The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Albee Messing - One of the best experts on this subject based on the ideXlab platform.
-
refining the concept of gfap toxicity in Alexander Disease
Journal of Neurodevelopmental Disorders, 2019Co-Authors: Albee MessingAbstract:Alexander Disease is caused by dominantly acting mutations in glial fibrillary acidic protein (GFAP), the major intermediate filament of astrocytes in the central nervous system. In addition to the sequence variants that represent the origin of Disease, GFAP accumulation also takes place, together leading to a gain-of-function that has sometimes been referred to as “GFAP toxicity.” Whether the nature of GFAP toxicity in patients, who have mixtures of both mutant and normal protein, is the same as that produced by simple GFAP excess, is not yet clear. The implications of these questions for the design of effective treatments are discussed.
-
tissue and cellular rigidity and mechanosensitive signaling activation in Alexander Disease
Nature, 2018Co-Authors: Liqun Wang, Tracy L. Hagemann, Albee Messing, Jing Xia, Jeffrey R Jones, Ernest Fraenkel, David A Weitz, Suchun Zhang, Mel B. FeanyAbstract:Glial cells have increasingly been implicated as active participants in the pathogenesis of neurological Diseases, but critical pathways and mechanisms controlling glial function and secondary non-cell autonomous neuronal injury remain incompletely defined. Here we use models of Alexander Disease, a severe brain disorder caused by gain-of-function mutations in GFAP, to demonstrate that misregulation of GFAP leads to activation of a mechanosensitive signaling cascade characterized by activation of the Hippo pathway and consequent increased expression of A-type lamin. Importantly, we use genetics to verify a functional role for dysregulated mechanotransduction signaling in promoting behavioral abnormalities and non-cell autonomous neurodegeneration. Further, we take cell biological and biophysical approaches to suggest that brain tissue stiffness is increased in Alexander Disease. Our findings implicate altered mechanotransduction signaling as a key pathological cascade driving neuronal dysfunction and neurodegeneration in Alexander Disease, and possibly also in other brain disorders characterized by gliosis.
-
Antisense suppression of glial fibrillary acidic protein as a treatment for Alexander Disease.
Annals of neurology, 2018Co-Authors: Tracy L. Hagemann, Berit Powers, Curt Mazur, Aneeza Kim, Steven Wheeler, Gene Hung, Eric E. Swayze, Albee MessingAbstract:Objective Alexander Disease is a fatal leukodystrophy caused by autosomal dominant gain-of-function mutations in the gene for glial fibrillary acidic protein (GFAP), an intermediate filament protein primarily expressed in astrocytes of the central nervous system. A key feature of pathogenesis is overexpression and accumulation of GFAP, with formation of characteristic cytoplasmic aggregates known as Rosenthal fibers. Here we investigate whether suppressing GFAP with antisense oligonucleotides could provide a therapeutic strategy for treating Alexander Disease. Methods In this study, we use GFAP mutant mouse models of Alexander Disease to test the efficacy of antisense suppression and evaluate the effects on molecular and cellular phenotypes and non-cell-autonomous toxicity. Antisense oligonucleotides were designed to target the murine Gfap transcript, and screened using primary mouse cortical cultures. Lead oligonucleotides were then tested for their ability to reduce GFAP transcripts and protein, first in wild-type mice with normal levels of GFAP, and then in adult mutant mice with established pathology and elevated levels of GFAP. Results Nearly complete and long-lasting elimination of GFAP occurred in brain and spinal cord following single bolus intracerebroventricular injections, with a striking reversal of Rosenthal fibers and downstream markers of microglial and other stress-related responses. GFAP protein was also cleared from cerebrospinal fluid, demonstrating its potential utility as a biomarker in future clinical applications. Finally, treatment led to improved body condition and rescue of hippocampal neurogenesis. Interpretation These results demonstrate the efficacy of antisense suppression for an astrocyte target, and provide a compelling therapeutic approach for Alexander Disease. Ann Neurol 2018;83:27-39.
