RESUMEN
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.
Asunto(s)
Señalización del Calcio/genética , Calcio/metabolismo , Homeostasis/fisiología , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Interferencia de ARN/fisiología , Adenosina Trifosfato/farmacología , Señalización del Calcio/efectos de los fármacos , Línea Celular Transformada , Quelantes/farmacología , Proteína Coatómero/metabolismo , Citoesqueleto/metabolismo , Ácido Egtácico/farmacología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/microbiología , Inhibidores Enzimáticos/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Homeostasis/efectos de los fármacos , Homeostasis/genética , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Microscopía Electrónica de Transmisión , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mitocondrias/ultraestructura , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/metabolismo , Mutación Missense/genética , Proteínas del Tejido Nervioso/genética , Transporte de Proteínas/genética , Receptores de Superficie Celular/metabolismo , Transfección , Proteínas de Unión al GTP rab/metabolismoRESUMEN
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.
Asunto(s)
Etanol/toxicidad , Aparato de Golgi/efectos de los fármacos , Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Fosfatos de Fosfatidilinositol/metabolismo , Animales , Células Cultivadas , Etanol/administración & dosificación , Femenino , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Hipocampo/metabolismo , Hipocampo/ultraestructura , Neuronas/metabolismo , Neuronas/ultraestructura , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Alcohol abuse and alcoholism can cause brain damage, loss of white matter, myelin fiber disruption, and even neuronal injury. The underlying mechanisms of these alterations remain elusive. We have shown that chronic ethanol intake, by activating glial toll-like receptor 4 (TLR4) receptors, triggers the production of inflammatory mediators and can cause brain damage. Because neuroinflammation may be associated with demyelination and neuronal damage, we evaluate whether the ethanol-induced TLR4-dependent proinflammatory environment in the brain could be involved in the myelin disruptions observed in alcoholics. Using brains from wild-type (WT) and TLR4 knockout (KO, TLR4(-/-) ) mice, we demonstrate that chronic ethanol treatment downregulated proteins involved in myelination [proteolipid protein (PLP), myelin basic protein (MBP), myelin-oligodendrocyte glycoprotein, 2,3-cyclic-nucleotide-3-phosphodiesterase, and myelin-associated glycoprotein], while increased chondroitin sulfate proteoglycan NG2 (NG2)-proteoglycan in several brain regions of ethanol-treated WT mice. The immunohistochemistry analysis also revealed that ethanol-treatment-altered myelin morphology reduced the number of MBP-positive fibers and caused oligodendrocyte death, as demonstrated by an increase in caspase-3-positive oligodendrocytes. The in vivo imaging system further confirmed that chronic ethanol intake markedly reduced the PLP in WT mice. Most myelin alterations were not observed in brains from ethanol-treated TLR4(-/-) mice. Electron microscopy studies revealed that although 41-47% of axons showed myelin sheath disarrangements in the cerebral cortex and corpus callosum of WT ethanol-treated mice, respectively, small focal fiber disruptions were noticed in these brain areas of ethanol-treated TLR4(-/-) mice. In summary, the present results suggest that ethanol-induced neuroinflammation might be involved in myelin disruptions and white matter loss observed in human alcoholics.
Asunto(s)
Alcoholismo/metabolismo , Alcoholismo/patología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de la Mielina/metabolismo , Vaina de Mielina/patología , Receptor Toll-Like 4/metabolismo , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Animales , Antígenos/metabolismo , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Depresores del Sistema Nervioso Central/administración & dosificación , Modelos Animales de Enfermedad , Etanol/administración & dosificación , Femenino , Fluoresceínas , Regulación de la Expresión Génica/genética , Indoles , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica , Proteínas de la Mielina/genética , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/metabolismo , Fibras Nerviosas Mielínicas/patología , Oligodendroglía/patología , Compuestos Orgánicos , Proteoglicanos/metabolismo , Receptor Toll-Like 4/deficienciaRESUMEN
In the present study, we analyze the effects of ethanol on the Golgi structure and membrane transport in differentiated PC12 cells, which are used as a model of neurons. Chronic exposure to moderate doses of ethanol induces Golgi fragmentation, a common characteristic of many neurodegenerative diseases. Alcohol impaired the lateral linking of stacks without causing microtubule damage. Extensive immunocytochemical and western blot analyses of representative Golgi proteins showed that few, but important, proteins are significantly affected. Thus, alcohol exposure induced a significant ER-to-Golgi transport delay, the retention of the GTPase Rab1 in the Golgi membranes and the accumulation of tethering factor p115 in the cytosol. These modifications would explain the observed fragmentation. The amount of p115 and the stacking protein GRASP65 increased in alcohol-treated cells, which might be a mechanism to reverse Golgi damage. Importantly, the overexpression of GTP-tagged Rab1 but not of a dominant-negative Rab1 mutant, restored the Golgi morphology, suggesting that this protein is the main target of alcohol. Taken together, our results support the view that alcohol and neurodegenerative diseases such as Parkinson have similar effects on intracellular trafficking and provide new clues on the neuropathology of alcoholism.
