1,720,983 research outputs found
ROLE OF SPHINGOSINE-1-PHOSPHATE RECEPTOR MODULATORS IN MOUSE CENTRAL NERVOUS SYSTEM: RELEVANCE TO AUTOIMMUNE DEMYELINATING DISEASES
No full textThe current PhD thesis focuses on the role of sphingosine-1-phophate receptors (S1PRs) and their modulators in mice central nervous system (CNS). Beside the well-recognized immunomodulatory effect of the compounds, new insights are now suggesting that these molecules may directly interact with S1PRs located on CNS cells. In this context, the project aims at i) investigating the presence and the activity of S1PRs in mouse cortical synaptosomes (isolated nerve endings) and gliosomes (astrocytic processes) in both control and EAE mice ii) studying the effect of siponimod (BAF312) on oligodendroglia cells differentiation. While the first part of the project was carried out at the University of Genoa (Italy), experiments on oligodendroglial cells were performed at the University of Hasselt (Belgium).
Firstly, we performed Western blot and confocal analyses which confirmed the existence of S1P1R and S1P3R in both glutamatergic cortical synaptosomes and gliosomes. Then, we performed functional studies (superfusion experiments) to investigate the effect of S1PRs activation in controlling glutamate transmission. By using different ligands (the broad-spectrum agonist sphingosine-1-phosphate (S1P), the S1P1R agonist CS-2100 and the selective S1P3R agonist CYM-5541) we demonstrated that presynaptic S1P1Rs are able to inhibit the 12 mM KCl-evoked release of preloaded [3H]D-aspartate (an analogue of glutamate) in cortical synaptosomes, while the activation of presynaptic S1P3Rs significantly enhances it. In contrast, the S1P1R agonist was ineffective in cortical gliosomes while CYM-5541 inhibited the 15 mM KCl-evoked [3H]D-aspartate exocytosis from cortical gliosomes. The opposite outcomes can be explained by the coupling of the receptors to different types of G-protein which, when activated, can generate opposite intracellular signals. The hypothesis is further support by the evidence that S1P failed to affect the [3H]D-aspartate release in both cortical preparations. The concomitant activation of both S1P1R and S1P3R avoid the possibility to observe a clear effect in glutamate transmission in presence of this agonist.
Then, we investigated the expression of both receptors in cortical synaptosomes and gliosomes from experimental autoimmune encephalomyelitis (EAE). In particular, S1P1R and S1P3R are significantly increased in cortical synaptosomal lysates from EAE mice at the acute stage of disease, but unchanged in the gliosomal preparation. Interestingly, oral therapeutic administration of fingolimod (FTY720; 0.03 mg/Kg/day for 14 days) recovered the receptor densities to almost physiological levels and also restored their release-regulating activity. Surprisingly, biotinylation studies performed in synaptosomes and gliosomes from healthy mice suggested that the receptors are not expressed in the outer layer on the cellular membrane, since the S1P1R and S1P3R immunopositivity was absent in the biotinylated samples from both preparations. This evidence suggests that S1P1R and S1P3R may be preferentially located intracellularly or in the inner side of the plasma membrane. Similarly, biotinylation studies performed on control and EAE mice unveiled no changes in the membrane expression of the receptors.The results obtained in this first part improve the knowledge on the existence and the release-regulating activity of S1PRs in components of the tripartite synapsis (namely the presynaptic and the astrocytic one). All experiments were performed on cortical samples and not on the spinal cord, where inflammatory infiltration is known to be much higher in EAE animal model. The choice of this specific area for our investigation allowed to study the effects of FTY720 beyond its immunomodulatory activity, adding new insights on the effect of this S1PRs modulator on glutamatergic transmission.
