PMID- 23384605 OWN - NLM STAT- MEDLINE DCOM- 20130923 LR - 20131121 IS - 1873-7544 (Electronic) IS - 0306-4522 (Linking) VI - 237 DP - 2013 May 1 TI - Spatiotemporal resolution of BDNF neuroprotection against glutamate excitotoxicity in cultured hippocampal neurons. PG - 66-86 LID - S0306-4522(13)00098-5 [pii] LID - 10.1016/j.neuroscience.2013.01.054 [doi] AB - Brain-derived neurotrophic factor (BDNF) protects hippocampal neurons from glutamate excitotoxicity as determined by analysis of chromatin condensation, through activation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3-K) signaling pathways. However, it is still unknown whether BDNF also prevents the degeneration of axons and dendrites, and the functional demise of synapses, which would be required to preserve neuronal activity. Herein, we have studied the time-dependent changes in several neurobiological markers, and the regulation of proteolytic mechanisms in cultured rat hippocampal neurons, through quantitative western blot and immunocytochemistry. Calpain activation peaked immediately after the neurodegenerative input, followed by a transient increase in ubiquitin-conjugated proteins and increased abundance of cleaved-caspase-3. Proteasome and calpain inhibition did not reproduce the protective effect of BDNF and caspase inhibition in preventing chromatin condensation. However, proteasome and calpain inhibition did protect the neuronal markers for dendrites (MAP-2), axons (Neurofilament-H) and the vesicular glutamate transporters (VGLUT1-2), whereas caspase inhibition was unable to mimic the protective effect of BDNF on neurites and synaptic markers. BDNF partially prevented the downregulation of synaptic activity measured by the KCl-evoked glutamate release using a Forster (Fluorescence) resonance energy transfer (FRET) glutamate nanosensor. These results translate a time-dependent activation of proteases and spatial segregation of these mechanisms, where calpain activation is followed by proteasome deregulation, from neuronal processes to the soma, and finally by caspase activation in the cell body. Moreover, PI3-K and PLCgamma small molecule inhibitors significantly blocked the protective action of BDNF, suggesting an activity-dependent mechanism of neuroprotection. Ultimately, we hypothesize that neuronal repair after a degenerative insult is initiated at the synaptic level. CI - Copyright (c) 2013 IBRO. Published by Elsevier Ltd. All rights reserved. FAU - Melo, C V AU - Melo CV AD - CNC - Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal. FAU - Okumoto, S AU - Okumoto S FAU - Gomes, J R AU - Gomes JR FAU - Baptista, M S AU - Baptista MS FAU - Bahr, B A AU - Bahr BA FAU - Frommer, W B AU - Frommer WB FAU - Duarte, C B AU - Duarte CB LA - eng GR - 1R01DK079109/DK/NIDDK NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20130204 PL - United States TA - Neuroscience JT - Neuroscience JID - 7605074 RN - 0 (Amino Acid Chloromethyl Ketones) RN - 0 (Brain-Derived Neurotrophic Factor) RN - 0 (Enzyme Inhibitors) RN - 0 (Nerve Tissue Proteins) RN - 0 (Neuroprotective Agents) RN - 0 (TRPC Cation Channels) RN - 0 (benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone) RN - 3KX376GY7L (Glutamic Acid) RN - EC 3.4.22.- (Calpain) SB - IM MH - Amino Acid Chloromethyl Ketones/pharmacology MH - Animals MH - Axons/drug effects/metabolism MH - Brain-Derived Neurotrophic Factor/*pharmacology MH - Calpain/metabolism MH - Cells, Cultured MH - Down-Regulation/drug effects MH - Drug Interactions MH - Embryo, Mammalian MH - Enzyme Inhibitors/pharmacology MH - Glutamic Acid/*toxicity MH - Hippocampus/*cytology MH - Nerve Tissue Proteins/metabolism MH - Neurons/cytology/*drug effects MH - Neuroprotective Agents/*pharmacology MH - Rats MH - Signal Transduction/drug effects MH - TRPC Cation Channels/metabolism MH - Time Factors EDAT- 2013/02/07 06:00 MHDA- 2013/09/24 06:00 CRDT- 2013/02/07 06:00 PHST- 2012/12/20 00:00 [received] PHST- 2013/01/28 00:00 [accepted] PHST- 2013/02/07 06:00 [entrez] PHST- 2013/02/07 06:00 [pubmed] PHST- 2013/09/24 06:00 [medline] AID - S0306-4522(13)00098-5 [pii] AID - 10.1016/j.neuroscience.2013.01.054 [doi] PST - ppublish SO - Neuroscience. 2013 May 1;237:66-86. doi: 10.1016/j.neuroscience.2013.01.054. Epub 2013 Feb 4.