WO2024186167A1 - Composition containing l1td1 protein for preventing or treating neurodegenerative diseases - Google Patents

Abstract

The present invention relates to a composition for preventing or treating neurodegenerative diseases, containing L1TD1 protein as an active ingredient. The L1TD1 protein according to the present invention alleviates disease symptoms by decomposing RNA granules produced by stress or mutant proteins that cause neurodegenerative diseases, and thus can be effectively used for preventing, treating or alleviating neurodegenerative diseases.

Description

Composition for preventing or treating neurodegenerative diseases comprising L1TD1 protein

The present invention relates to a composition for preventing or treating neurodegenerative diseases, comprising L1TD1 protein as an active ingredient.

Neurodegenerative diseases are mental function deterioration diseases caused by gradual structural and functional loss of nerve cells (neurons). Neurodegenerative diseases are accompanied by symptoms such as dementia, extrapyramidal abnormalities, cerebellar abnormalities, sensory disorders, and motor disorders due to the degeneration of nerve cells in specific parts of the nervous system, and symptoms may appear in combination as abnormalities occur in multiple parts at the same time. The disease is diagnosed based on the clinical appearance of the patient, but the symptoms appear diversely and many different diseases show common clinical symptoms, making diagnosis difficult.

Neurodegenerative diseases show signs of the disease gradually and often develop with aging. Once developed, the disease progresses continuously for years or even decades until death, resulting in a huge social burden. It is known that genetic influences based on family history have a significant effect on the cause of the disease, but acquired factors are also considered to play an important role. Neurodegenerative diseases are largely classified into progressive dementia (e.g. Alzheimer's disease), neurological abnormalities (e.g. Pick's disease), postural and motor abnormalities (e.g. Parkinson's disease), progressive ataxia, muscular atrophy and weakness, and sensory and motor disorders, depending on their clinical symptoms.

Several treatments for neurodegenerative diseases are being studied, but despite many studies, most of them only show effects to the extent of slowing the progression, and few cases show clear therapeutic efficacy. In particular, in the case of spinal muscular atrophy, correction of mRNA using antisense oligonucleotides (ASO) has been approved by the FDA, but its effectiveness is limited, and the gene therapy ZOLGENSMA is very expensive and has risks such as inducing mutations in the patient's genome.

Meanwhile, RNA granules are dynamically generated and resolved repeatedly within cells, and occur as part of a stress response process. In many neurodegenerative diseases, there are problems with the formation and resolution of RNA granules within nerve cells, and unresolved RNA granules can be toxic to cells and produce insoluble precipitates. In addition, it is known that proteins that induce these diseases through mutations are located in RNA granules and induce their production.

Therefore, intracellular RNA granules can be a new target for treating neurodegenerative diseases, and indirect methods such as decreasing intracellular mRNA translation or increasing autophagy are being attempted to promote the resolution of RNA granules. However, although some effects are observed at the laboratory level, considering the roles of translation and autophagy in cell activity, there are limits to obtaining practical effects through control of these processes.

Against this backdrop, the inventors of the present invention have conducted extensive research efforts to develop a substance for treating neurodegenerative diseases by dissolving intracellular RNA granules, and have confirmed that the L1TD1 protein can be applied as a substance for improving neurodegenerative diseases, thereby completing the present invention.

The present invention aims to solve the above-mentioned problems and other problems related thereto.

One exemplary object of the present invention is to provide a pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising as an active ingredient a L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain.

Another exemplary object of the present invention is to provide a food composition for preventing or improving a neurodegenerative disease, comprising as an effective ingredient a L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain.

Another exemplary object of the present invention is to provide a health functional food for preventing or improving neurodegenerative diseases, comprising as an effective ingredient L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain.

Another exemplary object of the present invention is to provide a pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising as an active ingredient a L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain.

Another exemplary object of the present invention is to provide a feed composition for preventing or improving neurodegenerative diseases, comprising as an effective ingredient L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain.

Another exemplary object of the present invention is to provide a method for improving, preventing or treating a neurodegenerative disease, which comprises administering to a subject in need thereof an effective amount of L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain, for improving, preventing or treating the neurodegenerative disease.

Another exemplary object of the present invention is to provide a use of L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain for improving, preventing or treating a neurodegenerative disease.

Another exemplary object of the present invention is to provide a use of a L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain, for the manufacture of a medicament for improving, preventing or treating a neurodegenerative disease.

The technical problems to be achieved according to the technical idea of the invention disclosed in this specification are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

This is explained specifically as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of this application cannot be considered limited by the specific description described below.

As one aspect for achieving the above purpose, the present invention provides a pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising, as an active ingredient, L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain.

The above L1TD1 is a protein expressed in stem cells and is known to be located in intracellular RNA granules. The amino acid sequence of the above L1TD1 is represented by sequence number 1 as NCBI reference No. NP_061952.3, and the gene (mRNA) sequence of the above L1TD1 is represented by sequence number 2 as NCBI reference No. NM_019079.5.

In the present invention, the L1TD1 protein may be a concept including a fragment of a peptide constituting the amino acid sequence or an analogue thereof.

In the present invention, the fragment or analogue may be included in the scope of the present invention if it has functional identity even if a part of the amino acid sequence of the L1TD1 is deleted, added, or substituted. Having the functional identity means that the L1TD1 defined by the sequence in the present specification can achieve the desired effect.

The term “domain” as used herein refers to a region of a protein having structural and functional characteristics, which corresponds to a continuous section of amino acid sequence that has an independent structure within the protein or can perform a specific function.

In the present invention, the domain constituting the L1TD1 may be at least one of an OH (ORF1-homology regions) 1 domain, an ER (glutamate-rich domain) domain, and an OH2 domain, preferably, it may include an ER domain and an OH2 domain, and more preferably, it may be a combination of an ER domain and an OH2 domain, or a fusion protein in which these are fused.

As a preferred example, the amino acid sequence of the OH1 domain is represented by SEQ ID NO: 4, and the gene (mRNA) sequence of the OH1 domain is represented by SEQ ID NO: 5.

