Biomarker composition for diagnosing sarcopenia caused by aging or alzheimer's dementia, comprising BACE1, β-amyloid, β-amyloid oligomer, water-soluble amyloid precursor protein-β, or combination thereof, and use thereof

Abstract

The present invention relates to a biomarker composition for diagnosing sarcopenia caused by aging or Alzheimer's dementia, comprising BACE1, β-amyloid, β-amyloid oligomer, water-soluble amyloid precursor protein-β, or a combination thereof, and to a use thereof. Specifically, the present invention relates to a composition for diagnosing sarcopenia caused by aging or Alzheimer's dementia, comprising, as an active ingredient, an agent for measuring the level of at least one protein or mRNA selected from the group consisting of BACE1, β-amyloid, β-amyloid oligomer, and water-soluble amyloid precursor protein-β, to a kit for diagnosing sarcopenia using the composition, and to a method for providing information for diagnosing sarcopenia.

Description

Biomarker composition for diagnosing sarcopenia caused by aging or Alzheimer's dementia, composed of BAC1, beta-amyloid, beta-amyloid oligomer, water-soluble amyloid precursor protein-beta, or a combination thereof, and the use thereof

[0001] The present invention relates to a biomarker composition for diagnosing sarcopenia caused by aging or Alzheimer's dementia, comprising BACE1, beta-amyloid, beta-amyloid oligomer, water-soluble amyloid precursor protein-beta, or a combination thereof, and the use thereof.

[0002] Sarcopenia is the degenerative loss of skeletal muscle mass, muscle quality, and muscle strength associated with aging and immobility, and generally affects older adults aged approximately 60 years or older. The rate of muscle loss depends on exercise levels, comorbidities, nutrition, and other factors, and the decline in muscle mass and strength in older adults often leads to a decrease in physical functional ability, lowering the quality of life and increasing the risk of adverse health events (e.g., falls and subsequent fractures).

[0003] With the recent increase in the number of patients with sarcopenia worldwide, it was recognized as an official disease with a diagnostic code in the United States (ICD 10-CM) in 2016, and in 2021, it also became an official disease with a diagnostic code in Korea (KCD-8).

[0004] The most common causes of sarcopenia are reduced protein intake and lack of exercise; in particular, the rate of sarcopenia is very high due to insufficient intake and absorption of essential amino acids. Another common cause is hormone deficiency associated with aging. Skeletal muscle is the largest organ in the human body, accounting for 40 to 50 percent of its mass. Skeletal muscle tends to decrease by about 1% annually starting from age 30, and declines rapidly after age 65. As aging progresses, the functional capacity of muscles also gradually decreases; sarcopenia is diagnosed when both muscle strength and function decline along with a decrease in muscle mass.

[0005] There are various biological mechanisms that induce sarcopenia, including inflammation, mitochondrial dysfunction in muscle cells, a decrease in the number of muscle stem cells and aging, an imbalance between protein synthesis and degradation, and autophagy abnormalities. Therefore, the development of therapeutic substances capable of promoting muscle cell differentiation and growth and inhibiting muscle atrophy by regulating these biological mechanisms can be useful as a method for preventing or treating sarcopenia caused by aging or disease.

[0006] It is important to detect and manage sarcopenia early before it progresses to a severe stage. When muscles weaken, bones also weaken, making osteoporosis more likely. Furthermore, a decrease in muscle mass leads to a reduction or overproduction of various factors produced by muscles, which hinders the formation of new blood vessels and nerves and can ultimately cause serious conditions such as cognitive decline. Additionally, early detection and management are crucial because sarcopenia is closely linked to conditions such as fatty liver, cardiac hypertrophy, pancreatic dysfunction, and diabetes.

[0007] Currently, the diagnosis of sarcopenia is made by assessing muscle mass, strength, and function. While muscle mass is measured using methods such as dual-energy X-ray absorptiometry, bioimpedance measurement, CT, and MRI, these methods have limitations as diagnostic tools due to the low correlation between skeletal muscle mass and strength. Muscle strength is measured by leg strength or grip strength, while muscle function is assessed by performing one or two tests among walking speed, the Short Physical Performance Battery (SPPB), the 400-meter walk test, and the 6-minute walk test. However, since normal values ​​for muscle mass vary depending on age, gender, height, body weight, and body fat mass, the criteria for evaluating strength and function are not precise. Furthermore, performing these tests poses a burden for the elderly.

[0008] Therefore, there is a need to develop new diagnostic technologies, such as diagnosing sarcopenia more objectively and accurately, and furthermore, discovering biomarkers capable of detecting changes in biological indicators to track the worsening or improvement of sarcopenia.

