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 ResultadosRESUMEN
We report that the neurotrophin receptor p75 contributes to sensory neuron survival through the regulation of cholesterol metabolism in Schwann cells. Selective deletion of p75 in mouse Schwann cells of either sex resulted in a 30% loss of dorsal root ganglia (DRG) neurons and diminished thermal sensitivity. P75 regulates Schwann cell cholesterol biosynthesis in response to BDNF, forming a co-receptor complex with ErbB2 and activating ErbB2-mediated stimulation of sterol regulatory element binding protein 2 (SREBP2), a master regulator of cholesterol synthesis. Schwann cells lacking p75 exhibited decreased activation of SREBP2 and a reduction in 7-dehydrocholesterol (7-DHC) reductase (DHCR7) expression, resulting in accumulation of the neurotoxic intermediate, 7-dehyrocholesterol in the sciatic nerve. Restoration of DHCR7 in p75 null Schwann cells in mice significantly attenuated DRG neuron loss. Together, these results reveal a mechanism by which the disruption of lipid metabolism in glial cells negatively influences sensory neuron survival, which has implications for a wide range of peripheral neuropathies.SIGNIFICANCE STATEMENT Although expressed in Schwann cells, the role of p75 in myelination has remained unresolved in part because of its dual expression in sensory neurons that Schwann cells myelinate. When p75 was deleted selectively among Schwann cells, myelination was minimally affected, while sensory neuron survival was reduced by 30%. The phenotype is mainly due to dysregulation of cholesterol biosynthesis in p75-deficient Schwann cells, leading to an accumulation of neurotoxic cholesterol precursor, 7-dehydrocholesterol (7-DHC). Mechanism-wise, we discovered that in response to BDNF, p75 recruits and activates ErbB2 independently of ErbB3, thereby stimulating the master regulator, sterol regulatory element binding protein 2 (SREBP2). These results together highlight a novel role of p75 in Schwann cells in regulating DRG neuron survival by orchestrating proper cholesterol metabolism.
Asunto(s)
Receptores de Factor de Crecimiento Nervioso/deficiencia , Receptores de Factor de Crecimiento Nervioso/genética , Células de Schwann/metabolismo , Células Receptoras Sensoriales/metabolismo , Animales , Supervivencia Celular/fisiología , Células Cultivadas , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas , Células de Schwann/ultraestructura , Células Receptoras Sensoriales/ultraestructuraRESUMEN
Polypharmacy, or the simultaneous use of multiple drugs to treat a single patient, is a common practice in psychiatry. Unfortunately, data on the health effects of commonly used combinations of medications are very limited. In this study, we therefore investigated the effects and interactions between two commonly prescribed psychotropic medications with sterol inhibiting side effects, trazodone (TRZ), an antidepressant, and aripiprazole (ARI), an antipsychotic. In vitro cell culture experiments revealed that both medications alone disrupted neuronal and astroglial sterol biosynthesis in dose-dependent manners. Furthermore, when ARI and TRZ were combined, exposure resulted in an additive 7-dehydrocholesterol (7-DHC) increase, as well as desmosterol (DES) and cholesterol decreases in both cell types. In adult mice, at baseline, we found that the three investigated sterols showed significant differences in distribution across the eight assessed brain regions. Furthermore, experimental mice treated with ARI or TRZ, or a combination of both medications for 8 days, showed strong sterol disruption across all brain regions. We show ARI or TRZ alone elevated 7-DHC and decreased DES levels in all brain regions, but with regional differences. However, the combined utilization of these two medications for 8 days did not lead to additive changes in sterol disturbances. Based on the complex roles of 7-DHC derived oxysterols, we conclude that individual and potentially simultaneous use of medications with sterol biosynthesis-inhibiting properties might have undesired side effects on the adult brain, with as yet unknown long-term consequences on mental or physical health.
