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Journal of Veterinary Diagnostic... Jul 2023In humans, allopregnanolone plays important roles in a number of different neurodegenerative disorders, and it has been proposed for use in some therapies. Horses are...
In humans, allopregnanolone plays important roles in a number of different neurodegenerative disorders, and it has been proposed for use in some therapies. Horses are commonly used as animal models for human neurodegenerative diseases, mental and behavioral disorders, and neuropsychiatric disorders, and there is interest in using hair as a biological sample to study hormones in these conditions. We validated the use of a commercial ELISA kit (DetectX allopregnanolone kit; Arbor Assays), which was designed for serum, plasma, feces, urine, and tissue samples, to assess allopregnanolone in hair samples from 30 humans and 63 horses. The ELISA kit had good precision (intra- and inter-assay CVs: 6.4% and 11.0% for equine hair; 7.3% and 11.0% for human hair, respectively), sensitivity (50.4 pg/mL for both equine and human hair), and accuracy (parallelism and recovery tests) in determining allopregnanolone concentrations in hair from both species. The allopregnanolone concentrations in human hair were 7.3-79.1 pg/mg; the concentrations were 286 ± 141 pg/mg (x̄ ± SD) in mares on the day of parturition and 16.9 ± 5.5 pg/mg in nonpregnant mares. The DetectX ELISA kit offered a simple and accessible analysis of allopregnanolone in human and equine hair samples.
Topics: Humans; Horses; Animals; Female; Pregnanolone; Enzyme-Linked Immunosorbent Assay; Feces; Hair
PubMed: 37114774
DOI: 10.1177/10406387231171045 -
Antioxidants (Basel, Switzerland) Apr 2023We obtained evidence that mouse BV2 microglia synthesize neurosteroids dynamically to modify neurosteroid levels in response to oxidative damage caused by rotenone....
We obtained evidence that mouse BV2 microglia synthesize neurosteroids dynamically to modify neurosteroid levels in response to oxidative damage caused by rotenone. Here, we evaluated whether neurosteroids could be produced and altered in response to rotenone by the human microglial clone 3 (HMC3) cell line. To this aim, HMC3 cultures were exposed to rotenone (100 nM) and neurosteroids were measured in the culture medium by liquid chromatography with tandem mass spectrometry. Microglia reactivity was evaluated by measuring interleukin 6 (IL-6) levels, whereas cell viability was monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. After 24 h (h), rotenone increased IL-6 and reactive oxygen species levels by approximately +37% over the baseline, without affecting cell viability; however, microglia viability was significantly reduced at 48 h ( < 0.01). These changes were accompanied by the downregulation of several neurosteroids, including pregnenolone, pregnenolone sulfate, 5α-dihydroprogesterone, and pregnanolone, except for allopregnanolone, which instead was remarkably increased ( < 0.05). Interestingly, treatment with exogenous allopregnanolone (1 nM) efficiently prevented the reduction in HMC3 cell viability. In conclusion, this is the first evidence that human microglia can produce allopregnanolone and that this neurosteroid is increasingly released in response to oxidative stress, to tentatively support the microglia's survival.
PubMed: 37107338
DOI: 10.3390/antiox12040963 -
Neuroscience and Biobehavioral Reviews Jun 2023Endogenous neurosteroids and synthetic neuroactive steroids (NAS) are important targets for therapeutic development in neuropsychiatric disorders. These steroids... (Review)
Review
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.
Topics: Humans; Neurosteroids; Brain; Receptors, GABA-A
PubMed: 37085023
DOI: 10.1016/j.neubiorev.2023.105191 -
Acta Obstetricia Et Gynecologica... Oct 2023The mechanism underlying endometriosis-related pain remains poorly understood. Previous studies have indicated that γ-aminobutyric acid (GABA) type A (GABA ) receptors...
INTRODUCTION
The mechanism underlying endometriosis-related pain remains poorly understood. Previous studies have indicated that γ-aminobutyric acid (GABA) type A (GABA ) receptors and GABAergic substances (eg endogenous neurosteroids) play important mechanistic roles in various pain conditions. Our primary objective was to compare GABA receptor function between women with endometriosis and healthy controls by performing a challenge test with diazepam, a GABA receptor agonist, using the saccadic eye velocity as the main outcome. The secondary objective was to investigate the relation between GABA receptor function and serum levels of allopregnanolone, an endogenous positive modulator of the GABA receptor, in the participating women.
