RESUMEN
BACKGROUND: Traffic-generated air pollution-exposure is associated with adverse effects in the central nervous system (CNS) in both human exposures and animal models, including neuroinflammation and neurodegeneration. While alterations in the blood brain barrier (BBB) have been implicated as a potential mechanism of air pollution-induced CNS pathologies, pathways involved have not been elucidated. OBJECTIVES: To determine whether inhalation exposure to mixed vehicle exhaust (MVE) mediates alterations in BBB permeability, activation of matrix metalloproteinases (MMP) -2 and -9, and altered tight junction (TJ) protein expression. METHODS: Apolipoprotein (Apo) E(-/-) and C57Bl6 mice were exposed to either MVE (100 µg/m(3) PM) or filtered air (FA) for 6 hr/day for 30 days and resulting BBB permeability, expression of ROS, TJ proteins, markers of neuroinflammation, and MMP activity were assessed. Serum from study mice was applied to an in vitro BBB co-culture model and resulting alterations in transport and permeability were quantified. RESULTS: MVE-exposed Apo E(-/-) mice showed increased BBB permeability, elevated ROS and increased MMP-2 and -9 activity, compared to FA controls. Additionally, cerebral vessels from MVE-exposed mice expressed decreased levels of TJ proteins, occludin and claudin-5, and increased levels of inducible nitric oxide synthase (iNOS) and interleukin (IL)-1ß in the parenchyma. Serum from MVE-exposed animals also resulted in increased in vitro BBB permeability and altered P-glycoprotein transport activity. CONCLUSIONS: These data indicate that inhalation exposure to traffic-generated air pollutants promotes increased MMP activity and degradation of TJ proteins in the cerebral vasculature, resulting in altered BBB permeability and expression of neuroinflammatory markers.
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Barrera Hematoencefálica/efectos de los fármacos , Permeabilidad Capilar/efectos de los fármacos , Exposición por Inhalación/efectos adversos , Metaloproteinasas de la Matriz/genética , Proteínas de Uniones Estrechas/genética , Emisiones de Vehículos/toxicidad , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/sangre , Animales , Apolipoproteínas E/genética , Barrera Hematoencefálica/enzimología , Barrera Hematoencefálica/metabolismo , Western Blotting , Técnicas de Cocultivo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inflamación Neurogénica/sangre , Inflamación Neurogénica/inducido químicamente , Inflamación Neurogénica/enzimología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuroglía/patologíaRESUMEN
Creatine transporter deficiency (CTD), a leading cause of intellectual disability is a result of the mutation in the gene encoding the creatine transporter SLC6A8, which prevents creatine uptake into the brain, causing mental retardation, expressive speech and language delay, autistic-like behavior and epilepsy. Preclinical in vitro and in vivo data indicate that dodecyl creatine ester (DCE) which increases the creatine brain content, might be a therapeutic option for CTD patients. To gain a better understanding of the pathophysiology and DCE treatment efficacy in CTD, this study focuses on the identification of biomarkers related to cognitive improvement in a Slc6a8 knockout mouse model (Slc6a8-/y) engineered to mimic the clinical features of CTD patients which have low brain creatine content. Shotgun proteomics analysis of 4,035 proteins in four different brain regions; the cerebellum, cortex, hippocampus (associated with cognitive functions) and brain stem, and muscle as a control, was performed in 24 mice. Comparison of the protein abundance in the four brain regions between DCE-treated intranasally Slc6a8-/y mice and wild type and DCE-treated Slc6a8-/y and vehicle group identified 14 biomarkers, shedding light on the mechanism of action of DCE. Integrative bioinformatics and statistical modeling identified key proteins in CTD, including KIF1A and PLCB1. The abundance of these proteins in the four brain regions was significantly correlated with both the object recognition and the Y-maze tests. Our findings suggest a major role for PLCB1, KIF1A, and associated molecules in the pathogenesis of CTD.
