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Tau is an intracellular protein but also known to be released into the extracellular fluid. Tau release mechanisms have drawn intense attention as these are known to play a key role in Alzheimer's disease (AD) pathology. However, tau can also be released under physiological conditions although its physiological function and release mechanisms have been poorly characterized, especially in human neuronal cells. We investigated endogenous tau release in ReNCell VM, a human neuroprogenitor cell line, under physiological conditions and found that tau is spontaneously released from cells. To study activity-dependent release of endogenous tau, human ReNCell VM culture was stimulated by 100µM AMPA or 50mM KCl for one-hour, tau was actively released to the culture medium. The released tau was highly phosphorylated at nine phosphorylation sites (pSites) detected by phospho-specific tau antibodies including AT270 (T175/T181), AT8 (S202/T205), AT100 (T212/S214), AT180 (T231), and PHF-1 (S396/S404), showing that these pSites are important for activity-dependent tau release from human ReNCell VM. Intracellular tau showed various phosphorylation status across these sites, with AT270 and PHF-1 highly phosphorylated while AT8 and AT180 were minimally phosphorylated, suggesting that AT8 and AT180 pSites exhibit a propensity for secretion rather than being retained intracellularly. This activity-dependent tau release was significantly decreased by inhibition of GSK-3ß, demonstrating that GSK3ß-dependent phosphorylation of tau plays an important role in its release by neuronal activity. In this study, we showed that ReNCell VM serves as a valuable model for studying endogenous physiological tau release. Further, ReNCell model can be also used to study pathological release of human tau that will contribute to our understanding of the progression of AD and related dementias.
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The intricate relationship between the dopaminergic system and olfactory associative learning in Drosophila has been an intense scientific inquiry. Leveraging the formidable genetic tools, we conducted a screening of 57 dopaminergic drivers, leading to the discovery of DAN-c1 driver, uniquely targeting the single dopaminergic neuron (DAN) in each brain hemisphere. While the involvement of excitatory D1-like receptors is well-established, the role of D2-like receptors (D2Rs) remains underexplored. Our investigation reveals the expression of D2Rs in both DANs and the mushroom body (MB) of third instar larval brains. Silencing D2Rs in DAN-c1 via microRNA disrupts aversive learning, further supported by optogenetic activation of DAN-c1 during training, affirming the inhibitory role of D2R autoreceptor. Intriguingly, D2R knockdown in the MB impairs both appetitive and aversive learning. These findings elucidate the distinct contributions of D2Rs in diverse brain structures, providing novel insights into the molecular mechanisms governing associative learning in Drosophila larvae.
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Parkinson's disease (PD) is a neurodegenerative disease showing uncontrollable motor symptoms that are primarily caused by the progressive loss of dopaminergic neurons in the brain. Currently no treatment exists to prevent PD progression. Therefore, discovery of new neuroprotective strategies still has great potential to benefit PD patients. A handful of studies show that activation of cAMP pathways is neuroprotective against PD progression. However, the neuroprotective role of this signaling cascade specifically in DA neurons has not been explored. In this study, fruit fly Drosophila melanogaster was used because of its sophisticated and powerful genetic approaches, especially with related to cAMP signaling pathway. We have investigated molecular mechanisms of neuroprotection in a fly larval model of PD by administering an environmental PD toxin rotenone. Increased cAMP signaling in the dunce mutant fly carrying defects in phosphodiesterase (PDE) gene, is neuroprotective against rotenone-induced locomotion deficits. Furthermore, the neuroprotective role of cAMP signaling specifically in DA neurons has been studied as it has not been explored. By using transgenic flies expressing designer receptors exclusively activated by designer drugs (DREADDs), we have shown that an increase of cAMP levels in DA neurons rescues rotenone-induced locomotion deficits. We also showed that this neuroprotection is mediated by activation of Gαs and PKA-C1 subunits. The results provide novel findings that expand our knowledge of neuroprotective mechanisms in DA neurons affecting PD progression, which could contribute to the development of new therapeutic treatments against PD. An important future study will explore downstream targets of cAMP-PKA signaling.
