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1.
bioRxiv ; 2024 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-39282298

RESUMEN

Protein aggregates are emerging therapeutic targets in rare monogenic causes of cardiomyopathy and amyloid heart disease, but their role in more prevalent heart failure syndromes remains mechanistically unexamined. We observed mis-localization of desmin and sarcomeric proteins to aggregates in human myocardium with ischemic cardiomyopathy and in mouse hearts with post-myocardial infarction ventricular remodeling, mimicking findings of autosomal-dominant cardiomyopathy induced by R120G mutation in the cognate chaperone protein, CRYAB. In both syndromes, we demonstrate increased partitioning of CRYAB phosphorylated on serine-59 to NP40-insoluble aggregate-rich biochemical fraction. While CRYAB undergoes phase separation to form condensates, the phospho-mimetic mutation of serine-59 to aspartate (S59D) in CRYAB mimics R120G-CRYAB mutants with reduced condensate fluidity, formation of protein aggregates and increased cell death. Conversely, changing serine to alanine (phosphorylation-deficient mutation) at position 59 (S59A) restored condensate fluidity, and reduced both R120G-CRYAB aggregates and cell death. In mice, S59D CRYAB knock-in was sufficient to induce desmin mis-localization and myocardial protein aggregates, while S59A CRYAB knock-in rescued left ventricular systolic dysfunction post-myocardial infarction and preserved desmin localization with reduced myocardial protein aggregates. 25-Hydroxycholesterol attenuated CRYAB serine-59 phosphorylation and rescued post-myocardial infarction adverse remodeling. Thus, targeting CRYAB phosphorylation-induced condensatopathy is an attractive strategy to counter ischemic cardiomyopathy.

2.
Mol Pharmacol ; 106(5): 264-277, 2024 Oct 17.
Artículo en Inglés | MEDLINE | ID: mdl-39214710

RESUMEN

The γ-aminobutyric acid type A (GABAA) receptor is modulated by a number of neuroactive steroids. Sulfated steroids and 3ß-hydroxy steroids inhibit, while 3α-hydroxy steroids typically potentiate the receptor. Here, we have investigated inhibition of the α1ß3γ2L GABAA receptor by the endogenous neurosteroid 3α-hydroxy-5ß-pregnan-20-one (3α5ßP) and the synthetic neuroactive steroid 3α-hydroxy-5α-androstane-17ß-carbonitrile (ACN). The receptors were expressed in Xenopus oocytes. All experiments were done using two-electrode voltage-clamp electrophysiology. In the presence of low concentrations of GABA, 3α5ßP and ACN potentiate the GABAA receptor. To reveal inhibition, we conducted the experiments on receptors activated by the combination of a saturating concentration of GABA and propofol to fully activate the receptors and mask potentiation, or on mutant receptors in which potentiation is ablated. Under these conditions, both steroids inhibited the receptor with IC50s of ∼13 µM and maximal inhibitory effects of 70-90%. Receptor inhibition by 3α5ßP was sensitive to substitution of the α1 transmembrane domain (TM) 2-2' residue, previously shown to ablate inhibition by pregnenolone sulfate. However, results of coapplication studies and the apparent lack of state dependence suggest that pregnenolone sulfate and 3α5ßP inhibit the GABAA receptor independently and through distinct mechanisms. Mutations to the neurosteroid binding sites in the α1 and ß3 subunits statistically significantly, albeit weakly and incompletely, reduced inhibition by 3α5ßP and ACN. SIGNIFICANCE STATEMENT: The heteromeric GABAA receptor is inhibited by sulfated steroids and 3ß-hydroxy steroids, while 3α-hydroxy steroids are considered to potentiate the receptor. We show here that 3α-hydroxy steroids have inhibitory effects on the α1ß3γ2L receptor, which are observed in specific experimental settings and are expected to manifest under different physiological conditions.


