RESUMO
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
Recent studies suggest that apolipoprotein A-I (ApoA-I), the major protein constituent of high-density lipoprotein particles, plays a critical role in preserving cerebrovascular integrity and reducing Alzheimer's risk. ApoA-I present in brain is thought to be primarily derived from the peripheral circulation. Although plasma-to-brain delivery of ApoA-I is claimed to be handled by the blood-cerebrospinal fluid barrier (BCSFB), a contribution by the blood-brain barrier (BBB), which serves as a major portal for protein delivery to brain, cannot be ruled out. In this study, we assessed the permeability-surface area product (PS) of radioiodinated ApoA-I (125I-ApoA-I) in various brain regions of wild-type rats after an intravenous bolus injection. The PS value at the cortex, caudate putamen, hippocampus, thalamus, brain stem, and cerebellum was found to be 0.39, 0.28, 0.28, 0.36, 0.69, and 0.76 (ml/g per second × 10-6), respectively. Solutes delivered into brain via the BCSFB are expected to show greater accumulation in the thalamus due to its periventricular location. The modest permeability for 125I-ApoA-I into the thalamus relative to other regions suggests that BCSFB transport accounts for only a portion of total brain uptake and thus BBB transport cannot be ruled out. In addition, we show that Alexa Flour 647-labeled ApoA-I (AF647-ApoA-I) undergoes clathrin-independent and cholesterol-mediated endocytosis in transformed human cerebral microvascular endothelial cells (hCMEC/D3). Further, Z-series confocal images of the hCMEC/D3 monolayers and Western blot detection of intact ApoA-I on the abluminal side demonstrated AF647-ApoA-I transcytosis across the endothelium. These findings implicate the BBB as a significant portal for ApoA-I delivery into brain.
Assuntos
Apolipoproteína A-I/metabolismo , Barreira Hematoencefálica/metabolismo , Colesterol/metabolismo , Clatrina/metabolismo , Endocitose , Animais , Apolipoproteína A-I/sangue , Barreira Hematoencefálica/citologia , Endotélio/metabolismo , Humanos , Masculino , Permeabilidade , Transporte Proteico , Ratos , Ratos Sprague-DawleyRESUMO
Accumulation of amyloid beta (Aß) peptides in the cerebral vasculature, referred to as cerebral amyloid angiopathy (CAA), is widely observed in Alzheimer's disease (AD) brain and was shown to accelerate cognitive decline. There is no effective method for detecting cerebrovascular amyloid (CVA) and treat CAA. The targeted nanoparticles developed in this study effectively migrated from the blood flow to the vascular endothelium as determined by using quartz crystal microbalance with dissipation monitoring (QCM-D) technology. We also improved the stability, and blood-brain barrier (BBB) transcytosis of targeted nanoparticles by coating them with a cationic BBB penetrating peptide (K16ApoE). The K16ApoE-Targeted nanoparticles demonstrated specific targeting of vasculotropic DutchAß40 peptide accumulated in the cerebral vasculature. Moreover, K16ApoE-Targeted nanoparticles demonstrated significantly greater uptake into brain and provided specific MRI contrast to detect brain amyloid plaques.
Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Imageamento por Ressonância Magnética/métodos , Nanopartículas/química , Animais , Barreira Hematoencefálica/metabolismo , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Cães , Humanos , Células Madin Darby de Rim CaninoRESUMO
Inhibition of mitochondrial axonal trafficking by amyloid beta (Aß) peptides has been implicated in early pathophysiology of Alzheimer's Disease (AD). Yet, it remains unclear whether the loss of motility inevitably induces the loss of mitochondrial function, and whether restoration of axonal trafficking represents a valid therapeutic target. Moreover, while some investigations identify Aß oligomers as the culprit of trafficking inhibition, others propose that fibrils play the detrimental role. We have examined the effect of a panel of Aß peptides with different mutations found in familial AD on mitochondrial motility in primary cortical mouse neurons. Peptides with higher propensity to aggregate inhibit mitochondrial trafficking to a greater extent with fibrils inducing the strongest inhibition. Binding of Aß peptides to the plasma membrane was sufficient to induce trafficking inhibition where peptides with reduced plasma membrane binding and internalization had lesser effect on mitochondrial motility. We also found that Aß peptide with Icelandic mutation A673T affects axonal trafficking of mitochondria but has very low rates of plasma membrane binding and internalization in neurons, which could explain its relatively low toxicity. Inhibition of mitochondrial dynamics caused by Aß peptides or fibrils did not instantly affect mitochondrial bioenergetic and function. Our results support a mechanism where inhibition of axonal trafficking is initiated at the plasma membrane by soluble low molecular weight Aß species and is exacerbated by fibrils. Since trafficking inhibition does not coincide with the loss of mitochondrial function, restoration of axonal transport could be beneficial at early stages of AD progression. However, strategies designed to block Aß aggregation or fibril formation alone without ensuring the efficient clearance of soluble Aß may not be sufficient to alleviate the trafficking phenotype.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Axônios/metabolismo , Membrana Celular/metabolismo , Mitocôndrias/metabolismo , Agregados Proteicos/fisiologia , Sequência de Aminoácidos , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/farmacologia , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/patologia , Células Cultivadas , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Gravidez , Agregados Proteicos/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologiaRESUMO
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
The formation of amyloid ß (Aß) peptide aggregates, oligomers, and fibrils is a dynamic process; however, the kinetics of their formation is not well understood. This study compares the time course of aggregate/fibril formation by transmission electron microscopy (TEM) analyses with that of oligomer/fibril formation by Western blot analysis under native and denaturing conditions. Efforts to deaggregate/defibrillate these peptides by using hexafluoroisopropanol, ammonium hydroxide, or dimethylsulfoxide did not change the nondenaturing polyacrylamide gel electrophoresis (PAGE) footprints or drive the peptides to a monomeric species. Regardless of the pretreatment protocol, TEM analyses reveal that all Aß peptides (Aß40, Aß42, Aß39E22Δ [Osaka], Aß40E22G [Arctic], Aß40E22Q [Dutch], and Aß40A2T [Icelandic]) immediately formed nonfibrillar, amorphous aggregates when first placed into solution with the Osaka mutation, quickly forming early-stage fibrils. The extent of fibril formation for other Aß peptides is time dependent, with the Arctic mutation forming fibrils at 1 hr, the Dutch and Icelandic at 4 hr, Aß42 at 8 hr, and Aß40 at 24 hr. In contrast, nondenaturing PAGE revealed unique footprints for the different Aß species. The rapidity of aggregate formation and the rapid transition to fibrils, particularly for the Osaka deletion, suggest an important role for aggregates/fibrils of Aß in the development of neuronal degeneration.
Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Amiloide/genética , Fragmentos de Peptídeos/genética , Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Humanos , Mutação , Fragmentos de Peptídeos/metabolismo , Deleção de SequênciaRESUMO
Cerebral amyloid angiopathy (CAA) is characterized by the deposition of amyloid beta (Aß) proteins within the walls of the cerebral vasculature with subsequent aggressive vascular inflammation leading to recurrent hemorrhagic strokes. The objective of the study was to develop theranostic nanovehicles (TNVs) capable of a) targeting cerebrovascular amyloid; b) providing magnetic resonance imaging (MRI) contrast for the early detection of CAA; and c) treating cerebrovascular inflammation resulting from CAA. The TNVs comprised of a polymeric nanocore made from Magnevist (MRI contrast agent) conjugated chitosan. The nanocore was also loaded with cyclophosphamide (CYC), an immunosuppressant shown to reduce the cerebrovascular inflammation in CAA. Putrescine modified F(ab')2 fragment of anti-amyloid antibody, IgG4.1 (pF(ab')24.1) was conjugated to the surface of the nanocore to target cerebrovascular amyloid. The average size of the control chitosan nanoparticles (conjugated with albumin and are devoid of Magnevist, CYC, and pF(ab')24.1) was 164±1.2 nm and that of the TNVs was 239±4.1 nm. The zeta potential values of the CCNs and TNVs were 21.6±1.7 mV and 11.9±0.5 mV, respectively. The leakage of Magnevist from the TNVs was a modest 0.2% over 4 days, and the CYC release from the TNVs followed Higuchi's model that describes sustained drug release from polymeric matrices. The studies conducted in polarized human microvascular endothelial cell monolayers (hCMEC/D3) in vitro as well as in mice in vivo have demonstrated the ability of TNVs to target cerebrovascular amyloid. In addition, the TNVs provided contrast for imaging cerebrovascular amyloid using MRI and single photon emission computed tomography. Moreover, the TNVs were shown to reduce pro-inflammatory cytokine production by the Aß challenged blood brain barrier (BBB) endothelium more effectively than the cyclophosphamide alone.
