Site-specific phosphorylation and caspase cleavage of GFAP are new markers of Alexander disease severity

Abstract

Alexander disease (AxD) is a fatal neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP), which supports the structural integrity of astrocytes. Over 70 GFAP missense mutations cause AxD, but the mechanism linking different mutations to disease-relevant phenotypes remains unknown. We used AxD patient brain tissue and induced pluripotent stem cell (iPSC)-derived astrocytes to investigate the hypothesis that AxD-causing mutations perturb key post-translational modifications (PTMs) on GFAP. Our findings reveal selective phosphorylation of GFAP-Ser13 in patients who died young, independently of the mutation they carried. AxD iPSC-astrocytes accumulated pSer13-GFAP in cytoplasmic aggregates within deep nuclear invaginations, resembling the hallmark Rosenthal fibers observed in vivo. Ser13 phosphorylation facilitated GFAP aggregation and was associated with increased GFAP proteolysis by caspase-6. Furthermore, caspase-6 was selectively expressed in young AxD patients, and correlated with the presence of cleaved GFAP. We reveal a novel PTM signature linking different GFAP mutations in infantile AxD.

eLife digest

Alexander disease is a rare and fatal neurodegenerative condition. It is caused by mutations in a gene that is crucial for the structure of astrocytes, a type of brain cell whose role is to support neurons. The gene codes for a protein called GFAP, which is made almost exclusively in astrocytes. In Alexander disease, mutated versions of the gene make GFAP collect in disordered clumps or aggregates, which interfere with the astrocytes’ normal activities.

All Alexander disease patients develop GFAP aggregates, but the type of mutation they have in the gene for GFAP does not predict how their illness will progress. The age of onset of disease, for example, can vary between less than one year old to more than 70 years old. Battaglia et al. sought to understand how GFAP aggregates form in the cells of Alexander disease patients. One way that GFAP can be altered in the cell is by a process called phosphorylation. Enzymes called kinases add phosphate groups to GFAP, which can regulate the protein’s activity, stability and interactions with other proteins.

Battaglia et al. found high levels of phosphorylation at one specific site in the GFAP protein in people who had very early onset of Alexander disease. This phosphorylation was not related to any particular mutation in the gene for GFAP. An added phosphate group at this location in the protein made GFAP more likely to be broken into two pieces by an enzyme called caspase-6. One of the breakdown products is already known to play a role in aggregation. Young patients with Alexander disease had high levels of GFAP breakdown products and caspase-6. The phosphorylated protein and this enzyme were found to accumulate in astrocyte aggregates.

The findings provide a basis for investigating new strategies to treat Alexander disease that target phosphorylation – as removing or preventing the addition of phosphate groups can be done with drugs. But before exploring how to do this, it will be necessary to find out which enzyme is responsible for phosphorylating GFAP at this particular position. These studies may also give a broader understanding of other GFAP-like proteins (called intermediate filament proteins), which are involved in over 70 human diseases.