Inclusion-body Myositis Muscle Fibers Research Articles

Association between TDP-43 and mitochondria in inclusion body myositis

Inclusion body myositis (IBM) is the most common cause of primary myopathy in individuals aged 50 years and over, and is pathologically characterized by protein aggregates of p62 and mislocalized cytoplasmic TDP-43, as well as mitochondrial abnormalities in affected muscle fibers. Our recent studies have shown the accumulation of TDP-43 in mitochondria in neurons from patients with amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD), and revealed mitochondria as critical mediators of TDP-43 neurotoxicity. In this study, we investigated the association between mitochondria and TDP-43 in biopsied skeletal muscle samples from IBM patients. We found that IBM pathological markers TDP-43, phosphorylated TDP-43, and p62 all coexisted with intensively stained key subunits of mitochondrial oxidative phosphorylation complexes I–V in the same skeletal muscle fibers of patients with IBM. Further immunoblot analysis showed increased levels of TDP-43, truncated TDP-43, phosphorylated TDP-43, and p62, but decreased levels of key subunits of mitochondrial oxidative phosphorylation complexes I and III in IBM patients compared to aged matched control subjects. This is the first demonstration of the close association of TDP-43 accumulation with mitochondria in degenerating muscle fibers in IBM and this association may contribute to the development of mitochondrial dysfunction and pathological protein aggregates.

Activation of the γ-secretase complex and presence of γ-secretase-activating protein may contribute to Aβ42 production in sporadic inclusion-body myositis muscle fibers

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-β 42 (Aβ42) and its cytotoxic oligomers. The exact mechanisms leading to Aβ42 production within s-IBM muscle fibers are not known.Aβ42 and Aβ40 are generated after the amyloid-precursor protein (AβPP) is cleaved by β-secretase and the γ-secretase complex. Aβ42 is considered more cytotoxic than Aβ40, 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 Aβ42 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 AβPP and increase of Aβ42. Since autophagy is impaired in biopsied s-IBM muscle, the same mechanism might be responsible for its having increased γ-secretase activity and Aβ42 production. Accordingly, improving lysosomal function might be a therapeutic strategy for s-IBM patients.

Novel demonstration of conformationally modified tau in sporadic inclusion-body myositis muscle fibers

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) Aβ42 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.

Inclusion Body Myositis: Laser Microdissection Reveals Differential Up-Regulation of IFN-γ Signaling Cascade in Attacked versus Nonattacked Myofibers

Sporadic inclusion body myositis (IBM) is a muscle disease with two separate pathogenic components, degeneration and inflammation. Typically, nonnecrotic myofibers are focally surrounded and invaded by CD8+ T cells and macrophages. Both attacked and nonattacked myofibers express high levels of human leukocyte antigen class I (HLA-I) molecules, a prerequisite for antigen presentation to CD8+ T cells. However, only a subgroup of HLA-I+ myofibers is attacked by immune cells. By using IHC, we classified myofibers from five patients with sporadic IBM as attacked (AIBM) or nonattacked (NIBM) and isolated the intracellular contents of myofibers separately by laser microdissection. For comparison, we isolated myofibers from control persons (HCTRL). The samples were analyzed by microarray hybridization and quantitative PCR. HLA-I up-regulation was observed in AIBM and NIBM, whereas HCTRL were negative for HLA-I. In contrast, the inducible chain of the interferon (IFN) γ receptor (IFNGR2) and several IFN-γ–induced genes were up-regulated in AIBM compared with NIBM and HCTRL fibers. Confocal microscopy confirmed segmental IFNGR2 up-regulation on the membranes of AIBM, which positively correlated with the number of adjacent CD8+ T cells. Thus, the differential up-regulation of the IFN-γ signaling cascade observed in the attacked fibers is related to local inflammation, whereas the ubiquitous HLA-I expression on IBM muscle fibers does not require IFNGR expression.

