RESUMO
ClC-3, ClC-4, and ClC-5 are electrogenic chloride/proton exchangers that can be found in endosomal compartments of mammalian cells. Although the association with genetic diseases and the severe phenotype of knock-out animals illustrate their physiological importance, the cellular functions of these proteins have remained insufficiently understood. We here study the role of two Clcn3 splice variants, ClC-3b and ClC-3c, in granular exocytosis and catecholamine accumulation of adrenal chromaffin cells using a combination of high-resolution capacitance measurements, amperometry, protein expression/gene knock out/down, rescue experiments, and confocal microscopy. We demonstrate that ClC-3c resides in immature as well as in mature secretory granules, where it regulates catecholamine accumulation and contributes to the establishment of the readily releasable pool of secretory vesicles. The lysosomal splice variant ClC-3b contributes to vesicle priming only with low efficiency and leaves the vesicular catecholamine content unaltered. The related Cl-/H+ antiporter ClC-5 undergoes age-dependent downregulation in wild-type conditions. Its upregulation in Clcn3-/- cells partially rescues the exocytotic mutant defect. Our study demonstrates how different CLC transporters with similar transport functions, but distinct localizations can contribute to vesicle functions in the regulated secretory pathway of granule secretion in chromaffin cells.SIGNIFICANCE STATEMENT Cl-/H+ exchangers are expressed along the endosomal/lysosomal system of mammalian cells; however, their exact subcellular functions have remained insufficiently understood. We used chromaffin cells, a system extensively used to understand presynaptic mechanisms of synaptic transmission, to define the role of CLC exchangers in neurosecretion. Disruption of ClC-3 impairs catecholamine accumulation and secretory vesicle priming. There are multiple ClC-3 splice variants, and only expression of one, ClC-3c, in double Cl-/H+ exchanger-deficient cells fully rescues the WT phenotype. Another splice variant, ClC-3b, is present in lysosomes and is not necessary for catecholamine secretion. The distinct functions of ClC-3c and ClC-3b illustrate the impact of expressing multiple CLC transporters with similar transport functions and separate localizations in different endosomal compartments.
Assuntos
Células Cromafins , Prótons , Animais , Catecolaminas/metabolismo , Cloretos/metabolismo , Células Cromafins/metabolismo , Exocitose/fisiologia , Mamíferos , Camundongos , Camundongos Knockout , Vesículas Secretórias/metabolismoRESUMO
BACKGROUND: Given that changes in brain water content are often correlated with disease, investigating water content non-invasively and in vivo could lead to a better understanding of the pathogenesis of several neurologic diseases. PURPOSE: To adapt a super-resolution-based technique, previously developed for humans, to the rat brain and report in vivo high-resolution (HR) water content maps in comparison with ex vivo wet/dry methods. STUDY TYPE: Prospective. ANIMAL MODEL: Eight healthy male Wistar rats. FIELD STRENGTH/SEQUENCE: 9.4-T, multi-echo gradient-echo (mGRE) sequence. ASSESSMENT: Using super-resolution reconstruction (SRR), a HR mGRE image (200 µm isotropic) was reconstructed from three low-resolution (LR) orthogonal whole-brain images in each animal, which was followed by water content mapping in vivo. The animals were subsequently sacrificed, the brains excised and divided into five regions (front left, front right, middle left, middle right, and cerebellum-brainstem regions), and the water content was measured ex vivo using wet/dry measurements as the reference standard. The water content values of the in vivo and ex vivo methods were then compared for the whole brain and also for the different regions separately. STATISTICAL TESTS: Friedman's non-parametric test was used to test difference between the five regions, and Pearson's correlation coefficient was used for correlation between in vivo and ex vivo measurements. A P-value <0.05 was considered statistically significant. RESULTS: Water content values derived from in vivo MR measurements showed strong correlations with water content measured ex vivo at a regional level (r = 0.902). Different brain regions showed significantly different water content values. Water content values were highest in the frontal brain, followed by the midbrain, and lowest in the cerebellum and brainstem regions. DATA CONCLUSION: An in vivo technique to achieve HR isotropic water content maps in the rat brain using SRR was adopted in this study. The MRI-derived water content values obtained using the technique showed strong correlations with water content values obtained using ex vivo wet/dry methods. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY: Stage 1.
