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1.
J Neuropathol Exp Neurol ; 74(6): 538-46, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25978849

ABSTRACT

Muscle fibers in patients with sporadic inclusion-body myositis (s-IBM),the most common age-associated myopathy, are characterized by autophagic vacuoles and accumulation of ubiquitinated and congophilic multiprotein aggregates that contain amyloid-ß and phosphorylated tau. Muscle fibers of autosomal-recessive hereditary inclusion-body myopathy caused by the GNE mutation (GNE-h-IBM) display similar pathologic features, except with less pronounced congophilia. Accumulation of unfolded/misfolded proteins inside the endoplasmic reticulum (ER) lumen leads to ER stress, which elicits the unfolded protein response (UPR) as a protective mechanism. Here we demonstrate for the first time that UPR is activated in s-IBM muscle biopsies, since there was 1) increased activating transcription factor 4 (ATF4) protein and increased mRNA of its target C/EBP homologous protein; 2) cleavage of the ATF6 and increased mRNA of its target glucose-regulated protein 78; and 3) an increase of the spliced form of X-box binding protein 1 and increased mRNA of ER degradation-enhancing α-mannosidase-like protein, target of heterodimer of cleaved ATF6 and spliced X-box binding protein 1. In contrast, we did not find similar evidence of the UPR induction in GNE-h-IBM patient muscle, suggesting that different intracellular mechanisms might lead to similar pathologic phenotypes. Interestingly, cultured GNE-h-IBM muscle fibers had a robust UPR response to experimental ER stress stimuli, suggesting that the GNE mutation per se is not responsible for the lack of UPR in GNE-h-IBM biopsied muscle.


Subject(s)
Distal Myopathies/pathology , Distal Myopathies/physiopathology , Muscle, Skeletal/metabolism , Myositis, Inclusion Body/pathology , Myositis, Inclusion Body/physiopathology , Unfolded Protein Response/physiology , Activating Transcription Factor 4/metabolism , Activating Transcription Factor 6/metabolism , Aged , Cadherins/metabolism , Cells, Cultured , DNA-Binding Proteins/metabolism , Distal Myopathies/genetics , Endoplasmic Reticulum Chaperone BiP , Enzyme Inhibitors/pharmacology , Female , Heat-Shock Proteins/metabolism , Humans , Male , Middle Aged , Multienzyme Complexes/genetics , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/pathology , Mutation/genetics , RNA, Messenger/metabolism , Regulatory Factor X Transcription Factors , Transcription Factors/metabolism , Unfolded Protein Response/genetics
3.
Biochim Biophys Acta ; 1852(4): 633-43, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25241263

ABSTRACT

Sporadic inclusion-body myositis (s-IBM) is the most common degenerative muscle disease in which aging appears to be a key risk factor. In this review we focus on several cellular molecular mechanisms responsible for multiprotein aggregation and accumulations within s-IBM muscle fibers, and their possible consequences. Those include mechanisms leading to: a) accumulation in the form of aggregates within the muscle fibers, of several proteins, including amyloid-ß42 and its oligomers, and phosphorylated tau in the form of paired helical filaments, and we consider their putative detrimental influence; and b) protein misfolding and aggregation, including evidence of abnormal myoproteostasis, such as increased protein transcription, inadequate protein disposal, and abnormal posttranslational modifications of proteins. Pathogenic importance of our recently demonstrated abnormal mitophagy is also discussed. The intriguing phenotypic similarities between s-IBM muscle fibers and the brains of Alzheimer and Parkinson's disease patients, the two most common neurodegenerative diseases associated with aging, are also discussed. This article is part of a Special Issue entitled: Neuromuscular Diseases: Pathology and Molecular Pathogenesis.


Subject(s)
Aging , Brain , Muscle Fibers, Skeletal , Myositis, Inclusion Body , Protein Aggregation, Pathological , Proteostasis Deficiencies , Aging/metabolism , Aging/pathology , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid beta-Peptides , Animals , Brain/metabolism , Brain/pathology , Humans , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Myositis, Inclusion Body/metabolism , Myositis, Inclusion Body/pathology , Parkinson Disease/metabolism , Parkinson Disease/pathology , Peptide Fragments , Protein Aggregation, Pathological/metabolism , Protein Aggregation, Pathological/pathology , Protein Biosynthesis , Protein Processing, Post-Translational , Proteostasis Deficiencies/metabolism , Proteostasis Deficiencies/mortality
4.
Neurobiol Dis ; 65: 93-101, 2014 May.
Article in English | MEDLINE | ID: mdl-24472798

