RESUMEN
Cholesterol metabolism dysregulation is associated with several neurological disorders. In Huntington's disease (HD), several enzymes involved in cholesterol metabolism are downregulated, among which the neuronal cholesterol 24-hydroxylase, CYP46A1, is of particular interest. The restoration of CYP46A1 expression in striatal neurons of HD mouse models is beneficial for motor behavior, cholesterol metabolism, transcriptomic activity, and alleviates neuropathological hallmarks induced by mHTT. Among the genes regulated after CYP46A1 restoration, those involved in cholesterol synthesis and efflux may explain the positive effect of CYP46A1 on cholesterol precursor metabolites. Since cholesterol homeostasis results from a fine-tuning between neurons and astrocytes, we quantified the distribution of key genes regulating cholesterol metabolism and efflux in astrocytes and neurons using in situ hybridization coupled with S100ß and NeuN immunostaining, respectively. Neuronal expression of CYP46A1 in the striatum of HD zQ175 mice increased key cholesterol synthesis driver genes (Hmgcr, Dhcr24), specifically in neurons. This effect was associated with an increase of the srebp2 transcription factor gene that regulates most of the genes encoding for cholesterol enzymes. However, the cholesterol efflux gene, ApoE, was specifically upregulated in astrocytes by CYP46A1, probably though a paracrine effect. In summary, the neuronal expression of CYP46A1 has a dual and specific effect on neurons and astrocytes, regulating cholesterol metabolism. The neuronal restoration of CYP46A1 in HD paves the way for future strategies to compensate for mHTT toxicity.
Asunto(s)
Enfermedad de Huntington , Ratones , Animales , Colesterol 24-Hidroxilasa/genética , Enfermedad de Huntington/metabolismo , Neuronas/metabolismo , Colesterol/metabolismo , Homeostasis , Modelos Animales de Enfermedad , Cuerpo Estriado/metabolismoRESUMEN
Efavirenz (EFV), an FDA-approved anti-HIV drug, has off-target binding to CYP46A1, the CNS enzyme which converts cholesterol to 24-hydroxycholesterol. At small doses, EFV allosterically activates CYP46A1 in mice and humans and mitigates some of the Alzheimer's disease manifestations in 5XFAD mice, an animal model. Notably, in vitro, all phase 1 EFV hydroxymetabolites activate CYP46A1 as well and bind either to the allosteric site for EFV, neurotransmitters or both. Herein, we treated 5XFAD mice with 8,14-dihydroxyEFV, the binder to the neurotransmitter allosteric site, which elicits the highest CYP46A1 activation in vitro. We found that treated animals of both sexes had activation of CYP46A1 and cholesterol turnover in the brain, decreased content of the amyloid beta 42 peptide, increased levels of acetyl-CoA and acetylcholine, and altered expression of the brain marker proteins. In addition, male mice had improved performance in the Barnes Maze test and increased expression of the acetylcholine-related genes. This work expands our knowledge of the beneficial CYP46A1 activation effects and demonstrates that 8,14-dihydroxyEFV crosses the blood-brain barrier and has therapeutic potential as a CYP46A1 activator.
Asunto(s)
Acetilcolina , Enfermedad de Alzheimer , Encéfalo , Colesterol 24-Hidroxilasa , Acetilcolina/análisis , Acetilcolina/metabolismo , Alquinos/metabolismo , Alquinos/farmacología , Alquinos/uso terapéutico , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Benzoxazinas/metabolismo , Benzoxazinas/farmacología , Benzoxazinas/uso terapéutico , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Colesterol/metabolismo , Colesterol 24-Hidroxilasa/genética , Colesterol 24-Hidroxilasa/metabolismo , Colesterol 24-Hidroxilasa/farmacología , Ciclopropanos/metabolismo , Ciclopropanos/farmacología , Ciclopropanos/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Masculino , RatonesRESUMEN
BACKGROUND: Patients with multiple sclerosis (MS) have significantly lower vitamin D levels. Cholesterol is known to be the precursor for vitamin D synthesis, and cholesterol removal is regulated by cholesterol 7α-hydroxylase (CYP7A1) in the liver and cholesterol 24S-hydroxylase (CYP46A1) in the brain. In this study, single nucleotide polymorphisms (SNPs) within the genes CYP7A1 (rs3808607) and CYP46A1 (rs754203) were investigated for their effects on serum lipid profiles, vitamin D levels, and the risk of developing MS. METHODS: Patients with MS (n = 191) and controls (n = 100) were tested using the PCR-RFLP method to determine their genotypes for rs3808607 and rs754203 SNPs. RESULTS: The minor (C) allele frequency for CYP7A1 rs3808607 variation was 0.380 in patients with MS and 0.305 in control subjects (P = .074). For CYP46A1 rs754203, the frequencies of the minor (C) allele were 0.272 and 0.250 in patients and control subjects, respectively (P = .563). Serum vitamin D (25(OH)D3) concentrations were significantly lower in patients than in control subjects (P = .002). The CYP46A1 rs754203 SNP was associated with total cholesterol levels in patients, whereas the CYP7A1 rs3808607 variant was not associated with serum lipid parameters or vitamin D levels in patients or control subjects. CONCLUSION: CYP7A1 rs3808607 and CYP46A1 rs754203 variations are not likely to confer an independent risk for MS development in the Turkish population. To the best of our knowledge, this is the first study to investigate the association between CYP46A1 rs754203 and MS risk.
