The human-specific duplicated α7 gene inhibits the ancestral α7, negatively regulating nicotinic acetylcholine receptor-mediated transmitter release

Gene duplication generates new functions and traits, enabling evolution. Human-specific duplicated genes in particular are primary sources of innovation during our evolution although they have very few known functions. Here we examine the brain function of one of these genes (CHRFAM7A) and its product (dupα7 subunit). This gene results from a partial duplication of the ancestral CHRNA7 gene encoding the α7 subunit that forms the homopentameric α7 nicotinic acetylcholine receptor (α7-nAChR). The functions of α7-nAChR in the brain are well defined, including the modulation of synaptic transmission and plasticity underlying normal attention, cognition, learning, and memory processes. However, the role of the dupα7 subunit remains unexplored at the neuronal level. Here, we characterize that role by combining immunoblotting, quantitative RT-PCR and FRET techniques with functional assays of α7-nAChR activity using human neuroblastoma SH-SY5Y cell variants with different dupα7 expression levels. Our findings reveal a physical interaction between dupα7 and α7 subunits in fluorescent protein-tagged dupα7/α7 transfected cells that negatively affects normal α7-nAChR activity. Specifically, in both single cells and cell populations, the [Ca2+]i signal and the exocytotic response induced by selective stimulation of α7-nAChR were either significantly inhibited by stable dupα7 overexpression or augmented after silencing dupα7 gene expression with specific siRNAs. These findings identify a new role for the dupα7 subunit as a negative regulator of α7-nAChR-mediated control of exocytotic neurotransmitter release. If this effect is excessive, it would result in an impaired synaptic transmission that could underlie the neurocognitive and neuropsychiatric disorders associated with α7-nAChR dysfunction.Ministerio de Economía Españ

Mast Cell Changes the Phenotype of Microglia via Histamine and ATP

Background/Aims: Microglia are the dynamic motile phagocytes of the brain considered the first line of defense against threats or disturbances to the Central Nervous System (CNS). Microglia help orchestrate the immunological response by interacting with others immune cells. Mast cells (MCs) are effector cells of the innate immune system distributed in all organs and vascularized tissues, brain included. Several molecular mechanisms for potential interactions between MCs and microglia have been determined. However, the effect of MCs on regulated exocytosis and phagocytic clearance in microglia has not been explored. Methods: Cocktails of MCs mediators (MCM) obtained at 37°C and 53°C were used to induce microglia activation. Changes in intracellular calcium [Ca2+]i and ATP release were studied by calcium and quinacrine fluorescence imaging. Fluorescent latex beads were used to assay phagocytosis in microglia after MCM treatment and compared to that measured in the presence of histamine, ATP and lipopolysaccharide (LPS). Iba-1 expression and area were quantified by immunofluorescence and histamine levels evaluated by ELISA techniques. Results: Local application onto microglia of the MC mediator cocktail elicited Ca2+ transients and exocytotic release associated with quinacrine dye de-staining. Ca2+ signals were mimicked by histamine and blocked by the H1 receptor (H1R) antagonist, cetirizine. Hydrolysis of ATP by apyrase also affected Ca2+ transients to a lesser extent. Iba-1 fluorescence, cell area and phagocytosis were enhanced by histamine through H1R. However, ATP prevented iba-1 expression and microglial phagocytosis. MCM showed combined effects of histamine and ATP, increasing the number of internalized microbeads per cell and area without raising iba1 expression. Conclusion: Our results highlight the relevance of MC-derived histamine and ATP in the modulation of secretory and phagocytic activities that would explain the heterogeneity of microglial responses in different pathological contexts.Agencia Estatal de Investigación/ProyectoJunta de Andalucí

PDA-Based Glyconanomicelles for Hepatocellular Carcinoma Cells Active Targeting Via Mannose and Asialoglycoprotein Receptors

