experimental-approaches-for-biochemical-analysis-glial-fibrillary

Experimental Approaches for Biochemical Analysis of Glial Fibrillary Acidic Protein and Its Disease-associated Variants

Glial Fibrillary Acidic Protein (GFAP) is a key intermediate filament protein critical for maintaining the structural integrity and function of astrocytes ...

National Tsing Hua University

Alexander disease (AxD) is a primary genetic disorder of astrocytes caused by dominant mutations in the gene encoding the intermediate filament (IF) protein GFAP. This disease is characterized by excessive accumulation of GFAP, known as Rosenthal fibers, within astrocytes. Abnormal GFAP aggregation also occurs in giant axon neuropathy (GAN), which is caused by recessive mutations in the gene encoding gigaxonin. Given that one of the functions of gigaxonin is to facilitate proteasomal degradation of several IF proteins, we sought to determine whether gigaxonin is involved in the degradation of GFAP. Using a lentiviral transduction system, we demonstrated that gigaxonin levels influence the degradation of GFAP in primary astrocytes and in cell lines that express this IF protein. Gigaxonin was similarly involved in the degradation of some but not all AxD-associated GFAP mutants. In addition, gigaxonin directly bound to GFAP, and inhibition of proteasome reversed the clearance of GFAP in cells achieved by overexpressing gigaxonin. These studies identify gigaxonin as an important factor that targets GFAP for degradation through the proteasome pathway. Our findings provide a critical foundation for future studies aimed at reducing or reversing pathological accumulation of GFAP as a potential therapeutic strategy for AxD and related diseases.

The role of gigaxonin in the degradation of the glial-specific intermediate filament protein GFAP.

Alexander disease (AxD) is a neurodegenerative disease caused by heterozygous mutations in the GFAP gene, which encodes the major intermediate filament protein of astrocytes. This disease is characterized by the accumulation of cytoplasmic protein aggregates, known as Rosenthal fibers. Antibodies specific to GFAP could provide invaluable tools to facilitate studies of the normal biology of GFAP and to elucidate the pathologic role of this IF protein in disease. While a large number of antibodies to GFAP are available, few if any of them have defined epitopes. Here we described the characterization of a panel of commonly used anti-GFAP antibodies, which recognized epitopes at regions extending across the rod domain of GFAP. We show that all of the antibodies are useful for immunoblotting and immunostaining, and identify a subset that preferentially recognized human GFAP. Using these antibodies, we demonstrate the presence of biochemically modified forms of GFAP in brains of human AxD patients and mouse AxD models. These data suggest that this panel of anti-GFAP antibodies will be useful for studies of animal and cell-based models of AxD and related diseases in which cytoskeletal defects associated with GFAP modifications occur.

Characterization of a panel of monoclonal antibodies recognizing specific epitopes on GFAP.

Neuroinflammation plays a central role in the pathophysiology of Alzheimer's disease (AD). Compounds that suppress neuroinflammation have been identified as potential therapeutic targets for AD. Rhinacanthin C (RC), a naphthoquinone ester found in  Kurz (Acanthaceae), is currently proposed as an effective molecule against inflammation. However, the exact role of RC on neuroinflammation remains to be elucidated. In the present study, we investigated RC effect on modulating lipopolysaccharides (LPS), amyloid- peptide (A), or interferon-- (IFN--) evoked pathological events in neurons and glia. Our findings demonstrated that RC prevented A-induced toxicity in rat hippocampal neurons and attenuated LPS-activated nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression, and NF-B signaling in rat glia. Likewise, RC suppressed LPS-induced neuroinflammation by reducing NO production and iNOS, IL-1, CCL-2, and CCL-5 mRNA levels in rat microglia. Further studies using BV-2 microglia revealed that RC inhibited LPS-, A-, and IFN--stimulated IL-6 and TNF- secretion. Of note, NF-B and ERK activation was abrogated by RC in BV-2 cell response to A or IFN-. Moreover, RC protected neurons from A-stimulated microglial conditioned media-dependent toxicity. Collectively, these data highlight the beneficial effects of RC on neuroprotection and support the therapeutic implications of RC to neuroinflammation-mediated conditions.

Rhinacanthin C Alleviates Amyloid- Fibrils' Toxicity on Neurons and Attenuates Neuroinflammation Triggered by LPS, Amyloid-, and Interferon- in Glial Cells.

