RESUMO
Mitochondrial deregulation has emerged as one of the earliest pathological events in Alzheimer's disease (AD), the most common age-related neurodegenerative disorder. Improvement of mitochondrial function in AD has been considered a relevant therapeutic approach. L-carnitine (LC), an amino acid derivative involved in the transport of long-chain fatty acids into mitochondria, was previously demonstrated to improve mitochondrial function, having beneficial effects in neurological disorders; moreover, acetyl-L-carnitine (ALC) is currently under phase 4 clinical trial for AD (ClinicalTrials.gov NCT01320527). Thus, in the present study, we investigated the impact of different forms of carnitines, namely LC, ALC and propionyl-L-carnitine (PLC) on mitochondrial toxicity induced by amyloid-beta peptide 1-42 oligomers (AßO; 1 µM) in mature rat hippocampal neurons. Our results indicate that 5 mM LC, ALC and PLC totally rescued the mitochondrial membrane potential and alleviated both the decrease in oxygen consumption rates and the increase in mitochondrial fragmentation induced by AßO. These could contribute to the prevention of neuronal death by apoptosis. Moreover, only ALC ameliorated AßO-evoked changes in mitochondrial movement by reducing the number of stationary mitochondria and promoting reversal mitochondrial movement. Data suggest that carnitines (LC, ALC and PLC) may act differentially to counteract changes in mitochondrial function and movement in neurons subjected to AßO, thus counteracting AD-related pathological phenotypes.
Assuntos
Acetilcarnitina/farmacologia , Doença de Alzheimer/tratamento farmacológico , Carnitina/análogos & derivados , Fármacos Neuroprotetores/farmacologia , Doença de Alzheimer/fisiopatologia , Animais , Apoptose/efeitos dos fármacos , Carnitina/farmacologia , Células Cultivadas , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/parasitologia , Fármacos Neuroprotetores/química , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Inhibition of the initial stages of amyloid-ß peptide self-assembly is a key approach in drug development for Alzheimer's disease, in which soluble and highly neurotoxic low molecular weight oligomers are produced and aggregate in the brain over time. Here we report a high-throughput method based on ion mobility mass spectrometry and multivariate statistical analysis to rapidly select statistically significant early-stage species of amyloid-ß1-40 whose formation is inhibited by a candidate theranostic agent. Using this method, we have confirmed the inhibition of a Zn-porphyrin-peptide conjugate in the early self-assembly of Aß40 peptide. The MS/MS fragmentation patterns of the species detected in the samples containing the Zn-porphyrin-peptide conjugate suggested a porphyrin-catalyzed oxidation at Met-35(O) of Aß40. We introduce ion mobility MS combined with multivariate statistics as a systematic approach to perform data analytics in drug discovery/amyloid research that aims at the evaluation of the inhibitory effect on the Aß early assembly in vitro models at very low concentration levels of Aß peptides.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Espectrometria de Mobilidade Iônica/métodos , Fragmentos de Peptídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Doença de Alzheimer/tratamento farmacológico , Humanos , Análise MultivariadaRESUMO
The cholesteryl-ester transfer protein (CETP) facilitates the transfer of cholesterol esters and triglycerides between lipoproteins in plasma where the critical site for its function is situated in the C-terminal domain. Our group has previously shown that this domain presents conformational changes in a non-lipid environment when the mutation D(470)N is introduced. Using a series of peptides derived from this C-terminal domain, the present study shows that these changes favor the induction of a secondary ß-structure as characterized by spectroscopic analysis and fluorescence techniques. From this type of secondary structure, the formation of peptide aggregates and fibrillar structures with amyloid characteristics induced cytotoxicity in microglial cells in culture. These supramolecular structures promote cell cytotoxicity through the formation of reactive oxygen species (ROS) and change the balance of a series of proteins that control the process of endocytosis, similar to that observed when ß-amyloid fibrils are employed. Therefore, a fine balance between the highly dynamic secondary structure of the C-terminal domain of CETP, the net charge, and the physicochemical characteristics of the surrounding microenvironment define the type of secondary structure acquired. Changes in this balance might favor misfolding in this region, which would alter the lipid transfer capacity conducted by CETP, favoring its propensity to substitute its physiological function.