Asunto(s)
Colesterol 24-Hidroxilasa , Colesterol 7-alfa-Hidroxilasa , Esclerosis Múltiple , Colesterol , Colesterol 24-Hidroxilasa/genética , Colesterol 7-alfa-Hidroxilasa/genética , Humanos , Intrones , Lípidos/sangre , Esclerosis Múltiple/epidemiología , Esclerosis Múltiple/genética , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas , Turquía/epidemiología , Vitamina D/sangreRESUMEN
Cholesterol and 24-hydroxycholesterol are the most abundant brain sterols and represent the substrate and product, respectively, of cytochrome P450 46A1 (CYP46A1), a CNS-specific enzyme. CYP46A1 controls cholesterol elimination and turnover in the brain, the two processes that determine the rate of brain sterol flux through the plasma membranes and thereby the properties of these membranes. Brain sterol flux is decreased in Cyp46a1-/- mice compared to wild-type mice and increased in 5XFAD mice (a model of Alzheimer's disease) when they are treated with a small dose of efavirenz, a CYP46A1 activator. Herein, we first assessed the brain proteome (synaptosomal fractions) and phospho-proteome (synaptosomal fractions and brain homogenates) of efavirenz-treated and control 5XFAD mice. Then, based on the pattern of protein abundance change, we conducted acetyl-CoA measurements (brain homogenates and mitochondria) and metabolic profiling (brain homogenates). The phospho-proteomics datasets were used for comparative analyses with the datasets obtained by us previously on mice with the same changes (efavirenz-treated and control 5XFAD mice from a different treatment paradigm) or with changes in the opposite direction (Cyp46a1-/- vs wild-type mice) in brain sterol flux. We found that CYP46A1 activity or the rate of brain sterol flux affects acetyl-CoA-related metabolic pathways as well as phosphorylation of cytoskeletal and other proteins. Knowledge of the key roles of acetyl-CoA and cytoskeletal phosphorylation in cell biology expands our understanding of the significance of CYP46A1-mediated cholesterol 24-hydroxylation in the brain and provides an additional explanation for why CYP46A1 activity modulations are beneficial in mouse models of different brain diseases.
Asunto(s)
Acetilcoenzima A/metabolismo , Encéfalo/metabolismo , Colesterol 24-Hidroxilasa/metabolismo , Proteínas del Citoesqueleto/metabolismo , Esteroles/metabolismo , Acetilcoenzima A/genética , Animales , Colesterol 24-Hidroxilasa/genética , Proteínas del Citoesqueleto/genética , Masculino , Metabolómica/métodos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosforilación/fisiologíaRESUMEN
The human microsomal cytochrome P450 enzyme CYP46A1 plays a crucial role in cholesterol elimination from the brain. It performs a 24-hydroxylation of cholesterol and is of outstanding significance for memory and cognition. This study demonstrates the catalytic activity of human CYP46A1 towards an anabolic androgenic steroid, oral turinabol (dehydrochloromethyltestosterone, 4-chloro-17ß-dihydroxy,17α-methylandrosta-1,4-dien-3-one), which is a doping substance. CYP46A1 is the first human microsomal steroid-converting P450 showing activity towards this xenobiotic compound. Furthermore, the inhibitory effect of oral turinabol on the cholesterol conversion has been investigated in vitro demonstrating competition of the two substrates on the active site of CYP46A1 which might be of importance for potential pathogenic effects of oral turinabol. The conversion of oral turinabol was found to be selective resulting in the formation of only one product, as shown by HPLC analysis. To produce sufficient amounts of this product for NMR analysis, a system expressing human full-length CYP46A1 and CPR on a bicistronic vector was successfully developed realizing the selective cholesterol 24-hydroxylation in E. coli in mg amounts. Using this novel whole-cell system, the conversion of oral turinabol was performed and the product of this conversion by CYP46A1 was isolated and identified as 16ß-hydroxy oral turinabol by NMR.
