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ER-mitochondria contacts and cholesterol metabolism are disrupted by disease-associated tau protein.EMBO Reports Aug 2023Abnormal tau protein impairs mitochondrial function, including transport, dynamics, and bioenergetics. Mitochondria interact with the endoplasmic reticulum (ER) via...
Abnormal tau protein impairs mitochondrial function, including transport, dynamics, and bioenergetics. Mitochondria interact with the endoplasmic reticulum (ER) via mitochondria-associated ER membranes (MAMs), which coordinate and modulate many cellular functions, including mitochondrial cholesterol metabolism. Here, we show that abnormal tau loosens the association between the ER and mitochondria in vivo and in vitro. Especially, ER-mitochondria interactions via vesicle-associated membrane protein-associated protein (VAPB)-protein tyrosine phosphatase-interacting protein 51 (PTPIP51) are decreased in the presence of abnormal tau. Disruption of MAMs in cells with abnormal tau alters the levels of mitochondrial cholesterol and pregnenolone, indicating that conversion of cholesterol into pregnenolone is impaired. Opposite effects are observed in the absence of tau. Besides, targeted metabolomics reveals overall alterations in cholesterol-related metabolites by tau. The inhibition of GSK3β decreases abnormal tau hyperphosphorylation and increases VAPB-PTPIP51 interactions, restoring mitochondrial cholesterol and pregnenolone levels. This study is the first to highlight a link between tau-induced impairments in the ER-mitochondria interaction and cholesterol metabolism.
Topics: tau Proteins; Mitochondria; Endoplasmic Reticulum; Protein Tyrosine Phosphatases; Cholesterol
PubMed: 37401859
DOI: 10.15252/embr.202357499 -
Steroids Jul 2016Steroids have been widely used in the clinical setting. They bind and activate nuclear receptors to regulate gene expression. In addition to activating genomic... (Review)
Review
Steroids have been widely used in the clinical setting. They bind and activate nuclear receptors to regulate gene expression. In addition to activating genomic transcription, steroids also exert nongenomic actions. The current article focuses on the nongenomic actions of neurosteroids, including pregnenolone (P5), 7α-hydroxypregnenolone, pregnenolone sulfate and allopregnanolone. Pregnenolone and its derivatives promote neuronal activity by enhancing learning and memory, relieving depression, enhancing locomotor activity, and promoting neuronal cell survival. They exert these effects by activating various target proteins located in the cytoplasm or cell membrane. Pregnenolone and its metabolites bind to receptors such as microtubule-associated proteins and neurotransmitter receptors to elicit a series of reactions including stabilization of microtubules, increase of ion flux into cells, and dopamine release. The wide actions of neurosteroids indicate that pregnenolone derivatives have great potential in future treatment of neurological diseases.
Topics: 17-alpha-Hydroxypregnenolone; Animals; Depression; Humans; Microtubules; Neurodegenerative Diseases; Neurotransmitter Agents; Pregnanolone; Pregnenolone
PubMed: 26844377
DOI: 10.1016/j.steroids.2016.01.017 -
Cell Metabolism Feb 2022Obesity and type 2 diabetes are associated with cognitive dysfunction. Because the hypothalamus is implicated in energy balance control and memory disorders, we...
Obesity and type 2 diabetes are associated with cognitive dysfunction. Because the hypothalamus is implicated in energy balance control and memory disorders, we hypothesized that specific neurons in this brain region are at the interface of metabolism and cognition. Acute obesogenic diet administration in mice impaired recognition memory due to defective production of the neurosteroid precursor pregnenolone in the hypothalamus. Genetic interference with pregnenolone synthesis by Star deletion in hypothalamic POMC, but not AgRP neurons, deteriorated recognition memory independently of metabolic disturbances. Our data suggest that pregnenolone's effects on cognitive function were mediated via an autocrine mechanism on POMC neurons, influencing hippocampal long-term potentiation. The relevance of central pregnenolone on cognition was also confirmed in metabolically unhealthy patients with obesity. Our data reveal an unsuspected role for POMC neuron-derived neurosteroids in cognition. These results provide the basis for a framework to investigate new facets of POMC neuron biology with implications for cognitive disorders.
Topics: Animals; Diabetes Mellitus, Type 2; Humans; Hypothalamus; Metabolic Diseases; Mice; Mice, Inbred C57BL; Pregnenolone; Pro-Opiomelanocortin
PubMed: 35108514
DOI: 10.1016/j.cmet.2021.12.023 -
Methods in Enzymology 2023Cytochrome P450 (P450) 17A1 plays a key role in steroidogenesis, in that this enzyme catalyzes the 17α-hydroxylation of both pregnenolone and progesterone, followed by...