Asunto(s)
Diferenciación Celular , Retículo Endoplásmico/metabolismo , Etanol/farmacología , Aparato de Golgi/metabolismo , Proteínas de Unión al GTP rab1/genética , Animales , Proteínas de la Matriz de Golgi , Proteínas de la Membrana/metabolismo , Células PC12 , Transporte de Proteínas , Ratas , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab1/metabolismoRESUMEN
N-substituted benztropine (BZT) analogs are molecules that display high affinity for the dopamine transporter (DAT), therapeutic-like effects in animal models of cocaine abuse, and psychopharmacological characteristics consistent with those of a substitute medication for cocaine addiction. Since amphetamine (Amph) and cocaine share mechanisms of action at the DAT, we evaluated the effectiveness of a BZT analog in animal models of Amph addiction. We tested in mice and rats the effects of the BZT derivative, 3α-[bis(4-fluorophenyl)methoxy]-tropane (AHN-1055), on Amph-induced conditioned place preference (CPP), locomotor activity, sensitization, self-administration and ΔFosB accumulation in the nucleus accumbens (NAc). The results showed that AHN-1055 did not produce rewarding, stimulant, or sensitized locomotor effects in mice when administered alone but it readily blocked the rewarding, stimulant, and sensitizing effects of repeated Amph exposure. Furthermore, in mice undergoing conditioning in the CPP paradigm, the BZT analog prevented the accumulation of ΔFosB protein induced in the NAc shell region by Amph treatment. Notably, treatment with AHN-1055 dose-dependently reduced Amph self-administration in rats with a steady history of voluntary Amph intake. These results provide a straightforward demonstration that a BZT derivative with binding affinity for DAT exhibits high efficacy in animal models of Amph abuse, suggesting that the novel generation of BZT analogs could have wider therapeutic applications in stimulant-spectrum disorders than those previously recognized.
Asunto(s)
Trastornos Relacionados con Anfetaminas/tratamiento farmacológico , Conducta Animal/efectos de los fármacos , Benzotropina/análogos & derivados , Inhibidores de Captación de Dopamina/farmacología , Anfetamina/farmacología , Animales , Benzotropina/farmacología , Condicionamiento Psicológico/efectos de los fármacos , Modelos Animales de Enfermedad , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/antagonistas & inhibidores , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/fisiología , Masculino , Ratones , Actividad Motora , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/fisiología , Ratas , Recompensa , AutoadministraciónRESUMEN
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 â¼30%. 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.