-
Alexander Disease: A Guide for Patients and Families
2017Co-Authors: Albee MessingAbstract:Abstract This book offers a comprehensive overview of Alexander Disease, a rare and devastating neurological disorder that often affects the white matter of the brain and spinal cord. Its distinctive neuropathology consists of abundant Rosenthal fibers within astrocytes (one of the four major cell types of the central nervous system). Nearly all cases are caused by variants in the gene encoding the intermediate filament protein GFAP, but how these changes in GFAP lead to the widespread manifestations of Disease is poorly understood. Astrocytes, while discovered over a century ago, are themselves still much of a mystery. They exhibit considerable diversity, defy precise definition, and yet actively regulate many aspects of nervous system functioning. We also have incomplete understanding of Rosenthal fibers, odd structures that contain GFAP as just one of many components. Whether they are toxic or protective is unknown. Moreover, Rosenthal fibers are not absolutely unique to Alexander Disease, and are seen...
-
glial fibrillary acidic protein exhibits altered turnover kinetics in a mouse model of Alexander Disease
Journal of Biological Chemistry, 2017Co-Authors: Laura R Moody, Gregory A Barrettwilt, Michael R Sussman, Albee MessingAbstract:Mutations in the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP) lead to the rare and fatal disorder, Alexander Disease (AxD). A prominent feature of the Disease is aberrant accumulation of GFAP. It has been proposed that this accumulation occurs because of an increase in gene transcription coupled with impaired proteasomal degradation, yet this hypothesis remains untested. We therefore sought to directly investigate GFAP turnover in a mouse model of AxD that is heterozygous for a Disease-causing point mutation (GfapR236H/+) (and thus expresses both wild-type and mutant protein). Stable isotope labeling by amino acids in cell culture, using primary cortical astrocytes, indicated that the in vitro half-lives of total GFAP in astrocytes from wild-type and mutant mice were similar at ∼3-4 days. Surprisingly, results obtained with stable isotope labeling of mammals revealed that, in vivo, the half-life of GFAP in mutant mice (15.4 ± 0.5 days) was much shorter than that in wild-type mice (27.5 ± 1.6 days). These unexpected in vivo data are most consistent with a model in which synthesis and degradation are both increased. Our work reveals that an AxD-causing mutation alters GFAP turnover kinetics in vivo and provides an essential foundation for future studies aimed at preventing or reducing the accumulation of GFAP. In particular, these data suggest that elimination of GFAP might be possible and occurs more quickly than previously surmised.
James E Goldman - One of the best experts on this subject based on the ideXlab platform.
-
Alexander Disease an astrocytopathy that produces a leukodystrophy
Brain Pathology, 2018Co-Authors: Alexander A Sosunov, Markel Olabarria, James E GoldmanAbstract:Alexander Disease (AxD) is a degenerative disorder caused by mutations in the GFAP gene, which encodes the major intermediate filament of astrocytes. As other cells in the CNS do not express GFAP, AxD is a primary astrocyte Disease. Astrocytes acquire a large number of pathological features, including changes in morphology, the loss or diminution of a number of critical astrocyte functions and the activation of cell stress and inflammatory pathways. AxD is also characterized by white matter degeneration, a pathology that has led it to be included in the "leukodystrophies." Furthermore, variable degrees of neuronal loss take place. Thus, the astrocyte pathology triggers alterations in other cell types. Here, we will review the neuropathology of AxD and discuss how a Disease of astrocytes can lead to severe pathologies in non-astrocytic cells. Our knowledge of the pathophysiology of AxD will also lead to a better understanding of how astrocytes interact with other CNS cells and how astrocytes in the gliosis that accompanies many neurological disorders can damage the function and survival of other cells.
