Research ArticleProperties of astrocytes cultured from GFAP over-expressing and GFAP mutant mice
Introduction
Glial fibrillary acidic protein (GFAP) is the major intermediate filament protein in astrocytes. Heterozygous missense mutations within the coding region of GFAP account for the majority of cases of Alexander disease, a fatal neurodegenerative disorder that typically affects young children [1]. Many patients suffer seizures and/or macrocephaly as their initial clinical sign, and then experience a variety of delays or regression in psychomotor development. MRI of patients with infantile onset reveals a frontal leukodystrophy with characteristic changes in periventricular regions [2]. From its initial description by Alexander [3], attention focused on astrocytes as the instigators of disease because of the hallmark proteinaceous aggregates found within their cytoplasm — Rosenthal fibers. More recent biochemical studies show that Rosenthal fibers are complex mixtures of GFAP, vimentin, αB-crystallin, HSP27, plectin, and p62 (and other unknown components) [4], [5], [6], [7], [8], and bear some resemblance to the neurofilament-containing Lewy bodies of neurons and the keratin-containing Mallory bodies of hepatocytes.
Whether Rosenthal fibers per se cause astrocyte dysfunction, and what the precise trigger(s) is for their formation, is not clear. These inclusions have long been known to occur in the context of chronic gliosis or up-regulation of GFAP expression of various causes. The first description of Rosenthal fibers was from a patient with syringomyelia [9], and subsequently they have been observed in a wide variety of conditions including multiple sclerosis [10] and pilocytic astrocytomas [11] (for a more complete review, see [12]). Transgenic studies clearly show that simply elevating levels of wild type GFAP to a sufficient degree will lead to Rosenthal fibers [13], and it is possible that reactive astrocytes (that also up-regulate GFAP) and Alexander disease astrocytes (expressing a mixture of mutant and wild type GFAP) have certain properties in common.
Precisely how mutations in GFAP lead to the pleiotropic manifestations of Alexander disease is not known [14], [15]. Nearly half of all patients carry mutations in either of two amino acids, R79 or R239, although it appears that mutations distributed throughout the protein produce essentially identical Rosenthal fibers and similar disease [16], [17]. A number of arguments point to the idea that the GFAP mutations, which are genetically dominant, act in a gain-of-function fashion, and that elevations of total GFAP levels are a major factor in pathogenesis. One way in which this issue has been studied is by transfection of cultured cells, where over-expression of either mutant or wild-type GFAP leads to the formation of cytoplasmic protein aggregates with recruitment of small stress proteins and shifts in GFAP solubility [18], [19], [20]. Multiple positive feedback loops act to further increase accumulation of GFAP, both by inhibition of proteasomal degradation and by increased expression. Activation of JNK and p38 also occurs, and may further contribute to GFAP accumulation [21]. However, the aggregates formed via transfection either fail to replicate the morphological features of Alexander disease Rosenthal fibers [18], or are studied in non-astrocytic cell lines [20]. In addition, the effects of GFAP alterations on cell lines may not be identical to changes that are induced in bona fide astrocytes.
Mouse models have been created via both transgenic and knock-in approaches that reproduce key aspects of the Alexander phenotype, particularly the formation of Rosenthal fibers identical to those found in the human disease [13], and increased seizure susceptibility [22], [23]. To provide new tools for investigating the nature of astrocyte dysfunction in Alexander disease, we have established primary astrocyte cultures from two of these mouse models (a knock-in at the endogenous mouse locus of the R236H mutation [22], and a transgenic over-expressing wild-type GFAP, termed TgGFAP-wt [13]),and studied their properties in culture. We find that mutant GFAP, as well as excess wild type GFAP, promotes formation of cytoplasmic inclusions, disrupts the cytoskeleton, decreases cell proliferation while increasing cell death, reduces proteasomal function, and compromises astrocyte resistance to stress.
Section snippets
Primary cortical astrocyte cultures
Cortical astrocyte cultures are prepared from 0–2 day old postnatal mice, either heterozygotes (for the R236H mice [22]), hemizygotes (for the TgGFAP-wt mice, 73.7 line [13]), or wild-type controls. To facilitate comparisons between these in vitro studies and ongoing in vivo experiments utilizing crosses between the R236H knock-in mice and the TgGFAP-wt mice [22], all cultures described here were derived from mice that are F1 hybrids between the two parental background strains (FVB/N and 129S6,
GFAP levels are increased in R236H and TgGFAP-wt astrocytes
The parental R236H line (a knock-in at the endogenous mouse locus) and TgGFAP-wt transgenic line (transgenics over-expressing wild type human GFAP) from which the primary astrocyte cultures are derived have increased levels of GFAP in brain [13], [24], [22]. To determine whether such an increase in expression is reflected in cultured astrocytes, we evaluated total GFAP immunoreactivity after 2 DIV (passage 2) in cell extracts using a sandwich ELISA (Fig. 1). By this assay the R236H astrocytes
Discussion
An increase in absolute levels of GFAP may be central to the pathogenesis of Alexander disease. Our mouse models employ two distinct genetic means by which such an increase is achieved, either by added copy number of a wild-type sequence, or by expression of a mutant allele. Expression of mutant protein apparently activates multiple downstream pathways, including p38, JNK, and MAPK, that impact GFAP turnover in various and sometimes opposing ways [21], [29]. The inhibition of proteasomal
Acknowledgments
We thank Denice Springman, Channi Kaur, and Benjamin August for technical support, and Michael Brenner, James Goldman, Tracy Hagemann, and Roy Quinlan for advice on the manuscript. This work was supported by NIH grants NS42803 and NS060120 (A.M.), and by HD03352 (Waisman Center). W.C. was supported in part by the Wayne and Jean Roper Wisconsin Distinguished Graduate Fellowship.
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2018, Brain Research BulletinCitation Excerpt :GFAP mutations that lead to GFAP overexpression, accumulation of disorganized intermediate filaments and formation of Rosenthal fibers are causative of Alexander disease (Brenner et al., 2001). In vitro, overexpression of GFAP leads to Rosenthal fiber formation, disruption of the cytoskeleton, decrease in astrocyte proliferation, increased cell death, reduction of proteasomal function, and impairment of astrocyte resistance to oxidative stress (Cho and Messing, 2009). Several drugs were reported to decrease GFAP expression in vitro and in vivo (Bargagna-Mohan et al., 2010; Cho et al., 2010; Middeldorp et al., 2009).
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2017, Journal of Biological ChemistryCitation Excerpt :Protein aggregation disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis are thought to challenge normal proteostasis, whereby aggregation-associated proteotoxicity overcomes the “normal” levels of protein degradation and clearance (10, 11). In AxD, work in cell culture models demonstrates impairment in proteasome activity (12, 13), although autophagy appears to be enhanced (6). At the same time, considerable evidence exists for a rise in levels of GFAP mRNA, which would suggest an increase in GFAP synthesis (9, 14, 15).