Jaime Renau-Piqueras

Ethanol Increases Cytochrome P4502E1 and Induces Oxidative Stress in Astrocytes

Abstract: 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 that importance of oxidative stress as a mechanism involved in alcohol‐induced neural and brain damage.

ACTIN MICROFILAMENTS ARE ESSENTIAL FOR THE CYTOLOGICAL POSITIONING AND MORPHOLOGY OF THE GOLGI COMPLEX

The organization and function of the Golgi complex was studied in normal rat kidney cells following disruption of the actin cytoskeleton induced by cytochalasin D. In cells treated with these reagents, the reticular and perinuclear Golgi morphology acquired a cluster shape restricted to the centrosome region. Golgi complex alteration affected all Golgi subcompartments as revealed by double fluorescence staining with antibodies to the cis/middle Mannosidase II and the trans-Golgi network TGN38 proteins or vital staining with the lipid derivate C6-NBD-ceramide. The ultrastructural and stereological analysis showed that the Golgi cisternae remained attached in a stacked conformation, but they were swollen and contained electron-dense intra-cisternal bodies. The Golgi complex cluster remained linked to microtubules since it was fragmented and dispersed after treatment with nocodazole. Moreover, the reassembly of Golgi fragments after the disruption of the microtubuli with nocodazole does not utilize the actin microfilaments. The actin microfilament requirement for the disassembly and reassembly of the Golgi complex and for the ER-Golgi vesicular transport were also studied. The results show that actin microfilaments are not needed for either the retrograde fusion of the Golgi complex with the endoplasmic reticulum promoted by brefeldin A or the anterograde reassembly after the removal of the drug, or the ER-Golgi transport of VSV-G glycoprotein. However, actin microfilaments are directly involved in the subcellular localization and the morphology of the Golgi complex.

Ethanol Exposure Affects Glial Fibrillary Acidic Protein Gene Expression and Transcription During Rat Brain Development

Abstract: Exposure to ethanol during fetal development reduces the astroglial‐specific marker glial fibrillary acidic protein (GFAP) and its mRNA levels in brains of fetal rats and in radial glia in primary culture, affecting the proliferation and differentiation of astrocytes. The objectives of this study were to evaluate the possible effect of ethanol on GFAP mRNA levels in astrocytes and to investigate the molecular mechanism(s) involved in ethanol‐induced changes in GFAP expression by analyzing the GFAP transcription rate, GFAP mRNA stability, and GFAP DNA methylation. We show here that prenatal exposure to ethanol reduces significantly GFAP immunoreactivity and its mRNA levels in both astrocytes in primary culture and brains of pups from alcohol‐fed mothers. Runoff experiments from nuclei of astrocytes indicate that ethanol exposure decreases GFAP transcription rate significantly and reduces GFAP mRNA stability slightly. DNA methylation analysis indicates that prenatal ethanol exposure induces a hypermethylated state of the GFAP DNA in fetal brains. Methylation‐mediated repression of GFAP transcription could be a mechanism involved in ethanol‐induced reduction of GFAP expression. Ethanol‐induced alterations in GFAP expression and astroglial development may underlie the CNS dysfunctions observed after prenatal alcohol exposure.

Actin Microfilaments Facilitate the Retrograde Transport from the Golgi Complex to the Endoplasmic Reticulum in Mammalian Cells

