Composition for preventing or treating mitochondrial-related diseases comprising Prdx3 as active ingredient, and use thereof

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating a mitochondrial-related disease comprising a Prdx3 peptide or an analog thereof as an active ingredient, and a use thereof. The pharmaceutical composition comprising a Prdx3 peptide or an analog thereof as an active ingredient can be used for the prevention and treatment of mitochondrial-related diseases by the elimination of mitochondrial autophagy, the elimination of alpha-synuclein aggregates, or the like.

Description

Technical Field

[0001] This invention relates to a composition for the prevention or treatment of mitochondrial-related diseases containing Prdx3 as an active ingredient, and its use therein. Background Technology

[0002] The heart is a tissue with a high number and density of mitochondria. In an adult heart, mitochondria account for about 30% of the total volume of cardiomyocytes and are mainly used to compensate for the high consumption of adenosine triphosphate (ATP) during heartbeat by supplying energy.

[0003] Even when subjected to reactive oxygen species (ROS)-induced mitochondrial damage, cardiac mitochondria maintain mitochondrial quality control (MQC) through persistent mitophagy. Therefore, mitochondria are the primary sites of energy and ROS generation in the heart, and MQC dysfunction can lead to diastolic dysfunction and is associated with heart failure.

[0004] MQC is a process essential for cell physiology and homeostasis. It is strictly controlled by preventing mitochondrial damage and clearing damaged mitochondria to maintain a healthy mitochondrial network.

[0005] Mitochondrial-specific peroxidase, peroxidase 3 (Prdx3), can scavenge reactive oxygen species within mitochondria, thereby participating in the prevention of mitochondrial damage through mechanisms such as preventing mitochondrial dysfunction. However, whether Prdx3 affects the clearance of damaged mitochondria through mechanisms such as mitophagy requires further investigation. Summary of the Invention

[0006] One aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of mitochondrial-related diseases, comprising Prdx3 peptide or an analogue thereof as an active ingredient.

[0007] Another aspect of the present invention provides a health functional food composition for the prevention or improvement of mitochondrial-related diseases, comprising Prdx3 peptide or its analogues as an active ingredient.

[0008] Another aspect of the present invention provides a pharmaceutical preparation for the prevention or treatment of mitochondrial-related diseases, comprising Prdx3 peptide or an analogue thereof as an active ingredient.

[0009] Another aspect of the present invention provides a method for the prevention or treatment of mitochondrial-related diseases, including the step of administering a pharmaceutical composition comprising a Prdx3 peptide or an analogue thereof to an individual.

[0010] The inventors have confirmed that Prdx3 peptide or its analogues can be used as active ingredients for the prevention or treatment of mitochondrial-related diseases, thus completing this invention.

[0011] The present invention provides a pharmaceutical composition for the prevention or treatment of mitochondrial-related diseases, comprising Prdx3 peptide or its analogues as an active ingredient.

[0012] In this specification, the term "Prdx3 (Peroxiredoxin 3)" refers to a member of the peroxiredoxin family that plays an important role in protecting cells from oxidative stress by detoxifying peroxides. As an oxidase, Prdx3 can be found in mitochondria, and more specifically, in tissues such as the heart, adrenal glands, liver, and brain.

[0013] In this invention, it was confirmed that Prdx3 can maintain mitochondrial activity or clear damaged mitochondria even without relying on peroxide detoxification effects. In one specific example, Prdx3 can act on the clearance of mitophagy, thereby exhibiting preventive or therapeutic effects against mitochondrial-related diseases.

[0014] In one embodiment of the present invention, the Prdx3 can regulate mitochondrial quality through dual antioxidant and molecular chaperone functions.

[0015] In this specification, "mitochondrial quality management" may include the protection of mitochondria from reactive oxygen species and mitophagy, the process of clearing damaged mitochondria.

[0016] In this specification, the term "peptide" refers to an amino acid polymer whose constituent elements may include not only natural amino acids but also non-protein amino acids.

[0017] In this specification, the term "peptide analog" can include analogs in which the side chain of an amino acid or the main chain of an α-amino acid is replaced by one or more other functional groups. Examples of peptide analogs with modified side chains or main chains include, but are not limited to, hydroxyproline or N-methylglycine "peptides" such as those with a pyrrolidine ring replaced by a hydroxyl group. The types of peptide analogs are well known in the art.

[0018] In one embodiment of the present invention, the Prdx3 analogue is a dominant-negative (DN) mutant of Prdx3. The dominant-negative Prdx3 mutant can be a form in which cysteine ​​residues at positions 108 and 229 of Prdx3 are replaced by serine residues. Through the mutations described above, even in the absence of Prdx3's ROS scavenging function, it will exhibit characteristics that regulate mitophagy.

[0019] In one embodiment of the present invention, the Prdx3 peptide may include a chain of 194 amino acids from position 64 to position 257 of the Prdx3 gene, and a transit peptide consisting of 63 amino acids from position 1 to position 63 of the Prdx3 gene.

[0020] In one embodiment of the invention, the Prdx3 peptide can eliminate mitophagy. In a specific example, the Prdx3 peptide can regulate the submitochondrial localization of PINK1 and prevent mitochondrial damage.

[0021] In this specification, the term "autophagy" refers to the activity by which cells obtain energy by degrading their own proteins or actively removing unwanted cellular components when they are in a state of nutrient deficiency. For example, it could be mitophagy.

[0022] In this specification, the term "mitophage" refers to the selective degradation of mitochondria through autophagy, a mitochondrial-specific response to arbitrary damage or stress. Its characteristic feature is that it prevents the accumulation of dysfunctional mitochondria that cause cellular degradation by promoting mitochondrial turnover, thereby maintaining mitochondria in a healthy state.

[0023] In other words, when mitophagy cannot proceed smoothly, mitochondrial-related diseases may occur due to abnormalities within the cell. Therefore, regulating mitophagy is beneficial for the prevention or treatment of these diseases.

[0024] The compositions of the present invention contain Prdx3 peptide or its analogues as active ingredients, and therefore have the effect of improving or treating diseases caused by autophagy disorders in mitochondria, thereby contributing to related research, and also have the advantage of being usable as various pharmaceuticals and health functional foods.

[0025] In one embodiment of the present invention, the absence of the Prdx3 peptide may cause cardiac dysfunction, such as myocardial infarction (MI).

[0026] In one embodiment of the present invention, the Prdx3 peptide can interact with the PINK1 peptide and can regulate mitophagy through the degradation of the PINK1 peptide.

[0027] In this specification, the term "PINK1 (PTEN-induced kinase 1)" refers to a protein located in the mitochondrial matrix whose N-terminus can be cleaved by mitochondrial serine proteases (PARL) and mitochondrial processing peptidases (MPP) through staged protein degradation. In response to mitochondrial damage, PINK1 can accumulate on the OMM of damaged mitochondria, thereby mediating mitophagy.

[0028] In one embodiment of the present invention, even if PINK1 is upregulated, the absence of the Prdx3 peptide may lead to a reduction in mitophagy.

[0029] In one embodiment of the present invention, the Prdx3 can inhibit the expression or function of Oma1 and maintain its stability by inhibiting the degradation of PINK1 caused by Oma1.

[0030] In one embodiment of the present invention, the Prdx3 peptide can influence the localization of Parkin and regulate PINK1-Parkin-mediated mitophagy.

[0031] In one embodiment of the present invention, the Prdx3 can clear alpha-synuclein aggregates.

[0032] In this invention, the term alpha-synuclein refers to a protein that is abundant in the human brain. When alpha-synuclein aggregates to form insoluble fibrils, it acts as a neurotoxin, thereby reducing dopamine and inducing Parkinson's disease.

[0033] The "mitochondrial-related disease" in this invention may be selected from one or more of the following groups: cardiovascular disease, lymphoma, glomerulonephritis, osteoporosis, motor neuron disease, muscular atrophy, Down syndrome, carcinoma, Pick disease, Parkinson's syndrome, and Alzheimer's disease.

[0034] In one embodiment of the invention, Prdx3 can simultaneously regulate both the protection of mitochondria from reactive oxygen species and mitophagy, which clears damaged mitochondria, thereby participating in mitochondrial quality management. Furthermore, in another embodiment, mice lacking Prdx3 were observed to contain damaged mitochondria in most tissues, confirming their potential use as an important mouse model for studying mitochondrial function in vivo.

[0035] The term "prevention" as used in this invention refers to all actions that suppress or delay the onset of mitochondrial-related diseases by administering the pharmaceutical compositions according to the invention.

[0036] The term "treatment" as used in this invention refers to all actions that improve or improve the symptoms of mitochondrial-related diseases by administering the pharmaceutical compositions according to the invention.

[0037] The compositions according to the present invention can be used alone or in combination with surgery, radiotherapy, chemotherapy and biological response modifiers for the prevention or treatment of mitochondrial-related diseases. Preferably, they can be used in combination with drugs that promote the prevention or treatment of mitochondrial-related diseases.

[0038] The compositions according to the present invention may further comprise pharmaceutically permissible carriers. These pharmaceutically permissible carriers are substances commonly used in formulation and may include, but are not limited to, physiological saline, sterile water, Ringer's solution, buffered physiological saline, cyclodextrin, glucose solution, maltodextrin solution, glycerol, ethanol, and liposomes. Further additions such as antioxidants and buffer solutions may be included as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the product into dosage forms such as aqueous solutions, suspensions, and emulsions for injection, infusion bags, sprays, pills, capsules, granules, or tablets. Suitable pharmaceutically permissible carriers and formulation methods can be used, based on the methods disclosed in the Remington Pharmaceutical Sciences literature, to appropriately formulate each component. The pharmaceutical formulations of the present invention are not particularly limited in their dosage form and can be formulated into injections, infusions, sprays, liquids, or topical preparations.

[0039] The compositions of the present invention can be administered orally or non-orally (e.g., including local administration via intravenous, subcutaneous, intraperitoneal, or ocular routes) according to the target method, and the dosage will vary depending on the patient's condition and weight, the severity of the disease, the form of the drug, the route of administration, and the time, which can be appropriately selected by relevant practitioners.

[0040] The compositions of this invention can be administered in pharmaceutically effective amounts. In this invention, a "pharmaceutically effective amount" means an amount sufficient to treat or diagnose a disease based on a reasonable benefit / risk ratio applicable to medical treatment or diagnosis. The effective dosage level can be determined based on factors including the patient's disease type, severity, drug activity, drug sensitivity, timing of administration, route of administration, and excretion rate, treatment duration, and concurrent medications, as well as other factors known in the medical field. The compositions according to the invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with existing therapeutic agents, or administered once or multiple times. It is important to comprehensively consider all the factors described above and administer the drug in the amount that achieves the maximum effect with the minimum dose without causing side effects; this can be easily determined by those skilled in the art.

