Composition for microglial rejuvenation

Abstract

A composition for microglial rejuvenation according to an aspect, in which the composition comprises, as an active ingredient, an expression of an ACE protein or a gene inhibitor encoding same, rejuvenates microglia rather than suppresses neuroinflammatory responses in order to treat, prevent, or alleviate brain aging-related diseases. Accordingly, the composition is expected to be widely used in the treatment of most brain aging-related diseases for which existing neuroinflammatory response suppression strategies have failed.

Description

Composition for microglial rejuvenation

[0001] It relates to a composition for rejuvenating microglial cells.

[0002] Average human life expectancy has been steadily increasing over the past several decades. According to the 2024 Global Health Statistics Report published by the World Health Organization (WHO), the global life expectancy is projected to reach 71.4 years in 2022. While the COVID-19 pandemic has slowed the pace of improvement, life expectancy continues to steadily increase.

[0003] In this aging society, interest in and demand for anti-aging and reverse aging is steadily increasing, and companies such as the newly launched Altos Lab and existing companies such as Turn Biotechnologies and ALKAHEST are attempting to develop rejuvenation technologies through cell reprogramming.

[0004] Among these, demand for brain aging treatments is exploding, but commercialization of foods or drugs designed to rejuvenate the brain remains challenging. Through dedicated efforts to rejuvenate the brain, the inventors have discovered a novel target protein capable of rejuvenating microglia.

[0005] One aspect is to provide a composition for rejuvenating microglial cells, comprising as an active ingredient an inhibitor of the expression of ACE protein or a gene encoding the same.

[0006] Another aspect is to provide a composition for treating or preventing a degenerative brain disease, comprising as an active ingredient an inhibitor of the expression of ACE protein or a gene encoding it.

[0007] Another aspect is to provide a health functional food composition for treating or improving a degenerative brain disease, comprising as an active ingredient an inhibitor of the expression of ACE protein or a gene encoding it.

[0008] Another aspect provides a method for screening a therapeutic agent for a degenerative brain disease, comprising the steps of: treating a candidate substance to a cell expressing an ACE protein; measuring the protein expression levels of p-mTOR protein and p-AMPK in the cell; and selecting a substance that reduces the expression level of p-mTOR and increases the expression level of p-AMPK compared to a group not treated with the candidate substance.

[0009]

[0010] However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the relevant technical field from the description below.

[0011] One aspect provides a composition for rejuvenating microglial cells, comprising as an active ingredient an inhibitor of the expression of ACE protein or a gene encoding the same.

[0012] The composition of the present invention can rejuvenate aged cells or delay or prevent cellular aging through an anti-aging effect that returns aged cells to a young cell state, such as by causing naturally or artificially aged brain cells to resume cell division or by reducing the accumulation of undesirable metabolic products due to cellular aging.

[0013] The composition may contain as an active ingredient an inhibitor of the expression of ACE protein or a gene encoding the same, and may be at least one selected from the group consisting of captopril, trandolapril, and lisinopril. Preferably, it may be captopril.

[0014] In particular, the composition of the present invention can provide a synergistic effect on rejuvenating senescent cells or delaying or preventing cellular aging through specific active ingredients. Furthermore, based on these effects, the composition of the present invention can effectively prevent, treat, or improve diseases associated with cellular aging, such as those caused by aging of brain cells, particularly microglia.

[0015] The disease caused by aging of the brain or microglial cells may be a degenerative brain disease, and may be one or more diseases selected from the group consisting of, but not limited to, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, stroke, multiple sclerosis, movement disorders, learning disorders, and memory impairment.

[0016] As used herein, rejuvenation of senescent cells refers to reverting various characteristics of senescent cells to those of or similar to those of young cells. For example, it may refer to reverse senescence, in which old cells that no longer proliferate resume active proliferation like young cells, and / or reverse senescence, in which the levels of beta-galactosidase, p16, p21, p53, or lipid droplets return to the levels of young cells (e.g., to the levels of expression observed in young cells). In one embodiment, the composition of the present invention has at least one activity selected from the group consisting of increasing cell proliferation, decreasing beta-galactosidase expression, and decreasing lipid droplet expression levels.

[0017] The rejuvenating composition may be treated for, but is not limited to, 12 to 240 hours, 24 to 144 hours, 24 to 120 hours, 24 to 96 hours, or 24 to 72 hours.

