Compositions for the prevention or treatment of mitochondrial-related diseases containing PRDX3 as an active ingredient and their uses

Abstract

This invention provides a pharmaceutical composition for the prevention or treatment of mitochondrial-related diseases, comprising PRDX3 peptide or its analogue as an active ingredient, and its uses. [Solution] A pharmaceutical composition containing PRDX3 peptide or its analogue as an active ingredient can be used for the prevention and treatment of mitochondrial-related diseases by removing mitophagy, removing α-synuclein aggregates, and the like.

Description

Technical Field

[0001] The present invention relates to a composition for preventing or treating mitochondrial-related diseases containing PRDX3 as an active ingredient and its use.

Background Art

[0002] The heart is an organ with a high number and density of mitochondria. Mitochondria in the adult heart account for approximately 30% of the total cardiomyocyte volume and mainly supply energy to compensate for the high consumption of adenosine triphosphate (ATP) due to the beating of the heart.

[0003] Despite the damage to mitochondria induced by reactive oxygen species (ROS), mitophagy is repeated to maintain mitochondrial quality control (MQC). Therefore, mitochondria are the main sites for energy and ROS production in the heart, and MQC dysfunction may cause diastolic dysfunction, which is associated with heart failure.

[0004] MQC is an essential process in cell physiology and homeostasis and is strictly regulated to maintain a healthy mitochondrial network by preventing mitochondrial damage and removing damaged mitochondria.

[0005] Peroxiredoxin 3 (PRDX3), a mitochondrial-specific peroxidase, is involved in preventing mitochondrial damage through protecting against mitochondrial dysfunction by removing reactive oxygen species within mitochondria. However, it is actually necessary to conduct further research on whether PRDX3 affects the removal of damaged mitochondria by mitophagy.

Summary of the Invention

Problems to be Solved by the Invention

[0006] According to one aspect of the present invention, a pharmaceutical composition for the prevention or treatment of mitochondrial-related diseases is provided, comprising PRDX3 peptide or an analogue as an active ingredient.

[0007] According to another aspect of the present invention, a health functional food composition for preventing or improving mitochondrial-related diseases is provided, comprising PRDX3 peptide or an analogue thereof as an active ingredient.

[0008] According to yet another aspect of the present invention, a pharmaceutical formulation for the prevention or treatment of mitochondrial-related diseases is provided, comprising PRDX3 peptide or an analogue thereof as an active ingredient.

[0009] According to another aspect of the present invention, a method for preventing or treating mitochondrial-related diseases is provided, comprising the step of administering a pharmaceutical composition containing PRDX3 peptide or an analog thereof to an individual. [Means for solving the problem]

[0010] The inventors of this invention have confirmed that PRDX3 peptide or its analogues can be used as active ingredients for the prevention or treatment of mitochondrial-related diseases, and have completed the present invention.

[0011] The present invention provides a pharmaceutical composition for the prevention or treatment of mitochondrial-related diseases, comprising PRDX3 peptide or an analogue as an active ingredient.

[0012] In this specification, the term "PRDX3 (Peroxiredoxin 3)" refers to a member of the peroxiredoxin family that plays a crucial role in detoxifying peroxides and protecting cells from oxidative stress. PRDX3 is an oxidase that can be found primarily in mitochondria, specifically in tissues such as the heart, adrenal glands, liver, and brain.

[0013] In this invention, it has been found that PRDX3 can maintain mitochondrial activity or remove damaged mitochondria without relying on the detoxification effect of peroxides. In one embodiment, PRDX3 can act to remove mitophagy, thereby producing preventive or therapeutic effects on mitochondrial-related diseases.

[0014] In one embodiment of the present invention, PRDX3 can regulate mitochondrial quality through dual antioxidant and molecular chaperone functions.

[0015] In this specification, "mitochondrial quality control" may include the action of protecting mitochondria from reactive oxygen species and the action of mitophagy, which removes damaged mitochondria.

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

[0017] In this specification, the term "peptide analogue" may include analogues in which the side chain of an amino acid or the backbone of an α-amino acid is substituted with one or more other functional groups. Examples of side-chain or back-chain modified peptide analogues include, but are not limited to, hydroxyproline, in which the pyrrolidine ring is substituted with a hydroxyl group, and N-methylglycine-based "peptoids." The types of peptide analogues are known in the art.

[0018] In one embodiment of the present invention, the PRDX3 analog is characterized by being a dominant-negative (DN) mutant of PRDX3. The dominant-negative mutant of PRDX3 may be in which the 108th and 229th cysteine ​​molecules of PRDX3 are replaced with serine molecules, and such a mutation exhibits the property of being able to regulate mitophagy even without the ROS removal function of PRDX3.

[0019] In one embodiment of the present invention, the PRDX3 peptide may consist of a chain of 194 amino acids from positions 64 to 257 of the PRDX3 gene and a transit peptide consisting of 194 amino acids from positions 1 to 63 of the PRDX3 gene.

[0020] In one embodiment, the PRDX3 peptide can remove mitophagy. In one embodiment, the PRDX3 peptide can regulate the mitochondrial localization of PINK1, thereby preventing mitochondrial damage.

[0021] In this specification, the term "autophagy" refers to the activity by which cells obtain energy when they are in a state of nutritional deficiency by breaking down their own proteins or by removing unnecessary cellular components. For example, this may also refer to mitophagy.

[0022] In this specification, the term "mitophagy" refers to the selective degradation of mitochondria by autophagy, a mitochondrial-specific reaction that occurs in response to any damage or stress. This reaction is characterized by promoting mitochondrial turnover, preventing the accumulation of dysfunctional mitochondria that cause cellular degradation, and maintaining mitochondria in a healthy state.

[0023] In other words, if mitophagy does not proceed smoothly, mitochondrial-related diseases can occur due to abnormal development within cells. Therefore, regulating mitophagy is useful for preventing or treating these diseases.

[0024] The composition of the present invention contains a PRDX3 peptide or its analog as an active ingredient, has an improving or therapeutic effect on diseases caused by autophagy disorders in mitochondria, can contribute to related research, and has the advantage of being able to be utilized as various pharmaceuticals, health functional foods, and the like.

[0025] In one embodiment of the present invention, when the PRDX3 peptide is deficient, it can cause cardiac dysfunction, for example, it can induce cardiac dysfunction due to myocardial infarction (MI).

[0026] In one embodiment of the present invention, the PRDX3 peptide can interact with the PINK1 peptide and regulate mitophagy by the degradation of the PINK1 peptide.

[0027] In this specification, the term "PINK1 (PTEN-induced kinase 1)" is localized in the mitochondrial matrix, and its N-terminus is cleaved by presenilin-related rhomboid-like protein (PARL) and mitochondrial processing peptidase (MPP) through sequential proteins. In response to mitochondrial damage, PINK1 accumulates on the OMM of damaged mitochondria, which can mediate mitophagy.

[0028] In one embodiment of the present invention, when the PRDX3 peptide is deficient, it may cause a decrease in mitophagy despite the upregulation of PINK1.

[0029] In one embodiment of the present invention, the PRDX3 can suppress the expression or action of Oma1, suppress the degradation of PINK1 by Oma1, and maintain its stability.

[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, PRDX3 can remove alpha-synuclein aggregates.

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

[0033] In the present invention, "mitochondrial-related disease" can be one or more selected from the group consisting of cardiovascular disease, lymphoma, glomerulonephritis, osteoporosis, motor neuron disease, muscular atrophy, Down syndrome, carcinoma, Pick's disease, Parkinson's syndrome, and Alzheimer's syndrome.

[0034] In one embodiment of the present invention, PRDX3 can contribute to mitochondrial quality control by regulating both the protective effect on mitochondria from reactive oxygen species and the mitophagy effect that removes damaged mitochondria. In another embodiment, it was observed that mice lacking PRDX3 contained damaged mitochondria in most tissues in their bodies, confirming that they can serve as an important model mouse for studying mitochondrial function in the body.

[0035] As used in this invention, the term "prevention" means all actions that suppress or delay the onset of mitochondrial-related diseases by administering the pharmaceutical composition according to the present invention.

[0036] As used in this invention, the term "treatment" means all actions that improve or favorably alter the symptoms of mitochondrial-related diseases by administering the pharmaceutical compositions according to the present 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, and preferably in combination with agents that promote the prevention or treatment of mitochondrial-related diseases.

[0038] The composition according to the present invention may further contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is one commonly used in formulation and may include, but is not limited to, physiological saline, sterile water, Ringer's solution, buffered physiological saline, cyclodextrin, glucose solution, maltodextrin solution, glycerol, ethanol, and liposomes, and may further contain other common additives such as antioxidants and buffers as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the composition as an injectable preparation such as an aqueous solution, suspension, or emulsion; an injectable preparation such as an infusion bag; an aerosol preparation such as a spray; a pill, capsule, granule, or tablet. Appropriate pharmaceutically acceptable carriers and formulations can be appropriately formulated according to each component using methods published in Remington's Pharmaceutical Science literature. The pharmaceutical formulations of the present invention are not particularly limited in dosage form, but can be formulated as injections, injectable preparations, sprays, liquids, or topical preparations.

[0039] The compositions of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically, including in the eyes) according to the method of the purpose, 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, and can be appropriately selected by those skilled in the art.

[0040] The compositions of the present invention can be administered in pharmaceutically effective amounts. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat or diagnose a disease in accordance with a reasonable benefit / risk ratio applicable to medical treatment or diagnosis, and the effective dose level is determined by factors including the type and severity of the patient’s disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and elimination rate, the duration of treatment and drugs used concurrently, and other factors known in the medical field. The compositions of the present invention can be administered as monotherapy or in combination with other therapeutic agents, can be administered sequentially or concurrently with existing therapeutic agents, and can be administered as a single or multiple doses. Considering all of the above factors comprehensively, it is important to administer the amount that provides the maximum effect with the minimum dose without side effects, which can be easily determined by those skilled in the art.

[0041] Specifically, the effective amount of the composition of the present invention varies 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 concomitant drugs. Generally, 0.001 to 150 mg, preferably 0.01 to 100 mg per kg of body weight, can be administered daily or every other day, or divided into 1 to 3 doses per day. However, the dosage can be increased or decreased depending on the route of administration, degree of obesity, sex, weight, age, etc., so the above dosage does not limit the scope of the present invention in any way.

[0042] In this specification, the concomitant administration can be used alternately with parallel administration, and the form of concomitant administration may include all forms in which a peptide or peptide analog is administered simultaneously with or separately from other compounds.

[0043] Furthermore, the present invention provides a health functional food composition for preventing or improving mitochondrial-related diseases, comprising PRDX3 peptide or its analogue as an active ingredient.

[0044] The term "improvement" may mean all actions that reduce, for example, the severity of symptoms, parameters related to the treatment state. In this case, the health functional food may be used simultaneously with or individually with therapeutic drugs before or after the onset of cerebrovascular disease for the prevention or improvement of the disease.

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

[0046] The aforementioned health functional food further contains one or more of a carrier, excipient, diluent, and additive, and can be formulated as one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquids. Foods to which the compound in one embodiment can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, complex vitamins, and health functional foods.

[0047] Specific examples of the carrier, excipient, diluent, and additive may be at least one selected from the group consisting of lactose, glucose, sucrose, sorbitol, mannitol, erythritol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, methylcellulose, water, syrup, methyl parahydroxybenzoate, propyl parahydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0048] In addition to containing the active ingredients, the aforementioned health functional foods may contain other essential ingredients without any special restrictions. For example, like general beverages, they may contain various flavorings or natural carbohydrates as additional ingredients. Examples of the natural carbohydrates include common sugars such as monosaccharides (e.g., glucose, fructose), disaccharides (e.g., maltose, sucrose), and polysaccharides (e.g., dextrin, cyclodextrin), as well as sugar alcohols such as xylitol, sorbitol, and erythritol. In addition to the flavorings mentioned above, natural flavorings (thaumatin, stevia extract (rebaudioside A and glycyrrhizic acid, etc.)) and synthetic flavorings (saccharin and aspartame, etc.) may also be used. The ratio of the natural carbohydrates can be appropriately determined by those skilled in the art.

[0049] In addition to those described above, a functional health food according to one embodiment may include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents, neutralizing agents (such as those used in cheese and chocolate), pectin acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. These components can be used individually or in combination, and the ratios of the additives can be appropriately selected by those skilled in the art.

[0050] In one embodiment of the present invention, the PRDX3 peptide or its analogue acts on autophagy and apoptosis in mitochondria, thereby producing a preventive or ameliorative effect on mitochondrial-related diseases.

[0051] Furthermore, the present invention provides a pharmaceutical formulation for the prevention or treatment of mitochondrial-related diseases, comprising PRDX3 peptide or its analogue as an active ingredient.

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

[0053] Furthermore, the present invention provides a method for preventing or treating mitochondrial-related diseases, comprising the step of administering a pharmaceutical composition containing PRDX3 peptide or an analog thereof to an individual.

[0054] In the present invention, "individual" means a subject requiring disease treatment, and more specifically, a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, or cow.

[0055] Furthermore, the present invention provides PRDX3 peptide or its analogues for use in the prevention or treatment of mitochondrial-related diseases.

[0056] Furthermore, the present invention provides compositions containing PRDX3 peptide or its analogues as active ingredients for use in the prevention or treatment of mitochondrial-related diseases.

[0057] Furthermore, the present invention provides applications for the PRDX3 peptide or its analogues for producing agents for the prevention or treatment of mitochondrial-related diseases.

[0058] Furthermore, the present invention provides applications for compositions containing the PRDX3 peptide or its analogue as an active ingredient for producing agents for the prevention or treatment of mitochondrial-related diseases.

[0059] The features disclosed herein can be used in combination, and the fact that each of these features is described in different dependent claims does not mean that they cannot be used in combination.

[0060] Furthermore, the present invention provides a PRDX3 gene deletion mouse model in which exons 1 to 4 of the 5'-UTR of the PRDX3 gene are deleted.

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

[0062] Furthermore, the present invention provides a method for producing a PRDX3 gene deletion mouse model, which includes the step of deleting exons 1 to 4 of the 5'-UTR of the PRDX3 gene. In one embodiment of the present invention, exons 1 to 6 of the PRDX3 gene consist of sequences represented by SEQ ID NOs: 7 to 12.

[0063] In one embodiment of the present invention, deletion of exons 1 to 4 of the 5'-UTR of the PRDX3 gene can be performed by homologous recombination.

[0064] In one embodiment of the present invention, the deletion step may be carried out by substituting the base sequence of exon 1 to exon 4 of the 5'-UTR of the PRDX3 gene with a construct containing the neomycin gene.

[0065] In the present invention, the construct may be formed by incorporating a portion of the intron of the 5'-UTR of exon 1 of the PRDX3 gene into the 5'-UTR of an exogenous gene, namely neomycin, and reconstructing the intron portion of the 3'-UTR of exon 4 of the PRDX3 gene into the 3'-UTR.

[0066] In one embodiment of the present invention, the replacement of the construct can be carried out by electroporation. [Effects of the Invention]

[0067] The pharmaceutical composition comprising the PRDX3 peptide or its analogues of the present invention can be used for the prevention and treatment of mitochondrial-related diseases (such as Parkinson's disease) by removing mitophagy and α-synuclein aggregates using the PRDX3 peptide or its analogues.

[0068] However, the effects of the present invention are not limited to those described above, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the configuration of the invention as described in the claims. [Brief explanation of the drawing]