[0041] Specifically, the effective amount of the composition of the present invention can vary depending on the patient's age, sex, condition, weight, absorption rate, inactivation rate, and excretion rate of the active ingredient in the body, the type of disease, and the drugs used in combination. Generally, it can be administered at a dose of 0.001 to 150 mg per kg of body weight, preferably 0.01 to 100 mg daily or every other day, or divided into one to three daily doses. However, it may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, and age, etc. Therefore, the dosage described does not limit the scope of the present invention in any way.

[0042] In this specification, the combination administration may be used alternately with parallel administration, and the combination administration form may include all forms of administration of peptides or peptide analogs simultaneously or separately with other compounds.

[0043] Furthermore, the present invention provides a health functional food composition for the prevention or improvement of mitochondrial-related diseases, in which Prdx3 peptide or its analogues are included as active ingredients.

[0044] The term "improvement" can refer to all behaviors that reduce at least one of the parameters related to the treatment state, such as the severity of symptoms. In this case, the health functional food can be used, simultaneously or separately from therapeutic agents, before or after the onset of the corresponding disease, for the prevention or improvement of cerebrovascular disease.

[0045] In the aforementioned health functional foods, the active ingredients can be added directly to the food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The amount of active ingredients mixed can be appropriately determined according to its intended use (prevention or improvement). Generally speaking, when manufacturing food or beverages, the aforementioned health functional foods can be added in an amount of approximately 15% by weight or less, more specifically, approximately 10% by weight or less, relative to the raw materials. However, for long-term intake for health and wellness purposes or for health regulation purposes, the amount can be below the aforementioned range.

[0046] The health functional food may further include one or more of a carrier, diluent, excipient, and additive, thereby being formulated into a form selected from the group consisting of tablets, pills, powders, granules, capsules, and liquids. Foods that may contain compounds according to one aspect include various food categories, powders, granules, tablets, capsules, syrups, beverages, chewing gum, tea, multivitamins, and other health functional foods.

[0047] As a specific example of the carrier, excipient, diluent, and additive, it may be derived from... At least one of the following is selected from the group consisting of lactose, glucose, sucrose, sorbitol, mannitol, erythritol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0048] In addition to the aforementioned active ingredients, the health functional food may also contain other ingredients as essential components without special restrictions. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients, just like a regular beverage. Examples of natural carbohydrates as described above include common sugars (such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as dextrin and cyclodextrin) and sugar alcohols such as xylitol, sorbitol, and erythritol. In addition to the flavoring agents described above, natural flavoring agents (such as sematriene and stevia extracts such as rebaudioside A and glycyrrhizin) and synthetic flavoring agents (such as saccharin and aspartame) may also be used. The proportion of the natural carbohydrates may be appropriately determined according to the choices of relevant practitioners.

[0049] In addition, depending on the specific health function food, it may also contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and flavor enhancers (such as cheese and chocolate), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, and carbonating agents used in carbonated beverages. The above-mentioned ingredients can be used independently or in combination, and the proportions of the additives described above can be appropriately selected by relevant professionals.

[0050] In one embodiment of the present invention, the Prdx3 peptide or its analogues can act on autophagy and apoptosis in mitochondria, thereby exhibiting preventive or ameliorative effects on mitochondrial-related diseases.

[0051] Furthermore, the present invention provides a pharmaceutical preparation for the prevention or treatment of mitochondrial-related diseases, in which Prdx3 peptide or its analogues are included as active ingredients.

[0052] As one embodiment of the present invention, the pharmaceutical preparation may be an injection, an infusion, a spray, or a liquid.

[0053] Furthermore, the present invention provides a method for the prevention or treatment of mitochondrial-related diseases, including the step of administering a pharmaceutical composition comprising Prdx3 peptide or an analogue thereof to an individual.

[0054] In this invention, "individual" refers to the object that needs to be treated for a disease, and more specifically, to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cattle.

[0055] Furthermore, the present invention provides a use for the prevention or treatment of mitochondrial-related diseases of Prdx3 peptide or its analogues.

[0056] Furthermore, the present invention provides a composition comprising Prdx3 peptide or an analogue thereof as an active ingredient for the prevention or treatment of mitochondrial-related diseases.

[0057] Furthermore, the present invention provides the use of Prdx3 peptide or analogues thereof for manufacturing pharmaceutical agents for the prevention or treatment of mitochondrial-related diseases.

[0058] Furthermore, the present invention provides the use of a composition comprising Prdx3 peptide or an analogue thereof and macrophages as an active ingredient in a pharmaceutical agent for the prevention or treatment of mitochondrial-related diseases.

[0059] The various features described in this application can be used in combination, and the fact that the various features are described in different dependent claims in the claims does not mean that the corresponding features cannot be used in combination.

[0060] In addition, the present invention provides a Prdx3 gene knockout mouse model in which exon 1 to exon 4 of the 5'-UTR of the Prdx3 gene are knocked out.

[0061] In one embodiment of the present invention, the mouse model may be a Parkinson's disease mouse model.

[0062] Furthermore, this invention provides a method for manufacturing a Prdx3 gene knockout mouse model, comprising the step of knocking out exon 1 to exon 4 of the 5'-UTR of the Prdx3 gene. In one specific example of this invention, exons 1 to 6 of the Prdx3 gene may be composed of sequences represented by sequence numbers 7 to 12.

[0063] In one embodiment of the present invention, the knockout of exon 1 to exon 4 of the 5'-UTR of the Prdx3 gene can be performed by homologous recombination.

[0064] In one embodiment of the present invention, the knockout step may be performed by replacing the base sequence of the 5'-UTR exon 1 to exon 4 of the Prdx3 gene with a construct containing the neomycin gene.

[0065] In this invention, the construct can be obtained by inserting a portion of the intron of the 5'-UTR of exon 1 of the Prdx3 gene into the 5'-UTR of the exogenous gene neomycin and recombining the intron portion of the 3'-UTR of exon 4 of the Prdx3 gene in the 3'-UTR.

[0066] In one embodiment of the present invention, the replacement of the construct may be performed by electroporation.

[0067] The pharmaceutical composition of the present invention, comprising Prdx3 peptide or its analogues, can achieve the clearance of mitophagy and α-synuclein aggregates through Prdx3 peptide or its analogues, thereby being used for the prevention and treatment of mitochondrial-related diseases (such as Parkinson's disease).

[0068] However, the effects of the present invention are not limited to those described, but should be understood to include all effects that can be derived from the invention as described in the detailed description of the invention or the invention as set forth in the claims. Attached Figure Description

[0069] Figure 1 is a schematic diagram confirming the large and damaged mitochondria and cardiac hypertrophy induced by the deletion of Peroxiredoxin 3 (Prdx3). Figure 1A illustrates the heart weight / body weight ratio between 10-week-old (n=5 / group) and 52-week-old (n=6 / group) Prdx3 wild-type (Prdx3 WT) and Prdx3-deficient (Prdx3 KO) mice. Figures 1B to 1D show the cardiac function analysis results using echocardiography in 10-week-old Prdx3 WT (n=6), 52-week-old Prdx3 WT (n=10), 10-week-old Prdx3 KO (n=6), and 52-week-old Prdx3-deficient mice (n=6). Stroke volume (SV) (1b), cardiac output (CO) (1c), and ejection fraction (EF) (1d) were measured in the long axis view of the left ventricle (LV) using the Simpson single-plane disk method. Figure 1E shows the results of hematoxylin and eosin staining of the hearts of Prdx3 WT and Prdx3 KO mice (scale bar, 1 mm). Figures 1F and 1G show the results of visualization of cell boundaries and quantification of LV fibrosis by wheat germ lectin staining of LV muscle sections (scale bar, 20 μm). Figures 1H to 1I show the results of Masson's trichrome staining of LV muscle sections and quantification of cardiac fibrosis (scale bar, 50 μm). Figures 1J and 1K are representative electron micrographs of the hearts of Prdx3 WT and Prdx3 KO mice and the quantitative results of damaged mitochondria (red arrows) (scale bar for 10-week-old Prdx3 WT and Prdx3 KO mice: 0.5 (boxed area) or 1 μm; scale bar for 52-week-old Prdx3 WT and Prdx KO mice: 1 (boxed area) or 2 μm; *P<0.05, **P<0.01 and ***P<0.001).

[0070] Figure 2 is a schematic diagram illustrating the transthoracic echocardiography results of peroxidase 3 (Prdx3) wild-type (Prdx3 WT) and Prdx3-deficient (Prdx3 KO) mice. Figure 2A illustrates the combined echocardiography results of 10-week-old Prdx3 WT (n=6), 52-week-old Prdx3 WT (n=10), 10-week-old Prdx3 KO (n=6), and 52-week-old aged Prdx3 KO mice (n=6). EF (ejection fraction), SV (stroke volume), FS (fractional shortening), and CO (cardiac output) are used. Figure 2B shows cardiac images (scale bar, 1 mm) of 10-week-old Prdx3 WT, 52-week-old Prdx3 WT, 10-week-old Prdx3 KO, and 52-week-old Prdx3 KO mice.

[0071] Figure 3 is a schematic diagram confirming the accelerated left ventricular (LV) remodeling and heart failure induced by Peroxiredoxin 3 (Prdx3) deficiency after myocardial infarction (MI). Figure 3A shows the cardiac function analysis results using echocardiography in Prdx3 wild-type (Prdx3 WT, n=7) and Prdx3-deficient (Prdx3 KO, n=8) mice 15 days after MI. The left ventricular end-diastolic volume (EDV) in Figure 3B and the end-systolic volume (ESV) in Figure 3C were measured in the long axis view of the LV using the Simpson single-plane disk method with ejaculation volume (SV) (Figure 3D) and ejection fraction (EF) (Figure 3E). Figure 3F is a schematic diagram illustrating serial sections of Masson trichrome stained hearts from Prdx3 WT and Prdx3 KO mice 15 days after MI (the percentage of fibrosis is calculated relative to the total area of ​​the left ventricle). Figure 3G shows representative electron micrographs of the hearts of wild-type and Prdx3 Ko mice 1 day after MI, with red arrows indicating damaged mitochondria (scale bar: 0.5 (boxed area) or 2 μm). Figure 3H shows the results of TUNEL (terminal transferase-mediated nick-end labeling) analysis of the infarcted regions of Prdx3 WT (n=5) and Prdx3 Ko mice (n=5) 1 day after MI (scale bar: 10 (boxed area) or 50 μm; *P<0.05, **P<0.01).

[0072] Figure 4 is a schematic diagram illustrating the transthoracic echocardiography results of wild-type peroxidase 3 (Prdx3) mice (Prdx3 WT) and Prdx3-deficient (Prdx3 KO) mice after myocardial infarction (MI). Figure 4A shows the meta-annual echocardiography data of Prdx3 WT (n=7) and Prdx3 KO mice (n=8). Figure 4B shows the results of left ventricular function assessment using VevoStrain software.