As a preferred example, the amino acid sequence of the ER domain is represented by SEQ ID NO: 6, and the gene (mRNA) sequence of the ER domain is represented by SEQ ID NO: 7.

As a preferred example, the amino acid sequence of the OH2 domain is represented by SEQ ID NO: 8, and the gene (mRNA) sequence of the OH2 domain is represented by SEQ ID NO: 9.

The above L1TD1 protein is known to be a protein derived from Long interspersed element 1 (LINE-1 or L1), and specifically, as confirmed in the examples of the present invention and FIG. 11, L1TD1 and L1 OFR1 (Open Reading Frame 1) share genetic homology. Specifically, among the two exons encoding L1TD1, the first coding exon conserves L1 OFR1 and the CTD (C-terminal domain) region, and the second coding exon conserves L1 OFR1 and the CC (coiled-coil motif), RRM (RNA recognition motif), and CTD (C-terminal domain) region.

The above OH1 domain, OH2 domain and ER domain may be a concept including a peptide fragment or an analogue thereof constituting each of the above amino acid sequences.

In the present invention, the fragment or analogue may be included in the scope of the present invention if it has functional identity even if a part of the amino acid sequence of the OH1 domain, OH2 domain and ER domain is deleted, added or substituted. Having the functional identity means that it can achieve the desired effect of the OH1 domain, OH2 domain and ER domain defined by the sequence in the present specification.

In the present invention, the OH1 domain, OH2 domain and ER domain or the gene encoding them may decompose RNA granules in nerve cells.

In the present invention, the L1TD1 protein or a gene encoding the same may decompose RNA granules within nerve cells.

The above RNA granules may be produced by mutant proteins that cause neurodegenerative diseases or by stress to cells.

In the present invention, the mutated protein may be, but is not limited to, FUS1, TDP-43, SOD-1 or SMN1.

In the present invention, the mutant protein is a protein containing a mutation in all or part of the protein, and may be, for example, FUS1-493X, a mutant protein of FUS1, or TDP-43-M337V, a mutant protein of TDP-43, but is not limited thereto.

In the present invention, the stress may be, but is not limited to, oxidative stress, osmotic stress or high temperature stress.

In the present invention, the neurodegenerative disease may be at least one selected from the group consisting of Lou Gehrig's disease, frontotemporal lobar degeneration, spinal muscular atrophy, Alzheimer's disease, Parkinson's disease, Huntington's chorea, spinocerebellar degeneration, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, vascular dementia, dementia with Lewy bodies, and dementia accompanying Parkinson's disease.

Specifically, in an embodiment of the present invention, when L1TD1 was introduced into a HeLa cell line and oxidative stress, osmotic stress, and heat stress were respectively applied, it was confirmed that the number of cells containing stress granules was significantly reduced compared to the control group to which L1TD1 was not introduced, and when L1TD1 was introduced into the cell line, it was confirmed that granules produced by a mutant protein that causes a neurodegenerative disease were significantly reduced compared to the control group to which L1TD1 was not introduced.

The term "prevention" of the present invention means any act of inhibiting or delaying a neurodegenerative disease by administering the composition of the present invention, and the term "treatment" of the present invention means any act of improving, alleviating, or beneficially altering a neurodegenerative disease that has developed by administering the composition of the present invention. The term "improvement" of the present invention means any act of improving a neurodegenerative disease by administering the composition of the present invention.

The term "pharmaceutical composition" of the present invention means a composition manufactured for the purpose of preventing or treating a disease, and each may be formulated and used in various forms according to conventional methods. For example, it may be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, depending on the route of administration, and may be formulated and used in the form of external preparations and sterile injectable solutions. Specifically, the administration route may be any appropriate route including a topical route, an oral route, an intravenous route, an intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be combined and used. An example of a combination of two or more routes is a case where two or more drug formulations are combined according to the route of administration, for example, a case where one drug is first administered via the intravenous route and another drug is secondarily administered via the topical route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specific examples can be found in the pharmacopoeias of each country, including the ‘Korean Pharmacopoeia.’

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent according to a conventional method. Pharmaceutically acceptable carriers are well known in the art depending on the administration route or formulation, and specifically, reference can be made to the pharmacopoeias of each country including the 'Korean Pharmacopoeia'. Carriers, excipients and diluents that may be included in the composition of the present invention include, but are not limited to, 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, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Additionally, carriers, excipients and diluents that may be included in the composition of the present invention may be non-natural carriers, but are not limited thereto.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injection solutions according to conventional methods. Specifically, when formulating, it can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations can be prepared by mixing the compound with at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, 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, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include withepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin. Specific formulations of pharmaceutical compositions are known in the art, and reference may be made to, for example, Remington's Pharmaceutical Sciences (19th ed., 1995). The above document is considered to be a part of the present specification.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not causing side effects, and the effective dosage level can be determined based on factors including the patient's health condition, the type and severity of the disease, the activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration, and the excretion rate, the treatment period, the drugs used in combination or simultaneously, and other factors well known in the medical field. The dosage and frequency of administration do not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, dogs, cats, cows, horses, pigs, and humans through various routes, and humans may be preferred. All modes of administration are conceivable, and include, but are not limited to, oral, intravenous, intramuscular, or subcutaneous injection.

The term "comprising as an effective ingredient" of the present invention means including an effective amount capable of exhibiting a preventive or therapeutic effect on a neurodegenerative disease as a pharmaceutical composition.

Another aspect of the present invention for achieving the above purpose provides a food composition for preventing or improving a neurodegenerative disease, comprising as an effective ingredient L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain.

The above L1TD1, domain and neurodegenerative disease are as described above.

The food composition of the present invention can be manufactured into various dosage forms, and unlike general drugs, it has the advantage of having no side effects that may occur when taking drugs for a long period of time since it uses food as a raw material. The food composition of the present invention can be manufactured into any form, and specifically, it can be at least one dosage form selected from the group consisting of health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, beverages, gums, and candies, but is not particularly limited thereto.

