Jaime Renau-piqueras - Academia.edu (original) (raw)

Papers by Jaime Renau-piqueras

Research paper thumbnail of Protein Traffic Is an Intracellular Target in Alcohol Toxicity

Pharmaceuticals, 2011

Eukaryotic cells comprise a set of organelles, surrounded by membranes with a unique composition,... more Eukaryotic cells comprise a set of organelles, surrounded by membranes with a unique composition, which is maintained by a complex synthesis and transport system. Cells also synthesize the proteins destined for secretion. Together, these processes are known as the secretory pathway or exocytosis. In addition, many molecules can be internalized by cells through a process called endocytosis. Chronic and acute alcohol (ethanol) exposure alters the secretion of different essential products, such as hormones, neurotransmitters and others in a variety of cells, including central nervous system cells. This effect could be due to a range of mechanisms, including alcohol-induced alterations in the different steps involved in intracellular transport, such as glycosylation and vesicular transport along cytoskeleton elements. Moreover, alcohol consumption during pregnancy disrupts developmental processes in the central nervous system. No single mechanism has proved sufficient to account for these effects, and multiple factors are likely involved. One such mechanism indicates that ethanol also perturbs protein trafficking. The purpose of this review is to summarize our understanding of how ethanol exposure alters the trafficking of proteins in different cell systems, especially in central nervous system cells (neurons and astrocytes) in adult and developing brains.

Research paper thumbnail of Ethanol Increases Cytochrome P4502E1 and Induces Oxidative Stress in Astrocytes

Journal of Neurochemistry, 2002

We demonstrate the presence of cytochrome P4502E1 (CYP2E1) in astrocytes in primary culture, its ... more We demonstrate the presence of cytochrome P4502E1 (CYP2E1) in astrocytes in primary culture, its induction by ethanol, and the concomitant generation of free radical species. Double immunofluorescence using anti-CYP2E1 and anti-glial fibrillary acidic protein showed that CYP2E1 was distributed over the cytoplasm and processes, although labeling was more pronounced over the nuclear membrane. Immunogold labeling confirmed this pattern of distribution. Addition of 25 mM ethanol to the astrocyte culture medium for 14 days resulted in an increase in the CYP2E1 content, as determined by confocal microscopy and dot blot. In addition, ethanol induced a dose-dependent increase in the formation of reactive oxygen species that was partially prevented by incubating the astrocytes with anti-CYP2E1. Alcohol also induced a dose-dependent increase in malonaldehyde and hydroxynonenal formation and a depletion of the glutathione (GSH) content. These results suggest that ethanol induces oxidative damage in astrocytes, which could explain some of the toxic effects of ethanol on these cells, such as cytoskeletal alterations. This assumption is supported here by the fact that an increase in GSH content prevents the deleterious effects of alcohol on the cytoskeleton of astrocytes. These results suggest the importance of oxidative stress as a mechanism involved in alcohol-induced neural and brain damage.

Research paper thumbnail of Extramedullary multiple myeloma

Virchows Archiv B Cell …, 1982

A case of multiple myeloma with a mediastinal tumor and other unusual findings is described. The ... more A case of multiple myeloma with a mediastinal tumor and other unusual findings is described. The findings included the presence of extramedullary tumor masses at multiple sites in addition to the medullary tumor and, more interestingly, the occurence of pleural and peritoneal effusions. The plasma cells from these effusions were analyzed using stereological morphometric methods. The plasma cells were also cultured and established as a continuous cell line and were studied using the same methods. Our results indicate that there are qualitative and quantitative morphologic differences between the pleural and peritoneal plasma cells. Comparison between plasma cells and cultured cells from the intraperitoneal exudate showed marked morphologic differences. The analysis of these differences indicated that mature plasma cells, when subjected to culture, were transformed into immature lymphoid cells.

Research paper thumbnail of Prenatal exposure to ethanol induces changes in the nerve growth factor and its receptor in proliferating astrocytes in primary culture

Brain Research, 1994

We have analyzed the effect of prenatal exposure to alcohol on the binding, internalization and s... more We have analyzed the effect of prenatal exposure to alcohol on the binding, internalization and secretion of NGF as well as on the content of the NGF receptor (NGFr) in cortical rat astrocytes in primary culture. Secretion of NGF was approximately 1.8-fold greater in 6,-day control astrocytes than in 13-day cells. Intracellular content of NGF was very low. Astrocytes in 6-day cultures from control fetuses expressed a relatively large number of NGFr on the cell surface with a steady-state constant in the low nanomolar range. NGF was internalized by astrocytes at a slow rate. Prenatal exposure to ethanol induces a moderate increase in the number of NGFr on the cell surface as well as an increase in the intracellular pool of both NGF and NGFr which is accompanied by an important reduction in the secretion of this factor. We speculate that this decrease in NGF secretion could alter the neuronal migration pattern during development, resulting in the presence of ectopic neurons in the cortex.

Research paper thumbnail of Neurotoxicity and persistent cognitive deficits induced by combined MDMA and alcohol exposure in adolescent rats

Addiction Biology, 2010

ABSTRACTa db_259 413..423 Recent trend assessments of drug consumption reveal an increase in the ... more ABSTRACTa db_259 413..423 Recent trend assessments of drug consumption reveal an increase in the simultaneous use of several drugs at raves, clubs and college settings among youngsters and young adults. We studied in adolescent rats the effects of repeated exposure to cocaine and 3,4-methylenedioxymethanphetamine (MDMA, ecstasy), given alone or in combination with alcohol, on memory performance, adult hippocampal neurogenesis and neurotoxicity. Rats were trained two weeks after the drug treatments in the radial arm maze. The results showed that only rats exposed to combinations of alcohol and MDMA exhibited significant memory deficits. Alcohol, MDMA and combinations thereof significantly decreased 5-bromodeoxyuridine labeling in the dentate gyrus (DG), indicating reduced survival of neuronal precursors. None of the treatments altered the length of the dendritic arbors of doublecortin (DCX)-positive neurons or the number and length of DCX-negative gaps in the DG. Thus, changes in adult neurogenesis were not causally related to the cognitive alterations induced by the treatments. Only the combination of alcohol and MDMA significantly decreased the population of mature granule neurons in the DG and increased the presence of cluster of differentiation 11b+ reactive microglia in the bordering areas of the subgranular zone. Critically, memory impairment was correlated with granule cell depletion. These observations demonstrate that exposure to alcohol and MDMA during adolescence, at doses that do not provoke apparent cognitive impairment when given separately, causes neurotoxic alterations affecting the DG region as well as persistent memory deficits. The findings highlight the elevated risk associated with the concurrent recreational use of alcohol and MDMA.

Research paper thumbnail of Protein Traffic Is an Intracellular Target in Alcohol Toxicity

Pharmaceuticals, 2011

Eukaryotic cells comprise a set of organelles, surrounded by membranes with a unique composition,... more Eukaryotic cells comprise a set of organelles, surrounded by membranes with a unique composition, which is maintained by a complex synthesis and transport system. Cells also synthesize the proteins destined for secretion. Together, these processes are known as the secretory pathway or exocytosis. In addition, many molecules can be internalized by cells through a process called endocytosis. Chronic and acute alcohol (ethanol) exposure alters the secretion of different essential products, such as hormones, neurotransmitters and others in a variety of cells, including central nervous system cells. This effect could be due to a range of mechanisms, including alcohol-induced alterations in the different steps involved in intracellular transport, such as glycosylation and vesicular transport along cytoskeleton elements. Moreover, alcohol consumption during pregnancy disrupts developmental processes in the central nervous system. No single mechanism has proved sufficient to account for these effects, and multiple factors are likely involved. One such mechanism indicates that ethanol also perturbs protein trafficking. The purpose of this review is to summarize our understanding of how ethanol exposure alters the trafficking of proteins in different cell systems, especially in central nervous system cells (neurons and astrocytes) in adult and developing brains.