Assuntos
Angiopatia Amiloide Cerebral/diagnóstico , Sistemas de Liberação de Medicamentos , Nanopartículas/uso terapêutico , Nanoestruturas/uso terapêutico , Placa Amiloide/patologia , Peptídeos beta-Amiloides , Animais , Barreira Hematoencefálica/metabolismo , Linhagem Celular , Angiopatia Amiloide Cerebral/terapia , Ciclofosfamida/administração & dosagem , Ciclofosfamida/uso terapêutico , Gadolínio/administração & dosagem , Humanos , Imunossupressores/administração & dosagem , Imunossupressores/uso terapêutico , Inflamação/diagnóstico , Inflamação/terapia , Imageamento por Ressonância Magnética , Camundongos , Nanopartículas/metabolismo , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
Cerebral amyloid angiopathy (CAA) results from the accumulation of Aß proteins primarily within the media and adventitia of small arteries and capillaries of the cortex and leptomeninges. CAA affects a majority of Alzheimer's disease (AD) patients and is associated with a rapid decline in cognitive reserve. Unfortunately, there is no pre-mortem diagnosis available for CAA. Furthermore, treatment options are few and relatively ineffective. To combat this issue, we have designed nanovehicles (nanoparticles-IgG4.1) capable of targeting cerebrovascular amyloid (CVA) and serving as early diagnostic and therapeutic agents. These nanovehicles were loaded with Gadolinium (Gd) based (Magnevist(®)) magnetic resonance imaging contrast agents or single photon emission computed tomography (SPECT) agents, such as (125)I. In addition, the nanovehicles carry either anti-inflammatory and anti-amyloidogenic agents such as curcumin or immunosuppressants such as dexamethasone, which were previously shown to reduce cerebrovascular inflammation. Owing to the anti-amyloid antibody (IgG4.1) grafted on the surface, the nanovehicles are capable of specifically targeting CVA deposits. The nanovehicles effectively marginate from the blood flow to the vascular wall as determined by using quartz crystal microbalance with dissipation monitoring (QCM-D) technology. They demonstrate excellent distribution to the brain vasculature and target CVA, thus providing MRI and SPECT contrast specific to the CVA in the brain. In addition, they also display the potential to carry therapeutic agents to reduce cerebrovascular inflammation associated with CAA, which is believed to trigger hemorrhage in CAA patients.