[0069] [Figure 1A]Figure 1 shows that peroxiredoxin 3 (PRDX3) deficiency induces cardiac hypertrophy along with enlarged and damaged mitochondria. Figure 1A shows the cardiac weight / body weight ratio between PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mice at 10 weeks of age (n=5 / group) and 52 weeks of age (n=6 / group). Figures 1B-1D show the results of echocardiographic cardiac function analysis 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 (n=6) mice. Stroke volume (SV) (Figure 1B), cardiac output (CO) (Figure 1C), and ejection fraction (EF) (Figure 1D) were measured using the Simpson method with a single-plane long-axis field of view via a disk approach. Figure 1E shows the hematoxylin and eosin staining results for PRDX3 WT and PRDX3 KO hearts (scale bar, 1 mm). Figures 1F and 1G show the visualization of cell boundaries and quantification of LV fibrosis by wheat germ lectin staining in the LV muscle region (scale bar, 20 μm). Figures 1H and 1I show the results of Masson trichrome staining and quantification of cardiac fibrosis in the LV muscle region (scale bar, 50 μm). Figures 1J and 1K show representative electron micrographs of the heart of PRDX3 WT and PRDX3 KO mice and quantification results of damaged mitochondria (arrows) (scale bars, 0.5 (box area) or 1 μm for 10-week-old PRDX WT and PRDX3 KO mice; scale bars, 1 (box area) or 2 μm for 52-week-old PRDX WT and PRDX KO mice *P<0.05, **P<0.01 and ***P<0.001). [Figure 1B]Figure 1 shows that peroxiredoxin 3 (PRDX3) deficiency induces cardiac hypertrophy along with enlarged and damaged mitochondria. Figure 1A shows the cardiac weight / body weight ratio between PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mice at 10 weeks of age (n=5 / group) and 52 weeks of age (n=6 / group). Figures 1B-1D show the results of echocardiographic cardiac function analysis 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 (n=6) mice. Stroke volume (SV) (Figure 1B), cardiac output (CO) (Figure 1C), and ejection fraction (EF) (Figure 1D) were measured using the Simpson method with a single-plane long-axis field of view via a disk approach. Figure 1E shows the hematoxylin and eosin staining results for PRDX3 WT and PRDX3 KO hearts (scale bar, 1 mm). Figures 1F and 1G show the visualization of cell boundaries and quantification of LV fibrosis by wheat germ lectin staining in the LV muscle region (scale bar, 20 μm). Figures 1H and 1I show the results of Masson trichrome staining and quantification of cardiac fibrosis in the LV muscle region (scale bar, 50 μm). Figures 1J and 1K show representative electron micrographs of the heart of PRDX3 WT and PRDX3 KO mice and quantification results of damaged mitochondria (arrows) (scale bars, 0.5 (box area) or 1 μm for 10-week-old PRDX WT and PRDX3 KO mice; scale bars, 1 (box area) or 2 μm for 52-week-old PRDX WT and PRDX KO mice *P<0.05, **P<0.01 and ***P<0.001). [Figure 1C]Figure 1 shows that peroxiredoxin 3 (PRDX3) deficiency induces cardiac hypertrophy along with enlarged and damaged mitochondria. Figure 1A shows the cardiac weight / body weight ratio between PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mice at 10 weeks of age (n=5 / group) and 52 weeks of age (n=6 / group). Figures 1B-1D show the results of echocardiographic cardiac function analysis 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 (n=6) mice. Stroke volume (SV) (Figure 1B), cardiac output (CO) (Figure 1C), and ejection fraction (EF) (Figure 1D) were measured using the Simpson method with a single-plane long-axis field of view via a disk approach. Figure 1E shows the hematoxylin and eosin staining results for PRDX3 WT and PRDX3 KO hearts (scale bar, 1 mm). Figures 1F and 1G show the visualization of cell boundaries and quantification of LV fibrosis by wheat germ lectin staining in the LV muscle region (scale bar, 20 μm). Figures 1H and 1I show the results of Masson trichrome staining and quantification of cardiac fibrosis in the LV muscle region (scale bar, 50 μm). Figures 1J and 1K show representative electron micrographs of the heart of PRDX3 WT and PRDX3 KO mice and quantification results of damaged mitochondria (arrows) (scale bars, 0.5 (box area) or 1 μm for 10-week-old PRDX WT and PRDX3 KO mice; scale bars, 1 (box area) or 2 μm for 52-week-old PRDX WT and PRDX KO mice *P<0.05, **P<0.01 and ***P<0.001). [Figure 1D]Figure 1 shows that peroxiredoxin 3 (PRDX3) deficiency induces cardiac hypertrophy along with enlarged and damaged mitochondria. Figure 1A shows the cardiac weight / body weight ratio between PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mice at 10 weeks of age (n=5 / group) and 52 weeks of age (n=6 / group). Figures 1B-1D show the results of echocardiographic cardiac function analysis 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 (n=6) mice. Stroke volume (SV) (Figure 1B), cardiac output (CO) (Figure 1C), and ejection fraction (EF) (Figure 1D) were measured using the Simpson method with a single-plane long-axis field of view via a disk approach. Figure 1E shows the hematoxylin and eosin staining results for PRDX3 WT and PRDX3 KO hearts (scale bar, 1 mm). Figures 1F and 1G show the visualization of cell boundaries and quantification of LV fibrosis by wheat germ lectin staining in the LV muscle region (scale bar, 20 μm). Figures 1H and 1I show the results of Masson trichrome staining and quantification of cardiac fibrosis in the LV muscle region (scale bar, 50 μm). Figures 1J and 1K show representative electron micrographs of the heart of PRDX3 WT and PRDX3 KO mice and quantification results of damaged mitochondria (arrows) (scale bars, 0.5 (box area) or 1 μm for 10-week-old PRDX WT and PRDX3 KO mice; scale bars, 1 (box area) or 2 μm for 52-week-old PRDX WT and PRDX KO mice *P<0.05, **P<0.01 and ***P<0.001). [Figure 1E]Figure 1 shows that peroxiredoxin 3 (PRDX3) deficiency induces cardiac hypertrophy along with enlarged and damaged mitochondria. Figure 1A shows the cardiac weight / body weight ratio between PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mice at 10 weeks of age (n=5 / group) and 52 weeks of age (n=6 / group). Figures 1B-1D show the results of echocardiographic cardiac function analysis 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 (n=6) mice. Stroke volume (SV) (Figure 1B), cardiac output (CO) (Figure 1C), and ejection fraction (EF) (Figure 1D) were measured using the Simpson method with a single-plane long-axis field of view via a disk approach. Figure 1E shows the hematoxylin and eosin staining results for PRDX3 WT and PRDX3 KO hearts (scale bar, 1 mm). Figures 1F and 1G show the visualization of cell boundaries and quantification of LV fibrosis by wheat germ lectin staining in the LV muscle region (scale bar, 20 μm). Figures 1H and 1I show the results of Masson trichrome staining and quantification of cardiac fibrosis in the LV muscle region (scale bar, 50 μm). Figures 1J and 1K show representative electron micrographs of the heart of PRDX3 WT and PRDX3 KO mice and quantification results of damaged mitochondria (arrows) (scale bars, 0.5 (box area) or 1 μm for 10-week-old PRDX WT and PRDX3 KO mice; scale bars, 1 (box area) or 2 μm for 52-week-old PRDX WT and PRDX KO mice *P<0.05, **P<0.01 and ***P<0.001). [Figure 1F]Figure 1 shows that peroxiredoxin 3 (PRDX3) deficiency induces cardiac hypertrophy along with enlarged and damaged mitochondria. Figure 1A shows the cardiac weight / body weight ratio between PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mice at 10 weeks of age (n=5 / group) and 52 weeks of age (n=6 / group). Figures 1B-1D show the results of echocardiographic cardiac function analysis 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 (n=6) mice. Stroke volume (SV) (Figure 1B), cardiac output (CO) (Figure 1C), and ejection fraction (EF) (Figure 1D) were measured using the Simpson method with a single-plane long-axis field of view via a disk approach. Figure 1E shows the hematoxylin and eosin staining results for PRDX3 WT and PRDX3 KO hearts (scale bar, 1 mm). Figures 1F and 1G show the visualization of cell boundaries and quantification of LV fibrosis by wheat germ lectin staining in the LV muscle region (scale bar, 20 μm). Figures 1H and 1I show the results of Masson trichrome staining and quantification of cardiac fibrosis in the LV muscle region (scale bar, 50 μm). Figures 1J and 1K show representative electron micrographs of the heart of PRDX3 WT and PRDX3 KO mice and quantification results of damaged mitochondria (arrows) (scale bars, 0.5 (box area) or 1 μm for 10-week-old PRDX WT and PRDX3 KO mice; scale bars, 1 (box area) or 2 μm for 52-week-old PRDX WT and PRDX KO mice *P<0.05, **P<0.01 and ***P<0.001). [Figure 1G]Figure 1 shows that peroxiredoxin 3 (PRDX3) deficiency induces cardiac hypertrophy along with enlarged and damaged mitochondria. Figure 1A shows the cardiac weight / body weight ratio between PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mice at 10 weeks of age (n=5 / group) and 52 weeks of age (n=6 / group). Figures 1B-1D show the results of echocardiographic cardiac function analysis 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 (n=6) mice. Stroke volume (SV) (Figure 1B), cardiac output (CO) (Figure 1C), and ejection fraction (EF) (Figure 1D) were measured using the Simpson method with a single-plane long-axis field of view via a disk approach. Figure 1E shows the hematoxylin and eosin staining results for PRDX3 WT and PRDX3 KO hearts (scale bar, 1 mm). Figures 1F and 1G show the visualization of cell boundaries and quantification of LV fibrosis by wheat germ lectin staining in the LV muscle region (scale bar, 20 μm). Figures 1H and 1I show the results of Masson trichrome staining and quantification of cardiac fibrosis in the LV muscle region (scale bar, 50 μm). Figures 1J and 1K show representative electron micrographs of the heart of PRDX3 WT and PRDX3 KO mice and quantification results of damaged mitochondria (arrows) (scale bars, 0.5 (box area) or 1 μm for 10-week-old PRDX WT and PRDX3 KO mice; scale bars, 1 (box area) or 2 μm for 52-week-old PRDX WT and PRDX KO mice *P<0.05, **P<0.01 and ***P<0.001). [Figure 1H]Figure 1 shows that peroxiredoxin 3 (PRDX3) deficiency induces cardiac hypertrophy along with enlarged and damaged mitochondria. Figure 1A shows the cardiac weight / body weight ratio between PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mice at 10 weeks of age (n=5 / group) and 52 weeks of age (n=6 / group). Figures 1B-1D show the results of echocardiographic cardiac function analysis 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 (n=6) mice. Stroke volume (SV) (Figure 1B), cardiac output (CO) (Figure 1C), and ejection fraction (EF) (Figure 1D) were measured using the Simpson method with a single-plane long-axis field of view via a disk approach. Figure 1E shows the hematoxylin and eosin staining results for PRDX3 WT and PRDX3 KO hearts (scale bar, 1 mm). Figures 1F and 1G show the visualization of cell boundaries and quantification of LV fibrosis by wheat germ lectin staining in the LV muscle region (scale bar, 20 μm). Figures 1H and 1I show the results of Masson trichrome staining and quantification of cardiac fibrosis in the LV muscle region (scale bar, 50 μm). Figures 1J and 1K show representative electron micrographs of the heart of PRDX3 WT and PRDX3 KO mice and quantification results of damaged mitochondria (arrows) (scale bars, 0.5 (box area) or 1 μm for 10-week-old PRDX WT and PRDX3 KO mice; scale bars, 1 (box area) or 2 μm for 52-week-old PRDX WT and PRDX KO mice *P<0.05, **P<0.01 and ***P<0.001). [Figure 1I]Figure 1 shows that peroxiredoxin 3 (PRDX3) deficiency induces cardiac hypertrophy along with enlarged and damaged mitochondria. Figure 1A shows the cardiac weight / body weight ratio between PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mice at 10 weeks of age (n=5 / group) and 52 weeks of age (n=6 / group). Figures 1B-1D show the results of echocardiographic cardiac function analysis 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 (n=6) mice. Stroke volume (SV) (Figure 1B), cardiac output (CO) (Figure 1C), and ejection fraction (EF) (Figure 1D) were measured using the Simpson method with a single-plane long-axis field of view via a disk approach. Figure 1E shows the hematoxylin and eosin staining results for PRDX3 WT and PRDX3 KO hearts (scale bar, 1 mm). Figures 1F and 1G show the visualization of cell boundaries and quantification of LV fibrosis by wheat germ lectin staining in the LV muscle region (scale bar, 20 μm). Figures 1H and 1I show the results of Masson trichrome staining and quantification of cardiac fibrosis in the LV muscle region (scale bar, 50 μm). Figures 1J and 1K show representative electron micrographs of the heart of PRDX3 WT and PRDX3 KO mice and quantification results of damaged mitochondria (arrows) (scale bars, 0.5 (box area) or 1 μm for 10-week-old PRDX WT and PRDX3 KO mice; scale bars, 1 (box area) or 2 μm for 52-week-old PRDX WT and PRDX KO mice *P<0.05, **P<0.01 and ***P<0.001). [Figure 1J]Figure 1 shows that peroxiredoxin 3 (PRDX3) deficiency induces cardiac hypertrophy along with enlarged and damaged mitochondria. Figure 1A shows the cardiac weight / body weight ratio between PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mice at 10 weeks of age (n=5 / group) and 52 weeks of age (n=6 / group). Figures 1B-1D show the results of echocardiographic cardiac function analysis 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 (n=6) mice. Stroke volume (SV) (Figure 1B), cardiac output (CO) (Figure 1C), and ejection fraction (EF) (Figure 1D) were measured using the Simpson method with a single-plane long-axis field of view via a disk approach. Figure 1E shows the hematoxylin and eosin staining results for PRDX3 WT and PRDX3 KO hearts (scale bar, 1 mm). Figures 1F and 1G show the visualization of cell boundaries and quantification of LV fibrosis by wheat germ lectin staining in the LV muscle region (scale bar, 20 μm). Figures 1H and 1I show the results of Masson trichrome staining and quantification of cardiac fibrosis in the LV muscle region (scale bar, 50 μm). Figures 1J and 1K show representative electron micrographs of the heart of PRDX3 WT and PRDX3 KO mice and quantification results of damaged mitochondria (arrows) (scale bars, 0.5 (box area) or 1 μm for 10-week-old PRDX WT and PRDX3 KO mice; scale bars, 1 (box area) or 2 μm for 52-week-old PRDX WT and PRDX KO mice *P<0.05, **P<0.01 and ***P<0.001). [Figure 1K]Figure 1 shows that peroxiredoxin 3 (PRDX3) deficiency induces cardiac hypertrophy along with enlarged and damaged mitochondria. Figure 1A shows the cardiac weight / body weight ratio between PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mice at 10 weeks of age (n=5 / group) and 52 weeks of age (n=6 / group). Figures 1B-1D show the results of echocardiographic cardiac function analysis 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 (n=6) mice. Stroke volume (SV) (Figure 1B), cardiac output (CO) (Figure 1C), and ejection fraction (EF) (Figure 1D) were measured using the Simpson method with a single-plane long-axis field of view via a disk approach. Figure 1E shows the hematoxylin and eosin staining results for PRDX3 WT and PRDX3 KO hearts (scale bar, 1 mm). Figures 1F and 1G show the visualization of cell boundaries and quantification of LV fibrosis by wheat germ lectin staining in the LV muscle region (scale bar, 20 μm). Figures 1H and 1I show the results of Masson trichrome staining and quantification of cardiac fibrosis in the LV muscle region (scale bar, 50 μm). Figures 1J and 1K show representative electron micrographs of the heart of PRDX3 WT and PRDX3 KO mice and quantification results of damaged mitochondria (arrows) (scale bars, 0.5 (box area) or 1 μm for 10-week-old PRDX WT and PRDX3 KO mice; scale bars, 1 (box area) or 2 μm for 52-week-old PRDX WT and PRDX KO mice *P<0.05, **P<0.01 and ***P<0.001).

[0070] [Figure 2A]Figure 2 shows the transthoracic echocardiographic results of peroxiredoxin 3 (PRDX3) wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mice. Figure 2A shows the integrated echocardiographic 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 to aged PRDX3 KO mice (n=6). (EF (ejection fraction), SV (stroke volume), FS (fractional shortening), CO (cardiac output)). Figure 2B shows cardiac images of 10-week-old PRDX3 WT, 52-week-old PRDX3 WT, 10-week-old PRDX3 KO, and 52-week-old PRDX3 KO mice (scale bar, 1 mm). [Figure 2B] Figure 2 shows the transthoracic echocardiographic results of peroxiredoxin 3 (PRDX3) wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mice. Figure 2A shows the integrated echocardiographic 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 to aged PRDX3 KO mice (n=6). (EF (ejection fraction), SV (stroke volume), FS (fractional shortening), CO (cardiac output)). Figure 2B shows cardiac images of 10-week-old PRDX3 WT, 52-week-old PRDX3 WT, 10-week-old PRDX3 KO, and 52-week-old PRDX3 KO mice (scale bar, 1 mm).

[0071] [Figure 3A]Figure 3 shows evidence that peroxiredoxin 3 (PRDX3) deficiency accelerates left ventricular (LV) remodeling and heart failure after myocardial infarction (MI). Figure 3A shows the results of echocardiographic function analysis in PRDX3 wild-type (PRDX3 WT, n=7) and PRDX KO (n=8) mice 15 days after MI. Figure 3B shows left ventricular end-diastolic volume (EDV), Figure 3C shows end-systolic volume (ESV), Figure 3D shows stroke volume (SV), and Figure 3E shows ejection fraction (EF), all measured in the LV long-axis field of view using the Simpson method with a single-plane long-axis field of view via a disk approach. Figure 3F shows the serial region of Masson trichrome-stained hearts in PRDX3 WT and PRDX KO mice 15 days after MI (percentage of fibrosis was calculated relative to the total left ventricular area). Figure 3G shows representative electron micrographs of the hearts of wild-type and PRDX KO mice 1 day after MI, with arrows indicating damaged mitochondria (scale bars, 0.5 (box area) or 2 μm). Figure 3H shows the results of TUNEL analysis of the infarcted area in PRDX3 WT (n=5) and PRDX KO mice (n=5) 1 day after MI (scale bars, 10 (box area) or 50 μm *P<0.05 and **P<0.01). [Figure 3B]Figure 3 shows evidence that peroxiredoxin 3 (PRDX3) deficiency accelerates left ventricular (LV) remodeling and heart failure after myocardial infarction (MI). Figure 3A shows the results of echocardiographic function analysis in PRDX3 wild-type (PRDX3 WT, n=7) and PRDX KO (n=8) mice 15 days after MI. Figure 3B shows left ventricular end-diastolic volume (EDV), Figure 3C shows end-systolic volume (ESV), Figure 3D shows stroke volume (SV), and Figure 3E shows ejection fraction (EF), all measured in the LV long-axis field of view using the Simpson method with a single-plane long-axis field of view via a disk approach. Figure 3F shows the serial region of Masson trichrome-stained hearts in PRDX3 WT and PRDX KO mice 15 days after MI (percentage of fibrosis was calculated relative to the total left ventricular area). Figure 3G shows representative electron micrographs of the hearts of wild-type and PRDX KO mice 1 day after MI, with arrows indicating damaged mitochondria (scale bars, 0.5 (box area) or 2 μm). Figure 3H shows the results of TUNEL analysis of the infarcted area in PRDX3 WT (n=5) and PRDX KO mice (n=5) 1 day after MI (scale bars, 10 (box area) or 50 μm *P<0.05 and **P<0.01). [Figure 3C]Figure 3 shows evidence that peroxiredoxin 3 (PRDX3) deficiency accelerates left ventricular (LV) remodeling and heart failure after myocardial infarction (MI). Figure 3A shows the results of echocardiographic function analysis in PRDX3 wild-type (PRDX3 WT, n=7) and PRDX KO (n=8) mice 15 days after MI. Figure 3B shows left ventricular end-diastolic volume (EDV), Figure 3C shows end-systolic volume (ESV), Figure 3D shows stroke volume (SV), and Figure 3E shows ejection fraction (EF), all measured in the LV long-axis field of view using the Simpson method with a single-plane long-axis field of view via a disk approach. Figure 3F shows the serial region of Masson trichrome-stained hearts in PRDX3 WT and PRDX KO mice 15 days after MI (percentage of fibrosis was calculated relative to the total left ventricular area). Figure 3G shows representative electron micrographs of the hearts of wild-type and PRDX KO mice 1 day after MI, with arrows indicating damaged mitochondria (scale bars, 0.5 (box area) or 2 μm). Figure 3H shows the results of TUNEL analysis of the infarcted area in PRDX3 WT (n=5) and PRDX KO mice (n=5) 1 day after MI (scale bars, 10 (box area) or 50 μm *P<0.05 and **P<0.01). [Figure 3D]Figure 3 shows evidence that peroxiredoxin 3 (PRDX3) deficiency accelerates left ventricular (LV) remodeling and heart failure after myocardial infarction (MI). Figure 3A shows the results of echocardiographic function analysis in PRDX3 wild-type (PRDX3 WT, n=7) and PRDX KO (n=8) mice 15 days after MI. Figure 3B shows left ventricular end-diastolic volume (EDV), Figure 3C shows end-systolic volume (ESV), Figure 3D shows stroke volume (SV), and Figure 3E shows ejection fraction (EF), all measured in the LV long-axis field of view using the Simpson method with a single-plane long-axis field of view via a disk approach. Figure 3F shows the serial region of Masson trichrome-stained hearts in PRDX3 WT and PRDX KO mice 15 days after MI (percentage of fibrosis was calculated relative to the total left ventricular area). Figure 3G shows representative electron micrographs of the hearts of wild-type and PRDX KO mice 1 day after MI, with arrows indicating damaged mitochondria (scale bars, 0.5 (box area) or 2 μm). Figure 3H shows the results of TUNEL analysis of the infarcted area in PRDX3 WT (n=5) and PRDX KO mice (n=5) 1 day after MI (scale bars, 10 (box area) or 50 μm *P<0.05 and **P<0.01). [Figure 3E]Figure 3 shows evidence that peroxiredoxin 3 (PRDX3) deficiency accelerates left ventricular (LV) remodeling and heart failure after myocardial infarction (MI). Figure 3A shows the results of echocardiographic function analysis in PRDX3 wild-type (PRDX3 WT, n=7) and PRDX KO (n=8) mice 15 days after MI. Figure 3B shows left ventricular end-diastolic volume (EDV), Figure 3C shows end-systolic volume (ESV), Figure 3D shows stroke volume (SV), and Figure 3E shows ejection fraction (EF), all measured in the LV long-axis field of view using the Simpson method with a single-plane long-axis field of view via a disk approach. Figure 3F shows the serial region of Masson trichrome-stained hearts in PRDX3 WT and PRDX KO mice 15 days after MI (percentage of fibrosis was calculated relative to the total left ventricular area). Figure 3G shows representative electron micrographs of the hearts of wild-type and PRDX KO mice 1 day after MI, with arrows indicating damaged mitochondria (scale bars, 0.5 (box area) or 2 μm). Figure 3H shows the results of TUNEL analysis of the infarcted area in PRDX3 WT (n=5) and PRDX KO mice (n=5) 1 day after MI (scale bars, 10 (box area) or 50 μm *P<0.05 and **P<0.01). [Figure 3F]Figure 3 shows evidence that peroxiredoxin 3 (PRDX3) deficiency accelerates left ventricular (LV) remodeling and heart failure after myocardial infarction (MI). Figure 3A shows the results of echocardiographic function analysis in PRDX3 wild-type (PRDX3 WT, n=7) and PRDX KO (n=8) mice 15 days after MI. Figure 3B shows left ventricular end-diastolic volume (EDV), Figure 3C shows end-systolic volume (ESV), Figure 3D shows stroke volume (SV), and Figure 3E shows ejection fraction (EF), all measured in the LV long-axis field of view using the Simpson method with a single-plane long-axis field of view via a disk approach. Figure 3F shows the serial region of Masson trichrome-stained hearts in PRDX3 WT and PRDX KO mice 15 days after MI (percentage of fibrosis was calculated relative to the total left ventricular area). Figure 3G shows representative electron micrographs of the hearts of wild-type and PRDX KO mice 1 day after MI, with arrows indicating damaged mitochondria (scale bars, 0.5 (box area) or 2 μm). Figure 3H shows the results of TUNEL analysis of the infarcted area in PRDX3 WT (n=5) and PRDX KO mice (n=5) 1 day after MI (scale bars, 10 (box area) or 50 μm *P<0.05 and **P<0.01). [Figure 3G]Figure 3 shows evidence that peroxiredoxin 3 (PRDX3) deficiency accelerates left ventricular (LV) remodeling and heart failure after myocardial infarction (MI). Figure 3A shows the results of echocardiographic function analysis in PRDX3 wild-type (PRDX3 WT, n=7) and PRDX KO (n=8) mice 15 days after MI. Figure 3B shows left ventricular end-diastolic volume (EDV), Figure 3C shows end-systolic volume (ESV), Figure 3D shows stroke volume (SV), and Figure 3E shows ejection fraction (EF), all measured in the LV long-axis field of view using the Simpson method with a single-plane long-axis field of view via a disk approach. Figure 3F shows the serial region of Masson trichrome-stained hearts in PRDX3 WT and PRDX KO mice 15 days after MI (percentage of fibrosis was calculated relative to the total left ventricular area). Figure 3G shows representative electron micrographs of the hearts of wild-type and PRDX KO mice 1 day after MI, with arrows indicating damaged mitochondria (scale bars, 0.5 (box area) or 2 μm). Figure 3H shows the results of TUNEL analysis of the infarcted area in PRDX3 WT (n=5) and PRDX KO mice (n=5) 1 day after MI (scale bars, 10 (box area) or 50 μm *P<0.05 and **P<0.01). [Figure 3H]Figure 3 shows evidence that peroxiredoxin 3 (PRDX3) deficiency accelerates left ventricular (LV) remodeling and heart failure after myocardial infarction (MI). Figure 3A shows the results of echocardiographic function analysis in PRDX3 wild-type (PRDX3 WT, n=7) and PRDX KO (n=8) mice 15 days after MI. Figure 3B shows left ventricular end-diastolic volume (EDV), Figure 3C shows end-systolic volume (ESV), Figure 3D shows stroke volume (SV), and Figure 3E shows ejection fraction (EF), all measured in the LV long-axis field of view using the Simpson method with a single-plane long-axis field of view via a disk approach. Figure 3F shows the serial region of Masson trichrome-stained hearts in PRDX3 WT and PRDX KO mice 15 days after MI (percentage of fibrosis was calculated relative to the total left ventricular area). Figure 3G shows representative electron micrographs of the hearts of wild-type and PRDX KO mice 1 day after MI, with arrows indicating damaged mitochondria (scale bars, 0.5 (box area) or 2 μm). Figure 3H shows the results of TUNEL analysis of the infarcted area in PRDX3 WT (n=5) and PRDX KO mice (n=5) 1 day after MI (scale bars, 10 (box area) or 50 μm *P<0.05 and **P<0.01).

[0072] [Figure 4A] Figure 4 shows the results of transthoracic echocardiography of peroxiredoxin 3 (PRDX3) wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mice after myocardial infarction (MI). Figure 4A shows pooled echocardiographic data from PRDX3 WT (n=7) and PRDX3 KO (n=8) mice. Figure 4B shows the results of left ventricular function assessment using VevoStrain software. [Figure 4B]Figure 4 shows the results of transthoracic echocardiography of peroxiredoxin 3 (PRDX3) wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mice after myocardial infarction (MI). Figure 4A shows pooled echocardiographic data from PRDX3 WT (n=7) and PRDX3 KO (n=8) mice. Figure 4B shows the results of left ventricular function assessment using VevoStrain software.

