RESUMEN
Cell adhesion molecules might play an important role in the inflammatory mechanisms associated with neurodegeneration. We have previously observed, in rats, that subcutaneous injection of complete Freund's adjuvant (CFA), a pro-inflammatory agent that induces a peripheral inflammatory stimulus, reduces the nigrostriatal degeneration and microglial activation caused by stereotaxic injection of 6-hydroxydopamine (6-OHDA). Here we further investigated the effects of CFA in 6-OHDA-lesioned rats by evaluating the expression of selected adhesion molecules, both at central and peripheral levels. Male, Sprague-Dawley rats received a subcutaneous injection of CFA followed, 10 days later, by intrastriatal injection of 6-OHDA. Animals were sacrificed at various time points and changes affecting intercellular (ICAM-1), vascular (VCAM-1), platelet endothelial (PECAM-1) and neural (NCAM-1) cell adhesion molecules were analyzed in striatum, ventral midbrain (containing the substantia nigra) and sera. Our results confirmed the protective effect of systemic CFA on 6-OHDA-induced nigrostriatal degeneration. Injection of 6-OHDA increased striatal ICAM-1 and PECAM-1 expression, while opposite changes (decreased expression) were detected in the ventral midbrain, particularly for VCAM-1 and NCAM-1. Pretreatment with CFA counteracted these changes. Nigrostriatal degeneration also affected peripheral immune function, with lesioned animals showing increased sPECAM levels with respect to intact animals. Also in this case, CFA pretreatment blocked the 6-OHDA induced increase of sPECAM. Our findings confirm that a pre-existing, peripheral pro-inflammatory condition reduces the neuroinflammatory response and associated neurodegeneration provoked by centrally-administered 6-OHDA, with a mechanism that seems to involve selected adhesion molecules. The link between peripheral and central immune responses may, therefore, represent a target for new therapeutic strategies aimed at reducing the neuroinflammatory component associated with neurodegeneration.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Adyuvante de Freund/farmacología , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Fármacos Neuroprotectores/metabolismo , Trastornos Parkinsonianos/tratamiento farmacológico , Trastornos Parkinsonianos/metabolismo , Animales , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Oxidopamina/toxicidad , Trastornos Parkinsonianos/patología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Ratas , Ratas Sprague-Dawley , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Simpaticolíticos/toxicidad , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Abnormal deposition of protein aggregates and increased susceptibility to apoptotic cell death may result from defects in the activity of the ubiquitin-proteasome system (UPS); neurotoxicity related to UPS defects seems to require dopamine to be fully expressed. The aim of this study was to investigate the pro-apoptotic effects caused by proteasomal activity inhibition, as well as the synergistic effect of dopaminergic stimulation in human lymphocytes isolated from healthy volunteers. Cells were incubated 20 h at 37 degrees C, with: (1) lactacystin, (2) increasing concentrations of dopamine or (3) mixture of dopamine and lactacystin. Activities of proteasome 20S and pro-apoptotic caspases-3 and -9 and levels of anti-apoptotic Bcl-2 were measured with fluorimetric or immunochemical assays, while a "DNA diffusion" assay was used to determine the apoptosis. Incubation of lymphocytes with lactacystin, which caused reduction of proteasomal activity, was associated with activation of caspases. A clear, dose-dependent reduction of proteasomal activity was also seen in the presence of increasing doses of dopamine, which was accompanied by a slight dose-dependent increase of caspases activities and Bcl-2 levels. Both effects on proteasome and caspase activities were enhanced when cells were simultaneously exposed to lactacystin and elevated concentrations of dopamine. Apoptosis was detected in all treated samples, but not in controls, without significant differences among the treatment groups; however, the association of dopamine and lactacystin induced a clear reduction in the number of cells being analyzed, pointing to marked cytotoxicity. Our data confirm the potentiation of cytotoxicity related to proteasome inhibition, in the presence of dopaminergic stimulation.
