RESUMEN
Enhanced brain apoptosis (neurons and glia) may be involved in major depression (MD) and schizophrenia (SZ), mainly through the activation of the intrinsic (mitochondrial) apoptotic pathway. In the extrinsic death pathway, pro-apoptotic Fas-associated death domain (FADD) adaptor and its non-apoptotic p-Ser194 FADD form have critical roles interacting with other death regulators such as phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) and extracellular signal-regulated kinase (ERK). The basal status of FADD (protein and messenger RNA (mRNA)) and the effects of psychotropic drugs (detected in blood/urine samples) were first assessed in postmortem prefrontal cortex of MD and SZ subjects (including a non-MD/SZ suicide group). In MD, p-FADD, but not total FADD (and mRNA), was increased (26%, n=24; all MD subjects) as well as p-FADD/FADD ratio (a pro-survival marker) in antidepressant-free MD subjects (50%, n=10). In contrast, cortical FADD (and mRNA), p-FADD, and p-FADD/FADD were not altered in SZ brains (n=21) regardless of antipsychotic medications (except enhanced mRNA in treated subjects). Similar negative results were quantified in the non-MD/SZ suicide group. In MD, the regulation of multifunctional PEA-15 (i.e., p-Ser116 PEA-15 blocks pro-apoptotic FADD and PEA-15 prevents pro-survival ERK action) and the modulation of p-ERK1/2 were also investigated. Cortical p-PEA-15 was not changed whereas PEA-15 was increased mainly in antidepressant-treated subjects (16-20%). Interestingly, cortical p-ERK1/2/ERK1/2 ratio was reduced (33%) in antidepressant-free when compared to antidepressant-treated MD subjects. The neurochemical adaptations of brain FADD (increased p-FADD and pro-survival p-FADD/FADD ratio), as well as its interaction with PEA-15, could play a major role to counteract the known activation of the mitochondrial apoptotic pathway in MD.
Asunto(s)
Encéfalo/metabolismo , Trastorno Depresivo Mayor/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Fosfoproteínas/metabolismo , Esquizofrenia/metabolismo , Adulto , Antidepresivos/uso terapéutico , Antipsicóticos/uso terapéutico , Proteínas Reguladoras de la Apoptosis , Trastorno Depresivo Mayor/tratamiento farmacológico , Proteína de Dominio de Muerte Asociada a Fas/genética , Femenino , Humanos , Immunoblotting , Masculino , Persona de Mediana Edad , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , ARN Mensajero/metabolismo , Esquizofrenia/tratamiento farmacológico , SuicidioRESUMEN
Introducción: Numerosos estudios han puesto de manifiesto que la alteración en la capacidad comunicativa es uno de los síntomas característicos de la enfermedad de Alzheimer (EA). Objetivo: Estudiar la organización temporal del habla en una tarea de lectura en un grupo de pacientes con EA y otro de controles sanos, utilizando un procedimiento semiautomático, así como valorar su capacidad para discriminar entre ambos grupos. Sujetos y método: Se aplicó una prueba de lectura a 70 sujetos: 35 con EA y otros tantos controles. Antes del registro del habla, los sujetos fueron sometidos a una batería de pruebas neuropsicológicas. Los grupos no diferían en edad, sexo o nivel de estudios. Resultados: Todas las variables analizadas se mostraron alteradas en el grupo con EA. Los resultados señalan que la lectura de los pacientes con EA se caracteriza por una reducida velocidad de elocución y articulación, una baja efectividad del tiempo de fonación, así como en un incremento del número y la proporción de las pausas. Los algoritmos de procesamiento de la señal aplicados a las grabaciones de fluidez lectora demostraron su capacidad para discriminar entre ambos grupos con una precisión del 80% (especificidad 74,2%; sensibilidad 77,1%) mediante la velocidad de elocución. Conclusión: El análisis de la fluidez de lectura oral puede representar una herramienta potencial para el análisis objetivo y la cuantificación de los déficits del lenguaje en la EA (AU)
Introduction: Many studies highlight that an impaired ability to communicate is one of the key clinical features of Alzheimer disease (AD). Objective: To study temporal organisation of speech in an oral reading task in patients with AD and in matched healthy controls using a semi-automatic method, and evaluate that method's ability to discriminate between the 2 groups. Subjects and methods: A test with an oral reading task was administered to 70 subjects, comprising 35 AD patients and 35 controls. Before speech samples were recorded, participants completed a battery of neuropsychological tests. There were no differences between groups with regard to age, sex, or educational level. Results: All of the study variables showed impairment in the AD group. According to the results, AD patients oral reading was marked by reduced speech and articulation rates, low effectiveness of phonation time, and increases in the number and proportion of pauses. Signal processing algorithms applied to reading fluency recordings were shown to be capable of differentiating between AD patients and controls with an accuracy of 80% (specificity 74.2%, sensitivity 77.1%) based on speech rate. Conclusion: Analysis of oral reading fluency may be useful as a tool for the objective study and quantification of speech deficits in AD (AU)
Asunto(s)
Humanos , Enfermedad de Alzheimer/fisiopatología , Lectura , Trastornos del Conocimiento/fisiopatología , Trastornos del Habla/epidemiología , Trastornos de la Articulación/epidemiologíaRESUMEN
