RESUMEN
The molecular and biochemical mode of cell death of dopaminergic neurons in Parkinson's disease (PD) is uncertain. In an attempt at further clarification we studied the effects of 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), on dopaminergic PC12 cells. In humans and nonhuman primates MPTP/MPP+ causes a syndrome closely resembling PD. MPP+ toxicity is thought to be mediated by the block of complex I of the mitochondrial electron transport chain. Treatment of undifferentiated PC12 cells with MPP+ primarily inhibited proliferation of PC12 cells and secondarily led to cell death after the depletion of all energy substrates by glycolysis. This cell death showed no morphological characteristics of apoptosis and was not blocked by treatment with caspase inhibitors. The inhibition of cell growth was not dependent on an inhibition of complex I activity since MPP+ also inhibited cell proliferation in SH-SY5Y cells lacking mitochondrial DNA and complex I activity (p0 cells). As shown by flow cytometric analysis, MPP+ induced a block in the G0/G1 to S phase transition that correlated with increased expression of the cyclin-dependent kinase inhibitor p21(WAF1/Cip1) and growth arrest. Since treatment with 1 microM MPP+ caused apoptotic cell death in p21(WAF1/Cip1)-deficient (p21(-/-)) but not in parental (p21(+/+)) mouse embryo fibroblasts, our data suggest that in an early phase MPP+-induced p21(WAF1/Cip1) expression leads to growth arrest and prevents apoptosis until energy depletion finally leads to a nonapoptotic cell death.
Asunto(s)
1-Metil-4-fenilpiridinio/toxicidad , Apoptosis , Ciclinas/biosíntesis , Dopaminérgicos/toxicidad , Neuronas/efectos de los fármacos , Transducción de Señal , 1-Metil-4-fenilpiridinio/metabolismo , Animales , División Celular , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , ADN/biosíntesis , Dopaminérgicos/metabolismo , Metabolismo Energético , Fase G1 , Ratones , Mitocondrias/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Neuronas/citología , Neuronas/metabolismo , Células PC12 , Ratas , Fase de Descanso del Ciclo Celular , Fase S , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
Glutathione (GSH) levels are supposed to determine the vulnerability of many cells towards a wide array of insults. We investigated the effects of chronic inhibition of GSH synthesis and acute depletion of GSH on cerebellar granule neurons in vitro and determined cytoplasmic and mitochondrial GSH with relation to mitochondrial function and generation of reactive oxygen intermediates (ROI). l-buthionine sulfoximine (BSO), which irreversibly blocks gamma-glutamyl-cysteine synthase, led to a time- and concentration-dependent loss of cytoplasmic GSH, while mitochondrial GSH was relatively preserved. No increased generation of ROI was detected over 48 h and the mitochondrial membrane potential was largely maintained. Neuronal degeneration occurred when mitochondrial GSH levels had fallen below 50% of control after 24-36 h. In contrast, direct conjugation of mitochondrial and cytoplasmic GSH with etacrynic acid (EA), resulted in immediate loss of mitochondrial GSH, a large increase of ROI within 2 h, subsequent collapse of the mitochondrial membrane potential and complete cell death within 4-8 h. Electron microscopy studies revealed an as yet unknown change of the chromatin structure to a homogeneous granular pattern after BSO, while EA resulted in typical necrotic changes. No typical features of apoptosis, i.e., no chromatin condensation or DNA fragmentation were detected after GSH depletion after BSO or EA treatment.
Asunto(s)
Apoptosis/fisiología , Glutatión/metabolismo , Mitocondrias/metabolismo , Neuronas/citología , Especies Reactivas de Oxígeno/metabolismo , Animales , Antimetabolitos/farmacología , Bisbenzimidazol , Butionina Sulfoximina/farmacología , Cerebelo/citología , Cicloheximida/farmacología , Fragmentación del ADN , Colorantes Fluorescentes , Glutatión/análogos & derivados , Glutatión/farmacología , Microscopía Electrónica , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
The inhibitor of apoptosis (IAP) family of antiapoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 mM potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 mM potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N-acetyl-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, but not at later time points, suggesting that IAPs delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 microM or 1 mM glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.
Asunto(s)
Adenoviridae , Apoptosis/fisiología , Técnicas de Transferencia de Gen , Neuronas/citología , Infecciones por Adenoviridae , Animales , Caspasa 3 , Caspasas/metabolismo , Células Cultivadas , Cerebelo/citología , Inhibidores de Cisteína Proteinasa/genética , Fragmentación del ADN , Activación Enzimática/fisiología , Regulación Viral de la Expresión Génica , Etiquetado Corte-Fin in Situ , Proteínas Inhibidoras de la Apoptosis , Neuronas/enzimología , Neurotoxinas/metabolismo , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Proteínas Virales/genéticaRESUMEN
Bcl-2 family proteins are principal regulators of cell death during normal development as well as in many disease states. Differentiated cerebellar granule neurons are protected from apoptosis by depolarizing concentrations of potassium. Further, these cells acquire resistance to glutamate-mediated excitotoxicity when pre-exposed to subtoxic concentrations of the glutamate receptor agonist, N-methyl-D-aspartate. Here, we report that the expression of bcl-2, bcl-xL, bcl-xS, bax and bad mRNA as well as of Bcl-2, Bax, Bcl-XL, Bcl-XS and Bag-1 proteins is not modulated in these two paradigms of neuronal cell death. However, mitochondrial release of cytochrome c, which is thought to be controlled by Bcl-2 family proteins, is detected 5 h after switching the neurons to low potassium conditions. Thus, there appears to be regulation of Bcl-2 family protein bioactivity in the absence of altered protein expression during potassium deprivation-induced apoptosis of cerebellar granule neurons.
Asunto(s)
Apoptosis , Cerebelo/citología , Grupo Citocromo c/metabolismo , Neuronas/citología , Potasio/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Células Cultivadas , Cerebelo/metabolismo , Medios de Cultivo , Regulación de la Expresión Génica/efectos de los fármacos , Genes bcl-2 , N-Metilaspartato/farmacología , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/metabolismo , RatasRESUMEN
Depolarizing concentrations of potassium (K+) promote maturation and survival of cerebellar granule neurons in vitro. Withdrawal of potassium from differentiated neurons induces morphological and biochemical features of apoptosis, including membrane blebbing, nuclear condensation, activation of caspases, and internucleosomal DNA fragmentation. Significant DNA fragmentation is detectable at 6 h after K+ withdrawal and slowly increases thereafter. Two observations indicate that endonucleolytic DNA degradation is neither required nor sufficient for K+ withdrawal-induced apoptosis in cerebellar granule neurons: (i) neurons are rescued from apoptosis by readdition of K+ up to 8 h after K+ withdrawal, when DNA fragmentation has already occurred. (ii) The endonuclease inhibitor, aurintricarboxylic acid, inhibits DNA fragmentation as assessed by quantitative DNA fluorometry, TUNEL staining, and DNA gel electrophoresis, but not cell death or chromatin condensation induced by K+ withdrawal.