Method for screening substance that suppresses intramuscular adipose tissue (IMAT) formation, and composition for suppressing IMAT formation

Abstract

The present invention provides, inter alia, a method for screening a substance that suppresses intramuscular adipose tissue (IMAT) formation. Provided are: a method for screening a substance that suppresses IMAT formation, the method comprising: (I) a step for bringing a Wnt5b protein, an Ror2 protein, and a test substance into contact with each other; and (II) a step for confirming that the test substance inhibits binding of the Wnt5b protein and Ror2 protein; and a composition for suppressing IMAT formation.

Description

Method for screening for inhibitors of intramuscular adipose tissue (IMAT) formation, and composition for inhibiting IMAT formation

[0001] The present disclosure relates to a method for screening for a substance that inhibits IMAT formation, a composition for inhibiting IMAT formation, and the like.

[0002] Skeletal muscle tissue stem cells, satellite cells (SCs) and mesenchymal progenitor cells (MPCs), play important roles in the maintenance and regeneration of skeletal muscle. After skeletal muscle injury, SCs proliferate, differentiate, and fuse to ultimately form myofibers, contributing to the repair of the injured area. Furthermore, MPCs, which reside in skeletal muscle, promote skeletal muscle regeneration by promoting the proliferation and differentiation of SCs. However, in pathological conditions of skeletal muscle, such as muscular dystrophy and sarcopenia, MPCs differentiate into adipocytes, forming IMATs, which are normally absent in skeletal muscle, and causing a significant decline in muscle function. Based on these findings, it is believed that SCs and MPCs are strictly regulated during skeletal muscle regeneration, but the underlying mechanisms remain unclear. Previously, it has been found that expression of the Ror family receptors Ror1 and Ror2, known as receptors for the secreted glycoprotein Wnt protein, increases in skeletal muscle after injury (Kamizaki K, et al., J. Biol. Chem., 2017, 292, 38). Ror1 (Receptor tyrosine kinase-like orphan receptor 1) is highly expressed in SCs and has been shown to contribute to skeletal muscle regeneration by promoting SC proliferation after skeletal muscle injury (Kamizaki K, et al., J. Biol. Chem., 2017, 292, 38). Ectopic fat accumulation Ectopic adipocytes

[0003] Aging (Albany NY). 2018 Apr 25;10(4):747-763. doi:10.18632 / aging. 101425. Cell Stem Cell. 2019 Mar 7;24(3):433-446. e7. doi:10.1016 / j. stem. 2018.12.014. Epub 2019 Jan 24. Nature. 2022 Jul;607(7920):790-798. doi:10.1038 / s41586-022-04924-6. Epub 2022 Jun 29. Nat Med. 2015 Jul;21(7):786-94. doi:10.1038 / nm. 3869. Epub 2015 Jun 8. Nat Cell Biol. 2018 Aug; 20(8):917-927. doi:10.1038 / s41556-018-0151-y. Epub 2018 Jul 26.

[0004] The molecular mechanisms that determine whether MPCs contribute to skeletal muscle maintenance and regeneration or promote IMAT formation are not fully understood. However, it is expected that maintaining the skeletal muscle maintenance and regeneration mechanisms of MPCs while suppressing their IMAT formation ability can inhibit skeletal muscle pathologies. Therefore, identifying substances that suppress IMAT formation would significantly contribute to improving the conditions of skeletal muscle diseases and age-related skeletal muscle disorders (e.g., locomotive syndrome, frailty), and would also significantly contribute to improving people's quality of life (QOL) and ADL (activity-related lifestyle) in Japan, which has become a super-aged society, and internationally. However, no substances with such effects have been discovered to date, and no methods for identifying or screening for them have been established. An objective of the present disclosure is to provide a method for screening for substances that suppress IMAT formation. Another objective of the present disclosure is to provide a composition for suppressing IMAT formation.

[0005] The present inventors have found that Ror2 (Receptor tyrosine kinase-like orphan receptor 2) is highly expressed in MPCs and functions as a receptor for Wnt5b, whose expression increases during IMAT formation, thereby inducing excessive proliferation of MPCs and contributing to the formation of IMAT. They have also found that the Wnt11-Ror2 signal contributes to the maintenance and regeneration of skeletal muscle by suppressing cellular senescence in MPCs, while activation of the Wnt5b-Ror2 signal in MPCs under pathological conditions may promote IMAT formation. Based on these unique findings, and by utilizing the in vitro and in vivo analytical methods used to accumulate these findings, the researchers were able to select substances that suppress Wnt5b-Ror2 signaling while maintaining Wnt11-Ror2 signaling in MPCs, thereby discovering a method for identifying (screening) substances that may significantly contribute to improving the condition of various skeletal muscle diseases and age-related skeletal muscle disorders (locomotor syndrome, frailty, etc.).

[0006] The present disclosure includes, for example, the subject matter described in the following paragraphs. Item 1. A method for screening for a substance that suppresses IMAT formation, comprising: (I) contacting a Wnt5b protein, a Ror2 protein, and a test substance; and (II) confirming that the test substance inhibits the binding of the Wnt5b protein and the Ror2 protein. Item 2. The method of Item 1, further comprising contacting a Wnt11 protein in step (I), and further confirming that the test substance does not inhibit the binding of the Wnt11 protein and the Ror2 protein in step (II). Item 3. A method for screening for a substance that suppresses IMAT formation, comprising: (1) contacting a test substance with at least one species selected from the group consisting of SCs, MPCs, and Schwann cells; and (2) confirming that the test substance suppresses the expression level of Wnt5b. Item 4. The method of Item 3, further comprising confirming that the test substance does not suppress the expression level of Wnt11 in step (2). Item 5. Item 6. A method for screening for a substance that inhibits IMAT formation, comprising: (A) contacting Wnt5b protein, Ror2 protein, p38 protein, and a test substance; and (B) confirming that the test substance inhibits phosphorylation of p38 protein. Item 7. A composition for inhibiting IMAT formation, comprising, as an active ingredient, a neutralizing antibody to Wnt5b protein or a nucleic acid that inhibits expression of the Wnt5b gene. Item 8. The composition for inhibiting IMAT formation according to Item 7, for use in inhibiting or treating a muscular disease or symptom in which IMAT accumulation is observed. Item 9. The composition for inhibiting IMAT formation according to Item 8, wherein the muscular disease or symptom in which IMAT accumulation is observed is muscular dystrophy, amyotrophic lateral sclerosis, sarcopenia, frailty, or locomotive syndrome.

[0007] The molecular mechanisms determining whether MPCs are involved in the maintenance and regeneration of skeletal muscle or contribute to the promotion of IMAT formation have not been fully elucidated. However, the present inventors have discovered that Wnt11-Ror2 signaling in MPCs is important for the maintenance and regeneration of skeletal muscle, while Wnt5b-Ror2 signaling is important for the formation of IMAT. These findings reveal that whether Ror2 functions as a receptor for Wnt11 or Wnt5b is crucial in determining the function of MPCs. Wnt5b, Wnt11, and Ror2 play important roles in tissue and organ formation during development and in the progression of cancer, but are rarely expressed in normal adult tissues. Therefore, targeting Wnt5b, Wnt11, and Ror2 as therapeutic targets for muscle diseases is expected to lead to the development of highly effective treatments with minimal side effects, and is a major advantage in discovering such treatments. However, because MPCs are also important for the maintenance and regeneration of skeletal muscle, simply inhibiting the function of MPCs is not sufficient; it is necessary to inhibit the ability to form IMAT while maintaining the skeletal muscle maintenance and regeneration mechanism. Based on the above-mentioned unique findings, the present inventors have established a method for selecting substances that inhibit Wnt5b-Ror2 signaling while maintaining Wnt11-Ror2 signaling, and can significantly contribute to improving the condition of various skeletal muscle diseases and age-related skeletal muscle disorders (e.g., locomotive syndrome, frailty). A method for screening for substances that inhibit IMAT formation is provided. Also provided is a composition for inhibiting IMAT formation.