[0009] Meanwhile, beta-amyloid (Abeta) is a protein known to be the causative agent of Alzheimer's disease; it is well known to accumulate primarily in the brain, inducing neurodegeneration and inflammation. Beta-amyloid is generated by cleaving the amyloid precursor protein APP through BACE1 (beta-secretase 1) and gamma-secretase, producing Abeta42 and Abeta40, which cause Alzheimer's disease. Abeta42 and Abeta40 are proteins with a beta sheet structure composed of 42 and 40 amino acids, respectively; they easily clump together to form oligomers, specifically beta-amyloid oligomers, which are well known to cause neurotoxicity. Furthermore, beta-amyloid oligomers gradually develop into fibrils and plaques, ultimately leading to increased accumulation of amyloid plaques in the brains of Alzheimer's patients.

[0010] However, the role of beta-amyloid in peripheral tissues is not yet well understood. While some studies have reported that the mislocation of APP in the mitochondria of adipocytes induces insulin resistance, and that obesity may lead to increased BACE1 expression and beta-amyloid production in adipose and muscle tissues, the association between beta-amyloid and sarcopenia caused by aging or Alzheimer's dementia has not yet been studied.

[0011] Accordingly, while conducting research to discover biomarkers capable of diagnosing sarcopenia caused by aging or Alzheimer's dementia, the inventors confirmed that both beta-amyloid oligomers and BACE1 were overexpressed in the muscle tissue of naturally aged mice. As they confirmed that this induces the overproduction of beta-amyloid and oligomers, which can act as a cause of sarcopenia, they completed the present invention by identifying that sarcopenia can be diagnosed by measuring the expression levels of beta-amyloid, beta-amyloid oligomers, BACE1, and soluble amyloid precursor protein-beta within the muscle tissue.

[0012] Therefore, the objective of the present invention is to provide a composition for diagnosing sarcopenia caused by aging or Alzheimer's dementia, comprising as an active ingredient a preparation for measuring the level of one or more proteins or mRNA selected from the group consisting of BACE1, beta-amyloid, beta-amyloid oligomer, and soluble amyloid precursor protein-beta.

[0013] Another objective of the present invention is to provide a kit for diagnosing sarcopenia caused by aging or Alzheimer's dementia, comprising the composition of the present invention.

[0014] Another objective of the present invention is to provide a method for providing information for diagnosing sarcopenia caused by aging or Alzheimer's dementia, comprising: (1) measuring the level of one or more proteins or mRNA selected from the group consisting of BACE1, beta-amyloid, beta-amyloid oligomer, and soluble amyloid precursor protein-beta in a biological sample separated from a subject; and (2) comparing the protein or mRNA level measured in step (1) with a control group.

[0015] Another objective of the present invention is to provide a method for diagnosing sarcopenia caused by aging or Alzheimer's dementia, comprising: (1) measuring the level of one or more proteins or mRNA selected from the group consisting of BACE1, beta-amyloid, beta-amyloid oligomer, and soluble amyloid precursor protein-beta in a biological sample separated from a subject; and (2) comparing the protein or mRNA level measured in step (1) with a control group.

[0016] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the present invention, and the present invention is defined only by the scope of the claims.

[0017] The terms used in this specification are for describing the embodiments and are not intended to limit the invention. In this specification, the singular form includes the plural form unless specifically stated otherwise in the text. The terms "comprises" and / or "comprising" used in this specification do not exclude the presence or addition of one or more other components in addition to the components mentioned. Throughout the specification, the same reference numerals refer to the same components, and "and / or" includes each of the mentioned components and all combinations of one or more. Although terms such as "first," "second," etc., are used to describe various components, these components are not limited by these terms. These terms are used merely to distinguish one component from another. Therefore, the first component mentioned below may be the second component within the technical scope of the invention.

[0018] Unless otherwise defined, all terms used herein (including technical and scientific terms) may be used in a meaning commonly understood by those skilled in the art to which the present invention pertains. Additionally, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0019] The present invention is a technology that provides a novel biomarker capable of diagnosing sarcopenia caused by aging or Alzheimer's dementia. Specifically, the present invention is characterized by providing a composition for diagnosing sarcopenia caused by aging or Alzheimer's dementia, comprising as an active ingredient a preparation that measures the level of one or more proteins or mRNA selected from the group consisting of BACE1, beta-amyloid, beta-amyloid oligomer, and soluble amyloid precursor protein-beta.

[0020] As previously mentioned in the prior art, current methods for diagnosing sarcopenia include verifying muscle mass, strength, and function; however, these methods have the problem of lacking objectivity because the diagnostic criteria can be subjective, and performing these tests is burdensome for the elderly.

[0021] Therefore, at a time when there is a need to develop biomarkers capable of diagnosing sarcopenia more objectively and accurately, and furthermore detecting changes in biological indicators for tracking the worsening or improvement of sarcopenia, the inventors confirmed that age-related sarcopenia can be diagnosed by measuring the expression levels of one or more selected from a group consisting of beta-amyloid, beta-amyloid oligomers, BACE1, and soluble amyloid precursor protein-beta in the muscle tissue of naturally aged mice.