Asunto(s)
Neuroesteroides , Receptores de GABA-A , Xenopus laevis , Receptores de GABA-A/metabolismo , Receptores de GABA-A/genética , Animales , Humanos , Neuroesteroides/metabolismo , Neuroesteroides/farmacología , Oocitos/metabolismo , Oocitos/efectos de los fármacos , Pregnanolona/farmacología , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacología , Femenino , Pregnenolona/farmacología
3.
Int J Mol Sci ; 25(16)2024 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-39201544

RESUMEN

5ß-Dihydrosteroids are produced by the reduction of Δ4-3-ketosteroids catalyzed by steroid 5ß-reductase (AKR1D1). By analogy with steroid 5α-reductase, genetic deficiency exists in AKR1D1 which leads to errors in newborn metabolism and in this case to bile acid deficiency. Also, like the 5α-dihydrosteroids (e.g., 5α-dihydrotestosterone), the 5ß-dihydrosteroids produced by AKR1D1 are not inactive but regulate ligand access to nuclear receptors, can act as ligands for nuclear and membrane-bound receptors, and regulate ion-channel opening. For example, 5ß-reduction of cortisol and cortisone yields the corresponding 5ß-dihydroglucocorticoids which are inactive on the glucocorticoid receptor (GR) and provides an additional mechanism of pre-receptor regulation of ligands for the GR in liver cells. By contrast, 5ß-pregnanes can act as neuroactive steroids at the GABAA and NMDA receptors and at low-voltage-activated calcium channels, act as tocolytic agents, have analgesic activity and act as ligands for PXR, while bile acids act as ligands for FXR and thereby control cholesterol homeostasis. The 5ß-androstanes also have potent vasodilatory properties and work through blockade of Ca2+ channels. Thus, a preference for 5ß-dihydrosteroids to work at the membrane level exists via a variety of mechanisms. This article reviews the field and identifies gaps in knowledge to be addressed in future research.


Asunto(s)
Ácidos y Sales Biliares , Humanos , Animales , Ácidos y Sales Biliares/metabolismo , Oxidorreductasas/metabolismo
4.
bioRxiv ; 2024 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-39091845

RESUMEN

Sterol transport proteins (STPs) play a pivotal role in cholesterol homeostasis and therefore are essential for healthy human physiology. Despite recent advances in dissecting functions of STPs in the human cell, there is still a significant knowledge gap regarding their specific biological functions and a lack of suitable selective probes for their study. Here, we profile fluorescent steroid-based probes across ten STPs, uncovering substantial differences in their selectivity, aiding the retrospective and prospective interpretation of biological results generated with those probes. These results guided the establishment of an STP screening panel combining diverse biophysical assays, enabling the evaluation of 41 steroid-based natural products and derivatives. Combining this with a thorough structural analysis revealed the molecular basis for STP specific selectivity profiles, leading to the uncovering of several new potent and selective Aster-B inhibitors, and supporting the role of this protein in steroidogenesis.

5.
Br J Pharmacol ; 181(21): 4229-4244, 2024 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-38978389

RESUMEN

BACKGROUND AND PURPOSE: Neurosteroids are allosteric modulators of GABAA currents, acting through several functional binding sites although their affinity and specificity for each site are unknown. The goal of this study was to measure steady-state binding affinities of various neurosteroids for specific sites on the GABAA receptor. EXPERIMENTAL APPROACH: Two methods were developed to measure neurosteroid binding affinity: (1) quenching of specific tryptophan residues in neurosteroid binding sites by the neurosteroid 17-methylketone group, and (2) FRET between MQ290 (an intrinsically fluorescent neurosteroid) and tryptophan residues in the binding sites. The assays were developed using ELIC-α1GABAAR, a chimeric receptor containing transmembrane domains of the α1-GABAA receptor. Tryptophan mutagenesis was used to identify specific interactions. KEY RESULTS: Allopregnanolone (3α-OH neurosteroid) was shown to bind at intersubunit and intrasubunit sites with equal affinity, whereas epi-allopregnanolone (3ß-OH neurosteroid) binds at the intrasubunit site. MQ290 formed a strong FRET pair with W246, acting as a site-specific probe for the intersubunit site. The affinity and site-specificity of several neurosteroid agonists and inverse agonists was measured using the MQ290 binding assay. The FRET assay distinguishes between competitive and allosteric inhibition of MQ290 binding and demonstrated an allosteric interaction between the two neurosteroid binding sites. CONCLUSIONS AND IMPLICATIONS: The affinity and specificity of neurosteroid binding to two sites in the ELIC-α1GABAAR were directly measured and an allosteric interaction between the sites was revealed. Adaptation of the MQ290 FRET assay to a plate-reader format will enable screening for high affinity agonists and antagonists for neurosteroid binding sites.