Research paper thumbnail of Endocytosis in Cultured Neurons Is Altered by Chronic Alcohol Exposure

Toxicological Sciences, 2010

Endocytosis is required for many cellular pivotal processes, including membrane recycling, nutrie... more Endocytosis is required for many cellular pivotal processes, including membrane recycling, nutrient uptake, and signal transduction. This complex process is particularly relevant in polarized cells, such as neurons. Previous studies have demonstrated that alcohol alters intracellular traffic, including endocytosis, in several cell types. However, information on the effect of chronic alcohol exposure on this process in neurons is scarce. As an approach, we investigated the effect of alcohol exposure on the internalization of two widely used endocytic markers, albumin and transferrin, in developing hippocampal neurons in primary culture. The effect of this treatment on the levels of several representative proteins involved in the endocytic process was also analyzed. Some of these proteins are also involved in the organization of the actin cytoskeleton. Pretreatment of cells with inhibitors chlorpromazine or nystatin indicates that albumin is internalized mainly by caveolin-dependent endocytosis. On the other hand, alcohol decreases the endocytosis of both markers, although no qualitative changes in the distribution of either of these molecules were observed. Finally, the effect of ethanol on the proteins analyzed was heterogeneous. Alcohol decreases the levels of clathrin, AP-2, SNX9, Rab5, Rab11, EEA1, Cdc42, or RhoA but increases the amount of Arf6. Moreover, alcohol does not affect the levels of caveolin1, dynamin1, Rab7, and LAMP2. This toxic effect of alcohol on endocytosis could affect some of the important neuronal activities, which depend on this process, including cell signaling. Our results in neurons also stress the notion that one of the main targets of ethanol is intracellular transport.

Research paper thumbnail of Chronic Ethanol Exposure Alters the Levels, Assembly, and Cellular Organization of the Actin Cytoskeleton and Microtubules in Hippocampal Neurons in Primary Culture

Toxicological Sciences, 2010

The organization and dynamics of microtubules (MTs) and the actin cytoskeleton are critical for t... more The organization and dynamics of microtubules (MTs) and the actin cytoskeleton are critical for the correct development and functions of neurons, including intracellular traffic and signaling. In vitro ethanol exposure impairs endocytosis, exocytosis, and nucleocytoplasmic traffic in astrocytes and alters endocytosis in cultured neurons. In astrocytes, these effects relate to changes in the organization and/or function of MTs and the actin cytoskeleton. To evaluate this possibility in hippocampal cultured neurons, we analyzed if chronic ethanol exposure affects the levels, assembly, and cellular organization of both cytoskeleton elements and the possible underlying mechanisms of these effects by morphological and biochemical methods. In the experiments described below, we provide the first evidence that chronic alcohol exposure decreases the amount of both filamentous actin and polymerized tubulin in neurons and that the number of MTs in dendrites lowers in treated cells. Alcohol also diminishes the MT-associated protein-2 levels, which mainly localizes in the somatodendritic compartment in neurons. Ethanol decreases the levels of total Rac, Cdc42, and RhoA, three small guanosine triphosphatases (GTPases) involved in the organization and dynamics of the actin cytoskeleton and MTs. Yet when alcohol decreases the levels of the active forms (GTP bound) of Rac1 and Cdc42, it does not affect the active form of RhoA. We also investigated the levels of several effector and regulator molecules of these GTPases to find that alcohol induces heterogeneous results. In conclusion, our results show that MT, actin cytoskeleton organization, and Rho GTPase signaling pathways are targets for the toxic effects of ethanol in neurons.

Research paper thumbnail of Protective Effects of Lysophosphatidic Acid (LPA) on Chronic Ethanol-Induced Injuries to the Cytoskeleton and on Glucose Uptake in Rat Astrocytes

Journal of Neurochemistry, 2003

Ethanol induces severe alterations in membrane trafficking in hepatocytes and astrocytes, the mol... more Ethanol induces severe alterations in membrane trafficking in hepatocytes and astrocytes, the molecular basis of which is unclear. One of the main candidates is the cytoskeleton and the molecular components that regulate its organization and dynamics. Here, we examine the effect of chronic exposure to ethanol on the organization and dynamics of actin and microtubule cytoskeletons and glucose uptake in rat astrocytes. Ethanol-treated cells cultured in either the presence or absence of fetal calf serum showed a significant increase in 2-deoxyglucose uptake. Ethanol also caused alterations in actin organization, consisting of the dissolution of stress fibres and the appearance of circular filaments beneath the plasma membrane. When lysophosphatidic acid (LPA), which is a normal constituent of serum and a potent intercellular lipid mediator with growth factor and actin rearrangement activities, was added to ethanol-treated astrocytes cultured without fetal calf serum, it induced the re-appearance of actin stress fibres and the normalization of 2-deoxyglucose uptake. Furthermore, ethanol also perturbed the microtubule dynamics, which delayed the recovery of the normal microtubule organization following removal of the microtubule-disrupting agent nocodazole. Again, pre-treatment with LPA prevented this alteration. Ethanol-treated rodent fibroblast NIH3T3 cells that constitutively express an activated Rho mutant protein (GTP-bound form) were insensitive to ethanol, as they showed no alteration either in actin stress-fibre organization or in 2-deoxyglucose uptake. We discuss the putative signalling targets by which ethanol could alter the cytoskeleton and hexose uptake and the cytoprotective effect of LPA against ethanol-induced damages. The latter opens the possibility that LPA or a similar non-hydrolysable lipid derivative could be used as a cytoprotective agent against the noxious effects of ethanol.

Research paper thumbnail of Lysophosphatidic Acid Rescues RhoA Activation and Phosphoinositides Levels in Astrocytes Exposed to Ethanol

Journal of Neurochemistry, 2007

Long-term ethanol treatment substantially impairs glycosylation and membrane trafficking in prima... more Long-term ethanol treatment substantially impairs glycosylation and membrane trafficking in primary cultures of rat astrocytes. Our previous studies indicated that these effects were attributable to a primary alteration in the dynamics and organization of the actin cytoskeleton, although the molecular mechanism(s) remains to be elucidated. As small Rho GTPases and phosphoinositides are involved in the actin cytoskeleton organization, we now explore the effects of chronic ethanol treatment on these pathways. We show that chronic ethanol treatment of rat astrocytes specifically reduced endogenous levels of active RhoA as a result of the increase of in the RhoGAP activity. Furthermore, ethanol-treated astrocytes showed reduced phosphoinositides levels. When lysophosphatidic acid was added to ethanol-treated astrocytes, it rapidly reverted actin cytoskeleton reorganization and raised active RhoA levels and phosphoinositides content to those observed in untreated astrocytes. Overall, our results indicate that the harmful effects of chronic exposure to ethanol on a variety of actin dynamics-associated cellular events are primarily because of alterations of activated RhoA and phosphoinositides pools.

