SIGNALLING CASCADES INVOLVED IN THE FK506-DEPENDENT NEURODIFFERENTIATION

MARÍA E. ROSBACO, CAMILA G. SZCZEPANIK, MARIO D. GALIGNIANA

Congreso; LXVIII REUNIÓN ANUAL DE SAIC; 2023

In previous works, we reported that the immunophilin ligand FK506 (tacrolimus) is a neurodifferentiating agent in vitro and in vivo. In this study, we aimed to evidence the signalling cascades involved in the early steps of neurodifferentiation in a model of mouse neuroblastoma Neuro-2a cells. Because neurons are dependent on insulin stimulation, whose mechanism of action involves the PI3K/AKT and MAPK pathways, the potential influence of this hormone on neuronal differentiation was also evaluated. Cells were incubated in a medium without other trophic factors (including serum) with either 1 M FK506 or 1 M retinoic acid (RA), a known differentiating agent. The neurite length was measured at different times. FK506 was more efficient than the standard agent RA, showing ∼35% longer neurites after 24 h incubation. Western blots of cell lysates showed that immunophilin-induced profile of phospho-FKBP51 and phospho-FKBP52 isoforms was different for both agents, suggesting dissimilar mechanisms of activation. FK506 was used in the subsequent assays. The presence of 10 nM insulin (simultaneous or preincubated for 24 h) did not affect neurodifferentiation. The cell pretreatment during 5 h with 10 M UO126, a MEK1-2 inhibitor, shortened the neurite length, implying a key role of the MAPK/ERK pathway. Then, the following inhibitors were assayed: 50 M PD98059 (PD) (for ERK), 1LY2940022 (LY) (for PI3K), and SP600125 (SP)(for JNK). As it was expected, PD confirmed the previous observation made with UO126 for the involvement of the MAPK/ERK pathway, whereas the PI3K and JNK inhibitors exhibited only a mild effect on neurite outgrowth. Nonetheless, the number of neurites per cell was significantly greater in the presence of the JNK inhibitor. It is concluded that FK506 is a more efficient neurodifferentiating agent than RA, and it is proposed that the FK506 mechanism of action requires the MAPK/ERK pathway, whereas the JNK inhibition appears to favour neuronal arborization.