The second part of the project focused on the effect of another S1PRs modulator (siponimod, BAF312) on OPC in vitro differentiation. Despite very preliminary, the results suggested that treatment with BAF312 (10 nM) seems to induce a partial OPC differentiation as we can observed by the outcomes emerged from the immunostaining images as well as by the qPCR analysis. In parallel, at the same concentration, the treatment with BAF312 seems to enhance both TrkB mRNA expression and p-TrkB protein density. As an increased release of neurotrophic factors has been observed in literature following FTY720 administration, the increased expression and activation of TrkB receptors could represent an indirect way in which the drug can exert its neuroprotective effect. Further experiments will be conducted to confirm the preliminary results and study the effective role of BAF312 in these cells
Anti-NMDA and Anti-AMPA Receptor Antibodies in Central Disorders: Preclinical Approaches to Assess Their Pathological Role and Translatability to Clinic
No full textAutoantibodies against NMDA and AMPA receptors have been identified in the central nervous system of patients suffering from brain disorders characterized by neurological and psychiatric symptoms. It has been demonstrated that these autoantibodies can affect the functions and/or the expression of the targeted receptors, altering synaptic communication. The importance to clarify, in preclinical models, the molecular mechanisms involved in the autoantibody-mediated effects has emerged in order to understand their pathogenic role in central disorders, but also to propose new therapeutic approaches for preventing the deleterious central consequences. In this review, we describe some of the available preclinical literature concerning the impact of antibodies recognizing NMDA and AMPA receptors in neurons. This review discusses the cellular events that would support the detrimental roles of the autoantibodies, also illustrating some contrasting findings that in our opinion deserve attention and further investigations before translating the preclinical observations to clinic
Differences in presynaptic hippocampal GABAergic terminals at the early stage of life in female and male mice: effect of an acute early inflammatory challenge
No full textGABA dictates the efficiency of synaptic connection, influencing its developmental complexity, but its role is tuned by developmental sex differences which affect the efficiency of its innervation. We investigated the efficiency of mechanisms of GABA storage and exocytosis in hippocampal terminals of male and female mice during the juvenile period (PND21), adolescence (PND36) or adulthood (PND90). The expression of mRNA encoding for the presynaptic GABA transporter type 1, (GAT1) and the vesicular GABA transporter (VGAT1) was analysed. A significant scaling-down in the GAT1 mRNA levels (SLC6A1) was detected at PND21 in both sexes until adulthood, while the SLC32A1-VGAT mRNA level was conserved. We also analysed the density of GAT1 and VGAT proteins. Western blot analysis unveiled the presence of a monomeric and an oligomeric form of GAT1. The density of the monomeric form was conserved at the different stages of development in both sexes. Differently, the oligomeric assembly was significantly overexpressed in hippocampal synaptosomal lysates from PND21 male and female mice, but recovered at PND36. VGAT density was largely conserved in PND21 and PND36 male hippocampal synaptosomal lysates when compared to adult particles, but significantly lower in PND21 female particles. Notably, these changes are consistent and support the altered vesicular storage of newly taken-up [3H]GABA detected in PND21 male and female hippocampal synaptosomes as well as the different responsiveness of GABAergic male and female synaptosomes to increasing depolarizing stimuli (12, 20 and 30 mM KCl-enriched solutions) measured as efficiency of the [3H]GABA exocytosis. Interstingly, an acute LPS treatment affects the efficiency of GABA exocytosis at PND36 in a sex-dependent manner. These results add new knowledge on the role of GABA as effector of central inhibitory plasticity at the early stage of development and its relevance in dimorphic adaptation in physio pathological conditions
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Presynaptic Release-Regulating Sphingosine 1-Phosphate 1/3 Receptors in Cortical Glutamatergic Terminals: Adaptations in EAE Mice and Impact of Therapeutic FTY720
No full textThis study provides evidence of the existence of presynaptic inhibitory sphingosine-1-phosphate receptor 1 (S1P1R) and facilitatory S1P3R in cortical nerve endings (synaptosomes) of healthy mice. The conclusion relies on the findings that (i) the S1P1R agonist CS-2100 (0.1-30 nM) inhibits the 12 mM KCl-evoked glutamate exocytosis (quantified as the release of [ H]D-aspartate) while the S1P3R allosteric agonist CYM-5541 potentiates it and (ii) these effects are inhibited by the S1P1R antagonist Ex 26 (30-300 nM) and the S1P3R antagonist TY-52156 (100-1000 nM), respectively. Confocal microscopy and western blot analysis confirmed the presence of S1P1R and S1P3R proteins in cortical glutamatergic synaptosomes, which were scarcely accessible to biotin in a biotinylation study. Then, we demonstrated that S1P1R and S1P3R densities and their release activity are amplified in cortical synaptosomes of mice suffering from experimental autoimmune encephalomyelitis (EAE), despite receptors maintain their preferential internal distribution. Receptor changes recover following chronic oral therapeutic FTY720 (0.03 mg/Kg/day). These results improve our knowledge of the role of presynaptic release-regulating S1P1Rs and S1P3Rs controlling glutamate transmission in the CNS also unravelling functional adaptations during EAE that recover following chronic FTY720. In a whole, these findings provide new information on the central neuroprotectant activities of FTY720
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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