73
Neuroscience Letters, 130 (1991) 73-76
© 1991 ElsevierScientificPublishers Ireland Ltd. 0304-3940/91/$03.50 ADONIS 030439409I00477G NSL 07997
Immunolocalization of ubiquitin in muscle biopsies of patients with inclusion body myositis and oculopharyngeal muscular dystrophy Valerie A s k a n a s , P i r a y e S e r d a r o g l u , W. K i n g Engel a n d R e n a t e B. A l v a r e z u s c Neuromuscular Center, University of Southern California School of Medicine, Los Angeles, CA 90017 (U.S.A.)
(Received 17 April 1991;Revisedversion received27 May 1991;Accepted27 May 1991) Key words: Ubiquitin; Inclusionbody myositis;Oculopharyngealmuscular dystrophy;Muscle biopsy; Cytoplasmictubulofilament
In 10/10 inclusion body myositis (IBM) patients and 2/2 oculopharyngealmuscular dystrophy (OPMD) patients, vacuolated muscle fibers contained darkly stained ubiquitin (Ub)-immunoreactivecytoplasmicinclusions. By electronmicroscopy,Ub-immunoreactivematerial was strictlylocalized to the 15-21 nm pathologic cytoplasmictubulofilaments(CTFs). None of 18 control muscle biopsies contained the Ub-immunoreactiveinclusions that are typical for IBM and OPMD. Thus, (a) finding that CTFs are ubiquitinated places their protein in the Ub-mediated turnover pathway and provides their first molecularmarker; (b) easy accessibility,as compared to the central nervoussystem,of muscle tissue containingubiquitinated inclusions should be advantageous for biochemicaland molecularstudies and may provide informationimportant to both systems.
Ubiquitin (Ub) is a 76-amino acid intracellular protein present in all eukaryocytes. It is held responsible for targeting a selective breakdown of abnormal or short-livednormal proteins by an ATP-dependent pathway (see reviews in refs. 8, 14, 17). Ubiquitination is of current interest in relation to its proposed role in the pathogenesis of neurodegenerative disorders [16, 26-28]. Ub is a component of neurofibrillary tangles and senile plaques in Alzheimer's disease brain [24, 31, 32], Lewy bodies in Parkinson's disease [4, 22, 28], and abnormal inclusions in lower motor neurons of patients with amyotrophic lateral sclerosis [19, 21, 25, 30] and infantile spinal muscular atrophy [18, 20]. Ub has received very little attention in muscle pathology. In one report, human muscle cytoplasmic bodies, which contain intermediate-filament desmin, were thought to stain with antibody against Ub [24]. Ub positivity was not detected in muscle of acid maltase deficiency and rod disease (nemaline myopathy) [28]. Reports regarding highly ubiquitinated filamentous inclusions in amyotrophic lateral sclerosis and in Alzheimer disease stimulated us to study whether cytoplasmic tubulofilamentous (CTFs) inclusions in inclusion body myositis (IBM) are also ubiquitinated. IBM is diagnosed by a combination of clinical and pathologic features [6, 7, 23, 35, 36]. Light-microscopic (LM) pathologic features include: various degrees of inflammation; muscle fibers with rimmed vacuoles that Correspondence: V. Askanas, USC Neuromuscular Center, 637 South Lucas Avenue, Los Angeles,CA 90017, U.S.A. Fax: (1)(213)481-1577.