Assuntos
Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Encéfalo/patologia , Animais , Encéfalo/metabolismo , Linhagem Celular , Angiopatia Amiloide Cerebral/metabolismo , Angiopatia Amiloide Cerebral/terapia , Quitosana/metabolismo , Humanos , Imageamento por Ressonância Magnética , Camundongos , Placa Amiloide , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
Amyloid-ß (Aß) deposition in the brain vasculature results in cerebral amyloid angiopathy (CAA), which occurs in about 80% of Alzheimer's disease (AD) patients. While Aß42 predominates parenchymal amyloid plaques in AD brain, Aß40 is prevalent in the cerebrovascular amyloid. Dutch mutation of Aß40 (E22Q) promotes aggressive cerebrovascular accumulation and leads to severe CAA in the mutation carriers; knowledge of how DutchAß40 drives this process more efficiently than Aß40 could reveal various pathophysiological events that promote CAA. In this study we have demonstrated that DutchAß40 shows preferential accumulation in the blood-brain-barrier (BBB) endothelial cells due to its inefficient blood-to-brain transcytosis. Consequently, DutchAß40 establishes a permeation barrier in the BBB endothelium, prevents its own clearance from the brain, and promotes the formation of amyloid deposits in the cerebral microvessels. The BBB endothelial accumulation of native Aß40 is not robust enough to exercise such a significant impact on its brain clearance. Hence, the cerebrovascular accumulation of Aß40 is slow and may require other copathologies to precipitate into CAA. In conclusion, the magnitude of Aß accumulation in the BBB endothelial cells is a critical factor that promotes CAA; hence, clearing vascular endothelium of Aß proteins may halt or even reverse CAA.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Barreira Hematoencefálica/metabolismo , Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/genética , Animais , Bovinos , Células Cultivadas , Angiopatia Amiloide Cerebral Familiar/genética , Angiopatia Amiloide Cerebral Familiar/metabolismo , Células Endoteliais/metabolismo , Humanos , Modelos Neurológicos , Proteínas Mutantes/sangue , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Transporte Proteico , TranscitoseRESUMO
Accumulation of amyloid ß-protein (Aß) in neurons has been demonstrated to precede its formation as amyloid plaques in the extracellular space in Alzheimer's disease (AD) patients. Consequently, intraneuronal Aß accumulation is thought to be a critical first step in the fatal cascade of events that leads to neuronal degeneration in AD. Understanding the structural basis of neuronal binding and uptake of Aß might lead to potential therapeutic targets that could block this binding and the subsequent neurodegeneration that leads to the pathogenesis of AD. Previously, we demonstrated that mutation of the two adjacent histidine residues of Aß40 (H13,14G) resulted in a significant decrease in its level of binding to PC12 cells and mouse cortical/hippocampal neurons. We now demonstrate that the weakened neuronal binding follows the mutation order of H13G < H14G < H13,14G, which suggests that the primary domain for neuronal binding of Aß40 involves histidine at position 13. A novel APP mutation (E693Δ) that produced a variant Aß lacking glutamate 22 (E22Δ) in Japanese pedigrees was recently identified to have AD-type dementia without amyloid plaque formation but with extensive intraneuronal Aß in transfected cells and transgenic mice expressing this deletion. Deletion of glutamate 22 of Aß40 resulted in a 6-fold enhancement of PC12 neuronal binding that was not decreased by the H13G mutation. The dose-dependent enhanced binding of E22Δ explains the high level of intraneuronal Aß seen in this pedigree. Fluorescence anisotropy experiments at room temperature showed very rapid aggregation with increased tyrosine rigidity of Aß39E22Δ, Aß41E22Δ, and Aß42 but not Aß40. This rigidity was decreased but not eliminated by prior treatment with dimethyl sulfoxide. Surprisingly, all peptides showed an aggregated state when evaluated by transmission electron microscopy, with Aß39E22Δ having early stage fibrils, which was also verified by atomic force microscopy. This aggregation was not affected by centrifugation or pretreatment with organic solvents. The enhanced neuronal binding of Aß, therefore, results from aggregate binding to neurons, which requires H13 for Aß40 but not for E22Δ or Aß42. These latter proteins display increased tyrosine rigidity that likely masks the H13 residue, or alternatively, the H13 residue is not required for neuronal binding of these proteins as it is for Aß40. Late state fibrils also showed enhanced neuronal binding for E22Δ but not Aß40 with subsequent intraneuronal accumulation in lysosomes. This suggests that there are multiple pathways of binding/internalization for the different Aß proteins and their aggregation states or fibrillar structure.