[0073] Figure 5 is a schematic diagram confirming the increased mitochondrial damage induced by reactive oxygen species (ROS) accumulation due to peroxidase (Prdx3) deficiency. Figures 5A and 5B are representative low- and high-magnification images of the boxed regions of Prdx3 wild-type (Prdx3 WT) and Prdx3-deficient (Prdx3 KO) mouse embryonic fibroblasts (MEFs). MEFs were infected with mitochondrial catalase adenovirus for 24 hours and immunostained for 25 minutes with either Tom20 (green) + 100 nM MitoTracker (red) (Figure 5A) or 150 nM MitoTracker (green) + 5 μM MitoSOX (red) (Figure 5B) antibodies (scale bar: 2 μm (boxed region) or 10 μm). Figures 5C and 5D show the results of quantification of depolarized (damaged) mitochondria (Figure 5C) or mitochondrial ROS levels (Figure 5D) in Prdx3 WT and Prdx3 KO MEF (n=25–33 cells). Figure 5E shows the results of immunoblotting of whole-cell lysates from Prdx3 WT and Prdx3 KO MEF cells infected with mitochondrial catalase adenovirus for 24 hours using catalase, Prdx3, and Tom20 antibodies (**P<0.01). Data (5A–5E) represent three independent experiments.

[0074] Figure 6 This is a schematic diagram confirming the increased mitochondrial damage induced by the loss of Peroxiredoxin (Prdx3). Figure 6 A and 6B are schematic diagrams illustrating representative electron micrographs of the liver, skeletal muscle (gastrocnemius), and brain (dentate gyrus region) tissues from 10-week-old Prdx3 wild-type (Prdx3 WT), 52-week-old Prdx3 WT, 10-week-old Prdx3-deficient (Prdx3 KO), and 52-week-old Prdx3 KO mice. Red arrows indicate damaged mitochondria (scale bar: 0.5 μm (boxed area) or 1 μm for 10-week-old mice, 1 μm (boxed area) or 2 μm for 52-week-old mice).

[0075] Figure 7 is a schematic diagram confirming the cardiac mitochondrial dysfunction induced by Prdx3 deficiency. Figure 7A is a schematic diagram comparing ATP production in wild-type (Prdx3 WT) and Prdx3-deficient (Prdx3 KO) cardiomyocytes. Figure 7B is a schematic diagram illustrating representative tracking results of oxygen consumption rate (OCR, pMoles / min) in wild-type (Prdx3 WT) and Prdx3-deficient (Prdx3 KO) cardiomyocytes. Arrows indicate the injection of oligomycin (Oligo, 1.5 μM), p-trifluoromethoxyphenylhydrazone carbonyl cyanide (FCCP, 1 μM), and Rot&AA (rotenone and antimycin A, 0.5 μM each) into 1×10 4 Time points per cell. Figure 7C shows the time points per 1 × 10⁻⁶ cells. 4 The OCR values ​​for basal respiration in individual cells are shown in Figure 7D, ATP-related respiration OCR values ​​in Figure 7E, proton leakage OCR values ​​in Figure 7F, maximal respiration OCR values ​​in Figure 7G, and reserve respiration volume OCR values ​​in Figure 7G (*P<0.05, **P<0.01). Data (7A to 7G) represent three independent experiments.

[0076] Figure 8 is a schematic diagram confirming the reduction in mitophagy in the heart induced by Peroxiredoxin 3 (Prdx3) deficiency. Figures 8A and 8B are representative images (Figure 8A) and graphs (Figure 8B) of mitophagy in the heart of mitochondrial-targeted Keima(mt)-Keima mice with mitochondrial targeting (Prdx3) in 10-week-old wild-type (Prdx3 WT, n=9), 52-week-old Prdx3 WT (n=6), 10-week-old Pinkl-deficient (Pinkl KO, n=7), 52-week-old Pink KO (n=6), 10-week-old Prdx3-deficient (Prdx3 KO, n=8), and 52-week-old Prdx3 KO (n=6) mice (scale bar: 10 μm). Figures 8C and 8D are representative images (Figure 8C) and quantitative graphs (Figure 8D) of mitophagy in the infarcted hearts of Prdx3 WT (n=5) and Prdx3 KO mt-Keima mice (n=7) one day after induction of myocardial infarction (MI) (scale bar: 10 μm; ***P<0.001).

[0077] Figure 9 is a schematic diagram confirming the reduction in mitophagy induced in vivo in the absence of peroxidase (Prdx3). Figures 9A to 9C illustrate representative images and quantitative graphs of mitophagy in the liver (Figure 9A), skeletal muscle (soleus muscle) (Figure 9B), and brain tissue (dentate gyrus region) (Figure 9C) of wild-type (n=9), Pink1-deficient (KO) mitochondrial-targeted Keima(mt)-Keima (n=7), and Prdx3-deficient (KO) mt-Keima mice (n=8) (scale bar: 20 μm for liver and skeletal muscle, 50 μm for brain tissue). Figure 9D shows representative images of infarcted hearts 4 hours after induced myocardial infarction (MI) in Prdx3 wild-type (Prdx3 WT, n=6), Prdx3-deficient (Prdx3 KO, n=5), Prdx3 WT (n=6), and Prdx3 KO mt-Keima mice (n=6), along with a quantitative graph of mitophagy (scale bar: 10 μm). Figure 9E shows representative confocal images illustrating the levels of mitophagy in adult fat bodies expressing White RNAi or Prdx3 RNAi (da-GAL4>mt-Keima; White RNAi, da-GAL4>mt-Keima; Prdx3 RNAi) and in the wing primordia of mt-Keima-Kei. The level of mitophagy was quantified using multiple tissue samples (e.g., [WhiteRNAi], n=8; adult fat body [WhiteRNAi], n=8; larvae [Prdx3RNAi], n=9; adult fat body [Prdx3RNAi], n=5) (scale bar: 20 μm or 50 μm; **P<0.01).

[0078] Figure 10 is a schematic diagram illustrating the need for Peroxiredoxin 3 (Prdx3) to confirm PINK1-Parkin-mediated mitophagy. Figures 10A and 10B show the results of Western blot analysis (Figure 10A) and quantification (Figure 10B) of PINK1 expression in mitochondrial components of Prdx3 wild-type (Prdx3 WT) and Prdx3-deficient (Prdx3 KO) mouse embryonic fibroblasts (MEFs) treated with dimethyl sulfoxide (DMSO), 20 μM MG132, or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. Immunoblot detection of these mitochondrial components was performed using PINK1 and Tom20 antibodies. Figure 10C shows the results of immunostaining of PINK1-GFP-infected Prdx3 WT and Prdx3 KO MEFs using Tom20 antibody (red). The graph illustrates the fluorescence intensity of Tom20 and PINK1-GFP measured at the dashed line (scale bar: 1 μm (boxed area) or 10 μm).

[0079] Figures 10D and 10E show the results of Western blot analysis (Figure 10D) and quantitative graphs (Figure 10E) of PINK1 expression in mitochondrial fractions of Prdx3 Wf and Prdx3 KO MEF treated with 10 μM CCCP or 2.5 μM oligomycin and 250 nM antimycin A (OA) for 4 hours. The mitochondrial fractions were detected using Western blot with PJNK1 and Tom20 antibodies. Hash symbols represent non-specific bands. Figures 10F to 10I show the results of immunostaining and quantitative graphs of PINK1-GFP (10F and 10G) and Parkin-GFP levels (10H and 10I) in Prdx3 WT and Prdx3 KO MEF. MEFs were infected with mitochondrial catalase adenovirus for 24 hours and transfected with PINK1-GFP or Parkin-GFP. They were then treated with DMSO or 10 μM CCCP for 4 hours, followed by immunostaining with Tom20 antibody (red) (scale bar: 10 μm; *P<0.05, **P<0.01). Data (10A to 10I) represent three independent experiments.

[0080] Figure 11 is a schematic diagram illustrating the regulation of PINK1 recruitment by Peroxiredoxin 3 (Prdx3) in the outer mitochondrial membrane (OMM) via the matrix and PINK1-Parkin-mediated mitophagy. Figure 11A shows the Western blot analysis and quantification results of PINK1 levels in cell lysates of Prdx3 wild-type (Prdx3 WT) and Prdx3-deficient (Prdx3 KO) mouse embryonic fibroblasts (MEF) treated with DMSO, 20 μM MG132, or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The cell lysates were immunoblot-detected using PINK1 and microtubule antibodies (*P<0.05, **P<0.01). Figure 11B shows the results of immunostaining of HeLa cells transfected with control group or PRDX3 siRNA using Tom20 antibody (red). The fluorescence intensities of Tom20 and PINK1-GFP measured at the dashed lines are illustrated in Figure 11C. In Figure 11C, Prdx3 WT and Prdx3 KO MEF cells were immunostained after transfection with PINK1-GFP or Parkin-GFP using Tom20 antibody (red) via DMSO or 2.5 μM oligomycin / 250 nM antimycin A (OA) (scale bar: 10 μm). Figure 11D shows the immunostaining results of HeLa cells transfected with PINK1-GFP or Parkin-GFP using Tom20 antibody (red) via DMSO or 10 μM CCCP for 4 hours, or with control siRNA for 4 hours (scale bar: 10 μm, boxed area scale bar: 1 μm).

[0081] Figure 12 is a schematic diagram confirming the interaction between peroxidase 3 (Prdx3) and PINK1. Figure 12A is a schematic diagram confirming the interaction between endogenous PINK1 and PRDX3. After immunoprecipitation of whole-cell lysates (WCL) from 293T cells using control immunoglobulin G (IgG) and anti-Prdx3 antibody, Western blot analysis was performed using anti-PINK1 antibody. Figure 12B shows the results of subsequent Western blot analysis of WCL from 293T cells co-transfected with different combinations of constructs expressing PINK1-GFP, PRDX3-Myc, L53V PRDX3-Myc, and PRDX3-Myc dominant-negative mutants (DN; Cys108Ser, Cys229Ser) using anti-Myc magnetic beads and GFP-specific antibodies. Hash symbols represent IgG. Figure 12C shows the results of subsequent Western blot analysis of WCL cells co-transfected with different combinations of constructs expressing PINK1-GFP, A93VPINK1-GFP, and PRDX3-Myc using anti-Myc magnetic beads and GFP-specific antibodies. Hash symbols represent IgG. Figure 12D shows the results of Western blot analysis using GST-... 1-40 PRDX3, GST- 1-40 PRDX3 L53V or GST- 1- 40 Immunoprecipitation of PRDX3 DN-conjugated magnetic beads and subsequent Western blot assays using PINK1 or GFP-specific antibodies were performed using WCLs from 293T cells transfected with a PINK1-GFP construct. Hash symbols represent non-specific bands. Figure 12E shows the use of GST- 1-40 In immunoprecipitation of PRDX3 and subsequent Western blot assays using PINK1- or GFP-specific antibodies, wCLs from 293T cells transfected with constructs expressing PINK1-GFP or A93V PINK1-GFP were used. Hash symbols represent non-specific bands. Figure 12F shows the use of wCLs from 293T cells transfected with Myc-His, 63-256 PRDX3-Myc-His, or 1-256 PRDX3-Myc-His expression vectors in Western blot assays using Prdx3 and tubulin antibodies. Figure 12G illustrates the mass spectrometry analysis and early sequence (residues 1-36 of the Prdx3 mitochondrial targeting sequence) of 1-256 PRDX3-Myc-His (red box in Figure f) and the predicted MPP cleavage site of mature Prdx3. Data (12A to 12E) represent three independent experiments.