usually contain red-staining material with the modified trichrome reaction [11]; and atrophic muscle fibers, indicative of lower motor neuron involvement. By electronmicroscopy (EM), abnormal muscle fibers contain CTFs, 15-21 nm external diameter and 3-6 nm internal diameter - - these are a diagnostic criterion of IBM [6, 7, 23, 35, 36]. Up to 3.5% of muscle nuclei can contain collections of tubulofilaments of the same diameter as CTFs [35]. Autosomal recessive 'familial IBM' designates rare patients with progressive muscle weakness and CTFs in vacuoles of abnormal muscle fibers [9, 13, 29]. Muscle fibers with rimmed vacuoles, atrophic denervated muscle fibers and CTFs with the same EM features also occur in 80% of patients with oculopharyngeal muscular dystrophy (OPMD) [10], which is an autosomaldominant, adult-onset, slowly progressive myopathy. Pathogeneses of these disorders, which involve muscle to a major degree and lower motor neurons to a minor degree, are unknown. Ub immunolocalization was performed on 5 - 1 0 / l m transverse sections of freshly frozen diagnostic muscle biopsies from 30 patients, ages 11-70 years, as follows: sporadic IBM 9; familial IBM 1; O P M D 2; controls 18 (including ones with normal muscle, polymyositis, 'vacuolar myopathy with rimmed vacuoles without CTFs', central core disease, hypokalemic periodic paralysis, neuromuscular disease with tubular aggregates, ragged-red-fiber mitochondrial myopathy, and various degrees of denervation). Diagnosis was based on clinical, laboratory, muscle biopsy histochemistry (18 reactions
74
C Fig. 1. Bright-fieldlight-microscopyof ubiquinated inclusions in IBM muscle biopsy, PAP reaction. Cytoplasmic inclusions ( × 1000) in: familial IBM (A); sporadicIBM (B); OPMD (C). Nuclearinclusions ( × 6250) in: familial IBM (D) and OPMD (E).
[12]), and EM data. Two patients with OPMD had OPMD-specific nuclear inclusions, 'Tom6 bodies' [34], by EM. Specific and well-characterized antibodies against Ub were generously provided by our colleagues. Monoclonal antibodies were: Ub-l, Ub-2 and Ub-4 (Gerry Shaw), 4-3H8,CC11 and 4-2D8,BA9 (Harry Smith), and DF-2 (Hiroshi Mori). Seven affinity-purified rabbit polyclonal antibodies were provided by Arthur Haas, Gerry Shaw, Lucila Autilio-Gambetti, and Nigel Leigh. Specificities of all the antibodies have been described previously [15, 21, 30-33]. For LM, Ub was localized by peroxidase-antiperoxidase (PAP) and fluorescence stainings following the same general procedures as we have described for desrain immunolocalization [2]. Primary antibodies against Ub were used in dilutions from 1:10 to 1:800, depending on their potency. For EM, Ub was localized on 10 #m unfixed frozen sections adhered to the bottom of 35 mm Petri dishes. Following incubation in a monoclonal antibody against Ub, sections were incubated in either 1:20 diluted goat anti-mouse-IgG serum conjugated to 10-nm gold (Amersham), or in 1:50 diluted goat anti-mouse IgG serum conjugated to H R P (Boehringer), and were embedded in situ in the Petri dish [3]. Desmin immunolocalization was achieved by PAP staining on sections consecutive to Ub localization, utilizing monoclonal anti-desmin antibody (Zymed), as described previously [2]. By LM, vacuolated muscle fibers of IBM (sporadic and familial) and O P M D patients contained very darkly immunoreactive inclusions within the vacuoles, and sometimes in vacuole-free cytoplasm (Fig. 1). In these positive patients, Ub-immunoreactive inclusions were present in nearly 100% of their vacuolated fibers. Ub-
positive inclusions had an amorphous or laminated pattern and were located internally or subsarcolemmally in the fibers. In the vacuolated fibers, all antibodies gave exactly the same Ub immunolocalization, although their staining intensities differed. When the primary antibody was either omitted or replaced by non-immunized rabbit serum, no staining was obtained. In all biopsies, muscle cytoplasm, sarcolemma and the perinuclear membrane were very faintly stained with all antibodies except Ub-2, 4-2DS,BA9 and 4-3H8,CCll. Ub-positive inclusions were not immunoreactive with the antibody against desmin. By EM, all patients with Ub-positive inclusions identified