Asunto(s)
Antipsicóticos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Trazodona , Animales , Antidepresivos , Aripiprazol , Encéfalo , Ratones , EsterolesRESUMEN
Ferroptosis is a type of programmed cell death caused by phospholipid peroxidation that has been implicated as a mechanism in several diseases resulting from ischemic-reperfusion injury. Most recently, ferroptosis has been identified as a possible key injury mechanism in neonatal hypoxic-ischemic brain injury (HIBI). This review summarizes the current literature regarding the different ferroptotic pathways, how they may be activated after neonatal HIBI, and which current or investigative interventions may attenuate ferroptotic cell death associated with neonatal HIBI.
Asunto(s)
Lesiones Encefálicas , Ferroptosis , Hipoxia-Isquemia Encefálica , Apoptosis , Muerte Celular , Humanos , Hipoxia-Isquemia Encefálica/metabolismo , Recién Nacido , Peroxidación de LípidoRESUMEN
Cariprazine (CAR) is a strong inhibitor of the Dhcr7 enzyme, the last enzyme in the cholesterol biosynthesis pathway. We assessed the effects of CAR on maternally exposed Dhcr7+/- and wild-type mouse offspring, and tested the biochemical effects of CAR in human serum samples. Dhcr7+/- and wild-type time-pregnant mice were exposed to vehicle or 0.2 mg/kg CAR from E12 to E19. Levels of CAR, CAR metabolites, sterols, and oxysterols were measured in the brain of maternally exposed offspring at various time points using LC-MS/MS. Embryonic exposure to CAR significantly increased levels of 7-DHC in all organs of exposed embryos, with a particularly strong effect in the brain. Detectable levels of CAR and elevated 7-DHC were observed in the brain of newborn pups 14 days after drug exposure. In addition, CAR altered sterol metabolism in all animals analyzed, with the strongest effect on the brain of Dhcr7+/- pups born to Dhcr7+/- dams. Furthermore, CAR elevated toxic oxysterols in the brain of maternally exposed Dhcr7+/- offspring to levels approaching those seen in a mouse model of Smith-Lemli-Opitz syndrome. Finally, we observed that patients taking CAR have elevated 7-DHC in their serum. In summary, maternal DHCR7 heterozygosity, combined with offspring DHCR7 heterozygosity might represent a vulnerability factor to medications that interfere with sterol biosynthesis. Due to the conserved sterol biosynthesis between mice and humans, we suggest that the 1-3% of patient population with single-allele DHCR7 mutations might not be ideal candidates for CAR use, especially if they are nursing, pregnant or plan to become pregnant.
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Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Colesterol/biosíntesis , Exposición Materna/efectos adversos , Piperazinas/administración & dosificación , Piperazinas/efectos adversos , Efectos Tardíos de la Exposición Prenatal/metabolismo , Animales , Antipsicóticos/administración & dosificación , Antipsicóticos/efectos adversos , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Cromatografía Liquida , Femenino , Humanos , Masculino , Ratones , Embarazo , Espectrometría de Masas en TándemRESUMEN
Mutations in both copies in the gene encoding 7-dehydrocholesterol reductase (DHCR7) cause Smith-Lemli-Opitz Syndrome (SLOS), which is characterized by a toxic elevation in 7-dehydrocholesterol (7-DHC). Aripiprazole (ARI) exposure, independent of genetic mutations, also leads to elevation of 7-DHC. We investigated the combined effect of a single-copy Dhcr7+/- mutation and maternal ARI exposure on the developing offspring brain. We generated a time-pregnant mouse model where WT and Dhcr7+/- embryos were maternally exposed to ARI or vehicle (VEH) from E12 to E19 (5 mg/kg). Levels of cholesterol, its precursors, ARI and its metabolites were measured at P0. We found that ARI and its metabolites were transported across the placenta and reached the brain of offspring. Maternal ARI exposure led to decreased viability of embryos and increased 7-DHC levels, regardless of maternal or offspring Dhcr7 genotype. In addition, Dhcr7+/- pups were more vulnerable to maternal ARI exposure than their WT littermates, and maternal Dhcr7+/- genotype also exacerbated offspring response to ARI treatment. Finally, both 7-DHC levels and 7-DHC/cholesterol ratio is the highest in Dhcr7+/- pups from Dhcr7+/- mothers exposed to ARI, underscoring a potentially dangerous interaction between maternal genotype×embryonic genotype×treatment. Our findings have important clinical implications. SLOS patients should avoid drugs that increase 7-DHC levels such as ARI, trazodone and haloperidol. In addition, treatment with 7-DHC elevating substances might be potentially unsafe for the 1-1.5% of population with single-allele disruptions of the DHCR7 gene. Finally, prenatal and parental genetic testing for DHCR7 should be considered before prescribing sterol-interfering medications during pregnancy.
