrations below 10 μM. • TRPM3-mediated vasodilation is partly driven by activation of K V channels in VSMC. • Endogenous TRPM3 agonists can contribute to vascular tone regulation. [ABSTRACT FROM AUTHOR]

Abstract Stimulation of transient receptor potential M3 (TRPM3) channels with the steroid pregnenolone sulfate increases the transcriptional activation potential of Elk-1, a transcription factor that regulates serum response element-mediated transcription. Here, we show that an influx of Ca2+ ions into the cells is essential for the activation of Elk-1 following stimulation of TRPM3. Using genetically encoded Ca2+ buffers, we show that a rise in cytoplasmic Ca2+ is required for the upregulation of the transcriptional activation potential of Elk-1, while buffering of Ca2+ in the nucleus had no inhibitory effect on the transcriptional activity of Elk-1. Pharmacological and genetic experiments showed that extracellular signal-regulated protein kinase (ERK1/2) functions as signal transducer connecting TRPM3 channels with the Elk-1 transcription factor. Accordingly, dephosphorylation of ERK1/2 in the nucleus by MAP kinase phosphatase attenuated TRPM3-mediated Elk-1 activation. Moreover, we show that the Ca2+/calmodulin-dependent protein phosphatase calcineurin is part of a shut-off-device for the signaling cascade connecting TRPM3 channels with the activation of Elk-1. The fact that TRPM3 channel stimulation activates Elk-1 connects TRPM3 with the biological functions of Elk-1, including the regulation of proliferation, differentiation, survival, transcription, and cell migration. [ABSTRACT FROM AUTHOR]

Abstract Positive modulators of NMDA receptors are important candidates for therapeutic development to treat psychiatric disorders including autism and schizophrenia. Sulfated neurosteroids have been studied as positive allosteric modulators of NMDA receptors for years, but we understand little about the cellular fate of these compounds, an important consideration for drug development. Here we focus on a visualizable sulfated neurosteroid analogue, KK-169. As expected of a pregnenolone sulfate analogue, the compound strongly potentiates NMDA receptor function, is an antagonist of GABA A receptors, exhibits occlusion with pregnenolone sulfate potentiation, and requires receptor domains important for pregnenolone sulfate potentiation. KK-169 exhibits somewhat higher potency than the natural parent, pregnenolone sulfate. The analogue contains a side-chain alkyne group, which we exploited for retrospective click labeling of neurons. Although the anionic sulfate group is expected to hinder cell entry, we detected significant accumulation of KK-169 in neurons with even brief incubations. Adding a photolabile diazirine group revealed that the expected plasma membrane localization of KK-169 is likely lost during fixation. Overall, our studies reveal new facets of the structure-activity relationship of neurosteroids at NMDA receptors, and their intracellular distribution suggests that sulfated neurosteroids could have unappreciated targets in addition to plasma membrane receptors. Highlights • KK-169 is a potent and efficacious positive allosteric modulator of NMDA receptors. • KK-169 is a clickable analogue of pregnenolone sulfate. • KK-169 reveals intracellular accumulation of sulfated steroids. [ABSTRACT FROM AUTHOR]

• PregS promotes synaptic transmission to dorsal horn neurons in acute spinal slices. • TRPM3 receptor activation increases action potential-dependent transmitter release. • Neuroendocrine and nociceptive signaling contribute to latent sensitization of LBP. Latent spinal sensitization is one key mechanism developing at the early stage of chronic low back pain (LBP). TRPM3-mediated calcium transients of dorsal root ganglia (DRG) neurons are considered critical presynaptic signals involved in this latent sensitization. However, postsynaptic consequences in input laminae of the spinal cord have not been addressed so far. Here, by electrophysiological recordings in acute spinal cord slices from adult rats, we show that perfusion of the TRPM3 agonist pregnenolone sulfate (PregS) induced a significant increase in the frequency but not amplitude of spontaneous postsynaptic currents in lamina I and II neurons. This frequency increase started slowly during PregS perfusion but was reversible following washout. This result is consistent with a presynaptic action of the neurosteroid PregS, indicating the presynaptic expression of functional TRPM3 in the superficial dorsal horn of adult rats. Thus, PregS-induced TRPM3 activation enhances spinal synaptic strength, implying a mediating role of TRPM3 between neuroendocrine and nociceptive signaling, which might as well exist in chronic LBP primed by chronic stress that promotes the biosynthesis of PregS. [ABSTRACT FROM AUTHOR]

