TLR4 mediates the impairment of ubiquitin-proteasome and autophagy-lysosome pathways induced by ethanol treatment in brain.

New evidence indicates the involvement of protein degradation dysfunctions in neurodegeneration, innate immunity response and alcohol hepatotoxicity. We recently demonstrated that ethanol increases brain proinflammatory mediators and causes brain damage by activating Toll-like receptor 4 (TLR4) signaling in glia. However, it is uncertain if the ubiquitin-proteasome and autophagy-lysosome pathways are involved in ethanol-induced brain damage and whether the TLR4 response is implicated in proteolytic processes. Using the cerebral cortex of WT and TLR4-knockout mice with and without chronic ethanol treatment, we demonstrate that ethanol induces poly-ubiquitinated proteins accumulation and promotes immunoproteasome activation by inducing the expression of ß2i, ß5i and PA28α, although it decreases the 20S constitutive proteasome subunits (α2, ß5). Ethanol also upregulates mTOR phosphorylation, leading to a downregulation of the autophagy-lysosome pathway (ATG12, ATG5, cathepsin B, p62, LC3) and alters the volume of autophagic vacuoles. Notably, mice lacking TLR4 receptors are protected against ethanol-induced alterations in protein degradation pathways. In summary, the present results provide the first evidence demonstrating that chronic ethanol treatment causes proteolysis dysfunctions in the mouse cerebral cortex and that these events are TLR4 dependent. These findings could provide insight into the mechanisms underlying ethanol-induced brain damage.

Ethanol affects calmodulin and the calmodulin-binding proteins neuronal nitric oxide synthase and alphaII-spectrin (alpha-fodrin) in the nucleus of growing and differentiated rat astrocytes in primary culture.

The distribution of calmodulin (CaM) and the CaM-binding proteins neuronal nitric oxide synthase (nNOS) and alphaII-spectrin (alpha-fodrin) in the nucleus of growing and differentiated astrocytes was analysed using immunogold electronmicroscopy. We also analysed the effect of moderate ethanol exposure on these proteins. For this, female Wistar rat were fed with an alcoholic liquid diet and exposed to males after several weeks. Pregnant rats were fed with this diet and, after birth, the foetuses brains were used to establish primary cultures of astrocytes. Astrocytes from control and ethanol-exposed rats foetuses were cultured in the absence or presence of ethanol (30 mM) for 7 days (growing cells) and 21 days (differentiated astrocytes). Our results indicate that all the proteins studied appeared mainly on the condensed chromatin of both control- and alcohol-exposed cells and that there are significant variations in the amount of these proteins between quiescent and dividing astrocytes. Altogether, we have not found a co-localisation between CaM and the CaM-binding proteins.

RhoA and lysophosphatidic acid are involved in the actin cytoskeleton reorganization of astrocytes exposed to ethanol.

Astroglial cells play an important role in maintaining neuronal function in the adult and in the developing nervous system. Ethanol exposure induces profound alterations in the astrogliogenesis process, affecting important cell functions, including intracellular protein trafficking. Because the actin cytoskeleton plays a crucial role in intracellular protein transport, the aim of the present study was to analyze the effects of ethanol on actin cytoskeleton organization and the involvement of the RhoA signaling pathway in these effects. We show that RhoA and lysophosphatidic acid (LPA), an upstream activator of RhoA, stimulate the formation of stress fibers and focal adhesion in cortical astrocytes in primary culture. Exposure of cultured astrocytes to different concentrations of ethanol profoundly disorganizes the actin cytoskeleton, leading to the formation of actin rings at the cell periphery and decreasing the content of focal adhesion proteins. Furthermore, LPA treatment or RhoA transfection revert the ethanol-induced actin alterations in astrocytes, whereas transfection with an inactive mutant of RhoA is unable to revert the actin ring organization. In addition, inhibition of endogenous RhoA by C3 exoenzyme effectively blocks ethanol-induced actin ring formation. These results suggest that the effects of alcohol on actin cytoskeleton organization are mediated by the RhoA signaling pathway. Disruptions in actin organization may impair important astrocyte functions, participating in ethanol-induced astroglial and brain damage during development.

Ethanol impairs monosaccharide uptake and glycosylation in cultured rat astrocytes.

