Composition comprising dihydrocostus lactone as active ingredient for preventing or treating diseases related to fused in sarcoma protein or TDP-43

Abstract

The present invention relates to a composition for preventing or treating diseases related to fused in sarcoma (FUS) protein or TAR DNA-binding protein 43 (TDP-43), the composition comprising dehydrocostus lactone as an active ingredient. Specifically, the present invention relates to a pharmaceutical composition and a health functional food for preventing or treating diseases related to FUS or TDP-43, the composition comprising dehydrocostus lactone as an active ingredient. In addition, the present invention relates to a method for inhibiting the overexpression or aggregation of FUS or TDP-43 in cells, the method comprising a step for treating cells with dehydrocostus lactone in vitro.

Description

A composition for preventing or treating a sarcoma fusion protein or Tdp forty-three-related disease comprising dehydrocostus lactone as an active ingredient

[0001] The present invention relates to a composition for preventing or treating a disease related to fused in sarcoma protein (FUS) or TDP-43 (TAR DNA-binding protein 43), which comprises dehydrocostus lactone as an active ingredient.

[0002] The FUS (Fused in sarcoma) protein is a DNA / RNA binding protein that plays a role in various stages of gene expression, including transcription, RNA splicing, mRNA transport, and DNA repair. Meanwhile, mutations and cytoplasmic aggregation of the FUS gene are known risk factors for neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and polyglutamine (polyQ) repeat disorders.

[0003] FUS has been reported to shuttle in the cytoplasm to regulate mRNA trafficking to dendrites in postmitotic neurons and to localize protein synthesis at synapses. FUS's RNA targets in the cerebral cortex have been identified, and it is known to regulate splicing of mRNA sequences important for neuronal integrity and signaling.

[0004] Meanwhile, FUS mutations have been found in frontotemporal dementia and amyotrophic lateral sclerosis, which are representative types of early-onset dementia caused by mutations in RNA-binding genes. In addition, several missense and nonsense mutations in FUS have been reported to be associated with amyotrophic lateral sclerosis, frontotemporal dementia, and hand tremor. Most of these mutations are located in the C-terminal domain, which is known to function as a nuclear localization signal (NLS). Therefore, FUS mutations are known to prevent FUS from translocating to the nucleus, causing it to abnormally localize in the cytoplasm and form FUS aggregates. These cytoplasmic FUS aggregates have been observed in the brains of people with amyotrophic lateral sclerosis or frontotemporal dementia, suggesting that FUS aggregates act as a pathogenic factor in these diseases.

[0005] Transactive response DNA-binding protein 43 (TDP-43) is encoded by TARDBP and is a ubiquitously expressed DNA / RNA-binding protein. TDP-43 contains two RNA recognition motifs, a nuclear localization sequence (NLS), a nuclear export signal, and a glycine-rich C-terminus that mediates protein-protein interactions.

[0006] TDP-43 is predominantly located in the nucleus, but it can be transported between nucleocytoplasms. TDP-43 plays a crucial role in regulating RNA splicing and microRNA biogenesis in the nucleus. It also provides an autoregulatory mechanism at the protein level, allowing it to control the stability of its own mRNA. Furthermore, TDP-43 regulates the splicing and stability of numerous other transcripts, influencing diverse cellular processes.

[0007] Furthermore, TDP-43 is a key component of dendrite and somatodendritic RNA transport granules in neurons, playing a crucial role in neuronal plasticity by regulating local protein synthesis in dendrites. TDP-43 is also involved in the formation of the cytoplasmic stress granule response—a protein complex that sequesters mRNAs unnecessary for survival, suggesting that TDP-43 function is particularly important under conditions of cellular stress.

[0008] Meanwhile, TDP-43 is mainly present in the nucleus, and it is known that TDP-43 proteinopathy is induced when TDP-43 aggregates accumulate in the cytoplasm. Therefore, inhibition of TDP-43 aggregates and improvement of mislocalization to the cytoplasm may be a method for preventing and treating TDP-43 proteinopathy such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and limbic predominant age-related TDP-43 encephalopathy (LATE). However, research on this is still insufficient.

[0009] Both FUS and TDP-43 are ubiquitously expressed, including in glial cells of the central nervous system, and their excessive accumulation in astrocytes and neurons is known to induce neuronal cell death in cell culture and mammalian brain, which may lead to the development of neurodegenerative diseases such as ALS, FTD, AD, and PD.

[0010] Meanwhile, there is still no development of therapeutic agents that can effectively prevent, improve, and treat neurodegenerative diseases through the regulation of FUS and TDP-43.

[0011] Accordingly, the present inventors have found that the dehydrocostus lactone compound can improve neurotoxicity induced by FUS and TDP-43, inhibit the aggregation of FUS and TDP-43, improve mislocalization of TDP-43, and improve damaged mitochondria.

[0012] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating a disease related to FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43), which comprises dehydrocostus lactone or a pharmaceutically acceptable salt thereof as an active ingredient.

[0013] Another object of the present invention is to provide a health functional food for preventing or improving diseases related to FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43), which comprises dehydrocostus lactone or an acceptable salt thereof as an active ingredient.

[0014] Another object of the present invention is to provide a method for inhibiting overexpression or aggregation of FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43) in a cell, comprising a step of treating the cell with dehydrocostus lactone or an acceptable salt thereof in a test tube.

[0015] To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating a disease related to FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43), comprising dehydrocostus lactone or a pharmaceutically acceptable salt thereof as an active ingredient.

[0016] In one embodiment of the present invention, the disease may be a disease caused by overexpression or aggregation of FUS; overexpression or aggregation of TDP-43; mislocalization of TDP-43 into the cytoplasm; neuroinflammation and neurotoxicity caused by overexpression or aggregation of FUS or TDP-43; or mitochondrial dysfunction caused by overexpression or aggregation of FUS or TDP-43.

[0017] In one embodiment of the present invention, the disease may be selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington's disease (HD), and limbic predominant age-related TDP-43 encephalopathy (LATE).

[0018] In one embodiment of the present invention, the dehydrocostus lactone may have an activity of inhibiting overexpression or aggregation of FUS or TDP-43; inhibiting neuroinflammation; inhibiting neurotoxicity; or improving mitochondrial dysfunction.

[0019] In addition, the present invention provides a health functional food for preventing or improving diseases related to FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43), which comprises dehydrocostus lactone or an acceptable salt thereof as an active ingredient.

[0020] In one embodiment of the present invention, the disease may be selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington's disease (HD), and limbic predominant age-related TDP-43 encephalopathy (LATE).

[0021] In one embodiment of the present invention, the dehydrocostus lactone may have an activity of inhibiting overexpression or aggregation of FUS or TDP-43; inhibiting neuroinflammation; inhibiting neurotoxicity; or improving mitochondrial dysfunction.

[0022] The present invention also provides a method for inhibiting overexpression or aggregation of FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43) in a cell, comprising a step of treating the cell with dehydrocostus lactone or an acceptable salt thereof in a test tube.

[0023] In one embodiment of the present invention, cells treated with dehydrocostus lactone may have reduced neurotoxicity and neuroinflammation caused by overexpression or aggregation of FUS or TDP-43.

