The Alexander disease-causing glial fibrillary acidic protein mutant, R416W, accumulates into Rosenthal fibers by a pathway that involves filament aggregation and the association of alpha B-crystallin and HSP27

Here, we describe the early events in the disease pathogenesis of Alexander disease. This is a rare and usually fatal neurodegenerative disorder whose pathological hallmark is the abundance of protein aggregates in astrocytes. These aggregates, termed "Rosenthal fibers," contain the protein chaperones alpha B-crystallin and HSP27 as well as glial fibrillary acidic protein (GFAP), an intermediate filament (IF) protein found almost exclusively in astrocytes. Heterozygous, missense GFAP mutations that usually arise spontaneously during spermatogenesis have recently been found in the majority of patients with Alexander disease. In this study, we show that one of the more frequently observed mutations, R416W, significantly perturbs in vitro filament assembly. The filamentous structures formed resemble assembly intermediates but aggregate more strongly. Consistent with the heterozygosity of the mutation, this effect is dominant over wild-type GFAP in coassembly experiments. Transient transfection studies demonstrate that R416W GFAP induces the formation of GFAP-containing cytoplasmic aggregates in a wide range of different cell types, including astrocytes. The aggregates have several important features in common with Rosenthal fibers, including the association of alpha B-crystallin and HSP27. This association occurs simultaneously with the formation of protein aggregates containing R416W GFAP and is also specific, since HSP70 does not partition with them. Monoclonal antibodies specific for R416W GFAP reveal, for the first time for any IF-based disease, the presence of the mutant protein in the characteristic histopathological feature of the disease, namely Rosenthal fibers. Collectively, these data confirm that the effects of the R416W GFAP are dominant, changing the assembly process in a way that encourages aberrant filament-filament interactions that then lead to protein aggregation and chaperone sequestration as early events in Alexander disease.     

Mutations in vimentin disrupt the cytoskeleton in fibroblasts and delay execution of apoptosis

To get new insights into the function of the intermediate filament (IF) protein vimentin in cell physiology, we generated two mutant cDNAs, one with a point mutation in the consensus motif in coil1A (R113C) and one with the complete deletion of coil 2B of the rod domain. In keratins and glia filament protein (GFAP), analogous mutations cause keratinopathies and Alexander disease, respectively. Both mutants prevented filament assembly in vitro and inhibited assembly of wild-type vimentin when present in equal amounts. In stably transfected preadipocytes, these mutants caused the complete disruption of the endogenous vimentin network, demonstrating their dominant-negative behaviour. Cytoplasmic vimentin aggregates colocalised with the chaperones alphaB-crystallin and HSP40. Moreover, vimR113C mutant cells were more resistant against staurosporine-induced apoptosis compared to controls. We hypothesise that mutations in the vimentin gene, like in most classes of IF genes, may contribute to distinct human diseases.     

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

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.     

Do the ends justify the mean? Proline mutations at the ends of the keratin coiled-coil rod segment are more disruptive than internal mutations

Intermediate filament (IF) assembly is remarkable, in that it appears to be self-driven by the primary sequence of IF proteins, a family (40-220 kd) with diverse sequences, but similar secondary structures. Each IF polypeptide has a central 310 amino acid residue alpha-helical rod domain, involved in coiled-coil dinner formation. Two short (approximately 10 amino acid residue) stretches at the ends of this rod are more highly conserved than the rest, although the molecular basis for this is unknown. In addition, the rod is segmented by three short nonhelical linkers of conserved location, but not sequence. To examine the degree to which different conserved helical and nonhelical rod sequences contribute to dimer, tetramer, and higher ordered interactions, we introduced proline mutations in residues throughout the rod of a type I keratin, and we removed existing proline residues from the linker regions. To further probe the role of the rod ends, we introduced more subtle mutations near the COOH-terminus. We examined the consequences of these mutations on (a) IF network formation in vivo, and (b) 10-nm filament assembly in vitro. Surprisingly, all proline mutations located deep in the coiled-coil rod segment showed rather modest effects on filament network formation and 10-nm filament assembly. In addition, removing the existing proline residues was without apparent effect in vivo, and in vitro, these mutants assembled into 10-nm filaments with a tendency to aggregate, but with otherwise normal appearance. The most striking effects on filament network formation and IF assembly were observed with mutations at the very ends of the rod. These data indicate that sequences throughout the rod are not equal with respect to their role in filament network formation and in 10-nm filament assembly. Specifically, while the internal rod segments seem able to tolerate considerable changes in alpha-helical conformation, the conserved ends seem to be essential for creating a very specific structure, in which even small perturbations can lead to loss of IF stability and disruption of normal cellular interactions. These findings have important implications for the disease Epidermolysis Bullosa Simplex, arising from point mutations in keratins K5 or K14.     

Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro

To investigate the sequences important for assembly of keratins into 10- nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins. Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro. Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency. Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly. Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo. In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation. For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype. Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred. Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro.     

Dynamics of mutated GFAP aggregates revealed by real-time imaging of an astrocyte model of Alexander disease

Alexander disease (AxD) is a rare neurodegenerative disorder characterized by large cytoplasmic aggregates in astrocytes and myelin abnormalities and caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), the main intermediate filament protein in astrocytes. We tested the effects of three mutations (R236H, R76H and L232P) associated with AxD in cells transiently expressing mutated GFAP fused to green fluorescent protein (GFP). Mutated GFAP-GFP expressed in astrocytes formed networks or aggregates similar to those found in the brains of patients with the disease. Time-lapse recordings of living astrocytes showed that aggregates of mutated GFAP-GFP may either disappear, associated with cell survival, or coalesce in a huge juxtanuclear structure associated with cell death. Immunolabeling of fixed cells suggested that this gathering of aggregates forms an aggresome-like structure. Proteasome inhibition and immunoprecipitation assays revealed mutated GFAP-GFP ubiquitination, suggesting a role of the ubiquitin-proteasome system in the disaggregation process. In astrocytes from wild-type-, GFAP-, and vimentin-deficient mice, mutated GFAP-GFP aggregated or formed a network, depending on qualitative and quantitative interactions with normal intermediate filament partners. Particularly, vimentin displayed an anti-aggregation effect on mutated GFAP. Our data indicate a dynamic and reversible aggregation of mutated GFAP, suggesting that therapeutic approaches may be possible.     

Caspase cleavage of GFAP produces an assembly-compromised proteolytic fragment that promotes filament aggregation

IF (intermediate filament) proteins can be cleaved by caspases to generate proapoptotic fragments as shown for desmin. These fragments can also cause filament aggregation. The hypothesis is that disease-causing mutations in IF proteins and their subsequent characteristic histopathological aggregates could involve caspases. GFAP (glial fibrillary acidic protein), a closely related IF protein expressed mainly in astrocytes, is also a putative caspase substrate. Mutations in GFAP cause AxD (Alexander disease). The overexpression of wild-type or mutant GFAP promotes cytoplasmic aggregate formation, with caspase activation and GFAP proteolysis. In this study, we report that GFAP is cleaved specifically by caspase 6 at VELD²²⁵ in its L12 linker domain in vitro. Caspase cleavage of GFAP at Asp²²⁵ produces two major cleavage products. While the C-GFAP (C-terminal GFAP) is unable to assemble into filaments, the N-GFAP (N-terminal GFAP) forms filamentous structures that are variable in width and prone to aggregation. The effect of N-GFAP is dominant, thus affecting normal filament assembly in a way that promotes filament aggregation. Transient transfection of N-GFAP into a human astrocytoma cell line induces the formation of cytoplasmic aggregates, which also disrupt the endogenous GFAP networks. In addition, we generated a neo-epitope antibody that recognizes caspase-cleaved but not the intact GFAP. Using this antibody, we demonstrate the presence of the caspase-generated GFAP fragment in transfected cells expressing a disease-causing mutant GFAP and in two mouse models of AxD. These findings suggest that caspase-mediated GFAP proteolysis may be a common event in the context of both the GFAP mutation and excess.     