Hepatocellular carcinoma (HCC) is the sixth most common neoplasia and the fourth most common cause of cancer-related mortality worldwide. Sorafenib is the first-line molecular therapy for patients in an advanced stage of HCC. However, the recommended clinical dose of Sorafenib is associated with several complications, which derive from its lack of cell specificity and its very low water solubility. To circumvent these drawbacks, in the present study we developed two sugar-coated polydiacetylene-based nanomicelles-Sorafenib carriers targeting mannose and asialoglycoprotein receptors (MR and ASGPR, respectively). The strategies allowed the inducement of apoptosis and reduction of cell proliferation at a nanomolar, instead of micromolar, range in liver cancer cells. The study showed that, contrary to literature data, Sorafenib included into the pMicMan (Man = mannose) vector (targeting MR) is more efficient than pMicGal (Gal = galactose) (targeting ASGPR). Indeed, pMicMan increased the endosomal incorporation with an increased intracellular Sorafenib concentration that induced apoptosis and reduced cell proliferation at a low concentration range (10-20 nM).Financial support was provided by the Spanish Ministry of Economy and Competitiveness (CTQ2016-78580-C2-1-R to N.K.) and the Institute of Health Carlos III (ISCIII) (PI13/00021, PI16/00090, and PI19/01266 to J.M.) both cofinanced by the European Regional Development Fund (ERDF) from FEDER and the European Social Fund (ESF), as well by the Andalusian Ministry of Economy, Science and Innovation (P07-FQM-2774 to N.K., CV20-04221 to N.K. P20_00882 to N.K. and CTS-6264 to J.M.), the PAIDI Program from the Andalusian Government (FQM-313 to N.K., CV20-04221 to N.K., P20_00882 to N.K., P20_00882 to N.K., and CTS-0664 to J.M.), the Andalusian Ministry of Health (PI-00025-2013, and PI-0198-2016 to J.M.), and the CSIC (CSIC–COV19-047). We thank the Biomedical Research Network Center for Liver and Digestive Diseases founded by the ISCIII and cofinanced by FEDER “A way to achieve Europe” and ERDF for their financial support. The COST action CA-18132 “Functional Glyconanomaterials for the Development of Diagnostic and Targeted Therapeutic Probe” is also acknowledged. E.R.B., C.C.A., and P. de la C.-O. were supported by FPU predoctoral fellowship (FPU15/04267, FPU17/00190, and FPU17/00026) from Spanish Ministry of Education, Culture and Sports. E.N.-V. was supported by the predoctoral i-PFIS IIS-enterprise contract in science and technologies in health (IFI18/00014) from ISCiii.Peer reviewe

Desarrollo y aplicación de la técnica de amerometría en parche al estudio de la exocitosis en células aisladas mastocitos y cromafines

Un problema aún peor por resolver en biología es cómo se produce la liberación del contenido de vesículas secretoras al espacio extracelular. Este mecanismo es fundamental para la comunicación química entre células. La exocitosis, proceso ... por el cual, la liberación tiene lugar, comprende una secuencia bien definida. La última y más desconocida etapa de esta secuencia es la fusión de la membrana de la vesícula con la membrana plasmática, la cual da vía libre a los productos secretores contenidos dentro de la vesícula. En los últimos años, dos nuevas técnicas han proporcionado significativos avances en este campo: Métodos electrofisiológicos, para monitorizar cambios en la capacidad de la membrana, cuando se funden las vesículas con la membrana plasmática, y métodos electroquímicos, para detectar los productos secretores liberados. Estas técnicas han contribuido con información única y complementaria, avanzando en el conocimiento sobre la cinética de fusión y captación de membranas y en la identificación y liberación de productos secretores. En este trabajo nos hemos propuesto utilizar estas dos técnicas simultáneamente sobre la misma célula, lo cual pensamos, nos aportaría información múltiple y complementaria. Además, también quisimos desarrollar y aplicar una nueva técnica, que denominamos amperometría en parche y que combina la medida de capacidad en la configuración in situ de la técnica de patch-clamp con la detección electoquímica por amperometría de los productos secretados. Esta técnica ha resultado ser una herramienta muy poderosa por su alta resolución, para poder estudiar fusiones de vesículas de tamaño de hasta mn de radio, permitiendo por vez primera, el estudio

Mechanisms of Granule Membrane Recapture followingExocytosis in Intact Mast Cells

