RESUMO
Blood-brain barrier dysfunction is recognized as a precursor of Alzheimer's disease development. Endothelial cells as structural basis of blood-brain barrier were observed tight junction failure in amyloid-ß(1-42)-stimulated environment. In this study, we found NOVA2, PPP2R3A were down-regulated while PART1, p-NFκB-p65 were up-regulated in amyloid-ß(1-42)-incubated endothelial cells. Knockdown of either NOVA2 or PPP2R3A and overexpression of PART1 all increased blood-brain barrier permeability. Lower blood-brain barrier permeability was observed in overexpression of NOVA2 and PPP2R3A and knockdown of PART1 and NFκB-p65. Same tendencies were found in the tight junction-related proteins expressions. Furthermore, overexpression and knockdown of NOVA2 and PART1 had no effect on cell viability. Mechanistically, NOVA2 overexpression was confirmed to reduce half-life of PART1. PART1 could destabilize PPP2R3A messenger RNA (mRNA) by interacting with STAU1. In addition, p-NFκB-p65 functioning as transcription factor reduced the expression of tight junction-related proteins, which was prompted by low protein level of PPP2R3A. Our study highlights the crucial role of NOVA2/PART1/PPP2R3A/p-NFκB-p65 pathway in amyloid-ß(1-42)-incubated endothelial cells to modulating blood-brain barrier permeability through STAU1-mediated messenger RNA degradation, implying a potential mechanism of lncRNA and protein interaction in pathogenesis of Alzheimer's disease.
Assuntos
Barreira Hematoencefálica/fisiologia , Proteínas do Citoesqueleto/metabolismo , Antígeno Neuro-Oncológico Ventral/genética , RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/farmacocinética , Barreira Hematoencefálica/efeitos dos fármacos , Células Cultivadas , Proteínas do Citoesqueleto/genética , Impedância Elétrica , Células Endoteliais , Regulação da Expressão Gênica , Humanos , Antígeno Neuro-Oncológico Ventral/metabolismo , Neuroglia , Fragmentos de Peptídeos/farmacocinética , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Estabilidade Proteica , Estabilidade de RNA , RNA não Traduzido/genética , Proteínas de Ligação a RNA/genética , Proteínas de Junções Íntimas/genética , Proteínas de Junções Íntimas/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
BACKGROUND: Age is the most common risk factor for Alzheimer's disease (AD), a neurodegenerative disorder characterized by the hallmarks of toxic amyloid-ß (Aß) plaques and hyperphosphorylated tau tangles. Moreover, sub-physiological brain insulin levels have emerged as a pathological manifestation of AD. OBJECTIVE: Identify age-related changes in the plasma disposition and blood-brain barrier (BBB) trafficking of Aß peptides and insulin in mice. METHODS: Upon systemic injection of 125I-Aß40, 125I-Aß42, or 125I-insulin, the plasma pharmacokinetics and brain influx were assessed in wild-type (WT) or AD transgenic (APP/PS1) mice at various ages. Additionally, publicly available single-cell RNA-Seq data [GSE129788] was employed to investigate pathways regulating BBB transport in WT mice at different ages. RESULTS: The brain influx of 125I-Aß40, estimated as the permeability-surface area product, decreased with age, accompanied by an increase in plasma AUC. In contrast, the brain influx of 125I-Aß42 increased with age, accompanied by a decrease in plasma AUC. The age-dependent changes observed in WT mice were accelerated in APP/PS1 mice. As seen with 125I-Aß40, the brain influx of 125I-insulin decreased with age in WT mice, accompanied by an increase in plasma AUC. This finding was further supported by dynamic single-photon emission computed tomography (SPECT/CT) imaging studies. RAGE and PI3K/AKT signaling pathways at the BBB, which are implicated in Aß and insulin transcytosis, respectively, were upregulated with age in WT mice, indicating BBB insulin resistance. CONCLUSION: Aging differentially affects the plasma pharmacokinetics and brain influx of Aß isoforms and insulin in a manner that could potentially augment AD risk.