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Enfermedades Neurodegenerativas , Fármacos Neuroprotectores , Enfermedad de Parkinson , Animales , Humanos , Enfermedad de Parkinson/metabolismo , Drosophila/metabolismo , Neuronas Dopaminérgicas/metabolismo , Drosophila melanogaster/metabolismo , Rotenona , Enfermedades Neurodegenerativas/metabolismo , Larva , AMP Cíclico/metabolismo , Transducción de Señal , Fármacos Neuroprotectores/metabolismo , Modelos Animales de EnfermedadRESUMEN
Synchrotron X-ray fluorescence microscopy (SXRF) presents a valuable opportunity to study the metallome of single cells because it simultaneously provides high-resolution subcellular distribution and quantitative cellular content of multiple elements. Different sample preparation techniques have been used to preserve cells for observations with SXRF, with a goal to maintain fidelity of the cellular metallome. In this case study, mouse pancreatic beta-cells have been preserved with optimized chemical fixation. We show that cell-to-cell variability is normal in the metallome of beta-cells due to heterogeneity and should be considered when interpreting SXRF data. In addition, we determined the impact of several immunofluorescence (IF) protocols on metal distribution and quantification in chemically fixed beta-cells and found that the metallome of beta-cells was not well preserved for quantitative analysis. However, zinc and iron qualitative analysis could be performed after IF with certain limitations. To help minimize metal loss using samples that require IF, we describe a novel IF protocol that can be used with chemically fixed cells after the completion of SXRF.
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Metales , Sincrotrones , Animales , Ratones , Rayos X , Espectrometría por Rayos X/métodos , Metales/análisis , Hierro/análisisRESUMEN
Neuronal activity can enhance tau release and thus accelerate tauopathies. This activity-dependent tau release can be used to study the progression of tau pathology in Alzheimer's disease (AD), as hyperphosphorylated tau is implicated in AD pathogenesis and related tauopathies. However, our understanding of the mechanisms that regulate activity-dependent tau release from neurons and the role that tau phosphorylation plays in modulating activity-dependent tau release is still rudimentary. In this study, Drosophila neurons in primary culture expressing human tau (hTau) were used to study activity-dependent tau release. We found that hTau release was markedly increased by 50 mM KCl treatment for 1 h. A similar level of release was observed using optogenetic techniques, where genetically targeted neurons were stimulated for 30 min using blue light (470 nm). Our results showed that activity-dependent release of phosphoresistant hTauS11A was reduced when compared with wildtype hTau. In contrast, release of phosphomimetic hTauE14 was increased upon activation. We found that released hTau was phosphorylated in its proline-rich and C-terminal domains using phosphorylation site-specific tau antibodies (e.g., AT8). Fold changes in detectable levels of total or phosphorylated hTau in cell lysates or following immunopurification from conditioned media were consistent with preferential release of phosphorylated hTau after light stimulation. This study establishes an excellent model to investigate the mechanism of activity-dependent hTau release and to better understand the role of phosphorylated tau release in the pathogenesis of AD since it relates to alterations in the early stage of neurodegeneration associated with increased neuronal activity.
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Enfermedad de Alzheimer/metabolismo , Mutación Missense , Neuronas/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Sustitución de Aminoácidos , Animales , Células Cultivadas , Drosophila melanogaster , Humanos , Luz , Fosforilación , Proteínas tau/genéticaRESUMEN
Pancreatic beta-cells synthesize and secrete insulin maintaining an organism's energy homeostasis. In humans, beta-cell dysfunction and death contribute to the pathogenesis of type 2 diabetes (T2D). Although the causes of beta-cell dysfunction are complex, obesity-induced low-grade systemic inflammation plays a role. For example, obese individuals exhibiting increased levels of proinflammatory cytokines IL-6 and IL-1beta have a higher risk of beta-cell dysfunction and T2D. Interestingly, obesity-induced inflammation changes the expression of several cellular metal regulating genes, prompting this study to examine changes in the beta-cell metallome after exposure to proinflammatory-cytokines. Primary mouse beta-cells were exposed to a combination of IL-6 and IL-1beta for 48 hours, were chemically fixed and imaged by synchrotron X-ray fluorescent microscopy. Quantitative analysis showed a surprising 2.4-fold decrease in the mean total cellular content of zinc from 158 ± 57.7 femtograms (fg) to 65.7 ± 29.7 fg; calcium decreased from 216 ± 67.4 to 154.3 ± 68.7 fg (control vs. cytokines, respectively). The mean total cellular iron content slightly increased from 30.4 ± 12.2 to 47.2 ± 36.4 fg after cytokine treatment; a sub-population of cells (38%) exhibited larger increases of iron density. Changes in the subcellular distributions of zinc and calcium were observed after cytokine exposure. Beta-cells contained numerous iron puncta that accumulated still more iron after exposure to cytokines. These findings provide evidence that exposure to low levels of cytokines is sufficient to cause changes in the total cellular content and/or subcellular distribution of several metals known to be critical for normal beta-cell function.