Asunto(s)
Anabolizantes/farmacología , Colesterol 24-Hidroxilasa/metabolismo , Testosterona/análogos & derivados , Encéfalo/enzimología , Colesterol 24-Hidroxilasa/genética , Escherichia coli/enzimología , Escherichia coli/genética , Humanos , Simulación del Acoplamiento Molecular , Oxandrolona/farmacología , Testosterona/farmacologíaRESUMEN
During brain development, Uncoordinated locomotion 5 (UNC5) receptors control axonal extension through their sensing of the guidance molecule Netrin-1. The correct positioning of receptors into cholesterol-enriched membrane raft microdomains is crucial for the efficient transduction of the recognized signals. However, whether such microdomains are required for the appropriate axonal guidance mediated by UNC5 receptors remains unknown. Here, we combine the use of confocal microscopy, live-cell FRAP analysis and single-particle tracking PALM to characterize the distribution of UNC5 receptors into raft microdomains, revealing differences in their membrane mobility properties. Using pharmacological and genetic approaches in primary neuronal cultures and brain cerebellar explants we further demonstrate that disrupting raft microdomains inhibits the chemorepulsive response of growth cones and axons against Netrin-1. Together, our findings indicate that the distribution of all UNC5 receptors into cholesterol-enriched raft microdomains is heterogeneous and that the specific localization has functional consequences for the axonal chemorepulsion against Netrin-1.
Asunto(s)
Microdominios de Membrana/metabolismo , Receptores de Netrina/metabolismo , Netrina-1/metabolismo , Animales , Axones/metabolismo , Células Cultivadas , Colesterol/metabolismo , Colesterol 24-Hidroxilasa/genética , Colesterol 24-Hidroxilasa/metabolismo , Femenino , Recuperación de Fluorescencia tras Fotoblanqueo , Células HEK293 , Humanos , Ratones , Receptores de Netrina/genética , Neuronas/citología , Neuronas/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismoRESUMEN
Cholesterol 24-hydroxylase (CH24H) is a brain-specific enzyme that converts cholesterol into 24S-hydroxycholesterol, the primary mechanism of cholesterol catabolism in the brain. The therapeutic potential of CH24H activation has been extensively investigated, whereas the effects of CH24H inhibition remain poorly characterized. In this study, the therapeutic potential of CH24H inhibition was investigated using a newly identified small molecule, soticlestat (TAK-935/OV935). The biodistribution and target engagement of soticlestat was assessed in mice. CH24H-knockout mice showed a substantially lower level of soticlestat distribution in the brain than wild-type controls. Furthermore, brain-slice autoradiography studies demonstrated the absence of [3H]soticlestat staining in CH24H-knockout mice compared with wild-type mice, indicating a specificity of soticlestat binding to CH24H. The pharmacodynamic effects of soticlestat were characterized in a transgenic mouse model carrying mutated human amyloid precursor protein and presenilin 1 (APP/PS1-Tg). These mice, with excitatory/inhibitory imbalance and short life-span, yielded a remarkable survival benefit when bred with CH24H-knockout animals. Soticlestat lowered brain 24S-hydroxycholesterol in a dose-dependent manner and substantially reduced premature deaths of APP/PS1-Tg mice at a dose lowering brain 24S-hydroxycholesterol by approximately 50%. Furthermore, microdialysis experiments showed that soticlestat can suppress potassium-evoked extracellular glutamate elevations in the hippocampus. Taken together, these data suggest that soticlestat-mediated inhibition of CH24H may have therapeutic potential for diseases associated with neural hyperexcitation.
Asunto(s)
Colesterol 24-Hidroxilasa/antagonistas & inhibidores , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Piperidinas/farmacología , Piridinas/farmacología , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encefalopatías/tratamiento farmacológico , Encefalopatías/metabolismo , Encefalopatías/fisiopatología , Colesterol 24-Hidroxilasa/deficiencia , Colesterol 24-Hidroxilasa/genética , Inhibidores Enzimáticos del Citocromo P-450/química , Inhibidores Enzimáticos del Citocromo P-450/farmacocinética , Modelos Animales de Enfermedad , Desarrollo de Medicamentos , Femenino , Humanos , Hidroxicolesteroles/metabolismo , Longevidad/efectos de los fármacos , Longevidad/genética , Longevidad/fisiología , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Piperidinas/química , Piperidinas/farmacocinética , Presenilina-1/genética , Presenilina-1/metabolismo , Piridinas/química , Piridinas/farmacocinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Abnormal lipid droplet (LD) metabolism causes a variety of disorders, especially to nonalcoholic fatty liver disease (NAFLD). But the mechanism of abnormal aggregation of LD is still not fully elucidated. Here, Genome-wide CRISPR-Cas9 knockout (GeCKO) screening was employed to identify candidate genes regulating LD metabolism in L02 cell. We analyzed simultaneously the transcriptomics of liver tissues of NAFLD to find potential genes involved in pathogenesis of NAFLD. After integration these data, we found that the expression of 43 candidate genes from the GeCKO screening was also decreased in tissues of NAFLD patients. Many of these 43 overlapping genes have been reported to play an important role in the formation of LD. Subsequently, we focused on CYP46A1, one of 43 candidate genes and mitochondria-related genes. We confirmed that the protein expression of CYP46A1 is deceased in tissues of NAFLD patients. Downregulation or overexpression of CYP46A1 affected LD accumulation in vitro. Deficiency of CYP46A1 impaired mitochondrial morphology and function, which may be responsible for the accumulation of LD. In summary, this study explored regulatory factors of LD accumulation at the whole-genome level, and demonstrated that CYP46A1 regulated LD formation involving in NAFLD pathogenesis. It provides new clues for studying the molecular mechanisms of diseases related to abnormal lipid metabolism.
