Rab11A Depletion in Microglia-Derived Extracellular Vesicle Proteome upon Beta-Amyloid Treatment.

Microglia, the macrophage-like glial cells, behave as sentinels against exogenous pathogens invading the neural tissue. Their commitment is not only confined to the defensive function, but they also perform balancing trophic activities such as neuronal postnatal development, remodeling and pruning of synapses. Likewise, microglia-derived extracellular vesicles (EVs) can play strategic roles in maintaining a healthy brain by modulating neuronal activity and by controlling neurite outgrowth as well as innate immune response. Nevertheless, strong evidence also points to their role in the development of neurodegenerative pathologies such as Alzheimer's disease (AD). Here, we explored EV protein content released by BV2 microglial cells in a resting state and after stimulation with beta-amyloid peptides (Aß), mimicking conditions occurring in AD. In the resting BV2 cells, we extended the list of proteins present in mouse microglia EV cargo with respect to those reported in the Vesiclepedia exosome database while, in amyloid-triggered microglia, we highlighted a pronounced drop in EV protein content. Focusing on Rab11A, a key factor in the recycling routes of amyloid species, we observed a dramatic decrease of this protein in Aß-treated microglia EV cargo with respect to the EVs from the untreated sample. This decrease might affect the delivery of Rab11A to neurons thus increasing the harmful amyloid burden in neuronal cells that eventually may lead to their death. We tentatively proposed that alterations observed in EVs derived from Aß-treated microglia may represent molecular features that, among others, shape the disease-associated microglial phenotype, a recently proposed subset of microglial population, present in neurodegenerative pathologies.

A Combined Proteomics, Metabolomics and In Vivo Analysis Approach for the Characterization of Probiotics in Large-Scale Production.

The manufacturing processes of commercial probiotic strains may be affected in different ways in the attempt to optimize yield, costs, functionality, or stability, influencing gene expression, protein patterns, or metabolic output. Aim of this work is to compare different samples of a high concentration (450 billion bacteria) multispecies (8 strains) formulation produced at two different manufacturing sites, United States of America (US) and Italy (IT), by applying a combination of functional proteomics, metabolomics, and in vivo analyses. Several protein-profile differences were detected between IT- and US-made products, with Lactobacillus paracasei, Streptococcus thermophilus, and Bifidobacteria being the main affected probiotics/microorganisms. Performing proton nuclear magnetic spectroscopy (1H-NMR), some discrepancies in amino acid, lactate, betaine and sucrose concentrations were also reported between the two products. Finally, we investigated the health-promoting and antiaging effects of both products in the model organism Caenorhabditis elegans. The integration of omics platforms with in vivo analysis has emerged as a powerful tool to assess manufacturing procedures.

Poly(ADP-ribosylated) proteins in ß-amyloid peptide-stimulated microglial cells.

Amyloid-treated microglia prime and sustain neuroinflammatory processes in the central nervous system activating different signalling pathways inside the cells. Since a key role for PARP-1 has been demonstrated in inflammation and in neurodegeneration, we investigated PARylated proteins in resting and in ß-amyloid peptide treated BV2 microglial cells. A total of 1158 proteins were identified by mass spectrometry with 117 specifically modified in the amyloid-treated cells. Intervention of PARylation on the proteome of microglia showed to be widespread in different cellular districts and to affect various cellular pathways, highlighting the role of this dynamic post-translational modification in cellular regulation. Ubiquitination is one of the more enriched pathways, encompassing PARylated proteins like NEDD4, an E3 ubiquitine ligase and USP10, a de-ubiquitinase, both associated with intracellular responses induced by ß-amyloid peptide challenge. PARylation of NEDD4 may be involved in the recruiting of this protein to the plasma membrane where it regulates the endocytosis of AMPA receptors, whereas USP10 may be responsible for the increase of p53 levels in amyloid stimulated microglia. Unfolded protein response and Endoplasmic Reticulum Stress pathways, strictly correlated with the Ubiquitination process, also showed enrichment in PARylated proteins. PARylation may thus represent one of the molecular switches responsible for the transition of microglia towards the inflammatory microglia phenotype, a pivotal player in brain diseases including neurodegenerative processes. The establishment of trials with PARP inhibitors to test their efficacy in the containment of neurodegenerative diseases may be envisaged.

Plasma Membrane Protein Profiling in Beta-Amyloid-Treated Microglia Cell Line.

