Modulation of Fas receptor proteins and dynamin during opiate addiction and induction of opiate withdrawal in rat brain

The Fas receptor is involved in the regulation of apoptosis but also can function as a non-apoptotic signal transducer. This study was mainly designed to quantitate Fas proteins in rat brain during heroin addiction and opiate withdrawal. In rat, mouse and human brains, and in SH-SY5Y cells, similar forms of Fas were immunodetected with different antibodies (i.e., 35kDa native Fas and 48- and 51-kDa glycosylated Fas). Acute (2h) treatments with the μ-opioid receptor agonists heroin (10mg/kg) and morphine (30mg/kg) increased the immunodensity of native Fas (124% and 36%) but not that of glycosylated Fas in the cerebral cortex. Chronic (5days) heroin (5-30mg/kg) and morphine (10-100mg/kg) were also associated with increased native Fas (76% and 45%) and with different expressions of glycosylated Fas. In heroin-dependent rats, opiate withdrawal (48h) resulted in a sustained increase in native Fas (107%) and in up-regulation of 51kDa glycosylated Fas (51%). Acute treatments with selective δ-receptor (SNC-80, 10mg/kg) or κ-receptor (U 50488-H, 10mg/kg) agonists did not alter the content of native or glycosylated Fas. Chronic pentazocine (10-80mg/kg, 5days), a mixed opiate drug and σ1 receptor agonist, decreased native (48%) and glycosylated (38-82%) Fas proteins. Similarly, the selective σ1 agonist (+)-SKF 10047 also decreased native Fas (37%) and the effect was blocked by the σ1 antagonist BD 1063. Brain dynamin was up-regulated by acute and/or chronic heroin (30-39%), morphine (47-85%), pentazocine (51%) and heroin withdrawal (74%). The main results indicate that chronic heroin/morphine treatment and heroin withdrawal are associated with up-regulation of 35kDa native Fas (and with different expressions of glycosylated Fas), and also with concomitant increases of dynamin in rat brai

Transcriptional Changes Common to Human Cocaine, Cannabis and Phencyclidine Abuse

A major goal of drug abuse research is to identify and understand drug-induced changes in brain function that are common to many or all drugs of abuse. As these may underlie drug dependence and addiction, the purpose of the present study was to examine if different drugs of abuse effect changes in gene expression that converge in common molecular pathways. Microarray analysis was employed to assay brain gene expression in postmortem anterior prefrontal cortex (aPFC) from 42 human cocaine, cannabis and/or phencyclidine abuse cases and 30 control cases, which were characterized by toxicology and drug abuse history. Common transcriptional changes were demonstrated for a majority of drug abuse cases (N = 34), representing a number of consistently changed functional classes: Calmodulin-related transcripts (CALM1, CALM2, CAMK2B) were decreased, while transcripts related to cholesterol biosynthesis and trafficking (FDFT1, APOL2, SCARB1), and Golgi/endoplasmic reticulum (ER) functions (SEMA3B, GCC1) were all increased. Quantitative PCR validated decreases in calmodulin 2 (CALM2) mRNA and increases in apolipoprotein L, 2 (APOL2) and semaphorin 3B (SEMA3B) mRNA for individual cases. A comparison between control cases with and without cardiovascular disease and elevated body mass index indicated that these changes were not due to general cellular and metabolic stress, but appeared specific to the use of drugs. Therefore, humans who abused cocaine, cannabis and/or phencyclidine share a decrease in transcription of calmodulin-related genes and increased transcription related to lipid/cholesterol and Golgi/ER function. These changes represent common molecular features of drug abuse, which may underlie changes in synaptic function and plasticity that could have important ramifications for decision-making capabilities in drug abusers

Development of Proteomics-Based Fungicides: New Strategies for Environmentally Friendly Control of Fungal Plant Diseases

Proteomics has become one of the most relevant high-throughput technologies. Several approaches have been used for studying, for example, tumor development, biomarker discovery, or microbiology. In this “post-genomic” era, the relevance of these studies has been highlighted as the phenotypes determined by the proteins and not by the genotypes encoding them that is responsible for the final phenotypes. One of the most interesting outcomes of these technologies is the design of new drugs, due to the discovery of new disease factors that may be candidates for new therapeutic targets. To our knowledge, no commercial fungicides have been developed from targeted molecular research, this review will shed some light on future prospects. We will summarize previous research efforts and discuss future innovations, focused on the fight against one of the main agents causing a devastating crops disease, fungal phytopathogens

A new non-classical transgenic animal model of Depression

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Deglycosylation of Fas receptor and chronic morphine treatment up-regulate high molecular mass Fas aggregates in rat brain