Research paper thumbnail of Chronic Ethanol Exposure Alters the Levels, Assembly, and Cellular Organization of the Actin Cytoskeleton and Microtubules in Hippocampal Neurons in Primary Culture

Toxicological Sciences, 2010

The organization and dynamics of microtubules (MTs) and the actin cytoskeleton are critical for t... more The organization and dynamics of microtubules (MTs) and the actin cytoskeleton are critical for the correct development and functions of neurons, including intracellular traffic and signaling. In vitro ethanol exposure impairs endocytosis, exocytosis, and nucleocytoplasmic traffic in astrocytes and alters endocytosis in cultured neurons. In astrocytes, these effects relate to changes in the organization and/or function of MTs and the actin cytoskeleton. To evaluate this possibility in hippocampal cultured neurons, we analyzed if chronic ethanol exposure affects the levels, assembly, and cellular organization of both cytoskeleton elements and the possible underlying mechanisms of these effects by morphological and biochemical methods. In the experiments described below, we provide the first evidence that chronic alcohol exposure decreases the amount of both filamentous actin and polymerized tubulin in neurons and that the number of MTs in dendrites lowers in treated cells. Alcohol also diminishes the MT-associated protein-2 levels, which mainly localizes in the somatodendritic compartment in neurons. Ethanol decreases the levels of total Rac, Cdc42, and RhoA, three small guanosine triphosphatases (GTPases) involved in the organization and dynamics of the actin cytoskeleton and MTs. Yet when alcohol decreases the levels of the active forms (GTP bound) of Rac1 and Cdc42, it does not affect the active form of RhoA. We also investigated the levels of several effector and regulator molecules of these GTPases to find that alcohol induces heterogeneous results. In conclusion, our results show that MT, actin cytoskeleton organization, and Rho GTPase signaling pathways are targets for the toxic effects of ethanol in neurons.

Research paper thumbnail of Polyphosphoinositide Metabolism and Golgi Complex Morphology in Hippocampal Neurons in Primary Culture is Altered by Chronic Ethanol Exposure

Alcohol and Alcoholism, 2013

Aims: Ethanol affects not only the cytoskeletal organization and activity, but also intracellular... more Aims: Ethanol affects not only the cytoskeletal organization and activity, but also intracellular trafficking in neurons in the primary culture. Polyphosphoinositide (PPIn) are essential regulators of many important cell functions, including those mentioned, cytoskeleton integrity and intracellular vesicle trafficking. Since information about the effect of chronic ethanol exposure on PPIn metabolism in neurons is scarce, this study analysed the effect of this treatment on three of these phospholipids. Methods: Phosphatidylinositol (PtdIns) levels as well as the activity and/or levels of enzymes involved in their metabolism were analysed in neurons chronically exposed to ethanol. The levels of phospholipases C and D, and phosphatidylethanol formation were also assessed. The consequence of the possible alterations in the levels of PtdIns on the Golgi complex (GC) was also analysed. Results: We show that phosphatidylinositol (4,5)-bisphosphate and phosphatidylinositol (3,4,5)-trisphosphate levels, both involved in the control of intracellular trafficking and cytoskeleton organization, decrease in ethanol-exposed hippocampal neurons. In contrast, several kinases that participate in the metabolism of these phospholipids, and the level and/or activity of phospholipases C and D, increase in cells after ethanol exposure. Ethanol also promotes phosphatidylethanol formation in neurons, which can result in the suppression of phosphatidic acid synthesis and, therefore, in PPIn biosynthesis. This treatment also lowers the phosphatidylinositol 4-phosphate levels, the main PPIn in the GC, with alterations in their morphology and in the levels of some of the proteins involved in structure maintenance. Conclusions: The deregulation of the metabolism of PtdIns may underlie the ethanol-induced alterations on different neuronal processes, including intracellular trafficking and cytoskeletal integrity.

Research paper thumbnail of Endocytosis in Cultured Neurons Is Altered by Chronic Alcohol Exposure

Toxicological Sciences, 2010

Endocytosis is required for many cellular pivotal processes, including membrane recycling, nutrie... more Endocytosis is required for many cellular pivotal processes, including membrane recycling, nutrient uptake, and signal transduction. This complex process is particularly relevant in polarized cells, such as neurons. Previous studies have demonstrated that alcohol alters intracellular traffic, including endocytosis, in several cell types. However, information on the effect of chronic alcohol exposure on this process in neurons is scarce. As an approach, we investigated the effect of alcohol exposure on the internalization of two widely used endocytic markers, albumin and transferrin, in developing hippocampal neurons in primary culture. The effect of this treatment on the levels of several representative proteins involved in the endocytic process was also analyzed. Some of these proteins are also involved in the organization of the actin cytoskeleton. Pretreatment of cells with inhibitors chlorpromazine or nystatin indicates that albumin is internalized mainly by caveolin-dependent endocytosis. On the other hand, alcohol decreases the endocytosis of both markers, although no qualitative changes in the distribution of either of these molecules were observed. Finally, the effect of ethanol on the proteins analyzed was heterogeneous. Alcohol decreases the levels of clathrin, AP-2, SNX9, Rab5, Rab11, EEA1, Cdc42, or RhoA but increases the amount of Arf6. Moreover, alcohol does not affect the levels of caveolin1, dynamin1, Rab7, and LAMP2. This toxic effect of alcohol on endocytosis could affect some of the important neuronal activities, which depend on this process, including cell signaling. Our results in neurons also stress the notion that one of the main targets of ethanol is intracellular transport.

Research paper thumbnail of Ethanol Reduces Zincosome Formation in Cultured Astrocytes

Alcohol and Alcoholism, 2011

Aims: Zinc is an ion that participates in basic cellular and tissular functions. Zinc deficiency ... more Aims: Zinc is an ion that participates in basic cellular and tissular functions. Zinc deficiency is present in many physiological and health problems affecting most body organs, including the brain. Among the circumstances involved in zinc deficiency, ethanol consumption is probably one of the most frequent. A dietary zinc supplement has been proposed as possibly being an efficient method to palliate zinc deficiency. Astrocytes form part of the hematoencephalic barrier, and they are apparently implicated in the homeostasis of the neuronal medium. In this work, we analyze the effect of ethanol on extracellular zinc management by rat astrocytes in culture. Methods: Intracellular levels of 'free zinc ions', in controls and 30 mM ethanol-treated astrocytes, were visualized by using the zinc fluorochrome TSQ. Cytoplasmic fluorescence and zincosome formation were measured after adding extracellular 50 µM ZnSO 4 to cell monolayers. Zincosomes were also observed at the electron microscopy level. Results: Exposure to ethanol for 7 days lowered the basal zinc levels of astrocytes by~30%. This difference was consistently maintained after the zinc pulse. Zinc ions were confined to bright fluorescent particles, the 'zincosomes', which appeared to be formed by the endocytic pathway. Zincosomes were less abundant in alcohol-treated cells, indicating a deficit in endocytoses as the origin of low zinc intake in astrocytes after ethanol treatment. Conclusions: Ethanol reduces both intracellular ionic zinc levels and extracellular zinc uptake, resulting in poorer zincosome formation. Given the endocytic nature of zincosomes, the effect of ethanol on membrane trafficking is apparently the origin of this deficit.

Research paper thumbnail of Protein Traffic Is an Intracellular Target in Alcohol Toxicity

Pharmaceuticals, 2011

Eukaryotic cells comprise a set of organelles, surrounded by membranes with a unique composition,... more Eukaryotic cells comprise a set of organelles, surrounded by membranes with a unique composition, which is maintained by a complex synthesis and transport system. Cells also synthesize the proteins destined for secretion. Together, these processes are known as the secretory pathway or exocytosis. In addition, many molecules can be internalized by cells through a process called endocytosis. Chronic and acute alcohol (ethanol) exposure alters the secretion of different essential products, such as hormones, neurotransmitters and others in a variety of cells, including central nervous system cells. This effect could be due to a range of mechanisms, including alcohol-induced alterations in the different steps involved in intracellular transport, such as glycosylation and vesicular transport along cytoskeleton elements. Moreover, alcohol consumption during pregnancy disrupts developmental processes in the central nervous system. No single mechanism has proved sufficient to account for these effects, and multiple factors are likely involved. One such mechanism indicates that ethanol also perturbs protein trafficking. The purpose of this review is to summarize our understanding of how ethanol exposure alters the trafficking of proteins in different cell systems, especially in central nervous system cells (neurons and astrocytes) in adult and developing brains.

