Single Cell Proteomics reveals partial uncoupling of the Glycerol-3-Phosphate shuttle to compensate for Electron Transport Chain dysfunction in Alexander Disease GFAP mutations

Alexander disease (AxD) is a rare genetic leukodystrophy caused by dominant mutations in the gene coding for the astrocytic intermediate filament protein Glial Fibrillary Acidic Protein (GFAP). Understanding disease mechanisms is complicated by the fact that mutant GFAP promotes further GFAP accumulation, making it difficult to distinguish mutation-specific effects from those caused by elevated GFAP levels. Here, we performed mass spectrometry-based single cell proteomics to investigate proteomic alterations associated with increasing levels of wildtype GFAP and a common AxD-causing mutant GFAP R239C. By analyzing individual cells spanning a broad range of GFAP expression levels, we separated dosage-dependent effects from mutation-specific responses. Cells expressing high levels of wildtype GFAP increasingly resembled R239C-expressing cells at the proteomic level, supporting a role for GFAP accumulation in disease pathogenesis. In contrast, mutant GFAP specifically correlated with increased mitochondrial antioxidant enzymes and remodeling of the proteasome tailored toward ATP- and ubiquitin-independent degradation of oxidized proteins. Integration with proteomics data from patient-derived cortical organoids revealed increased abundance of nuclear-encoded mitochondrial proteins and respiratory chain assembly factors without corresponding changes in mitochondrially encoded proteins, suggesting impaired electron transport chain (ETC) assembly. Consistent with this, AxD cells displayed reduced oxygen consumption, increased glycolytic activity, enhanced utilization of the cytosolic part of the glycerol-3-phosphate (G3P) shuttle, and accumulation of triacylglycerides and lipid droplets. We propose that ETC dysfunction promotes partial uncoupling of the G3P shuttle, allowing sustained regeneration of cytosolic NAD to support glycolysis while diverting G3P toward lipid synthesis and storage. By regenerating NAD through the G3P shuttle rather than lactate fermentation, AxD astrocytes may sustain glycolytic flux while preserving lactate production, an important metabolic substrate for neighboring neurons. Together, our findings identify ETC dysfunction as a central feature of AxD and suggest that metabolic adaptation through the G3P shuttle links mitochondrial dysfunction to glycolytic reprogramming and lipid accumulation. More broadly, these results provide new insight into how astrocyte dysfunction drives molecular pathways implicated in neurodegeneration.