[0024] In one embodiment of the present invention, cells treated with dehydrocostus lactone may have improved mitochondrial dysfunction caused by overexpression or aggregation of FUS or TDP-43.

[0025] In one embodiment of the present invention, improvement of mitochondrial dysfunction may be an increase in reduced cellular respiration rate and ATP production due to overexpression or aggregation of FUS or TDP-43.

[0026] The composition comprising dehydrocostus lactone as an active ingredient provided in the present invention can suppress overexpression or aggregation of FUS or TDP-43, suppress neuroinflammation and neurotoxicity caused by FUS or TDP-43, and improve mitochondrial dysfunction, and therefore can be usefully used in the manufacture of medicines and health functional foods that can effectively prevent, improve, and treat FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43)-related diseases such as atrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington's disease (HD), and limbic predominant age-related TDP-43 encephalopathy (LATE).

[0027] Figure 1 shows the inhibition of pathological FUS protein expression by dehydrocostus lactone (DHE) treatment in primary astrocytes expressing FUS P525L. A shows the analysis of cell viability through CCK-8 assay after treating primary mouse astrocytes with DHE (10 μM) for 24 hours. B to D show the results of immunoblotting analysis of the expression level of FUS protein after transfecting primary astrocytes with GFP or mutFUS P525L-GFP expressing DNA and treating them with DHE. Each sample was divided into whole cell lysate (B), soluble fraction, and insoluble fraction (C). D shows the result of immunoblotting analysis of the level of FUS protein after separating primary astrocytes into nuclear and cytoplasmic fractions, respectively.

[0028] Figure 2 shows the inhibitory activity of dehydrocostus lactone (DHE) on FUS P525L-induced inflammatory response through the NF-κB pathway. A and B are results of immunoblotting analysis of phosphorylated NF-κB (Ser536) and total NF-κB protein expression levels after treating mutFUS-GFP-transfected astrocytes with DHE (10 μM) for 24 hours, and the graph shows a comparison of the expression levels of phosphorylated NF-κB and total NF-κB. C is results of treating GFP- or mutFUS P525L-GFP-transfected astrocytes with DHE (10 μM) for 24 hours, and then obtaining astrocyte-conditioned medium (ACM). IL-1β, IL-6, IL-1β ... This shows the results of ELISA analysis of the levels of TNF-α and CXCL10.

[0029] Figure 3 shows that dehydrocostus lactone (DHE) can rescue neurotoxicity and mitochondrial dysfunction induced by FUS P525L in astrocytes. A shows the results of CCK-8 assay for neurotoxicity in primary cortical neurons stimulated with GFP ACM, mutFUS P525L ACM, or mutFUS P525L + DHE ACM for 5 days, and B shows the results of basal OCR, ATP production, maximal reserve, and respiratory capacity, which can determine mitochondrial function, after stimulating primary cortical neurons in XF24-well culture plates with GFP ACM, mutFUS P525L ACM, or mutFUS P525L + DHE ACM for 5 days. OCR was quantitatively normalized to total protein concentration (OD).

[0030] Figure 4 shows the effect of reducing neurotoxicity caused by FUS P525L by treatment with dehydrocostus lactone (DHE) in a fruit fly model. A shows the analysis of the level of climbing activity in fruit flies treated with Repo-Gal4, Repo, mutFUS 525L or Repo, mutFUS 525L / DHE at the indicated time points, B shows the analysis of the survival rate of fruit flies for each experimental group, and C shows the results of real-time PCR analysis of mRNA levels of Attc, DptB, Dif and Rel genes in head lysates of control or mutFUS P525L-expressing fruit flies.

[0031] Figure 5 shows that dehydrocostus lactone (DHE) can reduce cytoplasmic TDP-43 protein levels and insoluble TDP-43 protein levels in TDP-43-overexpressing primary astrocytes. A to C are the results of immunoblotting after treating astrocytes transfected with TDP-43-GFP and GFP, respectively, with DHE (10 μM) for 24 hours. A shows the TDP-43 protein levels detected in the whole cell lysate, B shows the TDP-43 protein levels detected in the soluble and insoluble fractions of primary astrocytes, and C shows the TDP-43 protein levels detected in each fraction after dividing primary astrocytes into nuclear and cytoplasmic fraction extracts.

[0032] Figure 6 shows the effect of dehydrocostus lactone (DHE) on NF-κB activity and neurotoxicity regulation by TDP-43. A shows the analysis of phosphorylated NF-κB (Ser536) and total NF-κB protein levels in GFP- or TDP-43-GFP-transfected cells treated with DHE (10 μM) for 24 hours and in the untreated group, and B shows the cell viability determined by CCK-8 assay after stimulating primary cortical neurons with GFP ACM, TDP-43 ACM, or TDP-43 + DHE ACM for 5 days, respectively.

[0033] Figure 7 shows the inhibitory effect of dehydrocostus lactone (DHE) treatment on TDP-43-induced neurotoxicity in Drosophila glial cells. A shows the results of climbing activity for groups of Drosophila treated with Repo-Gal4, Repo-TDP-43, or Repo-TDP-43+DHE, and B shows the survival rate of each group of Drosophila analyzed over time.

[0034] Figure 8 shows the inhibition of inflammation by dehydrocostus lactone (DHE) treatment in primary fibroblasts obtained from FUS-ALS patients treated with NaAsO₂ (0.5 mM). A and B are the results of immunoblotting analysis of FUS protein levels and phosphorylated NF-κB (Ser536) and total NF-κB protein levels in primary fibroblasts derived from normal individuals and FUS-ALS patients treated with NaAsO₂ (0.5 mM) for 12 hours, and C is the result of pretreatment of primary fibroblasts derived from normal individuals and FUS-ALS patients with 10 μM dehydrocostus lactone (DHE) for 30 minutes, followed by treatment with NaAsO₂ (0.5 mM), and then analysis of the protein levels of phosphorylated NF-κB (Ser536) and total NF-κB. Here, the expression of phosphorylated NF-κB was expressed as a ratio to total NF-κB and was used as a control for quantification standardization of β-actin protein.

[0035] The present invention is characterized by providing a pharmaceutical composition for preventing or treating a disease related to FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43), comprising dehydrocostus lactone or a pharmaceutically acceptable salt thereof as an active ingredient.

[0036] Dehydrocostus lactone is a natural sesquiterpene lactone extracted from many plant species, including Saussurea lappa, and is known to possess anticancer activity. Specifically, in breast cancer cells, dehydrocostus lactone is known to induce cell cycle arrest and apoptosis through the c-Myc / p53 and AKT signaling pathways. It is also known to inhibit glioma by targeting IKKβ and inhibiting the NF-κB / COX-2 signaling pathway.

[0037] Meanwhile, the relationship between dehydrocostus lactone and diseases caused by FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43) has not been previously known.

[0038] Accordingly, the present inventors, while researching to develop a new treatment for diseases caused by FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43), confirmed that the dehydrocostus lactone compound can suppress the overexpression or aggregation of FUS or TDP-43, and has the effect of suppressing neuroinflammation and neurotoxicity caused by them, as well as the effect of improving mitochondrial dysfunction, thereby elucidating that dehydrocostus lactone can be used as a new treatment for diseases related to FUS and / or TDP-43.

