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Accumulation of hippocampal cholinergic neurostimulating peptide (HCNP) precursor protein in muscle fibers in inclusion-body myositis

Etsuo Yamada1, Eiiti Katada1, Shigehisa Mitake1, Yasushi Otsuka1, Noriyuki Matsukawa1, Mina Maki1, Hiko Suzuki1, Manabu Morishita1, Hideka Nakazawa1, Osamu Fujimori2, Kou Sahashi3, Tatsu Ibi3, Ryuzou Ueda1, and Kosei Ojika4
1Second Department of Internal Medicine, 2Secand Department of Anatomy and 4Department of Neurology, Nagoya City University Medical School, Kawasumi, Mizuho-ku, Nagoya 467-8601, Japan. 3Department of Neurology, Aichi Medical University, Yazako, Nagakute, Aichi 480-1195, Japan.
Running title: HCNP precursor protein in muscle fibers in IBM
Key words: Inclusion-body myositis, hippocampal cholinergic neurostimulating peptide, HCNP, serine protease inhibitor, Raf-1 inhibitor
Address correspondence to: Dr. Kosei Ojika, Department of Neurology, Nagoya City University Medical School, Kawasumi, Mizuho-ku, Nagoya 467-8601, Japan.
Telephone number: +81-52-851-5511, E-mail: kojika@med.nagoya-cu.ac.jp
Footnote: IBM, inclusion myositis; HCNP, hippocampal cholinergic neurostimulating peptide; PM, polymyositis; bAPP, b-amyloid precursor protein; HBs, Hirano bodies; sIBM, sporadic IBM; MD, myotonic muscular dystrophy; ALS, amyotrophic lateral sclerosis; EDTA, ethylenediaminetetraacetic acid; APMSF, (4-amidinophenyl)-methylsulfonyl fluoride; SDS, sodium dodecyl sulphate; PAGE, polyacrylamide gel electrophoresis; ECL, enhanced chemi-luminescence; PE, phosphatidylethanolamine; ATP, adenosine triphosphate; and RKIP, Raf-1 kinase inhibitory protein.

Summary
In inclusion-body myositis (IBM), muscle fibers show abnormal accumulations of several molecules that are also deposited in the Alzheimer耼s disease brain. We have previously demonstrated that hippocampal cholinergic neurostimulating peptide (HCNP) enhances cholinergic phenotype development, and that HCNP-related antigen accumulates specifically in Hirano bodies. To examine whether HCNP precursor protein is involved in IBM molecular pathogenesis, we employed immunocytochemical and Western blot analysis. Immunocytochemical analysis showed that in all degenerated muscles from sIBM patients, but not in muscles from non-IBM disease controls, the antigens were specifically precipitated within the inclusions and cytoplasm of vacuolated or vacuole-free atrophic fibers, mostly co-localized with ubiquitin. Furthermore, Western blot analysis indicated that in sIBM muscle HCNP precursor protein was specifically increased in the insoluble fraction.