Neurosteroids, comprising pregnane, androstane, and sulfated steroids can alter neuronal excitability through interaction with ligand-gated ion channels and other receptors and have therefore a therapeutic potential in several brain disorders. They can be formed in brain cells or are synthesized by an endocrine gland and reach the brain by penetrating the blood–brain barrier (BBB). Especially sulfated steroids such as pregnenolone sulfate (PregS) and dehydroepiandrosterone sulfate (DHEAS) depend on transporter proteins to cross membranes. In this review, we discuss the involvement of ATP-binding cassette (ABC)- and solute carrier (SLC)-type membrane proteins in the transport of these compounds at the BBB and in the choroid plexus (CP), but also in the secretion from neurons and glial cells. Among the ABC transporters, especially BCRP (ABCG2) and several MRP/ABCC subfamily members (MRP1, MRP4, MRP8) are expressed in the brain and known to efflux conjugated steroids. Furthermore, several SLC transporters have been shown to mediate cellular uptake of steroid sulfates. These include members of the OATP/SLCO subfamily, namely OATP1A2 and OATP2B1, as well as OAT3 (SLC22A3), which have been reported to be expressed at the BBB, in the CP and in part in neurons. Furthermore, a role of the organic solute transporter OSTα-OSTβ (SLC51A/B) in brain DHEAS/PregS homeostasis has been proposed. This transporter was reported to be localized especially in steroidogenic cells of the cerebellum and hippocampus. To date, the impact of transporters on neurosteroid homeostasis is still poorly understood. Further insights are desirable also with regard to the therapeutic potential of these compounds.

Background and Purpose: GABA A receptors are regulated by numerous classes of allosteric modulators. However, regulation of receptor macroscopic desensitisation remains largely unexplored and may offer new therapeutic opportunities. Here, we report the emerging potential for modulating desensitisation with analogues of the endogenous inhibitory neurosteroid, pregnenolone sulfate., Experimental Approach: New pregnenolone sulfate analogues were synthesised incorporating various heterocyclic substitutions located at the C-21 position on ring D. The pharmacological profiles of these compounds were assessed using electrophysiology and recombinant GABA A receptors together with mutagenesis, molecular dynamics simulations, structural modelling and kinetic simulations., Key Results: All seven analogues retained a negative allosteric modulatory capability whilst exhibiting diverse potencies. Interestingly, we observed differential effects on GABA current decay by compounds incorporating either a six- (compound 5) or five-membered heterocyclic ring (compound 6) on C-21, which was independent of their potencies as inhibitors. We propose that differences in molecular charges, and the targeted binding of analogues to specific states of the GABA A receptor, are the most likely cause of the distinctive functional profiles., Conclusions and Implications: Our findings reveal that heterocyclic addition to inhibitory neurosteroids not only affected their potency and macroscopic efficacy but also affected innate receptor mechanisms that underlie desensitisation. Acute modulation of macroscopic desensitisation will determine the degree and duration of GABA inhibition, which are vital for the integration of neural circuit activity. Discovery of this form of modulation could present an opportunity for next-generation GABA A receptor drug design and development., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background Pregnenolone sulfate (PregS), an endogenous neurosteroid, which negatively and positively modulates gamma amino butyric acid subunit A (GABA A ) and N-methyl D-aspartate (NMDA) receptors (R) respectively, among other potential neuroplastic changes on synaptic processes, has shown some beneficial effects on treating cognitive impairment associated with schizophrenia (CIAS) and negative symptoms. Lurasidone (Lur), an atypical antipsychotic drug (AAPD), and tandospirone (Tan), a 5-HT 1A  R partial agonist, have also been reported to improve cognitive or negative symptoms, or both, in some schizophrenia patients. Methods We tested whether PregS, by itself, and in combination with Lur or Tan could rescue persistent deficits produced by subchronic treatment with the NMDAR antagonist, phencyclidine (PCP)-in episodic memory, executive functioning, and social behavior, using novel object recognition (NOR), operant reversal learning (ORL), and social interaction (SI) tasks, in male C57BL/6 J mice. Results PregS (10, but not 3 mg/kg) significantly rescued subchronic PCP-induced NOR and SI deficits. Co-administration of sub-effective doses (SEDs) of PregS (3 mg/kg) + Lur (0.1 mg/kg) or Tan (0.03 mg/kg) rescued scPCP-induced NOR and SI deficits. Further, PregS (30, but not 10 mg/kg) rescued PCP-induced ORL deficit, as did the combination of SED PregS (10 mg/kg) +SED Lur (1 mg/kg) or Tan (1 mg/kg). Conclusion PregS was effective alone and as adjunctive treatment for treating two types of cognitive impairments and negative symptoms in this schizophrenia model. Further study of the mechanisms by which PregS alone and in combination with AAPDs and 5-HT 1A  R partial agonists, rescues the deficits in cognition and SI in this preclinical model is indicated. [ABSTRACT FROM AUTHOR]