RESUMEN
Creatine transporter deficiency (CTD) is an X-linked disease caused by mutations in the SLC6A8 gene. The impaired creatine uptake in the brain results in intellectual disability, behavioral disorders, language delay, and seizures. In this work, we generated human brain organoids from induced pluripotent stem cells of healthy subjects and CTD patients. Brain organoids from CTD donors had reduced creatine uptake compared with those from healthy donors. The expression of neural progenitor cell markers SOX2 and PAX6 was reduced in CTD-derived organoids, while GSK3ß, a key regulator of neurogenesis, was up-regulated. Shotgun proteomics combined with integrative bioinformatic and statistical analysis identified changes in the abundance of proteins associated with intellectual disability, epilepsy, and autism. Re-establishment of the expression of a functional SLC6A8 in CTD-derived organoids restored creatine uptake and normalized the expression of SOX2, GSK3ß, and other key proteins associated with clinical features of CTD patients. Our brain organoid model opens new avenues for further characterizing the CTD pathophysiology and supports the concept that reinstating creatine levels in patients with CTD could result in therapeutic efficacy.
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Discapacidad Intelectual , Discapacidad Intelectual Ligada al Cromosoma X , Humanos , Discapacidad Intelectual/genética , Creatina/genética , Creatina/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Discapacidad Intelectual Ligada al Cromosoma X/genética , Discapacidad Intelectual Ligada al Cromosoma X/metabolismo , Encéfalo/metabolismo , Organoides/metabolismoRESUMEN
Brain penetration is characterized by its extent and rate and is influenced by drug physicochemical properties, plasma exposure, plasma and brain protein binding and BBB permeability. This raises questions related to physiology, interspecies differences and in vitro/in vivo extrapolation. We herein discuss the use of in vitro human and animal BBB model as a tool to improve CNS compound selection. These cell-based BBB models are characterized by low paracellular permeation, well-developed tight junctions and functional efflux transporters. A study of twenty drugs shows similar compound ranking between rat and human models although with a 2-fold higher permeability in rat. cLogP < 5, PSA < 120 Å, MW < 450 were confirmed as essential for CNS drugs. An in vitro/in vivo correlation in rat (R² = 0.67; P = 2 × 10â»4) was highlighted when in vitro permeability and efflux were considered together with plasma exposure and free fraction. The cell-based BBB model is suitable to optimize CNS-drug selection, to study interspecies differences and then to support human brain exposure prediction.
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Barrera Hematoencefálica/metabolismo , Animales , Transporte Biológico/fisiología , Encéfalo/metabolismo , Células Cultivadas , Descubrimiento de Drogas/métodos , Humanos , Masculino , Modelos Biológicos , Ratas , Uniones Estrechas/metabolismoRESUMEN
We have evaluated a novel in vitro cell-based human blood-brain barrier (BBB) model that could predict in vivo human brain penetration for compounds with different BBB permeabilities using the clinical positron emission tomography (PET) data. Comparison studies were also performed to demonstrate that the in vitro cell-based human BBB model resulted in better predictivity over the traditional permeability model in discovery organizations, Caco-2 cells. We evaluated the in vivo BBB permeability of [(18)F] and [(11)C]-compounds in humans by PET imaging. The in vivo plasma-brain exchange parameters used for comparison were determined in humans by PET using a kinetic analysis of the radiotracer binding. For each radiotracer, the parameters were determined by fitting the brain kinetics of the radiotracer using a two-tissue compartment model of the ligand-receptor interaction. Bidirectional transport studies with the same compounds as in in vivo studies were carried out using the in vitro cell-based human BBB model as well as Caco-2 cells. The in vitro cell-based human BBB model has important features of the BBB in vivo and is suitable for discriminating between CNS and non-CNS marketed drugs. A very good correlation (r(2) = 0.90; P < 0.001) was demonstrated between in vitro BBB permeability and in vivo permeability coefficient. In contrast, a poor correlation (r(2) = 0.17) was obtained between Caco-2 data and in vivo human brain penetration. This study highlights the potential of this in vitro cell-based human BBB model in drug discovery and shows that it can be an extremely effective screening tool for CNS programs.