In addition, the food composition of the present invention may contain food additives in addition to the effective ingredients. Food additives can be generally understood as substances that are added to, mixed in, or infiltrated into food during the manufacturing, processing, or preservation of food, and their safety must be guaranteed because they are consumed daily and for a long period of time with food. The food additives are classified into sweeteners, flavoring agents, preservatives, emulsifiers, acidulants, thickeners, etc. in terms of function, and are not particularly limited as long as they are consistent with the purpose to be achieved by the food composition of the present invention. In addition, the food composition of the present invention may contain, in addition to the food additives, physiologically active substances or minerals known in the art for the purpose of functionality and nutritional supplementation and whose safety as a food additive is guaranteed. The physiologically active substances or minerals are not particularly limited as long as they are consistent with the purpose to be achieved by the food composition of the present invention.

The food composition of the present invention may contain the aforementioned food additives in an effective amount that can achieve the purpose of addition depending on the product type, and with respect to other food additives that may be contained in the food composition of the present invention, reference may be made to the food codes or food additive codes of each country.

Another aspect of the present invention for achieving the above purpose provides a health functional food for preventing or improving a neurodegenerative disease, comprising as an effective ingredient L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain.

The above L1TD1, domain and neurodegenerative disease are as described above.

In the present invention, the term "health functional food" refers to a food manufactured and processed using raw materials or ingredients having functionality useful to the human body. The "functionality" above means obtaining a useful effect for health purposes such as regulating nutrients or physiological effects for the structure and function of the human body. The health functional food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art during manufacturing.

In addition, the formulation of the above health functional food can be manufactured without limitation as long as it is a formulation recognized as a health functional food, and non-limiting examples of the formulation include one or more formulations selected from the group consisting of health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, beverages, gums, and candies, but are not particularly limited thereto.

Another aspect of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising as an active ingredient a L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain.

The above L1TD1, domain and neurodegenerative disease are as described above.

The quasi-drug composition of the present invention may contain, in addition to the L1TD1 protein or the gene encoding it, components commonly used in quasi-drug compositions, and may include, for example, an abrasive, a wetting agent, a binder, a foaming agent, a sweetener, a preservative, a medicinal ingredient, a flavoring agent, a pigment, a solvent, a whitening agent, a solubilizer, or a pH adjuster, but is not limited thereto.

In the present invention, the type of the above-mentioned quasi-drug is not particularly limited, and may be a quasi-drug commonly used in the relevant technical field. Non-limiting examples of the above-mentioned quasi-drug may be at least one formulation selected from the group consisting of toothpaste, mouthwash, gum, candy, mouth spray, oral ointment, oral varnish, oral film, and gum massage cream. These may be used alone or in combination of two or more.

Another aspect of the present invention for achieving the above object provides a feed composition for preventing or improving a neurodegenerative disease, comprising as an effective ingredient a L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain.

The above feed composition may include a feed additive. The feed additive of the present invention corresponds to a supplementary feed under the Feed Management Act.

In the present invention, the term "feed" may mean any natural or artificial diet, meal, etc., or a component of said meal, especially for eating, ingesting, and digesting by an animal or suitable therefor.

The type of the above feed is not particularly limited, and feed commonly used in the relevant technical field can be used. Non-limiting examples of the above feed include plant feed such as grains, roots, food processing by-products, algae, fibers, pharmaceutical by-products, fats, starches, meal, or grain by-products; and animal feed such as proteins, inorganic substances, fats, minerals, fats, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

Another aspect of the present invention for achieving the above object provides a method for improving, preventing or treating a neurodegenerative disease, which comprises administering to a subject in need thereof an effective amount of L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain for improving, preventing or treating the neurodegenerative disease.

In the present invention, “effective amount” means an amount sufficient for the composition of the present invention described above to achieve the effect of improving, preventing, or treating a neurodegenerative disease.

The term "subject" in this specification is not particularly limited to, but may be, for example, a human, a monkey, a cow, a horse, a sheep, a pig, a chicken, a turkey, a quail, a cat, a dog, a mouse, a rat, a rabbit or a guinea pig, and preferably means a mammal, more preferably a human.

The term “administration” as used herein means providing a given substance to a subject by any suitable means.

The route of administration of the composition of the present invention may be oral or parenteral administration through any common route as long as it can reach the target tissue. In addition, the composition of the present invention may be administered using any device capable of delivering the active ingredient to the target cell, tissue or organ.

Another aspect of the present invention for achieving the above object provides the use of L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain for improving, preventing, or treating a neurodegenerative disease.

Another aspect of the present invention for achieving the above object provides a use of L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain, for the manufacture of a drug for improving, preventing, or treating a neurodegenerative disease.

The L1TD1 protein according to the present invention can be usefully used for the prevention, treatment or improvement of neurodegenerative diseases by alleviating the symptoms of the disease by decomposing mutant proteins causing neurodegenerative diseases or RNA granules generated by stress.

Figure 1 is a schematic diagram of the gene introduction of the present invention using CRISPR/Cas9 (A) and the result of confirming the expression of eIF3b protein coupled with GFP (B).

Figure 2 shows the results of confirming the formation of stress granules in HeLa-eIF3B-GFP cells by oxidative stress (addition of arsenite).

Figure 3 shows the results of confirming the formation of stress granules (A) and changes in the number of granule-containing cells when L1TD1 was introduced into a cell line containing granules generated by oxidative stress.

Figure 4 shows the results of confirming the formation of stress granules (A) and changes in the number of granule-containing cells (B) when L1TD1 was introduced into a cell line containing granules produced by osmotic stress.

Figure 5 shows the results of confirming the formation of stress granules (A) and changes in the number of granule-containing cells (B) when L1TD1 was introduced into a cell line containing granules produced by heat stress.

Figure 6 shows the location of eIF3B granules, G3BP1, G3BP2, DDX6, and L1TD1 produced in HeLa-eIF3B-GFP cell lines subjected to oxidative stress.

