Publications by authors named "Ming-Der Perng"

26 Publications

  • Page 1 of 1

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

FASEB J 2021 May;35(5):e21614

Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan.

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.
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http://dx.doi.org/10.1096/fj.202100087RDOI Listing
May 2021

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

Mov Disord 2020 09 6;35(9):1662-1667. Epub 2020 May 6.

Department of Neurology, Chang Gung University College of Medicine, Kaohsiung, Taiwan.

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

Objectives: The objective of this report is to characterize the clinical phenotype and identify the genetic mutation associated with adult-onset AxD.

Methods: A man presented with progressive unsteadiness since age 16. Magnetic resonance imaging findings revealed characteristic features of AxD. The GFAP gene was screened, and a candidate variant was functionally tested to evaluate causality.

Results: A homozygous c.197G > A (p.Arg66Gln) mutation was found in the proband, and his asymptomatic parents were heterozygous for the same mutation. This mutation affected GFAP solubility and promoted filament aggregation. The presence of the wild-type protein rescued mutational effects, consistent with the recessive nature of this mutation.

Conclusions: This study is the first report of AxD caused by a homozygous mutation in GFAP. The clinical implication is while examining patients with characteristic features on suspicion of AxD, GFAP screening is recommended even without a supportive family history. © 2020 International Parkinson and Movement Disorder Society.
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http://dx.doi.org/10.1002/mds.28099DOI Listing
September 2020

Extract Prevents Cisplatin-Induced Myelotoxicity and .

Oxid Med Cell Longev 2020 25;2020:7353618. Epub 2020 Jan 25.

Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute, 321 Kuang Fu 2 Road, Hsinchu 30011, Taiwan.

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.
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http://dx.doi.org/10.1155/2020/7353618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003270PMC
September 2020

Site-specific phosphorylation and caspase cleavage of GFAP are new markers of Alexander disease severity.

Elife 2019 11 4;8. Epub 2019 Nov 4.

Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, United States.

Alexander disease (AxD) is a fatal neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP), which supports the structural integrity of astrocytes. Over 70 GFAP missense mutations cause AxD, but the mechanism linking different mutations to disease-relevant phenotypes remains unknown. We used AxD patient brain tissue and induced pluripotent stem cell (iPSC)-derived astrocytes to investigate the hypothesis that AxD-causing mutations perturb key post-translational modifications (PTMs) on GFAP. Our findings reveal selective phosphorylation of GFAP-Ser13 in patients who died young, independently of the mutation they carried. AxD iPSC-astrocytes accumulated pSer13-GFAP in cytoplasmic aggregates within deep nuclear invaginations, resembling the hallmark Rosenthal fibers observed in vivo. Ser13 phosphorylation facilitated GFAP aggregation and was associated with increased GFAP proteolysis by caspase-6. Furthermore, caspase-6 was selectively expressed in young AxD patients, and correlated with the presence of cleaved GFAP. We reveal a novel PTM signature linking different GFAP mutations in infantile AxD.
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http://dx.doi.org/10.7554/eLife.47789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927689PMC
November 2019

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

Oxid Med Cell Longev 2017 18;2017:5414297. Epub 2017 Oct 18.

Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute, 321 Kuang Fu 2nd Road, Hsinchu 30011, Taiwan.

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.
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http://dx.doi.org/10.1155/2017/5414297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664341PMC
July 2018

Aggregation-prone GFAP mutation in Alexander disease validated using a zebrafish model.

BMC Neurol 2017 Sep 7;17(1):175. Epub 2017 Sep 7.

Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju, 501-759, Republic of Korea.

Background: Alexander disease (AxD) is an astrogliopathy that predominantly affects the white matter of the central nervous system (CNS), and is caused by a mutation in the gene encoding the glial fibrillary acidic protein (GFAP), an intermediate filament primarily expressed in astrocytes and ependymal cells. The main pathologic feature of AxD is the presence of Rosenthal fibers (RFs), homogeneous eosinophilic inclusions found in astrocytes. Because of difficulties in procuring patient' CNS tissues and the presence of RFs in other pathologic conditions, there is a need to develop an in vivo assay that can determine whether a mutation in the GFAP results in aggregation and is thus disease-causing.