Assuntos
Proteínas de Transferência de Ésteres de Colesterol/química , Peptídeos/química , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Camundongos , Fragmentos de Peptídeos/química , Peptídeos/síntese química , Peptídeos/toxicidade , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , TemperaturaRESUMO
SCOPE: Proteolysis-resistant gliadin peptides are intensely investigated in biomedical research relates to celiac disease and gluten-related disorders. Herein, the first integrated supramolecular investigation of pepsin-digested gliadin peptides (p-gliadin) is presented in combination with its functional behavior in the Caco-2 cell line. METHODS AND RESULTS: First, gliadins are degraded by pepsin at pH 3, and the physicochemical properties of p-gliadin are compared with gliadin. An integrated approach using interfacial, spectroscopic, and microscopic techniques reveals that the p-gliadin forms spontaneously soluble large supramolecular structures, mainly oligomers and fibrils, capable of binding amyloid-sensitive dyes. The self-assembly of p-gliadin starts at a concentration of 0.40 µg mL-1 . Second, the stimulation of Caco-2 cells with the p-gliadin supramolecular system is performed, and the mRNA expression levels of a panel of genes are tested. The experiments show that p-gliadin composed of supramolecular structures triggers significant mRNA up-regulation (p < 0.05) of pro-apoptotic biomarkers (ratio Bcl2/Bak-1), chemokines (CCL2, CCL3, CCL4, CCL5, CXCL8), and the chemokine receptor CXCR3. CONCLUSIONS: This work demonstrates that p-gliadin is interfacial active, forming spontaneously amyloid-type structures that trigger genes in the Caco-2 cell line involved in recruiting specialized immune cells.
Assuntos
Gliadina/química , Nanoestruturas , Pepsina A/metabolismo , Apoptose , Células CACO-2 , Doença Celíaca/imunologia , Fatores Quimiotáticos , Regulação da Expressão Gênica , Humanos , Inflamação , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , ProteóliseRESUMO
Superior to anodic photoelectrochemical (PEC) method, cathodic bioanalysis integrates merits of excellent anti-interference and high stability, representing a promising and competitive methodology in precise monitoring targets in complex matrices. However, serious consideration of photocathode is far behind the anodic one, developing high-performance photocathode for PEC biosensing is thus urgently desired. Herein, a high-performance cathodic PEC aptasensing platform for detection of amyloid-beta oligomers (AßO) was constructed by integrating CuO/g-C3N4 p-n heterojunction with MoS2 QDs@Cu NWs multifunction signal amplifier. The CuO/g-C3N4, exhibiting intense visible light-harvesting and high photoelectric conversion efficiency, was synthesized by in-situ pyrolysis of Cu-MOF and dicyandiamide. The MoS2 QDs@Cu NWs was obtained by electrostatical self-assembly, which acted not only as a sensitizer to boost PEC response, but also as a nanozyme for biocatalytic precipitation. The aptasensor was fabricated by DNA hybridization between the cDNA on photocathode and MoS2 QDs@Cu NWs-labeled aptamer. Based on "on-off-on" photocurrent response generated by multifunction signal amplification, ultrasensitive aptasensing of AßO was realized in a wider linear range from 10 fM to 0.5 µM with an ultralow detection limit of 5.79 fM. The feasibility of the sensor for AßO determination in human blood serum was demonstrated.
Assuntos
Técnicas Biossensoriais , Cobre , Técnicas Eletroquímicas , Eletrodos , Humanos , Limite de Detecção , MolibdênioRESUMO
The aggregation kinetics of Aß1-40 peptide was characterized using a synergistic approach by a combination of nuclear magnetic resonance, thioflavin-T fluorescence, transmission electron microscopy and dynamic light scattering. A major finding is the experimental detection of high molecular weight oligomers (HMWO) that converts into fibrils nuclei. Our observations are consistent with a mechanism of Aß1-40 fibrillogenesis that includes the following key steps: i) slow formation of HMWO (Rh ~ 20 nm); ii) conversion of the HMWO into more compact Rh ~ 10 nm fibrils nuclei; iii) fast formation of additional fibrils nuclei through fibril surface catalysed processes; and iv) growth of fibrils by addition of soluble Aß species. Moreover, NMR diffusion experiments show that at 37 °C soluble Aß1-40 remains intrinsically disordered and mostly in monomeric form despite evidences of the presence of dimers and/or other small oligomers. A mathematical model is proposed to simulate the aggregation kinetics of Aß1-40.