Asunto(s)
Colesterol 24-Hidroxilasa/metabolismo , Metabolismo de los Lípidos , Enfermedad del Hígado Graso no Alcohólico/genética , Adulto , Sistemas CRISPR-Cas , Colesterol 24-Hidroxilasa/genética , Femenino , Técnicas de Inactivación de Genes/métodos , Genómica/métodos , Humanos , Hígado/metabolismo , Masculino , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/metabolismo , TranscriptomaRESUMEN
Age-related macular degeneration (AMD) is a predominant cause of visual deficit in aged population. Abnormal accumulation of cholesterol, including oxidized low-density lipoprotein (oxLDL), underneath the retinal pigment epithelium (RPE) cells contributes to the development of AMD. Gypenosides (Gyp) are glycosides extracted from Gynostemma pentaphyllum and have demonstrated protective effects against inflammation and oxidative stress. To determine the therapeutic potential of Gyp for AMD, we investigated its effect on cholesterol trafficking and metabolism and assessed the protective function of Gyp against oxLDL-induced damage in RPE cells. Cholesterol efflux to high-density lipoprotein (HDL) and human serum was significantly increased in RPE cells treated with Gyp when compared to untreated control cells. Expression of cholesterol metabolism (CYP27A1, CYP46A1) and trafficking (TSPO, ABCA1 and ABCG1) genes was also markedly increased in Gyp-treated RPE cells. OxLDL-treated RPE cells had significantly increased cholesterol accumulation and lipid droplet formation. There were marked increases in reactive oxygen species (ROS) generation and proinflammatory cytokines via NF-κB activation in RPE cells treated with oxLDL, while incubation with Gyp rectified these changes. These findings provide pharmacological evidence that Gyp has the potential to treat patients with early onset AMD by promoting cellular cholesterol removal from RPE cells and inhibiting inflammation and oxidative stress.
Asunto(s)
Colesterol/metabolismo , Inflamación/metabolismo , Lipoproteínas LDL/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Transportador 1 de Casete de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/metabolismo , Western Blotting , Línea Celular , Colestanotriol 26-Monooxigenasa/genética , Colesterol 24-Hidroxilasa/genética , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica/fisiología , Gynostemma/química , Humanos , FN-kappa B/metabolismo , Extractos Vegetales/farmacología , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de GABA/metabolismo , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
Dysregulated cholesterol metabolism is a hallmark of many cancers, including glioblastoma (GBM), but its role in disease progression is not well understood. Here, we identified cholesterol 24-hydroxylase (CYP46A1), a brain-specific enzyme responsible for the elimination of cholesterol through the conversion of cholesterol into 24(S)-hydroxycholesterol (24OHC), as one of the most dramatically dysregulated cholesterol metabolism genes in GBM. CYP46A1 was significantly decreased in GBM samples compared with normal brain tissue. A reduction in CYP46A1 expression was associated with increasing tumour grade and poor prognosis in human gliomas. Ectopic expression of CYP46A1 suppressed cell proliferation and in vivo tumour growth by increasing 24OHC levels. RNA-seq revealed that treatment of GBM cells with 24OHC suppressed tumour growth through regulation of LXR and SREBP signalling. Efavirenz, an activator of CYP46A1 that is known to penetrate the blood-brain barrier, inhibited GBM growth in vivo. Our findings demonstrate that CYP46A1 is a critical regulator of cellular cholesterol in GBM and that the CYP46A1/24OHC axis is a potential therapeutic target.
