NEURONS AND ASTROCYTES SHED EXTRACELLULAR VESICLES THAT SEEM TO VEHICLE ANGIOGENIC FACTORS

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

A three-cell type in vitro-model of BBB

Il trasferimento di molecole tra cellule cerebrali è mediato anche da vescicole extracellulari

Correlation between polymorphism of TYMS gene and toxicity response to treatment with 5-fluoruracil and capecitabine

Tumorigenesis is a multiphasic process in which genetic alterations guide the progressive transformation in cancer cells1. In order to evaluate the possible correlation between some gene variants and the risk of the toxicity development onset, two of the polymorphisms of the thymidylate synthase (TYMS), rs34743033 (2R/3R) and rs16430 (DEL/INS) were investigated. We enrolled in our study 47 patients from the Hospital of Sicily. Our preliminary findings suggest that there could be a linkage between the genotypes discussed and the development of the toxicity following the chemotherapy treatment. These results need to be confirmed by further studies, however this short paper offers some initial…

A 3D‑scaffold of PLLA induces the morphological differentiation and migration of primary astrocytes and promotes the production of extracellular vesicles

The present study analyzed the ability of primary rat astrocytes to colonize a porous scaffold, mimicking the reticular structure of the brain parenchyma extracellular matrix, as well as their ability to grow, survive and differentiate on the scaffold. Scaffolds were prepared using poly-L-lactic acid (PLLA) via thermally-induced phase separation. Firstly, the present study studied the effects of scaffold morphology on the growth of astrocytes, evaluating their capability to colonize. Specifically, two different morphologies were tested, which were obtained by changing the polymer concentration in the starting solution. The structures were characterized by scanning electron microscopy, and a…

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.

Regulation of the expression of RNA-binding proteins in developing and adult rat brains: the role of thyroid hormones

EFFETTO DI TERRENI CONDIZIONATI DA NEURONI ED ASTROCITI SULLE PROPRIETÀ BARRIERA DI CELLULE DEI CAPILLARI CEREBRALI.

Neurons and astrocytes shed extracellular membrane vesicles containing angiogenic factors

Synergistic effects of neurons and astrocytes on the differentiation of brain capillary endothelial cells in culture

Brain capillary endothelial cells form a functional barrier between blood and brain, based on the existence of tight junctions that limit paracellular permeability. Occludin is one of the major transmembrane proteins of tight junctions and its peripheral localization gives indication of tight junction formation. We previously reported that RBE4.B cells (brain capillary endothelial cells), cultured on collagen IV, synthesize occludin and correctly localize it at the cell periphery only when cocultured with neurons. In the present study, we describe a three-cell type-culture system that allowed us to analyze the combined effects of neurons and astrocytes on differentiation of brain capillary …

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

RNA-binding proteins expressed in differentiating rat astrocytes

Replication-independent expression of H1˚ and H3.3 histone variants is probably regulated by different RNA-binding proteins

DNA in eukaryotes is wrapped around core histones to form nucleosomes, the basic units of chromatin. The linker histones H1 bind DNA where it enters and leaves the nucleosome, thus stabilizing higher order structures. Chromatin is a dynamic complex, modulated by different processes such as DNA-methylation, post-translational modifications of histones, and incorporation of specific histone variants. Throughout rat brain development, expression of H1° and H3.3 histone variants is mainly regulated at the post-transcriptional level. These proteins are of interest for their possible involvement in the replication-independent chromatin remodelling induced by extracellular stimuli. We previously c…

Thyroid hormones and the central nervous system of mammals (Review)

Abstract. The thyroid hormones (THs) L-thyroxine (T4) and L-triiodothyronine (T3) have a profound influence on the development and maturation of the mammalian brain, both before and after birth. Any impairment in the supply of THs to the developing nervous system leads to severe and irreversible changes in both the overall architecture and functions of the brain and causes, in humans, neurological and motor deficits known as cretinism. Pronounced neurological symptoms are also commonly observed in adult patients suffering from both hyperthyroidism and hypothyroidism, and it has recently emerged that certain symptoms might result from the reduced brain uptake, rather than the insufficient pr…

SHEDDING OF ANGIOGENIC FACTORS BY BRAIN CELLS

Aquaporin-4 distribution in control and stressed astrocytes in culture and in the cerebrospinal fluid of patients with traumatic brain injuries

Distribution of aquaporin-4 (AQP4) was studied by western analysis and immunofluorescence in rat astrocytes exposed to either hypothermic (30 °C) or hyperosmolar (0.45 M sucrose) stress, and in the cerebrospinal fluid (CSF) of patients who suffered traumatic brain injury (TBI). CSF was obtained from 5 healthy subjects and from 20 patients suffering from severe TBI. CSF samples were taken at admission and on days 3 and 5-7. Here we report that, in response to both hypothermia and hyperosmolar stress, AQP4 was markedly reduced in cultured astrocytes. We also found that AQP4 significantly increased in patients with severe brain injury in respect to healthy subjects (P < 0.002). AQP4 in CSF rem…

Physical Activity and Brain Health.

Physical activity (PA) has been central in the life of our species for most of its history, and thus shaped our physiology during evolution. However, only recently the health consequences of a sedentary lifestyle, and of highly energetic diets, are becoming clear. It has been also acknowledged that lifestyle and diet can induce epigenetic modifications which modify chromatin structure and gene expression, thus causing even heritable metabolic outcomes. Many studies have shown that PA can reverse at least some of the unwanted effects of sedentary lifestyle, and can also contribute in delaying brain aging and degenerative pathologies such as Alzheimer&#8217;s Disease, diabetes, and multiple s…

EFFECTS OF THYROID HORMONES ON TWO PUTATIVE RNA-BINDING PROTEINS EXPRESSED IN DEVELOPING RAT BRAIN.

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…

Toxic effects on astrocytes of extracellular vesicles from CSF of multiple sclerosis patients: a pilot in vitro study.

