Acta Neuropathol (2002) 104 : 297–304 DOI 10.1007/s00401-002-0559-z
R E G U L A R PA P E R
Dirk Fischer · Jens Matten · Jens Reimann · Carsten Bönnemann · Rolf Schröder
Expression, localization and functional divergence of αB-crystallin and heat shock protein 27 in core myopathies and neurogenic atrophy
Received: 30 October 2001 / Revised: 25 February 2002 / Accepted: 25 February 2002 / Published online: 8 June 2002 © Springer-Verlag 2002
Abstract αB-crystallin (αBC) and heat shock protein 27 (hsp 27) are members of the family of small heat shock proteins (shsps), which exert a role as molecular chaperones by binding unfolded or denatured proteins, thereby suppressing irreversible protein aggregation and consecutive cell damage. The essential role of shsps in human neuromuscular disorders is highlighted by the observation that a mutation of the human αBC gene causes an autosomal dominant ‘myofibrillar myopathy’ characterized by αBC and desmin accumulation. Furthermore, an aberrant immunostaining of αBC was recently reported in sporadic inclusion body myositis. In the present study we analyzed the expression and localization of αB-crystallin and hsp 27 in various congenital myopathies by means of indirect immunofluorescence, immunogold electron microscopy and Western blotting. We demonstrate an increased immunoreactivity of αBC and hsp 27 in central and minicore lesions as well as in target fibers, which renders both shsps as reliable, but nonspecific, markers for core and target structures. In contrast, Western blotting demonstrated a normal expression level of αBC and hsp 27, which indicates that the increased immunostaining is not the result of an enhanced protein expression. Furthermore, thiocyanate-induced degradation of actin filaments led to a dramatic decrease of hsp 27 immunostaining in core and target lesions, whereas the increased αBC and desmin immunostaining was found to be even more enhanced. The latter findings imply a functional diversity of both shsps with a preferential association of hsp 27 with the actin miThe first two authors contributed equally to this study. D. Fischer · J. Matten · J. Reimann · R. Schröder (✉) Department of Neurology, University Hospital Bonn, Rheinische Friedrich-Wilhelms-Universität, Sigmund-Freud-Str. 25, 53105 Bonn, Germany e-mail:
[email protected], Tel.: +49-228-2876805, Fax: +49-228-2876805 C. Bönnemann Division of Neurology, The Children’s Hospital of Philadelphia, Abramson 516A, 34th Street and Civic Center Blvd., Philadelphia, PA 10104, USA
crofilament system and αBC with the intermyofibrillar desmin cytoskeleton in human skeletal muscle. Keywords Small heat shock proteins · αB crystallin · Heat shock protein 27 · Central core myopathy · Minicore myopathy
Introduction αB-crystallin (αBC; 22 kDa) and hsp 27 (27 kDa) are members of the highly conserved family of small heat shock proteins (shsps) that are constitutively expressed in a wide variety of cells and tissues [5, 11, 14]. The expression of shsps is induced in response to heat or other nonphysiological conditions (i.e., anoxia), thereby protecting cells against damage, a phenomenon also termed stress tolerance [13, 17]. Molecular and structural analysis revealed that all shsps contain a conserved 80- to 100-amino acid sequence (the so-called α-crystallin core domain) residing in their C-terminal domains. These domains are essential for direct protein-protein interactions between individual αBC or hsp 27 molecules and the consecutive formation of dynamic, metastable multimeric shsps complexes [16]. Shsps exert an essential physiological role as molecular chaperones by controlled binding to and release from an otherwise unstable conformer of other proteins, thereby guiding the correct assembly, folding and functional state of individual proteins [8]. In particular, shsps have been implicated to play a central role in the structural and functional organization of the three-dimensional intermediate filament and the actin microfilament system [1, 3, 14]. Moreover, shsps were shown to bind unfolded polypeptides, and the up-regulation of shsps is considered to be an important mechanism of cellular defense against the formation of pathological protein aggregates, which are the classical morphological hallmark of various human disorders ranging from neurodegenerative diseases (i.e., Alzheimer’s disease, Parkinson’s disease, diffuse Lewy body disease, Huntington’s disease and Alexander’s disease) to the formation of cataracts as well as of Mallory bodies in hepatic cirrhosis [5, 15].
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The essential role of shsps in human skeletal muscle is highlighted by the observation that a mutation of the human αBC gene on chromosome 11q21 lead to an autosomal dominant myofibrillar myopathy with abnormal desmin accumulation in combination with cataracts [4, 20]. Furthermore, a recent study in sporadic inclusion body myositis (sIBM) demonstrated an enhanced αBC immunostaining in structurally abnormal fibers and in fibers without significant structural abnormalities (X-fibers) [2]. The latter finding has led to the hypothesis that an enhanced αBC expression is an upstream event in the pathogenesis of sIBM [2]. To gain insight into the role of αBC and hsp 27 in myopathies with morphologically distinct myofibrillar and/or intermyofibrillar abnormalities we studied the expression of both proteins in various congenital myopathies by means of indirect immunofluorescence, immunogold electron microscopy and Western blotting.