Asunto(s)
Aripiprazol/efectos adversos , Desarrollo Embrionario/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Alelos , Animales , Aripiprazol/metabolismo , Colesterol , Modelos Animales de Enfermedad , Desarrollo Embrionario/genética , Femenino , Masculino , Exposición Materna/efectos adversos , Ratones , Ratones Noqueados , Mutación , Neurogénesis/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Embarazo , Efectos Tardíos de la Exposición Prenatal , Síndrome de Smith-Lemli-OpitzRESUMEN
Cholesterol serves as a building material for cellular membranes and plays an important role in cellular metabolism. The brain relies on its own cholesterol biosynthesis, which starts during embryonic development. Cholesterol is synthesized from two immediate precursors, desmosterol and 7-dehydrocholesterol (7-DHC). Mutations in the DHCR24 enzyme, which converts desmosterol into cholesterol, lead to desmosterolosis, an autosomal recessive developmental disorder. In this study, we assessed the brain content of desmosterol, 7-DHC, and cholesterol from development to adulthood, and analyzed the biochemical, molecular, and anatomical consequences of Dhcr24 mutations on the sterol profile in a mouse model of desmosterolosis and heterozygous Dhcr24+/- carriers. Our HPLC-MS/MS studies revealed that by P0 desmosterol almost entirely replaced cholesterol in the Dhcr24-KO brain. The greatly elevated desmosterol levels were also present in the Dhcr24-Het brains irrespective of maternal genotype, persisting into adulthood. Furthermore, Dhcr24-KO mice brains showed complex changes in expression of lipid and sterol transcripts, nuclear receptors, and synaptic plasticity transcripts. Cultured Dhcr24-KO neurons showed increased arborization, which was also present in the Dhcr24-KO mouse brains. Finally, we observed a shared pathophysiological mechanism between the mouse models of desmosterolosis and Smith-Lemli-Opitz syndrome (a genetic disorder of conversion of 7-DHC to cholesterol).
Asunto(s)
Anomalías Múltiples/metabolismo , Encéfalo/metabolismo , Colesterol/biosíntesis , Errores Innatos del Metabolismo Lipídico/metabolismo , Anomalías Múltiples/genética , Animales , Membrana Celular/metabolismo , Deshidrocolesteroles/metabolismo , Desmosterol/metabolismo , Femenino , Homeostasis , Errores Innatos del Metabolismo Lipídico/genética , Masculino , Ratones , Mutación , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Síndrome de Smith-Lemli-Opitz , Esteroles/metabolismo , Espectrometría de Masas en TándemRESUMEN
While antipsychotic medications provide important relief from debilitating psychotic symptoms, they also have significant adverse side effects, which might have relevant impact on human health. Several research studies, including ours, have shown that commonly used antipsychotics such as haloperidol and aripiprazole affect cholesterol biosynthesis at the conversion of 7-dehydrocholesterol (7-DHC) to cholesterol. This transformation is promoted by the enzyme DHCR7 and its inhibition causes increases in plasma and tissue levels of 7-DHC. The inhibition of this enzymatic step by mutations in the Dhcr7 gene leads to Smith-Lemli-Opitz syndrome, a devastating human condition that can be replicated in rats by small molecule inhibitors of DHCR7. The fact that two compounds, brexpiprazole and cariprazine, that were recently approved by the FDA have substructural elements in common with the DHCR7 inhibitor aripiprazole, prompted us to evaluate the effect of brexpiprazole and cariprazine on cholesterol biosynthesis. We report that cariprazine affects levels of 7-DHC and cholesterol in cell culture incubations at concentrations as low as 5â¯nM. Furthermore, a common metabolite of cariprazine and aripiprazole, 2,3-(dichlorophenyl) piperazine, inhibits DHCR7 activity at concentrations comparable to those of the potent teratogen AY9944. The cell culture experiments were corroborated in mice in studies showing that treatment with cariprazine elevated 7-DHC in brain and serum. The consequences of sterol inhibition by antipsychotics in the developing nervous system and the safety of their use during pregnancy remains to be established.