Assuntos
Envelhecimento , Doença de Alzheimer , Peptídeos beta-Amiloides/farmacocinética , Barreira Hematoencefálica/metabolismo , Insulina/farmacocinética , Placa Amiloide/metabolismo , Fatores Etários , Envelhecimento/sangue , Envelhecimento/fisiologia , Doença de Alzheimer/sangue , Doença de Alzheimer/patologia , Animais , Encéfalo/irrigação sanguínea , Encéfalo/patologia , Modelos Animais de Doenças , Radioisótopos do Iodo/farmacocinética , Camundongos , Camundongos Transgênicos , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
We have previously developed a unique 8-amino acid Aß42 oligomer-Interacting Peptide (AIP) as a novel anti-amyloid strategy for the treatment of Alzheimer's disease. Our lead candidate has successfully progressed from test tubes (i.e., in vitro characterization of protease-resistant D-AIP) to transgenic flies (i.e., in vivo rescue of human Aß42-mediated toxicity via D-AIP-supplemented food). In the present study, we examined D-AIP in terms of its stability in multiple biological matrices (i.e., ex-vivo mouse plasma, whole blood, and liver S9 fractions) using MALDI mass spectrometry, pharmacokinetics using a rapid and sensitive LC-MS method, and blood brain barrier (BBB) penetrance in WT C57LB/6 mice. D-AIP was found to be relatively stable over 3 h at 37 °C in all matrices tested. Finally, label-free MALDI imaging showed that orally administered D-AIP can readily penetrate the intact BBB in both male and female WT mice. Based upon the favorable stability, pharmacokinetics, and BBB penetration outcomes for orally administered D-AIP in WT mice, we then examined the effect of D-AIP on amyloid "seeding" in vitro (i.e., freshly monomerized versus preaggregated Aß42). Complementary biophysical assays (ThT, TEM, and MALDI-TOF MS) showed that D-AIP can directly interact with synthetic Aß42 aggregates to disrupt primary and/or secondary seeding events. Taken together, the unique mechanistic and desired therapeutic potential of our lead D-AIP candidate warrants further investigation, that is, testing of D-AIP efficacy on the altered amyloid/tau pathology in transgenic mouse models of Alzheimer's disease.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Encéfalo , Fragmentos de Peptídeos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/farmacocinética , Peptídeos beta-Amiloides/farmacologia , Animais , Encéfalo/metabolismo , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/farmacocinética , Fragmentos de Peptídeos/farmacologiaRESUMO
BACKGROUND: The interactions between nanoparticles (NPs) and plasma proteins form a protein corona around NPs after entering the biological environment, which provides new biological properties to NPs and mediates their interactions with cells and biological barriers. Given the inevitable interactions, we regard nanoparticleâprotein interactions as a tool for designing protein corona-mediated drug delivery systems. Herein, we demonstrate the successful application of protein corona-mediated brain-targeted nanomicelles in the treatment of glioma, loading them with paclitaxel (PTX), and decorating them with amyloid ß-protein (Aß)-CN peptide (PTX/Aß-CN-PMs). Aß-CN peptide, like the Aß1-42 peptide, specifically binds to the lipid-binding domain of apolipoprotein E (ApoE) in vivo to form the ApoE-enriched protein corona surrounding Aß-CN-PMs (ApoE/PTX/Aß-CN-PMs). The receptor-binding domain of the ApoE then combines with low-density lipoprotein receptor (LDLr) and LDLr-related protein 1 receptor (LRP1r) expressed in the blood-brain barrier and glioma, effectively mediating brain-targeted delivery. METHODS: PTX/Aß-CN-PMs were prepared using a film hydration method with sonication, which was simple and feasible. The specific formation of the ApoE-enriched protein corona around nanoparticles was characterized by Western blotting analysis and LC-MS/MS. The in vitro physicochemical properties and in vivo anti-glioma effects of PTX/Aß-CN-PMs were also well studied. RESULTS: The average size and zeta potential of PTX/Aß-CN-PMs and ApoE/PTX/Aß-CN-PMs were 103.1 nm, 172.3 nm, 7.23 mV, and 0.715 mV, respectively. PTX was efficiently loaded into PTX/Aß-CN-PMs, and the PTX release from rhApoE/PTX/Aß-CN-PMs exhibited a sustained-release pattern in vitro. The formation of the ApoE-enriched protein corona significantly improved the cellular uptake of Aß-CN-PMs on C6 cells and human umbilical vein endothelial cells (HUVECs) and enhanced permeability to the blood-brain tumor barrier in vitro. Meanwhile, PTX/Aß-CN-PMs with ApoE-enriched protein corona had a greater ability to inhibit cell proliferation and induce cell apoptosis than taxol. Importantly, PTX/Aß-CN-PMs exhibited better anti-glioma effects and tissue distribution profile with rapid accumulation in glioma tissues in vivo and prolonged median survival of glioma-bearing mice compared to those associated with PMs without the ApoE protein corona. CONCLUSIONS: The designed PTX/Aß-CN-PMs exhibited significantly enhanced anti-glioma efficacy. Importantly, this study provided a strategy for the rational design of a protein corona-based brain-targeted drug delivery system. More crucially, we utilized the unfavorable side of the protein corona and converted it into an advantage to achieve brain-targeted drug delivery.