-
The origin of Rosenthal fibers and their contributions to astrocyte pathology in Alexander Disease
Acta Neuropathologica Communications, 2017Co-Authors: Alexander A Sosunov, Guy M Mckhann, James E GoldmanAbstract:Rosenthal fibers (RFs) are cytoplasmic, proteinaceous aggregates. They are the pathognomonic feature of the astrocyte pathology in Alexander Disease (AxD), a neurodegenerative disorder caused by heterozygous mutations in the GFAP gene, encoding glial fibrillary acidic protein (GFAP). Although RFs have been known for many years their origin and significance remain elusive issues. We have used mouse models of AxD based on the overexpression of human GFAP (transgenic, TG) and a point mutation in mouse GFAP (knock-in, KI) to examine the formation of RFs and to find astrocyte changes that correlate with the appearance of RFs. We found RFs of various sizes and shapes. The smallest ones appear as granular depositions on intermediate filaments. These contain GFAP and the small heat shock protein, alphaB-crystallin. Their aggregation appears to give rise to large RFs. The appearance of new RFs and the growth of previously formed RFs occur over time. We determined that DAPI is a reliable marker of RFs and in parallel with Fluoro-Jade B (FJB) staining defined a high variability in the appearance of RFs, even in neighboring astrocytes. Although many astrocytes in AxD with increased levels of GFAP and with or without RFs change their phenotype, only some cells with large numbers of RFs show a profound reconstruction of cellular processes, with a loss of fine distal processes and the appearance of large, lobulated nuclei, likely due to arrested mitosis. We conclude that 1) RFs appear to originate as small, osmiophilic masses containing both GFAP and alphaB-crystallin deposited on bundles of intermediate filaments. 2) RFs continue to form within AxD astrocytes over time. 3) DAPI is a reliable marker for RFs and can be used with immunolabeling. 4) RFs appear to interfere with the successful completion of astrocyte mitosis and cell division.
-
disorders of astrocytes Alexander Disease as a model
Annual Review of Pathology-mechanisms of Disease, 2017Co-Authors: Markel Olabarria, James E GoldmanAbstract:Astrocytes undergo important phenotypic changes in many neurological disorders, including strokes, trauma, inflammatory Diseases, infectious Diseases, and neurodegenerative Diseases. We have been studying the astrocytes of Alexander Disease (AxD), which is caused by heterozygous mutations in the GFAP gene, which is the gene that encodes the major astrocyte intermediate filament protein. AxD is a primary astrocyte Disease because GFAP expression is specific to astrocytes in the central nervous system (CNS). The accumulation of extremely large amounts of GFAP causes many molecular changes in astrocytes, including proteasome inhibition, stress kinase activation, mechanistic target of rapamycin (mTOR) activation, loss of glutamate and potassium buffering capacity, loss of astrocyte coupling, and changes in cell morphology. Many of these changes appear to be common to astrocyte reactions in other neurological disorders. Using AxD to illuminate common mechanisms, we discuss the molecular pathology of AxD astrocytes and compare that to astrocyte pathology in other disorders.
-
Composition of Rosenthal Fibers, the Protein Aggregate Hallmark of Alexander Disease.
Journal of proteome research, 2016Co-Authors: Michael R. Heaven, James E Goldman, Alexander A Sosunov, Daniel Flint, Shan M. Randall, Landon Wilson, Stephen Barnes, David C. Muddiman, Michael BrennerAbstract:Alexander Disease (AxD) is a neurodegenerative disorder characterized by astrocytic protein aggregates called Rosenthal fibers (RFs). We used mouse models of AxD to determine the protein composition of RFs to obtain information about Disease mechanisms including the hypothesis that sequestration of proteins in RFs contributes to Disease. A method was developed for RF enrichment, and analysis of the resulting fraction using isobaric tags for relative and absolute quantitation mass spectrometry identified 77 proteins not previously associated with RFs. Three of five proteins selected for follow-up were confirmed enriched in the RF fraction by immunobloting of both the AxD mouse models and human patients: receptor for activated protein C kinase 1 (RACK1), G1/S-specific cyclin D2, and ATP-dependent RNA helicase DDX3X. Immunohistochemistry validated cyclin D2 as a new RF component, but results for RACK1 and DDX3X were equivocal. None of these was decreased in the non-RF fractions compared to controls. A simila...