[0039] As previously mentioned in the prior art, FUS mutations are found in frontotemporal dementia (FTD), a type of early-onset dementia, and amyotrophic lateral sclerosis (ALS), and are characterized by abnormal localization and aggregate formation in the cytoplasm of FUS mutations.

[0040] In addition, TDP-43 protein (TAR DNA-binding protein 43, TDP43, TARDBP) is a protein encoded by the TARDBP gene, and TDP-43 is overexpressed in neuronal cells of approximately 95% of ALS patients and approximately 50% of FTD patients, and there are pathological characteristics in which abnormal accumulation (aggregation) of TDP-43 and mislocalization to the cytoplasm are observed.

[0041] Therefore, if we can suppress the overexpression of FUS and TDP-43 proteins and control their aggregation and mislocalization to the cytoplasm, we can prevent, improve, and treat neurodegenerative diseases caused by FUS and TDP-43.

[0042] In one embodiment of the present invention, it was investigated whether dehydrocostus lactone can suppress the expression and aggregation of FUS in FUS-overexpressing astrocytes and control inflammation caused by it. As a result, it was found that in the group in which dehydrocostus lactone was treated in astrocytes in which the FUS-P525L mutation was overexpressed, the expression and aggregation of FUS were reduced, and the aggregation of FUS was suppressed. In addition, it was confirmed that phosphorylation of NF-κB, a signaling pathway that induces neuronal cell death due to inflammatory inflammation, was suppressed, and translocation of NF-κB into the nucleus was suppressed, and thus it was found that the expression of inflammatory factors could be suppressed.

[0043] In addition, in another embodiment of the present invention, the expression levels of inflammatory cytokines and chemokines contained in the conditioned medium of FUS P525L-expressing astrocytes treated with dehydrocostus lactone and the conditioned medium of FUS P525L-expressing astrocytes not treated with dehydrocostus lactone were analyzed. As a result, the expression levels of IL-1β, IL-6, TNF-α, and CXCL10 were found to be increased in the conditioned medium of astrocytes expressing mutant FUS P525L compared to the control conditioned medium, whereas the expression levels of these inflammatory cytokines and chemokines were found to be significantly decreased in the conditioned medium of FUS P525L-expressing astrocytes treated with dehydrocostus lactone.

[0044] Through these results, the inventors were able to find that Dehydrocostus lactone can inhibit the overexpression and aggregation of FUS protein and suppress the neuroinflammatory response induced by FUS.

[0045] Overexpression and aggregation of FUS mutants (mutFUS) in astrocytes induces neurotoxicity, which induces apoptosis of neurons.

[0046] In the examples below, the ability of dehydrocostus lactone to inhibit such neurotoxicity and neuronal cell death was analyzed, and it was found that neurotoxicity was reduced in the group in which primary cortical neurons were treated with conditioned medium obtained from FUS P525L-overexpressing astrocytes treated with dehydrocostus lactone.

[0047] In addition, in another embodiment of the present invention, it was shown that overexpression and aggregation of FUS mutant (mutFUS) in astrocytes caused mitochondrial dysfunction in neurons, while it was confirmed that the dehydrocostus lactone of the present invention could improve such mitochondrial dysfunction.

[0048] In particular, since mitochondrial dysfunction is known to be a cause of amyotrophic lateral sclerosis (ALS), the inventors of the present invention were able to find that the dehydrocostus lactone of the present invention can prevent, improve, and treat diseases caused by mitochondrial dysfunction.

[0049] Furthermore, the present invention confirmed that dehydrocostus lactone can suppress the level of insoluble TDP-43 protein in astrocytes in which TDP-43 is overexpressed, and has the effect of improving the mislocalization of TDP-43 protein into the cytoplasm.

[0050] As previously mentioned in the prior art, TDP-43 is mainly present in the nucleus, but when TDP-43 aggregates are mislocalized and accumulated in the cytoplasm, it causes TDP-43 protein disease.

[0051] Accordingly, in one embodiment of the present invention, when the level of TDP-43 protein was analyzed after treating astrocytes in which TDP-43 protein was overexpressed with dehydrocostus lactone, it was found that the expression level of TDP-43 protein was significantly reduced in the group treated with dehydrocostus lactone compared to the group not treated, and in particular, the expression level of insoluble TDP-43 protein was found to be effectively suppressed, and the level of neurotoxic TDP-43 protein located in the cytoplasm was found to be reduced.

[0052] In addition, in another embodiment of the present invention, it was confirmed that dehydrocostus lactone can suppress or reduce neurotoxicity caused by accumulation (aggregation) of TDP-43 protein. While neuroinflammation was shown to increase in astrocytes in which TDP-43 was overexpressed, it was confirmed that the group treated with dehydrocostus lactone could suppress phosphorylation of NF-κB caused by TDP-43, which is related to inflammatory signal transduction, thereby inducing a reduction in neurotoxicity and inhibition of neuronal cell death.

[0053] Through these results, the inventors of the present invention were able to determine that a composition containing dehydrocostus lactone as an active ingredient can be used as a pharmaceutical composition capable of preventing or treating FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43)-related diseases.

[0054] Therefore, the present invention can provide a pharmaceutical composition for preventing or treating a disease related to FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43), which comprises dehydrocostus lactone or a pharmaceutically acceptable salt thereof as an active ingredient.

[0055] In the present invention, the FUS or TDP-43 related disease is a disease caused by overexpression or aggregation of FUS; overexpression or aggregation of TDP-43; mislocalization of TDP-43 to the cytoplasm; neuroinflammation and neurotoxicity caused by overexpression or aggregation of FUS or TDP-43; or mitochondrial dysfunction caused by overexpression or aggregation of FUS or TDP-43; and the diseases may include, but are not limited to, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington's disease (HD), and limbic predominant age-related TDP-43 encephalopathy (LATE).

[0056] Additionally, the dehydrocostus lactone compound of the present invention may be included in the composition of the present invention in the form of a 'pharmaceutically acceptable salt' or in the form of an 'acceptable salt'.

[0057] In the present invention, the term "pharmaceutically acceptable salt" refers to a salt that can be used pharmaceutically among salts, which are substances in which cations and anions are bonded by electrostatic attraction, and refers to any organic or inorganic addition salt of the dehydrocostus lactone compound at a concentration that is relatively non-toxic and harmless to the patient and in which side effects caused by the salt do not reduce the beneficial efficacy of the dehydrocostus lactone compound. Such salts include acid addition salts formed by pharmaceutically acceptable free acids or metal salts formed by bases.

[0058] Organic acids and inorganic acids can be used as free acids, and inorganic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, and tartaric acid can be used as inorganic acids, and organic acids such as methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, and hydroiodic acid can be used, but are not limited thereto.

[0059] Additionally, pharmaceutically or food-grade acceptable metal salts can be prepared using bases. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering out the undissolved compound salt, and evaporating and drying the filtrate. In this case, sodium, potassium, or calcium salts are particularly suitable for pharmaceutical or food applications, but are not limited thereto.

