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1.
Cancers (Basel) ; 16(15)2024 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-39123366

RESUMO

Glioblastoma (GBM) presents a significant public health challenge as the deadliest and most common malignant brain tumor in adults. Despite standard-of-care treatment, which includes surgery, radiation, and chemotherapy, mortality rates are high, underscoring the critical need for advancing GBM therapy. Over the past two decades, numerous clinical trials have been performed, yet only a small fraction demonstrated a benefit, raising concerns about the predictability of current preclinical models. Traditionally, preclinical studies utilize treatment-naïve tumors, failing to model the clinical scenario where patients undergo standard-of-care treatment prior to recurrence. Recurrent GBM generally exhibits distinct molecular alterations influenced by treatment selection pressures. In this review, we discuss the impact of treatment-surgery, radiation, and chemotherapy-on GBM. We also provide a summary of treatments used in preclinical models, advocating for their integration to enhance the translation of novel strategies to improve therapeutic outcomes in GBM.

2.
Cancers (Basel) ; 16(11)2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38893116

RESUMO

Glioblastoma (GBM) is the most aggressive brain cancer. To model GBM in research, orthotopic brain tumor models, including syngeneic models like GL261 and genetically engineered mouse models like TRP, are used. In longitudinal studies, tumor growth and the treatment response are typically tracked with in vivo imaging, including bioluminescence imaging (BLI), which is quick, cost-effective, and easily quantifiable. However, BLI requires luciferase-tagged cells, and recent studies indicate that the luciferase gene can elicit an immune response, leading to tumor rejection and experimental variation. We sought to optimize the engraftment of two luciferase-expressing GBM models, GL261 Red-FLuc and TRP-mCherry-FLuc, showing differences in tumor take, with GL261 Red-FLuc cells requiring immunocompromised mice for 100% engraftment. Immunohistochemistry and MRI revealed distinct tumor characteristics: GL261 Red-FLuc tumors were well-demarcated with densely packed cells, high mitotic activity, and vascularization. In contrast, TRP-mCherry-FLuc tumors were large, invasive, and necrotic, with perivascular invasion. Quantifying the tumor volume using the HALO® AI analysis platform yielded results comparable to manual measurements, providing a standardized and efficient approach for the reliable, high-throughput analysis of luciferase-expressing tumors. Our study highlights the importance of considering tumor engraftment when using luciferase-expressing GBM models, providing insights for preclinical research design.

3.
Fluids Barriers CNS ; 20(1): 70, 2023 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-37803468

RESUMO

BACKGROUND: Loss of P-glycoprotein (P-gp) at the blood-brain barrier contributes to amyloid-ß (Aß) brain accumulation in Alzheimer's disease (AD). Using transgenic human amyloid precursor protein (hAPP)-overexpressing mice (Tg2576), we previously showed that Aß triggers P-gp loss by activating the ubiquitin-proteasome pathway, which leads to P-gp degradation. Furthermore, we showed that inhibiting the ubiquitin-activating enzyme (E1) prevents P-gp loss and lowers Aß accumulation in the brain of hAPP mice. Based on these data, we hypothesized that repurposing the FDA-approved proteasome inhibitor, bortezomib (Velcade®; BTZ), protects blood-brain barrier P-gp from degradation in hAPP mice in vivo. METHODS: We treated hAPP mice with the proteasome inhibitor BTZ or a combination of BTZ with the P-gp inhibitor cyclosporin A (CSA) for 2 weeks. Vehicle-treated wild-type (WT) mice were used as a reference for normal P-gp protein expression and transport activity. In addition, we used the opioid receptor agonist loperamide as a P-gp substrate in tail flick assays to indirectly assess P-gp transport activity at the blood-brain barrier in vivo. We also determined P-gp protein expression by Western blotting, measured P-gp transport activity levels in isolated brain capillaries with live cell confocal imaging and assessed Aß plasma and brain levels with ELISA. RESULTS: We found that 2-week BTZ treatment of hAPP mice restored P-gp protein expression and transport activity in brain capillaries to levels found in WT mice. We also observed that hAPP mice displayed significant loperamide-induced central antinociception compared to WT mice indicating impaired P-gp transport activity at the blood-brain barrier of hAPP mice in vivo. Furthermore, BTZ treatment prevented loperamide-induced antinociception suggesting BTZ protected P-gp loss in hAPP mice. Further, BTZ-treated hAPP mice had lower Aß40 and Aß42 brain levels compared to vehicle-treated hAPP mice. CONCLUSIONS: Our data indicate that BTZ protects P-gp from proteasomal degradation in hAPP mice, which helps to reduce Aß brain levels. Our data suggest that the proteasome system could be exploited for a novel therapeutic strategy in AD, particularly since increasing Aß transport across the blood-brain barrier may prove an effective treatment for patients.