-
Astrocyte pathology in Alexander Disease causes a marked inflammatory environment
Acta Neuropathologica, 2015Co-Authors: Markel Olabarria, Maria Putilina, Ellen C. Riemer, James E GoldmanAbstract:Astrocytes and microglia are commonly involved in a wide variety of CNS pathologies. However, they are typically involved in a secondary response in which many cell types are affected simultaneously and therefore it is difficult to know their contributions to the pathology. Here, we show that pathological astrocytes in a mouse model of Alexander Disease (AxD; GFAP ^Tg; Gfap ^+/R236H) cause a pronounced immune response. We have studied the inflammatory response in the hippocampus and spinal cord of these mice and have found marked microglial activation, which follows that of astrocytes in a spatial pathological progression, as shown by increased levels of Iba1 and microglial cell (Iba1+) density. RNA sequencing and subsequent gene ontology (GO) analysis revealed that a majority of the most upregulated genes in GFAP ^Tg; Gfap ^+/R236H mice are directly associated with immune function and that cytokine and chemokine GO attributes represent nearly a third of the total immune attributes. Cytokine and chemokine analysis showed CXCL10 and CCL2 to be the most and earliest increased molecules, showing concentrations as high as EAE or stroke models. CXCL10 was localized exclusively to astrocytes while CCL2 was also present in microglia. Despite the high levels of CXCL10 and CCL2, T cell infiltration was mild and no B cells were found. Thus, mutations in GFAP are sufficient to trigger a profound inflammatory response. The cellular stress caused by the accumulation of GFAP likely leads to the production of inflammatory molecules and microglial activation. Examination of human AxD CNS tissues also revealed microglial activation and T cell infiltrates. Therefore, the inflammatory environment may play an important role in producing the neuronal dysfunction and seizures of AxD.
Michael Brenner - One of the best experts on this subject based on the ideXlab platform.
-
relative stabilities of wild type and mutant glial fibrillary acidic protein in patients with Alexander Disease
Journal of Biological Chemistry, 2019Co-Authors: Michael R. Heaven, Landon Wilson, Stephen Barnes, Michael BrennerAbstract:Alexander Disease (AxD) is an often fatal astrogliopathy caused by dominant gain-of-function missense mutations in the glial fibrillary acidic protein (GFAP) gene. The mechanism by which the mutations produce the AxD phenotype is not known. However, the observation that features of AxD are displayed by mice that express elevated levels of GFAP from a human WT GFAP transgene has contributed to the notion that the mutations produce AxD by increasing accumulation of total GFAP above some toxic threshold rather than the mutant GFAP being inherently toxic. A possible mechanism for accumulation of GFAP in AxD patients is that the mutated GFAP variants are more stable than the WT, an attribution abetted by observations that GFAP complexes containing GFAP variants are more resistant to solvent extraction. Here we tested this hypothesis by determining the relative levels of WT and mutant GFAP in three individuals with AxD, each of whom carried a common but different GFAP mutation (R79C, R239H, or R416W). Mass spectrometry analysis identified a peptide specific to the mutant or WT GFAP in each patient, and we quantified this peptide by comparing its signal to that of an added [15N]GFAP standard. In all three individuals, the level of mutant GFAP was less than that of the WT. This finding suggests that AxD onset is due to an intrinsic toxicity of the mutant GFAP instead of it acting indirectly by being more stable than WT GFAP and thereby increasing the total GFAP level.
-
Composition of Rosenthal Fibers, the Protein Aggregate Hallmark of Alexander Disease.