Forced expression of desmin and desmin mutants in cultured cells: impact of myopathic missense mutations in the central coiled-coil domain on network formation

We recently demonstrated that inherited disease-causing mutations clustered in the alpha-helical coiled-coil "rod" domain of the muscle-specific intermediate filament (IF) protein desmin display a wide range of inhibitory effects on regular in vitro assembly. In these studies, we showed that individual mutations exhibited phenotypes that were not, with respect to the severity of interference, predictable by our current knowledge of the structural design of IF proteins. Moreover, the behavior of some mutated proteins in a standard tissue culture cell expression system was found to be even more complex. Here, we systematically investigate the behavior of these disease mutants in four different cell types: three not containing desmin or the related IF protein vimentin and the standard fibroblast line 3T3, which has an extensive vimentin system. The ability of the mutants to form filaments in the vimentin-free cells varies considerably, and only the mutants forming IFs in vitro generate extended filamentous networks. Furthermore, these latter mutants integrate into the 3T3 vimentin network but all the others do not. Instead, they cause the endogenous network of 3T3 vimentin to reorganize into perinuclear bundles. In addition, most of these assembly-deficient mutant desmins completely segregate from the vimentin system. Instead, the small round to fibrillar particles formed distribute independently throughout the cytoplasm as well as between the collapsed vimentin filament arrays in the perinuclear area.     

Dual transgenic reporter mice as a tool for monitoring expression of glial fibrillary acidic protein

Glial fibrillary acidic protein (GFAP) is the major intermediate filament protein of astrocytes, and its expression changes dramatically during development and following injury. To facilitate study of the regulation of GFAP expression, we have generated dual transgenic mice expressing both firefly luciferase under the control of a 2.2 kb human GFAP promoter and Renilla luciferase under the control of a 0.5 kb human Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) promoter for normalization of the GFAP signal. The GFAP-fLuc was highly expressed in brain compared to other tissues, and was limited to astrocytes, whereas the GAPDH-RLuc was more widely expressed. Normalization of the GFAP signal to the GAPDH signal reduced the inter-individual variability compared to using the GFAP signal alone. The GFAP/GAPDH ratio correctly reflected the up-regulation of GFAP that occurs following retinal degeneration in FVB/N mice because of the rd mutation. Following kainic acid-induced seizures, changes in the GFAP/GAPDH ratio precede those in total GFAP protein. In knock-in mice expressing the R236H Alexander disease mutant, GFAP promoter activity is only transiently elevated and may not entirely account for the accumulation of GFAP protein that takes place.     

Infantile Alexander Disease: Spectrum of GFAP Mutations and Genotype-Phenotype Correlation

Heterozygous, de novo mutations in the glial fibrillary acidic protein (GFAP) gene have recently been reported in 12 patients affected by neuropathologically proved Alexander disease. We searched for GFAP mutations in a series of patients who had heterogeneous clinical symptoms but were candidates for Alexander disease on the basis of suggestive neuroimaging abnormalities. Missense, heterozygous, de novo GFAP mutations were found in exons 1 or 4 for 14 of the 15 patients analyzed, including patients without macrocephaly. Nine patients carried arginine mutations (four had R79H; four had R239C; and one had R239H) that have been described elsewhere, whereas the other five had one of four novel mutations, of which two affect arginine (2R88C and 1R88S) and two affect nonarginine residues (1L76F and 1N77Y). All mutations were located in the rod domain of GFAP, and there is a correlation between clinical severity and the affected amino acid. These results confirm that GFAP mutations are a reliable molecular marker for the diagnosis of infantile Alexander disease, and they also form a basis for the recommendation of GFAP analysis for prenatal diagnosis to detect potential cases of germinal mosaicism.     

GFAP and its role in Alexander disease

Here we review how GFAP mutations cause Alexander disease. The current data suggest that a combination of events cause the disease. These include: (i) the accumulation of GFAP and the formation of characteristic aggregates, called Rosenthal fibers, (ii) the sequestration of the protein chaperones alpha B-crystallin and HSP27 into Rosenthal fibers, and (iii) the activation of both Jnk and the stress response. These then set in motion events that lead to Alexander disease. We discuss parallels with other intermediate filament diseases and assess potential therapies as part of this review as well as emerging trends in disease diagnosis and other aspects concerning GFAP.     