The endocannabinoid system is implicated in the neurobiology of cocaine addiction. This study evaluated the status of cannabinoid (CB) CB1 and CB2 receptors, the endocytic cycle of CB1 receptors, G protein-coupled receptor regulatory kinases (GRK), and associated signaling (mammalian target of rapamicin (mTOR) and 70kDa ribosomal protein S6 kinase (p70S6K)) in brain cortices of drug abusers and cocaine- and cannabinoid-treated rodents. The main results indicate that in cocaine adddicts, but not in mixed cocaine/opiate or opiate abusers, CB1 receptor protein in the prefrontal cortex (PFC) was reduced (-44%, total homogenate) with a concomitant receptor redistribution and/or internalization (decreases in membranes and increases in cytosol). In cocaine addicts, the reductions of CB1 receptors and GRK2/3/5 (-26% to -30%) indicated receptor desensitization. CB2 receptor protein was not significantly altered in the PFC of cocacine addicts. Chronic cocaine in mice and rats also reduced CB1 receptor protein (-41% and -80%) in the cerebral cortex inducing receptor redistribution and/or internalization. The CB1 receptor agonist WIN55212-2 caused receptor downregulation (decreases in membranes and cytosol) and the antagonists rimonabant and AM281 induced opposite effects (receptor upregulation in membranes and cytosol). Rimonabant and AM281 also behaved as inverse agonists on the activation of mTOR and its target p70S6K. Chronic cocaine in mice was associated with tolerance to the acute activation of mTOR and p70S6K. In long-term cocaine addicts, mTOR and p70S6K activations were not altered when compared with controls, indicating that CB1 receptor signaling was dampened. The dysregulation of CB1 receptor, GRK2/3/5, and mTOR/p70S6K signaling by cocaine may contribute to alterations of neuroplasticity and/or neurotoxicity in brains of cocaine addicts.
Asunto(s)
Encéfalo/metabolismo , Trastornos Relacionados con Cocaína/metabolismo , Cocaína/farmacología , Receptor Cannabinoide CB1/metabolismo , Transducción de Señal/fisiología , Adulto , Animales , Encéfalo/efectos de los fármacos , Encéfalo/patología , Homólogo de la Proteína Chromobox 5 , Trastornos Relacionados con Cocaína/patología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Conducta Animal/fisiología , Encéfalo/metabolismo , Proteínas Munc18/metabolismo , Esquizofrenia/metabolismo , Animales , Caspasa 3/metabolismo , Modelos Animales de Enfermedad , Dopamina/metabolismo , Humanos , Peroxidación de Lípido/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microdiálisis , Actividad Motora/genética , Proteínas Munc18/genética , Fibras Nerviosas Amielínicas/patología , Tamaño de los Órganos/genética , Fenotipo , Reconocimiento en Psicología/fisiología , Reflejo de Sobresalto/genética , Proteínas SNARE/metabolismo , Conducta SocialRESUMEN
INTRODUCTION: Many studies highlight that an impaired ability to communicate is one of the key clinical features of Alzheimer disease (AD). OBJECTIVE: To study temporal organisation of speech in an oral reading task in patients with AD and in matched healthy controls using a semi-automatic method, and evaluate that method's ability to discriminate between the 2 groups. SUBJECTS AND METHODS: A test with an oral reading task was administered to 70 subjects, comprising 35 AD patients and 35 controls. Before speech samples were recorded, participants completed a battery of neuropsychological tests. There were no differences between groups with regard to age, sex, or educational level. RESULTS: All of the study variables showed impairment in the AD group. According to the results, AD patients' oral reading was marked by reduced speech and articulation rates, low effectiveness of phonation time, and increases in the number and proportion of pauses. Signal processing algorithms applied to reading fluency recordings were shown to be capable of differentiating between AD patients and controls with an accuracy of 80% (specificity 74.2%, sensitivity 77.1%) based on speech rate. CONCLUSION: Analysis of oral reading fluency may be useful as a tool for the objective study and quantification of speech deficits in AD.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Lectura , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Estudios Retrospectivos , HablaRESUMEN
RATIONALE: Regulator of G-protein signaling (RGS) proteins, RGS4 and RGS10, may be involved in the pathophysiology of schizophrenia. RGS4 has attracted special interest since the reports of genetic association between SNPs in RGS4 and schizophrenia. However, there is no information about the subcellular distribution of RGS4 and RGS10 proteins in psychiatric disorders. OBJECTIVES: Plasma membrane RGS4 and cytosolic RGS10 protein immunoreactivity in prefrontal cortex from schizophrenic subjects (n = 25), non-diagnosed suicides (n = 13), and control subjects (n = 35), matched by age, gender, and postmortem delay, was analyzed by western blot. A second group of depressed subjects (n = 25) and control subjects (n = 25) was evaluated. The effect of the antipsychotic or antidepressant treatments was also assessed. RESULTS: No significant differences in plasma membrane RGS4 and cytosolic RGS10 protein expression were observed between schizophrenic subjects, non-diagnosed suicides, and control subjects. However, RGS4 immunoreactivity was significantly higher (Δ = 33 ± 10 %, p < 0.05) in the antipsychotic-treated subgroup (n = 12) than in the antipsychotic-free subgroup (n = 13). Immunodensities of plasma membrane RGS4 and cytosolic RGS10 proteins did not differ between depressed and matched control subjects. CONCLUSIONS: Expression of RGS4 and RGS10 proteins at their predominant subcellular location was studied in the postmortem brain of subjects with psychiatric disorders. The results suggest unaltered membrane RGS4 and cytosolic RGS10 proteins levels in schizophrenia and major depression. Antipsychotic treatment seems to increase membrane RGS4 immunoreactivity. Further studies are needed to elucidate RGS4 and RGS10 functional status.