[0018] Another aspect provides a composition for treating or preventing a degenerative brain disease, comprising as an active ingredient an inhibitor of the expression of ACE protein or a gene encoding it.

[0019] The above-mentioned degenerative brain disease may be one or more diseases selected from the group consisting of dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, stroke, multiple sclerosis, movement disorders, learning disabilities, and memory impairment. The above-mentioned degenerative brain disease may be induced by dexamethasone treatment or by aging of the brain, particularly microglial cells. Additionally, it may be induced by mutations in the GRN (progranulin) gene or overexpression of the TDP-43 protein, but is not limited thereto.

[0020] A pharmaceutical composition for the prevention or treatment of a degenerative brain disease according to one aspect may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, according to a conventional method, and may include an appropriate carrier, excipient, or diluent conventionally used in the manufacture of pharmaceutical compositions for formulation.

[0021] The carrier or excipient or diluent may include various compounds or mixtures including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0022] When formulating, it can be manufactured using diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants that are commonly used.

[0023] Solid formulations for oral administration can be prepared by mixing the ACE protein expression or the gene inhibitor encoding it with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

[0024] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives.

[0025] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerol gelatin.

[0026] The preferred dosage of a pharmaceutical composition for the prevention or treatment of degenerative brain diseases according to the aspect may vary depending on the patient's condition, body weight, severity of the disease, drug form, route of administration, and duration of administration, but can be appropriately selected by those skilled in the art. However, for desirable effects, the dosage may be 0.0001 to 2,000 mg / kg per day, preferably 0.001 to 2,000 mg / kg. The dosage may be administered once a day or divided into several doses. However, the scope of the present invention is not limited by the above dosage.

[0027] Pharmaceutical compositions for the prevention or treatment of degenerative brain diseases according to their nature can be administered to mammals such as rats, mice, livestock, and humans via various routes. All modes of administration can be administered, for example, orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine, or intracerebroventricular injection.

[0028] Another aspect provides a health functional food composition for treating or improving a degenerative brain disease, comprising as an active ingredient an inhibitor of the expression of ACE protein or a gene encoding the same.

[0029] In a health functional food composition for improving degenerative brain diseases according to one aspect, when the ACE protein expression or the gene inhibitor encoding it is used as an additive to the health functional food, it can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredients can be appropriately determined depending on the intended use, such as prevention, health, or treatment.

[0030] The formulation of health functional foods can be in the form of powders, granules, pills, tablets, capsules, or any other form of general food or beverage.

[0031] There is no particular limitation on the types of the above foods, and examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and all foods in the conventional sense can be included.

[0032] In general, when manufacturing food or beverage, the effective ingredient may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, per 100 parts by weight of the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range. Among the health functional foods according to one aspect, beverages may contain various flavoring agents or natural carbohydrates as additional ingredients, like regular beverages. The natural carbohydrates mentioned above may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, a natural sweetener such as thaumatin and stevia extract, or a synthetic sweetener such as saccharin and aspartame may be used. The proportion of the above natural carbohydrates may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the beverage according to the present invention.

[0033] In addition to the above, the health functional food composition for preventing or improving degenerative brain diseases according to one aspect may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. In addition, the sleep improvement composition of the present invention may contain fruit pulp for producing natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in mixtures. The ratio of these additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight relative to 100 parts by weight of the health functional food of the present invention.

[0034] Another aspect provides a method for treating a degenerative brain disease, comprising administering the pharmaceutical composition to a subject.

[0035] The above term "subject" is interpreted to mean mammals including humans, primates including chimpanzees, pets such as dogs and cats, livestock such as cows, horses, sheep and goats, and rodents such as mice and rats that are likely to develop or have developed skin aging.

[0036] Another aspect provides a use of the pharmaceutical composition for treating degenerative brain diseases.

[0037] Another aspect provides a method for screening for a treatment agent for a degenerative brain disease, comprising the steps of: treating cells expressing ACE protein with a candidate substance; measuring the level of ACE protein expression in the cells; and selecting a substance that reduces the level of ACE protein expression compared to a group not treated with the candidate substance.