Asunto(s)
Antipsicóticos/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Piperazinas/farmacología , Animales , Anticolesterolemiantes/farmacología , Química Encefálica/efectos de los fármacos , Línea Celular , Colesterol/biosíntesis , Deshidrocolesteroles/metabolismo , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Smith-Lemli-Opitz syndrome is a recessive disorder caused by mutations in 7-dehydrocholesterol reductase (DHCR)7 with a heterozygous (HET) carrier frequency of 1-3%. A defective DHCR7 causes accumulation of 7-dehydrocholesterol (DHC), which is a highly oxidizable and toxic compound. Recent studies suggest that several antipsychotics, including the highly prescribed pharmaceuticals, aripiprazole (ARI) and trazodone (TRZ), increase 7-DHC levels in vitro and in humans. Our investigation was designed to compare the effects of ARI and TRZ on cholesterol (Chol) synthesis in fibroblasts from DHCR7+/- human carriers and controls (CTRs). Six matched pairs of fibroblasts were treated and their sterol profile analyzed by LC-MS. Significantly, upon treatment with ARI and TRZ, the total accumulation of 7-DHC was higher in DHCR7-HET cells than in CTR fibroblasts. The same set of experiments was repeated in the presence of 13C-lanosterol to determine residual Chol synthesis, revealing that ARI and TRZ strongly inhibit de novo Chol biosynthesis. The results suggest that DHCR7 carriers have increased vulnerability to both ARI and TRZ exposure compared with CTRs. Thus, the 1-3% of the population who are DHCR7 carriers may be more likely to sustain deleterious health consequences on exposure to compounds like ARI and TRZ that increase levels of 7-DHC, especially during brain development.
Asunto(s)
Aripiprazol/farmacología , Inhibidores Enzimáticos/farmacología , Mutación , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Trazodona/farmacología , Adulto , Colesterol/biosíntesis , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Síndrome de Smith-Lemli-Opitz/enzimología , Síndrome de Smith-Lemli-Opitz/genéticaRESUMEN
Lipid peroxidation is a well-known process that has been implicated in many diseases. Recent evidence has shown that mitochondrial cholesterol levels are increased under specific conditions, making it an important target for peroxidation inside the mitochondria. Cholesterol peroxidation generates, as primary products, several hydroperoxides (ChOOH), which can react with transition metals and metalloproteins. In this sense, cytochrome c (CYTC), a heme protein largely found in the mitochondria, becomes a candidate to react with ChOOH. Using CYTC associated with SDS micelles to mimic mitochondrial conditions, we show that ChOOH induces dose-dependent CYTC Soret band bleaching, indicating that it is using ChOOH as a substrate. This reaction leads to protein oligomerization, suggesting the formation of a protein radical that, subsequently, recombines, giving dimers, trimers, and tetramers. EPR experiments confirmed the production of carbon-centered radicals from both protein and lipid in the presence of ChOOH. Similar results were obtained with linoleic acid hydroperoxides (LAOOH). In addition, replacing SDS micelles by cardiolipin-containing liposomes as the mitochondrial mimetic led to similar results with either ChOOH or LAOOH. Importantly, kinetic experiments show that CYTC bleaching is faster with ChOOH than with H2O2, suggesting that these hydroperoxides could be relevant substrates for CYTC peroxidase-like activity in biological media. Altogether, these results show that CYTC induces homolytic cleavage of lipid-derived hydroperoxides, producing lipid and protein radicals.
