KR20210155932A - Method for screening therapeutic agents for muscle diseases using the mutual coupling of Nogo-A and Pilamine-C - Google Patents

Abstract

The present invention relates to a method for screening a therapeutic agent for muscle diseases using mutual coupling of Nogo-A and filamin-C, and specifically, to a method for predicting prognosis and screening a therapeutic agent for muscle diseases caused by muscle loss and muscle damage using the mutual coupling of Nogo-A and filamin-C by confirming that a level of Nogo-A increases at the time of muscle differentiation and the mutual coupling of Nogo-A and filamin-C is a major mechanism for muscle differentiation.

Description

Method for screening therapeutic agents for muscle diseases using the mutual coupling of Nogo-A and Pilamine-C

The present invention relates to a screening method for a therapeutic agent for a muscle disease using the mutual binding of Nogo-A and pilamine-C.

Skeletal muscle makes up 40% of the total body tissue and plays a major role in body metabolism, respiration, posture, and body movement. Muscle fibers are multinucleated muscle cells that make up contractile units that move through repeated contraction and relaxation. The physical performance of muscles is affected by physiological changes such as loss of motor units, muscle fiber atrophy, ischemia, heat stress, and reduced neuromuscular activation.

Muscular dystrophy (Muscular dystrophy) is a disease in which muscle fiber function is impaired due to a defect in muscle regeneration as well as weakening and damage of muscle fibers through inflammation. In particular, Duchenne muscular dystrophy (DMD) is a muscle disease related to the abrogated dystrophin gene, and increased damage to dystrophin is associated with increased damage to pro-inflammatory cytokines, mitochondrial dysfunction and satellite cells. caused by damage

The damaged muscle regenerates the muscle to restore its functional structure. The process of muscle regeneration is similar to the muscle development that occurs during embryonic development. Upon muscle injury, satellite cells are converted into myoblasts and finally differentiate into muscle fibers through the process of muscle development. Myogenesis involves several stages, including myoblast transformation of satellite cells, differentiation into confluent myoblasts, and multinuclear myotube fusion of myoblasts. This process is regulated by myogenic regulatory factors (MRF), myogenic factor-5 (Myf5), MyoD, and myogenin, resulting in the expression of muscle-specific genes.

The neurite growth inhibitor Nogo, also known as reticulon 4 (RTN4), is one of the RTN families encoded by the RTN4 gene. RTN4 cDNA has been independently cloned by three groups and has been shown to generate three Nogo isoforms, called Nogo-A, Nogo-B, and Nogo-C, by alternative splicing or operation of a unique promoter. All isoforms contain a common C-terminal region devoid of the conventional secretory signal peptide, but have two hydrophobic transmembrane domains flanked by a 66 hydrophilic amino acid region called Nogo-66. Nogo is a reticulon protein that is mainly localized in the endoplasmic reticulum (ER), but Nogo is also observed on the cell surface. Nogo-A is the longest isoform and acts as a potent inhibitor in the central nervous system (CNS). Nogo-C has a shorter N-terminus than the other two transport types, but all three have the same N-terminus.

Nogo-A is expressed in the brain, spinal cord, eye and skeletal muscle, and the function of Nogo-A is known as a neurite growth inhibitor. Cell surface Nogo-A acts through receptors NgR1, sphingosine-1-phosphate receptor-2 (S1PR2), and myelin-associated glycoprotein (MAG). The Nogo-66 region, which is a common C-terminal sequence among Nogo isoforms, is known to bind to NgR1 and exert an inhibitory effect on neurite glands through the Rho/Rho-associated kinase (ROCK) signaling pathway.

Amyotrophic lateral sclerosis (ALS) is a serious neurodegenerative disorder that leads to deterioration of motor neurons in the brain and spinal cord. The increased expression of Nogo-A in the skeletal muscle of ALS patients suggests that Nogo-A is involved in the pathophysiology of ALS. Nogo-C is identified as the predominant isomer in skeletal muscle, and it has been reported that Nogo-C may be an apoptosis inducer of myocardial cells during myocardial infarction. However, continuous research is needed on the activation mechanism of Nogo-A in relation to muscle regeneration and muscle development.