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Antipsicóticos/uso terapéutico , Trastorno Depresivo Mayor/metabolismo , Corteza Prefrontal/metabolismo , Proteínas RGS/biosíntesis , Esquizofrenia/metabolismo , Adulto , Antipsicóticos/sangre , Biopsia , Western Blotting , Membrana Celular/metabolismo , Citosol/metabolismo , Trastorno Depresivo Mayor/tratamiento farmacológico , Trastorno Depresivo Mayor/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Cambios Post Mortem , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/patología , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/patología , SuicidioRESUMEN
Cyclin-dependent kinase 5 (cdk5) participates in opioid receptor signalling through complex molecular mechanisms. The acute effects of selective µ-(fentanyl) and δ-(SNC-80) opioid receptor agonists, as well as the chronic effects of morphine (the prototypic opiate agonist mainly acting at µ-receptors), modulating cdk5 and activators p35/p25 and their interactions with neurotoxic/apoptotic factors, dopamine- and cAMP-regulated phosphoprotein of 32kDa (DARPP-32) and extracellular signal-regulated kinase (ERK) were quantified (Western Blot analyses) in the rat corpus striatum and/or cerebral cortex. To assess the involved mechanisms, MDL28170 was used to inhibit calpain activity and SL327 to disrupt MEK (ERK kinase)-ERK activation. Acute fentanyl (0.1mg/kg) and SNC-80 (10mg/kg) induced rapid (7-60 min) 2- to 4-fold increases of p25 content, without induction of cdk5/p25 pro-apoptotic c-Jun NH(2)-terminal protein kinase or aberrant cleavage of poly(ADP-ribose)-polymerase-1, a hallmark of apoptosis. In contrast, fentanyl and SNC-80 stimulated cdk5-mediated p-Thr75 DARPP-32 (+116-166%; PKA inhibition) and p-Thr286 MEK1 (+21-82%; MEK inactivation), and this latter effect resulted in uncoupling of MEK to ERK signals. Calpain inhibition with MDL28170 (cleavage of p35 to p25) attenuated fentanyl-induced p25 accumulation (-57%), but not the stimulation of p-Thr286 MEK1 or p-Thr75 DARPP-32. MEK-ERK inhibition with SL327 fully prevented fentanyl-induced p25 upregulation. Notably, chronic morphine treatment (10-100mg/kg for 6 days) also increased p25 content and p25/p35 ratio (and activated/inactivated MEK1) in rat brain cortex, which indicated that p25 upregulation persisted under the sustained stimulation of µ-opioid receptors. The results demonstrate that the acute stimulation of opioid receptors leads to upregulation of p25 activator through a MEK-ERK and calpain-dependent pathway, and to disruption of MEK-ERK signalling by a cdk5/p35-induced MEK1 inhibition. Moreover, the effects induced by the sustained stimulation of µ-receptors with morphine suggest the participation of cdk5/p25 complex in opiate-induced long-term neuroplasticity.
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Encéfalo/metabolismo , Quinasa 5 Dependiente de la Ciclina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fosfotransferasas/metabolismo , Receptores Opioides/metabolismo , Regulación hacia Arriba/fisiología , Análisis de Varianza , Animales , Benzamidas/farmacología , Encéfalo/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Fentanilo/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , MAP Quinasa Quinasa 1/metabolismo , Masculino , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Narcóticos/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Cocaine induces apoptotic effects in cultured cells and in the developing brain, but the aberrant activation of cell death in the adult brain remains inconclusive, especially in humans. This postmortem human brain study examined the status of canonical apoptotic pathways, signaling partners, and the cleavage of poly(ADP-ribose) polymerase-1 (PARP-1), a sensor of DNA damage, in prefrontal cortex (PFC) of a small but well-characterized cohort of cocaine abusers (n=10). For comparison, the chosen targets were also quantified in the cerebral cortex of cocaine-treated rats. In the PFC of cocaine abusers, FS7-associated cell surface antigen (Fas) receptor aggregates and Fas-associated death domain (FADD) adaptor were reduced (-26% and -66%, respectively) as well as the content of mitochondrial cytochrome c (-61%). In the same brain samples of cocaine abusers, the proteolytic cleavage of PARP-1 was increased (+39%). Nuclear PARP-1 degradation, possibly a consequence of increased mitochondrial oxidative stress, involved the activation of apoptosis-inducing factor (AIF) and not that of caspase-3. In the PFC of cocaine abusers, several signaling molecules associated with cocaine/dopamine and/or apoptotic pathways were not significantly altered, with the exception of anti-apoptotic truncated DARPP-32 (t-DARPP), a truncated isoform of dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), whose content was decreased (-28%). Chronic exposure to cocaine in rats, including withdrawal for 3 days, did not alter Fas-FADD receptor complex, cytochrome c, caspase-3/fragments, AIF, PARP-1 cleavage, and associated signaling in the cerebral cortex. Chronic cocaine and abstinence, however, increased the content of t-DARPP (+39% and +47%) in rat brain cortex. The major findings indicate that cocaine addiction in humans is not associated with abnormal activation of extrinsic and intrinsic apoptotic pathways in PFC. The downregulation of Fas-FADD receptor complex and cytochrome c could reflect the induction of contraregulatory adaptations or non-apoptotic (neuroplastic) actions induced by the psychostimulant. The enhanced degradation of nuclear PARP-1, a hallmark of apoptosis, indicates the possibility of aberrant cell death.
