1,354,181 research outputs found
Endocytic proteins: An expanding repertoire of presynaptic functions
No full textFrom a presynaptic perspective, neuronal communication mainly relies on two interdependent events: The fast Ca2+-triggered fusion of neurotransmitter-containing synaptic vesicles (SVs) and their subsequent high-fidelity reformation. To allow rapid neurotransmission, SVs have evolved into fascinating molecular nanomachines equipped with a well-defined set of proteins. However, upon exocytosis, SVs fully collapse into the presynaptic plasma membrane leading to the dispersal of their molecular components. While the canonical function of endocytic proteins at the presynapse was believed to be the retrieval of SV proteins via clathrin-mediated endocytosis, it is now evident that clathrin-independent endocytic mechanisms predominate. We will highlight in how far these mechanisms still rely on the classical endocytic machinery and discuss the emerging functions of endocytic proteins in release site clearance and SV reformation from endosomal-like vacuoles
Novel insights into botulinum neurotoxins mechanism of action and the discovery of prophylactic inhibitors against botulism
Get PDFBotulinum neurotoxins (BoNTs), the most poisonous substances identified so far, are protein toxins that cause botulism, a severe neuroparalytic disease. They are produced by different species of neurotoxigenic Clostridia and can be grouped into seven serotypes (BoNT/A to /G). Using genomic and proteomic approach, many novel BoNTs have been recently identified and are classified as subtypes, though they cannot be completely neutralized by currently available immunological methods. However, all BoNTs have a similar molecular architecture which reflects a conserved mechanism of action. Therefore this situation can be tackled by developing inhibitors targeting the BoNT intracellular intoxication process.
The BoNTs consist of two main chains linked by a unique inter-chain disulfide bond: the heavy chain (H, 100 kDa) and the catalytic light chain (L, 50 kDa). The C-terminal part of H (HC) is responsible for the neurospecific binding and the internalization within an endocytic compartment, whilst the N-terminal part (HN) is involved in the translocation of L across the endosome membrane. L is a Zn2+ dependent metalloprotease that targets specifically the SNARE proteins, the three proteins constituting the core of neuroexocytosis. This cleavage results in a prolonged inactivation of neurotransmitter release and causes the flaccid paralysis typical of botulism. To penetrate into neurons, BoNTs exploit synaptic vesicles (SV) recycling and their lumen acidification induces the HN-mediated membrane translocation of L. It has been demonstrated that, once on the cytosolic side, the L metalloprotease remains connected to H via the interchain disulphide bridge and the reduction of this bond is necessary to release the protease in the cytosol and enable their catalytic activity.
Using a series of well characterized inhibitors of Thioredoxin Reductase (TrxR)/Thioredoxin (Trx) system, we found that this redox system is involved in the cytosolic reduction of the interchain disulphide bond of BoNTs. In neuronal cultures, these molecules prevent the metalloproteolytic activity of all toxin serotypes without significantly affecting cell viability. Moreover, such compounds are very effective in vivo, lowering the severity and the duration of paralysis caused by a local BoNT injection. More importantly, one of these drugs elicits a remarkable protection in mice systemically injected with lethal doses of different serotypes. These results entail that the reduction of the interchain disulphide bond is a strict prerequisite for the activity of BoNTs and that this class of inhibitors can prevent the neurotoxicity regardless of their different immunogenicity. Intriguingly, we also found that the TrxR/Trx system is bound to the cytosolic side of the SV membrane and that it is enriched in those SV that are docked to active zones. We speculated that this redox system may play a role in maintaining SV protein function by controlling the redox state of the different SV protein disulfides.
Another step in BoNTs mechanism of action that might offer a good template for drug design is their trafficking. Recently, an inhibitor of different pathogens that require a passage through acidic endosomes to invade cells has been identified and dubbed EGA. We tested the effect of this molecule in neurons treated with BoNTs as also their neurotoxicity is strictly dependent on the passage through an intracellular acidic compartment. We focused our investigation on BoNT/A and BoNT/B, the two serotypes mainly associated with human botulism and used in therapy, and BoNT/D, that scarcely affects humans, but frequently causes botulism in animals. We found that EGA inhibits BoNTs activity on neuronal cultures, without interfering with any of the main steps characterising their cellular mechanism of intoxication. We speculated that, rather than having a direct effect on BoNTs, this compound impinges on an intracellular target which is responsible for their trafficking. Importantly, we found that EGA is not toxic per se in vivo, and is particularly efficacious in preventing botulism induced by BoNT/B and BoNT/D. Instead, in the case of BoNT/A the lethality was not reduced, but botulism symptoms developed later. We argued that the trafficking of the different BoNT types might be differently impacted by EGA and this compound may be used as a new tool for studying different intracellular routes exploited by BoNTs.
