ABSTRACT
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Subject(s)
Mythology , Symbolism , Information Services/standards , Information ServicesABSTRACT
Although numerous studies have demonstrated a neuroprotective and anti-apoptotic role of lithium in neuronal cell cultures, the precise mechanism by which this occurs, remains to be elucidated. In this study, we evaluated the lithium-mediated neuroprotection against colchicine-induced apoptosis in cultured cerebellar granule neurons. Previously, it has been demonstrated that colchicine mediates apoptosis in cerebellar granule neurons through cytoskeletal alteration and activation of an intrinsic pro-apoptotic pathway. Recently we also demonstrated a potential role of cyclin-dependent kinase 5 (cdk5) in this pathway. Here we report that colchicine induces dephosphorylation in Ser-9 and phosphorylation in Tyr-216, and thus activation, of glycogen synthase kinase-3beta in cerebellar granule neurons, and that this modification is inhibited by the presence of 5 mM lithium. However, the selective glycogen synthase kinase-3beta inhibitors SB-415286 and SB-216763 were unable to prevent colchicine-induced apoptosis in these cells, suggesting that the anti-apoptotic activity of lithium is not mediated by glycogen synthase kinase-3beta under these conditions. On the other hand, 5 mM lithium prevented the colchicine-induced increase in cdk5 expression and breakdown of cdk5/p35 to cdk5/p25. In addition, we show that up-regulation of cdk5/p25 is unrelated to inhibition of the activity of myocyte enhancer factor 2, a pro-survival transcription factor. These data suggest a previously undescribed neuroprotective mechanism of lithium associated with the modulation of cdk5/p35 or cdk5/p25 expression.
Subject(s)
Cerebellum/cytology , Cyclin-Dependent Kinases/metabolism , Lithium/administration & dosage , Neurons/drug effects , Neuroprotective Agents/administration & dosage , Aminophenols/pharmacology , Analysis of Variance , Animals , Animals, Newborn , Apoptosis/drug effects , Blotting, Western/methods , Caspases/metabolism , Cell Survival/drug effects , Cells, Cultured , Colchicine/pharmacology , Culture Media, Serum-Free/pharmacology , Cyclin-Dependent Kinase 5 , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Flow Cytometry/methods , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Indoles/pharmacology , MEF2 Transcription Factors , Maleimides/pharmacology , Microscopy, Electron, Transmission/methods , Myogenic Regulatory Factors , Neurons/ultrastructure , Potassium Deficiency , Rats , Rats, Sprague-Dawley , Serine/metabolism , Threonine/metabolism , Time Factors , Transcription Factors/metabolismABSTRACT
The mitochondrial peripheral benzodiazepine receptor (PBR) is involved in a functional structure designated as the mitochondrial permeability transition (MPT) pore, which controls apoptosis. PBR expression in nervous system has been reported in glial and immune cells. We now show expression of both PBR mRNA and protein, and the appearance of binding of a synthetic ligand fluo-FGIN-1-27 in mitochondria of rat cerebellar granule cells (CGCs). Additionally, the effect of PBR ligands on colchicine-induced apoptosis was investigated. Colchicine-induced neurotoxicity in CGCs was measured at 24 h. We show that, in vitro, PBR ligands 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4- benzodiazepin-2-one (Ro5-4864) and diazepam (25- 50 microM) enhanced apoptosis induced by colchicine, as demonstrated by viability experiments, flow cytometry and nuclear chromatin condensation. Enhancement of colchicine-induced apoptosis was characterized by an increase in mitochondrial release of cytochrome c and AIF proteins and an enhanced activation of caspase-3, suggesting mitochondrion dependent mechanism that is involved in apoptotic process. Our results indicate that exposure of neural cells to PBR ligands generates an amplification of apoptotic process induced by colchicine and that the MPT pore may be involved in this process.
Subject(s)
Apoptosis/drug effects , Benzodiazepinones/pharmacology , Diazepam/pharmacology , Isoquinolines/pharmacology , Neurons/drug effects , Receptors, GABA-A/drug effects , Animals , Animals, Newborn , Cell Survival/drug effects , Cells, Cultured , Cerebellum/cytology , Colchicine/toxicity , Ligands , Mitochondria/metabolism , Neurons/metabolism , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/metabolism , Time FactorsABSTRACT
Several lines of evidence show that cyclin-dependent kinases (CDKs) contribute to neurodegenerative disorders such as Alzheimer's and Parkinson's diseases, and amyotrophic lateral sclerosis. Given their role in the neuronal apoptosis, the inhibition of CDKs by specific drugs such as flavopiridol may be a valid therapeutic approach. Expression of CDKs was observed in rodent models of excitotoxicity and stroke, and CDK inhibitors showed neuroprotective effects. Flavopiridol may provide significant improvement in neurodegenerative diseases in humans.