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Calcio/metabolismo , Inflamación/metabolismo , Células Secretoras de Insulina/metabolismo , Hierro/metabolismo , Imagen Óptica/métodos , Sincrotrones , Zinc/metabolismo , Animales , Mediadores de Inflamación/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Interleucina-1beta/farmacología , Interleucina-6/farmacología , Masculino , Ratones , Fracciones Subcelulares/metabolismoRESUMEN
The belief that the vertebrate brain functions normally without classical lymphatic drainage vessels has been held for many decades. On the contrary, new findings show that functional lymphatic drainage does exist in the brain. The brain lymphatic drainage system is composed of basement membrane-based perivascular pathway, a brain-wide glymphatic pathway, and cerebrospinal fluid (CSF) drainage routes including sinus-associated meningeal lymphatic vessels and olfactory/cervical lymphatic routes. The brain lymphatic systems function physiological as a route of drainage for interstitial fluid (ISF) from brain parenchyma to nearby lymph nodes. Brain lymphatic drainage helps maintain water and ion balance of the ISF, waste clearance, and reabsorption of macromolecular solutes. A second physiological function includes communication with the immune system modulating immune surveillance and responses of the brain. These physiological functions are influenced by aging, genetic phenotypes, sleep-wake cycle, and body posture. The impairment and dysfunction of the brain lymphatic system has crucial roles in age-related changes of brain function and the pathogenesis of neurovascular, neurodegenerative, and neuroinflammatory diseases, as well as brain injury and tumors. In this review, we summarize the key component elements (regions, cells, and water transporters) of the brain lymphatic system and their regulators as potential therapeutic targets in the treatment of neurologic diseases and their resulting complications. Finally, we highlight the clinical importance of ependymal route-based targeted gene therapy and intranasal drug administration in the brain by taking advantage of the unique role played by brain lymphatic pathways in the regulation of CSF flow and ISF/CSF exchange.
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Encéfalo/fisiología , Encéfalo/fisiopatología , Sistema Linfático/fisiología , Sistema Linfático/fisiopatología , Enfermedades del Sistema Nervioso/fisiopatología , Animales , Encéfalo/anatomía & histología , Humanos , Sistema Linfático/anatomía & histologíaRESUMEN
Increasing evidence suggests that metal dyshomeostasis plays an important role in human neurodegenerative diseases. Although distinctive metal distributions are described for mature hippocampus and cortex, much less is known about metal levels and intracellular distribution in individual hippocampal neuronal somata. To solve this problem, we conducted quantitative metal analyses utilizing synchrotron radiation X-Ray fluorescence on frozen hydrated primary cultured neurons derived from rat embryonic cortex (CTX) and two regions of the hippocampus: dentate gyrus (DG) and CA1. Comparing average metal contents showed that the most abundant metals were calcium, iron, and zinc, whereas metals such as copper and manganese were less than 10% of zinc. Average metal contents were generally similar when compared across neurons cultured from CTX, DG, and CA1, except for manganese that was larger in CA1. However, each metal showed a characteristic spatial distribution in individual neuronal somata. Zinc was uniformly distributed throughout the cytosol, with no evidence for the existence of previously identified zinc-enriched organelles, zincosomes. Calcium showed a peri-nuclear distribution consistent with accumulation in endoplasmic reticulum and/or mitochondria. Iron showed 2-3 distinct highly concentrated puncta only in peri-nuclear locations. Notwithstanding the small sample size, these analyses demonstrate that primary cultured neurons show characteristic metal signatures. The iron puncta probably represent iron-accumulating organelles, siderosomes. Thus, the metal distributions observed in mature brain structures are likely the result of both intrinsic neuronal factors that control cellular metal content and extrinsic factors related to the synaptic organization, function, and contacts formed and maintained in each region.