[0008] Figure 1 shows the results of measuring the expression levels of Ror2 in SCs and MPCs. Figure 2 shows the results of measuring the proliferation ability, adipocyte differentiation ability, and cellular senescence of MPCs isolated from Ctx-administered tibialis anterior (TA) muscle. Figure 3 shows the results of measuring the weights of the TA muscle, gastrocnemius muscle, and soleus muscle, and the results of staining myofibers in the TA muscle in control mice and Ror2cKO mice. Figure 4 shows the results of staining myofibers and fat in the TA muscle in control mice and Ror2cKO mice. Figure 5 shows the results of measuring the expression levels of Wnt11 and cellular senescence in MPCs isolated from Ctx-administered TA muscle. Figure 6 shows the results of stress fiber and nuclear staining in MPCs isolated from Ctx-administered TA muscle. Figure 1 shows the expression levels of Wnt family genes in Ctx-administered muscles and glycerol-administered muscles, as well as the results of measuring cell proliferation when Wnt5a or Wnt5b was added to MPCs isolated from Ctx-administered TA muscles. Figure 2 shows the results of Western blot using MPCs isolated from Ctx-administered TA muscles, and the results of a WST-8 assay using MPCs isolated from Ctx-administered TA muscles. Figure 3 shows the results of whole-mount staining of EDL (Extensor digitorum longus) muscles 3 days after administration of Ctx or glycerol. Figure 4 shows the results of measuring the expression levels of Wnt5b in various cells isolated from TA muscles 3 days after administration of Ctx or glycerol. Figure 5 shows the results of measuring the expression levels of Wnt5b and cell proliferation in MPCs isolated from Ctx-administered TA muscles.

[0009] Each embodiment of the present disclosure will be described in more detail below. The present disclosure includes a method for screening for an IMAT formation inhibitor. In this specification, this method may be referred to as the "screening method of the present disclosure" or the like.

[0010] As used herein, IMAT (intramuscular adipose tissue) refers to ectopic adipose tissue found within skeletal muscle, a condition in which fat accumulates despite the fact that skeletal muscle is essentially devoid of fat.

[0011] The screening method of the present disclosure includes a step of contacting a Wnt5b protein, a Ror2 protein, and a test substance. In this specification, this step may be referred to as step (I) or the like.

[0012] Wnt (Wingless-type MMTV integration site family) proteins are secreted glycoproteins known to bind to Ror proteins and the like. Nineteen types of Wnt proteins have been identified in mammals, including Wnt5a, Wnt5b, and Wnt11. The amino acid sequences, nucleotide sequences, and the like of these proteins can be found from publicly known databases. For example, at the NCBI (National Center for Biotechnology Information), the accession number for the mRNA sequence of the human Wnt5b gene is NM_030775, and the accession number for the mRNA sequence of the human Wnt11 gene is XM_005274231. Examples of Wnt5b-producing cells include SCs, MPCs, Schwann cells, and the like. Examples of Wnt11-producing cells include MPCs, and the like.

[0013] Ror (Receptor tyrosine kinase-like orphan receptor) protein is a single-pass transmembrane receptor that functions as a receptor for Wnt proteins and is known to activate the non-canonical Wnt pathway. Examples of Ror proteins include Ror1 and Ror2. The amino acid sequence, nucleotide sequence, and the like of these proteins can be found from publicly known databases. For example, at NCBI, the accession number for the mRNA sequence of the human Ror2 gene is NM_001318204. Examples of Ror2-expressing cells include MPCs.

[0014] The test substance is not particularly limited. For example, it may be a compound or a composition. The compound may be, for example, a low molecular weight compound, a nucleic acid (e.g., DNA, RNA, etc.), a protein (e.g., an antibody or a portion thereof, etc.), or a polymeric compound such as a polymer. The composition may also be an extract obtained from an organism (e.g., an animal, a plant, a microorganism, etc.), or a combination of two or more compounds.

[0015] In step (I), the method for contacting the Wnt5b protein, Ror2 protein, and test substance is not particularly limited. For example, the Wnt5b protein, Ror2 protein, and test substance may be added to an appropriate solvent, or the Wnt5b protein and test substance may be added to cells expressing the Ror2 protein. Suitable solvents are not particularly limited. Examples include water and liquid cell culture media. The concentration of the Wnt5b protein is not particularly limited. For example, it can be about 0.1 to 4 μg / ml. The concentration of the Ror2 protein is not particularly limited. For example, it can be about 0.1 to 4 μg / ml. The concentration of the test substance can be appropriately set depending on the type of test substance. The temperature during contact is not particularly limited. For example, it can be about 25 to 40°C, preferably about 30 to 40°C. The contact time is not particularly limited. For example, it can be about 10 seconds to 3 hours, preferably about 3 minutes to 1 hour. During contact, the cells can be stirred, shaken, etc. as needed. Examples of cells that express Ror2 protein include MPCs.

[0016] The screening method of the present disclosure can further include contacting a Wnt11 protein in step (I). In other words, the screening method of the present disclosure can include contacting a Wnt5b protein, a Wnt11 protein, a Ror2 protein, and a test substance in step (I).

[0017] The method for contacting the Wnt5b protein, Wnt11 protein, Ror2 protein, and test substance is not particularly limited. For example, the Wnt5b protein, Wnt11 protein, Ror2 protein, and test substance may be added to an appropriate solvent, or the Wnt5b protein, Wnt11 protein, and test substance may be added to cells expressing Ror2 protein. The concentration of the Wnt11 protein is not particularly limited. For example, it can be about 0.1 to 4 μg / ml. For other contact methods, the above-mentioned description can be used.

[0018] The screening method of the present disclosure further includes a step of confirming that the test substance inhibits the binding between Wnt5b protein and Ror2 protein. In this specification, this step may be referred to as step (II) or the like.

[0019] In step (II), the method for confirming that the test substance inhibits the binding between the Wnt5b protein and the Ror2 protein is not particularly limited, and examples thereof include ELISA, AlphaScreen, and analysis using a Biacore system.

[0020] For example, after confirming that the binding between Wnt5b protein and Ror2 protein can be detected by various analytical techniques such as absorbance, luminescence, fluorescence, and surface plasmon resonance, it is preferable that the amount of binding between Wnt5b protein and Ror2 protein is reduced by 35% or more when the test substance is added, compared to when the test substance is not added. The upper or lower limit of this range may be, for example, approximately 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. Specifically, it may be approximately 35 to 100%.

[0021] According to the screening method of the present disclosure, for example, it is possible to screen for a substance that can inhibit the binding between Wnt5b protein and Ror2 protein when the test substance is added compared to when the test substance is not added.

