178 research outputs found
Potential roles of extracellular vesicles in brain cell-to-cell communication
No full textPotential roles of extracellular vesicles in brain cell-to-cell communication
Extracellular vesicles (EVs) are released into thè extracellular space from both cancer and normal
brain cells, and are probably able to modify thè phenotypic properties of receiving cells1. EVs
released from astrocytes and neurons contain FGF2 and VEGF2'3 and induce a 'blood-brain
barrier' (BBB) phenotype in cultured brain capillary endothelial cells (BCECs, unpublished results),
On thè other hand, EVs from G26/24 oligodendroglioma induce apoptosis in neurons and
astrocytes4-5. These effects are probably due to Fas Ligand and TRAIL, present in G26/24
vesicles4-5. Moreover, G26/24 EVs contain extracellular matrix remodeling proteases (such as
ADAMTS)6, H1.0 histone protein, and H1.0 mRNA7. In particular, we previously hypothesized that
G26/24 cells, and tumor cells in generai, can escape differentiation cues, and continue to
proliferate by eliminating proteins, such as thè H1° linker histone (and its mRNA)7, which could
otherwise block proliferation.
To study vesicle release in a System that can better resemble in vivo conditions, astrocytes and
BCECs were cultured on poly-L-lactic acid (PLLA) scaffolds and tested for their ability to grow and
survive on this three-dimensional structures. We analyzed in parallel thè celi growth in 2D and 3D
culture systems and observed thè differences in celi morphology by fluorescence analysis: threedimensional
scaffolds have thè ability to guide celi growth, provide support, encourage celi
adhesion and proliferation. Astrocytes8 and BCECs (unpublished results) adapted well to these
porous matrices, not only remaining on thè surface, but also penetrating inside thè scaffolds.
EVs released by astrocytes in these scaffolds are probably exosomes, as suggested by
transmission electron microscopy pictures, and by thè presence of intracellular structures
resembling multivesicular bodies. This 3D celi culture System could be further enriched to host
different brain celi types, in order to set, for example, an in vitro model of BBB, that may be useful
for drug delivery studies, and for thè formulation of new therapeutic strategies for thè treatment of
neurological diseases.
References
[1] Schiera, G., Di Liegro, C.M., Di Liegro I. Int J Mol Sci. 2017, 18(12). pii: E2774.
[2] Schiera, G., Proia, P., Alberti, C., Mineo, M., Savettieri, G., Di Liegro, I., 2007. J Celi Mol Med. 2007,
111(6), 1384-94.
[3] Proia, P., Schiera, G., Mineo, M., Ingrassia, A.M. Santoro, G., Savettieri, G., Di Liegro, I. Int J Mol Med.
2008, 21(1), 63-7.
[4] D'Agostino, S., Salamene, M., Di Liegro, I., Vittorelli, ML, Int J Oncol. 2006, 29(5), 1075-85.
[5] Lo Cicero, A., Schiera, G., Proia, P., Saladino, P., Savettieri, G., Di Liegro, C.M., Di Liegro, I. Int J Oncol.
2011,39(6): 1353-7.
[6] Lo Cicero, A., Majkowska, I., Nagase, H., Di Liegro, I., Troeberg, L., Matrix Biol. 2012, 31(4), 229-33.
[7] Schiera, G., Di Liegro, C.M., Saladino, P., Pitti, R., Savettieri, G., Proia, P., Di Liegro, I. Int J Oncol. 2013,
43(6), 1771-6.
[8] Carfì Pavia, F., Di Bella, M.A., Brucato, V., Blanda, V., Zummo, F., Vitrano, I., Di Liegro, C.M., Ghersi, G.,
Di Liegro, I., Schiera, G. Mol Med Rep. 2019 [Epub ahead of print].
[9] Di Bella MA, Zummo F., Carfì Pavia F., Brucato V., Di Liegro I., Schiera G. 2017, In: Microscopy and
Imaging Science: practical approaches to applied research and education, pp 260-264. Ed: A. Méndez-Vilas
Publisher, Formatex Research Center (Spain), ISBN-13, 978-84-942134-9-6
Developing rat brain as well as cultured astrocytes contain H1° mRNA-protein complexes
No full textRNA-binding proteins (RBPs) regulate intracellular transport, pre-localization, stability, and translation of mRNAs [1].