Assuntos
Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Mutação , Neurônios/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Amiloide/química , Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Animais , Dimetil Sulfóxido/química , Polarização de Fluorescência , Histidina/genética , Humanos , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Células PC12 , Estrutura Terciária de Proteína , RatosRESUMO
Mounting evidence suggests that the pathological hallmarks of Alzheimer's disease (AD), neurofibrillary tangles and parenchymal amyloid plaques, are downstream reflections of neurodegeneration caused by the intraneuronal accumulation of amyloid-ß proteins (Aß), particularly Aß42 and Aß40. While the neurotoxicity of more amyloidogenic but less abundant Aß42 is well documented, the effect of Aß40 on neurons has been understudied. The Aß40 expression in the presymptomatic AD brain is ten times greater than that of Aß42. However, the Aß40:42 ratio decreases with AD progression and coincides with increased amyloid plaque deposition in the brain. Hence, it is thought that Aß40 protects neurons from the deleterious effects of Aß42. The pathophysiological pathways involved in the neuronal uptake of Aß40 or Aß42 have not been clearly elucidated. Lack of such critical information obscures therapeutic targets and thwarts rational drug development strategies aimed at preventing neurodegeneration in AD. The current study has shown that fluorescein labeled Aß42 (F-Aß42) is internalized by neurons via dynamin dependent endocytosis and is sensitive to membrane cholesterol, whereas the neuronal uptake of F-Aß40 is energy independent and nonendocytotic. Following their uptake, both F-Aß40 and F-Aß42 did not accumulate in early/recycling endosomes; F-Aß42 but not F-Aß40 accumulated in late endosomes and in the vesicles harboring caveolin-1. Furthermore, F-Aß42 demonstrated robust accumulation in the lysosomes and damaged their integrity, whereas F-Aß40 showed only a sparse lysosomal accumulation. Such regulated trafficking along distinct pathways suggests that Aß40 and Aß42 exercise differential effects on neurons. These differences must be carefully considered in the design of a pharmacological agent intended to block the neurodegeneration triggered by Aß proteins.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Caveolina 1/metabolismo , Linhagem Celular Tumoral , Descoberta de Drogas/métodos , Endocitose/fisiologia , Endossomos/metabolismo , Lisossomos/metabolismo , Neurônios/metabolismo , Células PC12 , RatosRESUMO
BACKGROUND: The identification of early mechanisms underlying Alzheimer's Disease (AD) and associated biomarkers could advance development of new therapies and improve monitoring and predicting of AD progression. Mitochondrial dysfunction has been suggested to underlie AD pathophysiology, however, no comprehensive study exists that evaluates the effect of different familial AD (FAD) mutations on mitochondrial function, dynamics, and brain energetics. METHODS AND FINDINGS: We characterized early mitochondrial dysfunction and metabolomic signatures of energetic stress in three commonly used transgenic mouse models of FAD. Assessment of mitochondrial motility, distribution, dynamics, morphology, and metabolomic profiling revealed the specific effect of each FAD mutation on the development of mitochondrial stress and dysfunction. Inhibition of mitochondrial trafficking was characteristic for embryonic neurons from mice expressing mutant human presenilin 1, PS1(M146L) and the double mutation of human amyloid precursor protein APP(Tg2576) and PS1(M146L) contributing to the increased susceptibility of neurons to excitotoxic cell death. Significant changes in mitochondrial morphology were detected in APP and APP/PS1 mice. All three FAD models demonstrated a loss of the integrity of synaptic mitochondria and energy production. Metabolomic profiling revealed mutation-specific changes in the levels of metabolites reflecting altered energy metabolism and mitochondrial dysfunction in brains of FAD mice. Metabolic biomarkers adequately reflected gender differences similar to that reported for AD patients and correlated well with the biomarkers currently used for diagnosis in humans. CONCLUSIONS: Mutation-specific alterations in mitochondrial dynamics, morphology and function in FAD mice occurred prior to the onset of memory and neurological phenotype and before the formation of amyloid deposits. Metabolomic signatures of mitochondrial stress and altered energy metabolism indicated alterations in nucleotide, Krebs cycle, energy transfer, carbohydrate, neurotransmitter, and amino acid metabolic pathways. Mitochondrial dysfunction, therefore, is an underlying event in AD progression, and FAD mouse models provide valuable tools to study early molecular mechanisms implicated in AD.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Mitocôndrias/metabolismo , Amiloide/genética , Animais , Biomarcadores/metabolismo , Encéfalo/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Hipocampo/metabolismo , Humanos , Metabolômica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Neurônios/metabolismo , Presenilina-1/genética , Fatores de TempoRESUMO
The increasing prevalence of Alzheimer's disease (AD) has provided motivation for developing novel methods for assessing the disease and the effects of potential treatments. Magnetic resonance elastography (MRE) is an MRI-based method for quantitatively imaging the shear tissue stiffness in vivo. The objective of this research was to determine whether this new imaging biomarker has potential for characterizing neurodegenerative disease. Methods were developed and tested for applying MRE to evaluate the mouse brain, using a conventional large bore 3.0T MRI system. The technique was then applied to study APP-PS1 mice, a well-characterized model of AD. Five APP-PS1 mice and 8 age-matched wild-type mice were imaged immediately following sacrifice. Brain shear stiffness measurements in APP-PS1 mice averaged 22.5% lower than those for wild-type mice (P = .0031). The results indicate that mouse brain MRE is feasible at 3.0T, and brain shear stiffness has merit for further investigation as a potential new biomarker for Alzheimer's disease.
