Brain-derived neurotrophic factor-induced phosphorylation of neurofilament-H subunit in primary cultures of embryo rat cortical neurons

Phosphorylation of the neurofilament-H subunit (NF-H) was investigated in r at embryonic brain neurons in culture. A portion of the NF-H was phospphory lated in vivo at embryonic day 17 when brain neurons were prepared. When th e neurons were isolated and_cultured, the NF proteins disappeared once and then reappeared over the next several days in the following order: (1) NF-L /NF-M, (2) dephosphorylated NF-H and (3) phosphorylated NF-H, Phosphorylati on of NF-H began around 4 days after cell plating, at about the time of syn apse formation. Treatments that appeared to modulate the timing of synapse formation also affected the timing of NF-H phosphorylation: (1) earlier pho sphorylation was observed at higher neuronal cell density, (2) earlier phos phorylation was observed in neurons cultured on a coating substrate that pr omotes rapid neurite extension and (3) phosphorylation was suppressed when neurite extension was inhibited by brefeldin A, Three possible synapse form ation-induced events, excitation, cell-cell contact through adhesion protei ns and elevated concentrations of neurotrophic factors, were examined for t heir possible involvement in generating the signal for NF-H phosphorylation , Neither excitation nor cell contact enhanced NF-H phosphorylation, Neurot rophic factors, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) stimulated phosphorylation of NF-H, The BDNF-stimulated phosphorylat ion was inhibited by an anti-BDNF antibody and K252a, an inhibitor of BDNF receptor TrkB tyrosine kinase, Among known NF-H kinases of cyclin-dependent kinase 5 (CDK5), external signal-regulated protein kinase (ERK) and stress -activated protein kinase (SAPK), CDK5 and SAPK showed an increase in kinas e activity or an active form with a time course similar to NPH phosphorylat ion in control culture, On the other hand, BDNF stimulated the kinase activ ity of CDK5 and induced appearance of an active form of ERK transiently. Th ese results suggest a possibility that synapse formation induces NF-H phosp horylation, at least in part, through activation of CDK5 by BDNF.