Assuntos
Doença de Alzheimer , Humanos , Camundongos , Animais , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Barreira Hematoencefálica/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/farmacologia , Complexo de Endopeptidases do Proteassoma/uso terapêutico , Loperamida/metabolismo , Loperamida/farmacologia , Loperamida/uso terapêutico , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Inibidores de Proteassoma/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Camundongos Transgênicos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo
4.
Pharmacol Rev ; 75(5): 815-853, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-36973040

RESUMO

The drug efflux transporters ABCB1 and ABCG2 at the blood-brain barrier limit the delivery of drugs into the brain. Strategies to overcome ABCB1/ABCG2 have been largely unsuccessful, which poses a tremendous clinical problem to successfully treat central nervous system (CNS) diseases. Understanding basic transporter biology, including intracellular regulation mechanisms that control these transporters, is critical to solving this clinical problem.In this comprehensive review, we summarize current knowledge on signaling pathways that regulate ABCB1/ABCG2 at the blood-brain barrier. In Section I, we give a historical overview on blood-brain barrier research and introduce the role that ABCB1 and ABCG2 play in this context. In Section II, we summarize the most important strategies that have been tested to overcome the ABCB1/ABCG2 efflux system at the blood-brain barrier. In Section III, the main component of this review, we provide detailed information on the signaling pathways that have been identified to control ABCB1/ABCG2 at the blood-brain barrier and their potential clinical relevance. This is followed by Section IV, where we explain the clinical implications of ABCB1/ABCG2 regulation in the context of CNS disease. Lastly, in Section V, we conclude by highlighting examples of how transporter regulation could be targeted for therapeutic purposes in the clinic. SIGNIFICANCE STATEMENT: The ABCB1/ABCG2 drug efflux system at the blood-brain barrier poses a significant problem to successful drug delivery to the brain. The article reviews signaling pathways that regulate blood-brain barrier ABCB1/ABCG2 and could potentially be targeted for therapeutic purposes.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Barreira Hematoencefálica , Humanos , Barreira Hematoencefálica/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Encéfalo/metabolismo , Transporte Biológico , Proteínas de Neoplasias/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo
5.
BMC Cancer ; 22(1): 844, 2022 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-35922758

RESUMO

Glioblastoma (GBM) is one of the deadliest cancers. Treatment options are limited, and median patient survival is only several months. Translation of new therapies is hindered by a lack of GBM models that fully recapitulate disease heterogeneity. Here, we characterize two human GBM models (U87-luc2, U251-RedFLuc). In vitro, both cell lines express similar levels of luciferase and show comparable sensitivity to temozolomide and lapatinib exposure. In vivo, however, the two GBM models recapitulate different aspects of the disease. U87-luc2 cells quickly grow into large, well-demarcated tumors; U251-RedFLuc cells form small, highly invasive tumors. Using a new method to assess GBM invasiveness based on detecting tumor-specific anti-luciferase staining in brain slices, we found that U251-RedFLuc cells are more invasive than U87-luc2 cells. Lastly, we determined expression levels of ABC transporters in both models. Our findings indicate that U87-luc2 and U251-RedFLuc GBM models recapitulate different aspects of GBM heterogeneity that need to be considered in preclinical research.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Temozolomida/farmacologia , Temozolomida/uso terapêutico
6.
J Control Release ; 343: 434-442, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35104569

RESUMO

Since it has been known that in vitro cell lines for analyzing drug transport at the inner blood-retinal barrier (BRB) do not completely retain several in vivo functions, new ex vivo/in vitro methods to evaluate drug transport across the inner BRB help us understand the role of this barrier in maintaining the homeostasis of vision and regulating drug distribution to the retina. To expand the limitations of existing in vitro approaches, we established a protocol to isolate fresh rat retinal capillaries as ex vivo model of the inner BRB. Fresh retinal capillaries were prepared by applying serial filtration steps and using density gradient centrifugation. We performed mRNA and protein analyses by reverse transcription-polymerase chain reaction and immunostaining that indicated expression of marker proteins such as facilitative glucose transporter 1 and claudin-5 in freshly isolated rat retinal capillaries. We also used fluorescent transporter substrates to characterize functional activity of organic anion transporter (Oat) 3, P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-associated protein (Mrp) 4 in isolated retinal capillaries. Capillary luminal accumulation of fluorescent substrates of P-glycoprotein and Bcrp was decreased in the presence of transporter inhibitors. Moreover, luminal accumulation of the Oat3 and Mrp4 substrate, 8-(2-[fluoresceinyl]aminoethylthio) adenosine-3',5'-cyclic monophosphate (8-[fluo]-cAMP), was reduced by substrates/inhibitors of Oat3 and Mrp4. In conclusion, our study shows that freshly isolated retinal capillaries retain marker protein expression and transporter functional activity. It is suggested that isolated retinal capillaries are a useful tool to study transport across the inner BRB. Using freshly isolated retinal capillaries, we anticipate applying this approach to determine the role of transporters at the inner BRB during pathophysiological states of the eye and evaluate the drug delivery to the retina.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Capilares , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Capilares/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Ratos , Ratos Wistar , Retina/metabolismo
7.
Fluids Barriers CNS ; 18(1): 10, 2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33676539

