Role of Extracellular Vesicles in the Progression of Brain Tumors.

Brain tumors, and, in particular, glioblastoma (GBM), are among the most aggressive forms of cancer. In spite of the advancement in the available therapies, both diagnosis and treatments are still unable to ensure pathology-free survival of the GBM patients for more than 12-15 months. At the basis of the still poor ability to cope with brain tumors, we can consider: (i) intra-tumor heterogeneity; (ii) heterogeneity of the tumor properties when we compare different patients; (iii) the blood-brain barrier (BBB), which makes difficult both isolation of tumor-specific biomarkers and delivering of therapeutic drugs to the brain. Recently, it is becoming increasingly clear that cancer cells release large amounts of extracellular vesicles (EVs) that transport metabolites, proteins, different classes of RNAs, DNA, and lipids. These structures are involved in the pathological process and characterize any particular form of cancer. Moreover, EVs are able to cross the BBB in both directions. Starting from these observations, researchers are now evaluating the possibility to use EVs purified from organic fluids (first of all, blood and saliva), in order to obtain, through non-invasive methods (liquid biopsy), tumor biomarkers, and, perhaps, also for obtaining nanocarriers for the targeted delivering of drugs.

Role of Post-Transcriptional Regulation in Learning and Memory in Mammals.

After many decades, during which most molecular studies on the regulation of gene expression focused on transcriptional events, it was realized that post-transcriptional control was equally important in order to determine where and when specific proteins were to be synthesized. Translational regulation is of the most importance in the brain, where all the steps of mRNA maturation, transport to different regions of the cells and actual expression, in response to specific signals, constitute the molecular basis for neuronal plasticity and, as a consequence, for structural stabilization/modification of synapses; notably, these latter events are fundamental for the highest brain functions, such as learning and memory, and are characterized by long-term potentiation (LTP) of specific synapses. Here, we will discuss the molecular bases of these fundamental events by considering both the role of RNA-binding proteins (RBPs) and the effects of non-coding RNAs involved in controlling splicing, editing, stability and translation of mRNAs. Importantly, it has also been found that dysregulation of mRNA metabolism/localization is involved in many pathological conditions, arising either during brain development or in the adult nervous system.

Hormonal and psychological influences on performance anxiety in adolescent female volleyball players: a multi-approach study.

Background: The neuroendocrine system has important implications for affiliation behavior among humans and can be used to assess the correlation between social relationships, stress, and health. This can be influenced by social closeness; this aspect is the closeness towards another individual or a group of individuals such as a sports team. Sports performance anxiety is considered an unpleasant emotional reaction composed of physiological, cognitive, affective, and behavioral components. This motivates us to learn about the process that can influence the outcome of competition. Hormones and genetics would seem to influence outcome and performance. In this regard, many studies have focused on the exercise response as a function of ovarian hormones and it has been observed that progesterone is a hormone that plays a key role in reducing anxiety, and thus stress, in humans and other animals. On the other hand, high cortisol concentrations are known to contribute to increased anxiety levels. However, the salivary alpha-amylase (sAA) enzyme has been suggested as marker of acute stress than cortisol. Genetics also seem to influence anxiety and stress management as in the case of brain-derived neurotrophic factor (BDNF) and striatal dopamine transporter (DAT). Therefore, the study aims to investigate social closeness, as a measure of sports team cohesion that can influence athletes' performance results, and its ability to influence the secretion of hormones, such as progesterone and cortisol, that affect the management of sports anxiety while also taking into account genetic background during a volleyball match. Methods: Twenty-six female volleyball players who volunteered participated in this study (mean ± SD: age, 12.07 ± 0.7 years), and played in the final of the provincial volleyball championship in Palermo. All girls were during the ovarian cycle, in detail between the follicular and early ovulatory phases. Results: The results showed a significant decrease in salivary cortisol only in the winning group (p < 0.039). In fact, whilst in the latter the pre-match level was 7.7 ng/ml and then decreased to 4.5 ng/ml after the match, in the losers group change was not statistically significant (7.8 ng/ml vs 6.6 ng/ml pre- and post-match). As to the sAA concentration, the winning team showed a statistically significant variation between pre- and post-match than the losers (166.01 ± 250 U/ml vs 291.59 ± 241 U/ml) (p = 0.01). Conclusion: Analyzing the results of the SAS-2 psychological test it is highlighted that, on average, the loser group was more anxious than the winning group, and this contributed to the final result. In conclusion, there is strong evidence supporting the state of the art that many factors can affect performance anxiety and thus the performance itself.

Involvement of Astrocytes in the Formation, Maintenance, and Function of the Blood-Brain Barrier.