Asunto(s)
Neuroesteroides , Receptores de GABA-A , Receptores de GABA-A/metabolismo , Receptores de GABA-A/química , Receptores de GABA-A/genética , Sitios de Unión , Neuroesteroides/metabolismo , Animales , Pregnanolona/farmacología , Pregnanolona/metabolismo , Humanos , Transferencia Resonante de Energía de Fluorescencia , Xenopus laevis , Unión Proteica
6.
PLoS One ; 19(6): e0304481, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38875235

RESUMEN

Pro-inflammatory changes contribute to multiple neuropsychiatric illnesses. Understanding how these changes are involved in illnesses and identifying strategies to alter inflammatory responses offer paths to potentially novel treatments. We previously found that acute pro-inflammatory stimulation with high (µg/ml) lipopolysaccharide (LPS) for 10-15 min dampens long-term potentiation (LTP) in the hippocampus and impairs learning. Effects of LPS involved non-canonical inflammasome signaling but were independent of toll-like receptor 4 (TLR4), a known LPS receptor. Low (ng/ml) LPS also inhibits LTP when administered for 2-4 h, and here we report that this LPS exposure requires TLR4. We also found that effects of low LPS on LTP involve the oxysterol, 25-hydroxycholesterol, akin to high LPS. Effects of high LPS on LTP are blocked by inhibiting synthesis of 5α-reduced neurosteroids, indicating that neurosteroids mediate LTP inhibition. 5α-Neurosteroids also have anti-inflammatory effects, and we found that exogenous allopregnanolone (AlloP), a key 5α-reduced steroid, prevented effects of low but not high LPS on LTP. We also found that activation of TLR2, TLR3 and TLR7 inhibited LTP and that AlloP prevented the effects of TLR2 and TLR7, but not TLR3. The enantiomer of AlloP, a steroid that has anti-inflammatory actions but low activity at GABAA receptors, prevented LTP inhibition by TLR2, TLR3 and TLR7. In vivo, both AlloP enantiomers prevented LPS-induced learning defects. These studies indicate that neurosteroids play complex roles in network effects of acute neuroinflammation and have potential importance for development of AlloP analogues as therapeutic agents.


Asunto(s)
Hipocampo , Lipopolisacáridos , Potenciación a Largo Plazo , Neuroesteroides , Animales , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Lipopolisacáridos/farmacología , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Neuroesteroides/metabolismo , Receptores Toll-Like/metabolismo , Aprendizaje/efectos de los fármacos , Ratones , Plasticidad Neuronal/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Inflamación/metabolismo , Ratones Endogámicos C57BL , Hidroxicolesteroles/farmacología , Hidroxicolesteroles/metabolismo , Pregnanolona/farmacología , Pregnanolona/metabolismo
7.
Biochim Biophys Acta Biomembr ; 1866(7): 184350, 2024 10.
Artículo en Inglés | MEDLINE | ID: mdl-38806103