[0060] Pharmaceutically acceptable salts of the dehydrocostus lactone compound of the present invention include salts of acidic or basic groups that may be present in the dehydrocostus lactone compound, unless otherwise specified. For example, pharmaceutically or food-wise acceptable salts may include sodium, calcium and potassium salts of hydroxyl groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, and the like, and may be prepared by methods for preparing salts known in the art.

[0061] In addition, the dehydrocostus lactone of the present invention can prevent, improve, and treat diseases caused by FUS or TDP-43 through inhibition of overexpression or aggregation of FUS or TDP-43; inhibition of neuroinflammation; inhibition of neurotoxicity; and / or improvement of mitochondrial dysfunction; and activation.

[0062] In patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), overexpression, mutations, and cytoplasmic protein aggregation of FUS and TDP-43 have been found. Normally, they should be present in the nucleus, but under pathological conditions, they are located in the cytoplasm, resulting in cytoplasmic toxicity or loss of function in the nucleus, which has been revealed to be an important pathogenic factor in these diseases. It is known that abnormal cytoplasmic accumulation of TDP-43 and FUS is found in Alzheimer's disease (up to 30%), Parkinson's disease, and Huntington's disease in addition to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).

[0063] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The composition containing the pharmaceutically acceptable carrier may be administered orally or parenterally in various dosage forms. When formulated, it is prepared using commonly used diluents or excipients, such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0064] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.

[0065] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can 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 glycerogelatin.

[0066] The above pharmaceutical composition may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.

[0067] In addition, the composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined based on factors including the type and severity of the individual, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0068] The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Considering all of the above factors, it is important to administer an amount that achieves maximum efficacy with the minimum amount possible without causing side effects. A typical dosage for the pharmaceutical composition of the present invention is 0.001-100 mg / kg for adults.

[0069] The pharmaceutical composition may be administered via any conventional route as long as it can reach the target tissue. The composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonary, or rectally, depending on the intended purpose, but is not limited thereto. Furthermore, the composition may be administered via any device capable of transporting the active ingredient to target cells.

[0070] The composition of the present invention can be used alone or in combination with methods using surgery, hormone therapy, drug therapy, and biological response modifiers for the prevention and treatment of target diseases.

[0071] In addition, the present invention can provide a health functional food for preventing or improving diseases related to FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43), which contains dehydrocostus lactone or an acceptable salt thereof as an active ingredient.

[0072] The health functional food containing the dehydrocostus lactone of the present invention as an active ingredient for preventing or improving a disease related to FUS or TDP-43 can be easily utilized as a food effective in preventing and improving the symptoms of the disease, such as a main ingredient, auxiliary ingredient, food additive, functional food, or beverage of a food.

[0073] In this application, the term 'food' refers to a natural or processed product containing one or more nutrients, preferably one that has gone through a certain degree of processing to become directly edible, and in its general sense, includes all foods, food additives, functional foods, and beverages.

[0074] Foods to which the composition according to the present invention can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, functional foods, etc. In addition, foods in the present invention include, but are not limited to, special nutritional foods (e.g., formulated milk, infant and toddler food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g., ramen, noodles, etc.), bread, health supplements, seasoned foods (e.g., soy sauce, soybean paste, red pepper paste, mixed paste, etc.), sauces, confectionery (e.g., snacks), candies, chocolates, gum, ice cream, processed dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickled vegetables, etc.), beverages (e.g., fruit drinks, vegetable drinks, soy milk, fermented drinks, etc.), and natural seasonings (e.g., ramen soup, etc.). The above food, beverage or food additive can be manufactured by a conventional manufacturing method.

[0075] In addition, the above-mentioned 'functional food' refers to a food group or food composition that has been processed to sufficiently express the body's regulatory function regarding biological defense rhythm regulation, disease prevention and recovery, etc., by using physical, biochemical, or bioengineering techniques to give added value to the food so that the food's function can be performed and expressed for a specific purpose, and may be specifically a health functional food. The above-mentioned functional food may include food additives that are acceptable in terms of food science, and may further include appropriate carriers, excipients, and diluents that are commonly used in the manufacture of functional foods.

[0076] Additionally, in the present invention, the term "beverage" refers to a general term for anything consumed to quench thirst or enjoy a flavor, and includes functional beverages. The beverage, in addition to including a composition for preventing and improving the symptoms of the disease as an essential ingredient in the indicated proportions, has no particular limitations on other ingredients, and, like conventional beverages, may contain various flavorings or natural carbohydrates as additional ingredients.

[0077] Furthermore, in addition to those described above, a food containing the composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., and the above components may be used independently or in combination.

[0078] In a food containing the composition of the present invention, the amount of the composition of the present invention may be comprised in an amount of 0.001 wt% to 90 wt% of the total food weight, preferably 0.1 wt% to 40 wt%, and in the case of a beverage, it may be comprised in an amount of 0.001 g to 2 g, preferably 0.01 g to 0.1 g, based on 100 ml. 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, and since the active ingredient does not have any problems in terms of safety, it may be used in an amount greater than the above range, and thus is not limited to the above range.

[0079] In addition, the present invention can provide a method for inhibiting overexpression or aggregation of FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43) in a cell, which comprises a step of treating the cell with dehydrocostus lactone in a test tube.

[0080] Cells treated with dehydrocostus lactone exhibit reduced neurotoxicity and neuroinflammation caused by overexpression or aggregation of FUS or TDP-43, and improved mitochondrial dysfunction caused by overexpression or aggregation of FUS or TDP-43.

[0081] The improvement in the above mitochondrial dysfunction may be due to the restoration and increase of the reduced cellular respiration rate and ATP production caused by overexpression or aggregation of FUS or TDP-43.

[0082] Furthermore, the present invention can provide a method for treating a disease related to FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43), which comprises a step of administering a composition containing dehydrocostus lactone or an acceptable salt thereof as an active ingredient to a subject other than a human suffering from a disease caused by FUS or TDP-43.

[0083] The composition may be administered to a patient group suffering from a FUS or TDP-43-related disease caused by overexpression or aggregation of FUS; overexpression or aggregation of TDP-43; mislocalization of TDP-43 to the cytoplasm; neuroinflammation and neurotoxicity caused by overexpression or aggregation of FUS or TDP-43; or mitochondrial dysfunction caused by overexpression or aggregation of FUS or TDP-43.

[0084] Additionally, the FUS or TDP-43 related disease may be amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington's disease (HD), or limbic predominant age-related TDP-43 encephalopathy (LATE).

[0085] The present invention will now be described in more detail with reference to examples. These examples are intended merely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

[0086]

[0087] <Preparation example and experimental method>

[0088] reagent

[0089] Dehydrocostus lactone was purchased from Sigma and used, and sodium arsenite and ethanol were purchased from Merck-Millipore.

[0090]

[0091] antibodies

[0092] The antibodies used in this example are as follows: rabbit anti-FUS (A300-292A) [Bethyl Laboratories]; rabbit anti-TDP-43 (10782-2-AP) [Proteintech]; mouse anti-Lamin A / C (05-714) [EMD Millipore]; rabbit anti-NF-κB p65 (Ser536) (3033), rabbit anti-NF-κB (8242), and HRP-conjugated anti-α-tubulin (9099) [Cell Signaling Technology]; HRP-conjugated anti-rabbit IgG (sc-2004) and HRP-conjugated anti-mouse IgG (sc-2005) [Santa Cruz].