RESUMO
Multiple sources of evidence suggest that soluble amyloid ß (Aß)-oligomers are responsible for the development and progression of Alzheimer's disease (AD). In order to specifically eliminate these toxic Aß-oligomers, our group has developed a variety of all-d-peptides over the past years. One of them, RD2, has been intensively studied and showed such convincing in vitro and in vivo properties that it is currently in clinical trials. In order to further optimize the compounds and to elucidate the characteristics of therapeutic d-peptides, several rational drug design approaches have been performed. Two of these d-peptides are the linear tandem (head-to-tail) d-peptide RD2D3 and its cyclized form cRD2D3. Tandemization and cyclization should result in an increased in vitro potency and increase pharmacokinetic properties, especially crossing the blood-brain-barrier. In comparison, cRD2D3 showed a superior pharmacokinetic profile to RD2D3. This fact suggests that higher efficacy can be achieved in vivo at equally administered concentrations. To prove this hypothesis, we first established the in vitro profile of both d-peptides here. Subsequently, we performed an intraperitoneal treatment study. This study failed to provide evidence that cRD2D3 is superior to RD2D3 in vivo as in some tests cRD2D3 failed to show equal or higher efficacy.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Oligopeptídeos/química , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacocinéticaRESUMO
Neuroinflammation is a pathological hallmark of several neurodegenerative disorders and plays a key role in the pathogenesis of amyotrophic lateral sclerosis (ALS). It has been implicated as driver of disease progression and is observed in ALS patients, as well as in the transgenic SOD1G93A mouse model. Here, we explore and validate the therapeutic potential of the d-enantiomeric peptide RD2RD2 upon oral administration in SOD1G93A mice. Transgenic mice were treated daily with RD2RD2 or placebo for 10 weeks and phenotype progression was followed with several behavioural tests. At the end of the study, plasma cytokine levels and glia cell markers in brain and spinal cord were analysed. Treatment resulted in a significantly increased performance in behavioural and motor coordination tests and a decelerated neurodegenerative phenotype in RD2RD2-treated SOD1G93A mice. Additionally, we observed retardation of the average disease onset. Treatment of SOD1G93A mice led to significant reduction in glial cell activation and a rescue of neurons. Analysis of plasma revealed normalisation of several cytokines in samples of RD2RD2-treated SOD1G93A mice towards the levels of non-transgenic mice. In conclusion, these findings qualify RD2RD2 to be considered for further development and testing towards a disease modifying ALS treatment.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Neurônios Motores/enzimologia , Superóxido Dismutase/metabolismo , Administração Oral , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Peptídeos , Superóxido Dismutase/genéticaRESUMO
Understanding the physiopathology of Alzheimer's disease (AD) has improved substantially based on studies of mouse models mimicking at least one aspect of the disease. Many transgenic lines have been established, leading to amyloidosis but lacking neurodegeneration. The aim of the current study was to generate a novel mouse model that develops neuritic plaques containing the aggressive pyroglutamate modified amyloid-ß (pEAß) species in the brain. The TAPS line was developed by intercrossing of the pEAß-producing TBA2.1 mice with the plaque-developing line APPswe/PS1ΔE9. The phenotype of the new mouse line was characterized using immunostaining, and different cognitive and general behavioral tests. In comparison to the parental lines, TAPS animals developed an earlier onset of pathology and increased plaque load, including striatal pEAß-positive neuritic plaques, and enhanced neuroinflammation. In addition to abnormalities in general behavior, locomotion, and exploratory behavior, TAPS mice displayed cognitive deficits in a variety of tests that were most pronounced in the fear conditioning paradigm and in spatial learning in comparison to the parental lines. In conclusion, the combination of a pEAß- and a plaque-developing mouse model led to an accelerated amyloid pathology and cognitive decline in TAPS mice, qualifying this line as a novel amyloidosis model for future studies.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Disfunção Cognitiva/metabolismo , Doença de Alzheimer/patologia , Animais , Linhagem Celular , Disfunção Cognitiva/patologia , Modelos Animais de Doenças , CamundongosRESUMO