Asunto(s)
Colesterol 24-Hidroxilasa , Colesterol/metabolismo , Glioblastoma , Encéfalo/metabolismo , Colesterol 24-Hidroxilasa/genética , Colesterol 24-Hidroxilasa/metabolismo , Glioblastoma/metabolismo , Homeostasis , HumanosRESUMEN
While the presence and abundance of the major oxysterols and cholestenoic acids in the circulation is well established, minor cholesterol metabolites may also have biological importance and be of value to investigate. In this study by observing the metabolism of deuterium-labelled cholesterol in the pdgfbret/ret mouse, a mouse model with increased vascular permeability in brain, and by studying the sterol content of plasma from the CYP46A1 transgenic mouse overexpressing the human cholesterol 24S-hydroxylase enzyme we have been able to identify a number of minor cholesterol metabolites found in the circulation, make approximate-quantitative measurements and postulate pathways for their formation. These "proof of principle" data may have relevance when using mouse models to mimic human disease and in respect of the increasing possibility of treating human neurodegenerative diseases with pharmaceuticals designed to enhance the activity of CYP46A1 or by adeno-associated virus delivery of CYP46A1.
Asunto(s)
Colestenos/sangre , Colesterol 24-Hidroxilasa/genética , Oxiesteroles/sangre , Animales , Deuterio , Masculino , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Dysfunctions in brain cholesterol homeostasis have been extensively related to brain disorders. The main pathway for brain cholesterol elimination is its hydroxylation into 24S-hydroxycholesterol by the cholesterol 24-hydrolase, CYP46A1. Increasing evidence suggests that CYP46A1 has a role in the pathogenesis and progression of neurodegenerative disorders, and that increasing its levels in the brain is neuroprotective. However, the mechanisms underlying this neuroprotection remain to be fully understood. Huntington's disease is a fatal autosomal dominant neurodegenerative disease caused by an abnormal CAG expansion in huntingtin's gene. Among the multiple cellular and molecular dysfunctions caused by this mutation, altered brain cholesterol homeostasis has been described in patients and animal models as a critical event in Huntington's disease. Here, we demonstrate that a gene therapy approach based on the delivery of CYP46A1, the rate-limiting enzyme for cholesterol degradation in the brain, has a long-lasting neuroprotective effect in Huntington's disease and counteracts multiple detrimental effects of the mutated huntingtin. In zQ175 Huntington's disease knock-in mice, CYP46A1 prevented neuronal dysfunctions and restored cholesterol homeostasis. These events were associated to a specific striatal transcriptomic signature that compensates for multiple mHTT-induced dysfunctions. We thus explored the mechanisms for these compensations and showed an improvement of synaptic activity and connectivity along with the stimulation of the proteasome and autophagy machineries, which participate to the clearance of mutant huntingtin (mHTT) aggregates. Furthermore, BDNF vesicle axonal transport and TrkB endosome trafficking were restored in a cellular model of Huntington's disease. These results highlight the large-scale beneficial effect of restoring cholesterol homeostasis in neurodegenerative diseases and give new opportunities for developing innovative disease-modifying strategies in Huntington's disease.
Asunto(s)
Encéfalo/metabolismo , Colesterol 24-Hidroxilasa/uso terapéutico , Colesterol/metabolismo , Terapia Genética , Vectores Genéticos/uso terapéutico , Enfermedad de Huntington/terapia , Fármacos Neuroprotectores/uso terapéutico , Animales , Autofagia , Transporte Axonal , Factor Neurotrófico Derivado del Encéfalo/fisiología , Células Cultivadas , Corteza Cerebral/fisiopatología , Colesterol 24-Hidroxilasa/genética , Cuerpo Estriado/metabolismo , Cuerpo Estriado/fisiopatología , Dependovirus/genética , Endosomas/metabolismo , Técnicas de Sustitución del Gen , Vectores Genéticos/genética , Humanos , Proteína Huntingtina/genética , Enfermedad de Huntington/metabolismo , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Vías Nerviosas/fisiopatología , Fármacos Neuroprotectores/administración & dosificación , Oxiesteroles/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Agregación Patológica de Proteínas , Proteínas Tirosina Quinasas/fisiología , Prueba de Desempeño de Rotación con Aceleración Constante , Transmisión Sináptica , TranscriptomaRESUMEN
Cholesterol 24-hydroxylation is the major mechanism for cholesterol removal from the brain and the reaction catalyzed by cytochrome P450 46A1 (CYP46A1), a CNS-specific enzyme. This review describes CYP46A1 in the context of cholesterol homeostasis in the brain and summarizes available experimental data on CYP46A1 association with different neurologic diseases, including the mechanisms by which changes in the CYP46A1 activity in the brain could be beneficial for these diseases. The modulation of CYP46A1 activity by genetic and pharmacologic means is also presented along with a brief synopsis of the two clinical trials that evaluate CYP46A1 as a therapeutic target for Alzheimer's disease as well as Dravet and Lennox-Gastaut syndromes.