Research paper thumbnail of Chronic Alcohol Alters Dendritic Spine Development in Neurons in Primary Culture

Neurotoxicity Research, 2013

Dendritic spines are specialised membrane protrusions of neuronal dendrites that receive the majo... more Dendritic spines are specialised membrane protrusions of neuronal dendrites that receive the majority of excitatory synaptic inputs. Abnormal changes in their density, size and morphology have been associated with various neurological and psychiatric disorders, including those deriving from drug addiction. Dendritic spine formation, morphology and synaptic functions are governed by the actin cytoskeleton. Previous in vivo studies have shown that ethanol alters the number and morphology of spines, although the mechanisms underlying these alterations remain unknown. It has also been described how chronic ethanol exposure affects the levels, assembly and cellular organisation of the actin cytoskeleton in hippocampal neurons in primary culture. Therefore, we hypothesised that the ethanol-induced alterations in the number and shape of dendritic spines are due to alterations in the mechanisms regulating actin cytoskeleton integrity. The results presented herein show that chronic exposure to moderate levels of alcohol (30 mM) during the first 2 weeks of culture reduces dendritic spine density and alters the proportion of the different morphologies of these structures in hippocampal neurons, which affects the formation of mature spines. Apparently, these effects are associated with an increase in the G-actin/F-actin ratio due to a reduction of the F-actin fraction, leading to changes in the levels of the different factors regulating the organisation of this cytoskeletal component. The data presented herein indicate that these effects occur between weeks 1 and 2 of culture, an important period in dendritic spines development. These changes may be related to the dysfunction in the memory and learning processes present in children prenatally exposed to ethanol.

Research paper thumbnail of Chronic Alcohol Exposure Affects the Cell Components Involved in Membrane Traffic in Neuronal Dendrites

Neurotoxicity Research, 2014

The specific traffic of the membrane components in neurons is a major requirement to establish an... more The specific traffic of the membrane components in neurons is a major requirement to establish and maintain neuronal domains-the axonal and the somatodendritic domains-and their polarized morphology. Unlike axons, dendrites contain membranous organelles, which are involved in the secretory pathway, including the endoplasmic reticulum, the Golgi apparatus and post-Golgi apparatus carriers, the cytoskeleton, and plasma membrane. A variety of molecules and factors are also involved in this process. Previous studies have shown that chronic alcohol exposure negatively affects several of these cell components, such as the Golgi apparatus or cytoskeleton in neurons. Yet very little information is available on the possible effects of this exposure on the remaining cell elements involved in intracellular trafficking in neurons, particularly in dendrites. By qualitative and quantitative electron microscopy, immunofluorescence and immunoblotting, we herein show that chronic exposure to moderate levels (30 mM) of ethanol in cultured neurons reduces the volume and surface density of the rough endoplasmic reticulum, and increases the levels of GRP78, a chaperone involved in endoplasmic reticulum stress. Ethanol also significantly diminishes the proportion of neurons that show an extension of Golgi into dendrites and dendritic Golgi outposts, a structure present exclusively in longer, thicker apical dendrites. Both Golgi apparatus types were also fragmented into a large number of cells. We also investigated the effect of alcohol on the levels of microtubule-based motor proteins KIF5, KIF17, KIFC2, dynein, and myosin IIb, responsible for transporting different cargoes in dendrites. Of these, alcohol differently affects several of them by lowering dynein and raising KIF5, KIFC2, and myosin IIb. These results, together with other previously published ones, suggest that practically all the protein trafficking steps in dendrites are altered to a greater or lesser extent by chronic alcohol exposure in neuronal cells, which may have negative repercussions for the development and maintenance of their polarized morphology and function.

Research paper thumbnail of Chronic ethanol exposure alters the levels, assembly, and cellular organization of the actin cytoskeleton and microtubules in hippocampal neurons in primary culture

Toxicological sciences : an official journal of the Society of Toxicology, 2010

The organization and dynamics of microtubules (MTs) and the actin cytoskeleton are critical for t... more The organization and dynamics of microtubules (MTs) and the actin cytoskeleton are critical for the correct development and functions of neurons, including intracellular traffic and signaling. In vitro ethanol exposure impairs endocytosis, exocytosis, and nucleocytoplasmic traffic in astrocytes and alters endocytosis in cultured neurons. In astrocytes, these effects relate to changes in the organization and/or function of MTs and the actin cytoskeleton. To evaluate this possibility in hippocampal cultured neurons, we analyzed if chronic ethanol exposure affects the levels, assembly, and cellular organization of both cytoskeleton elements and the possible underlying mechanisms of these effects by morphological and biochemical methods. In the experiments described below, we provide the first evidence that chronic alcohol exposure decreases the amount of both filamentous actin and polymerized tubulin in neurons and that the number of MTs in dendrites lowers in treated cells. Alcohol al...

Research paper thumbnail of Fluorescent Analogues of Plasma Membrane Sphingolipids Are Sorted to Different Intracellular Compartments in Astrocytes; Harmful Effects of Chronic Ethanol Exposure on Sphingolipid Trafficking and Metabolism

FEBS Letters, 2004

Sphingolipids are basic constituents of cellular membranes and are essential for numerous functio... more Sphingolipids are basic constituents of cellular membranes and are essential for numerous functions such as intracellular signalling. They are transported along the exocytic and endocytic pathways in eukaryotic cells. After endocytosis, fluorescent-labelled sphingolipids are sorted to distinct intracellular organelles prior to recycling (via early/recycling endosomes) or degradation (late endosomes/lysosomes). Here we examine, in primary cultures of rat astrocytes, the internalisation routes followed by C(6)-NBD-glucosylceramide (NBD-GlcCer) and C(6)-NBD-sphingomyelin (NBD-SM) and the effects of ethanol on their endocytic trafficking. Endocytosed plasma membrane NBD-GlcCer and NBD-SM are diverted to the Golgi apparatus and lysosomes, respectively. These different internalisation pathways are maintained regardless of the differentiation stage of astrocytes. Chronic ethanol exposure did not alter this endocytic sorting, but delayed the internalisation of both NBD-sphingolipids. Moreover, ethanol also stimulated the in situ metabolism of NBD-ceramide to NBD-GlcCer and NBD-SM. We conclude that in rat astrocytes internalised plasma membrane NBD-sphingolipids are sorted to different subcellular compartments. The exposure to chronic ethanol perturbed the lipid endocytic process and stimulated the de novo synthesis of NBD-sphingolipids, shifting the balance of sphingolipid metabolism in favour of the sphingomyelin pathway.

Research paper thumbnail of Actin Filaments are Involved in the Maintenance of Golgi Cisternae Morphology and intra-Golgi pH

Cell Motility and the Cytoskeleton, 2006

Here we examine the contribution of actin dynamics to the architecture and pH of the Golgi comple... more Here we examine the contribution of actin dynamics to the architecture and pH of the Golgi complex. To this end, we have used toxins that depolymerize (cytochalasin D, latrunculin B, mycalolide B, and Clostridium botulinum C2 toxin) or stabilize (jasplakinolide) filamentous actin. When various clonal cell lines were examined by epifluorescence microscopy, all of these actin toxins induced compaction of the Golgi complex. However, ultrastructural analysis by transmission electron microscopy and electron tomography/three-dimensional modelling of the Golgi complex showed that F-actin depolymerization first induces perforation/fragmentation and severe swelling of Golgi cisternae, which leads to a completely disorganized structure. In contrast, F-actin stabilization results only in cisternae perforation/fragmentation. Concomitantly to actin depolymerization-induced cisternae swelling and disorganization, the intra-Golgi pH significantly increased. Similar ultrastructural and Golgi pH alkalinization were observed in cells treated with the vacuolar H+ -ATPases inhibitors bafilomycin A1 and concanamycin A. Overall, these results suggest that actin filaments are implicated in the preservation of the flattened shape of Golgi cisternae. This maintenance seems to be mediated by the regulation of the state of F-actin assembly on the Golgi pH homeostasis.