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterised by selective neuronal death in the brain stem and spinal cord. The cause is unknown, but an increasing amount of evidence has firmly certified that neuroinflammation plays a key role in ALS pathogenesis. Neuroinflammation is a pathological hallmark of several neurodegenerative disorders and has been implicated as driver of disease progression. Here, we describe a treatment study demonstrating the therapeutic potential of a tandem version of the well-known all-d-peptide RD2 (RD2RD2) in a transgenic mouse model of ALS (SOD1*G93A). Mice were treated intraperitoneally for four weeks with RD2RD2 vs. placebo. SOD1*G93A mice were tested longitudinally during treatment in various behavioural and motor coordination tests. Brain and spinal cord samples were investigated immunohistochemically for gliosis and neurodegeneration. RD2RD2 treatment in SOD1*G93A mice resulted not only in a reduction of activated astrocytes and microglia in both the brain stem and lumbar spinal cord, but also in a rescue of neurons in the motor cortex. RD2RD2 treatment was able to slow progression of the disease phenotype, especially the motor deficits, to an extent that during the four weeks treatment duration, no significant progression was observed in any of the motor experiments. Based on the presented results, we conclude that RD2RD2 is a potential therapeutic candidate against ALS.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Anti-Inflamatórios/uso terapêutico , Oligopeptídeos/uso terapêutico , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Anti-Inflamatórios/química , Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/patologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Destreza Motora/efeitos dos fármacos , Destreza Motora/fisiologia , Proteínas Mutantes/genética , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/fisiopatologia , Oligopeptídeos/química , Fenótipo , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Superóxido Dismutase/genética , Superóxido Dismutase-1/genéticaRESUMO
Alzheimer's disease, a multifactorial incurable disorder, is mainly characterised by progressive neurodegeneration, extracellular accumulation of amyloid-ß protein (Aß), and intracellular aggregation of hyperphosphorylated tau protein. During the last years, Aß oligomers have been claimed to be the disease causing agent. Consequently, development of compounds that are able to disrupt already existing Aß oligomers is highly desirable. We developed d-enantiomeric peptides, consisting solely of d-enantiomeric amino acid residues, for the direct and specific elimination of toxic Aß oligomers. The drug candidate RD2 did show high oligomer elimination efficacy in vitro and the in vivo efficacy of RD2 was demonstrated in treatment studies by enhanced cognition in transgenic mouse models of amyloidosis. Here, we report on the in vitro and in vivo efficacy of the compound towards pyroglutamate-Aß, a particular aggressive Aß species. Using the transgenic TBA2.1 mouse model, which develops pyroglutamate-Aß(3-42) induced neurodegeneration, we are able to show that oral RD2 treatment resulted in a significant deceleration of the progression of the phenotype. The in vivo efficacy against this highly toxic Aß species further validates RD2 as a drug candidate for the therapeutic use in humans.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Amiloide/metabolismo , Encéfalo/metabolismo , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/metabolismo , Administração Oral , Doença de Alzheimer/patologia , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Oligopeptídeos/administração & dosagem , Oligopeptídeos/química , Fenótipo , EstereoisomerismoRESUMO