Asunto(s)
Encefalopatías/prevención & control , Colesterol 24-Hidroxilasa/metabolismo , Colesterol/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Encéfalo/enzimología , Encéfalo/metabolismo , Encefalopatías/tratamiento farmacológico , Colesterol 24-Hidroxilasa/genética , Humanos , Enfermedad de Huntington/metabolismo , Hidroxilación , Enfermedad de Parkinson/metabolismoRESUMEN
24S-hydroxycholesterol (24HC) is the major metabolic breakdown product of cholesterol in the brain. Among its other effects on neurons, 24HC modulates N-methyl-d-aspartate (NMDA or GluN) receptors, but our understanding of this mechanism is poor. We used whole-cell patch clamp recordings and various pharmacological approaches in mouse brain slices to record isolated NMDAR-mediated (INMDA) tonic and evoked synaptic currents. 24HC (1 µΜ) significantly enhanced tonic, but not evoked, INMDA of dentate gyrus granule cells. The INMDA had both GluN2A and GluN2B-mediated components. Preincubation of the slices with PEAQX (a GluN2A antagonist) or Ro25-6981 (a GluN2B antagonist) dramatically changed the INMDA modulatory potential of 24HC. Ro25-6981 blocked the enhancing effect of 24HC on tonic INMDA, while preincubation with PEAQX had no effect. In cholesterol 24-hydroxylase (CYP46A1) knockout mice, in sharp contrast to WT, 24HC slightly decreased the tonic INMDA of granule cells. Furthermore, 24HC had no effect on tonic INMDA of dentate gyrus parvalbumin interneurons (PV-INs), known to express different GluN subunits than granule cells. Taken together, our results revealed a specific enhancement of GluN2B-containing NMDARs by 24HC, indicating a novel endogenous pathway to influence a subclass of NMDARs critically involved in cortical plasticity and in numerous neurological and psychiatric disorders.
Asunto(s)
Giro Dentado/fisiología , Hidroxicolesteroles/metabolismo , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Colesterol 24-Hidroxilasa/genética , Giro Dentado/efectos de los fármacos , Potenciales Evocados/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Interneuronas/fisiología , Masculino , Ratones , Ratones Noqueados , Neuronas/efectos de los fármacos , Neuronas/fisiología , Fenoles/farmacología , Piperidinas/farmacología , Quinoxalinas/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Potenciales Sinápticos/fisiologíaRESUMEN
Polymorphisms of the cholesterol-24S-hydroxylase (CYP46A1) and apolipoprotein E (APOE) genes are risk factors for Alzheimer's disease (AD). Plasma level of 24S-hydroxcholesterol (24-OHC), the metabolite of cholesterol, is thought to correlate with AD. The present study investigated the correlation between these genetic factors and blood 24-OHC and amyloid-beta (Aß) levels in AD patients. Association analysis, logistic regression, and linear regression were used to analyze the correlation of CYP46A1 and APOE genotypes with blood 24-OHC and Aß levels and AD risk. We found that the APOEε4 alleles were significantly higher in patients with AD and there was a potential synergistic interaction between the CYP46A1 C allele and APOEε4 allele in AD. Blood 24-OHC level and Aß level were significantly higher in AD patients than controls, indicating 24-OHC could be a marker in AD diagnosis. However, AD patients with the CYP46A1 TT, but not CC, genotype had higher 24-OHC levels, which indicated that there may be other mechanisms in the relationship between CYP46A1 polymorphisms and AD.
Asunto(s)
Alelos , Enfermedad de Alzheimer/genética , Apolipoproteínas E/genética , Colesterol 24-Hidroxilasa/genética , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple/genética , Anciano , Enfermedad de Alzheimer/sangre , Péptidos beta-Amiloides/sangre , Estudios de Casos y Controles , Demografía , Femenino , Estudios de Asociación Genética , Humanos , Hidroxicolesteroles/sangre , Masculino , Factores de Riesgo , Índice de Severidad de la EnfermedadRESUMEN
Cytochrome P450 46A1 (CYP46A1) converts cholesterol to 24-hydroxycholesterol and thereby controls the major pathways of cholesterol removal from the brain. Cyp46a1-/- mice have a reduction in the rate of cholesterol biosynthesis in the brain and significant impairments to memory and learning. To gain insights into the mechanisms underlying Cyp46a1-/- phenotype, we used Cyp46a1-/- mice and quantified their brain sterol levels and the expression of the genes pertinent to cholesterol homeostasis. We also compared the Cyp46a1-/- and wild type brains for protein phosphorylation and ubiquitination. The data obtained enable the following inferences. First, there seems to be a compensatory upregulation in the Cyp46a1-/- brain of the pathways of cholesterol storage and CYP46A1-independent removal. Second, transcriptional regulation of the brain cholesterol biosynthesis via sterol regulatory element binding transcription factors is not significantly activated in the Cyp46a1-/- brain to explain a compensatory decrease in cholesterol biosynthesis. Third, some of the liver X receptor target genes (Abca1) are paradoxically upregulated in the Cyp46a1-/- brain, possibly due to a reduced activation of the small GTPases RAB8, CDC42, and RAC as a result of a reduced phosphorylation of RAB3IP and PAK1. Fourth, the phosphorylation of many other proteins (a total of 146) is altered in the Cyp46a1-/- brain, including microtubule associated and neurofilament proteins (the MAP and NEF families) along with proteins related to synaptic vesicles and synaptic neurotransmission (e.g., SLCs, SHANKs, and BSN). Fifth, the extent of protein ubiquitination is increased in the Cyp46a1-/- brain, and the affected proteins pertain to ubiquitination (UBE2N), cognition (STX1B and ATP1A2), cytoskeleton function (TUBA1A and YWHAZ), and energy production (ATP1A2 and ALDOA). The present study demonstrates the diverse potential effects of CYP46A1 deficiency on brain functions and identifies important proteins that could be affected by this deficiency.