Korean Patent Publication No. 10-2016-0076312

The present invention relates to a method for screening a therapeutic agent for a muscle disease using the mutual binding of Nogo-A and pilamine-C. Specifically, the level of Nogo-A is increased at the time of muscle differentiation, and Nogo-A is pilamine- By confirming that cross-binding with C is a major mechanism for muscle differentiation, using the mutual binding of Nogo-A and pilamine-C to provide a prognostic prediction and therapeutic agent screening method for muscle diseases caused by muscle loss and muscle damage will be.

The present invention provides a kit for predicting prognosis for muscle disease, comprising a formulation measuring the mutual binding between Nogo-A and Filamin-C.

In addition, the present invention provides an information providing method for predicting the prognosis of a patient with a muscle disease, comprising the step of measuring the mutual binding between Nogo-A and Filamin-C in muscle tissue isolated from the patient.

In addition, the present invention provides a composition for screening a therapeutic agent for a muscle disease, comprising an agent for measuring the mutual binding between Nogo-A and Filamin-C.

In addition, the present invention provides a first step of measuring the mutual binding between Nogo-A and Filamin-C (Filamin-C) in a biological sample isolated from muscle tissue; And when the cross-binding between Nogo-A and Filamine-C (Filamin-C) increases after treatment with the test substance compared to the biological sample that is not treated with the test substance, the second judged as a preventive or therapeutic agent for muscle disease It provides a screening method of an agent for the prevention or treatment of muscle disease, including the step.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases, comprising a mutual binding promoter between Nogo-A and Filamin-C as an active ingredient.

According to the present invention, the level of Nogo-A increases at the time of muscle differentiation, and by confirming that the mutual binding of Nogo-A with pilamine-C is the main mechanism for muscle differentiation, Nogo-A and pilamine-C It is possible to provide a prognostic prediction and therapeutic agent screening method for muscle diseases caused by muscle loss and muscle damage by using the mutual coupling of

1 is a diagram showing a muscle differentiation model through the interaction of Nogo-A and filamine-C (Filamin-C).
Figure 2 is a photograph of sectioning gastrocnemius muscles isolated from an 8-week-old mouse, and staining Nogo-A (green) and MYH2 (red). Scale bar is 100 μm.
3 is a photograph of Nogo-A (red) and Calnexin (ER protein, green) stained in C2C12 myoblasts under growth conditions. Scale bar is 10 μm.
4 is a photograph of Nogo-A (red) and desmin (muscle-specific, type III intermediate filaments, green) stained in C2C12 myoblasts under differentiation conditions. Scale bar is 10 μm.
5 is a graph measuring the relative expression levels of Nogo-A related molecules in differentiated C2C12 myoblasts during culture days 0, 2, and 4;
6 is a graph measuring the relative expression level of Nogo-A related molecules in differentiated C2C12 myoblasts 3 days or 2 weeks after notexin damage.
7 shows the relative expression levels of Nogo-A related molecules when the Nogo-A gene is silenced with si-Nogo-A in C2C12 myoblasts and cultured in growth conditions (GM) and maturation conditions (DM) for 3 days. This is the measured graph.
8 is a photograph showing the results of immunoblot analysis using C2C12 myoblasts under growth conditions (GM) and maturation conditions (DM) using Filamin-C and Nogo-A specific antibodies.
Figure 9 shows the intensity of the results of immunoblot analysis using Filamin-C and Nogo-A specific antibodies in C2C12 myoblasts in growth condition (GM) and maturation condition (DM) using software. It is a graph quantified using

The terms used in this specification have been selected as currently widely used general terms as possible while considering the functions in the present invention, but these may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technology, and the like. In addition, in a specific case, there is a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than the name of a simple term.

Unless defined otherwise, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Numerical ranges are inclusive of the values defined in that range. Every maximum numerical limitation given throughout this specification includes all lower numerical limitations as if the lower numerical limitation were expressly written. Every minimum numerical limitation given throughout this specification includes all higher numerical limitations as if the higher numerical limitation were expressly written. All numerical limitations given throughout this specification will include all numerical ranges that are better within the broader numerical limits, as if the narrower numerical limitations were expressly written.

