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Microglia play decisive roles during the development of the central nervous system (CNS). Phagocytosis is one of the classical functions attributed to microglia, being involved in nearly all phases of the embryonic and postnatal development of the brain, such as rapid clearance of cell debris to avoid an inflammatory response, controlling the number of neuronal and glial cells or their precursors, contribution to axon guidance and to refinement of synaptic connections. To carry out all these tasks, microglial cells are equipped with a panoply of receptors, that convert microglia to the "professional phagocytes" of the nervous parenchyma. These receptors are modulated by spatiotemporal cues that adapt the properties of microglia to the needs of the developing CNS. Thus, in this chapter, we will discuss the role of microglial phagocytosis in all the aforementioned processes. First, we will explain the general phagocytic process, to describe afterward the performance of microglial cells in detail.
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Microglía , Fagocitosis , Microglía/metabolismo , Microglía/fisiología , Fagocitosis/fisiología , Humanos , Animales , Sistema Nervioso Central/metabolismo , EncéfaloRESUMEN
Microglia play an important protective role in the healthy nervous tissue, being able to react to a variety of stimuli that induce different intracellular cascades for specific tasks. Ca2+ signaling can modulate these pathways, and we recently reported that microglial functions depend on the endoplasmic reticulum as a Ca2+ store, which involves the Ca2+ transporter SERCA2b. Here, we investigated whether microglial functions may also rely on the Golgi, another intracellular Ca2+ store that depends on the secretory pathway Ca2+/Mn2+-transport ATPase isoform 1 (SPCA1). We found upregulation of SPCA1 upon lipopolysaccharide stimulation of microglia BV2 cells and primary microglia, where alterations of the Golgi ribbon were also observed. Silencing and overexpression experiments revealed that SPCA1 affects cell morphology, Golgi apparatus integrity, and phagocytic functions. Since SPCA1 is also an efficient Mn2+ transporter and considering that Mn2+ excess causes manganism in the brain, we addressed the role of microglial SPCA1 in Mn2+ toxicity. Our results revealed a clear effect of Mn2+ excess on the viability and morphology of microglia. Subcellular analysis showed Golgi fragmentation and subsequent alteration of SPCA1 distribution from early stages of toxicity. Removal of Mn2+ by washing improved the culture viability, although it did not effectively reverse Golgi fragmentation. Interestingly, pretreatment with curcumin maintained microglia cultures viable, prevented Mn2+-induced Golgi fragmentation, and preserved SPCA Ca2+-dependent activity, suggesting curcumin as a potential protective agent against Mn2+-induced Golgi alterations in microglia.
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Adenosina Trifosfatasas , Curcumina , Adenosina Trifosfatasas/metabolismo , Lipopolisacáridos/toxicidad , Microglía/metabolismo , ATPasas Transportadoras de Calcio/genética , ATPasas Transportadoras de Calcio/metabolismo , Vías Secretoras , Curcumina/metabolismo , Regulación hacia Arriba , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Proteínas de Transporte de Membrana/metabolismo , Isoformas de Proteínas/metabolismo , Calcio/metabolismoRESUMEN
Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis, a rapid and acute infection of the central nervous system with a fatal outcome in >97% of cases. Due to the infrequent report of cases and diagnostic gaps that hinder the possibility of recovering clinic isolates, studies related to pathogenesis of the disease are scarce. However, the secretion of cytolytic molecules has been proposed as a factor involved in the progression of the infection. Several of these molecules could be included in extracellular vesicles (EVs), making them potential virulence factors and even modulators of the immune response in this infection. In this work, we evaluated the immunomodulatory effect of EVs secreted by two clinic isolates of Naegleria fowleri using in vitro models. For this purpose, characterization analyses between EVs produced by both isolates were first performed, for subsequent gene transcription analyses post incubation of these vesicles with primary cultures from mouse cell microglia and BV-2 cells. Analyses of morphological changes induced in primary culture microglia cells by the vesicles were also included, as well as the determination of the presence of nucleic acids of N. fowleri in the EV fractions. Results revealed increased expression of NOS, proinflammatory cytokines IL-6, TNF-α, and IL-23, and the regulatory cytokine IL-10 in primary cultures of microglia, as well as increased expression of NOS and IL-13 in BV-2 cells. Morphologic changes from homeostatic microglia, with small cellular body and long processes to a more amoeboid morphology were also observed after the incubation of these cells with EVs. Regarding the presence of nucleic acids, specific Naegleria fowleri DNA that could be amplified using both conventional and qPCR was confirmed in the EV fractions. Altogether, these results confirm the immunomodulatory effects of EVs of Naegleria fowleri over microglial cells and suggest a potential role of these vesicles as biomarkers of primary acute meningoencephalitis.