[0082] Figure 13 is a schematic diagram illustrating the mapping of the PINK1 domain, which is essential for binding to peroxidase 3 (Prdx3). Figure 13A shows the domain boundaries of full-length PINK1, truncated PINK1, and full-length PRDX3 on the left and right sides, respectively. The PINK1 domain consists of a mitochondrial targeting sequence (MTS), a transmembrane domain (TMD), and a kinase domain. Prdx3 interacts with the N-terminus of PINK1 (positions 1-94) containing the MTS. Figure 13B is a schematic diagram illustrating the immunoprecipitation using anti-GFP magnetic beads in WCL and PRDX3-Myc in 293T cells co-transfected with various combinations of constructs expressing 287-581 PINK1-GFP, 1-286 PINK1-GFP, 1-156 PINK1-GFP, 1-110 PINK1-GFP, and 1-94 PINK1-GFP, and the subsequent immunoblotting results using Myc-specific antibodies. Hash symbols represent IgG. Figure 13C is a schematic diagram illustrating the results of subsequent immunoblotting in WCLs of 293T cells co-transfected with various combinations of constructs expressing 63-256 PRDX3-Myc, 37-256 PRDX3-Myc, PRDX3-Myc, and 1-110 PINK1-GFP, using anti-GFP magnetic beads and Myc-specific antibodies. Hash symbols represent IgG. Data (13B and 13C) represent three independent experiments.

[0083] Figure 14 is a schematic diagram confirming the regulation of PINK1 stability by peroxidase 3 (Prdx3). Figures 14A and 14B show the results of Western blot analysis (Figure 14A) and quantification (Figure 10B) of Pink1 and Prdx3 expression in mitochondrial components of mouse embryonic fibroblasts (MEFs) treated with dimethyl sulfoxide (DMSO) or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. Immunoblot detection of mitochondrial components was performed using PJNK1, Prdx3, and Cox4 antibodies. Figure 14C shows the results of immunostaining using Tom20 (green) and / or PRDX3 antibodies (red) in HeLa cells transfected with pDsRed2-Mito (MTX, mauix) and treated with DMSO or 10 μM CCCP for 4 hours. (Scale bar, 2 (boxed area) or 10 μm). Figure 14D shows the immunostaining results (scale bar, 2 (boxed area) or 10 μM CCCP) using Tom20 (green) and / or PRDX3 antibody (red) in HeLa cells infected with PINK1-GFP and treated with 10 μM CCCP for 4 hours. Figures 14E and 14F show the results of Western blot analysis (Figure 14E) and quantification (Figure 14F) of Pink1 in mitochondrial components of Prdx3 wild-type (Prdx3 WT) and Prdx3-deficient (Prdx3 KO) MEF cells infected with Omal siRNA and treated with 10 μM CCCP for 4 hours. The mitochondrial components were detected by Western blot using PJNK1, Omal, Prdx3, and Cox4 antibodies. Figures 14G and 14H show the results of immunostaining with Tom20 antibody in Prdx3WT and Prdx3 KO MEF simultaneously infected with OmalsiRNA and PINKl-GFP (Figure 14G) and the quantitative results of PINKl-GFP (Figure 14H) (scale bar, 10 μm; *P<0.05 and **P<0.01). Data (14A to 14H) represent three independent experiments.

[0084] Figure 15 This is a schematic diagram confirming the PINK1 stability regulation of Peroxiredoxin 3 (PRDX3). It shows the immunostaining results of the control group and HeLa cells transfected with PRDX3 siRNA or PRDX3 and OMA1 siRNA using Tom20 antibody (red) (scale bar, 10 μm).

[0085] Figure 16 is a schematic diagram illustrating the recruitment of damaged mitochondria to Parkin by PINK1 expression on the outer mitochondrial membrane (OMM) in the absence of peroxidase 3 (Prdx3). Figure 16A shows the expression of N-Mid49+ PINK1. 111-581 The model of the -GFP structure is illustrated. Figure 16B shows the structure after N-Mid49 + PINK1. 111 -581 Representative low- and high-magnification images (scale bar, 2 μm (boxed area) or 10 μm) of the boxed region of Prdx3-deficient (Prdx KO) mouse embryonic fibroblasts (MEF) transfected with GFP and immunostained with Tom20 antibody (red). Figure 16C shows the results after RFP-Parkin and N-Mid49+PINK1 immunostaining. 111-581 Representative images (scale bar, 10 μm) of Prdx3 wild-type (Prdx3 WT) and Prdx3 KO MEF transfected with GFP and treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 h. Figure 16D shows the control group and Prdx3 KO MEF after PRDX3 siRNA transfection with RFP-Parkin and N-Mid49+PINK1. 111-581 Representative images (scale bar, 10 μm) of HeLa cells converted with N-Mid49+ PINK1 and treated with DMSO or 10 μM CCCP for 4 hours. Figure 16E shows HeLa cells converted with N-Mid49+ PINK1. 111-581 Western blot analysis of mitochondrial components from Prdx3 WT and Prdx3 KO MEF transfected with GFP and treated with 10 μM CCCP for 4 hours. Immunoblot detection of mitochondrial components was performed using GFP, Prdx3, and Cox4 antibodies. Figure 16F shows the results after N-Mid49 + PINK1... 111-581 Immunostaining results of GFP-transfected Prdx3 KO MEF using MitoTracker staining (red) or Tom20 antibody (blue). Figure 16G shows the results after N-Mid49 + PINK1 immunostaining. 111-581 Immunostaining results (scale bar, 10 μm) of Prdx3 WT transfected with GFP and treated with 10 μM CCCP for 24 hours and Prdx3 KO MEF transfected with MitoTracker staining (red) or Tom20 antibody (blue).

[0086] Figure 17 is a schematic diagram confirming that Peroxiredoxin 3 (Prdx3) is a core regulator of mitochondrial quality control (MQC) in cardiomyocytes. Figure 17A shows the Tom20 (red) and Parkin (green) immunostaining results (scale bar, 2 μm (boxed area) or 10 μM) of carbonyl cyanide m-chlorophenylhydrazone (CCCP) from Prdx3 wild-type (Prdx3 WT) and Prdx3-deficient (Prdx3 KO) mice treated with DMSO or 10 μM for 4 hours. Figures 17B and 17C are representative images (Figure 17B) and quantitative results (Figure 17C) of mitophagy in cardiomyocytes of Prdx3 WT and Prdx3 KO mitochondrial-targeted Keima (mt-Keima) mice. Cardiomyocytes were infected with Prdx3 adenovirus or Prdx3 dominant-negative (DN) adenovirus for 24 hours and then with 10 μM CCCP for 4 hours (scale bar, 10 μm). Figures 17D and 17E are representative electron micrographs (Figure 17D) and quantitative results (Figure 17E) of damaged mitochondria (red arrows) in cardiomyocytes from Prdx3 WT and Prdx3KO mt-Keima mice. Cardiomyocytes were infected with Prdx3 or Prdx3 DN adenovirus for 24 hours. Arrows represent myofibrils, while hollow arrows represent the Z-line of sarcomeres (scale bar: 1 μm (boxed area) or 2 μm, *P<0.05, **P<0.01, ***P<0.001).

[0087] Figure 18 This is a schematic diagram illustrating the results of the isolation of new cardiomyocytes, showing the immunostaining results of cardiomyocytes extracted using myomesin (cardiomyocyte marker, green) and MitoTracker (red) pits (scale bar, 10 μm).

[0088] Figure 19 This is a schematic diagram illustrating the gene portion removed from Prdx3 conventional knockout (KO) mice.

[0089] Figure 20 This is a schematic diagram illustrating the confirmation of Prdx3 knockout mice via PCR.

[0090] Figure 21 The results are from an analysis of the substantia nigra, a brain tissue containing dopamine neurons, in 5-month-old mice.

[0091] Figure 22This is a schematic diagram confirming the reduction in mitochondrial function in the substantia nigra of the brain of Prdx3 knockout mice.

[0092] Figure 23 This is a schematic diagram illustrating the identification of alpha-synuclein aggregation and lipid droplets, which are observed in Parkinson's disease, in dopamine neurons of the substantia nigra of Prdx3 knockout mice.

[0093] Figure 24 This is a schematic diagram confirming the characteristic features of Parkinson's disease—the significant increase in lipid droplets and neuromelanin—in the dopamine neurons of the substantia nigra of Prdx3 knockout mice.

[0094] Figure 25 This diagram illustrates how inhibiting Prdx3 expression in SH-SY5Y neuron cultured cells reduces dopamine levels, while treatment with adenovirus-Prdx3 restores dopamine levels.

[0095] Figure 26 This diagram illustrates how inhibiting Prdx3 expression in SH-SY5Y neuron cells under reactive oxygen species stress (ROS) conditions induced by sodium arsenite treatment increases alpha-synuclein aggregation, while restoring Prdx3 expression decreases it.

[0096] Figure 27 This diagram illustrates the process of confirming the disappearance of alpha-synuclein aggregation (grass green) in mouse neurons treated with alpha-synuclein pre-formed fibrous fibers (PFFs) directly involved in the pathogenesis of Parkinson's disease by using cell immunostaining. The alpha-synuclein aggregation (grass green) disappeared when Prdx3 expression was increased using adenovirus-Prdx3.

[0097] Figure 28This is a schematic diagram illustrating the reduction of insoluble alpha-synuclein, representing alpha-synuclein aggregation, in mouse neurons that have formed alpha-synuclein aggregation by treatment with alpha-synuclein pre-formed fibroils (PFFs) directly involved in the pathogenesis of Parkinson's disease. The diagram is presented using Western blotting to confirm the reduction of insoluble alpha-synuclein, representing alpha-synuclein aggregation, when Prdx3 expression is increased using adenovirus-Prdx3. Detailed Implementation

[0098] The present invention will now be described in more detail through embodiments. However, these embodiments are merely illustrative and the scope of the invention is not limited to these embodiments.

[0099] Experiment Example 1: Materials and Methods 1.1. Animal Model To generate Prdx3-deficient mitochondrial-targeting Keima (mt-Keima) mice, Prdx3-deficient mice were mated with mt-Keima mice. To generate Pink1-deficient mt-Keima mice, Pink1-deficient mice (provided by Professor Han-woong Lee of Yonsei University) were mated with mt-Keima mice. The mice were backcrossed more than seven times in a C57BL / 6J background (Jackson Laboratory). All animal management and experimental procedures were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee of Ewha Womans University.

