Mecanismos molecurales y celulares de la neuroprotección y plasticidad inducida por factor neurotrófico derivado del cerebro
[EN]Brain-derived neurotrophic factor (BDNF) is a pro-survival protein, highly expressed in the hippocampus, with critical functions in both developing and adult neurons. BDNF preferentially binds to TrkB receptors activating, in parallel, the Ras-ERK, PI3K/AKT and PLCgamma signaling pathways. Under physiological conditions, BDNF regulates several mechanisms of synaptic plasticiy. Under pathological conditions, BDNF protects hippocampal neurons from glutamate excitotoxicity and ischemia. However, the precise molecular mechanisms BDNF triggers upon TrkB receptor activation to induce neuronal protection and/or recovery are not fully understood. This study sought to address these issues and further examine the possible link between BDNF-induced mechanisms of neuroprotection and/or recovery and mechanisms of synaptic plasticity. In the first part of this study we examined molecular alterations induced by excitotoxicity, focusing on the downregulation of glutamic acid decarboxylase, which likely affects inhibitory synaptic transmission. We found that excitotoxic stimulation of cultured hippocampal neurons with glutamate leads to a time-dependent N-terminal cleavage of glutamic acid decarboxylase isoforms GAD65 and GAD67, upon ubiquitination and degradation of an unknown binding partner by the proteasome. The characteristic punctate distribution of GAD65 along neurites of differentiated cultured hippocampal neurons and total GAD activity measured in cerebellum or cerebral cortex extracts were significantly reduced. The results showed the deregulation of GADs under excitotoxic conditions, which most likely affects GABAergic neurotransmission, upon UPS activity, in addition to other proteolytic systems previously implicated in glutamate-induced excitotoxicity (Please refer to chapter 1). We then further evaluated the differential activation of the three main proteolytic mechanisms (UPS, calpains and caspases) implicated in excitotoxicity. We sought to study the protective effect of BDNF in different neuronal compartments and time points towards best understanding the spatiotemporal activation of BDNF-induced mechanisms of neuroprotection. These results showed a time-dependent activation of proteases and spatial segregation of these mechanisms. Calpain activation was followed by proteasome deregulation, in synaptic terminals and neuronal processes. Caspase activation subsequetly occurred in the cell body and all proteolytic mechanisms were significantly decreased by BDNF pre-incubation. Furthermore, proteasome and calpain inhibitors were unable to mimic the protective effect of BDNF and caspase inhibition in preventing chromatin condensation. Conversely, proteasome and calpain inhibition did protect the neuronal markers for dendrites (MAP-2), axons (Neurofilament H) and the vesicular glutamate transporters (VGLUT1 and VGLUT2), whereas caspase inhibition failed to mimic the protective effect of BDNF on neurites and synaptic markers. BDNF also partly prevented the downregulation of synaptic activity measured by the KCl-evoked glutamate release using a FRET glutamate nanosensor. Moreover, PLC¿ chemical inhibitors significantly blocked the protective action of BDNF, suggesting an activity-dependent mechanism of neuroprotection. Thus, we hypothesize that neuronal repair after a degenerative insult may start at the synaptic level and BDNF most likely induces recovery through reactivation of mechanisms of synaptic plasticity involving de novo protein synthesis (Please refer to chapter 2). The hypothesis above was assessed testing the effect of BDNF on VGLUT expression as an experimental paradigm. Exogenous application of BDNF to cultured hippocampal neurons at DIV7 (days in vitro) rapidly increased VGLUT2 mRNA and protein levels, in a dose-dependent manner, while VGLUT1 expression was only transiently increased. However, at DIV14, BDNF stably increased VGLUT1 expression, whilst VGLUT2 levels remained low. Transcription and translation inhibition fully blocked BDNF-induced VGLUT upregulation. Fluorescence microscopy imaging upon BDNF incubation showed a transient upregulation of VGLUT1 axonal trafficking and redistribution of VGLUT2-positive vesicles. These results suggest that BDNF may also affect VGLUTs subcellular distribution during development. Moreover, inhibition of TrkB receptors and PLC¿ signaling precluded BDNF-induced VGLUT upregulation, suggesting that BDNF regulates VGLUT expression during development and its effect on VGLUT1 may contribute to enhance glutamate release in LTP (Please refer to chapter 3). Overall, these results indicate that BDNF neuroprotection is not restricted to the cell soma and attenuation of caspase activation, also significantly protecting neurons from the excitotoxicity-induced damage to axons, dendrites and synapses, which predominantly results from calpain activation and increased protein ubiquitination. Furthermore, BDNF activates overlapping mechanisms under physiological and pathological conditions. BDNF concomitantly promotes connectivity between neurons and neuroprotection, by attenuating proteolytic mechanisms and/or inducing de novo expression of key neuronal markers, namely VGLUT1 and VGLUT2. Thus, we propose that reactivating BDNF-mediated developmental mechanisms of neuronal plasticity may enable to attenuate neuronal damages. BDNF may decrease proteolytic activation and/or induce recovery of hippocampal neurons under conditions of neurodegeneration.
Neuroscience, Life sciences, Cell biology, Molecular Biology, Tesis y disertaciones académicas, Universidad de Salamanca (España), Academic dissertations, Neurociencias, Ciencias de la vida, Biología celular, Biología molecular
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