[0038] Another aspect provides a method for screening for a treatment agent for a degenerative brain disease, comprising the steps of: treating cells expressing ACE protein with a candidate substance; measuring the protein expression levels of p-mTOR protein and p-AMPK in the cells; and selecting a substance that reduces the expression level of p-mTOR and increases the expression level of p-AMPK compared to a group not treated with the candidate substance.

[0039] A composition for rejuvenating microglial cells, which comprises as an active ingredient an inhibitor of the expression of ACE protein or a gene encoding the same according to a daily aspect, can be helpful in treating, preventing and improving diseases related to brain aging by rejuvenating or anti-aging microglial cells that are vulnerable to brain aging.

[0040] Figure 1 is a diagram showing the expression level of siACE after treating microglial cells (BV2) isolated from a mouse with dexamethasone for 24 hours, and performing knockdown (silencing) of both ACE in the control and experimental groups.

[0041] Figure 2 shows the level of beta-galactosidase (SA-b-gal), a marker of aging, after primary cultured microglial cells were treated with dexamethasone for 24 hours, and ACE was knocked down in both the control and experimental groups.

[0042] Figure 3 shows the level of beta-galactosidase (SA-b-gal), a marker of aging, after primary cultured microglial cells were treated with dexamethasone for 72 hours and treated with ACE inhibitor drugs such as captopril (C), trandolapril (T), or lisinopril (L), which knocked down all ACE.

[0043] Figure 4 shows the level of beta-galactosidase (SA-b-gal) when primary cultured microglial cells were treated with dexamethasone for 72 hours and then treated with catopril.

[0044] Figure 5 shows the reduction level of lipid droplets after primary cultured microglial cells were treated with dexamethasone for 24 hours, and ACE of both the control and experimental groups was knocked down.

[0045] Figure 6 shows the level of lipid droplets after primary cultured microglia were aged by dexamethasone treatment or long-term culture, and after dexamethasone treatment for 3 days and then catopril treatment for 24 hours.

[0046] Figure 7 is a diagram showing primary cultured microglia treated with dexamethasone or cultured for a long period of time to induce senescence, and the level of senescence was confirmed by treating with catopril.

[0047] Figure 8 is a diagram showing the expression level of Lamin B, a marker of senescence, when primary cultured microglia were aged through long-term culture and treated with catopril.

[0048] Figure 9 is a diagram showing the expression of the senescence associated secretory phenotype (SASP) after primary cultured microglia were aged through long-term culture.

[0049] Figure 10 shows the expression levels of aging-related proteins after intraperitoneal injection of catopril into 13-week-old young mice (Young: Y) and 85-week-old aged mice (Aged, A).

[0050] Figure 11a is a diagram showing the process of preparing a mouse to confirm lipofuscin, an aging-related marker, and fat droplets in microglia isolated from the dentate gyrus, cortex, and hypothalamus of the mouse brain.

[0051] Figure 11b is a diagram showing the detection of lipofuscin, an aging-related marker, in microglia isolated from the dentate gyrus, cortex, and hypothalamus of the mouse brain.

[0052] Figure 11c is a diagram showing the presence of lipid droplets, a marker, in microglia isolated from the dentate gyrus, cortex, and hypothalamus of the mouse brain.

[0053] Figure 12a is a diagram showing the process of preparing a mouse to predict the biological age (aging clock) of the mouse.

[0054] Figures 12b and 12c are diagrams showing the biological age of a mouse using RNAAgeCalc, which was trained based on human multi-tissue RNA expression data (Multi-tissue RNA sequencing) to predict the biological age (Aging clock), after performing sequencing to predict the biological age (Aging clock).

[0055] Figure 13 is a diagram showing the expression levels of p-mTOR, p-AMPK, and p-Akt, which are signaling systems related to cell aging, after long-term culture of microglia to induce aging.

[0056] Figure 14 is a diagram showing the level of grn after transfecting BV2 cells with grn siRNA.

[0057] Figure 15 shows the results of transfecting BV2 and primary cultured microglia with grn siRNA and then checking the level of lipid droplets.

[0058] Figure 16a is a diagram showing the recovery of phagocytic function in microglia after primary cultured microglia were transfected with grn siRNA and then treated with a composition of one aspect, by visualizing the uptake of zymosan.

[0059] Figure 16b is a graph showing the recovery of phagocytic function in microglia over time when primary cultured microglia were transfected with grn siRNA and then treated with a composition of one aspect.

