Morphological Neurite Changes Induced by Porcupine Inhibition Are Rescued by Wnt Ligands

Abstract

Background: Wnt signaling plays key roles in cellular and physiological processes, including cell proliferation, differentiation and migration during development and tissue homeostasis in adults. This pathway can be defined as Wnt/β-catenin-dependent or β-catenin-independent or "non-canonical", both signaling are involved in neurite and synapse development/maintenance. Porcupine (PORCN), an acylase that o-acylates Wnt ligands, a major modification in secretion and interaction with its receptors. We use Wnt-C59, a specific PORCN inhibitor, to block the secretion of endogenous Wnts in young hippocampal neurons (DIV 4). Under these conditions, the morphology and length of the neurites and the complexity of the dendritic tree and axonal polarity were evaluated. Methods: Cultured primary young hippocampal neurons obtained from Sprague-Dawley rat fetuses (E18), were cultured until day in vitro (DIV) 4 (according to Banker´s protocol) and treated with Wnt-C59 for 24h, Wnt ligands were added to the cultures on DIV 3 for 24h. Dendritic arbors and neurites were analysis by fluorescence microscopy. Transfection with Lipofectamine 2000 on DIV 2 of plasmid expressing eGFP and KIF5-Cherry and neurons were fixed on DIV 4. Immunostaining was performed with MAP1B and Tau protein. Immunoblot analysis was carried out with Wnt3a, b-catenin and GSK-3b (p-Ser9). Quantitative analysis of dendrite morphology was carried out with ImageJ (NIH) software with Neuron J Plugin.Results: We report, here, that Wnt-C59 treatment changed the morphology of the dendritic arbors and neurites of young hippocampal neurons, with decreases -catenin and Wnt3a and an increase in GSK-3 (p-Ser9) levels No effect was observed on axonal polarity. In sister cultures, addition of exogenous Wnt3a, 5a and 7a ligands rescued the changes in neuronal morphology. Wnt3a restored the length of neurites to near that of the control, but Wnt7a increased the neurite length beyond that of the control. Wnt5a also restored the length of neurites relative Wnt concentrations. Conclusions: Our results indicated that all 3 Wnt ligands restored dendritic arbor complexity with recovery of secondary and tertiary projections in young hippocampal neurons. We proposed that PORCN is an emerging molecular target of interest in the search for preclinical options to study and treat neurological diseases.