GANGLIOSIDOME CHARACTERIZATION OF PURIFIED MURINE CORTEX MITOCHONDRIA

Gangliosides, sialic acid-containing membrane glycosphingolipids, are major determinants of the brain glycome. Predominantly located in the outer leaflet of the plasma membrane, they play key roles in signal transduction, immune response and cell communication, via direct or indirect protein binding and subsequent activity modulation. Recent studies revealed that patients with specific congenital disorders of glycosylation affecting gangliosides exhibit features of mitochondrial diseases. A currently unexplored perspective is that this phenotype could arise from a pathological cascade originating on the mitochondrial membrane, not only plasma membrane where gangliosides mostly reside and exert their effects, due to disrupted mitochondrial protein-ganglioside interactome. Therefore, the aim of our study was to revisit the characterization of mitochondrial membrane ganglioside composition. First, we developed a purification method to isolate highly purified mitochondria from murine cortex, with minimal to no contamination from plasma and organelle membranes, using Western blotting for purity confirmation with an array of plasma membrane and mitochondrial markers. Next, we purified the gangliosides by extraction in organic solvents and subsequent gel filtration. Composition of the purified gangliosides was then analyzed by mass spectrometry. Lastly, we visualized the viable purified mitochondria by confocal microscopy using ganglioside and mitochondrial markers. The results of both mass spectrometry and immunofluorescent imaging of highly purified mitochondria show that mitochondria contain gangliosides, and furthermore have a unique ganglioside profile. Our findings open the doors to new ganglioside-centered research of metabolism in neurodegenerative disorders, starting at the mitochondrial membrane interactome level.