Use of hydroxymethyltransferase in treatment of neurodegenerative diseases

Abstract

Provided are a hydroxymethyltransferase and a use of a cofactor or metabolic substrate thereof in preparation of drugs for treating neurodegenerative diseases. Specifically, a hydroxymethyl transfer reaction is performed on glycine residues in protein aggregates such as Poly-GA, Poly-GR, and TDP-43 by means of serine hydroxymethyltransferase 1 (SHMT1) and serine hydroxymethyltransferase 2 (SHMT2), so that the aggregates are degraded, thereby reducing pathological aggregation, and thus ameliorating the pathological condition of patients with neurodegenerative diseases such as ALS and FTD and delaying disease progression. A cofactor and a metabolic substrate of the hydroxymethyltransferase can promote the activity of the hydroxymethyltransferase, thereby enhancing the hydroxymethyl transfer effect on the aggregates such as Poly-GA, Poly-GR and TDP-43, and thus can also be used for treatment of the neurodegenerative diseases such as ALS and FTD.

Description

Application of hydroxymethyltransferase in the treatment of neurodegenerative diseases Technical Field

[0001] This invention relates to the field of medicine, specifically to the use of a class of hydroxymethyltransferases and their cofactors or metabolic substrates in the treatment of neurodegenerative diseases. Background Technology

[0002] With the accelerating aging of the population, neurodegenerative diseases are increasingly becoming a significant global public health challenge. These diseases not only severely impact patients' quality of life but also impose a heavy economic burden on families and society. Among the many neurodegenerative diseases, those related to pathological protein aggregation, such as amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTD), and Alzheimer's disease (AD), have received widespread attention due to their unique pathological mechanisms. The challenges in developing effective therapies for neurodegenerative diseases stem in part from the diversity of patients' genetic backgrounds and the limited knowledge we have about them. Early detection and intervention are crucial for improving patient prognosis and slowing disease progression.

[0003] Therefore, there is an urgent need in this field to develop new, safe, and effective drugs for the treatment of neurodegenerative diseases. Summary of the Invention

[0004] The purpose of this invention is to provide a class of active ingredients that are safe and effective in treating neurodegenerative diseases.

[0005] A first aspect of the present invention provides the use of an active ingredient for preparing a formulation or composition, wherein the active ingredient is selected from the group consisting of serine hydroxymethyltransferase 1 (SHMT1) or serine hydroxymethyltransferase 2 (SHMT2) and optionally activators, cofactors or metabolic substrates of SHMT1 or SHMT2.

[0006] And the formulation or composition is intended for use selected from the group consisting of:

[0007] (1) Convert glycine residues in proteins or aggregates rich in glycine residues into serine residues;

[0008] (2) Promotes the degradation of protein aggregates rich in glycine residues;

[0009] (3) Reduces the pathological aggregation and neurotoxicity of proteins or aggregates rich in glycine residues;

[0010] (4) Treatment and / or relief of neurodegenerative diseases.

[0011] In another preferred embodiment, the cofactor or metabolic substrate is selected from the group consisting of pyridoxal phosphate, 5,10-methylenetetrahydrofolate, folic acid, or 5-methylenetetrahydrofolate.

[0012] In another preferred embodiment, the protein or aggregate thereof rich in glycine residues is an insoluble protein aggregate.

[0013] In another preferred embodiment, the active ingredient is used to convert at least 1 / 10, preferably at least 1 / 5, and most preferably at least 1 / 2 of glycine (G) residues in a protein or aggregate rich in glycine residues into serine (S) residues.

[0014] In another preferred embodiment, at least 1 / 10 of the glycine (G) residues are converted to serine (S) residues, resulting in the substantial degradation of the glycine-rich protein aggregates.

[0015] In another preferred embodiment, the basic degradation refers to the degradation of at least 60%, more preferably at least 70%, more preferably at least 80%, and more preferably at least 90% of the protein aggregates rich in glycine residues.

