Abstract

Mutations in the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP) lead to the rare and fatal disorder, Alexander disease (AxD). A prominent feature of the disease is aberrant accumulation of GFAP. It has been proposed that this accumulation occurs because of an increase in gene transcription coupled with impaired proteasomal degradation, yet this hypothesis remains untested. We therefore sought to directly investigate GFAP turnover in a mouse model of AxD that is heterozygous for a disease-causing point mutation (GfapR236H/+) (and thus expresses both wild-type and mutant protein). Stable isotope labeling by amino acids in cell culture, using primary cortical astrocytes, indicated that the in vitro half-lives of total GFAP in astrocytes from wild-type and mutant mice were similar at ∼3-4 days. Surprisingly, results obtained with stable isotope labeling of mammals revealed that, in vivo, the half-life of GFAP in mutant mice (15.4 ± 0.5 days) was much shorter than that in wild-type mice (27.5 ± 1.6 days). These unexpected in vivo data are most consistent with a model in which synthesis and degradation are both increased. Our work reveals that an AxD-causing mutation alters GFAP turnover kinetics in vivo and provides an essential foundation for future studies aimed at preventing or reducing the accumulation of GFAP. In particular, these data suggest that elimination of GFAP might be possible and occurs more quickly than previously surmised.

Highlights

  • Mutations in the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP) lead to the rare and fatal disorder, Alexander disease (AxD)

  • In vitro analysis reveals no difference between GFAP؉/؉ and GFAPR236H/؉ turnover kinetics

  • Negligible differences were observed in isotopic envelope patterns when comparing GFAPϩ/ϩ and Alexander disease is a gain of function disorder that in part results from the accumulation of GFAP above a putative toxic threshold

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Summary

The abbreviations used are

AxD, Alexander disease; GFAP, glial fibrillary acidic protein; RIA, relative isotope abundance; SILAC, stable isotope labeling by amino acids in cell culture; SILAM, stable isotope labeling of mammals; RF, Rosenthal fiber; XIC, extracted ion chromatogram; RT, retention time. Using rat primary astrocytes, Morrison et al [19] found only monophasic decay with a half-life of 7.5 days In contrast to these in vitro studies, Eng and co-workers [20] used pulse labeling with radioactive tracers to examine degradation of GFAP in mouse spinal cord and found a half-life of ϳ9 weeks

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Results
Discussion
Experimental procedures

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