Novel demonstration of amyloid-β oligomers in sporadic inclusion-body myositis muscle fibers

Accumulation of amyloid-β (Aβ) within muscle fibers has been considered an upstream step in the development of the s-IBM pathologic phenotype. Aβ42, which is considered more cytotoxic than Aβ40 and has a higher propensity to oligomerize, is preferentially increased in s-IBM muscle fibers. In Alzheimer disease (AD), low-molecular weight Aβ oligomers and toxic oligomers, also referred to as "Aβ-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 Aβ-dimer, -trimer, and -tetramer are identifiable by immunoblots. While all the s-IBM samples we studied had Aβ-oligomers, their molecular weights and intensity varied between the patient samples. None of the control muscle biopsies had Aβ 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 Aβ oligomers. This novel demonstration of Aβ42 oligomers in s-IBM muscle biopsy provides additional evidence that intra-muscle fiber accumulation of Aβ42 oligomers in s-IBM may contribute importantly to s-IBM pathogenic cascade.

Increased BACE1 mRNA and noncoding BACE1-antisense transcript in sporadic inclusion-body myositis muscle fibers—Possibly caused by endoplasmic reticulum stress

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 AβPP, accumulation of amyloid-β (Aβ), and increased BACE1 protein. Aβ42 is prominently increased in AD brain and within s-IBM fibers, and its oligomers are putatively toxic to both tissues—accordingly, minimizing Aβ42 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 AβPP of Aβ42 and Aβ40, 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.

Role of cytokines and chemokines in idiopathic inflammatory myopathies

Cytokines and chemokines are essential players in the initiation and progression of the idiopathic inflammatory myopathies (IIMs). This review focuses on the most recent data and the new insight they provide for the disease mechanisms of dermatomyositis, polymyositis and sporadic inclusion body myositis. Interferon-alpha and beta are implicated in the innate immune responses underlying dermatomyositis, whereas interferon-gamma stands forward as a more general regulator of the IIMs, reflected by the induction of many interferon-gamma-inducible genes in patients. Interleukin-1beta and interleukin-18 are localized to the inflammatory cells present in IIM muscle, where they may focally induce further recruitment of immune cells. Lymphotoxins are implicated in the cytotoxic activities toward polymyositis and inclusion body myositis muscle fibers, and in the organization and antibody production by B-cells in dermatomyositis. The alpha-chemokines CXCL9, CXCL10 and the beta-chemokines CCL2, CCL3, CCL4, CCL19 and CCL21 are expressed in IIM muscle. The B-cell activator CXCL13 is particularly prominent in the larger perimysial infiltrates of dermatomyositis. The cytokine-chemokine patterns described in recent studies provide further evidence for predominance of Th1-mediated reactions in the different IIMs, inflammation-induced degenerative phenomena in inclusion body myositis, and a possible role for lymphoneogenesis in the sustained inflammatory response in dermatomyositis.

P62/SQSTM1 is overexpressed and prominently accumulated in inclusions of sporadic inclusion-body myositis muscle fibers, and can help differentiating it from polymyositis and dermatomyositis

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.

Decreased SIRT1 deacetylase activity in sporadic inclusion-body myositis muscle fibers

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.

In inclusion-body myositis muscle fibers Parkinson-associated DJ-1 is increased and oxidized

Sporadic inclusion-body myositis (s-IBM) is the most common muscle disease of older persons. The muscle-fiber molecular phenotype exhibits similarities to both Alzheimer-disease (AD) and Parkinson-disease (PD) brains, including accumulations of amyloid-β, phosphorylated tau, α-synuclein, and parkin, as well as evidence of oxidative stress and mitochondrial abnormalities. Early-onset autosomal-recessive PD can be caused by mutations in the DJ-1 gene, leading to its inactivation. DJ-1 has antioxidative and mitochondrial-protective properties. In AD and PD brains, DJ-1 is increased and oxidized. We studied DJ-1 in 17 s-IBM and 18 disease-control and normal muscle biopsies by: (1) immunoblots of muscle homogenates and mitochondrial fractions; (2) real-time PCR; (3) oxyblots evaluating DJ-1 oxidation; (4) light- and electron-microscopic immunocytochemistry. Compared to controls, in s-IBM muscle fibers DJ-1 was: (a) increased in the soluble fraction, monomer 2-fold ( P = 0.01), and dimer 2.8-fold ( P = 0.004); (b) increased in the mitochondrial fraction; (c) highly oxidized; and (d) aggregated in about 15% of the abnormal muscle fibers. DJ-1 mRNA was increased 3.5-fold ( P = 0.034). Accordingly, DJ-1 might play a role in human muscle disease, and thus not be limited to human CNS degenerations. In s-IBM muscle fibers, DJ-1 could be protecting these fibers against oxidative stress, including protection of mitochondria.