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Apoptosis/fisiología , Corteza Cerebral/metabolismo , Trastornos Relacionados con Cocaína/metabolismo , Cocaína/farmacología , Corteza Prefrontal/metabolismo , Transducción de Señal/fisiología , Adulto , Animales , Apoptosis/efectos de los fármacos , Factor Inductor de la Apoptosis/metabolismo , Caspasa 3/metabolismo , Corteza Cerebral/efectos de los fármacos , Modelos Animales de Enfermedad , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , Corteza Prefrontal/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Receptor fas/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Natural and synthetic cannabinoids (CBs) induce deleterious or beneficial actions on neuronal survival. The Fas-associated protein with death domain (FADD) promotes apoptosis, and its phosphorylated form (p-FADD) mediates non-apoptotic actions. The regulation of Fas/FADD, mitochondrial apoptotic proteins and other pathways by CB receptors was investigated in the mouse brain. EXPERIMENTAL APPROACH: Wild-type, CB(1) and CB(2) receptor knock-out (KO) mice were used to assess differences in receptor genotypes. CD1 mice were used to evaluate the effects of CB drugs on canonical apoptotic pathways and associated signalling systems. Target proteins were quantified by Western blot analysis. KEY RESULTS: In brain regions of CB(1) receptor KO mice, Fas/FADD was reduced, but p-Ser191 FADD and the p-FADD/FADD ratio were increased. In CB(2) receptor KO mice, Fas/FADD was increased, but the p-FADD/FADD ratio was not modified. In mutant mice, cleavage of poly(ADP-ribose)-polymerase (PARP) did not indicate alterations in brain cell death. In CD1 mice, acute WIN55212-2 (CB(1) receptor agonist), but not JWH133 (CB(2) receptor agonist), inversely modulated brain FADD and p-FADD. Chronic WIN55212-2 induced FADD down-regulation and p-FADD up-regulation. Acute and chronic WIN55212-2 did not alter mitochondrial proteins or PARP cleavage. Acute, but not chronic, WIN55212-2 stimulated activation of anti-apoptotic (ERK, Akt) and pro-apoptotic (JNK, p38 kinase) pathways. CONCLUSIONS AND IMPLICATIONS: CB(1) receptors appear to exert a modest tonic activation of Fas/FADD complexes in brain. However, chronic CB(1) receptor stimulation decreased pro-apoptotic FADD and increased non-apoptotic p-FADD. The multifunctional protein FADD could participate in the mechanisms of neuroprotection induced by CBs.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Encéfalo/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB2/agonistas , Transducción de Señal/efectos de los fármacos , Receptor fas/metabolismo , Animales , Benzoxazinas/farmacología , Encéfalo/efectos de los fármacos , Cannabinoides/farmacología , Muerte Celular/genética , Regulación hacia Abajo , Masculino , Ratones , Ratones Noqueados , Morfolinas/farmacología , Naftalenos/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB2/genética , Regulación hacia ArribaRESUMEN
Fas-associated protein with death domain (FADD) is a multifunctional protein that can induce both apoptotic and non-apoptotic actions. Recently, FADD was found downregulated in the prefrontal cortex of opiate abusers, which suggested an attenuation of Fas death signals in human addicts. Phosphorylation of FADD (Ser194) has been reported to regulate its non-apoptotic activity, which might include the induction of neuroplastic effects in the brain. This postmortem brain study examined the status of phosphorylated (p)-Ser194 FADD and signaling pathways involved in neuroplasticity in the prefrontal cortex (BA 9) of short-term (ST) and long-term (LT) heroin or methadone abusers. In these subjects, the content of monomeric p-FADD was significantly increased when compared with that in age-, gender-, and postmortem delay-matched controls (all addicts: 65%, n=26; ST abuse: 51%; n=11; LT abuse: 75%, n=15). Oligomeric p-FADD forms were modestly increased (11%-23%). At the subcellular level, opiate addiction upregulated the expression of monomeric p-FADD in the nucleus (110%) and that of p-oligomers in the cytosol (66%). In LT opiate addicts (but not ST abusers), a pronounced downregulation of p-extracellular signal-regulated kinase (ERK)1/2 (52%) and p-c-Jun NH(2)-terminal protein kinase (JNK)1/2 (51%), but not p-p38 mitogen-activated protein kinase (MAPK), was quantified in the prefrontal cortex (total homogenate and subcellular compartments). Similarly, the signaling pathway mediated by p-phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) protein and its phosphorylating kinase p-Akt1 was also downregulated in cortical homogenate (43% and 41%, respectively) and cytosolic preparations of chronic opiate addicts. The results indicate that opiate addiction in humans is associated with an altered balance between p-Ser194 FADD (increased) and total FADD (decreased) in brain, which may favor its neuroplastic actions. The interaction between p-FADD (upregulated) and neuronal pathways (downregulated) could play a relevant role in mediating specific forms of structural and behavioral neuroplasticity.