Asunto(s)
Astrocitos/metabolismo , Vesículas Citoplasmáticas/metabolismo , Etanol/farmacología , Zinc/deficiencia , Zinc/metabolismo , Animales , Astrocitos/efectos de los fármacos , Astrocitos/ultraestructura , Barrera Hematoencefálica/metabolismo , Células Cultivadas , Vesículas Citoplasmáticas/ultraestructura , Endocitosis/efectos de los fármacos , Homeostasis , Ratas , Zinc/química , Zinc/farmacologíaRESUMEN
Chronic vitamin A deficiency induces a substantial delay in the rates of weight and height gain in both humans and experimental animals. This effect has been associated with an impaired nutrient metabolism and loss of body protein. Therefore, we analyzed the effect of vitamin A deficiency on endogenous proteolysis and nitrogen metabolism and its reversibility with all-trans retinoic acid (RA). Male weanling rats, housed in pairs, were pair-fed a vitamin A-deficient (VAD) or control diet until they were 60 d old. A group of deficient rats were further treated with daily intraperitoneal injections of all-trans RA for 10 d. Final body and tissue (i.e. liver and heart) weights were significantly lower and tissue:body weight ratios were similar in VAD rats and in controls. Conversely, the epididymal white fat:body weight ratio and the plasma concentrations of alanine aminotransferase and adiponectin were significantly higher in VAD rats, which also had hepatic macrovesicular lipid accumulations. Plasma and gastrocnemius muscle 3-methylhistidine, urine nitrogen, and plasma and urine urea concentrations were all significantly higher in the VAD group. The expression of the genes encoding urea cycle enzymes and their activities increased in VAD livers. These changes were partially reverted by all-trans RA. We propose that fuel partitioning in vitamin A deficiency may shift from fatty acids to protein catabolism as an energy source. Our results emphasize the importance of vitamin A on the energy balance control system and they provide an explanation for the role of vitamin A in protein turnover, development, and growth.
Asunto(s)
Antioxidantes/uso terapéutico , Hígado/metabolismo , Tretinoina/uso terapéutico , Urea/metabolismo , Deficiencia de Vitamina A/metabolismo , Animales , Antioxidantes/farmacología , Inducción Enzimática , Peroxidación de Lípido/efectos de los fármacos , Hígado/enzimología , Hígado/ultraestructura , Masculino , Metilhistidinas/sangre , Metilhistidinas/metabolismo , Músculo Esquelético/metabolismo , Nitrógeno/metabolismo , Ratas , Retinoides/sangre , Retinoides/metabolismo , Tretinoina/farmacología , Triglicéridos , Deficiencia de Vitamina A/tratamiento farmacológico , Deficiencia de Vitamina A/enzimologíaRESUMEN
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.
Asunto(s)
Giro Dentado/efectos de los fármacos , Etanol/toxicidad , Aprendizaje por Laberinto/efectos de los fármacos , Recuerdo Mental/efectos de los fármacos , N-Metilaspartato/toxicidad , Neurogénesis/efectos de los fármacos , Neurotoxinas/toxicidad , Factores de Edad , Animales , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dendritas/efectos de los fármacos , Giro Dentado/patología , Proteína Doblecortina , Sinergismo Farmacológico , Masculino , Microglía/efectos de los fármacos , Orientación/efectos de los fármacos , Ratas , Ratas Long-Evans , Retención en Psicología/efectos de los fármacosRESUMEN
Nucleocytoplasmic transport is a crucial process for cell function. We assessed the general effect of chronic alcohol exposure on this transport in growing astrocytes for the first time. Import and export of proteins to the nucleus were examined by pulse-chase experiments using (3)H-methionine, and we showed that ethanol induces a delay in both processes. Furthermore, we took an approach to evaluate the mechanisms involved in this effect. Whereas alcohol did not affect the amount and the distribution of several representative proteins that participate in nuclear import, such as RanBP1, RanGAP1 and the importins alpha2 and beta3, it decreased the amount of Exp1/CRM1, which is a general export receptor involved in the nuclear export. In addition, the density and distribution of nuclear pore complexes, which contribute to nucleocytoplasmic transport, were also affected by ethanol. These effects can be related with changes found in the content of several proteins associated with the nuclear envelope and the nuclear pore complex structure such as lamins A/C, and nucleoporins p62 and RanBP2, respectively. These results suggest that ethanol could interfere with some of the important processes regulated by nucleocytoplasmic transport in astrocytes and support the idea that one of the main ethanol targets is intracellular transport.
Asunto(s)
Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Etanol/toxicidad , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/fisiología , Animales , Astrocitos/ultraestructura , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Núcleo Celular/ultraestructura , Células Cultivadas , RatasRESUMEN
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.