Assuntos
Doença de Alzheimer/patologia , Técnicas de Imagem por Elasticidade/métodos , Algoritmos , Animais , Modelos Animais de Doenças , Análise dos Mínimos Quadrados , Camundongos , Camundongos Transgênicos , Projetos Piloto , Estatísticas não ParamétricasRESUMO
Alzheimer's disease amyloid ß (Aß) proteins accumulate in the cerebral vasculature and cause cerebral amyloid angiopathy (CAA). The objective of this study was to resolve critical formulation issues in developing nanoparticles (NPs) capable of permeating the blood brain barrier (BBB) and targeting cerebrovascular Aß proteins. To achieve this objective we designed immuno-nanovehicles, which are chitosan-coated poly lactic-co-glycolic acid (PLGA) NPs conjugated with a novel anti-Aß antibody. Measurements made according to Derjaguin-Landau-Verwey-Overbeek (DLVO) theory indicated that the immuno-nanovehicles have a much lower propensity to aggregate than the control nanovehicles. Immuno-nanovehicles showed enhanced uptake at the BBB and better targeting of the Aß proteins deposited in the CAA model in vitro in comparison with the control nanovehicles. In addition, chitosan enhanced aqueous dispersibility and increased the stability of immuno-nanovehicles during lyophilization, thus transforming them into ideal vehicles for delivering therapeutic and diagnostic agents to the cerebral vasculature ridden with vascular amyloid. FROM THE CLINICAL EDITOR: In this study, the authors report the development of chitosan-coated PLGA nanoparticles conjugated with anti-amyloid antibody to be used as immuno-nanovehicles to image cerebral amyloid angiopathy deposits in vivo. This method enables delivering therapeutic and diagnostic agents to the cerebral vasculature ridden with vascular amyloid.
Assuntos
Peptídeos beta-Amiloides/imunologia , Anticorpos Anti-Idiotípicos/imunologia , Angiopatia Amiloide Cerebral/diagnóstico , Quitosana/química , Nanopartículas/química , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/imunologia , Peptídeos beta-Amiloides/metabolismo , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/metabolismo , Angiopatia Amiloide Cerebral/imunologia , Quitosana/metabolismo , Diagnóstico por Imagem/métodos , HumanosRESUMO
The relevance of cerebral amyloid angiopathy (CAA) to the pathogenesis of Alzheimer disease (AD) and dementia in general emphasizes the importance of developing novel targeting approaches for detecting and treating cerebrovascular amyloid (CVA) deposits. We developed a nanoparticle-based technology that uses a monoclonal antibody against fibrillar human amyloid-ß42 that is surface coated onto a functionalized phospholipid monolayer. We demonstrate that this conjugated nanoparticle binds to CVA deposits in arterioles of AD transgenic mice (Tg2576) after infusion into the external carotid artery using 3 different approaches. The first 2 approaches use a blood vessel enrichment of homogenized brain and a leptomeningeal vessel preparation from thin tangential brain slices from the surface of the cerebral cortex. Targeting of CVA by the antibody-coated nanoparticle was visualized using fluorescent lissamine rhodamine-labeled phospholipids in the nanoparticles, which were compared with fluorescent staining of the endothelial cells and amyloid deposits using confocal laser scanning microscopy. The third approach used high-field strength magnetic resonance imaging of antibody-coated iron oxide nanoparticles after infusion into the external carotid artery. Dark foci of contrast enhancement in cortical arterioles were observed in T2*-weighted images of ex vivo AD mouse brains that correlated histologically with CVA deposits. The targeting ability of these nanoparticles to CVA provides opportunities for the prevention and treatment of CAA.