The blood-brain barrier (BBB) is a fundamental structure that protects the composition of the brain by determining which ions, metabolites, and nutrients are allowed to enter the brain from the blood or to leave it towards the circulation. The BBB is structurally composed of a layer of brain capillary endothelial cells (BCECs) bound to each other through tight junctions (TJs). However, its development as well as maintenance and properties are controlled by the other brain cells that contact the BCECs: pericytes, glial cells, and even neurons themselves. Astrocytes seem, in particular, to have a very important role in determining and controlling most properties of the BBB. Here, we will focus on these latter cells, since the comprehension of their roles in brain physiology has been continuously expanding, even including the ability to participate in neurotransmission and in complex functions such as learning and memory. Accordingly, pathological conditions that alter astrocytic functions can alter the BBB's integrity, thus compromising many brain activities. In this review, we will also refer to different kinds of in vitro BBB models used to study the BBB's properties, evidencing its modifications under pathological conditions.

Involvement of the H3.3 Histone Variant in the Epigenetic Regulation of Gene Expression in the Nervous System, in Both Physiological and Pathological Conditions.

All the cells of an organism contain the same genome. However, each cell expresses only a minor fraction of its potential and, in particular, the genes encoding the proteins necessary for basal metabolism and the proteins responsible for its specific phenotype. The ability to use only the right and necessary genes involved in specific functions depends on the structural organization of the nuclear chromatin, which in turn depends on the epigenetic history of each cell, which is stored in the form of a collection of DNA and protein modifications. Among these modifications, DNA methylation and many kinds of post-translational modifications of histones play a key role in organizing the complex indexing of usable genes. In addition, non-canonical histone proteins (also known as histone variants), the synthesis of which is not directly linked with DNA replication, are used to mark specific regions of the genome. Here, we will discuss the role of the H3.3 histone variant, with particular attention to its loading into chromatin in the mammalian nervous system, both in physiological and pathological conditions. Indeed, chromatin modifications that mark cell memory seem to be of special importance for the cells involved in the complex processes of learning and memory.

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 downregulated and 101 upregulated in the clone. Moreover, 46 proteins are only expressed in the clone and 82 only in the normal cells. Notably, only 11 upregulated/unique proteins are encoded in the duplicated q arm of isochromosome 8 (i(8q)), which cytogenetically characterizes the clone. Since both normal and transformed brain cells release extracellular vesicles (EVs), which might induce epigenetic modifications in the neighboring cells, we also compared EVs released from transformed and normal astrocytes. Interestingly, we found that the clone releases EVs containing proteins, such as matrix metalloproteinase 3 (MMP3), that can modify the extracellular matrix, thus allowing invasion.

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 translation of the transcripts. Notably, most RBPs contain intrinsically disordered regions (IDRs) that are thought to be involved in the formation of membrane-less structures, probably due to liquid-liquid phase separation (LLPS). Such structures are evidenced as a variety of granules that contain proteins and different classes of RNAs. The other side of the peculiar properties of IDRs is, however, that, under altered cellular conditions, they are also prone to form aggregates, as observed in neurodegeneration. Interestingly, RBPs, as part of both normal and aggregated complexes, are also able to enter extracellular vesicles (EVs), and in doing so, they can also reach cells other than those that produced them.

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, psychological and hematochemicals parameters, and dietary habits being tested. The results obtained confirmed that lactacid exercise can influence brain-derived neurotrophic factor (BDNF) levels as well as dehydroepiandrosterone sulfate (DHEAS) levels. In addition, levels of baseline lactate, which could be best used as an energy substrate, showed a decrease after the protocol training. Self-efficacy regarding worries and concerns management significantly increased from T0 to T1. The eating attitudes test (EAT-26) did not highlight any eating disease in the patients with a normal diet enrolled in our study. Physical exercise also greatly influenced the patients psychologically and emotionally, increasing their self-esteem. Lactate threshold training, together with dietary habits, appears to exert synergic positive effects on inflammation, neural plasticity and neuroprotection, producing preventive effects on MS symptoms and progression.

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 apoptotic process, to determine possible down- or upregulation of mRNAs after the treatment on SJNKP and the N-Myc-amplified IMR5 cell lines with BSAO/SPM. The experiments were carried out considering the proapoptotic genes Tp53 and caspase-3. After treatment with BSAO/SPM, both cell lines displayed increased mRNA levels for all these proapoptotic genes. Western blotting analysis with PARP and caspase-3 antibody support that BSAO/SPM treatment induces high levels of apoptosis in cells. The major conclusion is that BSAO/SPM treatment leads to antiproliferative and cytotoxic activity of both NB cell lines, associated with activation of apoptosis.

Involvement of Thyroid Hormones in Brain Development and Cancer.

The development and maturation of the mammalian brain are regulated by thyroid hormones (THs). Both hypothyroidism and hyperthyroidism cause serious anomalies in the organization and function of the nervous system. Most importantly, brain development is sensitive to TH supply well before the onset of the fetal thyroid function, and thus depends on the trans-placental transfer of maternal THs during pregnancy. Although the mechanism of action of THs mainly involves direct regulation of gene expression (genomic effects), mediated by nuclear receptors (THRs), it is now clear that THs can elicit cell responses also by binding to plasma membrane sites (non-genomic effects). Genomic and non-genomic effects of THs cooperate in modeling chromatin organization and function, thus controlling proliferation, maturation, and metabolism of the nervous system. However, the complex interplay of THs with their targets has also been suggested to impact cancer proliferation as well as metastatic processes. Herein, after discussing the general mechanisms of action of THs and their physiological effects on the nervous system, we will summarize a collection of data showing that thyroid hormone levels might influence cancer proliferation and invasion.