[0060] Figure 16c is a graph showing the recovery of phagocytic function in microglia after primary cultured microglia were transfected with grn siRNA and then treated with a composition of one aspect for 400 minutes, as indicated by the phagocytic index.

[0061] Figure 17 is a diagram showing a method for producing a fruit fly model for in vivo experiments and confirming whether motility is restored by treatment with a composition of one aspect.

[0062] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0063] As used herein, the singular forms may include the plural forms unless the context clearly dictates otherwise. Furthermore, as used herein, the words “comprise” and “include” and / or “comprising” and “including” specify the presence of stated features, numbers, steps, operations, elements, elements, and / or groups thereof, but do not preclude the presence or addition of one or more other features, numbers, operations, elements, elements, and / or groups thereof.

[0064]

[0065] Example

[0066] 1. Preparation of experiments and reagents

[0067] 1-1. Cell culture

[0068] 13.5-day-old mouse embryos were isolated from the uterus, and the neuroepithelial layer of the head was dissected to prepare a single-cell suspension in HBSS (Gibco, 14170-112). The cells in HBSS were centrifuged at 1200 rpm for 3 minutes, and then the HBSS was removed. 1X Trypsin-EDTA (Gibco, 15400-054) was added, incubated in a water bath for 3 minutes, and then centrifuged again. The pelleted cells were resuspended in DMEM medium containing 10% FBS (Gibco, #16000-044), 0.1X GlutaMAX (Gibco, #11995-065), and 1% penicillin / streptomycin (Gibco, #15140122). Microglia were cultured for 2 weeks in 25T flasks coated with poly-D-lysine (Sigma, #P7280). Subcultures were then performed and cultured for an additional week. Finally, microglia were isolated using the MACS method using CD11b (Miltenyi, #130-093-636) beads and an MS column (Milteyni, #130-042-201).

[0069]

[0070] 1-2. Fat drop staining

[0071] Cells were fixed in 4% paraformaldehyde (Biosolution, #BP079a) for 10 minutes at room temperature, and then washed twice with PBS (Welgene, #LB004-01). IBA1 (Wako, #019-19741), a microglial marker, was stained overnight. After staining with secondary antibodies, BODIPY (Invitrogen, #D3922) solution was diluted 1:1000 to stain lipid droplets, and then the cells were washed twice with PBS. Mounting was performed using a mounting solution containing DAPI (Invitrogen, #P36931), and images of lipid droplets overlapping with microglia were captured and analyzed using a confocal microscope (TCSSP5 II, Leica microsystems).

[0072]

[0073] 1-3. Age-related beta-galactosidase staining

[0074] Cells were fixed in 4% paraformaldehyde solution for 10 minutes at room temperature, then washed twice with PBS. A staining solution prepared using a beta-galactosidase activation kit (Cell biolabs, #CBA-230) was added to the cells, followed by incubation at 37°C for 4 hours. The cells were washed with PBS, and the blue-stained cells were examined under a microscope.

[0075]

[0076] 1-4. Confirmation of the phagocytic function of microglia

[0077] To evaluate the phagocytic capacity of microglia, 12 mm coverslips were placed in a 24-well plate and 1 × 10 5Cells were seeded. The cells were incubated with 2 μl of Fluoresbrite microspheres (Sigma, #L3030) containing red fluorescent latex beads for 2 hours at 37°C. To stop phagocytosis, 2 ml of ice-cold PBS was added. The cells were washed twice and fixed with 4% paraformaldehyde solution. The cells were washed twice with PBS and stained overnight for the microglial marker IBA1. After staining with a secondary antibody, the cells were washed twice with PBS. After fixation using a mounting solution containing DAPI, the cells were analyzed using a confocal microscope. The level of phagocytosis was determined by the number of phagocytosed beads per cell.

[0078]

[0079] 1-5. Immunofluorescence staining

[0080] Cells were washed twice with PBS, fixed with 4% paraformaldehyde, and 25 μm mouse brain tissues in cryoprotectant solution were washed twice with PBS. Afterwards, the sections were blocked with PBS containing 0.5% Triton X-100 and 4% normal donkey serum for 1 hour. Immunofluorescence was performed using rabbit anti-IBA1 (wako, #019-19741), rabbit anti-GFAP (abcam, #ab7260), and mouse anti-NeuN (merck, #MAB377) as primary antibodies, and donkey anti-rabbit Alexa-488 (thermo, #A21206), donkey anti-rabbit Alexa-647 (thermo, #A31571) as secondary antibodies.

