RESUMEN
BACKGROUND: Activated microglia play an essential role in inflammatory responses elicited in the central nervous system (CNS). Microglia-derived extracellular vesicles (EVs) are suggested to be involved in propagation of inflammatory signals and in the modulation of cell-to-cell communication. However, there is a lack of knowledge on the regulation of EVs and how this in turn facilitates the communication between cells in the brain. Here, we characterized microglial EVs under inflammatory conditions and investigated the effects of inflammation on the EV size, quantity, and protein content. METHODS: We have utilized western blot, nanoparticle tracking analysis (NTA), and mass spectrometry to characterize EVs and examine the alterations of secreted EVs from a microglial cell line (BV2) following lipopolysaccharide (LPS) and tumor necrosis factor (TNF) inhibitor (etanercept) treatments, or either alone. The inflammatory responses were measured with multiplex cytokine ELISA and western blot. We also subjected TNF knockout mice to experimental stroke (permanent middle cerebral artery occlusion) and validated the effect of TNF inhibition on EV release. RESULTS: Our analysis of EVs originating from activated BV2 microglia revealed a significant increase in the intravesicular levels of TNF and interleukin (IL)-6. We also observed that the number of EVs released was reduced both in vitro and in vivo when inflammation was inhibited via the TNF pathway. Finally, via mass spectrometry, we identified 49 unique proteins in EVs released from LPS-activated microglia compared to control EVs (58 proteins in EVs released from LPS-activated microglia and 37 from control EVs). According to Gene Ontology (GO) analysis, we found a large increase of proteins related to translation and transcription in EVs from LPS. Importantly, we showed a distinct profile of proteins found in EVs released from LPS treated cells compared to control. CONCLUSIONS: We demonstrate altered EV production in BV2 microglial cells and altered cytokine levels and protein composition carried by EVs in response to LPS challenge. Our findings provide new insights into the potential roles of EVs that could be related to the pathogenesis in neuroinflammatory diseases.
Asunto(s)
Citocinas/metabolismo , Vesículas Extracelulares/patología , Infarto de la Arteria Cerebral Media/complicaciones , Inflamación/etiología , Microglía/patología , Animales , Línea Celular Transformada , Modelos Animales de Enfermedad , Etanercept/farmacología , Vesículas Extracelulares/ultraestructura , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Inmunosupresores/farmacología , Infarto de la Arteria Cerebral Media/patología , Inflamación/patología , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/deficiencia , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Mint/X11 is one of the four neuronal trafficking adaptors that interact with amyloid precursor protein (APP) and are linked with its cleavage to generate ß-amyloid peptide, a key player in the pathology of Alzheimer's disease. How APP switches between adaptors at different stages of the secretory pathway is poorly understood. Here, we show that tyrosine phosphorylation of Mint1 regulates the destination of APP. A canonical SH2-binding motif ((202) YEEI) was identified in the N-terminus of Mint1 that is phosphorylated on tyrosine by C-Src and recruits the active kinase for sequential phosphorylation of further tyrosines (Y191 and Y187). A single Y202F mutation in the Mint1 N-terminus inhibits C-Src binding and tyrosine phosphorylation. Previous studies observed that co-expression of wild-type Mint1 and APP causes accumulation of APP in the trans-Golgi. Unphosphorylatable Mint1 (Y202F) or pharmacological inhibition of Src reduced the accumulation of APP in the trans-Golgi of heterologous cells. A similar result was observed in cultured rat hippocampal neurons where Mint1(Y202F) permitted the trafficking of APP to more distal neurites than the wild-type protein. These data underline the importance of the tyrosine phosphorylation of Mint1 as a critical switch for determining the destination of APP. The regulation of amyloid precursor protein (APP) trafficking is poorly understood. We have discovered that the APP adapter, Mint1, is phosphorylated by C-Src kinase. Mint1 causes APP accumulation in the trans-Golgi network, whereas inhibition of Src or mutation of Mint1-Y202 permits APP recycling. The phosphorylation status of Mint1 could impact on the pathological trafficking of APP in Alzheimer's disease.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Tirosina/metabolismo , Familia-src Quinasas/metabolismo , Red trans-Golgi/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Precursor de Proteína beta-Amiloide/genética , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Femenino , Células HeLa , Humanos , Masculino , Ratones , Proteínas del Tejido Nervioso/genética , Fosforilación/fisiología , Transporte de Proteínas/fisiología , Ratas , Ratas Wistar , Tirosina/genética , Familia-src Quinasas/genética , Red trans-Golgi/genéticaRESUMEN
