Lysosomal function, resistance to stress and repair are compromised by expression of the Alexander disease GFAP R239C mutant

Abstract

Intermediate filaments are critical regulators of cell responses and organizers of cellular structures. GFAP (glial fibrillary acidic protein) is an intermediate filament protein mainly expressed in astrocytes. GFAP mutations are associated with Alexander disease (AxD), a type of leukodystrophy that causes degeneration of astrocytes and ultimately neurodegeneration. AxD astrocytes display protein aggregation, proteostasis defects and altered organelle homeostasis. We previously showed that expression of GFAP AxD mutants in astrocytes provoked mitochondrial alterations and oxidative stress. Here we have used an astrocytoma cell model to explore the impact of GFAP AxD mutants on the lysosomal degradation pathway. Expression of GFAP AxD mutants in this model elicits marked alterations in lysosomal distribution. Cells expressing the GFAP R239C mutant display defective lysosomal activity and intraluminal acidification. Lysosomes are primary sites of oxidative damage. Moreover, expression of GFAP R239C increases susceptibility of lysosomes to oxidative stress, resulting in a greater loss of lysosomal "mass" and compromised membrane integrity, as revealed by increased intraluminal recruitment of galectins, with respect to cells expressing GFAP wt. Notably, lysosomes in GFAP R239C expressing cells are also more vulnerable to chemically-induced rupture. Interestingly, lysosomes of cells expressing GFAP wt are able to rapidly recover after removal of the damaging agent. In sharp contrast, recovery of acidic vesicles is severely impaired in cells expressing GFAP R239C, suggesting a defect in lysosomal repair. Taken together, our results show that expression of the GFAP AxD mutant is sufficient to deeply perturb lysosomal distribution, function and repair. These alterations could contribute to proteostasis defects and cellular toxicity in AxD.