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Región CA1 Hipocampal/ultraestructura , Calcio/análisis , Giro Dentado/ultraestructura , Hierro/análisis , Neuronas/ultraestructura , Zinc/análisis , Animales , Región CA1 Hipocampal/metabolismo , Calcio/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Corteza Cerebral/metabolismo , Corteza Cerebral/ultraestructura , Cobre/análisis , Cobre/metabolismo , Giro Dentado/metabolismo , Embrión de Mamíferos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Congelación , Hierro/metabolismo , Manganeso/análisis , Manganeso/metabolismo , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Neuronas/metabolismo , Cultivo Primario de Células , Ratas , Espectrometría por Rayos X , Sincrotrones , Zinc/metabolismoRESUMEN
The divalent cation chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), often used to buffer physiological changes in cytosolic Ca(2+), also binds Zn(2+) with high affinity. In a recently published method (Lamboley et al. 2015. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201411250), the absorbance shift of BAPTA at 292 nm was successfully used to determine the total calcium concentrations of various skeletal muscle tissues. In the present study, we show that endogenous Zn(2+) in rat skeletal muscle tissue can be unknowingly measured as "Ca(2+)," unless appropriate measures are taken to eliminate Zn(2+) interference. We analyzed two rat skeletal muscle tissues, soleus and plantaris, for total calcium and zinc using either inductively coupled plasma mass spectrometry (ICP-MS) or the BAPTA method described above. ICP-MS analysis showed that total zinc contents in soleus and plantaris were large enough to affect the determination of total calcium by the BAPTA method (calcium = 1.72 ± 0.31 and 1.96 ± 0.14, and zinc = 0.528 ± 0.04 and 0.192 ± 0.01; mean ± standard error of the mean [SEM]; n = 5; mmole/kg, respectively). We next analyzed total calcium using BAPTA but included the Zn(2+)-specific chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) that buffers Zn(2+) without affecting Ca(2+)/BAPTA binding. We found that estimated concentrations of total calcium ([CaT]WM) in soleus and plantaris were reduced after TPEN addition ([CaT]WM = 3.71 ± 0.62 and 3.57 ± 0.64 without TPEN and 3.39 ± 0.64 and 3.42 ± 0.62 with TPEN; mean ± SEM; n = 3; mmole/kg, respectively). Thus, we show that a straightforward correction can be applied to the BAPTA method to improve the accuracy of the determination of total calcium that should be applicable to most any tissue studied. In addition, we show that using TPEN in combination with the BAPTA method allows one to make reasonable estimates of total zinc concentration that are in agreement with the direct determination of zinc concentration by ICP-MS.
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Calcio/metabolismo , Músculo Esquelético/metabolismo , Zinc/metabolismo , Animales , Quelantes/metabolismo , Citosol/metabolismo , Ácido Egtácico/análogos & derivados , Ácido Egtácico/metabolismo , Etilenodiaminas/metabolismo , Masculino , Ratas , Ratas Endogámicas F344RESUMEN
Correction for 'Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics' by Robert A. Colvin et al., Metallomics, 2015, 7, 1111-1123.
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The purpose of this study was to demonstrate how single cell quantitative and subcellular metallomics inform us about both the spatial distribution and cellular mechanisms of metal buffering and homeostasis in primary cultured neurons from embryonic rat brain, which are often used as models of human disease involving metal dyshomeostasis. The present studies utilized synchrotron radiation X-ray fluorescence (SRXRF) and focused primarily on zinc and iron, two abundant metals in neurons that have been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Total single cell contents for calcium, iron, zinc, copper, manganese, and nickel were determined. Resting steady state zinc showed a diffuse distribution in both soma and processes, best defined by the mass profile of the neuron with an enrichment in the nucleus compared with the cytoplasm. Zinc buffering and homeostasis was studied using two modes of cellular zinc loading - transporter and ionophore (pyrithione) mediated. Single neuron zinc contents were shown to statistically significantly increase by either loading method - ionophore: 160 million to 7 billion; transporter 160 million to 280 million atoms per neuronal soma. The newly acquired and buffered zinc still showed a diffuse distribution. Soma and processes have about equal abilities to take up zinc via transporter mediated pathways. Copper levels are distributed diffusely as well, but are relatively higher in the processes relative to zinc levels. Prior studies have observed iron puncta in certain cell types, but others have not. In the present study, iron puncta were characterized in several primary neuronal types. The results show that iron puncta could be found in all neuronal types studied and can account for up to 50% of the total steady state content of iron in neuronal soma. Although other metals can be present in iron puncta, they are predominantly iron containing and do not appear to be associated with ferritin cages or transferrin receptor endosomes. The iron content and its distribution in puncta were similar in all neuron types studied including primary dopaminergic neurons. In summary, quantitative measurements of steady state metal levels in single primary cultured neurons made possible by SRXRF analyses provide unique information on the relative levels of each metal in neuronal soma and processes, subcellular location of zinc loads, and have confirmed and extended the characterization of heretofore poorly understood cytoplasmic iron puncta.