by LM had CTFs in the vacuolated fibers. Their external and internal diameters were 15-21 and 3.5 nm, respectively. Immuno-EM revealed that Ub-immunoreactive deposits were exclusively on CTFs (Fig. 2AD). Under the staining conditions used, no other structures or organelles in the muscle fibers contained Ub immunoreactivity. For example, within the vacuoles, myeloid figures and various cellular debris were negative (Fig. 2C). In some of the muscle nuclei of O P M D and IBM patients, Ub-immunoreactive dark inclusions were also present by LM (Fig. 1D,E). The nuclear inclusions were especially large in OPMD, occasionally occupying the entire nucleoplasm (Fig. I E). Their ultrastructural analysis is in process. None of the biopsies of 5 patients with polymyositis (without rimmed-vacuoles) and none of the other patients' biopsies had the Ub-immunoreactive inclusions characteristic of IBM and OPMD. Portions of cores in central core disease were slightly more immunoreactive than the core-free muscle fiber cytoplasm with the antiUb antibodies, but much weaker than the inclusions of IBM and OPMD. Ub immunoreactivity of cores corre-
75
Fig. 2. ImmunoelectronmicroscopyofCTFs. A,C: HRP reaction; x 2,600 and 24,900; insert in C x 186,750. B,D: gold labeling; x 14,760 and 24,900. A-D emphasize the highly selectivelocalization of Ub-immunoreactivityto CTFs. A round myeloid-bodyin lower-leftof C is not Ub-immunoreactive. Inset in C demonstrates the tubular structure of CTFs.
sponded to the 'hot-spots' [5] of desmin immunoreactivity. Although the origin and nature of the pathologic C T F s in IBM and O P M D are not known, our immunocytochemical study should aid in their understanding. The CTFs did not stain with an antibody to the musclespecific intermediate filament desmin. CTFs, 15-21 nm in diameter, are larger than filaments of most of the other Ub-associated inclusions, which contain 10-15 nm filaments [24]. Therefore, it is unlikely that C T F s are derived from intermediate filaments as was postulated for other ubiquitinated cytoplasmic inclusions [24]. The fact that C T F s are ubiquitinated places their protein in the Ub-mediated turnover pathway, providing a focus for further studies to identify their composition and pathogenesis. It is currently not known whether abnormal accumulation of Ub-positive C T F s in IBM and O P M D reflects: (a) accumulation of an abnormal protein, or (b) an abnormal accumulation of a normal protein that cannot be further degraded because of a malfunction of the Ub-dependent proteolytic system, as were suggested for neurodegenerative diseases [16, 26, 27]. Because U b is covalently bound to proteins, affinity purification and molecular analysis of ubiquitinated CTFs should be possible. Since C T F s have been demonstrated in cultured familial-IBM muscle [1], this source could provide material for the purification studies. The
relationship between ubiquitinated C T F s and ubiquitinated inclusions in the neurodegenerative disorders is presently unknown. However, since the disorders in which we have found ubiquitinated C T F s in muscle also have lower m o t o r neuron involvement, future molecular studies of CTFs in muscle may help elucidate the origin of ubiquitinated inclusions in the central nervous system of various disorders. It is also possible that some other disorders with major ubiquitination in the central nervous system, such as Alzheimer's disease, might have a minor amount of abnormal-protein ubiquitination detectable in muscle by immunocytochemistry. I f so, muscle would be a readily accessible source of living tissue to study.
We are grateful to our colleagues for graciously providing their antibodies and for their helpful discussions, and to Burt Handelsman for technical assistance. Supported in part by M D A . 1 Alvarez, R.B., Fardeau, M., Askanas, V,, Engel, W.K., McFerrin, J. and Tom6, F.M.S., Characteristic filamentous inclusions reproduced in cultured innervated musclefibers from patients with familial 'inclusion body myositis', J. Neurol. Sci., 98 (1990) 178. 2 Askanas, V., Bornemann, A. and Engel, W.K., Immunocytochemical localization of desmin at human neuromuscular junctions, Neurology, 40 (1990) 949-953.