Methods: We found a GFAP mutation (c.382G > A, p.Asp128Asn) in a 68-year-old man with slowly progressive gait disturbance with tendency to fall. The patient was tentatively diagnosed with AxD based on clinical and radiological findings. To develop a vertebrate model to assess the aggregation tendency of GFAP, we expressed several previously reported mutant GFAPs and p.Asp128Asn GFAP in zebrafish embryos.

Results: The most common GFAP mutations in AxD, p.Arg79Cys, p.Arg79His, p.Arg239Cys and p.Arg239His, and p.Asp128Asn induced a significantly higher number of GFAP aggregates in zebrafish embryos than wild-type GFAP.

Conclusions: The p.Asp128Asn GFAP mutation is likely to be a disease-causing mutation. Although it needs to be tested more extensively in larger case series, the zebrafish assay system presented here would help clinicians determine whether GFAP mutations identified in putative AxD patients are disease-causing.
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http://dx.doi.org/10.1186/s12883-017-0938-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5590178PMC
September 2017

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

PLoS One 2017 10;12(7):e0180694. Epub 2017 Jul 10.

Institute of Molecular Medicine, College of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0180694PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5503259PMC
September 2017

αB-crystallin is a sensor for assembly intermediates and for the subunit topology of desmin intermediate filaments.

Cell Stress Chaperones 2017 07 3;22(4):613-626. Epub 2017 May 3.

Department of Biosciences and the Biophysical Sciences Institute, University of Durham, Durham, UK.

Mutations in the small heat shock protein chaperone CRYAB (αB-crystallin/HSPB5) and the intermediate filament protein desmin, phenocopy each other causing cardiomyopathies. Whilst the binding sites for desmin on CRYAB have been determined, desmin epitopes responsible for CRYAB binding and also the parameters that determine CRYAB binding to desmin filaments are unknown. Using a combination of co-sedimentation centrifugation, viscometric assays and electron microscopy of negatively stained filaments to analyse the in vitro assembly of desmin filaments, we show that the binding of CRYAB to desmin is subject to its assembly status, to the subunit organization within filaments formed and to the integrity of the C-terminal tail domain of desmin. Our in vitro studies using a rapid assembly protocol, C-terminally truncated desmin and two disease-causing mutants (I451M and R454W) suggest that CRYAB is a sensor for the surface topology of the desmin filament. Our data also suggest that CRYAB performs an assembly chaperone role because the assembling filaments have different CRYAB-binding properties during the maturation process. We suggest that the capability of CRYAB to distinguish between filaments with different surface topologies due either to mutation (R454W) or assembly protocol is important to understanding the pathomechanism(s) of desmin-CRYAB myopathies.
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http://dx.doi.org/10.1007/s12192-017-0788-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465037PMC
July 2017

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

Mol Biol Cell 2016 12 26;27(25):3980-3990. Epub 2016 Oct 26.

Institute of Molecular Medicine, College of Life Sciences, National Tsing Hua University, Hsinchu 300, Taiwan

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.
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http://dx.doi.org/10.1091/mbc.E16-06-0362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5156539PMC
December 2016

Purification of Protein Chaperones and Their Functional Assays with Intermediate Filaments.

Methods Enzymol 2016 9;569:155-75. Epub 2015 Sep 9.

Biophysical Sciences Institute, University of Durham, Durham, United Kingdom.

Intermediate filament (IF) scaffolds facilitate small heat shock protein (sHSP) function, while IF function is sHSP dependent. sHSPs interact with IFs and the importance of this interaction is to maintain the individuality of the IFs and to modulate interfilament interactions both in networks and in assembly intermediates. Mutations in both sHSPs and their interacting IF proteins phenocopy each other in the human diseases they cause. This establishes a key functional relationship between these two very distinct protein families, and it also evidences the role of this cytoskeleton-chaperone complex in the cellular stress response. In this chapter, we describe the detailed experimental protocols for the preparation of purified IF proteins and sHSPs to facilitate the study in vitro of their functional interactions. In addition, we describe the detailed biochemical procedures to assess the effect of sHSP on the assembly of IFs, the binding to IFs, and the prevention of noncovalent filament-filament interactions using in vitro cosedimentation, electron microscopy, and viscosity assays. These assays are valuable research tools to study and manipulate sHSP-IF complexes in vitro and therefore to determine the structure-function detail of this complex, and how it contributes to cellular, tissue, and organismal homeostasis and the in vivo stress response.
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http://dx.doi.org/10.1016/bs.mie.2015.07.025DOI Listing
October 2016

Granulocyte colony-stimulating factor reduces fibrosis in a mouse model of chronic pancreatitis.