RESUMO

BACKGROUND: Failure to clear Aß from the brain is partly responsible for Aß brain accumulation in Alzheimer's disease (AD). A critical protein for clearing Aß across the blood-brain barrier is the efflux transporter P-glycoprotein (P-gp). In AD, P-gp levels are reduced, which contributes to impaired Aß brain clearance. However, the mechanism responsible for decreased P-gp levels is poorly understood and there are no strategies available to protect P-gp. We previously demonstrated in isolated brain capillaries ex vivo that human Aß40 (hAß40) triggers P-gp degradation by activating the ubiquitin-proteasome pathway. In this pathway, hAß40 initiates P-gp ubiquitination, leading to internalization and proteasomal degradation of P-gp, which then results in decreased P-gp protein expression and transport activity levels. Here, we extend this line of research and present results from an in vivo study using a transgenic mouse model of AD (human amyloid precursor protein (hAPP)-overexpressing mice; Tg2576). METHODS: In our study, hAPP mice were treated with vehicle, nocodazole (NCZ, microtubule inhibitor to block P-gp internalization), or a combination of NCZ and the P-gp inhibitor cyclosporin A (CSA). We determined P-gp protein expression and transport activity levels in isolated mouse brain capillaries and Aß levels in plasma and brain tissue. RESULTS: Treating hAPP mice with 5 mg/kg NCZ for 14 days increased P-gp levels to levels found in WT mice. Consistent with this, P-gp-mediated hAß42 transport in brain capillaries was increased in NCZ-treated hAPP mice compared to untreated hAPP mice. Importantly, NCZ treatment significantly lowered hAß40 and hAß42 brain levels in hAPP mice, whereas hAß40 and hAß42 levels in plasma remained unchanged. CONCLUSIONS: These findings provide in vivo evidence that microtubule inhibition maintains P-gp protein expression and transport activity levels, which in turn helps to lower hAß brain levels in hAPP mice. Thus, protecting P-gp at the blood-brain barrier may provide a novel therapeutic strategy for AD and other Aß-based pathologies.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides , Precursor de Proteína beta-Amiloide/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo , Moduladores de Tubulina/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Peptídeos beta-Amiloides/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Ciclosporina/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Transgênicos , Nocodazol/farmacologia
8.
Pharm Res ; 38(1): 113-125, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33527223

RESUMO

PURPOSE: In this study, we investigated in detail the transport of phenytoin across the blood-brain barrier (BBB) to identify the transporter(s) involved in BBB-mediated phenytoin efflux from the brain. METHODS: We evaluated the brain-to-blood efflux transport of phenytoin in vivo by determining the brain efflux index (BEI) and uptake in brain slices. We additionally conducted brain perfusion experiments and BEI studies in P-glycoprotein (P-gp)-deficient mice. In addition, we determined the mRNA expression of monocarboxylate transporter (MCT) in isolated brain capillaries and performed phenytoin uptake studies in MCT-expressing Xenopus oocytes. RESULTS: [14C]Phenytoin brain efflux was time-dependent with a half-life of 17 min in rats and 31 min in mice. Intracerebral pre-administration of unlabeled phenytoin attenuated BBB-mediated phenytoin efflux transport, suggesting carrier-mediated phenytoin efflux transport across the BBB. Pre-administration of P-gp substrates in rats and genetic P-gp deficiency in mice did not affect BBB-mediated phenytoin efflux transport. In contrast, pre-administration of MCT8 inhibitors attenuated phenytoin efflux. Moreover, rat MCT8-expressing Xenopus oocytes exhibited [14C]phenytoin uptake, which was inhibited by unlabeled phenytoin. CONCLUSION: Our data suggest that MCT8 at the BBB participates in phenytoin efflux transport from the brain to the blood.