RESUMEN

Polyene macrolides are antifungal substances, which interact with cells in a sterol-dependent manner. While being widely used, their mode of action is poorly understood. Here, we employ ultraviolet-sensitive (UV) microscopy to show that the antifungal polyene natamycin binds to the yeast plasma membrane (PM) and causes permeation of propidium iodide into cells. Right before membrane permeability became compromised, we observed clustering of natamycin in the PM that was independent of PM protein domains. Aggregation of natamycin was paralleled by cell deformation and membrane blebbing as revealed by soft X-ray microscopy. Substituting ergosterol for cholesterol decreased natamycin binding and caused a reduced clustering of natamycin in the PM. Blocking of ergosterol synthesis necessitates sterol import via the ABC transporters Aus1/Pdr11 to ensure natamycin binding. Quantitative imaging of dehydroergosterol (DHE) and cholestatrienol (CTL), two analogues of ergosterol and cholesterol, respectively, revealed a largely homogeneous lateral sterol distribution in the PM, ruling out that natamycin binds to pre-assembled sterol domains. Depletion of sphingolipids using myriocin increased natamycin binding to yeast cells, likely by increasing the ergosterol fraction in the outer PM leaflet. Importantly, binding and membrane aggregation of natamycin was paralleled by a decrease of the dipole potential in the PM, and this effect was enhanced in the presence of myriocin. We conclude that ergosterol promotes binding and aggregation of natamycin in the yeast PM, which can be synergistically enhanced by inhibitors of sphingolipid synthesis.


Asunto(s)
Antifúngicos , Membrana Celular , Ergosterol , Natamicina , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Natamicina/farmacología , Natamicina/química , Natamicina/metabolismo , Ergosterol/metabolismo , Membrana Celular/metabolismo , Membrana Celular/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Antifúngicos/farmacología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Colesterol/metabolismo , Colesterol/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/química
8.
Steroids ; 205: 109391, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38437943

RESUMEN

A unified total synthesis route has been used to prepare 18- and 19-trideuterated testosterone, androstenedione and progesterone. The 18-trideuterated steroid synthetic method starts with the synthesis of 2-(methyl-d3)-1,3-cyclopentanedione from CD3I and 1,3-cyclopentanedione and is subsequently converted into the Hajos-Parrish ketone for synthesis of these trideuterated steroids. The 19-trideuterated steroid synthesis proceeds through non-deuterated Hajos-Parrish ketone with incorporation of the 19-methyl-d3 group from CD3I at a later stage of the same synthetic route. Utilization of CD3I at both the initial and later stages of the synthesis provides a route to 18,19-hexadeuterated steroids. The deuterated steroids are useful for studies of steroid biosynthesis and metabolism.


Asunto(s)
Androstenodiona , Progesterona , Androstenodiona/metabolismo , Progesterona/metabolismo , Testosterona/metabolismo , Esteroides , Cetonas
9.
ACS Chem Neurosci ; 15(5): 909-915, 2024 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-38386612

RESUMEN

Activation of the GABAA receptor is associated with numerous behavioral end points ranging from anxiolysis to deep anesthesia. The specific behavioral effect of a GABAergic compound is considered to correlate with the degree of its functional effect on the receptor. Here, we tested the hypothesis that a low-efficacy allosteric potentiator of the GABAA receptor may act, due to a ceiling effect, as a sedative with reduced and limited action. We synthesized a derivative, named (3α,5ß)-20-methyl-pregnane-3,20-diol (KK-235), of the GABAergic neurosteroid 5ß-pregnane-3α,20α-diol. Using electrophysiology, we showed that KK-235 is a low-efficacy potentiator of the synaptic-type α1ß2γ2L GABAA receptor. In the zebrafish larvae behavioral assay, KK-235 was found to only partially block the inverted photomotor response (PMR) and to weakly reduce swimming behavior, whereas the high-efficacy GABAergic steroid (3α,5α,17ß)-3-hydroxyandrostane-17-carbonitrile (ACN) fully blocked PMR and spontaneous swimming. Coapplication of KK-235 reduced the potentiating effect of ACN in an electrophysiological assay and dampened its sedative effect in behavioral experiments. We propose that low-efficacy GABAergic potentiators may be useful as sedatives with limited action.