Hereinafter, the present invention will be described in more detail.

The present inventors confirmed the phenomenon of increasing Nogo-A in pathological conditions including genetic, acute and chronic muscle damage, and studied that changes in Nogo-A expression are related to changes in muscle status. In addition, the present invention was completed by confirming that the increase in the interaction of Nogo-A and Filamin-C in the muscle during the pathological process is related to the regeneration of the muscle.

The Nogo-A was known as an inhibitor of axonal regeneration and neurite growth in the central nervous system (CNS) through the RhoA/ROCK signaling pathway through the phenomenon of improved axonal regeneration after spinal injury in Nogo-deficient mice. It is known that Nogo-A can act as a disease marker through its high expression in the muscles of patients with amyotrophic lateral sclerosis (ALS). The present invention confirmed that Nogo-A plays a major role in muscle regeneration and maintenance of muscle homeostasis.

The muscle regeneration process is regulated by muscle factors and forms biological regenerative units composed of muscle cells and satellite cells. Muscle regeneration initiates the activation of satellite cells and results in the formation of myotubes. In this process, it undergoes continuous expression of various muscle factors including Pax7, MyoD, and myogenin.

In the present invention, Nogo-A increases in the cytoskeleton in the process of muscle development, and candidate substances that interact with Nogo-A during the process of muscle development through IP-MS (Immunoprecipitation-Mass Spectrometry) analysis are shown in Table 1 below. same selection.

During the muscle differentiation process, the level of Filamin-C increases, and Filamin-C is localized in the cytoskeleton and interacts with Nogo-A. In addition, when the expression of Nogo-A was suppressed, it was shown that the expression of filamine-C (Filamin-C) was decreased.

The filamin-C is a muscle-specific pyramine, which is a group of actin-binding proteins and interacts with transmembrane proteins such as sarcoglycans. The filamin-C is known to interact with vinculin and F-actin in ILK-mediated signal transduction that induces cell motility.

In the present invention, it was confirmed that the level of Nogo-A and Filamin-C (Filamin-C) increased during differentiation of C2C12 myoblasts, and the mutual binding between Nogo-A and Filamin-C (Filamin-C) was increased. did

The cytoskeleton is a pivotal process in completing muscle development. As a result of analyzing the protein interacting with Nogo-A, it was confirmed that Nogo-A plays a major role in cell motility and cytoskeletal tissue through MYL. In particular, the increased mutual binding between Nogo-A and Filamin-C during myogenesis suggests that Nogo-A is involved in myogenesis by participating in cytoskeletal rearrangement.

The present invention suggests that Nogo plays a central role in maintaining muscle homeostasis by participating in muscle cell differentiation. In addition, the present invention suggests that muscle regeneration according to injury induces muscle regeneration by increasing the interaction between Nogo-A and Filamine-C.

The present invention provides a kit for predicting prognosis for muscle disease, comprising a formulation measuring the mutual binding between Nogo-A and Filamin-C.

The muscle disease is muscular dystrophy, muscular dystrophy, muscular dystrophy, dystrophinopathy, myopathy, sacopenia and Charcot Marie Tooth (CMT) from the group consisting of It may be one or more selected.

The agent capable of measuring the level of cross-binding is an antibody, peptide, aptamer or compound that specifically binds to Nogo-A or Filamin-C, and the antibody, peptide, aptamer or compound binds made of a fluorescent material.

In addition, the present invention provides an information providing method for predicting the prognosis of a patient with a muscle disease, comprising the step of measuring the mutual binding between Nogo-A and Filamin-C in muscle tissue isolated from the patient. In addition, the present invention provides a composition for screening a therapeutic agent for a muscle disease, comprising an agent for measuring the mutual binding between Nogo-A and Filamin-C.

In addition, the present invention provides a first step of measuring the mutual binding between Nogo-A and Filamin-C (Filamin-C) in a biological sample isolated from muscle tissue; And when the cross-binding between Nogo-A and Filamine-C (Filamin-C) increases after treatment with the test substance compared to the biological sample that is not treated with the test substance, the second judged as a preventive or therapeutic agent for muscle disease It provides a screening method of an agent for the prevention or treatment of muscle disease, including the step.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases, comprising a mutual binding promoter between Nogo-A and Filamin-C as an active ingredient.