Figure 7 shows the results of confirming the locations of L1TD1 protein and LC3 protein before and after abate treatment.

Figure 8 shows the results of confirming the amount of L1TD1 protein after abate treatment, abate treatment, and chloroquine treatment.

Figure 9 shows the results of confirming the effect of L1TD1 protein on granules produced by mutations causing neurodegenerative diseases (A: Confirmation of granule formation by expression of TDP-43-M337V protein and L1TD1, B: Confirmation of granule formation by expression of FUS1-493X protein and L1TD1, C: Change in the number of cells containing eIF3B granules when FUS1-493X and TDP-43-M337V were each expressed together with L1TD1).

Figure 10 shows the results of confirming the location of granules and L1TD1 protein produced by FUS1-493X and TDP-43-M337V proteins in the HeLa-eIF3B-GFP cell line (A: Confirmation of the same location of FUS1-493X and TDP-43-M337V proteins and eIF3B, B: Confirmation of the same location of FUS1-493X and TDP-43-M337V proteins and L1TD1).

Figure 11 shows a schematic diagram of the domain regions constituting the L1 ORF1p and L1TD1 proteins.

Figure 12 shows a schematic diagram of the deletion mutants of the L1TD1 protein, OH2, OH1-OH2, and ER-OH2.

Figure 13 shows the results of confirming the subcellular location (A) and changes in the number of cells containing stress granules (B) of L1 ORF1p and L1TD1 deletion mutants.

Figure 14 shows the results of confirming the subcellular localization (A) and changes in the number of cells containing stress granules (B) of L1 ORF1p and L1TD1 deletion mutants according to mutations causing neurodegenerative diseases.

Figure 15 shows the results of confirming the intracellular location of the ER-OH2 deletion mutant.

Figure 16 shows the results of confirming the gene expression levels of the full-length protein and deletion mutant of L1TD1.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are intended to exemplify the present invention, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of HeLa-eIF3B-GFP cell line

eIF3B is a stress granule marker that is known to be toxic to cells and produce insoluble precipitates when not resolved (degraded) in neurons of neurodegenerative diseases. It also forms stress granules with other proteins and RNA under various stress conditions. To facilitate observation of the formation and degradability of stress granules, a HeLa cell line (HeLa-eIF3B-GFP cell line) expressing a protein in the form of a fusion of the eIF3B gene and GFP (eIF3B-GFP) was created.

Specifically, pRGEN-CMV-Cas9 (Toolgen), a guide plasmid, and the target construct were introduced into HeLa cells using a Neon transfection system (Thermo), and clones with puromycin resistance were obtained. The target construct was prepared by obtaining the corresponding part of the EIF3B gene in the HeLa cell genome using PCR, inserting it into the pBluescript-SK+ vector, and then inserting the GFP-IRES-puro cassette in front of the stop codon. Figure 1A shows a gene targeting method using CRISPR/Cas9. Thereafter, electrophoresis was performed to confirm whether the HeLa cell line prepared by the above method was successfully produced. As a result, as shown in Fig. 1B, the HeLa cell line produced using CRISPR/Cas9 showed bands for eIF3B (eIF3B-GFP) and GFP (eIF3B-GFP), confirming that a HeLa cell line expressing a protein (eIF3B-GFP) in the form of a fusion of the eIF3B gene and GFP (HeLa-eIF3B-GFP cell line) was successfully produced.

Arsenite (0.5 mM sodium arsenite), which causes oxidative stress, was added to the manufactured HeLa-eIF3B-GFP cell line, and fluorescence image analysis was performed after 15 minutes.

For fluorescence image analysis, each cell line was fixed with cold methanol at -20°C for 5 minutes, washed with PBS, and images were acquired. For fluorescence staining, fixed cells were treated with PBS containing 0.1% Tween-20 and 2.5% BSA at room temperature for 40 minutes. Primary antibodies were diluted in PBS containing 0.1% Tween-20 and 0.5% BSA and treated at 4°C for 12 hours. After washing three times with PBS, they were treated with secondary antibodies for 1 hour at room temperature. After staining with DAPI (1 μg/mL) for 5 minutes at room temperature, they were mounted using VECTASHIELD Antifade Mounting Media (Vector Lab, #H1200). Image analysis was performed using a fluorescence microscope (EVOS FL Auto 2 Cell Imaging System) and a confocal microscope (ZEISS LSM 900). The antibodies used were as follows: anti-Human L1TD1 mouse monoclonal antibody (R&D, #MAB8317); anti-beta Actin mouse monoclonal antibody (SantCruz, #SC47778); anti-DDX6 rabbit polyclonal antibody (Bethyl, #A300-461A); anti-G3BP1 rabbit polyclonal antibody (Bethyl, #A302-034A); anti-rabbit Alexa Fluor™ 594 (Invitrogen, #A11037); anti-mouse Alexa Fluor™ 488 (Invitrogen, #A11029); anti-rabbit Alexa Fluor™ 488 (Invitrogen, #A11034).

In all experiments below, fluorescence image analysis was performed using the same method.

As a result, as shown in Fig. 2, it was confirmed that eIF3B granules were produced when oxidative stress was applied by adding arsenite compared to the control group to which arsenite was not added.

Example 2. Analysis of the efficacy of L1TD1 protein in improving neurodegenerative diseases

2-1. Analysis of the effect of L1TD1 on granules generated by stress

The effects of L1TD1 on granules produced by oxidative stress caused by the addition of arsenite, osmotic stress caused by the addition of sorbitol, and high temperature stress were analyzed.

First, a control group in which the pCAG-tRFP plasmid was introduced into the HeLa-eIF3B-GFP cell line manufactured in Example 1, and an experimental group in which the pCAG-L1TD1tRPF plasmid was introduced were manufactured. The L1TD1 gene was obtained from cDNA of H1 embryonic stem cells using PCR, and it was confirmed to be identical to the coding sequence (SEQ ID NO: 2) of NCBI reference No. NM_019079.5 through base sequence analysis. The pCAG vector is a plasmid containing the CAG promoter and the BGA polyA signal, and the CAG promoter is manufactured by combining several elements and has the base sequence of SEQ ID NO: 3.