Asunto(s)
Colesterol/análogos & derivados , Citocromos c/química , Radicales Libres/química , Animales , Bovinos , Colesterol/química , Peróxido de Hidrógeno/química , Cinética , Ácidos Linoleicos/química , Peroxidación de Lípido , Peróxidos Lipídicos/química , Liposomas , Micelas , Polimerizacion , Piridinas/química , Dodecil Sulfato de SodioRESUMEN
Cholesterol undergoes ozonolysis to afford a variety of oxysterol products, including cholesterol-5,6-epoxide (CholEp) and the isomeric aldehydes secosterol A (seco A) and secosterol B (seco B). These oxysterols display numerous important biological activities, including protein adduction; however, much remains to be learned about the identity of the reactive species and the range of proteins modified by these oxysterols. Here, we synthesized alkynyl derivatives of cholesterol-derived oxysterols and employed a straightforward detection method to establish secosterols A and B as the most protein-reactive of the oxysterols tested. Model adduction studies with an amino acid, peptides, and proteins provide evidence for the potential role of secosterol dehydration products in protein adduction. Hydrophobic separation methods-Folch extraction and solid phase extraction (SPE)-were successfully applied to enrich oxysterol-adducted peptide species, and LC-MS/MS analysis of a model peptide-seco adduct revealed a unique fragmentation pattern (neutral loss of 390 Da) for that species. Coupling a hydrophobic enrichment method with proteomic analysis utilizing characteristic fragmentation patterns facilitates the identification of secosterol-modified peptides and proteins in an adducted protein. More broadly, these improved enrichment methods may give insight into the role of oxysterols and ozone exposure in the pathogenesis of a variety of diseases, including atherosclerosis, Alzheimer's disease, Parkinson's disease, and asthma.
Asunto(s)
Colesterol/química , Ozono/química , Péptidos/química , Proteínas/química , Aldehídos/química , Secuencia de Aminoácidos , Biotina/química , Colesterol/análogos & derivados , Cromatografía Líquida de Alta Presión , Química Clic , Citocromos c/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Isomerismo , Datos de Secuencia Molecular , Péptidos/análisis , Albúmina Sérica/química , Extracción en Fase Sólida , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Estreptavidina/químicaRESUMEN
Neonatal hypoxic-ischemic brain injury (HIBI) results in part from excess reactive oxygen species and iron-dependent lipid peroxidation (i.e. ferroptosis). The vitamin D precursor 7-dehydrocholesterol (7-DHC) may inhibit iron-dependent lipid peroxidation. Primary neurons underwent oxygen and glucose deprivation (OGD) injury and treatment with 7-DHC-elevating medications such as cariprazine (CAR) or vehicle. Postnatal day 9 mice underwent sham surgery or carotid artery ligation and hypoxia and received intraperitoneal CAR. In neurons, CAR administration resulted in significantly increased cell survival compared to vehicle controls, whether administered 48 h prior to or 30 min after OGD, and was associated with increased 7-DHC. In the mouse model, malondialdehyde and infarct area significantly increased after HIBI in the vehicle group, which were attenuated by post-treatment with CAR and were negatively correlated with tissue 7-DHC concentrations. Elevating 7-DHC concentrations with CAR was associated with improved cellular and tissue viability after hypoxic-ischemic injury, suggesting a novel therapeutic avenue.