76 3 Askanas, V. and Engel, W.K., A technique of fiber selection from human muscle tissue cultures for histochemical-electronmicroscopic studies, J. Histochem. Cytochem., 23 (1975) 144-146. 4 Bancher, C., Lassmann, H., Budka, H., Jellinger, K., GrundkeIqbal, I., Iqbal, K., Wiche, G., Seitelberger, F. and Wisniewski, H.M., An antigenic profile of Lewy bodies: immunocytochemical indication for protein phosphorylation and ubiquitination, J. Neuropathol. Exp. Neurol., 48 (1989) 81-93. 5 Bornemann, A., Askanas, V. and Engel, W.K., Immunocytochemical localization of desmin and vimentin in human muscle biopsies, Neurology, 40 (1990) 208. 6 Carpenter, S., Karpati, G., Heller, I. and Eisen, A., Inclusion body myositis: a distinct variety of idiopathic inflammatory myopathy, Neurology, 28 (1978) 8-17. 7 Chou, S.-M., Myxovirus-like structures in a case of human chronic polymyositis, Science, 158 (1967) 1453-1455. 8 Ciechanover, A. and Schwartz, A.L., How are substrates recognized by the ubiquitin-mediated proteolytic system?, TIBS, 14 (1989) 483-488. 9 Cole, A.J., Kuzniecky, R., Karpati, G., Carpenter, S., Andermann, E. and Andermann, F., Familial myopathy with changes resembling inclusion body myositis and periventricular leukoencephalopathy, Brain, 111 (1988) 1025-1037. 10 Coquet, M., Vital, C. and Julien, J., Non-specificity of inclusion body myositis-like filaments: their occurrence in oculopharyngeal muscular dystrophy. Ultrastructural study of ten cases, J. Neurol. Sci., 98 (1990) 179. 11 Engel, W.K. and Cunningham, G.G., Rapid examination of muscle tissue - - an improved trichrome method for rapid diagnosis of muscle biopsy fresh-frozen sections, Neurology, 13 (1963) 919 -923. 12 Engel, W.K., Muscle biopsies in neuromuscular diseases, Pediatr. Clin. North. Am., 14 (1967) 963-996. 13 Fardeau, M., Askanas, V., Tomr, F.M.S., Engel, W.K., Alvarez, R., McFerrin, J. and Chevallay, M., Hereditary neuromuscular disorder with inclusion body myositis-like filamentous inclusions: clinical, pathological, and tissue culture studies, Neurology, 40 (1990) 120. 14 Finley, D. and Varshavsky, A., The ubiquitin system: functions and mechanisms, TIBS, 10 (1985) 343 347. 15 Fried, V.A., Smith, H.T., Hildebrandt, E. and Weiner, K., Ubiquitin has intrinsic proteolytic activity: implications for cellular regulation, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 3685 3689. 16 Gallo, J.M. and Anderton, B.H., Ubiquitous variations in nerves, Nature, 337 (1989) 687~88. 17 Hershko, A. and Ciechanover, A., The ubiquitin pathway for the degradation of intracellular proteins, Prog. Nucleic Acid Res. Mol. Biol., 33 (1986) 19--56. 18 Kato, S. and Hirano, A., Ubiquitin and phosphorylated neurofilament epitopes in ballooned neurons of the extraocular muscle nuclei in a case of Werdnig-Hoffmann disease, Acta Neuropathology, 80 (1990) 334-337. 19 Kato, T., Katagiri, T., Hirano, A., Kawanami, T. and Sasaki, H., Lewy body-like hyaline inclusions in sporadic motor neuron disease are ubiquitinated, Acta Neuropathol., 77 (1989) 391 396. 20 Lee, S., Park, Y.D., Yen, S.H.C., Ksiezak-Reding, H., Goldman. J.E. and Dickson, D.W., A study of infantile motor neuron disease with neurofilament and ubiquitin immunocytochemistry, Neuropediatrics, 20 (1989) 107-111.