All plasmids were constructed by introducing the CAG promoter into the BGH polyA site of the pCAG vector. The constructed plasmids were introduced into the HeLa-eIF3B-GFP cell line using Lipofectamine 3000 (Thermo) according to the manufacturer's protocol.

After 24 h, oxidative stress (0.5 mM sodium arsenite treatment for 15 min), osmotic stress (0.4 M sorbitol treatment for 1 h), and hyperthermia stress (42°C treatment for 30 min) were applied. The number of cells with stress granules in each control and experimental group was counted. The experiment was repeated three times, and 100 cell lines were analyzed for each.

As a result, it was confirmed that cells containing stress granules (SGs) generated by oxidative stress due to the addition of arsenite, osmotic stress due to the addition of sorbitol, and high temperature stress rapidly decreased in the presence of L1TD1 (Figs. 3 to 5).

Specifically, as shown in Fig. 3A, it was confirmed that about 40% of cells expressing only tRFP formed stress granules due to the conversion process or abnormal RNA expression even in the absence of stress treatment. On the other hand, in cells expressing a protein combining L1TD1-tRFP, it was confirmed that eIF3B protein was hardly present at a specific location within the cell. In addition, as shown in Fig. 3A and Fig. 3B, when oxidative stress was induced by the addition of arsenite, the number of cells containing stress granules increased by up to 100% in both tRFP and L1TD1-tRFP, and after 60, 75, and 90 minutes of recovery after treatment, the number of cells containing stress granules decreased in both tRFP and L1TD1-Trfp, but in the case of L1TD1-tRFP, it decreased to less than 40% after 60 minutes of recovery, confirming that stress granules were decomposed more quickly.

As shown in Fig. 4, when osmotic stress was applied by adding sorbitol, the number of cells containing stress granules increased by up to 100% in both tRFP and L1TD1-tRFP, and after 30 and 60 minutes of recovery after treatment, the number of cells containing stress granules decreased in both tRFP and L1TD1-tRFP. However, in the case of L1TD1-tRFP, the number decreased to less than 10% after 30 minutes of recovery, confirming that stress granules were decomposed more quickly.

As shown in Fig. 5, when heat stress was applied by applying heat, the number of cells containing stress granules increased by up to 100% in both tRFP and L1TD1-tRFP, and after 15 and 30 minutes of recovery after treatment, the number of cells containing stress granules decreased in both tRFP and L1TD1-tRFP, but in the case of L1TD1-tRFP, the number decreased to less than 10% after 30 minutes of recovery, confirming that stress granules are decomposed more quickly.

In addition, as shown in Fig. 6, when oxidative stress is induced using arsenite, L1TD1 is located in the same location as eIF3B granules, as well as in the same location as core components of stress granules, G3BP1 and G3BP2, and DDX6, an RNA-binding protein involved in RNA metabolism and subcellular localization, as confirmed through fluorescence image analysis.

In addition, the effect of L1TD1 on autophagy was analyzed. As a result, as shown in Fig. 7, it was confirmed through fluorescent staining that LC3, an autophagy marker, was co-located with L1TD1 even before stress application, and that it was also co-located with L1TD1 in stress granules formed after arsenite treatment. In addition, it was confirmed that L1TD1 decreased after arsenite treatment.

As described above, HeLa-eIF3B-GFP cells were introduced with the pCAG-L1TD1tRPF vector. Twelve hours later, 50 μM chloroquine was treated to inhibit autophagy, and then 12 hours later, 0.5 mM arsenite was treated for 15 minutes. The protein amount was then analyzed by Western blotting.

As a result, as shown in Fig. 8, when only arsenate was treated, L1TD1 decreased, but when chloroquine was treated to inhibit autophagy, the decrease was confirmed to be attenuated compared to when chloroquine was not treated, and this effect was not observed in the case of MG132, a proteasome inhibitor. Therefore, it is presumed that L1TD1 is degraded by autophagy under stress granule formation conditions, and this action is one of the causes of rapid resolution of stress granules.

2-2. Analysis of the effect of L1TD1 on granules produced by mutant proteins causing neurodegenerative diseases

We analyzed the effect of L1TD1 on granules produced by the mutant proteins FUS1-493X, which causes Lou Gehrig's disease, and TDP-43-M337V, which causes frontotemporal lobar degeneration.

FUS1-493X is known to be a short protein produced by a stop codon at amino acid position 493 due to a mutation. The FUS1 and TDP-43 (TARBP) genes were obtained from cDNA of H1 embryonic stem cells by PCR, and were confirmed to match the coding sequences of NCBI reference sequence NM_004960.4 and NM_007375.4, respectively, through base sequence analysis. After the 1-493 base sequence of FUS1 was obtained by PCR, it was conjugated with the tRFP gene and introduced into the pCAG vector to produce an expression plasmid (pCAG-FUS1-493X-tRFP). For the TDP-43-M337V mutation, the TDP sequence was cloned using PCR, and then a mutation was introduced at amino acid position 337, conjugated with the tRFP gene, and introduced into the pCAG vector to produce an expression plasmid (pCAG-TDP-43-M337V-tRFP). Each plasmid was introduced into the HeLa-eIF3B-GFP cell line prepared in Example 1 through transfection with the L1TD1 expression vector (pCAG-L1TD1tRPF) to produce an experimental group, and each plasmid was introduced into the HeLa-eIF3B-GFP cell line through transfection with the empty vector (pCAG) to produce a control group.

After 24 hours, the number of cells containing stress granules in each control and experimental group was counted. The experiment was repeated three times, and 100 cell lines were analyzed for each.