Multiple sclerosis (MS) is an autoimmune and degenerative disorder of the central nervous system (CNS) that causes a progressive loss of motor and cognitive perfor-mances. Moreover, since the earlier phases, axonal loss as well as neuronal degener-ation and a failure of oligodendrocytes to promote myelin repair have been demon-strated. In previous studies, it has been shown that the treatment of rat neuronal primary cultures with serum from MS patients can be toxic for neurons. Here we report a pilot investigation showing that CSF from patients contains extracellular vesicles (EVs) able to induce cell death in rat cultured astrocytes. Although these data are still preliminary, they suggest …

Extracellular Membrane Vesicles as Vehicles for Brain Cell-to-Cell Interactions in Physiological as well as Pathological Conditions.

Extracellular vesicles are involved in a great variety of physiological events occurring in the nervous system, such as cross talk among neurons and glial cells in synapse development and function, integrated neuronal plasticity, neuronal-glial metabolic exchanges, and synthesis and dynamic renewal of myelin. Many of these EV-mediated processes depend on the exchange of proteins, mRNAs, and noncoding RNAs, including miRNAs, which occurs among glial and neuronal cells. In addition, production and exchange of EVs can be modified under pathological conditions, such as brain cancer and neurodegeneration. Like other cancer cells, brain tumours can use EVs to secrete factors, which allow escaping…

Triiodothyronine-Induced Shortening of Chromatin Repeat Length in Neurons Cultured in a Chemically Denned Medium

Abstract: At the time of terminal differentiation, mammalian cortical neurons undergo a dramatic change in the structural organization of their chromatin: the nucleosomal repeat length shortens from ∼200 base pairs in fetuses to a value of 165 base pairs after birth. These events occur several days after the end of neuronal proliferation. Previously, we reported that rat cortical neurons cultured in a very selective synthetic medium were not yet programmed to these events at the end of mitotic cycles. Herein, we report that addition of triiodothyronine to neuronal cultures induces a shortening of the chromatin repeat length comparable to the natural one. Copyright © 1987, Wiley Blackwell. A…

Cellular mechanism of action of thyroid hormones.

Abstract It has emerged in the last decade that the molecular mechanism of action of thyroid hormones resembles that of steroids; thyroid hormones indeed exert their effects mainly by directly regulating gene expression, on association with specific chromatin-bound receptors. Of the two thyroid hormones, thyroxine (T4) appears to be a sort of prohormone, whereas triiodothyronine (T3) seems to be the active form; in this respect, T4-deiodination, which occurs at the level of the target tissues, may be crucial in the local homeostasis of T3. Moreover, many cellular compartments, other than the nucleus, can bind thyroid hormone, and at least some of these further sites might play some role in …

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…

Biological effects of inorganic arsenic on primary cultures of rat astrocytes

It is well established that inorganic arsenic induces neurotoxic effects and neurological defects in humans and laboratory animals. The cellular and molecular mechanisms of its actions, however, remain elusive. Herein we report the effects of arsenite (NaAsO2) on primary cultures of rat astrocytes. Cells underwent induction of heat shock protein 70 only at the highest doses of inorganic arsenic (30 and 60 microM), suggesting a high threshold to respond to stress. We also investigated arsenic genotoxicity with the comet assay. Interestingly, although cells treated with 10 microM arsenite for 24 h maintained >70% viability, with respect to untreated cells, high DNA damage was already observed…

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…

A low repeat length in oligodendrocyte chromatin

Abstract: The behavior of oligodendrocyte chromatin after micrococcal nuclease digestion of nuclei was assayed in brains of rats of four different ages. During oligodendrocyte differentiation, a decreasing sensitivity of the chromatin to enzymatic attack was observed. On the other hand, the nucleosomal repeat length showed a slight tendency to increase during development. It is worth noting that even the highest values reported here for “oligodendrocyte’ chromatin repeat lengths are significantly lower than 200 base pairs, the value previously reported by others for “non‐astrocytic glia.” Copyright © 1985, Wiley Blackwell. All rights reserved

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

RNA-binding ability of PIPP in requires the entire protein

Post-transcriptional fate of eukaryotic mRNAs depends on association with different classes of RNA-binding proteins (RBPs). Among these proteins, the cold-shock domain (CSD)-containing proteins, also called Y-box proteins, play a key role in controlling the recruitment of mRNA to the translational machinery, in response to environmental cues, both in development and in differentiated cells. We recently cloned a rat cDNA encoding a new CSD-protein that we called PIPPin. This protein also contains two putative double-stranded RNA-binding motifs (PIP(1) and PIP(2)) flanking the central CSD, and is able to bind mRNAs encoding H1 degrees and H3.3 histone variants. In order to clarify the role of…

RNA-Binding Proteins which interact with mRNAs for H1° and H3.3 histone variants

Oligodendroglioma cells synthesize the differentiation-specific linker histone H1˚ and release it into the extracellular environment through shed vesicles

Chromatin remodelling can be involved in some of the epigenetic modifications found in tumor cells. One of the mechanisms at the basis of chromatin dynamics is likely to be synthesis and incorporation of replacement histone variants, such as the H1° linker histone. Regulation of the expression of this protein can thus be critical in tumorigenesis. In developing brain, H1° expression is mainly regulated at the post-transcriptional level and RNA-binding proteins (RBPs) are involved. In the past, attention mainly focused on the whole brain or isolated neurons and little information is available on H1° expression in other brain cells. Even less is known relating to tumor glial cells. In this st…

H1.0 Linker Histone as an Epigenetic Regulator of Cell Proliferation and Differentiation

H1 linker histones are a class of DNA-binding proteins involved in the formation of supra-nucleosomal chromatin higher order structures. Eleven non-allelic subtypes of H1 are known in mammals, seven of which are expressed in somatic cells, while four are germ cell-specific. Besides having a general structural role, H1 histones also have additional epigenetic functions related to DNA replication and repair, genome stability, and gene-specific expression regulation. Synthesis of the H1 subtypes is differentially regulated both in development and adult cells, thus suggesting that each protein has a more or less specific function. The somatic variant H1.0 is a linker histone that was recognized…