[0073] [Figure 5A] Figure 5 shows evidence that increased mitochondrial damage is due to the accumulation of reactive oxygen species (ROS) caused by peroxiredoxin 3 (PRDX3) deficiency. Figures 5A and 5B show representative low-magnification and high-magnification images of box regions in PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mouse embryonic fibroblasts (MEFs). MEFs were infected with mitocatalase adenovirus for 24 hours and immunostained for 25 minutes with antibodies against Tom20 + 100 nM MitoTracker (Figure 5A) or 150 nM MitoTracker + 5 μM MitoSOX (Figure 5B) (scale bars, 2 μm (box region) or 10 μm). Figures 5C and 5D show the results of quantifying depolarized (damaged) mitochondria (Figure 5C) or mitochondrial ROS levels (Figure 5D) in PRDX3 WT and PRDX3 KO MEFs (n=25-33 cells). Figure 5E shows the results of immunoblotting of whole cell lysates of PRDX3 WT and PRDX3 KO MEF cells infected with adenovirus targeting mitochondrial catalase for 24 hours using catalase, PRDX3, and Tom20 antibodies (**P<0.01). The data (Figures 5A-5E) show three independent experiments. [Figure 5B]Figure 5 shows evidence that increased mitochondrial damage is due to the accumulation of reactive oxygen species (ROS) caused by peroxiredoxin 3 (PRDX3) deficiency. Figures 5A and 5B show representative low-magnification and high-magnification images of box regions in PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mouse embryonic fibroblasts (MEFs). MEFs were infected with mitocatalase adenovirus for 24 hours and immunostained for 25 minutes with antibodies against Tom20 + 100 nM MitoTracker (Figure 5A) or 150 nM MitoTracker + 5 μM MitoSOX (Figure 5B) (scale bars, 2 μm (box region) or 10 μm). Figures 5C and 5D show the results of quantifying depolarized (damaged) mitochondria (Figure 5C) or mitochondrial ROS levels (Figure 5D) in PRDX3 WT and PRDX3 KO MEFs (n=25-33 cells). Figure 5E shows the results of immunoblotting of whole cell lysates of PRDX3 WT and PRDX3 KO MEF cells infected with adenovirus targeting mitochondrial catalase for 24 hours using catalase, PRDX3, and Tom20 antibodies (**P<0.01). The data (Figures 5A-5E) show three independent experiments. [Figure 5C]Figure 5 shows evidence that increased mitochondrial damage is due to the accumulation of reactive oxygen species (ROS) caused by peroxiredoxin 3 (PRDX3) deficiency. Figures 5A and 5B show representative low-magnification and high-magnification images of box regions in PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mouse embryonic fibroblasts (MEFs). MEFs were infected with mitocatalase adenovirus for 24 hours and immunostained for 25 minutes with antibodies against Tom20 + 100 nM MitoTracker (Figure 5A) or 150 nM MitoTracker + 5 μM MitoSOX (Figure 5B) (scale bars, 2 μm (box region) or 10 μm). Figures 5C and 5D show the results of quantifying depolarized (damaged) mitochondria (Figure 5C) or mitochondrial ROS levels (Figure 5D) in PRDX3 WT and PRDX3 KO MEFs (n=25-33 cells). Figure 5E shows the results of immunoblotting of whole cell lysates of PRDX3 WT and PRDX3 KO MEF cells infected with adenovirus targeting mitochondrial catalase for 24 hours using catalase, PRDX3, and Tom20 antibodies (**P<0.01). The data (Figures 5A-5E) show three independent experiments. [Figure 5D]Figure 5 shows evidence that increased mitochondrial damage is due to the accumulation of reactive oxygen species (ROS) caused by peroxiredoxin 3 (PRDX3) deficiency. Figures 5A and 5B show representative low-magnification and high-magnification images of box regions in PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mouse embryonic fibroblasts (MEFs). MEFs were infected with mitocatalase adenovirus for 24 hours and immunostained for 25 minutes with antibodies against Tom20 + 100 nM MitoTracker (Figure 5A) or 150 nM MitoTracker + 5 μM MitoSOX (Figure 5B) (scale bars, 2 μm (box region) or 10 μm). Figures 5C and 5D show the results of quantifying depolarized (damaged) mitochondria (Figure 5C) or mitochondrial ROS levels (Figure 5D) in PRDX3 WT and PRDX3 KO MEFs (n=25-33 cells). Figure 5E shows the results of immunoblotting of whole cell lysates of PRDX3 WT and PRDX3 KO MEF cells infected with adenovirus targeting mitochondrial catalase for 24 hours using catalase, PRDX3, and Tom20 antibodies (**P<0.01). The data (Figures 5A-5E) show three independent experiments. [Figure 5E]Figure 5 shows evidence that increased mitochondrial damage is due to the accumulation of reactive oxygen species (ROS) caused by peroxiredoxin 3 (PRDX3) deficiency. Figures 5A and 5B show representative low-magnification and high-magnification images of box regions in PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mouse embryonic fibroblasts (MEFs). MEFs were infected with mitocatalase adenovirus for 24 hours and immunostained for 25 minutes with antibodies against Tom20 + 100 nM MitoTracker (Figure 5A) or 150 nM MitoTracker + 5 μM MitoSOX (Figure 5B) (scale bars, 2 μm (box region) or 10 μm). Figures 5C and 5D show the results of quantifying depolarized (damaged) mitochondria (Figure 5C) or mitochondrial ROS levels (Figure 5D) in PRDX3 WT and PRDX3 KO MEFs (n=25-33 cells). Figure 5E shows the results of immunoblotting of whole cell lysates of PRDX3 WT and PRDX3 KO MEF cells infected with adenovirus targeting mitochondrial catalase for 24 hours using catalase, PRDX3, and Tom20 antibodies (**P<0.01). The data (Figures 5A-5E) show three independent experiments.

[0074] [Figure 6] Figure 6 shows evidence that peroxiredoxin 3 (PRDX3) deficiency increases mitochondrial damage. Figures 6A and 6B show representative electron micrographs of liver, skeletal muscle (soleus muscle), and brain (dentate gyrus region) tissue 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. Arrows indicate damaged mitochondria (scale bars, 0.5 μm (box area) or 1 μm for 10-week-old mice, 1 μm (box area) or 2 μm for 52-week-old mice).

[0075] [Figure 7A]Figure 7 shows evidence that PRDX3 deficiency causes cardiac mitochondrial dysfunction. Figure 7A compares ATP production in wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) cardiomyocytes. Figure 7B shows representative follow-up results for oxygen consumption rate (OCR, pMoles / min) in wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) cardiomyocytes. The arrows indicate the time points at which oligomycin (Oligo, 1.5 μM), FCCP (1 μM), and Rot&AA (rotenone and antimycin A, 0.5 μM each) were injected into 1 × 10⁴ cells. Figure 7C shows the OCR value for basal respiration per 1 × 10⁴ cells. Figure 7D shows the OCR value for ATP-bound respiration. Figure 7E shows the OCR value for proton leak. Figure 7F shows the OCR value for maximal respiration. Figure 7G shows the OCR values ​​of respiratory reserve (*P<0.05 and **P<0.01). The data (Figures 7A-7G) represent three independent experiments. [Figure 7B] Figure 7 shows evidence that PRDX3 deficiency causes cardiac mitochondrial dysfunction. Figure 7A compares ATP production in wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) cardiomyocytes. Figure 7B shows representative follow-up results for oxygen consumption rate (OCR, pMoles / min) in wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) cardiomyocytes. The arrows indicate the time points at which oligomycin (Oligo, 1.5 μM), FCCP (1 μM), and Rot&AA (rotenone and antimycin A, 0.5 μM each) were injected into 1 × 10⁴ cells. Figure 7C shows the OCR value for basal respiration per 1 × 10⁴ cells. Figure 7D shows the OCR value for ATP-bound respiration. Figure 7E shows the OCR value for proton leak. Figure 7F shows the OCR value for maximal respiration. Figure 7G shows the OCR values ​​of respiratory reserve (*P<0.05 and **P<0.01). The data (Figures 7A-7G) represent three independent experiments. [Figure 7C]Figure 7 shows evidence that PRDX3 deficiency causes cardiac mitochondrial dysfunction. Figure 7A compares ATP production in wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) cardiomyocytes. Figure 7B shows representative follow-up results for oxygen consumption rate (OCR, pMoles / min) in wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) cardiomyocytes. The arrows indicate the time points at which oligomycin (Oligo, 1.5 μM), FCCP (1 μM), and Rot&AA (rotenone and antimycin A, 0.5 μM each) were injected into 1 × 10⁴ cells. Figure 7C shows the OCR value for basal respiration per 1 × 10⁴ cells. Figure 7D shows the OCR value for ATP-bound respiration. Figure 7E shows the OCR value for proton leak. Figure 7F shows the OCR value for maximal respiration. Figure 7G shows the OCR values ​​of respiratory reserve (*P<0.05 and **P<0.01). The data (Figures 7A-7G) represent three independent experiments. [Figure 7D] Figure 7 shows evidence that PRDX3 deficiency causes cardiac mitochondrial dysfunction. Figure 7A compares ATP production in wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) cardiomyocytes. Figure 7B shows representative follow-up results for oxygen consumption rate (OCR, pMoles / min) in wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) cardiomyocytes. The arrows indicate the time points at which oligomycin (Oligo, 1.5 μM), FCCP (1 μM), and Rot&AA (rotenone and antimycin A, 0.5 μM each) were injected into 1 × 10⁴ cells. Figure 7C shows the OCR value for basal respiration per 1 × 10⁴ cells. Figure 7D shows the OCR value for ATP-bound respiration. Figure 7E shows the OCR value for proton leak. Figure 7F shows the OCR value for maximal respiration. Figure 7G shows the OCR values ​​of respiratory reserve (*P<0.05 and **P<0.01). The data (Figures 7A-7G) represent three independent experiments. [Figure 7E]Figure 7 shows evidence that PRDX3 deficiency causes cardiac mitochondrial dysfunction. Figure 7A compares ATP production in wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) cardiomyocytes. Figure 7B shows representative follow-up results for oxygen consumption rate (OCR, pMoles / min) in wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) cardiomyocytes. The arrows indicate the time points at which oligomycin (Oligo, 1.5 μM), FCCP (1 μM), and Rot&AA (rotenone and antimycin A, 0.5 μM each) were injected into 1 × 10⁴ cells. Figure 7C shows the OCR value for basal respiration per 1 × 10⁴ cells. Figure 7D shows the OCR value for ATP-bound respiration. Figure 7E shows the OCR value for proton leak. Figure 7F shows the OCR value for maximal respiration. Figure 7G shows the OCR values ​​of respiratory reserve (*P<0.05 and **P<0.01). The data (Figures 7A-7G) represent three independent experiments. [Figure 7F] Figure 7 shows evidence that PRDX3 deficiency causes cardiac mitochondrial dysfunction. Figure 7A compares ATP production in wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) cardiomyocytes. Figure 7B shows representative follow-up results for oxygen consumption rate (OCR, pMoles / min) in wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) cardiomyocytes. The arrows indicate the time points at which oligomycin (Oligo, 1.5 μM), FCCP (1 μM), and Rot&AA (rotenone and antimycin A, 0.5 μM each) were injected into 1 × 10⁴ cells. Figure 7C shows the OCR value for basal respiration per 1 × 10⁴ cells. Figure 7D shows the OCR value for ATP-bound respiration. Figure 7E shows the OCR value for proton leak. Figure 7F shows the OCR value for maximal respiration. Figure 7G shows the OCR values ​​of respiratory reserve (*P<0.05 and **P<0.01). The data (Figures 7A-7G) represent three independent experiments. [Figure 7G]Figure 7 shows evidence that PRDX3 deficiency causes cardiac mitochondrial dysfunction. Figure 7A compares ATP production in wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) cardiomyocytes. Figure 7B shows representative follow-up results for oxygen consumption rate (OCR, pMoles / min) in wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) cardiomyocytes. The arrows indicate the time points at which oligomycin (Oligo, 1.5 μM), FCCP (1 μM), and Rot&AA (rotenone and antimycin A, 0.5 μM each) were injected into 1 × 10⁴ cells. Figure 7C shows the OCR value for basal respiration per 1 × 10⁴ cells. Figure 7D shows the OCR value for ATP-bound respiration. Figure 7E shows the OCR value for proton leak. Figure 7F shows the OCR value for maximal respiration. Figure 7G shows the OCR values ​​of respiratory reserve (*P<0.05 and **P<0.01). The data (Figures 7A-7G) represent three independent experiments.

[0076] [Figure 8A] Figure 8 shows evidence that peroxiredoxin 3 (PRDX3) deficiency reduces cardiac mitophagy. Figures 8A and 8B are representative images (Figure 8A) and a graph (Figure 8B) of mitophagy in the heart of mitochondrial-targeted Keima(mt)-Keima mice: 10-week-old PRDX3 wild-type (PRDX3 WT, n=9), 52-week-old PRDX3 WT (n=6), 10-week-old Pink1-deficient (Pink1 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 quantification 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 myocardial infarction (MI) induction (scale bar, 10 μm *** P < 0.001). [Figure 8B]Figure 8 shows evidence that peroxiredoxin 3 (PRDX3) deficiency reduces cardiac mitophagy. Figures 8A and 8B are representative images (Figure 8A) and a graph (Figure 8B) of mitophagy in the heart of mitochondrial-targeted Keima(mt)-Keima mice: 10-week-old PRDX3 wild-type (PRDX3 WT, n=9), 52-week-old PRDX3 WT (n=6), 10-week-old Pink1-deficient (Pink1 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 quantification 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 myocardial infarction (MI) induction (scale bar, 10 μm *** P < 0.001). [Figure 8C] Figure 8 shows evidence that peroxiredoxin 3 (PRDX3) deficiency reduces cardiac mitophagy. Figures 8A and 8B are representative images (Figure 8A) and a graph (Figure 8B) of mitophagy in the heart of mitochondrial-targeted Keima(mt)-Keima mice: 10-week-old PRDX3 wild-type (PRDX3 WT, n=9), 52-week-old PRDX3 WT (n=6), 10-week-old Pink1-deficient (Pink1 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 quantification 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 myocardial infarction (MI) induction (scale bar, 10 μm *** P < 0.001). [Figure 8D]Figure 8 shows evidence that peroxiredoxin 3 (PRDX3) deficiency reduces cardiac mitophagy. Figures 8A and 8B are representative images (Figure 8A) and a graph (Figure 8B) of mitophagy in the heart of mitochondrial-targeted Keima(mt)-Keima mice: 10-week-old PRDX3 wild-type (PRDX3 WT, n=9), 52-week-old PRDX3 WT (n=6), 10-week-old Pink1-deficient (Pink1 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 quantification 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 myocardial infarction (MI) induction (scale bar, 10 μm *** P < 0.001).

[0077] [Figure 9A]Figure 9 shows that mitophagy is reduced in vivo in the absence of peroxiredoxin 3 (PRDX3). Figures 9A to 9C show representative images and quantification 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 (liver and skeletal muscle) or 50 μm (brain)). Figure 9D shows representative images of infarcted hearts and quantification graphs of mitophagy 4 hours after induction of myocardial infarction (MI) in PRDX3 wild-type (PRDX3 WT, n=6), PRDX3 knockout (PRDX3 KO, n=5), PRDX3 WT (n=6), and PRDX3 KO mt-Keima mice (n=6) (scale bar, 10 μm). Figure 9E shows representative confocal images showing the level of mitophagy in adult fat bodies expressing white RNAi or Prx3RNAi (da-GAL4>mt-Keima; white RNAi, da-GAL4>mt-Keima; Prx3RNAi) and in the wing discs of mt-Keima-Kei. Mitophagy levels were quantified using multiple tissue samples (e.g., [white RNAi], n=8; adult fat body [white RNAi], n=8; larval [Prx3RNAi], n=9; adult fat body [Prx3RNAi], n=5) (scale bar, 20 or 50 μm; **P<0.01). [Figure 9B]Figure 9 shows that mitophagy is reduced in vivo in the absence of peroxiredoxin 3 (PRDX3). Figures 9A to 9C show representative images and quantification 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 (liver and skeletal muscle) or 50 μm (brain)). Figure 9D shows representative images of infarcted hearts and quantification graphs of mitophagy 4 hours after induction of myocardial infarction (MI) in PRDX3 wild-type (PRDX3 WT, n=6), PRDX3 knockout (PRDX3 KO, n=5), PRDX3 WT (n=6), and PRDX3 KO mt-Keima mice (n=6) (scale bar, 10 μm). Figure 9E shows representative confocal images showing the level of mitophagy in adult fat bodies expressing white RNAi or Prx3RNAi (da-GAL4>mt-Keima; white RNAi, da-GAL4>mt-Keima; Prx3RNAi) and in the wing discs of mt-Keima-Kei. Mitophagy levels were quantified using multiple tissue samples (e.g., [white RNAi], n=8; adult fat body [white RNAi], n=8; larval [Prx3RNAi], n=9; adult fat body [Prx3RNAi], n=5) (scale bar, 20 or 50 μm; **P<0.01). [Figure 9C]Figure 9 shows that mitophagy is reduced in vivo in the absence of peroxiredoxin 3 (PRDX3). Figures 9A to 9C show representative images and quantification 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 (liver and skeletal muscle) or 50 μm (brain)). Figure 9D shows representative images of infarcted hearts and quantification graphs of mitophagy 4 hours after induction of myocardial infarction (MI) in PRDX3 wild-type (PRDX3 WT, n=6), PRDX3 knockout (PRDX3 KO, n=5), PRDX3 WT (n=6), and PRDX3 KO mt-Keima mice (n=6) (scale bar, 10 μm). Figure 9E shows representative confocal images showing the level of mitophagy in adult fat bodies expressing white RNAi or Prx3RNAi (da-GAL4>mt-Keima; white RNAi, da-GAL4>mt-Keima; Prx3RNAi) and in the wing discs of mt-Keima-Kei. Mitophagy levels were quantified using multiple tissue samples (e.g., [white RNAi], n=8; adult fat body [white RNAi], n=8; larval [Prx3RNAi], n=9; adult fat body [Prx3RNAi], n=5) (scale bar, 20 or 50 μm; **P<0.01). [Figure 9D]Figure 9 shows that mitophagy is reduced in vivo in the absence of peroxiredoxin 3 (PRDX3). Figures 9A to 9C show representative images and quantification 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 (liver and skeletal muscle) or 50 μm (brain)). Figure 9D shows representative images of infarcted hearts and quantification graphs of mitophagy 4 hours after induction of myocardial infarction (MI) in PRDX3 wild-type (PRDX3 WT, n=6), PRDX3 knockout (PRDX3 KO, n=5), PRDX3 WT (n=6), and PRDX3 KO mt-Keima mice (n=6) (scale bar, 10 μm). Figure 9E shows representative confocal images showing the level of mitophagy in adult fat bodies expressing white RNAi or Prx3RNAi (da-GAL4>mt-Keima; white RNAi, da-GAL4>mt-Keima; Prx3RNAi) and in the wing discs of mt-Keima-Kei. Mitophagy levels were quantified using multiple tissue samples (e.g., [white RNAi], n=8; adult fat body [white RNAi], n=8; larval [Prx3RNAi], n=9; adult fat body [Prx3RNAi], n=5) (scale bar, 20 or 50 μm; **P<0.01). [Figure 9E]Figure 9 shows that mitophagy is reduced in vivo in the absence of peroxiredoxin 3 (PRDX3). Figures 9A to 9C show representative images and quantification 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 (liver and skeletal muscle) or 50 μm (brain)). Figure 9D shows representative images of infarcted hearts and quantification graphs of mitophagy 4 hours after induction of myocardial infarction (MI) in PRDX3 wild-type (PRDX3 WT, n=6), PRDX3 knockout (PRDX3 KO, n=5), PRDX3 WT (n=6), and PRDX3 KO mt-Keima mice (n=6) (scale bar, 10 μm). Figure 9E shows representative confocal images showing the level of mitophagy in adult fat bodies expressing white RNAi or Prx3RNAi (da-GAL4>mt-Keima; white RNAi, da-GAL4>mt-Keima; Prx3RNAi) and in the wing discs of mt-Keima-Kei. Mitophagy levels were quantified using multiple tissue samples (e.g., [white RNAi], n=8; adult fat body [white RNAi], n=8; larval [Prx3RNAi], n=9; adult fat body [Prx3RNAi], n=5) (scale bar, 20 or 50 μm; **P<0.01).

