title: Spatiotemporal resolution of BDNF neuroprotection against glutamate excitotoxicity in cultured hippocampal neurons.
abstract: 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 Förster (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 PLCγ 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.
authors: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.
authors: C V
authors: CV
authors: Melo
authors: S
authors: S
authors: Okumoto
authors: J R
authors: JR
authors: Gomes
authors: M S
authors: MS
authors: Baptista
authors: B A
authors: BA
authors: Bahr
authors: W B
authors: WB
authors: Frommer
authors: C B
authors: CB
authors: Duarte
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D001369:Axons / Q000187:drug effects / Q000378:metabolism; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology*; D002154:Calpain / Q000378:metabolism; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D004347:Drug Interactions; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018698:Glutamic Acid / Q000633:toxicity*; D006624:Hippocampus / Q000166:cytology*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D050052:TRPC Cation Channels / Q000378:metabolism; D013997:Time Factors
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D019208:Brain-Derived Neurotrophic Factor; D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; D018696:Neuroprotective Agents; D050052:TRPC Cation Channels; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D018698:Glutamic Acid; D002154:Calpain