Astrocyte and glial-neuron interactions have a critical role in brain development, which is partially mediated by glycoproteins, including adhesion molecules and growth factors. Ethanol affects the synthesis, intracellular transport, subcellular distribution and secretion of these glycoproteins, suggesting alterations in glycosylation. We analyzed the effect of long-term exposure to low doses of ethanol (30 mm) on glycosylation process in growing cultured astrocytes in vitro. Cells were incubated for short (5 min) and long (90 min) periods with several radioactively labeled carbohydrate precursors. The uptake, kinetics and metabolism of these precursors, as well as the radioactivity distribution in protein gels were analyzed. The levels of GLUT1 and mannosidase II were also determined. Ethanol increased the uptake of monosaccharides and the protein levels of GLUT1 but decreased those of mannosidase II. It altered the carbohydrate moiety of proteins and increased cell surface glycoproteins containing terminal non-reduced mannose. These results indicate that ethanol impairs glycosylation in rat astrocytes, thus disrupting brain development.

Ethanol exposure enhances cell death in the developing cerebral cortex: role of brain-derived neurotrophic factor and its signaling pathways.

Exposure to ethanol during fetal development induces brain damage, causing cell loss in several brain areas and affecting synaptic connections. Because neurotrophin signaling plays an important role in neuronal survival and differentiation, we have investigated the effect of ethanol exposure on cell death in the developing cerebral cortex and whether this effect correlates with alterations in brain-derived neurotrophic factor (BDNF) levels, expression of its receptors, TrkB, and its signaling. We report that chronic ethanol intake during gestation and lactation enhances natural cell death and induces cell necrosis, decreases BDNF levels, and increases the ratio of the truncated to full-length TrkB mRNA receptors during postnatal developing cerebral cortex. Furthermore, we provide evidence that during brain development BDNF activates the extracellular signal-regulated kinases (ERK1 and ERK2) and the phosphoinoside-3-kinase (PI-3-K/Akt) pathways. However, BDNF-induced cell signaling throughout the above-mentioned survival pathways is significantly reduced by ethanol exposure. These findings suggest that ethanol-induced alterations in BDNF availability and in its receptor function might impair intracellular signaling pathways involved in cell survival, growth, and differentiation, leading to enhanced natural cell death during cerebral cortex development.

Síndrome alcohólico fetal y corazón / Fetal alcohol syndrome and heart

El consumo materno de alcohol durante la gestación afecta severamente al feto en desarrollo causando anomalías importantes en su sistema nervioso central, hígado, corazón, huesos craneofaciales, etc. El síndrome alcohólico fetal (SAF) describe la consecuencia sobre el feto de dicho consumo y el conjunto de dismorfias físicas, retraso en el crecimiento intrauterino y en el desarrollo intelectual. Dicho consumo, además, puede inducir daño tanto en el tejido muscular del corazón como en el desarrollo de este órgano, de forma que aproximadamente un 30 per cent de los niños con SAF presentan defectos cardíacos congénitos. Los estudios en modelos animales como roedores y pollo, así como en modelos in vitro permiten profundizar en los mecanismos teratogénicos del etanol sobre el corazón. (AU)

Role of thyroid hormone in craniofacial and eye development using a rat model.

Thyroid hormones (TH) are essential for somatic and neural development. Epidemiological studies have pointed to TH-dependent craniofacial features occurring during development. In an attempt to elucidate the precise role of TH in the developing eyes and adnexa (orbit, lids, nasolacrimal structures), we analysed the craniofacial and eyeball developmental characteristics in a rat model of congenital-neonatal hypothyroidism (HG), induced by combined chemical-surgical thyroidectomy. The heads and eyeballs from control and HG animals were obtained at key developmental stages and processed for scanning electron, light and transmission electron microscopy. On embryological day 13 (E13), significantly reduced values for head parameters (25% less), optic primordia area (0.053 +/- 0.0085 vs. 0.111 +/- 0.012 microm(2); p < 0.05) and volume (3.96 +/- 0.141 vs. 8.09 +/- 0.123 microm(3); p < 0.05) were found in the HG with respect to the controls. In addition, a delayed prenatal eye closure and postnatal eye opening took place in the treated rats. The photoreceptor and ganglion cell layer thickness displayed significantly lower values (p < 0.001) in HG, at each developmental time point. Postnatally, a delay in photoreceptor outer segment morphogenesis (in relation to retarded disc formation) and significantly lower values for ganglion cell nuclear volumes (p < 0.001) and nuclear pore density (p < 0.01) were observed in the TH-deficient animals. All data suggest that TH play a pivotal role in the development of the face and eye. Therefore, a series of defects due to a loss of TH actions involved in anterior-posterior development of the head and face and the loss of TH-dependent signals crucial for cell differentiation, migration, proliferation and establishment of definitive cell phenotypes in the eyes may appear. Gestational and neonatal screenings for thyroid functioning are suggested to paediatricians and ophthalmologists in order to prevent craniofacial malformations and visual abnormalities.