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During development microglia colonize the central nervous system (CNS) and play an important role in programmed cell death, not only because of their ability to remove dead cells by phagocytosis, but also because they can promote the death of neuronal and glial cells. To study this process, we used as experimental systems the developing in situ quail embryo retina and organotypic cultures of quail embryo retina explants (QEREs). In both systems, immature microglia show an upregulation of certain inflammatory markers, e.g., inducible NO synthase (iNOS), and nitric oxide (NO) under basal conditions, which can be further enhanced with LPS-treatment. Hence, we investigated in the present study the role of microglia in promoting ganglion cell death during retinal development in QEREs. Results showed that LPS-stimulation of microglia in QEREs increases (i) the percentage of retinal cells with externalized phosphatidylserine, (ii) the frequency of phagocytic contacts between microglial and caspase-3-positive ganglion cells, (iii) cell death in the ganglion cell layer, and (iv) microglial production of reactive oxygen/nitrogen species, such as NO. Furthermore, iNOS inhibition by L-NMMA decreases cell death of ganglion cells and increases the number of ganglion cells in LPS-treated QEREs. These data demonstrate that LPS-stimulated microglia induce ganglion cell death in cultured QEREs by a NO-dependent mechanism. The fact that phagocytic contacts between microglial and caspase-3-positive ganglion cells increase suggests that this cell death might be mediated by microglial engulfment, although a phagocytosis-independent mechanism cannot be excluded.
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A fluorescent labeling protocol for hydroxylated natural compounds with promising antitumor properties has been used to synthesize, in yields of 72-86%, 12 derivatives having fluorescent properties and biological activity. The reagent used for the synthesis of these fluorescent derivatives was 7-nitrobenzo-2-oxa-1,3-diazole chloride (NBD-Cl). The linkers employed to bind the NBD-Cl reagent to the natural compounds were ω-amino acids (Aa) of different chain lengths. The natural triterpene compounds chosen were oleanolic and maslinic acid, as their corresponding 28-benzylated derivatives. Thus, 12 NBD-Aa-triterpene conjugates have been studied for their optical fluorescence properties and their biological activities against cell proliferation in three cancer cell lines (B16-F10, HT-29, and HepG2), compared with three nontumor cell lines (HPF, IEC-18, and WRL68) from different tissues. The results of the fluorescence study have shown that the best fluorescent labels are those in which the ω-amino acid chain is shorter, and the carboxylic group is not benzylated. Analysis by confocal microscopy showed that these compounds were rapidly incorporated into cells in all three cancer cell lines, with these same derivatives showing the highest toxicity against the cancer cell lines tested. Then, the fluorescent labeling of these NBD-Aa-triterpene conjugates enabled their uptake and subcellular distribution to be followed in order to probe in detail their biological properties at the cellular and molecular level.
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Triterpenos , Humanos , Transporte Biológico , Colorantes Fluorescentes/farmacología , Colorantes Fluorescentes/química , Células HT29 , Triterpenos/farmacología , Triterpenos/químicaRESUMEN
Microglia are the tissue-resident macrophages of the central nervous parenchyma. In mammals, microglia are thought to originate from yolk sac precursors and posteriorly maintained through the entire life of the organism. However, the contribution of microglial cells from other sources should also be considered. In addition to "true" or "bona-fide" microglia, which are of embryonic origin, the so-called "microglia-like cells" are hematopoietic cells of bone marrow origin that can engraft the mature brain mainly under pathological conditions. These cells implement great parts of the microglial immune phenotype, but they do not completely adopt the "true microglia" features. Because of their pronounced similarity, true microglia and microglia-like cells are usually considered together as one population. In this review, we discuss the origin and development of these two distinct cell types and their differences. We will also review the factors determining the appearance and presence of microglia-like cells, which can vary among species. This knowledge might contribute to the development of therapeutic strategies aiming at microglial cells for the treatment of diseases in which they are involved, for example neurodegenerative disorders like Alzheimer's and Parkinson's diseases.