Asunto(s)
Encéfalo/metabolismo , Colesterol 24-Hidroxilasa/metabolismo , Colesterol/aislamiento & purificación , Procesamiento Proteico-Postraduccional , Transcripción Genética , Animales , Colesterol/metabolismo , Colesterol 24-Hidroxilasa/genética , Femenino , Perfilación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfoproteínas/metabolismo , Reacción en Cadena de la Polimerasa , Reacción en Cadena en Tiempo Real de la Polimerasa , UbiquitinaciónRESUMEN
Cytochrome P450 46A1 (CYP46A1, cholesterol 24-hydroxylase) is the enzyme responsible for the majority of cholesterol elimination from the brain. Previously, we found that the anti-HIV drug efavirenz (EFV) can pharmacologically activate CYP46A1 in mice. Herein, we investigated whether CYP46A1 could also be activated by endogenous compounds, including major neurotransmitters. In vitro experiments with purified recombinant CYP46A1 indicated that CYP46A1 is activated by l-glutamate (l-Glu), l-aspartate, γ-aminobutyric acid, and acetylcholine, with l-Glu eliciting the highest increase (3-fold) in CYP46A1-mediated cholesterol 24-hydroxylation. We also found that l-Glu and other activating neurotransmitters bind to the same site on the CYP46A1 surface, which differs from the EFV-binding site. The other principal differences between EFV and l-Glu in CYP46A1 activation include an apparent lack of l-Glu binding to the P450 active site and different pathways of signal transduction from the allosteric site to the active site. EFV and l-Glu similarly increased the CYP46A1 kcat, the rate of the "fast" phase of the enzyme reduction by the redox partner NADPH-cytochrome P450 oxidoreductase, and the amount of P450 reduced. Spectral titrations with cholesterol, in the presence of EFV or l-Glu, suggest that water displacement from the heme iron can be affected in activator-bound CYP46A1. Moreover, EFV and l-Glu synergistically activated CYP46A1. Collectively, our in vitro data, along with those from previous cell culture and in vivo studies by others, suggest that l-Glu-induced CYP46A1 activation is of physiological relevance.
Asunto(s)
Acetilcolina/metabolismo , Ácido Aspártico/metabolismo , Colesterol 24-Hidroxilasa/metabolismo , Ácido Glutámico/metabolismo , Modelos Moleculares , Proteínas del Tejido Nervioso/agonistas , Ácido gamma-Aminobutírico/metabolismo , Acetilcolina/química , Alquinos , Regulación Alostérica/efectos de los fármacos , Sustitución de Aminoácidos , Fármacos Anti-VIH/química , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacología , Ácido Aspártico/química , Benzoxazinas/química , Benzoxazinas/metabolismo , Benzoxazinas/farmacología , Sitios de Unión , Biocatálisis/efectos de los fármacos , Colesterol 24-Hidroxilasa/química , Colesterol 24-Hidroxilasa/genética , Ciclopropanos , Medición de Intercambio de Deuterio , Activación Enzimática/efectos de los fármacos , Ácido Glutámico/química , Ligandos , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Ácido gamma-Aminobutírico/químicaRESUMEN
Increased evidence suggests that dysregulation of cholesterol metabolism may be a key event contributing to progression of multiple sclerosis (MS). Using an experimental autoimmune encephalomyelitis (EAE) model of MS we revealed specific changes in the mRNA and protein expression of key molecules involved in the maintaining of cholesterol homeostasis in the rat spinal cord: 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (HMGCR), apolipoprotein E (ApoE) and cholesterol 24-hydroxylase (CYP46A1) during the course of disease. The presence of myelin lipid debris was seen only at the peak of EAE in demyelination loci being efficiently removed during the recovery period. Since CYP46A1 is responsible for removal of cholesterol excess, we performed a detailed profiling of CYP46A1 expression and revealed regional and temporal specificities in its distribution. Double immunofluorescence staining demonstrated CYP46A1 localization with neurons, infiltrated macrophages, microglia and astrocytes in the areas of demyelination, suggesting that these cells play a role in cholesterol turnover in EAE. We propose that alterations in the regulation of cholesterol metabolism at the onset and peak of EAE may add to the progression of disease, while during the recovery period may have beneficial effects contributing to the regeneration of myelin sheath and restoration of neuronal function.
