Brain cells shed angiogenic and/or pro-apoptotic factors by extracellular vesicles

Metodo per la purificazione da sistemi di produzione batterici di proteine ricombinanti attive

Un metodo per la produzione e purificazione da sistemi di produzione batterici di proteine ricombinanti attive con pre-sequenza di sei istidine.

Neurons and astrocytes shed extracellular membrane vesicles containing angiogenic factors

In vitro models of blood-brain barrier and application in the study of the multiple sclerosis

Metformin decreases progerin expression and alleviates pathological defects of Hutchinson–Gilford progeria syndrome cells

Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disorder that causes systemic accelerated aging in children. This syndrome is due to a mutation in the LMNA gene that leads to the production of a truncated and toxic form of lamin A called progerin. Because the balance between the A-type lamins is controlled by the RNA-binding protein SRSF1, we have hypothesized that its inhibition may have therapeutic effects for HGPS. For this purpose, we evaluated the antidiabetic drug metformin and demonstrated that 48 h treatment with 5 mmol/l metformin decreases SRSF1 and progerin expression in mesenchymal stem cells derived from HGPS induced pluripotent stem cells (HGPS MSCs). The effect …

Extracellular vesicles shuffling intercellular messages: for good or for bad

The release of extracellular vesicles (EVs) is a highly conserved process exploited by diverse organisms as a mode of intercellular communication. Vesicles of sizes ranging from 30 to 1000. nm, or even larger, are generated by blebbing of the plasma membrane (microvesicles) or formed in multivesicular endosomes (MVEs) to be secreted by exocytosis as exosomes. Exosomes, microvesicles and other EVs contain membrane and cytosolic components that include proteins, lipids and RNAs, a composition that differs related to their site of biogenesis. Several mechanisms are involved in vesicle formation at the plasma membrane or in endosomes, which is reflected in their heterogeneity, size and composit…

Pathological modelling of pigmentation disorders associated with Hutchinson-Gilford Progeria Syndrome (HGPS) revealed an impaired melanogenesis pathway in iPS-derived melanocytes

AbstractHutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder that leads to premature aging. In this study, we used induced pluripotent stem cells to investigate the hypopigmentation phenotypes observed in patients with progeria. Accordingly, two iPS cell lines were derived from cells from HGPS patients and differentiated into melanocytes. Measurements of melanin content revealed a lower synthesis of melanin in HGPS melanocytes as compared to non-pathologic cells. Analysis of the melanosome maturation process by electron microscopy revealed a lower percentage of mature, fully pigmented melanosomes. Finally, a functional rescue experiment revealed the direct role of progerin…

Microvesicles shed by oligodendroglioma cells and rheumatoid synovial fibroblasts contain aggrecanase activity

Membrane microvesicle shedding is an active process and occurs in viable cells with no signs of apoptosis or necrosis. We report here that microvesicles shed by oligodendroglioma cells contain an ‘aggrecanase’ activity, cleaving aggrecan at sites previously identified as targets for adamalysin metalloproteinases with disintegrin and thrombospondin domains (ADAMTSs). Degradation was inhibited by EDTA, the metalloproteinase inhibitor GM6001 and by tissue inhibitor of metalloproteinases (TIMP)-3, but not by TIMP-1 or TIMP-2. This inhibitor profile indicates that the shed microvesicles contain aggrecanolytic ADAMTS(s) or related TIMP-3-sensitive metalloproteinase(s). The oligodendroglioma cells…

PEP-19 and LPI camstatins are RNA-binding proteins

Oligodendroglioma cells shed microvesicles which contain TRAIL as well as molecular chaperones and induce cell death in astrocytes.

Microvesicles (MVs) shed from G26/24 oligodendroglioma cells were previously reported to cause a reproducible, dose-dependent, inhibitory effect on neurite outgrowth, and eventually neuronal apoptosis, when added to primary cultures of rat cortical neurons. These effects were reduced but not abolished by functional monoclonal antibodies against Fas-L. In order to investigate whether MVs contain other factors able to induce cell death, we tested them for TRAIL and found clear evidence of its presence in the vesicles. This finding suggests the possibility that Fas-L and TRAIL cooperate in inducing brain cell death. Aimed at understanding the route through which the vesicles deliver their mess…

Pippin protein expression changes during cell differentiation

PIPPin is a CSD-containing protein with the ability to interact both with mRNAs encoding histone variants and chromatin. A major fraction of chromatin-bound PIPPin is sumoylated and sumoylation seems to be controlled by thyroid hormones, both in vivo and in vitro. We studied its expression in different tissues and cell lines and even in tumor cells and found that, even if more expressed in the brain respect to other tissues of the adult rat, it is also expressed in brain tumors and in cell lines as different as kidney NRK cells and PC12. The expression of the protein is strongly increased by treatments that induce differentiation, such as treatment of PC12 with NGF. We also found an increas…

EXPRESSION OF PIPPIN PROTEIN AND CELL DIFFERENTIATION.