ABSTRACT

Sporadic inclusion-body myositis (s-IBM) is a severe, progressive muscle disease for which there is no enduring treatment. Pathologically characteristic are vacuolated muscle fibers having: accumulations of multi-protein aggregates, including amyloid-ß(Aß) 42 and its toxic oligomers; increased γ-secretase activity; and impaired autophagy. Cultured human muscle fibers with experimentally-impaired autophagy recapitulate some of the s-IBM muscle abnormalities, including vacuolization and decreased activity of lysosomal enzymes, accompanied by increased Aß42, Aß42 oligomers, and increased γ-secretase activity. Sodium phenylbutyrate (NaPB) is an orally bioavailable small molecule approved by the FDA for treatment of urea-cycle disorders. Here we describe that NaPB treatment reverses lysosomal dysfunction in an in vitro model of inclusion-body myositis, involving cultured human muscle fibers. NaPB treatment improved lysosomal activity, decreased Aß42 and its oligomers, decreased γ-secretase activity, and virtually prevented muscle-fiber vacuolization. Accordingly, NaPB might be considered a potential treatment of s-IBM patients.


Subject(s)
Amyloid beta-Peptides/pharmacology , Muscle Fibers, Skeletal/drug effects , Peptide Fragments/pharmacology , Thiazines/pharmacology , Amyloid Precursor Protein Secretases/metabolism , Analysis of Variance , Cathepsin D/metabolism , Cell Culture Techniques , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Epoxy Compounds/metabolism , Gene Expression Regulation/drug effects , Green Fluorescent Proteins/genetics , Humans , Lysosomes/drug effects , Lysosomes/metabolism , Microtubule-Associated Proteins/genetics , Transfection , Tyrosine/analogs & derivatives , Tyrosine/metabolism
6.
Chembiochem ; 14(5): 607-16, 2013 Mar 18.
Article in English | MEDLINE | ID: mdl-23450708

ABSTRACT

Small hydrophobic ligands identifying intracellular protein deposits are of great interest, as protein inclusion bodies are the pathological hallmark of several degenerative diseases. Here we report that fluorescent amyloid ligands, termed luminescent conjugated oligothiophenes (LCOs), rapidly and with high sensitivity detect protein inclusion bodies in skeletal muscle tissue from patients with sporadic inclusion body myositis (s-IBM). LCOs having a conjugated backbone of at least five thiophene units emitted strong fluorescence upon binding, and showed co-localization with proteins reported to accumulate in s-IBM protein inclusion bodies. Compared with conventional amyloid ligands, LCOs identified a larger fraction of immunopositive inclusion bodies. When the conjugated thiophene backbone was extended with terminal carboxyl groups, the LCO revealed striking spectral differences between distinct protein inclusion bodies. We conclude that 1) LCOs are sensitive, rapid and powerful tools for identifying protein inclusion bodies and 2) LCOs identify a wider range of protein inclusion bodies than conventional amyloid ligands.


Subject(s)
Fluorescent Dyes/chemistry , Proteins/chemistry , Thiophenes/chemistry , Adaptor Proteins, Signal Transducing/analysis , Adaptor Proteins, Signal Transducing/chemistry , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/metabolism , Biosensing Techniques , Humans , Inclusion Bodies/chemistry , Inclusion Bodies/metabolism , Ligands , Microscopy, Fluorescence , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Myositis, Inclusion Body/metabolism , Myositis, Inclusion Body/pathology , Proteins/analysis , Sequestosome-1 Protein
7.
Autophagy ; 8(4): 445-544, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22966490

ABSTRACT

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.


Subject(s)
Autophagy , Biological Assay/methods , Animals , Autophagy/genetics , Humans , Models, Biological
8.
J Neuropathol Exp Neurol ; 71(8): 680-93, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22805774

ABSTRACT

The pathogenesis of sporadic inclusion-body myositis (s-IBM) is complex; it involves multidimensional pathways and the most critical issues are still unresolved. The onset of muscle fiber damage is age related and the disease is slowly, but inexorably, progressive. Muscle fiber degeneration and mononuclear cell inflammation are major components of s-IBM pathology, but which is precedent and how they interrelate is not known. There is growing evidence that aging of the muscle fiber associated with intramyofiber accumulation of conformationally modified proteins plays a primary pathogenic role leading to muscle fiber destruction. Here, we review the presumably most important known molecular abnormalities that occur in s-IBM myofibers and that likely contribute to s-IBM pathogenesis. Abnormal accumulation within the fibers of multiprotein aggregates (several of which are congophilic and, therefore, generically called "amyloid") may result from increased transcription of several proteins, their abnormal posttranslational modifications and misfolding, and inadequate protein disposal, that is, abnormal "myoproteostasis," which is combined with and may be provoked or abetted by an aging intracellular milieu. The potential cytotoxicity of accumulated amyloid ß protein (Aß42) and its oligomers, phosphorylated tau in the form of paired helical filaments and α-synuclein, and the putative pathogenic role and cause of the mitochondrial abnormalities and oxidative stress are reviewed. On the basis of our experimental evidence, potential interventions in the complex, interwoven pathogenic cascade of s-IBM are suggested.