[0022] As shown in the Examples below, it is believed that Wnt5b and Ror2 promote the proliferation of MPCs via p38, thereby inducing the excessive accumulation of MPCs in skeletal muscle tissue and contributing to the formation of IMAT. Without wishing to be bound by theory, it is speculated that an IMAT formation inhibitor obtained by the screening method of the present disclosure may inhibit the binding between Wnt5b protein and Ror2 protein, thereby suppressing the excessive proliferation of MPCs and suppressing the formation of IMAT.

[0023] When Wnt5b protein, Ror2 protein, Wnt11 protein, and a test substance are contacted in step (I), the screening method of the present disclosure further includes a step of confirming in step (II) that the test substance does not inhibit the binding of Wnt11 protein to Ror2 protein. In other words, the screening method of the present disclosure can confirm in step (II) that the test substance inhibits the binding of Wnt5b protein to Ror2 protein, but does not inhibit the binding of Wnt11 protein to Ror2 protein.

[0024] The method for confirming that a test substance does not inhibit the binding between Wnt11 protein and Ror2 protein is not particularly limited, and examples thereof include ELISA, AlphaScreen, and analysis using a Biacore system.

[0025] In step (II), it is possible to first confirm that the test substance inhibits the binding between Wnt5b protein and Ror2 protein, and then confirm that the test substance does not inhibit the binding between Wnt11 protein and Ror2 protein; it is also possible to first confirm that the test substance does not inhibit the binding between Wnt11 protein and Ror2 protein, and then confirm that the test substance inhibits the binding between Wnt5b protein and Ror2 protein; or it is also possible to simultaneously confirm that the test substance inhibits the binding between Wnt5b protein and Ror2 protein and does not inhibit the binding between Wnt11 protein and Ror2 protein.

[0026] As used herein, "does not inhibit" means that the binding between Wnt11 and Ror2 proteins is not inhibited completely or substantially. "Does not substantially inhibit" means that, after confirming that the binding between Wnt11 and Ror2 proteins is detectable by various analytical techniques, such as absorbance, luminescence, fluorescence, or surface plasmon resonance, the rate of decrease in the amount of binding between Wnt11 and Ror2 proteins when the test substance is added is 30% or less compared to when the test substance is not added. This decrease may be preferably about 20%, more preferably about 10%, even more preferably about 5%, even more preferably about 3%, and particularly preferably about 1% or less. Alternatively, it means that in cells (e.g., MPCs) co-expressing Wnt11 and Ror2 proteins, the rate of increase in actin stress fiber formation, abnormal nuclear morphology, etc. when the test substance is added is 30% or less compared to when the test substance is not added. This increase may be preferably about 20%, more preferably about 10%, even more preferably about 5%, even more preferably about 3%, and particularly preferably about 1% or less.

[0027] According to the screening method of the present disclosure, for example, it is possible to screen for a substance that can inhibit the binding between Wnt5b protein and Ror2 protein but cannot inhibit the binding between Wnt11 protein and Ror2 protein when the test substance is added, compared to when the test substance is not added.

[0028] As shown in the Examples below, Wnt5b and Ror2 promote the proliferation of MPCs via p38, thereby inducing the excessive accumulation of MPCs in skeletal muscle tissue and contributing to the formation of IMAT. Furthermore, as shown in the Examples below, Wnt11 and Ror2 suppress the formation of stress fibers in MPCs, thereby inhibiting nuclear morphological abnormalities and the associated activation of the cGas-Sting pathway, and as a result, are thought to contribute to the regeneration and maintenance of skeletal muscle by suppressing cellular senescence of MPCs. Without wishing to be bound by theory, it is speculated that an IMAT formation inhibitor obtained by the screening method of the present disclosure may inhibit the excessive proliferation of MPCs and suppress the formation of IMAT by inhibiting the binding between Wnt5b protein and Ror2 protein, while not inhibiting the binding between Wnt11 protein and Ror2 protein, thereby suppressing cellular senescence of MPCs and maintaining or regenerating skeletal muscle.

[0029] Furthermore, inhibiting the binding between Wnt5b and Ror2 in this manner can suppress the excessive proliferation of MPCs and the formation of IMAT. Therefore, inhibiting the expression level of Wnt5b can also reduce the binding between Wnt5b and Ror2, thereby suppressing the excessive proliferation of MPCs and the formation of IMAT. Examples of Wnt5b-producing cells include SCs, MPCs, Schwann cells, and the like. Therefore, the screening method of the present disclosure may include (1) the step of contacting at least one selected from the group consisting of SCs, MPCs, and Schwann cells with a test substance, and (2) the step of confirming that the test substance suppresses the expression level of Wnt5b. Furthermore, the Wnt5b-producing cells may also be cells that produce Ror2 and / or p38. Therefore, the screening method including the aforementioned steps (I) and (II) may or may not further include steps (1) and (2). Furthermore, a screening method including steps (1) and (2) may not include steps (I) and (II). While not limiting the present disclosure, for example, as described above, in step (I), MPCs or the like may be contacted with a test substance as cells expressing Ror2 protein, SCs, MPCs, and / or Schwann cells or the like as cells expressing Wnt5b protein, and / or MPCs or the like as cells expressing Wnt11 protein. When the inhibition of binding is confirmed in step (II), step (2) may be further performed to confirm that the test substance suppresses the expression level of Wnt5b. Step (2) may be performed before, simultaneously with, or after step (II).

[0030] In step (1), the method for contacting at least one cell selected from the group consisting of SCs, MPCs, and Schwann cells with the test substance is not particularly limited. For example, at least one cell selected from the group consisting of SCs, MPCs, and Schwann cells and the test substance may be added to an appropriate solvent, or the test substance may be added to a culture solution containing the cells. The appropriate solvent is not particularly limited, and the above-mentioned description can be used. SCs, MPCs, and Schwann cells may be used alone or in combination of two or more. The concentration of the cells is not particularly limited, and the cell concentration (total cell concentration) when contacted with the test substance may be, for example, 1 x 10 ４ ~1 x 10 ６ The concentration of the test substance can be set to about 1000 cells / ml. The concentration of the test substance can be set appropriately depending on the type of test substance. The test substance is not particularly limited, and the above description can be used. The temperature during contact is not particularly limited, and can be, for example, about 25 to 40°C, preferably about 30 to 40°C. The contact time is not particularly limited, and can be, for example, about 1 to 6 hours, preferably about 12 to 48 hours. During contact, stirring, shaking, etc. can be performed as necessary.

[0031] In step (2), the method for confirming that the test substance suppresses the expression level of Wnt5b is not particularly limited. The expression level of Wnt5b may be the expression level of the Wnt5b gene or the expression level of the Wnt5b protein. The method for confirming the suppression of the expression level of Wnt5b is not particularly limited, and examples thereof include quantitative RT-PCR, fluorescence in situ hybridization, Western blotting, immunostaining, and flow cytometry.

[0032] For example, after confirming that the expression level of Wnt5b can be measured by luminescence, fluorescence, or the like using various analytical techniques, it is preferable that the expression level of Wnt5b is reduced by 35% or more when the test substance is added compared to when the test substance is not added. The upper or lower limit of this range may be, for example, approximately 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. Specifically, it may be approximately 35 to 100%.

[0033] According to the screening method including steps (1) and (2) of the present disclosure, for example, it is possible to screen for a substance that can suppress the expression level of Wnt5b when the test substance is added compared to when the test substance is not added.