[0022] According to one embodiment of the present invention, when the differentiation pattern of muscle cells was analyzed for a group treated with beta-amyloid oligomers and a control group not treated, it was found that the group treated with beta-amyloid oligomers showed a decrease in the expression of MHC, a marker of muscle cells, and a decrease in the expression of muscle differentiation factors MyoD and myogenin compared to the control group, as well as a decrease in mitochondrial activity. In addition, it was found that the group treated with beta-amyloid oligomers in the differentiated muscle cells showed increased oxidative stress compared to the control group.

[0023] Through these results, the inventors have identified that beta-amyloid oligomers, known as causative agents of degenerative brain diseases such as Alzheimer's disease, act to inhibit the differentiation of muscle cells.

[0024] In addition, the inventors analyzed whether beta-amyloid and beta-amyloid oligomers could have a direct effect on aging and age-induced sarcopenia using a mouse animal model. As a result, it was found that the concentration of beta-amyloid in mice with accelerated aging (SAMP8) was significantly higher than in the control group (mice with normal aging), indicating an association between beta-amyloid and accelerated aging.

[0025] In addition, we confirmed that beta-amyloid oligomers were overexpressed in the muscles of an Alzheimer's-induced mouse model, and confirmed the characteristics of sarcopenia, such as decreased muscle function and decreased muscle weight.

[0026] These results indicate that beta-amyloid and beta-amyloid oligomers play a role not only in aging but also in the degeneration of muscle tissue and the induction of sarcopenia caused by Alzheimer's dementia.

[0027] Furthermore, the inventors analyzed whether there was a difference in the expression of beta-amyloid oligomers in the muscle tissues of naturally aged mice and young mice. As a result, it was confirmed that the expression of beta-amyloid oligomers was significantly overexpressed in aged mice compared to young mice, and in addition, the expression of BACE1 protein was also found to be overexpressed in aged mice. Moreover, as BACE1, an APP cleavage enzyme, was overexpressed in aged mice, it was confirmed that the expression of the N-terminal fragment of APP (APP-NT), which remains after cleavage by BACE1 and the soluble amyloid precursor protein-beta is detached, was also reduced in aged mice.

[0028] In addition, in mice that underwent natural aging, the expression of the muscle marker MHC was reduced compared to young mice, while the expression of the muscle atrophy markers MAFbx and MuRF1 was overexpressed, and the expression of the aging markers p21 and p16 was increased.

[0029] Based on the above results, the inventors were able to determine that the overexpression of BACE1, beta-amyloid, and beta-amyloid oligomers is a characteristic molecular expression that occurs during sarcopenia caused by aging or Alzheimer's dementia, and that the increased expression of soluble amyloid precursor protein-beta, which is generated by the cleavage of APP by BACE1, can also be a characteristic molecular expression that occurs during sarcopenia caused by aging or Alzheimer's dementia.

[0030] Therefore, in the present invention, it was found that BACE1, beta-amyloid, beta-amyloid oligomer, soluble amyloid precursor protein-beta, or a combination thereof can be used as a biomarker for diagnosing sarcopenia caused by aging or Alzheimer's dementia.

[0031] Accordingly, the present invention may provide a composition for diagnosing sarcopenia caused by aging or Alzheimer's dementia, comprising as an active ingredient a preparation for measuring the level of one or more proteins or mRNA selected from the group consisting of BACE1, beta-amyloid, beta-amyloid oligomer, and soluble amyloid precursor protein-beta.

[0032] In the present invention, BACE1 (beta-secretase 1) is also known as beta-site amyloid precursor protein cleavage enzyme 1, and is an enzyme encoded by the BACE1 gene in humans, and is known to be expressed mainly in neurons and oligodendrocytes. It is known that the expression of BACE1 is affected by inflammatory conditions, and it is known as a major beta-secreting enzyme for beta-amyloid production in neurons in relation to Alzheimer's disease. The amino acid sequence of the BACE1 protein according to the present invention is known in the art (NCBI accession number: NP_036236.1).

[0033] In addition, the above beta-amyloid is known to be the main component of amyloid plaques found in the brains of patients with Alzheimer's disease and refers to a 36-43 amino acid peptide that is critically involved in Alzheimer's disease. This peptide is produced when amyloid precursor protein (APP) is broken down by beta-secretase and gamma-secretase.

[0034] The above beta-amyloid oligomer is formed by the aggregation of beta-amyloid molecules and is known as a causative agent of Alzheimer's disease as an intermediate stage of the fibrillation of monomeric beta-amyloid.