Assuntos
Antineoplásicos/administração & dosagem , Apolipoproteínas E/administração & dosagem , Encéfalo/efeitos dos fármacos , Glioma/tratamento farmacológico , Nanopartículas/administração & dosagem , Coroa de Proteína , Peptídeos beta-Amiloides/administração & dosagem , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/farmacocinética , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Apolipoproteínas E/química , Apolipoproteínas E/farmacocinética , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Glioma/metabolismo , Humanos , Camundongos , Micelas , Nanopartículas/química , Paclitaxel/administração & dosagem , Paclitaxel/química , Paclitaxel/farmacocinética , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacocinética , Poliésteres/administração & dosagem , Poliésteres/química , Poliésteres/farmacocinética , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Coroa de Proteína/químicaRESUMO
Amyloid fibrils found in plaques in Alzheimer's disease (AD) brains are composed of amyloid-ß peptides. Oligomeric amyloid-ß 1-42 (Aß42) is thought to play a critical role in neurodegeneration in AD. Here, we determine how size and conformation affect neurotoxicity and internalisation of Aß42 assemblies using biophysical methods, immunoblotting, toxicity assays and live-cell imaging. We report significant cytotoxicity of Aß42 oligomers and their internalisation into neurons. In contrast, Aß42 fibrils show reduced internalisation and no toxicity. Sonicating Aß42 fibrils generates species similar in size to oligomers but remains nontoxic. The results suggest that Aß42 oligomers have unique properties that underlie their neurotoxic potential. Furthermore, we show that incubating cells with Aß42 oligomers for 24 h is sufficient to trigger irreversible neurotoxicity.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/toxicidade , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/toxicidade , Agregação Patológica de Proteínas , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amiloide/química , Amiloide/metabolismo , Amiloide/farmacologia , Amiloide/toxicidade , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacocinética , Sobrevivência Celular/efeitos dos fármacos , Humanos , Peso Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacocinética , Conformação Proteica , SonicaçãoRESUMO
Amyloid-ß peptides of 40 and 42 amino acid lengths, which are synthesized in neurons and degraded in the brain and liver, have the potential to aggregate and form neuritic plaques in Alzheimer disease. The kinetics of human amyloid-ß (hAß) 40 were examined in the rat pursuant to intravenous and intracerebroventricular administration after pretreatment with calcitriol, the active vitamin D receptor ligand (6.4 nmol·kg-1 in 0.3 ml corn oil every other day for four intraperitoneal doses) to induce P-glycoprotein (P-gp) and enhance hAß40 brain efflux. The interference of hAß40 by media matrix that suppressed absorbance readings in the ELISA assay was circumvented with use of different calibration curves prepared in Standard Dilution Buffer, undiluted, 10-10,000 or 5-fold diluted plasma, or artificial cerebrospinal fluid. Simultaneous fitting of hAß40 plasma and cerebrospinal fluid (CSF) data after intravenous and intracerebroventricular administration were described by catenary-mammillary models comprising of a central and two peripheral compartments, the brain, and one to four CSF compartments. The model with only one CSF compartment (model I) best fitted the intravenous data that showed a faster plasma decay t1/2 and slower equilibration between plasma and brain/CSF. Calcitriol induction increased the brain efflux rate constant, k41 (1.8-fold), at the blood-brain barrier when compared with the control group, as confirmed by the 2-fold (P < 0.05) increase in brain P-gp relative protein expression. SIGNIFICANCE STATEMENT: An accurate description of the kinetic behavior of human amyloid-ß (hAß) 40 is needed in defining the toxic peptide as a biomarker of Alzheimer disease. Modeling of hAß40 data after intravenous and intracerebroventricular administration to the rat revealed an initially faster plasma half-life that reflected faster peripheral distribution but slower equilibration between plasma and brain/cerebrospinal fluid even with calcitriol pretreatment that increased P-glycoprotein protein expression and enhanced efflux clearance from brain.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Peptídeos beta-Amiloides/farmacocinética , Barreira Hematoencefálica/metabolismo , Calcitriol/administração & dosagem , Fragmentos de Peptídeos/farmacocinética , Subfamília B de Transportador de Cassetes de Ligação de ATP/agonistas , Doença de Alzheimer/sangue , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/administração & dosagem , Animais , Humanos , Injeções Intravenosas , Injeções Intraventriculares , Masculino , Modelos Animais , Fragmentos de Peptídeos/administração & dosagem , RatosRESUMO
As an important neuropathological hallmark of Alzheimer's disease (AD), the oligomerization of amyloid-ß (Aß) peptides has been intensively investigated in both theoretical and experimental studies. However, the oligomerization space in terms of the kinetics, molecular mechanism, and oligomer structures remains mysterious to us. An equation that can quantitatively describe the time it takes for Aß oligomers to appear in the human brain at a given Aß monomer concentration is extremely vital for us to understand the development and disease progression of AD. In this study, we utilized molecular dynamics (MD) simulations to investigate the oligomerization of Aß42 peptides at five different monomer concentrations. We have elucidated the formation pathways of Aß tetramers, characterized the oligomer structures, estimated the oligomerization time for Aß dimers, trimers, and tetramers, and for the first-time derived equations that could quantitatively describe the relationship between the oligomerization time and the monomer concentration. Applying these equations, our prediction of oligomerization time agrees well with the experimental and clinical findings, in spite of the limitations of our oligomerization simulations. We have found that the Aß oligomerization time depends on the monomer concentration by a power of -2.4. The newly established equations will enable us to quantitatively estimate the risk score of AD, which is a function of age. Moreover, we have identified the most dominant pathway of forming Aß tetramers, probably the most important and toxic Aß oligomer. Our results have shown that the structures of Aß42 dimer, trimer, and tetramer, which are distinguishable from each other, depend on the monomer concentration at which the oligomers form. Representative oligomer structures, which can serve as potential drug targets, have been identified by clustering analysis. The MD sampling adequacy has been validated by the excellent agreement between the calculated and measured collisional cross section (CCS) parameters (the prediction errors are within 2%). In a conclusion, this study provides the kinetics and structure basis for developing inhibitors to decelerate the Aß oligomerization process.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/farmacocinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacocinética , Agregados Proteicos , Agregados Proteicos/fisiologia , Estrutura Secundária de ProteínaRESUMO