RNA binding proteins in brain cells differentiation

Extracellular Vesicles Shed by Melanoma Cells Contain a Modified Form of H1.0 Linker Histone and H1.0 mRNA-binding Proteins

Extracellular vesicles (EVs) are shed in the extracellular environment by both prokaryotes and eukaryotes. Although produced from both normal and cancer cells, malignant cells release a much higher amount of EVs, which also contain tumor-specific proteins and RNAs. We previously found that G26/24 oligodendroglioma cells shed EVs that contain the pro-apoptotic factors FasL and TRAIL1-2. Interestingly, G26/24 release, via EVs, extracellular matrix remodelling proteases3, and H1° histone protein4, and mRNA. To shed further light on the role of EVs in discarding proteins and mRNAs otherwise able to counteract proliferative signals, we studied a melanoma cell line (A375). We found that also thes…

Cell-to-cell communications among brain cells by extracellular vesicles

Rat PIPPin is probably part of a large complex of RNA-binding proteins

Throughout rat brain development, expression of histones variants is mainly regulated at the post-transcriptional level. We previously cloned two cDNAs encoding, respectively, PIPPin (or CSD-C2), a brain-enriched protein able to bind the 3’end of H1° and H3.3 mRNAs, and LPI (longer isoform of PEP-19). Both PEP-19 and LPI are brain-specific. By western blot, we found that PIPPin expression in PC12 cells is enhanced by NGF-induced differentiation. We investigated the RNA-binding properties of the three proteins using their 6 histidine-tagged recombinant fusions and found that they all bind H1° and H3.3 RNAs. Since PEP-19 and LPI are camstatins, we also analyzed whether calmodulin could interf…

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

Social closeness,salivary hormones and physical exercise

Introduction: Saliva collection and analysis is quickly becoming a useful and non-invasive tool for the evaluation of sport biomarkers. The aim of this study is to create a multidisciplinary assessment model, which can help to provide psychological and physiological responses, related to sport performances, social closeness and performance anxiety management in team sports. Materials and methods: We enrolled in our research 26 female volleyball players aged 13 ± 1 years old of three different teams (T1: 12 players; T2: 9 players; T3: 5 players). Saliva collection was carried out before and after the match for every team. Then we analyzed cortisol and progesterone concentrations through Elis…

Effects of thyroid hormones on RNA-binding proteins involved in the regulation of H1° and H3.3 histone variant expression

RNAbinding proteins involved in nerve cell differentiation.

EXTRACELLULAR FACTORS INVOLVED IN THE INDUCTION OF BLLOD-BRAIN BARRIER

Genetic and Epigenetic Modulation of Cell Functions by Physical Exercise

Since 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 Ex…

EFFECTS OF THYROID HORMONES ON RNA-BINDING PROTEINS EXPRESSED IN DEVELOPING RAT BRAIN

Proteine di fusione, metodo di preparazione, e relativi anticorpi per la diagnosi di patologie mitocondriali

SHEDDING OF EXTRACELLULAR VESICLES FROM BRAIN CELLS

INTEGRATIVE APPROACH BETWEEN GENE VARIANTS, NEUROENDOCRINE EFFECTORS AND ANXIETY RESPONSE: A PILOT STUDY

The aim of this study is to investigate the neurobiology of stress/emotionality, creating a multidisciplinary assessment model, which can help to provide psychological and physiological responses depending on the genetic background related to sport performances, social closeness and performance anxiety management in team sports. We enrolled 20 female volleyball players aged 13 ± 1 years old played in two different teams during a regional championship final. Saliva collection was carried out before and after the match. In order to evaluate the neuroendocrine effectors involved in stress and performance, we analyzed cortisol and progesterone levels through Elisa standard kit as well as HSP70 …

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

Identification of nuclear proteins which interact with H1° mRNA.

In developing rat brain the synthesis of H1° histone is mainly regulated at posttranscriptional level and probably depends on RNA-binding proteins (RBPs) (1). We previously identified RBPs apparently specific for this messenger (2) and cloned two novel proteins by screening an expression cDNA library by binding to radiolabeled RNA (3-10). Here we report the use of biotinylated H1° RNA as bait to isolate by chromatography nuclear proteins which interact with H1° mRNA. We identified by mass spectrometry abundant RBPs and molecular chaperones. By western blot we also evidenced, among the RNA-bound proteins, the cold shock domain-containing protein 2 (CSD-C2, also know as PIPPin), a brain-enric…

Effects of thyroid hormones on sumoylation of the RNA-binding protein PIPPin

STUDIO DELL’ESPRESSIONE DI GENI CODIFICANTI PROTEINE MITOCONDRIALI IN RATTUS NORVEGICUS.

Extracellular membrane vesicle shedding and the blood-brain barrier

Extracellular Vesicle‐Associated RNA as a Carrier of Epigenetic Information

Post-transcriptional regulation of messenger RNA (mRNA) metabolism and subcellular localization is of the utmost importance both during development and in cell differentiation. Besides carrying genetic information, mRNAs contain cis-acting signals (zip codes), usually present in their 5'- and 3'-untranslated regions (UTRs). By binding to these signals, trans-acting factors, such as RNA-binding proteins (RBPs), and/or non-coding RNAs (ncRNAs), control mRNA localization, translation and stability. RBPs can also form complexes with non-coding RNAs of different sizes. The release of extracellular vesicles (EVs) is a conserved process that allows both normal and cancer cells to horizontally tran…

Vesicles shed by G26/24 oligodendroglioma cells, added to fetal murin cortical neurons, inhibit neurite sprouting and induce neuronal death

Dietary Fatty Acids in Metabolic Syndrome, Diabetes and Cardiovascular Diseases

In the last few decades, the prevalence of overweight and essential obesity has been undergoing a fast and progressive worldwide increase. Obesity has been in turn linked to type II diabetes, with the total number of diabetic patients worryingly increasing, in the last fifteen years, suggesting a pandemic phenomenon. At the same time, an increase in the prevalence of cardiovascular diseases has been also recorded. Increasing evidence suggests that the diet is involved in such escalation. In particular, the progressive globalization of food industry allowed massive supply, at a relatively low price, of a great variety of pre-packed food and bakery products, with very high energy content. Mos…

