E258 residue modulates Gpm6a intracellular sorting and its role in neuronal morphogenesis

Functional membrane trafficking is critical for neuronal growth and differentiation. In this context, the neuronal membrane glycoprotein M6a (Gpm6a), a four-transmembrane-domain protein of the PLP/DM20 family, plays a major role in neuronal development. Gpm6a overexpression promotes filopodia formation, and its endocytic/recycling pathway regulates synapse formation and maintenance. However, the mechanisms targeting Gpm6a for recycling or degradation are still unknown. Here, we demonstrate that overexpressing the Gpm6a mutant E258A reduces filopodia formation and arborization complexity in rat hippocampal neurons, while compromising neurite outgrowth during N2a differentiation. By live-cell imaging of N2a cells, we show that the E258A mutation increases Gpm6a vesicle dynamics and their colocalization with Rab7 and Lamp1 positive compartments, while it does not alter Gpm6a association with Rab5 or Rab11 positive endosomes, indicating that this mutation redirects intracellular trafficking of Gpm6a towards late endosomal/lysosomal compartments. When LysoTracker labeling was used to confirm compartment identity, we observed that E258A accumulates within the lysosomes. Moreover, an increased association between E258A and GalNAc-T (a TGN marker) was observed. We conclude that E258A leads to enhanced Gpm6a sorting to the degradative pathway and propose the E258 residue as a critical switch regulating Gpm6a targeting for degradation, thereby influencing neuronal morphogenesis.