BACKGROUND: In recent years, argon has been shown to exert neuroprotective effects in an array of models. However, the mechanisms by which argon exerts its neuroprotective characteristics remain unclear. Accumulating evidence imply that argon may exert neuroprotective effects via modulating the activation and polarization of microglia/macrophages after ischemic stroke. In the present study, we analyzed the underlying neuroprotective effects of delayed argon application until 7 days after reperfusion and explored the potential mechanisms. METHODS: Twenty-one male Wistar rats underwent transient middle cerebral artery occlusion or sham surgery randomly for 2 h using the endoluminal thread model. Three hours after transient middle cerebral artery occlusion induction and 1 h after reperfusion, animals received either 50% vol Argon/50% vol O2 or 50% vol N2/50% vol O2 for 1 h. The primary outcome was the 6-point neuroscore from 24 h to d7 after reperfusion. Histological analyses including infarct volume, survival of neurons (NeuN) at the ischemic boundary zone, white matter integrity (Luxol Fast Blue), microglia/macrophage activation (Iba1), and polarization (Iba1/Arginase1 double staining) on d7 were conducted as well. Sample size calculation was performed using nQuery Advisor + nTerim 4.0. Independent t test, one-way ANOVA and repeated measures ANOVA were performed, respectively, for statistical analysis (SPSS 23.0). RESULTS: The 6-point neuroscore from 24 h to d7 after reperfusion showed that tMCAO Ar group displayed significantly improved neurological performance compared to tMCAO N2 group (p = 0.026). The relative numbers of NeuN-positive cells in the ROIs of tMCAO Ar group significantly increased compared to tMCAO N2 group (p = 0.010 for cortex and p = 0.011 for subcortex). Argon significantly suppressed the microglia/macrophage activation as revealed by Iba1 staining (p = 0.0076) and promoted the M2 microglia/macrophage polarization as revealed by Iba1/Arginase 1 double staining (p = 0.000095). CONCLUSIONS: Argon administration with a 3 h delay after stroke onset and 1 h after reperfusion significantly alleviated neurological deficit within the first week and preserved the neurons at the ischemic boundary zone 7 days after stroke. Moreover, argon reduced the excessive microglia/macrophage activation and promoted the switch of microglia/macrophage polarization towards the anti-inflammatory M2 phenotype. Studies making efforts to further elucidate the protective mechanisms and to benefit the translational application are of great value.
Assuntos
Argônio , Lesões Encefálicas , Encefalite , Acidente Vascular Cerebral , Animais , Masculino , Ratos , Análise de Variância , Argônio/farmacologia , Argônio/uso terapêutico , Lesões Encefálicas/etiologia , Lesões Encefálicas/patologia , Lesões Encefálicas/fisiopatologia , Modelos Animais de Doenças , Encefalite/fisiopatologia , Encefalite/prevenção & controle , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/fisiopatologia , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Distribuição Aleatória , Ratos Wistar/lesões , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/fisiopatologiaRESUMO
The assessment of cerebral gliomas using magnetic resonance imaging (MRI) provides excellent structural images but cannot solve all diagnostic problems satisfactorily. The differentiation of tumour tissue from non-neoplastic changes may be difficult especially in the post-treatment phase. In recent years, positron emission tomography (PET) using radiolabelled amino acids has gained considerable interest as an additional tool to improve the diagnosis of cerebral gliomas and brain metastases. A key step for this advancement was the development of the F-18 labelled amino acid O-(2-[18F]fluoroethyl)-L-tyrosine (FET) which has spread rapidly in the last decade and replaced carbon-11 labelled amino acid tracers such as 11C-methyl-L-methionine (MET) in many centres in Europe. FET can be produced with high efficiency and distributed in a satellite concept like 2-[18F]fluoro-2-deoxy-D-glucose (FDG). Furthermore, FET exhibits favourable properties such as high in vivo stability, high tumour to background contrast and tissue specific tracer kinetics, which provides additional information for tumour grading or differential diagnosis. The Response Assessment in Neuro-Oncology (RANO) working group - an international effort to develop new standardized response criteria for clinical trials in brain tumours - has recently recommended the additional use of amino acid PET imaging for brain tumour management. FET PET can provide important diagnostic information in crucial situations such as the definition of biopsy site, the delineation of cerebral gliomas for therapy planning, sensitive monitoring of treatment response and an improved differentiation of tumour recurrence from treatment-related changes. In this article the basic information, methodological aspects and the actual status of clinical application of FET PET are reviewed.
Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Tirosina/análogos & derivados , Animais , Humanos , Tomografia por Emissão de Pósitrons/tendências , Transporte Proteico/fisiologia , Tirosina/administração & dosagem , Tirosina/metabolismoRESUMO
The application of small peptides targeting amyloid beta (Aß) is one of many drug development strategies for the treatment of Alzheimer's disease (AD). We have previously identified several peptides consisting solely of D-enantiomeric amino acid residues obtained from mirror-image phage display selection, which bind to Aß in different assembly states and eliminate toxic Aß aggregates. Some of these D-peptides show both diagnostic and therapeutic potential in vitro and in vivo. Here we have analysed the similarity of the arginine-rich D-peptide D3 to the arginine-rich motif (ARM) of the human immunodeficiency virus type 1 transactivator of transcription (HIV-Tat) protein, and examined its in vivo blood-brain barrier (BBB) permeability using wild type mice and transgenic mouse models of Alzheimer's disease. We are able to demonstrate that D3 rapidly enters the brain where it can be found associated with amyloid plaques suggesting a direct penetration of BBB.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/química , Barreira Hematoencefálica/efeitos dos fármacos , Fármacos Neuroprotetores/farmacocinética , Oligopeptídeos/metabolismo , Administração Oral , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Permeabilidade Capilar , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/metabolismo , Oligopeptídeos/síntese química , Oligopeptídeos/farmacocinética , Alinhamento de Sequência , Produtos do Gene tat do Vírus da Imunodeficiência Humana/químicaRESUMO
PURPOSE: O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET) is an established tracer for the diagnosis of brain tumors with PET. This study investigates the influence of blood-brain barrier (BBB) permeability on 18F-FET uptake in two rat glioma models and one human xenograft model. METHODS: F98 glioma, 9L gliosarcoma or human U87 glioblastoma cells were implanted into the striatum of 56 Fischer or RNU rats. Thereafter, animals were divided into a control group and a group receiving injections of the glucocorticoid dexamethasone (Dex). After 12-13 days of tumor growth animals received injection of Evans blue dye (EBD) to visualize BBB disturbance and underwent 18F-FET PET followed by autoradiography. Time activity curves, standardized uptake values (SUV) and Tumor-to-brain ratios (TBR) of 18F-FET uptake [18-61 min post injection (p.i.)] were evaluated using a volume-of-Interest (VOI) analysis. BBB disturbance was quantitatively evaluated by EBD fluorescence. The membrane gaps of blood vessel endothelial tight junctions were measured using electron microscopy to visualize ultrastructural BBB alterations in one untreated and one Dex treated F98 glioma. Data were analyzed by two-way ANOVAs. RESULTS: In Dex treated animals EBD extravasation was significantly reduced in 9L (P < 0.001) and U87 (P = 0.008) models and showed a trend in F98 models (P = 0.053). In contrast, no significant differences of 18F-FET uptake were observed between Dex treated animals and control group except a decrease of the TBR in the 9L tumor model in PET (P < 0.01). Ultrastructural evaluation of tumor blood vessel endothelia revealed significant reduction of the cleft diameter between endothelial cells after Dex treatment in F98 model (P = 0.010). CONCLUSION: Despite a considerable reduction of BBB permeability in rat gliomas after Dex treatment, no relevant changes of 18F-FET uptake were noted in this experimental study. Thus, 18F-FET uptake in gliomas appears to be widely independent of the permeability of the BBB.
Assuntos
Barreira Hematoencefálica/diagnóstico por imagem , Permeabilidade Capilar , Glioma/diagnóstico por imagem , Compostos Radiofarmacêuticos/farmacocinética , Tirosina/análogos & derivados , Animais , Antineoplásicos Hormonais/efeitos adversos , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Linhagem Celular Tumoral , Dexametasona/efeitos adversos , Humanos , Masculino , Ratos , Junções Íntimas/metabolismo , Junções Íntimas/ultraestrutura , Tirosina/farmacocinéticaRESUMO
BACKGROUND: Focal cerebral ischemia induces distinct neuroinflammatory processes. We recently reported the extracellular phosphor-glyco-protein osteopontin (OPN) to directly affect primary microglia in vitro, promoting survival while shifting their inflammatory profile towards a more neutral phenotype. We here assessed the effects of OPN on microglia after stroke in vivo, with focus on infarct demarcation. METHODS: Animals underwent focal photothrombotic stroke and were injected intracerebroventricularly with 500 µg OPN or vehicle. Immunohistochemistry assessed neuronal damage and infarct volume, neovascularisation, glial scar formation, microglial activation, and M1 and M2 polarisation. RESULTS: After photothrombotic stroke, areas covered by M1 and M2 microglia substantially overlapped. OPN treatment reduced that overlap, with microglia appearing more spread out and additionally covering the infarct core. OPN additionally modulated the quantity of microglia subpopulations, reducing iNOS+ M1 cells while increasing M2 microglia, shifting the M1/M2 balance towards an M2 phenotype. Moreover, OPN polarized astrocytes towards the infarct. CONCLUSION: Microglial activation and M1 and M2 polarization have distinct but overlapping spatial patterns in permanent focal ischemia. Data suggest that OPN is involved in separating M1 and M2 subpopulations, as well as in shifting microglia polarization towards the M2 phenotype modulating beneficially inflammatory responses after focal infarction.