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Metales/metabolismo , Neuronas/metabolismo , Animales , Células Cultivadas , Homeostasis , Hierro/análisis , Hierro/metabolismo , Metales/análisis , Neuronas/citología , Ratas , Zinc/análisis , Zinc/metabolismoRESUMEN
ATP plays central roles in cancer metabolism and the Warburg effect. Intratumoral ATP concentrations are up to 10(4) times higher than those of interstitial ATP in normal tissues. However, extracellular ATP is not known to enter cancer cells. Here we report that human A549 lung cancer cells internalized extracellular ATP by macropinocytosis as demonstrated by colocalization of a nonhydrolyzable fluorescent ATP and a macropinocytosis tracer high-molecular-weight dextran, as well as by a macropinocytosis inhibitor study. Extracellular ATP also induced increase of intracellular ATP levels, without involving transcription and translation at significant levels, and cancer cells' resistance to ATP-competitor anticancer drugs, likely through the mechanism of ATP internalization. These findings, described for the first time, have profound implications in ATP-sharing among cancer cells in tumors and highlight a novel anticancer target.
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Adenosina Trifosfato/metabolismo , Antineoplásicos/farmacología , Neoplasias Pulmonares/metabolismo , Adenosina Trifosfato/farmacocinética , Adenosina Trifosfato/farmacología , Adenilato Quinasa/metabolismo , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Espacio Extracelular/metabolismo , Glucólisis , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Fosforilación Oxidativa , Fosforilación , Pinocitosis , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/metabolismoRESUMEN
Our knowledge of the molecular mechanisms of intracellular homeostatic control of zinc ions is now firmly grounded on experimental findings gleaned from the study of zinc proteomes and metallomes, zinc transporters, and insights from the use of computational approaches. A cell's repertoire of zinc homeostatic molecules includes cytosolic zinc-binding proteins, transporters localized to cytoplasmic and organellar membranes, and sensors of cytoplasmic free zinc ions. Under steady state conditions, a primary function of cytosolic zinc-binding proteins is to buffer the relatively large zinc content found in most cells to a cytosolic zinc(ii) ion concentration in the picomolar range. Under non-steady state conditions, zinc-binding proteins and transporters act in concert to modulate transient changes in cytosolic zinc ion concentration in a process that is called zinc muffling. For example, if a cell is challenged by an influx of zinc ions, muffling reactions will dampen the resulting rise in cytosolic zinc ion concentration and eventually restore the cytosolic zinc ion concentration to its original value by shuttling zinc ions into subcellular stores or by removing zinc ions from the cell. In addition, muffling reactions provide a potential means to control changes in cytosolic zinc ion concentrations for purposes of cell signalling in what would otherwise be considered a buffered environment not conducive for signalling. Such intracellular zinc ion signals are known to derive from redox modifications of zinc-thiolate coordination environments, release from subcellular zinc stores, and zinc ion influx via channels. Recently, it has been discovered that metallothionein binds its seven zinc ions with different affinities. This property makes metallothionein particularly well positioned to participate in zinc buffering and muffling reactions. In addition, it is well established that metallothionein is a source of zinc ions under conditions of redox signalling. We suggest that the biological functions of transient changes in cytosolic zinc ion concentrations (presumptive zinc signals) complement those of calcium ions in both spatial and temporal dimensions.