On the basis of the present knowledge about BoNTs mechanism of action, it is clear that once the LC has been released in the cytosol, the inhibitors tested here are no longer effective. Therefore, these drugs are to be considered as prophylactics. However, if given soon after diagnosis, these compounds could reduce symptoms severity by preventing the entry into neurons of circulating BoNTs, thus reducing the severity of poisoning and shortening the period of hospitalization that is related to the high costs of intensive care. Moreover, these molecules may be administered without knowing the BoNT serotype and subtype, therefore saving the time needed for toxin characterization
Preparation of Cerebellum Granule Neurons from Mouse or Rat Pups and Evaluation of Clostridial Neurotoxin Activity and Their Inhibitors by Western Blot and Immunohistochemistry
Get PDFCerebellar Granule Neurons (CGN) from post-natal rodents have been widely used as a model to study neuronal development, physiology and pathology. CGN cultured in vitro maintain the same features displayed in vivo by mature cerebellar granule cells, including the development of a dense neuritic network, neuronal activity, neurotransmitter release and the expression of neuronal protein markers. Moreover, CGN represent a convenient model for the study of Clostridial Neurotoxins (CNT), most notably known as Tetanus and Botulinum neurotoxins, as they abundantly express both CNT receptors and intraneuronal substrates, i.e., Soluble N-ethylmaleimide-sensitive factor activating protein receptors (SNARE proteins). Here, we describe a protocol for obtaining a highly pure culture of CGN from postnatal rats/mice and an easy procedure for their intoxication with CNT. We also illustrate handy methods to evaluate CNT activity and their inhibition
The complex web of membrane contact sites in brain aging and neurodegeneration
Get PDFTo sustain the essential biological functions required for life, eukaryotic cells rely on complex interactions between different intracellular compartments. Membrane contact sites (MCS), regions where organelles come into close proximity, have recently emerged as major hubs for cellular communication, mediating a broad range of physiological processes, including calcium signalling, lipid synthesis and bioenergetics. MCS are particularly abundant and indispensable in polarized and long-lived cells, such as neurons, where they support both structural and functional integrity. In this review, we explore the functional diversity, molecular composition, and dynamic regulation of key mammalian MCS: endoplasmic reticulum (ER)-plasma membrane, ER-mitochondria and contact sites involving lipid droplets. We highlight their central role in neuronal health and discuss how MCS dysfunction has increasingly been recognized as a hallmark of brain aging and various neurodegenerative diseases, most notably Alzheimer's disease, where altered MCS dynamics contribute to pathogenesis. Finally, we emphasize the therapeutic potential of targeting MCS and outline key unanswered questions to guide future research
Mouse-derived Synaptosomes Trypsin Cleavage Assay to Characterize Synaptic Protein Sub-localization
Get PDFNeurons communicate through neurotransmission at highly specialized junctions called synapses. Each neuron forms numerous synaptic connections, consisting of presynaptic and postsynaptic terminals. Upon the arrival of an action potential, neurotransmitters are released from the presynaptic site and diffuse across the synaptic cleft to bind specialized receptors at the postsynaptic terminal. This process is tightly regulated by several proteins at both presynaptic and postsynaptic sites. The localization, abundance, and function of these proteins are essential for productive neurotransmission and are often affected in neurological and neurodegenerative disorders. Here, we outline a method for purifying mouse synaptosomes and using limited tryptic digestion to assess the subcellular localization of synaptic proteins. During synaptosomes purification, presynaptic terminals reseal and are protected from proteolysis, while postsynaptic proteins remain susceptible to tryptic cleavage. These changes can easily be evaluated by western blot analysis. This approach offers a straightforward and reliable method to evaluate the subcellular localization of synaptic proteins based on their proteolytic sensitivity, providing valuable insights into synaptic physiology and pathology
Offering of active distribution network in real-time energy market by integrated energy management system and Volt-Var optimization
No full textThis paper considers an active distribution network (ADN) that can benefit from price-responsive demands, inverter-based photovoltaic (PV) generating units, and battery energy storage systems (BESSs) to participate in the real-time energy market. However, a challenging issue arises due to the coupling of energy offerings in the market and Volt-Var schedules of system devices. This paper develops an integrated framework of energy management system and Volt-Var optimization (EMS-VVO) in the ADN. This application is in bidirectional communication with the energy market and electricity assets. In the first stage and some minutes before each hour, the EMS-VVO unit makes the offering decisions in the energy market, as well as the schedules of demands and slow-acting devices (i.e., on-load tap changers (OLTCs) and capacitors) under uncertainties in the market prices, PV power production, and power loads. In the second stage, when the actual values of available PV productions and the updated market prices are known during the power delivery, this application controls the voltage in minutes by dispatching the fast-acting devices (i.e., PV generating units and BESSs) throughout the distribution network in minutes. A number of comparative out-of-sample simulations through a 69-node test system demonstrate how the offering and controlling decisions of the ADN can be modified under different PV penetration levels, voltage violation penalties, demand flexibility levels, and reactive power capabilities of fast-acting devices
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Variations on the Author
Get PDF“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
Get PDFWe 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
Get PDFWe 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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