Subject(s)
Aging/pathology , Muscle Proteins/metabolism , Myositis, Inclusion Body/metabolism , Myositis, Inclusion Body/pathology , Proteostasis Deficiencies/metabolism , Proteostasis Deficiencies/pathology , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/biosynthesis , Amyloid beta-Protein Precursor/genetics , Autophagy , Biopsy , Humans , Lithium Compounds/therapeutic use , Mitochondria, Muscle/metabolism , Muscle Fibers, Skeletal/pathology , Muscle, Skeletal/pathology , Myositis, Inclusion Body/diagnosis , Myositis, Inclusion Body/drug therapy , Peptide Fragments/metabolism , Phenylbutyrates/therapeutic use , Polyphenols/therapeutic use , Proteasome Endopeptidase Complex/metabolism , Proteostasis Deficiencies/diagnosis , Proteostasis Deficiencies/drug therapy , alpha-Synuclein/metabolism , tau Proteins/metabolism
9.
Neurobiol Dis ; 48(1): 141-9, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22750528

ABSTRACT

The muscle-fiber phenotype of sporadic inclusion-body myositis (s-IBM), the most common muscle disease associated with aging, shares several pathological abnormalities with Alzheimer disease (AD) brain, including accumulation of amyloid-ß 42 (Aß42) and its cytotoxic oligomers. The exact mechanisms leading to Aß42 production within s-IBM muscle fibers are not known. Aß42 and Aß40 are generated after the amyloid-precursor protein (AßPP) is cleaved by ß-secretase and the γ-secretase complex. Aß42 is considered more cytotoxic than Aß40, and it has a higher propensity to oligomerize, form amyloid fibrils, and aggregate. Recently, we have demonstrated in cultured human muscle fibers that experimental inhibition of lysosomal enzyme activities leads to Aß42 oligomerization. In s-IBM muscle, we here demonstrate prominent abnormalities of the γ-secretase complex, as evidenced by: a) increase of γ-secretase components, namely active presenilin 1, presenilin enhancer 2, nicastrin, and presence of its mature, glycosylated form; b) increase of mRNAs of these γ-secretase components; c) increase of γ-secretase activity; d) presence of an active form of a newly-discovered γ-secretase activating protein (GSAP); and e) increase of GSAP mRNA. Furthermore, we demonstrate that experimental inhibition of lysosomal autophagic enzymes in cultured human muscle fibers a) activates γ-secretase, and b) leads to posttranslational modifications of AßPP and increase of Aß42. Since autophagy is impaired in biopsied s-IBM muscle, the same mechanism might be responsible for its having increased γ-secretase activity and Aß42 production. Accordingly, improving lysosomal function might be a therapeutic strategy for s-IBM patients.


Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/metabolism , Muscle, Skeletal/metabolism , Myositis, Inclusion Body/metabolism , Peptide Fragments/metabolism , Proteins/metabolism , Aged , Aged, 80 and over , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Humans , Middle Aged , Muscle, Skeletal/pathology , Myositis, Inclusion Body/pathology , Polymyositis/metabolism , Polymyositis/pathology
10.
Acta Neuropathol ; 122(5): 627-36, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21935636

ABSTRACT

Intra-muscle fiber accumulation of ubiquitinated protein aggregates containing several conformationally modified proteins, including amyloid-ß and phosphorylated tau, is characteristic of the pathologic phenotype of sporadic inclusion-body myositis (s-IBM), the most common progressive degenerative myopathy of older persons. Abnormalities of protein-degradation, involving both the 26S proteasome and autophagic-lysosomal pathways, were previously demonstrated in s-IBM muscle. NBR1 is a ubiquitin-binding scaffold protein importantly participating in autophagic degradation of ubiquitinated proteins. Whereas abnormalities of p62, a ubiquitin-binding protein, were previously described in s-IBM, abnormalities of NBR1 have not been reported in s-IBM. We have now identified in s-IBM muscle biopsies that NBR1, by: (a) immunohistochemistry, was strongly accumulated within s-IBM muscle-fiber aggregates, where it closely co-localized with p62, ubiquitin, and phosphorylated tau; (b) immunoblots, was increased threefold (p < 0.001); and (c) immunoprecipitation, was associated with p62 and LC3. By real-time PCR, NBR1 mRNA was increased twofold (p < 0.01). None of the various disease- and normal-control muscle biopsies had any NBR1 abnormality. In cultured human muscle fibers, NBR1 also physically associated with both p62 and LC3, and experimental inhibition of either the 26S proteasome or the lysosomal activity resulted in NBR1 increase. Our demonstration of NBR1 abnormalities in s-IBM provides further evidence that altered protein degradation pathways may be critically involved in the s-IBM pathogenesis. Accordingly, attempts to unblock defective protein degradation might be a therapeutic strategy for s-IBM patients.