In secretory cells, several exocytosis-coupled forms of endocytosis have been proposed including clathrin-mediated endocytosis, kiss-and-run endocytosis, cavicapture, and bulk endocytosis. These forms of endocytosis can be induced under different conditions, but their detailed molecular mechanisms and functions are largely unknown. We studied exocytosis and endocytosis in mast cells with both perforated-patch and whole-cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric serotonin detection. We found that intact mast cells exhibit an early endocytosis that follows exocytosis induced by compound 48/80. Direct observation of individual exocytic and endocytic events showed a higher percentage of capacitance flickers (27.3%) and off-steps (11.4%) in intact mast cells than in dialyzed cells (5.4% and 2.9%, respectively). Moreover, we observed a type of endocytosis of large pieces of membrane that were likely formed by cumulative fusion of several secretory granules with the cell membrane. We also identified “large-capacitance flickers” that occur after large endocytosis events. Pore conductance analysis indicated that these transient events may represent “compound cavicapture,” most likely due to the flickering of a dilated fusion pore. Using fluorescence imaging of individual exocytic and endocytic events we observed that granules can fuse to granules already fused with the plasma membrane, and then the membranes and dense cores of fused granules are internalized. Altogether, our results suggest that stimulated exocytosis in intact mast cells is followed by several forms of compensatory endocytosis, including kiss-and-run endocytosis and a mechanism for efficient retrieval of the compound membrane of several secretory granules through a single membrane fission event. Background: When vesicles undergo exocytosis, the vesicle membrane must be retrieved by endocytosis to maintain a constant cell surface area. Results: Exocytosis in intact mast cells is followed by endocytosis. Conclusion: Mechanisms of granule membrane recapture in intact mast cells include kiss-and-run and “compound endocytosis.” Significance: Compound endocytosis may be a novel mechanism for efficiently compensating for the membrane excess caused by exocytosis.Ministerio de Ciencia e Innovación ISCIIIand Fondos FEDER Grant PI11/0025

Myosin II contributes to fusion pore expansion during exocytosis

During exocytosis, the fusion pore expands to allow release of neurotransmitters and hormones to the extracellular space. To understand the process of synaptic transmission, it is of outstanding importance to know the properties of the fusion pore and how these properties affect the release process. Many proteins have been implicated in vesicle fusion; however, there is little evidence for proteins involved in fusion pore expansion. Myosin II has been shown to participate in the transport of vesicles and, surprisingly, in the final phases of exocytosis, affecting the kinetics of catecholamine release in adrenal chromaffin cells as measured by amperometry. Here, we have studied single vesicle exocytosis in chromaffin cells overexpressing an unphosphorylatable form (T18AS19A RLC-GFP) of myosin II that produces an inactive protein by patch amperometry. This method allows direct determination of fusion pore expansion by measuring its conductance, whereas the release of catecholamines is recorded simultaneously by amperometry. Here we demonstrated that the fusion pore is of critical importance to control the release of catecholamines during single vesicle secretion in chromaffin cells. We proved that myosin II acts as a molecular motor on the fusion pore expansion by hindering its dilation when it lacks the phosphorylation sites. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported by Instituto de Salud Carlos III Grant 05/0675 (to E. A.) and Ministerio de Educacion y Ciencia Grants BFU2006/13647 (to G. A. T.) and BFU U2005/02154 (to L. M. G.).Peer Reviewe

Exocytosis of catecholamine (CA)-containing and CA-free granules in chromaffin cells

Recent evidence suggests that endocytosis in neuroendocrine cells and neurons can be tightly coupled to exocytosis, allowing rapid retrieval from the plasma membrane of fused vesicles for future use. This can be a much faster mechanism for membrane recycling than classical clathrin-mediated endocytosis. During a fast exo-endocytotic cycle, the vesicle membrane does not fully collapse into the plasma membrane; nevertheless, it releases the vesicular contents through the fusion pore. Once the vesicle is depleted of transmitter, its membrane is recovered without renouncing its identity. In this report, we show that chromaffin cells contain catecholamine-free granules that retain their ability to fuse with the plasma membrane. These catecholaminefree granules represent 7% of the total population of fused vesicles, but they contributed to 47% of the fusion events when the cells were treated with reserpine for several hours. We propose that rat chromaffin granules that transiently fuse with the plasma membrane preserve their exocytotic machinery, allowing another round of exocytosis