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 beta/gamma-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.

Neural cell adhesion molecule is endocytosed via a clathrin-dependent pathway.

Neural cell adhesion molecule (NCAM) constitutes a group of cell surface glycoproteins that regulate cell-cell interactions in the developing and adult brain. Endocytosis is a mechanism which dynamically controls the amount of cell surface NCAM expression and may involve the rapid changes occurring in NCAM expression under certain physiological or pathological conditions. However, the endocytic pathway of NCAM is presently unknown. Using astrocytes in culture and immunofluorescence we show that NCAM is internalized and that the immunolabelling presents a high degree of colocalization with clathrin, alpha-adaptin and transferrin, suggesting that NCAM is endocytosed by a clathrin-dependent pathway. Potassium depletion which disrupts clathrin-mediated endocytosis, inhibited internalization of NCAM. Electron microscopy and immunogold studies also demonstrate that the surface of clathrin-coated vesicles are also immunolabelled for both alpha-adaptin and PSA-NCAM, the highly sialylated isoform of NCAM. Furthermore, immunoprecipation studies demonstrate that NCAM is associated with both clathrin and alpha-adaptin, a component of adaptor complex AP-2, in brain, neurons and astrocytes. These findings indicate that NCAM is mainly endocytosed via clathrin-coated vesicles, suggesting a possible mechanism that may contribute to the rapid changes in NCAM expression at the cell surface.

Glia and fetal alcohol syndrome.

Glial cells and their interactions with neurons play vital roles during the ontogeny of the nervous system and in the adult brain. Alcohol intake during pregnancy can cause mental retardation and neurobehavioral disorders as well as fetal alcohol syndrome (FAS). Clinical and experimental evidence indicate that in utero alcohol exposure induces structural and functional abnormalities in gliogenesis and in glial-neuronal interactions, suggesting a potential role of glial cells on ethanol-induced developmental brain abnormalities. In vivo studies have shown ethanol-associated alterations in the migration of neurons and radial glial as well as in astrogliogenesis and myelin development. In astrocytes in primary culture, ethanol has been found to (1) impair cell growth and differentiation, (2) decrease the levels of glialfibrillary acidic protein or GFAP (an astrocyte marker) and its gene expression and (3) interfere with the stimulatory effect of trophic factors affecting their release and receptor expression. Evidence also suggests that ethanol affects intracellular protein trafficking, which may mediate some effects of ethanol on astroglial cells. These findings suggest that glial cells are target of ethanol toxicity during brain development and may underlie the neurodevelopmental abnormalities observed after in utero alcohol exposure and in FAS.

Alcohol exposure alters the expression pattern of neural cell adhesion molecules during brain development.

Neural cell adhesion molecules (NCAMs) play critical roles during development of the nervous system. The aim of this study is to investigate the possible effect of ethanol exposure on the pattern of expression and sialylation of NCAM isoforms during postnatal rat brain development because alterations in NCAM content and distribution have been associated with defects in cell migration, synapse formation, and memory consolidation, and deficits in these processes have been observed after in utero alcohol exposure. The expression of NCAM isoforms in the developing cerebral cortex of pups from control and alcohol-fed mothers was assessed by western blotting, ribonuclease protection assay, and immunocytochemistry. The highly sialylated form of NCAM [polysialic acid (PSA)-NCAM] is mainly expressed during the neonatal period and then is down-regulated in parallel with the appearance of NCAM 180 and NCAM 140. Ethanol exposure increases PSA-NCAM levels during the neonatal period, delays the loss of PSA-NCAM, decreases the amount of NCAM 180 and NCAM 140 isoforms, and reduces sialyltransferase activity during postnatal brain development. Neuraminidase treatment of ethanol-exposed neonatal brains leads to more intense band degradation products, suggesting a higher content of NCAM polypeptides carrying PSA in these samples. However, NCAM mRNA levels are not changed by ethanol. Immunocytochemical analysis demonstrates that ethanol triggers an increase in PSA-NCAM immunolabeling in the cytoplasm of astroglial cells, accompanied by a decrease in immunogold particles over the plasma membrane. These findings indicate that ethanol exposure during brain development alters the pattern of NCAM expression and suggest that modification of NCAM could affect neuronal-glial interactions that might contribute to the brain defects observed after in utero alcohol exposure.