Research paper thumbnail of Protein Traffic Is an Intracellular Target in Alcohol Toxicity

Pharmaceuticals, 2011

Eukaryotic cells comprise a set of organelles, surrounded by membranes with a unique composition,... more Eukaryotic cells comprise a set of organelles, surrounded by membranes with a unique composition, which is maintained by a complex synthesis and transport system. Cells also synthesize the proteins destined for secretion. Together, these processes are known as the secretory pathway or exocytosis. In addition, many molecules can be internalized by cells through a process called endocytosis. Chronic and acute alcohol (ethanol) exposure alters the secretion of different essential products, such as hormones, neurotransmitters and others in a variety of cells, including central nervous system cells. This effect could be due to a range of mechanisms, including alcohol-induced alterations in the different steps involved in intracellular transport, such as glycosylation and vesicular transport along cytoskeleton elements. Moreover, alcohol consumption during pregnancy disrupts developmental processes in the central nervous system. No single mechanism has proved sufficient to account for these effects, and multiple factors are likely involved. One such mechanism indicates that ethanol also perturbs protein trafficking. The purpose of this review is to summarize our understanding of how ethanol exposure alters the trafficking of proteins in different cell systems, especially in central nervous system cells (neurons and astrocytes) in adult and developing brains.

Research paper thumbnail of Ethanol Increases Cytochrome P4502E1 and Induces Oxidative Stress in Astrocytes

Journal of Neurochemistry, 2002

We demonstrate the presence of cytochrome P4502E1 (CYP2E1) in astrocytes in primary culture, its ... more We demonstrate the presence of cytochrome P4502E1 (CYP2E1) in astrocytes in primary culture, its induction by ethanol, and the concomitant generation of free radical species. Double immunofluorescence using anti-CYP2E1 and anti-glial fibrillary acidic protein showed that CYP2E1 was distributed over the cytoplasm and processes, although labeling was more pronounced over the nuclear membrane. Immunogold labeling confirmed this pattern of distribution. Addition of 25 mM ethanol to the astrocyte culture medium for 14 days resulted in an increase in the CYP2E1 content, as determined by confocal microscopy and dot blot. In addition, ethanol induced a dose-dependent increase in the formation of reactive oxygen species that was partially prevented by incubating the astrocytes with anti-CYP2E1. Alcohol also induced a dose-dependent increase in malonaldehyde and hydroxynonenal formation and a depletion of the glutathione (GSH) content. These results suggest that ethanol induces oxidative damage in astrocytes, which could explain some of the toxic effects of ethanol on these cells, such as cytoskeletal alterations. This assumption is supported here by the fact that an increase in GSH content prevents the deleterious effects of alcohol on the cytoskeleton of astrocytes. These results suggest the importance of oxidative stress as a mechanism involved in alcohol-induced neural and brain damage.

Research paper thumbnail of Extramedullary multiple myeloma

Virchows Archiv B Cell …, 1982

A case of multiple myeloma with a mediastinal tumor and other unusual findings is described. The ... more A case of multiple myeloma with a mediastinal tumor and other unusual findings is described. The findings included the presence of extramedullary tumor masses at multiple sites in addition to the medullary tumor and, more interestingly, the occurence of pleural and peritoneal effusions. The plasma cells from these effusions were analyzed using stereological morphometric methods. The plasma cells were also cultured and established as a continuous cell line and were studied using the same methods. Our results indicate that there are qualitative and quantitative morphologic differences between the pleural and peritoneal plasma cells. Comparison between plasma cells and cultured cells from the intraperitoneal exudate showed marked morphologic differences. The analysis of these differences indicated that mature plasma cells, when subjected to culture, were transformed into immature lymphoid cells.

Research paper thumbnail of Prenatal exposure to ethanol induces changes in the nerve growth factor and its receptor in proliferating astrocytes in primary culture

Brain Research, 1994

We have analyzed the effect of prenatal exposure to alcohol on the binding, internalization and s... more We have analyzed the effect of prenatal exposure to alcohol on the binding, internalization and secretion of NGF as well as on the content of the NGF receptor (NGFr) in cortical rat astrocytes in primary culture. Secretion of NGF was approximately 1.8-fold greater in 6,-day control astrocytes than in 13-day cells. Intracellular content of NGF was very low. Astrocytes in 6-day cultures from control fetuses expressed a relatively large number of NGFr on the cell surface with a steady-state constant in the low nanomolar range. NGF was internalized by astrocytes at a slow rate. Prenatal exposure to ethanol induces a moderate increase in the number of NGFr on the cell surface as well as an increase in the intracellular pool of both NGF and NGFr which is accompanied by an important reduction in the secretion of this factor. We speculate that this decrease in NGF secretion could alter the neuronal migration pattern during development, resulting in the presence of ectopic neurons in the cortex.

Research paper thumbnail of Neurotoxicity and persistent cognitive deficits induced by combined MDMA and alcohol exposure in adolescent rats

Addiction Biology, 2010

ABSTRACTa db_259 413..423 Recent trend assessments of drug consumption reveal an increase in the ... more ABSTRACTa db_259 413..423 Recent trend assessments of drug consumption reveal an increase in the simultaneous use of several drugs at raves, clubs and college settings among youngsters and young adults. We studied in adolescent rats the effects of repeated exposure to cocaine and 3,4-methylenedioxymethanphetamine (MDMA, ecstasy), given alone or in combination with alcohol, on memory performance, adult hippocampal neurogenesis and neurotoxicity. Rats were trained two weeks after the drug treatments in the radial arm maze. The results showed that only rats exposed to combinations of alcohol and MDMA exhibited significant memory deficits. Alcohol, MDMA and combinations thereof significantly decreased 5-bromodeoxyuridine labeling in the dentate gyrus (DG), indicating reduced survival of neuronal precursors. None of the treatments altered the length of the dendritic arbors of doublecortin (DCX)-positive neurons or the number and length of DCX-negative gaps in the DG. Thus, changes in adult neurogenesis were not causally related to the cognitive alterations induced by the treatments. Only the combination of alcohol and MDMA significantly decreased the population of mature granule neurons in the DG and increased the presence of cluster of differentiation 11b+ reactive microglia in the bordering areas of the subgranular zone. Critically, memory impairment was correlated with granule cell depletion. These observations demonstrate that exposure to alcohol and MDMA during adolescence, at doses that do not provoke apparent cognitive impairment when given separately, causes neurotoxic alterations affecting the DG region as well as persistent memory deficits. The findings highlight the elevated risk associated with the concurrent recreational use of alcohol and MDMA.

Research paper thumbnail of Protein Traffic Is an Intracellular Target in Alcohol Toxicity

Pharmaceuticals, 2011

Eukaryotic cells comprise a set of organelles, surrounded by membranes with a unique composition,... more Eukaryotic cells comprise a set of organelles, surrounded by membranes with a unique composition, which is maintained by a complex synthesis and transport system. Cells also synthesize the proteins destined for secretion. Together, these processes are known as the secretory pathway or exocytosis. In addition, many molecules can be internalized by cells through a process called endocytosis. Chronic and acute alcohol (ethanol) exposure alters the secretion of different essential products, such as hormones, neurotransmitters and others in a variety of cells, including central nervous system cells. This effect could be due to a range of mechanisms, including alcohol-induced alterations in the different steps involved in intracellular transport, such as glycosylation and vesicular transport along cytoskeleton elements. Moreover, alcohol consumption during pregnancy disrupts developmental processes in the central nervous system. No single mechanism has proved sufficient to account for these effects, and multiple factors are likely involved. One such mechanism indicates that ethanol also perturbs protein trafficking. The purpose of this review is to summarize our understanding of how ethanol exposure alters the trafficking of proteins in different cell systems, especially in central nervous system cells (neurons and astrocytes) in adult and developing brains.