The pharmaceutical composition may be formulated in the form of one or more external preparations selected from the group consisting of creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastas, and cataplasmas.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier and diluent for formulation. The pharmaceutically acceptable carriers and diluents include starch, sugar, and excipients such as mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose, hydroxypropylcellulose, gelatin, alginate, and polyvinyl blood. binders such as rolidone, lubricants such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone and crospovidone, surfactants such as polysorbates, cetyl alcohol, and glycerol does not The pharmaceutically acceptable carrier and diluent may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents, and/or dispersion media.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) according to a desired method, and is preferably administered orally. In addition, the dosage of the pharmaceutical composition of the present invention varies depending on the subject's body weight, age, sex, health status, diet, administration time, administration method, excretion rate and severity of disease, but is not limited thereto.

Hereinafter, experimental examples and examples will be described in detail to help the understanding of the present invention. However, the following experimental examples and examples are only to illustrate the content of the present invention, the scope of the present invention is not limited to the following experimental examples and examples. Experimental examples and examples of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Experimental example> Experimental material and method

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Tissue Samples

The samples of this experiment were prepared in the same way as in the previous study. Muscle tissue was fixed in 4% paraformaldehyde in PBS for 16 hours, and then treated with 30% and 35% sucrose PBS solutions for each step for 6 hours and 16 hours, respectively. Then, the muscle was blocked with OCT (optimal cutting temperature compound, Leica), stored at -70°C, cut into 4-5 μm slides with a Leica cryostat, and dried at room temperature for 30 minutes. The prepared sections were stored at -70°C.

2. Immunofluorescence Staining of Muscle Tissue

Prepared sections were hydrated in PBS, boiled with 10 mM citric acid buffer (pH 6.0) to remove antigen, cooled at room temperature for 30 minutes, washed with PBS, and 0.1% Triton-X (Triton-X) for 5 minutes ) was diluted in PBS. Then, the sections were blocked with 5% donkey serum in PBS for 1 hour, and mixed with the primary antibody in blocking buffer for half a day at 4°C and incubated. After washing with PBS, the fluorescent-secondary antibody was treated at room temperature for 1 hour. After washing with PBS, the sections or cells were treated with an antifade mounting solution containing DAPI (cell signaling), and imaged using a confocal laser scanning microscope (Carl Zeiss). Images were analyzed using the ZEN 2.1 Lite software provided by the manufacturer.

3. Cell Culture and Differentiation

C2C12 myoblasts, an immortal mouse myoblast cell line, were prepared in DMEM medium (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum (FBS) and antibodies (penicillin and streptomycin 100 U/mL) at 37°C and 5% CO _{2 humidified condition.} incubated in To induce myotube differentiation, cells were washed with PBS and cultured in DMEM medium containing 2% horse serum and 100 U/mL penicillin and streptomycin for 3 days.

4. Immunofluorescence Staining of Cells

The medium was removed from the cultured cells, washed with PBS, and fixed with 4% paraformaldehyde for 10 minutes. For permeabilization, cells were incubated in cold methanol for 10 min and washed with PBS. The cells were blocked using 5% donkey serum, diluted in a block solution mixed with the primary antibody, and incubated at 4°C for half a day. Mixed with fluorescent-conjugated secondary antibody and incubated for 1 hour. The coverslips were treated with an antifade mounting solution containing DAPI and imaged using a confocal laser scanning microscope (Carl Zeiss). Images were analyzed using the ZEN 2.1 Lite software provided by the manufacturer.

5. Immunofluorescence Image Analysis

Stained cells and muscles were photographed with a confocal laser scanning microscope (LSM700, Carl Zeiss). Five non-overlapping images were obtained from each slide at 100x, 200x or 400x magnification. Pinhole size, image resolution, color depth, scan speed, and average (number of scans per image) were set the same in all experiments. Images were adjusted and analyzed using Zen 2.1 lite (Carl Zeiss). The number of integrated nuclei and the total number of nuclei in myotube for each region were quantified. The level of fusion was calculated as the percentage of integrated nuclei in myotubes.