[0016] In another preferred embodiment, the glycine-rich protein includes, but is not limited to, Poly-GA protein, Poly-GR protein, Poly-GP protein, Poly-G protein, and TAR DNA-binding protein 43 (TDP-43).

[0017] In another preferred embodiment, the formulation or composition is administered to a subject.

[0018] In another preferred embodiment, the subject's nervous system has a high content of proteins or aggregates thereof rich in glycine residues.

[0019] In another preferred embodiment, the above-mentioned high content refers to a content significantly higher than the benchmark value.

[0020] In another preferred embodiment, the benchmark value is the corresponding content in normal subjects.

[0021] In another preferred embodiment, the content is significantly higher than the reference value of ≥6 / 5, preferably ≥5 / 4, more preferably ≥4 / 3, and even more preferably ≥3 / 2.

[0022] In another preferred embodiment, the subject is a mammal, more preferably a rodent (such as a mouse or rat) or a human.

[0023] In another preferred embodiment, the neurodegenerative disease includes, but is not limited to, amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTD), and Alzheimer's disease (AD).

[0024] In another preferred embodiment, the formulation or composition includes a carrier containing a nucleic acid molecule encoding a protein or fragment thereof encoding the serine hydroxymethyltransferase 1 (SHMT1) or serine hydroxymethyltransferase 2 (SHMT2).

[0025] In another preferred embodiment, the vector includes liposomes, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, or combinations thereof.

[0026] In another preferred embodiment, the serine hydroxymethyltransferase SHMT1 or SHMT2 protein or fragments thereof further include peptides that specifically target protein aggregates rich in glycine residues.

[0027] A second aspect of the present invention provides an active ingredient combination, the active ingredient combination comprising:

[0028] (i) a first active ingredient, wherein the first active ingredient is a protein or fragment thereof selected from the group consisting of: serine hydroxymethyltransferase 1 (SHMT1), serine hydroxymethyltransferase 2 (SHMT2), or a combination thereof; and

[0029] (ii) A second active ingredient, which is selected from the group consisting of cofactors and metabolic substrates of SHMT1 or SHMT2.

[0030] In another preferred embodiment, the cofactor or metabolic substrate is selected from the group consisting of pyridoxal phosphate, 5,10-methylenetetrahydrofolate, folic acid, or 5-methylenetetrahydrofolate.

[0031] In another preferred embodiment, the combination further includes an activator of SHMT1 or SHMT2, or a compound capable of increasing SHMT expression levels.

[0032] A third aspect of the invention provides the use of the active ingredient combination of the second aspect of the invention for preparing a formulation or composition, said formulation or composition for:

[0033] (1) Convert glycine residues in proteins or aggregates rich in glycine residues into serine residues;

[0034] (2) Promotes the degradation of protein aggregates rich in glycine residues;

[0035] (3) Reduces the pathological aggregation and neurotoxicity of proteins or aggregates rich in glycine residues;

[0036] (4) Treatment and / or relief of neurodegenerative diseases.

[0037] A fourth aspect of the present invention provides a pharmaceutical composition comprising: (A) a combination of active ingredients as described in the second aspect of the present invention, and (B) a pharmaceutically acceptable carrier.

[0038] In another preferred embodiment, the content of the active ingredient combination in the pharmaceutical composition is 0.01-99 wt%, more preferably 0.1-90 wt%, based on the total weight of the pharmaceutical composition.

[0039] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of oral preparations and injections.

[0040] In another preferred embodiment, the oral preparation is selected from the group consisting of capsules, tablets, pills, powders, granules, emulsions, solutions, suspensions, syrups, and tinctures.

[0041] In another preferred embodiment, the injectable is a liquid formulation or a lyophilized formulation.

[0042] In another preferred embodiment, the method of administration of the pharmaceutical composition is selected from the group consisting of oral, intravenous, intramuscular, parenteral, transdermal, intraperitoneal, or combinations thereof.