[0100] 1.2. Fruit fly (Drosophila strain) The mt-Keima transgenic fruit fly (UAS-mt-Keima) had previously been generated. The blank RNAi (blank GD14981) strain was purchased from the Vienna Fruit Fly Resource Center. The da-GAL4 and Prx3 RNAi (Prx3HMJ22845) strains were purchased from the Bloomington Stock Center (Indiana University, Bloomington, IN, USA).

[0101] 1.3. Mammalian cell cultures Primary mouse embryonic fibroblasts (MEFs) were isolated from wild-type and Prdx3-deficient embryos on day 13.5 of the embryonic period. Primary MEFs, HeLa cells, and 293T cells were maintained in Durbecco modified Eagle medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 100 U / ml penicillin, and 100 μg / ml streptomycin (Invitrogen). Cardiomyocytes were isolated from wild-type and Prdx3-deficient mice on day 3 after birth using the Pierce™ Primary Cardiomyocyte Isolation Kit, following the manufacturer's instructions. The isolated cells were cultured for 7 days prior to analysis to confirm normal cell morphology. To restore Prdx3 expression, Prdx3-deficient cardiomyocytes were infected with either Prdx3 adenovirus or dominant-negative Prdx3 (PrdX-DN) adenovirus (Sirion).

[0102] 1.4. Plasmids and RNAi oligonucleotides The Prdx3-Myc plasmid was generated by PCR amplification of the full-length PRDX3 (NCBI accession number NM_006793.5) and cloning the product into the EcoRI-XhoI site of the pcDNA3.1 / Myc-His(-)A plasmid (Invitrogen). The construct encoding PINK1-GFP was generated by PCR amplification of the full-length PINK1 (NCBI accession number NM_032409.3) and subcloning (Clontech) the product into the EcoRI-BamHI site of the pEGFP-N3 plasmid. The construct encoding Mid49-GFP was generated by PCR amplification of the full-length Mid49 (isoform 1; NCBI accession number NM_139162.3) and cloning (Clontech) the product into the EcoRI-BamHI site of the pEGFP-N3 plasmid. The N-terminal sequence of Mid49 (amino acids 1-126, N-Mid49) or amino acids 111-581 of PINK1 (PINK1) 111-581 The structures encoded by N-Mid49 and PINK1 are generated through PCR amplification of either Mid49 or PINK1. To generate N-Mid49 and PINK1... 111-581 The fusion construct first uses Mid49 1-126 Subcloning (Clontech) into the XhoI-EcoRI site of the pEGFP-N3 plasmid, followed by PINK1 111-581Subcloning was performed into the EcoRI-BamHI site of the N-mid49 pEGPF-N3 plasmid. (For expression of PRDX3) L53V and PRDX3 DN (of) PRDx3 and (used to express PINK1) A93V The point mutation in PINK1 was generated using a fusion site-directed mutagenesis kit according to the manufacturer's protocol (Thermo Fisher Scientific). The construct encoding PRKN-GFP was generated by PCR amplification of the full-length PRKN (isotype 1; NCBI accession number NM_004562.3) and subcloning the product (Clontech) into the EcoRI-BamHI site of the pERFP-C1 plasmid. pDsRed2-Mito (Clontech) was used for mitochondrial matrix staining. GST- Δ1-40 The construct encoding PRDX3 was generated by cloning the product into the EcoRI-XhoI site of the pGEM-4T-1 plasmid (GE Healthcare). The siRNA was synthesized by GenePharma. The target sequence for human siPRDX3 is 5'-AAG CCA AGT CCA GCT GCTTCC-3' (Sequence No. 1), the target sequence for human siOMA1 is 5'-GAA GTG CTT TGT CAT CTA ATT-3' (Sequence No. 2), and the target sequence for mouse siOma1 is 5'-GGA TAC AGT CAA AGT TGC AGG-3' (Sequence No. 3). As a control, Silencer Negative Control siRNA (GenePharma) was used.

[0103] 1.5. Transfection and drug treatment To overexpress mitochondrial catalase, Prdx3-deficient MEFs or Prdx3-depleted HeLa cells were infected with mitochondrial catalase adenovirus and cultured in DMEM supplemented with 10% FBS for 24 hours. Cells (2 × 10⁶ cells / year) were then cultured. 5 Cells (per well) were seeded into 6-well plates and cultured for 18 hours prior to transfection. Plasmid transfection was performed for 4 hours in OPTI-MEM medium (Invitrogen) containing 3 μl of Lipofectamine 2000 or in the NEON™ transfection system (Invitrogen). Next, cells were seeded at approximately 0.5 × 10⁻⁶ cells / well. 5Cells were seeded at a density of 1 / ml into microscope culture dishes with coverslip bottoms (SPL). For RNAi transfection, cells were seeded into 6-well plates at 30% to 40% confluency (or equivalent density). Next, 4 μl of Lipofectamine 2000 (Invitrogen) or 5 μl of RNAiMAX (Invitrogen) and 40 nM of RNAi oligonucleotides were added to each well. After 24 hours, cells were transfected again with 3 μl of Lipofectamine 2000 and an appropriate amount of plasmid DNA. For mitochondrial staining, cells were treated in DMEM for 20 minutes prior to fixation with 100 nM MitoTracker Red CMXRos (Invitrogen), a live-cell mitochondrial marker that exhibits membrane potential-dependent accumulation. For live-cell imaging of mitochondria, cells were treated in DMEM for 20 minutes with 150 nM MitoTracker Green (Invitrogen) and 5 μM MitoSOX (Invitrogen) (as an indicator of mitochondrial superoxide in live cells). For treatment with carbonyl cyanide m-chlorophenylhydrazone (CCCP, Sigma), oligomycin (Sigma), antimycin A (Sigma), or MG132 (Sigma), cells were seeded on poly-L-lysine-coated coverslips (0.1 mg / ml, Sigma) and incubated in serum-containing medium with 10 μM CCCP for 4 to 24 hours, with 2.5 μM oligomycin and (plus) 250 nM antimycin A (OA), or with 20 μM MG132 for 4 hours. DMSO was used as a vehicle control.

[0104] 1.6. Electron Microscope To prepare cellular transmission electron microscopy (TEM) samples, heart, liver, skeletal muscle, and brain tissues were isolated from 10-week-old wild-type and Prdx3-deficient mice. The samples were then fixed in 0.1 M phosphate-buffered saline (PBS, pH 7.4) with 2% paraformaldehyde for 2 hours, followed by three 30-minute washes in 0.1 M PBS (pH 7.4, 1 mM). The tissues were then post-fixed in 1% OsO4 dissolved in 0.1 M PBS (pH 7.4) for 2 hours, dehydrated in a gradient of ethanol (50%, 60%, 70%, 80%, 90%, 95%, and 100%), and then co-incubated with propylene oxide. The specimens were embedded using the Poly / Bed 812 kit (Polysciences, USA). After embedding the samples in pure fresh resin at 60°C for 24 hours in an electron microscope oven (TD-700, DOSAKA, Japan), sections with a thickness of 300 nm were first cut, stained with toluidine blue, and observed using an optical microscope (Olympus BX40, Japan). Next, sections with a thickness of 80 nm were double-stained with 7% uranium acetate and lead citrate as a control staining (20 minutes). The sections were then cut using a Leica Ultracut UCT microtome (Leica Microsystems, Austria). All samples were observed using TEM (JEM-1011, JEOL, Japan) at an accelerating voltage of 80 kV.

[0105] 1.7. Measurement of Mitophagy Levels As described above, mitophagy levels were investigated using the pH-dependent fluorescent probe mt-Keima via confocal microscopy. To analyze the mt-Keima fluorescence signal, mt-Keima mouse and Drosophila tissue samples were examined using a Carl Zeiss LSM800 confocal microscope equipped with Plan-Apochromat 10x / 0.45M27, Plan-Apochromat 20x / 0.8 M27, and C-Apochromat 40x / 1.20W Korr lenses. mt-Keima fluorescence was imaged using two sequential excitation lasers (488 and 555 nm) and an emission bandwidth of 595 to 700 nm. As described above, quantification of mitophagy based on mt-Keima confocal images was performed pixel-by-pixel using ZeissZen software. Mitophagy level (% of mitophagy) was defined as the number of pixels with a high red / green ratio divided by the total number of pixels. To quantify the level of mitochondrial autophagy in the heart, at least five tissue samples were used for quantification, and the mean was calculated. All imaging parameters were kept consistent across all confocal microscopy analyses, with gain levels adjusted only to prevent saturation of arbitrary pixels. Results are expressed as mean ± standard deviation (SD).

[0106] 1.8. Isolation of mitochondria Mitochondria were isolated from lysates of MEF, 293T, and HeLa cells using a mitochondrial isolation kit for cells cultured according to the manufacturer’s protocol (Thermo Fisher Scientific, Inc.).

[0107] 1.9. ATP level measurement and respiration measurement To perform ATP analysis by determining ATP levels in cardiomyocytes, mitochondria were isolated from cardiomyocyte lysates using a mitochondrial isolation kit (Thermo Fisher Scientific, Inc.). Relative ATP levels were calculated by dividing the measured ATP concentration by 20 μg of mitochondria. ATP concentration was determined using the ENLITEN ATP Assay System Bioluminescence Detection Kit for ATP Measurement (Promega, USA), as described above. Oxygen consumption rate (OCR) of cardiomyocytes was determined using the XFp Analyzer (Agilent, USA), as described above.

[0108] 1.10. Immunoprecipitation and Western blotting For immunoprecipitation experiments, both untransfected and transfected cells were lysed in whole-cell extraction buffer (10 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) [pH 7.9], 400 mM NaCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 5% glycerol, 1 mM dithiothreitol (DTT), and a protease inhibitor). After adding untreated cell lysates or anti-PRDX3 antibody (1 μg) to the lysates, anti-Myc-tagged agarose (MBL) or protein A agarose (UpstateBiotech) was added to tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid-glycerol (TEG) reaction buffer (20 mM tris-hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 7.4, 1 mM EDTA, 10% glycerol, 1 mM DTT, and 150 mM NaCl), and the mixture was stirred at 4°C for 3 hours or overnight. Immunoprecipitates were washed with TEG wash buffer (TEG reaction buffer containing 0.1% Triton X-100). For Western blotting, cells were lysed in whole-cell extraction buffer or homogenized using a MICRA D-8 homogenizer (ART Moderne Labortechnik) in protein extraction buffer (20 mM HEPES [pH 7.9], 300 mM NaCl, 10 mM EDTA, 0.1% NP40, 100 mM KCl, and protease inhibitors). Total protein was separated on a sodium dodecyl sulfate-polyacrylamide gel and transferred to a nitrocellulose membrane (Amersham Biosciences). Primary antibodies against the following proteins were used: PRDX3 (1:100, AbFrontier, LF PA0044), catalase (1:100, Labfrontier, BC 100494), PRKN (1:100, Santa Cruz Biotechnology, sc-32282), GFP (1:1100, Santa Cruz Biotechnology, sc-9976), TOM20 (1:100100, Santa Cruz Biotechnology, sc-9976). Biotechnology, sc-11415, tubulin (1:300, Sigma, T6199), OMA1 (1:1000, Proteintech, 17116-1-AP), PRKN (1:1100, Abcam, ab77924), Myc (1:1200, Abcam, ab32), COX4 (1:1008, Abcam, ab33985), myosin (1:1000, DSHB, B4-S), and PINK1 (1:150, Novus, BC 10494).The secondary antibodies were fluorescein-conjugated anti-rabbit IgG, anti-mouse IgG, and anti-goat IgG (Invitrogen), as well as anti-rabbit, anti-mouse, and anti-goat horseradish peroxidase (HRP) conjugate antibodies (Zymed Laboratories). Protein-antibody complexes were detected using the ECL Plus system (Amersham Biosciences).