Subject(s)
Autophagy/physiology , Muscle Fibers, Skeletal/metabolism , Myositis, Inclusion Body/metabolism , Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Aged , Aged, 80 and over , Biopsy , Case-Control Studies , Cells, Cultured , Humans , Intracellular Signaling Peptides and Proteins , Microtubule-Associated Proteins/metabolism , Middle Aged , Muscle Fibers, Skeletal/pathology , Muscle, Skeletal , Myositis, Inclusion Body/pathology , RNA, Messenger/metabolism , Sequestosome-1 Protein
11.
Neurosci Lett ; 503(3): 229-33, 2011 Oct 10.
Article in English | MEDLINE | ID: mdl-21896314

ABSTRACT

s-IBM is the most common muscle disease of older persons. Its muscle fiber molecular phenotype has close similarities to Alzheimer disease (AD) brain, including intra-muscle-fiber accumulations of (a) Aß42 and its oligomers, and (b) large, squiggly or linear, clusters of paired-helical filaments (PHFs) that are immunoreactive with various antibodies directed against several epitopes of phosphorylated tau (p-tau), and thereby strongly resembling neurofibrillary tangles of AD brain. In AD brain, conformational changes of tau, including its modifications detectable with specific antibodies TG3 (recognizing phosphorylated-Thr231), and Alz50 and MC1 (both recognizing amino acids 5-15 and 312-322) are considered early and important modifications leading to tau's abnormal folding and assembly into PHFs. We have now identified conformationally modified tau in 14 s-IBM muscle biopsies by (a) light-and electron-microscopic immunohistochemistry, (b) immunoblots, and (c) dot-immunoblots, using TG3, Alz50 and MC1 antibodies. Our double-immunolabeling on the light- and electron-microscopic levels, which combined an antibody against p62 that recognizes s-IBM clusters of PHFs, revealed that TG3 immunodecorated, abundantly and exclusively, all p62 immunopositive clusters, while Alz50 labeling was less abundant, and MC1 was mainly diffusely immunoreactive. Interestingly, in the very atrophic degenerating fibers, TG3 co-localized with PHF-1 antibody that recognizes tau phosphorylated at Ser396/404, which is considered a later change in the formation of PHFs; however, most of TG3-positive inclusions in non-atrophic fibers were immunonegative with PHF-1. None of the 12 normal- and disease-control muscle biopsies contained conformational or PHF-1 immunoreactive tau. This first demonstration of conformational tau in s-IBM, because of its abundance in non-atrophic muscle fibers, suggests that it might play an early role in s-IBM PHFs formation and thus be pathogenically important.


Subject(s)
Muscle Fibers, Skeletal/metabolism , Myositis, Inclusion Body/metabolism , tau Proteins/metabolism , Aged , Alzheimer Disease/pathology , Antibodies, Monoclonal , Antibody Specificity , Biopsy , Blotting, Western , Endoplasmic Reticulum/chemistry , Endoplasmic Reticulum/metabolism , Female , Fluorescent Antibody Technique , Humans , Immunohistochemistry , Male , Microscopy, Immunoelectron , Middle Aged , Muscle Fibers, Skeletal/ultrastructure , Phosphorylation , Protein Conformation , tau Proteins/chemistry
12.
Presse Med ; 40(4 Pt 2): e219-35, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21392932

ABSTRACT

The pathogenesis of sporadic inclusion-body myositis (s-IBM), the most common muscle disease of older persons, is complex and multifactorial. Both the muscle fiber degeneration and the mononuclear-cell inflammation are components of the s-IBM pathology, but how each relates to the pathogenesis remains unsettled. We consider that the intramuscle fiber degenerative component plays the primary and the major pathogenic role leading to muscle fiber destruction and clinical weakness. In this article we review the newest research advances that provide a better understanding of the s-IBM pathogenesis. Cellular abnormalities occurring in s-IBM muscle fibers are discussed, including: several proteins that are accumulated in the form of aggregates within muscle fibers, including amyloid-ß42 and its oligomers, and phosphorylated tau in the form of paired helical filaments, and we consider their putative detrimental influence; cellular mechanisms leading to protein misfolding and aggregation, including evidence of their inadequate disposal; pathogenic importance of endoplasmic reticulum stress and the unfolded protein response demonstrated in s-IBM muscle fibers; and decreased deacetylase activity of SIRT1. All these factors are combined with, and perhaps provoked by, an ageing intracellular milieu. Also discussed are the intriguing phenotypic similarities between s-IBM muscle fibers and the brains of Alzheimer and Parkinson's disease patients, the two most common neurodegenerative diseases associated with ageing. Muscle biopsy diagnostic criteria are also described and illustrated.


