簡易檢索 / 詳目顯示

研究生: 簡腕萱
Jian, Wan-Syuan
論文名稱: 膠質纖維酸性蛋白質在亞歷山大氏症中的病理性修飾
Pathological modification of glial fibrillary acidic protein in Alexander disease
指導教授: 彭明德
Perng, Ming-Der
口試委員: 張壯榮
Chang, Chuang-Rung
吳宗遠
Wu, Tzong-Yuan
黃建銘
Huang, Jian-Ming
學位類別: 碩士
Master
系所名稱: 生命科學暨醫學院 - 分子醫學研究所
Institute of Molecular Medicine
論文出版年: 2024
畢業學年度: 112
語文別: 英文
論文頁數: 107
中文關鍵詞: 亞歷山大氏症星狀細胞中間型蛋白絲羅森塔爾纖維泛素
外文關鍵詞: Alexander disease, Astrocyte, Glial fibrillary acidic protein, Rosenthal fiber, Ubiquitin
相關次數: 點閱:51下載:0
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報
  • 亞歷山大氏症 (Alexander disease, AxD) 是一種罕見的中樞神經系統 (Central nervous system, CNS) 退化性疾病,已知主要致病原因為表現在星狀細胞 (astrocyte) 內的中間型蛋白絲 (intermediate filament, IF) ,也就是膠質纖維酸性蛋白質 (glial fibrillary acidic protein, GFAP) 發生基因突變,影響細胞骨架支持星狀細胞基本型態的功能,進而失去維持中樞神經系統恆定的能力。先前的研究發現,當GFAP在細胞中大量表現時,會導致在星狀細胞中產生大量且不正常的蛋白質聚集,稱為羅森塔爾纖維 (Rosenthal fiber, RF) ,是亞歷山大氏症主要的病理特徵,由GFAP、泛素 (ubiquitin) 、αB-crystallin以及其他蛋白質組成並堆積於組織中。本研究利用體外聚合實驗和以及慢病毒轉導實驗,探討與AxD相關的突變如何影響GFAP在體外的聚合和星狀細胞中間型蛋白絲網絡的形成,以及轉譯後修飾,例如泛素-蛋白酶體系統,是如何促進蛋白質病理性聚集的相關致病機轉。透過生化數據表明,從病患檢體分離出的羅森塔爾纖維中,可以偵測到泛素化的GFAP、透過雙硫鍵結合的高分子量GFAP,以及經由蛋白酶降解的蛋白質片段。根據此研究,我們提出了一個潛在的病理模型,以突變後的GFAP為引起疾病的首要條件,由於突變改變了GFAP聚合的特性,誘導GFAP被泛素修飾,進而導致蛋白質異常聚集。另一個可能的致病途徑為,突變後的異常GFAP聚集,會抑制蛋白酶體的功能,導致進一步被泛素化,最終引發疾病。
    本篇碩士論文中,已有部分發表於我最近刊登於期刊之文章(Lin et al., 2024),並對內容進行修改和更新,而該文章部分結果來自於就讀國立清華大學(NTHU)碩士在學期間,並且由國家科學及技術委員會(NSTC)提供計畫資助。


    Alexander disease (AxD) is a rare degenerative disorder of the central nervous system (CNS), which mainly caused by mutations in the gene encoding for glial fibrillary acidic protein (GFAP), an intermediate filament (IF) protein expressed in astrocytes. These mutations disrupt the cytoskeletal system of astrocytes, affecting their ability to maintain CNS homeostasis. Previous studies have shown that overexpression of GFAP leads to the formation of abnormal protein aggregates in astrocytes, known as Rosenthal fibers (RFs), which are a hallmark of AxD. RF is primarily composed of GFAP, ubiquitin, αB-crystallin and other proteins. This study uses in vitro assembly assay and lentivirus transduction experiment to investigate how AxD-associated mutations affect the assembly of GFAP in vitro and the formation of IF networks in astrocytes, as well as the role of ubiquitination of GFAP in promoting pathological protein aggregation. Biochemical data indicate that RFs isolated from AxD patient brains contain ubiquitinated GFAP, high molecular weight (HMW) GFAP modified by crosslinking through disulfide bonds, and proteolytic fragments. Based on this study, we propose a potential pathogenic model in which mutated GFAP is a primary trigger of the disease. The mutations alter assembly properties of GFAP, leading to the induction of GFAP ubiquitination, which causes further aggregation. Another circuit of abnormal GFAP aggregation inhibits the function of the proteasome, causing additional ubiquitination and ultimately contributing to disease progression.
    Some parts of this thesis are modified and updated from my recent publication (Lin et al., 2024), which was supported by grants from National Science and Technology Council (NSTC) and is in partial fulfillment for the requirement of the Master degree in NTHU.

