Non synonymous polymorphisms (nsSNPSs) in GPM6A´s transmembrane domain coding region: In silico analysis and in vitro study of the effect of point mutations in synapse formation, selfinteraction and protein stability

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

Mar del Plata

Congreso; Reunion Anual de la Sociedad Argentina de Investigación en Neurociencias; 2015

SAN

M6a is a membrane glycoprotein that together with M6b and PLP/DM20 belongs to theproteolipid protein family. We have found that M6a induces neurite and filopodia/spineformation and increases the motility of filopodial protrusions, probably aiding synapseformation, but the mechanism of action remains unknown. We have found that M6a´stransmembrane domains (TMDs) drive M6a induced filopodia formation by its selfassociationand protein stabilization. Here we further analyze whether 3 nsSNPs present inthe coding region of M6a´s-TMD affect synapse formation, TMD self-interaction andprotein stability. Cultured hippocampal neurons at 10-12 days in vitro expressing GFP aloneor M6a or its mutants fused to GFP were labeled with synaptophysin (pre-synaptic marker)and NMDA-R1 (post-synaptic marker). The number of functional synapses (points of triplecolocalization) was quantified using the Image J plugin Puncta Analyser. The number ofsynapses in M6a expressing neurons was significantly higher compared with GFPexpressing neurons. On the contrary, in neurons expressing M6a´s TMD mutants thenumber of synapses was similar to control. We also showed that these mutants impair M6astability and self-interaction. In this work we provide evidence that the self-interaction ofM6a´s TMDs, as well as the stability and folding of the protein, is critical for filopodium andsynapse formation in cultured hippocampal neurons.