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Barrera Hematoencefálica/fisiología , Encéfalo/diagnóstico por imagen , Modelos Neurológicos , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Encéfalo/metabolismo , Células CACO-2 , Claudinas/genética , Claudinas/metabolismo , Descubrimiento de Drogas , Humanos , Cinética , Permeabilidad , Tomografía de Emisión de Positrones , ARN Mensajero/genética , ARN Mensajero/metabolismo , RadiofármacosRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Neurodegenerative diseases, including Alzheimer's and Parkinson's disease, are characterized by increased protein aggregation in the brain, progressive neuronal loss, increased inflammation, and neurogenesis impairment. We analyzed the effects of a new purine derivative drug, PDD005, in attenuating mechanisms involved in the pathogenesis of neurodegenerative diseases, using both in vivo and in vitro models. We show that PDD005 is distributed to the brain and can rescue cognitive deficits associated with aging in mice. Treatment with PDD005 prevents impairment of neurogenesis by increasing sex-determining region Y-box 2, nestin, and also enhances synaptic function through upregulation of synaptophysin and postsynaptic density protein 95. PDD005 treatment also reduced neuro-inflammation by decreasing interleukin-1ß expression, activation of astrocytes, and microglia. We identified prohibitin as a potential target in mediating the therapeutic effects of PDD005 for the treatment of cognitive deficit in aging mice. Additionally, in the current study, glycogen synthase kinase appears to attenuate tau pathology.
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Trastornos del Conocimiento/prevención & control , Hipocampo/efectos de los fármacos , Terapia Molecular Dirigida , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Fármacos Neuroprotectores/farmacología , Proteínas Represoras/antagonistas & inhibidores , Tauopatías/prevención & control , Envejecimiento/psicología , Animales , Barrera Hematoencefálica , Encéfalo/metabolismo , Células Cultivadas , Trastornos del Conocimiento/tratamiento farmacológico , Donepezilo/farmacología , Evaluación Preclínica de Medicamentos , Células Endoteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/biosíntesis , Glucógeno Sintasa Quinasa 3 beta/genética , Interleucina-1beta/biosíntesis , Interleucina-1beta/genética , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/efectos de los fármacos , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Neurogénesis/efectos de los fármacos , Neuroglía/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Fármacos Neuroprotectores/farmacocinética , Fosforilación/efectos de los fármacos , Prohibitinas , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Tauopatías/tratamiento farmacológico , Proteínas tau/metabolismoRESUMEN
Efflux proteins have been shown to greatly affect the uptake of antiretroviral drugs by cells and to hamper their access to the human immunodeficiency virus type 1 replication site. This study evaluated the factors that may lead to drug-drug interactions between emtricitabine (FTC), tenofovir (TFV), and efavirenz (EFV), including the modulation of efflux transporter expression and function. Peripheral blood mononuclear cells from healthy volunteers were used to determine whether or not an interaction between antiretroviral drugs and target cells occurred in any combination of FTC, TFV, EFV, FTC-TFV, TFV-EFV, or FTC-TFV-EFV. Following 20 h of treatment, intracellular drug concentrations were measured by liquid chromatography-tandem mass spectrometry. Efflux transporter functionality and inhibitor drug properties were assessed by measuring fluorescent dye efflux. ABCB1 (P-glycoprotein), ABCC 1 to 6 (multidrug resistance-associated protein), and OAT (organic anion transporter) expression in response to the treatments was quantified by semiquantitative real-time PCR. Cells treated with a double combination (FTC-TFV or TFV-EFV) or the triple combination (FTC-TFV-EFV) produced higher FTC and TFV intracellular concentrations than cells treated with FTC or TFV alone. However, no change in the EFV intracellular concentration was observed. FTC tended to induce abcc5 mRNA expression and EFV tended to induce abcc1 and abcc6 mRNA expression, whereas TFV tended to reduce mdr1, abcc1, abcc5, and abcc6 mRNA expression. Under these conditions, a decrease in the functionality of ABCC was observed, and this decrease was associated with the direct inhibitory actions of these drugs. This in vitro study reveals a benefit of the combination FTC-TFV-EFV in terms of the intracellular FTC and TFV concentrations and highlights the pharmacological mechanisms that lead to this effect.