[eng] This study was designed to immunodetect and characterize Fas receptor aggregates (oligomerization) in the brain and to assess its possible modulation in opiate addiction. High molecular mass, sodium dodecyl sulfate (SDS)- and β-mercaptoethanol-resistant Fas aggregates (∼110/120 and ∼203 kDa specific peptides) were immunodetected with a cytoplasmic domain-specific antibody in brain tissue (rat, mouse and human) and SH-SY5Y cells by Western blot analysis. Preincubation of rat cortical membranes with N-ethylmaleimide (NEM; 1 mM for 1 h at 37 °C) reduced the immunodensity of ∼203 kDa Fas aggregates (51%) and increased that of 35 kDa native Fas (172%) and 51/48 kDa glycosylated Fas (47%), indicating that disulfide bonds are involved in Fas dimerization. Enzymatic N-deglycosylation of Fas receptor increased the content of Fas aggregates (∼110/120 kDa: five- to sixfold, and ∼203 kDa: two- to threefold), suggesting that Fas glycosylation is involved in regulating receptor dimerization. Chronic (10-100 mg/kg for 5 days), but not acute (30 mg/kg for 2 h), treatment with morphine (a μ-opioid peptide receptor agonist) induced up-regulation of Fas aggregates in the brain (∼110/120 kDa: 39%, and ∼203 kDa: 89%). The acute and/or chronic treatments with δ- and κ-opioid peptide receptor agonists and with a σ1-receptor agonist did not readily alter the content of Fas aggregates in the rat brain. The results indicate that Fas aggregates are natively expressed in the brain and that its density is regulated by the state of Fas glycosylation. These forms of Fas (receptor homodimerization) are functionally relevant because they were up-regulated in the brain of morphine-dependent rats

Long-term regulation of signaling components of adenylyl cyclase and mitogen-activated protein kinases in the pre-frontal cortex of human opiate addicts.

[eng] Opiate addiction involves the development of chronic adaptive changes in µ‐opioid receptors and associated pathways (e.g. cAMP signalling) which lead to neuronal plasticity in the brain. This study assessed the status of cAMP and mitogen‐activated protein kinase (MAPK) pathways in brains (pre‐frontal cortex) of chronic opiate addicts. In these subjects (n = 24), the immunodensities of adenylyl cyclase‐I, PKA Cα, total and phosphorylated CREB were not different from those in sex‐, age‐ and PMD‐matched controls. Moreover, the ratio pCREB/tCREB was similar in opiate addicts (0.74) and controls (0.76), further indicating that opiate addiction in humans is not associated with an upregulation of several key components of cAMP signalling in the pre‐frontal cortex. In contrast, the components of MAPK cascade (Ras/c‐Raf‐1/MEK/ERK) were decreased in the same brains. Notably, pronounced downregulations of phosphorylated MEK (85%) and ERK1/2 (pERK1: 81%; pERK2: 80%) were quantitated in brains of opiate addicts. Chronic morphine treatment in rats (10-100 mg/kg for 5 days) was also associated with decreases of pERK1/2 (59-68%) in the cortex. In SH‐SY5Y cells, morphine also stimulated the activity of pERK1/2 (2.5‐fold) and the MEK inhibitor PD98059 blocked this effect (90%). The abnormalities of MAPK signalling might have important consequences in the long term development of various forms of neural plasticity associated with opiate addiction in humans

Modulation of Fas receptor proteins and dynamin during opiate addiction and induction of opiate withdrawal in rat brain

[eng] The Fas receptor is involved in the regulation of apoptosis but also can function as a non-apoptotic signal transducer. This study was mainly designed to quantitate Fas proteins in rat brain during heroin addiction and opiate withdrawal. In rat, mouse and human brains, and in SH-SY5Y cells, similar forms of Fas were immunodetected with different antibodies (i.e., 35 kDa native Fas and 48- and 51-kDa glycosylated Fas). Acute (2 h) treatments with the μ-opioid receptor agonists heroin (10 mg/kg) and morphine (30 mg/kg) increased the immunodensity of native Fas (124% and 36%) but not that of glycosylated Fas in the cerebral cortex. Chronic (5 days) heroin (5-30 mg/kg) and morphine (10-100 mg/kg) were also associated with increased native Fas (76% and 45%) and with different expressions of glycosylated Fas. In heroin-dependent rats, opiate withdrawal (48 h) resulted in a sustained increase in native Fas (107%) and in up-regulation of 51 kDa glycosylated Fas (51%). Acute treatments with selective δ-receptor (SNC-80, 10 mg/kg) or κ-receptor (U 50488-H, 10 mg/kg) agonists did not alter the content of native or glycosylated Fas. Chronic pentazocine (10-80 mg/kg, 5 days), a mixed opiate drug and σ1 receptor agonist, decreased native (48%) and glycosylated (38-82%) Fas proteins. Similarly, the selective σ1 agonist (+)-SKF 10047 also decreased native Fas (37%) and the effect was blocked by the σ1 antagonist BD 1063. Brain dynamin was up-regulated by acute and/or chronic heroin (30-39%), morphine (47-85%), pentazocine (51%) and heroin withdrawal (74%). The main results indicate that chronic heroin/morphine treatment and heroin withdrawal are associated with up-regulation of 35 kDa native Fas (and with different expressions of glycosylated Fas), and also with concomitant increases of dynamin in rat brain