[0081] Specifically, the primary antibody was diluted in PBS containing 0.5% Triton X-100 and 1% normal donkey serum and incubated overnight at 4°C. After washing three times with PBS for 5 minutes each, the secondary antibody was used. The cells were fixed using a mounting solution containing DAPI, and after fixation, they were analyzed using a confocal microscope.

[0082]

[0083] 1-6. Real-time PCR performance

[0084] Total RNA was extracted using TRIzol (Invitrogen, #15596026) reagent. cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (thermo, #K1621). The expression of genes related to the senescence-associated secretory phenotype (SASP) was analyzed. cDNA was amplified using Power SYBR Green PCR Master Mix and primers on an Applied QuantStudio1 system. The amplification conditions were set to 95°C for 10 min, followed by 50 cycles of 95°C for 15 s, and 60°C for 1 min. Melting curves were generated to confirm the specificity of the amplification. Relative quantitation (RQ) levels were calculated using the 2ΔΔCt method using GAPDH as an internal standard control.

[0085]

[0086] 1-7. Preparation of the Drosophila Animal Model

[0087] For in vivo animal experiments, we prepared transgenic fruit flies designed to drive the Gal4 transcription factor by the Repo (Reverse Polarity) promoter, which is specifically expressed in glial cells of Drosophila melanogaster. This fruit fly model overexpresses Gal4 only in glial cells due to the specificity of the Repo promoter. To create this model, Repo-Gal4 flies and UAS-TDP43WT flies were crossed to produce a disease model fruit fly that overexpresses human TDP-43 protein specifically in glial cells. The offspring generated through this crossing have the Repo-Gal4 / + ; UAS-TDP43WT / + genotype, and the Gal4 transcription factor is expressed in glial cells, which induces transcription of the TDP-43WT gene located downstream of the UAS sequence, resulting in glial cell-specific overexpression of the TDP-43 protein.

[0088]

[0089] Example 2. Confirmation of ACE expression levels according to aging in mice.

[0090] The neural epithelium of the head of 13.5-day-old mouse embryos was cultured for 2 weeks. Microglia alone (primary cultured microglia) were isolated from the cultured cells using CD11b beads. Primary cultured microglia and the microglia cell line BV2 cells were treated with dexamethasone (DEX) for 24 hours (50 nM, 20 nM, vehicle), followed by control and ACE knockdown (silencing, si). After 24 hours, the expression level of siACE was confirmed using real-time PCR. As shown in Figure 1, siACE expression was reduced in all experimental groups compared to the control group.

[0091]

[0092] Example 3. Confirmation of the expression level of aging factors according to the inhibition of ACE expression.

[0093] 3-1. Confirmation of aging factor expression level

[0094] Primary cultured microglia were treated with dexamethasone for 24 hours to induce senescence. The ACE gene was then knocked down and cultured for an additional 24 hours. Cells were then fixed with 4% paraformaldehyde and stained for the senescence marker beta-galactosidase (b-gal) to assess senescence levels. As shown in Figure 3, knocking down the ACE gene in dexamethasone-induced senescence in microglia resulted in a decrease in beta-galactosidase expression.

[0095] Primary cultured microglia were treated with dexamethasone for 72 hours to induce senescence, and then treated with ACE inhibitors to evaluate whether the same effects as the siRNA used for ACE knockdown were observed. The ACE inhibitors used were limited to captopril (C), trandolapril (T), and lisinopril (L), which can cross the blood-brain barrier. The group treated with captopril for 1 day was designated as C1 (captopril treatment for 1 day).

[0096] As a result, as shown in Fig. 4, the dexamethasone-treated group showed an increase in aging-related beta-galactosidase expression compared to the control group. However, when treated with an ACE inhibitor for 24 hours after dexamethasone treatment, beta-galactosidase expression decreased in the order of catopril, lisinopril, and trandolapril. Among these, catopril was confirmed to exhibit the most effective anti-aging and rejuvenating effects on microglia.