[0035] In addition, the above-mentioned soluble amyloid precursor protein-beta is generated through the degradation process of amyloid precursor protein (APP), in which the amyloid precursor protein is cleaved by beta-secretase to form a beta-amyloid type amino-terminal, and a soluble amino-terminal fragment, soluble amyloid precursor protein-beta, is produced. It is known that if this soluble amyloid precursor protein-beta accumulates excessively, it can act as a toxic agent to the brain.

[0036] In the present invention, “sarcopenia” refers to a condition in which muscle strength and physical function decline due to a decrease in skeletal muscle mass caused by aging. Patients with sarcopenia exhibit various physical functional abnormalities as muscle strength declines; for example, their gait slows down, muscle endurance decreases, and osteoporosis, falls, and fractures occur easily. Furthermore, as the blood and hormone buffering function of muscles decreases in patients with sarcopenia, their basal metabolic rate decreases, making it difficult to control chronic diseases, and diabetes and cardiovascular diseases can easily worsen. Skeletal muscle is important not only as an organ for movement but also affects the entire body, including bones, blood vessels, the heart, and nerves; therefore, sarcopenia resulting from a decrease in skeletal muscle can lead to diseases such as fatty liver, cardiac hypertrophy, pancreatic dysfunction, diabetes, and cognitive decline.

[0037] In the present invention, sarcopenia may include sarcopenia caused by aging as well as sarcopenia caused by other diseases, such as, for example, obesity, infectious diseases caused by viruses or bacteria, diabetes, cancer, neurodegenerative diseases (dementia, etc.), menopause, and degenerative diseases (spinal stenosis, etc.), but any disease that can cause a decrease in muscle mass, a decrease in muscle strength, and / or a decrease in muscle function is included without limitation.

[0038] Specifically, the sarcopenia mentioned above may include, but is not limited to, muscular dystrophy, useless muscle atrophy, spinal muscular atrophy, muscular dystrophy, muscle rigidity, hypotonia, muscle weakness, muscle degenerative atrophy, amyotrophic lateral sclerosis, spinal bulbar muscle atrophy, and myasthenia gravis.

[0039] In the present invention, "diagnosis" means confirming the existence or characteristics of a pathological condition, and may include confirming the presence or absence of sarcopenia, the onset of sarcopenia, the possibility of onset of sarcopenia (risk of onset), the prognosis of sarcopenia, etc.

[0040] More specifically, the term “diagnosis” as used in the present invention may include determining the susceptibility of an object to a specific disease or condition, determining whether an object currently has a specific disease or condition, determining the prognosis of an object with a specific disease or condition, or therametrics (e.g., monitoring the condition of an object to provide information on therapeutic efficacy).

[0041] In the present invention, the level of one or more proteins or mRNA selected from the group consisting of BACE1, beta-amyloid, beta-amyloid oligomer, and soluble amyloid precursor protein-beta is increased in individuals with sarcopenia compared to normal individuals.

[0042] An increase in the aforementioned protein or mRNA levels means that something that was not previously detected is now being detected, or that the detected amount is relatively higher than the normal level.

[0043] Furthermore, in the present invention, the term "measurement" includes both measuring and confirming the presence (expression) of a target substance (in the case of the present invention, the mRNA or protein of a sarcopenia biomarker gene) and measuring and confirming a change in the level of presence (expression level) of the target substance. That is, measuring the expression level of the protein means measuring whether it is expressed (i.e., measuring the presence or absence of expression), or measuring the level of qualitative or quantitative change of the protein.

[0044] The above measurements may be performed without limitation, including both qualitative (analytical) and quantitative methods. The types of qualitative and quantitative methods for measuring protein levels are well known in the art, and the experimental methods described herein are included therein. Specific methods for comparing protein levels for each method are well known in the art. Therefore, the detection of the target protein means detecting the presence of a biomarker protein or confirming an increase (upregulation) in the protein level.

[0045] If the above composition is intended to measure the level of mRNA, the agent for measuring the level of mRNA may be a primer set or probe that specifically binds to mRNA. The composition of the present invention, comprising a primer set or probe specific to the mRNA of biomarker genes according to the present invention, may further comprise an agent necessary for a known method of detecting RNA. By using a known method of detecting RNA with the composition of the present invention without limitation, the level of mRNA of the biomarkers in a subject can be measured.

[0046] In the present invention, the 'primer' is a fragment that recognizes a target gene sequence and includes a forward and reverse primer pair, but preferably is a primer pair that provides analysis results having specificity and sensitivity. High specificity can be conferred when the nucleic acid sequence of the primer is a sequence that is inconsistent with the non-target sequence present in the sample, so that it amplifies only the target gene sequence containing the complementary primer binding site and does not induce non-specific amplification.