[0109] 1.11. Thoracic echocardiography and MI surgery Thoracic echocardiography was performed in wild-type and Prdx3-deficient mice (10 weeks and 49–55 weeks old) using a Vevo2100 system (VisualSonics) with a 25–55 MHz linear array probe. After anesthesia with 1.5–2% isoflurane, echocardiography was performed while maintaining the mice's heart rate within the physiological range (>450 bpm) under anesthesia, and the body temperature was maintained at 37°C using a warming pad during the echocardiography. Left ventricular (LV) parameters, such as ejection fraction (EF), fractional shortening (FS), SV, cardiac output (CO), end-diastolic volume, and end-systolic volume, were analyzed using the Simpson monoplane method (provided by the manufacturer) and VevoStrain software in a long-axis view. Cardiac surgery was performed in wild-type and Prdx3-deficient mice (10–12 weeks old) under isoflurane anesthesia. The left anterior descending coronary artery was permanently sutured using 7-0 silk sutures. Echocardiography was performed on day 15 postoperatively using a Vevo2100 system (VisualSonics) with a 25-55 MHz linear array probe. Anesthesia was administered and maintained by inhalation of 1.5-2% isoflurane, and body temperature was monitored during the echocardiography. Left ventricular parameters (LV variables), such as EF, SV, end-diastolic volume, and end-systolic volume, were analyzed using the manufacturer-supplied VevoStrain software via the Simpson monoplane method with a long-axis view of the disc method.

[0110] 1.12. Cardiac fibrosis analysis Left ventricular (LV) fibrosis was assessed using serial sections of the heart stained with Masson's trichrome. Paraffin-embedded LV samples were partitioned into 6 μm thick sections and stained using a Masson's trichrome staining kit (BBC Biosciences) according to the manufacturer's instructions. Tissue sections were scanned using the Vectra Polaris Automated Quantitative Pathology Imaging System (PerkinElmer), and the percentage of fibrosis relative to LV area was determined using Form Advanced Imaging Analysis Software (PerkinElmer).

[0111] 1.13. Live-cell imaging and confocal microscopy Live-cell imaging was performed using a spinning disc confocal system (A1C; Nikon). For live cells, imaging was performed in an LCI chamber (Carmelid TC; LCI) at 37°C and 5% CO2, with images captured using an oil immersion lens with a numerical aperture (NA) of 1.4 x 60. For fixed cells, imaging was performed using a laser scanning confocal microscope (LSM880; Carl Zeiss) and a structured illumination microscope (ELYRA S.1; Carl Zeiss). Images were captured using an oil immersion lens with a NA of 1.4 x 63. Images were analyzed using Carl Zeiss Elements, Photoshop (Adobe), IMARIS (Bitplane AG), or ImageJ (US National Institutes of Health) software. Scale bars were generated using Carl Zeiss Elements and ImageJ.

[0112] 1.14. Damaged mitochondria and mitochondrial ROS analysis To determine depolarized (damaged) mitochondria, quantification was performed using ImageJ software on only contiguous z-plane images of mitochondria-associated MitoTracker Red CMXRos (Invitrogen). For mitochondrial ROS analysis, quantification was performed using ImageJ software on only contiguous z-plane images of mitochondrial-associated MitoSOX (Invitrogen). Statistical analysis was performed using SigmaPlot (SysStat Software, Inc.). Data are expressed as mean ± standard error (SEM) from at least three experiments.

[0113] 1.15. Immunofluorescence and histology Cultured cells were fixed in 4% formaldehyde in PBS for 20 minutes at room temperature, washed with PBS, and then permeabilized with 0.1% Triton X-100 for 15 minutes. Cells were washed with PBS and blocked with 3% bovine serum albumin (BSA) in PBS for 1 hour, followed by incubation with primary antibody in PBS at RT for approximately 1 to 3 hours. After washing with PBS, cells were incubated with secondary antibody at RT for 1 hour. Cells were then stained with 10 μM 4,6-diamidinyl-2-phenylindole (DAPI) and mounted using Vectashield (VECTOR). Paraffin-embedded sections of the heart (6 μm thick) were fixed in 10% buffered formaldehyde and used for TUNEL assays and immunofluorescence analysis.

[0114] 1.16. Mass Spectrometry Analysis The PRDX3 band in the gel was desalted and the resulting peptides were extracted after trypsin digestion. The peptides were then analyzed using nanoAcquity™ UPLC / ESI / q-TOF MS / MS (SYNAPT). TM G2Si HDMS TM The peptides were analyzed by Waters Co., UK.

[0115] 1.17. TUNEL Analysis 24 hours after MI, apoptosis in the heart of myocardial infarction was assessed using the TACS 2 TdT fluorescein in situ apoptosis detection kit (Trevigen) according to the manufacturer's protocol.

[0116] 1.18. Statistical Analysis Differences between the two experimental groups were analyzed using the Student t-test or the Mann-Whitney U test. One-way ANOVA with one-way correction was used to compare three or more groups. A p-value < 0.05 was considered statistically significant.

[0117] Experiment Example 2: Results 2.1. Cardiac hypertrophy and dysfunction induced by Prdx3 deficiency Mitochondria contribute significantly to the homeostasis of the cardiovascular system, and subtle adjustments made to them by MQCs are crucial for the survival of cardiovascular cells and the maintenance of physiological cardiac function. Disruption of the MQC is closely associated with cardiac defects.

[0118] To determine the role of Prdx3 in MQC, cardiac phenotypes were investigated in wild-type and Prdx3-deficient mice at 10 and 52 weeks of age. Prdx3-deficient mice exhibited cardiac hypertrophy, including a significantly increased heart-to-body weight ratio, decreased cardiac output (CO) at 52 weeks of age, and decreased cardiac septum (SV) at both 10 and 52 weeks of age without changes in cardiac ejection fraction (EF) (Figures 1A-1D and 2A). These results are consistent with previous reports that mitochondrial dysfunction is associated with heart failure due to diastolic dysfunction, leading to decreased SV and CO. Therefore, Prdx3 deficiency-induced LV remodeling and cardiac hypertrophy indicate that mitochondrial dysfunction is a significant risk factor for heart failure such as human HFpEF.

[0119] Furthermore, although Prdx3-deficient mice had similar heart size to 10-week-old wild-type mice, they exhibited cardiac hypertrophy, and at 52 weeks of age, their cardiomyocyte size and LV fibrosis were significantly increased (Figs. 1E to 1I and 2B). In addition, damaged mitochondria were observed in Prdx3-deficient cardiomyocytes of 10-week-old mice, and this increase was significant at 52 weeks of age. The mice exhibited morphologically abnormal, extremely large, and damaged mitochondria (Figs. 1J and 1K). These results indicate that Prdx3 deficiency-induced MQC dysfunction leads to mitochondrial dysfunction and the accumulation of large mitochondria, resulting in cardiac dysfunction.

[0120] 2.2. Prdx3 The absence of this feature leads to worsening of cardiac dysfunction associated with myocardial infarction (MI). Myocardial infarction is characterized by mitochondrial damage induced by oxidative stress, which promotes excessive loss of cardiomyocytes and LV remodeling.

[0121] To assess whether Prdx3 deficiency-induced mitochondrial dysfunction and reduced mitophagy contribute to worsening heart failure after myocardial infarction (MI), cardiac function was evaluated using echocardiography 15 days after MI induction (Fig. 4A). The ejection fraction (EF) and venous velocity (SV) of Prdx3-deficient mice were significantly lower than those of wild-type mice. Although the end-systolic volume of the left ventricle (LV) was significantly increased in Prdx3-deficient mice, there was no significant difference in end-diastolic volume between wild-type and Prdx3-deficient mice (Figs. 3A–3E). These results suggest that Prdx3 deficiency contributes to worsening cardiac dysfunction after MI. In particular, Masson's trichrome staining indicated that Prdx3 deficiency leads to cardiac fibrosis and worsened LV remodeling (Fig. 3F). Furthermore, it was confirmed that the number of damaged mitochondria with multiple vesicles was higher in the infarcted hearts of Prdx3-deficient mice than in wild-type mice (Fig. 3G). Disruption of MQC-induced clearance of damaged mitochondria will induce malignant cell death characterized by activation of the apoptotic cascade.

[0122] Furthermore, in situ apoptosis analysis was performed using infarcted hearts from wild-type and Prdx3-deficient mice after MI. The results confirmed a significant increase in apoptotic cell death in the infarcted hearts of Prdx3-deficient mice (Figure 3H). Although 10-week-old Prdx3-deficient mice did not exhibit significant cardiac dysfunction under physiological conditions (Figure 4B), the increased number of damaged mitochondria due to impaired MQCs could explain the incidence of heart failure after MI. Therefore, this indicates that Prdx3 deficiency leads to increased mitochondrial damage in the infarcted heart under MI conditions, ultimately resulting in apoptosis.

[0123] 2.3. Prdx3 Caused by missing In vivo Mitochondrial autophagy inhibition To further determine whether mitochondrial damage caused by Prdx3 deficiency is due to mitochondrial ROS accumulation, mitochondrial phenotypes were confirmed in Prdx3-deficient mice. Analysis of mitochondrial membrane potential in MEFs isolated from Prdx3-deficient mice revealed an increase in the number of damaged or depolarized mitochondria (Fig. 5A and Fig. 5C). Next, analysis of mitochondrial ROS in Prdx3-deficient MEFs confirmed that Prdx3 deficiency induces increased mitochondrial ROS levels (Fig. 5B and Fig. 5D). After confirming the main molecular function of Prdx3 in preventing ROS accumulation, the study investigated whether overexpression of mitochondrial catalase, a ROS scavenger, could alleviate mitochondrial damage associated with Prdx3 deficiency. The results showed that overexpression of catalase reduced Prdx3 deficiency-induced mitochondrial damage (Fig. 5A to Fig. 5E). These results indicate that Prdx3 deficiency-induced mitochondrial damage is closely related to abnormal ROS accumulation. Furthermore, Prdx3 deficiency in vivo leads to mitochondrial damage in the heart and other tissues of Prdx3-deficient mice. Figure 6 A and Figure 6 B). In Prdx3-deficient cardiomyocytes, ATP levels and oxygen consumption rate (OCR) were significantly reduced, indicating that Prdx3 deficiency induces cardiac mitochondrial dysfunction (Figs. 7A to 7G). In the heart, liver, skeletal muscle, and brain of 52-week-old Prdx3-deficient mice, the number of damaged mitochondria was increased (Figs. 1J and 1K). Figure 6 A and Figure 6 (B) This indicates that Prdx3-deficient mice are a suitable animal model for studying MQC.