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Neurological disorders, including neurodegenerative diseases, are often characterized by neuroinflammation, which is largely driven by microglia, the resident immune cells of the central nervous system (CNS). Under these conditions, microglia are able to secrete neurotoxic substances, provoking neuronal cell death. However, microglia in the healthy brain carry out CNS-supporting functions. This is due to the ability of microglia to acquire different phenotypes that can play a neuroprotective role under physiological conditions or a pro-inflammatory, damaging one during disease. Therefore, therapeutic strategies focus on the downregulation of these neuroinflammatory processes and try to re-activate the neuroprotective features of microglia. Mesenchymal stem cells (MSC) of different origins have been shown to exert such effects, due to their immunomodulatory properties. In recent years, MSC derived from adipose tissue have been made the center of attention because of their easy availability and extraction methods. These cells induce a neuroprotective phenotype in microglia and downregulate neuroinflammation, resulting in an improvement of clinical symptoms in a variety of animal models for neurological pathologies, e.g., Alzheimer's disease, traumatic brain injury and ischemic stroke. In this review, we will discuss the application of adipose tissue-derived MSC and their conditioned medium, including extracellular vesicles, in neurological disorders, their beneficial effect on microglia and the signaling pathways involved.
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Vesículas Extracelulares , Células Madre Mesenquimatosas , Enfermedades Neurodegenerativas , Animales , Células Madre Mesenquimatosas/metabolismo , Microglía/metabolismo , Enfermedades Neurodegenerativas/metabolismo , NeuroprotecciónRESUMEN
Recent evidence has shown that inflammation can contribute to all tumorigenic states. We have investigated the anti-inflammatory effects of a diamine-PEGylated derivative of oleanolic acid (OADP), in vitro and in vivo with inflammation models. In addition, we have determined the sub-cytotoxic concentrations for anti-inflammatory assays of OADP in RAW 264.7 cells. The inflammatory process began with incubation with lipopolysaccharide (LPS). Nitric oxide production levels were also determined, exceeding 75% inhibition of NO for a concentration of 1 µg/mL of OADP. Cell-cycle analysis showed a reversal of the arrest in the G0/G1 phase in LPS-stimulated RAW 264.7 cells. Furthermore, through Western blot analysis, we have determined the probable molecular mechanism activated by OADP; the inhibition of the expression of cytokines such as TNF-α, IL-1ß, iNOS, and COX-2; and the blocking of p-IκBα production in LPS-stimulated RAW 264.7 cells. Finally, we have analyzed the anti-inflammatory action of OADP in a mouse acute ear edema, in male BL/6J mice treated with OADP and tetradecanoyl phorbol acetate (TPA). Treatment with OADP induced greater suppression of edema and decreased the ear thickness 14% more than diclofenac. The development of new derivatives such as OADP with powerful anti-inflammatory effects could represent an effective therapeutic strategy against inflammation and tumorigenic processes.
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Antiinflamatorios/farmacología , Enfermedades del Oído/tratamiento farmacológico , Edema/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Ácido Oleanólico/análogos & derivados , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Células RAW 264.7RESUMEN
Activation of microglia is an early immune response to damage in the brain. Although a key role for Ca2+ as trigger of microglial activation has been considered, little is known about the molecular scenario for regulating Ca2+ homeostasis in these cells. Taking into account the importance of the endoplasmic reticulum as a cellular Ca2+ store, the sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA2b) is an interesting target to modulate intracellular Ca2+ dynamics. We found upregulation of SERCA2b in activated microglia of human brain with Alzheimer's disease and we further studied the participation of SERCA2b in microglial functions by using the BV2 murine microglial cell line and primary microglia isolated from mouse brain. To trigger microglia activation, we used the bacterial lipopolysaccharide (LPS), which is known to induce an increase of cytosolic Ca2+ . Our results showed an upregulated expression of SERCA2b in LPS-induced activated microglia likely associated to an attempt to restore the increased cytosolic Ca2+ concentration. We analyzed SERCA2b contribution in microglial migration by using the specific SERCA inhibitor thapsigargin in scratch assays. Microglial migration was strongly stimulated with thapsigargin, even more than with LPS-induction, but delayed in time. However, phagocytic capacity of microglia was blocked in the presence of the SERCA inhibitor, indicating the importance of a tight control of cytosolic Ca2+ in these processes. All together, these results provide for the first time compelling evidence for SERCA2b as a major player regulating microglial functions, affecting migration and phagocytosis in an opposite manner.