Immunoelectron microscopy Skeletal muscle tissue was fixed in a solution of 4% paraformaldehyde and 0.1% glutaraldehyde with HEPES buffer (0.1 M, pH 7.5), washed three times for 10 min with the same buffer, dehydrated with series of ethanol and embedded in LR White. Ultrathin sections were rinsed on drops for 10 min, etched for 15 min with saturated sodium metaperiodate, followed by a rinse in water and 10 min in 0.1 M HCl. Following this, grids were rinsed with water for 10 min and preincubated in 5% normal goat serum in washing buffer (PBS, 0.5% BSA, 0.1% fish gelatin) for 35 min at room temperature. The grids were washed 10 min with washing buffer and then incubated in a wet chamber at 4°C. The primary antibodies against desmin and αB-crystallin were diluted 1:15 and 1:350, respectively. After overnight incubation with the primary antibody the grids were washed with washing buffer and incubated with a secondary antibody coupled to colloidal gold (diluted 1:20; 10 nm particles) overnight at 4°C in a wet chamber. After incubation the grids were washed with washing buffer (four times for 3 min), followed by four washes in PBS and postfixed with 1% glutaraldehyde in PBS. After another wash with distilled water (twice for 5 min), the preparations were contrasted using conventional techniques. Ultrathin sections were examined in a Zeiss 900 electron microscope (Zeiss, Jena, Germany).
Materials and methods Muscle biopsies Cryostatic sections were obtained from diagnostic muscle biopsy material of 21 patients with one of the following neuromuscular disorders: central core diseases (CCD, n=5), minicore disease (MCD, n=1), nemaline myopathy (NM, n=5), myopathy with tubular aggregates (MTA, n=3) and neurogenic atrophy with multiple target fibers (NA/TF, n=5). Diagnoses were based on combined clinical, electrophysiological and conventional histopathological criteria. Histological criteria for cores included absence of oxidative enzyme reactivity and a predilection of cores for type I fibers, while criteria for target fibers included a composition of three distinct zones: a central zone devoid of oxidative enzyme activity, a densely stained intermediate zone with increased oxidative enzyme activity and a normal peripheral zone of intermediate activity [7]. Normal control specimens (N) were obtained from 3 patients who underwent muscle biopsy for diagnosis of neuromuscular complaints, but who were ultimately deemed to be normal by means of combined clinical, electrophysiological and histological criteria. Antibodies, immunofluorescence microscopy and sodium thiocyanate The following primary antibodies were used: a polyclonal rabbit antibody directed against residues 1–10 of human αB-crystallin (Chemicon, USA; applied dilution 1:200); a monoclonal mouse antibody against the purified hsp 27 isolated from MCF-7, a human breast tumor cell line (StressGen, Canada; 1:100); a monoclonal mouse antibody against desmin (DAKO, Denmark; clone D33; 1:300); and a monoclonal mouse antibody against actin (Sigma Aldrich, Munich, Germany; clone AC-40; 1:50). Isotype-specific secondary antibodies conjugated with fluorescein isothiocyanate or Texas Red were applied according to the recommendations of the manufacturers (Sigma Aldrich; Dianova, Hamburg, Germany). Bleed-through of one of the conjugates in the complementary channel was never observed. All specimens were examined and pictures were digitally acquired using a Nikon Eclipse E 800 microscope (Nikon, Düsseldorf, Germany). Indirect immunofluorescence of 6-µm-thick cryostat sections was performed as described previously [18]. Removal of actin filaments from muscle tissue was performed by incubation of freshly cut, unfixed cryostat sections with sodium thiocyanate (1 M NaSCN in PBS, pH 7,4) for 15 min at room temperature [12].
Immunoblotting Total protein extracts from fresh frozen human skeletal muscle were prepared as described previously [18]. SDS-PAGE using 12% polyacrylamide gels, protein transfer and visualization of proteins on membranes were also carried out as described [19]. Immunodetection of αB-crystallin and hsp 27 using rabbit polyclonal antiserum (1:2,000) and mouse monoclonal antiserum (1:500) were performed with peroxidase-coupled secondary antibodies (Dianova; 1:30.000) and the Supersignal enhanced chemiluminescence system (Pierce, Rockford, Ill.).