PLoS One 2014 31;9(12):e116229. Epub 2014 Dec 31.

Department of Gastroenterology, Linkou Chang Gung Memorial Hospital, Taoyuan, Taiwan; College of Medicine, Chang Gung University, Taoyuan, Taiwan.

Background: Chronic pancreatitis (CP) is a necroinflammatory process resulting in extensive pancreatic fibrosis. Granulocyte colony-stimulating factor (G-CSF), a hematopoietic stem cell mobilizer, has been shown to exert an anti-fibrotic effect partly through the enrichment of bone marrow (BM) cells in fibrotic organ. We aimed to test the effect of G-CSF on fibrosis in a mouse model of CP.

Methods: CP was induced in C57Bl/6J mice by consecutive cerulein injection (50 µg/kg/day, 2 days a week) for 6 weeks. Mice were then treated with G-CSF (200 µg/kg/day, 5 day a week) or normal saline for 1 week, and sacrificed at week 7 or week 9 after first cerulein injection. Pancreatic histology, pancreatic matrix metallopeptidase 9 (MMP-9), MMP-13 and collagen expression were examined. Pancreatic myofibroblasts were isolated and cultured with G-CSF. Collagen, MMP-9 and MMP-13 expression by myofibroblasts was examined. The BM-mismatched mice model was used to examine the change of BM-derived myofibroblasts and non-myofibroblastic BM cells by G-CSF in the pancreas.

Results: The pancreatic collagen expression were significantly decreased in the G-CSF-treated group sacrificed at week 9. While collagen produced from myofibroblasts was not affected by G-CSF, the increase of MMP13 expression was observed in vitro. There were no effect of G-CSF in the number of myofibroblasts and BM-derived myofibroblasts. However, the number of non-myofibroblastic BM cells and macrophages were significantly increased in the pancreata of cerulein- and G-CSF-treated mice, suggesting a potential anti-fibrotic role of non-myofibroblastic BM cells and macrophages stimulated by G-CSF.

Conclusions: Our data indicated that G-CSF contributed to the regression of pancreatic fibrosis. The anti-fibrotic effects were possibly through the stimulation of MMP-13 from myofibroblasts, and the enhanced accumulation of non-myofibroblastic BM cells and macrophages in the pancreas.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0116229PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4281240PMC
May 2016

Identification of a novel nonsense mutation in the rod domain of GFAP that is associated with Alexander disease.

Eur J Hum Genet 2015 Jan 23;23(1):72-8. Epub 2014 Apr 23.

Department of Neurology, Chonnam National University Medical School, Gwangju, Republic of Korea.

Alexander disease (AxD) is an astrogliopathy that primarily affects the white matter of the central nervous system (CNS). AxD is caused by mutations in a gene encoding GFAP (glial fibrillary acidic protein). The GFAP mutations in AxD have been reported to act in a gain-of-function manner partly because the identified mutations generate practically full-length GFAP. We found a novel nonsense mutation (c.1000 G>T, p.(Glu312Ter); also termed p.(E312*)) within a rod domain of GFAP in a 67-year-old Korean man with a history of memory impairment and leukoencephalopathy. This mutation, GFAP p.(E312*), removes part of the 2B rod domain and the whole tail domain from the GFAP. We characterized GFAP p.(E312*) using western blotting, in vitro assembly and sedimentation assay, and transient transfection of human adrenal cortex carcinoma SW13 (Vim(+)) cells with plasmids encoding GFAP p.(E312*). The GFAP p.(E312*) protein, either alone or in combination with wild-type GFAP, elicited self-aggregation. In addition, the assembled GFAP p.(E312*) aggregated into paracrystal-like structures, and GFAP p.(E312*) elicited more GFAP aggregation than wild-type GFAP in the human adrenal cortex carcinoma SW13 (Vim(+)) cells. Our findings are the first report, to the best of our knowledge, on this novel nonsense mutation of GFAP that is associated with AxD and paracrystal formation.
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http://dx.doi.org/10.1038/ejhg.2014.68DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4266748PMC
January 2015

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

ASN Neuro 2013 Nov 19;5(5):e00125. Epub 2013 Nov 19.