Asunto(s)
Neuroesteroides , Receptores de GABA-A , Animales , Pez Cebra , Esteroides/farmacología , Pregnanos
10.
Nat Struct Mol Biol ; 31(4): 667-677, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38326651

RESUMEN

The orphan G protein-coupled receptor (GPCR) GPR161 plays a central role in development by suppressing Hedgehog signaling. The fundamental basis of how GPR161 is activated remains unclear. Here, we determined a cryogenic-electron microscopy structure of active human GPR161 bound to heterotrimeric Gs. This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, a sterol that binds adjacent to transmembrane helices 6 and 7 stabilizes a GPR161 conformation required for Gs coupling. Mutations that prevent sterol binding to GPR161 suppress Gs-mediated signaling. These mutants retain the ability to suppress GLI2 transcription factor accumulation in primary cilia, a key function of ciliary GPR161. By contrast, a protein kinase A-binding site in the GPR161 C terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the role of GPR161 function in other signaling pathways.


Asunto(s)
Proteínas Hedgehog , Transducción de Señal , Humanos , Proteínas Hedgehog/genética , Receptores Acoplados a Proteínas G/metabolismo , Mutación , Cilios/metabolismo
11.
Nature ; 626(7998): 401-410, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38297129

RESUMEN

Ferroptosis is a form of cell death that has received considerable attention not only as a means to eradicate defined tumour entities but also because it provides unforeseen insights into the metabolic adaptation that tumours exploit to counteract phospholipid oxidation1,2. Here, we identify proferroptotic activity of 7-dehydrocholesterol reductase (DHCR7) and an unexpected prosurvival function of its substrate, 7-dehydrocholesterol (7-DHC). Although previous studies suggested that high concentrations of 7-DHC are cytotoxic to developing neurons by favouring lipid peroxidation3, we now show that 7-DHC accumulation confers a robust prosurvival function in cancer cells. Because of its far superior reactivity towards peroxyl radicals, 7-DHC effectively shields (phospho)lipids from autoxidation and subsequent fragmentation. We provide validation in neuroblastoma and Burkitt's lymphoma xenografts where we demonstrate that the accumulation of 7-DHC is capable of inducing a shift towards a ferroptosis-resistant state in these tumours ultimately resulting in a more aggressive phenotype. Conclusively, our findings provide compelling evidence of a yet-unrecognized antiferroptotic activity of 7-DHC as a cell-intrinsic mechanism that could be exploited by cancer cells to escape ferroptosis.


Asunto(s)
Linfoma de Burkitt , Deshidrocolesteroles , Ferroptosis , Neuroblastoma , Animales , Humanos , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , Supervivencia Celular , Deshidrocolesteroles/metabolismo , Peroxidación de Lípido , Trasplante de Neoplasias , Neuroblastoma/metabolismo , Neuroblastoma/patología , Oxidación-Reducción , Fenotipo , Reproducibilidad de los Resultados
12.
Sci Rep ; 14(1): 1042, 2024 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-38200205

RESUMEN

Neuropsychiatric and neurodegenerative disorders are correlated with cellular stress. Macroautophagy (autophagy) may represent an important protective pathway to maintain cellular homeostasis and functionality, as it targets cytoplasmic components to lysosomes for degradation and recycling. Given recent evidence that some novel psychiatric treatments, such as the neuroactive steroid (NAS) allopregnanolone (AlloP, brexanolone), may induce autophagy, we stably transfected human embryonic kidney 293 (HEK) cells with a ratiometric fluorescent probe to assay NAS effects on autophagy. We hypothesized that NAS may modulate autophagy in part by the ability of uncharged NAS to readily permeate membranes. Microscopy revealed a weak effect of AlloP on autophagic flux compared with the positive control treatment of Torin1. In high-throughput microplate experiments, we found that autophagy induction was more robust in early passages of HEK cells. Despite limiting studies to early passages for maximum sensitivity, a range of NAS structures failed to reliably induce autophagy or interact with Torin1 or starvation effects. To probe NAS in a system where AlloP effects have been shown previously, we surveyed astrocytes and again saw minimal autophagy induction by AlloP. Combined with other published results, our results suggest that NAS may modulate autophagy in a cell-specific or context-specific manner. Although there is merit to cell lines as a screening tool, future studies may require assaying NAS in cells from brain regions involved in neuropsychiatric disorders.