Asunto(s)
Deshidrocolesteroles , Ferroptosis , Hipoxia-Isquemia Encefálica , Animales , Ratones , Animales Recién Nacidos , Encéfalo , Hipoxia/complicaciones , Oxígeno/uso terapéutico , Isquemia/complicaciones , Hierro/uso terapéuticoRESUMEN
Lipid modifications aid in regulating (and misregulating) protein function and localization. However, efficient methods to screen for a lipid's ability to modify proteins are not readily available. We present a strategy to identify protein-reactive lipids and apply it to a neurodevelopmental disorder, Smith-Lemli-Opitz syndrome (SLOS). Alkynyl surrogates were synthesized for polyunsaturated fatty acids, phospholipids, cholesterol, 7-dehydrocholesterol (7-DHC), and a 7-DHC-derived oxysterol. To probe for protein-reactive lipids, we used click chemistry to biotinylate the alkynyl tag and detected the lipid-adducted proteins with streptavidin Western blotting. In Neuro2a cells, the trend in amount of protein adduction followed known rates of lipid peroxidation (7-DHC >> arachidonic acid > linoleic acid >> cholesterol), with alkynyl-7-DHC producing the most adduction among alkynyl lipids. 7-DHC reductase-deficient cells, which cannot properly metabolize 7-DHC, exhibited significantly more alkynyl-7-DHC-protein adduction than control cells. Model studies demonstrated that a 7-DHC peroxidation product covalently modifies proteins. We hypothesize that 7-DHC generates electrophiles that can modify the proteome, contributing to SLOS's complex pathology. These probes and methods would allow for analysis of lipid-modified proteomes in SLOS and other disorders exhibiting 7-DHC accumulation. More broadly, the alkynyl lipid library would facilitate exploration of lipid peroxidation's role in specific biological processes in numerous diseases.
Asunto(s)
Procesamiento Proteico-Postraduccional , Síndrome de Smith-Lemli-Opitz/metabolismo , Línea Celular Tumoral , Citocromos c/química , Citocromos c/metabolismo , Deshidrocolesteroles/química , Deshidrocolesteroles/metabolismo , Ácidos Grasos Insaturados/química , Ácidos Grasos Insaturados/metabolismo , Humanos , Lipoilación , Oxidación-Reducción , Fosfolípidos/química , Fosfolípidos/metabolismo , Proteoma/metabolismo , Síndrome de Smith-Lemli-Opitz/diagnósticoRESUMEN
Mitochondrial cholesterol has been reported to be increased under specific pathological conditions associated with enhanced oxidative stress parameters. In this scenario, cholesterol oxidation would be increased, leading to the production of reactive aldehydes, including cholesterol carboxyaldehyde (ChAld). By using SDS micelles as a mitochondrial mimetic model, we have demonstrated that ChAld covalently modifies cytochrome c (cytc), a protein known to participate in electron transport and apoptosis signaling. This mimetic model induces changes in cytc structure in the same way as mitochondrial membranes do. Tryptic digestion of the cytc-ChAld adduct followed by MALDI-TOF/TOF analyses revealed that modifications occur at Lys residues (K22) localized at cytc site L, a site involved in protein-protein and protein-membrane interactions. Interestingly, ChAld ligation prevented cytc detachment from liposomes even under high ionic strength conditions. Overall, it can be concluded that ChAld ligation to Lys residues at site L creates a hydrophobic tail at cytc, which promotes cytc anchoring to the membrane. Although not investigated in detail in this study, cytc adduction to cholesterol derived aldehydes could have implications in cytc release from mitochondria under apoptotic stimuli.
Asunto(s)
Aldehídos/química , Materiales Biomiméticos/metabolismo , Colesterol/análogos & derivados , Colesterol/química , Citocromos c/metabolismo , Liposomas/metabolismo , Mitocondrias/metabolismo , Aldehídos/toxicidad , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Materiales Biomiméticos/química , Bovinos , Citocromos c/química , Transporte de Electrón/efectos de los fármacos , Luz , Liposomas/química , Lisina/química , Modelos Moleculares , Miocardio/metabolismo , Oxidación-Reducción , Péptidos/análisis , Péptidos/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Tripsina/metabolismoRESUMEN
Medications having the unwanted side effect of inhibiting 7-dehydrocholesterol reductase (DHCR7), one of the last enzymes in the cholesterol biosynthesis pathway, account for about 300 million yearly prescriptions in the United States. Many of these drugs are currently prescribed to pregnant women. Many DHCR7-inhibiting medications share chemical similarities, which can be the active substructure responsible for the medication affinity to the enzyme. This work highlights a computational strategy to identify enriched fragments in a set of DHCR7-inhibiting medications. The computational approach used here involves systematic fragmentation of molecules using the molBLOCKS tool, followed by enrichment analysis. The results of this approach highlight putative pharmacophores that might be responsible for the DHCR7-inhibiting activity of some of these medications. The identification of DHCR7-inhibiting substructures is an important step toward knowledge-based drug development and can improve the neurodevelopmental safety of medications.