[0047] In the present invention, the term 'probe' refers to a substance capable of specifically binding to a target substance to be detected within a sample, and refers to a substance capable of specifically confirming the presence of the target substance within the sample through said binding. The type of probe is not limited to substances commonly used in the industry, but preferably may be PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA, or DNA. The probe may be a biomaterial derived from an organism or similar, or manufactured in vitro. Examples include enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, nerve cells, DNA, and RNA. DNA includes cDNA, genomic DNA, and oligonucleotides, RNA includes genomic RNA, mRNA, and oligonucleotides, and examples of proteins may include antibodies, antigens, enzymes, peptides, etc.

[0048] Since the information (amino acid sequence, nucleic acid sequence, etc.) of the biomarker (protein or gene) according to the present invention is known, a person skilled in the art can easily design primers, probes, or antisense nucleotides that specifically bind to the gene encoding the said protein based thereon. Primers or probes can be chemically synthesized using the phosphoramidite solid support synthesis method or other widely known methods and include all functional equivalents. In addition, primers or probes can be modified in various ways according to methods known in the art to the extent that they do not interfere with hybridization with mRNA. Examples of such modifications include methylation, capping, substitution with one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoromidate, carbamate, etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), and the binding of fluorescent or enzymatic labeling materials.

[0049] In cases where the composition of the present invention is intended to measure the level of protein, the agent for measuring the level of protein may be an antibody or aptamer specific to said protein.

[0050] As used herein, the term "antibody" refers to a specific protein molecule indicated for an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein and includes polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. Furthermore, any part of a whole antibody that possesses antigen-antibody binding ability is included in the antibodies of the present invention, and all types of immunoglobulin antibodies that specifically bind to the proteins of the present invention are included. For example, this includes not only complete antibodies having two full-length light chains and two full-length heavy chains, but also functional fragments of antibody molecules, namely Fab, F(ab'), F(ab')2, and Fv, which have antigen-binding functions. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies and chimeric antibodies, as well as recombinant antibodies, provided that they can specifically bind to the proteins of the present invention.

[0051] As used herein, the term 'aptamer' refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) having a stable tertiary structure that can specifically bind to an analyte to be detected in a sample, and can specifically confirm the presence of a target protein in a sample. The preparation of an aptamer may be carried out according to a general method for preparing aptamers, by determining and synthesizing a sequence of an oligonucleotide that has selective and high binding affinity to the target protein to be confirmed, and then modifying the 5' end or 3' end of the oligonucleotide with -SH, -COOH, -OH, or NH2 so that it can bind to the functional group of the aptamer chip, but is not limited thereto.

[0052] The composition of the present invention may additionally include a preparation necessary for a method of detecting the biomarker protein, and the expression level of a protein in a subject (or a biological sample obtained from a subject) can be measured using a known method of detecting a protein without limitation using the composition.

[0053] The composition according to the present invention can be used for the analysis of biological samples isolated from an individual requiring diagnosis.

[0054] In the present invention, the term 'biological sample' may be included without limitation as long as it is collected from a subject for whom sarcopenia is to be diagnosed or for whom the risk of onset thereof is to be predicted. For example, the biological sample may be selected from blood, whole blood, plasma, urine, saliva, tissue, muscle tissue, cells, organs, bone marrow, fine needle aspiration specimens, core needle biopsy specimens, and vacuum aspiration biopsy specimens.

[0055] In addition, the present invention can provide a kit for diagnosing sarcopenia comprising a composition according to the present invention.

[0056] In the present invention, the term "kit" refers to a diagnostic device or set, etc., capable of diagnosing sarcopenia by measuring the amount of mRNA or protein of biomarkers according to the present invention contained in a sample, and is not limited to any form that can measure the expression levels of said genes from a biological sample isolated from an individual. For example, the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a Rapid kit, a Mass Spectrometry kit, or an MRM (Multiple Reaction Monitoring) kit, but is not limited thereto. Accordingly, the kit of the present invention may include not only antibodies that recognize target proteins as markers or primer sets and probes that recognize mRNA as markers, but also one or more other constituent compositions, solutions, or devices suitable for the analysis method. At this time, the substance for detecting the expression level of said gene, mRNA, or protein may be applied one or more times without limitation on the number of times, and there is no restriction on the order in which each substance is applied, and the application of each substance may proceed simultaneously or in stages. In addition, the kit according to the present invention may further include an instruction manual describing a diagnostic method, etc. according to the present invention.

[0057] Furthermore, the present invention may provide a method for diagnosing sarcopenia or a method for providing information for diagnosis, comprising the following steps.

[0058] The method for diagnosing sarcopenia or the method for providing information for diagnosis provided in the present invention comprises: (1) a step of measuring the level of one or more proteins or mRNA selected from the group consisting of BACE1, beta-amyloid, beta-amyloid oligomer, and soluble amyloid precursor protein-beta in a biological sample separated from a subject; and (2) a step of comparing the level of proteins or mRNA measured in step (1) with a control group.