[0043] In another preferred embodiment, the preparation is a laboratory preparation.

[0044] A fifth aspect of the present invention provides a medicine box, the medicine box comprising:

[0045] (C1) A first formulation located in a first container, the first formulation comprising a first active ingredient, the first active ingredient being a protein or fragment thereof selected from the group consisting of: serine hydroxymethyltransferase 1 (SHMT1), serine hydroxymethyltransferase 2 (SHMT2), or a combination thereof.

[0046] (C2) A second formulation located within a second container, the second formulation comprising a second active ingredient selected from the group consisting of: cofactors and metabolic substrates of SHMT1 or SHMT2 and / or activators of SHMT1 or SHMT2, or compounds capable of increasing SHMT expression levels; and

[0047] (C3) Instructions for use, which state that the kit is used to treat diseases caused by protein aggregates rich in glycine residues and / or to delay the onset and / or progression of diseases caused by protein aggregates rich in glycine residues.

[0048] A sixth aspect of the present invention provides a method for promoting the degradation of glycine-rich protein aggregates in vitro, comprising the steps of:

[0049] Contacting cells with the active ingredient or an expression vector that overexpresses the active ingredient promotes the degradation of the glycine-rich protein aggregates.

[0050] The active ingredient is selected from the group consisting of serine hydroxymethyltransferase 1 (SHMT1) or serine hydroxymethyltransferase 2 (SHMT2) and optionally activators of SHMT1 or SHMT2 or compounds, cofactors or metabolic substrates that can enhance SHMT expression levels.

[0051] In another preferred embodiment, the active ingredient is used to convert at least 1 / 10, preferably at least 1 / 5, and most preferably at least 1 / 2 of glycine (G) residues in a protein or aggregate rich in glycine residues into serine (S) residues.

[0052] In another preferred embodiment, at least 1 / 10 of the glycine (G) residues are converted to serine (S) residues, resulting in the substantial degradation of the glycine-rich protein aggregates.

[0053] In another preferred embodiment, the basic degradation refers to the degradation of at least 60%, more preferably at least 70%, more preferably at least 80%, and more preferably at least 90% of the protein aggregates rich in glycine residues.

[0054] In another preferred embodiment, the glycine-rich protein includes, but is not limited to, Poly-GA protein, Poly-GR protein, Poly-GP protein, Poly-G protein, and TAR DNA-binding protein 43 (TDP-43).

[0055] In another preferred embodiment, the cell is a cell that highly expresses a protein rich in glycine residues.

[0056] In another preferred embodiment, the concentration of the active ingredient is from 0.001 mM to 10 M; preferably, from 0.01 mM to 1 M; more preferably, from 0.1 mM to 0.1 M; even more preferably, from 0.5 mM to 10 mM.

[0057] A seventh aspect of the present invention provides a method for treating neurodegenerative diseases, comprising: administering to a subject in need a therapeutically effective amount of the combination of active ingredients of the second aspect of the present invention or the pharmaceutical composition of the fourth aspect of the present invention.

[0058] In another preferred embodiment, the neurodegenerative diseases include, but are not limited to, amyotrophic lateral sclerosis (ALS), frontotemporal lobe degeneration (FTD), and Alzheimer's disease (AD).

[0059] In another preferred embodiment, the subjects include rodents (such as mice and rats) and primates (such as humans).

[0060] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0061] Figure 1 shows the corresponding interaction mass spectrometry results for Poly-GA and Poly-GR.

[0062] Figure 2 shows the results of immunoprecipitation experiments verifying the interaction between GA100 and SHMT1 and SHMT2.

[0063] Figure 3 shows that SHMT1 overexpression reduces the size of Poly-GA aggregates.

[0064] Figure 4 shows that SHMT1 overexpression reduces the area of ​​Poly-GA aggregates.

[0065] Figure 5 shows that SHMT1 overexpression and combined treatment with its cofactors and metabolites reduce Poly-GA aggregates.

