BACKGROUND: Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The extracellular-signal-regulated kinase (ERK) signaling pathway plays a crucial role in almost all cell functions, including proliferation, differentiation, survival, and death. However, the effect of ERK inhibition on oxysterol-induced apoptosis remains uncertain. METHODS: This study assessed the effect of ERK inhibition on the apoptotic effect of 7-ketocholesterol. RESULTS: Treatment with 7-ketocholesterol increased phosphorylated-ERK1/2 levels in differentiated PC12 cells, while the total amount of ERK was not altered. 7-Ketocholesterol decreased Bid and Bcl-2 levels, increased Bax and p53 levels, and promoted cytochrome c release, which elicits the activation of caspases (-8, -9, and -3), nuclear damage, and cell death. ERK and farnesyltransferase inhibitors inhibited the 7-ketocholesterol-induced phosphorylation of ERK1/2, activation of apoptosis-related proteins, and cell death in PC12 cells. CONCLUSIONS: The ERK and farnesyltransferase inhibitors, which did not exhibit toxicity, may inhibit the 7-ketocholesterol toxicity on differentiated PC12 cells by suppressing the activation of the caspase-8-dependent pathway as well as activation of the mitochondria-mediated cell-death pathway, leading to the activation of caspases. The inhibition of ERK may confer a beneficial protective effect against the neuronal cell injury induced by cholesterol oxidation products.
Animals ; Apoptosis ; Caspases ; Cell Death ; Cholesterol ; Cytochromes c ; Farnesyltranstransferase ; Ketocholesterols ; Neurons ; PC12 Cells ; Phosphorylation ; Phosphotransferases ; Proteins

BACKGROUND: Formation of cholesterol oxidation products is a suggested mechanism of neurodegenerative disorders. Neuronal cell death is mediated by an increased release of excitotoxic glutamate from the presynaptic nerve endings. Tyrosine-specific protein kinases modulate neurotransmitter release at the nerve terminals. Tyrphostin AG126 has anti-inflammatory and cytoprotective effects. However, it remains uncertain whether tyrphostin AG126 has a preventive effect on the alteration of nerve terminal function induced by cholesterol oxidation products. METHODS: The present study was performed to assess the effect of cholesterol oxidation products against nerve terminal function using synaptosomes isolated from rat cerebrum. We determined the preventive effect of tyrphostin AG126 against oxysterol toxicity by measuring the effects on the glutamate release, depolarization of the membrane potential, changes in Ca2+ levels, and Na+/K+-ATPase activity. RESULTS: Synaptosomes treated with 7-ketocholesterol or 25-hydroxycholesterol exhibited a sustained release of glutamate, depolarization of membrane potential, early rapid increase in cellular Ca2+ levels and decrease in Na+/K+-ATPase activity. Those responses were concentration-dependent. Treatment of tyrphostin AG126 interfered with alteration of synaptosomal functions and decrease in Na+/K+-ATPase activity induced by 7-ketocholesterol or 25-hydroxycholesterol. CONCLUSIONS: The results show that 7-ketocholesterol and 25-hydroxycholesterol seem to cause the release of glutamate by inducing depolarization of the membrane potential and early rapid increase in cellular Ca2+ levels and by inactivating Na+/K+-ATPase in the cerebral synaptosomes. Treatment of tyrphostin AG126 may prevent the oxysterol-induced nerve terminal dysfunction.
Animals ; Brain ; Cell Death ; Cerebrum ; Cholesterol ; Glutamic Acid ; Hydroxycholesterols ; Ketocholesterols ; Membrane Potentials ; Neurodegenerative Diseases ; Neurons ; Neurotransmitter Agents ; Presynaptic Terminals ; Protein-Tyrosine Kinases ; Rats ; Synaptosomes ; Tyrphostins

BACKGROUND: It has been shown that defects in mitochondrial function are involved in the induction of neuronal cell injury. Prostanoids such as prostaglandin E2 (PGE2) are thought to play an important role in inflammation and neurologic disorders. However, the effect of PGE2 on cholesterol-oxidation-product-induced neuronal cell injury remains uncertain. METHODS: The effect of PGE2 on toxicity of 7-ketocholesterol (7-KCS) was assessed in PC12 cells that were differentiated following treatment with nerve growth factor. The mitochondria-mediated apoptotic process was evaluated by examining the inhibitory effect of PGE2 on 7-KCS-induced toxicity. RESULTS: 7-KCS induced BID cleavage, increased the production of proapoptotic Bax protein, decreased antiapoptotic Bcl-2, increased p53, and promoted cytochrome c release in the cytosolic fraction, which subsequently elicited the activation of caspase-3, DNA fragmentation, and cell death. Treatment with PGE2 inhibited this 7-KCS-induced apoptotic process and cell death. CONCLUSIONS: The results show that PGE2 inhibits 7-KCS-induced toxicity in differentiated PC12 cells by suppressing the mitochondria-mediated apoptotic process. PGE2 may protect against cholesterol-oxidation-product-induced neuronal cell injury.
Animals ; bcl-2-Associated X Protein ; Caspase 3 ; Cell Death ; Cytochromes c ; Cytosol ; Dinoprostone ; DNA Fragmentation ; Inflammation ; Ketocholesterols ; Mitochondria ; Nerve Growth Factor ; Nervous System Diseases ; Neurons ; PC12 Cells ; Prostaglandins