21 Leigh, P.N., Anderton, B.H., Dodson, A., Gallo, J.M., Swash, M. and Power, D.M., Ubiquitin deposits in anterior horn cells in motor neurone disease, Neurosci. Lett., 93 (1988) 197-203. 22 Lennox, G., Lowe, J., Morrell, K., Landon, M. and Mayer, R.J., Anti-ubiquitin immunocytochemistry is more sensitive than conventional techniques in the detection of diffuse Lewy body disease, J. Neurol. Neurosurg. Psychiatry, 52 (1989) 67-71. 23 Lotz, B.P., Engel, A.G., Nishino, H., Stevens, J.C. and Litchy, W.J., Inclusion body myositis: observations in 40 patients, Brain, 112 (1989) 727-747. 24 Lowe, J., Blanchard, A., Morrell, K., Lennox, G., Reynolds, L., Billet, M., Landon, M. and Mayer, R.J., Ubiquitin is a common factor in intermediate filament inclusion bodies of diverse type in man, including those of Parkinson's disease, Pick's disease, and Alzheimer's diesease, as well as Rosenthal fibres in cerebellar astrocytomas, cytoplasmic bodies in muscle, and Mallory bodies in alcoholic liver disease, J. Pathol., 155 (1988) 9-15. 25 Lowe, J., Lennox, G., Jefferson, D., Morrell, K., McQuire, D., Gray, T., Landon, M., Doherty, F. and Mayer, R.J., A filamentous inclusion body within anterior horn neurones in motor neurone disease defined by immunocytochemical localization of ubiquitin, Neurosci. Lett., 94 (1988) 203-210. 26 Lowe, J. and Mayer, J., Ubiquitin. New insights into chronic degenerative diseases, Br. J. Hosp. Med., 42 (1989) 462-466. 27 Lowe, J. and Mayer, R.J., Ubiquitin, cell stress and diseases of the nervous system, Neuropathol. Appl. Neurobiol., 16 (1990) 281 291. 28 Manetto, V., Perry, G., Tabaton, M., Bullvihill, P., Fried, V.A.. Smith, H.T., Gambetti, P. and Autilio-Gambetti, L., Ubiquitin is associated with abnormal cytoplasmic filaments characteristic of neurodegenerative diseases, Proc. Natl. Acad. Sci. U.S.A., 25 (1988) 4501-4505. 29 Massa, R., Weller, B., Karpati, G., Shonbridge, E. and Carpenter, S., Familial inclusion body myosites among Kurdish-lranian Jews, Arch. Neurol., 48 (1991) 519-522. 30 Migheli, A., Autilio-Gambetti, L., Gambetti, P., Mocellini, C., Vigtiani, M.C. and Schiffer, D., Ubiquitinated filamentous inclusions in spinal cord of patients with motor neuron disease, Neurosci. Lett., 114 (1990) 5 10. 31 Mori, H., Kondo, J. and lhara, Y., Ubiquitin is a component of paired helical filaments in Alzheimer's disease, Science, 235 (1987) 1641 1644. 32 Perry, G., Friedman, R., Shaw, G. and Chau, V., Ubiquitin is detected in neurofibrillary tangles and senile plaque neurites of Alzheimer disease brains, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 3033 3036. 33 Riley, D.A., Bain, J.L.W., Ellis, S. and Haas, A.L., Quantitation and immunocytochemical localization of ubiquitin conjugates within rat red and white skeletal muscles, J. Histochem. Cytochem., 36 (1988) 621-632. 34 Tom& F.M.S. and Fardeau, M., Nuclear inclusion in oculopharyngeal dystrophy, Acta Neuropathol., 49 (1980) 85-87. 35 Tom& F.M.S., Fardeau, M., Lebon, P. and Chevallay, M., Inclusion body myositis, Acta Neuropathol., 7 (1981) 287-291. 36 Yunis, E.J. and Samaha. F.J., Inclusion body myositis, Lab. Invest., 25 (1971) 240-248.