    Abstract-----------Ι 摘要---------------Ⅱ 致謝---------------Ⅲ Abbreviation-------Ⅳ Chapter 1 Introduction---------1 1.1 Intermediate filaments and the function of GFAP in Astrocyte-1 1.2 Genetics of Ax-------------4 1.3 Mechanisms of disease------5 1.4 Gain or loss of function---7 Chapter 2 Material and Methods-9 2.1 Plasmid construction and site direct mutagenesis---------------9 2.2 Bacterial expression and purification of recombinant GFAPs-----9 2.3 In vitro assembly assay and sedimentation assay----------------11 2.4 Negative staining and transmission electron microscopy---------12 2.5 Rodent models and genotyping-----------------------------------12 2.6 Primary astrocyte culture--------------------------------------13 2.7 Cell line culture----------------------------------------------14 2.8 Lentiviral production and transduction-------------------------14 2.9 Subcellular fractionation--------------------------------------15 2.10 Immunoblotting------------------------------------------------16 2.11 Immunofluorescence microscopy---------------------------------17 2.12 Preparation of Rosenthal fiber fraction from brain tissue-----18 Chapter 3 Results--------------------------------------------------19 3.1 Scope and outline of this study--------------------------------19 Figure 1. Outline of experiments in this study---------------------20 3.2 Expression and purification of recombinant human GFAP----------21 Figure 2. A schematic view summarized the structural organization of GFAP and the localization of mutations causing AxD used in this study--22 Figure 3. Inclusion body preparation of GFAP------------------------------23 Figure 4. Purification of recombinant human GFAP by column chromatography-24 3.3 In vitro assembly studies---------------------------------------------25 Figure 5. A Schematic diagram illustrates the in vitro assembly process and subsequent analysis----------------27 Figure 6. Effects of GFAP mutation upon the in vitro filament assembly-----------------------------------------28 3.4 Effects of GFAP mutants on IF network formation in Primary astrocytes--------------------------------------30 Figure 7. A flowchart for primary cell preparation and lentiviral transduction---------------------------------31 Figure 8. Characterization of primary astrocytes derived from GFAP KO rats-------------------------------------32 Figure 9. GFAP mutants formed aggregates when transduced into GFAP KO astrocytes-------------------------------36 Figure 10. Effect of GFAP mutations on IF network formation in GFAP KO astrocytes------------------------------37 Figure 11. Solubility properties of GFAP mutants in GFAP KO astrocytes-----------------------------------------39 Figure 12. Ubiquitin was colocalized with aggregates of mutant GFAP--------------------------------------------41 3.5 Mutant GFAPs alter anti-GFAP antibody binding sites on GFAP------------------------------------------------43 Figure 13. Epitope mapping of anti-GFAP antibody---------------------------------------------------------------44 3.6 Effects of mutant GFAP on the endogenous GFAP networks in primary WT astrocyte-----------------------------46 Figure 14. Effect of GFAP mutation of the endogenous GFAP on the aggregation process in primary rat astrocytes-48 Figure 15. Effect of GFAP mutation on the aggregation process in GFAP heterozygous KO astrocytes---------------50 Figure 16. Solubility properties of GFAP mutation in GFAP WT astrocytes----------------------------------------52 3.7 Ubiquitination of GFAP in SW13 (Vim-) cells----------------------------------------------------------------54 Figure 17. Solubility properties and ubiquitination of mutant GFAP in SW13(Vim-)-------------------------------56 Figure 18. Aggregates formed by mutant GFAP was co-stained with GFAP and ubiquitin-----------------------------57 3.8 GFAP is ubiquitinated in mouse AxD model and human AxD patients--------------------------------------------59 Figure 19. GFAP was modified by ubiquitination in the enriched RF fraction in Tg mice brain--------------------61 Figure 20. GFAP was modified by ubiquitination in the enriched RF fraction in human patient of AxD-------------63 Figure 21. Oxidative modification of GFAP in human AxD brains--------------------------------------------------65 3.9 Sequences required for GFAP assembly in vitro--------------------------------------------------------------66 Figure 22. A schematic view of GFAP structure and localization the truncated GFAP------------------------------68 Figure 23. N-terminal truncated form of GFAP disrupted the filament assembly in vitro--------------------------69 Figure 24. Analysis of the solubility properties of N- or C-terminal deleted GFAP------------------------------70 Figure 25. C-terminal deletion affected GFAP assembly in vitro-------------------------------------------------73 Figure 26. Structures of C-terminal deletion variants before assembly in low ionic strength buffer-------------76 Chapter 4 Discussion-------------------------------------------------------------------------------------------77 4.1 Purification of recombinant human GFAP---------------------------------------------------------------------77 4.2 Ubiquitination of GFAP in AxD------------------------------------------------------------------------------78 4.3 A pathogenic cycle initiated by GFAP mutations-------------------------------------------------------------82 Figure 27. Pathogenic cycle of disease progression in AxD------------------------------------------------------84 4.4 GFAP segments required for filament assembly---------------------------------------------------------------85 4.5 Concluding Remarks-----------------------------------------------------------------------------------------88 Tables---------------------------------------------------------------------------------------------------------91 Table 1. Major types of intermediate filaments-----------------------------------------------------------------91 Table 2. GFAP isoforms and their characteristic----------------------------------------------------------------91 Table 3. Classification of AxD and its clinical features-------------------------------------------------------92 Table 4. List of Rosenthal fiber components--------------------------------------------------------------------92 Table 5. Different types of genetic mutation in AxD and their location-----------------------------------------93 Table 6. Primers used in this study----------------------------------------------------------------------------94 Table 7. Primary antibodies used in this study-----------------------------------------------------------------94 Table 8. Details of human post-mortem tissue samples analyzed by immnoblotting---------------------------------95 Table 9. List of GFAP mutation used for in this study----------------------------------------------------------95 Table 10. GFAP purification of column chromatography-----------------------------------------------------------96 References-----------------------------------------------------------------------------------------------------97

    Albers, K., and E. Fuchs. (1987). The expression of mutant epidermal keratin cDNAs transfected in simple epithelial and squamous cell carcinoma lines. J Cell Biol. 105:791-806. doi:10.1083/jcb.105.2.791
    Allaman, I., Bélanger, M., & Magistretti, P. J. (2011). Astrocyte-neuron metabolic relationships: for better and for worse. Trends Neurosci, 34(2), 76-87. doi:10.1016/j.tins.2010.12.001
    Balcarek, J. M., & Cowan, N. J. (1985). Structure of the mouse glial fibrillary acidic protein gene: implications for the evolution of the intermediate filament multigene family. Nucleic Acids Research, 13(15), 5527-5543. doi:10.1093/nar/13.15.5527
    Bar, H., S. Sharma, H. Kleiner, N. Mucke, H. Zentgraf, H.A. Katus, U. Aebi, and H. Herrmann. (2009). Interference of amino-terminal desmin fragments with desmin filament formation. Cell Motil Cytoskeleton. 66:986-999. doi: 10.1002/cm.20396
    Battaglia, R. A., Beltran, A. S., Delic, S., Dumitru, R., Robinson, J. A., Kabiraj, P., . . . Snider, N. T. (2019). Site-specific phosphorylation and caspase cleavage of GFAP are new markers of Alexander disease severity. Elife, 8. doi:10.7554/eLife.47789
    Bongcam-Rudloff, E., Nistér, M., Betsholtz, C., Wang, J. L., Stenman, G., Huebner, K., . . . Westermark, B. (1991). Human glial fibrillary acidic protein: complementary DNA cloning, chromosome localization, and messenger RNA expression in human glioma cell lines of various phenotypes. Cancer Res, 51(5), 1553-1560.