[0034] As shown in the Examples below, it is believed that Wnt5b and Ror2 promote the proliferation of MPCs via p38, thereby inducing excessive accumulation of MPCs in skeletal muscle tissue and contributing to the formation of IMAT. Without wishing to be bound by theory, it is speculated that an IMAT formation inhibitor obtained by the screening method comprising steps (1) and (2) of the present disclosure may suppress the excessive proliferation of MPCs and the formation of IMAT by suppressing the expression level of Wnt5b.

[0035] Furthermore, for example, MPCs are also Wnt11-producing cells. Therefore, as described above, when, for example, MPCs are contacted with a test substance in step (1), step (2) may further include a step of confirming that the test substance does not suppress the expression level of Wnt11. In other words, in step (2), it can be confirmed that the test substance suppresses the expression level of Wnt5b but does not suppress the expression level of Wnt11.

[0036] The method for confirming that a test substance does not suppress the expression level of Wnt11 is not particularly limited. The expression level of Wnt11 may be the expression level of the Wnt11 gene or the expression level of the Wnt11 protein. The method for confirming that a test substance does not suppress the expression level of Wnt11 is not particularly limited, and examples thereof include quantitative RT-PCR, fluorescence in situ hybridization, Western blotting, immunostaining, and flow cytometry.

[0037] In step (2), it is possible to first confirm that the test substance suppresses the expression level of Wnt5b, and then confirm that the test substance does not suppress the expression level of Wnt11, or to first confirm that the test substance does not suppress the expression level of Wnt11, and then confirm that the test substance suppresses the expression level of Wnt5b, or to simultaneously confirm that the test substance suppresses the expression level of Wnt5b and does not suppress the expression level of Wnt11.

[0038] As used herein, "does not suppress" means that the expression level of Wnt11 is not suppressed completely or substantially. "Does not substantially suppress" means, for example, that it has been confirmed that the expression level of Wnt11 can be measured by various analytical techniques, such as luminescence or fluorescence analysis, and that the rate of decrease in the expression level of Wnt11 when the test substance is added is 30% or less compared to when the test substance is not added. This decrease may be preferably about 20%, more preferably about 10%, even more preferably about 5%, even more preferably about 3%, and particularly preferably about 1% or less. Alternatively, it means that in cells (e.g., MPCs) co-expressing Wnt11 and Ror2 proteins, the rate of increase in actin stress fiber formation, abnormal nuclear morphology, etc. when the test substance is added is 30% or less compared to when the test substance is not added. This increase may be preferably about 20%, more preferably about 10%, even more preferably about 5%, even more preferably about 3%, and particularly preferably about 1% or less.

[0039] Therefore, according to the screening method including steps (1) and (2) of the present disclosure, it is further possible to screen for a substance that can suppress the expression level of Wnt5b but cannot suppress the expression level of Wnt11 when the test substance is added, compared to when the test substance is not added.

[0040] As described above, Wnt5b and Ror2 promote the proliferation of MPCs via p38, thereby inducing excessive accumulation of MPCs in skeletal muscle tissue and contributing to the formation of IMAT. Furthermore, as shown in the Examples below, Wnt11 and Ror2 inhibit the formation of stress fibers in MPCs, thereby suppressing nuclear morphological abnormalities and the associated activation of the cGas-Sting pathway, and as a result, are thought to contribute to the regeneration and maintenance of skeletal muscle by inhibiting cellular senescence of MPCs. Without wishing to be bound by theory, it is speculated that an IMAT formation inhibitor obtained by the screening method comprising steps (1) and (2) of the present disclosure may inhibit the excessive proliferation of MPCs and the formation of IMAT by suppressing the expression level of Wnt5b, while not inhibiting the expression level of Wnt11, thereby inhibiting cellular senescence of MPCs and maintaining or regenerating skeletal muscle.

[0041] Furthermore, as described above, it can be said that Wnt5b and Ror2 promote the proliferation of MPCs via p38. Therefore, in the screening method of the present disclosure, p38 protein may be further present when Wnt5b and Ror2 are contacted with a test substance (hereinafter, this step may be referred to as step (A)). While not limiting the present disclosure, a preferred example of step (A) is contacting the cells (e.g., MPCs) expressing Ror2 protein and p38 protein with Wnt5b protein and the test substance. The contact in step (A) may be performed in the same manner as described in step (I) or (1) above, except that p38 protein is contacted with the test substance. The concentration of p38 protein is not particularly limited and can be, for example, approximately 0.1 to 4 μg / ml. In this case, the method may also include a step of confirming that the test substance inhibits the phosphorylation of p38 protein (hereinafter, this step may be referred to as step (B)). For this reason, step (A) may be further carried out in step (I) and / or (1). Furthermore, step (B) may be further carried out together with step (II) and / or (2). Step (B) may be carried out before step (II) and / or (2), simultaneously with step (II) and / or (2), or after step (II) and / or (2). From the test examples described below, it can be said that phosphorylation of p38 protein is Wnt5b-dependent. Therefore, it can be said that confirmation of inhibition of phosphorylation can confirm inhibition of p38 protein phosphorylation by Wnt5b protein.

[0042] As described above, Wnt5b and Ror2 can be said to promote the proliferation of MPCs via p38, and therefore, substances that can inhibit the phosphorylation of p38 protein induced by Wnt5b stimulation can also be said to be preferred as IMAT formation inhibitors. Therefore, candidate substances that are capable of inhibiting the binding and / or suppressing the expression level and inhibiting p38 protein phosphorylation can be said to be substances that can inhibit Wnt5b protein- and Ror2 protein-dependent p38 protein phosphorylation. From this perspective, when confirming that a test substance inhibits p38 protein phosphorylation, it may be possible to confirm in advance that the test substance inhibits the binding of Wnt5b protein and Ror2 protein and / or suppresses the expression level of Wnt5b according to the above steps (II) and / or (2). In this way, when a test substance that has been previously confirmed to be capable of inhibiting binding and / or suppressing expression levels is used, and it is further confirmed that phosphorylation of p38 protein is inhibited, the candidate substance can be said to be a substance that can inhibit Wnt5b protein- and Ror2 protein-dependent phosphorylation of p38 protein.

[0043] The p38 protein, also known as p38 MAP kinase (mitogen-activated protein kinase, MAPK), is known to be involved in signal transduction cascades that control cellular responses to cytokines and stress. Four types of p38 protein have been identified in mammals: p38-α (MAPK14), p38-β (MAPK11), p38-γ (MAPK12 / ERK6), and p38-δ (MAPK13 / SAPK4). The amino acid sequence, nucleotide sequence, etc. of the protein can be found from publicly known databases. For example, at NCBI, the accession number for the mRNA sequence of p38-α (MAPK14) is NM_139012.3.

[0044] The method for confirming that a test substance inhibits the phosphorylation of p38 protein is not particularly limited, and examples thereof include Western blotting, immunofluorescence staining, and flow cytometry. As shown in the Examples below, a preferred example is a method using an anti-phosphorylated p38 antibody.

[0045] The confirmation of inhibition of phosphorylation of p38 protein may be carried out before, simultaneously with, or after the confirmation of the inhibition of binding and / or the confirmation of the suppression of expression level.