Neuroscience Letters, 130 (1991) 73-76
© 1991 ElsevierScientificPublishers Ireland Ltd. 0304-3940/91/$03.50 ADONIS 030439409I00477G NSL 07997
Immunolocalization of ubiquitin in muscle biopsies of patients with inclusion body myositis and oculopharyngeal muscular dystrophy Valerie A s k a n a s , P i r a y e S e r d a r o g l u , W. K i n g Engel a n d R e n a t e B. A l v a r e z u s c Neuromuscular Center, University of Southern California School of Medicine, Los Angeles, CA 90017 (U.S.A.)
(Received 17 April 1991;Revisedversion received27 May 1991;Accepted27 May 1991) Key words: Ubiquitin; Inclusionbody myositis;Oculopharyngealmuscular dystrophy;Muscle biopsy; Cytoplasmictubulofilament
In 10/10 inclusion body myositis (IBM) patients and 2/2 oculopharyngealmuscular dystrophy (OPMD) patients, vacuolated muscle fibers contained darkly stained ubiquitin (Ub)-immunoreactivecytoplasmicinclusions. By electronmicroscopy,Ub-immunoreactivematerial was strictlylocalized to the 15-21 nm pathologic cytoplasmictubulofilaments(CTFs). None of 18 control muscle biopsies contained the Ub-immunoreactiveinclusions that are typical for IBM and OPMD. Thus, (a) finding that CTFs are ubiquitinated places their protein in the Ub-mediated turnover pathway and provides their first molecularmarker; (b) easy accessibility,as compared to the central nervoussystem,of muscle tissue containingubiquitinated inclusions should be advantageous for biochemicaland molecularstudies and may provide informationimportant to both systems.
Ubiquitin (Ub) is a 76-amino acid intracellular protein present in all eukaryocytes. It is held responsible for targeting a selective breakdown of abnormal or short-livednormal proteins by an ATP-dependent pathway (see reviews in refs. 8, 14, 17). Ubiquitination is of current interest in relation to its proposed role in the pathogenesis of neurodegenerative disorders [16, 26-28]. Ub is a component of neurofibrillary tangles and senile plaques in Alzheimer's disease brain [24, 31, 32], Lewy bodies in Parkinson's disease [4, 22, 28], and abnormal inclusions in lower motor neurons of patients with amyotrophic lateral sclerosis [19, 21, 25, 30] and infantile spinal muscular atrophy [18, 20]. Ub has received very little attention in muscle pathology. In one report, human muscle cytoplasmic bodies, which contain intermediate-filament desmin, were thought to stain with antibody against Ub [24]. Ub positivity was not detected in muscle of acid maltase deficiency and rod disease (nemaline myopathy) [28]. Reports regarding highly ubiquitinated filamentous inclusions in amyotrophic lateral sclerosis and in Alzheimer disease stimulated us to study whether cytoplasmic tubulofilamentous (CTFs) inclusions in inclusion body myositis (IBM) are also ubiquitinated. IBM is diagnosed by a combination of clinical and pathologic features [6, 7, 23, 35, 36]. Light-microscopic (LM) pathologic features include: various degrees of inflammation; muscle fibers with rimmed vacuoles that Correspondence: V. Askanas, USC Neuromuscular Center, 637 South Lucas Avenue, Los Angeles,CA 90017, U.S.A. Fax: (1)(213)481-1577.