We previously described a CSD-containing protein that seemed to bind mRNAs encoding histone variants and was present both in the nucleus and in the cytoplasm of specific populations of brain cells. Since other CSD-containing proteins have the ability to interact both with RNA and chromatin, we investigated the possibility that PIPPin binds to chromatin and indeed found that about 50% of nuclear PIPPin cannot be extracted from nuclei with salt and is instead extracted with acid, together with histones. Interestingly, a major fraction of chromatin-bound PIPPin is sumoylated and sumoylation seems to be controlled by thyroid hormones, both in vivo and in vitro. In order to study the functions o…

Effect of the serum from multiple sclerosis patients on an in vitro model of blood-brain barrier.

Multiple sclerosis (MS) is characterized by focal inflammatory demyelination, largely due to autoimmune responses against different components of the myelin sheet. It is also generally accepted that the pathogenesis of MS consists of inflammatory and neurodegenerative phases, where demyelination should produce partially reversible clinical deficits that can remit, due to limited remyelination, while axonal degeneration produces permanent non-remitting clinical damage. It is also assumed that nervous system inflammation is initiated by autoreactive, myelin-specific T cells that permeate the blood-brain barrier and trigger a series of events leading to tissue destruction. In addition to antib…

RNA-binding CSD-C2 protein and its interactors in nerve cell differentiation

In vitro model of blood-brain barrier and application on the study of the multiple sclerosis.

Characterization of a nuclear factor associated to the chromatin of sea urchin histone genes

RNAbinding proteins involved in nerve cell differentiation.

Vertebrate Hedgehog is secreted on two types of extracellular vesicles with different signaling properties

Hedgehog (Hh) is a secreted morphogen that elicits differentiation and patterning in developing tissues. Multiple proposed mechanisms to regulate Hh dispersion includes lipoprotein particles and exosomes. Here we report that vertebrate Sonic Hedgehog (Shh) is secreted on two types of extracellular-vesicles/exosomes, from human cell lines and primary chick notochord cells. Although largely overlapping in size as estimated from electron micrographs, the two exosomal fractions exhibited distinct protein and RNA composition. We have probed the functional properties of these vesicles using cell-based assays of Hh-elicited gene expression. Our results suggest that while both Shh-containing exo-ve…

ATTIVITÀ RNA LEGANTE DELLA PROTEINA CSD-C2 RICOMBINANTE PRODOTTA IN ESCHERICHIA COLI RNA BINDING ACTIVITY OF RECOMBINANT CSD-C2 PROTEIN EXPRESSED IN ESCHERICHIA COLI

Extracellular membrane vesicle shedding and the blood-brain barrier

Apolipoprotein E Regulates Amyloid Formation within Endosomes of Pigment Cells.

International audience; Accumulation of toxic amyloid oligomers is a key feature in the pathogenesis of amyloid-related diseases. Formation of mature amyloid fibrils is one defense mechanism to neutralize toxic prefibrillar oligomers. This mechanism is notably influenced by apolipoprotein E variants. Cells that produce mature amyloid fibrils to serve physiological functions must exploit specific mechanisms to avoid potential accumulation of toxic species. Pigment cells have tuned their endosomes to maximize the formation of functional amyloid from the protein PMEL. Here, we show that ApoE is associated with intraluminal vesicles (ILV) within endosomes and remain associated with ILVs when th…

Exosomes released by keratinocytes modulate melanocyte pigmentation

Cells secrete extracellular vesicles (EVs), exosomes and microvesicles, which transfer proteins, lipids and RNAs to regulate recipient cell functions. Skin pigmentation relies on a tight dialogue between keratinocytes and melanocytes in the epidermis. Here we report that exosomes secreted by keratinocytes enhance melanin synthesis by increasing both the expression and activity of melanosomal proteins. Furthermore, we show that the function of keratinocyte-derived exosomes is phototype-dependent and is modulated by ultraviolet B. In sum, this study uncovers an important physiological function for exosomes in human pigmentation and opens new avenues in our understanding of how pigmentation is…

EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

Brain-specific RNA-binding protein

Angiogenic and/or pro-apoptotic factors are shed from brain cells via extracellular vesicles

We set a three-cell type coculture system in which neurons and astrocytes synergistically induce brain capillary endothelial cells to form a monolayer with permeability properties resembling those of the physiological blood-brain barrier (BBB) (Schiera et al., 2003; Schiera et al., 2005). On the basis of immunofluorescence, scanner electron microscopy and western blot analyses, we also suggested that both astrocytes and neurons in culture shed extracellular vesicles that contain FGF-2 and VEGF, as well as beta1-integrin, a membrane protein that can be considered a marker of shedding (Schiera et al, 2007; Proia et al., 2008). In addition, it was already known that transformed glial cells (ol…

Proapoptotic effects of oligodendroglioma cells on primary brain cells in culture.