In the responsiveness of microglia to toxic stimuli, plasma membrane proteins play a key role. In this study we treated with a synthetic beta amyloid peptide murine microglial cells metabolically differently labelled with stable isotope amino acids (SILAC). The plasma membrane was selectively enriched by a multi-stage aqueous two-phase partition system. We were able to identify by 1D-LC-MS/MS analyses 1577 proteins, most of them are plasma membrane proteins according to the Gene Ontology annotation. An unchanged level of amyloid receptors in this data set suggests that microglia preserve their responsiveness capability to the environment even after 24-h challenge with amyloid peptides. On the other hand, 14 proteins were observed to change their plasma membrane abundance to a statistically significant extent. Among these, we proposed as reliable biomarkers of the inflammatory microglia phenotype in AD damaged tissues MAP/microtubule affinity-regulating kinase 3 (MARK3), Interferon-induced transmembrane protein 3 (IFITM3), Annexins A5 and A7 (ANXA5, ANXA7) and Neuropilin-1 (NRP1), all proteins known to be involved in the inflammation processes and in microtubule network assembly rate.

PAR1 activation affects the neurotrophic properties of Schwann cells.

Protease-activated receptor-1 (PAR1) is the prototypic member of a family of four G-protein-coupled receptors that signal in response to extracellular proteases. In the peripheral nervous system, the expression and/or the role of PARs are still poorly investigated. High PAR1 mRNA expression was found in the rat dorsal root ganglia and the signal intensity of PAR1 mRNA increased in response to sciatic nerve transection. In the sciatic nerve, functional PAR1 receptor was reported at the level of non-compacted Schwann cell myelin microvilli of the nodes of Ranvier. Schwann cells are the principal population of glial cells of the peripheral nervous system which myelinate axons playing an important role during axonal regeneration and remyelination. The present study was undertaken in order to determine if the activation of PAR1 affects the neurotrophic properties of Schwann cells. Our results suggest that the stimulation of PAR1 could potentiate the Schwann cell ability to favour nerve regeneration. In fact, the conditioned medium obtained from Schwann cell cultures challenged with a specific PAR1 activating peptide (PAR1 AP) displays increased neuroprotective and neurotrophic properties with respect to the culture medium from untreated Schwann cells. The proteomic analysis of secreted proteins in untreated and PAR1 AP-treated Schwann cells allowed the identification of factors differentially expressed in the two samples. Some of them (such as macrophage migration inhibitory factor, matrix metalloproteinase-2, decorin, syndecan 4, complement C1r subcomponent, angiogenic factor with G patch and FHA domains 1) appear to be transcriptionally regulated after PAR1 AP treatment as shown by RT-PCR.

Bioenergetic Impairment in Animal and Cellular Models of Alzheimer's Disease: PARP-1 Inhibition Rescues Metabolic Dysfunctions.

Amyloid-beta peptide accumulation in the brain is one of the main hallmarks of Alzheimer's disease. The amyloid aggregation process is associated with the generation of free radical species responsible for mitochondrial impairment and DNA damage that in turn activates poly(ADP-ribose)polymerase 1 (PARP-1). PARP-1 catalyzes the poly(ADP-ribosylation), a post-translational modification of proteins, cleaving the substrate NAD+ and transferring the ADP-ribose moieties to the enzyme itself or to an acceptor protein to form branched polymers of ADP-ribose. In this paper, we demonstrate that a mitochondrial dysfunction occurs in Alzheimer's transgenic mice TgCRND8, in SH-SY5Y treated with amyloid-beta and in 7PA2 cells. Moreover, PARP-1 activation contributes to the functional energetic decline affecting cytochrome oxidase IV protein levels, oxygen consumption rates, and membrane potential, resulting in cellular bioenergetic deficit. We also observed, for the first time, an increase of pyruvate kinase 2 expression, suggesting a modulation of the glycolytic pathway by PARP-1. PARP-1 inhibitors are able to restore both mitochondrial impairment and pyruvate kinase 2 expression. The overall data here presented indicate a pivotal role for this enzyme in the bioenergetic network of neuronal cells and open new perspectives for investigating molecular mechanisms underlying energy charge decline in Alzheimer's disease. In this scenario, PARP-1 inhibitors might represent a novel therapeutic intervention to rescue cellular energetic metabolism.

Extracellular Matrix Molecular Remodeling in Human Liver Fibrosis Evolution.

Chronic liver damage leads to pathological accumulation of ECM proteins (liver fibrosis). Comprehensive characterization of the human ECM molecular composition is essential for gaining insights into the mechanisms of liver disease. To date, studies of ECM remodeling in human liver diseases have been hampered by the unavailability of purified ECM. Here, we developed a decellularization method to purify ECM scaffolds from human liver tissues. Histological and electron microscopy analyses demonstrated that the ECM scaffolds, devoid of plasma and cellular components, preserved the three-dimensional ECM structure and zonal distribution of ECM components. This method has been then applied on 57 liver biopsies of HCV-infected patients at different stages of liver fibrosis according to METAVIR classification. Label-free nLC-MS/MS proteomics and computation biology were performed to analyze the ECM molecular composition in liver fibrosis progression, thus unveiling protein expression signatures specific for the HCV-related liver fibrotic stages. In particular, the ECM molecular composition of liver fibrosis was found to involve dynamic changes in matrix stiffness, flexibility and density related to the dysregulation of predominant collagen, elastic fibers and minor components with both structural and signaling properties. This study contributes to the understanding of the molecular bases underlying ECM remodeling in liver fibrosis and suggests new molecular targets for fibrolytic strategies.