Research paper thumbnail of Endocytosis in Cultured Neurons Is Altered by Chronic Alcohol Exposure

Toxicological Sciences, 2010

Endocytosis is required for many cellular pivotal processes, including membrane recycling, nutrie... more Endocytosis is required for many cellular pivotal processes, including membrane recycling, nutrient uptake, and signal transduction. This complex process is particularly relevant in polarized cells, such as neurons. Previous studies have demonstrated that alcohol alters intracellular traffic, including endocytosis, in several cell types. However, information on the effect of chronic alcohol exposure on this process in neurons is scarce. As an approach, we investigated the effect of alcohol exposure on the internalization of two widely used endocytic markers, albumin and transferrin, in developing hippocampal neurons in primary culture. The effect of this treatment on the levels of several representative proteins involved in the endocytic process was also analyzed. Some of these proteins are also involved in the organization of the actin cytoskeleton. Pretreatment of cells with inhibitors chlorpromazine or nystatin indicates that albumin is internalized mainly by caveolin-dependent endocytosis. On the other hand, alcohol decreases the endocytosis of both markers, although no qualitative changes in the distribution of either of these molecules were observed. Finally, the effect of ethanol on the proteins analyzed was heterogeneous. Alcohol decreases the levels of clathrin, AP-2, SNX9, Rab5, Rab11, EEA1, Cdc42, or RhoA but increases the amount of Arf6. Moreover, alcohol does not affect the levels of caveolin1, dynamin1, Rab7, and LAMP2. This toxic effect of alcohol on endocytosis could affect some of the important neuronal activities, which depend on this process, including cell signaling. Our results in neurons also stress the notion that one of the main targets of ethanol is intracellular transport.

Research paper thumbnail of Chronic Ethanol Exposure Alters the Levels, Assembly, and Cellular Organization of the Actin Cytoskeleton and Microtubules in Hippocampal Neurons in Primary Culture

Toxicological Sciences, 2010

The organization and dynamics of microtubules (MTs) and the actin cytoskeleton are critical for t... more The organization and dynamics of microtubules (MTs) and the actin cytoskeleton are critical for the correct development and functions of neurons, including intracellular traffic and signaling. In vitro ethanol exposure impairs endocytosis, exocytosis, and nucleocytoplasmic traffic in astrocytes and alters endocytosis in cultured neurons. In astrocytes, these effects relate to changes in the organization and/or function of MTs and the actin cytoskeleton. To evaluate this possibility in hippocampal cultured neurons, we analyzed if chronic ethanol exposure affects the levels, assembly, and cellular organization of both cytoskeleton elements and the possible underlying mechanisms of these effects by morphological and biochemical methods. In the experiments described below, we provide the first evidence that chronic alcohol exposure decreases the amount of both filamentous actin and polymerized tubulin in neurons and that the number of MTs in dendrites lowers in treated cells. Alcohol also diminishes the MT-associated protein-2 levels, which mainly localizes in the somatodendritic compartment in neurons. Ethanol decreases the levels of total Rac, Cdc42, and RhoA, three small guanosine triphosphatases (GTPases) involved in the organization and dynamics of the actin cytoskeleton and MTs. Yet when alcohol decreases the levels of the active forms (GTP bound) of Rac1 and Cdc42, it does not affect the active form of RhoA. We also investigated the levels of several effector and regulator molecules of these GTPases to find that alcohol induces heterogeneous results. In conclusion, our results show that MT, actin cytoskeleton organization, and Rho GTPase signaling pathways are targets for the toxic effects of ethanol in neurons.

Research paper thumbnail of Protective Effects of Lysophosphatidic Acid (LPA) on Chronic Ethanol-Induced Injuries to the Cytoskeleton and on Glucose Uptake in Rat Astrocytes

Journal of Neurochemistry, 2003

Ethanol induces severe alterations in membrane trafficking in hepatocytes and astrocytes, the mol... more Ethanol induces severe alterations in membrane trafficking in hepatocytes and astrocytes, the molecular basis of which is unclear. One of the main candidates is the cytoskeleton and the molecular components that regulate its organization and dynamics. Here, we examine the effect of chronic exposure to ethanol on the organization and dynamics of actin and microtubule cytoskeletons and glucose uptake in rat astrocytes. Ethanol-treated cells cultured in either the presence or absence of fetal calf serum showed a significant increase in 2-deoxyglucose uptake. Ethanol also caused alterations in actin organization, consisting of the dissolution of stress fibres and the appearance of circular filaments beneath the plasma membrane. When lysophosphatidic acid (LPA), which is a normal constituent of serum and a potent intercellular lipid mediator with growth factor and actin rearrangement activities, was added to ethanol-treated astrocytes cultured without fetal calf serum, it induced the re-appearance of actin stress fibres and the normalization of 2-deoxyglucose uptake. Furthermore, ethanol also perturbed the microtubule dynamics, which delayed the recovery of the normal microtubule organization following removal of the microtubule-disrupting agent nocodazole. Again, pre-treatment with LPA prevented this alteration. Ethanol-treated rodent fibroblast NIH3T3 cells that constitutively express an activated Rho mutant protein (GTP-bound form) were insensitive to ethanol, as they showed no alteration either in actin stress-fibre organization or in 2-deoxyglucose uptake. We discuss the putative signalling targets by which ethanol could alter the cytoskeleton and hexose uptake and the cytoprotective effect of LPA against ethanol-induced damages. The latter opens the possibility that LPA or a similar non-hydrolysable lipid derivative could be used as a cytoprotective agent against the noxious effects of ethanol.

Research paper thumbnail of Lysophosphatidic Acid Rescues RhoA Activation and Phosphoinositides Levels in Astrocytes Exposed to Ethanol

Journal of Neurochemistry, 2007

Long-term ethanol treatment substantially impairs glycosylation and membrane trafficking in prima... more Long-term ethanol treatment substantially impairs glycosylation and membrane trafficking in primary cultures of rat astrocytes. Our previous studies indicated that these effects were attributable to a primary alteration in the dynamics and organization of the actin cytoskeleton, although the molecular mechanism(s) remains to be elucidated. As small Rho GTPases and phosphoinositides are involved in the actin cytoskeleton organization, we now explore the effects of chronic ethanol treatment on these pathways. We show that chronic ethanol treatment of rat astrocytes specifically reduced endogenous levels of active RhoA as a result of the increase of in the RhoGAP activity. Furthermore, ethanol-treated astrocytes showed reduced phosphoinositides levels. When lysophosphatidic acid was added to ethanol-treated astrocytes, it rapidly reverted actin cytoskeleton reorganization and raised active RhoA levels and phosphoinositides content to those observed in untreated astrocytes. Overall, our results indicate that the harmful effects of chronic exposure to ethanol on a variety of actin dynamics-associated cellular events are primarily because of alterations of activated RhoA and phosphoinositides pools.