The effect of cadmium on brain cells in culture

Cadmium is a long-living heavy metal, abundantly present in the environment, which accumulates in the body. In this study, we investigated the effects of cadmium on the expression of molecular chaperones, and of certain cell-specific proteins, in a variety of brain cell types in culture, namely primary cultures of rat cortical neurons and astrocytes, a brain capillary endothelial cell line (RB4E.B cells), and pheochromocytoma cells (PC12), induced or not to differentiate by NGF treatment. The metal induces a dose-dependent increase of Hsp70 in all cell types. Responses to the metal are cell-specific in the case of Hsc70 and Hsp90: i) in astrocytes, as well as in PC12 cells, cadmium has no s…

EFFETTO DEGLI ORMONI TIROIDEI SULL’ESPRESSIONE DELLE PROTEINE PIPPIN ED LPI NEL CERVELLO DI RATTO IN SVILUPPO.

DEVELOPMENT OF A HOME-BASED TRAINING PROGRAM FOR PATIENTS WITH PARKINSON DISEASE: NEUROBIOLOGICAL AND MOTOR SKILLS EFFECT

Neurodegenerative diseases are inherited diseases of the central nervous system, which cause progressive damage to specific populations of neurons and lead to a deterioration in the quality of life (1,2). Parkinson disease (PD) is a progressive neurodegenerative disease and is the second most common after AD, and is characterized by postural instability, tremor and rigidity. Moreover, physical activity can reduces risk of other geriatric diseases such as diabetes, hypertension, osteoporosis and cardiovascular disease, which may also contribute to PD pathogenesis (3). We enrolled 12 subjects (age: 62.74 ± 4.94; height: 175,5cm ± 7,41 cm; weight: 75,5 ± 17,95 kg) affected by PD. An home-based…

Analysis of interaction between messenger RNA encoding H3.3 histone variant and pippin protein by biolayer interferometry

Genomic and non-genomic mechanisms of action of thyroid hormones and their catabolite 3,5-diiodo-l-thyronine in Mammals

Since the realization that the cellular homologs of a gene found in the retrovirus that contributes to erythroblastosis in birds (v-erbA), i.e. the proto-oncogene c-erbA encodes the nuclear receptors for thyroid hormones (THs), most of the interest for THs focalized on their ability to control gene transcription. It was found, indeed, that, by regulating gene expression in many tissues, these hormones could mediate critical events both in development and in adult organisms. Among their effects, much attention was given to their ability to increase energy expenditure, and they were early proposed as anti-obesity drugs. However, their clinical use has been strongly challenged by the concomita…

Neuronal and BBB damage induced by sera from patients with secondary progressive multiple sclerosis.

An important component of the pathogenic process of multiple sclerosis (MS) is the blood-brain barrier (BBB) damage. We recently set an in vitro model of BBB, based on a three-cell-type co-culture system, in which rat neurons and astrocytes synergistically induce brain capillary endothelial cells to form a monolayer with permeability properties resembling those of the physiological BBB. Herein we report that the serum from patients with secondary progressive multiple sclerosis (SPMS) has a damaging effect on isolated neurons. This finding suggests that neuronal damaging in MS could be a primary event and not only secondary to myelin damage, as generally assumed. SPMS serum affects the perme…

In vitro models of blood-brain barrier set by co-culturing primary pericytes, astrocytes and brain capillary endothelial cells

The blood-brain barrier (BBB) is formed by brain capillary endothelial cells (BCECs) under the effects of the brain microenvironment. BCECs are sealed together by tight junctions (TJs) [2]. We previously found that occludin, one of the main TJ components, is correctly localized at the cell periphery only if BCECs are co-cultured with neurons/astrocytes [1-2]. Moreover, pericytes induce PLA2 protein expression through activation of PKCalpha and the MAPK/ERK cascade in immortalized GP8.3 endothelial cells [3]. In the present work, we analyzed the effects of pericytes and/or astrocytes on survival and differentiation of primary endothelial cells, in the presence of a serum-free medium. To test…

Two immortalized rat astrocyte cell lines as in vitro model for specific cell proliferation studies: cytogenetic and epigenomic characterization and diversification

Here we report differences between: 1) a heterogeneous population of primary rat brain astrocytes (Primary), in culture since several years ago, and 2) a cloned cell line (Clone), obtained from the Primary cells. Both populations maintain astrocyte morphology but, according to cytogenetic and epigenomic characterization, differ for the chromosomal asset from rat normal cells (42 chromosomes): Primary cells show mostly a bimodal karyotype with 41 or 43 chromosomes, and Clone has a unique-modal karyotype of 43 chromosomes. Interestingly, we also found that both cell lines show genome-wide DNA hypomethylation, with Clone showing even more pronounced demethylation respect to Primary cells. Thes…

Isolation and characterization of a Paracentrotus lividus cDNA encoding a stress-inducible chaperonin

Chaperonins are ubiquitous proteins that facilitate protein folding in an adenosine triphosphate–dependent manner. Here we report the isolation of a sea urchin cDNA (Plhsp60) coding for mitochondrial chaperonin (Cpn60), whose basal expression is further enhanced by heat shock. The described cDNA corresponds to a full-length mRNA encoding a protein of 582 amino acids, the first 32 of which constitute a putative mitochondrial targeting leader sequence. Comparative analysis has demonstrated that this protein is highly conserved in evolution.

Tipi diversi di cellule cerebrali rilasciano fattori di crescita attraverso vescicole extracellulari.

Histones and non-histone proteins during sea urchin development

Abstract Chromatin from blastulae and plutei of Paracentrotus lividus was fractionated through a hydroxyapatite column. The ratio of histones versus non-histone proteins decreases from blastula to pluteus stage.