MATERIAL AND METHODS
15 women with pelvic pain and laparoscopically confirmed endometriosis and 10 healthy, symptom-free, control women, aged 18-40 years, underwent the diazepam challenge test during the follicular phase of the menstrual cycle. Basal serum allopregnanolone levels were measured prior to diazepam injection.
RESULTS
Compared with healthy controls, women with pelvic pain and confirmed endometriosis had a significantly smaller change in saccadic eye velocity after GABA receptor stimulation with diazepam, indicating lower sensitivity to diazepam. The saccadic eye velocity response was not correlated with the serum allopregnanolone levels.
CONCLUSIONS
Women with painful endometriosis show altered GABA receptor function, depicted as a muted response to an exogenous GABA receptor agonist.
Topics: Female; Humans; Receptors, GABA-A; Pregnanolone; Endometriosis; gamma-Aminobutyric Acid; Diazepam; Gonadal Steroid Hormones; Pelvic Pain
PubMed: 36944570
DOI: 10.1111/aogs.14559 -
Epilepsia Open Jun 2023The neurosteroid allopregnanolone (ALLO) is under investigation as a treatment for benzodiazepine-refractory status epilepticus (SE). Here, we assess the cardiopulmonary...
The neurosteroid allopregnanolone (ALLO) is under investigation as a treatment for benzodiazepine-refractory status epilepticus (SE). Here, we assess the cardiopulmonary safety of intravenous ALLO by itself and after a clinically recommended dose of midazolam (MDZ) in two healthy adult beagles. Each dog received ALLO (1 mg/kg, IV), and after a washout period of 2 weeks, each dog was dosed with MDZ (0.2 mg/kg, IV) followed 10 minutes later by ALLO. Behavioral state, vital signs, arterial blood gases, blood chemistries, and plasma ALLO concentrations were monitored for up to 6 hours after dosing. The dogs appeared sleepy but were fully responsive after both treatments. No depression of mean arterial pressure or respiratory rate was noted. Blood gas measurements failed to show evidence of drug-induced acute respiratory acidosis. Estimated maximum plasma ALLO concentrations were in the range of 1500 to 3000 ng/ml. The results indicate that intravenous ALLO can be used safely to treat benzodiazepine-refractory SE, even when administered shortly after a benzodiazepine.
Topics: Dogs; Animals; Midazolam; Pregnanolone; Status Epilepticus; Administration, Intravenous
PubMed: 36919379
DOI: 10.1002/epi4.12723 -
Current Opinion in Endocrine and... Aug 2022PTSD is associated with deficits in synthesis of progesterone metabolites such as allopregnanolone and pregnanolone that potently facilitate gamma-amino-butyric acid...
PTSD is associated with deficits in synthesis of progesterone metabolites such as allopregnanolone and pregnanolone that potently facilitate gamma-amino-butyric acid (GABA) effects at GABA receptors. These neurosteroids modulate neuronal firing rate, regional brain connectivity, and activation of amygdala-mediated autonomic nervous system, hypothalamic-pituitary-adrenal axis, and behavioral reactions to unconditioned and conditioned threat. They also play critical roles in learning and memory processes such as extinction and extinction retention and inhibit toll-like receptor activation of intracellular pro-inflammatory pathways. Deficient synthesis of these neurosteroids thus may contribute to individually variable PTSD clinical phenotypes encompassing symptom severity, capacity for PTSD recovery, and vulnerability to common PTSD-comorbidities such as major depression, chronic pain, alcohol and nicotine dependence, cardiovascular disease, metabolic syndrome, reproductive disorders, and autoimmune conditions.
PubMed: 36909842
DOI: 10.1016/j.coemr.2022.100359 -
Epilepsy Research Mar 2023Protocadherin-19 (PCDH19)-clustering epilepsy is a distinct developmental and epileptic encephalopathy characterized by early-onset seizures that are often treatment... (Randomized Controlled Trial)
Randomized Controlled Trial
INTRODUCTION
Protocadherin-19 (PCDH19)-clustering epilepsy is a distinct developmental and epileptic encephalopathy characterized by early-onset seizures that are often treatment refractory. Caused by a mutation of the PCDH19 gene on the X chromosome, this rare epilepsy syndrome primarily affects females with seizure onset commonly in the first year of life. A global, randomized, double-blind, placebo-controlled, phase 2 trial was conducted to evaluate the efficacy, safety, and tolerability of ganaxolone compared with placebo as adjunctive therapy to a standard antiseizure medication regimen in patients with PCDH19-clustering epilepsy (VIOLET; NCT03865732).