*Institute of Molecular Medicine, College of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan.

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.
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http://dx.doi.org/10.1042/AN20130032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3833455PMC
November 2013

The specificity of the interaction between αB-crystallin and desmin filaments and its impact on filament aggregation and cell viability.

Philos Trans R Soc Lond B Biol Sci 2013 May 25;368(1617):20120375. Epub 2013 Mar 25.

School of Biological and Biomedical Sciences, The University of Durham, South Road, Durham DH1 3LE, UK.

CRYAB (αB-crystallin) is expressed in many tissues and yet the R120G mutation in CRYAB causes tissue-specific pathologies, namely cardiomyopathy and cataract. Here, we present evidence to demonstrate that there is a specific functional interaction of CRYAB with desmin intermediate filaments that predisposes myocytes to disease caused by the R120G mutation. We use a variety of biochemical and biophysical techniques to show that plant, animal and ascidian small heat-shock proteins (sHSPs) can interact with intermediate filaments. Nevertheless, the mutation R120G in CRYAB does specifically change that interaction when compared with equivalent substitutions in HSP27 (R140G) and into the Caenorhabditis elegans HSP16.2 (R95G). By transient transfection, we show that R120G CRYAB specifically promotes intermediate filament aggregation in MCF7 cells. The transient transfection of R120G CRYAB alone has no significant effect upon cell viability, although bundling of the endogenous intermediate filament network occurs and the mitochondria are concentrated into the perinuclear region. The combination of R120G CRYAB co-transfected with wild-type desmin, however, causes a significant reduction in cell viability. Therefore, we suggest that while there is an innate ability of sHSPs to interact with and to bind to intermediate filaments, it is the specific combination of desmin and CRYAB that compromises cell viability and this is potentially the key to the muscle pathology caused by the R120G CRYAB.
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http://dx.doi.org/10.1098/rstb.2012.0375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3638400PMC
May 2013

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.

Exp Cell Res 2011 Oct 2;317(16):2252-66. Epub 2011 Jul 2.

Institute of Molecular Medicine, College of Life Sciences, National Tsing Hua University, Hsinchu 300, Taiwan.

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.
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http://dx.doi.org/10.1016/j.yexcr.2011.06.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4308095PMC
October 2011

Stochastically determined directed movement explains the dominant small-scale mitochondrial movements within non-neuronal tissue culture cells.

FEBS Lett 2009 Apr 3;583(8):1267-73. Epub 2009 Mar 3.

Biophysical Sciences Institute, Department of Physics, South Road, Durham University, Durham DH1 3LE, UK.

The apparently stationary phase of mitochondrial motion was investigated in epithelial cells by spinning disk confocal light microscopy combined with image correlation based single particle tracking using custom software producing sub-pixel accuracy measurements (approximately 5 nm) at 10-12 Hz frame-rates. The analysis of these data suggests that the previously described stationary, or anchored phase, in mitochondrial movement actually comprise Brownian diffusion, interspersed with frequent and brief motor-driven events whose duration are stochastically determined. We have therefore discovered a new aspect of mitochondrial behavior, which we call stochastically determined, directed movement.
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http://dx.doi.org/10.1016/j.febslet.2009.02.041DOI Listing
April 2009

Glial fibrillary acidic protein filaments can tolerate the incorporation of assembly-compromised GFAP-delta, but with consequences for filament organization and alphaB-crystallin association.

Mol Biol Cell 2008 Oct 6;19(10):4521-33. Epub 2008 Aug 6.

School of Biological and Biomedical Sciences, The University of Durham, Durham DH1 3LE, United Kingdom.