[0097]

[0098] 3-2. Confirmation of aging factors and fat droplet expression levels according to catopril treatment

[0099] When primary cultured microglia were treated with DEX for 72 hours or aged by long-term culture and then treated with catopril, the expression of beta-galactosidase, a senescence-related factor, was confirmed. When treated with dexamethasone for 3 days and then treated with catopril for 24 hours, the expression of beta-galactosidase associated with senescence was confirmed to be reduced. When compared to aged microglia (DIV10) by long-term culture (DIV, day in vitro (numbers indicate days)), when catopril was treated for 72 hours (3 days) at DIV7 (DIV7+CAP3), the expression of beta-galactosidase associated with senescence was confirmed to be reduced compared to DIV10, as shown in Fig. 5.

[0100] In addition, an experiment was conducted to determine changes in lipid droplet levels following catopril treatment. Primary microglia were treated with dexamethasone for 24 hours, and lipid droplet levels were determined in response to ACE knockdown. As shown in Figure 6, ACE knockdown resulted in a decrease in the expression of aging-associated beta-galactosidase.

[0101] In addition, primary cultured microglia were aged through 72-hour DEX treatment or long-term culture, and then treated with catopril to determine the expression level of lipid droplets. As a result, as shown in Figure 6, compared to long-term cultured aged microglia (DIV10), it was confirmed that lipid droplets were reduced when catopril was treated for 1 day (24 hours) and 3 days (72 hours). In addition, it was confirmed that when catopril was treated for 3 days, lipid droplets were reduced more than in the 1-day treatment group.

[0102]

[0103] Example 4. Confirmation of microglia function at the cellular level according to aging.

[0104] 4-1. Confirmation of predation function

[0105] We aimed to investigate the effect of catopril on phagocytosis, a key function of microglia. As shown in Figure 7, microglia aged through dexamethasone treatment or long-term culture (DIV10) showed a decrease in phagocytosis compared to the control group. However, when catopril was treated in aged microglia for 1 or 3 days, phagocytosis was restored.

[0106]

[0107] 4-2. Confirmation of the expression level of Lamin B1, an aging-related factor.

[0108] An experiment was conducted to determine the effect of catopril on the expression level of lamin B1, a marker of cellular senescence. As shown in Figure 8, lamin B1 expression decreased in long-term cultured senescent microglia (DIV10). However, after 3 days of catopril treatment, lamin B1 expression was restored.

[0109]

[0110] 4-3. Confirmation of the senescence-associated secretory phenotype

[0111] The level of senescence in aged cells was determined by examining the levels of various factors secreted by aged cells. To this end, real-time PCR was used to determine the expression of senescence-associated secretory phenotypes in long-term cultured aged microglia (DIV10), microglia treated with catopril for 1 day at DIV9, and microglia treated with catopril for 3 days at DIV7. As a result, as shown in Figure 9, expression of p16, p19, and MCP1 increased in aged microglia. However, when catopril was treated for 3 days, mRNA expression of these genes decreased.

[0112]

[0113] Example 5. Confirmation of rejuvenation effect at the mouse level

[0114] An experiment was conducted to determine whether the rejuvenating effects observed in Examples 3 and 4 above also occurred at the mouse level. For this purpose, 13-week-old young mice (Young, Y) and 85-week-old aged mice (Aged, A) were selected as experimental groups. The aged mice were injected intraperitoneally with catopril at a dose of 10 mg / kg or 20 mg / kg daily for 3 weeks (21 days), and the expression of aging-related proteins was analyzed in the hippocampus of the aged mice.

[0115] As a result, as shown in Figure 10, p16 and p53 expression increased in aged mice compared to young mice. However, when aged mice were injected with 20 mg / kg of catopril for 3 weeks, p16, p21, and p53 expression decreased. In contrast, when 10 mg / kg of catopril was administered, no significant difference was observed compared to aged mice, and the same trend was confirmed in qPCR analysis.

[0116]

[0117] Example 6. Determination of lipofuscin and lipid droplet levels in mouse brain.

[0118] Immunofluorescence (IF) staining was performed to evaluate the degree of lipofuscin and lipid droplet accumulation in the hippocampus (dentate gyrus), cortex, and hypothalamus of mice. As shown in Figures 11a to 11c, lipofuscin and lipid droplet accumulation increased in microglia (IBA-1 positive) of aged mice, and treatment with catopril confirmed that these accumulation levels were reduced.