We previously identified a set of proteins which interact with mRNAs encoding H1° and H3.3 histones [2-5]. All these proteins are probably part of a ribonucleoprotein particle [6]. Here we report the results of a more detailed study on the expression and intracellular localization of some of these RBPs, such as hnRNP K and A1, and Hsc70, during rat brain development and in cultured rat astrocytes. We also investigated the presence in the complexes of PIPPin/CSD-C2 protein.
Affinity chromatography was performed as already described [6]. Preparation of total lysates and cellular sub-fractions was done as reported in [3]. Possible co-localization of Hsc70 with CSD-C2 in cultured astrocytes was analysed by immunofluorescence microscopy.
The presence of Hsc70 chaperone in the already identified ribonucleoprotein complex [6] was confirmed by affinity chromatography. We also found that the complex itself is present not only in the nuclear extracts, but also in the cytoplasmic fraction. Moreover, A1 hnRNP, previously found in the complexes, was found to be differentially expressed and localized during rat brain maturation. In particular, we found that nuclear levels of A1 increase in cultured astrocytes grown on a fibronectin-containing substrate, but decrease when cells are fed with Maat medium [7].
We finally report that sumoylated PIPPin, already found in neurons, is also present in the nuclei of cultured astrocytes.
[1] Di Liegro et al. 2014, Int J Mol Med 33:747-62.
[2] Scaturro et al. 1998, J Biol Chem 273:22788-91.
[3] Nastasi et al. 1999, J Biol Chem 274:24087-93.
[4] Sala et al. 2007, Int J Mol Med 19:501-9.
[5] Saladino et al. 2012, Int J Mol Med 29:141-5
[6] Di Liegro et al. 2013, Neuroscience 229:71-6.
[7] Cestelli et al., 1985, Brain Res 354:219-27
H1° mRNA-containing complexes in rat brain cells. In: Proceedings of the Abstracts
No full textPost-transcriptional regulation of gene expression depends on RNA-binding proteins (RBPs), which are able to regulate translation, stability and subcellular localization of mRNAs [1]. RNA-protein complexes start to be built up since transcription; some proteins remain then bound to the transcript, while others behave as only transient components. In the developing nervous system of mammals, the postnatal production of the histone variants H1° and H3.3 is mainly regulated at the post-transcriptional level. Synthesis and incorporation into chromatin of the two histone proteins has been suggested to be involved in the epigenetic regulation of gene expression, both in normal brain development and brain cancer.
We previously identified a set of proteins which interact with mRNAs encoding H1° and H3.3 mRNA [2-5]. Recently, we demonstrated that these proteins are probably part of a ribonucleoprotein particle which contains both unspecific and specific RBPs [6]. Here we confirm, by affinity chromatography, the presence of Hsc70 chaperone in the complex and, in addition, we report that the complex is present not only in the nuclear extracts, as previously shown, but also in a membrane-containing cytoplasmic pellet, obtained by centrifugation at 105.000 x g (P105). In parallel, we demonstrated that sumoylated PIPPin, already found in neurons, is present also in the nuclei of cultured astrocytes. Finally, we found that two of the hnRNPs previously found in the complexes show a differential expression and localization during rat brain maturation.
[1] Di Liegro CM et al. 2014, Int J Mol Med 33:747-62.
[2] Scaturro M et al. 1998, J Biol Chem 273:22788-91.
[3] Nastasi T et al. 1999, J Biol Chem 274:24087-93.
[4] Sala A et al. 2007, Int J Mol Med 19:501-9.
[5] Saladino P et al. 2012, Int J Mol Med 29:141-5
[6] Di Liegro CM et al. 2013, Neuroscience 229:71-6
Expression and intracellular localization of H1° mRNA-containing complexes in developing rat brain and astrocytes
No full textINTRODUCTION: Post-transcriptional regulation of gene expression relies on RNA-binding proteins
(RBPs), which regulate intracellular transport, stability, and translation of mRNAs [1]. We previously identified
a set of proteins which interact with mRNAs encoding H1° and H3.3 histones [2-5]. All these proteins
are probably part of a ribonucleoprotein particle [6]. Here we report more details on the expression and
intracellular localization of some of these RBPs, during rat brain development and in isolated rat astrocytes.