[0066] Figure 6 shows that SHMT1 inactivation mutations and overexpression of SHMT1 and SHMT2 reduce the level of Poly-GA aggregates.

[0067] Figure 7 shows that hydroxymethylation of glycine residues in Poly-GA reduces the formation of protein aggregates.

[0068] Figure 8 shows the interaction of SHMT1 and SHMT2 with the glycine-rich domains in the TDP-43 protein.

[0069] Figure 9 shows that SHMT2 can be recruited around TDP-43 protein aggregates and reduce aggregate formation.

[0070] Figure 10 shows a schematic diagram of the domains of the TDP-43 protein. Detailed Implementation

[0071] Through extensive and in-depth research, this invention has discovered that serine hydroxymethyltransferase 1 (SHMT1) and serine hydroxymethyltransferase 2 (SHMT2) can degrade protein aggregates by performing a small-scale hydroxymethyl transfer reaction on glycine residues (approximately 1 / 10 of the glycine residues) in various glycine-rich protein aggregates, thereby reducing the pathological aggregation of glycine-rich proteins, thus improving the pathological state of patients with neurodegenerative diseases such as ALS and FTD and delaying disease progression. The cofactors and metabolic substrates of these hydroxymethyltransferases (pyridoxal phosphate, 5,10-methylenetetrahydrofolate, folic acid, etc.) can promote the activity of these hydroxymethyltransferases, thereby enhancing the hydroxymethyl transfer effect on aggregates such as Poly-GA, Poly-GR, and TDP-43, and therefore can also be used for the treatment of neurodegenerative diseases such as ALS and FTD. This invention was completed based on these findings.

[0072] the term

[0073] As used herein, the term "first active ingredient" means a protein product, peptide product, mRNA product, or DNA product that can express SHMT1 or / and SHMT2 protein or fragments thereof.

[0074] As used herein, the term "second active ingredient" refers to a protein product, peptide product, mRNA product, or DNA product that can express SHMT1 or SHMT2 protein or fragments thereof that have signal peptides targeting protein aggregates such as Poly-GA, Poly-GR, and TDP-43.

[0075] As used herein, the term "third active ingredient" refers to a third active ingredient selected from the group consisting of cofactors and metabolic substrates of SHMT1 / 2, including (pyridoxal phosphate, 5,10-methylenetetrahydrofolate, folic acid or 5-methylenetetrahydrofolate, etc., or pharmaceutically acceptable salts thereof, or combinations thereof).

[0076] As used herein, the term "fourth active ingredient" refers to a fourth active ingredient selected from the group consisting of: SHMT1 or SHMT2 activators, or compounds that can enhance SHMT expression levels, or combinations thereof.

[0077] As used herein, the terms “combination of active ingredients of the present invention”, “combination of the present invention”, and “combination of pharmaceutical ingredients of the present invention” are used interchangeably to refer to the combination of the first / second active ingredient and the third active ingredient described above.

[0078] As used herein, the term "pharmaceutical composition of the present invention" refers to a composition comprising or containing a first active ingredient (or a first formulation containing the first active ingredient), a second active ingredient (or a second formulation containing the second active ingredient), a third active ingredient (or a third formulation containing the third active ingredient), and a fourth active ingredient (or a fourth formulation containing the fourth active ingredient). The first, second, third, and fourth formulations may be the same or different formulations. Furthermore, the first, second, third, and fourth formulations may be the same formulation or separate formulations.

[0079] Proteins rich in glycine residues

[0080] Proteins rich in glycine residues are characterized by a high glycine content in their sequence and the presence of conserved segments, including glycine-containing structural motifs composed of repeating amino acid residues, including but not limited to Poly-GA protein, Poly-GR protein, Poly-GP protein, Poly-G protein, TAR DNA-binding protein 43 (TDP-43), etc.