usually contain red-staining material with the modified trichrome reaction [11]; and atrophic muscle fibers, indicative of lower motor neuron involvement. By electronmicroscopy (EM), abnormal muscle fibers contain CTFs, 15-21 nm external diameter and 3-6 nm internal diameter - - these are a diagnostic criterion of IBM [6, 7, 23, 35, 36]. Up to 3.5% of muscle nuclei can contain collections of tubulofilaments of the same diameter as CTFs [35]. Autosomal recessive 'familial IBM' designates rare patients with progressive muscle weakness and CTFs in vacuoles of abnormal muscle fibers [9, 13, 29]. Muscle fibers with rimmed vacuoles, atrophic denervated muscle fibers and CTFs with the same EM features also occur in 80% of patients with oculopharyngeal muscular dystrophy (OPMD) [10], which is an autosomaldominant, adult-onset, slowly progressive myopathy. Pathogeneses of these disorders, which involve muscle to a major degree and lower motor neurons to a minor degree, are unknown. Ub immunolocalization was performed on 5 - 1 0 / l m transverse sections of freshly frozen diagnostic muscle biopsies from 30 patients, ages 11-70 years, as follows: sporadic IBM 9; familial IBM 1; O P M D 2; controls 18 (including ones with normal muscle, polymyositis, 'vacuolar myopathy with rimmed vacuoles without CTFs', central core disease, hypokalemic periodic paralysis, neuromuscular disease with tubular aggregates, ragged-red-fiber mitochondrial myopathy, and various degrees of denervation). Diagnosis was based on clinical, laboratory, muscle biopsy histochemistry (18 reactions
74
C Fig. 1. Bright-fieldlight-microscopyof ubiquinated inclusions in IBM muscle biopsy, PAP reaction. Cytoplasmic inclusions ( × 1000) in: familial IBM (A); sporadicIBM (B); OPMD (C). Nuclearinclusions ( × 6250) in: familial IBM (D) and OPMD (E).
[12]), and EM data. Two patients with OPMD had OPMD-specific nuclear inclusions, 'Tom6 bodies' [34], by EM. Specific and well-characterized antibodies against Ub were generously provided by our colleagues. Monoclonal antibodies were: Ub-l, Ub-2 and Ub-4 (Gerry Shaw), 4-3H8,CC11 and 4-2D8,BA9 (Harry Smith), and DF-2 (Hiroshi Mori). Seven affinity-purified rabbit polyclonal antibodies were provided by Arthur Haas, Gerry Shaw, Lucila Autilio-Gambetti, and Nigel Leigh. Specificities of all the antibodies have been described previously [15, 21, 30-33]. For LM, Ub was localized by peroxidase-antiperoxidase (PAP) and fluorescence stainings following the same general procedures as we have described for desrain immunolocalization [2]. Primary antibodies against Ub were used in dilutions from 1:10 to 1:800, depending on their potency. For EM, Ub was localized on 10 #m unfixed frozen sections adhered to the bottom of 35 mm Petri dishes. Following incubation in a monoclonal antibody against Ub, sections were incubated in either 1:20 diluted goat anti-mouse-IgG serum conjugated to 10-nm gold (Amersham), or in 1:50 diluted goat anti-mouse IgG serum conjugated to H R P (Boehringer), and were embedded in situ in the Petri dish [3]. Desmin immunolocalization was achieved by PAP staining on sections consecutive to Ub localization, utilizing monoclonal anti-desmin antibody (Zymed), as described previously [2]. By LM, vacuolated muscle fibers of IBM (sporadic and familial) and O P M D patients contained very darkly immunoreactive inclusions within the vacuoles, and sometimes in vacuole-free cytoplasm (Fig. 1). In these positive patients, Ub-immunoreactive inclusions were present in nearly 100% of their vacuolated fibers. Ub-
positive inclusions had an amorphous or laminated pattern and were located internally or subsarcolemmally in the fibers. In the vacuolated fibers, all antibodies gave exactly the same Ub immunolocalization, although their staining intensities differed. When the primary antibody was either omitted or replaced by non-immunized rabbit serum, no staining was obtained. In all biopsies, muscle cytoplasm, sarcolemma and the perinuclear membrane were very faintly stained with all antibodies except Ub-2, 4-2DS,BA9 and 4-3H8,CCll. Ub-positive inclusions were not immunoreactive with the antibody against desmin. By EM, all patients with Ub-positive inclusions identified by LM had CTFs in the