The morphology and subcellular positioning of the Golgi complex depend on both microtubule and actin cytoskeletons. In contrast to microtubules, the role of actin cytoskeleton in the secretory pathway in mammalian cells has not been clearly established. Using cytochalasin D, we have previously shown that microfilaments are not involved in the endoplasmic reticulum–Golgi membrane dynamics. However, it has been reported that, unlike botulinum C2 toxin and latrunculins, cytochalasin D does not produce net depolymerization of actin filaments. Therefore, we have reassessed the functional role of actin microfilaments in the early steps of the biosynthetic pathway using C2 toxin and latrunculin B. The anterograde endoplasmic reticulum‐to‐Golgi transport monitored with the vesicular stomatitis virus‐G protein remained unaltered in cells treated with cytochalasin D, latrunculin B or C2 toxin. Conversely, the brefeldin A‐induced Golgi membrane fusion into the endoplasmic reticulum, the Golgi‐to‐endoplasmic reticulum transport of a Shiga toxin mutant form, and the subcellular distribution of the KDEL receptor were all impaired when actin microfilaments were depolymerized by latrunculin B or C2 toxin. These findings, together with the fact that COPI‐coated and uncoated vesicles contain β/γ‐actin isoforms, indicate that actin microfilaments are involved in the endoplasmic reticulum/Golgi interface, facilitating the retrograde Golgi‐to‐endoplasmic reticulum membrane transport, which could be mediated by the orchestrated movement of transport intermediates along microtubule and microfilament tracks.

PTOV1 Enables the Nuclear Translocation and Mitogenic Activity of Flotillin-1, a Major Protein of Lipid Rafts

ABSTRACT PTOV1 is a mitogenic protein that shuttles between the nucleus and the cytoplasm in a cell cycle-dependent manner. It consists of two homologous domains arranged in tandem that constitute a new class of protein modules. We show here that PTOV1 interacts with the lipid raft protein flotillin-1, with which it copurifies in detergent-insoluble floating fractions. Flotillin-1 colocalized with PTOV1 not only at the plasma membrane but, unexpectedly, also in the nucleus, as demonstrated by immunocytochemistry and subcellular fractionation of endogenous and exogenous flotillin-1. Flotillin-1 entered the nucleus concomitant with PTOV1, shortly before the initiation of the S phase. Protein levels of PTOV1 and flotillin-1 oscillated during the cell cycle, with a peak in S. Depletion of PTOV1 significantly inhibited nuclear localization of flotillin-1, whereas depletion of flotillin-1 did not affect nuclear localization of PTOV1. Depletion of either protein markedly inhibited cell proliferation under basal conditions. Overexpression of PTOV1 or flotillin-1 strongly induced proliferation, which required their localization to the nucleus, and was dependent on the reciprocal protein. These observations suggest that PTOV1 assists flotillin-1 in its translocation to the nucleus and that both proteins are required for cell proliferation.

Glial fibrillary acidic protein expression in rat brain and in radial glia culture is delayed by prenatal ethanol exposure.

Abstract: The alterations in astrocyte proliferation and differentiation induced by prenatal exposure to alcohol (PEA) suggest that ethanol exposure affects the radial glial cells, the main astrocytic precursors. We have investigated the effects of ethanol on the early stages of astrogliogenesis by analyzing the developmental pattern of vimentin and glial fibrillary acidic protein (GFAP) immunoreactivity and their mRNA levels during embryonic/fetal brain development and in radial glia in primary culture. GFAP appeared late in gestation and at day 5 of culture of radial glial, whereas GFAP mRNA was first detected on fetal day 15 and increased in content on fetal day 21. In contrast, the levels of vimentin and its mRNA were high at fetal day 15 but decreased on day 21. Alcohol exposure delays the appearance of GFAP and its mRNA and significantly decreases the GFAP expression in fetal brain and in primary culture of radial glial. In addition, some morphological alterations were observed in PEA glial cells in culture. These results demonstrate that astroglial precursor cells are damaged by prenatal exposure to ethanol and suggest that abnormalities in the astrogliogenesis may underlie the disruption in neuronal migration and other CNS alterations observed after prenatal ethanol exposure.

Immunocytochemical and biochemical demonstration of formaldhyde dehydrogenase (class III alcohol dehydrogenase) in the nucleus.