Asunto(s)
Biomarcadores de Tumor/fisiología , Núcleo Celular/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/fisiología , Transporte Activo de Núcleo Celular/fisiología , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Ciclo Celular/fisiología , Núcleo Celular/química , Proliferación Celular , Células Cultivadas , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/metabolismo , Sustancias de Crecimiento/fisiología , Humanos , Microdominios de Membrana/química , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/genética , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
We have previously reported that actin filaments are involved in protein transport from the Golgi complex to the endoplasmic reticulum. Herein, we examined whether myosin motors or actin comets mediate this transport. To address this issue we have used, on one hand, a combination of specific inhibitors such as 2,3-butanedione monoxime (BDM) and 1-[5-isoquinoline sulfonyl]-2-methyl piperazine (ML7), which inhibit myosin and the phosphorylation of myosin II by the myosin light chain kinase, respectively; and a mutant of the nonmuscle myosin II regulatory light chain, which cannot be phosphorylated (MRLC2(AA)). On the other hand, actin comet tails were induced by the overexpression of phosphatidylinositol phosphate 5-kinase. Cells treated with BDM/ML7 or those that express the MRLC2(AA) mutant revealed a significant reduction in the brefeldin A (BFA)-induced fusion of Golgi enzymes with the endoplasmic reticulum (ER). This delay was not caused by an alteration in the formation of the BFA-induced tubules from the Golgi complex. In addition, the Shiga toxin fragment B transport from the Golgi complex to the ER was also altered. This impairment in the retrograde protein transport was not due to depletion of intracellular calcium stores or to the activation of Rho kinase. Neither the reassembly of the Golgi complex after BFA removal nor VSV-G transport from ER to the Golgi was altered in cells treated with BDM/ML7 or expressing MRLC2(AA). Finally, transport carriers containing Shiga toxin did not move into the cytosol at the tips of comet tails of polymerizing actin. Collectively, the results indicate that 1) myosin motors move to transport carriers from the Golgi complex to the ER along actin filaments; 2) nonmuscle myosin II mediates in this process; and 3) actin comets are not involved in retrograde transport.
Asunto(s)
Actinas/química , Diacetil/análogos & derivados , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Miosinas/química , Transporte de Proteínas , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Actinas/metabolismo , Animales , Transporte Biológico , Brefeldino A/farmacología , Calcio/metabolismo , Línea Celular , ADN Complementario/metabolismo , Diacetil/farmacología , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente Indirecta , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/metabolismo , Miosinas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Proteínas Recombinantes/metabolismo , Toxina Shiga/farmacología , Factores de Tiempo , Proteínas del Envoltorio Viral/metabolismo , Quinasas Asociadas a rhoRESUMEN
Chronic alcohol consumption may cause neurodevelopmental and neurodegenerative disorders. Alcohol neurotoxicity is associated with the production of acetaldehyde and reactive oxygen species that induce oxidative DNA damage. However, the molecular mechanisms by which ethanol disturbs the DNA damage response (DDR), resulting in a defective DNA repair, remain unknown. Here, we have used cultured primary cortical neurons exposed to 50 or 100 mM ethanol for 7 days to analyze the ethanol-induced DDR. Ethanol exposure produced a dose-dependent generation of double strand breaks and the formation of DNA damage foci immunoreactive for the histone γH2AX, a DNA damage marker, and for the ubiquitylated H2A, which is involved in chromatin remodeling at DNA damage sites. Importantly, these DNA damage foci failed to recruit the protein 53BP1, a crucial DNA repair factor. This effect was associated with a drop in 53BP1 mRNA and protein levels and with an inhibition of global transcription. Moreover, ethanol-exposed neurons treated with ionizing radiation (2 Gy) also failed to recruit 53BP1 at DNA damage foci and exhibited a greater vulnerability to DNA lesions than irradiated control neurons. Our results support that defective DNA repair, mediated by the deficient expression and recruitment of 53BP1 to DNA damage sites, represents a novel mechanism involved in ethanol neurotoxicity. The design of therapeutic strategies that increase or stabilize 53BP1 levels might potentially promote DNA repair and partially compensate alcohol neurotoxicity.
Asunto(s)
Depresores del Sistema Nervioso Central/farmacología , Corteza Cerebral/citología , Reparación del ADN/efectos de los fármacos , Etanol/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neuronas/efectos de los fármacos , Animales , Cromatina/efectos de los fármacos , Cromatina/ultraestructura , Ensayo Cometa , Daño del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de la radiación , Hipocampo/citología , Histonas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/ultraestructura , Ratas , Factores de Tiempo , Activación Transcripcional/efectos de los fármacos , Rayos X/efectos adversosRESUMEN
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.
