M6A TRANSMEMBRANE DOMAINS SELF-INTERACTION CONTRIBUTES TO SYNAPSES FORMATION IN HIPPOCAMPAL NEURONS

KARINA FORMOSO; MICAELA D GARCIA; ALBERTO C C FRASCH; CAMILA SCORTICATI

Rio de Janeiro

Congreso; IBRO 9th World Congress; 2015

INTERNATIONAL BRAIN RESEARCH ORGANIZATION (IBRO)

M6a is a membrane glycoprotein that together with M6b, PLP and its splice variant DM20 belongs to the proteolipid protein (PLP) family. M6a is composed of 278 amino acids that form four transmembrane domains (TMDs), two external loops and the N- and C-terminal regions facing the cell cytoplasm. This protein is mainly expressed in neurons of the central nervous system. We have found that M6a induces neurite, filopodia and spine formation and increases the motility of filopodial protusions, probably aiding synapse formation, but the mechanism of action remains unknown. Recently, we have determined that the self-interaction of the TMDs of M6a might be driving M6a dimerization in neuronal membranes. We also established that mutations on M6a which affected TMDs interaction impaired filopodia formation in neurons. These mutations include a triple glycine substitution in TMD2 (G87/96/103A), a double glycine substitution in TMD4 (G220/222) and two non-synonymous polymorphisms from the NCBI data base (dbSNP) located in the TMD2?s coding region of the GPM6A (F93C here SNP1 and I97S here SNP2). In the present work we further analyse whether M6a TMDs self-interaction is involved in synapse formation. Cultured hippocampal neurons at 10-12 days in vitro expressing GFP alone as a control or M6a or its mutants fused to GFP were fixed and labelled with synaptophysin (as a presynaptic marker) and N-methyl-D-aspartate receptor type 1, NMDA-R1 (as a post synaptic marker). The number of functional synapses, points of triple colocalization, was quantified in 25 µm of dendrite using the Image J plugin Puncta Analyser.  The number of synapses in M6a expressing neurons was significantly higher compared with GFP expressing neurons. We found that neurons expressing SNP1, SNP2, G87/96/103A or G220/222A mutant drastically reduced the number of synapses showing levels similar to control. Taken together, in this work we determined that M6a positively contributes to synapses formation. Moreover, the results presented here lead us to speculate that the self-interaction of M6a TMDs could be involved in the pathway leading to synapse formation.