Results αBC and hsp 27 show a marked immunostaining of central core, minicore and target lesions Indirect immunofluorescence assays using antibodies against αBC and hsp 27 resulted in a faint cytoplasmic staining in cross sections of normal human skeletal muscle. While the cases of nemaline myopathy and the cases of myopathy with tubular aggregates showed no aberrant αBC or hsp 27 immunoreactivity, the analysis of skeletal muscle tissue from the patient with MCD (Fig. 1) as well as of three of the five cases with CCD (Fig. 2) revealed a markedly intensified anti-αBC (Figs. 1, 2E) and anti-hsp 27 (Figs. 1, 2C) immunoreactivity of core lesions. This pattern was also observed using antibodies against actin Fig. 1A–H Indirect immunofluorescence of skeletal muscle tissue sections from the patient with MCD. All core structures showed strong immunoreactivities with antibodies against αBC (E) and hsp 27 (C). A similar pattern was observed with antibodies directed against actin (A) and desmin (G). In contrast, cytoplasmic areas surrounding the core lesions showed a faint immunoreactivity. Furthermore, thiocyanate-induced degradation of actin filaments (B) decreases hsp 27 (D) immunostaining within and outside the core structures, whereas the increased αBC (F) and desmin (H) immunolabeling became even more pronounced (MCD minicore disease, αBC αB-crystallin). Bar 100 µm
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(Figs. 1, 2A) and desmin (Figs. 1, 2G). In contrast, the staining intensity of fibers without cores or cytoplasmic areas surrounding core lesions remained unchanged when compared to normal controls. Furthermore, using an LR-White post-embedding protocol in conjunction with immunogold electron microscopy, we analyzed the distribution of αBC and desmin at the ultrastructural level in muscle fibers with abundant central core structures. Here, cores were found to be devoid of myofibrils and mitochondria but contained highly unordered filamentous structures, which were densely labeled by antibodies directed against αBC and desmin (Fig. 3A, B). In the remaining two cases of CCD only a very faint αBC, hsp 27, desmin and actin immunoreactivity of core structures was observed. To address the question whether the observed immunoreactivity is restricted to central core and minicore lesions we further analyzed the αBC and hsp 27 staining in five samples of neurogenic atrophy with abundant target fibers (NA/TF, Fig. 4). In all biopsy specimens we observed a markedly intensified αBC and hsp 27 immunostaining of all target structures (Fig. 4E, C). Similarly, an enhanced αBC expression in target fibers also had been observed by others [2]. However, in contrast to the findings with αBC, hsp 27 and desmin, our immunohistochemical analysis using an antibody against actin showed a marked immunolabeling of the central aspects of target and targetoid lesions, whereas the periphery of these lesions showed a decreased immunoreaction. Sodium thiocyanate induced degradation of actin filaments leads to a markedly reduced hsp 27 immunoreactivity of core and target lesions Previous studies indicated that αBC and hsp 27 exert a role in the structural and functional organization of the actin microfilament and desmin intermediate filament system [1, 3, 14]. Incubation of freshly cut, unfixed cryostat sections with sodium thiocyanate leads to degradation and subsequent removal of actin filaments from muscle tissue [12]. To analyze the effect of this treatment on central cores, minicores and target lesions, muscle sections from three cases with CCD, from the case with MCD as well as three cases with NA/TF were analyzed upon incubation with 1 M sodium thiocyanate. Immunostaining with antibodies against αBC, hsp 27, actin and desmin revealed that thiocyanate treatment led to a markedly reduced actin (Figs. 1, 2, 4B) and hsp 27 (Figs. 1, 2, 4D) immunoreactivity in both, normal appearing cytoplasmic regions as well as core and target lesions. In contrast, the immunoreactivity of αBC (Figs. 1, 2, 4F) and desmin Fig. 2A–H Skeletal muscle tissue sections from a patient with CCD. Using antibodies directed against actin (A), hsp 27 (C), αBC (E) and desmin (G) the central zones of the core structures showed strong immunoreactivities. Thiocyanate-induced degradation of actin filaments (B) reduced hsp 27 (D) immunostaining but increased αBC (F) and desmin (H) immunostaining (CCD central core disease, hsp heat shock protein). Bar 100 µm
(Figs. 1, 2, 4H) became even more prominent both in normal and pathological muscle fibers. The increased αBC/ hsp 27 immunostaining is not paralleled by an increased αBC/ hsp 27 protein expression Since immunofluorescence is not suitable to quantify protein expression, we performed Western blotting of total protein extracts from muscle biopsy specimens to compare the protein expression levels from normal human skeletal muscle, with that in MCD, CCD with abundant core lesions and neurogenic atrophy with a high degree of target fibers (at least 20% of fibers containing core or target lesions). As illustrated in Fig. 5 the signal intensities of the specific bands for both shsps did not differ in normal and diseased muscle tissue arguing against an up-regulation of αBC (Fig. 5A) and hsp 27 (Fig. 5B) protein expression.
