Properties of astrocytes cultured from GFAP over-expressing and GFAP mutant mice

Abstract

Alexander disease is a fatal leukoencephalopathy caused by dominantly-acting coding mutations in GFAP. Previous work has also implicated elevations in absolute levels of GFAP as central to the pathogenesis of the disease. However, identification of the critical astrocyte functions that are compromised by mis-expression of GFAP has not yet been possible. To provide new tools for investigating the nature of astrocyte dysfunction in Alexander disease, we have established primary astrocyte cultures from two mouse models of Alexander disease, a transgenic that over-expresses wild type human GFAP, and a knock-in at the endogenous mouse locus that mimics a common Alexander disease mutation. We find that mutant GFAP, as well as excess wild type GFAP, promotes formation of cytoplasmic inclusions, disrupts the cytoskeleton, decreases cell proliferation, increases cell death, reduces proteasomal function, and compromises astrocyte resistance to stress.

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.