[0093]

[0094] primary cell cultures

[0095] Primary cultures of isolated cerebral cortical neurons were obtained from 16-day-old C57BL / 6 mouse embryos. The brains were rapidly removed from the mouse embryos and placed in culture dishes containing HBSS (Gibco). The cortex was then isolated, transferred to a tube, and washed twice with HBSS (Gibco). The cerebral cortex tissue was then enzymatically treated with papain (20 units / ml) (Worthington Biochemical Corporation) and DNase I (0.005%) at 37°C for 30 min. The tissue was then mechanically dissociated using 1,000 μl and 200 μl pipette tips to ensure complete tissue homogenization. Cortical cells were centrifuged at 130 × g for 10 min at room temperature, and the dissociated cells were seeded onto poly-d-lysine (Sigma-Aldrich)-coated plates supplemented with neurobasal media containing 2 mM glutamine (Gibco), N2 supplement (Gibco), B27 supplement (Gibco), and 50 μg / ml penicillin-streptomycin (P / S, Gibco). The culture media was replaced after 5 days and then every 3 days thereafter, and the cells were used after culturing for 14–21 days.

[0096] Primary astrocyte culture was performed as follows. Brains were isolated from 1-2 day-old C57BL / 6 mice, and the isolated whole brains were homogenized and passed through a 70-μm strainer. The cells were then seeded in T75 culture flasks and cultured at 37°C in a humidified atmosphere containing 5% CO2. The medium was changed after 5 days of culture, and thereafter every 2 days. The cells were cultured for 14-21 days before use. Secondary pure astrocyte cultures were obtained by shaking the mixed glial cell cultures at 250 rpm for 4 hours, and the culture medium was discarded. The obtained astrocytes were dissociated using trypsin-EDTA (Life Technologies) and centrifuged at 800 × g for 30 minutes. Afterwards, the astrocytes were seeded onto plates containing DMEM (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 50 μg / ml P / S. In addition, the animals used in this experiment were cared for in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

[0097]

[0098] Transfection

[0099] 6-well plate (80x10 4Primary astrocytes cultured in 100 μl / well (10 cells / well) were transfected with 6 μg of GFP (pCMV6-AC-GFP, Origene Technologies, PS100010), human TDP-43 (pCMV6-AC-TDP-43-GFP, Origene Technologies, RG210639), or human FUS P525L (pCMV6-AC-FUS-GFP, Origene Technologies, RG201808, mutants were generated by site-directed mutagenesis) vectors using the Neon transfection system (Thermo Fisher Scientific) according to the manufacturer's instructions. Two days after transfection, cells were treated with DHE (10 μM) or ethanol for 24 h.

[0100]

[0101] Cytotoxicity test

[0102] Primary astrocytes (5 x 10 4 Cells / well) were cultured in 96-well plates and treated with DHE (10 μM) for 24 hours. The same volume of ethanol was used as a control group. Cytotoxicity was measured using Cell Counting Kit-8 (CCK-8; Enzo Life Science). 10 μl of CCK-8 reagent was added to each well, incubated at 37 °C for 2 hours, and then the absorbance was measured at 450 nm using a microplate reader (Tecan). Cell viability was expressed as a percentage of the control group, and all experiments were repeated three times.

[0103]

[0104] Astrocyte-conditioned media (ACM)

[0105] For the preparation of astrocyte-conditioned medium (ACM) treated with dehydrocostus lactone (DHE), live primary astrocytes (15x10) transfected with GFP / FUS P525L / TDP-43 obtained via FACS 4 (10 cells / well) were adapted in DMEM for 24 h. Primary astrocytes transfected with GFP, FUS P525L, or TDP-43 were treated with DHE (10 μM) or ethanol for 24 h. Primary astrocytes were then washed twice with PBS and cultured in fresh DMEM medium for an additional 24 h. ACM was then collected, centrifuged at 200 × g for 10 min to remove cell debris, and stored at -80°C.

[0106]

[0107] Neuronal cultures treated with astrocyte-conditioned medium

[0108] For the culture of neurons using ACM, primary cortical neurons were stimulated for 5 days with (1) GFP transfection + EtOH-treated ACM [GFP ACM], (2) FUS P525L transfection + EtOH-treated ACM [FUS P525L ACM], (3) FUS P525L transfection + Dehydrococcus lactone (DHE)-treated ACM [FUS P525L+DHE ACM], (4) TDP-43 transfection + EtOH-treated ACM [TDP-43 ACM], and (5) TDP-43 transfection + Dehydrococcus lactone (DHE)-treated ACM [TDP-43+DHE ACM], and then these cells were used for CCK-8 assay.

[0109]

[0110] Quantitative RT-PCR

[0111] Total RNA was extracted from cells and the head region of Drosophila using TRIzol reagent (Life Technologies), and analyzed using the RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions. cDNA synthesis was performed using 100 ng of RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems) at 37°C for 120 min. Quantitative RT-PCR was performed using TB Green Premix Ex Taq™ (Tli RNaseH Plus) (Takara Bio Inc.) according to the manufacturer's instructions, and detection was performed using an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems). Gapdh and 18S rRNA were used as internal controls, and the relative differences in gene expression determined by real-time PCR experiments were calculated using the 2-ΔΔCt method.

[0112]

[0113] Immunoblot analysis

[0114] For total protein extraction, cells were homogenized using cell lysis buffer (Cell Signaling Technology) or 1× LDS sample buffer (Invitrogen™) containing a cocktail of protease and phosphatase inhibitors (Roche). Protein concentrations of the cell lysates were determined using the BCA Protein Assay Kit (Thermo Scientific™). Protein extracts were mixed with 4× Bolt™ LDS sample buffer (Invitrogen™) and 10× Bolt™ Sample Reducing Agent buffer (Invitrogen), incubated at 95 °C for 5 minutes, and equal amounts of protein from each sample were separated by electrophoresis on Bolt™ 4-12% Bis-Tris gels (Thermo Fisher Scientific) or Bolt 3-8% Tris-acetate gels (Thermo Fisher Scientific), and then transferred to an iBlot™ 2 Transfer stack (PVDF; Invitrogen™) using an iBlot™ 2 Dry Blotting System (Invitrogen™). After blocking the membrane with 5% skim milk, the primary antibody and HRP-conjugated secondary antibodies (anti-rabbit IgG and anti-mouse IgG) were sequentially reacted, and proteins were detected with ECL Prime Western Blotting Protection Reagents (Cytiva). Relative expression levels were analyzed using Fusion-FX software (Vilber Lourmat).

[0115]

[0116] Preparation of nuclear and cytoplasmic fraction extracts

[0117] Astrocytes (15 x 10 ) transfected with FUS P525L or TDP-43 4(10 cells / well) were fractionated using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific). Nuclear and cytoplasmic fractions were obtained by centrifugation at 16,000 x g for 5 min at 4°C using cold CER I and CER II buffers. The supernatant containing the cytoplasmic extract was collected, and the pellet was dissolved in cold NER buffer. The supernatant was then vortexed, and the nuclear fraction extract of the supernatant was obtained by centrifugation at 16,000 x g for 10 min at 4°C. Each extract of the nuclear and cytoplasmic fractions was mixed with 4× Bolt™ LDS sample buffer and 10× Bolt™ sample reducing agent buffer, and then heated at 95°C for 5 min before use in the experiments.