[0119]

[0120] Example 7. Determining the biological age of mice through RNA transcriptome analysis.

[0121] We conducted an experiment to predict the biological age (aging clock) of mice using RNA transcriptome analysis. Biological age was calculated using RNAAgeCalc, which was trained on human multi-tissue RNA sequencing data. Mouse gene names were converted to human homologs and output as predicted human RNA age. Drug efficacy was evaluated by scaling this value, and data from young cerebrospinal fluid (YCSF), young plasma, and dasatinib and quercetin (Dasatinib+Quercetin), which have been proven to have anti-aging effects in previous studies, were used as a comparison standard.

[0122] According to previous reports, a rejuvenating effect based on the aging clock was reported to be 33.33% using young cerebrospinal fluid, 30.56% using young plasma, and 40% using dasatinib and quercetin.

[0123] In this experiment, RNA was isolated from the hippocampi of young, aged, and aged mice injected with catopril (20 mg / kg, 21 days), and sequenced. As shown in Figures 12a to 12c, a 23.86% decrease was observed in aged mice treated with catopril compared to aged mice. The heatmap reveals that the hierarchy is closer to Y (young), with AC (aged + cap) clustering closer to A (aged) than to A (aged).

[0124]

[0125] Example 8. Confirmation of the mechanism of the rejuvenating effect

[0126] To confirm the mechanism of the rejuvenating effect identified above, we conducted an experiment. Specifically, we examined the levels of p-mTOR, p-AMPK, and p-Akt, signaling systems associated with cell aging, after one-day treatment with catopril at DIV9 (DIV9+CAP1) in a long-term cultured aged microglia model. As a result, as confirmed in Figure 13, catopril reduced the increased levels of p-mTOR in aged microglia and re-increased the decreased levels of p-AMPK.

[0127]

[0128] Example 9. Confirmation of the therapeutic effect on neurodegenerative brain diseases.

[0129] 9-1. Confirmation of gene levels related to neuroinflammation

[0130] To confirm whether the composition of the above-mentioned efficacy actually has a therapeutic effect on the disease, additional experiments were conducted. BV2 cells or primary cultured microglia were transfected with non-targeting siRNA for the control group and grn siRNA for the experimental group. Specifically, BV2 cells were transfected with a pre-designed siGENOME mouse grn siRNA smart pool or non-targeting siRNA for the control group (Dharmacon, M-062705-01-0005; D-001210-02-05). Cells were transfected with a total of 50 nM siRNA using Lipofectamine 3000, and primary microglia were treated with 30 nM. Afterwards, grn mRNA was measured according to the qPCR procedure. As a result, as shown in Figure 14, it was confirmed that grn was reduced by more than 50% in BV2 cells.

[0131]

[0132] 9-2. Checking the level of fat droplets

[0133] Additionally, to determine the level of lipid droplet accumulation, the control group was transfected with non-targeting siRNA, and the experimental group was transfected with grn siRNA. The control group (siCON) was treated with vehicle, and the experimental group (siGRN) was treated with 1 mM captopril for 24 hours. As a result of staining and observing lipid droplets, it was confirmed that lipid droplet accumulation was reduced in both BV2 cells and primary cultured microglia, as shown in Fig. 14. Considering that lipid droplet accumulation is associated with the pathological mechanism that induces inflammation in degenerative neurodegenerative diseases such as Alzheimer's disease and Lou Gehrig's disease, this suggests that the present composition may be effective in treating neurodegenerative diseases by significantly reducing neuroinflammation-related genes (grn) and lipid droplet accumulation.

[0134]

[0135] 9-3. Confirmation of the phagocytic function of microglia

[0136] To evaluate phagocytosis, primary cultured microglia were transfected with non-targeting siRNA as a control group and grn siRNA as an experimental group. Subsequently, the siCON group was treated with vehicle, and the siGRN group was treated with captopril at concentrations of 1 mM, 2 nM, and 3 nM for 24 h. For live imaging evaluation of phagocytosis, primary cultured microglia were cultured in 48-well plates at a density of 6 × 10^4 cells / ml. Then, the cells were treated with 4 μL of pHrodo-conjugated zymosan, and real-time imaging was performed at 5-min intervals for a total of 400 min using a Juli Stage instrument. Since pHrodo-zymosan expresses red fluorescence in the low-pH environment of autolysosomes after being endocytosed, the phagocytic index was calculated as the proportion of cells exhibiting red fluorescence. As a result, as shown in Fig. 15, it was confirmed that catopril had the effect of restoring the phagocytic function of microglia reduced by grn siRNA treatment in a concentration-dependent manner.