Assuntos
Anticonvulsivantes/farmacocinética , Barreira Hematoencefálica/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Fenitoína/farmacocinética , Simportadores/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Anticonvulsivantes/administração & dosagem , Feminino , Meia-Vida , Masculino , Camundongos , Camundongos Transgênicos , Modelos Animais , Fenitoína/administração & dosagem , Ratos
9.
Fluids Barriers CNS ; 17(1): 56, 2020 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-32928243

RESUMO

David S. Miller was Acting Scientific Director of the Division of Intramural Research at the National Institute of Environmental Health Sciences, National Institutes of Health, and Head of the Intracellular Regulation Group in the Laboratory of Toxicology and Pharmacology before he retired in 2016. David received his Ph.D. in biochemistry from the University of Maine in 1973. David was a Group Leader at the Michigan Cancer Foundation before joining the NIEHS in 1985. His research covered a wide range from renal excretory transport mechanisms to regulation of transporters at the blood-CSF and blood-brain barriers, from fish, amphibians and birds to mammals. David was an outstanding scientist with irresistible enthusiasm for science and an incredible ability to think outside the box while being an exceptional mentor and friend.


Assuntos
Neurociências/história , Barreira Hematoencefálica/fisiologia , História do Século XX , História do Século XXI , Humanos , Masculino , Mentores , National Institutes of Health (U.S.) , Farmacologia/história , Transporte Proteico/fisiologia , Toxicologia/história , Estados Unidos
10.
FASEB J ; 33(12): 13966-13981, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31638830

RESUMO

The cause of antiseizure drug (ASD) resistance in epilepsy is poorly understood. Here, we focus on the transporter P-glycoprotein (P-gp) that is partly responsible for limited ASD brain uptake, which is thought to contribute to ASD resistance. We previously demonstrated that cyclooxygenase-2 (COX-2) and the prostaglandin E receptor, prostanoid E receptor subtype 1, are involved in seizure-mediated P-gp up-regulation. Thus, we hypothesized that inhibiting microsomal prostaglandin E2 (PGE2) synthase-1 (mPGES-1), the enzyme generating PGE2, prevents blood-brain barrier P-gp up-regulation after status epilepticus (SE). To test our hypothesis, we exposed isolated brain capillaries to glutamate ex vivo and used a combined in vivo-ex vivo approach by isolating brain capillaries from humanized mPGES-1 mice to study P-gp levels. We demonstrate that glutamate signaling through the NMDA receptor, cytosolic phospholipase A2, COX-2, and mPGES-1 increases P-gp protein expression and transport activity levels. We show that mPGES-1 is expressed in human, rat, and mouse brain capillaries. We show that BI1029539, an mPGES-1 inhibitor, prevented up-regulation of P-gp expression and transport activity in capillaries exposed to glutamate and in capillaries from humanized mPGES-1 mice after SE. Our data provide key signaling steps underlying seizure-induced P-gp up-regulation and suggest that mPGES-1 inhibitors could potentially prevent P-gp up-regulation in epilepsy.-Soldner, E. L. B., Hartz, A. M. S., Akanuma, S.-I., Pekcec, A., Doods, H., Kryscio, R. J., Hosoya, K.-I., Bauer, B. Inhibition of human microsomal PGE2 synthase-1 reduces seizure-induced increases of P-glycoprotein expression and activity at the blood-brain barrier.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Barreira Hematoencefálica/metabolismo , Dinoprostona/metabolismo , Microssomos/metabolismo , Prostaglandina-E Sintases/metabolismo , Convulsões/metabolismo , Animais , Transporte Biológico/fisiologia , Encéfalo/metabolismo , Capilares/metabolismo , Ciclo-Oxigenase 2/metabolismo , Epilepsia/metabolismo , Feminino , Ácido Glutâmico/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia
11.
Front Aging Neurosci ; 10: 186, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29997495