The success of the targeting of amyloid-ß (Aß) oligomers through immunotherapy in Alzheimer's disease (AD) mouse models has not been translated into the clinics. The use of single-chain variable fragments (scFvs) has been proposed to prevent the potential severe effects of full-length mAbs by precluding crystallizable fraction-mediated microglia activation. The efficacy of scFv-h3D6, a bapineuzumab-derived anti-Aß scFv, has been extensively proven. In this work, we compared scFv-h3D6-EL, an elongated variant of the scFv-h3D6, with its original version to assess whether its characteristic higher thermodynamic stability improved its pharmacokinetic parameters. Although scFv-h3D6-EL had a longer half-life than its original version, its absorption from the peritoneal cavity into the systemic compartment was lower than that of the original version. Moreover, we attempted to determine the mechanism underlying the protective effect of scFv-h3D6. We found that scFv-h3D6 showed compartmental distribution and more interestingly crossed the blood-brain barrier. In the brain, scFv-h3D6 was engulfed by glial cells or internalized by Aß peptide-containing neurons in the early phase post-injection, and was colocalized with the Aß peptide almost exclusively in glial cells in the late phase post-injection. Aß peptide levels in the brain decreased simultaneously with an increase in scFv-h3D6 levels. This observation in addition to the increased tumor necrosis factor-α levels in the late phase post-injection suggested that the engulfment of Aß peptide/scFv-h3D6 complex extruded from large neurons by phagocytic cells was the mechanism underlying Aß peptide withdrawal. The mechanism of action of scFv-h3D6 demonstrates the effectivity of Aß-immunotherapy and lays the background for other studies focused on the finding of a treatment for AD.
Assuntos
Doença de Alzheimer/imunologia , Peptídeos beta-Amiloides/imunologia , Fragmentos de Imunoglobulinas/imunologia , Anticorpos de Cadeia Única/imunologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/farmacocinética , Peptídeos beta-Amiloides/farmacologia , Animais , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacocinética , Modelos Animais de Doenças , Humanos , Fragmentos de Imunoglobulinas/farmacologia , Camundongos , Neurônios/metabolismo , Sinais Direcionadores de Proteínas/genética , Estabilidade Proteica , Anticorpos de Cadeia Única/farmacocinética , Anticorpos de Cadeia Única/farmacologia , TermodinâmicaRESUMO
Neuronal amyloid ß1-42 (Aß1-42) accumulation is considered an upstream event in Alzheimer's disease pathogenesis. Here we report the mechanism on synaptic activity-independent Aß1-42 uptake in vivo. When Aß1-42 uptake was compared in hippocampal slices after incubating with Aß1-42, In vitro Aß1-42 uptake was preferentially high in the dentate granule cell layer in the hippocampus. Because the rapid uptake of Aß1-42 with extracellular Zn2+ is essential for Aß1-42-induced cognitive decline in vivo, the uptake mechanism was tested in dentate granule cells in association with synaptic activity. In vivo rapid uptake of Aß1-42 was not modified in the dentate granule cell layer after co-injection of Aß1-42 and tetrodotoxin, a Na+ channel blocker, into the dentate gyrus. Both the rapid uptake of Aß1-42 and Zn2+ into the dentate granule cell layer was not modified after co-injection of CNQX, an AMPA receptor antagonist, which blocks extracellular Zn2+ influx, Both the rapid uptake of Aß1-42 and Zn2+ into the dentate granule cell layer was not also modified after either co-injection of chlorpromazine or genistein, an endocytic repressor. The present study suggests that Aß1-42 and Zn2+ are synaptic activity-independently co-taken up into dentate granule cells in the normal brain and the co-uptake is preferential in dentate granule cells in the hippocampus. We propose a hypothesis that Zn-Aß1-42 oligomers formed in the extracellular compartment are directly incorporated into neuronal plasma membranes and form Zn2+-permeable ion channels.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Giro Denteado/metabolismo , Fragmentos de Peptídeos/metabolismo , Sinapses/metabolismo , Zinco/metabolismo , 6-Ciano-7-nitroquinoxalina-2,3-diona/administração & dosagem , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Peptídeos beta-Amiloides/farmacocinética , Animais , Transporte Biológico/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , Antagonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/metabolismo , Masculino , Microscopia Confocal , Fragmentos de Peptídeos/farmacocinética , Ratos Wistar , Bloqueadores dos Canais de Sódio/administração & dosagem , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/administração & dosagem , Tetrodotoxina/farmacologia , Zinco/farmacocinéticaRESUMO
Reactive oxygen species (ROS) generated after exposure to ionizing radiation and toxic peptides, in mitochondrial metabolism and during aging contribute to damage of cell's structural and functional components and can lead to diseases. Monomers and small oligomers of amyloid beta (Aß) peptide, players in Alzheimer's disease, are recently suggested to be involved in damaging of neurons, instead of extracellular Aß plaques. We demonstrate that externally applied disaggregated Aß1-42 peptide interacts preferentially with acidic compartments (lysosomes). We compared standard cell cultivation (21% O2) to more physiological cell cultivation (5% O2). Cells did not exhibit a dramatic increase in ROS and change in glutathione level upon 4 µM Aß peptide treatment, whereas exposure to 2 Gy X-rays increased ROS and changed glutathione level and ATP concentration. The occurrence of the 4977 bp deletion in mtDNA and significant protein carbonylation were specific effects of IR and more pronounced at 21% O2. An increase in cell death after Aß peptide treatment or irradiation was unexpectedly restored to the control level or below when both were combined, particularly at 5% O2. Therefore, Aß peptide at low concentration can trigger neuroprotective mechanisms in cells exposed to radiation. Oxygen concentration is an important modulator of cellular responses to stress.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Estresse Oxidativo/fisiologia , Oxigênio/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeos beta-Amiloides/administração & dosagem , Peptídeos beta-Amiloides/farmacocinética , Animais , Linhagem Celular Tumoral , Humanos , Inflamação/metabolismo , Camundongos , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/farmacocinéticaRESUMO