Assuntos
Microglia/efeitos dos fármacos , Osteopontina/uso terapêutico , Animais , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Imuno-Histoquímica , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Microglia/metabolismo , Ratos , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/metabolismoRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is associated with the aggregation of the amyloid ß protein (Aß). Aß oligomers are currently thought to be the major neurotoxic agent responsible for disease development and progression. Thus, their elimination is highly desirable for therapy development. Our therapeutic approach aims at specific and direct elimination of toxic Aß oligomers by stabilizing Aß monomers in an aggregation-incompetent conformation. We have proven that our lead compound "D3", an all d-enantiomeric-peptide, specifically eliminates Aß oligomers in vitro. In vivo, D3 enhances cognition and reduces plaque load in several transgenic AD mouse models. Here, we performed a large-scale oral proof of concept efficacy study, in which we directly compared four of the most promising D3-derivatives in transgenic mice expressing human amyloid precursor protein with Swedish and London mutations (APPSL), transgenic mice, to identify the most effective compound. RD2 and D3D3, both derived from D3 by rational design, were discovered to be the most effective derivatives in improving cognition in the Morris water maze. The performance of RD2- and D3D3-treated mice within the Morris water maze was significantly better than placebo-treated mice and, importantly, nearly as good as those of non-transgenic littermates, suggesting a complete reversal of the cognitive deficit of APPSL mice.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Oligopeptídeos/uso terapêutico , Administração Oral , Doença de Alzheimer/metabolismo , Doença de Alzheimer/psicologia , Precursor de Proteína beta-Amiloide/genética , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Transtornos Cognitivos/tratamento farmacológico , Feminino , Humanos , Camundongos Transgênicos , Estrutura Molecular , Placa Amiloide/metabolismo , Estudo de Prova de Conceito , Agregação Patológica de Proteínas/metabolismo , EstereoisomerismoRESUMO
PURPOSE: Positron emission tomography (PET) using O-(2-(18)F-fluoroethyl)-L-tyrosine ((18)F-FET) is a well-established method for the diagnostics of brain tumors. This study investigates reproducibility of (18)F-FET uptake kinetics in rat gliomas and the influence of the frequently used dexamethasone (Dex) therapy. METHODS: F98 glioma or 9L gliosarcoma cells were implanted into the striatum of 31 Fischer rats. After 10-11 days of tumor growth, the animals underwent dynamic PET after injection of (18)F-FET (baseline). Thereafter, animals were divided into a control group and a group receiving Dex injections, and all animals were reinvestigated 2 days later. Tumor-to-brain ratios (TBR) of (18)F-FET uptake (18-61 min p.i.) and the slope of the time-activity-curves (TAC) (18-61 min p.i.) were evaluated using a Volume-of-Interest (VOI) analysis. Data were analyzed by two-way repeated measures ANOVA and reproducibility by the intraclass correlation coefficient (ICC). RESULTS: The slope of the tumor TACs showed high reproducibility with an ICC of 0.93. A systematic increase of the TBR in the repeated scans was noted (3.7 ± 2.8 %; p < 0.01), and appeared to be related to tumor growth as indicated by a significant correlation of TBR and tumor volume (r = 0.77; p < 0.0001). After correction for tumor growth TBR showed high longitudinal stability with an ICC of 0.84. Dex treatment induced a significant decrease of the TBR (-8.2 ± 6.1 %; p < 0.03), but did not influence the slope of the tumor TAC. CONCLUSION: TBR of (18)F-FET uptake and tracer kinetics in brain tumors showed high longitudinal stability. Dex therapy may induce a minor decrease of the TBR; this needs further investigation.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Dexametasona/farmacologia , Glioma/tratamento farmacológico , Glioma/metabolismo , Tirosina/análogos & derivados , Animais , Transporte Biológico/efeitos dos fármacos , Neoplasias Encefálicas/diagnóstico por imagem , Dexametasona/uso terapêutico , Modelos Animais de Doenças , Glioma/diagnóstico por imagem , Cinética , Masculino , Tomografia por Emissão de Pósitrons , Ratos , Reprodutibilidade dos Testes , Tirosina/metabolismoRESUMO