Asunto(s)
Neuroesteroides , Humanos , Autofagia , Macroautofagia , Riñón , Lisosomas
13.
RSC Adv ; 13(51): 36484-36492, 2023 Dec 08.
Artículo en Inglés | MEDLINE | ID: mdl-38099258

RESUMEN

The trifluoromethylphenyl diazirine (TPD) group is widely used in photoaffinity labeling studies. The TPDYNE group (TPD with an additional alkyne substituent on the phenyl ring) enables the use of click chemistry in conjunction with photoaffinity labeling and expands the utility of the TPD group. New methods for preparing previously known as well as new TPDYNE reagents are reported. Additional methods for preparation of a TPDYNE precursor from which the TPDYNE group can be generated once the precursor is attached to the molecule of interest are also described. Procedures for attaching the TPDYNE or TPDYNE precursor to carboxyl, amino, hydroxyl and alkyne groups are demonstrated using steroids as examples.

14.
BBA Adv ; 4: 100102, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37691996

RESUMEN

Natamycin is an antifungal polyene macrolide that is used as a food preservative but also to treat fungal keratitis and other yeast infections. In contrast to other polyene antimycotics, natamycin does not form ion pores in the plasma membrane, but its mode of action is poorly understood. Using nuclear magnetic resonance (NMR) spectroscopy of deuterated sterols, we find that natamycin slows the mobility of ergosterol and cholesterol in liquid-ordered (Lo) membranes to a similar extent. This is supported by molecular dynamics (MD) simulations, which additionally reveal a strong impact of natamycin dimers on sterol dynamics and water permeability. Interference with sterol-dependent lipid packing is also reflected in a natamycin-mediated increase in membrane accessibility for dithionite, particularly in bilayers containing ergosterol. NMR experiments with deuterated sphingomyelin (SM) in sterol-containing membranes reveal that natamycin reduces phase separation and increases lipid exchange in bilayers with ergosterol. In ternary lipid mixtures containing monounsaturated phosphatidylcholine, saturated SM, and either ergosterol or cholesterol, natamycin interferes with phase separation into Lo and liquid-disordered (Ld) domains, as shown by NMR spectroscopy. Employing the intrinsic fluorescence of natamycin in ultraviolet-sensitive microscopy, we can visualize the binding of natamycin to giant unilamellar vesicles (GUVs) and find that it has the highest affinity for the Lo phase in GUVs containing ergosterol. Our results suggest that natamycin specifically interacts with the sterol-induced ordered phase, in which it disrupts lipid packing and increases solvent accessibility. This property is particularly pronounced in ergosterol containing membranes, which could underlie the selective antifungal activity of natamycin.

15.
bioRxiv ; 2023 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-37292845

RESUMEN

The orphan G protein-coupled receptor (GPCR) GPR161 is enriched in primary cilia, where it plays a central role in suppressing Hedgehog signaling1. GPR161 mutations lead to developmental defects and cancers2,3,4. The fundamental basis of how GPR161 is activated, including potential endogenous activators and pathway-relevant signal transducers, remains unclear. To elucidate GPR161 function, we determined a cryogenic-electron microscopy structure of active GPR161 bound to the heterotrimeric G protein complex Gs. This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, we identify a sterol that binds to a conserved extrahelical site adjacent to transmembrane helices 6 and 7 and stabilizes a GPR161 conformation required for Gs coupling. Mutations that prevent sterol binding to GPR161 suppress cAMP pathway activation. Surprisingly, these mutants retain the ability to suppress GLI2 transcription factor accumulation in cilia, a key function of ciliary GPR161 in Hedgehog pathway suppression. By contrast, a protein kinase A-binding site in the GPR161 C-terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how unique structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the broader role of GPR161 function in other signaling pathways.

