Interaction of MT-I/II with megalin accelerates AKT-1 phosphorylation in cortical neurons and contributes to neurogenesis in cuprizone model of de- and remyelination

Background. Copper chelator cuprizone (CPZ) is neurotoxicant, which selectively disrupts oligodendroglial respiratory chain, leading to oxidative stress and subsequent apoptosis. Demyelination is, however, followed by spontaneous remyelination owing to the activation of intrinsic CNS repair mechanisms. To the later contribute also cysteine rich metallothioneins (MT), which through free radical scavenging and intracellular Zn/Cu regulation provide cytoprotection. Besides, it has been postulated that secreted MT-I/II might be bind on surface receptors belonging to the family of low-density lipoprotein receptor related proteins (LRP), such as LRP-2/megalin and LRP-1, which in turn activate the signal transduction pathways that support neurite outgrowth and survival. Aim. The goal of this study was to visualize MT/ megalin interaction in the brain tissue of mice affected by CPZ and to determine if this binding leads to the activation of serine/threonine-protein kinase-AKT-1/Protein kinase B signaling cascade. Methods. Experiments were performed in female C57BL/6 mice fed with 0.25% CPZ during 5 weeks. MT/megalin co-localization and interactions were examined by double immunofluorescence and proximity ligation assay (PLA), which enables in situ recognation of two potentially interacting proteins, respectively. The post-translational modifications in target cells were evaluated by the presence of phosphorylated AKT-1 (pAKT-1 ; phospho threonine 308). Results. CPZ-induced demyelination was followed by high astrogliosis and enhanced expression of MTs and megalin in white and gray matter of the brain. PLA clearly showed that MT-I/II interacted with megalin in cortical tissue and in hippocampal subgranular zone of dentate gyrus. Moreover, in most of megalin expressing cortical NeuN+ neurons nuclear expression of pAKT-1 was found. Conclusion. The data imply that astrocyte-derived MT-I/II modulates cell signaling and neuronal repair through direct contact with megalin and suggest that internalization of MT-I/II and accelerated phosphorylation of AKT-1 contribute to activation of signal transduction pathways that protect cortical neurons and neuronal progenitors against toxic effects of CPZ