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Microglía , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Animales , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Lipopolisacáridos/toxicidad , Ratones , Microglía/metabolismo , Fagocitosis , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Tapsigargina/farmacologíaRESUMEN
Calcium ions (Ca2+) are prominent cell signaling effectors that regulate a wide variety of cellular processes. Among the different players in Ca2+ homeostasis, primary active Ca2+ transporters are responsible for keeping low basal Ca2+ levels in the cytosol while establishing steep Ca2+ gradients across intracellular membranes or the plasma membrane. This review summarizes our current knowledge on the three types of primary active Ca2+-ATPases: the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps, the secretory pathway Ca2+- ATPase (SPCA) isoforms, and the plasma membrane Ca2+-ATPase (PMCA) Ca2+-transporters. We first discuss the Ca2+ transport mechanism of SERCA1a, which serves as a reference to describe the Ca2+ transport of other Ca2+ pumps. We further highlight the common and unique features of each isoform and review their structure-function relationship, expression pattern, regulatory mechanisms, and specific physiological roles. Finally, we discuss the increasing genetic and in vivo evidence that links the dysfunction of specific Ca2+-ATPase isoforms to a broad range of human pathologies, and highlight emerging therapeutic strategies that target Ca2+ pumps.
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Señalización del Calcio , ATPasas Transportadoras de Calcio/metabolismo , Enfermedad/etiología , Animales , HumanosRESUMEN
The disruption of Ca2+ signaling in neurons, together with a failure to keep optimal intracellular Ca2+ concentrations, have been proposed as significant factors for neuronal dysfunction in the Ca2+ hypothesis of Alzheimer's disease (AD). Tau is a protein that plays an essential role in axonal transport and can form abnormal structures such as neurofibrillary tangles that constitute one of the hallmarks of AD. We have recently shown that plasma membrane Ca2+-ATPase (PMCA), a key enzyme in the maintenance of optimal cytosolic Ca2+ levels in cells, is inhibited by tau in membrane vesicles. In the present study we show that tau inhibits synaptosomal PMCA purified from pig cerebrum, and reconstituted in phosphatidylserine-containing lipid bilayers, with a Ki value of 1.5±0.2nM tau. Noteworthy, the inhibitory effect of tau is dependent on the charge of the phospholipid used for PMCA reconstitution. In addition, nanomolar concentrations of calmodulin, the major endogenous activator of PMCA, protects against inhibition of the Ca2+-ATPase activity by tau. Our results in a cellular model such as SH-SY5Y human neuroblastoma cells yielded an inhibition of PMCA by nanomolar tau concentrations and protection by calmodulin against this inhibition similar to those obtained with purified synaptosomal PMCA. Functional studies were also performed with native and truncated versions of human cerebral PMCA4b, an isoform that has been showed to be functionally regulated by amyloid peptides, whose aggregates constitutes another hallmark of AD. Kinetic assays point out that tau binds to the C-terminal tail of PMCA, at a site distinct but close to the calmodulin binding domain. In conclusion, PMCA can be seen as a molecular target for tau-induced cytosolic calcium dysregulation in synaptic terminals. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
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Calmodulina/metabolismo , Fosfolípidos/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/antagonistas & inhibidores , Proteínas tau/metabolismo , Animales , Línea Celular Tumoral , Humanos , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , PorcinosRESUMEN
Ca2+-ATPases are plasma membrane and intracellular membrane transporters that use the energy of ATP hydrolysis to pump cytosolic Ca2+ out of the cell (PMCA) or into internal stores. These pumps are the main high-affinity Ca2+ systems involved in the maintenance of intracellular free Ca2+ at the properly low level in eukaryotic cells. The failure of neurons to keep optimal intracellular Ca2+ concentrations is a common feature of neurodegeneration by aging and aging-linked neuropathologies, such as Alzheimer's disease (AD). This disease is characterized by the accumulation of ß-amyloid senile plaques and neurofibrillary tangles of tau, a protein that plays a key role in axonal transport. Here we show a novel inhibition of PMCA activity by tau which is concentration-dependent. The extent of inhibition significantly decreases with aging in mice and control human brain membranes, but inhibition profiles were similar in AD-affected brain membrane preparations, independently of age. No significant changes in PMCA expression and localization with aging or neuropathology were found. These results point out a link between Ca2+-transporters, aging and neurodegeneration mediated by tau protein.
