BDNF-TrkB signaling promotes synaptic GluN2A-NMDA receptor expression and network hyperexcitability in cultured hippocampal neurons and during status epilepticus

Abstract

Brain-derived neurotrophic factor (BDNF) is a key modulator of synaptic function, acting through activation of TrkB receptors. This neurotrophic factor mediates synaptic plasticity and plays an important role in epileptogenesis, but the underlying molecular mechanisms have not been fully elucidated. In this work, we investigated the role of BDNF-TrkB signaling in the regulation of synaptic GluN2A-containing NMDA receptors (NMDAR), and the impact on network synchronization in cultured hippocampal neurons. Incubation with BDNF increased the synaptic surface expression of GluN2A-containing NMDAR in rat hippocampal synaptoneurosomes and in cultured hippocampal neurons. The effect in the latter preparation was time-dependent and required new protein synthesis. Mechanistically, we identified a signaling cascade involving hnRNPK, the non-receptor tyrosine kinase Pyk2, and protein kinase C (PKC) as essential for mediating BDNF-induced upregulation in the synaptic expression of GluN2A-containing NMDAR. Knockdown of hnRNPK or Pyk2, pharmacological inhibition of PKC, or expression of a phosphorylation -deficient Pyk2 mutant abolished BDNF-induced synaptic surface accumulation of Glu2A. Moreover, Pyk2 phosphorylation at Y402 was necessary for both basal and BDNF-induced synaptic GluN2A expression. Functional multielectrode array (MEA) recordings showed that endogenous BDNF and GluN2A-containing NMDAR contributed to the increase in network activity in cultured hippocampal neurons evoked by stimulation with a cocktail including bicuculline, 4-aminopyridine, and glycine. Importantly, BDNF-TrkB signaling also mediated the upregulation in the hippocampal synaptic surface expression of GluN2A-containing NMDAR, as determined in rats subjected to the pilocarpine model of temporal lobe epilepsy, where increased GluN2A synaptic expression was observed and shown to be TrkB-dependent. These findings unveil a crucial BDNF/TrkB-PKC-Pyk2-hnRNPK signaling axis that regulates synaptic GluN2A levels and network excitability, offering novel insights into the molecular basis of synaptic plasticity.