RESUMO

One characteristic of Alzheimer's disease (AD) is excessive accumulation of amyloid-ß (Aß) in the brain. Aß brain accumulation is, in part, due to a reduction in Aß clearance from the brain across the blood-brain barrier. One key element that contributes to Aß brain clearance is P-glycoprotein (P-gp) that transports Aß from brain to blood. In AD, P-gp protein expression and transport activity levels are significantly reduced, which impairs Aß brain clearance. The mechanism responsible for reduced P-gp expression and activity levels is poorly understood. We recently demonstrated that Aß40 triggers P-gp degradation through the ubiquitin-proteasome pathway. Consistent with these data, we show here that ubiquitinated P-gp levels in brain capillaries isolated from brain samples of AD patients are increased compared to capillaries isolated from brain tissue of cognitive normal individuals. We extended this line of research to in vivo studies using transgenic human amyloid precursor protein (hAPP)-overexpressing mice (Tg2576) that were treated with PYR41, a cell-permeable, irreversible inhibitor of the ubiquitin-activating enzyme E1. Our data show that inhibiting P-gp ubiquitination protects the transporter from degradation, and immunoprecipitation experiments confirmed that PYR41 prevented P-gp ubiquitination. We further found that PYR41 treatment prevented reduction of P-gp protein expression and transport activity levels and substantially lowered Aß brain levels in hAPP mice. Together, our findings provide in vivo proof that the ubiquitin-proteasome system mediates reduction of blood-brain barrier P-gp in AD and that inhibiting P-gp ubiquitination prevents P-gp degradation and lowers Aß brain levels. Thus, targeting the ubiquitin-proteasome system may provide a novel therapeutic approach to protect blood-brain barrier P-gp from degradation in AD and other Aß-based pathologies.

12.
J Neurosci ; 38(18): 4301-4315, 2018 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-29632167

RESUMO

The blood-brain barrier is dysfunctional in epilepsy, thereby contributing to seizure genesis and resistance to antiseizure drugs. Previously, several groups reported that seizures increase brain glutamate levels, which leads to barrier dysfunction. One critical component of barrier dysfunction is brain capillary leakage. Based on our preliminary data, we hypothesized that glutamate released during seizures mediates an increase in matrix-metalloproteinase (MMP) expression and activity levels, thereby contributing to barrier leakage. To test this hypothesis, we exposed isolated brain capillaries from male Sprague Dawley rats to glutamate ex vivo and used an in vivo/ex vivo approach of isolated brain capillaries from female Wistar rats that experienced status epilepticus as an acute seizure model. We found that exposing isolated rat brain capillaries to glutamate increased MMP-2 and MMP-9 protein and activity levels, and decreased tight junction protein levels, which resulted in barrier leakage. We confirmed these findings in vivo in rats after status epilepticus and in brain capillaries from male mice lacking cytosolic phospholipase A2 Together, our data support the hypothesis that glutamate released during seizures signals an increase in MMP-2 and MMP-9 protein expression and activity levels, resulting in blood-brain barrier leakage.SIGNIFICANCE STATEMENT The mechanism leading to seizure-mediated blood-brain barrier dysfunction in epilepsy is poorly understood. In the present study, we focused on defining this mechanism in the brain capillary endothelium. We demonstrate that seizures trigger a pathway that involves glutamate signaling through cytosolic phospholipase A2, which increases MMP levels and decreases tight junction protein expression levels, resulting in barrier leakage. These findings may provide potential therapeutic avenues within the blood-brain barrier to limit barrier dysfunction in epilepsy and decrease seizure burden.


Assuntos
Barreira Hematoencefálica/patologia , Epilepsia/patologia , Metaloproteinases da Matriz/metabolismo , Animais , Capilares/efeitos dos fármacos , Feminino , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Convulsões/patologia , Estado Epiléptico/metabolismo , Estado Epiléptico/patologia , Proteínas de Junções Íntimas/metabolismo
13.
Sci Rep ; 8(1): 6670, 2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29703936

RESUMO

Neurovascular integrity, including cerebral blood flow (CBF) and blood-brain barrier (BBB) function, plays a major role in determining cognitive capability. Recent studies suggest that neurovascular integrity could be regulated by the gut microbiome. The purpose of the study was to identify if ketogenic diet (KD) intervention would alter gut microbiome and enhance neurovascular functions, and thus reduce risk for neurodegeneration in young healthy mice (12-14 weeks old). Here we show that with 16 weeks of KD, mice had significant increases in CBF and P-glycoprotein transports on BBB to facilitate clearance of amyloid-beta, a hallmark of Alzheimer's disease (AD). These neurovascular enhancements were associated with reduced mechanistic target of rapamycin (mTOR) and increased endothelial nitric oxide synthase (eNOS) protein expressions. KD also increased the relative abundance of putatively beneficial gut microbiota (Akkermansia muciniphila and Lactobacillus), and reduced that of putatively pro-inflammatory taxa (Desulfovibrio and Turicibacter). We also observed that KD reduced blood glucose levels and body weight, and increased blood ketone levels, which might be associated with gut microbiome alteration. Our findings suggest that KD intervention started in the early stage may enhance brain vascular function, increase beneficial gut microbiota, improve metabolic profile, and reduce risk for AD.