As a result, it was confirmed that the number of cell lines containing eIF3B stress granules was significantly reduced in the experimental groups in which FUS1-493X and TDP-43-M337V were each expressed together with L1TD1 compared to the control group (Fig. 9, A: Confirmation of granule formation by expression of TDP-43-M337V protein and L1TD1, B: Confirmation of granule formation by expression of FUS1-493X protein and L1TD1, C: Change in the number of cells containing eIF3B granules when FUS1-493X and TDP-43-M337V were each expressed together with L1TD1).

In addition, fluorescence image analysis confirmed that eIF3B and L1TD1, which are granules produced by FUS1-493X and TDP-43-M337V proteins in the HeLa-eIF3B-GFP cell line, were colocalized (Fig. 10, A: Confirmation of colocalization of eIF3B with FUS1-493X and TDP-43-M337V proteins, B: Confirmation of colocalization of L1TD1 with FUS1-493X and TDP-43-M337V proteins).

Example 3. Functional analysis of each domain of the L1TD1 protein

3-1. Domain identification of L1TD1 protein and production of fusion protein

As shown in Fig. 11, the L1TD1 protein is composed of two coding exons (coding exon 1, 2), and each coding exon was confirmed to have a conserved region with L1 ORF1. Specifically, the CTD region was conserved in coding exon 1, and the CC, RRM, and CTD regions were conserved in coding exon 2, confirming that coding exon 2 is more conserved than coding exon 1.

In order to confirm the function of each domain constituting the L1TD1 full-length protein consisting of coding exons 1 and 2, the L1TD1 full-length protein was divided into three domains, OH1 domain, ER domain, and OH2 domain, based on the results of IUPred2A and CIDER v2.0, and fusion proteins consisting of OH2, OH1-OH2, and ER-OH2 were produced through deletion mutation of L1TD1, as shown in Fig. 12. Specifically, OH2 was produced by deleting the HpaI/AflII portion of the L1TD1 coding sequence, making the cleavage site blunt-ended using nuclease P1, and self-ligating it to produce a mutant in which the OH1 and ER portions were deleted. OH1-OH2 and ER-OH2 deletion mutants were created by deleting the HpaI/AflII portion of the L1TD1 coding sequence and inserting OH1 and ER amplified by PCR into the corresponding portion.

The amino acid sequences and mRNA sequences of the OH1 domain, ER domain, and OH2 domain are as shown in Table 1 below.