Alzheimer's disease (AD) pathogenesis leads to the appearance of senile plaques due to the production and deposition of the ß-amyloid peptide (Aß). Superparamagnetic iron oxide nanoparticles (SPIONs) have potential role in the detection and imaging of Aß plaques in AD. SPIONs have shown appropriate potential in the diagnosis and treatment of AD. In the present study, the pharmacokinetics of SPIONs and its effect in the biochemical pathway of AD were analyzed using collected information. During analysis, the interaction of SPIONs with amyloid beta-42 (Aß42), a biomarker for AD progression, has been shown. Nodes represent the entities and edges represent the relation (interactions) of one node to another node. Aß42 and their interaction with other entities making up biochemical network are involved in AD mechanism in presence of SPION. The kinetic simulation was done to investigate pharmacokinetics of SPIONs for AD, where concentration was assigned of nanoparticles and other entities were applied as a kinetic irreversible simple Michaelis-Menten or mass action kinetics. Simulation was done in presence and absence of SPIONs to investigate pharmacokinetic effect in AD and explore the mechanism of Aß42 in presence of SPIONs. This study may lead to better understanding, which is required to target the metabolism of Aß42 peptide, a pivotal player in this pathology.
Assuntos
Peptídeos beta-Amiloides/farmacocinética , Dextranos/química , Nanopartículas de Magnetita/química , Fragmentos de Peptídeos/farmacocinética , Transdução de Sinais , Biologia de Sistemas , Doença de Alzheimer , Ouro/química , Humanos , Simulação de Acoplamento MolecularRESUMO
AIMS: To investigate restorative effects of the receptor for advanced glycation end products (RAGE)-specific inhibitor FPS-ZM1 on abnormal amyloid ß (Aß) influx across the blood brain-barrier (BBB) and cognitive deficits in db/db mice. METHODS: Aß influx across the BBB was determined by intra-arterial infusion of 125I-Aß1-40. Receptor for advanced glycation end products (RAGE), Aß, NF-κB p65, caspase-3, Bax, Bcl-2, PSD-95 and synaptophysin were assayed by Western blot, immunohistochemistry or RT-PCR. Apoptosis was quantified by TUNEL assay. In vivo hippocampal long term potentiation (LTP) recording, Golgi Staining, Morris water maze (MWM) task and Y-maze test were performed. RESULTS: FPS-ZM1 (1.0â¯mg/kg i.p.) inhibited Aß influx across the BBB and expression of RAGE participating in Aß influx, consequently decreased hippocampal Aß1-40 and Aß1-42 in db/db mice. After FPS-ZM1 treatment, NF-κB signaling was inhibited, and neuronal apoptosis was reduced, which revealed by less TUNEL + cells, reduced caspase-3 activity and higher ratio of Bcl-2/Bax. In addition, FPS-ZM1 improved hippocampal plasticity evidenced by enhanced in vivo LTP and the restoration of spine deficit and increased PSD-95 expression in hippocampal neuron. Further studies found that FPS-ZM1 treatment alleviated cognitive deficits shown by better performance in behavioral tests, without significant metabolic effects on blood glucose, insulin and cerebral AGEs. CONCLUSION: Downregulation of abnormal Aß influx across the BBB by FPS-ZM1 at higher dosage contributes to reduced neuronal apoptosis, improved hippocampal plasticity and cognitive impairment in db/db mice.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Benzamidas/uso terapêutico , Barreira Hematoencefálica/fisiologia , Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/etiologia , Diabetes Mellitus Tipo 2/complicações , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Peptídeos beta-Amiloides/farmacocinética , Animais , Benzamidas/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/ultraestrutura , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/ultraestrutura , Caspase 3/metabolismo , Diabetes Mellitus Tipo 2/genética , Modelos Animais de Doenças , Comportamento Exploratório/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Microvasos/efeitos dos fármacos , Microvasos/metabolismo , Fragmentos de Peptídeos/farmacocinética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptor para Produtos Finais de Glicação Avançada/antagonistas & inibidores , Receptor para Produtos Finais de Glicação Avançada/genética , Receptores para Leptina/deficiência , Receptores para Leptina/genética , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Impaired brain clearance of amyloid-beta peptides (Aß) 40 and 42 across the blood-brain barrier (BBB) is believed to be one of the pathways responsible for Alzheimer's disease (AD) pathogenesis. Hyperinsulinemia prevalent in type II diabetes was shown to damage cerebral vasculature and increase Aß accumulation in AD brain. However, there is no clarity on how aberrations in peripheral insulin levels affect Aß accumulation in the brain. This study describes, for the first time, an intricate relation between plasma insulin and Aß transport at the BBB. Upon peripheral insulin administration in wild-type mice: the plasma clearance of Aß40 increased, but Aß42 clearance reduced; the plasma-to-brain influx of Aß40 increased, and that of Aß42 reduced; and the clearance of intracerebrally injected Aß40 decreased, whereas Aß42 clearance increased. In hCMEC/D3 monolayers (in vitro BBB model) exposed to insulin, the luminal uptake and luminal-to-abluminal permeability of Aß40 increased and that of Aß42 reduced; the abluminal-to-luminal permeability of Aß40 decreased, whereas Aß42 permeability increased. Moreover, Aß cellular trafficking machinery was altered. In summary, Aß40 and Aß42 demonstrated distinct distribution kinetics in plasma and brain compartments, and insulin differentially modulated their distribution. Cerebrovascular disease and metabolic disorders may disrupt this intricate homeostasis and aggravate AD pathology.