Introduction
Inclusion-body myositis (IBM) is a progressive myopathy usually occurring sporadically and in patients over the age of 55 years, and although it has clinical and pathological similarities to polymyositis (PM), it is either unresponsive or only poorly responsive to immunosuppressive treatment (1). Light-microscopic findings in IBM muscle-biopsy specimens include mononuclear cell infiltration (varying from abundant to none), atrophic muscle fibers, and muscle fibers with rimmed vacuoles that usually contain material stained red in the modified trichrome reaction (1). Immunohistochemical (1, 2) and molecular biological (3) studies have shown that within the vacuolated muscle fibers seen in IBM, there are abnormal accumulations and an increased gene expression of several proteins typically accumulated in the AD brain. Moreover, overexpressing 汕-amyloid precursor protein (汕APP) in muscle (4) and brain (5) leads to the development of similar morphological alterations in IBM muscles and AD brain. Although these findings suggest that IBM and AD may share a common pathogenetic mechanism (1), the etiology and mechanisms underlying the pathogenesis of IBM remain uncertain.
We previously showed that crude extracts of rat hippocampus enhance acetylcholine synthesis and morphological development in medial septal nuclei explant cultures (6). These effects are partly mediated by a novel undecapeptide purified from the hippocampus of young rats, and designated as hippocampal cholinergic neurostimulating peptide (HCNP) (7). Subsequent studies have shown (a) that while the antigens related to this peptide and their mRNAs are ubiquitous in their expression, the highest expressions are found in the hippocampus (8), and (b) that in the hippocampus, the peptide appears to be involved in the phenotype development of the septo-hippocampal system (8). HCNP precursor protein seems to have multiple functions (8): not only is the precursor of HCNP cleaved by a unique chymotrypsin-like thiol protease (9), but the protein itself interacts with other molecules (8), including phosphatidylethanolamine (10), Raf-1 kinase (11), and serine proeases (12). Interestingly, HCNP and/or its precursor protein may also have important roles in memory formation in adult animals. For example, cholinergic and glutamatergic stimulation reciprocally regulate the expression of HCNP precursor-protein gene in the hippocampus (13), and HCNP-related antigens are increased specifically in the hippocampus of aged senescence-acceleration-prone mice, which show a remarkable age-accelerated deterioration in their ability to learn tasks (14). In humans, HCNP antigens are at a high level in the CSF of some patients with AD (8), and they accumulate in all types of Hirano bodies (HBs), but not in normal tissues or in other abnormal structures in the brain (15,16). HBs are intraneuronal inclusions and represent an important neuropathology in the hippocampus of patients showing degenerative dementia, especially AD, although they can be found to some extent or other in the brain of normal elderly individuals (17).
In this study, we examined the muscles of sporadic IBM (sIBM) patients by immunocytochemical methods. The results show that HCNP precursor protein is accumulated in all abnormal structures within the degenerated muscle fibers. It is speculated that the accumulation of HCNP is involved in the sIBM muscle degeneration.

Material and Methods
Patients
Studies involving immunological assays of muscle fibers were performed using biopsy muscle specimens obtained for diagnosis from 33 patients, aged 25-72 years, with the following diagnoses: sIBM, 12; non-IBM controls 21 [PM, 7; myotonic muscular dystrophy (MD), 7; and amyotrophic lateral sclerosis (ALS), 7]. The mean age of the sIBM patients was 60 ﹢18 years and that of the non-IBM controls was 61 ﹢15 years. Diagnosis of all patients was based on clinical and laboratory studies, including the histopathology of muscle biopsy specimens. The specimens from all sIBM patients exhibited vacuolated muscle fibers on modified trichrome staining; muscle fibers containing interior and subsarcolemmal vacuoles, with or without a slight mononuclear cell infiltration adjacent to the abnormal fibers (Fig. 1A), Congo red-positive amyloid (Fig. 1B), ubiquitinated inclusions (Fig. 1C).
The study was approved by the Ethics Committee of the Medical School, Nagoya City University, and those of the affiliated institutions involved in this study. Informed consent was obtained from all subjects after the nature of the study had been fully explained.

Antibodies
Two previously characterized polyclonal antibodies (8) against HCNP and its precursor protein were used: 1) affinity-purified rabbit antibody to human deacetylated-HCNP (Pro-Val-Asp-Leu-Ser-Lys-Trp-Ser-Gly-Pro-Leu), diluted 1:1000; and 2) affinity-purified rabbit antibody to peptide_63-79 (the common amino acid sequence at positions 63-79 of rat and human HCNP-precursor protein), diluted 1:2000. The methods used for peptide synthesis, and for the generation and purification of these antibodies have been described previously (7, 8). Rabbit polyclonal anti-human ubiquitin antibody (DAKO) was used at a dilution of 1:100 to identify abnormal accumulations of ubiquitin in IBM muscle.
Western blotting analysis
A piece of muscle tissues (~0.1g wet weight) from patients with the various diseases were obtained at muscle biopsy, and homogenized (using a micro tissue grinder; Funakosi, Japan) in ice-cold buffer (50 mM Tris-HCl, pH 6.8, 2 mM EDTA, 2 米g/ml of aprotinin, 10 米g/ml of E-64, and 40 米g/ml of APMSF). Total homogenates (120 米g protein from each muscle sample) were centrifuged at 4∵ for 15 min at 20,000 ﹣g, and the supernatants (soluble extracts) and sediments were collected. The sediments were carefully dissolved in 200 米l of 70% formic acid, then centrifuged at 4∵ for 15 min at 20,000﹣g, and the supernatants were collected, evaporated, and then dissolved in 20 米l of 8 M urea, 2% SDS, and 5% 汕-mercaptoethanol. Aliquots (30 米g protein) of both supernatants and the formic-acid extracts of the sediments were electrophoresed through polyacrylamide gels containing sodium dodecyl sulphate (SDS-PAGE) and transferred to nitrocellulose membranes. Then, after non-specific binding had been blocked with 5% skim-milk, the membranes were incubated for 4 hr with the first antibodies at room temperature. The signal was detected using HRP-conjugated goat anti-rabbit IgG (Cappel) and the enhanced chemi-luminescence (ECL) method (following the protocol recommended by the manufacturer, Amersham). Appropriate molecular weight standards were included in each run. The protein concentrations in the muscle extracts were measured by the method of Bradford (18), using bovine serum albumin as a standard. Levels of intensity in Western blotting were analyzed using an image analyzer, as described previously (14), and the mean relative intensities were statistically analyzed using a Student's t-test.