[0124] Next, to confirm whether there is a potential synergistic effect between the two MQC processes—preventing mitochondrial damage by utilizing mitochondrial-specific Prdx3 to regulate ROS and clearing damaged mitochondria via PINK1-mediated mitophagy—mitochondrial phenotypes were compared in the hearts of Prdx3-deficient and Pink1-deficient mice. Although mitochondrial damage was increased in the hearts and other tissues of Prdx3-deficient mice (Fig. 1J and Fig. 1K), Figure 6 A and Figure 6 B), but mitophagy was significantly lower in the heart and other tissues of Prdx3-deficient mice compared with wild-type and Pink1-deficient mice (Fig. 8A and Fig. 8B, Fig. 9A to Fig. 9C).

[0125] Next, we confirmed the pathophysiological outcomes following myocardial infarction (MI), particularly whether Prdx3 affected the mitophagy pattern. Using Prdx3-deficient mt-Keima mice with MI, we characterized the function of Prdx3 in cardiac mitophagy. We confirmed that 4 hours after MI, mitophagy was significantly increased in the hearts of wild-type mt-Keima mice, but significantly reduced in the hearts of Prdx3-deficient mt-Keima mice, independent of MI (Fig. 9D). Furthermore, we confirmed that Prdx3 deficiency significantly attenuated mitophagy in cardiac tissue within 24 hours after MI (Figs. 8C and 8D). Similarly, depletion of Prdx3, the Drosophila ortholog, led to reduced mitophagy in the wing primordia of the fat bodies of Drosophila larvae and adults (Fig. 9E).

[0126] The results described above suggest that, in addition to its fundamental function of preventing mitochondrial damage by regulating ROS levels, Prdx3 may also play a potential role in the regulation of mitophagy.

[0127] 2.4. Prdx3 regulates mitophagy through Prdx3-dependent localization and PINK1 degradation in mitochondria. To confirm the role of Prdx3 in mitophagy, the question of whether PINK1 protein levels change due to Prdx3 deficiency was investigated. Therefore, PINK1 protein levels were compared between wild-type and Prdx3-deficient MEFs. PINK1 protein levels were higher in Prdx3-deficient MEFs and in Prdx3-deficient MEFs treated with the proteasome inhibitor MG132 than in wild-type MEFs (Fig. 10A, 10B, and 11A). Since even upregulation of PINK1 in Prdx3 deficiency leads to a reduction in mitophagy, it was necessary to confirm whether the submitochondrial localization of PINK1 is altered by Prdx3 deficiency. GFP-labeled PINK1 was overexpressed in the control group and Prdx3-deficient MEFs, and its localization in mitochondria labeled with the OMM marker Tom20 was compared using Airyscan super-resolution microscopy. In Prdx3-deficient MEF or Prdx3-depleted HeLa cells, PINK1-GFP co-localizes with Tom20 in the OMM, but in wild-type MEF and HeLA cells, PINK1-GFP preferentially localizes in the matrix (Fig. 10C and Fig. 11B). These results suggest that Prdx3 influences the submitochondrial localization of PINK1 and thereby regulates its degradation.

[0128] Next, we evaluated whether Prdx3 affects PINK1 expression under mitophagy-mediated conditions. Wild-type and Prdx3-deficient MEFs were treated with mitochondrial uncoupling agents (CCCP) or mitochondrial respiration inhibitors (oligomycin and antimycin A, OA), and PINK1 protein levels were confirmed. Historical studies have shown that CCCP or OA treatment significantly increases PINK1 accumulation in mitochondria, thereby inducing mitophagy. However, in this invention, the accumulation of PINK1 in mitochondria induced by CCCP or OA treatment was almost completely blocked due to the absence of Prdx3 (Figures 10A, 10B, 10D, 10E).

[0129] Similarly, when PINK1-GFP or Parkin-GFP was overexpressed in MEFs, although ROS-induced mitochondrial damage was mitigated by Prdx3-deficient mitochondrial catalase, CCCP-dependent mitochondrial accumulation of PINK1-GFP and Parkin-GFP in Prdx3-deficient MEFs was still significantly inhibited (Figs. 10F to 10I). Furthermore, when PINK1-GFP or Parkin-GFP was overexpressed in MEFs and HeLa cells, CCCP or OA-dependent mitochondrial accumulation of PINK1-GFP and Parkin-GFP was inhibited in Prdx3-deficient MEFs or PRDX3-deficient HeLa cells (Figs. 11C and 11D). These results indicate that Prdx3 can regulate the submitochondrial localization of PINK1 and mitochondrial degradation, thereby acting as a PINK1-Parkin-mediated regulator of mitophagy.

[0130] 2.5. Mitochondrial targeting sequence interaction between Prdx3 and PINK1 The co-immunoprecipitation assay was used to test whether there was an interaction between PRDX3 and PINK1, thereby confirming that endogenous PRDX3 binds to endogenous PINK1 (Figure 12A).

[0131] To identify the key binding site between PRDX3 and PINK1, a truncated point mutant structure of PRDX3 and PINK1 was constructed, thus confirming that the N-terminal domain of PINK1 is essential for binding with PRDX3 (Figures 13A and 13B). Further investigation was conducted to determine the binding site with PINK1. 1-110 The N-terminal structure of PRDX3 is an important part of the combination, using PRDX3 63-256 PRDX3 37-256 PRDX3 1-256 And PINK1 1-110Perform immunoprecipitation (IP) assay. PINK1 was detected. 1-110 With PRDX3 37-256 and PRDX3 1-256 Combined but not with PRDX3 63-256 This indicates that the N-short sequence (amino acids 37-62) of PRDX3 is essential for binding to PINK1 (Fig. 13C). Using the PRDX3Leu53Val and PINK1Ala93Val mutants, it was confirmed that Leu53 in PRDX3 and Ala93 in PINK1 are crucial for the binding between PRDX3 and PINK1 (Fig. 12B and Fig. 12C). The dominant-negative PRDx3 Cys108Ser / Cys229Ser, which blocks PRDX3-mediated ROS scavenging, can still bind to PINK1, indicating that ROS dynamic activity in dominant-negative PRDX3 can be functionally preserved (Fig. 12B). GST fusion protein sedimentation experiments confirmed that Leu53 in PRDX3 and Ala93 in PINK1 are essential for the direct binding between the two (Fig. 12D and Fig. 12E).

[0132] Previous reports have indicated that the N-terminal sequence of PRDX3 (amino acids 1 to 62) acts as a mitochondrial targeting signal. Mass spectrometry analysis used to further clarify the mitochondrial targeting signal sequence of PRDX3 predicted that the N-terminal sequence of PRDX3 (amino acids 1 to 36) is essential for mitochondrial targeting, and mature PRDX3 was found to contain Leu53, which ensures its binding to PINK1 (Figures 12F and 12G). Crystallographic studies of mitochondrial Prdx (Leishmania infantumortholog of Prdx3) showed that the N-terminal sequence outside the mitochondrial targeting sequence is crucial for the formation of structurally stable mitochondrial Prdx, thus confirming that the N-terminal sequence of PRDX3 (amino acids 37 to 62) can bind to the mitochondrial targeting signal sequence of PINK1.

[0133] 2.6. Regulation of PINK1 stability by Prdx3 through Oma1 inhibition in mitophagy The study aimed to confirm whether Prdx3 protein expression changes when PINK1 levels increase in damaged mitochondria. It was confirmed that Prdx3 protein levels also increase in damaged mitochondria, mimicking the upregulation of PINK1 (Fig. 14A and Fig. 14B). Furthermore, the study investigated whether the mitochondrial localization of Prdx3, upon binding to PINK1 under CCCP-induced mitochondrial damage conditions, translocates from the matrix to the OMM. It was confirmed that in co-immunostained Tom20 and Prdx3, Prdx3 is confined to the mitochondrial matrix in healthy mitochondria, while co-localization with Tom20 or PINK1 confirms its translocation to the OMM in damaged mitochondria (Fig. 14C and Fig. 14D).

[0134] Various cellular stresses can induce mitochondrial dysfunction, which in turn promotes the activation of the stress-induced protease OMA1, residing in IMMs. When activated in damaged mitochondria, OMA1 cleaves Dele1 and triggers a stress-induced protein degradation cascade. Recent studies have confirmed that OMA1 can degrade Parkinson's disease-associated PINK1 mutants that have entered damaged mitochondria. Prdx, including Prdx3, can function as a peroxidase under normal conditions and as a molecular chaperone exposed to various stresses. Human PRDX3 can act as a catalytically active self-assembling molecular chaperone under stress conditions.

[0135] To determine whether PINK1 is degraded by OMA1 regardless of the presence or absence of Prdx3, the stability of PINK1 in wild-type and Prdx3-deficient MEF damaged mitochondria was investigated after OmA1 depletion and CCCP treatment. In damaged mitochondria undergoing mitophagy, PINK1 was not degraded by OMA1 in the presence of Prdx3 (Figs. 14E and 14F). Furthermore, PINK1 was degraded by Oma1 in the absence of Prdx3, but its level was restored upon OmA1 depletion using siRNA (siOma1). PINK1-GFP was completely lost in Prdx3-deficient MEF and PRDX3-depleted HeLa cells after CCCP treatment. However, PINK1-GFP levels were significantly restored in CCCP-treated cells upon Oma1 depletion using siOma1 (Figs. 14G and 14H). Figure 15 Therefore, when Prdx3 binds to the N-terminus of PINK1, it protects the latter from degradation by preventing Oma1 from cleaving it. This suggests that Prdx3 can act as a chaperone protein for PINK1.