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Transportadoras de Casetes de Unión a ATP/genética , Adenina/análogos & derivados , Antivirales/farmacología , Benzoxazinas/farmacología , Desoxicitidina/análogos & derivados , Organofosfonatos/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Adenina/metabolismo , Adenina/farmacología , Alquinos , Antivirales/metabolismo , Benzoxazinas/metabolismo , Células Cultivadas , Ciclopropanos , Desoxicitidina/metabolismo , Desoxicitidina/farmacología , Interacciones Farmacológicas/genética , Quimioterapia Combinada , Emtricitabina , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Organofosfonatos/metabolismo , ARN Mensajero/metabolismo , Tenofovir , Factores de TiempoRESUMEN
The development of effective central nervous system (CNS) drugs has been hampered by the lack of robust strategies to mimic the blood-brain barrier (BBB) and cerebrovascular impairments in vitro. Recent technological advancements in BBB modeling using induced pluripotent stem cells (iPSCs) allowed to overcome some of these obstacles, nonetheless the pertinence for their use in drug permeation study remains to be established. This mandatory information requires a cross comparison of in vitro and in vivo pharmacokinetic data in the same species to avoid failure in late clinical drug development. Here, we measured the BBB permeabilities of 8 clinical positron emission tomography (PET) radioligands with known pharmacokinetic parameters in human brain in vivo with a newly developed in vitro iPSC-based human BBB (iPSC-hBBB) model. Our findings showed a good correlation between in vitro and in vivo drug brain permeability (R2 = 0.83; P = 0.008) which contrasted with the limited correlation between in vitro apparent permeability for a set of 18 CNS/non-CNS compounds using the in vitro iPSCs-hBBB model and drug physicochemical properties. Our data suggest that the iPSC-hBBB model can be integrated in a flow scheme of CNS drug screening and potentially used to study species differences in BBB permeation.
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Barrera Hematoencefálica/química , Encéfalo/diagnóstico por imagen , Células Madre Pluripotentes Inducidas/citología , Neuroglía/citología , Animales , Barrera Hematoencefálica/diagnóstico por imagen , Encéfalo/metabolismo , Diferenciación Celular , Células Cultivadas , Evaluación Preclínica de Medicamentos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Modelos Biológicos , Neuroglía/metabolismo , Permeabilidad , Tomografía de Emisión de Positrones , Prueba de Estudio Conceptual , RatasRESUMEN
Human mini-brains (MB) are cerebral organoids that recapitulate in part the complexity of the human brain in a unique three-dimensional in vitro model, yielding discrete brain regions reminiscent of the cerebral cortex. Specific proteins linked to neurodegenerative disorders are physiologically expressed in MBs, such as APP-derived amyloids (Aß), whose physiological and pathological roles and interactions with other proteins are not well established in humans. Here, we demonstrate that neuroectodermal organoids can be used to study the Aß accumulation implicated in Alzheimer's disease (AD). To enhance the process of protein secretion and accumulation, we adopted a chemical strategy of induction to modulate post-translational pathways of APP using an Amyloid-ß Forty-Two Inducer named Aftin-5. Secreted, soluble Aß fragment concentrations were analyzed in MB-conditioned media. An increase in the Aß42 fragment secretion was observed as was an increased Aß42/Aß40 ratio after drug treatment, which is consistent with the pathological-like phenotypes described in vivo in transgenic animal models and in vitro in induced pluripotent stem cell-derived neural cultures obtained from AD patients. Notably in this context we observe time-dependent Aß accumulation, which differs from protein accumulation occurring after treatment. We show that mini-brains obtained from a non-AD control cell line are responsive to chemical compound induction, producing a shift of physiological Aß concentrations, suggesting that this model can be used to identify environmental agents that may initiate the cascade of events ultimately leading to sporadic AD. Increases in both Aß oligomers and their target, the cellular prion protein (PrPC), support the possibility of using MBs to further understand the pathophysiological role that underlies their interaction in a human model. Finally, the potential application of MBs for modeling age-associated phenotypes and the study of neurological disorders is confirmed.