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Envejecimiento/patología , Enfermedad de Alzheimer/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Proteínas tau/metabolismo , Anciano , Anciano de 80 o más Años , Envejecimiento/metabolismo , Enfermedad de Alzheimer/patología , Animales , Células COS , Chlorocebus aethiops , Humanos , Ratones , Persona de Mediana EdadRESUMEN
BACKGROUND: Because of lack of worldwide standardization of influenza virus surveillance, comparison between countries of impact of a pandemic is challenging. For that, other approaches to allow internationally comparative serosurveys are welcome. OBJECTIVES: Here we explore the use of neonatal screening dried blood spots to monitor the trends of the 2009 influenza A (H1N1) pdm virus by the use of a protein microarray. STUDY DESIGN: We contacted colleagues from neonatal screening laboratories and asked for their willingness to participate in a study by testing anonymized neonatal screening bloodspots collected during the course of the pandemic. In total, 7749 dried blood spots from 13 countries in 5 continents where analyzed by using a protein microarray containing HA1 recombinant proteins derived from pandemic influenza A (H1N1) 2009 as well as seasonal influenza viruses. RESULTS: Results confirm the early start of the pandemic with extensive circulation in the US and Canada, when circulation of the new virus was limited in other parts of the world. The data collected from sites in Mexico suggested limited circulation of the virus during the early pandemic phase in this country. In contrast and to our surprise, an increase in seroprevalence early in 2009 was noted in the dataset from Argentina, suggestive of much more widespread circulation of the novel virus in this country than in Mexico. CONCLUSIONS: We conclude that this uniform serological testing of samples from a highly standardized screening system offers an interesting opportunity for monitoring population level attack rates of widespread diseases outbreaks and pandemics.
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Anticuerpos Antivirales/sangre , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/diagnóstico , Gripe Humana/epidemiología , Pandemias , Análisis por Matrices de Proteínas , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Femenino , Salud Global , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Tamizaje Neonatal , Embarazo , Adulto JovenRESUMEN
Excess Mn(2+) in humans causes a neurological disorder known as manganism, which shares symptoms with Parkinson's disease. However, the cellular mechanisms underlying Mn(2+) -neurotoxicity and the involvement of Mn(2+) -transporters in cellular homeostasis and repair are poorly understood and require further investigation. In this work, we have analyzed the effect of Mn(2+) on neurons and glia from mice in primary cultures. Mn(2+) overload compromised survival of both cell types, specifically affecting cellular integrity and Golgi organization, where the secretory pathway Ca(2+) /Mn(2+) -ATPase is localized. This ATP-driven Mn(2+) transporter might take part in Mn(2+) accumulation/detoxification at low loads of Mn(2+) , but its ATPase activity is inhibited at high concentration of Mn(2+) . Glial cells appear to be significantly more resistant to this toxicity than neurons and their presence in cocultures provided some protection to neurons against degeneration induced by Mn(2+) . Interestingly, the Mn(2+) toxicity was partially reversed upon Mn(2+) removal by wash out or by the addition of EDTA as a chelating agent, in particular in glial cells. These studies provide data on Mn(2+) neurotoxicity and may contribute to explore new therapeutic approaches for reducing Mn(2+) poisoning.
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ATPasas Transportadoras de Calcio/metabolismo , Aparato de Golgi/patología , Manganeso/toxicidad , Neuroglía/patología , Neuronas/patología , Animales , Apoptosis , Western Blotting , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Inmunohistoquímica , Manganeso/metabolismo , Ratones , Neuroglía/metabolismo , Neuronas/metabolismo , Neurotoxinas/toxicidad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vías Secretoras/efectos de los fármacosRESUMEN
The synaptosomal plasma membrane Ca(2+)-ATPase (PMCA) plays an essential role in regulating intracellular Ca(2+) concentration in brain. We have recently found that PMCA is the only Ca(2+) pump in brain which is inhibited by amyloid-ß peptide (Aß), a neurotoxic peptide implicated in the pathology of Alzheimer's disease (AD) [1], but the mechanism of inhibition is lacking. In the present study we have characterized the inhibition of PMCA by Aß. Results from kinetic assays indicate that Aß aggregates are more potent inhibitors of PMCA activity than monomers. The inhibitory effect of Aß could be blocked by pretreating the purified protein with Ca(2+)-calmodulin, the main endogenous activator of PMCA, and the activity of truncated PMCA lacking the calmodulin binding domain was not affected by Aß. Dot-overlay experiments indicated a physical association of Aß with PMCA and also with calmodulin. Thus, calmodulin could protect PMCA from inhibition by Aß by burying exposed sites on PMCA, making them inaccessible to Aß, and also by direct binding to the peptide. These results suggest a protective role of calmodulin against neuronal Ca(2+) dysregulation by PMCA inhibition induced by Aß.