Inclusion-body myositis and Alzheimer disease

Inclusion-body myositis (IBM) is an age-related, slowly progressive degenerative disorder of the skeletal musculature with no effective treatment. Clinically, the disease is characterized by the atrophy of both proximal and distal muscle groups, leading to a gradually advancing weakness.1 The muscle fibers from IBM patients display characteristic vacuolizations and contain heterogeneous, filamentous inclusions.2 Although initially characterized as a form of polymyositis, the disease is only weakly responsive to corticosteroid treatment or other forms of immunomodulation and is now recognized as a distinct disease entity. Aspects of IBM pathology share intriguing similarities to that of Alzheimer disease (AD), the most common form of elderly dementia. The severe cognitive dysfunction in AD is brought about by massive neurodegenerative changes to the brain. The bulk of available evidence implicates a small protein fragment, the amyloid-β peptide (Aβ), as the major causative element in the neurodegenerative process.3,4 In the AD brain, Aβ is deposited extracellularly as amyloid, and is the major proteinaceous component of the neuritic plaques that can be found throughout the cortex. For over a decade, it has been known that Aβ—or larger protein fragments that contain it—also accumulate as part of the characteristic intracellular lesions of IBM.2,5,6 What is unknown is whether or not this is important for IBM pathogenesis. Strong evidence for a role of the amyloid β-protein precursor (APP), APP fragments, Aβ, or all three, in the pathogenesis of IBM comes from several sources.6,7 The appearance of Aβ-positive, noncongophilic deposits precedes vacuolization in IBM muscle fibers.8 Further, APP mRNA is increased in the disease state,9 suggesting that the over expression of the precursor protein may be sufficient to cause pathology. As expected, the in vitro over expression of APP in muscle cells leads to a range of degenerative changes resembling various facets …

Myostatin is increased and complexes with amyloid-β within sporadic inclusion-body myositis muscle fibers

Myostatin is a negative regulator of muscle mass and strength. Sporadic inclusion-body myositis (s-IBM) is the most common degenerative muscle disease of older persons and is characterized by pronounced muscle wasting. s-IBM is of unknown etiology and pathogenesis, and it lacks definitive treatment. We have now demonstrated in samples from 12 s-IBM biopsies that: (1) by light and electron microscopic immunocytochemistry, myostatin/myostatin precursor is accumulated within muscle fibers and co-localized with amyloid-beta (Abeta); (2) by immunoblots, both myostatin and myostatin precursor are increased; and (3) by immunoprecipitation, myostatin precursor complexes with Abeta. Our study suggests that myostatin/myostatin precursor, either alone, or bound to Abeta, may play a novel role in the pathogenesis of s-IBM.

Mutant ubiquitin UBB+1 is accumulated in sporadic inclusion-body myositis muscle fibers.

Mutant ubiquitin (UBB+1), a product of "molecular misreading," is toxic to cells because its ubiquitinated form inhibits the proteasome, contributing to accumulation of misfolded proteins and their ensuing toxicity. The authors demonstrate in 10 sporadic inclusion body myositis (s-IBM) muscle biopsies that UBB+1 is accumulated in aggregates containing amyloid-beta and phosphorylated-tau. In s-IBM, UBB+1 may be pathogenic by inhibiting proteasome, thereby promoting accumulation of cytotoxic misfolded amyloid-beta and phosphorylated-tau.

Proteasomal expression, induction of immunoproteasome subunits, and local MHC class I presentation in myofibrillar myopathy and inclusion body myositis.