Asunto(s)
Acetilcisteína/análogos & derivados , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/toxicidad , Dopamina/metabolismo , Linfocitos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Acetilcisteína/toxicidad , Adulto , Caspasa 3/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 9/efectos de los fármacos , Caspasa 9/metabolismo , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Femenino , Genes bcl-2/efectos de los fármacos , Humanos , Técnicas In Vitro , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Inhibidores de Proteasoma , Ubiquitina/metabolismo , Proteína X Asociada a bcl-2RESUMEN
Wilson's disease (WD) is an inherited disorder, characterized by selective copper deposition in liver and brain, chronic hepatitis and extra-pyramidal signs. In this study, we investigated changes of biochemical markers of oxidative stress and apoptosis in liver, striatum and cerebral cortex homogenates from Long-Evans Cinnamon (LEC) rats, a mutant strain isolated from Long Evans (LE) rats, in whom spontaneous hepatitis develops shortly after birth. LEC and control (LE) rats at 11 and 14 weeks of age were used. We determined tissue levels of glutathione (GSH/GSSG ratio), lipid peroxides, protein-thiols (P-SH), nitric oxide metabolites, activities of caspase-3 and total superoxide-dismutase (SOD), striatal levels of monoamines and serum levels of hepatic amino-transferases. We observed a decrease of protein-thiols, GSH/GSSG ratio and nitrogen species associated to increased lipid peroxidation in the liver and striatum - but not in the cerebral cortex - of LEC rats, accompanied by dramatic increase in serum amino-transferases and decrease of striatal catecholamines. Conversely, SOD and caspase-3 activity increased consistently only in the cortex of LEC rats. Hence, we assume that enhanced oxidative stress may play a central role in the cell degeneration in WD, at the main sites of copper deposition, with discrete pro-apoptotic conditions developing in distal areas.
Asunto(s)
Apoptosis/fisiología , Encéfalo/metabolismo , Hepatitis Crónica/fisiopatología , Degeneración Hepatolenticular/fisiopatología , Hígado/metabolismo , Estrés Oxidativo/fisiología , Análisis de Varianza , Animales , Monoaminas Biogénicas/metabolismo , Caspasa 3 , Caspasas/metabolismo , Cromatografía Líquida de Alta Presión , Glutatión/metabolismo , Hepatitis Crónica/etiología , Degeneración Hepatolenticular/complicaciones , Peróxidos Lipídicos/metabolismo , Óxido Nítrico/metabolismo , Ratas , Ratas Endogámicas LEC , Compuestos de Sulfhidrilo/metabolismo , Superóxido Dismutasa/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico , Transaminasas/sangreRESUMEN
Long-term treatment of Parkinson disease (PD) is frequently associated with l-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesias (LIDs). L-DOPA-induced dyskinesias are likely due to changes in the signal transduction pathways, at the striatal level, related to pulsatile stimulation of dopamine receptors. We investigated whether markers of this phenomenon can also be detected peripherally. We analyzed mRNA expression for D5 (D1-like) and D3 (D2-like) receptors and levels of second messengers, such as cAMP and free intracellular Ca2+ ([Ca2+]i), in peripheral blood lymphocytes of PD patients with (LID+) or without LIDs (LID-). Patients with PD showed depressed [Ca2+]i rise in response to mitogen-induced activation. The defect was more pronounced in LID+ (-33% with respect to healthy controls) than in LID- patients (-20%). Peripheral blood lymphocyte levels of cAMP were decreased in both LID+ (3.8 +/- 2.9 pmol/10 cells) and LID- patients (4.2 +/- 2.4 pmol/10(6) cells), with respect to controls (6 +/- 2.6 pmol/10(6) cells). No differences were found in dopamine receptor mRNA expression. Our results demonstrate that second messenger levels are altered in the peripheral blood lymphocytes of PD patients treated with dopaminergic agents and that patients with LIDs show further alterations in the regulation of [Ca2+]i homeostasis. This may represent a distinctive trait of patients prone to develop dyskinetic movements.