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Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Calmodulina/metabolismo , Membrana Celular/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/antagonistas & inhibidores , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Sitios de Unión , Encéfalo/patología , Células COS , Calcio/metabolismo , Bovinos , Línea Celular , Chlorocebus aethiops , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Unión Proteica , Transducción de Señal , PorcinosRESUMEN
AD (Alzheimer's disease) is an age-associated neurodegenerative disorder where the accumulation of neurotoxic Aß (amyloid ß-peptide) in senile plaques is a typical feature. Recent studies point out a relationship between Aß neurotoxicity and Ca2+ dyshomoeostasis, but the molecular mechanisms involved are still under discussion. The PMCAs (plasma membrane Ca2+-ATPases) are a multi-isoform family of proteins highly expressed in brain that is implicated in the maintenance of low intraneural Ca2+ concentration. Therefore the malfunction of this pump may also be responsible for Ca2+ homoeostasis failure in AD. We have found that the Ca2+-dependence of PMCA activity is affected in human brains diagnosed with AD, being related to the enrichment of Aß. The peptide produces an inhibitory effect on the activity of PMCA which is isoform-specific, with the greatest inhibition of PMCA4. Besides, cholesterol blocked the inhibitory effect of Aß, which is consistent with the lack of any Aß effect on PMCA4 found in cholesterol-enriched lipid rafts isolated from pig brain. These observations suggest that PMCAs are a functional component of the machinery that leads to Ca2+ dysregulation in AD and propose cholesterol enrichment in rafts as a protector of the Aß-mediated inhibition on PMCA.
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Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/fisiopatología , ATPasas Transportadoras de Calcio/metabolismo , Membrana Celular/metabolismo , Enfermedad de Alzheimer/patología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/fisiopatología , Calcio/metabolismo , Colesterol/metabolismo , Humanos , Isoenzimas/metabolismo , Microdominios de Membrana/química , Placa Amiloide/patologíaRESUMEN
Calcium signaling is used by neurons to control a variety of functions, including cellular differentiation, synaptic maturation, neurotransmitter release, intracellular signaling and cell death. This review focuses on one of the most important Ca(2+) regulators in the cell, the plasma membrane Ca(2+)-ATPase (PMCA), which has a high affinity for Ca(2+) and is widely expressed in brain. The ontogeny of PMCA isoforms, linked to specific requirements of Ca(2+) during development of different brain areas, is addressed, as well as their function in the adult tissue. This is based on the high diversity of variants in the PMCA family in brain, which show particular kinetic differences possibly related to specific localizations and functions of the cell. Conversely, alterations in the activity of PMCAs could lead to changes in Ca(2+) homeostasis and, consequently, to neural dysfunction. The involvement of PMCA isoforms in certain neuropathologies and in brain ageing is also discussed.
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Neural cell differentiation involves a complex regulatory signal transduction network in which Ca(2+) ions and the secretory pathway play pivotal roles. The secretory pathway Ca(2+)-ATPase isoform 1 (SPCA1) is found in the Golgi apparatus where it is actively involved in the transport of Ca(2+) or Mn(2+) from the cytosol to the Golgi lumen. Its expression during brain development in different types of neurons has been documented recently, which raises the possibility that SPCA1 contributes to neuronal differentiation. In the present study, we investigated the potential impact of SPCA1 on neuronal polarization both in a cell line and in primary neuronal culture. In N2a neuroblastoma cells, SPCA1 was immunocytochemically localized in the juxtanuclear Golgi. Knockdown of SPCA1 by RNA interference markedly delayed the differentiation in these cells. The cells retarded in differentiation showed increased numbers of neurites of reduced length compared with control cells. Ca(2+) imaging assays showed that the lack of SPCA1 impaired Golgi Ca(2+) homeostasis and resulted in disturbed trafficking of different classes of proteins including normally Golgi-localized cameleon GT-YC3.3, bearing a Golgi-specific galactosyltransferase N terminus, and a normally plasma membrane-targeted, glycosyl phosphatidyl inositol-anchored cyan fluorescent protein construct. Also in hippocampal primary neurons, which showed a differential distribution of SPCA1 expression in Golgi stacks depending on differentiation stage, partial silencing of SPCA1 resulted in delayed differentiation, whereas total suppression drastically affected the cell survival. The disturbed overall cellular Ca(2+) homeostasis and/or the altered targeting of organellar proteins under conditions of SPCA1 knockdown highlight the importance of SPCA1 function for normal neural differentiation.