COMPOSITION AND EFFECTS OF EXTRACELLULAR VESICLES SHED BY OLIGODENDROGLIOMA CELLS

EFFECT OF SERA FROM PATIENTS WITH MULTIPLE SCLEROSIS ON A BLOOD-BRAIN BARRIER IN VITRO MODEL.

Histone RNA-binding proteins in the rat brain

In vitro models of BBB: a tool for the analysis of cell to cell communication in the brain

Many researchers have been trying to set in vitro models of the blood-brain barrier (BBB) aimed at analyzing, in simplified terms, the molecular mechanisms responsible for formation, maintenance and functioning of the BBB, as well as the capability of specific drugs and pro-drugs to cross BBB. We did it, starting with a simpler system of co-culture that allowed us to analyze the effects of neurons on differentiation of brain capillary endothelial cells (RBE4.B cells) in culture, and setting then a more complex model, that includes three cell types (endothelial cells, neurons and astrocytes). The reciprocal geometrical organization of brain cells in this model system is similar to the one ob…

A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells

AbstractHutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process an…

Shedding of extracellular membrane vesicles from both normal and tumor cells in culture

Tumor cells of different origins shed extracellular membrane vesicles (MVs), that contain angiogenetic- and pro-apoptotic-factors as well as matrix metalloproteases (MMPs). In addition, also neurons and astrocytes in culture produce VEGF- and FGF2- containing MVs, while oligodendroglioma (G26/24) cells release FasL-containing MVs that inhibit neurite sprouting and cause neuronal apoptosis. Starting from these observations, we have been analyzing composition of MVs produced by both normal and tumor cells in culture. We found that MVs from G26/24 cells contain TRAIL, Hsp70, and VEGF. We also traced the route of shed MVs, by adding vesicles that contain 35S-labeled proteins to unlabeled neuron…

RNA-binding activity of the rat calmodulin-binding PEP-19 protein and of the long PEP-19 isoform

Synthesis of H1˚ histone protein, in the developing rat brain, seems to be regulated mainly at the post-transcriptional level. Since regulation of RNA metabolism depends on a series of RNA-binding proteins, we have been searching for RNA-binding proteins involved in the post-transcriptional regulation of the H1˚ gene. We recently reported isolation, from a cDNA expression library, of an insert encoding a novel protein, the C-terminal half of which is identical to that of PEP-19, a brain-specific protein involved in calcium metabolism. The novel protein was called long PEP-19 isoform (LPI). Herein we show that LPI, as well as PEP-19, can bind H1˚ RNA. Moreover, in order to improve production…

The abietane diterpene taxodione contributes to the antioxidant activity of rosemary by-product in muscle tissue

International audience; Research on rosemary antioxidant activity and its potential use in human health and food applications is focused on rosemary leaves and two main bioactive compounds carnosic acid and carnosol. However, many other, not-yet identified molecules could be present, especially in rosemary by-products. In this study, we first showed that rosemary stem extract was the most efficient in protecting human skeletal muscle cells against oxidation. Then, using bioassay-guided fractionation, we identified taxodione, an abietane diterpene, as the main bioactive molecule in the rosemary stem extract. We demonstrated that taxodione protects skeletal muscle cells from hydrogen peroxide…

Pluripotent stem cells to model Hutchinson-Gilford progeria syndrome (HGPS): Current trends and future perspectives for drug discovery

Progeria, or Hutchinson-Gilford progeria syndrome (HGPS), is a rare, fatal genetic disease characterized by an appearance of accelerated aging in children. This syndrome is typically caused by mutations in codon 608 (p.G608G) of the LMNA, leading to the production of a mutated form of lamin A precursor called progerin. In HGPS, progerin accumulates in cells causing progressive molecular defects, including nuclear shape abnormalities, chromatin disorganization, damage to DNA and delays in cell proliferation. Here we report how, over the past five years, pluripotent stem cells have provided new insights into the study of HGPS and opened new original therapeutic perspectives to treat the disea…

Shedding of extracellular membrane vesicles from brain cells in culture