[0081] Amyotrophic Lateral Sclerosis

[0082] Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that primarily affects motor neurons in the brain and spinal cord, leading to muscle weakness, atrophy, and loss of neurological function. The pathological features of the disease include the progressive degeneration of motor neurons, ultimately resulting in the loss of voluntary motor function and respiratory function, typically leading to death within a few years of diagnosis. The exact cause of the disease is not fully understood, but it is likely related to both genetic and environmental factors.

[0083] Frontotemporal lobe degeneration

[0084] Frontotemporal dementia (FTD) is a group of diseases characterized by the degeneration of neurons in the frontal and temporal lobes, primarily manifesting as significant changes in behavior, emotion, and language function. These diseases can lead to personality changes, decreased language ability, and social dysfunction. They are common in middle-aged individuals and have a genetic predisposition. This category includes frontal lobe degeneration, certain forms of Alzheimer's disease, and other related dementias.

[0085] Poly-GA

[0086] Poly-GA refers to poly-glycine-alanine, which is produced by the aberrant translation of an extended GGGGCC repeat sequence in C9orf72 and is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia.

[0087] TDP-43

[0088] TAR DNA-binding protein 43 is an important RNA-binding protein containing the polyglycine polyG structure. It is mainly involved in RNA splicing and transport, and its abnormal accumulation is closely related to a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration.

[0089] Serine hydroxymethyltransferase 1

[0090] Serine hydroxymethyltransferase 1 (SMT1) is a key enzyme involved in folate metabolism, catalyzing the reversible conversion of serine and glycine. This enzyme plays a crucial role in DNA synthesis and repair.

[0091] Serine hydroxymethyltransferase 2

[0092] Serine hydroxymethyltransferase 2 (SHMT2) is an enzyme similar to SHMT1, primarily functioning in the mitochondria of cells. It plays a crucial role in maintaining one-carbon metabolism and energy metabolism within the cell. Another isoform, SHMT2α, which lacks the mitochondrial signal peptide, is located in the cytoplasm and nucleus.

[0093] Pyridoxal phosphate

[0094] Pyridoxal phosphate (PLP) is the active form of vitamin B6 and plays a crucial role in amino acid metabolism and neurotransmitter synthesis as a coenzyme for many enzymes. PLP levels and its metabolism may have significant implications for the development of neurological disorders.

[0095] 5,10-Methylenetetrahydrofolate

[0096] 5,10-Methylenetetrahydrofolate is an active form of folic acid that participates in methylation reactions and amino acid metabolism in the body, playing a particularly important role in the synthesis of thymidine nucleotides and the conversion of homocysteine, and is essential for DNA synthesis and repair.

[0097] folic acid

[0098] Folic acid is an important water-soluble B vitamin that participates in nucleic acid synthesis and cell division. Folic acid deficiency can lead to neural tube defects, anemia, and may be associated with the pathological mechanisms of neurodegenerative diseases.

[0099] Combination of active ingredients, pharmaceutical composition

[0100] This invention provides the use of hydroxymethyltransferase and its cofactors or metabolic substrates in the preparation of pharmaceuticals for treating neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD).

[0101] Based on the above components, the present invention provides an active ingredient combination that can be used for:

[0102] (1) Convert glycine residues in proteins or aggregates rich in glycine residues into serine residues;

[0103] (2) Promotes the degradation of protein aggregates rich in glycine residues;

[0104] (3) Reduces the pathological aggregation and neurotoxicity of proteins or aggregates rich in glycine residues;

[0105] (4) Treatment and / or relief of neurodegenerative diseases.

[0106] The present invention also provides a pharmaceutical composition comprising a combination of active ingredients within a safe and effective range, and a pharmaceutically acceptable carrier.

[0107] "Pharmaceutically acceptable carriers" refer to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here refers to the ability of the components in the composition to interact with and incorporate the active ingredient of the invention without significantly reducing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, saline, buffer solutions, glucose, water, glycerol, polysorbate, ethanol, etc.