Sulfonated steroids are increasingly recognized as a circulating reservoir of precursors for the local production of active steroids in certain target tissues. As an alternative to sulfonation of unconjugated steroids by cytosolic sulfotransferases, their direct formation from sulfonated precursors has been described. However, productivity and physiological relevance of this sulfate pathway of steroidogenesis are still widely unclear. Applying the porcine testis as a model, conversion of pregnenolone sulfate (P5S, sulfate pathway) by CYP17A1 was assessed in comparison to the parallel conversions of pregnenolone (P5, Δ5-pathway) and progesterone (P4, Δ4-pathway). To characterize conversions in the virtual absence of competing enzyme activities, in a first series of experiments porcine recombinant CYP17A1 was incubated with the respective substrate in the presence of bovine recombinant cytochrome P450 oxidoreductase (CPR) and cytochrome b5 (b5). Moreover, porcine testicular microsomal fractions were used as a source of homologous CYP17A1, CPR and b5. Invariably 17α-hydroxylation of P5S was, if at all, only minimal and no formation of dehydroepiandrosterone sulfate from P5S was detectable. Consistent with earlier studies porcine CYP17A1 efficiently metabolized P4 and P5 in both assay systems. Metabolism of P4 and P5 by testicular microsomal protein varied substantially between the five animals tested. In conclusion, a physiologically relevant sulfate pathway for the production of C19-steroids from P5S via CYP17A1 is very unlikely in the porcine testis. [ABSTRACT FROM AUTHOR]

Neurosteroids, comprising pregnane, androstane, and sulfated steroids can alter neuronal excitability through interaction with ligand-gated ion channels and other receptors and have therefore a therapeutic potential in several brain disorders. They can be formed in brain cells or are synthesized by an endocrine gland and reach the brain by penetrating the blood-brain barrier (BBB). Especially sulfated steroids such as pregnenolone sulfate (PregS) and dehydroepiandrosterone sulfate (DHEAS) depend on transporter proteins to cross membranes. In this review, we discuss the involvement of ATP-binding cassette (ABC)- and solute carrier (SLC)-type membrane proteins in the transport of these compounds at the BBB and in the choroid plexus (CP), but also in the secretion from neurons and glial cells. Among the ABC transporters, especially BCRP (ABCG2) and several MRP/ABCC subfamily members (MRP1, MRP4, MRP8) are expressed in the brain and known to efflux conjugated steroids. Furthermore, several SLC transporters have been shown to mediate cellular uptake of steroid sulfates. These include members of the OATP/SLCO subfamily, namely OATP1A2 and OATP2B1, as well as OAT3 (SLC22A3), which have been reported to be expressed at the BBB, in the CP and in part in neurons. Furthermore, a role of the organic solute transporter OSTα-OSTβ (SLC51A/B) in brain DHEAS/PregS homeostasis has been proposed. This transporter was reported to be localized especially in steroidogenic cells of the cerebellum and hippocampus. To date, the impact of transporters on neurosteroid homeostasis is still poorly understood. Further insights are desirable also with regard to the therapeutic potential of these compounds. [ABSTRACT FROM AUTHOR]