Inclusion body myositis (IBM) and myofibrillar myopathy (MM) are diseases characterized by the abnormal accumulation of proteins in muscle fibers, including desmin, alphaB-crystallin, gelsolin, actin, kinases, and phospho-tau, along with ubiquitin in muscle fibers, suggesting abnormal protein degradation as a possible cause of the surplus myopathy. Since the ubiquitin-proteasome system plays a crucial role in non-lysosomal protein degradation, the present study has examined by immunohistochemistry the expression of components of the catalytic core of 20S proteasomes and its regulators: 19S and PA28alpha/beta, and the expression of immunoproteasome subunits LMP2, LMP7, and MECL1 in 8 patients with MM and 10 patients with IBM. The patients with MM were from 6 unrelated families, 2 sporadic cases, I with autosomal recessive and 5 with autosomal dominant inheritance. One sporadic patient had a de novo R406W mutation in the desmin gene, and 1 patient with autosomal dominant MM had a single amino acid deletion at position 366 in the desmin gene. Increased immunoreactivity to 20S, 19S, and PA28alpha/beta colocalizing abnormal protein deposits, as revealed in consecutive serial sections, was seen in all cases with MM and IBM. In all cases, the subunits of the immunoproteasome LMP2, LMP7, and MECL1 colocalized with proteasomal immunoreactivity and abnormal protein accumulation. Immunohistochemistry revealed focal MHC class I immunoreactivity in the cytoplasmic membrane of muscle fibers in IBM and in association with protein aggregates in IBM, and to a lesser degree, in MM. The present findings provide a link between abnormal protein accumulation and altered proteasomal expression in IBM and MM.

Expression of Bcl-2 in inclusion body myositis.

On the background of the possible role of the anti-apoptotic protein Bcl-2 to inhibit apoptosis induced by the Fas/Fas ligand system in inflammatory myopathies we investigated the expression of Bcl-2 in inclusion body myositis (IBM). We examined muscle tissue from seven IBM patients and controls by immunocytochemistry using antibodies against Bcl-2, Fas (a member of the tumor necrosis factor receptor family) and the regeneration marker, neural cell adhesion molecule (N-CAM). We also investigated the occurrence of DNA fragmentation by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL)-method. Both Bcl-2 and Fas were up-regulated in muscle fibers in IBM and disease controls. Bcl-2 was expressed by regenerating muscle fibers while Fas was expressed by non-regenerating muscle fibers associated with inflammatory cell infiltrates. Bcl-2 and Fas were also expressed by inflammatory cells. There were scattered TUNEL positive nuclei and most of these appeared to be inflammatory cells. The low occurrence of apoptotic myonuclei is not related to Bcl-2 expression, which is confined to regenerating muscle cells in IBM and other myopathies.

Novel cytoplasmic immunolocalization of RNA polymerase II in inclusion-body myositis muscle.

Sporadic inclusion-body myositis (IBM) is a progressive degenerative muscle disease of older persons. Abnormalities of gene-expression and RNA metabolism have recently been proposed to contribute to the IBM pathogenic cascade. We now demonstrate, using well characterized, epitope-specific antibodies, that the largest subunit of RNA polymerase II is abnormally accumulated in the cytoplasm of IBM muscle fibers, where it is co-localized with phosphorylated tau on IBM paired helical filaments. Since RNA polymerase II is a crucial nuclear factor involved in both transcription and mRNA processing, our results support the hypothesis that abnormality of either or both of those processes might be caused, in part, by pathological trafficking of RNA polymerase II, and that abnormal trafficking might be an important factor in the IBM pathogenic cascade.

Cultured inclusion-body myositis muscle fibers do not accumulate beta-amyloid precursor protein and can be innervated.

Cultured muscle fibers from patients with sporadic inclusion-body myositis (s-IBM), similar to normal control muscle fibers, 1) did not have beta-amyloid precursor protein (betaAPP) immunoreactivity; and 2) became normally innervated, as evidenced by the following features: full cross-striation, continuous contraction, and acetylcholinesterase and acetylcholine receptors accumulated only at neuromuscular junctions. Thus, factors responsible for betaAPP accumulation and denervation-like changes in s-IBM muscle biopsies are not operative in the relatively short-term, non-aged, cultured s-IBM muscle fibers.