Subject(s)
Amyloid beta-Peptides/metabolism , Endoplasmic Reticulum/physiology , Lysosomes/metabolism , Muscle Fibers, Skeletal/physiology , Myositis, Inclusion Body/physiopathology , Peptide Fragments/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Conformation , tau Proteins/metabolism , Age Factors , Autophagy/physiology , Biopsy , Endoplasmic Reticulum/pathology , Humans , Lysosomes/pathology , Microscopy, Electron , Muscle Fibers, Skeletal/pathology , Muscles/pathology , Muscles/physiopathology , Myositis, Inclusion Body/pathology , Myostatin/metabolism , Phosphorylation , Proteostasis Deficiencies/pathology , Proteostasis Deficiencies/physiopathology , Sirtuin 1/metabolism , Ubiquitin-Protein Ligases/metabolism , alpha-Synuclein/metabolism
13.
Acta Neuropathol ; 120(5): 661-6, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20711838

ABSTRACT

Accumulation of amyloid-ß (Aß) within muscle fibers has been considered an upstream step in the development of the s-IBM pathologic phenotype. Aß42, which is considered more cytotoxic than Aß40 and has a higher propensity to oligomerize, is preferentially increased in s-IBM muscle fibers. In Alzheimer disease (AD), low-molecular weight Aß oligomers and toxic oligomers, also referred to as "Aß-Derived Diffusible Ligands" (ADDLs), are considered strongly cytotoxic and proposed to play an important pathogenic role. ADDLs have been shown to be increased in AD brain. We now report for the first time that in s-IBM muscle biopsies Aß-dimer, -trimer, and -tetramer are identifiable by immunoblots. While all the s-IBM samples we studied had Aß-oligomers, their molecular weights and intensity varied between the patient samples. None of the control muscle biopsies had Aß oligomers. Dot-immunoblots using highly specific anti-ADDL monoclonal antibodies also showed highly increased ADDLs in all s-IBM biopsies studied, while controls were negative. By immunofluorescence, in some of the abnormal s-IBM muscle fibers ADDLs were accumulated in the form of plaque-like inclusions, and were often increased diffusely in very small fibers. Normal and disease-controls were negative. By gold-immuno-electron microscopy, ADDL-immunoreactivities were in close proximity to 6-10 nm amyloid-like fibrils, and also were immunodecorating amorphous and floccular material. In cultured human muscle fibers, we found that inhibition of autophagy led to the accumulation of Aß oligomers. This novel demonstration of Aß42 oligomers in s-IBM muscle biopsy provides additional evidence that intra-muscle fiber accumulation of Aß42 oligomers in s-IBM may contribute importantly to s-IBM pathogenic cascade.


Subject(s)
Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/metabolism , Muscle, Skeletal/chemistry , Muscle, Skeletal/metabolism , Myositis, Inclusion Body/metabolism , Blotting, Western , Humans , Immunoblotting , Immunohistochemistry , Microscopy, Immunoelectron , Muscle, Skeletal/pathology , Myositis, Inclusion Body/pathology
14.
Am J Pathol ; 177(3): 1377-87, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20616343

ABSTRACT

The hallmark pathologies of sporadic inclusion-body myositis (s-IBM) muscle fibers are autophagic vacuoles and accumulation of ubiquitin-positive multiprotein aggregates that contain amyloid-beta or phosphorylated tau in a beta-pleated sheet amyloid configuration. Endoplasmic reticulum stress (ERS) and 26S proteasome inhibition, also associated with s-IBM, putatively aggrandize the accumulation of misfolded proteins. However, autophagosomal-lysosomal pathway formation and function, indicated by autophagosome maturation, have not been previously analyzed in this system. Here we studied the autophagosomal-lysosomal pathway using 14 s-IBM and 30 disease control and normal control muscle biopsy samples and our cultured human muscle fibers in a microenvironment modified to resemble aspects of s-IBM pathology. We report for the first time that in s-IBM, lysosomal enzyme activities of cathepsin D and B were decreased 60% (P < 0.01) and 40% (P < 0.05), respectively. We also detected two indicators of increased autophagosome maturation, the presence of LC3-II and decreased mammalian target of rapamycin-mediated phosphorylation of p70S6 kinase. Moreover, in cultured human muscle fibers, ERS induction significantly decreased activities of cathepsins D and B, increased levels of LC3-II, decreased phosphorylation of p70S6 kinase, and decreased expression of VMA21, a chaperone for assembly of lysosomal V-ATPase. We conclude that in s-IBM muscle, decreased lysosomal proteolytic activity might enhance accumulation of misfolded proteins, despite increased maturation of autophagosomes, and that ERS is a possible cause of s-IBM-impaired lysosomal function. Thus, unblocking protein degradation in s-IBM muscle fibers may be a desirable therapeutic strategy.