    Brenner, M., Goldman, J., Quinlan, R., & Messing, A. (2009). Astrocytes in (patho) physiology of the nervous system. Alexander disease: a genetic disorder of astrocytes, 591-648. doi: 10.1007/978-0-387-79492-1_24
    Brenner, M., Johnson, A. B., Boespflug-Tanguy, O., Rodriguez, D., Goldman, J. E., & Messing, A. (2001). Mutations in GFAP, encoding glial fibrillary acidic protein, are associated with Alexander disease. Nat Genet, 27(1), 117-120. doi:10.1038/83679
    Chen, M. H., Hagemann, T. L., Quinlan, R. A., Messing, A., & Perng, M. D. (2013). Caspase cleavage of GFAP produces an assembly-compromised proteolytic fragment that promotes filament aggregation. ASN Neuro, 5(5), e00125. doi:10.1042/an20130032
    Chen, W.J., and R.K. Liem. (1994). The endless story of the glial fibrillary acidic protein. J Cell Sci. 107(Pt 8):2299-2311. doi: 10.1242/jcs.107.8.2299
    Chen, Y. S., Lim, S. C., Chen, M. H., Quinlan, R. A., & Perng, M. D. (2011). 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, 317(16), 2252-2266. doi:10.1016/j.yexcr.2011.06.017
    Ching, G. Y., & Liem, R. K. (1999). Analysis of the roles of the head domains of type IV rat neuronal intermediate filament proteins in filament assembly using domain-swapped chimeric proteins. J Cell Sci, 112(Pt 13), 2233-2240. doi:10.1242/jcs.112.13.2233
    Cores, Á., Piquero, M., Villacampa, M., León, R., & Menéndez, J. C. (2020). NRF2 Regulation Processes as a Source of Potential Drug Targets against Neurodegenerative Diseases. Biomolecules, 10(6). doi:10.3390/biom10060904
    Coulombe, P.A., Y.M. Chan, K. Albers, and E. Fuchs. (1990). Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro. J Cell Biol. 111:3049-3064. doi: 10.1083/jcb.111.6.3049
    Cui, C., Stambrook, P. J., & Parysek, L. M. (1995). Peripherin assembles into homopolymers in SW13 cells. J Cell Sci, 108(Pt 10), 3279-3284. doi:10.1242/jcs.108.10.3279
    de Reus, A., Basak, O., Dykstra, W., van Asperen, J. V., van Bodegraven, E. J., & Hol, E. M. (2024). GFAP-isoforms in the nervous system: Understanding the need for diversity. Curr Opin Cell Biol, 87, 102340. doi:10.1016/j.ceb.2024.102340
    Djabali, K., Portier, M. M., Gros, F., Blobel, G., & Georgatos, S. D. (1991). Network antibodies identify nuclear lamin B as a physiological attachment site for peripherin intermediate filaments. Cell, 64(1), 109-121. doi:10.1016/0092-8674(91)90213-i
    Eckelt, A., H. Herrmann, and W.W. Franke. (1992). Assembly of a tail-less mutant of the intermediate filament protein, vimentin, in vitro and in vivo. Eur J Cell Biol. 58:319-330. https://europepmc.org/article/med/1425769
    Eliasson, C., Sahlgren, C., Berthold, C. H., Stakeberg, J., Celis, J. E., Betsholtz, C., . . . Pekny, M. (1999). Intermediate filament protein partnership in astrocytes. J Biol Chem, 274(34), 23996-24006. doi:10.1074/jbc.274.34.23996
    Eng, L. F., Ghirnikar, R. S., & Lee, Y. L. (2000). Glial fibrillary acidic protein: GFAP-thirty-one years (1969-2000). Neurochem Res, 25(9-10), 1439-1451. doi:10.1023/a:1007677003387
    Eriksson, J. E., Dechat, T., Grin, B., Helfand, B., Mendez, M., Pallari, H. M., & Goldman, R. D. (2009). Introducing intermediate filaments: from discovery to disease. J Clin Invest, 119(7), 1763-1771. doi:10.1172/jci38339
    Eroglu, C., & Barres, B. A. (2010). Regulation of synaptic connectivity by glia. Nature, 468(7321), 223-231. doi:10.1038/nature09612
    Fickert, P., Trauner, M., Fuchsbichler, A., Stumptner, C., Zatloukal, K., & Denk, H. (2003). Mallory body formation in primary biliary cirrhosis is associated with increased amounts and abnormal phosphorylation and ubiquitination of cytokeratins. J Hepatol, 38(4), 387-394. doi:10.1016/s0168-8278(02)00439-7
    Flint, D., Li, R., Webster, L. S., Naidu, S., Kolodny, E., Percy, A., . . . Brenner, M. (2012). Splice site, frameshift, and chimeric GFAP mutations in Alexander disease. Human mutation, 33(7), 1141-1148. doi:10.1002/humu.22094
    Fuchs, E. (1996). The cytoskeleton and disease: genetic disorders of intermediate filaments. Annu Rev Genet. 30:197-231. doi: 10.1146/annurev.genet.30.1.197