RESUMEN
Hypoxic-ischemic brain injury (HIBI) leads to depletion of ATP, mitochondrial dysfunction, and enhanced oxidant formation. Measurement of acylcarnitines may provide insight into mitochondrial dysfunction. Plasma acylcarnitine levels are altered in neonates after an HIBI, but individual acylcarnitine levels in the brain have not been evaluated. Additionally, it is unknown if plasma acylcarnitines reflect brain acylcarnitine changes. In this study, postnatal day 9 CD1 mouse pups were randomized to HIBI induced by carotid artery ligation, followed by 30 min at 8% oxygen, or to sham surgery and normoxia, with subgroups for tissue collection at 30 min, 24 h, or 72 h after injury (12 animals/group). Plasma, liver, muscle, and brain (dissected into the cortex, cerebellum, and striatum/thalamus) tissues were collected for acylcarnitine analysis by LC-MS. At 30 min after HIBI, acylcarnitine levels were significantly increased, but the differences resolved by 24 h. Palmitoylcarnitine was increased in the cortex, muscle, and plasma, and stearoylcarnitine in the cortex, striatum/thalamus, and cerebellum. Other acylcarnitines were elevated only in the muscle and plasma. In conclusion, although plasma acylcarnitine results in this study mimic those seen previously in humans, our data suggest that the plasma acylcarnitine profile was more reflective of muscle changes than brain changes. Acylcarnitine metabolism may be a target for therapeutic intervention after neonatal HIBI, though the lack of change after 30 min suggests a limited therapeutic window.
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Cholesterol is ubiquitous in cells; it plays a critical role in membrane structure and transport as well as in intracellular trafficking processes. There are suggestions that cholesterol metabolism is linked to innate immunity with inhibitors of DHCR7, the last enzyme in the cholesterol pathway, suggested to have potential as viral therapeutics nearly a decade ago. In fact, there are a number of highly prescribed pharmaceuticals that are off-target inhibitors of DHCR7, causing increased cellular levels of 7-dehydrodesmosterol (7-DHD) and 7-dehydrocholesterol (7-DHC). We report here dose-response studies of six such inhibitors on late-stage cholesterol biosynthesis in Neuro2a cells as well as their effect on infection of vesicular stomatitis virus (VSV). Four of the test compounds are FDA-approved drugs (cariprazine, trazodone, metoprolol, and tamoxifen), one (ifenprodil) has been the object of a recent Phase 2b COVID trial, and one (AY9944) is an experimental compound that has seen extensive use as a DHCR7 inhibitor. The three FDA-approved drugs inhibit replication of a GFP-tagged VSV with efficacies that mirror their effect on DHCR7. Ifenprodil and AY9944 have complex inhibitory profiles, acting on both DHCR7 and DHCR14, while tamoxifen does not inhibit DHCR7 and is toxic to Neuro2a at concentrations where it inhibits the Δ7-Δ8 isomerase of the cholesterol pathway. VSV itself affects the sterol profile in Neuro2a cells, showing a dose-response increase of dehydrolathosterol and lathosterol, the substrates for DHCR7, with a corresponding decrease in desmosterol and cholesterol. 7-DHD and 7-DHC are orders of magnitude more vulnerable to free radical chain oxidation than other sterols as well as polyunsaturated fatty esters, and the effect of these sterols on viral infection is likely a reflection of this fact of Nature.