Aggregation of the amyloid-ß (Aß) peptide into plaques is believed to play a crucial role in Alzheimer's disease. Amyloid plaques consist of fibrils of full length Aß peptides as well as N-terminally truncated species. ß-Site amyloid precursor protein-cleaving enzyme (BACE1) cleaves amyloid precursor protein in the first step in Aß peptide production and is an attractive therapeutic target to limit Aß generation. Inhibition of BACE1, however, induces a unique pattern of Aß peptides with increased levels of N-terminally truncated Aß peptides starting at position 5 (Aß5-X), indicating that these peptides are generated through a BACE1-independent pathway. Here we elucidate the aggregation mechanism of Aß5-42 and its influence on full-length Aß42. We find that, compared to Aß42, Aß5-42 is more aggregation prone and displays enhanced nucleation rates. Aß5-42 oligomers cause nonspecific membrane disruption to similar extent as Aß42 but appear at earlier time points in the aggregation reaction. Noteworthy, this implies similar toxicity of Aß42 and Aß5-42 and the toxic species are generated faster by Aß5-42. The increased rate of secondary nucleation on the surface of existing fibrils originates from a higher affinity of Aß5-42 monomers for fibrils, as compared to Aß42: an effect that may be related to the reduced net charge of Aß5-42. Moreover, Aß5-42 and Aß42 peptides coaggregate into heteromolecular fibrils and either species can elongate existing Aß42 or Aß5-42 fibrils but Aß42 fibrils are more catalytic than Aß5-42 fibrils. Our findings highlight the importance of the N-terminus for surface-catalyzed nucleation and thus the production of toxic oligomers.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Fragmentos de Péptidos/metabolismo , Placa Amiloide/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Humanos , Cinética , Nanopartículas/metabolismoRESUMEN
N2-Src is a poorly understood neuronal splice variant of the ubiquitous C-Src tyrosine kinase, containing a 17 amino acid insert in its Src homology 3 (SH3) domain. To characterise the properties of N2-Src we directly compared its SH3 domain specificity and kinase activity with C- and N1-Src in vitro. N2- and N1-Src had a similar low affinity for the phosphorylation of substrates containing canonical C-Src SH3 ligands and synaptophysin, an established neuronal substrate for C-Src. N2-Src also had a higher basal kinase activity than N1- and C-Src in vitro and in cells, which could be explained by weakened intramolecular interactions. Therefore, N2-Src is a highly active kinase that is likely to phosphorylate alternative substrates to C-Src in the brain.
Asunto(s)
Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Empalme del ARN , Dominios Homologos src , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular Tumoral , Ligandos , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Proteínas Proto-Oncogénicas pp60(c-src)/química , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Ratas , Homología de Secuencia de AminoácidoRESUMEN
α-Synuclein is a key protein in Parkinson disease. Not only is it the major protein component of Lewy bodies, but it is implicated in several cellular processes that are disrupted in Parkinson disease. Misfolded α-synuclein has also been shown to spread from cell-to-cell and, in a prion-like fashion, trigger aggregation of α-synuclein in the recipient cell. In this mini-review we explore the evidence that misfolded α-synuclein underlies the spread of pathology in Parkinson disease and discuss why it should be considered a prion-like protein.
Asunto(s)
Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Priones/metabolismo , alfa-Sinucleína/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Humanos , Cuerpos de Lewy/metabolismo , Cuerpos de Lewy/patología , Mitocondrias/metabolismo , Mitocondrias/patología , Estrés Oxidativo , Priones/análisis , Pliegue de Proteína , alfa-Sinucleína/análisisRESUMEN
Parkinson's disease is the second most prevalent neurodegenerative disease, yet despite this, very little is known about the underlying cellular mechanisms. Initially it was thought to be a disease primarily involving loss of dopaminergic neurons in the substantia nigra pars compacta. Recent studies, however, have focused on observations that aggregated α-synuclein protein, the major component of Lewy bodies, is found throughout the nervous system. It is speculated that misfolded α-synuclein transfers between cells in a prion-like manner, thereby mediating the spread of the neuropathology. In this review, we discuss the staging (according to Braak) of Parkinson pathology and the concept describing the disease progression from one region of the brain to the other. We highlight how α-synuclein might be responsible for the spread of the disease. We compare the idea of a prion-like mechanism contributing to Parkinson's disease to emerging concepts that other proteins participate in similar processes in other neurodegenerative diseases. We then examine the future implications of a critical role in disease pathogenesis of α-synuclein for the classification, diagnosis and treatment of Parkinson's disease in the future.