Asunto(s)
Acatisia Inducida por Medicamentos/etiología , Antiparkinsonianos/efectos adversos , Calcio/metabolismo , Homeostasis/efectos de los fármacos , Levodopa/efectos adversos , Enfermedad de Parkinson/metabolismo , Anciano , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Estudios de Casos y Controles , AMP Cíclico/metabolismo , Femenino , Humanos , Ionóforos/farmacología , Lactonas/farmacología , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Masculino , Persona de Mediana Edad , Mitógenos/farmacología , Enfermedad de Parkinson/tratamiento farmacológico , Fitohemaglutininas/farmacología , ARN Mensajero/metabolismo , Receptores de Dopamina D3/genética , Receptores de Dopamina D3/metabolismo , Receptores de Dopamina D5/genética , Receptores de Dopamina D5/metabolismo , Sesquiterpenos/farmacologíaRESUMEN
Altered glutamatergic neurotransmission is central to the expression of Parkinson's disease (PD) symptoms and may underlie l-DOPA-induced dyskinesias. Drugs acting on glutamate metabotropic receptors (mGluR) of group I can modulate subthalamic nucleus (STN) overactivity, which plays a pivotal role in these phenomena, and may counteract dyskinesias. To address these issues, we investigated the effects of a 3-week treatment with mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP), or of a subthalamic lesion, on abnormal involuntary movements (AIMs) and associated striatal expression of transcription factor FosB/Delta FosB caused by chronic l-DOPA administration, in rats with a nigrostriatal lesion. MPEP virtually abolished AIMs and reduced, dramatically, striatal expression of FosB/Delta FosB. Reduced FosB/Delta FosB expression, coupled with nonsignificant reduction of AIMs, was also observed in STN-lesioned rats. Our data confirm the role of glutamatergic neurotransmission in the pathogenesis of dyskinesias and the potential of mGluR5 antagonists in the treatment of l-DOPA-induced dyskinesias.
Asunto(s)
Antiparkinsonianos/efectos adversos , Discinesia Inducida por Medicamentos/tratamiento farmacológico , Discinesia Inducida por Medicamentos/etiología , Antagonistas de Aminoácidos Excitadores/efectos adversos , Levodopa/efectos adversos , Piridinas/efectos adversos , Núcleo Subtalámico/fisiología , Adrenérgicos/toxicidad , Análisis de Varianza , Animales , Conducta Animal , Modelos Animales de Enfermedad , Discinesia Inducida por Medicamentos/cirugía , Antagonistas de Aminoácidos Excitadores/uso terapéutico , Masculino , Oxidopamina/toxicidad , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/etiología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Piridinas/uso terapéutico , Ratas , Núcleo Subtalámico/lesiones , Factores de Tiempo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Despite the progressive development of innovative animal models for Parkinson's disease, the intracerebral infusion of neurotoxin 6-hydroxydopamine (6-OHDA) remains the most widely used means to induce an experimental lesion of the nigrostriatal pathway in the animal, due to its relatively low complexity and cost, coupled with the high reproducibility of the lesion obtained. To gain new information from such a classic model, we studied the time-course of the nigrostriatal damage, metabolic changes in the basal ganglia nuclei (cytochrome oxidase activity) and behavioural modifications (rotational response to apomorphine) following unilateral injection of 6-OHDA into the corpus striatum of rat, over a 4-week period. Striatal infusion of 6-OHDA caused early damage of dopaminergic terminals, followed by a slowly evolving loss of dopaminergic cell bodies in the substantia nigra pars compacta, which became apparent during the second week post-injection and peaked at the 28th day post-infusion; the rotational response to apomorphine was already present at the first time point considered (Day 1), and remained substantially stable throughout the 4-week period of observation. The evolution of the nigrostriatal lesion was accompanied by complex changes in the metabolic activity of the other basal ganglia nuclei investigated (substantia nigra pars reticulata, entopeduncular nucleus, globus pallidus and subthalamic nucleus), which led, ultimately, to a generalized, metabolic hyperactivity, ipsilaterally to the lesion. However, peculiar patterns of metabolic activation, or inhibition, characterized the post-lesional responses of each nucleus, in the early and intermediate phases, with peculiar response profiles that varied closely related to the functional position occupied within the basal ganglia circuitry.