The glial fibrillary acidic protein (GFAP) gene is alternatively spliced to give GFAP-alpha, the most abundant isoform, and seven other differentially expressed transcripts including GFAP-delta. GFAP-delta has an altered C-terminal domain that renders it incapable of self-assembly in vitro. When titrated with GFAP-alpha, assembly was restored providing GFAP-delta levels were kept low (approximately 10%). In a range of immortalized and transformed astrocyte derived cell lines and human spinal cord, we show that GFAP-delta is naturally part of the endogenous intermediate filaments, although levels were low (approximately 10%). This suggests that GFAP filaments can naturally accommodate a small proportion of assembly-compromised partners. Indeed, two other assembly-compromised GFAP constructs, namely enhanced green fluorescent protein (eGFP)-tagged GFAP and the Alexander disease-causing GFAP mutant, R416W GFAP both showed similar in vitro assembly characteristics to GFAP-delta and could also be incorporated into endogenous filament networks in transfected cells, providing expression levels were kept low. Another common feature was the increased association of alphaB-crystallin with the intermediate filament fraction of transfected cells. These studies suggest that the major physiological role of the assembly-compromised GFAP-delta splice variant is as a modulator of the GFAP filament surface, effecting changes in both protein- and filament-filament associations as well as Jnk phosphorylation.
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http://dx.doi.org/10.1091/mbc.e08-03-0284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2555932PMC
October 2008

Insights into the beaded filament of the eye lens.

Exp Cell Res 2007 Jun 6;313(10):2180-8. Epub 2007 Apr 6.

School of Biological and Biomedical Sciences, The University of Durham, DH1 3LE, UK.

Filensin (BFSP1) and CP49 (BFSP2) represent two members of the IF protein superfamily that are thus far exclusively expressed in the eye lens. Mutations in both proteins cause lens cataract and careful consideration of the detail of these cataract phenotypes alerts us to several interesting features concerning the function of filensin (BFSP1) and CP49 (BFSP2) in the lens. With the first filensin (BFSP1) mutation now having been reported to cause a recessive cataract phenotype, there is the suggestion that the mutation could predispose heterozygote carriers to the early onset of age-related nuclear cataract. In the case of CP49 (BFSP2), there are now three unrelated families who have been identified with a common E233 Delta mutation. Very interestingly this is linked to myopia in one family. Despite the apparent phenotypic differences of the filensin (BFSP1) and CP49 (BFSP2) mutations, the data are still consistent with the beaded filament proteins being essential for lens function and specifically contributing to the optical properties of the lens. The fact that none of the mutations thus far reported affect either the conserved LNDR or TYRKLLEGE motifs that flank the central rod domain supports the view that this pair of IF proteins have unusual structural features and a distinctive assembly mechanism. The multiple sequence divergences suggest these proteins have been adapted to the specific functional requirements of lens fibre cells, a function that can be traced from squid to man.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5073188PMC
http://dx.doi.org/10.1016/j.yexcr.2007.04.005DOI Listing
June 2007

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.

Am J Hum Genet 2006 Aug 12;79(2):197-213. Epub 2006 Jun 12.

School of Biological and Biomedical Sciences, The University of Durham, Durham, United Kingdom.

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.
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http://dx.doi.org/10.1086/504411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1559481PMC
August 2006

Alexander-disease mutation of GFAP causes filament disorganization and decreased solubility of GFAP.

J Cell Sci 2005 May 19;118(Pt 9):2057-65. Epub 2005 Apr 19.

Department of Pathology and the Center for Neurobiology and Behavior, Columbia University, New York, NY 10032, USA.

Alexander disease is a fatal neurological illness characterized by white-matter degeneration and the formation of astrocytic cytoplasmic inclusions called Rosenthal fibers, which contain the intermediate filament glial fibrillary acidic protein (GFAP), the small heat-shock proteins HSP27 and alphaB-crystallin, and ubiquitin. Many Alexander-disease patients are heterozygous for one of a set of point mutations in the GFAP gene, all of which result in amino acid substitutions. The biological effects of the most common alteration, R239C, were tested by expressing the mutated protein in cultured cells by transient transfection. In primary rat astrocytes and Cos-7 cells, the mutant GFAP was incorporated into filament networks along with the endogenous GFAP and vimentin, respectively. In SW13Vim(-) cells, which have no endogenous cytoplasmic intermediate filaments, wild-type human GFAP frequently formed filamentous bundles, whereas the R239C GFAP formed 'diffuse' and irregular patterns. Filamentous bundles of R239C GFAP were sometimes formed in SW13Vim(-) cells when wild-type GFAP was co-transfected. Although the presence of a suitable coassembly partner (vimentin or GFAP) reduced the potential negative effects of the R239C mutation on GFAP network formation, the mutation affected the stability of GFAP in cells in a dominant fashion. Extraction of transfected SW13Vim(-) cells with Triton-X-100-containing buffers showed that the mutant GFAP was more resistant to solubilization at elevated KCl concentrations. Both wild-type and R239C GFAP assembled into 10 nm filaments with similar morphology in vitro. Thus, although the R239C mutation does not appear to affect filament formation per se, the mutation alters the normal solubility and organization of GFAP networks.
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http://dx.doi.org/10.1242/jcs.02339DOI Listing
May 2005