Endocytosis and transcytosis in growing astrocytes in primary culture. Possible implications in neural development.

Endocytosis constitutes an essential process in the regulation of the expression of cell surface molecules and receptors and, therefore, could participate in the neural-glial interactions occurring during brain development. However, the relationship between endocytic pathways in astroglial cells under physiological and pathological conditions remains poorly understood. We analyzed the endocytosis and transcytosis processes in growing astrocytes and the possible effect of ethanol on these processes. Evidence demonstrates that ethanol affects endocytosis in the liver and we showed that ethanol exposure during brain development alters astroglial development changing plasma membrane receptors and surface glycoprotein composition. To study these processes we use several markers for receptor-mediated endocytosis, fluid phase endocytosis and non-specific endocytosis. These markers were labeled for fluorescence microscopy and electron microscopy. 125I-BSA was used to study the effect of ethanol on the internalization and recycling of this macromolecule. The distribution of several proteins involved in endocytosis (caveolin, clathrin, rab5 and beta-COP) was analyzed using immunofluorescence, immunoelectron microscopy and immunoblotting. Our results indicate that growing astrocytes have a developed endocytic system mainly composed of caveolae, clathrin coated pits and vesicles, tubulo-vesicular and spheric endosomes, multivesicular bodies and lysosomes. Ethanol exposure induces a fragmentation of tubular endosomes, decreases the internalization of 125I-BSA, alters the processing of internalized BSA, and decreases the levels of caveolin, clathrin, rab5 and beta-COP. These results indicate that ethanol alters the endocytosis and transcytosis processes and impairs protein trafficking in astrocytes, which could perturb astrocyte surface expression of molecules involved in neuronal migration and maturation during brain development.

Colocalization of aldehyde dehydrogenases and Fe/NADPH-induced lipid peroxidation in tissue sections of rat retina.

Epidemiological and experimental studies suggest the involvement of lipid peroxidation (LPO) in retinal diseases. Clinicians usually prescribe antioxidants to help in the treatment of proliferative diabetic vitreoretinopathy and age-related macular degeneration. In spite of this, these processes inexorably induce visual impairment and may progress towards blindness. In addition to other pathogenic mechanisms not fully understood, it may be that peroxidic aldehydes from LPO occurring in the eyes, acting as cytotoxic chemicals, mediate in these chronic disorders. To test the mechanisms of removing peroxidic aldehydes from retinal cells and in an attempt to understand long-lasting changes induced by LPO, the distribution and activity of aldehyde dehydrogenases (ALDH) in the rat retina were studied and compared with the LPO sites induced by iron/nicotine adenine dinucleotide phosphate. Histochemical and immunocytochemical assays revealed the colocalization of LPO and ALDH, mainly in the photoreceptors and inner retinal layers. This suggests the involvement of ALDH in detoxifying peroxidic aldehydes from the retina. Any change in ALDH retinal expression and distribution might be of crucial importance in assessing the paths of LPO-mediated vitreoretinopathies. Further research is needed to evaluate these findings and their application to new ophthalmic therapy.

The golgi-associated COPI-coated buds and vesicles contain beta/gamma -actin.