Subject(s)
Autophagy/physiology , Endoplasmic Reticulum/metabolism , Muscle Fibers, Skeletal/metabolism , Myositis, Inclusion Body/metabolism , Aged , Blotting, Western , Cathepsin B/metabolism , Cathepsin D/metabolism , Cells, Cultured , Endoplasmic Reticulum/pathology , Humans , Immunohistochemistry , Lysosomes/metabolism , Lysosomes/pathology , Middle Aged , Myositis, Inclusion Body/pathology
15.
Neurosci Lett ; 474(3): 140-143, 2010 May 03.
Article in English | MEDLINE | ID: mdl-20236612

ABSTRACT

Sporadic inclusion-body myositis (s-IBM) is the most common muscle disease of older persons. Its muscle-fiber phenotype shares several molecular similarities with Alzheimer-disease (AD) brain, including increased AbetaPP, accumulation of amyloid-beta (Abeta), and increased BACE1 protein. Abeta42 is prominently increased in AD brain and within s-IBM fibers, and its oligomers are putatively toxic to both tissues--accordingly, minimizing Abeta42 production can be a therapeutic objective in both tissues. The pathogenic development of s-IBM is unknown, including the mechanisms of BACE1 protein increase. BACE1 is an enzyme essential for production from AbetaPP of Abeta42 and Abeta40, which are proposed to be detrimental within s-IBM muscle fibers. Novel noncoding BACE1-antisense (BACE1-AS) was recently shown (a) to be increased in AD brain, and (b) to increase BACE1 mRNA and BACE1 protein. We studied BACE1-AS and BACE1 transcripts by real-time PCR (a) in 10 s-IBM and 10 age-matched normal muscle biopsies; and (b) in our established ER-Stress-Human-Muscle-Culture-IBM Model, in which we previously demonstrated increased BACE1 protein. Our study demonstrated for the first time that (a) in s-IBM biopsies BACE1-AS and BACE1 transcripts were significantly increased, suggesting that their increased expression can be responsible for the increase of BACE1 protein; and (b) experimental induction of ER stress significantly increased both BACE1-AS and BACE1 transcripts, suggesting that ER stress can participate in their induction in s-IBM muscle. Accordingly, decreasing BACE1 through a targeted downregulation of its regulatory BACE1-AS, or reducing ER stress, might be therapeutic strategies in s-IBM, assuming that it would not impair any normal cellular functions of BACE1.


Subject(s)
Amyloid Precursor Protein Secretases/biosynthesis , Aspartic Acid Endopeptidases/biosynthesis , DNA, Antisense/biosynthesis , Endoplasmic Reticulum/metabolism , Myositis, Inclusion Body/metabolism , Aged , Endoplasmic Reticulum/pathology , Humans , Middle Aged , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Myositis, Inclusion Body/genetics , Myositis, Inclusion Body/pathology , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Stress, Physiological/physiology
16.
J Neurochem ; 112(2): 389-96, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19878439

ABSTRACT

Muscle fiber degeneration in sporadic inclusion-body myositis (s-IBM) is characterized by accumulation of multiprotein aggregates, including aggregated amyloid-beta (Abeta)-precursor protein 751 (AbetaPP751), Abeta, phosphorylated tau, and other 'Alzheimer-characteristic' proteins. Proteasome inhibition is an important component of the s-IBM pathogenesis. In brains of Alzheimer's disease (AD) patients and AD transgenic-mouse models, phosphorylation of neuronal AbetaPP695 (p-AbetaPP) on Thr668 (equivalent to T724 of AbetaPP751) is considered detrimental because it increases generation of cytotoxic Abeta and induces tau phosphorylation. Activated glycogen synthase kinase3beta (GSK3beta) is involved in phosphorylation of both AbetaPP and tau. Lithium, an inhibitor of GSK3beta, was reported to reduce levels of both the total AbetaPP and p-AbetaPP in AD animal models. In relation to s-IBM, we now show for the first time that (1) In AbetaPP-overexpressing cultured human muscle fibers (human muscle culture IBM model: (a) proteasome inhibition significantly increases GSK3beta activity and AbetaPP phosphorylation, (b) treatment with lithium decreases (i) phosphorylated-AbetaPP, (ii) total amount of AbetaPP, (iii) Abeta oligomers, and (iv) GSK3beta activity; and (c) lithium improves proteasome function. (2) In biopsied s-IBM muscle fibers, GSK3beta is significantly activated and AbetaPP is phosphorylated on Thr724. Accordingly, treatment with lithium, or other GSK3beta inhibitors, might benefit s-IBM patients.