[0078] [Figure 10A]Figure 10 shows confirmation that peroxiredoxin 3 (PRDX3) is required for 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 the mitochondrial fraction of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mouse embryonic fibroblasts (MEFs) treated with DMSO, 20 μM MG132, or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The mitochondrial fraction was immunoblotted with PINK1 and Tom20 antibodies. Figure 10C shows the results of immunostaining of PRDX3 WT and PRDX3 KO MEFs infected with PINK1-GFP using Tom20 antibody. In the graphs, the dashed lines indicate the measured fluorescence intensities of Tom20 and PINK1-GFP (scale bars, 1 μm (box area) or 10 μm). Figures 10D and 10E show the results of Western blot analysis (Figure 10D) and quantification graphs (Figure 10E) of Pink1 expression in the mitochondrial fractions of PRDX3 WT 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 immunoblotted with PINK1 and Tom20 antibodies. Hashes show nonspecific bands. Figures 10F–10I show the results of immunostaining and quantification graphs of PINK1-GFP (Figures 10F and 10G) and Parkin-GFP levels (Figures 10H and 10I) in PRDX3 WT and PRDX3 KO MEF. MEFs were infected with mitochondrial-targeting catalase adenovirus for 24 hours, transfected with PINK1-GFP or Parkin-GFP, treated with DMSO or 10 μM CCCP for 4 hours, and then immunostained with Tom20 antibody (scale bar, 10 μm * P < 0.05 and ** P < 0.01). Data (Figures 10A-10I) show three independent experiments. [Figure 10B]Figure 10 shows confirmation that peroxiredoxin 3 (PRDX3) is required for 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 the mitochondrial fraction of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mouse embryonic fibroblasts (MEFs) treated with DMSO, 20 μM MG132, or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The mitochondrial fraction was immunoblotted with PINK1 and Tom20 antibodies. Figure 10C shows the results of immunostaining of PRDX3 WT and PRDX3 KO MEFs infected with PINK1-GFP using Tom20 antibody. In the graphs, the dashed lines indicate the measured fluorescence intensities of Tom20 and PINK1-GFP (scale bars, 1 μm (box area) or 10 μm). Figures 10D and 10E show the results of Western blot analysis (Figure 10D) and quantification graphs (Figure 10E) of Pink1 expression in the mitochondrial fractions of PRDX3 WT 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 immunoblotted with PINK1 and Tom20 antibodies. Hashes show nonspecific bands. Figures 10F–10I show the results of immunostaining and quantification graphs of PINK1-GFP (Figures 10F and 10G) and Parkin-GFP levels (Figures 10H and 10I) in PRDX3 WT and PRDX3 KO MEF. MEFs were infected with mitochondrial-targeting catalase adenovirus for 24 hours, transfected with PINK1-GFP or Parkin-GFP, treated with DMSO or 10 μM CCCP for 4 hours, and then immunostained with Tom20 antibody (scale bar, 10 μm * P < 0.05 and ** P < 0.01). Data (Figures 10A-10I) show three independent experiments. [Figure 10C]Figure 10 shows confirmation that peroxiredoxin 3 (PRDX3) is required for 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 the mitochondrial fraction of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mouse embryonic fibroblasts (MEFs) treated with DMSO, 20 μM MG132, or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The mitochondrial fraction was immunoblotted with PINK1 and Tom20 antibodies. Figure 10C shows the results of immunostaining of PRDX3 WT and PRDX3 KO MEFs infected with PINK1-GFP using Tom20 antibody. In the graphs, the dashed lines indicate the measured fluorescence intensities of Tom20 and PINK1-GFP (scale bars, 1 μm (box area) or 10 μm). Figures 10D and 10E show the results of Western blot analysis (Figure 10D) and quantification graphs (Figure 10E) of Pink1 expression in the mitochondrial fractions of PRDX3 WT 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 immunoblotted with PINK1 and Tom20 antibodies. Hashes show nonspecific bands. Figures 10F–10I show the results of immunostaining and quantification graphs of PINK1-GFP (Figures 10F and 10G) and Parkin-GFP levels (Figures 10H and 10I) in PRDX3 WT and PRDX3 KO MEF. MEFs were infected with mitochondrial-targeting catalase adenovirus for 24 hours, transfected with PINK1-GFP or Parkin-GFP, treated with DMSO or 10 μM CCCP for 4 hours, and then immunostained with Tom20 antibody (scale bar, 10 μm * P < 0.05 and ** P < 0.01). Data (Figures 10A-10I) show three independent experiments. [Figure 10D]Figure 10 shows confirmation that peroxiredoxin 3 (PRDX3) is required for 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 the mitochondrial fraction of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mouse embryonic fibroblasts (MEFs) treated with DMSO, 20 μM MG132, or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The mitochondrial fraction was immunoblotted with PINK1 and Tom20 antibodies. Figure 10C shows the results of immunostaining of PRDX3 WT and PRDX3 KO MEFs infected with PINK1-GFP using Tom20 antibody. In the graphs, the dashed lines indicate the measured fluorescence intensities of Tom20 and PINK1-GFP (scale bars, 1 μm (box area) or 10 μm). Figures 10D and 10E show the results of Western blot analysis (Figure 10D) and quantification graphs (Figure 10E) of Pink1 expression in the mitochondrial fractions of PRDX3 WT 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 immunoblotted with PINK1 and Tom20 antibodies. Hashes show nonspecific bands. Figures 10F–10I show the results of immunostaining and quantification graphs of PINK1-GFP (Figures 10F and 10G) and Parkin-GFP levels (Figures 10H and 10I) in PRDX3 WT and PRDX3 KO MEF. MEFs were infected with mitochondrial-targeting catalase adenovirus for 24 hours, transfected with PINK1-GFP or Parkin-GFP, treated with DMSO or 10 μM CCCP for 4 hours, and then immunostained with Tom20 antibody (scale bar, 10 μm * P < 0.05 and ** P < 0.01). Data (Figures 10A-10I) show three independent experiments. [Figure 10E]Figure 10 shows confirmation that peroxiredoxin 3 (PRDX3) is required for 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 the mitochondrial fraction of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mouse embryonic fibroblasts (MEFs) treated with DMSO, 20 μM MG132, or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The mitochondrial fraction was immunoblotted with PINK1 and Tom20 antibodies. Figure 10C shows the results of immunostaining of PRDX3 WT and PRDX3 KO MEFs infected with PINK1-GFP using Tom20 antibody. In the graphs, the dashed lines indicate the measured fluorescence intensities of Tom20 and PINK1-GFP (scale bars, 1 μm (box area) or 10 μm). Figures 10D and 10E show the results of Western blot analysis (Figure 10D) and quantification graphs (Figure 10E) of Pink1 expression in the mitochondrial fractions of PRDX3 WT 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 immunoblotted with PINK1 and Tom20 antibodies. Hashes show nonspecific bands. Figures 10F–10I show the results of immunostaining and quantification graphs of PINK1-GFP (Figures 10F and 10G) and Parkin-GFP levels (Figures 10H and 10I) in PRDX3 WT and PRDX3 KO MEF. MEFs were infected with mitochondrial-targeting catalase adenovirus for 24 hours, transfected with PINK1-GFP or Parkin-GFP, treated with DMSO or 10 μM CCCP for 4 hours, and then immunostained with Tom20 antibody (scale bar, 10 μm * P < 0.05 and ** P < 0.01). Data (Figures 10A-10I) show three independent experiments. [Figure 10F]Figure 10 shows confirmation that peroxiredoxin 3 (PRDX3) is required for 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 the mitochondrial fraction of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mouse embryonic fibroblasts (MEFs) treated with DMSO, 20 μM MG132, or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The mitochondrial fraction was immunoblotted with PINK1 and Tom20 antibodies. Figure 10C shows the results of immunostaining of PRDX3 WT and PRDX3 KO MEFs infected with PINK1-GFP using Tom20 antibody. In the graphs, the dashed lines indicate the measured fluorescence intensities of Tom20 and PINK1-GFP (scale bars, 1 μm (box area) or 10 μm). Figures 10D and 10E show the results of Western blot analysis (Figure 10D) and quantification graphs (Figure 10E) of Pink1 expression in the mitochondrial fractions of PRDX3 WT 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 immunoblotted with PINK1 and Tom20 antibodies. Hashes show nonspecific bands. Figures 10F–10I show the results of immunostaining and quantification graphs of PINK1-GFP (Figures 10F and 10G) and Parkin-GFP levels (Figures 10H and 10I) in PRDX3 WT and PRDX3 KO MEF. MEFs were infected with mitochondrial-targeting catalase adenovirus for 24 hours, transfected with PINK1-GFP or Parkin-GFP, treated with DMSO or 10 μM CCCP for 4 hours, and then immunostained with Tom20 antibody (scale bar, 10 μm * P < 0.05 and ** P < 0.01). Data (Figures 10A-10I) show three independent experiments. [Figure 10G]Figure 10 shows confirmation that peroxiredoxin 3 (PRDX3) is required for 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 the mitochondrial fraction of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mouse embryonic fibroblasts (MEFs) treated with DMSO, 20 μM MG132, or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The mitochondrial fraction was immunoblotted with PINK1 and Tom20 antibodies. Figure 10C shows the results of immunostaining of PRDX3 WT and PRDX3 KO MEFs infected with PINK1-GFP using Tom20 antibody. In the graphs, the dashed lines indicate the measured fluorescence intensities of Tom20 and PINK1-GFP (scale bars, 1 μm (box area) or 10 μm). Figures 10D and 10E show the results of Western blot analysis (Figure 10D) and quantification graphs (Figure 10E) of Pink1 expression in the mitochondrial fractions of PRDX3 WT 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 immunoblotted with PINK1 and Tom20 antibodies. Hashes show nonspecific bands. Figures 10F–10I show the results of immunostaining and quantification graphs of PINK1-GFP (Figures 10F and 10G) and Parkin-GFP levels (Figures 10H and 10I) in PRDX3 WT and PRDX3 KO MEF. MEFs were infected with mitochondrial-targeting catalase adenovirus for 24 hours, transfected with PINK1-GFP or Parkin-GFP, treated with DMSO or 10 μM CCCP for 4 hours, and then immunostained with Tom20 antibody (scale bar, 10 μm * P < 0.05 and ** P < 0.01). Data (Figures 10A-10I) show three independent experiments. [Figure 10H]Figure 10 shows confirmation that peroxiredoxin 3 (PRDX3) is required for 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 the mitochondrial fraction of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mouse embryonic fibroblasts (MEFs) treated with DMSO, 20 μM MG132, or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The mitochondrial fraction was immunoblotted with PINK1 and Tom20 antibodies. Figure 10C shows the results of immunostaining of PRDX3 WT and PRDX3 KO MEFs infected with PINK1-GFP using Tom20 antibody. In the graphs, the dashed lines indicate the measured fluorescence intensities of Tom20 and PINK1-GFP (scale bars, 1 μm (box area) or 10 μm). Figures 10D and 10E show the results of Western blot analysis (Figure 10D) and quantification graphs (Figure 10E) of Pink1 expression in the mitochondrial fractions of PRDX3 WT 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 immunoblotted with PINK1 and Tom20 antibodies. Hashes show nonspecific bands. Figures 10F–10I show the results of immunostaining and quantification graphs of PINK1-GFP (Figures 10F and 10G) and Parkin-GFP levels (Figures 10H and 10I) in PRDX3 WT and PRDX3 KO MEF. MEFs were infected with mitochondrial-targeting catalase adenovirus for 24 hours, transfected with PINK1-GFP or Parkin-GFP, treated with DMSO or 10 μM CCCP for 4 hours, and then immunostained with Tom20 antibody (scale bar, 10 μm * P < 0.05 and ** P < 0.01). Data (Figures 10A-10I) show three independent experiments. [Figure 10I]Figure 10 shows confirmation that peroxiredoxin 3 (PRDX3) is required for 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 the mitochondrial fraction of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mouse embryonic fibroblasts (MEFs) treated with DMSO, 20 μM MG132, or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The mitochondrial fraction was immunoblotted with PINK1 and Tom20 antibodies. Figure 10C shows the results of immunostaining of PRDX3 WT and PRDX3 KO MEFs infected with PINK1-GFP using Tom20 antibody. In the graphs, the dashed lines indicate the measured fluorescence intensities of Tom20 and PINK1-GFP (scale bars, 1 μm (box area) or 10 μm). Figures 10D and 10E show the results of Western blot analysis (Figure 10D) and quantification graphs (Figure 10E) of Pink1 expression in the mitochondrial fractions of PRDX3 WT 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 immunoblotted with PINK1 and Tom20 antibodies. Hashes show nonspecific bands. Figures 10F–10I show the results of immunostaining and quantification graphs of PINK1-GFP (Figures 10F and 10G) and Parkin-GFP levels (Figures 10H and 10I) in PRDX3 WT and PRDX3 KO MEF. MEFs were infected with mitochondrial-targeting catalase adenovirus for 24 hours, transfected with PINK1-GFP or Parkin-GFP, treated with DMSO or 10 μM CCCP for 4 hours, and then immunostained with Tom20 antibody (scale bar, 10 μm * P < 0.05 and ** P < 0.01). Data (Figures 10A-10I) show three independent experiments.

[0079] [Figure 11A]Figure 11 shows that peroxiredoxin 3 (PRDX3) modulates the recruitment of PINK1 at the outer mitochondrial membrane (OMM) via substrate and PINK1-Parkin mediated mitophagy. Figure 11A shows the results of Western blot analysis and quantification of Pink1 levels in cell lysates of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mouse embryonic fibroblasts (MEFs) treated with DMSO, 20 μM MG132, or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The cell lysates were immunoblotted with antibodies against PINK1 and tubulin (*P<0.05 and **P<0.01). Figure 11B shows the results of immunostaining of control or PRDX3 siRNA-transfected HeLa cells using Tom20 antibody. In the graph, the dashed lines indicate the measured fluorescence intensities of Tom20 and PINK1-GFP. Figure 11C shows immunostaining of PRDX3 WT and PRDX3 KO MEF cells transfected with PINK1-GFP or Parkin-GFP and treated with DMSO or 2.5 μM oligomycin / 250 nM antimycin (OA) using Tom20 antibody (scale bar, 10 μm). Figure 11D shows immunostaining results of PRDX3 siRNA or control siRNA-treated HeLa cells transfected with PINK1-GFP or Parkin-GFP and treated with DMSO or 10 μM CCCP for 4 hours using Tom20 antibody (scale bar, 10 μm, box area scale bar, 1 μm). [Figure 11B]Figure 11 shows that peroxiredoxin 3 (PRDX3) modulates the recruitment of PINK1 at the outer mitochondrial membrane (OMM) via substrate and PINK1-Parkin mediated mitophagy. Figure 11A shows the results of Western blot analysis and quantification of Pink1 levels in cell lysates of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mouse embryonic fibroblasts (MEFs) treated with DMSO, 20 μM MG132, or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The cell lysates were immunoblotted with antibodies against PINK1 and tubulin (*P<0.05 and **P<0.01). Figure 11B shows the results of immunostaining of control or PRDX3 siRNA-transfected HeLa cells using Tom20 antibody. In the graph, the dashed lines indicate the measured fluorescence intensities of Tom20 and PINK1-GFP. Figure 11C shows immunostaining of PRDX3 WT and PRDX3 KO MEF cells transfected with PINK1-GFP or Parkin-GFP and treated with DMSO or 2.5 μM oligomycin / 250 nM antimycin (OA) using Tom20 antibody (scale bar, 10 μm). Figure 11D shows immunostaining results of PRDX3 siRNA or control siRNA-treated HeLa cells transfected with PINK1-GFP or Parkin-GFP and treated with DMSO or 10 μM CCCP for 4 hours using Tom20 antibody (scale bar, 10 μm, box area scale bar, 1 μm). [Figure 11C]Figure 11 shows that peroxiredoxin 3 (PRDX3) modulates the recruitment of PINK1 at the outer mitochondrial membrane (OMM) via substrate and PINK1-Parkin mediated mitophagy. Figure 11A shows the results of Western blot analysis and quantification of Pink1 levels in cell lysates of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mouse embryonic fibroblasts (MEFs) treated with DMSO, 20 μM MG132, or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The cell lysates were immunoblotted with antibodies against PINK1 and tubulin (*P<0.05 and **P<0.01). Figure 11B shows the results of immunostaining of control or PRDX3 siRNA-transfected HeLa cells using Tom20 antibody. In the graph, the dashed lines indicate the measured fluorescence intensities of Tom20 and PINK1-GFP. Figure 11C shows immunostaining of PRDX3 WT and PRDX3 KO MEF cells transfected with PINK1-GFP or Parkin-GFP and treated with DMSO or 2.5 μM oligomycin / 250 nM antimycin (OA) using Tom20 antibody (scale bar, 10 μm). Figure 11D shows immunostaining results of PRDX3 siRNA or control siRNA-treated HeLa cells transfected with PINK1-GFP or Parkin-GFP and treated with DMSO or 10 μM CCCP for 4 hours using Tom20 antibody (scale bar, 10 μm, box area scale bar, 1 μm). [Figure 11D]Figure 11 shows that peroxiredoxin 3 (PRDX3) modulates the recruitment of PINK1 at the outer mitochondrial membrane (OMM) via substrate and PINK1-Parkin mediated mitophagy. Figure 11A shows the results of Western blot analysis and quantification of Pink1 levels in cell lysates of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) mouse embryonic fibroblasts (MEFs) treated with DMSO, 20 μM MG132, or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The cell lysates were immunoblotted with antibodies against PINK1 and tubulin (*P<0.05 and **P<0.01). Figure 11B shows the results of immunostaining of control or PRDX3 siRNA-transfected HeLa cells using Tom20 antibody. In the graph, the dashed lines indicate the measured fluorescence intensities of Tom20 and PINK1-GFP. Figure 11C shows immunostaining of PRDX3 WT and PRDX3 KO MEF cells transfected with PINK1-GFP or Parkin-GFP and treated with DMSO or 2.5 μM oligomycin / 250 nM antimycin (OA) using Tom20 antibody (scale bar, 10 μm). Figure 11D shows immunostaining results of PRDX3 siRNA or control siRNA-treated HeLa cells transfected with PINK1-GFP or Parkin-GFP and treated with DMSO or 10 μM CCCP for 4 hours using Tom20 antibody (scale bar, 10 μm, box area scale bar, 1 μm).