    Fuchs, E., & Cleveland, D. W. (1998). A structural scaffolding of intermediate filaments in health and disease. Science, 279(5350), 514-519. doi:10.1126/science.279.5350.514
    Fuchs, E., & Weber, K. (1994). Intermediate filaments: structure, dynamics, function, and disease. Annu Rev Biochem, 63, 345-382. doi:10.1146/annurev.bi.63.070194.002021
    Goldman, J.E., and E. Corbin. (1988). Isolation of a major protein component of Rosenthal fibers. Am J Pathol. 130:569-578. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1880665/
    Goldman, J.E., and E. Corbin. 1991. Rosenthal fibers contain ubiquitinated alpha B-crystallin. Am J Pathol. 139:933-938. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1886295/
    Gomi, H., Yokoyama, T., Fujimoto, K., Ikeda, T., Katoh, A., Itoh, T., & Itohara, S. (1995). Mice devoid of the glial fibrillary acidic protein develop normally and are susceptible to scrapie prions. Neuron, 14(1), 29-41. doi:10.1016/0896-6273(95)90238-4
    Green, L., Berry, I. R., Childs, A. M., McCullagh, H., Jose, S., Warren, D., . . . Livingston, J. H. (2018). Whole Exon Deletion in the GFAP Gene Is a Novel Molecular Mechanism Causing Alexander Disease. Neuropediatrics, 49(2), 118-122. doi:10.1055/s-0037-1608921
    Haass, C., & Selkoe, D. J. (2007). Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide. Nat Rev Mol Cell Biol, 8(2), 101-112. doi:10.1038/nrm2101
    Hagemann, T. L., Boelens, W. C., Wawrousek, E. F., & Messing, A. (2009). Suppression of GFAP toxicity by alphaB-crystallin in mouse models of Alexander disease. Hum Mol Genet, 18(7), 1190-1199. doi:10.1093/hmg/ddp013
    Hagemann, T. L., Connor, J. X., & Messing, A. (2006). Alexander disease-associated glial fibrillary acidic protein mutations in mice induce Rosenthal fiber formation and a white matter stress response. J Neurosci, 26(43), 11162-11173. doi:10.1523/jneurosci.3260-06.2006
    Hagemann, T. L., Gaeta, S. A., Smith, M. A., Johnson, D. A., Johnson, J. A., & Messing, A. (2005). Gene expression analysis in mice with elevated glial fibrillary acidic protein and Rosenthal fibers reveals a stress response followed by glial activation and neuronal dysfunction. Hum Mol Genet, 14(16), 2443-2458. doi:10.1093/hmg/ddi248
    Hagemann, T. L., Powers, B., Lin, N. H., Mohamed, A. F., Dague, K. L., Hannah, S. C., . . . Messing, A. (2021). Antisense therapy in a rat model of Alexander disease reverses GFAP pathology, white matter deficits, and motor impairment. Sci Transl Med, 13(620), eabg4711. doi:10.1126/scitranslmed.abg4711
    Hagemann, T.L. (2022). Alexander disease: models, mechanisms, and medicine. Current opinion in neurobiology. 72:140-147. https://doi.org/10.1016/j.conb.2021.10.002
    Haj-Yasein, N. N., Vindedal, G. F., Eilert-Olsen, M., Gundersen, G. A., Skare, Ø., Laake, P., . . . Nagelhus, E. A. (2011). Glial-conditional deletion of aquaporin-4 (Aqp4) reduces blood-brain water uptake and confers barrier function on perivascular astrocyte endfeet. Proc Natl Acad Sci U S A, 108(43), 17815-17820. doi:10.1073/pnas.1110655108
    Heaven, M. R., Flint, D., Randall, S. M., Sosunov, A. A., Wilson, L., Barnes, S., . . . Brenner, M. (2016). Composition of Rosenthal Fibers, the Protein Aggregate Hallmark of Alexander Disease. J Proteome Res, 15(7), 2265-2282. doi:10.1021/acs.jproteome.6b00316
    Heaven, M. R., Wilson, L., Barnes, S., & Brenner, M. (2019). Relative stabilities of wild-type and mutant glial fibrillary acidic protein in patients with Alexander disease. J Biol Chem, 294(43), 15604-15612. doi:10.1074/jbc.RA119.009777
    Heins, S., Wong, P. C., Müller, S., Goldie, K., Cleveland, D. W., & Aebi, U. (1993). The rod domain of NF-L determines neurofilament architecture, whereas the end domains specify filament assembly and network formation. J Cell Biol, 123(6 Pt 1), 1517-1533. doi:10.1083/jcb.123.6.1517
    Herrmann, H., and U. Aebi. (2004). Intermediate filaments: molecular structure, assembly mechanism, and integration into functionally distinct intracellular Scaffolds. Annu Rev Biochem. 73:749-789. https://doi.org/10.1146/annurev.biochem.73.011303.073823
    Herrmann, H., and U. Aebi. (2016). Intermediate Filaments: Structure and Assembly. Cold Spring Harbor perspectives in biology. 8.