Subject(s)
Amyloid beta-Protein Precursor/metabolism , Glycogen Synthase Kinase 3/metabolism , Lithium Chloride/pharmacology , Muscle Fibers, Skeletal/drug effects , Myositis, Inclusion Body/pathology , Proteasome Endopeptidase Complex/metabolism , Aged , Aged, 80 and over , Amyloid beta-Protein Precursor/genetics , Case-Control Studies , Endoplasmic Reticulum/drug effects , Enzyme Inhibitors/pharmacology , Female , Glycogen Synthase Kinase 3 beta , Humans , Immunoprecipitation/methods , Male , Middle Aged , Muscle Fibers, Skeletal/metabolism , Oxidative Stress/physiology , Phosphorylation/drug effects , Proteasome Endopeptidase Complex/drug effects , Proteasome Inhibitors , Protein Isoforms/metabolism , Threonine/metabolism , Tissue Culture Techniques
17.
Neurobiol Aging ; 31(9): 1637-48, 2010 Sep.
Article in English | MEDLINE | ID: mdl-18922603

ABSTRACT

SIRT1 belongs to the sirtuin family of NAD(+)-dependent histone/protein deacetylases. Experimentally, increased activity of SIRT1 facilitates calorie-restricted longevity, and decreases NF-kappaB activation and the amount of the amyloid-beta (Abeta). We studied SIRT1 in an aging-associated muscle disease, sporadic inclusion-body myositis (s-IBM), whose muscle fibers contain increased NF-kappaB activation and abnormal accumulation of Abeta. We show that, as compared to the age-matched controls, in s-IBM muscle fibers: (1) SIRT1 activity and deacetylation of SIRT1 targets, H4, NF-kappaB and p53 were decreased; (2) SIRT1 mRNA and protein were significantly increased; (3) in the cytoplasm, SIRT1 protein was accumulated in the form of cytoplasmic aggregates; (4) in the nuclei, SIRT1 protein was decreased. To our knowledge, this is the first demonstration of SIRT1 abnormalities, including decreased SIRT1 deacetylase activity, in human disease associated with aging. We propose that in s-IBM muscle fibers, inadequate activity of SIRT1 may be detrimental by increasing NF-kappaB activation and contributing to abnormal Abeta accumulation. Improving SIRT1 action by treatment with known SIRT1 activators might benefit s-IBM patients.


Subject(s)
Muscle Fibers, Skeletal/enzymology , Myositis, Inclusion Body/enzymology , Sirtuin 1/metabolism , Aged , Aged, 80 and over , Down-Regulation , Enzyme Activation , Female , Humans , Male , Middle Aged , Tissue Distribution
18.
Brain Pathol ; 19(3): 493-506, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19563541

ABSTRACT

Sporadic inclusion body myositis (s-IBM), the most common muscle disease of older persons, is of unknown cause, and there is no enduring treatment. Abnormal accumulation of intracellular multi-protein inclusions is a characteristic feature of the s-IBM phenotype, and as such s-IBM can be considered a "conformational disorder," caused by protein unfolding/misfolding combined with the formation of inclusion bodies. Abnormal intracellular accumulation of unfolded proteins may lead to their aggregation and inclusion body formation. The present article is focusing on the multiple proteins that are accumulated in the form of aggregates within s-IBM muscle fibers, and it explores the most recent research advances directed toward a better understanding of mechanisms causing their impaired degradation and abnormal aggregation. We illustrate that, among other factors, abnormal misfolding, accumulation and aggregation of proteins are associated with their inadequate disposal-and these factors are combined with, and perhaps provoked by, an aging intracellular milieu. Other concurrent and possibly provocative phenomena known within s-IBM muscle fibers are: endoplasmic reticulum stress and unfolded protein response, mitochondrial abnormalities, proteasome inhibition, lysosome abnormality and endodissolution. Together, these appear to lead to the s-IBM-specific vacuolar degeneration, and muscle fiber atrophy, concluding with muscle fiber death.


