The effect of GH on rat neurogenesis in the dentate gyrus of hypocampus
We previously showed that GH administration to rats with induced brain injury produces a clear proliferation of neural precursors within DG and a significant reduction of apoptosis. In this study, we tried to delineate, in vitro, the signaling pathways by which GH produces these effects on neurogenesis.
Methods: Neural stem cells were obtained from the Subgranular Zone of 9 days old mice and then plated on petri dishes under proliferation conditions (DMEM+EGF+FGF2) or in multiwells with differentiation medium (DMEM−EGF−FGF2). We first analysed whether in these cells there is GH and GH-R expression. The effects of the hormone in the presence or absence of selective inhibitors of GH signaling pathways was studied by using: SP600125 (20 μM) to inhibit P-JNK, rapamycin (20 nM) to inhibit mTor, and U0126 (20 μM) to inhibit Erk. Apoptosis was evaluated by TUNEL, while proliferation was evaluated by BrdU incorporation (10 μM). After fixing the cells in PFA results were evaluated by immunohistochemistry.
Results: GH and GH-R were showed to be present in neurospheres on proliferation medium, and in different cell populations migrating from neurospheres when they had been cultured in differentiation medium. Adding GH (500 ng/ml) to a differentiation medium did not increase BrdU (10 μM) incorporation, as it did when the hormone was added under proliferation conditions. GH administration significantly decreased apoptosis in basal differentiation conditions. Adding GH-signaling inhibitors significantly increased basal Apoptosis; this effect was reverted when GH was added to U0126- and rapamycin-tretaed cells, but not when added to SP600125-treated cells.
Conclusions: For the first time we demonstrate here the presence of GH and its receptor in stem cells from SGZ of DG. This may explicate the effects of the hormone on neurogenesis: increasing proliferation and decreasing apoptosis. Interestingly, the anti-apoptotic pathway at this level seems to be regulated by Erk and mTor. Or results open a new way for investigating the effects of GH in CNS regenerative therapies, corroborating our pioneer results in human patients.