Assuntos
Peptídeos beta-Amiloides/farmacocinética , Química Encefálica/efeitos dos fármacos , Insulina/farmacologia , Doença de Alzheimer , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/sangue , Animais , Barreira Hematoencefálica/metabolismo , Linhagem Celular , Humanos , Camundongos , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/farmacocinética , Transporte Proteico , Distribuição Tecidual/efeitos dos fármacosRESUMO
Alzheimer's disease (AD) is increasingly viewed as a disease of synapses. Loss of synapses correlates better with cognitive decline than amyloid plaques and neurofibrillary tangles, the hallmark neuropathological lesions of AD. Soluble forms of amyloid-ß (Aß) have emerged as mediators of synapse dysfunction. Aß binds to, accumulates, and aggregates in synapses. However, the anatomical and neurotransmitter specificity of Aß and the amyloid-ß protein precursor (AßPP) in AD remain poorly understood. In addition, the relative roles of Aß and AßPP in the development of AD, at pre- versus post-synaptic compartments and axons versus dendrites, respectively, remain unclear. Here we use immunogold electron microscopy and confocal microscopy to provide evidence for heterogeneity in the localization of Aß/AßPP. We demonstrate that Aß binds to a subset of synapses in cultured neurons, with preferential binding to glutamatergic compared to GABAergic neurons. We also highlight the challenge of defining pre- versus post-synaptic localization of this binding by confocal microscopy. Further, endogenous Aß42 accumulates in both glutamatergic and GABAergic AßPP/PS1 transgenic primary neurons, but at varying levels. Moreover, upon knock-out of presenilin 1 or inhibition of γ-secretase AßPP C-terminal fragments accumulate both pre- and post-synaptically; however earlier pre-synaptically, consistent with a higher rate of AßPP processing in axons. A better understanding of the synaptic and anatomical selectivity of Aß/AßPP in AD can be important for the development of more effective new therapies for this major disease of aging.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Neurônios/citologia , Sinapses/metabolismo , Peptídeos beta-Amiloides/farmacocinética , Precursor de Proteína beta-Amiloide/genética , Animais , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Diaminas/farmacologia , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glutamato Descarboxilase/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação/genética , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Neuropeptídeos/metabolismo , Fragmentos de Peptídeos/farmacocinética , Densidade Pós-Sináptica/efeitos dos fármacos , Densidade Pós-Sináptica/metabolismo , Densidade Pós-Sináptica/ultraestrutura , Presenilina-1/genética , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Sinapses/efeitos dos fármacos , Sinapses/genética , Sinapses/ultraestrutura , Sinapsinas/metabolismo , Sinaptofisina/metabolismo , Tiazóis/farmacologia , Fatores de Tempo , Proteínas tau/metabolismoRESUMO
The highly rigid and planar scaffold with π-conjugated systems has been widely considered to be indispensable for Aß binding probes. However, the flexible benzyloxybenzene derivative [(125)I]BOB-4 represents an excellent lead candidate for targeting Aß in AD brains. Based on that, we designed two pairs of more flexible and optically pure diphenoxy derivatives with a chiral center as novel Aß probes. These compounds possessed high affinity (Ki = 15.8-45.0 nM) for Aß1-42 aggregates, and (R)-enantiomers showed slightly better binding ability than (S)-enantiomers. In addition, the competition binding assay implied that the optically pure diphenoxy derivatives with more flexible geometry shared the same binding site as IMPY, a classical rigid and planar Aß probe. For (125)I-radiolabeled enantiomers, (S)-[(125)I]5 and (R)-[(125)I]5, specific plaque labeling on brain sections of Tg mice and AD patients were observed in in vitro autoradiography, persuasively proving the excellent affinity of the probes. In biodistribution, (S)-[(125)I]5 and (R)-[(125)I]5 with relatively low lipophilicity exhibited moderate initial brain uptake (4.37% and 3.72% ID/g at 2 min, respectively) and extremely fast washout from normal mice brain (brain2min/brain60min = 19.0 and 17.7, respectively). In summary, the separate enantiomers displayed similar properties in vitro and in vivo, and (S/R)-[(123)I]5 may be potential SPECT probes for recognizing Aß plaques in AD brains.
Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Placa Amiloide/química , Placa Amiloide/metabolismo , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/farmacocinética , Peptídeos beta-Amiloides/farmacologia , Compostos de Anilina/farmacocinética , Compostos de Anilina/farmacologia , Animais , Autorradiografia , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Cristalografia por Raios X , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Fragmentos de Peptídeos/farmacocinética , Fragmentos de Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Piridinas/farmacocinética , Piridinas/farmacologia , Estilbenos/farmacocinética , Estilbenos/farmacologia , Glândula Tireoide/diagnóstico por imagem , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/metabolismo , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/genéticaRESUMO
In the absence of conventional lymphatics, drainage of interstitial fluid and solutes from the brain parenchyma to cervical lymph nodes is along basement membranes in the walls of cerebral capillaries and tunica media of arteries. Perivascular pathways are also involved in the entry of CSF into the brain by the convective influx/glymphatic system. The objective of this study is to differentiate the cerebral vascular basement membrane pathways by which fluid passes out of the brain from the pathway by which CSF enters the brain. Experiment 1: 0.5 µl of soluble biotinylated or fluorescent Aß, or 1 µl 15 nm gold nanoparticles was injected into the mouse hippocampus and their distributions determined at 5 min by transmission electron microscopy. Aß was distributed within the extracellular spaces of the hippocampus and within basement membranes of capillaries and tunica media of arteries. Nanoparticles did not enter capillary basement membranes from the extracellular spaces. Experiment 2: 2 µl of 15 nm nanoparticles were injected into mouse CSF. Within 5 min, groups of nanoparticles were present in the pial-glial basement membrane on the outer aspect of cortical arteries between the investing layer of pia mater and the glia limitans. The results of this study and previous research suggest that cerebral vascular basement membranes form the pathways by which fluid passes into and out of the brain but that different basement membrane layers are involved. The significance of these findings for neuroimmunology, Alzheimer's disease, drug delivery to the brain and the concept of the Virchow-Robin space are discussed.
Assuntos
Membrana Basal/metabolismo , Vasos Sanguíneos/citologia , Hipocampo/metabolismo , Actinas/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacocinética , Animais , Membrana Basal/efeitos dos fármacos , Membrana Basal/ultraestrutura , Biotinilação , Líquido Cefalorraquidiano/efeitos dos fármacos , Líquido Cefalorraquidiano/metabolismo , Cisterna Magna/efeitos dos fármacos , Cisterna Magna/metabolismo , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Corantes Fluorescentes/farmacocinética , Hipocampo/efeitos dos fármacos , Hipocampo/ultraestrutura , Laminina/metabolismo , Masculino , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacocinéticaRESUMO
This Editorial highlights a study by Jana and coworkers published in the current issue of Journal of Neurochemistry, in which the authors performed a detailed, quantitative analysis to identify the Aß oligomer causing neuronal cell death. While most studies so far aimed to determine the Aß oligomer with highest toxicity using preformed and characterized Aß oligomers added to cell cultures, this study established an approach to analyze Aß oligomers bound to primary neurons. This may shed new light on how oligomeric status changes at the cell surface and if minor oligomeric species may account for measured effects. The authors' procedure allows to monitor the effects of different Aß oligomers in parallel, constituting an important advancement in the research field. Read the highlighted article 'Membrane bound tetramer and trimer Aß oligomeric species correlate with toxicity towards cultured neurons' on page 594.
Assuntos
Peptídeos beta-Amiloides/farmacocinética , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacocinética , Animais , Feminino , MasculinoRESUMO
Amyloid beta (Aß) peptide is the major constituent of the extracellular amyloid plaques deposited in the brains of Alzheimer's disease patients and is central to the pathogenic pathway causing this disease. The identity of the neurotoxic Aß species remains elusive. We previously reported that Aß toxicity correlates strongly with its neuronal cell binding leading us to hypothesize that neuronal cell death is caused by the binding of a specific oligomeric Aß species. To identify the specific oligomeric Aß species that is associated with cell death, we treated mouse cortical neuronal cultures with synthetic Aß40 and Aß42 peptides and identified that the cellular Aß binding and neurotoxicity were time and concentration dependent. We found a significant correlation between the amount of trimer and tetramer species bound to neurons with increasing neurotoxicity. We prepared Aß40 oligomers (up to tetramers) using photo-induced cross-linking of unmodified peptides to confirm this oligomer-specific neurotoxic activity. Our results identify the Aß tetramer, followed by the trimer, as the most toxic low-order oligomers Aß species. Our findings suggested that binding of amyloid-ß (Aß) tetramer and trimer, not monomer or dimer, to neurons is critical to induce neuronal cell death associated with Alzheimer's Disease. We proposed that Aß trimer and tetramer are the potential neurotoxic Aß species. This would provide more specific therapeutic target for Alzheimer's Disease.