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Arteríolas/patologia , Compostos Férricos , Nanopartículas , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/patologia , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/imunologia , Precursor de Proteína beta-Amiloide/genética , Animais , Arteríolas/ultraestrutura , Benzotiazóis , Modelos Animais de Doenças , Humanos , Imunoglobulina G , Imageamento por Ressonância Magnética/métodos , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão/métodos , Fragmentos de Peptídeos/imunologia , Placa Amiloide/ultraestrutura , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , TiazóisRESUMO
A major objective in the treatment of Alzheimer's disease is amyloid plaque reduction. Transgenic mouse models of Alzheimer's disease provide a controlled and consistent environment for studying amyloid plaque deposition in Alzheimer's disease. Magnetic resonance imaging is an attractive tool for longitudinal studies because it offers non-invasive monitoring of amyloid plaques. Recent studies have demonstrated the ability of magnetic resonance imaging to detect individual plaques in living mice. This review discusses the mouse models, MR pulse sequences, and parameters that have been used to image plaques and how they can be optimized for future studies.
RESUMO
BACKGROUND: Therapeutic intervention of numerous brain-associated disorders currently remains unrealized due to serious limitations imposed by the blood-brain-barrier (BBB). The BBB generally allows transport of small molecules, typically <600 daltons with high octanol/water partition coefficients, but denies passage to most larger molecules. However, some receptors present on the BBB allow passage of cognate proteins to the brain. Utilizing such receptor-ligand systems, several investigators have developed methods for delivering proteins to the brain, a critical requirement of which involves covalent linking of the target protein to a carrier entity. Such covalent modifications involve extensive preparative and post-preparative chemistry that poses daunting limitations in the context of delivery to any organ. Here, we report creation of a 36-amino acid peptide transporter, which can transport a protein to the brain after routine intravenous injection of the transporter-protein mixture. No covalent linkage of the protein with the transporter is necessary. APPROACH: A peptide transporter comprising sixteen lysine residues and 20 amino acids corresponding to the LDLR-binding domain of apolipoprotein E (ApoE) was synthesized. Transport of beta-galactosidase, IgG, IgM, and antibodies against amyloid plques to the brain upon iv injection of the protein-transporter mixture was evaluated through staining for enzyme activity or micro single photon emission tomography (micro-SPECT) or immunostaining. Effect of the transporter on the integrity of the BBB was also investigated. PRINCIPAL FINDINGS: The transporter enabled delivery to the mouse brain of functional beta-galactosidase, human IgG and IgM, and two antibodies that labeled brain-associated amyloid beta plaques in a mouse model of Alzheimer's disease. SIGNIFICANCE: The results suggest the transporter is able to transport most or all proteins to the brain without the need for chemically linking the transporter to a protein. Thus, the approach offers an avenue for rapid clinical evaluation of numerous candidate drugs against neurological diseases including cancer. (299 words).