    Herrmann, H., Bär, H., Kreplak, L., Strelkov, S. V., & Aebi, U. (2007). Intermediate filaments: from cell architecture to nanomechanics. Nat Rev Mol Cell Biol, 8(7), 562-573. doi:10.1038/nrm2197
    Herrmann, H., M. Haner, M. Brettel, S.A. Muller, K.N. Goldie, B. Fedtke, A. Lustig, W.W. Franke, and U. Aebi. (1996). Structure and assembly properties of the intermediate filament protein vimentin: the role of its head, rod and tail domains. J Mol Biol. 264:933-953. https://doi.org/10.1006/jmbi.1996.0688
    Ho, C. L., Chin, S. S., Carnevale, K., & Liem, R. K. (1995). Translation initiation and assembly of peripherin in cultured cells. Eur J Cell Biol, 68(2), 103-112. https://europepmc.org/article/med/8575457
    Hol, E. M., & Capetanaki, Y. (2017). Type III Intermediate Filaments Desmin, Glial Fibrillary Acidic Protein (GFAP), Vimentin, and Peripherin. Cold Spring Harb Perspect Biol, 9(12). doi:10.1101/cshperspect.a021642
    Hol, E. M., & Pekny, M. (2015). Glial fibrillary acidic protein (GFAP) and the astrocyte intermediate filament system in diseases of the central nervous system. Curr Opin Cell Biol, 32, 121-130. doi:10.1016/j.ceb.2015.02.004
    Inagaki, M., Y. Gonda, K. Nishizawa, S. Kitamura, C. Sato, S. Ando, K. Tanabe, K. Kikuchi, S. Tsuiki, and Y. Nishi. (1990). Phosphorylation sites linked to glial filament disassembly in vitro locate in a non-alpha-helical head domain. J Biol Chem. 265:4722-4729. https://doi.org/10.1016/S0021-9258(19)39622-X
    Inagaki, M., Y. Nakamura, M. Takeda, T. Nishimura, and N. Inagaki. (1994). Glial fibrillary acidic protein: dynamic property and regulation by phosphorylation. Brain pathology. 4:239-243. https://doi.org/10.1111/j.1750-3639.1994.tb00839.x
    Jany, P. L., Hagemann, T. L., & Messing, A. (2013). GFAP expression as an indicator of disease severity in mouse models of Alexander disease. ASN Neuro, 5(1), e00109. doi:10.1042/an20130003
    Jing, R., Wilhelmsson, U., Goodwill, W., Li, L., Pan, Y., Pekny, M., & Skalli, O. (2007). Synemin is expressed in reactive astrocytes in neurotrauma and interacts differentially with vimentin and GFAP intermediate filament networks. J Cell Sci, 120(Pt 7), 1267-1277. doi:10.1242/jcs.03423
    Jones, J. R., Kong, L., Hanna, M. G. t., Hoffman, B., Krencik, R., Bradley, R., . . . Zhang, S. C. (2018). Mutations in GFAP Disrupt the Distribution and Function of Organelles in Human Astrocytes. Cell Rep, 25(4), 947-958.e944. doi:10.1016/j.celrep.2018.09.083
    Kaiser, P., and C. Tagwerker. (2005). Is this protein ubiquitinated? Methods Enzymol. 399:243-248. https://doi.org/10.1016/S0076-6879(05)99016-2
    Kamphuis, W., Mamber, C., Moeton, M., Kooijman, L., Sluijs, J. A., Jansen, A. H., . . . Hol, E. M. (2012). GFAP isoforms in adult mouse brain with a focus on neurogenic astrocytes and reactive astrogliosis in mouse models of Alzheimer disease. PloS one, 7(8), e42823. doi:10.1371/journal.pone.0042823
    Kim, W., E.J. Bennett, E.L. Huttlin, A. Guo, J. Li, A. Possemato, M.E. Sowa, R. Rad, J. Rush, M.J. Comb, J.W. Harper, and S.P. Gygi. (2011). Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol Cell. 44:325-340. https://doi.org/10.1016/j.molcel.2011.08.025
    Koster, S., D.A. Weitz, R.D. Goldman, U. Aebi, and H. Herrmann. (2015). Intermediate filament mechanics in vitro and in the cell: from coiled coils to filaments, fibers and networks. Curr Opin Cell Biol. 32:82-91. https://doi.org/10.1016/j.ceb.2015.01.001
    Ku, N.O., and M.B. Omary. (2000). Keratins turn over by ubiquitination in a phosphorylation-modulated fashion. J Cell Biol. 149:547-552. https://doi.org/10.1083/jcb.149.3.547
    Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680-685. doi:10.1038/227680a0
    Li, R., Johnson, A. B., Salomons, G., Goldman, J. E., Naidu, S., Quinlan, R., . . . Brenner, M. (2005). Glial fibrillary acidic protein mutations in infantile, juvenile, and adult forms of Alexander disease. Ann Neurol, 57(3), 310-326. doi:10.1002/ana.20406
    Liedtke, W., Edelmann, W., Bieri, P. L., Chiu, F. C., Cowan, N. J., Kucherlapati, R., & Raine, C. S. (1996). GFAP is necessary for the integrity of CNS white matter architecture and long-term maintenance of myelination. Neuron, 17(4), 607-615. doi:10.1016/s0896-6273(00)80194-4
    Lin, N. H., Huang, Y. S., Opal, P., Goldman, R. D., Messing, A., & Perng, M. D. (2016). The role of gigaxonin in the degradation of the glial-specific intermediate filament protein GFAP. Mol Biol Cell, 27(25), 3980-3990. doi:10.1091/mbc.E16-06-0362
    Lin, N. H., Jian, W. S., Snider, N., & Perng, M. D. (2024). Glial fibrillary acidic protein is pathologically modified in Alexander disease. J Biol Chem, 107402. doi:10.1016/j.jbc.2024.107402
    Lin, N. H., Messing, A., & Perng, M. D. (2017). Characterization of a panel of monoclonal antibodies recognizing specific epitopes on GFAP. PloS one, 12(7), e0180694. doi:10.1371/journal.pone.0180694