[0118]

[0119] Preparation of soluble and insoluble cell extracts

[0120] Cells were homogenized with 1X cell lysis buffer (Cell signaling technology, #9803) containing protease and phosphatase inhibitor cocktails (Roche, 11836153001, 04906837001). Soluble and insoluble fractions were obtained by centrifugation at 100,000 x g for 30 min at 4°C. After centrifugation, the soluble fraction of the supernatant was obtained, and the insoluble fraction was used as the fraction obtained by solubilizing the pellet with 2% SDS buffer. After sonication, the cell lysate was mixed with 4x Bolt™ LDS sample buffer (Invitrogen, B0007) and 10x Bolt™ sample reducing agent buffer (Invitrogen, B0009) and then heated at 95°C for 5 min.

[0121]

[0122] ELISA

[0123] For analysis of protein levels of IL-1β, IL-6, and TNF-α, astrocytes (15 x 10 4Supernatants from 100 cells / well were analyzed using a mouse ELISA Development kit (R&D Systems) for each cytokine or chemokine. Specifically, a 96-well ELISA plate was coated with capture antibodies, and after blocking the antibodies, samples, recombinant cytokines, or chemokine standards were added. For detection, biotinylated detection antibodies were added, followed by the addition of streptavidin-HRP and substrate (R&D Systems) according to the manufacturer's instructions and reaction. The absorbance was then measured at 450 nm and 540 nm using a microplate reader (Tecan).

[0124]

[0125] Mitochondrial activity analysis

[0126] To analyze neuronal mitochondrial dysfunction, primary cortical neurons cultured in XF24-well cell culture plates (Seahorse Bioscience) were stimulated for 5 days with GFP ACM, FUS P525L ACM, or ACM treated with FUS P525L / DHE. Cells were then washed twice with XF basal medium supplemented with 2 mM L-glutamine, 10 mM D-glucose, and 1 mM sodium pyruvate (pH 7.4) and incubated for 1 h at 37°C in a non-CO2 incubator. Mitochondrial dysfunction was assessed using the XF Cell Mito Stress Test Kit (Seahorse Bioscience) according to the manufacturer's instructions and measured using the XF24 Extracellular Flux Analyser (Seahorse Bioscience). 24-well utility plates were hydrated and treated with 2 μM oligomycin, 1 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and 0.5 μM antimycin A+rotenone, and then calibrated using the analyzer. Basal oxygen consumption rate (OCR), ATP production, maximal reserve, and respiratory capacity were measured and averaged from five wells per condition in each individual experiment. OCR was normalized to total protein concentration (OD). After the seahorse assay, the plates were centrifuged at 280 × g for 5 minutes. The medium was removed, washed twice with PBS, and the cells were lysed in buffer. The protein concentration of the cell lysate was measured using the BCA assay kit.

[0127]

[0128] Obtaining patient-derived primary human fibroblasts and culturing of primary human fibroblasts

[0129] Human primary fibroblasts were collected from an ALS patient with a FUS mutation (p.G504Wfs*12, age: 34, female) and a healthy normal person (age: 35, female).

[0130] Primary human fibroblasts obtained from skin biopsies of patients with FUS G504Wfs*12 and healthy individuals were cultured in DMEM (Life Technologies) supplemented with 10% heat-inactivated FBS, 1X MEM-NEAA (Gibco), and 100 μg / ml Normocin (InvivoGen). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2, and passage-matched fibroblasts (passages 7–10) were used in all experiments.

[0131]

[0132] Drosophila staining

[0133] Drosophila stocks were maintained at 24°C on standard cornmeal agar medium unless otherwise specified. UAS-TDP-43 constructs, Repo-Gal4 / + and Repo-Gal4 / UAS-TDP-43, were described previously. Repo-Gal4 / UAS-FUS P525L was obtained from Dr. Nancy M. Bonini (University of Pennsylvania). Females harboring UAS-TDP-43 or UAS-FUS P525L were crossed with males harboring the Repo-Gal4 (pan-glial) driver to overexpress target genes in the entire glial cell population.

[0134]

[0135] Drosophila lifespan and climbing assays of adult Drosophila

[0136] Longevity and climbing assays were performed using progeny flies derived from crosses of UAS-FUS P525L or UAS-TDP-43 with the Repo-Gal4 strain. Adult males (0–1 day old) were isolated and transferred to laboratory vials containing fly medium supplemented with or without DHE (10 μM) at a density of 20 (for longevity assay) or 25 (for climbing assay) flies per vial. The number of dead flies was counted daily, and flies were transferred to fresh medium every other day. Analysis of adult locomotor function was performed using a conventional climbing assay, and 125 flies per genotype were analyzed per time point in all experiments.

[0137]

[0138] Statistical processing

[0139] Data were analyzed using Student's t test (Vassar Stats, www.vassarstats.net) or one-way ANOVA followed by Bonferroni's multiple comparison test (GraphPad Prism Software, La Jolla, CA). Differences were considered significant when p < 0.05 and are expressed as follows: *p < 0.05; **p < 0.005; ***p < 0.001; or NS, not significant.

[0140]

[0141] <Example 1>

[0142] Dehydrocostus lactone inhibits FUS aggregation and improves inflammatory responses in FUS-overexpressing astrocytes.

[0143] Most disease-associated FUS mutations cluster near the nuclear localization signal at the C terminus. The P525L mutation in FUS is one of the most frequently found mutations in amyotrophic lateral sclerosis (ALS) patients and is also associated with the rapid-onset form of ALS. Furthermore, increased insoluble FUS is known to be a hallmark pathological feature of FUS-induced neurodegeneration.

[0144] Accordingly, the inventors of the present invention treated astrocytes overexpressing FUS mutant (mutFUS; FUS-P525L) with dehydrocostus lactone and analyzed the effect of dehydrocostus lactone on FUS-induced neurodegeneration.

[0145] Prior to this analysis, primary mouse astrocytes were treated with dehydrocostus lactone at various concentrations, and then cytotoxicity was analyzed.

[0146] As a result, it was confirmed that a treatment concentration of 10 μM did not induce apoptosis of astrocytes, so in the following experiment, dehydrocostus lactone was used at a concentration of 10 μM (Fig. 1A).

[0147]

[0148] <1-1> Analysis of FUS protein expression and its effect on NF-κB

[0149] We analyzed whether dehydrocostus lactone affects FUS protein expression.

[0150] The level of FUS protein was measured from a group treated with and not treated with dehydrocostus lactone in astrocytes overexpressing the FUS-P525L mutant, and soluble and insoluble fractions were obtained from each experimental cell group. In addition, nuclear fractions and cytoplasmic fractions were obtained, respectively, and the level of FUS protein in each fraction was measured.

[0151] As a result, as shown in Figures 1B to 1D, the level of FUS protein increased by overexpression of FUS P525L in all experimental cell groups was found to be reduced by treatment with dehydrocostus lactone.