[0059] In the present invention, the "subject" is an individual requiring diagnosis of sarcopenia, the likelihood of developing sarcopenia, muscle mass, and / or muscle function. In the present invention, the "target individual" refers to a mammal including humans, and may be selected from a group consisting of, for example, humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cattle, horses, pigs, sheep, and goats; preferably, it may be a human, but is not limited thereto. The subject may refer to a person who has developed sarcopenia or is suspected of developing sarcopenia, and may mean a patient who requires or is expected to require appropriate treatment for sarcopenia, but is not limited thereto.

[0060] In one embodiment of the present invention, the method may further include the step of diagnosing sarcopenia when the level of the marker protein or mRNA of the present invention measured in step (1) is higher than that of a control group; or determining that there is a risk of developing sarcopenia (high likelihood of developing sarcopenia).

[0061] In the above method, protein levels can be determined by one or more methods selected from the group consisting of Western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chips.

[0062] In addition, in the above method, mRNA levels can be determined using one or more methods selected from the group consisting of PCR, RNase protection assay, northern blotting, southern blotting, in situ hybridization, DNA chips, and RNA chips.

[0063] The biomarkers provided in the present invention are proteins that show a close correlation with sarcopenia. In particular, they exhibit the characteristic that the expression of the biomarker proteins of the present invention increases upon the onset of sarcopenia caused by aging or Alzheimer's dementia. Therefore, these biomarkers can be used for the diagnosis of sarcopenia and further have the potential to serve as targets for sarcopenia treatments.

[0064] Therefore, agents that inhibit the expression or activity of biomarkers provided in the present invention can be used for the prevention, improvement, or treatment of sarcopenia caused by aging or Alzheimer's dementia.

[0065] Unlike conventional methods that measured muscle mass and muscle function, the biomarkers provided in this invention for diagnosing sarcopenia caused by aging or Alzheimer's dementia allow for the objective and quantitative analysis of the marker proteins present in biological samples through molecular diagnosis, thereby enabling more accurate and rapid diagnosis of sarcopenia. Furthermore, the biomarkers discovered in this invention can be usefully utilized as targets for the development of treatments for sarcopenia.

[0066] Figure 1 shows the results of confirming the muscle cell differentiation inhibitory activity of beta-amyloid oligomers by analyzing the expression levels of MHC (myosin heavy chain), a muscle cell marker, in a group treated with beta-amyloid oligomers and a control group not treated with beta-amyloid oligomers in C2C12 muscle cell lines.

[0067] Figure 2 shows the changes in the number and diameter of myotubes observed after treating differentiated muscle cells with beta-amyloid oligomers at different concentrations (A), the oxygen uptake rate of mitochondria measured (B), the expression levels of muscle differentiation factors confirmed by Western blotting (C), and the results of ATPlite analysis (D).

[0068] Figure 3 shows the results of confirming reactive oxygen species levels according to beta-amyloid treatment concentration in differentiated muscle cells using the DCF-DA assay (A), the results of confirming the expression levels of iNOS, HO-1, IL-6, IL-1β, and TNF-α (B), and beta-amyloid (Aβ) in muscle tissues of aging-accelerated mice (SAMP8) and control mice (SAMR1). 42 This shows the results of measuring the concentration of ) (C).

[0069] Figure 4 shows the results of morphological observation (A) and analysis of the expression levels of beta-amyloid oligomers (B) and muscle differentiation-related factors (C) in muscle tissue, as well as the results of analysis of the morphological functions of the muscle (D).

[0070] Figure 5 shows the results of analyzing the expression levels of beta-amyloid oligomers, BACE1, APP-NT, MHC, MAFbx, MuRF1, P21, and P16 in naturally aged mice (OC) and young mice (YC) (top figure) and the results of quantitatively comparing the expression levels of these proteins in a graph (bottom figure).

[0071] The contents of the present invention will be explained in more detail below through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, but includes variations of equivalent technical concepts.

[0072] <Example 1>

[0073] Confirmation of the effect of beta-amyloid oligomers on muscle cell differentiation

[0074] <1-1> Confirmation of Inhibition of Muscle Cell Differentiation by Beta-Amyloid Oligomers

[0075] The inventors performed the following experiment to determine whether beta-amyloid oligomers (Aβ oligomers) affect the differentiation of muscle cells.

[0076] After treating the C2C12 muscle cell line with beta-amyloid oligomers, the expression level of MHC (myosin heavy chain), a marker of muscle cells, was analyzed using immunofluorescence.

[0077] As a result, as shown in Figure 1, it was found that the expression level of MHC, a marker of muscle cells, was significantly reduced in muscle cells treated with beta-amyloid oligomers compared to the control group not treated with beta-amyloid oligomers.