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Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Plasticidad Neuronal , Trastornos Relacionados con Opioides/metabolismo , Fosfoproteínas/fisiología , Corteza Prefrontal/metabolismo , Proteínas Proto-Oncogénicas c-akt/fisiología , Adulto , Proteínas Reguladoras de la Apoptosis , Femenino , Humanos , Masculino , Fosforilación , Serina/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
Opiate addiction is a chronic medical disorder characterized by drug tolerance and dependence, behavioral sensitization, vulnerability to compulsive relapse, and high mortality. In laboratory animals, the potential effect of opiate drugs to induce cell death by apoptosis is a controversial topic. This postmortem human brain study examined the status of the extrinsic and intrinsic apoptotic pathways in the prefrontal cortex of a large group of well-characterized heroin or methadone abusers. In these subjects (n=36), the immunocontent of apoptosis-1 protein (Fas) death receptor did not differ from that in age-, gender-, and postmortem delay-matched controls. In contrast, Fas-associated protein with death domain (FADD), the mediator of the death signal, was significantly decreased in the same brain samples (all addicts: 30%, n=36; short-term abuse (ST): 31%, n=15; long-term abuse (LT): 29%, n=21). The initiator caspase-8 was not altered, but FLIP(L) (Fas-associated protein with death domain-like interleukin-1beta-converting enzyme-inhibitory protein), a dominant inhibitor of caspase-8, was increased in LT addicts (19%). In the intrinsic pathway, the pro-apoptotic mitochondrial proteins Bax (Bcl-2-associated X protein) and AIF (apoptosis-inducing factor) remained unchanged, but cytochrome c was decreased (all addicts: 25%; ST: 31%; LT: 20%) and anti-apoptotic B-cell leukemia 2 (Bcl-2) increased in LT addicts (24%). The content of executioner caspase-3 and the pattern of cleavage of the nuclear enzyme poly-(ADP-ribose)-polymerase-1 (PARP-1) were similar in opiate addicts and control subjects. Taken together, the data revealed that the extrinsic and intrinsic canonical apoptotic pathways are not abnormally activated in the prefrontal cortex of opiate abusers. Instead, the chronic modulation of some of their components (downregulation of FADD and cytochrome c; upregulation of FLIP(L) and Bcl-2) suggests the induction of non-apoptotic actions by opiate drugs related to phenomena of synaptic plasticity in the brain. These neurochemical adaptations could play a major role in the development of opiate tolerance, sensitization and relapse in human addicts.
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Apoptosis/fisiología , Trastornos Relacionados con Opioides/patología , Corteza Prefrontal/patología , Transducción de Señal/fisiología , Enfermedad Aguda , Adulto , Envejecimiento/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Western Blotting , Química Encefálica , Enfermedad Crónica , Femenino , Cabello/química , Humanos , Masculino , Persona de Mediana Edad , Narcóticos/análisis , Narcóticos/sangre , Plasticidad Neuronal/fisiología , Trastornos Relacionados con Opioides/metabolismo , Corteza Prefrontal/metabolismo , Caracteres Sexuales , Sinapsis/fisiología , Adulto JovenRESUMEN
Opiate addiction involves the development of chronic adaptive changes in micro -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 Calpha, 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.
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Adenilil Ciclasas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Morfina/farmacología , Trastornos Relacionados con Opioides/metabolismo , Corteza Prefrontal/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Adolescente , Adulto , Animales , Línea Celular , Enfermedad Crónica , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteína Quinasa 1 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Trastornos Relacionados con Opioides/patología , Corteza Prefrontal/metabolismo , Corteza Prefrontal/patología , Proteínas Proto-Oncogénicas c-raf/efectos de los fármacos , Proteínas Proto-Oncogénicas c-raf/metabolismo , Ratas , Ratas Sprague-Dawley , Tiempo , Proteínas ras/efectos de los fármacos , Proteínas ras/metabolismoRESUMEN
Opiate addiction is associated with abnormalities of neurofilament (NF) proteins and upregulation of cAMP signaling in the brain, which may modulate neuronal plasticity. This study investigated, using gene-targeted mice lacking mu-, delta- or kappa-opioid receptors, the role of these receptors in modulating the basal activity and the chronic effects of morphine on both intracellular targets. In WT mice, chronic treatment (5 days) with morphine (20-100 mg/kg) resulted in decreases in the immunodensity of neurofilament (NF)-L in the cerebral cortex (14-23%). In contrast, chronic morphine did not decrease NF-L in cortices of mu-, delta-, and kappa-KO mice, suggesting the involvement of the three types of opioid receptors in this effect of morphine. Also, the marked increase in phosphorylated NF-H induced by chronic morphine in WT mice (two-fold) was abolished in mu -KO mice. In cortex and/or striatum of mu-, delta- and kappa-KO mice, the basal immunodensities of Galphai1/2 proteins, the catalytic isoform (Calpha) of protein kinase A (PKA) and the total content of cAMP response element-binding protein (CREB, the nuclear target of PKA) were not different from those of WT mice. In contrast, phosphorylated CREB (the active form of this transcription factor) was reduced in cortex and/or striatum (23-26%) of mu- and delta-KO mice, but not in kappa-KO animals. These results suggest that the endogenous opioid tone acting on mu-/delta-receptors tonically stimulate CREB activation in the brain. In cortex and/or striatum of WT mice, chronic morphine did not induce upregulation of the main components of the cAMP signaling pathway. In contrast, chronic morphine treatment in mu-KO mice, but not in delta- or kappa-KO, resulted in a paradoxical upregulation of Galphai1/2 (12-19%), PKA (19-21%,) and phosphorylated CREB (21-73%), but not total CREB, in cortex and/or striatum. The induction of heterologous receptor adaptations in mu-KO mice may explain this paradoxical effect of morphine.