Subject(s)
Myositis, Inclusion Body/metabolism , Myositis, Inclusion Body/pathology , Humans , Protein Folding , Proteins/metabolism
19.
Acta Neuropathol ; 118(3): 407-13, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19557423

ABSTRACT

p62, also known as sequestosome1, is a shuttle protein transporting polyubiquitinated proteins for both the proteasomal and lysosomal degradation. p62 is an integral component of inclusions in brains of various neurodegenerative disorders, including Alzheimer disease (AD) neurofibrillary tangles (NFTs) and Lewy bodies in Parkinson disease. In AD brain, the p62 localized in NFTs is associated with phosphorylated tau (p-tau). Sporadic inclusion-body myositis (s-IBM) is the most common progressive muscle disease associated with aging, and its muscle tissue has several phenotypic similarities to AD brain. Abnormal accumulation of intracellular multiprotein inclusions, containing p-tau in the form of paired helical filaments, amyloid-beta, and several other "Alzheimer-characteristic proteins", is a characteristic feature of the s-IBM muscle fiber phenotype. Diminished proteasomal and lysosomal protein degradation appear to play an important role in the formation of intra-muscle-fiber inclusions. We now report that: (1) in s-IBM muscle fibers, p62 protein is increased on both the protein and the mRNA levels, and it is strongly accumulated within, and as a dense peripheral shell surrounding, p-tau containing inclusions, by both the light- and electron-microscopy. Accordingly, our studies provide a new, reliable, and simple molecular marker of p-tau inclusions in s-IBM muscle fibers. The prominent p62 immunohistochemical positivity and pattern diagnostically distinguish s-IBM from polymyositis and dermatomyositis. (2) In normal cultured human muscle fibers, experimental inhibition of either proteasomal or lysosomal protein degradation caused substantial increase of p62, suggesting that similar in vivo mechanisms might contribute to the p62 increase in s-IBM muscle fibers.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Myositis, Inclusion Body/metabolism , Adaptor Proteins, Signal Transducing/genetics , Aged , Aged, 80 and over , Biomarkers , Blotting, Western , Cells, Cultured , Dermatomyositis/diagnosis , Diagnosis, Differential , Gene Expression Profiling , Humans , Immunohistochemistry , Lysosomes/metabolism , Microscopy, Fluorescence , Microscopy, Immunoelectron , Middle Aged , Muscle Fibers, Skeletal/cytology , Myositis, Inclusion Body/diagnosis , Myositis, Inclusion Body/genetics , Phosphorylation , Polymyositis/diagnosis , Proteasome Endopeptidase Complex/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequestosome-1 Protein , tau Proteins/metabolism
20.
Acta Neuropathol ; 117(5): 569-74, 2009 May.
Article in English | MEDLINE | ID: mdl-19280202

ABSTRACT

Sporadic inclusion-body myositis (s-IBM) is the only muscle disease in which accumulation of amyloid-beta (Abeta) in abnormal muscle fibers appears to play a key pathogenic role. Increased amyloid-beta precursor protein (AbetaPP) and Abeta accumulation have been reported to be upstream steps in the development of the s-IBM pathologic phenotype, based on cellular and animal models. Abeta is released from AbetaPP as a 40 or 42 aminoacid peptide. Abeta42 is considered more cytotoxic than Abeta40, and it has a higher propensity to aggregate and form amyloid fibrils. Using highly specific antibodies, we evaluated in s-IBM muscle biopsies intra-muscle fiber accumulation of Abeta40 and Abeta42-immunoreactive aggregates by light- and electron-microscopic immunocytochemistry, and quantified their amounts by ELISA. In s-IBM, 80-90% of the vacuolated muscle fibers and 5-20% of the non-vacuolated muscle fibers contained plaque-like Abeta42-immunoreactive inclusions, while only 69% of those fibers also contained Abeta40 deposits. By immuno-electronmicroscopy, Abeta42 was associated with 6-10 nm amyloid-like fibrils, small electron-dense floccular clumps and larger masses of amorphous material. Abeta40 was present only on small patches of floccular clumps and amorphous material; it was not associated with 6-10 nm amyloid fibrils. By ELISA, in s-IBM muscle biopsies Abeta42 was present in values 8.53-44.7 pg/ml, while Abeta40 was not detectable; normal age-matched control biopsies did not have any detectable Abeta42 or Abeta40. Thus, in s-IBM muscle fibers, Abeta42 is accumulated more than Abeta40. We suggest that Abeta42 oligomers and their cytotoxicity may play an important role in the s-IBM pathogenesis.


Subject(s)
Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Myositis, Inclusion Body/metabolism , Myositis, Inclusion Body/pathology , Peptide Fragments/metabolism , Biopsy , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Humans , Immunohistochemistry , Inclusion Bodies/metabolism , Inclusion Bodies/pathology , Microscopy, Immunoelectron , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Muscle, Skeletal/ultrastructure , Myositis, Inclusion Body/etiology
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