[0078] These results showed that beta-amyloid oligomers act to inhibit the differentiation of muscle cells.

[0079] <1-2> Confirmation of the effects of beta-amyloid oligomers on myogenesis and the expression of muscle differentiation factors in differentiated muscle cells

[0080] Next, the inventors analyzed the effects on muscle development and the expression of muscle differentiation factors when treated with beta-amyloid oligomers in differentiated muscle cells. After treating differentiated muscle cells with beta-amyloid oligomers at 2 μM and 5 μM, respectively, the number and diameter of myotubes were analyzed, and the oxygen uptake rate of mitochondria was measured.

[0081] As a result, as shown in Figure 2, the group treated with beta-amyloid oligomers showed a decrease in the number and diameter of myotubes in a concentration-dependent manner compared to the control group not treated with it (A), and the oxygen uptake rate of mitochondria also showed a significant decrease, indicating a decrease in mitochondrial function (B and D).

[0082] In addition, the effects on the expression levels of muscle differentiation factors were analyzed by Western blot. As shown in Figure 2, the expression levels of the muscle differentiation factors MyoD and myogenin were found to be reduced by beta-amyloid oligomer treatment, and the expression levels of p21 and the mitochondrial fusion proteins MFN-1 and MFN-2 were also found to be reduced by beta-amyloid oligomer treatment. On the other hand, the expression of the atrophy factors MurF1 and MSTN and the apoptosis factor cleaved caspase 3 was found to be increased by beta-amyloid oligomer treatment (C).

[0083] Through these results, the inventors found that beta-amyloid oligomers in differentiated muscle cells inhibit the formation of myoducts, reduce mitochondrial function, and suppress the expression levels of factors involved in muscle differentiation, ultimately causing adverse effects on muscle formation and function.

[0084] <Example 2>

[0085] Confirmation of increased oxidative stress caused by beta-amyloid oligomers in differentiated muscle cells and increased beta-amyloid concentration in aged muscle tissue

[0086] To determine whether beta-amyloid oligomers act as a cause of increased oxidative stress in muscle cells, differentiated C2C12 cells were treated with beta-amyloid oligomers at concentrations of 2 μM and 5 μM, respectively, and the levels of reactive oxygen species were analyzed using DCF-DA analysis.

[0087] As a result, as shown in Figure 3, beta-amyloid oligomers were found to increase the production of reactive oxygen species in differentiated muscle cells in a concentration-dependent manner, indicating that beta-amyloid oligomers increase oxidative stress in muscle cells (A). In addition, in differentiated muscle cells treated with beta-amyloid oligomers, the expression levels of iNOS, HO-1, and IL-6 were found to increase compared to the control group not treated with beta-amyloid oligomers, while the expression of IL-1β and TNF-α showed no significant change (B).

[0088] In addition, when analyzing the concentration of beta-amyloid 42 in the muscle tissue of 8-month-old aging-accelerated SAMP8 (Senescence Accelerated Mouse-Prone 8) mice and control SAMR1 mice that underwent normal aging compared to SAMP8, it was confirmed that the concentration of beta-amyloid 42 was significantly increased in the aging-accelerated SAMP8 mice compared to the SAMR1 mouse group (C).

[0089] Through these results, the inventors found that beta-amyloid oligomers can act as factors causing oxidative stress in muscle cells, and that the concentration of beta-amyloid 42 in muscle tissue increases as aging is accelerated.

[0090] <Example 3>

[0091] Confirmation of overexpression of beta-amyloid oligomers in muscle tissue of an Alzheimer's animal model and confirmation of symptoms corresponding to sarcopenia

[0092] Muscle tissue was isolated from each mouse in 3xTg mice and normal mice (Non-Tg), which are Alzheimer's disease animal models, and the expression levels of beta-amyloid oligomers in each muscle tissue were analyzed using Western blot and immunohistochemistry with 6E10 antibodies.

[0093] As a result, as shown in Figure 4, the expression level of beta-amyloid oligomers in the muscle tissue of Alzheimer's mice was found to be significantly increased compared to normal mice, and immunohistochemistry results also confirmed that the accumulation of beta-amyloid was significantly increased in the Alzheimer's mouse group compared to normal mice (B).

[0094] In addition, the expression levels of factors affecting muscle cell differentiation, muscle weight, and findings corresponding to sarcopenia were analyzed in 3xTg mice, an Alzheimer's animal model, and normal mice (Non-Tg). It was found that the expression level of myogenin, a muscle differentiation factor, was significantly reduced in the Alzheimer's mouse group compared to the normal mouse group (Fig. 4C), and morphological functions of the muscle, such as body weight and muscle tissue weight, were reduced (Fig. 4D).

