Synergistic effects of the SAPK/JNK and the proteasome pathway on glial fibrillary acidic protein (GFAP) accumulation in Alexander disease

Protein aggregates in astrocytes that contain glial fibrillary acidic protein (GFAP), small heat shock proteins, and ubiquitinated proteins are termed Rosenthal fibers and characterize Alexander disease, a leukodystrophy caused by heterozygous mutations in GFAP. The mechanisms responsible for the massive accumulation of GFAP in Alexander disease remain unclear. In this study, we show that overexpression of both wild type and R239C mutant human GFAP led to cytoplasmic inclusions. GFAP accumulation also led to a decrease of proteasome activity and an activation of the MLK2-JNK pathway. In turn, the expression of activated mixed lineage kinases (MLKs) induced JNK activation and increased GFAP accumulation, whereas blocking the JNK pathway decreased GFAP accumulation. Activated MLK also inhibited proteasome function. A direct inhibition of proteasome function pharmacologically further activated JNK. Our data suggest a synergistic interplay between the proteasome and the SAPK/JNK pathway in the context of GFAP accumulation. Feedback interactions among GFAP accumulation, SAPK/JNK activation, and proteasomal hypofunction cooperate to produce further protein accumulation and cellular stress responses.     

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.     

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.     

Alexander Disease: A Leukodystrophy Caused by a Mutation in GFAP

Alexander disease, a rare fatal disorder of the central nervous system, causes progressive loss of motor and mental function. Until recently it was of unknown etiology, almost all cases were sporadic, and there was no effective treatment. It was most common in an infantile form, somewhat less so in a juvenile form, and was rarely seen in an adult-onset form. A number of investigators have now shown that almost all cases of Alexander disease have a dominant mutation in one allele of the gene for glial fibrillary acidic protein (GFAP) that causes replacement of one amino acid for another. Only in very rare cases of the adult-onset form is the mutation present in either parent. Thus, in almost all cases, the mutation arises as a spontaneous event, possibly in the germ cell of one parent.     

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.     

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.     

重组反义GFAP逆转录病毒对星形胶质细胞在胶质瘢痕增生中作用的研究

本文研究了脑穿刺损伤后伤灶组织中大胶质细胞的变化、性激素对脑损伤后星形胶质细胞反应的影响,以及反义胶质原纤维酸性蛋白（ＧＦＡＰ）逆转录病毒表达载体对Ａｓｔ形态结构,反应性胶质化及胶质瘢痕形成的作用。结果表明,胶质瘢痕中增生的大胶质细胞主要是Ａｓｔ,ＧＦＡＰ对维持Ａｓｔ的形态结构及功能具有重要作用;少突胶质细胞在胶质瘢痕形成过程中不是反应活跃的细胞成分;性激素对Ａｓｔ的反应性胶质化有一定程度的抑制作     

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.     

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.     

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.     

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.     

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.     

Purity,cell viability,expression of GFAP and bystin in astrocytes cultured by different procedures

Primary astrocyte cultures are the most commonly used in vitro model for neurobiological studies. We speculated that different protocols might induce differences not only in the percentage of astrocytes but also in their biological characteristics. In this study, we investigated the effects of four major protocols on the purity of astrocytes, cell viability, expression of glial fibrillary acidic protein (GFAP) and bystin of cultured astrocytes using MTT assay, immunocytochemical staining, and Western blot analysis. We demonstrated that the purity of astrocytes (98.9%) generated by the subculture (SC) procedure is significantly higher than those generated by primary culture (PC), shaken once culture (SK‐1) or shaken twice culture (SK‐2). We also showed that expressions of GFAP and bystin in astrocytes that are purified by the SK‐2 or SK‐1 procedures are significantly higher than those in astrocytes prepared by PC or SC. In addition, astrocytes cultured by SK‐2 or SK‐1 have a higher level of cell viabilities at most time points after ischemia compared with astrocytes cultured by PC or SC. These suggested that physical stimulation induced by “shaken” or culture operation might be able to activate astrocytes and implied that different procedures induce differences not only in the purity but also in the biological characteristics of astrocytes, such as the percentage of activated astrocytes, GFAP, and bystin expressions and responses to ischemia. A more detailed analysis about the effect of “culture protocol factor” on the biological characteristics of astrocytes is absolutely needed. J. Cell. Biochem. 109: 30–37, 2010. © 2009 Wiley‐Liss, Inc.     

Genetic Background Affects Human Glial Fibrillary Acidic Protein Promoter Activity

The human glial fibrillary acidic protein (hGFAP) promoter has been used to generate numerous transgenic mouse lines, which has facilitated the analysis of astrocyte function in health and disease. Here, we evaluated the expression levels of various hGFAP transgenes at different ages in the two most commonly used inbred mouse strains, FVB/N (FVB) and C57BL/6N (B6N). In general, transgenic mice maintained on the B6N background displayed weaker transgene expression compared with transgenic FVB mice. Higher level of transgene expression in B6N mice could be regained by crossbreeding to FVB wild type mice. However, the endogenous murine GFAP expression was equivalent in both strains. In addition, we found that endogenous GFAP expression was increased in transgenic mice in comparison to wild type mice. The activities of the hGFAP transgenes were not age-dependently regulated. Our data highlight the importance of proper expression analysis when non-homologous recombination transgenesis is used.     

S100B-Mediated Inhibition of the Phosphorylation of GFAP Is Prevented by TRTK-12

S100B belongs to a family of calcium-binding proteins involved in cell cycle and cytoskeleton regulation. We observed an inhibitory effect of S100B on glial fibrillary acidic protein (GFAP) phosphorylation, when stimulated by cAMP or Ca2+/calmodulin, in a cytoskeletal fraction from primary astrocyte cultures. We found that S100B has no direct effect on CaM KII activity, the major kinase in this cytoskeletal fraction able to phosphorylate GFAP. The inhibition of GFAP phosphorylation is most likely due to the binding of S100B to the phosphorylation sites on this protein and blocking the access of these sites to the protein kinases. This inhibition was dependent on Ca2+. However, Zn2+ could substitute for Ca2+. The inhibitory effect of S100B was prevented by TRTK-12, a peptide that blocks S100B interaction with several target proteins including glial fibrillary acidic protein. These data suggest a role for S100B in the assembly of intermediate filaments in astrocytes.