16.
Exp Biol Med (Maywood) ; 248(7): 578-587, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-37309730

RESUMEN

Preclinical studies have established that neonatal exposure to contemporary sedative/hypnotic drugs causes neurotoxicity in the developing rodent and primate brains. Our group recently reported that novel neuroactive steroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH) induced effective hypnosis in both neonatal and adult rodents but did not cause significant neurotoxicity in vulnerable brain regions such as subiculum, an output region of hippocampal formation particularly sensitive to commonly used sedatives/hypnotics. Despite significant emphasis on patho-morphological changes, little is known about long-term effects on subicular neurophysiology after neonatal exposure to neuroactive steroids. Hence, we explored the lasting effects of neonatal exposure to 3ß-OH on sleep macrostructure as well as subicular neuronal oscillations in vivo and synaptic plasticity ex vivo in adolescent rats. At postnatal day 7, we exposed rat pups to either 10 mg/kg of 3ß-OH over a period of 12 h or to volume-matched cyclodextrin vehicle. At weaning age, a cohort of rats was implanted with a cortical electroencephalogram (EEG) and subicular depth electrodes. At postnatal day 30-33, we performed in vivo assessment of sleep macrostructure (divided into wake, non-rapid eye movement, and rapid eye movement sleep) and power spectra in cortex and subiculum. In a second cohort of 3ß-OH exposed animals, we conducted ex vivo studies of long-term potentiation (LTP) in adolescent rats. Overall, we found that neonatal exposure to 3ß-OH decreased subicular delta and sigma oscillations during non-rapid eye movement sleep without altering sleep macrostructure. Furthermore, we observed no significant changes in subicular synaptic plasticity. Interestingly, our previous study found that neonatal exposure to ketamine increased subicular gamma oscillations during non-rapid eye movement sleep and profoundly suppressed subicular LTP in adolescent rats. Together these results suggest that exposure to different sedative/hypnotic agents during a critical period of brain development may induce distinct functional changes in subiculum circuitry that may persist into adolescent age.


Asunto(s)
Neuroesteroides , Ratas , Animales , Neuroesteroides/farmacología , Ratas Sprague-Dawley , Hipocampo , Plasticidad Neuronal , Hipnóticos y Sedantes/farmacología
17.
Neurosci Biobehav Rev ; 149: 105191, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37085023

RESUMEN

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.


Asunto(s)
Neuroesteroides , Humanos , Neuroesteroides/metabolismo , Encéfalo/metabolismo , Receptores de GABA-A/metabolismo
18.
Biomolecules ; 13(2)2023 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-36830708

RESUMEN

The neurosteroid allopregnanolone (ALLO) and pregnanolone (PREG), are equally effective positive allosteric modulators (PAMs) of GABAA 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 ent-ALLO potentiates GABA-elicited currents in α1ß3 GABAA receptors with lower potency and efficacy than ALLO, PREG or ent-PREG. The small PAM effect of ent-ALLO was prevented by the α1(Q242L) mutation in the intersubunit neurosteroid binding site between the ß3 and α1 subunits. Consistent with this result, neurosteroid analogue photolabeling with mass spectrometric readout, showed that ent-ALLO binds weakly to the ß3-α1 intersubunit binding site in comparison to ALLO, PREG and ent-PREG. Rigid body docking predicted that ent-ALLO binds in the intersubunit site with a preferred orientation 180° different than ALLO, PREG or ent-PREG, potentially explaining its weak binding and effect. Photolabeling studies did not identify differences between ALLO and ent-ALLO binding to the α1 or ß3 intrasubunit binding sites that also mediate neurosteroid modulation of GABAA receptors. The results demonstrate that differential binding of ent-ALLO and ent-PREG to the ß3-α1 intersubunit site accounts for the difference in enantioselectivity between ALLO and PREG.