[0080] [Figure 12A]Figure 12 shows evidence of peroxiredoxin 3 (PRDX3) interacting with PINK1. Figure 12A shows evidence of interaction between endogenous PINK1 and PRDX3. Whole cell lysates (WCL) of 293T cells were immunoprecipitated with control immunoglobulin G (IgG) and anti-PRDX3 antibody, and then blotted with anti-PINK1 antibody. Figure 12B shows subsequent immunoblotting results of WCL of 293T cells co-transfected with diverse combinations of constructs expressing PINK1-GFP, PRDX3-Myc, L53VPRDX3-Myc, and PRDX3-Myc dominant-negative (DN;Cys108Ser, Cys229Ser) using anti-Myc beads and GFP-specific antibody. Hashes indicate IgG. Figure 12C shows the results of subsequent immunoblotting of WCLs of 293T cells co-transfected with various combinations of constructs expressing PINK1-GFP, A93VPINK1-GFP, and PRDX3-Myc, using anti-Myc beads and GFP-specific antibodies. The hash indicates IgG. Figure 12D shows the results of subsequent immunoblotting of WCLs of 293T cells transfected with a construct expressing PINK1-GFP, using immunoprecipitation with GST1-40PRDX3, GST1-40PRDX3L53V, or GST1-40PRDX3DN-conjugated beads and PINK1 or GFP-specific antibodies. The hash indicates nonspecific bands. Figure 12E shows WCLs of 293T cells transfected with constructs expressing PINK1-GFP or A93VPINK1-GFP, used for immunoprecipitation with GST1-40PRDX3 and subsequent immunoblotting with PINK1 or GFP-specific antibodies. The hash shows a nonspecific band. Figure 12F shows WCLs of 293T cells transfected with Myc-His, 63-256PRDX3-Myc-His, or 1-256PRDX3-Myc-His expression vectors, used for immunoblotting with PRDX3 and tubulin antibodies.Figure 12G shows the mass spectrometry analysis of 1-256PRDX3-Myc-His (box in Figure 12F) and the predicted MPP cleavage sites of the immature sequence (residues 1-36 of the PRDX3 mitochondrial targeting sequence) and mature PRDX3. The data (Figures 12A-12E) show three independent experiments. [Figure 12B]Figure 12 shows evidence of peroxiredoxin 3 (PRDX3) interacting with PINK1. Figure 12A shows evidence of interaction between endogenous PINK1 and PRDX3. Whole cell lysates (WCL) of 293T cells were immunoprecipitated with control immunoglobulin G (IgG) and anti-PRDX3 antibody, and then blotted with anti-PINK1 antibody. Figure 12B shows subsequent immunoblotting results of WCL of 293T cells co-transfected with diverse combinations of constructs expressing PINK1-GFP, PRDX3-Myc, L53VPRDX3-Myc, and PRDX3-Myc dominant-negative (DN;Cys108Ser, Cys229Ser) using anti-Myc beads and GFP-specific antibody. Hashes indicate IgG. Figure 12C shows the results of subsequent immunoblotting of WCLs of 293T cells co-transfected with various combinations of constructs expressing PINK1-GFP, A93VPINK1-GFP, and PRDX3-Myc, using anti-Myc beads and GFP-specific antibodies. The hash indicates IgG. Figure 12D shows the results of subsequent immunoblotting of WCLs of 293T cells transfected with a construct expressing PINK1-GFP, using immunoprecipitation with GST1-40PRDX3, GST1-40PRDX3L53V, or GST1-40PRDX3DN-conjugated beads and PINK1 or GFP-specific antibodies. The hash indicates nonspecific bands. Figure 12E shows WCLs of 293T cells transfected with constructs expressing PINK1-GFP or A93VPINK1-GFP, used for immunoprecipitation with GST1-40PRDX3 and subsequent immunoblotting with PINK1 or GFP-specific antibodies. The hash shows a nonspecific band. Figure 12F shows WCLs of 293T cells transfected with Myc-His, 63-256PRDX3-Myc-His, or 1-256PRDX3-Myc-His expression vectors, used for immunoblotting with PRDX3 and tubulin antibodies.Figure 12G shows the mass spectrometry analysis of 1-256PRDX3-Myc-His (box in Figure 12F) and the predicted MPP cleavage sites of the immature sequence (residues 1-36 of the PRDX3 mitochondrial targeting sequence) and mature PRDX3. The data (Figures 12A-12E) show three independent experiments. [Figure 12C]Figure 12 shows evidence of peroxiredoxin 3 (PRDX3) interacting with PINK1. Figure 12A shows evidence of interaction between endogenous PINK1 and PRDX3. Whole cell lysates (WCL) of 293T cells were immunoprecipitated with control immunoglobulin G (IgG) and anti-PRDX3 antibody, and then blotted with anti-PINK1 antibody. Figure 12B shows subsequent immunoblotting results of WCL of 293T cells co-transfected with diverse combinations of constructs expressing PINK1-GFP, PRDX3-Myc, L53VPRDX3-Myc, and PRDX3-Myc dominant-negative (DN;Cys108Ser, Cys229Ser) using anti-Myc beads and GFP-specific antibody. Hashes indicate IgG. Figure 12C shows the results of subsequent immunoblotting of WCLs of 293T cells co-transfected with various combinations of constructs expressing PINK1-GFP, A93VPINK1-GFP, and PRDX3-Myc, using anti-Myc beads and GFP-specific antibodies. The hash indicates IgG. Figure 12D shows the results of subsequent immunoblotting of WCLs of 293T cells transfected with a construct expressing PINK1-GFP, using immunoprecipitation with GST1-40PRDX3, GST1-40PRDX3L53V, or GST1-40PRDX3DN-conjugated beads and PINK1 or GFP-specific antibodies. The hash indicates nonspecific bands. Figure 12E shows WCLs of 293T cells transfected with constructs expressing PINK1-GFP or A93VPINK1-GFP, used for immunoprecipitation with GST1-40PRDX3 and subsequent immunoblotting with PINK1 or GFP-specific antibodies. The hash shows a nonspecific band. Figure 12F shows WCLs of 293T cells transfected with Myc-His, 63-256PRDX3-Myc-His, or 1-256PRDX3-Myc-His expression vectors, used for immunoblotting with PRDX3 and tubulin antibodies.Figure 12G shows the mass spectrometry analysis of 1-256PRDX3-Myc-His (box in Figure 12F) and the predicted MPP cleavage sites of the immature sequence (residues 1-36 of the PRDX3 mitochondrial targeting sequence) and mature PRDX3. The data (Figures 12A-12E) show three independent experiments. [Figure 12D]Figure 12 shows evidence of peroxiredoxin 3 (PRDX3) interacting with PINK1. Figure 12A shows evidence of interaction between endogenous PINK1 and PRDX3. Whole cell lysates (WCL) of 293T cells were immunoprecipitated with control immunoglobulin G (IgG) and anti-PRDX3 antibody, and then blotted with anti-PINK1 antibody. Figure 12B shows subsequent immunoblotting results of WCL of 293T cells co-transfected with diverse combinations of constructs expressing PINK1-GFP, PRDX3-Myc, L53VPRDX3-Myc, and PRDX3-Myc dominant-negative (DN;Cys108Ser, Cys229Ser) using anti-Myc beads and GFP-specific antibody. Hashes indicate IgG. Figure 12C shows the results of subsequent immunoblotting of WCLs of 293T cells co-transfected with various combinations of constructs expressing PINK1-GFP, A93VPINK1-GFP, and PRDX3-Myc, using anti-Myc beads and GFP-specific antibodies. The hash indicates IgG. Figure 12D shows the results of subsequent immunoblotting of WCLs of 293T cells transfected with a construct expressing PINK1-GFP, using immunoprecipitation with GST1-40PRDX3, GST1-40PRDX3L53V, or GST1-40PRDX3DN-conjugated beads and PINK1 or GFP-specific antibodies. The hash indicates nonspecific bands. Figure 12E shows WCLs of 293T cells transfected with constructs expressing PINK1-GFP or A93VPINK1-GFP, used for immunoprecipitation with GST1-40PRDX3 and subsequent immunoblotting with PINK1 or GFP-specific antibodies. The hash shows a nonspecific band. Figure 12F shows WCLs of 293T cells transfected with Myc-His, 63-256PRDX3-Myc-His, or 1-256PRDX3-Myc-His expression vectors, used for immunoblotting with PRDX3 and tubulin antibodies.Figure 12G shows the mass spectrometry analysis of 1-256PRDX3-Myc-His (box in Figure 12F) and the predicted MPP cleavage sites of the immature sequence (residues 1-36 of the PRDX3 mitochondrial targeting sequence) and mature PRDX3. The data (Figures 12A-12E) show three independent experiments. [Figure 12E]Figure 12 shows evidence of peroxiredoxin 3 (PRDX3) interacting with PINK1. Figure 12A shows evidence of interaction between endogenous PINK1 and PRDX3. Whole cell lysates (WCL) of 293T cells were immunoprecipitated with control immunoglobulin G (IgG) and anti-PRDX3 antibody, and then blotted with anti-PINK1 antibody. Figure 12B shows subsequent immunoblotting results of WCL of 293T cells co-transfected with diverse combinations of constructs expressing PINK1-GFP, PRDX3-Myc, L53VPRDX3-Myc, and PRDX3-Myc dominant-negative (DN;Cys108Ser, Cys229Ser) using anti-Myc beads and GFP-specific antibody. Hashes indicate IgG. Figure 12C shows the results of subsequent immunoblotting of WCLs of 293T cells co-transfected with various combinations of constructs expressing PINK1-GFP, A93VPINK1-GFP, and PRDX3-Myc, using anti-Myc beads and GFP-specific antibodies. The hash indicates IgG. Figure 12D shows the results of subsequent immunoblotting of WCLs of 293T cells transfected with a construct expressing PINK1-GFP, using immunoprecipitation with GST1-40PRDX3, GST1-40PRDX3L53V, or GST1-40PRDX3DN-conjugated beads and PINK1 or GFP-specific antibodies. The hash indicates nonspecific bands. Figure 12E shows WCLs of 293T cells transfected with constructs expressing PINK1-GFP or A93VPINK1-GFP, used for immunoprecipitation with GST1-40PRDX3 and subsequent immunoblotting with PINK1 or GFP-specific antibodies. The hash shows a nonspecific band. Figure 12F shows WCLs of 293T cells transfected with Myc-His, 63-256PRDX3-Myc-His, or 1-256PRDX3-Myc-His expression vectors, used for immunoblotting with PRDX3 and tubulin antibodies.Figure 12G shows the mass spectrometry analysis of 1-256PRDX3-Myc-His (box in Figure 12F) and the predicted MPP cleavage sites of the immature sequence (residues 1-36 of the PRDX3 mitochondrial targeting sequence) and mature PRDX3. The data (Figures 12A-12E) show three independent experiments. [Figure 12F]Figure 12 shows evidence of peroxiredoxin 3 (PRDX3) interacting with PINK1. Figure 12A shows evidence of interaction between endogenous PINK1 and PRDX3. Whole cell lysates (WCL) of 293T cells were immunoprecipitated with control immunoglobulin G (IgG) and anti-PRDX3 antibody, and then blotted with anti-PINK1 antibody. Figure 12B shows subsequent immunoblotting results of WCL of 293T cells co-transfected with diverse combinations of constructs expressing PINK1-GFP, PRDX3-Myc, L53VPRDX3-Myc, and PRDX3-Myc dominant-negative (DN;Cys108Ser, Cys229Ser) using anti-Myc beads and GFP-specific antibody. Hashes indicate IgG. Figure 12C shows the results of subsequent immunoblotting of WCLs of 293T cells co-transfected with various combinations of constructs expressing PINK1-GFP, A93VPINK1-GFP, and PRDX3-Myc, using anti-Myc beads and GFP-specific antibodies. The hash indicates IgG. Figure 12D shows the results of subsequent immunoblotting of WCLs of 293T cells transfected with a construct expressing PINK1-GFP, using immunoprecipitation with GST1-40PRDX3, GST1-40PRDX3L53V, or GST1-40PRDX3DN-conjugated beads and PINK1 or GFP-specific antibodies. The hash indicates nonspecific bands. Figure 12E shows WCLs of 293T cells transfected with constructs expressing PINK1-GFP or A93VPINK1-GFP, used for immunoprecipitation with GST1-40PRDX3 and subsequent immunoblotting with PINK1 or GFP-specific antibodies. The hash shows a nonspecific band. Figure 12F shows WCLs of 293T cells transfected with Myc-His, 63-256PRDX3-Myc-His, or 1-256PRDX3-Myc-His expression vectors, used for immunoblotting with PRDX3 and tubulin antibodies.Figure 12G shows the mass spectrometry analysis of 1-256PRDX3-Myc-His (box in Figure 12F) and the predicted MPP cleavage sites of the immature sequence (residues 1-36 of the PRDX3 mitochondrial targeting sequence) and mature PRDX3. The data (Figures 12A-12E) show three independent experiments. [Figure 12G]Figure 12 shows evidence of peroxiredoxin 3 (PRDX3) interacting with PINK1. Figure 12A shows evidence of interaction between endogenous PINK1 and PRDX3. Whole cell lysates (WCL) of 293T cells were immunoprecipitated with control immunoglobulin G (IgG) and anti-PRDX3 antibody, and then blotted with anti-PINK1 antibody. Figure 12B shows subsequent immunoblotting results of WCL of 293T cells co-transfected with diverse combinations of constructs expressing PINK1-GFP, PRDX3-Myc, L53VPRDX3-Myc, and PRDX3-Myc dominant-negative (DN;Cys108Ser, Cys229Ser) using anti-Myc beads and GFP-specific antibody. Hashes indicate IgG. Figure 12C shows the results of subsequent immunoblotting of WCLs of 293T cells co-transfected with various combinations of constructs expressing PINK1-GFP, A93VPINK1-GFP, and PRDX3-Myc, using anti-Myc beads and GFP-specific antibodies. The hash indicates IgG. Figure 12D shows the results of subsequent immunoblotting of WCLs of 293T cells transfected with a construct expressing PINK1-GFP, using immunoprecipitation with GST1-40PRDX3, GST1-40PRDX3L53V, or GST1-40PRDX3DN-conjugated beads and PINK1 or GFP-specific antibodies. The hash indicates nonspecific bands. Figure 12E shows WCLs of 293T cells transfected with constructs expressing PINK1-GFP or A93VPINK1-GFP, used for immunoprecipitation with GST1-40PRDX3 and subsequent immunoblotting with PINK1 or GFP-specific antibodies. The hash shows a nonspecific band. Figure 12F shows WCLs of 293T cells transfected with Myc-His, 63-256PRDX3-Myc-His, or 1-256PRDX3-Myc-His expression vectors, used for immunoblotting with PRDX3 and tubulin antibodies.Figure 12G shows the mass spectrometry analysis of 1-256PRDX3-Myc-His (box in Figure 12F) and the predicted MPP cleavage sites of the immature sequence (residues 1-36 of the PRDX3 mitochondrial targeting sequence) and mature PRDX3. The data (Figures 12A-12E) show three independent experiments.

[0081] [Figure 13A] Figure 13 shows the PINK1 domain mapping essential for binding to peroxiredoxin 3 (PRDX3). In Figure 13A, the domain boundaries of full-length PINK1 and cleaved PINK1 and full-length PRDX3 are shown on the left and right, respectively. The PINK1 domain consists of a mitochondrial target sequence (MTS), a transmembrane domain (TMD), and a kinase domain. PRDX3 interacts with the N-terminus of PINK1(1-94), including the MTS. Figure 13B shows the WCL and PRDX3-Myc of 293T cells co-transfected with diverse combinations of constructs expressing 287-581PINK1-GFP, 1-286PINK1-GFP, 1-156PINK1-GFP, 1-110PINK1-GFP, and 1-94PINK1-GFP, followed by immunoblotting results using anti-GFP beads and Myc-specific antibodies. Hashes indicate IgG. Figure 13C shows subsequent immunoblotting results of WCLs of 293T cells co-transfected with diverse combinations of constructs expressing 63-256 PRDX3-Myc, 37-256 PRDX3-Myc, PRDX3-Myc, and 1-110 PINK1-GFP, using anti-GFP beads and Myc-specific antibodies. Hashes indicate IgG. Data (Figures 13B and 13C) show three independent experiments. [Figure 13B]Figure 13 shows the PINK1 domain mapping essential for binding to peroxiredoxin 3 (PRDX3). In Figure 13A, the domain boundaries of full-length PINK1 and cleaved PINK1 and full-length PRDX3 are shown on the left and right, respectively. The PINK1 domain consists of a mitochondrial target sequence (MTS), a transmembrane domain (TMD), and a kinase domain. PRDX3 interacts with the N-terminus of PINK1(1-94), including the MTS. Figure 13B shows the WCL and PRDX3-Myc of 293T cells co-transfected with diverse combinations of constructs expressing 287-581PINK1-GFP, 1-286PINK1-GFP, 1-156PINK1-GFP, 1-110PINK1-GFP, and 1-94PINK1-GFP, followed by immunoblotting results using anti-GFP beads and Myc-specific antibodies. Hashes indicate IgG. Figure 13C shows subsequent immunoblotting results of WCLs of 293T cells co-transfected with diverse combinations of constructs expressing 63-256 PRDX3-Myc, 37-256 PRDX3-Myc, PRDX3-Myc, and 1-110 PINK1-GFP, using anti-GFP beads and Myc-specific antibodies. Hashes indicate IgG. Data (Figures 13B and 13C) show three independent experiments. [Figure 13C]Figure 13 shows the PINK1 domain mapping essential for binding to peroxiredoxin 3 (PRDX3). In Figure 13A, the domain boundaries of full-length PINK1 and cleaved PINK1 and full-length PRDX3 are shown on the left and right, respectively. The PINK1 domain consists of a mitochondrial target sequence (MTS), a transmembrane domain (TMD), and a kinase domain. PRDX3 interacts with the N-terminus of PINK1(1-94), including the MTS. Figure 13B shows the WCL and PRDX3-Myc of 293T cells co-transfected with diverse combinations of constructs expressing 287-581PINK1-GFP, 1-286PINK1-GFP, 1-156PINK1-GFP, 1-110PINK1-GFP, and 1-94PINK1-GFP, followed by immunoblotting results using anti-GFP beads and Myc-specific antibodies. Hashes indicate IgG. Figure 13C shows subsequent immunoblotting results of WCLs of 293T cells co-transfected with diverse combinations of constructs expressing 63-256 PRDX3-Myc, 37-256 PRDX3-Myc, PRDX3-Myc, and 1-110 PINK1-GFP, using anti-GFP beads and Myc-specific antibodies. Hashes indicate IgG. Data (Figures 13B and 13C) show three independent experiments.

[0082] [Figure 14A]Figure 14 shows confirmation that peroxiredoxin 3 (PRDX3) modulates the stability of PINK1. Figures 14A and 14B show the results of Western blot analysis (Figure 14A) and quantification (Figure 14B) of Pink1 and PRDX3 expression in the mitochondrial fraction of mouse embryonic fibroblasts (MEFs) treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The mitochondrial fraction was immunoblotted with PINK1, PRDX3, and Cox4 antibodies. Figure 14C shows the results of immunostaining using Tom20 and / or PRDX3 antibodies in HeLa cells transfected with pDsRed2-Mito (MTX, matrix) and treated with DMSO or 10 μM CCCP for 4 hours (scale bar, 2 (box region) or 10 μm). Figure 14D shows the results of immunostaining using Tom20 and / or PRDX3 antibodies in HeLa cells infected with PINK1-GFP and treated with 10 μM CCCP for 4 hours (scale bar, 2 (box region) or 10 μm). Figures 14E and 14F show the results of Western blot analysis (Figure 14E) and quantification (Figure 14F) of Pink1 in the mitochondrial fractions of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) MEFs infected with Oma1 siRNA and treated with 10 μM CCCP for 4 hours. The mitochondrial fractions were immunoblotted with PINK1, Oma1, PRDX3, and Cox4 antibodies. Figures 14G and 14H show the results of immunostaining with Tom20 antibody in PRDX3 WT and PRDX3 KO MEF infected with Oma1 siRNA and PINK1-GFP (Figure 14G), and the quantification results of PINK1-GFP (Figure 14H) (scale bars, 10 μm * P < 0.05 and ** P < 0.01). The data (Figures 14A to 14H) show three independent experiments. [Figure 14B]Figure 14 shows confirmation that peroxiredoxin 3 (PRDX3) modulates the stability of PINK1. Figures 14A and 14B show the results of Western blot analysis (Figure 14A) and quantification (Figure 14B) of Pink1 and PRDX3 expression in the mitochondrial fraction of mouse embryonic fibroblasts (MEFs) treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The mitochondrial fraction was immunoblotted with PINK1, PRDX3, and Cox4 antibodies. Figure 14C shows the results of immunostaining using Tom20 and / or PRDX3 antibodies in HeLa cells transfected with pDsRed2-Mito (MTX, matrix) and treated with DMSO or 10 μM CCCP for 4 hours (scale bar, 2 (box region) or 10 μm). Figure 14D shows the results of immunostaining using Tom20 and / or PRDX3 antibodies in HeLa cells infected with PINK1-GFP and treated with 10 μM CCCP for 4 hours (scale bar, 2 (box region) or 10 μm). Figures 14E and 14F show the results of Western blot analysis (Figure 14E) and quantification (Figure 14F) of Pink1 in the mitochondrial fractions of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) MEFs infected with Oma1 siRNA and treated with 10 μM CCCP for 4 hours. The mitochondrial fractions were immunoblotted with PINK1, Oma1, PRDX3, and Cox4 antibodies. Figures 14G and 14H show the results of immunostaining with Tom20 antibody in PRDX3 WT and PRDX3 KO MEF infected with Oma1 siRNA and PINK1-GFP (Figure 14G), and the quantification results of PINK1-GFP (Figure 14H) (scale bars, 10 μm * P < 0.05 and ** P < 0.01). The data (Figures 14A to 14H) show three independent experiments. [Figure 14C]Figure 14 shows confirmation that peroxiredoxin 3 (PRDX3) modulates the stability of PINK1. Figures 14A and 14B show the results of Western blot analysis (Figure 14A) and quantification (Figure 14B) of Pink1 and PRDX3 expression in the mitochondrial fraction of mouse embryonic fibroblasts (MEFs) treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The mitochondrial fraction was immunoblotted with PINK1, PRDX3, and Cox4 antibodies. Figure 14C shows the results of immunostaining using Tom20 and / or PRDX3 antibodies in HeLa cells transfected with pDsRed2-Mito (MTX, matrix) and treated with DMSO or 10 μM CCCP for 4 hours (scale bar, 2 (box region) or 10 μm). Figure 14D shows the results of immunostaining using Tom20 and / or PRDX3 antibodies in HeLa cells infected with PINK1-GFP and treated with 10 μM CCCP for 4 hours (scale bar, 2 (box region) or 10 μm). Figures 14E and 14F show the results of Western blot analysis (Figure 14E) and quantification (Figure 14F) of Pink1 in the mitochondrial fractions of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) MEFs infected with Oma1 siRNA and treated with 10 μM CCCP for 4 hours. The mitochondrial fractions were immunoblotted with PINK1, Oma1, PRDX3, and Cox4 antibodies. Figures 14G and 14H show the results of immunostaining with Tom20 antibody in PRDX3 WT and PRDX3 KO MEF infected with Oma1 siRNA and PINK1-GFP (Figure 14G), and the quantification results of PINK1-GFP (Figure 14H) (scale bars, 10 μm * P < 0.05 and ** P < 0.01). The data (Figures 14A to 14H) show three independent experiments. [Figure 14D]Figure 14 shows confirmation that peroxiredoxin 3 (PRDX3) modulates the stability of PINK1. Figures 14A and 14B show the results of Western blot analysis (Figure 14A) and quantification (Figure 14B) of Pink1 and PRDX3 expression in the mitochondrial fraction of mouse embryonic fibroblasts (MEFs) treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The mitochondrial fraction was immunoblotted with PINK1, PRDX3, and Cox4 antibodies. Figure 14C shows the results of immunostaining using Tom20 and / or PRDX3 antibodies in HeLa cells transfected with pDsRed2-Mito (MTX, matrix) and treated with DMSO or 10 μM CCCP for 4 hours (scale bar, 2 (box region) or 10 μm). Figure 14D shows the results of immunostaining using Tom20 and / or PRDX3 antibodies in HeLa cells infected with PINK1-GFP and treated with 10 μM CCCP for 4 hours (scale bar, 2 (box region) or 10 μm). Figures 14E and 14F show the results of Western blot analysis (Figure 14E) and quantification (Figure 14F) of Pink1 in the mitochondrial fractions of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) MEFs infected with Oma1 siRNA and treated with 10 μM CCCP for 4 hours. The mitochondrial fractions were immunoblotted with PINK1, Oma1, PRDX3, and Cox4 antibodies. Figures 14G and 14H show the results of immunostaining with Tom20 antibody in PRDX3 WT and PRDX3 KO MEF infected with Oma1 siRNA and PINK1-GFP (Figure 14G), and the quantification results of PINK1-GFP (Figure 14H) (scale bars, 10 μm * P < 0.05 and ** P < 0.01). The data (Figures 14A to 14H) show three independent experiments. [Figure 14E]Figure 14 shows confirmation that peroxiredoxin 3 (PRDX3) modulates the stability of PINK1. Figures 14A and 14B show the results of Western blot analysis (Figure 14A) and quantification (Figure 14B) of Pink1 and PRDX3 expression in the mitochondrial fraction of mouse embryonic fibroblasts (MEFs) treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The mitochondrial fraction was immunoblotted with PINK1, PRDX3, and Cox4 antibodies. Figure 14C shows the results of immunostaining using Tom20 and / or PRDX3 antibodies in HeLa cells transfected with pDsRed2-Mito (MTX, matrix) and treated with DMSO or 10 μM CCCP for 4 hours (scale bar, 2 (box region) or 10 μm). Figure 14D shows the results of immunostaining using Tom20 and / or PRDX3 antibodies in HeLa cells infected with PINK1-GFP and treated with 10 μM CCCP for 4 hours (scale bar, 2 (box region) or 10 μm). Figures 14E and 14F show the results of Western blot analysis (Figure 14E) and quantification (Figure 14F) of Pink1 in the mitochondrial fractions of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) MEFs infected with Oma1 siRNA and treated with 10 μM CCCP for 4 hours. The mitochondrial fractions were immunoblotted with PINK1, Oma1, PRDX3, and Cox4 antibodies. Figures 14G and 14H show the results of immunostaining with Tom20 antibody in PRDX3 WT and PRDX3 KO MEF infected with Oma1 siRNA and PINK1-GFP (Figure 14G), and the quantification results of PINK1-GFP (Figure 14H) (scale bars, 10 μm * P < 0.05 and ** P < 0.01). The data (Figures 14A to 14H) show three independent experiments. [Figure 14F]Figure 14 shows confirmation that peroxiredoxin 3 (PRDX3) modulates the stability of PINK1. Figures 14A and 14B show the results of Western blot analysis (Figure 14A) and quantification (Figure 14B) of Pink1 and PRDX3 expression in the mitochondrial fraction of mouse embryonic fibroblasts (MEFs) treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The mitochondrial fraction was immunoblotted with PINK1, PRDX3, and Cox4 antibodies. Figure 14C shows the results of immunostaining using Tom20 and / or PRDX3 antibodies in HeLa cells transfected with pDsRed2-Mito (MTX, matrix) and treated with DMSO or 10 μM CCCP for 4 hours (scale bar, 2 (box region) or 10 μm). Figure 14D shows the results of immunostaining using Tom20 and / or PRDX3 antibodies in HeLa cells infected with PINK1-GFP and treated with 10 μM CCCP for 4 hours (scale bar, 2 (box region) or 10 μm). Figures 14E and 14F show the results of Western blot analysis (Figure 14E) and quantification (Figure 14F) of Pink1 in the mitochondrial fractions of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) MEFs infected with Oma1 siRNA and treated with 10 μM CCCP for 4 hours. The mitochondrial fractions were immunoblotted with PINK1, Oma1, PRDX3, and Cox4 antibodies. Figures 14G and 14H show the results of immunostaining with Tom20 antibody in PRDX3 WT and PRDX3 KO MEF infected with Oma1 siRNA and PINK1-GFP (Figure 14G), and the quantification results of PINK1-GFP (Figure 14H) (scale bars, 10 μm * P < 0.05 and ** P < 0.01). The data (Figures 14A to 14H) show three independent experiments. [Figure 14G]Figure 14 shows confirmation that peroxiredoxin 3 (PRDX3) modulates the stability of PINK1. Figures 14A and 14B show the results of Western blot analysis (Figure 14A) and quantification (Figure 14B) of Pink1 and PRDX3 expression in the mitochondrial fraction of mouse embryonic fibroblasts (MEFs) treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The mitochondrial fraction was immunoblotted with PINK1, PRDX3, and Cox4 antibodies. Figure 14C shows the results of immunostaining using Tom20 and / or PRDX3 antibodies in HeLa cells transfected with pDsRed2-Mito (MTX, matrix) and treated with DMSO or 10 μM CCCP for 4 hours (scale bar, 2 (box region) or 10 μm). Figure 14D shows the results of immunostaining using Tom20 and / or PRDX3 antibodies in HeLa cells infected with PINK1-GFP and treated with 10 μM CCCP for 4 hours (scale bar, 2 (box region) or 10 μm). Figures 14E and 14F show the results of Western blot analysis (Figure 14E) and quantification (Figure 14F) of Pink1 in the mitochondrial fractions of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) MEFs infected with Oma1 siRNA and treated with 10 μM CCCP for 4 hours. The mitochondrial fractions were immunoblotted with PINK1, Oma1, PRDX3, and Cox4 antibodies. Figures 14G and 14H show the results of immunostaining with Tom20 antibody in PRDX3 WT and PRDX3 KO MEF infected with Oma1 siRNA and PINK1-GFP (Figure 14G), and the quantification results of PINK1-GFP (Figure 14H) (scale bars, 10 μm * P < 0.05 and ** P < 0.01). The data (Figures 14A to 14H) show three independent experiments. [Figure 14H]Figure 14 shows confirmation that peroxiredoxin 3 (PRDX3) modulates the stability of PINK1. Figures 14A and 14B show the results of Western blot analysis (Figure 14A) and quantification (Figure 14B) of Pink1 and PRDX3 expression in the mitochondrial fraction of mouse embryonic fibroblasts (MEFs) treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours. The mitochondrial fraction was immunoblotted with PINK1, PRDX3, and Cox4 antibodies. Figure 14C shows the results of immunostaining using Tom20 and / or PRDX3 antibodies in HeLa cells transfected with pDsRed2-Mito (MTX, matrix) and treated with DMSO or 10 μM CCCP for 4 hours (scale bar, 2 (box region) or 10 μm). Figure 14D shows the results of immunostaining using Tom20 and / or PRDX3 antibodies in HeLa cells infected with PINK1-GFP and treated with 10 μM CCCP for 4 hours (scale bar, 2 (box region) or 10 μm). Figures 14E and 14F show the results of Western blot analysis (Figure 14E) and quantification (Figure 14F) of Pink1 in the mitochondrial fractions of PRDX3 wild-type (PRDX3 WT) and PRDX3-deficient (PRDX3 KO) MEFs infected with Oma1 siRNA and treated with 10 μM CCCP for 4 hours. The mitochondrial fractions were immunoblotted with PINK1, Oma1, PRDX3, and Cox4 antibodies. Figures 14G and 14H show the results of immunostaining with Tom20 antibody in PRDX3 WT and PRDX3 KO MEF infected with Oma1 siRNA and PINK1-GFP (Figure 14G), and the quantification results of PINK1-GFP (Figure 14H) (scale bars, 10 μm * P < 0.05 and ** P < 0.01). The data (Figures 14A to 14H) show three independent experiments.

