Copper complexes of synthetic peptides mimicking neurotrophin-3 enhance neurite outgrowth and CREB phosphorylation

In this work we report on the synthesis and physiochemical/biological characterization of a peptide encompassing the first thirteen residues of neurotrophin-3 (NT-3). The protein capability to promote neurite outgrowth and axonal branching by a downstream mechanism that involves the increase of the cAMP response element-binding level (CREB) was found for the NT3(1-13) peptide, thus validating its protein mimetic behaviour. Since copper ions are also involved in neurotransmission and their internalization may be an essential step in neuron differentiation and CREB phosphorylation, the peptide and its copper complexes were characterized by potentiometric and spectroscopic techniques, including UV-visible, CD and EPR. To have a detailed picture of the coordination features of the copper complexes with NT3(1-13), we also scrutinized the two peptide fragments encompassing the shorter sequences 1-5 and 5-13, respectively, showing that the amino group is the main anchoring site for Cu(ii) at physiological pH. The peptide activity increased in the presence of copper ions. The effect of copper(ii) addition is more marked for NT3(1-13) than the other two peptide fragments, in agreement with its higher affinity for metal ions. Confocal microscopy measurements carried out on fluorescently labelled NT3(1-13) indicated that copper ions increase peptide internalization