order
(N-ter → C-ter
or 5' → 3') Sequence number OH1 Amino acid sequence MSDVSTSVQSKFARLAKKKENITYMKREQLTETDKDIAPVLDLKCKDVSAIMNKFKVLMEIQDLMFEEMRETLKNDLKAVLGGKATIPEVKNSENSSSRTEFQQIINLALQKTGMVGKIEGENSKIGDDNENLTFKLEVNELSGKLDNTNEYNSNDGKKLPQGESRSYEVMGSMEETLCNIDDRDGNRNVHLEFTERESRKDGEDEFVK EMREERKFQKLKNKEEVLKASREEKVLMDEGAVLTLVADLSSATLDISKQWSNVFNILRENDFEPKFLLCEVKLAFKCDGEIKTFSDLQSLRKFASQKSSVKELLKDVLPQKEEINQGGRKYGIQEKR 4 mRNA sequence ATGTCTGATGTATCTACTAGTGTACAATCAAAATTTGCTAGACTTGCAAAGAAAAAGGAAAATATCACCTATATGAAAAGAGAGCAGTTAACAGAAACTGATAAGGACATAGCTCCGGTATTAGATTTAAAATGCAAGGACGTATCAGCAATTATGAATAAGTTTAAGGTCTTAATGGAAATTCAAGACCTGATGTTTGAGGAGATGAGGGAAACTCTTAAAAATGACCTAAAAGCAGTTTTAGGGGGAAAA GCTACAATACCTGAGGTAAAGAATTCAGAGAACTCCAGTAGTAGGACAGAGTTTCAGCAAATAATCAATTTAGCATTACAAAAAACAGGGATGGTAGGGAAAATAGAAGGAGAAAACTCTAAAATAGGTGATGATAATGAAAATTTAACCTTTAAATTAGAAGTAAATGAGCTGAGTGGTAAATTAGACAACACTAACGAATACAATAGTAATGATGGTAAGAAATTACCCCAGGGTGAATCACGAAGTTAC GAAGTCATGGGAAGTATGGAAGAAACCTTATGCAATATAGATGACAGAGATGGAAATCGCAAATGTCCATTTAGAATTTACAGAAAGAGAGAGTAGGAAGGATGGAGAGGATGAATTTGTCAAAGAAATGAGAGAGGAAAGAAAATTTCAGAAATTGAAGAATAAAGAGGAGGTTTTAAAAGCCTCCAGAGAAGAAAAAGTGTTGATGGATGAAGGAGCAGTACTTACCCTGGTAGCCGACCTTTCATCAGCA ACACTGGATATTAGTAAGCAATGGAGTAATGTCTTCAACATTCTGAGAGAAAATGATTTTGAACCTAAATTTCTGTGTGAAGTTAAAATTAGCATTTAAATGTGATGGTGAAATAAAGACATTTTCAGATCTGCAAAGCCTTAGAAAATTTGCCAGCCAAAAATCTTCTGTGAAAGAATTACTGAAAGATGTACTCCCACAAAAGGAAGAAATAAATCAAGGAGGAAGAAAATATGGAATTCAAGAAAAAAGG 5 ER Amino acid sequence DKTLIDSKHRAGEITSDGLSFLFLKEVKVAKPEEMKNLETQEEEFSELEELDEEAASGMEDDEDTSGLEEEEEEPSGLEEEEEEEASGLEEDEASGLEEEEEQTSEQDSTFQGHTLLVDAKHEVEITSDGMETTFIDSVEDSESEEEEEGKSSETGKVKTTSLTEKKASRRQKEIPFSYLVGDSGKKKLVKHQVVHKTQEEEETAVPTSQ 6 mRNA sequence GATAAAACCCTAATAGACTCAAAGCATAGAGCTGGAGAAATAACCAGTGATGGCTTGAGCTTCCTATTTCTTAAAGAAGTAAAAGTTGCTAAGCCAGAGGAGATGAAAAACTTAGAGACTCAAGAGGAAGAGTTTTCCGAGCTAGAGGAGCTGGATGAAGAGGCCTCAGGGATGGAGGATGATGAAGATACCTCAGGGCTGGAGGAGGAGGAGGAAGAGCCCTCAGGGCTGGAGGAGGAAGAAGAAGAAGGC TTCAGGGGTTGGAGGAGGATGAGGCCTCAGGGGCTAGAGGAGGAAGAGGAACAGACTTCA GAACAGGACTCAACCTTTCAGGGGTCATACTTTGGTAGATGCAAAGCATGAAGTTGAGATAACCAGTGATGGCATGGAAACTACTTTCATTGACTCTGTAGAGGATTCTGAATCAGAGGAGGAAGAAGAAGGAAAGAGCTCTGAAACAGGAAAGGTAAAGACTACCTCCCTGACTGAGAAAAAAGCCTCACGTAGACAAAAAGAAATTCCCTTTAGTTATTTGGTTGGGGACTCTGGGAAGAAAAAGTTGGTGAAACACCAGG TGGTGCACAAAACCCAGGAGGGAAGAGGAAACAGCTGTCCCACAAGTCAA 7 OH2 Amino acid sequence GTGTPCLTLCLASPSKSLEMSHDEHKKHSHTNLSISTGVTKLKKTEEKKHRTLHTEELTSKEADLTEETEENLRSSVINSIREIKEEIGNLKSSHSGVLEIENSVDDLSSRMDILEERIDSLEDQIEEFSKDTMQMTKQIISKERQRDIEERSRSCNIRLIGIPEKESYENRAEDIIKEIIDENFAELKKGSSLEIVSACRVPSKIDEKRLTPRHILVKFWNSSDKEK IIRASRERREITYQGTRIRLTADLSLDTLDARSKWSNVFKVLLEKGFNPRILYPAKMAFDFRGKTKVFLSIEEFRDYVLHMPTLRELLGNNIP 8 mRNA sequence GGAACTGGCACACCCTGTCTGACCTTATGTTTGGCCTCTCCCTCAAAGTCACTAGAGATGAGTCATGATGAGCATAAAAAGCATTCACATACAAATTTGAGTATTTCAACAGGAGTCACCAAACTTAAGAAAACAGAAGAAAAGAAACACAGAACTCTGCACACAGAAGAACTAACATCCAAAGAAGCAGACTTAACAGAGGAAACAGAAGAAAACTTGAGAAGTAGTGTGATTAATAGCATCAGAGAGATAAAAGAGGAGATTGG AAATTTGAAAAGTTCCCATTCAGGTGTCTTGGAAATTGAAAATTCAGTAGATGATCTGAGTAGCAGAATGGACATACTTGAAGAAAGAATAGACAGTCTAGAAGATCAAATTGAAGAATTCTCTAAGGATACAATGCAAATGACCAAACAGATAATTAGTAAAGAAAGGCAAAGAGATATAGAGGAGAGATCTAGAAGTTGCAACATTCGTTTGA TAGGAATTCCAGAAAAGGAGAGTTATGAGAATAGGGCAGAGGACATAATTAAAGAAATAATTGATGAAAACTTTGCAGAACTAAAGAAAGGTTCAAGTCTTGAGATTGTCAGTGCTTGTCGAGTACCTAGTAAAATTGATGAAAAGAGACTGACTCCTAGACACATCTTGGTGAAATTTTGGAATTCTAGTGATAAAGAGAAAATAATAAGGGCTTCTAGAGAGAGAAGAGAAAATTACCTACCAAGGAACAAGAATCAGGTTGACA GCAGACTTATCACTGGACACACTGGATGCTAGAAGTAAAATGGAGCAATGTCTTCAAAGTTCTGCTGGAAAAAGGCTTTAATCCTAGAATCCTATATCCAGCCAAAATGGCATTTGATTTTAGGGGTAAAACAAAGGTATTTCTTAGTATTGAAGAATTTAGAGATTATGTTTTGCATATGCCCACCTTGAGAGAATTACTGGGGAATAATATACCT 9

3-2. Analysis of the effects of each domain of L1TD1 on granules generated by stress

Since the resolution of stress granules is achieved through dispersion of components and autophagy, in order to confirm the intracellular distribution of the fusion protein consisting of OH2, OH1-OH2, and ER-OH2 through a deletion mutant of L1TD1 and to count the number of cells containing stress granules, a control group in which the pCAG-tRFP plasmid was introduced into the HeLa-eIF3B-GFP cell line prepared in Example 1, and an experimental group in which the pCAG-L1ORF1-tRPF, pCAG-L1TD1-tRPF, pCAG-ER-OH2-tRPF, pCAG-E1-OH2-tRPF, and pCAG-OH2-tRPF plasmids were introduced were prepared. The specific preparation method is the same as that described in Example 2-1.

In addition, the intracellular distribution was confirmed through fluorescence image analysis in the same manner as Example 1, and the number of cells containing stress granules in each control group (tRFP) and experimental group was counted after 24 hours. The experiment was repeated three times, and 100 cell lines were analyzed for each.

As a result, as shown in Fig. 13A, L1 ORF1-tRFP was distributed in a similar location to eIF3B and OH2-tRFP in cells where stress granules exist, OH1-OH2-tRFP showed no change in its intracellular distribution compared to L1TD1 that was not mutated, and ER-OH2 was distributed dispersed in the cytoplasm.

In addition, as shown in Figure 13B, OH2-tRFP did not significantly reduce the number of cells containing stress granules compared to the control group (tRFP), but ER-OH2-tRFP was found to have the smallest number of cells containing stress granules.