Cisplatin chemotherapy causes myelosuppression and often limits treatment duration and dose escalation in patients. Novel approaches to circumvent or lessen myelotoxicity may improve clinical outcome and quality of life in these patients.  (CS) is a freshwater unicellular green alga and exhibits encouraging efficacy in immunomodulation and anticancer in preclinical studies. However, the efficacy of CS on chemoprotection remains unclear. We report here, for the first time, that CS extract (CSE) could protect normal myeloid cells and PBMCs from cisplatin toxicity. Also, cisplatin-induced apoptosis in HL-60 cells was rescued through reservation of mitochondrial function, inhibition of cytochrome c release to cytosol, and suppression of caspase and PARP activation. Intriguingly, cotreatment of CSE attenuated cisplatin-evoked hypocellularity of bone marrow in mice. Furthermore, we observed the enhancement of CSF-GM activity in bone marrow and spleen in mice administered CSE and cisplatin, along with increased CD11b levels in spleen. In conclusion, we uncovered a novel mechanism of CSE on myeloprotection, whereby potentially supports the use of CSE as a chemoprotector against cisplatin-induced bone marrow toxicity. Further clinical investigation of CSE in combination with cisplatin is warranted.

 Extract Prevents Cisplatin-Induced Myelotoxicity  and .

Alexander disease (AxD) is an autosomal-dominant leukodystrophy caused by heterozygous mutations in the glial fibrillary acidic protein (GFAP) gene.

Recessively-Inherited Adult-Onset Alexander Disease Caused by a Homozygous Mutation in the GFAP Gene.

Alexander disease (AxD) caused by mutations in the coding region of GFAP is a neurodegenerative disease characterized by astrocyte dysfunction, GFAP aggregation, and Rosenthal fiber accumulation. Although how GFAP mutations cause disease is not fully understood, Rosenthal fibers could be induced by forced overexpression of human GFAP and this could be lethal in mice implicate that an increase in GFAP levels is central to AxD pathogenesis. Our recent studies demonstrated that intronic GFAP mutations cause disease by altering GFAP splicing, suggesting that an increase in GFAP isoform expression could lead to protein aggregation and astrocyte dysfunction that typify AxD. Here we test this hypothesis by establishing primary astrocyte cultures from transgenic mice overexpressing human GFAP. We found that GFAP-δ and GFAP-κ were disproportionately increased in transgenic astrocytes and both were enriched in Rosenthal fibers of human AxD brains. In vitro assembly studies showed that while the major isoform GFAP-α self-assembled into typical 10-nm filaments, minor isoforms including GFAP-δ, -κ, and -λ were assembly-compromised and aggregation prone. Lentiviral transduction showed that expression of these minor GFAP isoforms decreased filament solubility and increased GFAP stability, leading to the formation of Rosenthal fibers-like aggregates that also disrupted the endogenous intermediate filament networks. The aggregate-bearing astrocytes lost their normal morphology and glutamate buffering capacity, which had a toxic effect on neighboring neurons. In conclusion, our findings provide evidence that links elevated GFAP isoform expression with GFAP aggregation and impaired glutamate transport, and suggest a potential non-cell-autonomous mechanism underlying neurodegeneration through astrocyte dysfunction.

Elevated GFAP isoform expression promotes protein aggregation and compromises astrocyte function.

Alexander disease (AxD) is a devastating leukodystrophy caused by gain-of-function mutations in , and the only available treatments are supportive. Recent advances in antisense oligonucleotide (ASO) therapy have demonstrated that transcript targeting can be a successful strategy for human neurodegenerative diseases amenable to this approach. We have previously used mouse models of AxD to show that -targeted ASO suppresses protein accumulation and reverses pathology; however, the mice have a mild phenotype with no apparent leukodystrophy or overt clinical features and are therefore limited for assessing functional outcomes. In this report, we introduce a rat model of AxD that exhibits hallmark pathology with GFAP aggregation in the form of Rosenthal fibers, widespread astrogliosis, and white matter deficits. These animals develop normally during the first postnatal weeks but fail to thrive after weaning and develop severe motor deficits as they mature, with about 14% dying of unknown cause between 6 and 12 weeks of age. In this model, a single treatment with -targeted ASO provides long-lasting suppression, reverses GFAP pathology, and, depending on age of treatment, prevents or mitigates white matter deficits and motor impairment. In this report, we characterize an improved animal model of AxD with myelin pathology and motor impairment, recapitulating prominent features of the human disease, and use this model to show that ASO therapy has the potential to not only prevent but also reverse many aspects of disease.

Antisense therapy in a rat model of Alexander disease reverses GFAP pathology, white matter deficits, and motor impairment.

