GFAP mutants cause imbalance of selective lipid classes in a cellular model of Alexander disease

Alexander disease (AxD) is a rare neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP), primarily affecting astrocytes. Although these mutations alter astrocyte structure and metabolism, their impact on the lipidome remains understudied. Here, wild-type GFP-GFAP and AxD-associated mutants (R239C, R239G, R79C, and E373K) were expressed in U-87 MG astrocytoma cells to evaluate GFAP organization through confocal microscopy and lipidomic alterations quantified by mass spectrometry. Membrane fluidity and lipid storage were complementarily assessed. Expression of AxD-associated GFAP mutants induced distinct lipidomic alterations, with R239C eliciting the most pronounced changes, including enrichment of sphingomyelins, trihexosylceramides, acylcarnitines, triglycerides, cholesteryl esters, and ether-linked lipids. These alterations suggest adaptive mechanisms to preserve membrane integrity, lipid raft organization, bioenergetics, redox homeostasis, and cell survival. Overall, in the AxD astrocytoma cell model, different GFAP mutations induce extensive and non-overlapping remodeling of lipids involved in membrane structure and organization, antioxidant defense, and bioenergetic pathways.