EIF2B5 mutations compromise GFAP+ astrocyte generation in vanishing white matter leukodystrophy

Vanishing white matter disease (VWM) is a heritable leukodystrophy linked to mutations in translation initiation factor 2B (eIF2B). Although the clinical course of this disease has been relatively well described, the cellular consequences of EIF2B mutations on neural cells are unknown. Here we have established cell cultures from the brain of an individual with VWM carrying mutations in subunit 5 of eIF2B (encoded by EIF2B5). Despite the extensive demyelination apparent in this VWM patient, normal-appearing oligodendrocytes were readily generated in vitro. In contrast, few GFAP-expressing (GFAP+) astrocytes were present in primary cultures, induction of astrocytes was severely compromised, and the few astrocytes generated showed abnormal morphologies and antigenic phenotypes. Lesions in vivo also lacked GFAP+ astrocytes. RNAi targeting of EIF2B5 severely compromised the induction of GFAP+ cells from normal human glial progenitors. This raises the possibility that a deficiency in astrocyte function may contribute to the loss of white matter in VWM leukodystrophy.     

Mutations in Drosophila myosin rod cause defects in myofibril assembly

The roles of myosin during muscle contraction are well studied, but how different domains of this protein are involved in myofibril assembly in vivo is far less understood. The indirect flight muscles (IFMs) of Drosophila melanogaster provide a good model for understanding muscle development and function in vivo. We show that two missense mutations in the rod region of the myosin heavy-chain gene, Mhc, give rise to IFM defects and abnormal myofibrils. These defects likely result from thick filament abnormalities that manifest during early sarcomere development or later by hypercontraction. The thick filament defects are accompanied by marked reduction in accumulation of flightin, a myosin binding protein, and its phosphorylated forms, which are required to stabilise thick filaments. We investigated with purified rod fragments whether the mutations affect the coiled-coil structure, rod aggregate size or rod stability. No significant changes in these parameters were detected, except for rod thermodynamic stability in one mutation. Molecular dynamics simulations suggest that these mutations may produce localised rod instabilities. We conclude that the aberrant myofibrils are a result of thick filament defects, but that these in vivo effects cannot be detected in vitro using the biophysical techniques employed. The in vivo investigation of these mutant phenotypes in IFM development and function provides a useful platform for studying myosin rod and thick filament formation generically, with application to the aetiology of human myosin rod myopathies.     

Alexander disease: models,mechanisms, and medicine

Alexander disease is a primary disorder of astrocytes caused by gain-of-function mutations in the gene for glial fibrillary acidic protein (GFAP), which lead to protein aggregation and a reactive astrocyte response, with devastating effects on the central nervous system. Over the past two decades since the discovery of GFAP as the culprit, several cellular and animal models have been generated, and much has been learned about underlying mechanisms contributing to the disease. Despite these efforts, many aspects of Alexander disease have remained enigmatic, particularly the initiating events in GFAP accumulation and astrocyte pathology, the relation between astrocyte dysfunction and myelin deficits, and the variability in age of onset and disease severity. More recent work in both old and new models has begun to address these complex questions and identify new therapeutics that finally offer the promise of effective treatment.     

Beneficial effects of curcumin on GFAP filament organization and down-regulation of GFAP expression in an in vitro model of Alexander disease

Heterozygous mutations of the GFAP gene are responsible for Alexander disease, a neurodegenerative disorder characterized by intracytoplasmic Rosenthal fibers (RFs) in dystrophic astrocytes. In vivo and in vitro models have shown co-localization of mutant GFAP proteins with the small heat shock proteins (sHSPs) HSP27 and alphaB-crystallin, ubiquitin and proteasome components. Results reported by several recent studies agree on ascribing an altered cytoskeletal pattern to mutant GFAP proteins, an effect which induces mutant proteins accumulation, leading to impaired proteasome function and autophagy induction. On the basis of the protective role shown by both these small heat shock proteins (sHSPs), and on the already well established neuroprotective effects of curcumin in several diseases, we have investigated the effects of this compound in an in vitro model of Alexander disease, consisting in U251-MG astrocytoma cells transiently transfected with a construct encoding for GFAP carrying the p.R239C mutation in frame with the reporter green fluorescent protein (GFP). In particular, depending on the dose used, we have observed that curcumin is able to induce both HSP27 and alphaB-crystallin, to reduce expression of both RNA and protein of endogenous GFAP, to induce autophagy and, finally, to rescue the filamentous organization of the GFAP mutant protein, thus suggesting a role of this spice in counteracting the pathogenic effects of GFAP mutations.     

