Iron overload impairs the migratory ability of a model of immature and migratory GnRH neurons Steffani L, Di Nitto F, °Dongiovanni P, °Rametta R, °Valenti L, °Fargion S, Magni P., Ruscica M. Dep. of Pharmacological and Biomelecular Sciences and °Dep. of Pathophysiology and Transplantation, Università degli Studi di Milano liliana.steffani@unimi.it, paolo.magni@unimi.it, massimiliano.ruscica@unimi.it BACKGROUND Iron is essential for proper brain development in the fetal and early neonatal period. Iron represents a micronutrient for cellular metabolism and aerobic respiration, but cellular iron overload produces toxic build-up in many organs (including the brain) via free radical formation. In thalassaemic patients with pubertal failure, iron overload is the most important factor afflicting the hypothalamic-pituitary axis, leading to hypogonadotrophic hypogonadism and growth failure. AIM - To investigate the mechanisms of iron toxicity in in vitro GN-11 cells, a model of immature and migratory GnRH neurons METHODS Mouse GN-11 cells (immature GnRH neurons with migratory ability) were used. Hepcidin, ferritin and transferrin receptor gene expression was evaluated by PCR. GN-11 chemotaxis was assessed by Boyden chamber assay. Activation of chemomigration-related cell signaling (extracellular signal-regulated kinase (ERK), 5' adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC)) was evaluated by Western blot analysis (WB). RESULTS 1. GN-11 cells express hepcidin (Hep), ferritin and transferrin receptor (TFR) genes ✓Time-course experiments showed that 150 µM FAC was able to phosphorylate both ERK and AMPK after 10 min treatment TFR Hep OD pERK/ERK GT1-7 GN-11 GT1-7 GN-11 GT1-7 GN-11 GT1-7 Ferritin OD pAMPK/AMPK 400 GN-11 300 200 100 membrane chemotatic agent 75 *** 50 25 0 Migrated cells ✓ Pre-treatment with 100 µM deferoxamine (DFO), a specific iron chelator, rescued the FAC effect on cell-migration 250 200 150 100 50 Control 150 µM FAC - 5’ 10’ 75 *** 10' 30' 150 µM FAC incubation time (min) 250 200 * 60' Control 150 µM FAC 10 µM CMPC + 150 µM FAC * 150 100 50 30’ - 60’ 5’ 10’ 30’ 60’ 150 µM FAC incubation time (min) 150 µM FAC incubation time (min) (Mean ± SEM of six independent experiments) ✓ CMPC (10 µM) counteracted FAC-driven phosphorylation of acetylCoA carboxylase (ACC), an AMPK downstream protein 100 µM DFO 100 5' 0 OD pACC/ACC 1% FBS-induced migration (% of control) 125 Control 150 µM FAC 10 µM U0126 + 150 µM FAC 0 1% FBS ### - 10' 30' 60' 150 µM FAC incubation time (min) ✓ Specific ERK and AMPK inhibitors, U0126 and Compound C (CMPC), respectively, abolished FAC-mediated signaling Control 150 µM FAC 100 150 (Mean ± SEM of six independent experiments) OD pAMPK/AMPK cells 1% FBS-induced migration (% of control) 125 250 5' OD pERK/ERK 1. 150 µM ferric ammonium citrate (FAC) treatment inhibited (-35%, p<0.05) FBS-induced chemo-migration of GN-11 cells * 0 0 - Boyden’s chamber 350 * 1.2×107 1.0×107 8.0×106 * Control 150 µM FAC 10 µM CMPC + 150 µM FAC 6.0×106 4.0×106 2.0×10 6 50 0 - 25 0 1% FBS 5’ 10’ 30’ 150 µM FAC incubation time (min) 60’ (Mean ± SEM of six independent experiments) CONCLUSIONS - Iron negatively affects neuron migration via ERK and AMPK. Among the consequences of this event, iron overload may impair migration of GnRH neurons from the olfactory placode into forebrain and hypothalamus, where they promote reproductive competence.