6. Sample preparation and LC-MS/MS analysis

Cell lysate and IP protein concentrations in growth conditions (GM) and maturation conditions (DM) were determined by BCA assay (Pierce). The protein was reduced with 10 mM DTT at 56° C. and alkylated with 50 mM IAA at room temperature in the dark. Thereafter, the protein was diluted with 100 mM AMBIC, trypsin (Promega) was added at an enzyme:protein ratio of 1:50, and cultured at 37°C for half a day. After digestion, samples were acidified with 10% TFA and desalted on tC18 plates. tC18 plates were activated with 0.1% TFA, 80% ACN and equilibrated with 0.1% TFA. The trypticized peptide was loaded onto the plate and washed 3 times with 0.1% TFA. Peptides were eluted with 0.1% TFA, 80% ACN and lyophilized by vacuum centrifugation.

About 1 μg of peptide was loaded onto an EASY-Spray™ LC column (PepMap™ RSLC C18, 2 μm, 100 Å, 50 μm x 15 cm) and organic gradient (5-40% B, A = 0.1% FA, B) for 40 min. = ACN containing 0.1% FA) was used for gradient elution with a Q-Exactive PLUS mass spectrometer (Thermo Scientific). The electrospray voltage was set to 2.2 kV. The Q-Exactive PLUS mass spectrometer was programmed to operate in data-dependent mode with up to 20 precursors in each MS scan (AGC target = 3E6, maximum fill time = 100 ms, and resolution = 70 K), and high-energy collisional (HCD) dissociation) (separation = 1.7 Da, normalized collision energy = 27%, resolution = 7.5 K, AGC target = 1E5, maximum injection = 50 ms, and intensity threshold = 1.6E5). Dynamic exclusion was set with a repeat count of 1 and an exclusion period of 30 seconds.

7. Mass Spectrometry Data Processing

Native RAW data files of the Q-Exactive PLUS mass spectrometer were processed using Proteome Discoverer v3.0. All data were retrieved against a forward-reverse mouse database collected from the SWISSPROT database. For isotopically removed HCD spectra, the precursor mass tolerance was set at 10 ppm and the MS/MS papen ion tolerance was set at 0.02 Da. Search parameters include trypsin specificity with up to two missing cleavages, fixed carbamidomethylation of Cys (C, +57 Da) and variable oxidation of Met (M, +16 Da). Reported peptide sequences were filtered based on 1% FDR.

8. Statistical Analysis

Statistical differences between control and treated samples were assessed by student's unpaired t-test (two-tailed). All results were expressed as mean±standard deviation (SEM). A case with a P-value of less than 0.05 was considered statistically significant.

Example 1. Cellular localization of Nogo-A in muscle formation

In fully developed skeletal muscle, Nogo-A is measured in mouse gastrocnemius via co-staining with MYH2, which is involved in the structural and functional properties of skeletal muscle fibers. As shown in FIG. 2 , it was found that Nogo-A and MYH2 were commonly localized and expressed in mature muscle. Nogo-A is known to mainly reside in the endoplasmic reticulum (ER), bind to receptors on oligodendrocytes, and inhibit neurite growth of cell membranes.

To evaluate the function of Nogo-A during myogenesis, the intracellular localization of Nogo-A in myoblasts in the growth or differentiation state was determined by calnexin indicating the localization of the ER or desmin indicating the cytoskeletal localization. was determined through co-staining with As shown in FIG. 3 , in C2C12 myoblasts in a growth state or a differentiated state, Nogo-A was not observed at the same position as calnexin indicating the position of the ER. On the other hand, as shown in FIG. 4, desmin staining appeared around the nucleus in C2C12 myoblasts in the growth state, but desmin staining in the C2C12 myoblasts in the differentiated state appeared in the elongated cytoskeleton. , Nogo-A staining also appeared at the same location. Thus, the above results demonstrated that Nogo-A interacts with the cytoskeletal network during myogenesis.