It has been shown previously that the morphology and subcellular positioning of the Golgi complex is controlled by actin microfilaments. To further characterize the association between actin microfilaments and the Golgi complex, we have used the Clostridium botulinum toxins C2 and C3, which specifically inhibit actin polymerization and cause depolymerization of F-actin in intact cells by the ADP ribosylation of G-actin monomers and the Rho small GTP-binding protein, respectively. Normal rat kidney cells treated with C2 showed that disruption of the actin and the collapse of the Golgi complex occurred concomitantly. However, when cells were treated with C3, the actin disassembly was observed without any change in the organization of the Golgi complex. The absence of the involvement of Rho was further confirmed by the treatment with lysophosphatidic acid or microinjection with the constitutively activated form of RhoA, both of which induced the stress fiber formation without affecting the Golgi complex. Immunogold electron microscopy in normal rat kidney cells revealed that beta- and gamma-actin isoforms were found in Golgi-associated COPI-coated buds and vesicles. Taken together, the results suggest that the Rho signaling pathway does not directly regulate Golgi-associated actin microfilaments, and that beta- and gamma-actins might be involved in the formation and/or transport of Golgi-derived vesicular or tubular intermediates.

Effects of vitamin A deficiency on mitochondrial function in rat liver and heart.

The aim of this study was to investigate comparative effects of vitamin A deficiency on respiratory activity and structural integrity in liver and heart mitochondria. Male rats were fed a liquid control diet (control rats) or a liquid vitamin A-deficient diet (vitamin A-deficient rats) for 50 days. One group of vitamin-A deficient rats was refed a control diet for 15 days (vitamin A-recovered rats). To assess the respiratory function of mitochondria the contents of coenzyme Q (ubiquinone, CoQ), cytochrome c and the activities of the whole electron transport chain and of each of its respiratory complexes were evaluated. Chronic vitamin A deficiency promoted a significant increase in the endogenous coenzyme Q content in liver and heart mitochondria when compared with control values. Vitamin A deficiency induced a decrease in the activity of complex I (NADH-CoQ reductase) and complex II (succinate-CoQ reductase) and in the levels of complex I and cytochrome c in heart mitochondria. However, NADH and succinate oxidation rates were maintained at the control levels due to an increase in the CoQ content in accordance with the kinetic behaviour of CoQ as an homogeneous pool. On the contrary, the high CoQ content did not affect the electron-transfer rate in liver mitochondria, whose integrity was preserved from the deleterious effects of the vitamin A deficiency. Ultrastructural assessment of liver and heart showed that vitamin A deficiency did not induce appreciable alterations in the morphology of their mitochondria. After refeeding the control diet, serum retinol, liver and heart CoQ content and the activity of complex I and complex II in heart mitochondria returned to normality. However, the activities of both whole electron transfer chain and complex I in liver were increased over the control values. The interrelationships between physiological antioxidants in biological membranes and the beneficial effects of their administration in mitochondrial diseases are discussed.

Lipid peroxidation in proliferative vitreoretinopathies.

PURPOSE: To study the lipid hydroperoxide activity in vasoproliferative and fibroproliferative retinal disorders. METHODS: Vitreous body samples from patients undergoing vitrectomy because of proliferative vitreoretinopathy (PVR; n = 12) or proliferative diabetic retinopathy (PDR; n = 15), and rhegmatogenous retinal detachment/macular hole/epiretinal membranes as the comparison group (CG; n = 14), were analysed for protein content and basal and induced lipid peroxidation (LPO), as determined by the thiobarbituric acid reactive substances (TBARS) test and LPO 586 commercial kit. The antioxidant activity for superoxide dismutase (SOD) and catalase (CAT) was also assayed. RESULTS: Malondialdehyde (MDA)-like metabolites and 4-hydroxynonenal (4-HNE) mean values were first measured to assess basal LPO, and found to be significantly higher in the PVR and PDR cases than in the CG (p < or = 0.0001). LPO induced by nicotine adenine dinucleotide phosphate iron (NADPH-Fe) was then assayed and the data showed that MDA mean values were 5-fold greater for the PVR and PDR eyes than in the case of basal LPO (p < or = 0.0001). SOD activity was significantly smaller in the PVR (p = 0.0010) and PDR (p < or = 0.0001) groups than in the CG. CAT levels displayed significantly lower values in the PVR and PDR cases than in the CG (p < or = 0.0001). No significant differences in free radical (FR) formation and antioxidant status between PVR and PDR patients were observed. CONCLUSIONS: Fibrovascular proliferative vitreoretinopathies correlate with increased FR formation and decreased antioxidant activity in the human vitreous body.

N-Ras induces alterations in Golgi complex architecture and in constitutive protein transport.