[0152]

[0153] In addition, the present inventors have confirmed whether the dehydrocostus lactone of the present invention is involved in the NF-κB signaling pathway, as it is known that the expression of the FUS mutant protein associated with ALS in astrocytes induces neuronal cell death through the NF-κB pathway.

[0154] As a result, cells overexpressing FUS P525L showed increased phosphorylation of serine 536 residue of NF-κB compared to cells transfected with GFP alone, whereas FUS P525L-overexpressing cells treated with dehydrocostus lactone showed decreased phosphorylation of serine 536 residue of NF-κB (Fig. 2A). In addition, NF-κB was observed in the nuclear location in astrocytes overexpressing FUS P525L, whereas translocation of NF-κB into the nucleus was inhibited in the group treated with dehydrocostus lactone (Fig. 2B).

[0155]

[0156] <1-2> Analysis of the effect on the expression of inflammatory cytokines and chemokines

[0157] Next, to determine whether dehydrocostus lactone also affects the expression of inflammatory cytokines and chemokines in astrocytes, the inventors measured the levels of IL-1β, IL-6, TNF-α, and CXCL10 using ELISA kits in GFP-expressing astrocytes, FUS P525L-expressing astrocytes, and FUS P525L-expressing astrocytes treated with dehydrocostus lactone. The analysis was performed on ACM (astrocytic conditioned medium) obtained from each cell population.

[0158] As a result, the expression levels of IL-1β, IL-6, TNF-α, and CXCL10 were found to be increased in the ACM of astrocytes overexpressing FUS P525L compared to GFP-expressing astrocytes, whereas the expression levels of IL-1β, IL-6, TNF-α, and CXCL10 were found to be significantly reduced in the FUS P525L-overexpressing astrocyte group treated with dehydrocostus lactone (Fig. 2C).

[0159]

[0160] These results suggest that changes in FUS expression in astrocytes can affect the activation of the NF-κB pathway, thereby inducing an inflammatory response. Furthermore, we found that dehydrocostus lactone has the effect of alleviating FUS protein aggregation and FUS-induced inflammatory response in astrocytes.

[0161]

[0162] <Example 2>

[0163] Confirmation of the effect of Dehydrocostus lactone on reducing FUS-induced neurotoxicity and improving mitochondrial dysfunction.

[0164] We investigated whether dehydrocostus lactone could ameliorate neurotoxicity induced by FUS mutation (mutFUS) in astrocytes.

[0165] As a result, when ACM obtained from FUS P525L-overexpressing astrocytes was treated with primary cortical neurons, the neurotoxicity was analyzed, and it was found that neurotoxicity was induced. On the other hand, in the group in which primary cortical neurons were treated with ACM obtained from FUS P525L-overexpressing astrocytes treated with dehydrocostus lactone, neurotoxicity was found to be reduced (Fig. 3A).

[0166]

[0167] Furthermore, to investigate whether FUS P525L overexpression in astrocytes could induce mitochondrial dysfunction in neurons, the cellular oxygen consumption rate (OCR) was measured using a Seahorse XF24 extracellular flux analyzer and mitochondrial stress test kit (Seahorse Bioscience) in primary cortical neurons treated with ACM. The group treated with FUS P525L-ACM in primary cortical neurons showed a decreased cellular oxygen consumption rate, whereas the group treated with ACM obtained from FUS P525L-overexpressing astrocytes treated with dehydrocostus lactone showed an improvement in the decreased cellular oxygen consumption rate (Fig. 3B).

[0168] Furthermore, basal mitochondrial respiration, ATP production, maximal respiration, and reserve respiratory capacity were significantly reduced by FUS P525L-ACM treatment in primary cortical neurons, indicating mitochondrial dysfunction, whereas the group treated with ACM obtained from FUS P525L-overexpressing astrocytes treated with dehydrocostus lactone showed significantly improved mitochondrial dysfunction (Fig. 3B).

[0169] These results suggest that dehydrocostus lactone can effectively prevent, improve, and treat neuronal mitochondrial dysfunction induced by FUS mutations in astrocytes.

[0170]

[0171] <Example 3>

[0172] Confirmation of the amelioration of FUS-induced glial neurotoxicity by dehydrocostus lactone treatment in a Drosophila model.

[0173] We analyzed the effects of dehydrocostus lactone on the lifespan reduction and climbing defects induced by FUS mutations using a Drosophila model expressing human FUS P525L in all glial cell types.

[0174] As a result, fruit flies expressing FUS P525L showed significantly reduced climbing ability and lifespan. Meanwhile, flies expressing FUS P525L reared on a medium supplemented with dehydrocostus lactone showed recovery of the reduced lifespan and climbing defects (Figs. 4A and 4B).

[0175] Furthermore, we investigated whether Dehydrocostus lactone could modulate inflammatory responses through the NF-κB pathway in a Drosophila model. To this end, real-time PCR analysis was performed to analyze the effects on the expression of AMP genes (Attc and DptB) and the Toll and IMD pathways (Dif and Rel). These pathways are homologous to the NF-κB pathway in Drosophila.

[0176] As a result, it was found that the expression of four genes, Attc, DptB, Dif, and Rel, were all up-regulated in Drosophila expressing human FUS P525L, whereas in the dihydrocostus lactone-treated group, the expression of these up-regulated genes was down-regulated (Fig. 4C).

[0177]

[0178] <Example 4>

[0179] Dehydrocostus lactone induces a decrease in the level of insoluble TDP-43 protein and improves its mislocalization.

[0180] Overexpression of TDP-43 in astrocytes is known to cause the accumulation of insoluble aggregates and lead to TDP-43 mislocalization, resulting in neurotoxicity. Therefore, we performed immunoblotting to determine whether dehydrocostus lactone affects TDP-43 protein levels.

[0181] As a result, when dehydrocostus lactone was treated in astrocytes overexpressing TDP-43, the total protein level of TDP-43 was found to decrease (Fig. 5A). In addition, it was confirmed that the level of insoluble TDP-43 protein in TDP-43-overexpressing cells was also significantly reduced by dehydrocostus lactone treatment. Meanwhile, the level of soluble TDP-43 protein was not significantly affected by dehydrocostus lactone treatment (Fig. 5B). In addition, dehydrocostus lactone decreased both cytoplasmic and nuclear TDP-43 protein levels in astrocytes overexpressing TDP-43 (Fig. 5C).

[0182]

[0183] These results suggest that dehydrocostus lactone can reduce the level of insoluble TDP-43 protein in astrocytes overexpressing TDP-43, reduce the level of cytoplasmically mislocalized TDP-43, and ultimately suppress and reduce the level of TDP-43 protein that causes cytotoxicity.

[0184]

[0185] <Example 5>

[0186] Confirmation of the improved neurotoxicity caused by TDP-43 accumulation in astrocytes following treatment with dehydrocostus lactone.

[0187] We investigated whether Dehydrocostus lactone could modulate NF-κB pathway activation and neurotoxicity under conditions of TDP-43 overexpression in primary astrocytes. It is known that TDP-43 overexpression in astrocytes induces an inflammatory response via the NF-κB pathway, leading to the release of proinflammatory cytokines and chemokines, ultimately resulting in neuronal cell death.