Assuntos
Bactérias/metabolismo , Fatores Biológicos/metabolismo , Vasos Sanguíneos/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Dieta Cetogênica , Microbioma Gastrointestinal/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Camundongos , Óxido Nítrico Sintase/análise , Transporte Proteico , Serina-Treonina Quinases TOR/análise
14.
Mol Pharm ; 15(6): 2327-2337, 2018 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-29688723

RESUMO

Gabapentin is an antiseizure drug that is known to also have beneficial effects on the retinal cells. To use gabapentin in retinal pharmacotherapy, it is critical to understand gabapentin distribution in the retina. The purpose of this study was to clarify the kinetics of gabapentin influx transport across the inner and outer blood-retinal barrier (BRB), which regulates the exchange of compounds/drugs between the circulating blood and the retina. In vivo blood-to-retina gabapentin transfer was evaluated by the rat carotid artery injection technique. In addition, gabapentin transport was examined using in vitro models of the inner (TR-iBRB2 cells) and outer BRB (RPE-J cells). The in vivo [3H]gabapentin transfer to the rat retina across the BRB was significantly reduced in the presence of unlabeled gabapentin, suggesting transporter-mediated blood-to-retina distribution of gabapentin. Substrates of the Na+-independent l-type amino acid transporter 1 (LAT1), such as 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), also significantly inhibited the in vivo [3H]gabapentin transfer. [3H]Gabapentin uptake in TR-iBRB2 and RPE-J cells exhibited Na+-independent and saturable kinetics with a Km of 735 and 507 µM, respectively. Regarding the effect of various transporter substrates/inhibitors on gabapentin transport in these cells, LAT1 substrates significantly inhibited [3H]gabapentin uptake in TR-iBRB2 and RPE-J cells. In addition, preloaded [3H]gabapentin release from TR-iBRB2 and RPE-J cells was trans-stimulated by LAT1 substrates through the obligatory exchange mechanism as LAT1. Immunoblot analysis indicates the protein expression of LAT1 in TR-iBRB2 and RPE-J cells. These results imply that LAT1 at the inner and outer BRB takes part in gabapentin transport between the circulating blood and retina. Moreover, treatment of LAT1-targeted small interfering RNA to TR-iBRB2 cells significantly reduced both the level of LAT1 protein expression and [3H]gabapentin uptake activities in TR-iBRB2 cells. In conclusion, data from the present study indicate that LAT1 at the inner BRB is involved in retinal gabapentin transfer, and also suggest that LAT1 mediates gabapentin transport in the RPE cells.


Assuntos
Barreira Hematorretiniana/metabolismo , Gabapentina/farmacocinética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Animais , Linhagem Celular , Endotélio Vascular/citologia , Gabapentina/uso terapêutico , Masculino , Modelos Animais , Ratos Wistar , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/patologia
15.
Sci Signal ; 10(508)2017 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-29208681

RESUMO

Innate immune cells sense danger through a plethora of germline-encoded receptors that recognize pathogen-associated molecular patterns (PAMPs) or cellular molecules that are exposed only by stressed, infected, malignant, or dead cells. Many of these danger-sensing receptors belong to the C-type lectin-like superfamily (CLSF) and therefore are called C-type lectin-like receptors (CTLRs). Certain activating CTLRs, namely, CLEC-2, Dectin-1, DNGR-1, NKp80, and NKp65, which are encoded by genes that are clustered together in a subregion of the mammalian natural killer gene complex (NKC), use a single copy tyrosine signaling module termed the hemi-immunoreceptor tyrosine-based activation motif (hemITAM). These hemITAM-bearing CTLRs are present on myeloid cells and innate lymphocytes and stimulate various functions, such as phagocytosis, cytokine production, and cytotoxicity. Proximal signaling mechanisms involve the tyrosine phosphorylation of the hemITAM and the subsequent activation of the kinase Syk. Signaling and Syk recruitment by the hemITAM appear to be tuned by variable amino acids within or near the hemITAM, which give rise to differences in downstream signaling events and diverging functional outcomes among hemITAM-bearing receptors.