[0083] [Figure 15]Figure 15 shows the results of immunostaining using Tom20 antibody on HeLa cells transfected with a control group, PRDX3 siRNA, or PRDX3 and OMA1 siRNA, confirming that peroxiredoxin 3 (PRDX3) modulates the stability of PINK1 (scale bar, 10 μm).

[0084] [Figure 16A]Figure 16 shows that in the outer mitochondrial membrane (OMM), PINK1 expression recruits Parkin to damaged mitochondria in the absence of peroxiredoxin 3 (PRDX3). Figure 16A shows a model of the N-Mid49+PINK1111-581GFP structure. Figure 16B shows representative low-magnification and high-magnification images of the box region of PRDX3-deficient (PRDX KO) mouse embryonic fibroblasts (MEFs) transfected with N-Mid49+PINK1111-581GFP and immunostained with Tom20 antibody (scale bar, 2 μm (box region) or 10 μm). Figure 16C shows representative images of PRDX3 wild-type (PRDX3 WT) and PRDX3 KO MEF cells transfected with RFP-Parkin and N-Mid49+PINK1111-581GFP and treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours (scale bar, 10 μm). Figure 16D shows representative images of HeLa cells from the control group and cells transfected with PRDX3 siRNA, then transfected with RFP-Parkin and N-Mid49+PINK1111-581GFP and treated with DMSO or 10 μM CCCP for 4 hours (scale bar, 10 μm). Figure 16E shows the results of Western blot analysis of the mitochondrial fractions of PRDX3 WT and PRDX3 KO MEF transfected with N-Mid49+PINK1111-581GFP and treated with 10 μM CCCP for 4 hours. The mitochondrial fractions were immunoblotted with antibodies against GFP, PRDX3, and Cox4. Figure 16F shows the results of immunostaining using MitoTracker staining or Tom20 antibody in PRDX3 KO MEF transfected with N-Mid49+PINK1111-581GFP.Figure 16G shows the results of immunostaining using MitoTracker staining or Tom20 antibody in PRDX3 WT and PRDX3 KO MEF cells transfected with N-Mid49+PINK1111-581GFP and treated with 10 μM CCCP for 24 hours (scale bar, 10 μm). [Figure 16B]Figure 16 shows that in the outer mitochondrial membrane (OMM), PINK1 expression recruits Parkin to damaged mitochondria in the absence of peroxiredoxin 3 (PRDX3). Figure 16A shows a model of the N-Mid49+PINK1111-581GFP structure. Figure 16B shows representative low-magnification and high-magnification images of the box region of PRDX3-deficient (PRDX KO) mouse embryonic fibroblasts (MEFs) transfected with N-Mid49+PINK1111-581GFP and immunostained with Tom20 antibody (scale bar, 2 μm (box region) or 10 μm). Figure 16C shows representative images of PRDX3 wild-type (PRDX3 WT) and PRDX3 KO MEF cells transfected with RFP-Parkin and N-Mid49+PINK1111-581GFP and treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours (scale bar, 10 μm). Figure 16D shows representative images of HeLa cells from the control group and cells transfected with PRDX3 siRNA, then transfected with RFP-Parkin and N-Mid49+PINK1111-581GFP and treated with DMSO or 10 μM CCCP for 4 hours (scale bar, 10 μm). Figure 16E shows the results of Western blot analysis of the mitochondrial fractions of PRDX3 WT and PRDX3 KO MEF transfected with N-Mid49+PINK1111-581GFP and treated with 10 μM CCCP for 4 hours. The mitochondrial fractions were immunoblotted with antibodies against GFP, PRDX3, and Cox4. Figure 16F shows the results of immunostaining using MitoTracker staining or Tom20 antibody in PRDX3 KO MEF transfected with N-Mid49+PINK1111-581GFP.Figure 16G shows the results of immunostaining using MitoTracker staining or Tom20 antibody in PRDX3 WT and PRDX3 KO MEF cells transfected with N-Mid49+PINK1111-581GFP and treated with 10 μM CCCP for 24 hours (scale bar, 10 μm). [Figure 16C]Figure 16 shows that in the outer mitochondrial membrane (OMM), PINK1 expression recruits Parkin to damaged mitochondria in the absence of peroxiredoxin 3 (PRDX3). Figure 16A shows a model of the N-Mid49+PINK1111-581GFP structure. Figure 16B shows representative low-magnification and high-magnification images of the box region of PRDX3-deficient (PRDX KO) mouse embryonic fibroblasts (MEFs) transfected with N-Mid49+PINK1111-581GFP and immunostained with Tom20 antibody (scale bar, 2 μm (box region) or 10 μm). Figure 16C shows representative images of PRDX3 wild-type (PRDX3 WT) and PRDX3 KO MEF cells transfected with RFP-Parkin and N-Mid49+PINK1111-581GFP and treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours (scale bar, 10 μm). Figure 16D shows representative images of HeLa cells from the control group and cells transfected with PRDX3 siRNA, then transfected with RFP-Parkin and N-Mid49+PINK1111-581GFP and treated with DMSO or 10 μM CCCP for 4 hours (scale bar, 10 μm). Figure 16E shows the results of Western blot analysis of the mitochondrial fractions of PRDX3 WT and PRDX3 KO MEF transfected with N-Mid49+PINK1111-581GFP and treated with 10 μM CCCP for 4 hours. The mitochondrial fractions were immunoblotted with antibodies against GFP, PRDX3, and Cox4. Figure 16F shows the results of immunostaining using MitoTracker staining or Tom20 antibody in PRDX3 KO MEF transfected with N-Mid49+PINK1111-581GFP.Figure 16G shows the results of immunostaining using MitoTracker staining or Tom20 antibody in PRDX3 WT and PRDX3 KO MEF cells transfected with N-Mid49+PINK1111-581GFP and treated with 10 μM CCCP for 24 hours (scale bar, 10 μm). [Figure 16D]Figure 16 shows that in the outer mitochondrial membrane (OMM), PINK1 expression recruits Parkin to damaged mitochondria in the absence of peroxiredoxin 3 (PRDX3). Figure 16A shows a model of the N-Mid49+PINK1111-581GFP structure. Figure 16B shows representative low-magnification and high-magnification images of the box region of PRDX3-deficient (PRDX KO) mouse embryonic fibroblasts (MEFs) transfected with N-Mid49+PINK1111-581GFP and immunostained with Tom20 antibody (scale bar, 2 μm (box region) or 10 μm). Figure 16C shows representative images of PRDX3 wild-type (PRDX3 WT) and PRDX3 KO MEF cells transfected with RFP-Parkin and N-Mid49+PINK1111-581GFP and treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours (scale bar, 10 μm). Figure 16D shows representative images of HeLa cells from the control group and cells transfected with PRDX3 siRNA, then transfected with RFP-Parkin and N-Mid49+PINK1111-581GFP and treated with DMSO or 10 μM CCCP for 4 hours (scale bar, 10 μm). Figure 16E shows the results of Western blot analysis of the mitochondrial fractions of PRDX3 WT and PRDX3 KO MEF transfected with N-Mid49+PINK1111-581GFP and treated with 10 μM CCCP for 4 hours. The mitochondrial fractions were immunoblotted with antibodies against GFP, PRDX3, and Cox4. Figure 16F shows the results of immunostaining using MitoTracker staining or Tom20 antibody in PRDX3 KO MEF transfected with N-Mid49+PINK1111-581GFP.Figure 16G shows the results of immunostaining using MitoTracker staining or Tom20 antibody in PRDX3 WT and PRDX3 KO MEF cells transfected with N-Mid49+PINK1111-581GFP and treated with 10 μM CCCP for 24 hours (scale bar, 10 μm). [Figure 16E]Figure 16 shows that in the outer mitochondrial membrane (OMM), PINK1 expression recruits Parkin to damaged mitochondria in the absence of peroxiredoxin 3 (PRDX3). Figure 16A shows a model of the N-Mid49+PINK1111-581GFP structure. Figure 16B shows representative low-magnification and high-magnification images of the box region of PRDX3-deficient (PRDX KO) mouse embryonic fibroblasts (MEFs) transfected with N-Mid49+PINK1111-581GFP and immunostained with Tom20 antibody (scale bar, 2 μm (box region) or 10 μm). Figure 16C shows representative images of PRDX3 wild-type (PRDX3 WT) and PRDX3 KO MEF cells transfected with RFP-Parkin and N-Mid49+PINK1111-581GFP and treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours (scale bar, 10 μm). Figure 16D shows representative images of HeLa cells from the control group and cells transfected with PRDX3 siRNA, then transfected with RFP-Parkin and N-Mid49+PINK1111-581GFP and treated with DMSO or 10 μM CCCP for 4 hours (scale bar, 10 μm). Figure 16E shows the results of Western blot analysis of the mitochondrial fractions of PRDX3 WT and PRDX3 KO MEF transfected with N-Mid49+PINK1111-581GFP and treated with 10 μM CCCP for 4 hours. The mitochondrial fractions were immunoblotted with antibodies against GFP, PRDX3, and Cox4. Figure 16F shows the results of immunostaining using MitoTracker staining or Tom20 antibody in PRDX3 KO MEF transfected with N-Mid49+PINK1111-581GFP.Figure 16G shows the results of immunostaining using MitoTracker staining or Tom20 antibody in PRDX3 WT and PRDX3 KO MEF cells transfected with N-Mid49+PINK1111-581GFP and treated with 10 μM CCCP for 24 hours (scale bar, 10 μm). [Figure 16F]Figure 16 shows that in the outer mitochondrial membrane (OMM), PINK1 expression recruits Parkin to damaged mitochondria in the absence of peroxiredoxin 3 (PRDX3). Figure 16A shows a model of the N-Mid49+PINK1111-581GFP structure. Figure 16B shows representative low-magnification and high-magnification images of the box region of PRDX3-deficient (PRDX KO) mouse embryonic fibroblasts (MEFs) transfected with N-Mid49+PINK1111-581GFP and immunostained with Tom20 antibody (scale bar, 2 μm (box region) or 10 μm). Figure 16C shows representative images of PRDX3 wild-type (PRDX3 WT) and PRDX3 KO MEF cells transfected with RFP-Parkin and N-Mid49+PINK1111-581GFP and treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours (scale bar, 10 μm). Figure 16D shows representative images of HeLa cells from the control group and cells transfected with PRDX3 siRNA, then transfected with RFP-Parkin and N-Mid49+PINK1111-581GFP and treated with DMSO or 10 μM CCCP for 4 hours (scale bar, 10 μm). Figure 16E shows the results of Western blot analysis of the mitochondrial fractions of PRDX3 WT and PRDX3 KO MEF transfected with N-Mid49+PINK1111-581GFP and treated with 10 μM CCCP for 4 hours. The mitochondrial fractions were immunoblotted with antibodies against GFP, PRDX3, and Cox4. Figure 16F shows the results of immunostaining using MitoTracker staining or Tom20 antibody in PRDX3 KO MEF transfected with N-Mid49+PINK1111-581GFP.Figure 16G shows the results of immunostaining using MitoTracker staining or Tom20 antibody in PRDX3 WT and PRDX3 KO MEF cells transfected with N-Mid49+PINK1111-581GFP and treated with 10 μM CCCP for 24 hours (scale bar, 10 μm). [Figure 16G]Figure 16 shows that in the outer mitochondrial membrane (OMM), PINK1 expression recruits Parkin to damaged mitochondria in the absence of peroxiredoxin 3 (PRDX3). Figure 16A shows a model of the N-Mid49+PINK1111-581GFP structure. Figure 16B shows representative low-magnification and high-magnification images of the box region of PRDX3-deficient (PRDX KO) mouse embryonic fibroblasts (MEFs) transfected with N-Mid49+PINK1111-581GFP and immunostained with Tom20 antibody (scale bar, 2 μm (box region) or 10 μm). Figure 16C shows representative images of PRDX3 wild-type (PRDX3 WT) and PRDX3 KO MEF cells transfected with RFP-Parkin and N-Mid49+PINK1111-581GFP and treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours (scale bar, 10 μm). Figure 16D shows representative images of HeLa cells from the control group and cells transfected with PRDX3 siRNA, then transfected with RFP-Parkin and N-Mid49+PINK1111-581GFP and treated with DMSO or 10 μM CCCP for 4 hours (scale bar, 10 μm). Figure 16E shows the results of Western blot analysis of the mitochondrial fractions of PRDX3 WT and PRDX3 KO MEF transfected with N-Mid49+PINK1111-581GFP and treated with 10 μM CCCP for 4 hours. The mitochondrial fractions were immunoblotted with antibodies against GFP, PRDX3, and Cox4. Figure 16F shows the results of immunostaining using MitoTracker staining or Tom20 antibody in PRDX3 KO MEF transfected with N-Mid49+PINK1111-581GFP.Figure 16G shows the results of immunostaining using MitoTracker staining or Tom20 antibody in PRDX3 WT and PRDX3 KO MEF cells transfected with N-Mid49+PINK1111-581GFP and treated with 10 μM CCCP for 24 hours (scale bar, 10 μm).

[0085] [Figure 17A] Figure 17 shows evidence that peroxiredoxin 3 (PRDX3) is a core regulator of the mitochondrial quality control (MQC) process in cardiomyocytes. Figure 17A shows immunostaining results for Tom20 and Parkin in cardiomyocytes from PRDX3 wild-type (PRDX3 WT) and PRDX3 knockout (PRDX3 KO) mice treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours (scale bars, 2 μm (box region) or 10 μm). Figures 17B and 17C show representative images (Figure 17B) and quantification results (Figure 17C) of mitophagy in cardiomyocytes from 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 quantification results (Figure 17E) of damaged mitochondria (arrows) in cardiomyocytes from PRDX3 WT and PRDX3 KO mt-Keima mice. Cardiomyocytes were infected with PRDX3 or PRDX3DN adenovirus for 24 hours. Arrows indicate muscle fibers, and open arrows indicate Z-lines of sarcomeres (scale bar, 1 μm (box region) or 2 μm, *P<0.05, **P<0.01 and ***P<0.001). [Figure 17B]Figure 17 shows evidence that peroxiredoxin 3 (PRDX3) is a core regulator of the mitochondrial quality control (MQC) process in cardiomyocytes. Figure 17A shows immunostaining results for Tom20 and Parkin in cardiomyocytes from PRDX3 wild-type (PRDX3 WT) and PRDX3 knockout (PRDX3 KO) mice treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours (scale bars, 2 μm (box region) or 10 μm). Figures 17B and 17C show representative images (Figure 17B) and quantification results (Figure 17C) of mitophagy in cardiomyocytes from 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 quantification results (Figure 17E) of damaged mitochondria (arrows) in cardiomyocytes from PRDX3 WT and PRDX3 KO mt-Keima mice. Cardiomyocytes were infected with PRDX3 or PRDX3DN adenovirus for 24 hours. Arrows indicate muscle fibers, and open arrows indicate Z-lines of sarcomeres (scale bar, 1 μm (box region) or 2 μm, *P<0.05, **P<0.01 and ***P<0.001). [Figure 17C]Figure 17 shows evidence that peroxiredoxin 3 (PRDX3) is a core regulator of the mitochondrial quality control (MQC) process in cardiomyocytes. Figure 17A shows immunostaining results for Tom20 and Parkin in cardiomyocytes from PRDX3 wild-type (PRDX3 WT) and PRDX3 knockout (PRDX3 KO) mice treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours (scale bars, 2 μm (box region) or 10 μm). Figures 17B and 17C show representative images (Figure 17B) and quantification results (Figure 17C) of mitophagy in cardiomyocytes from 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 quantification results (Figure 17E) of damaged mitochondria (arrows) in cardiomyocytes from PRDX3 WT and PRDX3 KO mt-Keima mice. Cardiomyocytes were infected with PRDX3 or PRDX3DN adenovirus for 24 hours. Arrows indicate muscle fibers, and open arrows indicate Z-lines of sarcomeres (scale bar, 1 μm (box region) or 2 μm, *P<0.05, **P<0.01 and ***P<0.001). [Figure 17D]Figure 17 shows evidence that peroxiredoxin 3 (PRDX3) is a core regulator of the mitochondrial quality control (MQC) process in cardiomyocytes. Figure 17A shows immunostaining results for Tom20 and Parkin in cardiomyocytes from PRDX3 wild-type (PRDX3 WT) and PRDX3 knockout (PRDX3 KO) mice treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours (scale bars, 2 μm (box region) or 10 μm). Figures 17B and 17C show representative images (Figure 17B) and quantification results (Figure 17C) of mitophagy in cardiomyocytes from 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 quantification results (Figure 17E) of damaged mitochondria (arrows) in cardiomyocytes from PRDX3 WT and PRDX3 KO mt-Keima mice. Cardiomyocytes were infected with PRDX3 or PRDX3DN adenovirus for 24 hours. Arrows indicate muscle fibers, and open arrows indicate Z-lines of sarcomeres (scale bar, 1 μm (box region) or 2 μm, *P<0.05, **P<0.01 and ***P<0.001). [Figure 17E]Figure 17 shows evidence that peroxiredoxin 3 (PRDX3) is a core regulator of the mitochondrial quality control (MQC) process in cardiomyocytes. Figure 17A shows immunostaining results for Tom20 and Parkin in cardiomyocytes from PRDX3 wild-type (PRDX3 WT) and PRDX3 knockout (PRDX3 KO) mice treated with DMSO or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 hours (scale bars, 2 μm (box region) or 10 μm). Figures 17B and 17C show representative images (Figure 17B) and quantification results (Figure 17C) of mitophagy in cardiomyocytes from 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 quantification results (Figure 17E) of damaged mitochondria (arrows) in cardiomyocytes from PRDX3 WT and PRDX3 KO mt-Keima mice. Cardiomyocytes were infected with PRDX3 or PRDX3DN adenovirus for 24 hours. Arrows indicate muscle fibers, and open arrows indicate Z-lines of sarcomeres (scale bar, 1 μm (box region) or 2 μm, *P<0.05, **P<0.01 and ***P<0.001).