Asunto(s)
Colesterol/metabolismo , Encefalomielitis Autoinmune Experimental/etiología , Encefalomielitis Autoinmune Experimental/metabolismo , Metabolismo de los Lípidos/genética , Médula Espinal/metabolismo , Transcriptoma , Animales , Astrocitos/inmunología , Astrocitos/metabolismo , Biomarcadores , Colesterol 24-Hidroxilasa/genética , Colesterol 24-Hidroxilasa/metabolismo , Enfermedades Desmielinizantes/genética , Enfermedades Desmielinizantes/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Expresión Génica , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Microglía/inmunología , Microglía/metabolismo , Microglía/patología , Esclerosis Múltiple/etiología , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Neuronas/metabolismo , Fenotipo , Ratas , Índice de Severidad de la Enfermedad , Médula Espinal/patologíaRESUMEN
BACKGROUND: The involvement of genetic factors like gene polymorphisms has been found to contribute significantly to the development and progression of type 2 diabetes (T2DM). Thousands of single nucleotide polymorphisms in various genes have been found to be associated with risk of T2DM. The present study was aimed to investigate association of Multidrug resistance 1 (MDR1) (rs1045642) and CYP46A1 (rs754203) genes polymorphism with T2DM. SUBJECTS & METHODS: Study includes 333 subjects, 183 T2DM cases and 150 healthy controls. Single nucleotide polymorphism was evaluated by PCR-PFLP. Alleles and genotype frequencies between cases and controls were compared using χ2 and Student's t-tests. Odds ratios and 95% confidence intervals were calculated by logistic regression to assess the relative association between disease and genotypes. RESULTS: In case of CYP46A1 gene, CC (p < 0.001) and CT (p = 0.001) genotypes and C allele (p < 0.001) were found to be a positive risk factor and TT genotype (p < 0.001) and T allele (p < 0.001) as negative risk factor for T2DM whereas, no association of MDR1 gene was found with T2DM (P values of all genotypes and alleles were greater than 0.001). MDR1 (rs1045642) and CYP46A1 (rs754203) genes polymorphism was not found associated with Fasting Blood Sugar (FBS), Diastolic Blood Pressure (DBP) and Systolic Blood Pressure (SBP). CONCLUSION: CYP46A1 gene polymorphism is associated with the risk of T2DM whereas, MDR1 gene polymorphism was not found to confer any risk of T2DM in North Indian Ethnic group.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Colesterol 24-Hidroxilasa/genética , Diabetes Mellitus Tipo 2/genética , Polimorfismo de Nucleótido Simple , Glucemia , Presión Sanguínea/genética , Humanos , India , Oportunidad Relativa , Factores de RiesgoRESUMEN
Alzheimer's disease (AD) is a gradually debilitating disease that leads to dementia. The molecular mechanisms underlying AD are still not clear, and at present no reliable biomarkers are available for the early diagnosis. In the last several years, together with oxidative stress and neuroinflammation, altered cholesterol metabolism in the brain has become increasingly implicated in AD progression. A significant body of evidence indicates that oxidized cholesterol, in the form of oxysterols, is one of the main triggers of AD. The oxysterols potentially most closely involved in the pathogenesis of AD are 24-hydroxycholesterol and 27-hydroxycholesterol, respectively deriving from cholesterol oxidation by the enzymes CYP46A1 and CYP27A1. However, the possible involvement of oxysterols resulting from cholesterol autooxidation, including 7-ketocholesterol and 7ß-hydroxycholesterol, is now emerging. In a systematic analysis of oxysterols in post-mortem human AD brains, classified by the Braak staging system of neurofibrillary pathology, alongside the two oxysterols of enzymatic origin, a variety of oxysterols deriving from cholesterol autoxidation were identified; these included 7-ketocholesterol, 7α-hydroxycholesterol, 4ß-hydroxycholesterol, 5α,6α-epoxycholesterol, and 5ß,6ß-epoxycholesterol. Their levels were quantified and compared across the disease stages. Some inflammatory mediators, and the proteolytic enzyme matrix metalloprotease-9, were also found to be enhanced in the brains, depending on disease progression. This highlights the pathogenic association between the trends of inflammatory molecules and oxysterol levels during the evolution of AD. Conversely, sirtuin 1, an enzyme that regulates several pathways involved in the anti-inflammatory response, was reduced markedly with the progression of AD, supporting the hypothesis that the loss of sirtuin 1 might play a key role in AD. Taken together, these results strongly support the association between changes in oxysterol levels and AD progression.