Alexander disease (AxD) is a neurodegenerative astrogliopathy caused by mutation in the glial fibrillary acidic protein (GFAP) gene. A 42-year-old Korean man presented with temporary gait disturbance and psychiatric regression after a minor head trauma in the absence of bulbar symptoms and signs. Magnetic resonance images of the brain and spinal cord showed significant atrophy of the medulla oblongata and the entire spinal cord as well as contrast-enhanced T2 hypointensity in the basal ganglia. DNA sequencing revealed a novel 33-bp in-frame deletion mutation (p.Glu138_Leu148del) within the 1B rod domain of GFAP, which was predicted to be deleterious by PROVEAN analysis. To test whether the deletion mutant is disease-causing, we performed in vitro GFAP assembly and sedimentation assays, and GFAP aggregation assays in human adrenal carcinoma SW13 (Vim) cells and rat primary astrocytes. All the assays revealed that GFAP p.Glu138_Leu148del is aggregation prone. Based on these findings, we diagnosed the patient with Type II AxD. This is a report that demonstrates the pathogenicity of InDel mutation of GFAP through functional studies. This patient's atypical presentation as well as the discrepancy between clinical symptoms and radiologic findings may extend the scope of AxD.

A novel in-frame GFAP p.E138_L148del mutation in Type II Alexander disease with atypical phenotypes.

Alexander disease is a primary genetic disorder of astrocytes caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP). How single-amino-acid changes can lead to cytoskeletal catastrophe and brain degeneration remains poorly understood. In this study, we have analyzed 14 missense mutations located in the GFAP rod domain to investigate how these mutations affect in vitro filament assembly. Whereas the internal rod mutants assembled into filaments that were shorter than those of wild type, the rod end mutants formed structures with one or more of several atypical characteristics, including short filament length, irregular width, roughness of filament surface, and filament aggregation. When transduced into primary astrocytes, GFAP mutants with in vitro assembly defects usually formed cytoplasmic aggregates, which were more resistant to biochemical extraction. The resistance of GFAP to solubilization was also observed in brain tissues of patients with Alexander disease, in which a significant proportion of insoluble GFAP were accumulated in Rosenthal fiber fractions. These findings provide clinically relevant evidence that link GFAP assembly defects to disease pathology at the tissue level and suggest that altered filament assembly and properties as a result of GFAP mutation are critical initiating factors for the pathogenesis of Alexander disease.

Effects of Alexander disease-associated mutations on the assembly and organization of GFAP intermediate filaments.

Alzheimer's disease (AD) is a multifactorial disorder characterized by cognitive decline. Current available therapeutics for AD have limited clinical benefit. Therefore, preventive therapies for interrupting the development of AD are critically needed. Molecules targeting multifunction to interact with various pathlogical components have been considered to improve the therapeutic efficiency of AD. In particular, herbal medicines with multiplicity of actions produce cognitive benefits on AD. Bugu-M is a multi-herbal extract composed of  (Antler form),  Gaertn.,  Mill., and  with the ability of its various components to confer resilience to cognitive deficits.

Neuroprotective Effects of a Multi-Herbal Extract on Axonal and Synaptic Disruption  and Cognitive Impairment .

Here, we describe pathological events potentially involved in the disease pathogenesis of Alexander disease (AxD). This is a primary genetic disorder of astrocyte caused by dominant gain-of-function mutations in the gene coding for an intermediate filament protein glial fibrillary acidic protein (GFAP). Pathologically, this disease is characterized by the upregulation of GFAP and its accumulation as Rosenthal fibers. Although the genetic basis linking GFAP mutations with Alexander disease has been firmly established, the initiating events that promote GFAP accumulation and the role of Rosenthal fibers (RFs) in the disease process remain unknown. Here, we investigate the hypothesis that disease-associated mutations promote GFAP aggregation through aberrant posttranslational modifications. We found high molecular weight GFAP species in the RFs of AxD brains, indicating abnormal GFAP crosslinking as a prominent pathological feature of this disease. In vitro and cell-based studies demonstrate that cystine-generating mutations promote GFAP crosslinking by cysteine-dependent oxidation, resulting in defective GFAP assembly and decreased filament solubility. Moreover, we found GFAP was ubiquitinated in RFs of AxD patients and rodent models, supporting this modification as a critical factor linked to GFAP aggregation. Finally, we found that arginine could increase the solubility of aggregation-prone mutant GFAP by decreasing its ubiquitination and aggregation. Our study suggests a series of pathogenic events leading to AxD, involving interplay between GFAP aggregation and abnormal modifications by GFAP ubiquitination and oxidation. More important, our findings provide a basis for investigating new strategies to treat AxD by targeting abnormal GFAP modifications.

Glial fibrillary acidic protein is pathologically modified in Alexander disease.