[0086] [Figure 18] Figure 18 shows the results of the isolation of new cardiomyocytes, and illustrates the immunostaining results of cardiomyocytes using antibodies against myomesin (cardiomyocyte marker, green) and MitoTracker (scale bar, 10 μm).

[0087] [Figure 19]Figure 19 shows the gene region deleted from a PRDX3 conventional knockout (KO) mouse.

[0088] [Figure 20] Figure 20 shows the PRDX3 conventional knockout mouse identified via PCR.

[0089] [Figure 21] Figure 21 shows the results of an analysis of the substantia nigra (substantia nigra) of the brain containing dopamine neurons in 5-month-old mice.

[0090] [Figure 22] Figure 22 shows the results of a study confirming reduced mitochondrial function in the substantia nigra of the brain of PRDX3 knockout mice.

[0091] [Figure 23] Figure 23 shows the presence of alpha-synuclein aggregations and lipid droplets, which are characteristic of Parkinson's disease, in dopamine neurons of the substantia nigra of PRDX3 knockout mice.

[0092] [Figure 24] Figure 24 shows that in the dopamine neurons of the substantia nigra of PRDX3 knockout mice, lipid droplets and neuromelanin, which are characteristic of Parkinson's disease, are greatly increased.

[0093] [Figure 25] Figure 25 shows that suppressing PRDX3 expression in cultured SH-SY5Y neurons reduces dopamine levels, and treating them with adenovirus-PRDX3 restores dopamine levels.

[0094] [Figure 26]Figure 26 shows that when PRDX3 expression is suppressed in cultured SH-SY5Y neurons treated with sodium arsenite to induce reactive oxygen species stress, α-synuclein aggregates increase, and when PRDX3 expression is restored, α-synuclein aggregates decrease.

[0095] [Figure 27] Figure 27 shows, via immunohistochemical staining, that when alpha-synuclein pre-formed fibers (PFFs), which are directly involved in the development of Parkinson's disease, are treated with adenovirus-PRDX3 to increase PRDX3 expression in mouse neurons where alpha-synuclein aggregates have formed, the alpha-synuclein aggregates disappear.

[0096] [Figure 28] Figure 28 shows that in mouse neurons in which α-synuclein aggregates were formed by treating α-synuclein pre-formed fibers (PFFs), which are directly involved in the development of Parkinson's disease, Western blotting confirmed that increasing PRDX3 expression by treating the neurons with adenovirus-PRDX3 reduced the amount of insoluble α-synuclein that forms α-synuclein aggregates. [Modes for carrying out the invention]

[0097] The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative and do not limit the scope of the present invention to these examples.

[0098] Experimental Example 1: Materials and Methods

[0099] 1.1. Animal Models

[0100] To generate PRDX3-deficient mitochondrial target Keima (mt-Keima) mice, PRDX3-deficient mice were crossed with mt-Keima mice. To generate Pink1-deficient mt-Keima mice, Pink1-deficient mice (provided by Professor Lee Han-eun of Yonsei University) were crossed with Mt-Keima mice. The mice were backcrossed more than seven times in C57BL / 6J background (Jackson Laboratory). All animal management and experimental procedures were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee of Ewha Womans University.

[0101] 1.2. Drosophila strains

[0102] We previously produced Mt-Keima transgenic Drosophila (UAS-mt-Keima). We purchased the white RNAi (white GD14981) line from the Vienna Drosophila Resource Center. We purchased the da-GAL4 and Prx3RNAi (Prx3HMJ22845) lines from the Bloomington Stock Center (Indiana University, Bloomington, IN, USA).

[0103] 1.3. Mammalian cell cultures

[0104] Primary mouse embryonic fibroblasts (MEFs) were isolated from wild-type and PRDX3-deficient embryos at embryonic day 13.5. Primary MEFs, HeLa cells, and 293T cells were maintained in Dulbecco's modified Eagle medium (DMEM, Invitrogen) supplemented with 10% FBS (Invitrogen), 100 U / ml penicillin, and 100 μg / ml streptomycin (Invitrogen). Myocytes were isolated from wild-type and PRDX3-deficient mice at postnatal day 3 using the Pierce™ Primary Cardiomyocyte Isolation Kit, according to the manufacturer's instructions. Isolated cells were cultured for 7 days prior to analysis to confirm proper cell morphology. To restore PRDX3 expression, PrdX3-deficient cardiomyocytes were infected with PRDX3 adenovirus or dominant-negative PRDX3 (PrdX-DN) adenovirus (Sirion).

[0105] 1.4. Plasmids and RNAi oligonucleotides

[0106] The PRDX3-Myc plasmid was generated by PCR amplification of full-length PRDX3 (NCBI registry number: NM_006793.5) and cloning of the product to the EcoRI-XhoI site of the pcDNA.3.1 / MyC-His(-)A plasmid (Invitrogen). The construct encoding PINK1-GFP was generated by PCR amplification of full-length PINK1 (NCBI registry number: NM_032409.3) and subcloning of the product pEGFP-N3 plasmid to the EcoRI-BamHI site (Clontech). The construct encoding Mid49-GFP was generated by PCR amplification of full-length Mid49 (isoform 1; NCBI registry number: NM_139162.3) and cloning of the product pEGFP-N3 plasmid to the EcoRI-BamHI site (Clontech). Constructs encoding the N-terminal sequence of Mid49 (amino acids 1-126, N-Mid49) or amino acids 111-581 of PINK1 (PINK1111-581) were generated through PCR amplification of full-length Mid49 or PINK1, respectively. To generate fusion products of N-Mid49 and PINK1111-581, Mid491-126 was subcloned into the XhoI-EcoRI site of the pEGFP-N3 plasmid (Clontech), followed by subcloning of PINK1111-581 into the EcoRI-BamHI site of the N-mid49pEGPF-N3 plasmid. Point mutations in PRDX3 (for expressing PRDX3L53V and PRDX3DN) and PINK1 (for expressing PINK1A93V) were generated using fusion-site-directed mutagenesis kits following the manufacturer's protocol (Thermo Fisher Scientific). The construct encoding PRKN-GFP was generated by PCR amplification of full-length PRKN (isoform 1; NCBI registry number: NM_004562.3) and subcloning of the product into the EcoRI-BamHI site of the pERFP-C1 plasmid (Clontech). pDsRed2-Mito (Clontech) was used for mitochondrial matrix staining.The construct encoding GST-Δ1-40PRDX3 was generated by cloning the product into the EcoRI-XhoI site of the pGEM-4T-1 plasmid (GE Healthcare). siRNA was synthesized by GenePharma. The human siPRDX3 target sequence is 5'-AAG CCA AGT CCA GCT GCT TCC-3' (SEQ ID NO: 1), the human siOMA1 target sequence is 5'-GAA GTG CTT TGT CAT CTA ATT-3' (SEQ ID NO: 2), and the mouse siOma1 target sequence is 5'-GGA TAC AGT CAA AGT TGC AGG-3' (SEQ ID NO: 3). Silencer Negative Control siRNA (GenePharma) was used as a control group.

[0107] 1.5. Transfection and drug therapy

[0108] To overexpress mitochondrial-targeted catalase, PRDX3-deficient MEF or PRDX3-depleted HeLa cells were infected with adenovirus containing mitochondrial-targeted catalase and cultured for 24 hours in DMEM supplemented with 10% FBS. (2 × 10⁶ cells) 5 Cells (per well) were seeded into a 6-well plate 18 hours prior to transfection. Plasmid transfection was performed for 4 hours in OPTI-MEM medium (Invitrogen) containing 3 μl of lipofectamine 2000, or using the NEON™ transfection system (Invitrogen). Subsequently, the cells were reduced to approximately 0.5 × 10⁶. 5Cells were seeded in a microscope dish with a coverslip bottom (SPL) at a density of cells / ml. For RNAi transfection, cells were seeded in a 6-well plate at 30-40% confluence (or equivalent density). Then, 4 μl of Lipofectamine 2000 (Invitrogen) or 5 μl of RNAiMAX (Invitrogen) and 40 nM RNAi oligonucleotide were added to each well. Cells were re-transfected after 24 hours with 3 μl of Lipofectamine 2000 and appropriate plasmid DNA. For mitochondrial staining, cells were treated for 20 minutes before fixation with 100 nM MitoTracker Red CMXRos (Invitrogen), a live mitochondrial-labeling fluorescent dye that accumulates in a membrane potential-dependent manner in DMEM. For mitochondrial live cell imaging, cells were treated with 150 nM MitoTracker Green (Invitrogen) and 5 μM MitoSOX (Invitrogen) (a mitochondrial superoxide indicator in live cells) in DMEM for 20 minutes. For treatment with carbonylcyanide 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 4–24 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.

[0109] 1.6. Electron Microscope

[0110] To prepare samples for cell transmission electron microscopy (TEM), 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 2% glutaraldehyde paraformaldehyde in 0.1 M PBS (pH 7.4) for 2 hours and washed three times in 0.1 M PBS (pH 7.4, 1 mM) for 30 minutes each. Subsequently, the tissues were fixed in 1% OsO4 dissolved in 0.1 M PBS (pH 7.4) for 2 hours, dehydrated with a stepwise series of ethanol (50, 60, 70, 80, 90, 95, and 100%), and incubated with propylene oxide. The samples were packed using a 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), 300 nm thick sections were first cut, stained with toluidine blue, and observed under an optical microscope (Olympus BX40, Japan). Next, 80 nm thick sections were double-stained with 7% uranyl acetate and lead citrate for control staining (20 minutes). Subsequently, these sections were cut using a Leica UltraCut UCT ultramicrotome (Leica Microsystems, Austria). All samples were observed using a TEM (JEM-1011, JEOL, Japan) at an accelerating voltage of 80 kV.

[0111] 1.7. Measurement of mitophagy levels

[0112] Mitophagy levels were investigated using a pH-dependent fluorescent probe, mt-Keima, with a confocal microscope, as described above. To analyze the fluorescence signal of mt-Keima, mt-Keima mouse and Drosophila tissue samples were examined using a Zeiss LSM 800 confocal microscope (Carl Zeiss) equipped with plan-Apochromat 10x / 0.45 M27, Plan-Asopchromat 20x / 0.8 M27, and c-Aspchromat 40x / 1.20W Korr lenses. Mt-Keima fluorescence was imaged using two consecutive excitation lasers (488 and 555 nm) and an emission bandwidth of 595–700 nm. Mitophagy quantification based on mt-Keima confocal images was performed pixel-wise using Zeiss Zen software, as described above. The mitophagy level (mitophagy ratio) was defined as the number of pixels with a high red / green ratio divided by the total number of pixels. To quantify mitophagy levels in the heart, at least five tissue samples were used for quantification, and the mean value was calculated. In all confocal microscopy analyses, all imaging parameters were kept constant, with only the gain level adjusted to avoid saturation of any pixel. Results are presented as mean ± SD.

[0113] 1.8. Isolation of Mitochondria

[0114] Mitochondria were isolated from MEF, 293T cells, and HeLa cell lysates using a mitochondrial isolation kit for cultured cells, following the manufacturer's protocol (Thermo Fisher Scientific, Inc.).

[0115] 1.9. Measurement of ATP levels and respiration

[0116] To measure the ATP levels of cardiomyocytes for ATP analysis, 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 measured using the ENLITEN ATP Assay System Bioluminescence Detection Kit for ATP Measurement (Promega, USA). Cardiomyocyte oxygen consumption rate (OCR) was measured using the XFp Analyzer (Agilent, USA).

[0117] 1.10. Immunoprecipitation and Western blot

[0118] For immunoprecipitation experiments, untransfected and transfected cells were lysed in whole cell extraction buffer (10 mM HEPES [pH 7.9], 400 mM NaCl, 0.1 mM EDTA, 5% glycerol, 1 mM DTT, and a protease inhibitor). After adding either the untreated cell lysate or anti-PRDX3 (1 μg) to the lysate, anti-Myc-tagged agarose (MBL) or protein A agarose (Upstate Biotech) in TEG reaction buffer (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 10% glycerol, 1 mM DTT, and 150 mM NaCl) was added, and the mixture was stirred at 4°C for 3 hours or overnight. The immunoprecipitation was washed with TEG washing buffer (TEG reaction buffer containing 0.1% Triton X-100). For Western blotting, cells were lysed in whole cell extraction buffer or homogenized in protein extraction buffer (20 mM HEPES [pH 7.9], 300 mM NaCl, 10 mM EDTA, 0.1% NP40, 100 mM KCl, and a protease inhibitor) using a MICRA D-8 homogenizer (ART Moderne Labortechnik). Total protein was fractionated 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, LF PA0044, AbFrontier), catalase (1:100, BC 100494, Labfrontier), PRKN (1:100, sc-32282, Santa Cruz Biotechnology), GFP (1:1100, sc-9976, Santa Cruz Biotechnology), TOM20 (1:100100, sc-11415, Santa Cruz Biotechnology), tubulin (1:300, The antibodies used were T6199 (Sigma), OMA1 (1:1000, 17116-1-AP, Proteintech), PRKN (1:1100, ab77924, Abcam), Myc (1:1200, ab32, Abcam), COX4 (1:1008, ab33985, Abcam), Myomesin (1:1000, B4-S, DSHB), and PINK1 (1:150, BC 10494, NOVUS). Secondary antibodies were fluorescein-conjugated anti-rabbit IgG, anti-mouse IgG, and anti-goat IgG (Invitrogen), and anti-rabbit, anti-mouse, and anti-goat HRP-conjugated antibodies (Zymed Laboratories). Protein-antibody complexes were detected using the ECL Plus system (Amersham Biosciences).

[0119] 1.11. Thoracic echocardiography and MI surgery

[0120] Thoracic echocardiography was performed in wild-type and PRDX3-deficient mice (10 and 49–55 weeks old) using a Vevo2100 system (VisualSonics) equipped with 25–55 MHz linear array transducers. After inducing anesthesia with 1.5–2% isoflurane, echocardiography was performed while maintaining the mouse heart rate within the physiological range (>450 bpm) under anesthesia, and body temperature was maintained at 37°C using a constant temperature pad during the echocardiography. Left ventricular (LV) variables such as ejection fraction (EF), left ventricular diameter shortening (FS), stroke volume (SV), cardiac output (CO), end-diastolic volume, and end-systolic volume were analyzed in the long-axis field using a disk approach with the monoplane Simpson method provided by the manufacturer and VevoStrain software. Wild-type and PRDX3-deficient mice (10–12 weeks old) were subjected to cardiac surgery under isoflurane anesthesia. The left anterior descending coronary artery was permanently occluded with 7-0 silk sutures. On postoperative day 15, echocardiography was performed using a Vevo2100 system (VisualSonics) with a 25–55 MHz linear array transducer. Anesthesia was administered by inhalation of 1.5–2% isoflurane, and body temperature was monitored during the ultrasound examination. LV variables such as EF, SV, end-diastolic volume, and end-systolic volume were analyzed using the Simpson method for single-plane long-axis views with a disk approach, using VevoStrain software provided by the manufacturer.

[0121] 1.12. Cardiac fibrosis analysis

[0122] Left ventricular (LV) fibrosis was evaluated using a series of Masson trichrome-stained cardiac sections. Paraffin-embedded LV samples were divided into 6 μm thick slices and stained using the Masson trichrome staining kit (BBC Biochemical) according to the manufacturer's guidelines. Tissue sections were scanned using the Vectra Polaris automated quantitative pathology imaging system (PerkinElmer), and the percentage of fibrosis relative to the LV area was measured using Form Advanced Imaging Analysis Software (PerkinElmer).

[0123] 1.13. Live Imaging and Confocal Microscopy

[0124] Live cell imaging was performed using a spinning disc confocal system (A1C, Nikon). For live cells, imaging was performed in an LCI chamber (Chamlide TC, LCI) at 37°C and 5% CO2, using an x60 oil immersion objective lens with a numerical aperture (NA) of 1.4. 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 acquired with an x63 oil immersion objective lens (NA 1.4). Images were analyzed using Carl Zeiss Elements, Photoshop (Adobe), IMARIS (Bitplane AG), or ImageJ (National Institutes of Health) software. Scale bars were generated using Carl Zeiss Elements and ImageJ.

[0125] 1.14. Damaged Mitochondria and ROS Analysis of Mitochondria

[0126] To measure depolarized (damaged) mitochondria, only mitochondrial-related MitoTracker Red CMXRos (Invitrogen) images on a continuous z-plane were quantified using ImageJ software. To analyze mitochondrial ROS, only mitochondrial-related MitoSOX (Invitrogen) images on a continuous z-plane were quantified using ImageJ software. Statistical analysis was performed using SigmaPlot (Systat Software). Data are presented as the mean ± SEM of at least three experiments.

[0127] 1.15. Immunofluorescence and Histology

[0128] Cultured cells were fixed in 4% formaldehyde in PBS for 20 minutes at room temperature (RT), washed with PBS, and permeabilized with 0.1% Triton X-100 for 15 minutes. Cells were washed with PBS, blocked with 3% BSA in PBS for 1 hour, and incubated with primary antibody in PBS for approximately 1–3 hours at RT. After washing with PBS, cells were incubated with secondary antibody at RT for 1 hour. Subsequently, cells were stained with 10 μM 4,6-diamidino-2-phenylindole and mounted in Vectashield (Vector Laboratories). Paraffin-embedded compartments (6 μm thick) of the heart were fixed in 10% buffered formaldehyde and used for TUNEL assay and immunofluorescence analysis.

[0129] 1.16. Mass spectrometry

[0130] The PRDX3 band in the gel was desalted, digested with trypsin, and the resulting peptide was extracted and analyzed using nanoAcquity™ UPLC / ESI / q-TOF MS / MS (SYNAPT™ G2Si HDMS™, Waters Co., UK).

[0131] 1.17. TUNEL analysis

[0132] 24 hours after MI, apoptosis in myocardial infarction hearts was evaluated using the TACS 2 TdT fluorescein-based apoptosis detection kit (Trevigen), according to the manufacturer's protocol.

[0133] 1.18. Statistical analysis

[0134] Differences between two experimental groups were analyzed using Student's t-test or Mann-Whitney's U test. One-way analysis of variance (ANOVA) and one-way correction were used to compare three or more groups. P<0.05 was considered statistically significant.

[0135] Experimental Example 2: Results

[0136] 2.1. Induction of cardiac hypertrophy and dysfunction due to PRDX3 deficiency

[0137] Mitochondria contribute significantly to cardiovascular homeostasis, and their fine-tuning via MQCs is crucial for the survival of cardiovascular cells and the maintenance of physiological cardiac function. Disruption of MQCs is closely associated with cardiac defects.

[0138] To determine the role of PRDX3 in myocardial MQC, cardiac phenotypes were examined in wild-type and PRDX3-deficient mice at 10 and 52 weeks of age. PRDX3-deficient mice exhibited cardiac hypertrophy, including a significant increase in cardiac weight-to-body weight ratio, decreased CO at 52 weeks of age, and decreased SV at both 10 and 52 weeks of age without any change in EF (Figures 1A–1D and 2A). These results are consistent with previous reports that mitochondrial dysfunction reduces SV and CO in association with heart failure caused by diastolic dysfunction. Therefore, PRDX3-deficient-induced LV remodeling and cardiac hypertrophy suggest that mitochondrial dysfunction is a significant risk factor for heart failure reminiscent of human HFpEF.

[0139] Furthermore, PRDX3-deficient mice had a similar heart size to 10-week-old wild-type mice, but showed cardiac hypertrophy, and at 52 weeks of age, cardiomyocyte size and LV fibrosis were significantly increased (Figures 1E-1I, 2B). Damaged mitochondria were observed in PRDX3-deficient cardiomyocytes in 10-week-old mice and increased significantly at 52 weeks of age, with the mice exhibiting giant, damaged mitochondria with morphologically distinctive morphology and extremely large size (Figures 1J and 1K). These results indicate that MQC dysfunction induced by PRDX3 deficiency leads to dysfunction and accumulation of giant mitochondria, resulting in cardiac dysfunction.

[0140] 2.2. Worsening of myocardial infarction (MI)-related cardiac dysfunction due to PRDX3 deficiency

[0141] Mitochondrial damage caused by oxidative stress, a characteristic of infarct cardiomyopathy, promotes excessive loss of cardiomyocytes and LV remodeling.

[0142] To evaluate whether mitochondrial dysfunction and reduced mitophagy caused by PRDX3 deficiency exacerbate heart failure after MI, cardiac function was assessed using echocardiography 15 days after MI induction (Figure 4A). EF and SV were significantly lower in PRDX3-deficient mice than in wild-type mice. LV end-systolic volume was significantly increased in PRDX3-deficient mice, but there was no significant difference in LV end-diastolic volume between wild-type and PRDX3-deficient mice (Figures 3A-3E). These results indicate that PRDX3 deficiency exacerbates cardiac dysfunction after MI. Furthermore, Masson's trichrome staining showed that PRDX3 deficiency exacerbates cardiac fibrosis and LV remodeling (Figure 3F). We also 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 (Figure 3G). Destruction of damaged mitochondria by MQC-induced removal induces malignant cell death characterized by activation of the apoptotic cascade.

[0143] Following MI, in situ apoptosis analysis was performed using the infarcted hearts of wild-type and PRDX3-deficient mice. The results confirmed a significant increase in apoptotic cell death in the infarcted hearts of PRDX3-deficient mice (Figure 3H). While 10-week-old PRDX3-deficient mice did not exhibit obvious cardiac dysfunction under physiological conditions (Figure 4B), the increase in damaged mitochondria due to damaged MQCs can explain the prevalence of heart failure after MI. Therefore, under MI conditions, PRDX3 deficiency leads to increased mitochondrial damage and subsequent apoptosis in the infarcted heart.