METHODS: Affinity chromatography was performed as already described [6]. Preparation of total lysates
and cellular sub-fractions was done as reported in [3]. Possible co-localization of Hsc70 with CSD-C2 in cultured
astrocytes was analysed by immunofluorescence microscopy.
RESULTS: The presence of Hsc70 chaperone in the already identified ribonucleoprotein complex [6] was
confirmed by affinity chromatography. We also found that the complex itself is present not only in the nuclear
extracts, but also in the cytoplasmic fraction. Moreover, A1 and K hnRNPs, previously found in the complexes,
were found to be differentially expressed and localized during rat brain maturation; an increase in A1
expression was also demonstrated in cultured astrocytes grown on a fibronectin-containing substrate.
We finally report that sumoylated PIPPin, already found in neurons, is also present in the nuclei of cultured
astrocytes.
CONCLUSIONS: We confirmed the existence of a group of proteins able to interact with H1°/H3.3 mRNAs.
These proteins are, however, differentially expressed during brain maturation and also show different subcellular
localization.
[1] Di Liegro et al. 2014, Int J Mol Med 33:747-62. ; [2] Scaturro et al. 1998, J Biol Chem 273:22788-91.; [3]
Nastasi et al. 1999, J Biol Chem 274:24087-93. [4] Sala et al. 2007, Int J Mol Med 19:501-9. [5] Saladino et al.
2012, Int J Mol Med 29:141-5 [6] Di Liegro et al. 2013, Neuroscience 229:71-6
Melanoma cells release extracellular vesicles which contain H1° RNA and RNA-binding proteins
No full textG26/24 oligodendroglioma cells produce EVs that contain pro-apoptotic proteins, such as FasL and TRAIL, able to induce neuronal- [1] and astrocytic- [2] death. Cancer cells release EVs [3] through which transferring proteins, such as extracellular matrix remodelling proteases [4], and H1°, a differentiation-specific histone [5]. By releasing H1°, cells could escape differentiation cues [5]. To verify the role of EVs in releasing specific proteins and mRNAs, in this study we used A375 melanoma cells.
EVs were purified from cell culture media as previously reported [1, 2]. T1 RNase-protection assays were performed on total cell lysates and EVs, as described elsewhere [6]. RNA-binding proteins (RBPs) were isolated by using a biotinylated H1° RNA as a bait [7]. Melanoma cells were found to synthesize H1° and secrete it via EVs. Moreover, EVs also contain H1° mRNA. Interestingly, H1° histone sorted to vesicles seems to be sumoylated. By T1 RNase-protection assay, we evidenced in EVs three main H1° RNA-protein complexes, the most abundant of which has a molecular mass of around 65 kDa. By using as a bait biotinylated H1° RNA, we isolated a few proteins, then analyzed by mass spectrometry. The most abundant protein was myelin expression factor-2 (MYEF2), which has a molecular mass of about 60 kDa. Finally, we confirmed MYEF2 presence in EVs by western blot.
[1] D’Agostino et al. 2006, Int J Oncol 29:1075-85.
[2] Lo Cicero A et al. 2011, Int J Oncol 39:1353-7
[3] Di Liegro et al. 2015, Biomed Res Int, 2015, Article ID 152926.
[4] Lo Cicero A et al. 2012, Matrix Biol 31:229-33
[5] Schiera G et al. 2013, Int J Oncol 43:1771-6
[6] Scaturro et al. 1998, J Biol Chem 273:22788-91
[7] Scaturro et al. 2003, Int J Mol Med 11:509-51
STUDIO DELL’ESPRESSIONE DI GENI CODIFICANTI PROTEINE MITOCONDRIALI IN RATTUS NORVEGICUS.