PURPOSE: It has been shown that amyloid ß (Aß) oligomers play an important role in the pathology of Alzheimer's disease (AD). D3, a peptide consisting solely of D-enantiomeric amino acid residues, was developed to specifically eliminate Aß oligomers and is therapeutically active in transgenic AD mice. D-peptides have several advantages over L-peptides, but little is known about their pharmacokinetic potential in vivo. Here, we analysed the pharmacokinetic properties of RD2, a rationally designed and potent D3 derivative. METHODS: The pharmacokinetic analysis was performed using (3)H-RD2 after administration via several routes in mice. The time dependent amount of radiolabelled RD2 was measured in plasma and several organ homogenates by liquid scintillation counting. Furthermore, binding to plasma proteins was estimated. RESULTS: RD2 penetrates into the brain, where it is thought to implement its therapeutic function. All administration routes result in a maximal brain concentration per dose (Cmax/D) of 0.06 (µg/g)/(mg/kg) with brain/plasma ratios ranging between 0.7 and 1.0. RD2 shows a small elimination constant and a long terminal half-life in plasma of more than 2 days. It also exhibits high bioavailability after i.p., s.c. or p.o. administration. CONCLUSIONS: These excellent pharmacokinetic properties confirm that RD2 is a very promising drug candidate for AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/antagonistas & inibidores , Encéfalo/metabolismo , Peptídeos/farmacocinética , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Peptídeos/sangue , Peptídeos/farmacologiaRESUMO
BACKGROUND AND PURPOSE: Neuropathic pain affects up to 10% of the global population and is caused by an injury or a disease affecting the somatosensory, peripheral, or central nervous system. NP is characterized by chronic, severe and opioid-resistant properties. Therefore, its clinical management remains very challenging. The N-type voltage-gated calcium channel, Cav2.2, is a validated target for therapeutic intervention in chronic and neuropathic pain. The conotoxin ziconotide (Prialt®) is an FDA-approved drug that blocks Cav2.2 channel but needs to be administered intrathecally. Thus, although being principally efficient, the required application route is very much in disfavour. EXPERIMENTAL APPROACH AND KEY RESULTS: Here, we describe an orally available drug candidate, RD2, which competes with ziconotide binding to Cav2.2 at nanomolar concentrations and inhibits Cav2.2 almost completely reversible. Other voltage-gated calcium channel subtypes, like Cav1.2 and Cav3.2, were affected by RD2 only at concentrations higher than 10 µM. Data from sciatic inflammatory neuritis rat model demonstrated the in vivo proof of concept, as low-dose RD2 (5 mg·kg-1) administered orally alleviated neuropathic pain compared with vehicle controls. High-dose RD2 (50 mg·kg-1) was necessary to reduce pain sensation in acute thermal response assessed by the tail flick test. CONCLUSIONS AND IMPLICATIONS: Taken together, these results demonstrate that RD2 has antiallodynic properties. RD2 is orally available, which is the most convenient application form for patients and caregivers. The surprising and novel result from standard receptor screens opens the room for further optimization into new promising drug candidates, which address an unmet medical need.