Alexander disease (AxD) is a rare neurological disorder caused by dominant gain-of-function mutations in the gene for glial acidic fibrillary protein (). Expression of mutant protein results in astrocyte dysfunction that ultimately leads to developmental delay, failure to thrive, and intellectual and motor impairment. The disease is typically fatal, and at present there are no preventative or effective treatments. To gain a better understanding of the link between astrocyte dysfunction and behavioral deficits in AxD we recently developed a rat model that recapitulates many of the clinical features of the disease, including failure to thrive, motor impairment, and white matter deficits. In the present study, we show that both male and female AxD model rats exhibit a neurodegenerative profile with a progressive neuroinflammatory response combined with reduced expression of synaptic and mitochondrial proteins. Consistent with these results AxD rats show reduced hippocampal long-term potentiation and are cognitively impaired, as demonstrated by poor performance in the Barnes maze and novel object recognition tests. The AxD rat provides a novel model in which to investigate the impact of astrocyte pathology on central nervous system function and provides an essential platform for further development of effective treatments for AxD and potentially other neurodegenerative diseases with astrocyte pathology. Alexander disease (AxD) is a fatal neurodegenerative disorder caused by gain-of-function  mutations. We recently developed a   rat model which demonstrates hallmark astrocyte pathology, myelin deficits, and motor impairment. Here, we show that   rats also exhibit reduced synaptic plasticity and cognitive deficits as additional clinically relevant phenotypes, further demonstrating its utility as a model. Hippocampal transcriptomic analysis in young adult animals reveals a neurodegenerative signature with an innate immune response and loss of synaptic and metabolic gene expression, features that are typically associated with chronic diseases of aging. These results reveal mechanisms by which astrocyte dysfunction leads to learning and memory deficits in AxD and perhaps contributes to other diseases such as Alzheimer's and Parkinson's.

GFAP mutation and astrocyte dysfunction lead to a neurodegenerative profile with impaired synaptic plasticity and cognitive deficits in a rat model of Alexander disease.

Glial fibrillary acidic protein (GFAP) is classified as a type III intermediate filament protein predominantly expressed in mature astrocytes. It has the ability to self-assemble into 10 nm filaments in vitro, making it particularly valuable for elucidating the sequences essential for filament assembly. In this study, we created a series of deletion mutants targeting sequences in the N-terminal, C-terminal, and central rod domains to explore the sequences critical for the assembly of GFAP into 10 nm filaments. The impact of these deletions on filament formation was evaluated through in vitro assembly studies and transduction assays conducted with primary astrocytes. Our data revealed that deletions at the carboxy end resulted in abnormalities in either filament diameter calibration or lateral association, whereas deletions at the amino-terminal end significantly disrupted the filament assembly process, particularly restricting filament elongation. Furthermore, we discovered that the filament-forming sequences within the rod domain varied in their contributions to filament assembly and network formation. These findings enhance our understanding of the GFAP assembly process in vitro and provide a detailed mapping of the essential regions required for GFAP assembly. These insights hold significant implications for Alexander disease arising from deletion mutations in GFAP.

Article	Year
The role of gigaxonin in the degradation of the glial-specific intermediate filament protein GFAP. Molecular biology of the cell\| PubMed ID: 27798231	2016
Characterization of a panel of monoclonal antibodies recognizing specific epitopes on GFAP. PloS one\| PubMed ID: 28700643	2017
Rhinacanthin C Alleviates Amyloid- Fibrils' Toxicity on Neurons and Attenuates Neuroinflammation Triggered by LPS, Amyloid-, and Interferon- in Glial Cells. Oxidative medicine and cellular longevity\| PubMed ID: 29181126	2017
Extract Prevents Cisplatin-Induced Myelotoxicity and . Oxidative medicine and cellular longevity\| PubMed ID: 32047579	2020
Recessively-Inherited Adult-Onset Alexander Disease Caused by a Homozygous Mutation in the GFAP Gene. Movement disorders : official journal of the Movement Disorder Society\| PubMed ID: 32374915	2020
Elevated GFAP isoform expression promotes protein aggregation and compromises astrocyte function. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 33908669	2021
Antisense therapy in a rat model of Alexander disease reverses GFAP pathology, white matter deficits, and motor impairment. Science translational medicine\| PubMed ID: 34788075	2021
A novel in-frame GFAP p.E138_L148del mutation in Type II Alexander disease with atypical phenotypes. European journal of human genetics : EJHG\| PubMed ID: 35246666	2022
Effects of Alexander disease-associated mutations on the assembly and organization of GFAP intermediate filaments. Molecular biology of the cell\| PubMed ID: 35511821	2022
Neuroprotective Effects of a Multi-Herbal Extract on Axonal and Synaptic Disruption and Cognitive Impairment . Journal of Alzheimer's disease reports\| PubMed ID: 36777330	2023
Glial fibrillary acidic protein is pathologically modified in Alexander disease. The Journal of biological chemistry\| PubMed ID: 38782207	2024
GFAP mutation and astrocyte dysfunction lead to a neurodegenerative profile with impaired synaptic plasticity and cognitive deficits in a rat model of Alexander disease. eNeuro\| PubMed ID: 40064497	2025
Deletions in Glial Fibrillary Acidic Protein Leading to Alterations in Intermediate Filament Assembly and Network Formation. International journal of molecular sciences\| PubMed ID: 40076540	2025

Ni-Hsuan Lin

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