    Lin, N. H., Yang, A. W., Chang, C. H., & Perng, M. D. (2021). Elevated GFAP isoform expression promotes protein aggregation and compromises astrocyte function. Faseb j, 35(5), e21614. doi:10.1096/fj.202100087R
    Mahammad, S., S.N. Murthy, A. Didonna, B. Grin, E. Israeli, R. Perrot, P. Bomont, J.P. Julien, E. Kuczmarski, P. Opal, and R.D. Goldman. (2013). Giant axonal neuropathy-associated gigaxonin mutations impair intermediate filament protein degradation. J Clin Invest. 123:1964-1975. https://www.jci.org/articles/view/66387
    Marceau, N., Schutte, B., Gilbert, S., Loranger, A., Henfling, M. E., Broers, J. L., . . . Ramaekers, F. C. (2007). Dual roles of intermediate filaments in apoptosis. Exp Cell Res, 313(10), 2265-2281. doi:10.1016/j.yexcr.2007.03.038
    McCall, M. A., Gregg, R. G., Behringer, R. R., Brenner, M., Delaney, C. L., Galbreath, E. J., . . . Messing, A. (1996). Targeted deletion in astrocyte intermediate filament (Gfap) alters neuronal physiology. Proc Natl Acad Sci U S A, 93(13), 6361-6366. doi:10.1073/pnas.93.13.6361
    Messing, A. (2018). Alexander disease. Handb Clin Neurol, 148, 693-700. doi:10.1016/b978-0-444-64076-5.00044-2
    Messing, A. (2019). Refining the concept of GFAP toxicity in Alexander disease. J Neurodev Disord. 11:27. doi:10.1186/s11689-019-9290-0
    Messing, A., & Brenner, M. (2003). GFAP: Functional implications gleaned from studies of genetically engineered mice. Glia, 43(1), 87-90. doi:https://doi.org/10.1002/glia.10219
    Messing, A., & Brenner, M. (2020). GFAP at 50. ASN Neuro, 12, 1759091420949680. doi:10.1177/1759091420949680
    Messing, A., Head, M. W., Galles, K., Galbreath, E. J., Goldman, J. E., & Brenner, M. (1998). Fatal encephalopathy with astrocyte inclusions in GFAP transgenic mice. Am J Pathol, 152(2), 391-398. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1857948/
    Mikhaylova, V. V., Eronina, T. B., Chebotareva, N. A., Shubin, V. V., Kalacheva, D. I., & Kurganov, B. I. (2020). Effect of Arginine on Chaperone-Like Activity of HspB6 and Monomeric 14-3-3ζ. Int J Mol Sci, 21(6). doi:10.3390/ijms21062039
    Minkel, H. R., Anwer, T. Z., Arps, K. M., Brenner, M., & Olsen, M. L. (2015). Elevated GFAP induces astrocyte dysfunction in caudal brain regions: A potential mechanism for hindbrain involved symptoms in type II Alexander disease. Glia, 63(12), 2285-2297. doi:10.1002/glia.22893
    Nakamura, Y., M. Takeda, S. Aimoto, S. Hariguchi, S. Kitajima, and T. Nishimura. (1993). Acceleration of bovine neurofilament L assembly by deprivation of acidic tail domain. Eur J Biochem. 212:565-571. https://doi.org/10.1111/j.1432-1033.1993.tb17694.x
    Nawashiro, H., Messing, A., Azzam, N., & Brenner, M. (1998). Mice lacking GFAP are hypersensitive to traumatic cerebrospinal injury. Neuroreport, 9(8), 1691-1696. doi:10.1097/00001756-199806010-00004
    Nicholl, I. D., & Quinlan, R. A. (1994). Chaperone activity of alpha-crystallins modulates intermediate filament assembly. Embo j, 13(4), 945-953. doi:10.1002/j.1460-2075.1994.tb06339.x
    Nishibayashi, F., Kawashima, M., Katada, Y., Murakami, N., & Nozaki, M. (2013). Infantile-onset Alexander disease in a child with long-term follow-up by serial magnetic resonance imaging: a case report. J Med Case Rep, 7, 194. doi:10.1186/1752-1947-7-194
    Omary, M.B. (2009). "IF-pathies": a broad spectrum of intermediate filament-associated diseases. J Clin Invest. 119:1756-1762. doi:10.1172/JCI39894
    Omary, M.B., N.O. Ku, G.Z. Tao, D.M. Toivola, and J. Liao. (2006). "Heads and tails" of intermediate filament phosphorylation: multiple sites and functional insights. Trends Biochem Sci. 31:383-394. doi: 10.1016/j.tibs.2006.05.008
    Otani, N., Nawashiro, H., Fukui, S., Ooigawa, H., Ohsumi, A., Toyooka, T., . . . Brenner, M. (2006). Enhanced hippocampal neurodegeneration after traumatic or kainate excitotoxicity in GFAP-null mice. J Clin Neurosci, 13(9), 934-938. doi:10.1016/j.jocn.2005.10.018
    Parry, D.A., and P.M. Steinert. (1999). Intermediate filaments: molecular architecture, assembly, dynamics and polymorphism. Q Rev Biophys. 32:99-187. doi:10.1017/s0033583500003516
    Pekny, M., Eliasson, C., Siushansian, R., Ding, M., Dixon, S. J., Pekna, M., . . . Hamberger, A. (1999). The impact of genetic removal of GFAP and/or vimentin on glutamine levels and transport of glucose and ascorbate in astrocytes. Neurochem Res, 24(11), 1357-1362. doi:10.1023/a:1022572304626
    Pérez-Sala, D., & Quinlan, R. A. (2024). The redox-responsive roles of intermediate filaments in cellular stress detection, integration and mitigation. Curr Opin Cell Biol, 86, 102283. doi:10.1016/j.ceb.2023.102283
    Perng, M., M. Su, S.F. Wen, R. Li, T. Gibbon, A.R. Prescott, M. Brenner, and R.A. Quinlan. (2006). 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. 79:197-213. doi:10.1086/504411