Asunto(s)
Morfina/administración & dosificación , Proteínas de Neurofilamentos/metabolismo , Receptores Opioides delta/agonistas , Receptores Opioides kappa/agonistas , Receptores Opioides mu/agonistas , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , AMP Cíclico/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Neurofilamentos/genética , Receptores Opioides delta/deficiencia , Receptores Opioides delta/genética , Receptores Opioides kappa/deficiencia , Receptores Opioides kappa/genética , Receptores Opioides mu/deficiencia , Receptores Opioides mu/genéticaRESUMEN
Abnormalities in the density of neuroreceptors that regulate norepinephrine and serotonin release have been repeatedly reported in brains of suicide victims with mood disorders. Recently, the modulation of the [(35)S]GTPgammaS binding to G-proteins has been introduced as a suitable measure of receptor activity in postmortem human brain. The present study sought to evaluate the function of several G-protein coupled receptors in postmortem brain of suicide victims with mood disorders. Concentration-response curves of the [(35)S]GTPgammaS binding stimulation by selective agonists of alpha(2)-adrenoceptors, 5-HT(1A) serotonin, mu-opioid, GABA(B), and cholinergic muscarinic receptors were performed in frontal cortical membranes from 28 suicide victims with major depression or bipolar disorder and 28 subjects who were matched for gender, age and postmortem delay. The receptor-independent [(35)S]GTPgammaS binding stimulation by mastoparan and the G-protein density were also examined. The alpha(2A)-adrenoceptor-mediated stimulation of [(35)S]GTPgammaS binding with the agonist UK14304 displayed a 4.6-fold greater sensitivity in suicide victims than in controls, without changes in the maximal stimulation. No significant differences were found in parameters of 5-HT(1A) serotonin receptor and other receptor-mediated [(35)S]GTPgammaS binding stimulations. The receptor-independent activation of G-proteins was similar in both groups. Immunoreactive densities of G(alphai1/2)-, G(alphai3)-, G(alphao)-, and G(alphas)-proteins did not differ between suicide victims and controls. In conclusion, alpha(2A)-adrenoceptor sensitivity is increased in the frontal cortex of suicide victims with mood disorders. This receptor supersensitivity is not related to an increased amount or enhanced intrinsic activity of G-proteins. The new finding provides functional support to the involvement of alpha(2)-adrenoceptors in the pathogenesis of mood disorders.
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Encéfalo/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Trastornos del Humor/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Suicidio , Adulto , Anciano , Femenino , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Trastornos del Humor/diagnóstico , Trastornos del Humor/tratamiento farmacológico , Psicotrópicos/uso terapéutico , Ensayo de Unión Radioligante , Receptores de Serotonina/metabolismo , Receptores de Serotonina 5-HT1 , Radioisótopos de Azufre , Tubulina (Proteína)/metabolismoRESUMEN
The homologous regulation of receptors is mediated by G protein-coupled receptor kinases (GRKs) which phosphorylate the agonist-activated receptor. This study was designed to assess the in vivo indirect activation of adrenoceptors or 5-HT receptors by the reuptake blocker desipramine or fluoxetine on the cellular distribution of GRK 2/3 in rat brain. Immunoblot analysis (frontal cortex) with a GRK 2 antibody revealed a unique 80 kDa protein (mixed GRK 2/3) in total homogenate (H) and in membrane (P2) and cytosolic (S2) fractions. The proportion of GRK 2/3 in each fraction, relative to that of H, was: P2/H=0.11 and S2/H=0.45. Acute desipramine (noradrenaline reuptake blocker) increased in a dose- (1-30 mg/kg, i.p.) and time- (1-6 h) dependent manner the content of GRK 2/3 in the membrane (P2/H ratios increased by 37-164%). This effect vanished with a prolonged desipramine (30 mg/kg) exposure (24 h). Desipramine did not alter the content of GRK 2/3 in the cytosol (S2/H ratios). Chronic desipramine (10 mg/kg every 24 h) for 14 days did not change significantly the immunodensity of GRK 2/3 in the membrane or the cytosol. The acute administration (2 h) of fluoxetine (5-HT reuptake blocker; 3-30 mg/kg) did not induce significant changes in the content of GRK 2/3 in the membrane (P2/H ratio) or cytosol (S2/H ratio). The results indicate that the in vivo activation of adrenoceptors by desipramine is associated with a time-dependent modulation of membrane-associated GRK 2/3 (i.e. an acute increase in the kinase content which is followed by a return to the basal expression upon repeated treatment). In contrast, the acute in vivo activation of 5-HT receptors induced by fluoxetine does not result in modulation of GRK 2/3.