METHODS
Females aged 1-17 years with a molecularly confirmed pathogenic or likely pathogenic PCDH19 variant who were experiencing ≥12 seizures during a 12-week screening period were stratified by baseline allopregnanolone sulfate (Allo-S) levels (low: ≤2.5 ng/mL; high: >2.5 ng/mL) at screening and randomized 1:1 within each strata to receive ganaxolone (maximum daily dose of 63 mg/kg/day if ≤28 kg or 1800 mg/day if >28 kg) or matching placebo in addition to their standard antiseizure treatment for the 17-week double-blind phase. The primary efficacy endpoint was the median percentage change in 28-day seizure frequency from baseline to the 17-week double-blind phase. Treatment-emergent adverse events (TEAEs) were tabulated by overall, system organ class, and preferred term.
RESULTS
Of the 29 patients screened, 21 (median age, 7.0 years; IQR, 5.0-10.0 years) were randomized to receive either ganaxolone (n = 10) or placebo (n = 11). After the 17-week double-blind phase, the median (IQR) percentage change in 28-day seizure frequency from baseline was - 61.5% (-95.9% to -33.4%) among patients in the ganaxolone group and - 24.0% (-88.2% to -4.9%) among patients in the placebo group (Wilcoxon rank-sum test, p = 0.17). TEAEs were reported by 7 of 10 (70.0%) patients in the ganaxolone group and 11 of 11 (100%) patients in the placebo group. Somnolence was the most common TEAE (40.0% ganaxolone vs 27.3% placebo); serious TEAEs were more common in the placebo group (10.0% ganaxolone vs 45.5% placebo); and 1 (10.0%) patient in the ganaxolone group discontinued the study versus none in the placebo group.
CONCLUSIONS
Ganaxolone was generally well tolerated and led to a greater reduction in the frequency of PCDH19-clustering seizures compared to placebo; however, the trend did not reach statistical significance. Novel trial designs are likely needed to evaluate the effectiveness of antiseizure treatments for PCDH19-clustering epilepsy.
Topics: Female; Humans; Child; Anticonvulsants; Pregnanolone; Treatment Outcome; Seizures; Epilepsy, Generalized; Cluster Analysis; Protocadherins
PubMed: 36870093
DOI: 10.1016/j.eplepsyres.2023.107112 -
Aging Feb 2023Premenstrual dysphoric disorder (PMDD) is a common mental health challenge among women of reproductive age. Allopregnanolone (3α, 5α-THP; ALLO) mediated functional...
BACKGROUND
Premenstrual dysphoric disorder (PMDD) is a common mental health challenge among women of reproductive age. Allopregnanolone (3α, 5α-THP; ALLO) mediated functional alterations of GABAA receptors (GABAA-R) are involved in PMDD pathogenesis, however, the specific mechanism remains unknown. Therefore, we investigated the role of ALLO mediated GABAA-Rα4 in the pathophysiology of PMDD.
PURPOSE
We determined whether the pathogenesis of PMDD is associated with ALLO mediated GABAA-Rα4 expression changes in different brain regions.
METHODS
Rat models of PMDD liver-qi invasion syndrome (PMDD-LIS) were established via the resident intruder paradigm. Behavioral changes of rats were assessed by aggressive behavior tests, EPM and OFT. The levels of progesterone and ALLO in serum as well as brain areas were determined by ELISA. Variations in GABAA-Rα4 levels in brain regions were assessed by immunofluorescence and RT-PCR. Medicated serum was used to interfere with rat hippocampal neurons, and changes in Cl current were recorded through electrophysiology.
RESULTS
Premenstrual anxiety and irritability of PMDD-LIS patients can be simulated in PMDD-LIS rat models. Exogenous ALLO significantly improved the anxiety behaviors of PMDD-LIS rats. Changes in ALLO among different brain regions varied. GABAA-Rα4 expressions were low in the amygdala and abnormally high in the hippocampus, however, ALLO alleviated these deviations. Whole-cell patch clamp recording technique showed a weaker Cl current intensity of PMDD-LIS rats, reduced neuroinhibitory functions and increased Cl current intensity in the ALLO group drug serum intervention and enhanced emotional inhibition function.