Example 2. Number of Molecules Interacting with Nogo-A During Muscle Development

To analyze molecules interacting with Nogo-A during myogenesis, immunoprecipitation-mass spectrometry analysis was performed using Nogo-A specific antibody in C2C12 myoblasts in growth or differentiation state.

Interactions of known differentiation state-specific interacting molecules with Nogo-A were analyzed, and the locations of the analyzed molecules differed according to the extracellular matrix, plasma membrane, cytoskeleton, cytoplasm, ER, mitochondria and nucleus.

As shown in Table 2 below, in the analysis of the standard pathway, MYL, a protein that interacts with Nogo-A, was investigated to participate in pathways regulating cell movement and cytoskeletal tissue.

Among the molecules interacting with Nogo-A, 7 molecules of Cavin1, Cavin4, Flnc, Pdlim2, Hsp90b1, Naca, and Ybx3 were selected, and the expression levels of these 7 molecules were measured during differentiation of C2C12 myoblasts. As shown in FIG. 5 , during differentiation of C2C12 myoblasts, Cavin4, Flnc, Pdlim2, and Ybx3 were shown to steadily increase, and Cavin1 was shown to decrease.

In addition, as shown in FIG. 7 , as a result of suppressing the expression of Nogo-A through si-Nogo-A in C2C12 myoblasts in the growth or differentiation state, the expression of Flnc was decreased. The above results demonstrate that Nogo-A affects the level of Flnc during muscle development.

The interaction between Nogo-A and the Flnc gene product, Filamin-C, during muscle development was evaluated through a Nogo-A-specific antibody-mediated immunoprecipitation assay. As shown in FIGS. 8 and 9 , in C2C12 myoblasts in a differentiated state, the levels of Nogo-A and Filamine-C (Filamin-C) increased, and the interaction was increased compared to C2C12 myoblasts in a grown state. .

Filamine (Filamin) is known to affect cell motility through the binding of F-actin (F-actin) and vinculin (vinculin) in the integrin-linked kinase (ILK) mediated pathway. These results suggest that Nogo-A interacts with Filamin-C and plays a major role in muscle differentiation.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (8)

A kit for predicting prognosis for muscle diseases, comprising a formulation measuring the mutual binding between Nogo-A and Filamine-C. According to claim 1, wherein the muscle disease is muscular dystrophy, muscular dystrophy (muscular dystrophy), muscular dystrophy, dystrophinopathy, myopathy (myopathy), sacopenia and Charcot Marie Tooth (Charcot Marie Tooth, CMT) kit for prognosis prediction for muscle disease, characterized in that at least one selected from the group consisting of. According to claim 1, wherein the agent capable of measuring the level of cross-binding is an antibody, peptide, aptamer or compound that specifically binds to Nogo-A or Filamin-C and the antibody, peptide, A prognostic kit for muscle disease, characterized in that it consists of a fluorescent material bound to an aptamer or a compound. A method of providing information for predicting the prognosis of a patient with muscle disease, comprising measuring the mutual binding between Nogo-A and Filamin-C in muscle tissue isolated from the patient. A composition for screening therapeutic agents for muscle diseases, comprising a formulation for measuring the mutual binding between Nogo-A and Filamin-C. A first step of measuring the mutual binding between Nogo-A and Filamin-C in a biological sample isolated from muscle tissue; and
After treatment with the test substance, if the mutual binding between Nogo-A and Filamine-C is increased compared to the biological sample that is not treated with the test substance, the second step of determining as an agent for the prevention or treatment of muscle disease ; Screening method of a formulation for the prevention or treatment of muscle disease, including. 7. The method of claim 6, wherein the muscle disease is muscular dystrophy, muscular dystrophy, muscular dystrophy, dystrophinopathy, myopathy, sacopenia and Charcot Marie Tooth, CMT) screening method for a formulation for the prevention or treatment of muscle disease, characterized in that at least one selected from the group consisting of. A pharmaceutical composition for the prevention or treatment of muscle diseases, comprising a mutual binding promoter between Nogo-A and Filamine-C (Filamin-C) as an active ingredient.

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