Asunto(s)
Dendritas/efectos de los fármacos , Dendritas/ultraestructura , Etanol/farmacología , Transporte de Proteínas/efectos de los fármacos , Animales , Estrés del Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico Rugoso/efectos de los fármacos , Retículo Endoplásmico Rugoso/ultraestructura , Etanol/administración & dosificación , Femenino , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/ultraestructura , Proteínas de Choque Térmico/metabolismo , Proteínas Motoras Moleculares/efectos de los fármacos , Proteínas Motoras Moleculares/metabolismo , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Ratas WistarRESUMEN
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.
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Astrocitos/metabolismo , Membrana Celular/metabolismo , Etanol/farmacología , Membranas Intracelulares/metabolismo , Esfingolípidos/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Astrocitos/citología , Transporte Biológico/efectos de los fármacos , Brefeldino A/farmacología , Compartimento Celular/efectos de los fármacos , Diferenciación Celular , Membrana Celular/química , Células Cultivadas , Endocitosis/efectos de los fármacos , Feto , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Aparato de Golgi/metabolismo , Lisosomas/metabolismo , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Nocodazol/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Esfingolípidos/química , Factores de TiempoRESUMEN
Vitamin A is essential for lung development and pulmonary cell differentiation. Its deficiency leads to altered lung structure and function and to basement membrane architecture and composition disturbances. Previously, we showed that lack of retinoids thickens the alveolar basement membrane and increases collagen IV, which are reversed by retinoic acid, the main biologically active vitamin A form. This study analyzed how vitamin A deficiency affects the subunit composition of collagen IV and laminin of lung basement membranes and pulmonary matrix metalloproteinase content, plus the recovering effect of all-trans-retinoic acid. Male weanling pups were fed a retinol-adequate/-deficient diet until 60 days old. A subgroup of vitamin-A-deficient pups received daily intraperitoneal all-trans-retinoic acid injections for 10 days. Collagen IV and laminin chain composition were modified in vitamin-A-deficient rats. The protein and mRNA contents of chains α1(IV), α3(IV) and α4(IV) increased; those of chains α2(IV) and α5(IV) remained unchanged; and the protein and mRNA contents of laminin chains α5, ß1 and γ1 decreased. The mRNA of laminin chains α2 and α4 also decreased. Matrix metalloproteinases 2 and 9 decreased, but the tissue inhibitors of metalloproteinases 1 and 2 did not change. Treating vitamin-A-deficient rats with retinoic acid reversed all alterations, but laminin chains α2, α4 and α5 and matrix metalloproteinase 2 remained low. In conclusion, vitamin A deficiency alters the subunit composition of collagen IV and laminin and the lung's proteolytic potential, which are partly reverted by retinoic acid. These alterations could contribute to impaired lung function and predispose to pulmonary disease.
Asunto(s)
Colágeno Tipo IV/metabolismo , Laminina/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Tretinoina/farmacología , Deficiencia de Vitamina A/metabolismo , Animales , Membrana Basal/metabolismo , Colágeno Tipo IV/genética , Femenino , Expresión Génica , Laminina/genética , Masculino , Ratas , Ratas Wistar , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Vitamina A/sangre , Deficiencia de Vitamina A/tratamiento farmacológicoRESUMEN
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.
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Citoesqueleto de Actina/efectos de los fármacos , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/ultraestructura , Etanol/toxicidad , Acetilcolinesterasa/metabolismo , Actinas/metabolismo , Animales , Células Cultivadas , Homólogo 4 de la Proteína Discs Large , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Ratas , Ratas Wistar , Receptores Ionotrópicos de Glutamato/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismoRESUMEN
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.