    Pollard, T. D., & Cooper, J. A. (1982). Methods to characterize actin filament networks. Methods Enzymol, 85 Pt B, 211-233. doi:10.1016/0076-6879(82)85022-2
    Quinlan, R. A., Moir, R. D., & Stewart, M. (1989). Expression in Escherichia coli of fragments of glial fibrillary acidic protein: characterization, assembly properties and paracrystal formation. J Cell Sci, 93 (Pt 1), 71-83. doi:10.1242/jcs.93.1.71
    Quinlan, R.A., and M. Stewart. (1991). Molecular interactions in intermediate filaments. Bioessays. 13:597-600. doi:10.1002/bies.950131110
    Quinlan, R.A., M. Hatzfeld, W.W. Franke, A. Lustig, T. Schulthess, and J. Engel. (1986). Characterization of dimer subunits of intermediate filament proteins. J Mol Biol. 192:337-349. doi:10.1016/0022-2836(86)90369-4
    Raats, J.M., F.R. Pieper, W.T. Vree Egberts, K.N. Verrijp, F.C. Ramaekers, and H. Bloemendal. (1990). Assembly of amino-terminally deleted desmin in vimentin-free cells. J Cell Biol. 111:1971-1985. doi:10.1083/jcb.111.5.1971
    Sharma, S., Sarkar, S., Paul, S. S., Roy, S., & Chattopadhyay, K. (2013). A small molecule chemical chaperone optimizes its unfolded state contraction and denaturant like properties. Sci Rep, 3, 3525. doi:10.1038/srep03525
    Snider, N.T., and M.B. Omary. (2014). Post-translational modifications of intermediate filament proteins: mechanisms and functions. Nat Rev Mol Cell Biol. 15:163-177. doi:10.1038/nrm3753
    Snyder, N.A., and G.M. Silva. (2021). Deubiquitinating enzymes (DUBs): Regulation, homeostasis, and oxidative stress response. J Biol Chem. 297:101077. doi: 10.1016/j.jbc.2021.101077
    Soellner, P., R.A. Quinlan, and W.W. Franke. (1985). Identification of a distinct soluble subunit of an intermediate filament protein: tetrameric vimentin from living cells. Proc Natl Acad Sci U S A. 82:7929-7933. doi:10.1073/pnas.82.23.7929
    Sofroniew, M. V. (2020). Astrocyte Reactivity: Subtypes, States, and Functions in CNS Innate Immunity. Trends Immunol, 41(9), 758-770. doi:10.1016/j.it.2020.07.004
    Sofroniew, M. V., & Vinters, H. V. (2010). Astrocytes: biology and pathology. Acta Neuropathol, 119(1), 7-35. doi:10.1007/s00401-009-0619-8
    Steinert, P.M., Y.H. Chou, V. Prahlad, D.A. Parry, L.N. Marekov, K.C. Wu, S.I. Jang, and R.D. Goldman. (1999). A high molecular weight intermediate filament-associated protein in BHK-21 cells is nestin, a type VI intermediate filament protein. Limited co-assembly in vitro to form heteropolymers with type III vimentin and type IV alpha-internexin. J Biol Chem. 274:9881-9890. doi:10.1074/jbc.274.14.9881
    Stenzel, W., Soltek, S., Schlüter, D., & Deckert, M. (2004). The intermediate filament GFAP is important for the control of experimental murine Staphylococcus aureus-induced brain abscess and Toxoplasma encephalitis. J Neuropathol Exp Neurol, 63(6), 631-640. doi:10.1093/jnen/63.6.631
    Stewart, M., Quinlan, R. A., & Moir, R. D. (1989). Molecular interactions in paracrystals of a fragment corresponding to the alpha-helical coiled-coil rod portion of glial fibrillary acidic protein: evidence for an antiparallel packing of molecules and polymorphism related to intermediate filament structure. J Cell Biol, 109(1), 225-234. doi:10.1083/jcb.109.1.225
    Sullivan, S.M., R.K. Sullivan, S.M. Miller, Z. Ireland, S.T. Bjorkman, D.V. Pow, and P.B. Colditz. (2012). Phosphorylation of GFAP is associated with injury in the neonatal pig hypoxic-ischemic brain. Neurochem Res. 37:2364-2378. doi:10.1007/s11064-012-0774-5
    Szeverenyi, I., Cassidy, A. J., Chung, C. W., Lee, B. T. K., Common, J. E. A., Ogg, S. C., . . . Lane, E. B. (2008). The Human Intermediate Filament Database: comprehensive information on a gene family involved in many human diseases. Human mutation, 29(3), 351-360. doi:10.1002/humu.20652
    Takemura, M., H. Gomi, E. Colucci-Guyon, and S. Itohara. (2002). Protective role of phosphorylation in turnover of glial fibrillary acidic protein in mice. J Neurosci. 22:6972-6979. doi:10.1523/JNEUROSCI.22-16-06972.2002
    Tang, G., Perng, M. D., Wilk, S., Quinlan, R., & Goldman, J. E. (2010). Oligomers of mutant glial fibrillary acidic protein (GFAP) Inhibit the proteasome system in alexander disease astrocytes, and the small heat shock protein alphaB-crystallin reverses the inhibition. J Biol Chem, 285(14), 10527-10537. doi:10.1074/jbc.M109.067975
    Tang, G., Xu, Z., & Goldman, J. E. (2006). Synergistic effects of the SAPK/JNK and the proteasome pathway on glial fibrillary acidic protein (GFAP) accumulation in Alexander disease. J Biol Chem, 281(50), 38634-38643. doi:10.1074/jbc.M604942200
    Tang, G., Yue, Z., Talloczy, Z., & Goldman, J. E. (2008). Adaptive autophagy in Alexander disease-affected astrocytes. Autophagy, 4(5), 701-703.