Reversible redox modifications in the microglial proteome challenged by beta amyloid.

Microglia are resident macrophages in the central nervous system, whose participation against exogenous injuries and infections is mainly marked by an immediate release of inflammatory cytokines along with a toxic efflux of superoxide radicals. Indeed, many lines of evidence indicate that persistent activation of these cells turns their neuroprotective phenotype into a neurotoxic one, which contributes to destroy neuronal activity and induces neuronal loss in several neurodegeneration processes, such as Alzheimer's disease. In this study we attempted to fill-in the gap in our knowledge about redox regulation of amyloid activated microglia. With this aim, we carried out a robust and comprehensive characterization of the reversibly redox modified proteome both at the level of resting and amyloid-activated BV2 cells, an immortalised cell line of murine microglia. The approach we used combined the selective enrichment of reversible redox modified proteins through a biotin bait with nanoscale liquid chromatography tandem mass spectrometry of their proteolytic peptides. By this reliable approach, we identified 60 proteins changing the redox status of their selective cysteine residues upon treatment with the amyloidogenic Aß25-35 peptide. These results assessed that in microglia stimulated by amyloids, redox modifications of the proteome specifically target proteins involved in crucial cell processes, i.e. those involved in the protein synthesis. In particular, for peroxiredoxin-6 (Prdx6) and Ras-related C3 botulinum toxin substrate 1 (Rac1) we suggest mechanisms through which reversible redox modifications could affect the peculiar role of microglia in amyloidogenic injury, which at the same time reinforce the oxidative burst and resist toward it. Moreover, the redox modulation we observed on chloride intracellular channel protein 1 (CLIC1) strengthens the structural and functional relationship between the oxidative stress and the metamorphic transition of this protein from a soluble form to an integral membrane form. The redox signatures we determined might also provide neurologists with more specific and reliable biomarkers to distinguish the diverse microglia status in neurodegeneration and then to drive targeted drug design.

Acetylation and phosphorylation of STAT3 are involved in the responsiveness of microglia to beta amyloid.

Microglia are macrophages within the central nervous system playing a central role in neurodegenerative disorders. Although the initial engagement of microglia seems to be neuroprotective, many lines of evidence indicate that its persistent activation contributes to dismantle neuronal activity and to induce neuronal loss. The molecular pathways that lead from amyloid interaction with membrane receptors to the microglial activation have been extensively investigated, although a definitive picture is not yet at hand. In this work, primary and immortalized microglial cells were treated with a synthetic form of Aß peptides, and relative abundance of acetylated and phosphorylated STAT3 were assayed. Results highlight, for the first time, three distinctive sequential events: i) an earlier event marked by the increase in the level of STAT3 acetylated species, followed by ii) a later increase in the level of STAT3 phosphorylated form, and finally iii) an involvement of phosphorylated STAT3 in the increase in expression of the 14-3-3 epsilon, a protein frequently associated with neurodegenerative diseases and known to be a marker of Aß-activated microglia. These data outline a complex, time-dependent modification of STAT3 signalling triggered by amyloid in the microglial compartments, that once confirmed by in vivo experiments will broaden the knowledge of the molecular basis of amyloid neurotoxicity.

14-3-3ε marks the amyloid-stimulated microglia long-term activation.

Microglia-mediated inflammation in the central nervous system is a hallmark of the pathogenesis of several neurodegenerative diseases including Alzheimer's disease. Microglial cells activation follows the deposition of amyloid ß fibrils and it is generally considered a triggering factor in the early steps of the onset of Alzheimer's disease. Although the initial engagement of microglia seems to play a neuroprotective role, many lines of evidence indicate that a persistent activation with the production of proinflammatory molecules contributes to dismantle neuronal activity and to induce neuronal loss occurring in neurodegenerative diseases. To date, limited proteomic data are available on activated microglial cells in response to extracellular amyloidogenic peptides. In this study, murine microglial cells have been employed to investigate the effects of amyloid ß peptides in triggering microglial activation. The response was monitored at the proteome level through a two-dimensional gel electrophoresis-based approach. Results show only a limited number of differentially expressed proteins, among these a more acidic species of the cytosolic actin, and the 14-3-3ε protein, found significantly upregulated in Aß-activated cells. 14-3-3ε belongs to a regulatory protein family involved in important cellular processes, including those leading to neurodegenerative diseases, and thus its increased expression suggests a role of this protein in tuning microglia activation.