[0108] There are no particular limitations on the administration of the active ingredient combination or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc.

[0109] As used herein, the term “effective amount” or “effective dose” refers to an amount that is functional or active in humans and / or animals and / or cells and is acceptable to humans and / or animals.

[0110] When using pharmaceutical preparations, safe and effective amounts of active ingredients are combined and administered to mammals.

[0111] It should be understood that the effective amount of the active ingredient described in this invention may vary depending on the administration method and the severity of the disease. The preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters such as bioavailability, metabolism, and half-life; disease severity; patient weight; patient immune status; and route of administration.

[0112] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0113] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. Besides these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0114] In addition to the active ingredient, the suspension may contain suspending agents, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0115] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0116] The main advantages of this invention include:

[0117] (1) The present invention discovers a hydroxymethyltransferase SHMT1 and SHMT2, which can perform non-classical functions, perform hydroxymethyl transfer reactions on glycine residues in proteins rich in glycine residues, turning them into serine residues, thereby degrading pathological protein aggregates, improving the pathological state of patients with neurodegenerative diseases such as ALS and FTD and delaying disease progression.

[0118] (2) The present invention uses SHMT1 / 2 and its cofactors or metabolic substrates in combination to achieve synergistic therapeutic effects.

[0119] (3) The active ingredient SHMT1 / 2 used in this invention is delivered to the relevant concurrent areas using technologies such as adeno-associated virus (AAV) or liposome encapsulation, which is safe and efficient. The cofactor or metabolic substrate of the other active ingredient SHMT1 / 2 used in this invention is a metabolite that is already present in the human body and has high safety for the human body.

[0120] (4) This invention is applicable to a variety of neurodegenerative diseases, including but not limited to amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTD), and Alzheimer's disease (AD), and can achieve the goal of "treating different diseases with the same method".

[0121] Example 1. The interaction mass spectrometry of Poly-GA and Poly-GR could identify SHMT1 and SHMT2 proteins.

[0122] GA100 (poly(GA)) was purified from SY-5Y cells that stably expressed GA100-GFP-Flag or GR100-GFP-Flag. 100 Protein) and GR100 (poly(GR)) 100 Proteins were analyzed, and their interacting proteins were detected using a mass spectrometer.

[0123] As shown in Figure 1, both SHMT1 and SHMT2 are identified in the mass spectrometry results of Poly-GA or Poly-GR.

[0124] Example 2. SHMT1 and SHMT2 are both Poly-GA interacting proteins.

[0125] The Flag-GA100 plasmid or its empty vector control was transiently transfected into SY-5Y and 293T cells, respectively. After 48 h, the cells were collected, lysed with 0.5% IP lysis buffer, and immunoprecipitation was performed using Flag beads to verify the interaction between GA100 and SHMT1 and SHMT2.

[0126] As shown in Figure 2, GA100 can interact with endogenous SHMT1 and SHMT2 in SY-5Y and 293T cells.

[0127] Example 3. Overexpression of SHMT1 reduces the size of Poly-GA aggregates.

[0128] In two wells of a 24-well array, plasmids expressing GFP-GA100 and SHMT1 were transiently transfected into SY-5Y cells, followed by immunofluorescence staining 24 h later.

[0129] As shown in Figure 3, the Poly-GA100 aggregates in cells overexpressing SHMT1 were smaller than those in cells without SHMT1 expression, suggesting that SHMT1 can reduce the formation of Poly-GA aggregates.

[0130] Example 4. Overexpression of SHMT1 reduces the area of ​​Poly-GA aggregates.

[0131] Transiently transfect SY-5Y cells with plasmids expressing GFP-GA100 and SHMT1 or their empty vector controls, and perform immunofluorescence staining 24 h later.

[0132] Statistical results (Figure 4) showed that the area of ​​Poly-GA100 aggregates in cells treated with SHMT1 was significantly reduced compared to the control group, further indicating that SHMT1 can reduce the formation of Poly-GA aggregates.