Journal of proteome research, 2016Co-Authors: Michael R. Heaven, James E Goldman, Alexander A Sosunov, Daniel Flint, Shan M. Randall, Landon Wilson, Stephen Barnes, David C. Muddiman, Michael BrennerAbstract:Alexander Disease (AxD) is a neurodegenerative disorder characterized by astrocytic protein aggregates called Rosenthal fibers (RFs). We used mouse models of AxD to determine the protein composition of RFs to obtain information about Disease mechanisms including the hypothesis that sequestration of proteins in RFs contributes to Disease. A method was developed for RF enrichment, and analysis of the resulting fraction using isobaric tags for relative and absolute quantitation mass spectrometry identified 77 proteins not previously associated with RFs. Three of five proteins selected for follow-up were confirmed enriched in the RF fraction by immunobloting of both the AxD mouse models and human patients: receptor for activated protein C kinase 1 (RACK1), G1/S-specific cyclin D2, and ATP-dependent RNA helicase DDX3X. Immunohistochemistry validated cyclin D2 as a new RF component, but results for RACK1 and DDX3X were equivocal. None of these was decreased in the non-RF fractions compared to controls. A simila...
-
A new mutation in GFAP widens the spectrum of Alexander Disease.
European journal of human genetics : EJHG, 2014Co-Authors: Michael Brenner, Albee MessingAbstract:In this issue Nam et al1 describe a novel case of Alexander Disease that markedly expands the candidate patient population for this often fatal neurodegenerative disorder of astrocyte dysfunction. Astrocytes are a predominate cell type in the central nervous system; yet, despite their multiple critical activities, Alexander Disease is the only genetic disorder presently known attributable to a primary defect in these cells (reviewed in Brenner et al2). The Disease, defined by the abundant presence in astrocytes of protein aggregates termed Rosenthal fibers, was first described as a childhood leukodystrophy featuring the striking clinical signs of megalencephaly, seizures and psychomotor delay accompanied by massive white matter deficits in the frontal lobes. Subsequently, a later-onset form was added based on sharing the abundance of Rosenthal fibers, but with clinical signs that could be quite different, such as difficulty swallowing and speaking, autonomic dysfunction and ataxia. In addition, the radiologic abnormalities of the later-onset cases involve more caudal central nervous system regions, such as the cerebellum, brainstem and cervical spinal cord (see Prust et al3 for a description of the two forms). A common etiology for the two forms was established by finding that about 95% of both are caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP), an intermediate filament protein expressed strongly and predominantly in astrocytes.4, 5 All initial mutations discovered were heterozygous missense changes predicting production of both full-length mutant and wild-type proteins. This, together with the finding that GFAP null mice do not display signs of Alexander Disease, led to the conclusion that the mutations cause Disease by a dominant gain of toxic function.
-
Alexander Disease and astrotherapeutics
2014Co-Authors: Michael Brenner, Albee MessingAbstract:Alexander Disease is a protein aggregation disorder resulting from mutations in the intermediate filament protein, GFAP. Progress in the past 15 years has defined numerous aspects of astrocyte function that are impacted by these mutations, and that might be amenable to correction. Since reversal of astrocyte dysfunction is likely to be valuable in a wide variety of conditions, Alexander Disease offers unique opportunities for exploring the newly emerging field of astrotherapeutics.
-
archetypal and new families with Alexander Disease and novel mutations in gfap
JAMA Neurology, 2012Co-Authors: Albee Messing, Marjo S Van Der Knaap, Sakkubai Naidu, Daniel Flint, Paul J Taylor, Lital Silverman, Michael BrennerAbstract:Objective To describe genetic analyses of the 2 most thoroughly studied, historically seminal multigenerational families with Alexander Disease described prior to the identification of GFAP as the related gene, as well as 1 newly discovered family. Design Clinical histories were obtained and DNA was analyzed from blood, cheek epithelial cells, or fixed paraffin-embedded surgical samples. Subjects Affected and unaffected adult members of 3 families and affected children were included. Main Outcome Measures Mutations in GFAP and behavior of mutant protein in cellular transfection assays. Results Family A contains 4 siblings in whom we found a novel p.Ser247Pro mutation that was paternally inherited. The phenotypes of these siblings include 1 unaffected adult, 1 individual with juvenile-onset Disease, and 2 individuals with adult-onset Disease. Family B spans 4 generations, including the first described patient with adult-onset Disease originally reported in 1968. Analysis of members of the later generations revealed a novel p.Asp417Ala mutation. Family C contains 3 generations. We detected a novel p.Gln426Leu mutation that, to our knowledge, is the farthest C-terminal mutation known. Conclusions These families display clear evidence of variable phenotypes but do not support recessive inheritance. While germline mosaicism cannot be excluded for 1 family (A), we propose that for genetic counseling purposes the risk of germline mosaicism should be described as less than 1%.