Research paper thumbnail of Chronic Ethanol Exposure Alters the Levels, Assembly, and Cellular Organization of the Actin Cytoskeleton and Microtubules in Hippocampal Neurons in Primary Culture

Toxicological Sciences, 2010

The organization and dynamics of microtubules (MTs) and the actin cytoskeleton are critical for t... more The organization and dynamics of microtubules (MTs) and the actin cytoskeleton are critical for the correct development and functions of neurons, including intracellular traffic and signaling. In vitro ethanol exposure impairs endocytosis, exocytosis, and nucleocytoplasmic traffic in astrocytes and alters endocytosis in cultured neurons. In astrocytes, these effects relate to changes in the organization and/or function of MTs and the actin cytoskeleton. To evaluate this possibility in hippocampal cultured neurons, we analyzed if chronic ethanol exposure affects the levels, assembly, and cellular organization of both cytoskeleton elements and the possible underlying mechanisms of these effects by morphological and biochemical methods. In the experiments described below, we provide the first evidence that chronic alcohol exposure decreases the amount of both filamentous actin and polymerized tubulin in neurons and that the number of MTs in dendrites lowers in treated cells. Alcohol also diminishes the MT-associated protein-2 levels, which mainly localizes in the somatodendritic compartment in neurons. Ethanol decreases the levels of total Rac, Cdc42, and RhoA, three small guanosine triphosphatases (GTPases) involved in the organization and dynamics of the actin cytoskeleton and MTs. Yet when alcohol decreases the levels of the active forms (GTP bound) of Rac1 and Cdc42, it does not affect the active form of RhoA. We also investigated the levels of several effector and regulator molecules of these GTPases to find that alcohol induces heterogeneous results. In conclusion, our results show that MT, actin cytoskeleton organization, and Rho GTPase signaling pathways are targets for the toxic effects of ethanol in neurons.

Research paper thumbnail of Polyphosphoinositide Metabolism and Golgi Complex Morphology in Hippocampal Neurons in Primary Culture is Altered by Chronic Ethanol Exposure

Alcohol and Alcoholism, 2013

Aims: Ethanol affects not only the cytoskeletal organization and activity, but also intracellular... more Aims: Ethanol affects not only the cytoskeletal organization and activity, but also intracellular trafficking in neurons in the primary culture. Polyphosphoinositide (PPIn) are essential regulators of many important cell functions, including those mentioned, cytoskeleton integrity and intracellular vesicle trafficking. Since information about the effect of chronic ethanol exposure on PPIn metabolism in neurons is scarce, this study analysed the effect of this treatment on three of these phospholipids. Methods: Phosphatidylinositol (PtdIns) levels as well as the activity and/or levels of enzymes involved in their metabolism were analysed in neurons chronically exposed to ethanol. The levels of phospholipases C and D, and phosphatidylethanol formation were also assessed. The consequence of the possible alterations in the levels of PtdIns on the Golgi complex (GC) was also analysed. Results: We show that phosphatidylinositol (4,5)-bisphosphate and phosphatidylinositol (3,4,5)-trisphosphate levels, both involved in the control of intracellular trafficking and cytoskeleton organization, decrease in ethanol-exposed hippocampal neurons. In contrast, several kinases that participate in the metabolism of these phospholipids, and the level and/or activity of phospholipases C and D, increase in cells after ethanol exposure. Ethanol also promotes phosphatidylethanol formation in neurons, which can result in the suppression of phosphatidic acid synthesis and, therefore, in PPIn biosynthesis. This treatment also lowers the phosphatidylinositol 4-phosphate levels, the main PPIn in the GC, with alterations in their morphology and in the levels of some of the proteins involved in structure maintenance. Conclusions: The deregulation of the metabolism of PtdIns may underlie the ethanol-induced alterations on different neuronal processes, including intracellular trafficking and cytoskeletal integrity.

Research paper thumbnail of Endocytosis in Cultured Neurons Is Altered by Chronic Alcohol Exposure

Toxicological Sciences, 2010

Endocytosis is required for many cellular pivotal processes, including membrane recycling, nutrie... more Endocytosis is required for many cellular pivotal processes, including membrane recycling, nutrient uptake, and signal transduction. This complex process is particularly relevant in polarized cells, such as neurons. Previous studies have demonstrated that alcohol alters intracellular traffic, including endocytosis, in several cell types. However, information on the effect of chronic alcohol exposure on this process in neurons is scarce. As an approach, we investigated the effect of alcohol exposure on the internalization of two widely used endocytic markers, albumin and transferrin, in developing hippocampal neurons in primary culture. The effect of this treatment on the levels of several representative proteins involved in the endocytic process was also analyzed. Some of these proteins are also involved in the organization of the actin cytoskeleton. Pretreatment of cells with inhibitors chlorpromazine or nystatin indicates that albumin is internalized mainly by caveolin-dependent endocytosis. On the other hand, alcohol decreases the endocytosis of both markers, although no qualitative changes in the distribution of either of these molecules were observed. Finally, the effect of ethanol on the proteins analyzed was heterogeneous. Alcohol decreases the levels of clathrin, AP-2, SNX9, Rab5, Rab11, EEA1, Cdc42, or RhoA but increases the amount of Arf6. Moreover, alcohol does not affect the levels of caveolin1, dynamin1, Rab7, and LAMP2. This toxic effect of alcohol on endocytosis could affect some of the important neuronal activities, which depend on this process, including cell signaling. Our results in neurons also stress the notion that one of the main targets of ethanol is intracellular transport.

Research paper thumbnail of Ethanol Reduces Zincosome Formation in Cultured Astrocytes

Alcohol and Alcoholism, 2011

Aims: Zinc is an ion that participates in basic cellular and tissular functions. Zinc deficiency ... more Aims: Zinc is an ion that participates in basic cellular and tissular functions. Zinc deficiency is present in many physiological and health problems affecting most body organs, including the brain. Among the circumstances involved in zinc deficiency, ethanol consumption is probably one of the most frequent. A dietary zinc supplement has been proposed as possibly being an efficient method to palliate zinc deficiency. Astrocytes form part of the hematoencephalic barrier, and they are apparently implicated in the homeostasis of the neuronal medium. In this work, we analyze the effect of ethanol on extracellular zinc management by rat astrocytes in culture. Methods: Intracellular levels of 'free zinc ions', in controls and 30 mM ethanol-treated astrocytes, were visualized by using the zinc fluorochrome TSQ. Cytoplasmic fluorescence and zincosome formation were measured after adding extracellular 50 µM ZnSO 4 to cell monolayers. Zincosomes were also observed at the electron microscopy level. Results: Exposure to ethanol for 7 days lowered the basal zinc levels of astrocytes by~30%. This difference was consistently maintained after the zinc pulse. Zinc ions were confined to bright fluorescent particles, the 'zincosomes', which appeared to be formed by the endocytic pathway. Zincosomes were less abundant in alcohol-treated cells, indicating a deficit in endocytoses as the origin of low zinc intake in astrocytes after ethanol treatment. Conclusions: Ethanol reduces both intracellular ionic zinc levels and extracellular zinc uptake, resulting in poorer zincosome formation. Given the endocytic nature of zincosomes, the effect of ethanol on membrane trafficking is apparently the origin of this deficit.

Research paper thumbnail of Protein Traffic Is an Intracellular Target in Alcohol Toxicity

Pharmaceuticals, 2011

Eukaryotic cells comprise a set of organelles, surrounded by membranes with a unique composition,... more Eukaryotic cells comprise a set of organelles, surrounded by membranes with a unique composition, which is maintained by a complex synthesis and transport system. Cells also synthesize the proteins destined for secretion. Together, these processes are known as the secretory pathway or exocytosis. In addition, many molecules can be internalized by cells through a process called endocytosis. Chronic and acute alcohol (ethanol) exposure alters the secretion of different essential products, such as hormones, neurotransmitters and others in a variety of cells, including central nervous system cells. This effect could be due to a range of mechanisms, including alcohol-induced alterations in the different steps involved in intracellular transport, such as glycosylation and vesicular transport along cytoskeleton elements. Moreover, alcohol consumption during pregnancy disrupts developmental processes in the central nervous system. No single mechanism has proved sufficient to account for these effects, and multiple factors are likely involved. One such mechanism indicates that ethanol also perturbs protein trafficking. The purpose of this review is to summarize our understanding of how ethanol exposure alters the trafficking of proteins in different cell systems, especially in central nervous system cells (neurons and astrocytes) in adult and developing brains.