Alcohol dehydrogenase (ADH), the major enzyme catalyzing the biological oxidation of ethanol in mammals, includes four classes with very different capacities for ethanol oxidation. Class III ADH is present in all the tissues and is well conserved throughout evolution. This enzyme has a low activity with ethanol, is specific for the glutathione-dependent oxidation of formaldehyde, and is therefore a formaldehyde dehydrogenase (FALDH). Until now there have been few and conflicting studies concerning its intracellular distribution, which is important for the understanding of its role in cell function. In the present work we used biochemical and immunocytochemical methods to assess the distribution of FALDH in rat hepatocytes and astroglial cells. With the glutathione-dependent formaldehyde dehydrogenase assay, we found the highest activity in the cytosol of hepatocytes and brain cells (12 and 2.6 mU/mg protein, respectively), but nuclei also exhibited significant activity (1.16 and 2.1 mU/mg protein, respectively). The immunocytochemical results showed the presence of FALDH binding sites in both the cytoplasm and the nucleus of the different cell types studied. Whereas no specific gold particle labeling was seen associated with any cytoplasmic component, in the nucleus the particles were found mainly over condensed chromatin and interchromatin regions. Finally, the gold particle density over both the nucleus and cytoplasm was greater in differentiated than in proliferating astrocytes in primary culture. In contrast, class I ADH, primarily responsible for ethanol metabolism, was found only in the cytoplasm of hepatocytes. We propose that one of the functions of FALDH is to protect cell structures, including DNA, from the toxic effects of endogenous formaldehyde, which is an intermediate in many metabolic process.

Ethanol perturbs the secretory pathway in astrocytes.

Ethanol exposure induces retention of glycoproteins in growing astrocytes. We examined the intracellular sites at which this retention occurs and investigated whether this effect is accompanied by alterations in the Golgi complex and microtubular system. We studied the effects of ethanol on the Golgi complex structure, as well as on the secretory pathway functionality by monitoring both the transport of the VSV-G protein and the protein levels of several molecules involved in the regulation of this pathway. Ethanol was found to delay VSV-G transport, modify Golgi complex morphology, and reduce the number of secretory vesicles. Moreover, ethanol affected the levels of mannosidase II, p58, betaCOP, rbet1, and several Rab GTPases. It also affected microtubule organization and polymerization and the levels of the motor proteins kinesin and dynein. Most of these effects were dose-dependent. These alterations, together with those previously reported concerning biosynthesis of glycoconjugates, provide novel insights into how ethanol impairs brain development.

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

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.

Silencing of the Charcot-Marie-Tooth disease-associated gene GDAP1 induces abnormal mitochondrial distribution and affects Ca2+ homeostasis by reducing store-operated Ca2+ entry.

GDAP1 is an outer mitochondrial membrane protein that acts as a regulator of mitochondrial dynamics. Mutations of the GDAP1 gene cause Charcot-Marie-Tooth (CMT) neuropathy. We show that GDAP1 interacts with the vesicle-organelle trafficking proteins RAB6B and caytaxin, which suggests that GDAP1 may participate in the mitochondrial movement within the cell. GDAP1 silencing in the SH-SY5Y cell line induces abnormal distribution of the mitochondrial network, reduces the contact between mitochondria and endoplasmic reticulum (ER) and alters the mobilization of mitochondria towards plasma membrane upon depletion of ER-Ca(2+) stores. GDAP1 silencing does not affect mitochondrial Ca(2+) uptake, ER-Ca(2+), or Ca(2+) flow from ER to mitochondria, but reduces Ca(2+) inflow through store-operated Ca(2+) entry (SOCE) following mobilization of ER-Ca(2+) and SOCE-driven Ca(2+) entry in mitochondria. Our studies suggest that the pathophysiology of GDAP1-related CMT neuropathies may be associated with abnormal distribution and movement of mitochondria throughout cytoskeleton towards the ER and subplasmalemmal microdomains, resulting in a decrease in SOCE activity and impaired SOCE-driven Ca(2+) uptake in mitochondria.