Discussion In the present study, we have analyzed the expression of αBC and hsp 27 in various congenital myopathies. Using indirect immunofluorescence assays, we noted a markedly increased αBC, hsp 27, actin and desmin staining of core lesions in skeletal muscle tissue of a patient with MCD and in three out of five cases with CCD. Since both shsps also strongly labeled target fibers in cases with NA/TF, these findings imply that αBC and hsp 27 are sensitive, but nonspecific, markers for lesions with focal loss or disorganization of the myofibrillar structure. These immunofluorescence findings were mirrored at the ultrastructural level. Using an LR-White post-embedding protocol in conjunction with immunogold electron microscopy we found an abundant αBC and desmin labeling of all core lesions in skeletal muscle from a patient with CCD. At higher magnification, these cores were devoid of myofibrils or mitochondria (so-called unstructured cores) and contained highly unordered filamentous material, which was densely labeled by gold particles. The desmin labeling indicates that the filamentous backbone of unstructured cores is predominantly composed of remnants of the desmin intermediate filament cytoskeleton, which normally interlines the Z-discs of neighboring myofibrils of intact striated muscle. Beyond αBC, hsp 27, desmin and actin, a wide variety of other proteins have been documented as components of central and minicore lesions [6]. Since indirect immunofluorescence demonstrated a markedly increased shsps staining, we performed Western blot analysis to examine a possibly increased αBC and/or hsp 27 expression. However, no changes in the expression levels of both shsps were detected, even though samples used had a high content of central cores, minicores or target fibers, respectively. Although not performed at the single-fiber level, these results indicate that increased immunofluorescence signal intensities not necessarily indicate an up-regulation or excess of protein expression. As an alternative expla-
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303 Fig. 3 NA/TF. All target structures showed strong immunoreactivities with antibodies against actin (A), hsp 27 (C), αBC (E) and desmin (G). Thiocyanate treatment resulted in, as in MCD and CCD, decreased hsp 27 (D) immunostaining, but increased αBC (F) and desmin (H) immunolabeling (NA/TF Neurogenic atrophy with abundant target fibers). Bar 100 µm
Fig. 5 Western blot analyses of αBC (A) and hsp 27 (B) expression. The specific staining signal demonstrates the presence of αBC (22 kDa) and hsp 27 (27 kDa) in all samples. Compared with total protein extracts from normal muscle tissue (N) neither increased or decreased expression could be demonstrated in muscle tissue of CCD, MCD or NA/TF
Fig. 4 Ultrathin sections of skeletal muscle of a case of CCD with abundant cores. Immunogold electron microscopy revealed that these cores are devoid of myofibrils and mitochondria but contain highly unordered filamentous structures, which are labeled by antibodies directed against αBC (A) and desmin (B). Bar 1 µm
nation one should consider changes in the solubility and subcellular distribution of αBC and hsp 27 in response to cellular stress. Furthermore, increased immunolabeling of filamentous structures within cores or target fibers may be explained by an increased accessibility of αBC, hsp 27 or other epitopes, which may be attributed to loss of myofibrils or changes in the macromolecular composition and structure of intermyofibrillar filaments, i.e., due to disturbances of ionic balance and/or the pH of the cytoplasmic solvent within these lesions. This hypothesis is further substantiated by our observation that sodium thiocyanate-induced removal of actin filaments leads to an increased desmin labeling in normal as in core or target lesion-containing muscle fibers. Considering these observations, it is tempting to speculate that in structured lesions the presence of a normal myofibrillar apparatus interferes with the binding of various antibodies to intermyofibrillar cytoskeletal epitopes. Recent studies convincingly demonstrated the deleterious effect of a mutation of the αBC gene on the intermyofibrillar desmin cytoskeleton, thereby leading to an autosomal dominant myofibrillar myopathy characterized by the presence of intermyofibrillar desmin- and αBC-containing aggregates [4, 20]. The close functional and structural relationship between αBC and desmin, which colocalize at the periphery of Z-discs in normal striated muscle, is further emphasized by the observation that accumulations of αBC together with desmin are also found in myopathies caused by desmin mutations [9, 10]. Interestingly these aggregates are also intensely stained with antibodies against hsp 27 (unpublished observation, data not shown) and the question arises to what extent hsp 27, which has primarily been thought to chaperone the actin microfilament system, is connected to intermediate filament pathology. We addressed this issue by incubating normal and diseased muscle specimen with sodium thiocyanate, which leads to degradation and subsequent removal of actin from the muscle cryosections. Following this treatment we observed a markedly decreased hsp 27 staining of normal muscle fibers as well as of core and target lesions. In contrast, the removal of actin filaments did not result in a diminished αBC or desmin staining. Instead, an increased immunolabeling of both proteins was observed.
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Beyond a better epitope accessibility for desmin and αBC antibodies, these findings are in keeping with the concept of a functional divergence of these two shsps: hsp 27 is closely associated with the actin microfilament system, whereas αBC is primarily associated with the intermediate filament system.
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