Seeing is believing! The optical properties of the eye lens are dependent upon a functional intermediate filament cytoskeleton.

Exp Cell Res 2005 Apr 26;305(1):1-9. Epub 2005 Jan 26.

School of Biological and Biomedical Sciences, The University, South Road Science Site, Durham DH1 3LE, UK.

Beaded filaments are the major cytoskeletal element of the eye lens and they are essential to the optical properties of the eye lens. They were discovered in 1972 by Harry Maisel and Margaret Perry and have since been found to comprise two novel intermediate filament proteins, CP49 and filensin. These proteins possess unique structure features and unusual assembly characteristics, which distinguish them from canonical IF proteins. Whilst CP49 is completely tailless, filensin has a rather short rod domain and extremely large C-terminal tail domain. In vitro, CP49 and filensin do not form IFs on their own. In vitro studies suggest that CP49 and filensin have a distinct coassembly mechanism. Whilst CP49 self-assembles into thick bundles of filaments, filensin only forms short fibrils, but when combined together they form filaments. The generation of gene knockouts by the targeted deletion of Bfsp1 and Bfsp2 that encode filensin and CP49, respectively, have been made to explore the function of beaded filaments in the lens. Our results suggest that the lens-specific beaded filaments are the key cytoskeletal element in organising and maintaining lens fibre cell architecture and are a key factor in determining the optical properties of the lens. We have also found that some common mouse strains contain a natural mutation in Bfsp2 that will effectively generate a CP49 knockout. This finding has important implications for lens research involving other gene knockouts maintained on a 129 background. It has also been observed that mutations in Bfsp2 are the genetic basis of inherited human cataract. Collectively, these data demonstrate that beaded filaments are fundamental to lens function.
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http://dx.doi.org/10.1016/j.yexcr.2004.11.021DOI Listing
April 2005

R120G alphaB-crystallin promotes the unfolding of reduced alpha-lactalbumin and is inherently unstable.

FEBS J 2005 Feb;272(3):711-24

Department of Chemistry, University of Wollongong, NSW, Australia.

alpha-Crystallin is the principal lens protein which, in addition to its structural role, also acts as a molecular chaperone, to prevent aggregation and precipitation of other lens proteins. One of its two subunits, alphaB-crystallin, is also expressed in many nonlenticular tissues, and a natural missense mutation, R120G, has been associated with cataract and desmin-related myopathy, a disorder of skeletal muscles [Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D, Chapon F, Tome F, Dupret JM, Paulin D & Fardeau M (1998) Nat Genet20, 92-95]. In the present study, real-time 1H-NMR spectroscopy showed that the ability of R120G alphaB-crystallin to stabilize the partially folded, molten globule state of alpha-lactalbumin was significantly reduced in comparison with wild-type alphaB-crystallin. The mutant showed enhanced interaction with, and promoted unfolding of, reduced alpha-lactalbumin, but showed limited chaperone activity for other target proteins. Using NMR spectroscopy, gel electrophoresis, and MS, we observed that, unlike the wild-type protein, R120G alphaB-crystallin is intrinsically unstable in solution, with unfolding of the protein over time leading to aggregation and progressive truncation from the C-terminus. Light scattering, MS, and size-exclusion chromatography data indicated that R120G alphaB-crystallin exists as a larger oligomer than wild-type alphaB-crystallin, and its size increases with time. It is likely that removal of the positive charge from R120 of alphaB-crystallin causes partial unfolding, increased exposure of hydrophobic regions, and enhances its susceptibility to proteolysis, thus reducing its solubility and promoting its aggregation and complexation with other proteins. These characteristics may explain the involvement of R120G alphaB-crystallin with human disease states.
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http://dx.doi.org/10.1111/j.1742-4658.2004.04507.xDOI Listing
February 2005

The intermediate filament systems in the eye lens.