Assuntos
Motivo de Ativação do Imunorreceptor Baseado em Tirosina , Subfamília C de Receptores Semelhantes a Lectina de Células NK/química , Tirosina/metabolismo , Animais , Citocinas/metabolismo , Citotoxicidade Imunológica , Humanos , Imunidade Inata , Linfócitos/citologia , Linfócitos/imunologia , Camundongos , Células Mieloides/citologia , Células Mieloides/imunologia , Fagocitose/imunologia , Fosforilação , Quinase Syk/imunologia
16.
Front Neurol ; 8: 301, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28729850

RESUMO

Epilepsy is a common neurological disorder that affects over 70 million people worldwide. Despite the recent introduction of new antiseizure drugs (ASDs), about one-third of patients with epilepsy have seizures refractory to pharmacotherapy. Early identification of patients who will become refractory to ASDs could help direct such patients to appropriate non-pharmacological treatment, but the complexity in the temporal patterns of epilepsy could make such identification difficult. The target hypothesis and transporter hypothesis are the most cited theories trying to explain refractory epilepsy, but neither theory alone fully explains the neurobiological basis of pharmacoresistance. This review summarizes evidence for and against several major theories, including the pharmacokinetic hypothesis, neural network hypothesis, intrinsic severity hypothesis, gene variant hypothesis, target hypothesis, and transporter hypothesis. The discussion is mainly focused on the transporter hypothesis, where clinical and experimental data are discussed on multidrug transporter overexpression, substrate profiles of ASDs, mechanism of transporter upregulation, polymorphisms of transporters, and the use of transporter inhibitors. Finally, future perspectives are presented for the improvement of current hypotheses and the development of treatment strategies as guided by the current understanding of refractory epilepsy.

17.
J Biol Chem ; 292(8): 3213-3223, 2017 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-28082678

RESUMO

NKp65 is an activating human C-type lectin-like receptor (CTLR) triggering cellular cytotoxicity and cytokine secretion upon high-affinity interaction with the cognate CTLR keratinocyte-associated C-type lectin (KACL) selectively expressed by human keratinocytes. Previously, we demonstrated that NKp65-mediated cellular cytotoxicity depends on tyrosine 7, located in a cytoplasmic sequence motif of NKp65 resembling a hemi-immunoreceptor tyrosine-based activation motif (hemITAM). HemITAMs have been reported for a few activating myeloid-specific CTLRs, including Dectin-1 and CLEC-2, and consist of a single tyrosine signaling unit preceded by a triacidic motif. Upon receptor engagement, the hemITAM undergoes phosphotyrosinylation and specifically recruits spleen tyrosine kinase (Syk), initiating cellular activation. In this study, we addressed the functionality of the putative hemITAM of NKp65. We show that NKp65 forms homodimers and is phosphorylated at the hemITAM-embedded tyrosine 7 upon engagement by antibodies or KACL homodimers. HemITAM phosphotyrosinylation initiates a signaling pathway involving and depending on Syk, leading to cellular activation and natural killer (NK) cell degranulation. However, although NKp65 utilizes Syk for NK cell activation, a physical association of Syk with the NKp65 hemITAM could not be detected, unlike shown previously for the hemITAM of myeloid CTLR. Failure of NKp65 to recruit Syk is not due to an alteration of the triacidic motif, which rather affects the efficiency of hemITAM phosphotyrosinylation. In summary, NKp65 utilizes a hemITAM-like motif for cellular activation that requires Syk, although Syk appears not to be recruited to NKp65.


Assuntos
Motivo de Ativação do Imunorreceptor Baseado em Tirosina , Células Matadoras Naturais/imunologia , Receptores Semelhantes a Lectina de Células NK/imunologia , Quinase Syk/imunologia , Degranulação Celular , Linhagem Celular , Humanos , Imunidade Inata , Células Matadoras Naturais/citologia , Multimerização Proteica , Receptores Semelhantes a Lectina de Células NK/análise , Quinase Syk/análise
18.
Aging (Albany NY) ; 8(11): 2814-2826, 2016 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-27829242

RESUMO

Neurovascular integrity plays an important role in protecting cognitive and mental health in aging. Lifestyle interventions that sustain neurovascular integrity may thus be critical on preserving brain functions in aging and reducing the risk for age-related neurodegenerative disorders. Here we show that caloric restriction (CR) had an early effect on neurovascular enhancements, and played a critical role in preserving vascular, cognitive and mental health in aging. In particular, we found that CR significantly enhanced cerebral blood flow (CBF) and blood-brain barrier function in young mice at 5-6 months of age. The neurovascular enhancements were associated with reduced mammalian target of rapamycin expression, elevated endothelial nitric oxide synthase signaling, and increased ketone bodies utilization. With age, CR decelerated the rate of decline in CBF. The preserved CBF in hippocampus and frontal cortex were highly correlated with preserved memory and learning, and reduced anxiety, of the aging mice treated with CR (18-20 months of age). Our results suggest that dietary intervention started in the early stage (e.g., young adults) may benefit cognitive and mental reserve in aging. Understanding nutritional effects on neurovascular functions may have profound implications in human brain aging and age-related neurodegenerative disorders.


