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Unformatted text preview: Journal o f Neurochemistry LippincottRaven Publishers, Philadelphia 1998 International Society for Neurochemistry Neurotrophins Rescue Cerebellar Granule Neurons from Oxidative Stress-Mediated Apoptotic Death: Selective Involvement of Phosphatidylinositol 3-Kinase and the Mitogen-Activated Protein Kinase Pathway Stephen D. Skaper, Maura Floreani, *Alessandro Negro, Laura Facci, and Pietro Giusti Dipartimento di Farmacologia and *Dipartimento di Chimica Biologica, CRIBI, Universit di Padova, Padova, Italy Abstract: Cerebellar granule neurons maintained in me- dium containing serum and 25 mM K ~reliably undergo anapoptotic death when switched to serum-free medium with 5 mM K ~ .New mRNA and protein synthesis and formation of reactive oxygen intermediates are required steps inK ~deprivation-induced apoptosis of these neu- rons. Here we show that neurotrophins, members of the nerve growth factor gene family, protect from K~/serum deprivation-inducedapoptotic death of cerebellargranule neurons in atemporally distinct manner. Switching gran- ule neurons, on day in vitro (DIV) 4, 10, 20, 30, or 40, from high-K~to low-K~/serum-freemedium decreased viability by >50% when measured after 30 h. Treatment of low-K~granule neurons at DIV 4 with nerve growth factor, brain-derived neurotrophic factor (BDNF), neuro- trophin-3, or neurotrophin-4/5 (NT-4/5) demonstrated concentration-dependent (1100 ng/ml) protective ef- fects only for BDNF and NT-4/5. Between DIV 10 and 20, K ~-deprived granule neurons showed decreasing sensitivityto BDNF and no response to NT-4/5. Cerebel- lar granule neuron death induced by K ~withdrawal at DIV30 and 40was blocked onlyby neurotrophin-3. BDNF and NT-4/5 alsocircumvented glutamate-induced oxida- tive death in DIV 12 granule neurons. Granule neuron death caused by K ~withdrawal or glutamate-triggered oxidative stress was, moreover, limited by free radical scavengers like melatonin. Neurotrophin-protective ef- fects, but not those of antioxidants, were blocked by se- lective inhibitors of phosphatidylinositol 3-kinase or the mitogen-activated protein kinase pathway, depending on the nature of the oxidant stress. These observations indi- cate that the survival-promoting effects of neurotrophins for central neurons, whose cellular antioxidant defenses are challenged, require activation of distinct signal trans- duction pathways. Key Words: Oxidative stress ApoptosisNeurotrophinsNeuroprotection Signal transductionMelatoninGlutathione. J. Neurochem. 70, 18591868 (1998). The death of central neurons is widely recognized as a normal feature of vertebrate development. Neuronal programmed cell death (PCD) is thought to serve the removal of neuronal precursors that fail to establish appropriate synaptic connections (Oppenheim, 1991)....
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