[0046] As used herein, "inhibiting phosphorylation" means completely or substantially inhibiting phosphorylation. "Substantially inhibiting" preferably means that, after confirming that p38 protein phosphorylation can be measured by, for example, luminescence, fluorescence, or other analysis using various analytical techniques, p38 protein phosphorylation is reduced by 35% or more when the test substance is added compared to when the test substance is not added. The upper or lower limit of this range may be, for example, approximately 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. Specifically, it may be approximately 35 to 100%.

[0047] Therefore, according to the screening method of the present disclosure, which includes the steps (A) and (B), it is further possible to screen for substances that can inhibit the phosphorylation of p38 protein when the test substance is added, compared to when the test substance is not added.

[0048] The present disclosure also preferably includes compositions for inhibiting IMAT formation, the active ingredient of which is a neutralizing antibody to Wnt5b protein or a nucleic acid (e.g., siRNA, antisense oligonucleotide) that suppresses expression of the Wnt5b gene. The present disclosure also includes compositions for inhibiting IMAT formation that contain, in addition to these, for example, low-molecular-weight compounds, peptides, proteins, etc. as active ingredients. In this specification, such compositions for inhibiting IMAT formation may be referred to as "compositions of the present disclosure," etc.

[0049] Neutralizing antibodies to Wnt5b protein can be obtained, for example, by preparing antibodies that recognize Wnt5b protein by known methods or methods that can be easily derived from known methods. Nucleic acids that suppress Wnt5b gene expression (e.g., siRNA, antisense oligonucleotides) can be designed and synthesized, for example, based on the Wnt5b gene sequence by known methods or methods that can be easily derived from known methods. Furthermore, antibodies that recognize Wnt5b protein or nucleic acids that suppress Wnt5b gene expression obtained in this manner can be screened by the screening method of the present disclosure described above to obtain particularly preferred ones.

[0050] Although not limiting the present disclosure, when the nucleic acid that suppresses the expression of the Wnt5b gene is siRNA, the number of bases in either the sense strand or the antisense strand is not particularly limited, as long as it has the function of suppressing the expression of the Wnt5b gene, and preferably each is 18 to 25 bases, more preferably 20 to 22 bases. Similarly, when the nucleic acid that suppresses the expression of the Wnt5b gene is an antisense oligonucleotide, the number of bases is also not particularly limited, as long as it has the function of suppressing the expression of the Wnt5b gene, and preferably is 12 to 28 bases, more preferably is 15 to 25 bases.

[0051] Furthermore, although not limiting the present disclosure, examples of nucleic acids that suppress Wnt5b gene expression include, for example, siRNAs represented by SEQ ID NOs: 9 and 10, and 11 and 12. As long as the siRNA has the function of suppressing Wnt5b gene expression, it may have a protruding sequence (overhang) at its 5' and / or 3' end, or may have a mutation in its base sequence (e.g., substitution, deletion, or addition of 1, 2, or 3 bases), and the base lengths of the sense strand and antisense strand may be the same or different. In the present disclosure, having the function of suppressing Wnt5b gene expression is preferably exemplified by a decrease in the expression level of Wnt5b by 35% or more when the nucleic acid is added, compared to when the nucleic acid is not added, according to the above-mentioned steps (1) and (2). The suppression of expression, etc., is explained as above.

[0052] The composition of the present disclosure can be prepared by a conventional method by combining the above-mentioned active ingredient with other ingredients as needed. Examples of such other ingredients include pharmacologically acceptable bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, surfactants, antioxidants, preservatives, coating agents, colorants, flavorings, pH adjusters, and other drugs such as gastric mucosa protectants. These ingredients can be used alone or in combination of two or more.

[0053] In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure encompasses any and all combinations of the constituent elements described in this specification.

[0054] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to identify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein.

[0055] The contents of the present disclosure will be specifically explained using the following experimental examples. However, the present disclosure is not limited to these examples. In the following, unless otherwise specified, experiments were performed under atmospheric pressure and room temperature conditions. Furthermore, unless otherwise specified, "%" means "volume %."

[0056] Animal Experiments Male mice (C57BL / 6N) aged 8 to 12 weeks were purchased from Japan SLC (Shizuoka). Cardiotoxin (Ctx) (Latoxan, Valence, France) or glycerol (Nacalai Tesque, Kyoto) was used to induce injury to the tibialis anterior (TA) muscle. TA muscle injury was induced by injecting Ctx (2.5 μl of 10 μM Ctx / g body weight) or glycerol (50 μl of 50% (v / v) glycerol / TA muscle) into the TA muscle. Two weeks later, the injured TA muscle was harvested and subjected to immunofluorescence staining to analyze the cross-sectional area (CSA) of muscle fibers and the percentage of perilipin-positive areas. Note that Ctx induces injury repair without fat accumulation, whereas glycerol induces injury repair accompanied by fat accumulation. Ror2 ｆｌｏｘ／ｆｌｏｘ Mice were generated as previously described (Endo M. et al., Glia, 2017, 65, 1). To analyze the number of distal Schwann cells, Ctx or glycerol was injected into the extensor digitorum longus (EDL) muscle and analyzed by fluorescent staining.

[0057] To investigate the role of Wnt5b and Wnt11 in IMAT accumulation, recombinant Wnt5b (4 μg / ml, R&D System, Minneapolis, MN) and Wnt11 stably expressing cells in conditioned medium (details described below) were injected into the TA muscle after Ctx treatment. ＣｒｅＥＲ／＋ Mice were obtained from Jackson Laboratory (Stock No. 018280, Bar Harbor, ME). Ror2 mice were 10-14 weeks old. ｆｌｏｘ／ｆｌｏｘ ;Pdgfrα ＣｒｅＥＲ／＋ Mice were intraperitoneally injected with tamoxifen (100 μg / g body weight) (Sigma-Aldrich, St. Louis, MO) once daily for a total of 9 days to suppress Ror2 expression in MPCs. All animal experiments in this study were conducted at the Institute for Experimental Animals, Kobe University Graduate School of Medicine, in accordance with the Kobe University Animal Experimentation Regulations and with approval from the Institutional Animal Care and Use Committee (Permit Numbers: P171011-R1, P220904).

[0058] Isolation of SCs and MPCs. Bilateral TA, soleus, gastrocnemius, quadriceps, and gluteus maximus muscles were digested in DMEM / F-12 (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) containing 0.5% (w / v) collagenase type II (Worthington, Lakewood, NJ) for 90 minutes at 37°C. The digested skeletal muscles were filtered through a 40 μm nylon mesh (pluriSelect Life Science, Leipzig, Germany). The resulting cell suspension was washed with ice-cold PBS containing 2% fetal bovine serum (FBS). SCs and MPCs were separated from the cell suspension using a FACS Aria III (BD Biosciences, Franklin Lakes, NJ) with anti-CD31, CD45, Sca-1, and SM / C-2.6 antibodies (the antibody list is shown in Table 1). SCs can be identified as SM / C-2.6-positive, CD31-, CD45-, and Sca-1-negative cells. MPCs can be identified as Sca-1-positive, CD31-, CD45-, and SM / C-2.6-negative cells.