Withaferin A Targets Intermediate Filaments Glial Fibrillary Acidic Protein and Vimentin in a Model of Retinal Gliosis

Gliosis is a biological process that occurs during injury repair in the central nervous system and is characterized by the overexpression of the intermediate filaments (IFs) glial fibrillary acidic protein (GFAP) and vimentin. A common thread in many retinal diseases is reactive Müller cell gliosis, an untreatable condition that leads to tissue scarring and even blindness. Here, we demonstrate that the vimentin-targeting small molecule withaferin A (WFA) is a novel chemical probe of GFAP. Using molecular modeling studies that build on the x-ray crystal structure of tetrameric vimentin rod 2B domain we reveal that the WFA binding site is conserved in the corresponding domain of tetrameric GFAP. Consequently, we demonstrate that WFA covalently binds soluble recombinant tetrameric human GFAP at cysteine 294. In cultured primary astrocytes, WFA binds to and down-regulates soluble vimentin and GFAP expression to cause cell cycle G₀/G₁ arrest. Exploiting a chemical injury model that overexpresses vimentin and GFAP in retinal Müller glia, we demonstrate that systemic delivery of WFA down-regulates soluble vimentin and GFAP expression in mouse retinas. This pharmacological knockdown of soluble IFs results in the impairment of GFAP filament assembly and inhibition of cell proliferative response in Müller glia. We further show that a more severe GFAP filament assembly deficit manifests in vimentin-deficient mice, which is partly rescued by WFA. These findings illustrate WFA as a chemical probe of type III IFs and illuminate this class of withanolide as a potential treatment for diverse gliosis-dependent central nervous system traumatic injury conditions and diseases, and for orphan IF-dependent pathologies.     

A new splice variant of glial fibrillary acidic protein,GFAP epsilon,interacts with the presenilin proteins

We describe a new human isoform, GFAP epsilon, of the intermediary filament protein GFAP (glial fibrillary acidic protein). GFAP epsilon mRNA is the result of alternative splicing and a new polyadenylation signal, and thus GFAP epsilon has a new C-terminal protein sequence. This provides GFAP epsilon with the capacity for specific binding of presenilin proteins in yeast and in vitro. Our observations suggest a direct link between the presenilins and the cytoskeleton where GFAP epsilon is incorporated. Mutations in GFAP and presenilins are associated with Alexander disease and Alzheimer's disease, respectively. Accordingly, GFAP epsilon should be taken into consideration when studying neurodegenerative diseases.     

GFAP-Deficient Astrocytes Are Capable of Stellationin VitroWhen Cocultured with Neurons and Exhibit a Reduced Amount of Intermediate Filaments and an Increased Cell Saturation Density

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein predominantly expressed in cells of astroglial origin. To allow for the study of the biological functions of GFAP we have previously generated GFAP-negative mice by gene targeting [Peknyet al.(1995)EMBO J.14, 1590–1598]. Astrocytes in culture, similar to reactive astrocytesin vivo,express three intermediate filament proteins: GFAP, vimentin, and nestin. Using primary astrocyte-enriched cultures from GFAP-negative mice, we now report on the effect of GFAP absence on (i) the synthesis of other intermediate filament proteins in astrocytes, (ii) intermediate filament formation, (iii) astrocyte process formation (stellation) in response to neurons in mixed cerebellar astrocyte/neuron cultures, and (iv) saturation cell densityin vitro.GFAP−/− astrocytes were found to produce both nestin and vimentin. At the ultrastructural level, the amount of intermediate filaments as revealed by transmission electron microscopy was reduced in GFAP−/− astrocytes compared to that in GFAP+/+ astrocytes. GFAP−/− astrocytes retained the ability to form processes in response to neurons in mixed astrocyte/neuron cultures from the cerebellum. GFAP−/− astrocyte-enriched primary cultures exhibited an increased final cell saturation density. The latter leads us to speculate that the loss of GFAP expression observed focally in a proportion of human malignant gliomas may reflect tumor progression toward a more rapidly growing and malignant phenotype.