Background: Stunting affects ~25% of children <5 y of age and is associated with impaired cognitive and motor development and increased morbidity and mortality. The pathogenesis of stunting is poorly understood. Objective: The purpose of this study was to identify altered metabolic pathways associated with child stunting. Design: We measured 677 serum metabolites using liquid chromatography-tandem mass spectrometry in a cross-sectional study of 400 Malawian children aged 12-59 mo, of whom 62% were stunted. Results: A low height-for-age z score (HAZ) was associated with lower serum concentrations of 1) ω-3 (n-3) and ω-6 (n-6) polyunsaturated fatty acids (PUFAs), 2) sulfated neurosteroids, which play a role in brain development, 3) carnitine, a conditionally essential nutrient with an important role in the carnitine shuttle for the metabolism of fatty acids and energy production, and 4) g-glutamyl amino acids, which represent an altered γ-glutamyl cycle of glutathione metabolism. A low HAZ was associated with significantly higher serum concentrations of 5 biomarkers related to cigarette smoke exposure. Conclusions: This metabolomics study shows a cross-sectional association between stunting and low serum ω-3 and ω-6 long-chain PUFAs, which are essential for growth and development; low sulfated neurosteroids, which play a role in brain development; low carnitine, which is essential for β-oxidation of fatty acids; alterations in glutathione metabolism; and increased serum metabolites that are associated with secondhand tobacco smoke exposure. [ABSTRACT FROM AUTHOR]

The transient receptor potential melastatin-3 (TRPM3) channel belongs to the family of transient receptor potential (TRP) cation channels that are expressed in a variety of tissues and cell types, including dorsal root ganglia, cardiomyocytes and pancreatic beta-cells. Although its natural ligands are currently unknown, TRPM3 channels can be activated by the neurosteroid pregnenolone sulfate, the synthetic ligand CIM0216, and by noxious heat. TRPM3 channels are regulated by phosphoinositides, and perhaps by calmodulin. Stimulation of TRPM3 induces an intracellular signaling cascade involving a rise in intracellular Ca 2+ , activation of the protein kinases Raf, ERK and JNK, and the activation of the stimulus-responsive transcription factors AP-1, CREB, Egr-1, and Elk-1. Functionally, stimulation of TRPM3 channels is connected with heat sensation by somatosensory neurons, insulin secretion by pancreatic beta-cells, regulation of neurotransmitter release, iris constriction, and tumor promotion. With the development of highly specific activators and inhibitors of TRPM3 channels, we expect that additional tissue-specific functions of TRPM3 channels will be discovered, establishing TRPM3 channels as a new therapeutic target. [ABSTRACT FROM AUTHOR]

Patients affected by protocadherin 19 ( PCDH19)-female limited epilepsy ( PCDH19- FE) present a remarkable reduction in allopregnanolone blood levels. However, no information is available on other neuroactive steroids and the steroidogenic response to hormonal stimulation. For this reason, we evaluated allopregnanolone, pregnanolone, and pregnenolone sulfate by liquid chromatographic procedures coupled with electrospray tandem mass spectrometry in 12 unrelated patients and 15 age-matched controls. We also tested cortisol, estradiol, progesterone, and 17 OH-progesterone using standard immunoassays. Apart from estradiol and progesterone, all the considered hormones were evaluated in basal condition and after stimulation with adrenocorticotropic hormone ( ACTH). A generalized decrease in blood levels of almost all measured neuroactive steroids was found. When considering sexual development, cortisol and pregnenolone sulfate basal levels were significantly reduced in postpubertal girls affected by PCDH19- FE. Of interest, ACTH administration did not recover pregnenolone sulfate serum levels but restored cortisol to control levels. In prepubertal girls with PCDH19- FE, by challenging adrenal function with ACTH we disclosed defects in the production of cortisol, pregnenolone sulfate, and 17 OH-progesterone, which were not apparent in basal condition. These findings point to multiple defects in peripheral steroidogenesis associated with and potentially relevant to PCDH19- FE. Some of these defects could be addressed by stimulating adrenocortical activity. [ABSTRACT FROM AUTHOR]