Methods Cell Biol 2004 ;78:597-624

School of Biological and Biomedical Sciences, The University of Durham, Durham DH1 3LE, UK.

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http://dx.doi.org/10.1016/s0091-679x(04)78021-8DOI Listing
March 2005

Comparison of the small heat shock proteins alphaB-crystallin, MKBP, HSP25, HSP20, and cvHSP in heart and skeletal muscle.

Histochem Cell Biol 2004 Nov 12;122(5):415-25. Epub 2004 Oct 12.

Institute of Anatomy and Cell Biology, University of Würzburg, Koellikerstrasse 6, 97070, Würzburg, Germany.

Seven members of the small heat shock protein (sHSP) family are exceptional with respect to their constitutive high abundance in muscle tissue. It has been suggested that sHSPs displaying chaperone-like properties may stabilize myofibrillar proteins during stress conditions and prevent them from loss of function. In the present study five sHSPs (alphaB-crystallin, MKBP, HSP25, HSP20, and cvHSP) were investigated with respect to similarities and differences of their expression in heart and skeletal muscle under normal and ischemic conditions. In ischemic heart and skeletal muscle these five sHSPs translocated from cytosol to the Z-/I-area of myofibrils. Myofibrillar binding of all sHSPs was very tight and resisted for the most part extraction with 1 M NaSCN or 1 M urea. MKBP and HSP20 became extracted by 1 M NaSCN to a significant extent indicating that these two sHSPs may bind partially to actin-associated proteins which were completely extracted by this treatment. Ultrastructural localization of alphaB-crystallin showed diffuse distribution of immunogold label throughout the entire I-band in skeletal muscle fibers whereas in cardiomyocytes alphaB-crystallin was preferentially located at the N-line position of the I-band. These observations indicate different myofibrillar binding sites of alphaB-crystallin in cardiomyocytes versus skeletal muscle fibers. Further differences of the properties of sHSPs could be observed regarding fiber type distribution of sHSPs. Thus sHSPs form a complex stress-response system in striated muscle tissue with some common as well as some distinct functions in different muscle types.
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http://dx.doi.org/10.1007/s00418-004-0711-zDOI Listing
November 2004

Neuronal diseases: small heat shock proteins calm your nerves.

Curr Biol 2004 Aug;14(15):R625-6

School of Biological and Biomedical Science, South Road Science Site, The University, Durham DH1 3LE, UK.

Mutations in HSPB1 and HSPB8, members of the small heat shock protein family, have recently been shown to cause some distal motor neuropathies. Their function in motor neurones is now under scrutiny.
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http://dx.doi.org/10.1016/j.cub.2004.07.047DOI Listing
August 2004

Desmin aggregate formation by R120G alphaB-crystallin is caused by altered filament interactions and is dependent upon network status in cells.

Mol Biol Cell 2004 May 5;15(5):2335-46. Epub 2004 Mar 5.

School of Biological and Biomedical Sciences, The University of Durham, Durham DH1 3LE, United Kingdom.

The R120G mutation in alphaB-crystallin causes desmin-related myopathy. There have been a number of mechanisms proposed to explain the disease process, from altered protein processing to loss of chaperone function. Here, we show that the mutation alters the in vitro binding characteristics of alphaB-crystallin for desmin filaments. The apparent dissociation constant of R120G alphaB-crystallin was decreased while the binding capacity was increased significantly and as a result, desmin filaments aggregated. These data suggest that the characteristic desmin aggregates seen as part of the disease histopathology can be caused by a direct, but altered interaction of R120G alphaB-crystallin with desmin filaments. Transfection studies show that desmin networks in different cell backgrounds are not equally affected. Desmin networks are most vulnerable when they are being made de novo and not when they are already established. Our data also clearly demonstrate the beneficial role of wild-type alphaB-crystallin in the formation of desmin filament networks. Collectively, our data suggest that R120G alphaB-crystallin directly promotes desmin filament aggregation, although this gain of a function can be repressed by some cell situations. Such circumstances in muscle could explain the late onset characteristic of the myopathies caused by mutations in alphaB-crystallin.
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http://dx.doi.org/10.1091/mbc.e03-12-0893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC404027PMC
May 2004