Subject(s)
Apoptosis/drug effects , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Flavoproteins/administration & dosage , Models, Neurological , Neurodegenerative Diseases/drug therapy , Neurodegenerative Diseases/metabolism , Neurons/metabolism , Animals , Antineoplastic Agents/administration & dosage , Clinical Trials as Topic , Humans , Neurons/drug effects , Neuroprotective Agents/administration & dosageABSTRACT
OBJECTIVES: Here we evaluated the neuroprotective effects of two well-known mood stabilizers, lithium and valproic acid (VPA), against colchicine neurotoxicity in cerebellar granule cells (CGNs). METHODS: The CGNs were differentiated for 7 days, pretreated with lithium or VPA for 24 h and after colchicine 1 microM was added. Cellular damage was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) method and apoptosis in CGNs was characterized by chromatin condensation and DNA fragmentation. RESULTS: Incubation with lithium (1-5 mM) attenuated this apoptosis markedly, in a dose-dependent way however, the addition of VPA (0.5-2 mM) did not protect CGNs. Colchicine-induced apoptosis is mediated through the activation of caspase-3. An increase in caspase-3 activity was detected within 18 h and was blocked in presence of lithium 5 mM. CONCLUSIONS: Our data indicate that lithium treatment is selectively neuroprotective; however, in our experimental conditions VPA did not protect CGNs from apoptosis induced by colchicine. Our results support the hypothesis that distinct pathways mediate the neuroprotective effects of lithium and VPA.
Subject(s)
Apoptosis/drug effects , Cerebellum/drug effects , Cerebellum/pathology , Colchicine/antagonists & inhibitors , Colchicine/pharmacology , Gout Suppressants/pharmacology , Lithium Carbonate/pharmacology , Neurons/drug effects , Neurons/pathology , Neuroprotective Agents/pharmacology , Animals , Colchicine/administration & dosage , Colorimetry , Flow Cytometry , Gout Suppressants/administration & dosage , Lithium Carbonate/administration & dosage , Neuroprotective Agents/administration & dosage , Rats , Rats, Sprague-DawleyABSTRACT
Acetylcholinesterase inhibitors (AChEI) are among the drugs most widely used in the treatment of Alzheimer's disease. They increase the levels of acetylcholine and thus improve the cognitive symptoms that are impaired. We tested whether specific AChEI show additional neuroprotective properties against colchicine-induced apoptosis in cerebellar granule neurons (CGNs), a well established apoptotic model mediated by neuronal cytoskeleton alteration. Colchicine-induced apoptosis is due to an increase in the activity of GSK-3beta and CDK5, two enzymes involved in cytoskeletal alteration. Furthermore, the intrinsic apoptotic pathway is activated by colchicines, as revealed by cytochrome c release and Bax translocation. Tacrine, (-)-huperzine A and (+/-)-huprine Y, the AChEI tested in the study, did not reverse the loss of neuronal viability induced by colchicine. Moreover, the increase in apoptotic features induced by colchicine treatment, as measured by flow cytometry and nuclear chromatin condensation, was not prevented by these AChEI. Although some of these drugs are of interest to treat Alzheimer's disease, their lack of efficacy in the prevention of colchicine-induced apoptosis in CGNs suggests that they cannot prevent neuronal loss due to cytoskeleton alteration.
Subject(s)
Aminoquinolines/pharmacology , Apoptosis/drug effects , Cholinesterase Inhibitors/pharmacology , Cytoskeleton/drug effects , Heterocyclic Compounds, 4 or More Rings/pharmacology , Neuroprotective Agents/pharmacology , Sesquiterpenes/pharmacology , Tacrine/pharmacology , Alkaloids , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Aminoquinolines/administration & dosage , Animals , Animals, Newborn , Cerebellum/drug effects , Cerebellum/metabolism , Cerebellum/pathology , Cholinesterase Inhibitors/administration & dosage , Colchicine/adverse effects , Colchicine/antagonists & inhibitors , Cytoskeleton/pathology , Disease Models, Animal , Flow Cytometry , Glycogen Synthase Kinase 3/drug effects , Glycogen Synthase Kinase 3/metabolism , Heterocyclic Compounds, 4 or More Rings/administration & dosage , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Neuroprotective Agents/administration & dosage , Rats , Rats, Sprague-Dawley , Sesquiterpenes/administration & dosage , Tacrine/administration & dosageABSTRACT
The mechanisms underlying selective neuronal cell death in kainic acid-mediated neurodegeneration are not fully understood. We have recently demonstrated that in cerebellar granule neurons, kainic acid induces the expression of proteins associated with cell-cycle progression. In the present study we show that 3-amino thioacridone (3-ATA), a selective cyclin-dependent kinase 4 inhibitor, attenuates kainic acid-induced apoptosis in cerebellar granule neurons. When neurons were pre-treated with 3-ATA 10 microM for 24 h, they were less susceptible to damage induced by kainic acid 500 microM, since the number of dead cells decreased significantly. In flow cytometry studies using propidium iodide staining, 3-ATA also reduced the ratio of apoptotic cells induced by kainic acid. Moreover, 3-ATA decreased the proportion of cells with a condensed nucleus from 55% to 22%. Our data suggest that the cell cycle pathway is involved in the mechanism of apoptosis mediated by kainic acid and that cyclin-dependent kinase 4 plays a prominent role in this process. 3-ATA may to prevent the apoptosis associated with neurodegenerative disorders without the over-activation of excitatory amino acid receptors.
