Betulinic acid increases brain-derived neurotrophic factor expression via the PKA, NMDA receptor, and calcineurin pathways in cultured cortical neurons.

Brain-derived neurotrophic factor (BDNF) is a crucial neurotrophin that plays a vital role in neuronal survival, synaptic plasticity, and cognitive function. Reduced BDNF expression has been implicated in neurodegenerative and neuropsychiatric disorders. Thus, compounds that enhance Bdnf expression may have therapeutic potential for these disorders. In this study, we screened 48 compounds, including those from Morus alba L. (Moraceae) branches and structurally similar compounds, for their ability to induce Bdnf expression in primary cultured cortical neurons derived from Bdnf-Luciferase transgenic mice. M. alba-derived compounds failed to increase Bdnf expression; however, among the structurally similar compounds, betulinic acid (BA), moronic acid, and oleanolic acid elevated Bdnf mRNA levels. Further mechanistic analysis, particularly focusing on BA, revealed that the BA-induced Bdnf expression was inhibited by the protein kinase A (PKA) inhibitor H89, N-methyl-D-aspartate receptor (NMDAR) antagonist APV, and the calcineurin inhibitor FK506. Furthermore, BA promoted the nuclear translocation of cAMP-response element-binding protein (CREB)-regulated transcriptional coactivator 1 (CRTC1), which translocates to the nucleus upon dephosphorylation by calcineurin. The BA-induced nuclear translocation of CRTC1 was completely inhibited by H89, APV, and FK506. Although BA also increased CREB phosphorylation, the phosphorylation was prevented by H89 and APV, but not by FK506. These results suggest that CREB phosphorylation and CRTC1 nuclear translocation contribute to BA-induced Bdnf expression. Our findings indicate that BA enhances Bdnf expression in a manner dependent on PKA, NMDAR, and calcineurin signaling pathways, highlighting its potential as a modulator of activity-dependent BDNF transcription.