Light microscopic immunocytochemistry
Fresh-frozen muscle biopsies were sectioned at 10 米m, dried, and then fixed with acetone. Immunocytochemical staining was performed on transverse sections using a streptavidin-biotin-peroxidase method, following the protocol recommended by the manufacturer (DAKO; LSAB kit). Incubation with all primary antibodies was performed for 16 hr at 4∵, and was followed by a 60-min incubation with an appropriate secondary antiserum at room temperature.
Control experiments were performed to establish immunostaining specificity. These included the incubation of sections either 1) with antibodies for HCNP and for peptide_63-79preabsorbed using an excess of a synthetic form of the appropriate immunogens, or 2) with immunoglobulins prepared from non-immune rabbit serum. To block nonspecific binding of antibody to Fc receptors, sections were preincubated with 1:10-diluted normal goat serum.

Results
Western blotting analysis
Soluble extracts of muscles from patient with sIBM, PM, MD, or ALS were separated by 12.5% SDS-PAGE, transferred onto membranes, and probed with affinity-purified anti-HCNP antibody (each diluted 1:2000), or anti-peptide_63-79 antibody (1:5000). Each antibody specifically recognized a 23 kDa HCNP precursor protein. The staining intensities of the bands in the blots were essentially similar among the muscle extracts prepared from biopsy specimens obtained from sIBM patients and non-IBM control subjects (Fig. 2A). When formic acid extracts of the sediments were analyzed by Western blotting using 12.5% SDS-PAGE and anti-peptide_63-79 antibody, all sediments were also found to contain a 23 kDa component (Fig. 2B). The intensity levels of the bands for patients with sIBM (4 patients) and non-IBM (2 patients with ALS and PM) were 3049.0 ﹢793.0 and 1573.5 ﹢1063.5, respectively, and the levels were significantly (p<0.05) higher in sIBM patients than in the non-IBM controls.

Light microscopic immunocytochemistry
Use of antibodies against HCNP (Fig. 3A) and peptide_63-79 (Fig. 3D) in the immunohistochemical analysis of consecutive sections of sIBM muscle revealed almost the same abnormal accumulation pattern of their antigens. These antigens were distributed within the cytoplasm and/or in the subsarcolemma of all degenerated muscle fibers in specimens from patients with sIBM, but this was not the case in control muscles or in the apparently normal muscles in sIBM specimens (Figs. 1D and 3). The accumulation pattern of both HCNP (Figs. 3A, B, C) and peptide_63-79 (Figs. 3D, E, F) antigens in sIBM muscle could be classified into four types. [1] In muscle fibers with small vacuoles (associated with, or without small inclusions) in the cytoplasm as well as in the peripheral subsarcolemma, the antigens accumulated in close proximity to each other, and they rimmed the vacuoles (Figs. 3B, E). In muscle fibers with large vacuole(s) associated either [2] with small inclusions (Figs. 3A, D) or [3] with large inclusions (Figs. 3C, F), the antigens were diffusely accumulated in the degenerated muscle fibers and within the inclusions. [4] Some very abnormally degenerated vacuole-free, atrophic fibers also showed strong immunopositivity within the cytoplasm (Figs. 3A, B).
To compare the localization of HCNP-related components with that of ubiquitin, consecutive sections of IBM muscle biopsies were used. The immunocytochemical distribution patterns of the antigens probed with anti-ubiquitin antibody (Fig. 1C) and anti-HCNP (Fig. 1D) were similar, suggesting a co-localization of HCNP antigen with ubiquitin antigen.
No reaction-product deposits were seen in sections incubated without the primary antibodies, with antibodies preabsorbed with their respective antigens, or with nonimmune rabbit serum. None of the biopsy specimens from patients with polymyositis or from the other non-IBM control patients showed HCNP- or peptide_63-79-immunoreactivities like those exhibited by sIBM patients.