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Citosol/metabolismo , Metaloproteínas/metabolismo , Zinc/metabolismo , Animales , Citosol/química , Homeostasis , Humanos , Metaloproteínas/química , Proteoma , Transducción de Señal , Zinc/químicaRESUMEN
As humans age, cognitive performance decreases differentially across individuals. This age-related decline in otherwise healthy individuals is likely due to the interaction of multiple factors including genetics and environment. We hypothesized that altered spatial memory performance in genetically similar mice could be in part due to differential gene expression patterns in the hippocampus. To investigate this we utilized Morris water maze (MWM) testing in a group of young (3 months) and aged (24 months) C57BL/J male mice. Two sub-groups were identified in the aged animals; one in which MWM performance was not significantly different when compared to the young animals (aged-unimpaired; "AU") and one in which performance was significantly different by 1.5 standard deviations from the mean (aged-impaired; "AI"). One week after testing was completed the entire hippocampus was collected from six each of AU, AI and young mice and their gene expression profiles were compared using Affymetrix microarrays. Benjamini and Hochberg FDR correction at p<0.05 identified 18 genes differentially expressed between the AI and AU mice. The correlation between behavioral deficits and gene expression patterning allows a better understanding of how altered gene expression in the hippocampus contributes to accelerated age-related cognitive decline and delineates between gene expression changes associated with normal aging vs. memory performance.
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Envejecimiento/genética , Trastornos del Conocimiento/genética , Expresión Génica , Hipocampo/fisiología , Aprendizaje por Laberinto , Análisis de Varianza , Animales , Conducta Animal , Perfilación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Programas InformáticosRESUMEN
Zinc dyshomeostasis in brain might be involved in the pathogenesis of a series of brain diseases such as Alzheimer's disease and stroke. It is essential that the level of intracellular free Zn2+ in neurons is tightly controlled to maintain a narrow window of optimal concentration. The plasma membrane bound transporter ZnT1 is suggested to lower intracellular Zn2+ concentration. In this study, the function of ZnT1 in cultured cortical neurons was studied. Using vector-based shRNA interference, the expression of this protein was reduced approximately 40% in cultured rat cortical neurons when measured by immunofluorescence using a ZnT1 antibody. Changes in intracellular Zn2+ levels were tracked in individual neurons by microfluorometry using the Zn2+ selective fluorophore, FluoZin3. Unopposed Zn2+ efflux was measured by first loading cultured cortical neurons with Zn2+ then reducing extracellular Zn2+ to near zero by addition of EDTA. Reducing ZnT1 expression caused Zn2+ efflux to decrease compared with the Zn2+ efflux measured in nonsense transfected neurons, suggesting that ZnT1 plays a direct role in Zn2+ efflux. ZnT1 dependent Zn2+ efflux rate was higher in the first 10 min than at later time periods suggesting that ZnT1-mediated efflux was heavily dependent on the intracellular free Zn2+ concentration and/or required an outwardly directed Zn2+ gradient.
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Corteza Cerebral/citología , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Zinc/metabolismo , Animales , Proteínas de Transporte de Catión , Células Cultivadas , Embrión de Mamíferos , Indoles , Modelos Lineales , Proteínas de la Membrana/genética , Neuronas/efectos de los fármacos , ARN Interferente Pequeño/farmacología , RatasRESUMEN
Zinc dyshomeostasis in brain might be involved in the pathogenesis of brain diseases such as Alzheimer's disease and stroke. Resting neurons tightly regulate and maintain low to subnanomolar levels of intracellular free Zn2+, but mechanisms of normal Zn2+ homeostasis are poorly understood. In this study, the mechanisms of transporter-mediated Zn2+extrusion across the plasma membrane of cultured cortical neurons were studied. Changes in intracellular free Zn2+ levels were tracked in individual neurons by microfluorometry using a Zn2+ selective fluorophore, FluoZin3. Unopposed Zn2+efflux was measured by first loading cultured cortical neurons with Zn2+ then reducing extracellular Zn2+ to near zero by addition of EDTA. Studies revealed that the primary means of Zn2+ efflux in cortical neurons required both extracellular Na+ and Ca2+. The actions of either Na+ or Ca2+ on Zn2+ efflux were blunted in the absence of the other cation. Reversed Na+ gradients could induce Zn2+ uptake. The Na+ dependence of Zn2+ efflux was not affected by a small pHo shift (7.6-8);whereas an effect of Ca2+ was not observed at pHo 8. In summary, a Na+, Ca2+/Zn2+ exchanger mechanism is proposed to be the primary transport mechanism that extrudes Zn2+ when neuronal intracellular free Zn2+ levels rise.