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Antidepresivos Tricíclicos/administración & dosificación , Encéfalo/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/biosíntesis , Desipramina/administración & dosificación , Fluoxetina/administración & dosificación , Proteínas Serina-Treonina Quinasas/biosíntesis , Animales , Encéfalo/enzimología , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Quinasa 3 del Receptor Acoplado a Proteína-G , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Masculino , Ratas , Ratas Sprague-Dawley , Quinasas de Receptores Adrenérgicos betaRESUMEN
1. This study was designed to assess the influence of activation and blockade of the endogenous opioid system in the brain on two key proteins involved in the regulation of programmed cell death: the pro-apoptotic Fas receptor and the anti-apoptotic Bcl-2 oncoprotein. 2. The acute treatment of rats with the mu-opioid receptor agonist morphine (3-30 mg x kg(-1), i.p., 2 h) did not modify the immunodensity of Fas or Bcl-2 proteins in the cerebral cortex. Similarly, the acute treatment with low and high doses of the antagonist naloxone (1 and 100 mg x kg(-1), i.p., 2 h) did not alter Fas or Bcl-2 protein expression in brain cortex. These results discounted a tonic regulation through opioid receptors on Fas and Bcl-2 proteins in rat brain. 3. Chronic morphine (10-100 mg x kg(-1), 5 days, and 10 mg x kg(-1), 13 days) induced marked increases (47-123%) in the immunodensity of Fas receptor in the cerebral cortex. In contrast, chronic morphine (5 and 13 days) decreased the immunodensity of Bcl-2 protein (15-30%) in brain cortex. Chronic naloxone (10 mg x kg(-1), 13 days) did not alter the immunodensities of Fas and Bcl-2 proteins in the cerebral cortex. 4. The concurrent chronic treatment (13 days) of naloxone (10 mg x kg(-1)) and morphine (10 mg x kg(-1)) completely prevented the morphine-induced increase in Fas receptor and decrease in Bcl-2 protein immunoreactivities in the cerebral cortex. 5. The results indicate that morphine, through the sustained activation of opioid receptors, can promote abnormal programmed cell death by enhancing the expression of pro-apoptotic Fas receptor protein and damping the expression of anti-apoptotic Bcl-2 oncoprotein.
Asunto(s)
Morfina/farmacología , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Narcóticos/farmacología , Proteínas de Neurofilamentos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptor fas/metabolismo , Animales , Apoptosis , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/inmunología , Corteza Cerebral/metabolismo , Regulación hacia Abajo , Quimioterapia Combinada , Masculino , Proteínas de Neurofilamentos/química , Trastornos Relacionados con Opioides/tratamiento farmacológico , Trastornos Relacionados con Opioides/metabolismo , Ratas , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
Increased amounts of phosphorylated neurofilaments (pNF-H and pNF-M) are found in postmortem brains of opioid addicts. Because of the potential relevance of aberrant pNF in opioid addiction (alterations of neuronal cytoskeleton and associated functions), the effects of opiate drugs on pNF-H were investigated in rat brain. Acute morphine (30 mg/kg, 2 h) induced a marked increase in the immunodensity of pNF-H in the cerebral cortex (93%). Chronic morphine (10-100 mg/kg for 5 days) followed by opiate withdrawal resulted in a time-dependent decline in pNF-H (induction of tolerance). Thus, 2 h after the last dose of morphine, the abundance of pNF-H was still increased (27%), which was followed (6-24 h) by down-regulation of pNF-H (5% increase at 6 h; 5% decrease at 12 h, and 29% decrease at 24 h). The acute (10 mg/kg for 2 h) and chronic (2 x 10 mg/kg for 14 days) treatments with naloxone, an opioid receptor antagonist, did not alter pNF-H in the cerebral cortex, suggesting that the opioid receptors (probably the mu-type) modulating the phosphorylation state of NF-H are not tonically activated by endogenous opioids. The results indicate that morphine addiction is associated with an aberrant hyperphophorylation of NF-H in the rat brain.
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Morfina/farmacología , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Narcóticos/farmacología , Proteínas de Neurofilamentos/efectos de los fármacos , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Masculino , Proteínas de Neurofilamentos/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
The components of cyclic AMP signaling cascade (catalytic (Calpha) subunit of cyclic AMP-dependent protein kinase (PKA) and cyclic AMP response element binding protein (CREB)) were quantitated by Western blotting in the prefrontal cortex of depressed suicide victims (n=23) and their matched controls (n=14). There was a significant increase in the levels of CREB, both in total (tCREB; 121+/-8% (mean+/-S.E.M.), P<0.02) and phosphorylated (pCREB; 128+/-9%, P<0.01) forms, but not in PKA Calpha levels (109+/-9%, ns), in brains of depressed suicides compared to those in control subjects. The increases in CREB were specifically observed in antidepressant drug-free subjects (tCREB: 137+/-11%, P<0.01; pCREB: 136+/-12%, P<0.02; n=9), but not in the antidepressant-treated subjects (tCREB: 108+/-18%, ns; pCREB: 111+/-17%, ns; n=8). There were significant correlations between the levels of PKA and those of tCREB and pCREB in the prefrontal cortex of depressed suicides. These results indicate that the components of cyclic AMP signaling are upregulated in a coordinated manner in brains of depressed suicides and that this alteration is not related to antidepressant treatment.