Discussion
In the present study, we employed two different antibodies to detect HCNP precursor protein, each allowed to bind to the N-terminal (HCNP) and the middle domain (peptide_63-79). As shown previously by Ojika et al (8) and corroborated by the present study, these antibodies specifically recognize the 21 kDa HCNP precursor protein, which in Western blots is detected as a 23 kDa band. The 1.1 kDa HCNP proper could not be detected by anti-HCNP antibody because of its low binding to the transfer membrane (15). The immunohistochemical assays demonstrated that the two antibodies recognized the same abnormal structures, and that the distribution patterns of HCNP-related immunopositive components in the degenerated muscle fibers of sIBM patients were identical; the two antibodies were equal in their recognition of all abnormally degenerated sIBM muscles. Further, from the consistently negative immunostaining results obtained in the extensive control experiments carried out in this study, it is evident that the positive reaction observed with the antibodies is highly specific. These results strongly suggest that the accumulations of HCNP-related components seen in degenerated sIBM muscles consist of HCNP precursor protein. Further, the idea that these were precursor-protein accumulations was substantiated by the results of the Western blotting analysis. In this analytic modality, the level of HCNP precursor protein in the formic acid extracts of the sediments obtained by centrifugation of muscle homogenates was greater for sIBM patients than for the non-IBM controls, suggesting its aggregation or deposition as material scarcely soluble in neutral solution. Moreover, in the present study HCNP-related antigen accumulation was co-localized with ubiquitin, the important small protein that bears degradation signals recognized by the 26S proteasome (19). This suggests that HCNP precursor protein and ubiquitin may form a complex and deposit in the same subcellular organelles in the course of degradation. If this is in fact the case, the accumulation of HCNP antigen seen in sIBM muscles may be ascribed to its decreased degradation.
The significance of the putative accumulation of HCNP precursor protein in the pathogenesis of degenerated sIBM muscles is uncertain. HCNP precursor protein has multi-functional properties, and this is the binding protein of opioides (20), phosphatidylethanolamine (PE) (8, 10), and ATP (8, 21). Furthermore, a partial cDNA sequence of a rat protein that suppresses Raf-1 kinase activity (RKIP) is identical to the HCNP-precursor protein (11), and that the precursor protein acts as a member of a novel family of serine protease inhibitors in the brain (12). Members of this HCNP precursor protein family have been suggested to play important roles in the development and modulation of biological activities in many organisms (22). They include that a number of HCNP-antigen-bearing neurons is significantly increased in the perinatal period in the human hippocampus (23), HCNP itself enhances cholinergic phenotype-development in vitro (7, 8), and mRNA for HCNP precursor protein expressed in the hippocampus of adult rats changes with neuronal activity (13). Moreover, the expression of the precursor protein is significantly increased during certain periods of degeneration of skeletal muscles after traumatic injury, as well as during their regeneration (24). On the other hand, it is also noted that the HCNP-related antigen is hardly detectable by immunohistochemical assay in the normal adult brain (14, 15, 16, 20) or normal skeletal muscles (as shown in this study).
In contrast to the situation described above, it has been shown in a variety of disorders of the central nervous system that HCNP-related antigen specifically accumulates in all types of HBs (solitary and intermingled with senile plaques or neurofibrillary tangles) (15, 16). These represent an important cytoskeletal alteration in the hippocampus of patients with degenerative dementia, their number in the pyramidal layer of the AD brain being greater than in that of the other brains examined (16, 17). Accumulation of HCNP-related antigen in the degenerated muscles of sIBM patients could be a consequence of the muscle injury, as mentioned above. However, interestingly none of the muscles from the diseased controls examined in this study showed antigen accumulation, suggesting that the accumulation is not associated with muscle injury per se, and is relatively specific to the degenerated muscles of sIBM. These data obtained by our present and previous studies pertaining to HCNP-related antigen-accumulation strongly support the notion that sIBM and degenerative dementia, especially AD, share molecular-pathologic similarities (1).
The nature of the mechanisms involving HCNP precursor protein in the pathogenesis of the two different neuromuscular disorders, AD and sIBM, is unclear. However, it is possible that a change in the amount of HCNP precursor protein and/or in its activity as a serine protease inhibitor (12) may have a direct bearing on the development of specific morphologies by these two disorders, because the balance between serine proteases and their inhibitor has been suggested to be an important determinant of cell fate during development and following injury (25). Moreover, a change in the RKIP activity of the precursor protein might allow subsequent activation of Raf/MEK/ERK cascades (11) and cyclin-dependent kinases (26), which have indeed been shown to be abnormally activated and/or expressed in IBM muscles (27, 28) as well as in the AD brain (29,30). These observations may indicate that HCNP precursor protein is involved as a key molecule in the pathogenesis of these two disorders. However, verification of the putative functional role of HCNP precursor protein in the pathogenetic mechanisms underlying sIBM will require further experiments. We will need, for instance, to elucidate the precise function of HCNP precursor protein in muscle, as well as the nature of the mechanism responsible for its aggregation and deposition in this disease state.