Assuntos
Bloqueadores dos Canais de Cálcio , Canais de Cálcio Tipo N , Neuralgia , Animais , Humanos , Masculino , Camundongos , Ratos , Administração Oral , Bloqueadores dos Canais de Cálcio/administração & dosagem , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Tipo N/metabolismo , Canais de Cálcio Tipo N/efeitos dos fármacos , Relação Dose-Resposta a Droga , Camundongos Endogâmicos C57BL , Neuralgia/tratamento farmacológico , ômega-Conotoxinas/administração & dosagem , ômega-Conotoxinas/farmacologia , ômega-Conotoxinas/uso terapêutico , Ratos Endogâmicos LewRESUMO
The ubiquitin-proteasome pathway is a major protein degradation pathway whose dysfunction is now widely accepted as a cause of neurodegenerative diseases, including Alzheimer's disease. Here we demonstrate that the F-box and leucine rich repeat protein2 (FBL2), a component of the E3 ubiquitin ligase complex, regulates amyloid precursor protein (APP) metabolism through APP ubiquitination. FBL2 overexpression decreased the amount of secreted amyloid ß (Aß) peptides and sAPPß, whereas FBL2 mRNA knockdown by siRNA increased these levels. FBL2 overexpression also decreased the amount of intracellular Aß in Neuro2a cells stably expressing APP with Swedish mutation. FBL2 bound with APP specifically at its C-terminal fragment (CTF), which promoted APP/CTF ubiquitination. FBL2 overexpression also accelerated APP proteasome-dependent degradation and decreased APP protein localization in lipid rafts by inhibiting endocytosis. These effects were not observed in an F-box-deleted FBL2 mutant that does not participate in the E3 ubiquitin ligase complex. Furthermore, a reduced insoluble Aß and Aß plaque burden was observed in the hippocampus of 7-month-old FBL2 transgenic mice crossed with double-transgenic mice harboring APPswe and PS1(M146V) transgenes. These findings indicate that FBL2 is a novel and dual regulator of APP metabolism through FBL2-dependent ubiquitination of APP.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Endocitose , Proteínas F-Box/fisiologia , Inibição Neural , Ubiquitinação , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/genética , Animais , Linhagem Celular Tumoral , Células Cultivadas , Endocitose/genética , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Inibição Neural/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/genética , Ubiquitinação/genéticaRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting upper and lower motor neurons, thus, progressing to complete muscle loss until the patient dies from respiratory arrest. The disease is not curable, and patients die approximately 2-5 years after diagnosis. Studying the underlying disease mechanisms to get access to new treatment options is, therefore, essential for patients' benefit. However, so far, only three drugs that alleviate the symptoms have been approved by the U.S. Food and Drug Administration (FDA). A new drug candidate for the treatment of ALS is the all-d-enantiomeric peptide RD2RD2. In this study, we investigated the therapeutic effect of RD2RD2 in two setups. First, we analyzed disease progression and survival in 7 week-old B6.Cg-Tg(SOD1*G93A)1Gur/J mice. Second, we confirmed the result of the survival analysis in the B6SJL-Tg(SOD1*G93A)1Gur/J mouse line. Shortly before disease onset, the mice were treated daily with an oral dose of 50 mg/kg body weight. Treatment with RD2RD2 led to a delayed disease onset and reduced motor phenotype as shown using the SHIRPA test, the splay reflex test, and the pole test, but did not affect survival. In conclusion, RD2RD2 has the ability to delay the onset of symptoms.
RESUMO
[This corrects the article DOI: 10.3389/fncel.2022.920075.].
RESUMO
Positron emission tomography (PET) imaging with radiotracers that bind to fibrillary amyloid ß (Aß) deposits is an important tool for the diagnosis of Alzheimer's disease (AD) and for the recruitment of patients into clinical trials. However, it has been suggested that rather than the fibrillary Aß deposits, it is smaller, soluble Aß aggregates that exert a neurotoxic effect and trigger AD pathogenesis. The aim of the current study is to develop a PET probe that is capable of detecting small aggregates and soluble Aß oligomers for improved diagnosis and therapy monitoring. An 18F-labeled radioligand was prepared based on the Aß-binding d-enantiomeric peptide RD2, which is currently being evaluated in clinical trials as a therapeutic agent to dissolve Aß oligomers. 18F-labeling was carried out using palladium-catalyzed S-arylation of RD2 with 2-[18F]fluoro-5-iodopyridine ([18F]FIPy). Specific binding of [18F]RD2-cFPy to brain material from transgenic AD (APP/PS1) mice and AD patients was demonstrated with in vitro autoradiography. In vivo uptake and biodistribution of [18F]RD2-cFPy were evaluated using PET analyses in wild-type and transgenic APP/PS1 mice. Although brain penetration and brain wash-out kinetics of the radioligand were low, this study provides proof of principle for a PET probe based on a d-enantiomeric peptide binding to soluble Aß species.