Abstract. We previously 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. Moreover, we recently found that neurons produce fibroblast growth factor-2 and vascular endothelial growth factor and secrete them at least in part by shedding extracellular vesicles. In this study, on the basis of immunofluorescence, scanner electron microscopy and Western blot analyses, we concluded that also astrocytes in culture shed extracellular vesicles that contain the same angiogenic factors, as well as ß1-integrin, a membrane protein that is considered a marker of shedding. Vesicles released by astrocytes are smaller than the ones produced by neurons and have an average size of 150-500 nm. IntroductionWe previously found that neurons and astrocytes cooperate in controlling occludin expression and permeability in brain capillary endothelial cells (BCECs), in a three-cell-type in vitro model of the blood-brain barrier (BBB) (1-3). Since in this culture system physical contacts among the different cell types are not allowed, it is likely that neurons and astrocytes affect endothelial cells by releasing soluble factors. In support of this hypothesis it should be noted that other authors have reported that, in vivo, astrocytes not only form direct contacts with BCECs through astrocyte feet but also release soluble factors, such as basic fibroblast growth factor (bFGF or FGF-2) and vascular endothelial growth factor (VEGF) (4), which find receptors on BCECs. FGF-2 is a protein that lacks a standard signal sequence and cannot be sorted to the endoplasmic reticulum (5). It has been reported to be secreted by tumor cells through an unusual way, which involves shedding of extracellular vesicles from the plasma membrane (5). FGF-2 is a potent inducer of blood vessel formation (angiogenesis), with a fundamental role in the development and differentiation of various tissues (6,7), including the nervous system (7). In addition to a main polypeptide of 18 kDa, other FGF-2 isoforms have been described, ranging in size from 22 to 34 kDa (7).VEGF, another factor with well-known actions on endothelial cells, has also been reported to be associated with extracellular vesicles released by tumor cells (5,8). VEGF exists as a homodimer of isoforms of different sizes (i.e. 121, 145, 165, 189, 206), derived from alternative splicing of the same primary transcript (9). In addition to these smaller forms, a larger isoform, with an N-terminal extension of 200 amino acids and an apparent mass of 45 kDa has been described in the human and the mouse (10,11).As it has been recently found that some glial tumor cells (oligodendroglioma cells) (12) as well as primary neurons in culture (13) are able to release extracellular vesicles, we investigated the possibility that astrocytes can do the same. We found that indeed astrocytes produce extracellular structures that contain FGF-2...

The evolutionarily conserved ATP-dependent nucleosome remodelling factor ISWI can space nucleosomes affecting a variety of nuclear processes. In Drosophila, loss of ISWI leads to global transcriptional defects and to dramatic alterations in higher-order chromatin structure, especially on the male X chromosome. In order to understand if chromatin condensation and gene expression defects, observed in ISWI mutants, are directly correlated with ISWI nucleosome spacing activity, we conducted a genome-wide survey of ISWI binding and nucleosome positioning in wild-type and ISWI mutant chromatin. Our analysis revealed that ISWI binds both genic and intergenic regions. Remarkably, we found that ISWI binds genes near their promoters causing specific alterations in nucleosome positioning at the level of the Transcription Start Site, providing an important insights in understanding ISWI role in higher eukaryote transcriptional regulation. Interestingly, differences in nucleosome spacing, between wild-type and ISWI mutant chromatin, tend to accumulate on the X chromosome for all ISWIbound genes analysed. Our study shows how in higher eukaryotes the activity of the evolutionarily conserved nucleosome remodelling factor ISWI regulates gene expression and chromosome organization genome-wide.

The complexity in composition and function of the eukaryotic nucleus is achieved through its organization in specialized nuclear compartments. The Drosophila chromatin remodeling ATPase ISWI plays evolutionarily conserved roles in chromatin organization. Interestingly, ISWI genetically interacts with the hsrω gene, encoding multiple non-coding RNAs (ncRNA) essential, among other functions, for the assembly and organization of the omega speckles. The nucleoplasmic omega speckles play important functions in RNA metabolism, in normal and stressed cells, by regulating availability of hnRNPs and some other RNA processing proteins. Chromatin remodelers, as well as nuclear speckles and their associated ncRNAs, are emerging as important components of gene regulatory networks, although their functional connections have remained poorly defined. Here we provide multiple lines of evidence showing that the hsrω ncRNA interacts in vivo and in vitro with ISWI, regulating its ATPase activity. Remarkably, we found that the organization of nucleoplasmic omega speckles depends on ISWI function. Our findings highlight a novel role for chromatin remodelers in organization of nucleoplasmic compartments, providing the first example of interaction between an ATP-dependent chromatin remodeler and a large ncRNA.