SMAD pathway mediation of BDNF and TGF2 regulation of proliferation and differentiation of hippocampal granule neurons

Hippocampal granule cells self-renew throughout life, whereas their cerebellar counterparts become post-mitotic during early postnatal development, suggesting that locally acting, tissue-specific factors may regulate the proliferative potential of each cell type. Confirming this, we show that conditioned medium from hippocampal cells (CM_Hippocampus) stimulates proliferation in cerebellar cultures and, vice versa, that mitosis in hippocampal cells is inhibited by CM_Cerebellum. The anti-proliferative effects of CM_Cerebellum were accompanied by increased expression of the cyclin-dependent kinase inhibitors p21 and p27, as well as markers of neuronal maturity/differentiation. CM_Cerebellum was found to contain peptide-like factors with distinct anti-proliferative/differentiating and neuroprotective activities with differing chromatographic properties. Preadsorption of CM_Cerebellum with antisera against candidate cytokines showed that TGF2 and BDNF could account for the major part of the anti-proliferative and pro-differentiating activities, an interpretation strengthened by studies involving treatment with purified TGF2 and BDNF. Interference with signaling pathways downstream of TGF and BDNF using dominant-negative forms of their respective receptors (TGF2-RII and TRKB) or of dominant-negative forms of SMAD3 and co-SMAD4 negated the anti-proliferative/differentiating actions of CM_Cerebellum. Treatment with CM_Cerebellum caused nuclear translocation of SMAD2 and SMAD4, and also transactivated a TGF2-responsive gene. BDNF actions were shown to depend on activation of ERK1/2 and to converge on the SMAD signaling cascade, possibly after stimulation of TGF2 synthesis/secretion. In conclusion, our results show that the regulation of hippocampal cell fate in vitro is regulated through an interplay between the actions of BDNF and TGF.