Asunto(s)
Ganglios Basales , Conducta Animal/efectos de los fármacos , Oxidopamina , Enfermedad de Parkinson Secundaria , Sustancia Negra/patología , Animales , Ganglios Basales/efectos de los fármacos , Ganglios Basales/metabolismo , Ganglios Basales/patología , Conducta Animal/clasificación , Muerte Celular/efectos de los fármacos , Citocromos c/metabolismo , Modelos Animales de Enfermedad , Inmunohistoquímica/métodos , Masculino , Enfermedad de Parkinson Secundaria/inducido químicamente , Enfermedad de Parkinson Secundaria/patología , Enfermedad de Parkinson Secundaria/fisiopatología , Ratas , Ratas Sprague-Dawley , Sustancia Negra/efectos de los fármacos , Factores de Tiempo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Complete Freund's adjuvant (CFA), a pro-inflammatory agent, was inoculated, subcutaneously, to Sprague-Dawley rats prior to the intrastriatal injection of 6-hydroxydopamine (6-OHDA). Animals were sacrificed 7 and 28 days following 6-OHDA injection; neuronal damage, glial activation and cytokine levels, within the nigrostriatal system, were then investigated. Nigrostriatal degeneration induced by 6-OHDA was accompanied by early microglial and astroglial activation, which preceded the onset of dopaminergic cell loss, in the SNc, without significant changes in cytokine levels. CFA pretreatment markedly reduced the SNc neuronal loss and associated microglial activation, as well as the rotational response to apomorphine. These changes were associated with moderate, transient increases in the nigrostriatal levels of glial-cell-derived neurotrophic factor (GDNF) and pro-inflammatory cytokines, including interleukin (IL)-1alpha, IL-1beta and IL-6. Our results show that prior delivery of a peripheral, pro-inflammatory stimulus induces neuroprotection, in a rodent model of Parkinson's disease, possibly through the modulation of cytokine production at the nigrostriatal level.
Asunto(s)
Adyuvante de Freund/inmunología , Degeneración Nerviosa/inmunología , Neuroglía/inmunología , Fármacos Neuroprotectores/inmunología , Trastornos Parkinsonianos/inmunología , Adyuvantes Inmunológicos/uso terapéutico , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Adyuvante de Freund/uso terapéutico , Masculino , Actividad Motora/fisiología , Neostriado/efectos de los fármacos , Neostriado/inmunología , Neostriado/patología , Degeneración Nerviosa/inducido químicamente , Neuroglía/patología , Fármacos Neuroprotectores/uso terapéutico , Oxidopamina , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/patología , Trastornos Parkinsonianos/prevención & control , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Sustancia Negra/inmunología , Sustancia Negra/patologíaRESUMEN
Homocysteine (Hcy) is a nonprotein-forming sulphur amino acid that plays an important role in remethylation and trans-sulphuration processes. In recent years, it has been suggested that increased levels of plasma Hcy may play a role in the pathogenesis of various diseases, particularly at the cardiovascular level. The pathogenic mechanism of hyperhomocysteinemia, however, has not been clarified. Because oxygen radicals can be generated by the auto-oxidation of this amino acid, it has been suggested that Hcy may cause cellular damage through oxidative mechanisms, ultimately leading to apoptotic cell death. In this study, we sought to investigate the effects of Hcy on oxidative damage and antioxidant agent levels, as well as on apoptosis-related proteins and apoptosis occurrence in human cells. For this purpose, we measured levels of Bcl-2, caspase-3 and caspase-9 activity, Cu/Zn superoxide dismutase, reduced glutathione, lipid peroxidation [malondialdehyde and 4-hydroxy-2 (E)-nonenal concentrations], apoptotic single-stranded DNA and nuclear changes in human isolated lymphocytes exposed to increasing concentrations of Hcy. Incubation with Hcy did not induce significant changes in any of these biomarkers. Therefore, our results do not support the existence of a direct link between increased levels of Hcy and the occurrence of a pro-apoptotic state mediated by enhanced oxidative stress.