Assuntos
Amiloide/imunologia , Anticorpos/administração & dosagem , Imunoglobulina M/imunologia , beta-Galactosidase/administração & dosagem , Animais , Barreira Hematoencefálica , Camundongos , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
A key question in understanding AD is whether extracellular Abeta deposition of parenchymal amyloid plaques or intraneuronal Abeta accumulation initiates the AD process. Amyloid precursor protein (APP) is endocytosed from the cell surface into endosomes where it is cleaved to produce soluble Abeta which is then released into the brain interstitial fluid. Intraneuronal Abeta accumulation is hypothesized to predominate from the neuronal uptake of this soluble extracellular Abeta rather than from ER/Golgi processing of APP. We demonstrate that substitution of the two adjacent histidine residues of Abeta40 results in a significant decrease in its binding with PC12 cells and mouse cortical/hippocampal neurons. These substitutions also result in a dramatic enhancement of both thioflavin-T positive fibril formation and binding to preformed Abeta fibrils while maintaining its plaque-binding ability in AD transgenic mice. Hence, alteration of the histidine domain of Abeta prevented neuronal binding and drove Abeta to enhanced fibril formation and subsequent amyloid plaque deposition--a potential mechanism for removing toxic species of Abeta. Substitution or even masking of these Abeta histidine residues might provide a new therapeutic direction for minimizing neuronal uptake and subsequent neuronal degeneration and maximizing targeting to amyloid plaques.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Córtex Cerebral/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Animais , Córtex Cerebral/patologia , Hipocampo/patologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Células PC12 , Ratos , Espectrometria de Fluorescência , Ressonância de Plasmônio de SuperfícieRESUMO
Several different monoclonal antibodies (mAbs) have been actively developed in the field of Alzheimer's disease (AD) for basic science and clinical applications; however, the binding kinetics of many of the mAbs with the beta-amyloid peptides (Abeta) are poorly understood. A panel of mAbs with different Abeta recognition sites, including our plaque-binding antibody (IgG4.1), a peptide-capturing antibody (11A50), and two classical mAbs (6E10 and 4G8) used for immunohistochemistry, were chosen for characterization of their kinetics of binding to monomeric and fibrillar forms of Abeta40 using surface plasmon resonance and their amyloid plaque binding ability in AD mouse brain sections using immunohistochemistry. The plaque-binding antibody (IgG4.1) with epitope specificity of Abeta(2-10) showed a weaker affinity (512 nM) for monomeric Abeta40 but a higher affinity (1.5 nM) for Abeta40 fibrils and labeled dense core plaques better than 6E10 as determined by immunohistochemistry. The peptide-capturing antibody (11A50) showed preferential affinity (32.5 nM) for monomeric Abeta40 but did not bind to Abeta40 fibrils, whereas antibodies 6E10 and 4G8 had moderate affinity for monomeric Abeta40 (22.3 and 30.1 nM, respectively). 4G8, which labels diffuse plaques better than 6E10, had a higher association rate constant than 6E10 but showed similar association and dissociation kinetics compared to those of 11A50. Enzymatic digestion of IgG4.1 to the F(ab')(2)4.1 fragments or their polyamine-modified derivatives that enhance blood-brain barrier permeability did not affect the kinetic properties of the antigen binding site. These differences in kinetic binding to monomeric and fibrillar Abeta among various antibodies could be utilized to distinguish mAbs that might be useful for immunotherapy or amyloid plaque imaging versus those that could be utilized for bioanalytical techniques.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/imunologia , Peptídeos beta-Amiloides/metabolismo , Anticorpos Monoclonais/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Doença de Alzheimer/imunologia , Animais , Anticorpos Monoclonais/imunologia , Mapeamento de Epitopos , Imuno-Histoquímica , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação ProteicaRESUMO
One of the hallmark pathologies of Alzheimer's disease (AD) is amyloid plaque deposition. Plaques appear hypointense on T(2)-weighted and T(2)*-weighted MR images probably due to the presence of endogenous iron, but no quantitative comparison of various imaging techniques has been reported. We estimated the T(1), T(2), T(2)*, and proton density values of cortical plaques and normal cortical tissue and analyzed the plaque contrast generated by a collection of T(2)-weighted, T(2)*-weighted, and susceptibility-weighted imaging (SWI) methods in ex vivo transgenic mouse specimens. The proton density and T(1) values were similar for both cortical plaques and normal cortical tissue. The T(2) and T(2)* values were similar in cortical plaques, which indicates that the iron content of cortical plaques may not be as large as previously thought. Ex vivo plaque contrast was increased compared to a previously reported spin-echo sequence by summing multiple echoes and by performing SWI; however, gradient echo and SWI were found to be impractical for in vivo imaging due to susceptibility interface-related signal loss in the cortex.