Tracy L. Hagemann - One of the best experts on this subject based on the ideXlab platform.
-
type ii Alexander Disease caused by splicing errors and aberrant overexpression of an uncharacterized gfap isoform
Human Mutation, 2020Co-Authors: Ming-der Perng, Tracy L. Hagemann, Guy Helman, Asako Takanohashi, Marzena Walkiewicz, Sarah Woidill, Sunetra Sase, Zachary Cross, Ling ZhaoAbstract:Alexander Disease results from gain-of-function mutations in the gene encoding glial fibrillary acidic protein (GFAP). At least eight GFAP isoforms have been described, however, the predominant alpha isoform accounts for ∼90% of GFAP protein. We describe exonic variants identified in three unrelated families with Type II Alexander Disease that alter the splicing of GFAP pre-messenger RNA (mRNA) and result in the upregulation of a previously uncharacterized GFAP lambda isoform (NM_001363846.1). Affected members of Family 1 and Family 2 shared the same missense variant, NM_001363846.1:c.1289G>A;p.(Arg430His) while in Family 3 we identified a synonymous variant in the adjacent nucleotide, NM_001363846.1:c.1290C>A;p.(Arg430Arg). Using RNA and protein analysis of brain autopsy samples, and a mini-gene splicing reporter assay, we demonstrate both variants result in the upregulation of the lambda isoform. Our approach demonstrates the importance of characterizing the effect of GFAP variants on mRNA splicing to inform future pathophysiologic and therapeutic study for Alexander Disease.
-
type ii Alexander Disease caused by splicing errors and aberrant overexpression of an uncharacterized gfap isoform
bioRxiv, 2019Co-Authors: Ming-der Perng, Tracy L. Hagemann, Guy Helman, Asako Takanohashi, Marzena Walkiewicz, Sarah Woidill, Sunetra Sase, Zachary Cross, Ling ZhaoAbstract:Alexander Disease results from gain of function mutations in the gene encoding glial fibrillary acidic protein (GFAP), an intermediate filament protein expressed in astrocytes. At least eight GFAP isoforms have been described, however, the predominant alpha isoform accounts for approximately 90% of GFAP protein in the central nervous system. Here we describe exonic variants identified in three unrelated families with Type II Alexander Disease that alter the splicing of GFAP pre-mRNA and result in upregulation of a previously uncharacterised GFAP lambda isoform (NM_001363846.1). Affected members of Family 1 and Family 2 shared the same missense variant, NM_001363846.1:c.1289G>A;p.(Arg430His) while in Family 3 we identified a synonymous variant in the adjacent nucleotide, NM_001363846.1:c.1290C>A;p.(Arg430Arg). Using RNA and protein analysis of brain autopsy samples, and a mini-gene splicing reporter assay, we demonstrate both variants result in upregulation of the lambda isoform. We assessed other GFAP variants in the ClinVar database for predicted aberrant splicing and, using the same assay, demonstrated significant changes to splicing for two selected variants. In one case, we found that altered splicing due to a +5 intronic variant resulted in the inclusion of the GFAP kappa isoform. Our approach demonstrates the importance of characterizing the effect of GFAP variants on mRNA splicing in order to inform future pathophysiologic and therapeutic study for Alexander Disease.