    Tang, G., Yue, Z., Talloczy, Z., Hagemann, T., Cho, W., Messing, A., . . . Goldman, J. E. (2008). Autophagy induced by Alexander disease-mutant GFAP accumulation is regulated by p38/MAPK and mTOR signaling pathways. Hum Mol Genet, 17(11), 1540-1555. doi:10.1093/hmg/ddn042
    Thibaudeau, T.A., R.T. Anderson, and D.M. Smith. (2018). A common mechanism of proteasome impairment by neurodegenerative disease-associated oligomers. Nature communications. 9:1097. doi:10.1038/s41467-018-03509-0
    Thomsen, R., Daugaard, T. F., Holm, I. E., & Nielsen, A. L. (2013). Alternative mRNA splicing from the glial fibrillary acidic protein (GFAP) gene generates isoforms with distinct subcellular mRNA localization patterns in astrocytes. PloS one, 8(8), e72110. doi:10.1371/journal.pone.0072110
    Tian, R., Gregor, M., Wiche, G., & Goldman, J. E. (2006). Plectin regulates the organization of glial fibrillary acidic protein in Alexander disease. Am J Pathol, 168(3), 888-897. doi:10.2353/ajpath.2006.051028
    Valentim, L.M., C.B. Michalowski, S.P. Gottardo, L. Pedroso, L.G. Gestrich, C.A. Netto, C.G. Salbego, and R. Rodnight. (1999). Effects of transient cerebral ischemia on glial fibrillary acidic protein phosphorylation and immunocontent in rat hippocampus. Neuroscience. 91:1291-1297. doi:10.1016/s0306-4522(98)00707-6
    Verkhratsky, A., & Nedergaard, M. (2018). Physiology of Astroglia. Physiol Rev, 98(1), 239-389. doi:10.1152/physrev.00042.2016
    Vermeire, P. J., Lilina, A. V., Hashim, H. M., Dlabolová, L., Fiala, J., Beelen, S., . . . Strelkov, S. V. (2023). Molecular structure of soluble vimentin tetramers. Sci Rep, 13(1), 8841. doi:10.1038/s41598-023-34814-4
    Viedma-Poyatos, Á., González-Jiménez, P., Pajares, M. A., & Pérez-Sala, D. (2022). Alexander disease GFAP R239C mutant shows increased susceptibility to lipoxidation and elicits mitochondrial dysfunction and oxidative stress. Redox Biol, 55, 102415. doi:10.1016/j.redox.2022.102415
    Viedma-Poyatos, A., P. Gonzalez-Jimenez, M.A. Pajares, and D. Perez-Sala. (2022). Alexander disease GFAP R239C mutant shows increased susceptibility to lipoxidation and elicits mitochondrial dysfunction and oxidative stress. Redox Biol. 55:102415. doi: 10.1016/j.redox.2022.102415
    Walker, A. K., Daniels, C. M., Goldman, J. E., Trojanowski, J. Q., Lee, V. M., & Messing, A. (2014). Astrocytic TDP-43 pathology in Alexander disease. J Neurosci, 34(19), 6448-6458. doi:10.1523/jneurosci.0248-14.2014
    Wang, L., Colodner, K. J., & Feany, M. B. (2011). Protein misfolding and oxidative stress promote glial-mediated neurodegeneration in an Alexander disease model. J Neurosci, 31(8), 2868-2877. doi:10.1523/jneurosci.3410-10.2011
    Wessel, D., & Flügge, U. I. (1984). A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem, 138(1), 141-143. doi:10.1016/0003-2697(84)90782-6
    Wu, K.C., J.T. Bryan, M.I. Morasso, S.I. Jang, J.H. Lee, J.M. Yang, L.N. Marekov, D.A. Parry, and P.M. Steinert. (2000). Coiled-coil trigger motifs in the 1B and 2B rod domain segments are required for the stability of keratin intermediate filaments. Mol Biol Cell. 11:3539-3558. doi:10.1091/mbc.11.10.3539
    Yang, A. W., Lin, N. H., Yeh, T. H., Snider, N., & Perng, M. D. (2022). Effects of Alexander disease-associated mutations on the assembly and organization of GFAP intermediate filaments. Mol Biol Cell, 33(8), ar69. doi:10.1091/mbc.E22-01-0013

    QR CODE