RESUMEN
Pancreatic cancer (PC) is characterized by metabolic deregulations that often manifest as deviations in metabolite levels and aberrations in their corresponding metabolic genes across the clinical specimens and preclinical PC models. Cholesterol is one of the critical metabolites supporting PC, synthesized or acquired by PC cells. Nevertheless, the significance of the de novo cholesterol synthesis pathway has been controversial in PC, indicating the need to reassess this pathway in PC. We utilized preclinical models and clinical specimens of PC patients and cell lines and utilized mass spectrometry-based sterol analysis. Further, we also performed in silico analysis to corroborate the significance of de novo cholesterol synthesis pathway in PC. Our results demonstrated alteration in free sterol levels, including free cholesterol, across in vitro, in vivo, and clinical specimens of PC. Especially, our sterol analyses established consistent alterations in free cholesterol across the different PC models. Overall, this study demonstrates the significance and consistency in deviation of cholesterol synthesis pathway in PC while showing the aberrations in sterol metabolite intermediates and the related genes using preclinical models, in silico platforms, and the clinical specimens.
RESUMEN
Genes and environment interact during intrauterine life, and potentially alter the developmental trajectory of the brain. This can result in life-long consequences on brain function. We have previously developed two transgenic mouse lines that suppress Gad1 expression in parvalbumin (PVALB) and neuropeptide Y (NPY) expressing interneuron populations using a bacterial artificial chromosome (BAC)-driven miRNA-based silencing technology. We were interested to assess if maternal immune activation (MIA), genetic interneuronal inhibition, and the combination of these two factors disrupt and result in long-term changes in neuroinflammatory gene expression, sterol biosynthesis, and acylcarnitine levels in the brain of maternally exposed offspring. Pregnant female WT mice were given a single intraperitoneal injection of saline or polyinosinic-polycytidilic acid [poly(I:C)] at E12.5. Brains of offspring were analyzed at postnatal day 90. We identified complex and persistent neuroinflammatory gene expression changes in the hippocampi of MIA-exposed offspring, as well in the hippocampi of Npy/Gad1 and Pvalb/Gad1 mice. In addition, both MIA and genetic inhibition altered the post-lanosterol sterol biosynthesis in the neocortex and disrupted the typical acylcarnitine profile. In conclusion, our findings suggest that both MIA and inhibition of interneuronal function have long-term consequences on critical homeostatic mechanisms of the brain, including immune function, sterol levels, and energy metabolism.
Asunto(s)
Mediadores de Inflamación/inmunología , Interneuronas/inmunología , Neuroinmunomodulación/fisiología , Efectos Tardíos de la Exposición Prenatal/genética , Efectos Tardíos de la Exposición Prenatal/inmunología , Animales , Femenino , Glutamato Descarboxilasa/deficiencia , Glutamato Descarboxilasa/genética , Hipocampo/efectos de los fármacos , Hipocampo/inmunología , Hipocampo/metabolismo , Mediadores de Inflamación/metabolismo , Inductores de Interferón/toxicidad , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neocórtex/efectos de los fármacos , Neocórtex/inmunología , Neocórtex/metabolismo , Neuroinmunomodulación/efectos de los fármacos , Poli I-C/toxicidad , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Efectos Tardíos de la Exposición Prenatal/metabolismoRESUMEN
The last step of cholesterol biosynthesis is the conversion of 7-dehydrocholesterol (7-DHC) into cholesterol, a reaction catalyzed by dehydrocholesterol reductase 7 (DHCR7). Investigation of the effect of Dhcr7 single-allele mutations on the metabolism of aripiprazole (ARI) and cariprazine (CAR) in maternally exposed transgenic pups revealed that ARI, CAR, and their active metabolites were decreased in the liver and brain of Dhcr7 +/- . This difference in the drug and metabolite levels resulted in an increased turnover of ARI and CAR in tissues from Dhcr7 +/- animals, indicating an enhanced metabolism, which was at least partially due to increased levels of Cyp2d6 in the liver of Dhcr7 +/- mice. Finally, experiments with both WT and DHCR7 +/- human fibroblasts revealed lower drug levels in DHCR7 +/- heterozygous cells. Our findings have potential clinical implications, as DHCR7 heterozygosity is present in 1-3% in the human population, and these individuals might have reduced therapeutic levels of Cyp2d6-metabolized medications and are putatively more susceptible to unwanted side effects.