Cytochrome P450 (P450) 17A1 plays a key role in steroidogenesis, in that this enzyme catalyzes the 17α-hydroxylation of both pregnenolone and progesterone, followed by a lyase reaction to cleave the C-20 land C-21 carbons from each steroid. The reactions are important in the production of both glucocorticoids and androgens. The enzyme is critical in humans but is also a drug target in treatment of prostate cancer. Detailed methods are described for the heterologous expression of human P450 17A1 in bacteria, purification of the recombinant enzyme, reconstitution of the enzyme system in the presence of cytochrome b, and chromatographic procedures for sensitive analyses of reaction products. Historic assay approaches are reviewed. Some information is also provided about outstanding questions in the research field, including catalytic mechanisms and searches for selective inhibitors.
Topics: Humans; Lyases; Progesterone; Steroids; Steroid 17-alpha-Hydroxylase
PubMed: 37802581
DOI: 10.1016/bs.mie.2023.04.001 -
Psychopharmacology Sep 2014The neurosteroid pregnenolone sulfate (PregS) acts as a cognitive enhancer and modulator of neurotransmission, yet aligning its pharmacological and physiological effects... (Review)
Review
RATIONALE
The neurosteroid pregnenolone sulfate (PregS) acts as a cognitive enhancer and modulator of neurotransmission, yet aligning its pharmacological and physiological effects with reliable measurements of endogenous local concentrations and pharmacological and therapeutic targets has remained elusive for over 20 years.
OBJECTIVES
New basic and clinical research concerning neurosteroid modulation of the central nervous system (CNS) function has emerged over the past 5 years, including important data involving pregnenolone and various neurosteroid precursors of PregS that point to a need for a critical status update.
RESULTS
Highly specific actions of PregS affecting excitatory N-methyl-D-aspartate receptor (NMDAR)-mediated synaptic transmission and the pharmacological effects of PregS on various receptors and ion channels are discussed. The discovery of a high potency (nanomolar) signal transduction pathway for PregS-induced NMDAR trafficking to the cell surface via a Ca(2+)- and G protein-coupled receptor (GPCR)-dependent mechanism and a potent (EC50 ~ 2 pM) direct enhancement of intracellular Ca(2+) levels is discussed in terms of its agonist effects on long-term potentiation (LTP) and memory. Lastly, preclinical and clinical studies assessing the promnestic effects of PregS and pregnenolone toward cognitive dysfunction in schizophrenia, and altered serum levels in epilepsy and alcohol dependence, are reviewed.
CONCLUSIONS
PregS is present in human and rodent brain at physiologically relevant concentrations and meets most of the criteria for an endogenous neurotransmitter/neuromodulator. PregS likely plays a significant role in modulation of glutamatergic excitatory synaptic transmission underlying learning and memory, yet the molecular target(s) for its action awaits identification.
Topics: Animals; Humans; Neuronal Plasticity; Neurotransmitter Agents; Nootropic Agents; Pregnanolone; Synapses
PubMed: 24997854
DOI: 10.1007/s00213-014-3643-x -
Molecules (Basel, Switzerland) Sep 2021The CB1 cannabinoid receptor is a G-protein coupled receptor highly expressed throughout the central nervous system that is a promising target for the treatment of... (Review)
Review
The CB1 cannabinoid receptor is a G-protein coupled receptor highly expressed throughout the central nervous system that is a promising target for the treatment of various disorders, including anxiety, pain, and neurodegeneration. Despite the wide therapeutic potential of CB1, the development of drug candidates is hindered by adverse effects, rapid tolerance development, and abuse potential. Ligands that produce biased signaling-the preferential activation of a signaling transducer in detriment of another-have been proposed as a strategy to dissociate therapeutic and adverse effects for a variety of G-protein coupled receptors. However, biased signaling at the CB1 receptor is poorly understood due to a lack of strongly biased agonists. Here, we review studies that have investigated the biased signaling profile of classical cannabinoid agonists and allosteric ligands, searching for a potential therapeutic advantage of CB1 biased signaling in different pathological states. Agonist and antagonist bound structures of CB1 and proposed mechanisms of action of biased allosteric modulators are used to discuss a putative molecular mechanism for CB1 receptor activation and biased signaling. Current studies suggest that allosteric binding sites on CB1 can be explored to yield biased ligands that favor or hinder conformational changes important for biased signaling.
Topics: Allosteric Site; Cannabinoid Receptor Agonists; Central Nervous System; Humans; Indoles; Ligands; Models, Molecular; Piperidines; Pregnenolone; Protein Binding; Protein Conformation; Receptor, Cannabinoid, CB1; Signal Transduction
PubMed: 34500853
DOI: 10.3390/molecules26175413 -
The Journal of Biological Chemistry Aug 2023Neurosteroids, which are steroids synthesized by the nervous system, can exert neuromodulatory and neuroprotective effects via genomic and nongenomic pathways. The...