[0133] Example 5. Overexpression of SHMT1 and combined treatment with its cofactors and metabolic substrates further reduced Poly-GA aggregates.

[0134] Transiently transfect GA100 into SY-5Y cells that stably express SHMT1 and its corresponding empty vector. After 24 hours, SHMT1 cofactors and metabolic substrates were added. After 48 hours, the fluorescence of GA100 was photographed, and cells were collected for immunoblotting.

[0135] As shown in Figure 5, compared to the control plasmid, SHMT1 expression significantly reduced the fluorescence of GFP-GA100. Furthermore, the addition of SHMT1 cofactors and metabolic substrates also dose-dependently reduced the expression of GFP-GA100 aggregates. Further experiments separating soluble and insoluble proteins revealed that insoluble Poly-GA aggregates were significantly reduced after SHMT1 overexpression or the addition of SHMT1 cofactors and metabolic substrates.

[0136] Example 6. Overexpression of SHMT1 and SHMT2 reduced the level of Poly-GA aggregates, while SHMT1 inactivation mutations did not have this effect.

[0137] Transiently transfected GA100 into SY-5Y cells stably expressing SHMT1, SHMT1 enzyme inactivation mutant, SHMT2, and the corresponding empty vector, cells were collected 48 h later for immunoblotting to detect insoluble Poly-GA aggregates.

[0138] As shown in Figure 6, compared to the control plasmid, the expression of both SHMT1 and SHMT2 significantly reduced the level of insoluble PolyGA aggregates, while the expression of SHMT1-DN with lost enzyme activity did not have this effect. This suggests that SHMT1 / 2 may reduce PolyGA aggregation by converting glycine residues in Poly-GA into serine residues through their enzymatic activity.

[0139] Example 7. Hydroxymethylation of glycine residues in Poly-GA reduces protein aggregate formation.

[0140] Poly-GA100-GFP or its mutant plasmids with 1 / 10, 1 / 5, and 1 / 2 glycine (G) residues converted to serine (S) residues were stably expressed in SY-5Y cells. Cells were fixed for immunofluorescence experiments after 24 hours.

[0141] As shown in Figure 7, Poly-GA100-GFP can form obvious aggregates in cells, while Poly-GA100 proteins with G-to-S mutations at ratios of 1 / 2 and 1 / 5 do not form aggregates. Poly-GA100 proteins with G-to-S mutations at ratios of 1 / 10 still form aggregates, but the proportion of aggregates formed is significantly reduced. These findings indicate that the conversion of G to S after hydroxymethylation in Poly-GA100 protein can prevent or reduce the formation of protein aggregates.

[0142] Example 8. Interactions between SHMT1 and SHMT2 with glycine-rich domains in the TDP43 protein

[0143] Transiently transfect SY-5Y cells with the glycine-rich TDP-43-LCD plasmid containing the GFP tag and SHMT1 or SHMT2 and their corresponding control plasmids. After 48 hours, cells were collected for immunoprecipitation experiments.

[0144] As shown in Figure 8, both SHMT1 and SHMT2 can interact with the TDP-43-LCD protein.

[0145] Example 9. SHMT2 can be recruited to the vicinity of TDP43 protein aggregates and reduce aggregate formation.

[0146] Transiently transfect SY-5Y cells with the glycine-rich TDP-43-LCD plasmid containing the GFP tag and SHMT2 and its corresponding control plasmids. Cells were collected after 48 hours for immunofluorescence experiments.

[0147] As shown in Figure 9, SHMT2 can be recruited to the vicinity of TDP-43-LCD in SHMT2-expressing cells. Furthermore, compared to cells expressing the empty Flag vector, SHMT2-expressing cells showed significantly reduced aggregate formation. This suggests that SHMT2 can also modify the glycine-rich domains of TDP-43 to reduce aggregate formation.

