Year: 2016
Type: dissertation
Abstract: Brain derived neurotrophic factor (BDNF) plays an important role in long-term synaptic potentiation \n(LTP) in the hippocampus, partially due to the upregulation of translation activity. However, the \nmolecular mechanisms are not fully understood. In this work we investigated the effect of BDNF of \nthe surface expression of N-methyl-D-aspartate receptors, which play a key role in synaptic \nplasticity. Stimulation of cultured hippocampal neurons with BDNF induced the synaptic \naccumulation of GluN2B-containing NMDAR, as determined by immunocytochemistry with an \nantibody against the N-terminal region of the receptor subunit and by colocalization with pre- and \npost-synaptic markers, the vesicular glutamate transporter type 1 (vGlut1) and postsynaptic density \nprotein 95 (PSD-95), respectively. The effect of BDNF on the amplitude and frequency of NMDAR \nmediated miniature excitatory postsynaptic currents (mEPSCs) was also evaluated under the same \nconditions by voltage-clamp recordings. Incubation of cultured hippocampal neurons with BDNF \nupregulated the amplitude of NMDA receptor-mediated mEPSC, but did not change the rise and \ndecay time. The enhancement in the amplitude of the currents was abrogated by the GluN2B \ncompetitive antagonist Conantokin G, in agreement with the results obtained in the \nimmunocytochemistry experiments. Antibody-feeding experiments showed no significant alteration \nin the rate of internalization of GluN2B-containing NMDA receptors following incubation of the \nneurotrophin, suggesting that a different mechanism is involved. The BDNF-evoked upregulation of \nGluN2B surface expression, as well as the increase in the amplitude of mEPSC, were abrogated by \nthe protein synthesis inhibitor cycloheximide, suggesting a role for local translation activity. Based \non unpublished observations from the laboratory showing a key role for the hnRNP K \n(heterogeneous nuclear ribonucleoprotein K) RNA binding protein in the regulation of protein \nsynthesis by BDNF, we characterized the role of this protein in the neurotrophin-induced \nupregulation of NMDA receptor mediated mEPSC. Transfection of hippocampal neurons with an \nshRNA against hnRNP K suppressed the effects of BDNF on the amplitude of mEPSC. Considering the \navailable evidence showing that BDNF upregulates Pyk2 protein levels in hippocampal neurons by a \nmechanism dependent on hnRNP K, we hypothesized that de novo synthesis of the kinase could play \na role in the regulation of the synaptic expression of GluN2B-containing NMDA receptors by BDNF. \nTransfection of hippocampal neurons with an shRNA specific for Pyk2 abrogated the BDNF-evoked \nincrease in the amplitude of the NMDA receptor-mediated mEPSC, showing a role for the kinase as a \nmediator of the TrkB-BDNF signaling to regulate the activity of NMDA receptors. BDNF also increased the frequency of NMDA receptor mediated mEPSC in cultured hippocampal \nneurons, by a mechanism sensitive to cycloheximide and possibly dependent on the presence of \nhnRNP K. This shows a presynaptic response to BDNF in glutamatergic synapses, which requires \nprotein synthesis. \nTogether, the results show a novel effect of BDNF in the regulation of the synaptic expression of \nNMDA receptors that is mediated by the RNA binding protein hnRNP K, as well as by the Pyk2 \ntyrosine kinase. These alterations in the surface expression of NMDA receptors may play an \nimportant role in LTP in the hippocampus.
Author: Luca, Pasqualino De
Language: English
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W6996161318