Neurosteroids, which are steroids synthesized by the nervous system, can exert neuromodulatory and neuroprotective effects via genomic and nongenomic pathways. The neurosteroid and major steroid precursor pregnenolone has therapeutical potential in various diseases, such as psychiatric and pain disorders, and may play important roles in myelination, neuroinflammation, neurotransmission, and neuroplasticity. Although pregnenolone is synthesized by CYP11A1 in peripheral steroidogenic organs, our recent study showed that pregnenolone must be synthesized by another mitochondrial cytochrome P450 (CYP450) enzyme other than CYP11A1 in human glial cells. Therefore, we sought to identify the CYP450 responsible for pregnenolone production in the human brain. Upon screening for CYP450s expressed in the human brain that have mitochondrial localization, we identified three enzyme candidates: CYP27A1, CYP1A1, and CYP1B1. We found that inhibition of CYP27A1 through inhibitors and siRNA knockdown did not negatively affect pregnenolone synthesis in human glial cells. Meanwhile, treatment of human glial cells with CYP1A1/CYP1B1 inhibitors significantly reduced pregnenolone production in the presence of 22(R)-hydroxycholesterol. We performed siRNA knockdown of CYP1A1 or CYP1B1 in human glial cells and found that only CYP1B1 knockdown significantly decreased pregnenolone production. Furthermore, overexpression of mitochondria-targeted CYP1B1 significantly increased pregnenolone production under basal conditions and in the presence of hydroxycholesterols and low-density lipoprotein. Inhibition of CYP1A1 and/or CYP1B1 via inhibitors or siRNA knockdown did not significantly reduce pregnenolone synthesis in human adrenal cortical cells, implying that CYP1B1 is not a major pregnenolone-producing enzyme in the periphery. These data suggest that mitochondrial CYP1B1 is involved in pregnenolone synthesis in human glial cells.
Topics: Humans; Brain; Cholesterol Side-Chain Cleavage Enzyme; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Hydroxycholesterols; Mitochondria; Neuroglia; Pregnenolone; RNA, Small Interfering; Steroids
PubMed: 37442234
DOI: 10.1016/j.jbc.2023.105035 -
Neuropharmacology Jul 2021Pregnenolone is a neurosteroid that modulates glial growth and differentiation, neuronal firing, and several brain functions, these effects being attributed to...
Pregnenolone is a neurosteroid that modulates glial growth and differentiation, neuronal firing, and several brain functions, these effects being attributed to pregnenolone actions on the neurons and glial cells themselves. Despite the vital role of the cerebral circulation for brain function and the fact that pregnenolone is a vasoactive agent, pregnenolone action on brain arteries remain unknown. Here, we obtained in vivo concentration response curves to pregnenolone on middle cerebral artery (MCA) diameter in anesthetized male and female C57BL/6J mice. In both male and female animals, pregnenolone (1 nM-100 μM) constricted MCA in a concentration-dependent manner, its maximal effect reaching ~22-35% decrease in diameter. Pregnenolone action was replicated in intact and de-endothelialized, in vitro pressurized MCA segments with pregnenolone evoking similar constriction in intact and de-endothelialized MCA. Neurosteroid action was abolished by 1 μM paxilline, a selective blocker of Ca - and voltage-gated K channels of large conductance (BK). Cell-attached, patch-clamp recordings on freshly isolated smooth muscle cells from mouse MCAs demonstrated that pregnenolone at concentrations that constricted MCAs in vitro and in vivo (10 μM), reduced BK activity (NPo), with an average decrease in NPo reaching 24.2%. The concentration-dependence of pregnenolone constriction of brain arteries and inhibition of BK activity in intact cells were paralleled by data obtained in cell-free, inside-out patches, with maximal inhibition reached at 10 μM pregnenolone. MCA smooth muscle BKs include channel-forming α (slo1 proteins) and regulatory β subunits, encoded by KCNMA1 and KCNMB1, respectively. However, pregnenolone-driven decrease in NPo was still evident in MCA myocytes from KCNMB1 mice. Following reconstitution of slo1 channels into artificial, binary phospholipid bilayers, 10 μM pregnenolone evoked slo1 NPo inhibition which was similar to that seen in native membranes. Lastly, pregnenolone failed to constrict MCA from KCNMA1 mice. In conclusion, pregnenolone constricts MCA independently of neuronal, glial, endothelial and circulating factors, as well as of cell integrity, organelles, complex membrane cytoarchitecture, and the continuous presence of cytosolic signals. Rather, this action involves direct inhibition of SM BK channels, which does not require β subunits but is mediated through direct sensing of the neurosteroid by the channel-forming α subunit.