Ketotifen is a microglial stabilizer by inhibiting secretory vesicle acidification

Aims: Microglia survey the brain environment by sensing alarm signals to provide the first line of defense against injury or infection after which they acquire an activated phenotype, but they also respond to chemical signals sent from brain mast cells, sentinels of the immune system, when these are degranulated in response to noxious agents. Nevertheless, excessive microglia activation damages the surrounding healthy neural tissue causing progressive loss of neurons and inducing chronic inflammation. Thus, it would be of intense interest the development and application of agents which prevent mast cell mediator release and inhibit the actions of such mediators once released on microglia. Main methods: Fluorescence measurements of fura-2 and quinacrine were used to measure intracellular Ca2+ signaling and exocytotic vesicle fusion in resting and activated microglia. Key findings: We show that treatment of microglia with a cocktail of mast cell mediators induces microglia activation, phagocytosis, and exocytosis, and reveal by the first-time microglia undergo a phase of vesicular acidification just before the exocytotic fusion occurs. This acidification is an important process for vesicular maturation and contributes with ~25 % to the content that the vesicle can store and later release by exocytosis. Pre-incubation with ketotifen, a mast cell stabilizer and H1R antagonist completely abolished histaminemediated calcium signaling and acidification of microglial organelles, and concomitantly reduced the discharge of vesicle contents. Significance: These results highlight a key role for vesicle acidification in microglial physiology and provide a potential therapeutic target for diseases related to mast cell and microglia-mediated neuroinflammation

A choline-evoked [Ca2+]c signal causes catecholamine release and hyperpolarization of chromaffin cells

In bovine chromaffin cells fast-superfused with Krebs-HEPES solution containing 1–2 mM Ca2+, 5 s pulses of choline (1–10 mM), elicited catecholamine secretory responses that were only ~10% of those evoked by ACh (0.01–0.1 mM). However, in high-Ca2+ solutions (10–20 mM) the size of the choline secretory responses approached those of ACh. The choline responses (10 mM choline in 20 mM Ca2+, 10Cho/20Ca2+) tended to decline upon repetitive pulsing, whereas those of ACh were well maintained. The confocal [Ca2+]c increases evoked by 10Cho/20Ca2+ were similar to those of ACh. Whereas 10Cho/20Ca2+ caused mostly hyperpolarization of chromaffin cells, 0.1ACh/20 Ca2+ caused first depolarization and then hyperpolarization; in regular solutions (2 mM Ca2+), the hyperpolarizing responses did not show up. In Xenopus oocytes injected with mRNA for bovine α7 nicotinic receptors (nAChRs), 10Cho/20 Ca2+ fully activated an inward current; in oocytes expressing α3β4, however, the inward current elicited by choline amounted to only 4% of the size of α7 current. Our results suggest that choline activates the entry of Ca2+ through α7 nAChRs; this leads to a cytosolic concentration of calcium ([Ca2+]c) rise that causes the activation of nearby Ca2+-dependent K+ channels and the hyperpolarization of the chromaffin cell. This response, which could be unmasked provided that cells were stimulated with high-Ca2+ solutions, may be the underlying mechanism through which choline exerts a modulatory effect on the electrical activity of the chromaffin cell and on neurotransmitter release at cholinergic synapses.This study has been supported by grants to A.G.G. from Programa III PRICIT grupos estratégicos de la Comunidad de Madrid/UAM; DGICYT Nº PM99-0005 and PM99-0004; FIS No. 01/0183, Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo; MCYT BFI 2003- 02722; Cartonajes La Huerta, Molina de Segura, Murcia; Spain; and Fundación La Caixa, Barcelona, Spain. M.C. was supported by grants from the Spanish Ministry of Science and Technology, BMC2002-00972, and Generalitat Valenciana, CTIDIB/2002/138 and GRUPOS03/038. J.F is a fellow of MECD, Spain, and is also supported by Fundación Teófilo Hernando, Spain.Peer reviewe