[0144] 2.3. Inhibition of in vivo mitophagy due to PRDX3 deficiency

[0145] To further determine whether mitochondrial damage caused by PRDX3 deficiency is due to mitochondrial ROS accumulation, we examined the mitochondrial phenotype in PRDX3-deficient mice. Mitochondrial membrane potential was analyzed in MEFs isolated from PRDX3-deficient mice, showing an increased number of damaged or depolarized mitochondria (Figures 5A and 5C). Next, analysis of mitochondrial ROS in PRDX3-deficient MEFs confirmed that PRDX3 deficiency induces increased mitochondrial ROS levels (Figures 5B and 5D). After confirming the primary molecular function of PRDX3 in preventing ROS accumulation, we investigated whether overexpression of mitochondrial catalase, a mitochondrial ROS scavenger, could mitigate mitochondrial damage associated with PRDX3 deficiency. Overexpression of mitochondrial catalase was shown to reduce mitochondrial damage caused by PRDX3 deficiency (Figures 5A-5E). These results indicate that mitochondrial damage induced by PRDX3 deficiency is closely related to abnormal ROS accumulation. Furthermore, PRDX3 deficiency in vivo causes mitochondrial damage in the heart and other tissues of PRDX3-deficient mice (Figures 6A and 6B). ATP levels and oxygen consumption rate (OCR) are also significantly reduced in PRDX3-deficient cardiomyocytes, indicating that PRDX3 deficiency causes cardiac mitochondrial dysfunction (Figures 7A-7G). The number of damaged mitochondria is increased in the heart, liver, skeletal muscle, and brain of 52-week-old PRDX3-deficient mice (Figures 1J and 1K, 6A and 6B), indicating that PRDX3-deficient mice are a suitable animal model for MQC studies.

[0146] Next, to confirm the potential for modulation between two MQC processes—prevention of mitochondrial damage by regulation of ROS by mitochondrial-specific PRDX3 and removal of damaged mitochondria by PINK1-mediated mitophagy—we compared the mitochondrial phenotypes in the hearts of PRDX3-deficient and Pink1-deficient mice. Mitochondrial damage was increased in the hearts and other tissues of PRDX3-deficient mice (Figures 1J and 1K, 6A and 6B), but mitophagy was significantly lower in the hearts and other tissues of PRDX3-deficient mice than in wild-type and Pink1-deficient mice (Figures 8A and 8B, 9A-9C).

[0147] Next, we examined the pathophysiological outcomes after MI, particularly whether the mechanism of mitophagy is affected by PRDX3. Characterizing the cardiac function of PRDX3 in mitophagy using PRDX3-deficient mt-Keima mice with MI, we found that mitophagy was significantly increased in the hearts of wild-type mt-Keima mice 4 hours post-MI, but significantly decreased in the hearts of PRDX3-deficient mt-Keima mice regardless of MI (Figure 9D). We also confirmed that PRDX3 deficiency significantly weakened mitophagy in cardiac tissue 24 hours post-MI (Figures 8C and 8D). Consistently, depletion of PRDX3, the Drosophila ortholog of PRDX3, reduced mitophagy in the wing disc of the fat body in Drosophila larvae and adults (Figure 9E).

[0148] These results suggest a potential role for PRDX3 in mitophagy regulation, in addition to its essential role in regulating ROS levels and preventing mitochondrial damage.

[0149] 2.4 Regulation of mitophagy by PRDX3-dependent localization of PRDX3 and PINK1 degradation in mitochondria.

[0150] To confirm the role of PRDX3 in mitophagy regulation, we investigated whether PINK1 protein levels are altered by PRDX3 deficiency. For this purpose, we compared PINK1 protein levels between wild-type and PRDX3-deficient MEFs. PINK1 protein levels were higher in PRDX3-deficient MEFs and PRDX3-deficient MEFs treated with the proteasome inhibitor MG132 than in wild-type MEFs (Figures 10A and 10B, Figure 11A). Since PRDX3 deficiency causes a decrease in mitophagy despite PINK1 upregulation, we investigated whether the submitochondrial localization of PINK1 is altered by PRDX3 deficiency. GFP-tagged PINK1 was overexpressed in the control group and PRDX3-deficient MEFs, and its localization in OMM-marker Tom20-tagged mitochondria was compared using Airyscan super-resolution microscopy. In PRDX3-deficient MEFs or PRDX3-depleted HeLa cells, PINK1-GFP co-localized with Tom20 in the OMM, whereas in wild-type MEFs and HeLa cells, PINK1-GFP preferentially localized to the matrix (Figure 10C, Figure 11B). These results indicate that PRDX3 influences the submitochondrial localization of PINK1, thereby regulating its degradation.

[0151] Next, we evaluated whether PRDX3 affects PINK1 expression under precursor-mitophagy conditions. Wild-type and PRDX3-deficient MEFs were treated with the mitochondrial uncoupling agent CCCP or the mitochondrial respiratory inhibitors oligomycin and antimycin A (OA), and PINK1 protein levels were examined. Previous studies have shown that CCCP or OA treatment strongly increases PINK1 accumulation in mitochondria and induces mitophagy; however, in this invention, the induction of PINK1 accumulation in mitochondria by CCCP or OA treatment was almost completely abolished by PRDX3 deficiency (Figures 10A, 10B, 10D, and 10E).

[0152] Consistently, overexpression of PINK1-GFP or Parkin-GFP in MEFs strongly suppressed ROS-mediated mitochondrial damage in PRDX3-deficient MEFs, even though CCCP-dependent mitochondrial accumulation of PINK1-GFP and Parkin-GFP was mediated by PRDX3-deficient mitochondrial catalase (Figures 10F-10I). Furthermore, overexpression of PINK1-GFP or Parkin-GFP in MEFs and HeLa cells suppressed CCCP- or OA-dependent mitochondrial accumulation of PINK1-GFP and Parkin-GFP in PRDX3-deficient MEFs or PRDX3-deficient HeLa cells (Figures 11C and 11D). These results suggest that PRDX3 functions as a regulator of PINK1-Parkin-mediated mitophagy by regulating the submitochondrial localization and mitochondrial degradation of PINK1.

[0153] 2.5. Interaction between PRDX3 and PINK1 mitochondrial target sequences

[0154] We tested whether PRDX3 interacts with PINK1 using co-immunoprecipitation experiments and confirmed that endogenous PRDX3 binds to endogenous PINK1 (Figure 12A).

[0155] To identify the crucial binding site between PRDX3 and PINK1, we created cleaved point mutant structures of PRDX3 and PINK1 and confirmed that the N-terminal domain of PINK1 is necessary for binding to PRDX3 (Figures 13A and 13B). To further identify the N-terminal domain of PRDX3 that is important for binding to PINK11-110, we performed IP experiments using PRDX363-256, PRDX337-256, PRDX31-256, and PINK11-110. We found that PINK11-110 binds to PRDX337-256 and PRDX31-256 but not to PRDX363-256, which suggests that the N-terminal sequence of PRDX3 (amino acids 37-62) is necessary for binding to PINK1 (Figure 13C). Using PRDX3Leu53Val and PINK1Ala93Val mutants, we confirmed that Leu53 in PRDX3 and Ala93 in PINK1 are important for binding between PRDX3 and PINK1 (Figures 12B and 12C). Dominant-negative PRDX3Cys108Ser / Cys229Ser, which inhibits ROS scavenging by PRDX3, was still able to bind to PINK1, suggesting that ROS dynamic activity was functionally well maintained in dominant-negative PRDX3 (Figure 12B). Through GST pull-down experiments, we confirmed that Leu53 in PRDX3 and Ala93 in PINK1 are important for direct binding between these proteins (Figures 12D and 12E).

[0156] The N-terminal sequence (amino acids 1-62) of PRDX3 has been reported to function as a mitochondrial targeting signal. Mass spectrometry to further clarify the mitochondrial targeting signal sequence of PRDX3 predicted that the N-terminal sequence (amino acids 1-36) of PRDX3 is essential for mitochondrial targeting, and revealed that mature PRDX3 contains Leu53, which ensures its binding to PINK1 (Figures 12F and 12G). Crystallography of mitochondrial PRDX (the Leishmania infantum ortholog of PRDX3) suggested that the N-terminal sequence outside the mitochondrial targeting sequence is important for the formation of structurally stable mitochondrial PRDX, thus confirming that the N-terminal sequence (amino acids 37-62) of PRDX3 binds to the mitochondrial targeting sequence of PINK1.

[0157] 2.6. Regulation of PINK1 stability by suppression of Oma1 in PRDX3 mitophagy.

[0158] We investigated whether PRDX3 protein expression changes as PINK1 levels increase in damaged mitochondria. We confirmed that PRDX3 protein levels also increase in damaged mitochondria, mimicking PINK1 upregulation (Figures 14A and 14B). We also investigated whether mitochondrial localization of PRDX3 leads to translocation from the mitochondrial matrix to the OMM upon binding with PINK1 under CCCP-induced mitochondrial injury. Co-immunostaining of Tom20 and PRDX3 confirmed that PRDX3 was limited to the mitochondrial matrix in healthy mitochondria, but translocated to the OMM in damaged mitochondria, as confirmed by co-localization with Tom20 or PINK1 (Figures 14C and 14D).

[0159] Various cellular stresses can cause mitochondrial dysfunction, which in turn can promote the activation of OMA1, a stress-inducible protease that resides in the IMM. When activated in damaged mitochondria, OMA1 cleaves Dele1, initiating a stress-inducible proteolytic cascade. Recent studies have shown that OMA1 degrades Parkinson's disease-associated PINK1 mutants that infiltrate damaged mitochondria. PRDX, including PRDX3, can perform a dual function: as a peroxidase under normal conditions and as a chaperone under various stresses. Human PRDX3 functions as a catalytically active self-organizing chaperone under stress conditions.

[0160] To determine whether PINK1 is degraded by Oma1 regardless of the presence of PRDX3, we examined the stability of PINK1 in damaged mitochondria in wild-type and PRDX3-deficient MEFs after Oma1 depletion and CCCP treatment. In damaged mitochondria via mitophagy, PINK1 was not degraded by Oma1 in the presence of PRDX3 (Figures 14E and 14F). Furthermore, PINK1 was degraded by Oma1 in the absence of PRDX3, but its levels were restored by depleting Oma1 using siRNA (siOma1). PINK1-GFP also completely disappeared in PRDX3-deficient MEFs and PRDX3-depleted HeLa cells after CCCP treatment. However, Oma1 depletion using siOma1 dramatically restored PINK1-GFP levels in CCCP-treated cells (Figures 14G and 14H, Figure 15). Therefore, binding of PRDX3 to the N-terminus of PINK1 can protect the latter protein from degradation by blocking its cleavage by Oma1. This indicates that PRDX3 functions as a chaperone for PINK1.

[0161] To confirm that PRDX3 regulates PINK1-Parkin-mediated mitophagy by regulating PINK1 stability in damaged mitochondria, PINK1 recruitment / reaction experiments were performed. Mid49 is an OMM protein that regulates mitochondrial recruitment by Drp1, which controls fission in mitochondrial dynamics, and possesses an N-terminal mitochondrial target sequence and an OMM domain. Using N-Mid49, which contains both the mitochondrial target sequence and the OMM domain, a mitochondrial targeting domain-cleaved PINK1 (PINK1111-581) was constructed lacking both the PRDX3 binding region and the OMA1 cleavage site, resulting in persistent localization to the OMM (Figure 16A). When N-Mid49-PINK1111-581 is overexpressed in PRDX3-deficient MEFs, the synthesized product localizes normally to mitochondria independently of CCCP-induced mitochondrial damage (Figure 16B). When N-Mid49-PINK1111-581 is overexpressed in PRDX3-deficient MEFs or PRDX3-depleted HeLa cells, the preparation recruits Parkin to damaged mitochondria simply after CCCP treatment (Figures 16C and 16D). N-Mid49-PINK1111-581 was stably expressed in damaged mitochondria regardless of the presence of PRDX3 and CCCP-induced mitochondrial damage (Figure 16E). Furthermore, overexpression of N-Mid49-PINK1111-581 in PRDX3-deficient MEFs resulted in reduced mitochondrial depolarization due to normal mitophagy development compared to observations in PRDX3-deficient MEFs (Figure 16F). To induce mitochondrial depolarization in most cells, PRDX3-deficient MEFs and N-Mid49-PINK1111-581-overexpressing PRDX3-deficient MEFs were treated with CCCP for 24 hours. However, mitochondrial depolarization after CCCP treatment was reduced in N-Mid49-PINK1111-581 overexpressing PRDX3-deficient MEFs, suggesting the removal of damaged mitochondria by mitophagy mediated by N-Mid49-PINK1111-581 (Figure 16G).Therefore, PRDX3 acts as a chaperone, binding to proteins to maintain the stability of PINK1 in damaged mitochondria and protect it from cleavage by activated Oma1.

[0162] 2.7. Regulation of the MQC process in cardiomyocytes by PRDX3

[0163] To determine whether the suppression of mitophagy in PRDX3-deficient hearts has a functional connection to PINK1-Parkin-mediated mitophagy, we investigated the localization of Parkin in damaged mitochondria in wild-type and PRDX3-deficient cardiomyocytes after CCCP treatment. We confirmed that endogenous Parkin is limited to damaged mitochondria in wild-type cardiomyocytes but not in PRDX3-deficient cardiomyocytes (Figures 17A and 18), suggesting that PRDX3 modulates the PINK1-Parkin-mediated pathway in cardiomyocytes.

[0164] Next, we investigated whether PRDX3 or PRDX3-DN adenovirus could rescue mitophagy under CCCP-induced mitophagy. PRDX3-deficient cardiomyocytes were treated with CCCP and then infected with adenoviruses containing PRDX3 or PRDX3-DN. PRDX3-DN is defective for ROS scavenging but functional for PINK1-mediated mitophagy. Expression of PRDX3 or PRDX3-DN restored mitophagy in PRDX3-deficient cardiomyocytes to levels similar to those in wild-type cardiomyocytes (Figures 17B and 17C). Furthermore, the number of damaged mitochondria in PRDX3-deficient cardiomyocytes was reduced by adenovirus PRDX3 or PRDX3-DN transfection, suggesting that mitochondrial damage increased by PRDX3 deficiency can be reduced by activated functional mitophagy through PRDX3-DN, which is defective for ROS scavenging (Figures 17D and 17E).

[0165] As a result, it was found that PRDX3 acts as a major regulator through the interaction between the prevention of mitochondrial damage and the removal of damaged mitochondria, which is essential for cardiac function.

[0166] 2.8. Confirmation of the efficacy of PRDX3 in Parkinson's disease

[0167] 2.8.1. Generation of PRDX3 gene knockout mice

[0168] The PRDX3 gene is located on mouse chromosome 19 and, through gene translation from exon 1 to exon 7, is composed of a protein consisting of 257 amino acids. We confirmed that exon 1 of the PRDX3 gene contains a mitochondrial targeting signal, and that cysteine ​​residues that remove reactive oxygen species are present at exon 109 of exon 4 and exon 230 of exon 6. The PRDX3 gene is located in the mitochondrial matrix and has the function of removing reactive oxygen species in mitochondria.

[0169] Conventional knockout mice for PRDX3 were created by incorporating a portion of the intron of exon 1'-UTR of the PRDX3 gene into the 5'-UTR of the exogenous gene neomycin, and reconstructing the intron of exon 4'-UTR of the PRDX3 gene into the 3'-UTR. This construct was then injected into mouse embryonic stem cells using electroporation. Homologous recombination was then used to induce deletion of exons 1-4 of the PRDX3 gene. After selecting exon 1-4-deficient mouse embryonic stem cells in neomycin-containing medium, they were microinjected into mouse blastocysts to produce PRDX3 gene knockout mice (see Figure 19). In PRDX3 wild-type (WT) mice, as shown in Figure 20, 1) a PRDX3-forward primer (cag gaa atg tca ata agt gtc tac, SEQ ID NO: 4) was fabricated in the left intron region of exon 1, and 2) a PRDX3-reverse primer (cca gag gcc act tgt gta gc, SEQ ID NO: 5) was fabricated in exon 1. The results of a 390-base pair polymerase chain reaction (PCR) were then confirmed.PRDX3 knockout mice were identified as follows, as shown in Figure 20: 1) a PRDX3-forward primer (cag gaa atg tca ata agt gtc tac, SEQ ID NO: 4) was prepared using the left intron region of exon 1, and 3) a PRDX3-neo-reverse primer (cga gga cca gag caa cct tc, SEQ ID NO: 6) was prepared using neomycin, an exogenous gene introduced to delete exons 1 to 4 (SEQ ID NOs: 7 to 10) of the PRDX3 gene. The results of 300 base pairs were then confirmed by PCR.

[0170] Using this method, PRDX3 knockout mice were identified, and the absence of PRDX3 expression in MEF (Mouse Embryonic Fibroblast) cells derived from PRDX3 knockout mice was confirmed through Western blot results compared to PRDX3 wild-type mice (see Figure 20).

[0171] 2.8.2. Confirmation of Parkinson's disease development in PRDX3 gene knockout mice

[0172] Analysis of the substantia nigra (substantia nigra) of dopamine neurons in 5-month-old mice revealed a significant decrease in dopamine neurons in the substantia nigra of PRDX3 knockout mice compared to PRDX3 wild-type mice (see Figure 21). Reduced motility and decreased mitochondrial function in the substantia nigra of PRDX3 knockout mice were also confirmed (see Figure 22). Furthermore, alpha-synuclein aggregation and lipid droplets, characteristic of Parkinson's disease, were observed in the dopamine neurons of the substantia nigra of PRDX3 knockout mice (see Figure 23). Electron microscopy analysis confirmed a significant increase in lipid droplets and neuromelanin, characteristic of Parkinson's disease, in the dopamine neurons of the substantia nigra of PRDX3 knockout mice (see Figure 24).

[0173] The results described above suggest that the PRDX3 knockout mouse according to the present invention is a suitable Parkinson's disease model mouse for studying the onset and treatment of Parkinson's disease.

[0174] 2.8.3. Confirmation of the therapeutic effect of PRDX3 in Parkinson's disease

[0175] We confirmed that suppressing PRDX3 expression in SH-SY5Y neuronal culture cells, which are widely used in Parkinson's disease research, reduces dopamine levels, and treating them with adenovirus-PRDX3 restores dopamine levels (see Figure 25).

[0176] When α-synuclein-stable SH-SY5Y neuronal culture cells, a Parkinson's disease development cell model, were treated with sodium arsenite to induce reactive oxygen species stress, α-synuclein aggregation, which is directly involved in the development of Parkinson's disease, occurred. However, it was confirmed that suppressing PRDX3 expression increased α-synuclein aggregation, and restoring PRDX3 expression by treating with adenovirus-PRDX3 decreased α-synuclein aggregation (see Figure 26).

[0177] Furthermore, we confirmed that treating neurons extracted from mouse brains with alpha-synuclein pre-formed fibrils (PFFs), which are directly involved in the development of Parkinson's disease, induced neuronal degeneration while simultaneously causing alpha-synuclein aggregation, thus forming an in vitro Parkinson's disease model. We confirmed by cell immunostaining that increasing PRDX3 expression in these neurons with alpha-synuclein aggregation eliminated alpha-synuclein aggregation (see Figure 27).

[0178] Furthermore, through Western blotting experiments, we confirmed that increasing PRDX3 expression by treating adenovirus-PRDX3 reduced insoluble α-synuclein, which exhibits α-synuclein aggregation. This suggests that decreased PRDX3 expression may be involved in the formation of α-synuclein aggregates, which are a key cause of Parkinson's disease development, and that PRDX3 itself may play an important role in the treatment of Parkinson's disease (see Figure 28).

[0179] These results suggest that PRDX3 can be usefully used as a treatment for Parkinson's disease.

Claims

1. A pharmaceutical composition for the prevention or treatment of mitochondrial-related diseases, comprising PRDX3 peptide or its analogue as an active ingredient.

2. The pharmaceutical composition according to claim 1, wherein the PRDX3 analog is a PRDX3 dominant-negative (DN) variant.

3. The pharmaceutical composition according to claim 1, characterized in that the PRDX3 peptide removes mitophagy.

4. The pharmaceutical composition according to claim 3, characterized in that the PRDX3 peptide regulates mitophagy by degrading the PINK1 peptide.

5. The pharmaceutical composition according to claim 1, characterized in that the PRDX3 peptide removes alpha-synuclein aggregates.

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

7. A health functional food composition for preventing or improving mitochondrial-related diseases, comprising PRDX3 peptide or its analogue as an active ingredient.

8. A pharmaceutical preparation for the prevention or treatment of mitochondrial-related diseases, comprising PRDX3 peptide or its analogue as an active ingredient.

9. A method for preventing or treating mitochondrial-related diseases, comprising the step of administering a pharmaceutical composition containing PRDX3 peptide or an analog thereof to an individual.

10. A PRDX3 gene deletion mouse model in which the 5'-UTR exon 1 to exon 4 of the PRDX3 gene are deleted.

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 producing a PRDX3 gene deletion mouse model, comprising the step of deleting 5'-UTR exon 1 to exon 4 of the PRDX3 gene.

13. A method for producing a PRDX3 gene-deficient mouse model according to claim 12, wherein the deletion of 5'-UTR exon 1 to exon 4 of the PRDX3 gene is due to homologous recombination.

14. The method for producing a PRDX3 gene deletion mouse model according to claim 12, wherein the deletion step is performed by substituting a base sequence including the 5'-UTR exon 1 to exon 4 portion of the PRDX3 gene with a construct containing the neomycin gene.

15. A method for producing a PRDX3 gene deletion mouse model according to claim 14, wherein the substitution of the construct is performed by electroporation.

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