Alexander disease causing mutations in the C-terminal domain of GFAP are deleterious both to assembly and network formation with the potential to both activate caspase 3 and decrease cell viability

Abstract

Alexander disease is a primary genetic disorder of astrocyte caused by dominant mutations in the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP). While most of the disease-causing mutations described to date have been found in the conserved α-helical rod domain, some mutations are found in the C-terminal non-α-helical tail domain. Here, we compare five different mutations (N386I, S393I, S398F, S398Y and D417M14X) located in the C-terminal domain of GFAP on filament assembly properties in vitro and in transiently transfected cultured cells. All the mutations disrupted in vitro filament assembly. The mutations also affected the solubility and promoted filament aggregation of GFAP in transiently transfected MCF7, SW13 and U343MG cells. This correlated with the activation of the p38 stress-activated protein kinase and an increased association with the small heat shock protein (SHSP) chaperone, αB-crystallin. Of the mutants studied, D417M14X GFAP caused the most significant effects both upon filament assembly in vitro and in transiently transfected cells. This mutant also caused extensive filament aggregation coinciding with the sequestration of αB-crystallin and HSP27 as well as inhibition of the proteosome and activation of p38 kinase. Associated with these changes were an activation of caspase 3 and a significant decrease in astrocyte viability. We conclude that some mutations in the C-terminus of GFAP correlate with caspase 3 cleavage and the loss of cell viability, suggesting that these could be contributory factors in the development of Alexander disease.

Identification of a novel nonsense mutation in the rod domain of GFAP that is associated with Alexander disease

“…WB with antibody 1 detected both WT GFAP and GFAP p.(E312*) with the respective expected molecular weight, yet the band intensity of GFAP p.(E312*) was lower than that of WT GFAP (Figure 1h, upper panel). As expected, antibodies 2 and 3 did not detect GFAP p.(E312*) as these antibodies recognize the C-terminal fragment of GFAP 24 that is absent in GFAP p.(E312*) (Figure 1h, middle and lower panels). Taken together, these findings demonstrate that the p.(E312*) mutation truncates the GFAP and lowers expression levels of GFAP.…”

“…This cell line was derived from the human adrenal cortex carcinoma, features a vimentin filament network, expresses no endogenous GFAP and is well-suited for assessing mutant GFAP properties. 17,24 Transfection efficiency of SW13 (Vim þ ) cells was B26% in our hands (data not shown). Expressed WT GFAP assembled in 80% of the transfected cells into filamentous networks that partially coaligned with the vimentin IF networks (Figure 3a and d) that is consistent with previous observations.…”

“…Expressed WT GFAP assembled in 80% of the transfected cells into filamentous networks that partially coaligned with the vimentin IF networks (Figure 3a and d) that is consistent with previous observations. 17,24 In contrast, most of the cells (73%) expressing GFAP p.(E312*) formed GFAP-rich aggregates that often collapsed the vimentin IF networks (Figure 3c and d). Cotransfection with equimolar ratio of plasmids individually encoding WT GFAP and GFAP p.(E312*) resulted in GFAP aggregation with a frequency closer to that seen for cells transfected with GFAP p.(E312*) alone (Figure 3b and d).…”

Glial fibrillary acidic protein exhibits altered turnover kinetics in a mouse model of Alexander disease

“…We also do not distinguish between the GFAP-␣ and GFAP-␦ isoforms, although ␣ likely represents more than ϳ90% of the total in the CNS (42,43). Differences in solubility exist between the GFAP isoforms and between mutant and wild-type protein (39,41,44,45), although not under the conditions used for extraction in the experiments reported here.…”

The future of blood‐based biomarkers for Alzheimer's disease

“…In addition, similar processing steps involving combinations of brain proteases and brain proteins appear to occur in a series of other proteins related to neuronal pathologies, such as trans-activation response (TAR) DNA-binding protein 43, α-synuclein, and more [106–108] (Table 3) [109–119]. A recent finding indicated that a fragment of tau was present in serum samples, and that it correlated with cognitive function in a small clinical study, highlighting the potential of this approach [120].…”