[0059]

[0060] Cell culture: Cos7 cells were cultured in DMEM containing 10% FBS at 5% CO ２ and 20% O ２ The cells were cultured at 37°C under a 10:1 ratio of pCMV5-mWnt11 and a plasmid encoding a puromycin resistance gene (pBabe-Puro), and puromycin-resistant cells constitutively expressing mWnt11 were selected (Cos7-Wnt11). Cos7 cells transfected with an empty vector (pCMV5) together with pBabe-Puro were also selected by puromycin treatment (Cos7-mock). To prepare the conditioned medium, 5 × 10 ５Cos7-Wnt11 or Cos7-mock cells were cultured in 0% FBS DMEM in four φ10 dishes for 2 days. The medium was collected and centrifuged at 500 × g for 5 minutes to remove dead cells, and then concentrated by centrifugation at 2000 × g for 60 minutes using a Vivaspin 20-10K (Cytiva, Marlborough, MA). The concentrated conditioned medium was administered to the injured TA muscle.

[0061] Three days after Ctx administration, MPCs were isolated from both TA, soleus, and gastrocnemius muscles by FACS as described above and cultured in DMEM (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% FBS, 50 U / ml Penicillin (Meiji Seika Pharma, Tokyo, Japan), 50 μg / ml Kanamycin (Fujifilm Wako Pure Chemical Industries, Ltd.), and 10 ng / ml bFGF (Fujifilm Wako Pure Chemical Industries, Ltd.) under 5% CO ２ , 3% O ２ The MPCs were cultured at 37°C for 3 to 4 days. After that, the MPCs were used for the following in vitro experiments. In vitro experiments using MPCs were performed in a 5% CO atmosphere. ２ and 20% O ２ The reaction was carried out at 37°C under

[0062] To examine the percentage of Ki67-positive MPCs, siRNA was introduced into MPCs (the siRNA sequences are Ror2#1 and Ror2#2 in Table 2) and cultured in DMEM containing 10% FBS for 3 days. The cells were then fixed with 4% (w / v) paraformaldehyde (PFA) for 10 minutes at room temperature. The fixed MPCs were then stained with anti-Ki67 antibody and DAPI to assess the percentage of Ki67-positive MPCs.

[0063]

[0064] To examine the percentage of EdU-positive MPCs, MPCs were transfected with siRNA (Ror2#1, Ror2#2, Wnt5b#1, or Wnt5b#2 listed in Table 2) and cultured in DMEM containing 10% FBS for 3 days. Subsequently, MPCs were treated with EdU (10 μM, Thermo Fisher Scientific, Waltham, MA) for 30 minutes and then fixed with 4% (w / v) PFA. EdU incorporation was detected using the Click-it EdU imaging kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0065] For adipogenic differentiation, MPCs were cultured in adipogenic induction medium (DMEM containing 10% FBS, 0.5 mM IBMX (Sigma-Aldrich), 0.25 μM dexamethasone (Sigma-Aldrich), and 10 μg / ml insulin (Sigma-Aldrich)) for 3 days, and then treated with adipogenic maintenance medium (DMEM containing 10% FBS and 10 μg / ml insulin) for 3 days.

[0066] To detect senescent cells, MPCs transfected with siRNA (Ror2#1, Ror2#2, Wnt11#1, or Wnt11#2 listed in Table 2) were cultured in 10% FBS DMEM for 3 days and then stained with SPiDER-βGal (Dojindo Laboratories, Kumamoto) according to the manufacturer's instructions.

[0067] Oil Red O staining To stain adipocytes, cells were treated with 4% (w / v) PFA at room temperature for 10 minutes and fixed. The fixed cells were washed with 60% (v / v) isopropanol. Next, they were stained with Oil Red O (Fujifilm Wako Pure Chemical Industries, Ltd.) at room temperature for 30 minutes. The stained cells were washed with 60% (v / v) isopropanol and then treated with 100% isopropanol for 20 minutes to extract the Oil Red O taken up into the adipocytes. The absorbance of the Oil Red O in the supernatant was then measured at 490 nm.

[0068] WST-8 Assay. MPCs were seeded in 96-well plates (1,000 cells per well) and transfected with siRNA (Wnt5b#1 or Wnt5b#2, as listed in Table 2). After culturing for the desired period, WST-8 reagent (Cell Counting Kit-8, Dojindo Laboratories) was added to the culture medium, followed by incubation for 90 minutes. Absorbance at 450 nm was measured. To assess the involvement of p38 in Wnt5b-induced proliferation, a p38 inhibitor (10 μM SB203580, Cayman Chemical, Ann Arbor, MI) and recombinant Wnt5b (200 ng / ml, R&D System) were added to the culture medium one day after seeding MPCs in 96-well plates. WST-8 assays were then performed as described above.

[0069] Immunofluorescence staining. Isolated TA muscles were frozen in isopentane cooled with liquid nitrogen and sectioned using a cryostat (Leica CM1860, Wetzlar, Germany). Frozen sections were permeabilized with 0.2% (v / v) Triton X-100. The sections were then blocked with 3% (w / v) BSA in PBS and incubated with primary antibodies overnight at 4°C. The sections were washed with PBS, incubated with secondary antibodies at room temperature for 1 hour, and then mounted using Fluor-KEEPER (Nacalai Tesque). The mounted sections were observed under a fluorescence microscope (BZ-X700, Keyence, Osaka, Japan). The percentage of perilipin-positive areas and CSA in the TA muscle were analyzed using ImageJ (National Institutes of Health, Bethesda, MD). For whole-mount immunofluorescence staining, isolated EDL muscles were fixed with 4% (w / v) PFA for 30 minutes and then permeabilized with 0.2% (v / v) Triton X-100. After washing with PBS, the EDL muscles were blocked with 3% (w / v) BSA in PBS for 1 hour and then incubated with primary antibodies overnight at 4°C. The EDL muscles were then washed with PBS and incubated with secondary antibodies at room temperature for 1 hour. Stained EDL muscles were placed on glass-bottom dishes (Matsunami Glass Industry, Osaka) and observed using a laser scanning confocal imaging system (LSM700, Carl Zeiss, Oberkochen, Germany). Cultured cells were fixed with 4% (w / v) PFA for 10 minutes and then permeabilized with 0.2% (v / v) Triton X-100. After washing with PBS, they were blocked with 3% (w / v) BSA in PBS for 1 hour and then incubated with primary antibodies overnight at 4°C. Next, the cells were washed with PBS and incubated with secondary antibodies at room temperature for 1 hour. Stained cells were mounted with Fluor-KEEPER (Nacalai Tesque). The mounted cells were observed using a laser scanning confocal imaging system (LSM700, Carl Zeiss).

[0070] Western blotting Cells were lysed in lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% (v / v) Nonidet P-40, 1 mM EDTA, 10 mM NaF, 1 mM Na ３ VO ４ Proteins were solubilized in a buffer containing 10 μg / ml of PBS (10 μg / ml aprotinin, 10 μg / ml leupeptin, and 1 μM p-APMSF). Proteins (10 μg total per lane) were separated by SDS-PAGE and transferred onto an Immobilon-P membrane (Merck Millipore, Darmstadt, Germany). The membrane was blocked with 1% (v / w) BSA in TBST for 1 hour and then blotted overnight at 4°C with primary antibodies (listed in Table 1). After treatment with the primary antibody, the membrane was washed with TBST for 30 minutes at room temperature and then blotted with secondary antibodies (listed in Table 1) for 1 hour at room temperature. Finally, the membrane was washed with TBST for 30 minutes at room temperature. ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) and LAS 1000 (GE Healthcare, Chicago, IL) were used for analysis. Relative band intensities were determined using ImageJ software.