Behavioral, neurochemical and morphological changes induced by the overexpression of munc18-1a in brain of mice: relevance to schizophrenia

This work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License.-- et al.Overexpression of the mammalian homolog of the unc-18 gene (munc18-1) has been described in the brain of subjects with schizophrenia. Munc18-1 protein is involved in membrane fusion processes, exocytosis and neurotransmitter release. A transgenic mouse strain that overexpresses the protein isoform munc18-1a in the brain was characterized. This animal displays several schizophrenia-related behaviors, supersensitivity to hallucinogenic drugs and deficits in prepulse inhibition that reverse after antipsychotic treatment. Relevant brain areas (that is, cortex and striatum) exhibit reduced expression of dopamine D(1) receptors and dopamine transporters together with enhanced amphetamine-induced in vivo dopamine release. Magnetic resonance imaging demonstrates decreased gray matter volume in the transgenic animal. In conclusion, the mouse overexpressing brain munc18-1a represents a new valid animal model that resembles functional and structural abnormalities in patients with schizophrenia. The animal could provide valuable insights into phenotypic aspects of this psychiatric disorder.The study was supported by an intramural Grant from Centro de Investigación Biomédica en Red de Salud Mental, Instituto de Salud Carlos III (CIBERSAM), Spanish MICINN and FEDER (SAF2009-08460 to JJM; SAF2010-21948 to JLMMM; AGL2009-11358 to AG-A, SAF2011-29918 to JAGS; PI10/02986, CP08/00017 and CEN-20101014 to MD), Basque Government (S-PR10UN01 to JEO and IT-199/07 to JJM), University of the Basque Country (UPV/EHU) and Complutense University of Madrid (UCM GR42/10-962075 to JLMM).Peer Reviewe

Expresión de la aquaporina 4 en muestras post-mortem de corteza cerebral humana de sujetos con diferentes trastornos psiquiátricos

Trabajo presentado al XIII Congreso de la Sociedad Española de Neurociencia celebrado en Tarragona del 16 al 19 de septiembre de 2009.Las aquaporinas son canales transportadores responsables del mantenimiento de la homeostasis de agua e iones, siendo las isoformas 1 y 4 (AQP1 y AQP4) las predominantes en el sistema nervioso central de mamíferos. Se ha descrito la participación de la AQP4 en los mecanismos responsables de la generación del edema cerebral, así como su implicación en la migración de los astrocitos, y en el control de la excitabilidad neuronal. Éstos y otros datos, han convertido a la AQP4 en una diana importante para el tratamiento de enfermedades como el edema cerebral, la epilepsia del lóbulo temporal medial y una variante de esclerosis múltiple. Además, se ha descrito su sobre-expresión asociada con el trastorno bipolar y la depresión mayor; y recientemente, se ha establecido que su expresión es necesaria para el efecto antidepresivo de la fluoxetina. Nuestro objetivo ha sido evaluar la densidad de la proteína AQP4 en muestras post-mortem de corteza prefrontal de sujetos con un diagnóstico previo de depresión mayor (MD, n=15), trastorno bipolar (BD, n=19) o esquizofrenia (SCH, n=22). Se establecieron además, tres grupos de muestras controles emparejadas con cada sujeto objeto de estudio para los parámetros de género, edad e intervalo post-mortem. Por último, se incluyó un grupo de sujetos víctimas de suicidio sin diagnóstico ante-mortem de trastorno mental (n=12). La detección de las dos isoformas de AQP4 (34 y 32 kDa) se realizó simultáneamente con la del control interno beta-actina. También se cuantificó la AQP1 en los mismos grupos de muestras y controles para determinar la especificidad de los resultados. Los niveles de densidad óptica relativa obtenidos para cada muestra fueron normalizados y expresados como porcentaje sobre su control. Para AQP4 se obtuvo un incremento de la densidad de sus dos isoformas tanto en MD, como en BD y SCH, mientras no se observaron cambios en el grupo suicidio. Estos cambios fueron estadísticamente significativos para ambas isoformas en BD (34 kDa: 118%, p0,05) ni en el grupo suicidio (91%, p>0,05). Estos resultados sugieren una posible implicación de las aquaporinas en la biología de las enfermedades psiquiátricas.Financiado por el Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red de Salud Mental, CIBERSAM; el MICINN (SAF2004/2784, FIS04/0190) y fondos FEDER.Peer Reviewe