No full textMelanoma cells release extracellular vesicle which contain H1° linker histone as well as RNA-binding proteins which bind to the H1° mRNA
No full textWe previously demonstrated that G26/24 oligodendroglioma cells release EVs that contain proteins, such as FasL and TRAIL, which induce apoptosis in rat cortical neurons [1] and astrocytes [2]. We also reported that cancer cells use EVs for transferring, into the environment [3], proteins such as extracellular matrix remodelling proteases [4], and H1°, a differentiation-specific histone [5]. In particular, by releasing H1°, cells could escape differentiation cues [5]. To verify the role of EVs in releasing specific proteins and mRNAs, in this study we used as a model A375 melanoma cells.
METHODS
EVs were purified from cell culture media as previously reported [1, 2]. T1 RNase-protection assays were performed on total cell lysates and EVs, as described elsewhere [6]. RNA-binding proteins (RBPs) were isolated by using a biotinylated H1° RNA as a bait.
RESULTS
We found that also melanoma cells synthesize H1° and secrete it via EVs. Interestingly, H1° histone sorted to vesicles is probably sumoylated. By T1 RNase-protection assay, we evidenced in EVs three main H1° RNA-protein complexes, the most abundant of which has a molecular mass of around 65 kDa. By using as a bait biotinylated H1° RNA, we isolated a few proteins, then analyzed by mass spectrometry. The most abundant protein was myelin expression factor-2 (MYEF2), which has a molecular mass of about 60 kDa. Finally, we confirmed MYEF2 presence in EVs by western blot.
CONCLUSIONS
We demonstrated that EVs released from melanoma cells contain the H1° linker histone, which is probably sumoylated before sorting to the vesicles. In addition, EVs contain H1° RNA-binding proteins, one of which seems to be MYEF2.
[1] D’Agostino et al. 2006, Int J Oncol 29:1075-85.
[2] Lo Cicero A et al. 2011, Int J Oncol 39:1353-7
[3] Di Liegro et al. 2015, Biomed Res Int, in press
[4] Lo Cicero A et al. 2012, Matrix Biol 31:229-33
[5] Schiera G et al. 2013, Int J Oncol 43:1771-6
[6] Scaturro et al. 1998, J Biol Chem 273:22788-9
Metodo per la purificazione da sistemi di produzione batterici di proteine ricombinanti attive
No full textUn metodo per la produzione e purificazione da sistemi di produzione batterici di proteine ricombinanti attive con pre-sequenza di sei istidine
Molecular Determinants of Malignant Brain Cancers: From Intracellular Alterations to Invasion Mediated by Extracellular Vesicles
No full textMalignant glioma cells invade the surrounding brain parenchyma, by migrating along the blood vessels, thus promoting cancer growth. The biological bases of these activities are grounded in profound alterations of the metabolism and the structural organization of the cells, which consequently acquire the ability to modify the surrounding microenvironment, by altering the extracellular matrix and affecting the properties of the other cells present in the brain, such as normal glial-, endothelial- and immune-cells. Most of the effects on the surrounding environment are probably exerted through the release of a variety of extracellular vesicles (EVs), which contain many different classes of molecules, from genetic material to defined species of lipids and enzymes. EV-associated molecules can be either released into the extracellular matrix (ECM) and/or transferred to neighboring cells: as a consequence, both deep modifications of the recipient cell phenotype and digestion of ECM components are obtained, thus causing cancer propagation, as well as a general brain dysfunction. In this review, we first analyze the main intracellular and extracellular transformations required for glioma cell invasion into the brain parenchyma; then we discuss how these events may be attributed, at least in part, to EVs that, like the pawns of a dramatic chess game with cancer, open the way to the tumor cells themselves
Genetic and epigenetic modulation of cell functions by physical exercise
No full textSince ancient times, the importance of physical activity (PA) and of a wholesome diet for human health has been clearly recognized. However, only recently, it has been acknowledged that PA can reverse at least some of the unwanted effects of a sedentary lifestyle, contributing to the treatment of pathologies such as hypertension and diabetes, to the delay of aging and neurodegeneration, and even to the improvement of immunity and cognitive processes. At the same time, the cellular and molecular bases of these effects are beginning to be uncovered. The original research articles and reviews published in this Special Issue on “Genetic and Epigenetic Modulation of Cell Functions by Physical Exercise” focus on different aspects of the genetics and molecular biology of PA effects on health and, in addition, on the effects of different genotypes on the ability to perform PA. All authors have read and agreed to the published version of the manuscript
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