Neuroactive steroids are increasingly considered as relevant modulators of neuronal activity. Especially allopregnanolone ( AP) and pregnenolone sulfate ( PS) have been shown to possess, respectively, anticonvulsant or proconvulsant properties. In view of the potential role of these steroids, we aimed at evaluating AP and PS levels in cerebrospinal fluid ( CSF) and blood samples obtained from patients with status epilepticus ( SE). To this purpose, we enrolled 41 patients affected by SE and 41 subjects investigated for nonepileptic neurologic disorders. Liquid chromatographic procedures coupled with electrospray tandem mass spectrometry and routine laboratory investigations were performed. Significantly lower AP levels were found in the CSF of patients affected by SE (−30%; p < 0.05, Mann-Whitney test). Notably, AP was not detectable in 28 of 41 patients affected by SE (p < 0.01 vs. controls, Fisher's exact test). In serum, AP levels did not differ in the two considered groups. Conversely, PS was present at similar levels in the investigated groups. Finally, differences in AP levels could not be explained by a variation in CSF albumin content. These findings indicate that AP is defective in the CSF of patients affected by SE. This phenomenon was not dependent on carriers for steroids, such as albumin. [ABSTRACT FROM AUTHOR]

Endogenous neurosteroids and synthetic neuroactive steroids (NAS) are important targets for therapeutic development in neuropsychiatric disorders. These steroids modulate major signaling systems in the brain and intracellular processes including inflammation, cellular stress and autophagy. In this review, we describe studies performed using unnatural enantiomers of key neurosteroids, which are physiochemically identical to their natural counterparts except for rotation of polarized light. These studies led to insights in how NAS interact with receptors, ion channels and intracellular sites of action. Certain effects of NAS show high enantioselectivity, consistent with actions in chiral environments and likely direct interactions with signaling proteins. Other effects show no enantioselectivity and even reverse enantioselectivity. The spectrum of effects of NAS enantiomers raises the possibility that these agents, once considered only as tools for preclinical studies, have therapeutic potential that complements and in some cases may exceed their natural counterparts. Here we review studies of NAS enantiomers from the perspective of their potential development as novel neurotherapeutics. • Neuroactive steroids (NAS) are an important class of potential neurotherapeutics. • Certain effects of NAS show high, no, or reverse enantioselectivity. • Enantiomers of progesterone, allopregnanolone and estradiol are neuroprotective. • The enantiomer of pregenolone sulfate has cognitive enhancing effects. • NAS enantiomers could represent novel treatments for neuropsychiatric illnesses. [ABSTRACT FROM AUTHOR]

Pregnenolone sulfate is a steroid metabolite with a plethora of actions and functions. As a neurosteroid, pregnenolone sulfate modulates a variety of ion channels, transporters, and enzymes. Interestingly, as a sulfated steroid, pregnenolone sulfate is not the final- or waste-product of pregnenolone being sulfated via a phase II metabolism reaction and renally excreted, as one would presume from the pharmacology textbook knowledge. Pregnenolone sulfate is also the source and thereby the starting point for subsequent steroid synthesis pathways. Most recently, pregnenolone sulfate has been functionally “upgraded” from modulator of ion channels to an activating ion channel ligand. This review will focus on molecular aspects of the neurosteroid, pregnenolone sulfate, its metabolism, concentrations in serum and tissues and last not least will summarize the functional data.