Aberrant glycosylation of proteins and lipids is a common feature of many tumor cell types, and is often accompanied by alterations in membrane traffic and an anomalous localization of Golgi-resident proteins and glycans. These observations suggest that the Golgi complex is a key organelle for at least some of the functional changes associated with malignant transformation. To gain insight into this possibility, we have analyzed changes in the structure and function of the Golgi complex induced by the conditional expression of the transforming N-Ras(K61) mutant in the NRK cell line. A remarkable and specific effect associated with this N-Ras-induced transformation was a conspicuous rearrangement of the Golgi complex into a collapsed morphology. Ultrastructural and stereological analyses demonstrated that the Golgi complex was extensively fragmented. The collapse of the Golgi complex was also accompanied by a disruption of the actin cytoskeleton. Functionally, N-Ras-transformed KT8 cells showed an increase in the constitutive protein transport from the trans-Golgi network to the cell surface, and did not induce the appearance of aberrant cell surface glycans. The Golgi complex collapse, the actin disassembly, and the increased constitutive secretion were all partially inhibited by the phospholipase A2 inhibitor 4-bromophenylacyl bromide. The results thus suggest the involvement of the actin cytoskeleton in the shape of the Golgi complex, and intracellular phospholipase A2 in its architecture and secretory function.

Distribution of neurofilaments in the telencephalon and mesencephalon of the adult and developing Gallotia galloti lizard.

The location and chronology during development of the immunoreactivity due to the presence of neurofilaments (NF) in telencephalon and mesencephalon of the lizard Gallotia galloti has been studied. For this purpose we have used two antibodies recognizing both phosphorylated and non phosphorylated neurofilaments (NF), a polyclonal Ab (NF 005), and a commercial monoclonal antibody (NF-200). The study was completed by using the Bielschowsky technique. During ontogeny, the anti-NF 005 immunoreactivity appeared at E40 in some tracts in mesencephalon and increased in intensity in isolated nerve fibers, tracts and commissurae till adult. However, a weak staining appeared in some neurons. In telencephalon, the reactivity was detected only in adult specimens. It was clearly more abundant in mesencephalon than in telencephalon, which could indicate that a greater complexity and functional importance exist in the lizard midbrain in relation to other primitive regions as the basal nuclei and cortical areas. In contrast to young specimens, the monoclonal anti-NF 200 was detected in neuronal perikarya, dendrites and axons in adults. Thus, in lizards, both antibodies highly recognized phosphorylated and non-phosphorylated forms of proteins of NF (NF-H). In mammals, these forms of proteins are implicated in axonal maturation. The presence of these NF in reptiles, identified for the first time, proved to be phylogenetically stable. The anti-NF immunoreactivity distribution occurs both caudo-rostrally and from the ventral to the dorsal regions.

Intracellular location, temporal expression, and polysialylation of neural cell adhesion molecule in astrocytes in primary culture.

Neural cell adhesion molecules (NCAMs) constitute a group of cell surface glycoproteins that control cell-cell interactions and play important morphoregulatory roles in the developing and regenerating nervous system. NCAMs exist in a variety of isoforms differing in the cytoplasmic domain and/or their content in sialic acid. The highly sialylated form (PSA-NCAM) is expressed by neurons, whereas it is believed that the less sialylated NCAM forms are synthesised by astrocytes. Moreover, little is known about the molecular sequence of the events that contribute to its expression at the cell surface. Here we report that during the proliferation of cortical astrocytes, at 4 days in primary culture, these cells expressed PSA-NCAM as well as NCAM 180. Then, during cell differentiation these isoforms progressively disappeared and the NCAM 140 became predominant. By immunofluorescence and immunocytochemistry studies we also show that PSA-NCAM and NCAM are first observed in small cytoplasmic spots or vesicles, located in or near the Golgi apparatus, as demonstrated by their co-localization with labelled wheat germ agglutinin (WGA) in this cell organelle. Thereafter, immunostained cytoplasmic NCAM gradually disappeared and became detectable at the cell surface of differentiating astrocytes. We also describe for the first time sialyltransferase activity in these cells and report that the levels of this activity correlated with the decrease in PSA-NCAM expression during the differentiation of astrocytes. These results will contribute to our understanding of the PSA and NCAM intracellular transport pathways and their expression at the cell surface. Moreover, the presence of PSA-NCAM in astrocytes suggests their possible role in nerve branching, fasciculation, and synaptic plasticity.

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.