-
tissue and cellular rigidity and mechanosensitive signaling activation in Alexander Disease
Nature, 2018Co-Authors: Liqun Wang, Tracy L. Hagemann, Albee Messing, Jing Xia, Jeffrey R Jones, Ernest Fraenkel, David A Weitz, Suchun Zhang, Mel B. FeanyAbstract:Glial cells have increasingly been implicated as active participants in the pathogenesis of neurological Diseases, but critical pathways and mechanisms controlling glial function and secondary non-cell autonomous neuronal injury remain incompletely defined. Here we use models of Alexander Disease, a severe brain disorder caused by gain-of-function mutations in GFAP, to demonstrate that misregulation of GFAP leads to activation of a mechanosensitive signaling cascade characterized by activation of the Hippo pathway and consequent increased expression of A-type lamin. Importantly, we use genetics to verify a functional role for dysregulated mechanotransduction signaling in promoting behavioral abnormalities and non-cell autonomous neurodegeneration. Further, we take cell biological and biophysical approaches to suggest that brain tissue stiffness is increased in Alexander Disease. Our findings implicate altered mechanotransduction signaling as a key pathological cascade driving neuronal dysfunction and neurodegeneration in Alexander Disease, and possibly also in other brain disorders characterized by gliosis.
-
Antisense suppression of glial fibrillary acidic protein as a treatment for Alexander Disease.
Annals of neurology, 2018Co-Authors: Tracy L. Hagemann, Berit Powers, Curt Mazur, Aneeza Kim, Steven Wheeler, Gene Hung, Eric E. Swayze, Albee MessingAbstract:Objective Alexander Disease is a fatal leukodystrophy caused by autosomal dominant gain-of-function mutations in the gene for glial fibrillary acidic protein (GFAP), an intermediate filament protein primarily expressed in astrocytes of the central nervous system. A key feature of pathogenesis is overexpression and accumulation of GFAP, with formation of characteristic cytoplasmic aggregates known as Rosenthal fibers. Here we investigate whether suppressing GFAP with antisense oligonucleotides could provide a therapeutic strategy for treating Alexander Disease. Methods In this study, we use GFAP mutant mouse models of Alexander Disease to test the efficacy of antisense suppression and evaluate the effects on molecular and cellular phenotypes and non-cell-autonomous toxicity. Antisense oligonucleotides were designed to target the murine Gfap transcript, and screened using primary mouse cortical cultures. Lead oligonucleotides were then tested for their ability to reduce GFAP transcripts and protein, first in wild-type mice with normal levels of GFAP, and then in adult mutant mice with established pathology and elevated levels of GFAP. Results Nearly complete and long-lasting elimination of GFAP occurred in brain and spinal cord following single bolus intracerebroventricular injections, with a striking reversal of Rosenthal fibers and downstream markers of microglial and other stress-related responses. GFAP protein was also cleared from cerebrospinal fluid, demonstrating its potential utility as a biomarker in future clinical applications. Finally, treatment led to improved body condition and rescue of hippocampal neurogenesis. Interpretation These results demonstrate the efficacy of antisense suppression for an astrocyte target, and provide a compelling therapeutic approach for Alexander Disease. Ann Neurol 2018;83:27-39.
-
nitric oxide mediates glial induced neurodegeneration in Alexander Disease
Nature Communications, 2015Co-Authors: Liqun Wang, Tracy L. Hagemann, Albee Messing, Hermann Kalwa, Thomas Michel, Mel B. FeanyAbstract:Glia play critical roles in maintaining the structure and function of the nervous system; however, the specific contribution that astroglia make to neurodegeneration in human Disease states remains largely undefined. Here we use Alexander Disease, a serious degenerative neurological disorder caused by astrocyte dysfunction, to identify glial-derived NO as a signalling molecule triggering astrocyte-mediated neuronal degeneration. We further find that NO acts through cGMP signalling in neurons to promote cell death. Glial cells themselves also degenerate, via the DNA damage response and p53. Our findings thus define a specific mechanism for glial-induced non-cell autonomous neuronal cell death, and identify a potential therapeutic target for reducing cellular toxicity in Alexander Disease, and possibly other neurodegenerative disorders with glial dysfunction.
Ming-der Perng - One of the best experts on this subject based on the ideXlab platform.