[0188] Herein, we analyzed whether dehydrocostus lactone could regulate the activation of the NF-κB pathway and neurotoxicity under TDP-43 overexpression conditions. As a result, we confirmed that the group treated with dehydrocostus lactone showed a significant decrease in phosphorylation of NF-κB at serine 536 by TDP-43 in primary astrocytes (Fig. 6A). In addition, ACM obtained from TDP-43-overexpressing astrocytes treated with dehydrocostus lactone showed lower neurotoxicity in primary cortical neuron cultures than ACM obtained from TDP-43-overexpressing astrocytes (Fig. 6B).

[0189]

[0190] Furthermore, the present inventors analyzed the effects of dehydrocostus lactone on TDP-43-induced lifespan reduction and climbing function impairment using a Drosophila model expressing human TDP-43 in all glial cell types, including astrocyte-like glia, and analyzed lifespan and climbing function according to the ingestion of dehydrocostus lactone.

[0191] As a result, it was confirmed that the phenomenon of lifespan reduction and climbing defect induced by overexpression of TDP-43 was significantly improved in the group that was fed with dehydrocostus lactone in the medium (Figures 7A, 7B).

[0192]

[0193] <Example 6>

[0194] Confirmation of the effect of treatment with dehydrocostus lactone on improving inflammatory responses in fibroblasts derived from ALS patients.

[0195] Furthermore, the inventors of the present invention conducted experiments to determine the effect of dehydrocostus lactone on inflammatory responses using primary fibroblasts obtained from patients with ALS, a disease associated with FUS or TDP-43. To this end, fibroblasts from healthy individuals and FUS-ALS patients harboring the p.G504Wfs*12 mutation were treated with NaAsO2 to induce stress conditions. Then, phosphorylation of NF-κB at serine 536 and expression levels of FUS protein were analyzed. It is known that NaAsO2 is toxic to cellular responses including stress granule formation, oxidative stress, inflammation, and apoptosis.

[0196]

[0197] As a result of the analysis, it was confirmed that the phosphorylation at serine 536 residue of NF-κB and the expression level of FUS were significantly increased in ALS patient fibroblasts compared to the normal control group treated with NaAsO2 (Figures 8A and 8B), whereas when dehydrocostus lactone was treated, it was confirmed that the phosphorylation at serine 536 residue of NF-κB increased in FUS-ALS patient-derived fibroblasts was significantly decreased (Figure 8C).

[0198] These results indicate that the dehydrocostus lactone of the present invention can inhibit the activation of NF-κB induced by NaAsO2 in ALS patient fibroblasts, thereby reducing neuroinflammation, and thus preventing, improving, and treating neurodegenerative diseases caused by FUS or TDP-43.

[0199]

[0200] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

[0201]

[0202] The present invention is the result of research conducted with the support of the following national research and development project.

[0203] [Project ID] 2502030000

[0204] [Assignment Number] 25-BR-02-03

[0205] [Ministry Name] Ministry of Science and ICT

[0206] [Name of Project Management (Specialist) Institution] Korea Brain Research Institute

[0207] [Research Project Name] Korea Brain Research Institute Research Operational Expenses Support

[0208] [Research Project Title] Advancing the Rapid Verification System for Candidate Genes for Degenerative Brain Diseases and Using It to Precisely Analyze Pathogenesis

[0209] [Contribution rate] 1 / 3

[0210] [Name of the project performing organization: Korea Brain Research Institute

[0211] [Research Period] January 1, 2025 - December 31, 2030

[0212]

[0213] [Project ID] 2710083601

[0214] [Project Number] RS-2024-00343239

[0215] [Ministry Name] Ministry of Science and ICT

[0216] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0217] [Research Project Name] Individual Basic Research (Ministry of Science and ICT) (R&D)

[0218] [Research Project Title] Development of a Diagnostic and Treatment Strategy for ALS / FTD by Identifying Kinase-Mediated Interactions between Neuroinflammation and the Protein Quality Control System

[0219] [Contribution rate] 1 / 3

[0220] [Name of the project performing organization] Korea Brain Research Institute

[0221] [Research Period] May 1, 2024 - April 30, 2029

[0222]

[0223] [Project ID] 2710018794

[0224] [Project Number] RS-2024-00454715

[0225] [Ministry Name] Ministry of Science and ICT

[0226] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0227] [Research Project Name] Individual Basic Research (Ministry of Science and ICT)

[0228] [Research Project Title] Elucidating the Neuropathological Mechanism of TDP-43 Protein Disease through Cellular Region-Specific Interactome Analysis

[0229] [Contribution rate] 1 / 3

[0230] [Name of the project performing organization] Korea Brain Research Institute

[0231] Research Period: September 1, 2024 - August 31, 2027

Claims

1. A pharmaceutical composition for the prevention or treatment of a disease related to FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43), comprising dehydrocostus lactone or a pharmaceutically acceptable salt thereof as an active ingredient.

2. In paragraph 1, The above disease is, Overexpression or aggregation of FUS; Overexpression or aggregation of TDP-43; Mislocalization of TDP-43 to the cytoplasm; A pharmaceutical composition for preventing or treating a disease associated with FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43), characterized in that the disease is caused by neuroinflammation and neurotoxicity due to overexpression or aggregation of FUS or TDP-43; or mitochondrial dysfunction due to overexpression or aggregation of FUS or TDP-43.

3. In paragraph 2, A pharmaceutical composition for preventing or treating a disease related to FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43), characterized in that the disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington's disease (HD), and limbic predominant age-related TDP-43 encephalopathy (LATE).

4. In paragraph 1, The above dehydrocostus lactone is, Overexpression or aggregation inhibition of FUS or TDP-43; Inhibits neuroinflammation; Inhibition of neurotoxicity; or A pharmaceutical composition for the prevention or treatment of diseases related to FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43), characterized by improving mitochondrial dysfunction; and having activity.

5. A health functional food containing dehydrocostus lactone or an acceptable salt thereof as an active ingredient for preventing or improving diseases related to FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43).

6. In paragraph 5, A health functional food for preventing or improving a disease related to FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43), characterized in that the disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington's disease (HD), and limbic predominant age-related TDP-43 encephalopathy (LATE).

7. In paragraph 5, The above dehydrocostus lactone is, Overexpression or aggregation inhibition of FUS or TDP-43; Inhibits neuroinflammation; Inhibition of neurotoxicity; or A health functional food for the prevention or improvement of diseases related to FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43), characterized by improving mitochondrial dysfunction and having activity.

8. A step of treating cells with dehydrocostus lactone or an acceptable salt thereof in a test tube, A method for inhibiting overexpression or aggregation of FUS (Fused in sarcoma) or TDP-43 (TAR DNA-binding protein 43) within a cell.

9. In paragraph 8, A method wherein cells treated with dehydrocostus lactone exhibit reduced neurotoxicity and neuroinflammation due to overexpression or aggregation of FUS or TDP-43.

10. In paragraph 8, A method characterized in that cells treated with dehydrocostus lactone exhibit improved mitochondrial dysfunction caused by overexpression or aggregation of FUS or TDP-43.

11. In paragraph 10, A method for improving mitochondrial dysfunction characterized by increased cellular respiration rate and ATP production due to overexpression or aggregation of FUS or TDP-43.

Images