Topics: Animals; Brain; Cells, Cultured; Cerebral Arteries; Dose-Response Relationship, Drug; Female; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Large-Conductance Calcium-Activated Potassium Channels; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurosteroids; Pregnenolone; Vasoconstriction
PubMed: 34023335
DOI: 10.1016/j.neuropharm.2021.108603 -
Biochemistry. Biokhimiia Sep 2022Cholesterol oxidase is a highly demanded enzyme used in medicine, pharmacy, agriculture, chemistry, and biotechnology. It catalyzes oxidation of 3β-hydroxy-5-ene- to...
Cholesterol oxidase is a highly demanded enzyme used in medicine, pharmacy, agriculture, chemistry, and biotechnology. It catalyzes oxidation of 3β-hydroxy-5-ene- to 3-keto-4-ene- steroids with the formation of hydrogen peroxide. Here, we expressed 6xHis-tagged mature form of the extracellular cholesterol oxidase (ChO) from the actinobacterium Nocardioides simplex VKM Ac-2033D (55.6 kDa) in Escherichia coli cells. The recombinant enzyme (ChO) was purified using affinity chromatography. ChO proved to be functional towards cholesterol, cholestanol, phytosterol, pregnenolone, and dehydroepiandrosterone. Its activity depended on the structure and length of the aliphatic side chain at C17 atom of the steroid nucleus and was lower with pregnenolone and dehydroepiandrosterone. The enzyme was active in a pH range of 5.25÷6.5 with the pH optimum at 6.0. Kinetic assays and storage stability tests demonstrated that the characteristics of ChO were generally comparable with or superior to those of commercial ChO from Streptomyces hygroscopicus (ChO). The results contribute to the knowledge on microbial ChOs and evidence that ChO from N. simplex VKM Ac-2033D is a promising agent for further applications.
Topics: Actinobacteria; Cholestanols; Cholesterol Oxidase; Dehydroepiandrosterone; Hydrogen Peroxide; Phytosterols; Pregnenolone; Steroids
PubMed: 36180991
DOI: 10.1134/S0006297922090048 -
Experimental Neurology May 2023Growing preclinical and clinical evidence highlights neurosteroid pathway imbalances in Parkinson's Disease (PD) and L-DOPA-induced dyskinesias (LIDs). We recently...
Growing preclinical and clinical evidence highlights neurosteroid pathway imbalances in Parkinson's Disease (PD) and L-DOPA-induced dyskinesias (LIDs). We recently reported that 5α-reductase (5AR) inhibitors dampen dyskinesias in parkinsonian rats; however, unraveling which specific neurosteroid mediates this effect is critical to optimize a targeted therapy. Among the 5AR-related neurosteroids, striatal pregnenolone has been shown to be increased in response to 5AR blockade and decreased after 6-OHDA lesions in the rat PD model. Moreover, this neurosteroid rescued psychotic-like phenotypes by exerting marked antidopaminergic activity. In light of this evidence, we investigated whether pregnenolone might dampen the appearance of LIDs in parkinsonian drug-naïve rats. We tested 3 escalating doses of pregnenolone (6, 18, 36 mg/kg) in 6-OHDA-lesioned male rats and compared the behavioral, neurochemical, and molecular outcomes with those induced by the 5AR inhibitor dutasteride, as positive control. The results showed that pregnenolone dose-dependently countered LIDs without affecting L-DOPA-induced motor improvements. Post-mortem analyses revealed that pregnenolone significantly prevented the increase of validated striatal markers of dyskinesias, such as phospho-Thr-34 DARPP-32 and phospho-ERK, as well as D-D receptor co-immunoprecipitation in a fashion similar to dutasteride. Moreover, the antidyskinetic effect of pregnenolone was paralleled by reduced striatal levels of BDNF, a well-established factor associated with the development of LIDs. In support of a direct pregnenolone effect, LC/MS-MS analyses revealed that striatal pregnenolone levels strikingly increased after the exogenous administration, with no significant alterations in downstream metabolites. All these data suggest pregnenolone as a key player in the antidyskinetic properties of 5AR inhibitors and highlight this neurosteroid as an interesting novel tool to target LIDs in PD.
Topics: Male; Rats; Animals; Levodopa; Parkinson Disease; Dutasteride; Oxidopamine; Neurosteroids; Rats, Sprague-Dawley; Dyskinesia, Drug-Induced; Corpus Striatum; Antiparkinson Agents; Disease Models, Animal
PubMed: 36878398
DOI: 10.1016/j.expneurol.2023.114370