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Corteza Cerebral/citología , Neuronas/metabolismo , Zinc/metabolismo , Análisis de Varianza , Animales , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Quelantes/farmacología , Citofotometría/métodos , Relación Dosis-Respuesta a Droga , Etilenodiaminas/farmacología , Líquido Extracelular/efectos de los fármacos , Líquido Extracelular/metabolismo , Concentración de Iones de Hidrógeno , Modelos Lineales , Magnesio/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Ratas , Sodio/metabolismo , Sodio/farmacología , Factores de TiempoRESUMEN
To understand the mechanisms of neuronal Zn2+ homeostasis better, experimental data obtained from cultured cortical neurons were used to inform a series of increasingly complex computational models. Total metals (inductively coupled plasma-mass spectrometry), resting metallothionein, (65)Zn2+ uptake and release, and intracellular free Zn2+ levels using ZnAF-2F were determined before and after neurons were exposed to increased Zn2+, either with or without the addition of a Zn2+ ionophore (pyrithione) or metal chelators [EDTA, clioquinol (CQ), and N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine]. Three models were tested for the ability to match intracellular free Zn2+ transients and total Zn2+ content observed under these conditions. Only a model that incorporated a muffler with high affinity for Zn2+, trafficking Zn2+ to intracellular storage sites, was able to reproduce the experimental results, both qualitatively and quantitatively. This "muffler model" estimated the resting intracellular free Zn2+ concentration to be 1.07 nM. If metallothionein were to function as the exclusive cytosolic Zn2+ muffler, the muffler model predicts that the cellular concentration required to match experimental data is greater than the measured resting concentration of metallothionein. Thus Zn2+ buffering in resting cultured neurons requires additional high-affinity cytosolic metal binding moieties. Added CQ, as low as 1 microM, was shown to selectively increase Zn2+ influx. Simulations reproduced these data by modeling CQ as an ionophore. We conclude that maintenance of neuronal Zn2+ homeostasis, when challenged with Zn2+ loads, relies heavily on the function of a high-affinity muffler, the characteristics of which can be effectively studied with computational models.
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Corteza Cerebral/metabolismo , Simulación por Computador , Modelos Neurológicos , Neuronas/metabolismo , Zinc/metabolismo , Animales , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/embriología , Quelantes/farmacología , Clioquinol/farmacología , Ácido Edético/farmacología , Etilenodiaminas/farmacología , Homeostasis , Cinética , Espectrometría de Masas/métodos , Metalotioneína/metabolismo , Neuronas/efectos de los fármacos , Unión Proteica , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Espectrometría de Fluorescencia , Tionas/farmacologíaRESUMEN
Several studies have shown intracellular Zn(2+) release and concomitant cell death after prolonged exposure to exogenous NO. In the present study, we investigated whether cortical neurons briefly exposured to exogenous NO would demonstrate similar levels of intracellular Zn(2+) release and subsequent cell death. Cortical neurons were loaded with the Zn(2+) selective fluorophore FluoZin-3 and treated with various concentrations of the NO generator, spermine NONOate. Fluorescence microscopy was used to detect and quantify intracellular Zn(2+) levels. Concomitant EDTA perfusion was used to eliminate potential effects of extracellular Zn(2+). Neurons were perfused with the heavy metal chelator TPEN to selectively eliminate Zn(2+) induced fluorescence changes. A significant increase of intracellular fluorescence was detected during a 5 min perfusion with spermine NONOate. The increase in intracellular Zn(2+) release appeared to peak at 1 microM spermine NONOate (123.8 +/- 28.5%, increase above control n = 20, P < 0.001). Further increases in spermine NONOate levels as high as 1 mM failed to further increase detectable intracellular Zn(2+) levels. The NO scavenger hemoglobin blocked the effects of spermine NONOate and the inactive analog of the spermine NONOate, spermine, was without effect. No evidence of cell death induced by any of the brief treatments with exogenous NO was observed; only prolonged incubation with much larger amounts of exogenous NO resulted in significant cell death. These data suggest that in vivo release of NO may cause elevations of intracellular Zn(2+) in cortical neurons. The possibility that release of intracellular Zn(2+) in response to NO could play a role in intracellular signaling is discussed.