[0071] Quantitative RT-PCR. Total RNA was extracted from cultured cells or TA muscle using Sepasol-RNA I SuperG (Nacalai Tesque). cDNA was synthesized from this RNA using PrimeScript RT Reagent (Takara Bio, Shiga). Expression of each target gene was quantified using a LightCycler 480 II system (Roche, Basel, Switzerland). The amount of target mRNA was normalized to the amount of 18S ribosomal RNA. The sequences of the primer pairs are listed in Table 3.

[0072]

[0073] Statistical Analysis Data were analyzed using BellCurve for Excel (Social Survey Research Information Co., Ltd., Tokyo) and GraphPad Prism 9.0 (GraphPad Software, Boston, MA). Data are presented as mean ± standard deviation (S.D.). Significance was determined using an unpaired two-tailed Student's t test when comparing two groups, or one-way ANOVA with Holm's test or Dunnett's test when analyzing three or more groups, with *P<0.05, **P<0.01, or ***P<0.001.

[0074] The TA, gastrocnemius, soleus, quadriceps, and gluteus maximus muscles of mice were collected, and SCs and MPCs were isolated using FACS AriaIII. After RNA extraction, expression analysis was performed using quantitative RT-PCR.

[0075] As shown in Figure 1, it was confirmed that Ror2 is selectively expressed in MPCs.

[0076] MPCs isolated from Ctx-injected TA muscles were incubated in 5% CO ２ , 3% O ２ After culturing for 3 days at 5% CO, siRNA (Ror2#1 or Ror2#2 listed in Table 2) was introduced under normal oxygen concentration. The next day, the medium was replaced with fresh medium. Three days after siRNA introduction, the cells were fixed and stained with Ki67 antibody. MPCs isolated from the Ctx-administered TA muscle were cultured under 5% CO ２ , 3% O ２ After culturing for 3 days at 5% CO, siRNA (Ror2#1 or Ror2#2 listed in Table 2) was introduced under normal oxygen concentration. The next day, the medium was replaced with fresh medium. Three days after siRNA introduction, the cells were cultured in adipogenic induction medium for 3 days, followed by culture in adipogenic maintenance medium for 3 days to induce differentiation into adipocytes. After differentiation induction, Oil Red O staining was performed. MPCs isolated from Ctx-administered TA muscles were cultured at 5% CO ２ , 3% O ２After culturing for 3 days at 4°C, siRNA (Ror2#1 or Ror2#2 listed in Table 2) was introduced under normal oxygen concentration. The next day, the medium was replaced with fresh medium. Three days after siRNA introduction, senescent cells were stained with SPiDER-βGal.

[0077] As shown in Figure 2, it was confirmed that suppressing Ror2 expression in MPCs isolated and cultured from mouse skeletal muscle reduced the proliferation and differentiation ability of MPCs into adipocytes and accelerated cellular senescence. Since it has been reported that MPCs experience a decline in proliferation and differentiation ability into adipocytes when they undergo cellular senescence (Non-Patent Documents 1 and 2), it was suggested that Ror2 may control the proliferation and differentiation ability of MPCs into adipocytes by suppressing cellular senescence.

[0078] Tamoxifen was administered to 12- to 16-week-old control mice and Ror2cKO mice, and the mice were then housed for six months. Skeletal muscle weights were then compared and analyzed. Frozen sections of the TA muscle were prepared and stained with anti-Laminin antibodies to measure the cross-sectional area of ​​each muscle fiber.

[0079] As shown in Figure 3, the weights of the TA muscle, gastrocnemius muscle, and soleus muscle were reduced in Ror2cKO mice, and muscle fibers in the TA muscle were atrophied. This suggests that Ror2 is important for maintaining skeletal muscle.

[0080] After administering tamoxifen to 12- to 16-week-old control mice and Ror2cKO mice, Ctx or glycerol was administered to the TA muscle. Two weeks after administration, the TA muscles were harvested and stained with laminin or perilipin. The cross-sectional area of ​​each muscle fiber in the TA muscle was measured. In addition, the degree of fat accumulation was compared and analyzed by measuring the percentage of perilipin-positive area in the muscle tissue cross-sectional area using stained images of the glycerol-treated muscles.

[0081] As shown in Figure 4, muscle fiber thickness was smaller in Ror2cKO mice two weeks after Ctx administration than in control mice, indicating delayed skeletal muscle regeneration, suggesting that Ror2 is important for skeletal muscle regeneration. Furthermore, fat accumulation was reduced in Ror2cKO mice after glycerol administration, indicating that Ror2 is also important for IMAT formation.

[0082] MPCs isolated from Ctx-injected TA muscles were incubated in 5% CO ２ , 3% O ２ After culturing for 3 days at 4°C, siRNA (Wnt11#1 or Wnt11#2 listed in Table 2) was introduced under normal oxygen concentration. The next day, the medium was replaced with fresh medium. Three days after siRNA introduction, RNA was extracted and expression analysis was performed using quantitative RT-PCR. In parallel, SPiDER-βGal staining was also performed, and the staining intensity was compared and analyzed using flow cytometry.

[0083] As shown in FIG. 5, it was revealed that suppressing the expression of Wnt11 in MPCs induces cellular senescence in MPCs.

[0084] MPCs isolated from Ctx-injected TA muscles were incubated in 5% CO ２ , 3% O ２ After culturing for 3 days at 5% CO, siRNA (Ror2#1, Ror2#2, Wnt11#1, or Wnt11#2 listed in Table 2) was introduced under normal oxygen concentration. The next day, the medium was replaced with fresh medium. Two days after siRNA introduction, the cells were stained with phalloidin and DAPI. MPCs isolated from the Ctx-administered TA muscle were cultured under 5% CO ２ , 3% O ２ After culturing for 3 days at 4°C, siRNA (Ror2#1, Ror2#2, Wnt11#1, or Wnt11#2 listed in Table 2) was introduced under normal oxygen concentration. The next day, the medium was replaced with fresh medium. Three days after siRNA introduction, the cells were fixed and stained with LaminB1 and DAPI.

[0085] As shown in Figure 6, suppression of Wnt11 or Ror2 expression in MPCs was observed to promote the formation of actin stress fibers. Abnormalities in nuclear morphology were also observed. It has been reported that DNA leakage into the cytoplasm associated with abnormal nuclear morphology activates the cGas (cyclic GMP-AMP synthase)-mediated signal transduction pathway (cGAS-Sting pathway) (Non-Patent Document 3). Based on these findings, it is believed that Wnt11 and Ror2 suppress the formation of stress fibers in MPCs, thereby suppressing nuclear morphology abnormalities and the associated activation of the cGas-Sting pathway, thereby inhibiting cellular senescence and contributing to the regeneration and maintenance of skeletal muscle.

[0086] Next, to clarify the molecular mechanism by which Ror2 is involved in the formation of IMAT, a comparative analysis of gene expression was performed in Ctx- and glycerol-administered muscles. Ctx or glycerol was administered to the TA muscles of 8- to 12-week-old wild-type mice, and the TA muscles were collected after 0 (no administration), 3, and 7 days. Expression analysis of Wnt family genes that may function as Ror2 ligands was performed using quantitative RT-PCR. MPCs isolated from Ctx-administered TA muscles were incubated at 5% CO ２ , 3% O ２ After culturing for 3 days at 4°C, siRNA (Ror2#1 or Ror2#2 listed in Table 2) was introduced under normal oxygen concentration. The next day, the medium was replaced with one containing Wnt5a or Wnt5b. After 3 days, proliferation analysis was performed using EdU or Ki67 staining.