3-3. Analysis of the effect of each domain of L1TD1 on granules produced by mutant proteins causing neurodegenerative diseases

In the same manner as in Example 2-2, the 1-493 base sequence of FUS1 was obtained by PCR, conjugated with the tRFP gene, and introduced into the pCAG vector to prepare an expression plasmid (pCAG-FUS1-493X-tRFP). The plasmid was transfected with the expression vector, L1 ORF1-tRFP, L1TD1-tRFP, and vectors expressing the deletion mutants ER-OH2-tRFP, OH1-OH2-tRFP, and OH2-tRFP, respectively, into the HeLa-eIF3B-GFP cell line prepared in Example 1 to prepare an experimental group, and each plasmid was transfected with the empty vector (pCAG) into the HeLa-eIF3B-GFP cell line to prepare a control group. The intracellular distribution was confirmed through fluorescence image analysis in the same manner as in Example 1, and the number of cells with stress granules in each control group and experimental group was counted after 24 hours. The experiment was repeated three times, and 100 cell lines were analyzed each time.

As a result, as shown in Figure 14A, most of the fusion proteins were confirmed to be individually located in FUS-infected cells.

As shown in Figure 14B, OH2-tRFP did not significantly reduce the number of cells containing stress granules compared to the control group (tRFP), but ER-OH2-tRFP was found to have the lowest number of cells containing stress granules.

Therefore, it was expected that the ER-OH2 domain among the L1TD1 protein domains would have excellent stress granule decomposition activity.

Additionally, to confirm the characteristics of ER-OH2, the intracellular distribution of each protein was confirmed through fluorescence image analysis. First, the intracellular distribution of L1TD1-tRFP and ER-OH2 was confirmed according to the untreated and arsenite-treated (oxidative stress treatment) conditions. As shown in Fig. 15, ER-OH2-tRFP was confirmed to be distributed and dispersed in the cytoplasm both under untreated and arsenite-treated conditions.

Additionally, the isoelectric point of the ER domain was determined using the IPC 2.0 tool. The ER domain is highly acidic with a predicted isoelectric point of approximately 4.33, and thus is expected to have a high repulsion force with other negatively charged molecules such as RNP granules and RNA.

In addition, as a result of analyzing the expression of L1TD1 full-length protein and deletion mutants (ER-OH2, OH1-OH2, OH2) through electrophoresis, as shown in Fig. 16, the L1TD1 full-length protein showed the thickest band, indicating that the L1TD1 full-length protein had the highest activity. That is, since ER has a strong negative charge and induces dispersion of negatively charged RNA molecules, etc., ER-OH2 showed a dispersed distribution, and it was predicted that OH1 interacts with other components of stress granules to offset the repulsive force by ER, inhibiting dispersion, and causing OH2 to move to SG through RNA binding.

That is, it was predicted that the OH1 domain, another domain region of L1TD1, would help in the degradation of stress granules by keeping the full-length L1TD1 protein from being degraded in RNP granules through additional interactions.

Therefore, among the L1TD1 protein domains, the ER-OH2 domain showed considerably excellent stress granule decomposition activity, but in the case of the L1TD1 full-length protein, the OH1 domain, ER domain, and OH2 domain each complemented each other's functions to show stress granule decomposition activity, and it was confirmed that the L1TD1 full-length protein containing all of the OH1 domain, ER domain, and OH2 domains showed the greatest activity.

From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical idea or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. The scope of the present invention should be interpreted as including all changes or modifications derived from the meaning and scope of the patent claims described below rather than the above detailed description and their equivalent concepts within the scope of the present invention.

Claims (18)

A pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising as an active ingredient a L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain. In the first paragraph, A pharmaceutical composition, wherein the domain is at least one selected from the group consisting of an OH1 domain, an ER domain, and an OH2 domain. In the first paragraph, A pharmaceutical composition, wherein the above domain is a combination of an ER domain and an OH2 domain. In the first paragraph, A pharmaceutical composition characterized in that the L1TD1 protein, the domain, or a gene encoding the protein or domain decomposes RNA granules in nerve cells. In paragraph 4, A pharmaceutical composition, wherein the above RNA granules are produced by a mutant protein that causes a neurodegenerative disease. In paragraph 5, A pharmaceutical composition wherein the above-mentioned mutated protein is FUS1, TDP-43, SOD-1 or SMN1. In the first paragraph, A pharmaceutical composition, wherein the neurodegenerative disease is at least one selected from the group consisting of Lou Gehrig's disease, frontotemporal lobar degeneration, spinal muscular atrophy, Alzheimer's disease, Parkinson's disease, Huntington's chorea, spinocerebellar degeneration, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, vascular dementia, dementia with Lewy bodies, and dementia accompanying Parkinson's disease. A food composition for preventing or improving a neurodegenerative disease, comprising as an effective ingredient a L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain. In Article 8, A food composition characterized in that the L1TD1 protein, the domain, or a gene encoding the protein or domain decomposes RNA granules in nerve cells. A health functional food for preventing or improving neurodegenerative diseases, comprising as an active ingredient L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain. In Article 10, A health functional food characterized in that the L1TD1 protein, the domain, or a gene encoding the protein or domain decomposes RNA granules in nerve cells. A pharmaceutical composition for the prevention or treatment of a neurodegenerative disease, comprising as an active ingredient a L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain. In Article 12, A pharmaceutical composition characterized in that the L1TD1 protein, the domain, or a gene encoding the protein or domain decomposes RNA granules in nerve cells. A feed composition for preventing or improving neurodegenerative diseases, comprising as an active ingredient L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain. In Article 14, A feed composition characterized in that the L1TD1 protein, the domain, or a gene encoding the protein or domain decomposes RNA granules in nerve cells. A method for improving, preventing, or treating a neurodegenerative disease, comprising administering to a subject in need thereof a composition comprising, as an active ingredient, a L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain, in an amount effective for improving, preventing, or treating the neurodegenerative disease. Use of L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting said protein; or a gene encoding said protein or domain, for improving, preventing or treating a neurodegenerative disease. Use of a L1TD1 (LINE1 type transposase domain containing 1) protein or at least one domain constituting the protein; or a gene encoding the protein or domain, for the manufacture of a medicament for improving, preventing or treating a neurodegenerative disease.

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