Transfer of beta-amyloid precursor protein gene using adenovirus vector causes mitochondrial abnormalities in cultured normal human muscle.

As in Alzheimer-disease (AD) brain, vacuolated muscle fibers of inclusion-body myositis (IBM) contain abnormally accumulated beta-amyloid precursor protein (beta APP), including its beta-amyloid protein epitope, and increased beta APP-751 mRNA. Other similarities between IBM muscle and AD brain phenotypes include paired helical filaments, hyperphosphorylated tau protein, apolipoprotein E, and mitochondrial abnormalities, including decreased cytochrome-c oxidase (COX) activity. The pathogenesis of these abnormalities in IBM muscle and AD brain is not known. We now report that direct transfer of the beta APP gene, using adenovirus vector, into cultured normal human muscle fibers causes structural abnormalities of mitochondria and decreased COX activity. In this adenovirus-mediated beta APP gene transfer, we demonstrated that beta APP overproduction can induce mitochondrial abnormalities. The data suggest that excessive beta APP may be responsible for mitochondrial and COX abnormalities in IBM muscle and perhaps AD brain.

Beta-Amyloid precursor epitopes in muscle fibers of inclusion body myositis.

Sporadic inclusion body myositis (IBM) and hereditary inclusion body myopathy (hIBM) are severe and progressive muscle diseases, characterized pathologically by vacuolated muscle fibers that contain 15- to 21-nm cytoplasmic tubulofilaments (CTFs). Those vacuolated muscle fibers also contain abnormally accumulated ubiquitin and beta-amyloid protein (A beta), and they contain amyloid in beta-pleated sheets as indicated by Congo red and crystal violet positivity. Using several well-characterized antibodies, we have now demonstrated that, in addition to A beta, two other epitopes, N-terminal and C-terminal, of the beta-amyloid precursor protein (beta PP) are abnormally accumulated in IBM vacuolated muscle fibers and similarly in hIBM. At the light microscopy level, immunoreactivities of N- and C-epitopes of beta PP closely colocalized with A beta and ubiquitin immunoreactivities. However, by immunogold electronmicroscopy, even though N-, C-, and A beta epitopes of beta PP and ubiquitin colocalized at the amorphous and dense floccular structures, only A beta was localized to the 6- to 10-nm amyloid-like fibrils and only ubiquitin was localized to CTFs. beta PP immunoreactive structures were often in proximity to CTFs, but CTFs themselves never contained beta PP immunoreactivities. The fact that A beta but not C- or N-terminal epitopes of beta PP localized to the 6- to 10-nm amyloid-like fibrils suggests that free A beta might be generated during beta PP processing and, after aggregation, may be responsible for the amyloid present within IBM muscle fibers. Our study demonstrates that three epitopes of beta PP accumulate abnormally in diseased human muscle, and therefore this phenomenon is not unique to Alzheimer's disease, Down's syndrome brain, and Dutch-type cerebrovascular amyloidosis.

Strong immunoreactivity of alpha 1-antichymotrypsin co-localizes with beta-amyloid protein and ubiquitin in vacuolated muscle fibers of inclusion-body myositis.

In 10 of 10 inclusion-body myositis (IBM) patients, including 1 hereditary case, vacuolated muscle fibers contained large or small cytoplasmic inclusions immunoreactive for alpha 1-antichymotrypsin (alpha 1-ACT). All IBM muscle biopsies had characteristic cytoplasmic tubulo-filaments by electron microscopy. None of 17 control muscle biopsies contained the alpha 1-ACT immunoreactive inclusions characteristic of IBM. In vacuolated muscle fibers, alpha 1-ACT immunoreactive inclusions colocalized with beta-amyloid protein and ubiquitin immunoreactivities. Our study provides the first demonstration of alpha 1-ACT accumulations in abnormal human muscle, and it suggest that, as in Alzheimer's disease and Down's syndrome, alpha 1-ACT may be involved in the pathogenesis of IBM.