Histone H1° and H3.3 RNA-binding proteins identified in the developing rat brain

Histone H1° RNA-binding proteins in developing rat brain.

Lactate Threshold Training Program on Patients with Multiple Sclerosis: A Multidisciplinary Approach

Physical activity could play a key role in improving the quality of life, particularly in patients with nervous system diseases such as multiple sclerosis (MS). Through lactacid anaerobic training, this study aims to investigate the effects at a bio-psycho-physical level to counteract the chronic fatigue associated with the pathology, and to improve mental health at a psychological and neurotrophic level. Eight subjects (age: 34.88 ± 4.45 years) affected by multiple sclerosis were involved. A lactate threshold training program was administered biweekly for 12 weeks at the beginning of the study (T0), at the end of the study (T1) and at 9 months after the end of the study (T2), with physical…

ANALISI DELLA PERMEABILITÀ DI UNO STRATO DI CELLULE ENDOTELIALI DEI CAPILLARI CEREBRALI, CO-COLTIVATE CON NEURONI ED ASTROCITI.

ASTROCYTES SHED EXTRACELLULAR VESICLES THAT CONTAIN FIBROBLAST GROWTH FACTOR-2 AND VASCULAR ENDOTHELIAL GROWTH FACTOR.

An important component of the pathogenic process of multiple sclerosis (MS) is the blood-brain barrier (BBB) damage. We recently set an in vitro model of BBB, based on a three-cell-type co-culture system, in which rat neurons and astrocytes synergistically induce brain capillary endothelial cells to form a monolayer with permeability properties resembling those of the physiological BBB. Herein we report that the serum from patients with secondary progressive multiple sclerosis (SPMS) has a damaging effect on isolated neurons. This finding suggests that neuronal damaging in MS could be a primary event and not only secondary to myelin damage, as generally assumed. SPMS serum affects the perme…

DOPAMINE PRO-DRUGS FOR CNS TARGETING: SYNTHESIS, CHARACTERIZATION AND HYDROLYSIS BY BRAIN ENZYMES.

CLONING OF RAT-SPECIFIC LONG PCP4/PEP19 ISOFORM (LPI)

We report the identification of a cDNA that encodes a putative protein of 94 amino acids and expected molecular weight of 10.7 kDa, the C-terminal half of which is identical to that of PEP19, a small, brain-specific protein involved in Ca++/calmodulin signaling. The novel rat-specific protein, tentatively named long PEP19 isoform (LPI), is the product of alternative splicing of the rat PCP4 gene encoding PEP19. We found that antibodies raised against the first 13 N-terminal amino acids of LPI, not present in PEP19, recognize a protein enriched in the developing rat brain.

Vescicles shed by G26/24 oligodendroglioma cells, added to fetal murin cortical neurons, inhibit neurite sprouting and induce neural death.

RNA-Binding Proteins as Epigenetic Regulators of Brain Functions and Their Involvement in Neurodegeneration.

A central aspect of nervous system development and function is the post-transcriptional regulation of mRNA fate, which implies time- and site-dependent translation, in response to cues originating from cell-to-cell crosstalk. Such events are fundamental for the establishment of brain cell asymmetry, as well as of long-lasting modifications of synapses (long-term potentiation: LTP), responsible for learning, memory, and higher cognitive functions. Post-transcriptional regulation is in turn dependent on RNA-binding proteins that, by recognizing and binding brief RNA sequences, base modifications, or secondary/tertiary structures, are able to control maturation, localization, stability, and tr…

Analysis of cytochrome c oxidase subunits III and IV in developing rat brain

Analysis of cytochrome oxidase subunits III and IV expression in developing rat brain.

NOVEL ROUTES OF CELL TO CELL COMUNICATIONS AMONG BRAIN CELLS

Valutazione della didattica nel corso di laurea "Tecniche della prevenzione nell'ambiente e nei luoghi di lavoro"

Brain-specific RNA-binding protein

Extracellular vesicles as vehicles of angiogenic factors in brain cell cultures

The interaction of Fe(III), adriamycin and daunomycin with nucleotides and DNA and their effects on cell growth of fibroblasts (NIH-3T3)

The interactions of the iron complexes of the anthracycline antitumour drugs daunomycin (DN) and adriamycin (ADM) with the mononucleotide AMP, herring sperm DNA, plasmic pBR322 and immortalized 3T3 fibroblasts were studied. By means of Mössbauer spectroscopy it was demonstrated that DNA is a powerful ferric iron chelator as compared with AMP, which is not able to compete with DN or acetohydroxamic acid for ferric iron. The difference between AMP and DNA is postulated to be based on the chelate effect. The Mössbauer spectra of the ternary Fe-anthracycline-DNA systems differ from Fe-anthracycline binary complexes, indicating rearrangement reactions. Dialysis experiments clearly disclose the f…

3D cultures of primary astrocytes on Poly-L-lactic acid scaffolds

Tissue engineering is an emerging multidisciplinary field that aims at reproducing in vitro tissues with morphological and functional features similar to the biological tissue of the human body. Polymeric materials can be used in contact with biological systems in replacing destroyed tissue by transplantation [1]. Several biopolymers, including poly L (lactic acid) (PLLA), have been used in biomedical applications to set scaffolds with ductile proprieties and biodegradation kinetics [2]. In particular, the PLLA scaffold topography mimics the natural extracellular matrix and makes it a good candidate for neural tissue engineering. We report about of 3D system the PLLA porous scaffolds prepar…

Co-culture of rat brain cells as a tool for studying cell-cell interactions

Brain capillary endothelial cells (BCECs) form the blood-brain barrier (BBB) in response to interaction with other brain cells (astrocytes, pericytes and neurons). BCECs are characterized by tight junctions (TJ), maturation and stabilization of which require different proteins, such as occludin. When co-cultured with astrocytes and neurons, BCECs were found to form a monolayer resembling the natural BBB: paracellular flux of dopamine and sucrose (i.e. compounds which are unable to cross the BBB in vivo) significantly decreased (1), while the transendothelial electrical resistance (TEER) increased. In these conditions, BCECs produced a larger amount of occludin and tended to localize it at t…

Neuronal factors secreted through unusual pathways are probably involved in blood brain barrier (BBB) formation

Cloning of a rat-specific long PCP4/PEP19 isoform

We report the identification of a cDNA that encodes a putative protein of 94 amino acids and expected molecular weight of 10.7 kDa, the C-terminal half of which is identical to that of PEP19, a small, brain-specific protein involved in Ca++/calmodulin signaling. The novel rat-specific protein, tentatively named long PEP19 isoform (LPI), is the product of alternative splicing of the rat PCP4 gene encoding PEP19. We found that antibodies raised against the first 13 N-terminal amino acids of LPI, not present in PEP19, recognize a protein enriched in the developing rat brain.