Asunto(s)
Neuroesteroides , Receptores de GABA-A , Receptores de GABA-A/metabolismo , Estereoisomerismo , Pregnanolona/farmacología , Ácido gamma-Aminobutírico
19.
Br J Anaesth ; 130(2): 154-164, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36428160

RESUMEN

BACKGROUND: The novel synthetic neuroactive steroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH) blocks T-type calcium channels but does not directly modulate neuronal γ-aminobutyric acid type A (GABAA) currents like other anaesthetic neurosteroids. As 3ß-OH has sex-specific hypnotic effects in adult rats, we studied the mechanism contributing to sex differences in its effects. METHODS: We used a combination of behavioural loss of righting reflex, neuroendocrine, pharmacokinetic, in vitro patch-clamp electrophysiology, and in vivo electrophysiological approaches in wild-type mice and in genetic knockouts of the CaV3.1 T-type calcium channel isoform to study the mechanisms by which 3ß-OH and its metabolite produces sex-specific hypnotic effects. RESULTS: Adult male mice were less sensitive to the hypnotic effects of 3ß-OH compared with female mice, and these differences appeared during development. Adult males had higher 3ß-OH brain concentrations despite being less sensitive to its hypnotic effects. Females metabolised 3ß-OH into the active GABAA receptor positive allosteric modulator (3α,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3α-OH) to a greater extent than males. The 3α-OH metabolite has T-channel blocking properties with sex-specific hypnotic and pharmacokinetic effects. Sex-dependent suppression of the cortical electroencephalogram is more pronounced with 3α-OH compared with 3ß-OH. CONCLUSIONS: The sex-specific differences in the hypnotic effect of 3ß-OH in mice are attributable to differences in its peripheral metabolism into the more potent hypnotic metabolite 3α-OH.


Asunto(s)
Canales de Calcio Tipo T , Neuroesteroides , Ratas , Ratones , Femenino , Masculino , Animales , Hipnóticos y Sedantes/farmacología , Esteroides/farmacología , Receptores de GABA-A
20.
Neural Regen Res ; 18(5): 1004-1008, 2023 May.
Artículo en Inglés | MEDLINE | ID: mdl-36254981

RESUMEN

Neurosteroids are rapidly emerging as important new therapies in neuropsychiatry, with one such agent, brexanolone, already approved for treatment of postpartum depression, and others on the horizon. These steroids have unique properties, including neuroprotective effects that could benefit a wide range of brain illnesses including depression, anxiety, epilepsy, and neurodegeneration. Over the past 25 years, our group has developed ex vivo rodent models to examine factors contributing to several forms of neurodegeneration in the retina. In the course of this work, we have developed a model of acute closed angle glaucoma that involves incubation of ex vivo retinas under hyperbaric conditions and results in neuronal and axonal changes that mimic glaucoma. We have used this model to determine neuroprotective mechanisms that could have therapeutic implications. In particular, we have focused on the role of both endogenous and exogenous neurosteroids in modulating the effects of acute high pressure. Endogenous allopregnanolone, a major stress-activated neurosteroid in the brain and retina, helps to prevent severe pressure-induced retinal excitotoxicity but is unable to protect against degenerative changes in ganglion cells and their axons under hyperbaric conditions. However, exogenous allopregnanolone, at a pharmacological concentration, completely preserves retinal structure and does so by combined effects on gamma-aminobutyric acid type A receptors and stimulation of the cellular process of macroautophagy. Surprisingly, the enantiomer of allopregnanolone, which is inactive at gamma-aminobutyric acid type A receptors, is equally retinoprotective and acts primarily via autophagy. Both enantiomers are also equally effective in preserving retinal structure and function in an in vivo glaucoma model. These studies in the retina have important implications for the ongoing development of allopregnanolone and other neurosteroids as therapeutics for neuropsychiatric illnesses.

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