[0095] <Example 4>

[0096] Confirmation of overexpression of beta-amyloid oligomers and BACE1 in naturally aging mice

[0097] Furthermore, the inventors analyzed the expression levels of beta-amyloid oligomers and BACE1 in the muscle tissue of mice aged 22 months with natural aging corresponding to a human in their 60s (OC mice with signs of sarcopenia and YC mice without aging) using Western blot, and compared the expression levels of each protein using a quantitative graph.

[0098] As a result, as shown in Figure 5, the expression levels of beta-amyloid oligomers and BACE1 were found to be significantly increased in the mouse group exhibiting sarcopenia due to natural aging compared to the young mouse group. Conversely, the expression of the N-terminal fragment of APP (APP-NT), which remains on the cell membrane after leaching by BACE1 and the release of the soluble amyloid precursor protein beta, was found to be decreased. Additionally, the expression of the muscle marker MHC (myosin heavy chain) was found to be decreased compared to the young mouse group, while the expression of the muscle atrophy markers MAFbx and MuRF1 was found to be overexpressed in the naturally aged mouse group. Furthermore, the expression of the aging markers p21 and p16 was also found to be increased, indicating that muscle aging had progressed (see Figure 5).

[0099] Based on the experimental results above, the inventors found that the overexpression of beta-amyloid, beta-amyloid oligomers, and BACE1 can be a cause of sarcopenia caused by aging or Alzheimer's dementia, and therefore, if their overexpression can be suppressed, sarcopenia caused by aging or Alzheimer's dementia can be prevented, improved, or treated. Furthermore, the inventors found that sarcopenia can be diagnosed by measuring the expression levels of beta-amyloid, beta-amyloid oligomers, BACE1, and soluble amyloid precursor protein-beta.

[0100] Although embodiments of the present invention have been described above, the present invention is not limited to the above embodiments and can be manufactured in various different forms, and those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical concept or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A composition for diagnosing sarcopenia caused by aging or Alzheimer's dementia, comprising as an active ingredient a preparation for measuring the level of one or more proteins or mRNA selected from the group consisting of beta-amyloid, beta-amyloid oligomer, and soluble amyloid precursor protein-beta.

2. In Paragraph 1, A composition for diagnosing sarcopenia caused by aging or Alzheimer's dementia, characterized in that the levels of the protein or mRNA are increased in individuals with sarcopenia compared to normal individuals.

3. In Paragraph 1, A composition for diagnosing sarcopenia caused by aging or Alzheimer's dementia, characterized in that the preparation for measuring the protein level is an antibody or aptamer specific to the protein.

4. In Paragraph 1, A composition for diagnosing sarcopenia caused by aging or Alzheimer's dementia, characterized in that the preparation for measuring the mRNA expression level is a primer set or probe that specifically binds to the mRNA.

5. In Paragraph 1, A composition for diagnosing sarcopenia caused by aging or Alzheimer's dementia, characterized in that the protein or mRNA level is measured using a sample selected from the group consisting of blood, whole blood, plasma, lymph fluid, urine, saliva, tissue, muscle tissue, cell, organ, bone marrow, fine needle aspiration specimen, core needle biopsy specimen, and vacuum aspiration biopsy specimen.

6. A kit for diagnosing sarcopenia caused by aging or Alzheimer's dementia, comprising a composition of any one of claims 1 to 5.

7. (1) A step of measuring the level of one or more proteins or mRNA selected from the group consisting of BACE1, beta-amyloid, beta-amyloid oligomer, and soluble amyloid precursor protein-beta in a biological sample separated from a subject; and (2) A step of comparing the protein or mRNA level measured in step (1) above with a control group, Method for providing information for the diagnosis of sarcopenia caused by aging or Alzheimer's dementia.

8. In Paragraph 7, A method for providing information for the diagnosis of sarcopenia caused by aging or Alzheimer's dementia, characterized by further including a step of determining that sarcopenia has developed or is at risk of developing when the protein or mRNA level measured in step (1) above is higher than that of a control group.

9. In Paragraph 7, A method for providing information for the diagnosis of sarcopenia caused by aging or Alzheimer's dementia, characterized in that the biological sample is selected from the group consisting of blood, whole blood, plasma, lymph fluid, urine, saliva, tissue, muscle tissue, cell, organ, bone marrow, fine needle aspiration specimen, core needle biopsy specimen, and vacuum aspiration biopsy specimen.

10. In Paragraph 7, A method for providing information for the diagnosis of sarcopenia caused by aging or Alzheimer's dementia, characterized by performing the above protein level using one or more methods selected from the group consisting of Western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip.

11. In Paragraph 7, A method for providing information for the diagnosis of sarcopenia caused by aging or Alzheimer's dementia, characterized in that the mRNA level is determined by one or more methods selected from the group consisting of PCR, RNase protection assay, northern blotting, southern blotting, in situ hybridization, DNA chips, and RNA chips.

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