[0136] Furthermore, to confirm whether Prdx3 can regulate PINK1-Parkin-mediated mitophagy by modulating PINK1 stability in damaged mitochondria, a PINK1 recruitment / response experiment was performed. Mid49 is an OMM protein that regulates mitochondrial recruitment of Drp1, which controls cell division in mitochondrial dynamics, and possesses an N-terminal mitochondrial targeting sequence and an OMM domain. Using N-Mid49 containing the mitochondrial targeting sequence and OMM domain, a PINK1 morphology was generated with both the PRDX3 binding region and the OMA1 cleavage site simultaneously deleted, and the mitochondrial targeting domain, which is persistently confined to the OMM, being cleaved. 111-581 (Figure 16A). When N-Mid49-PINK1 111-581 In the case of Prdx3-deficient MEF overexpression, the construct localized normally to mitochondria regardless of CCCP-induced mitochondrial damage (Fig. 16B). When N-Mid49-PINK1 111-581 In HeLa cells overexpressed with Prdx3-deficient MEF or PRDX3 depletion, the construct recruited Parkin only in CCCP-damaged mitochondria (Fig. 16C and Fig. 16D). Regardless of the presence of Prdx3 and CCCP-induced mitochondrial damage, N-Mid49-PINK1... 111-581 It is stably expressed in mitochondria (Fig. 16E). In particular, N-Mid49-PINK1 is overexpressed in Prdx3-deficient MEFs. 111-581 In this case, compared with the findings in Prdx3-deficient MEF, mitochondrial depolarization was reduced through the occurrence of normal mitophagy (Fig. 16F). To induce mitochondrial depolarization in most cells, Prdx3-deficient MEF and N-Mid49-PINK were used. 111-581 Prdx3-deficient MEFs were treated with CCCP for 24 hours. However, mitochondrial depolarization occurred in N-Mid49-PINK1 after CCCP treatment. 111-581 The expression of Prdx3 was reduced in depleted MEFs, which suggests that it is transmitted through N-Mid49-PINK1. 111-581 Mediated mitophagy can clear damaged mitochondria (Fig. 16G). Therefore, Prdx3 can act as a molecular chaperone and bind to the protein to maintain the stability of PINK1 in damaged mitochondria and thereby protect it from cleavage by activated Oma1.

[0137] 2.7. Regulation of MQC processes in Prdx3 cardiomyocytes To confirm whether the inhibition of mitophagy in Prdx3-deficient hearts is functionally associated with PINK1-Parkin-mediated mitophagy, the localization of Parkin on damaged mitochondria was investigated in both wild-type and Prdx3-deficient cardiomyocytes after CCCP treatment. Although endogenous Parkin was confined to damaged mitochondria in wild-type cardiomyocytes, it was not limited in Prdx3-deficient cardiomyocytes (Figure 17A and 17B). Figure 18 This indicates that Prdx3 regulates PINK1-Parkin-mediated mitophagy in cardiomyocytes.

[0138] Next, we investigated whether Prdx3 or Prdx3-DN adenovirus could restore mitophagy under CCCP-induced mitophagy. Prdx3-deficient cardiomyocytes were treated with CCCP and then infected with adenoviruses containing Prdx3 or Prdx3-DN. Although Prdx3-DN is defective in ROS clearance, it still functions for PINK1-mediated mitophagy. Prdx3 or Prdx3-DN expression in Prdx3-deficient cardiomyocytes restored mitophagy to levels similar to wild-type cardiomyocytes (Figs. 17B and 17C). Furthermore, transfection with adenovirus Prdx3 or Prdx3-DN reduced the number of damaged mitochondria in Prdx3-deficient cardiomyocytes, suggesting that increased mitochondrial damage due to Prdx deficiency can be reduced by functional mitophagy activated by Prdx3-DN, which is defective in ROS clearance (Figs. 17D and 17E).

[0139] In summary, Prdx3 plays a key regulatory role by preventing mitochondrial damage and clearing interactions between damaged mitochondria, making it essential for heart function.

[0140] 2.8. Confirm the efficacy of Prdx3 in Parkinson's disease. 2.8.1. Creating Prdx3 gene knockout mice The Prdx3 gene is located on mouse chromosome 19 and is a 257-amino acid protein produced through the translation of exons 1 through 7. A mitochondrial targeting signal has been confirmed in exon 1 of the Prdx3 gene, while cysteine ​​residues for scavenging reactive oxygen species are present at position 109 of exon 4 and position 230 of exon 6. The Prdx3 gene is present in the mitochondrial matrix and has the function of scavenging reactive oxygen species from mitochondria.

[0141] Prdx3 conventional knockout mice are created by electroporation of a construct in which a portion of the intron of the 5'-UTR of exon 1 of the Prdx3 gene is inserted into the 5'-UTR of the exogenous gene neomycin, and the intron of the 3'-UTR of exon 4 of the Prdx3 gene is recombined in the 3'-UTR. This construct is then injected into mouse embryonic stem cells (ESSCs). This induces the deletion of exons 1 through 4 through homologous recombination of the introns of exon 1 and exon 4 of the Prdx3 gene. After screening for Prdx3 gene exon 1 through 4 deleted ESCs in a neomycin-containing medium, these ESCs are microinjected into mouse blastocysts to create Prdx3 knockout mice (see [link to documentation]). Figure 19 Prdx3 wild-type (WT) mice, such as Figure 20 As shown, ① the Prdx3 forward primer (cag gaa atg tca ata agt gtc tac, sequence number 4) was designed in the intron region to the left of exon 1, and ② the Prdx3 reverse primer (ccarag gcc act tgt gta gc, sequence number 5) was designed in exon 1. The product of 390 base pairs was confirmed by polymerase chain reaction (PCR). The identification of Prdx3 knockout mice is as follows. Figure 20As shown, ① the Prdx3-forward primer (cag gaa atg tca ata agtgtc tac, sequence number 4) was designed in the intron region to the left of exon 1, and ③ the Prdx3-neo-reverse primer (cga gga cca gag caa cct tc, sequence number 6) was designed in the exogenous gene neomycin introduced to delete exons 1 to 4 (sequence numbers 7 to 10) of the Prdx3 gene, and the product was deleting 300 base pairs by PCR.

[0142] Prdx3 knockout mice were identified using the methods described above, and compared with wild-type (Wt) Prdx3 mice in MEF (mouse embryonic fibroblast) cells derived from Prdx3 knockout mice. Western blot results confirmed the absence of Prdx3 expression in the knockout mice (see [link to article]). Figure 20 ).

[0143] 2.8.2. Confirm the pathogenesis of Parkinson's disease in Prdx3 gene knockout mice. Analysis of the substantia nigra of 5-month-old mice with dopamine neurons confirmed that, compared with Prdx3 wild-type mice, Prdx3 knockout mice had a significant reduction in dopamine neurons in the substantia nigra (see [reference]). Figure 21 It was confirmed that Prdx3 knockout mice exhibited decreased motor function and reduced mitochondrial function in the substantia nigra of the brain (see [reference]). Figure 22 Furthermore, alpha-synuclein aggregation and lipid droplets, which are present in Parkinson's disease, were observed in the dopamine neurons of the substantia nigra of Prdx3 knockout mice (see [reference]). Figure 23 Electron microscopy analysis confirmed a significant increase in Parkinson's disease-characteristic features, namely alpha-synuclein aggregation and lipid droplets, in the dopamine neurons of the substantia nigra of Prdx3 knockout mice (see [reference]). Figure 24 ).

[0144] The results indicate that the Prdx3 knockout mouse according to the present invention is a suitable mouse model for studying the pathogenesis and treatment of Parkinson's disease.

[0145] 2.8.3. Confirm the therapeutic effect of Prdx3 in Parkinson's disease It was confirmed that suppressing Prdx3 expression in SH-SY5Y neuron cultures, which are widely used in Parkinson's disease research, reduces dopamine levels, while treatment with adenovirus-Prdx3 restores dopamine levels (see [link to study]). Figure 25 ).

[0146] When sodium arsenite is used to treat cultured cells of a Parkinson's disease pathogenesis model—specifically, alpha-synuclein stable SH-SY5Y neurons—alpha-synuclein aggregation, which is directly involved in the pathogenesis of Parkinson's disease, is generated. Inhibition of Prdx3 expression increases alpha-synuclein aggregation, while treatment with adenovirus-Prdx3 to restore Prdx3 expression reduces alpha-synuclein aggregation (see [reference]). Figure 26 ).

[0147] Furthermore, when neurons extracted from the mouse brain were treated with alpha-synuclein pre-formed fibrous fibers (PFFs), which are directly involved in the pathogenesis of Parkinson's disease, alpha-synuclein aggregation was generated, thereby inducing neuronal degeneration and establishing an in vitro Parkinson's disease model. Cellular immunostaining confirmed that when Prdx3 expression was increased by treating neurons that had already formed the corresponding alpha-synuclein aggregation (grass green) with adenovirus-Prdx3, the alpha-synuclein aggregation (grass green) disappeared (see [reference]). Figure 27 ).

[0148] Furthermore, Western blot experiments confirmed that increasing Prdx3 expression through treatment with adenovirus-Prdx3 reduced the amount of alpha-synuclein expressing alpha-synuclein aggregation. This confirms that decreased Prdx3 expression may be involved in the formation of alpha-synuclein aggregation, a crucial pathogenesis of Parkinson's disease, and that Prdx3 itself may be effectively involved in the treatment of Parkinson's disease (see [reference needed]). Figure 28 ).

[0149] The results described above demonstrate that Prdx3 can be effectively used as a treatment for Parkinson's disease.

Claims

1. A pharmaceutical composition for the prevention or treatment of mitochondrial-related diseases. The active ingredient includes Prdx3 peptide or its analogues.

2. The pharmaceutical composition according to claim 1, The Prdx3 analogue is a dominant-negative (DN) mutant of Prdx3.

3. The pharmaceutical composition according to claim 1, characterized in that: The Prdx3 peptide clears mitophagy.

4. The pharmaceutical composition according to claim 3, characterized in that: The Prdx3 peptide regulates mitophagy through the degradation of the PINK1 peptide.

5. The pharmaceutical composition according to claim 1, The Prdx3 peptide clears alpha-synuclein aggregates.

6. The pharmaceutical composition according to claim 1, The mitochondrial-related diseases are selected from one or more of the following groups: cardiovascular disease, lymphoma, glomerulonephritis, osteoporosis, motor neuron disease, muscular atrophy, Down syndrome, carcinoma, Pick disease, Parkinson's syndrome, and Alzheimer's disease.

7. A health functional food composition for the prevention or improvement of mitochondrial-related diseases. The active ingredient includes Prdx3 peptide or its analogues.

8. A pharmaceutical preparation for the prevention or treatment of mitochondrial-related diseases. The active ingredient includes Prdx3 peptide or its analogues.

9. A method for the prevention or treatment of mitochondrial-related diseases, comprising: The steps of administering a pharmaceutical composition containing Prdx3 peptide or an analogue to an individual.

10. A Prdx3 gene knockout mouse model, The 5'-UTR exon 1 through exon 4 of the Prdx3 gene were knocked out.

11. The mouse model according to claim 10, characterized in that: The mouse model is a Parkinson's disease mouse model.

12. A method for manufacturing a Prdx3 gene knockout mouse model, comprising: The steps to knock out exon 1 through exon 4 of the 5'-UTR of the Prdx3 gene.

13. The method for manufacturing the Prdx3 gene knockout mouse model according to claim 12, The knockout of exon 1 through exon 4 of the 5'-UTR of the Prdx3 gene was performed via homologous recombination.

14. The method for manufacturing the Prdx3 gene knockout mouse model according to claim 12, The knockout step is performed by replacing the base sequence of exon 1 to exon 4 of the 5'-UTR containing the Prdx3 gene with a construct containing the neomycin gene.

15. The method for manufacturing the Prdx3 gene knockout mouse model according to claim 14, The replacement of the construct was performed by electroporation.

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