An In Vitro Model of Glioma Development

Gliomas are the prevalent forms of brain cancer and derive from glial cells. Among them, astrocytomas are the most frequent. Astrocytes are fundamental for most brain functions, as they contribute to neuronal metabolism and neurotransmission. When they acquire cancer properties, their functions are altered, and, in addition, they start invading the brain parenchyma. Thus, a better knowledge of transformed astrocyte molecular properties is essential. With this aim, we previously developed rat astrocyte clones with increasing cancer properties. In this study, we used proteomic analysis to compare the most transformed clone (A-FC6) with normal primary astrocytes. We found that 154 proteins are…

NEURONAL FACTOR SECRETED THROUGH UNUSUAL PATHWAYS ARE PROBABLY INVOLVED IN BLOOD BRAIN BARRIER (BBB) FORMATION.

Effects of thyroid hormones on RNA-binding proteins involved the regulation on H1° and H3,3 histone variant expression.

Neurobiology of performance anxiety:A new approach

The aim of this study is to investigate the neurobiology of stress/emotionality, creating a multidisciplinary assessment model, which can help to provide psychological and physiological responses depending on the genetic background related to sport performances, social closeness and performance anxiety management in team sports. We enrolled 20 female volleyball players aged 13 ± 1 years old played in two different teams during a regional championship final. Saliva collection was carried out before and after the match. In order to evaluate the neuroendocrine effectors involved in stress and performance, we analyzed cortisol and progesterone levels through Elisa standard kit as well as HSP70 …

Extracellular membrane vesicles can mediate intercellular transfer of molecules

Many eukaryotic cell types, including neural cells, release into the extracellular environment vesicles of different sizes and composition. Neurons and astrocytes shed extracellular vesicles which contain FGF2 and VEGF and could be involved in interaction with endothelial cells, to form the blood-brain barrier. Also brain tumor cells, such as glioblastomas, release vesicles in the extracellular space. Microvesicles (MVs) shed from G26/24 oligodendro¬glioma cells were previously reported to contain FAS-L and to cause a reproducible, dose-dependent, inhibitory effect on neurite outgrowth, and neuronal apoptosis, when added to primary cultures of rat cortical neurons. More recently, they were …

Migration of brain capillary endothelial cells inside poly (lactic acid) 3D scaffolds

The brain capillary endothelial cells (BCECs) form the blood brain barrier (BBB) under the effects of the brain microenvironment. BCECs are sealed together by tight junctions (TJs) that are responsible for the barrier phenotype. In these junctions, molecules such as JAM (junctional adhesion molecules), occludin and claudins are present. Threedimensional scaffolds are used to grow cells in order to obtain in vitro engineered tissues. On the base of these considerations, the aim of this work was to understand whether the endothelial cells were able to grow and survive on a new three-dimensional structure. If yes, indeed, this system could be further enriched and used to set a three-dimensiona…

Novel routes of cell to cell communications among brain cells

EXTRACELLULAR FACTORS INVOLVED IN THE INDUCTION OF BLOOD BRAIN BARRIER (BBB)

Effects of thyroid hormones on the expression of two rat brain proteins: PIPPin and the long PEP-19 isoform (LPI)

Cell-to-Cell Communication in Learning and Memory: From Neuro- and Glio-Transmission to Information Exchange Mediated by Extracellular Vesicles

Most aspects of nervous system development and function rely on the continuous crosstalk between neurons and the variegated universe of non-neuronal cells surrounding them. The most extraordinary property of this cellular community is its ability to undergo adaptive modifications in response to environmental cues originating from inside or outside the body. Such ability, known as neuronal plasticity, allows long-lasting modifications of the strength, composition and efficacy of the connections between neurons, which constitutes the biochemical base for learning and memory. Nerve cells communicate with each other through both wiring (synaptic) and volume transmission of signals. It is by now…

Extracellulare membrane vesicles as vehicles for cell-to-cell and cell-to-extracellular matrix functional interactions

Cloning and analysis of cDNA for rat histone H1°

Extracellular microvesicles as brain cell-to-cell communication carriers

POST-TRANSCRIPTIONAL REGULATION BY THYROID HORMONES OF SOME BRAIN-SPECIFIC PROTEINS PROBABLY INVOLVED IN RNA METABOLISM.

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.

Molecular Determinants of Malignant Brain Cancers: From Intracellular Alterations to Invasion Mediated by Extracellular Vesicles

Malignant 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 mol…

CHARACTERIZATION OF TRANSFORMED CELL LINES OBTAINED FROM PRIMARY RAT CORTICAL ASTROCYTES

Brain cancers are complex and heterogeneous; most of them derive from glial cells[1], and are called gliomas, further subdivided into astrocytomas, oligodendrogliomas, ependymomas and glioastrocytomas[2]. The malignant cells undergo modifications of their metabolism and behaviour, and acquire the ability to migrate along the blood vessels in small groups (model of the guerrilla war)[3], thus invading the surrounding brain parenchyma. Most important, they have the capacity to affect the surrounding microenvironment, by altering both the extracellular matrix and the properties of the normal cells present in the brain, including glial-, endothelial-, and immune-cells, further promoting cancer …

ROLE OF THYROID HORMONES IN DEVELOPING RAT BRAIN AND IN CULTURED NEURONS

Novel ways of cell-to-cell communications among brain cells

Thyroid hormones induce sumoylation of the cold shock domain-containing protein PIPPin in developing rat brain and in cultured neurons.