Research paper thumbnail of Chronic Alcohol Alters Dendritic Spine Development in Neurons in Primary Culture

Neurotoxicity Research, 2013

Dendritic spines are specialised membrane protrusions of neuronal dendrites that receive the majo... more Dendritic spines are specialised membrane protrusions of neuronal dendrites that receive the majority of excitatory synaptic inputs. Abnormal changes in their density, size and morphology have been associated with various neurological and psychiatric disorders, including those deriving from drug addiction. Dendritic spine formation, morphology and synaptic functions are governed by the actin cytoskeleton. Previous in vivo studies have shown that ethanol alters the number and morphology of spines, although the mechanisms underlying these alterations remain unknown. It has also been described how chronic ethanol exposure affects the levels, assembly and cellular organisation of the actin cytoskeleton in hippocampal neurons in primary culture. Therefore, we hypothesised that the ethanol-induced alterations in the number and shape of dendritic spines are due to alterations in the mechanisms regulating actin cytoskeleton integrity. The results presented herein show that chronic exposure to moderate levels of alcohol (30 mM) during the first 2 weeks of culture reduces dendritic spine density and alters the proportion of the different morphologies of these structures in hippocampal neurons, which affects the formation of mature spines. Apparently, these effects are associated with an increase in the G-actin/F-actin ratio due to a reduction of the F-actin fraction, leading to changes in the levels of the different factors regulating the organisation of this cytoskeletal component. The data presented herein indicate that these effects occur between weeks 1 and 2 of culture, an important period in dendritic spines development. These changes may be related to the dysfunction in the memory and learning processes present in children prenatally exposed to ethanol.

Research paper thumbnail of Chronic Alcohol Exposure Affects the Cell Components Involved in Membrane Traffic in Neuronal Dendrites

Neurotoxicity Research, 2014

The specific traffic of the membrane components in neurons is a major requirement to establish an... more The specific traffic of the membrane components in neurons is a major requirement to establish and maintain neuronal domains-the axonal and the somatodendritic domains-and their polarized morphology. Unlike axons, dendrites contain membranous organelles, which are involved in the secretory pathway, including the endoplasmic reticulum, the Golgi apparatus and post-Golgi apparatus carriers, the cytoskeleton, and plasma membrane. A variety of molecules and factors are also involved in this process. Previous studies have shown that chronic alcohol exposure negatively affects several of these cell components, such as the Golgi apparatus or cytoskeleton in neurons. Yet very little information is available on the possible effects of this exposure on the remaining cell elements involved in intracellular trafficking in neurons, particularly in dendrites. By qualitative and quantitative electron microscopy, immunofluorescence and immunoblotting, we herein show that chronic exposure to moderate levels (30 mM) of ethanol in cultured neurons reduces the volume and surface density of the rough endoplasmic reticulum, and increases the levels of GRP78, a chaperone involved in endoplasmic reticulum stress. Ethanol also significantly diminishes the proportion of neurons that show an extension of Golgi into dendrites and dendritic Golgi outposts, a structure present exclusively in longer, thicker apical dendrites. Both Golgi apparatus types were also fragmented into a large number of cells. We also investigated the effect of alcohol on the levels of microtubule-based motor proteins KIF5, KIF17, KIFC2, dynein, and myosin IIb, responsible for transporting different cargoes in dendrites. Of these, alcohol differently affects several of them by lowering dynein and raising KIF5, KIFC2, and myosin IIb. These results, together with other previously published ones, suggest that practically all the protein trafficking steps in dendrites are altered to a greater or lesser extent by chronic alcohol exposure in neuronal cells, which may have negative repercussions for the development and maintenance of their polarized morphology and function.

Research paper thumbnail of Chronic ethanol exposure alters the levels, assembly, and cellular organization of the actin cytoskeleton and microtubules in hippocampal neurons in primary culture

Toxicological sciences : an official journal of the Society of Toxicology, 2010

The organization and dynamics of microtubules (MTs) and the actin cytoskeleton are critical for t... more The organization and dynamics of microtubules (MTs) and the actin cytoskeleton are critical for the correct development and functions of neurons, including intracellular traffic and signaling. In vitro ethanol exposure impairs endocytosis, exocytosis, and nucleocytoplasmic traffic in astrocytes and alters endocytosis in cultured neurons. In astrocytes, these effects relate to changes in the organization and/or function of MTs and the actin cytoskeleton. To evaluate this possibility in hippocampal cultured neurons, we analyzed if chronic ethanol exposure affects the levels, assembly, and cellular organization of both cytoskeleton elements and the possible underlying mechanisms of these effects by morphological and biochemical methods. In the experiments described below, we provide the first evidence that chronic alcohol exposure decreases the amount of both filamentous actin and polymerized tubulin in neurons and that the number of MTs in dendrites lowers in treated cells. Alcohol al...

Research paper thumbnail of Fluorescent Analogues of Plasma Membrane Sphingolipids Are Sorted to Different Intracellular Compartments in Astrocytes; Harmful Effects of Chronic Ethanol Exposure on Sphingolipid Trafficking and Metabolism

FEBS Letters, 2004

Sphingolipids are basic constituents of cellular membranes and are essential for numerous functio... more Sphingolipids are basic constituents of cellular membranes and are essential for numerous functions such as intracellular signalling. They are transported along the exocytic and endocytic pathways in eukaryotic cells. After endocytosis, fluorescent-labelled sphingolipids are sorted to distinct intracellular organelles prior to recycling (via early/recycling endosomes) or degradation (late endosomes/lysosomes). Here we examine, in primary cultures of rat astrocytes, the internalisation routes followed by C(6)-NBD-glucosylceramide (NBD-GlcCer) and C(6)-NBD-sphingomyelin (NBD-SM) and the effects of ethanol on their endocytic trafficking. Endocytosed plasma membrane NBD-GlcCer and NBD-SM are diverted to the Golgi apparatus and lysosomes, respectively. These different internalisation pathways are maintained regardless of the differentiation stage of astrocytes. Chronic ethanol exposure did not alter this endocytic sorting, but delayed the internalisation of both NBD-sphingolipids. Moreover, ethanol also stimulated the in situ metabolism of NBD-ceramide to NBD-GlcCer and NBD-SM. We conclude that in rat astrocytes internalised plasma membrane NBD-sphingolipids are sorted to different subcellular compartments. The exposure to chronic ethanol perturbed the lipid endocytic process and stimulated the de novo synthesis of NBD-sphingolipids, shifting the balance of sphingolipid metabolism in favour of the sphingomyelin pathway.

Research paper thumbnail of Actin Filaments are Involved in the Maintenance of Golgi Cisternae Morphology and intra-Golgi pH

Cell Motility and the Cytoskeleton, 2006

Here we examine the contribution of actin dynamics to the architecture and pH of the Golgi comple... more Here we examine the contribution of actin dynamics to the architecture and pH of the Golgi complex. To this end, we have used toxins that depolymerize (cytochalasin D, latrunculin B, mycalolide B, and Clostridium botulinum C2 toxin) or stabilize (jasplakinolide) filamentous actin. When various clonal cell lines were examined by epifluorescence microscopy, all of these actin toxins induced compaction of the Golgi complex. However, ultrastructural analysis by transmission electron microscopy and electron tomography/three-dimensional modelling of the Golgi complex showed that F-actin depolymerization first induces perforation/fragmentation and severe swelling of Golgi cisternae, which leads to a completely disorganized structure. In contrast, F-actin stabilization results only in cisternae perforation/fragmentation. Concomitantly to actin depolymerization-induced cisternae swelling and disorganization, the intra-Golgi pH significantly increased. Similar ultrastructural and Golgi pH alkalinization were observed in cells treated with the vacuolar H+ -ATPases inhibitors bafilomycin A1 and concanamycin A. Overall, these results suggest that actin filaments are implicated in the preservation of the flattened shape of Golgi cisternae. This maintenance seems to be mediated by the regulation of the state of F-actin assembly on the Golgi pH homeostasis.