Quantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer-based precipitation and size exclusion chromatography.

The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer-based precipitation and size exclusion chromatography (Pre-SEC), we analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early- and late-eluting EV fractions, we performed a quantitative proteomic analysis of MDA-MB-468-derived EVs. We identified 286 exclusive proteins in early-eluting fractions and 148 proteins with a differential concentration between early- and late-eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, we found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional studies.

Differential distribution of HP1 proteins after trichostatin a treatment influences chromosomal stability in HCT116 and WI-38 cells.

BACKGROUND: Heterochromatin protein 1 (HP1) is important in the establishment, propagation, and maintenance of constitutive heterochromatin, especially at the pericentromeric region. HP1 might participate in recruiting and directing Mis12 to the centromere during interphase, and HP1 disruption or abrogation might lead to the loss of Mis12 incorporation into the kinetochore. Therefore, the centromere structure and kinetochore relaxation that are promoted in the absence of Mis12 could further induce chromosome instability (CIN) by reducing the capacity of the kinetochore to anchor microtubules. The aim of this study was to determine whether alterations in the localization of HP1 proteins induced by trichostatin A (TSA) modify Mis12 and Centromere Protein A (CENP-A) recruitment to the centromere and whether changes in the expression of HP1 proteins and H3K9 methylation at centromeric chromatin increase CIN in HCT116 and WI-38 cells. METHODS: HCT116 and WI-38 cells were cultured and treated with TSA to evaluate CIN after 24 and 48 h of exposure. Immunofluorescence, Western blot, ChIP, and RT-PCR assays were performed in both cell lines to evaluate the localization and abundance of HP1α/ß, Mis12, and CENP-A and to evaluate chromatin modifications during interphase and mitosis, as well as after 24 and 48 h of TSA treatment. RESULTS: Our results show that the TSA-induced reduction in heterochromatic histone marks on centromeric chromatin reduced HP1 at the centromere in the non-tumoral WI-38 cells and that this reduction was associated with cell cycle arrest and CIN. However, in HCT116 cells, HP1 proteins, together with MIS12 and CENP-A, relocated to centromeric chromatin in response to TSA treatment, even after H3K9me3 depletion in the centromeric nucleosomes. The enrichment of HP1 and the loss of H3K9me3 were associated with an increase in CIN, suggesting a response mechanism at centromeric and pericentromeric chromatin that augments the presence of HP1 proteins in those regions, possibly ensuring chromosome segregation despite serious CIN. Our results provide new insight into the epigenetic landscape of centromeric chromatin and the role of HP1 proteins in CIN.

Oxidative stress promotes JNK-dependent amyloidogenic processing of normally expressed human APP by differential modification of alpha-, beta- and gamma-secretase expression.

The pathogenesis of Alzheimer disease (AD) is complex and is certain to involve diverse etiological factors, but a central role has been strongly suggested for amyloid beta-protein (Abeta), based on genetic, biochemical and neurotoxicological evidence. In contrast with the well-documented effect of genetic mutations in Abeta overproduction, not much is known about the mechanisms involved in sporadic AD (SAD) which account for more than 95% of cases. Extensive data from patients and in vivo animal models indicate that oxidative stress is one of the cardinal factors most frequently associated with this neurodegenerative disease. The aim of the present study was to explore the effect of oxidative stress on the normally expressed wild-type amyloid precursor protein (APP) in human neuroblastoma cells, which represents a more physiological model of neuronal Abeta generation. Since H(2)O(2) is the main source of the highly reactive hydroxyl radical in the brain, and FeCl(2) can stimulate oxidative stress, including the formation of the hydroxyl radical from H(2)O(2), in the present work we studied the effect of these two pro-oxidant molecules on the levels and processing of human APP by alpha-, beta- and gamma-secretase, and the role of the stress-activated kinase c-jun N-terminal kinase (JNK). We provide evidence for a dual modulation of amyloid precursor protein metabolism in differentiated human neuroblastoma cells related with a down-regulation of alpha-secretase and up-regulation of gamma-secretase, and particularly of beta-secretase and also a JNK depending Abeta generation.

Role of oxidative stress on beta-amyloid neurotoxicity elicited during impairment of energy metabolism in the hippocampus: protection by antioxidants.

Age-associated oxidative stress has been implicated in neuronal damage linked with Alzheimer's disease (AD). In addition to the role of beta-amyloid peptide (Abeta) in the pathogenesis of AD, reduced glucose oxidative metabolism and decreased mitochondrial activity have been suggested as associated factors. However, the relationship between Abeta toxicity, metabolic impairment, and oxidative stress is far from being understood. In vivo neurotoxicity of Abeta25-35 peptide has been conflicting. However, in previous studies, we have shown that Abeta25-35 consistently induces synaptic toxicity and neuronal death in the hippocampus in vivo, when administered during moderate glycolytic or mitochondrial inhibition. In the present study, we have investigated whether enhancement of Abeta neurotoxicity during these conditions involves oxidative stress. Results show increased lipoperoxidation (LPO) when Abeta is administered in the hippocampus of rats previously treated with the glycolysis inhibitor, iodoacetate. Neuronal damage and LPO are efficiently prevented by vitamin E, while the spin trapper, alpha-phenyl-N-tert-butyl nitrone, shows partial protection. Abeta stimulates LPO in synaptosomes, but toxicity is only observed in the presence of metabolic inhibitors. Damage and LPO are efficiently prevented by vitamin E. The present results suggest an interaction between oxidative stress and metabolic impairment in the Abeta neurotoxic cascade.

beta-Amyloid neurotoxicity is exacerbated during glycolysis inhibition and mitochondrial impairment in the rat hippocampus in vivo and in isolated nerve terminals: implications for Alzheimer's disease.

Senile plaques composed mainly by beta-amyloid (Abeta) protein are one of the pathological hallmarks of Alzheimer's disease (AD). In vitro, Abeta and its active fragment 25-35 have been shown either to be directly neurotoxic or to exacerbate the damaging effect of other neurotoxic insults. However, the attempts to replicate Abeta neurotoxicity in vivo have yielded conflicting results. One of the most consistent alterations in AD is a reduced resting glucose utilization. Important evidence suggests that impairment of brain energy metabolism can lead to neuronal damage or facilitate the deleterious effects of some neurotoxic agents. In the present study we have investigated the influence of glycolysis inhibition induced by iodoacetate, and mitochondrial impairment induced by 3-nitropropionic acid (3-NP), in the toxicity of Abeta. We have studied Abeta neurotoxicity during energy deficiency both in vivo in the dentate gyrus of the hippocampal formation and in presynaptic terminals isolated from neocortex and hippocampus. Results show that during metabolic inhibition an enhanced vulnerability of hippocampal neurons to Abeta peptide toxicity occurs, probably resulting from decreased glucose metabolism and mitochondrial ATP production. Synaptosomal response to energy impairment and Abeta toxicity was evaluated by the MTT assay. Results suggest that synapses may be particularly sensitive to metabolic perturbation, which in turn exacerbates Abeta toxicity. The present data provide experimental support to the hypothesis that certain risk factors such as metabolic dysfunction and amyloid accumulation may interact to exacerbate AD, and that metabolic substrates such as pyruvate may play a role as a therapeutic tool.