We previously identified a cold shock domain (CSD)-containing protein (PIPPin), expressed at high level in brain cells. PIPPin has the potential to undergo different post-translational modifications and might be a good candidate to regulate the synthesis of specific proteins in response to extracellular stimuli. Here we report the effects of thyroid hormone (T3) on PIPPin expression in developing rat brain. We found that a significant difference among euthyroid- and hypothyroid- newborn rats concerns sumoylation of nuclear PIPPin, that is abolished by hypothyroidism. Moreover, T3-dependence of PIPPin sumoylation has been confirmed in cortical neurons purified from brain cortices and culture…

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

Purification by affinity chromatography of H1 RNA-Binding Proteins from rat brain

Post-transcriptional regulation of mRNA metabolism is involved in processes as different as cell fate specification in development and cell response to a large variety of environmental cues. Regulation of all steps of RNA metabolism depends on RNA-binding proteins (RBPs). By using a T1 RNase protection assay, we previously identified three H1° RNA-binding factors (p40, p70 and p110), highly expressed in the rat brain. Here we report enrichment of these factors from brain extracts, obtained by affinity chromatography of biotinylated H1° RNA-protein complexes on streptavidin-conjugated paramagnetic particles. The purified proteins maintain RNA-binding ability and preference for histone messag…

The dynamic properties of neuronal chromatin are modulated by triiodothyronine.

The effect of triiodothyronine (T3) on the rate of synthesis of nuclear proteins was studied during terminal differentiation of rat cortical neurons cultured in a serum-free medium. To this aim total and acid soluble nuclear proteins were analyzed by different electrophoretic techniques. Our results show that: 1) during maturation in vitro, neuronal nuclei undergo a dramatic change in the rate at which different classes of histones and high mobility group (HMG) proteins are synthesized; the synthetic activity, measured as incorporation of radioactive precursors into nuclear proteins, slows indeed down with age: especially evident is the decrease in core histones synthesis; at day 15, on the…

AN IN VITRO MODEL OF BLOOD BRAIN BARRIER (BBB).

Aquaporins and Brain Tumors

Brain primary tumors are among the most diverse and complex human cancers, and they are normally classified on the basis of the cell-type and/or the grade of malignancy (the most malignant being glioblastoma multiforme (GBM), grade IV). Glioma cells are able to migrate throughout the brain and to stimulate angiogenesis, by inducing brain capillary endothelial cell proliferation. This in turn causes loss of tight junctions and fragility of the blood–brain barrier, which becomes leaky. As a consequence, the most serious clinical complication of glioblastoma is the vasogenic brain edema. Both glioma cell migration and edema have been correlated with modification of the expression/localization …

Lactate as a Metabolite and a Regulator in the Central Nervous System

More than two hundred years after its discovery, lactate still remains an intriguing molecule. Considered for a long time as a waste product of metabolism and the culprit behind muscular fatigue, it was then recognized as an important fuel for many cells. In particular, in the nervous system, it has been proposed that lactate, released by astrocytes in response to neuronal activation, is taken up by neurons, oxidized to pyruvate and used for synthesizing acetyl-CoA to be used for the tricarboxylic acid cycle. More recently, in addition to this metabolic role, the discovery of a specific receptor prompted a reconsideration of its role, and lactate is now seen as a sort of hormone, even invol…

Histone RNA-binding proteins in the rat brain

Microscopic evidence of the primary astrocytes' morphological differentiation and migration inside porous Poly-L-lactic acid 3D‑scaffolds

Tissue engineering is an emerging multidisciplinary field that aims at reproducing in vitro and/or in vivo tissues with morphological and functional features similar to the biological tissue of the human body [1]. In the attempt to construct suitable tissue models, a critical step is the setting of 3D scaffolds that mimic the supportive structures of a natural extracellular matrix microenvironment into which cells are normally embedded. In this context, the generation of 3D cultures of brain cells is of particular interest. For instance, the poly L‐lactic acid (PLLA) polymer is wildly used because of its biocompatible and biodegradable potential; the PLLA scaffold topography simulates the n…

Involvement of Thyroid Hormones in Brain Development and Cancer

Simple Summary Development and function of the mammalian brain clearly require precise regulation of gene expression at both the transcriptional and post-transcriptional level. Thyroid hormones have been recognized to play a fundamental role in these processes, by acting at multiple levels and in different brain cell types, through direct effects on transcription, mediated by nuclear receptors, and also by triggering transduction pathways at the plasma membrane. At the same time, due to their effects on proliferation, differentiation, and cell metabolism, thyroid hormones may have a critical role in different kinds of cancer, including brain cancer. Abstract The development and maturation o…

Identificazione di proteine che interagiscono con il messaggero dell’istone H1° nel cervello di ratto in sviluppo

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…

NEURONS PRODUCE FGF-2 AND VEGF SECRETE THEM AT LEST IN PART BY SHEDDING EXTRACELLULAR VESCICLES

Abstract We previously found that neurons are able to affect the ability of brain capillary endothelial cells to form in vitro a monolayer with properties resembling the blood-brain barrier. We then looked, by immunofluorescence and western analysis, for factors, produced by neurons, with the potential to influence growth and differentiation of endothelial cells. In the present paper, we report that neurons produce both vascular endothelial growth factor and fibroblast growth factor 2, two well-known angiogenic factors. More interestingly, we gained evidence that both factors are released by neurons, at least in part, by shedding of extracellular vesicles, that contain β1 integrin, a membra…

Regulation of mRNA transport, localization and translation in the nervous system of mammals (Review).