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 cytogenetic and epigenetic level. In parallel, we also studied the expression of the differentiation-related H1.0 linker histone variant to evaluate its possible modification in relation with transformation. We found that the most modified astrocytes (A-FC6) have epigenetic and chromosomal alterations typical of cancer, and that the other two clones (A-GS1 and A-VV5) have intermediate properties. Surprisingly, the differentiation-specific somatic histone H1.0 steadily increases from the normal astrocytes to the most transformed ones. As a whole, our results suggest that these three cell lines, together with the starting primary cells, constitute a potential model for studying glioma development.

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 insight into the relationships between genetic background and the better outcome for patients.

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 concomitant onset of toxic effects, especially on the heart. Notably, it has been clearly demonstrated that, besides their direct action on transcription (genomic effects), THs also have non-genomic effects, mediated by cell membrane and/or mitochondrial binding sites, and sometimes triggered by their endogenous catabolites. Among these latter molecules, 3,5-diiodo-L-thyronine (3,5-T2) has been attracting increasing interest because some of its metabolic effects are similar to those induced by T3, but it seems to be safer. The main target of 3,5-T2 appears to be the mitochondria, and it has been hypothesized that, by acting mainly on mitochondrial function and oxidative stress, 3,5-T2 might prevent and revert tissue damages and hepatic steatosis induced by a hyper-lipid diet, while concomitantly reducing the circulating levels of low density lipoproteins (LDL) and triglycerides. Besides a summary concerning general metabolism of THs, as well as their genomic and non-genomic effects, herein we will discuss resistance to THs and the possible mechanisms of action of 3,5-T2, also in relation to its possible clinical use as a drug.

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's Disease, diabetes, and multiple sclerosis. Most importantly, PA improves cognitive processes and memory, has analgesic and antidepressant effects, and even induces a sense of wellbeing, giving strength to the ancient principle of "menssanain corporesano" (i.e., a sound mind in a sound body). In this review we will discuss the potential mechanisms underlying the effects of PA on brain health, focusing on hormones, neurotrophins, and neurotransmitters, the release of which is modulated by PA, as well as on the intra- and extra-cellular pathways that regulate the expression of some of the genes involved.

A 3D­scaffold 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 poly­L­lactic acid (PLLA) via thermally­induced 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 pore size of 20 µm (defined as the average distance between the pore walls) was detected. For comparison, astrocytes were also cultured in the traditional 2D culture system that we have been using since 2003. Then the effects of different substrates, such as collagen I and IV, and fibronectin were analyzed. The results revealed that the PLLA scaffolds, coated with collagen IV, served as very good matrices for astrocytes, which were observed to adhere, grow and colonize the matrix, acquiring their typical morphology. In addition, under these conditions, they secreted extracellular vesicles (EVs) that were compatible in size with exosomes. Their ability to produce exosomes was also suggested by transmission electron microscopy pictures which revealed both EVs and intracellular structures that could be interpreted as multivesicular bodies. The fact that these cells were able to adapt to the PLLA scaffold, together with our previous results, which demonstrated that brain capillary endothelial cells can grow and differentiate on the same scaffold, could support the future use of 3D brain cell co­culture systems.

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 clear that glial cells, and in particular astrocytes, also play critical roles in both modes by releasing different kinds of molecules (e.g., D-serine secreted by astrocytes). On the other hand, neurons produce factors that can regulate the activity of glial cells, including their ability to release regulatory molecules. In the last fifteen years it has been demonstrated that both neurons and glial cells release extracellular vesicles (EVs) of different kinds, both in physiologic and pathological conditions. Here we discuss the possible involvement of EVs in the events underlying learning and memory, in both physiologic and pathological conditions.

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 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.

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 since long ago to be involved in cell differentiation. Moreover, it has been recently found to affect generation of epigenetic and functional intra-tumor heterogeneity. Interestingly, H1.0 or post-translational forms of it have been also found in extracellular vesicles (EVs) released from cancer cells in culture, thus suggesting that these cells may escape differentiation at least in part by discarding H1.0 through the EV route. In this review we will discuss the role of H1.0 in development, differentiation, and stem cell maintenance, also in relation with tumorigenesis, and EV production.

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 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.

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 transfer molecules, and hence properties, to neighboring cells. By interacting with proteins that are specifically sorted to EVs, mRNAs as well as ncRNAs can be transferred from cell to cell. In this review, we discuss the mechanisms underlying the sorting to EVs of different classes of molecules, as well as the role of extracellular RNAs and the associated proteins in altering gene expression in the recipient cells. Importantly, if, on the one hand, RBPs play a critical role in transferring RNAs through EVs, RNA itself could, on the other hand, function as a carrier to transfer proteins (i.e., chromatin modifiers, and transcription factors) that, once transferred, can alter the cell's epigenome.