[0137]

[0138] 9-4. Confirmation of motility in fruit fly model

[0139] To achieve tissue-specific gene overexpression, a fruit fly model was prepared using the UAS-GAL4 system in a Drosophila model. By crossing the Repo-GAL4 strain, which induces gene expression specifically in glial cells, with the UAS-TDP-43 strain, which expresses the human TDP-43 gene, a fruit fly model overexpressing human TDP-43 protein in glial cells was created (Figs. 16a to 16c).

[0140] Approximately 10 days after mating, when adults emerged, they were reared for 7 and 14 days, respectively, while providing diets containing 0.3% DMSO (vehicle) or captopril (5 mM, 0.3% DMSO in DPBS). Afterwards, a climbing assay was performed to evaluate the locomotor ability of the fruit flies. This test is based on the ability of fruit flies to move against gravity, and motility was measured by quantifying the number of fruit flies that moved to the top of the container within a certain time.

[0141] In the case of the Repo-GAL4 / UAS-TDP-43 fruit fly model that overexpresses TDP-43, motor ability is significantly reduced and almost no movement is shown. Therefore, the effect was analyzed based on the number of fruit flies that moved upward to evaluate whether motor ability was recovered according to drug treatment. The Repo-GAL4 only fruit fly strain was used as a control. As a result, as shown in Figures 16a to 16c, in the fruit fly model that overexpresses TDP-43, the group treated with captopril showed a statistically significant increase in climbing activity at 7 days compared to the group treated with 0.3% DMSO (vehicle). These results suggest that captopril can have a positive effect on improving motor impairment caused by TDP-43 overexpression.

Claims

1. A composition for rejuvenating microglial cells, comprising as an active ingredient an inhibitor of the expression of ACE protein or a gene encoding the same.

2. In claim 1, A composition for rejuvenating microglial cells, wherein the expression of the ACE protein or the gene inhibitor encoding it is at least one selected from the group consisting of captopril, trandolapril, and lisinopril.

3. In claim 2, A composition for rejuvenating microglial cells, wherein the expression of the ACE protein or the gene encoding it is suppressed by captopril.

4. In claim 1, The above rejuvenating composition is a composition for rejuvenating microglial cells that reduces the expression level of beta-galactosidase.

5. In claim 1, The above rejuvenating composition is a composition for rejuvenating microglial cells that reduces the expression level of lipid droplets.

6. In claim 1, A composition for rejuvenating microglial cells, wherein the composition for rejuvenation is treated for 24 to 72 hours.

7. A composition for treating or preventing degenerative brain nerve diseases, comprising as an active ingredient an inhibitor of the expression of ACE protein or a gene encoding the same.

8. In claim 7, A composition for treating or preventing a degenerative brain disease, wherein the degenerative brain disease is at least one disease selected from the group consisting of dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, stroke, multiple sclerosis, movement disorder, learning disorder, and memory impairment.

9. In claim 7, A composition for treating or preventing a degenerative brain disease, wherein the above degenerative brain disease is induced by dexamethasone.

10. In claim 7, A composition for treating or preventing a degenerative brain disease, wherein the above degenerative brain disease is caused by aging of microglial cells.

11. In claim 7, A composition for treating or preventing a degenerative brain disease, wherein the above degenerative brain disease is caused by a mutation in the GRN (progranulin) gene.

12. In claim 7, A composition for treating or preventing a degenerative brain disease, wherein the above degenerative brain disease is caused by overexpression of TDP-43 protein.

13. A health functional food composition for treating or improving degenerative brain diseases, comprising as an active ingredient an inhibitor of the expression of ACE protein or a gene encoding the same.

14. A method for screening a therapeutic agent for a degenerative brain disease, comprising the following steps: A step of treating a candidate substance to cells expressing ACE protein; A step of measuring the protein expression levels of p-mTOR protein and p-AMPK of the above cells; and A step for selecting a substance that reduces the expression level of p-mTOR and increases the expression level of p-AMPK compared to the untreated group.

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