FK506 modulates Akt activity and promotes neurite initiation but not elongation in Neuro2a cells

Abstract

Previous reports have shown that the immunosuppressant FK506 accelerates nerve regeneration in vivo and exerts neurotrophic effects in vitro. We aimed to assess the involvement of the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signalling pathway in the neuritogenic effect of FK506 in vitro. For that purpose, Neuro2a cells (n=3) were treated with 0.1% DMSO (vehicle) or 1μM FK506 during both 4 and 24h or simultaneously treated with 1μM FK506 plus 10μM LY294002 (PI3K inhibitor) for 24h. After treatment, microphotographs of fields containing 200-250 cells were analyzed using NeuronJ (an ImageJ plugin) to quantify the percentage of Neurite Bearing Cells (%NBC) and the Mean Neurite Length (MNL), parameters that reflect neurite initiation and elongation, respectively. Cell viability was measured by MTT assay. Nitrocellulose membranes containing lysate proteins derived from cells treated with 10μM LY294002 for 90 min or 1μM FK506 for 15, 30 or 60 min were incubated with anti-phospho/total Akt and anti-GADPH antibodies to evaluate Akt phosphorylation levels by Western Blotting. The 4h treatment with FK506 increased 2x the %NBC without altering the MNL. In contrast, 24h treatment with FK506 didn’t alter the %NBC nor the MNL, perhaps due to a plateau effect. Nevertheless, FK506+LY294002 24h incubation reduced by 50% the %NBC without altering the MNL. If compared to the vehicle, FK506 treatments for 15 and 30 min reduced Akt phosphorylation levels by 20% and 10%, respectively. In addition, none of the treatments altered cell viability. In conclusion, FK506 promotes neurite initiation in a short period of time (4h). As an early event (15-30’), FK506 reduces the Akt phosphorylation which indicates a lower activity of this kinase and might explain the lack of changes in MNL. In a larger time-window (24h), PI3K inhibition reduces the neurite initiation. However, further studies are required to clarify the role of PI3K in FK506-induced neuritogenesis.