[0148] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0149] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of an active ingredient, characterized in that, For use in the preparation of a formulation or composition, wherein the active ingredient is selected from the group consisting of serine hydroxymethyltransferase 1 (SHMT1) or serine hydroxymethyltransferase 2 (SHMT2) and optionally an activator, cofactor or metabolic substrate of SHMT1 or SHMT2. And the formulation or composition is intended for use selected from the group consisting of: (1) Convert glycine residues in proteins or aggregates rich in glycine residues into serine residues; (2) Promotes the degradation of protein aggregates rich in glycine residues; (3) Reduces the pathological aggregation and neurotoxicity of proteins or aggregates rich in glycine residues; (4) Treatment and / or relief of neurodegenerative diseases.

2. The use as described in claim 1, characterized in that, The cofactor or metabolic substrate is selected from the group consisting of: pyridoxal phosphate, 5,10-methylenetetrahydrofolate, folic acid, or 5-methylenetetrahydrofolate.

3. The use as described in claim 1, characterized in that, The proteins rich in glycine residues include, but are not limited to, Poly-GA protein, Poly-GR protein, Poly-GP protein, Poly-G protein, and TAR DNA-binding protein 43 (TDP-43).

4. The use as described in claim 1, characterized in that, The neurodegenerative diseases mentioned include, but are not limited to, amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTD), and Alzheimer's disease (AD).

5. The use as described in claim 1, characterized in that, The formulation or composition includes a carrier that contains a nucleic acid molecule encoding a protein or fragment thereof encoding the serine hydroxymethyltransferase 1 (SHMT1) or serine hydroxymethyltransferase 2 (SHMT2).

6. A combination of active ingredients, characterized in that, The combination of active ingredients includes: (i) a first active ingredient, wherein the first active ingredient is a protein or fragment thereof selected from the group consisting of: serine hydroxymethyltransferase 1 (SHMT1), serine hydroxymethyltransferase 2 (SHMT2), or a combination thereof; and (ii) A second active ingredient, which is selected from the group consisting of cofactors and metabolic substrates of SHMT1 or SHMT2.

7. Use of the active ingredient combination as described in claim 2, for preparing a formulation or composition, said formulation or composition being used for: (1) Convert glycine residues in proteins or aggregates rich in glycine residues into serine residues; (2) Promotes the degradation of protein aggregates rich in glycine residues; (3) Reduces the pathological aggregation and neurotoxicity of proteins or aggregates rich in glycine residues; (4) Treatment and / or relief of neurodegenerative diseases.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (A) the active ingredient combination as described in claim 6, and (B) a pharmaceutically acceptable carrier.

9. A medicine box, characterized in that, The medicine box includes: (C1) A first formulation located in a first container, the first formulation comprising a first active ingredient, the first active ingredient being a protein or fragment thereof selected from the group consisting of: serine hydroxymethyltransferase 1 (SHMT1), serine hydroxymethyltransferase 2 (SHMT2), or a combination thereof. (C2) A second formulation located within a second container, the second formulation comprising a second active ingredient selected from the group consisting of: cofactors and metabolic substrates of SHMT1 or SHMT2 and / or activators of SHMT1 or SHMT2, or compounds capable of increasing SHMT expression levels; and (C3) Instructions for use, which state that the kit is used to treat diseases caused by protein aggregates rich in glycine residues and / or to delay the onset and / or progression of diseases caused by protein aggregates rich in glycine residues.

10. A method for promoting the degradation of glycine-rich protein aggregates in vitro, comprising the steps of: Contacting cells with the active ingredient or an expression vector that overexpresses the active ingredient promotes the degradation of the glycine-rich protein aggregates. The active ingredient is selected from the group consisting of serine hydroxymethyltransferase 1 (SHMT1) or serine hydroxymethyltransferase 2 (SHMT2) and optionally activators of SHMT1 or SHMT2 or compounds, cofactors or metabolic substrates that can enhance SHMT expression levels.

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