Assuntos
Envelhecimento/metabolismo , Ansiedade/metabolismo , Barreira Hematoencefálica/metabolismo , Restrição Calórica , Circulação Cerebrovascular/fisiologia , Memória/fisiologia , Animais , Comportamento Animal/fisiologia , Cognição/fisiologia , Lobo Frontal/metabolismo , Hipocampo/metabolismo , Masculino , Camundongos , Óxido Nítrico Sintase Tipo III/metabolismo , Transdução de Sinais/fisiologia
19.
J Cereb Blood Flow Metab ; 36(9): 1481-507, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27323783

RESUMO

Matrix metalloproteinases are versatile endopeptidases with many different functions in the body in health and disease. In the brain, matrix metalloproteinases are critical for tissue formation, neuronal network remodeling, and blood-brain barrier integrity. Many reviews have been published on matrix metalloproteinases before, most of which focus on the two best studied matrix metalloproteinases, the gelatinases MMP-2 and MMP-9, and their role in one or two diseases. In this review, we provide a broad overview of the role various matrix metalloproteinases play in brain disorders. We summarize and review current knowledge and understanding of matrix metalloproteinases in the brain and at the blood-brain barrier in neuroinflammation, multiple sclerosis, cerebral aneurysms, stroke, epilepsy, Alzheimer's disease, Parkinson's disease, and brain cancer. We discuss the detrimental effects matrix metalloproteinases can have in these conditions, contributing to blood-brain barrier leakage, neuroinflammation, neurotoxicity, demyelination, tumor angiogenesis, and cancer metastasis. We also discuss the beneficial role matrix metalloproteinases can play in neuroprotection and anti-inflammation. Finally, we address matrix metalloproteinases as potential therapeutic targets. Together, in this comprehensive review, we summarize current understanding and knowledge of matrix metalloproteinases in the brain and at the blood-brain barrier in brain disorders.


Assuntos
Barreira Hematoencefálica/enzimologia , Encefalopatias/enzimologia , Metaloproteinases da Matriz/fisiologia , Barreira Hematoencefálica/metabolismo , Humanos , Masculino , Metaloproteinases da Matriz/metabolismo , Terapia de Alvo Molecular
20.
J Neurosci ; 36(6): 1930-41, 2016 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-26865616

RESUMO

Failure to clear amyloid-ß (Aß) from the brain is in part responsible for Aß brain accumulation in Alzheimer's disease (AD). A critical protein for clearing Aß across the blood-brain barrier is the efflux transporter P-glycoprotein (P-gp) in the luminal plasma membrane of the brain capillary endothelium. P-gp is reduced at the blood-brain barrier in AD, which has been shown to be associated with Aß brain accumulation. However, the mechanism responsible for P-gp reduction in AD is not well understood. Here we focused on identifying critical mechanistic steps involved in reducing P-gp in AD. We exposed isolated rat brain capillaries to 100 nm Aß40, Aß40, aggregated Aß40, and Aß42. We observed that only Aß40 triggered reduction of P-gp protein expression and transport activity levels; this occurred in a dose- and time-dependent manner. To identify the steps involved in Aß-mediated P-gp reduction, we inhibited protein ubiquitination, protein trafficking, and the ubiquitin-proteasome system, and monitored P-gp protein expression, transport activity, and P-gp-ubiquitin levels. Thus, exposing brain capillaries to Aß40 triggers ubiquitination, internalization, and proteasomal degradation of P-gp. These findings may provide potential therapeutic targets within the blood-brain barrier to limit P-gp degradation in AD and improve Aß brain clearance. SIGNIFICANCE STATEMENT: The mechanism reducing blood-brain barrier P-glycoprotein (P-gp) in Alzheimer's disease is poorly understood. In the present study, we focused on defining this mechanism. We demonstrate that Aß40 drives P-gp ubiquitination, internalization, and proteasome-dependent degradation, reducing P-gp protein expression and transport activity in isolated brain capillaries. These findings may provide potential therapeutic avenues within the blood-brain barrier to limit P-gp degradation in Alzheimer's disease and improve Aß brain clearance.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Peptídeos beta-Amiloides/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Ubiquitina/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Capilares/efeitos dos fármacos , Capilares/metabolismo , Relação Dose-Resposta a Droga , Técnicas In Vitro , Masculino , Ratos , Ratos Sprague-Dawley , Ubiquitinação/efeitos dos fármacos
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