Post-transcriptional control of mRNA trafficking and metabolism plays a critical role in the actualization and fine tuning of the genetic program of cells, both in development and in differentiated tissues. Cis-acting signals, responsible for post-transcriptional regulation, reside in the RNA message itself, usually in untranslated regions, 5' or 3' to the coding sequence, and are recognized by trans-acting factors: RNA-binding proteins (RBPs) and/or non-coding RNAs (ncRNAs). ncRNAs bind short mRNA sequences usually present in the 3'-untranslated (3'-UTR) region of their target messages. RBPs recognize specific nucleotide sequences and/or secondary/tertiary structures. Most RBPs assemble on…

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…

PIPPIN SUMOYLATION IS CONTROLLED BY THYROID HORMONES

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…

Membrane vesicles shed by oligodendroglioma cells induce neuronal apoptosis.

In order to investigate the mechanism by which oligodendrogliomas cause neuronal damage, media conditioned by G26/24 oligodendroglioma cells, were fractionated into shed vesicles and vesicle-free supernatants, and added to primary cultures of rat fetal cortical neurons. After one night treatment with vesicles, a reproducible, dose-dependent, inhibitory effect on neurite outgrowth was already induced and, after 48-72 h of incubation, neuronal apoptosis was evident. Vesicle-free supernatants and vesicles shed by NIH-3T3 cells had no inhibitory effects on neurons. Western blot analyses showed that treated neurons expressed a decreased amount of neurofilament (NF), growth-associated protein (GA…

Functional feature of a novel model of blood brain barrier: Studies on permeation of test compounds

Drug delivery to the central nervous system (CNS) is subject to the permeability limitations imposed by the blood-brain barrier (BBB). Several systems in vitro have been described to reproduce the physical and biochemical behavior of intact BBB, most of which lack the feature of the in vivo barrier. We developed a fully formed monolayer of RBE4.B immortalized rat brain microvessel endothelial cells (ECs), grown on top of polycarbonate filter inserts with cortical neuronal cells grown on the outside. Neurons induce ECs to synthesize and sort occludin to the cell periphery. Occludin localization is regulated by both compositions of the substratum and soluble signals released by cortical co-cu…

Establishment and Preliminary Characterization of Three Astrocytic Cells Lines Obtained from Primary Rat Astrocytes by Sub-Cloning.

Gliomas are complex and heterogeneous tumors that originate from the glial cells of the brain. The malignant cells undergo deep modifications of their metabolism, and acquire the capacity to invade the brain parenchyma and to induce epigenetic modifications in the other brain cell types. In spite of the efforts made to define the pathology at the molecular level, and to set novel approaches to reach the infiltrating cells, gliomas are still fatal. In order to gain a better knowledge of the cellular events that accompany astrocyte transformation, we developed three increasingly transformed astrocyte cell lines, starting from primary rat cortical astrocytes, and analyzed them at the cytogenet…

Role of RNA-binding proteins in the replication-independent expression of H1° and H3.3 histone variants

Permeability properties of a three-cell type in vitro model of blood-brain barrier.

We previously found that RBE4.B brain capillary endothelial cells (BCECs) form a layer with blood‐brain barrier (BBB) properties if co‐cultured with neurons for at least one week. As astrocytes are known to modulate BBB functions, we further set a culture system that included RBE4.B BCECs, neurons and astrocytes. In order to test formation of BBB, we measured the amount of (3)H‐sucrose able to cross the BCEC layer in this three‐cell type model of BBB. Herein we report that both neurons and astrocytes induce a decrease in the permeability of the BCEC layer to sucrose. These effects are synergic as if BCECs are cultured with both neurons and astrocytes for 5 days, permeability to sucrose decr…

Shedding of extracellular membrane vesicles from brain cells in culture

Effect of inorganic arsenic on rat cortical astrocytes in culture

Although inorganic arsenic is a well known poisonous metalloid, the cellular and molecular mechanisms of its action remain elusive. The present study was aimed at analyzing the effects of NaAsO2 on primary cultures of rat astrocytes by determining DNA damage by comet assay, and by evaluating possible changes of the concentration of some conserved heat shock proteins. Cells treated with inorganic arsenic underwent induction of Hsp70, demonstrating a state of stress. Moreover, although micromolar NaAsO2 treatments (60 μM) only reduced cell viability to 60% respect to untreated cells, high DNA damage was already observed after 24h treatment with 10 μM arsenite. Since arsenic is known to be not…

Expression of synapsin I gene in primary cultures of differentiating rat cortical neurons

Synapsin I is a neuron-specific protein which is present in two isoforms, Ia and Ib. In the last few years this protein has been demonstrated to play a central role in the regulation of neurotransmitter release and synaptic plasticity. In this paper the developmental expression of this protein has been investigated in primary neuronal cultures from fetal rat brain cortices. The presence of thyroid hormone in the culture medium stimulates an early expression of the protein without exerting any effect at the level of mRNA transcription and accumulation. These observations implicate a T3-dependent regulation of this neuron-specific gene at the level of mRNA translation. © 1995 Plenum Publishin…

Enzymatic Spermine Metabolites Induce Apoptosis Associated with Increase of p53, caspase-3 and miR-34a in Both Neuroblastoma Cells, SJNKP and the N-Myc-Amplified Form IMR5

Neuroblastoma (NB) is a common malignant solid tumor in children and accounts for 15% of childhood cancer mortality. Amplification of the N-Myc oncogene is a well-established poor prognostic marker in NB patients and strongly correlates with higher tumor aggression and resistance to treatment. New therapies for patients with N-Myc-amplified NB need to be developed. After treating NB cells with BSAO/SPM, the detection of apoptosis was determined after annexin V-FITC labeling and DNA staining with propidium iodide. The mitochondrial membrane potential activity was checked, labeling cells with the probe JC-1 dye. We analyzed, by real-time RT-PCR, the transcript of genes involved in the apoptot…

REGULATION OF THE EXPRESSION OF RNA-BINDING PROTEINS IN DEVELOPING AND ADULT RAT BRAINS: ROLE OF THYROID HORMONES