-
type ii Alexander Disease caused by splicing errors and aberrant overexpression of an uncharacterized gfap isoform
Human Mutation, 2020Co-Authors: Ming-der Perng, Tracy L. Hagemann, Guy Helman, Asako Takanohashi, Marzena Walkiewicz, Sarah Woidill, Sunetra Sase, Zachary Cross, Ling ZhaoAbstract:Alexander Disease results from gain-of-function mutations in the gene encoding glial fibrillary acidic protein (GFAP). At least eight GFAP isoforms have been described, however, the predominant alpha isoform accounts for ∼90% of GFAP protein. We describe exonic variants identified in three unrelated families with Type II Alexander Disease that alter the splicing of GFAP pre-messenger RNA (mRNA) and result in the upregulation of a previously uncharacterized GFAP lambda isoform (NM_001363846.1). Affected members of Family 1 and Family 2 shared the same missense variant, NM_001363846.1:c.1289G>A;p.(Arg430His) while in Family 3 we identified a synonymous variant in the adjacent nucleotide, NM_001363846.1:c.1290C>A;p.(Arg430Arg). Using RNA and protein analysis of brain autopsy samples, and a mini-gene splicing reporter assay, we demonstrate both variants result in the upregulation of the lambda isoform. Our approach demonstrates the importance of characterizing the effect of GFAP variants on mRNA splicing to inform future pathophysiologic and therapeutic study for Alexander Disease.
-
type ii Alexander Disease caused by splicing errors and aberrant overexpression of an uncharacterized gfap isoform
bioRxiv, 2019Co-Authors: Ming-der Perng, Tracy L. Hagemann, Guy Helman, Asako Takanohashi, Marzena Walkiewicz, Sarah Woidill, Sunetra Sase, Zachary Cross, Ling ZhaoAbstract:Alexander Disease results from gain of function mutations in the gene encoding glial fibrillary acidic protein (GFAP), an intermediate filament protein expressed in astrocytes. At least eight GFAP isoforms have been described, however, the predominant alpha isoform accounts for approximately 90% of GFAP protein in the central nervous system. Here we describe exonic variants identified in three unrelated families with Type II Alexander Disease that alter the splicing of GFAP pre-mRNA and result in upregulation of a previously uncharacterised GFAP lambda isoform (NM_001363846.1). Affected members of Family 1 and Family 2 shared the same missense variant, NM_001363846.1:c.1289G>A;p.(Arg430His) while in Family 3 we identified a synonymous variant in the adjacent nucleotide, NM_001363846.1:c.1290C>A;p.(Arg430Arg). Using RNA and protein analysis of brain autopsy samples, and a mini-gene splicing reporter assay, we demonstrate both variants result in upregulation of the lambda isoform. We assessed other GFAP variants in the ClinVar database for predicted aberrant splicing and, using the same assay, demonstrated significant changes to splicing for two selected variants. In one case, we found that altered splicing due to a +5 intronic variant resulted in the inclusion of the GFAP kappa isoform. Our approach demonstrates the importance of characterizing the effect of GFAP variants on mRNA splicing in order to inform future pathophysiologic and therapeutic study for Alexander Disease.
-
Identification of a novel nonsense mutation in the rod domain of GFAP that is associated with Alexander Disease
European Journal of Human Genetics, 2015Co-Authors: Tai-seung Nam, Jin Hee Kim, Chi-hsuan Chang, Woong Yoon, Yoon Seok Jung, Sa-yoon Kang, Boo Ahn Shin, Ming-der Perng, Seok-yong Choi, Myeong-kyu KimAbstract: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.
-
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
Experimental Cell Research, 2011Co-Authors: Yisong Chen, Roy A. Quinlan, Suhciuan Lim, Meihsuan Chen, Ming-der PerngAbstract: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.
-
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 αb crystallin and hsp27
American Journal of Human Genetics, 2006Co-Authors: Ming-der Perng, Michael Brenner, Shu Fang Wen, Terry Gibbon, Alan R Prescott, Roy A. QuinlanAbstract: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 αB-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 αB-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.