Asunto(s)
Química Encefálica/fisiología , AMP Cíclico/metabolismo , Depresión/metabolismo , Corteza Prefrontal/metabolismo , Transducción de Señal/fisiología , Suicidio , Regulación hacia Arriba/fisiología , Adulto , Antidepresivos/farmacocinética , Biomarcadores/análisis , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Depresión/tratamiento farmacológico , Depresión/fisiopatología , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Fosforilación/efectos de los fármacos , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/fisiopatologíaRESUMEN
Interactions between brain alpha2- and beta-adrenoceptors are of interest in physiological (aging) and pathological (major depression) processes involving both receptors. In this study, total beta-adrenoceptors and beta1/2-subtypes were quantitated in postmortem human brains to investigate their relationships with alpha2A-adrenoceptors and specific G proteins during the process of aging and in brains of suicide victims. Analysis of [3H]CGP12177 binding, in the presence of CGP20712A (beta1-antagonist), indicated that the predominant beta-adrenoceptor in the frontal cortex is the beta1-subtype (65-75%). The density of total beta- (r=-0.60, n=44) or beta1-adrenoceptors (r=-0.78, n=22), but not the beta2-subtype, declined with aging (3-80 years). The density of total beta- or beta1-adrenoceptors, but not the beta2-subtype, correlated with the number of alpha2-adrenoceptors quantitated in the same brains with the agonist [3H]UK14304 (r=0.71-0.81) or the antagonist [3H]RX821002 (r=0.61-0.66). Interestingly, the ratios alpha2/beta- or alpha2/beta1-adrenoceptors did not correlate with the age of the subject at death, indicating that the proportion of alpha2/beta-adrenoceptors in brain remains rather constant during the process of aging. The density of beta-adrenoceptors correlated with the immunodensity of G(alpha)s (r=0.55) and Gbeta (r=0.61) proteins, and that of alpha2-adrenoceptors with those of G(alpha)i1/2 (r=0.88) and Gbeta (r=0.65). In brains of suicides, compared to controls, the ratio between alpha2- and beta- or beta1-adrenoceptors (alpha2-full agonist sites/beta-sites) was greater (1.3- to 2.0-fold; P<0.05). The results demonstrate a close interdependence between brain alpha2- and beta-adrenoceptors during aging, and in brains of suicides. The quantitation of the alpha2A/beta-adrenoceptor ratio could represent a relevant neurochemical index in the study of brain pathologies in which both receptors are involved.
Asunto(s)
Envejecimiento/metabolismo , Encéfalo/metabolismo , Depresión/metabolismo , Proteínas de Unión al GTP/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Adrenérgicos beta/metabolismo , Suicidio , Adolescente , Agonistas alfa-Adrenérgicos/farmacocinética , Antagonistas Adrenérgicos alfa/farmacocinética , Antagonistas Adrenérgicos beta/farmacocinética , Adulto , Anciano , Anciano de 80 o más Años , Química Encefálica/fisiología , Niño , Preescolar , Depresión/fisiopatología , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Propanolaminas/farmacocinética , Ensayo de Unión RadioliganteRESUMEN
1. This study was designed to assess the molecular and cellular events involved in the up-regulation (and receptor supersensitivity) of brain alpha(2)-adrenoceptors as a result of chronic depletion of noradrenaline (and other monoamines) by reserpine. 2. Chronic reserpine (0.25 mg kg(-1) s.c., every 48 h for 6 - 14 days) increased significantly the density (B(max) values) of cortical alpha(2)-adrenoceptor agonist sites (34 - 48% for [(3)H]-UK14304, 22 - 32% for [(3)H]-clonidine) but not that of antagonist sites (11 - 18% for [(3)H]-RX821002). Competition of [(3)H]-RX821002 binding by (-)-adrenaline further indicated that chronic reserpine was associated with up-regulation of the high-affinity state of alpha(2)-adrenoceptors. 3. In cortical membranes of reserpine-treated rats (0.25 mg kg(-1) s.c., every 48 h for 20 days), the immunoreactivities of various G proteins (Galphai(1/2), Galphai(3), Galphao and Galphas) were increased (25 - 34%). Because the high-affinity conformation of the alpha(2)-adrenoceptor is most probably related to the complex with Galphai(2) proteins, these results suggested an increase in signal transduction through alpha(2)-adrenoceptors (and other monoamine receptors) induced by chronic reserpine. 4. After alpha(2)-adrenoceptor alkylation, the analysis of receptor recovery (B(max) for [(3)H]-UK14304) indicated that the increased density of cortical alpha(2)-adrenoceptors in reserpine-treated rats was probably due to a higher appearance rate constant of the receptor ((Delta)r=57%) and not to a decreased disappearance rate constant ((Delta)k=7%). 5. Northern- and dot-blot analyses of RNA extracted from the cerebral cortex of saline- and reserpine-treated rats (0.25 mg kg(-1), s.c., every 48 h for 20 days) revealed that reserpine markedly increased the expression of alpha(2a)-adrenoceptor mRNA in the brain (125%). This transcriptional activation of the receptor gene expression appears to be the cellular mechanism by which reserpine induces up-regulation in the density of brain alpha(2)-adrenoceptors.