Acknowledgments:
The authors wish to thank Dr. Hiroshi Inagaki of the Second Department of Pathology, Nagoya City University Medical School, Dr. Tatuo Shirataki of the Section of Clinical Laboratory, Nagoya City Rehabilitation Center. Part of this work was supported by a grant-in-aid for research from Sumitomo Pharmaceutical Co. Ltd.

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Figure Legends
Fig. 1. Light microscopic co-localization of HCNP-related antigen and molecules commonly deposited in sIBM muscles. (A) Abnormal muscle fibers stained by the modified trichrome method contain interior and subsarcolemmal vacuoles with a slight mononuclear cell infiltration. Scale bar, 20 米m. (B) Amyloid deposits (arrow) in an abnormal muscle fiber (Congo red staining viewed in polarized light). Scale bar, 40 米m. Serial consecutive sections were used to identify the co-localization. The immunoreactive pattern of anti-ubiquitin antibody (C) is similar to that of anti-HCNP antibody (D). Hematoxylin was used as counterstain. Scale bar, 40 米m.
Fig 2. Western blotting analysis of muscle extracts. Muscle tissues obtained by biopsy were homogenized, then separated by centrifugation. (A) Aliquots (30 米g protein) of supernatants prepared from muscles [ALS (lane 1), MD (lane 4), PM (lane 2,5), and sIBM (lane 3,6)] were separated by 12.5% SDS-PAGE, transferred onto membranes, and probed with antibodies to HCNP (lanes 1,2,3) and peptide_63-79 (lanes 4,5,6). (B) Sediments of the muscle homogenates were re-extracted using formic acid. Aliquots (30 米g protein) of the extracts [sIBM (lane 1,3,5,7), ALS (lane 2,6), and PM (lane 4,8)] were analyzed by Western blotting using 12.5% SDS-PAGE and the antibody to peptide_63-79.
Fig 3. Light microscopic immunocytochemistry of the N-terminal (HCNP) and the middle domain (peptide_63-79) of HCNP precursor protein in abnormal muscle fibers of sIBM. The streptavidin-biotin-peroxidase method was used to detect antibody binding. Antibodies against human HCNP (A, B, C) and peptide_63-79 (D, E, F) revealed similar antigen distribution patterns. By the use of consecutive sections (A, D), both antigens can be seen to be equally accumulated in degenerated muscle fibers with a small inclusion in a large vacuole (arrow head) and in a vacuole-free, atrophic fiber (arrow), but not accumulated in normal muscle fibers. In a muscle fiber with small vacuoles (containing small inclusions), the antigens localized in close proximity to each other in the cytoplasm as well as in the peripheral region (the subsarcolemma), and they rim the vacuoles (B, E). Both antigens are diffusely distributed in the cytoplasm and in an inclusion body in a muscle fiber containing a large inclusion body within a large vacuole (arrow) (C, F). Scale bars = 30 米m

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