CONCLUSION
We established that ALLO regulation of the GABAA-Rα4 subunit in the amygdala and hippocampus is involved in PMDD-LIS pathogenesis.
Topics: Humans; Female; Rats; Animals; Premenstrual Dysphoric Disorder; Pregnanolone; Qi; Liver Diseases; Hippocampus; Amygdala
PubMed: 36842096
DOI: 10.18632/aging.204541 -
Biomolecules Feb 2023The neurosteroid allopregnanolone (ALLO) and pregnanolone (PREG), are equally effective positive allosteric modulators (PAMs) of GABA receptors. Interestingly, the PAM...
The neurosteroid allopregnanolone (ALLO) and pregnanolone (PREG), are equally effective positive allosteric modulators (PAMs) of GABA receptors. Interestingly, the PAM effects of ALLO are strongly enantioselective, whereas those of PREG are not. This study was aimed at determining the basis for this difference in enantioselectivity. The oocyte electrophysiology studies showed that -ALLO potentiates GABA-elicited currents in αβ GABA receptors with lower potency and efficacy than ALLO, PREG or -PREG. The small PAM effect of -ALLO was prevented by the α(Q242L) mutation in the intersubunit neurosteroid binding site between the β and α subunits. Consistent with this result, neurosteroid analogue photolabeling with mass spectrometric readout, showed that -ALLO binds weakly to the β-α intersubunit binding site in comparison to ALLO, PREG and -PREG. Rigid body docking predicted that -ALLO binds in the intersubunit site with a preferred orientation 180° different than ALLO, PREG or -PREG, potentially explaining its weak binding and effect. Photolabeling studies did not identify differences between ALLO and -ALLO binding to the α or β intrasubunit binding sites that also mediate neurosteroid modulation of GABA receptors. The results demonstrate that differential binding of -ALLO and -PREG to the β-α intersubunit site accounts for the difference in enantioselectivity between ALLO and PREG.
Topics: Receptors, GABA-A; Neurosteroids; Stereoisomerism; Pregnanolone; gamma-Aminobutyric Acid
PubMed: 36830708
DOI: 10.3390/biom13020341 -
Biological Psychiatry Aug 2023Chronic stress is a major risk factor for psychiatric illnesses, including depression. However, the pathophysiological mechanisms whereby stress leads to mood disorders...
BACKGROUND
Chronic stress is a major risk factor for psychiatric illnesses, including depression. However, the pathophysiological mechanisms whereby stress leads to mood disorders remain unclear. Allopregnanolone acts as a positive allosteric modulator preferentially on δ subunit-containing GABA (gamma-aminobutyric acid A) receptors. Accumulating clinical and preclinical evidence supports the antidepressant effects of exogenous administration of allopregnanolone analogs; yet, the role of endogenous allopregnanolone in the pathophysiology of depression remains unknown.
METHODS
We utilized a chronic unpredictable stress (CUS) mouse model, followed by behavioral and biochemical assays, to examine whether altered neurosteroid signaling contributes to behavioral outcomes following CUS. We subsequently performed in vivo CRISPR (clustered regularly interspaced short palindromic repeats) knockdown of rate-limiting enzymes involved in allopregnanolone synthesis, 5α-reductase type 1 and 2 (5α1/2), in addition to lentiviral overexpression of 5α1/2 in the basolateral amygdala (BLA) of mice that underwent CUS to assess the impact of 5α1/2 on behavioral outcomes.
RESULTS
The expression of δ subunit-containing GABA receptors and endogenous levels of allopregnanolone were reduced in the BLA following CUS. Treatment with an exogenous allopregnanolone analog, SGE-516, was sufficient to increase allopregnanolone levels in the BLA following CUS. Knockdown of 5α1/2 in the BLA mimicked the behavioral outcomes associated with CUS. Conversely, overexpression of 5α1/2 in the BLA improved behavioral outcomes following CUS.
CONCLUSIONS
Our findings demonstrate that chronic stress impairs endogenous neurosteroid signaling in the BLA, which is sufficient to induce behavioral deficits. Further, these studies suggest that allopregnanolone-based treatments may directly target the underlying pathophysiology of mood disorders suggesting that targeting endogenous neurosteroidogenesis may offer a novel therapeutic strategy.
Topics: Mice; Animals; Pregnanolone; Receptors, GABA-A; Neurosteroids; Signal Transduction; gamma-Aminobutyric Acid
PubMed: 36736870
DOI: 10.1016/j.biopsych.2023.01.022