vacuolated fibers. Their external and internal diameters were 15-21 and 3.5 nm, respectively. Immuno-EM revealed that Ub-immunoreactive deposits were exclusively on CTFs (Fig. 2AD). Under the staining conditions used, no other structures or organelles in the muscle fibers contained Ub immunoreactivity. For example, within the vacuoles, myeloid figures and various cellular debris were negative (Fig. 2C). In some of the muscle nuclei of O P M D and IBM patients, Ub-immunoreactive dark inclusions were also present by LM (Fig. 1D,E). The nuclear inclusions were especially large in OPMD, occasionally occupying the entire nucleoplasm (Fig. I E). Their ultrastructural analysis is in process. None of the biopsies of 5 patients with polymyositis (without rimmed-vacuoles) and none of the other patients' biopsies had the Ub-immunoreactive inclusions characteristic of IBM and OPMD. Portions of cores in central core disease were slightly more immunoreactive than the core-free muscle fiber cytoplasm with the antiUb antibodies, but much weaker than the inclusions of IBM and OPMD. Ub immunoreactivity of cores corre-
75
Fig. 2. ImmunoelectronmicroscopyofCTFs. A,C: HRP reaction; x 2,600 and 24,900; insert in C x 186,750. B,D: gold labeling; x 14,760 and 24,900. A-D emphasize the highly selectivelocalization of Ub-immunoreactivityto CTFs. A round myeloid-bodyin lower-leftof C is not Ub-immunoreactive. Inset in C demonstrates the tubular structure of CTFs.
sponded to the 'hot-spots' [5] of desmin immunoreactivity. Although the origin and nature of the pathologic C T F s in IBM and O P M D are not known, our immunocytochemical study should aid in their understanding. The CTFs did not stain with an antibody to the musclespecific intermediate filament desmin. CTFs, 15-21 nm in diameter, are larger than filaments of most of the other Ub-associated inclusions, which contain 10-15 nm filaments [24]. Therefore, it is unlikely that C T F s are derived from intermediate filaments as was postulated for other ubiquitinated cytoplasmic inclusions [24]. The fact that C T F s are ubiquitinated places their protein in the Ub-mediated turnover pathway, providing a focus for further studies to identify their composition and pathogenesis. It is currently not known whether abnormal accumulation of Ub-positive C T F s in IBM and O P M D reflects: (a) accumulation of an abnormal protein, or (b) an abnormal accumulation of a normal protein that cannot be further degraded because of a malfunction of the Ub-dependent proteolytic system, as were suggested for neurodegenerative diseases [16, 26, 27]. Because U b is covalently bound to proteins, affinity purification and molecular analysis of ubiquitinated CTFs should be possible. Since C T F s have been demonstrated in cultured familial-IBM muscle [1], this source could provide material for the purification studies. The
relationship between ubiquitinated C T F s and ubiquitinated inclusions in the neurodegenerative disorders is presently unknown. However, since the disorders in which we have found ubiquitinated C T F s in muscle also have lower m o t o r neuron involvement, future molecular studies of CTFs in muscle may help elucidate the origin of ubiquitinated inclusions in the central nervous system of various disorders. It is also possible that some other disorders with major ubiquitination in the central nervous system, such as Alzheimer's disease, might have a minor amount of abnormal-protein ubiquitination detectable in muscle by immunocytochemistry. I f so, muscle would be a readily accessible source of living tissue to study.
We are grateful to our colleagues for graciously providing their antibodies and for their helpful discussions, and to Burt Handelsman for technical assistance. Supported in part by M D A . 1 Alvarez, R.B., Fardeau, M., Askanas, V,, Engel, W.K., McFerrin, J. and Tom6, F.M.S., Characteristic filamentous inclusions reproduced in cultured innervated musclefibers from patients with familial 'inclusion body myositis', J. Neurol. Sci., 98 (1990) 178. 2 Askanas, V., Bornemann, A. and Engel, W.K., Immunocytochemical localization of desmin at human neuromuscular junctions, Neurology, 40 (1990) 949-953.
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