Assuntos
Peptídeos beta-Amiloides/farmacocinética , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacocinética , Peptídeos beta-Amiloides/toxicidade , Animais , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Feminino , Masculino , Camundongos , Fragmentos de Peptídeos/toxicidade , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Estrutura Secundária de Proteína , Fatores de Tempo , Proteínas tau/metabolismoRESUMO
According to the neurovascular hypothesis, impairment of low-density lipoprotein receptor-related protein-1 (LRP1) in brain capillaries of the blood-brain barrier (BBB) contributes to neurotoxic amyloid-ß (Aß) brain accumulation and drives Alzheimer's disease (AD) pathology. However, due to conflicting reports on the involvement of LRP1 in Aß transport and the expression of LRP1 in brain endothelium, the role of LRP1 at the BBB is uncertain. As global Lrp1 deletion in mice is lethal, appropriate models to study the function of LRP1 are lacking. Moreover, the relevance of systemic Aß clearance to AD pathology remains unclear, as no BBB-specific knockout models have been available. Here, we developed transgenic mouse strains that allow for tamoxifen-inducible deletion of Lrp1 specifically within brain endothelial cells (Slco1c1-CreER(T2) Lrp1(fl/fl) mice) and used these mice to accurately evaluate LRP1-mediated Aß BBB clearance in vivo. Selective deletion of Lrp1 in the brain endothelium of C57BL/6 mice strongly reduced brain efflux of injected [125I] Aß(1-42). Additionally, in the 5xFAD mouse model of AD, brain endothelial-specific Lrp1 deletion reduced plasma Aß levels and elevated soluble brain Aß, leading to aggravated spatial learning and memory deficits, thus emphasizing the importance of systemic Aß elimination via the BBB. Together, our results suggest that receptor-mediated Aß BBB clearance may be a potential target for treatment and prevention of Aß brain accumulation in AD.
Assuntos
Peptídeos beta-Amiloides/farmacocinética , Barreira Hematoencefálica , Células Endoteliais/fisiologia , Fragmentos de Peptídeos/farmacocinética , Receptores de LDL/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Encéfalo/metabolismo , Transtornos Cognitivos/etiologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico , TranscitoseRESUMO
UNLABELLED: ATP binding cassette transporter A1 (encoded by ABCA1) regulates cholesterol efflux from cells to apolipoproteins A-I and E (ApoA-I and APOE; encoded by APOA1 and APOE, respectively) and the generation of high density lipoproteins. In Abca1 knockout mice (Abca1(ko)), high density lipoproteins and ApoA-I are virtually lacking, and total APOE and APOE-containing lipoproteins in brain substantially decreased. As the ε4 allele of APOE is the major genetic risk factor for late-onset Alzheimer's disease, ABCA1 role as a modifier of APOE lipidation is of significance for this disease. Reportedly, Abca1 deficiency in mice expressing human APP accelerates amyloid deposition and behaviour deficits. We used APP/PS1dE9 mice crossed to Apoe and Apoa1 knockout mice to generate Apoe/Apoa1 double-knockout mice. We hypothesized that Apoe/Apoa1 double-knockout mice would mimic the phenotype of APP/Abca1(ko) mice in regards to amyloid plaques and cognitive deficits. Amyloid pathology, peripheral lipoprotein metabolism, cognitive deficits and dendritic morphology of Apoe/Apoa1 double-knockout mice were compared to APP/Abca1(ko), APP/PS1dE9, and single Apoa1 and Apoe knockouts. Contrary to our prediction, the results demonstrate that double deletion of Apoe and Apoa1 ameliorated the amyloid pathology, including amyloid plaques and soluble amyloid. In double knockout mice we show that (125)I-amyloid-ß microinjected into the central nervous system cleared at a rate twice faster compared to Abca1 knockout mice. We tested the effect of Apoe, Apoa1 or Abca1 deficiency on spreading of exogenous amyloid-ß seeds injected into the brain of young pre-depositing APP mice. The results show that lack of Abca1 augments dissemination of exogenous amyloid significantly more than the lack of Apoe. In the periphery, Apoe/Apoa1 double-knockout mice exhibited substantial atherosclerosis and very high levels of low density lipoproteins compared to APP/PS1dE9 and APP/Abca1(ko). Plasma level of amyloid-ß42 measured at several time points for each mouse was significantly higher in Apoe/Apoa1 double-knockout then in APP/Abca1(ko) mice. This result demonstrates that mice with the lowest level of plasma lipoproteins, APP/Abca1(ko), have the lowest level of peripheral amyloid-ß. Unexpectedly, and independent of amyloid pathology, the deletion of both apolipoproteins worsened behaviour deficits of double knockout mice and their performance was undistinguishable from those of Abca1 knockout mice. Finally we observed that the dendritic complexity in the CA1 region of hippocampus but not in CA2 is significantly impaired by Apoe/Apoa1 double deletion as well as by lack of ABCA1. IN CONCLUSION: (i) plasma lipoproteins may affect amyloid-ß clearance from the brain by the 'peripheral sink' mechanism; and (ii) deficiency of brain APOE-containing lipoproteins is of significance for dendritic complexity and cognition.