[0087] As shown in Figure 7, we found that Wnt5b expression increased after glycerol administration. This suggests that Wnt5b may contribute to IMAT formation. Furthermore, adding Wnt5b to cultured MPCs promoted their proliferation, while suppressing Ror2 expression inhibited the proliferation promotion induced by Wnt5b addition. Furthermore, adding Wnt5a, the best-known Ror2 ligand, did not affect MPC proliferation.

[0088] MPCs isolated from Ctx-injected TA muscles were incubated in 5% CO２ , 3% O ２ After culturing for 3 days at 5% CO, siRNA (Ror2#1 or Ror2#2 listed in Table 2) was introduced under normal oxygen concentration. The next day, the medium was replaced with fresh medium. Two days after siRNA introduction, the medium was replaced with OPTI-MEM and the cells were cultured overnight. Wnt5b was then added at 200 ng / ml, and after 1 hour, proteins were extracted from the cells using lysis buffer and subjected to Western blotting. MPCs isolated from the Ctx-administered TA muscle were incubated at 5% CO ２ , 3% O ２ After culturing for 3 days at 4°C, siRNA (Ror2#1 or Ror2#2 listed in Table 2) was introduced under normal oxygen concentration. The next day, the medium was replaced with one containing Wnt5b (200 ng / ml) or SB203580 (10 μM). At this time, cells without medium replacement were used as day 0 samples and analyzed by WST-8 assay. Three days after the medium replacement, the number of cells in each well was measured by WST-8 assay.

[0089] As shown in Figure 8, analysis of cultured MPCs revealed that Wnt5b phosphorylates p38 (reflecting activation) in a Ror2-dependent manner. Furthermore, treatment with the p38 inhibitor SB203580 suppressed Wnt5b-dependent MPC proliferation. These findings demonstrate that Wnt5b, Ror2, and p38 promote MPC proliferation. It has been suggested that when IMAT and fibrosis occur in skeletal muscle, MPCs accumulate excessively within the muscle and differentiate into adipocytes or fibroblasts (Non-Patent Documents 4 and 5). Our analysis suggests that Wnt5b, Ror2, and p38 promote MPC proliferation, thereby inducing excessive accumulation of MPCs in skeletal muscle tissue and contributing to the formation of IMAT.

[0090] Three days after the administration of Ctx or glycerol, the EDL muscles were subjected to whole mount staining.

[0091] As shown in Figure 9, we found that Wnt5b is expressed in Schwann cells (S100β-positive cells) and that the proliferation of Wnt5b-positive Schwann cells is enhanced upon administration of glycerol, suggesting that Schwann cells may be one of the Wnt5b-producing cells.

[0092] Ctx or glycerol was administered to the TA muscle of 8- to 12-week-old male mice, and the TA muscle was collected 3 days later. SCs, MPCs, CD31-positive and CD45-positive cells (a mixed population of hematopoietic cells and endothelial cells), and other cell populations were isolated using FACS AriaIII. RNA was extracted, and expression analysis was performed using quantitative RT-PCR.

[0093] As shown in Figure 10, we found that Wnt5b was expressed in SCs and MPCs in the TA muscle, indicating that the Wnt5b-producing cells in the TA muscle are SCs and MPCs.

[0094] MPCs isolated from Ctx-injected TA muscles were incubated in 5% CO ２ , 3% O ２ After culturing for 3 days at 5% CO, siRNA (Wnt5b#1 or Wnt5b#2 shown in Table 2) was introduced under normal oxygen concentration. The next day, the medium was replaced with fresh medium. Three days after siRNA introduction, RNA was extracted and expression analysis was performed using quantitative RT-PCR. MPCs isolated from the Ctx-administered TA muscle were cultured under 5% CO ２ , 3% O ２ After culturing for 3 days at 5% CO, siRNA (Wnt5b#1 or Wnt5b#2 shown in Table 2) was introduced under normal oxygen concentration. The next day, the medium was replaced with a new one. At this time, cells without medium replacement were analyzed by WST-8 assay as day 0 samples. Four days after medium replacement, the number of cells in each well was measured by WST-8 assay. MPCs isolated from Ctx-administered TA muscles were incubated at 5% CO ２ , 3% O ２ After culturing for 3 days at 4°C, siRNA (Wnt5b#1 or Wnt5b#2 shown in Table 2) was introduced under normal oxygen concentration. The next day, the medium was replaced with fresh medium. After 3 days, proliferation analysis using EdU was performed.

[0095] As shown in Figure 11, it was revealed that Wnt5b expression was suppressed by siRNA. Furthermore, analysis using cultured MPCs revealed that suppression of Wnt5b expression by siRNA inhibited the proliferation ability of MPCs.

[0096] These results suggest that under physiological conditions, Wnt11 and Ror2 suppress cellular senescence in MPCs and contribute to the maintenance and regeneration of skeletal muscle, but that under pathological conditions, Wnt5b, in addition to Wnt11, also functions as a ligand for Ror2, promoting excessive proliferation of MPCs and IMAT formation.

[0097] Examples of muscle diseases and / or conditions in which IMAT accumulation is observed include various skeletal muscle diseases such as muscular dystrophies (Duchenne muscular dystrophy, etc.) and muscular atrophy including amyotrophic lateral sclerosis (ALS), and various conditions associated with aging in skeletal muscles such as sarcopenia, frailty, and locomotive syndrome. The present disclosure can be utilized in the development of novel treatment methods that are effective with few side effects for skeletal muscle diseases and pathologies including muscular dystrophies in which IMAT accumulation is observed and sarcopenia that occurs with aging.

Claims

1. A method for screening a substance that inhibits IMAT formation, comprising: (I) the step of contacting Wnt5b protein, Ror2 protein, and a test substance; and (II) the step of confirming that the test substance inhibits the binding between Wnt5b protein and Ror2 protein.

2. The method according to claim 1, further comprising contacting with Wnt11 protein in step (I), and further confirming in step (II) that the test substance does not inhibit the binding between Wnt11 protein and Ror2 protein.

3. A method for screening a substance that inhibits IMAT formation, comprising: (1) a step of contacting at least one type selected from the group consisting of SCs, MPCs, and Schwann cells with a test substance; and (2) a step of confirming that the test substance inhibits the expression level of Wnt5b.

4. The method according to claim 3, wherein in step (2), it is confirmed that the test substance does not suppress the expression level of Wnt11.

5. A method for screening a substance that inhibits IMAT formation, comprising: (A) contacting Wnt5b protein, Ror2 protein, p38 protein, and a test substance; and (B) confirming that the test substance inhibits phosphorylation of p38 protein.

6. The method according to any one of claims 1 to 5, wherein the inhibition of IMAT formation is due to inhibition of proliferation of MPCs.

7. A composition for inhibiting IMAT formation, which comprises as an active ingredient a neutralizing antibody to Wnt5b protein or a nucleic acid that inhibits the expression of the Wnt5b gene.

8. A composition for inhibiting IMAT formation as described in claim 7 for use in inhibiting or treating a muscular disease or condition in which accumulation of IMAT is observed.

9. A composition for inhibiting IMAT formation as described in claim 8, wherein the muscle disease or condition in which accumulation of IMAT is observed is muscular dystrophy, amyotrophic lateral sclerosis, sarcopenia, frailty or locomotive syndrome.

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