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Depresores del Sistema Nervioso Central/toxicidad , Citoesqueleto/efectos de los fármacos , Etanol/toxicidad , Hipocampo/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Neuronas/efectos de los fármacos , Actinas/metabolismo , Animales , Células Cultivadas , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Femenino , Hipocampo/metabolismo , Hipocampo/patología , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Neuronas/metabolismo , Neuronas/ultraestructura , Ratas , Transducción de Señal/efectos de los fármacos , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Vitamin A is essential for lung development and pulmonary cell differentiation and its deficiency results in alterations of lung structure and function. Basement membranes (BMs) are also involved in those processes, and retinoic acid, the main biologically active form of vitamin A, influences the expression of extracellular matrix macromolecules. Therefore, we have analyzed the ultrastructure and collagen content of lung alveolar BM in growing rats deficient in vitamin A and the recovering effect of all-trans retinoic acid. Male weanling pups were fed a retinol-adequate or -deficient diet until they were 60 days old. A group of vitamin A-deficient pups were recovered by daily intraperitoneal injections of all-trans retinoic acid for 10 days. Alveolar BM in vitamin A-deficient rats doubled its thickness and contained irregularly scattered collagen fibrils. Immunocytochemistry revealed that these fibrils were composed of collagen I. Total content of both collagen I protein and its mRNA was greater in vitamin-deficient lungs. In agreement with the greater size of the BM the amount of collagen IV was also increased. Proinflammatory cytokines, IL-1alpha, IL-1beta and TNF-alpha, did not change, but myeloperoxidase and TGF-beta1 were increased. Treatment of vitamin A-deficient rats with retinoic acid reversed all the alterations, but the BM thickness recovered only partially. Retinoic acid recovering activity occurred in the presence of increasing oxidative stress. In conclusion, vitamin A deficiency results in alterations of the structure and composition of the alveolar BM which are probably mediated by TGF-beta1 and reverted by retinoic acid. These alterations could contribute to the impairment of lung function and predispose to pulmonary disease.
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Membrana Basal/efectos de los fármacos , Alveolos Pulmonares/efectos de los fármacos , Tretinoina/uso terapéutico , Deficiencia de Vitamina A/patología , Deficiencia de Vitamina A/fisiopatología , Animales , Membrana Basal/ultraestructura , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo IV/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Inmunohistoquímica , Interleucinas/metabolismo , Pulmón/metabolismo , Pulmón/patología , Masculino , Malondialdehído/metabolismo , Estrés Oxidativo/efectos de los fármacos , Peroxidasa/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Alveolos Pulmonares/ultraestructura , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Factor de Crecimiento Transformador beta1/metabolismo , Tretinoina/efectos adversos , Tretinoina/sangre , Tretinoina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Deficiencia de Vitamina A/tratamiento farmacológicoRESUMEN
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.
Asunto(s)
Depresores del Sistema Nervioso Central/toxicidad , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Etanol/toxicidad , Neuronas/efectos de los fármacos , Albúminas/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Biomarcadores/metabolismo , Células Cultivadas , Depresores del Sistema Nervioso Central/metabolismo , Clatrina/metabolismo , Endocitosis/fisiología , Endosomas/metabolismo , Etanol/metabolismo , Femenino , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/embriología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/patología , Neuronas/fisiología , Ratas , Transferrina/metabolismoRESUMEN
AIMS: Glycoproteins, such as adhesion molecules and growth factors, participate in the regulation of nervous system development. Ethanol affects the synthesis, intracellular transport, distribution, and secretion of N-glycoproteins in different cell types, including astrocytes and hepatocytes, suggesting alterations in the glycosylation process. We analysed the effect of exposure to low doses of ethanol (30 mm, 7 days) on glycosylation in cultured hippocampal neurons. METHODS: Neurons were incubated for short (5 min) and long (90 min) periods with the radioactively labelled carbohydrate precursors 2-deoxy-glucose, N-acetyl-D-mannosamine and mannose. The uptake and metabolism of these precursors, as well as the radioactivity distribution in protein gels, were analysed. The levels of the glucose transporters GLUT1 and GLUT3 were also determined. RESULTS: Ethanol exposure reduces the synthesis of proteins, DNA and RNA and decreased the uptake of mannose, but not of 2-deoxy-glucose and N-acetyl-D-mannosamine, and it increased the protein levels of both glucose transporters. Moreover, it altered the carbohydrate moiety of several proteins. Finally, alcohol treatment results in an increment of cell surface glycoconjugates containing terminal non-reduced mannose. CONCLUSIONS: Alcohol-induced alterations in glycosylation of proteins in neurons could be a key mechanism involved in the teratogenic effects of alcohol exposure on brain development.