Diagnostic markers and therapeutic compositions for neurodegenerative diseases

Abstract

To provide a pharmaceutical composition for preventing or treating neurodegenerative diseases.SOLUTION: A pharmaceutical composition comprises GSTO1 (omega class glutathione transferase 1) or GstO2 (omega class glutathione transferase 2) genes, which are factors inducing deglutathionylation of a FUS protein, or a protein encrypted by the genes as an active ingredient.SELECTED DRAWING: Figure 2b

Description

[Technical field]

[0001] The present invention relates to a diagnostic marker for nervous system degenerative diseases and uses thereof. More specifically, the present invention relates to a diagnostic marker composition for nervous system degenerative diseases comprising a glutathionylated FUS protein, a diagnostic composition for nervous system degenerative diseases comprising a preparation for measuring the glutathionylation level of FUS protein, a diagnostic kit for nervous system degenerative diseases comprising the composition, and a method for providing information for diagnosing nervous system degenerative diseases using the same.

[0002] The present invention also relates to a composition for treating degenerative diseases of the nervous system, comprising GstO2, and more specifically, to a composition for preventing or treating degenerative diseases of the nervous system, comprising GstO2 as an active ingredient, which induces deglutathionylation of FUS protein. [Background technology]

[0003] Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset degenerative neurological disease characterized by the gradual degeneration of motor neurons. ALS leads to progressive muscle weakness, eventually leading to fatal muscle atrophy and paralysis, with death occurring within 3 to 5 years after the onset of the disease. ALS can be classified into sporadic ALS, which has an unknown etiology, and familial ALS, which is caused by genetic defects in proteins directly related to the pathology, such as superoxide dismutase 1 (SOD1), transactive response DNA-binding protein-43 (TDP-43), fused in sarcoma (FUS), or TATA-binding protein-associated factor 15 (TAF15). Mutant forms of TDP-43 are toxic in neurons, and cytoplasmic mislocalization of the protein has been shown to be associated with the pathogenesis of amyotrophic lateral sclerosis.

[0004] Protein aggregates are also characteristically found in age-related neurodegenerative diseases (ND), such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease).

[0005] FUS (Fused in Sarcoma) is an RNA and DNA binding protein, and the N-terminus of the FUS protein is related to transcriptional activity, while the C-terminus is known to be involved in protein and RNA binding. The FUS protein contains recognition sites for the transcription factors AP2, GCF, and Sp1, and recent studies have identified various forms of mutant FUS protein in patients with amyotrophic lateral sclerosis and frontotemporal dementia. It has been revealed that these mutant FUS proteins are displaced from their original location in the nucleus and then exit the nucleus, forming aggregates in stress granules.

[0006] In addition, FUS mutations have been confirmed in most patients with amyotrophic lateral sclerosis (Non-Patent Document 1), and FUS P525L Mutations in FUS-mutated proteins show cytoplasmic mislocalization and are associated with acute FUS-induced amyotrophic lateral sclerosis (Non-Patent Document 2), and the FUS wild type and its variant, FUS P525L is known to show the same phenotype in Drosophila, such as rough eyes and reduced motility (Non-Patent Document 3; Non-Patent Document 4).

[0007] Amyotrophic lateral sclerosis cannot be diagnosed by magnetic resonance imaging (MRI) or blood tests, but can be diagnosed through a physical examination based on the patient's symptoms and by a medical staff with a wealth of experience. Although there have been attempts to study a method for diagnosing amyotrophic lateral sclerosis by measuring the expression levels or protein activity of the MARCH5 and MFN2 genes (Patent Document 1), there is still insufficient research on diagnostic markers and diagnostic compositions for amyotrophic lateral sclerosis associated with FUS protein and FUS protein aggregates.

[0008] In addition, little is known about the mechanism of the abnormal location of FUS mutant proteins and the accumulation of aggregates found in patients with amyotrophic lateral sclerosis and frontotemporal dementia, and there are no treatments based on this. As mentioned above, various studies have been attempted to identify the formation of FUS aggregates and their mechanism of action to develop therapeutic drugs (Patent Document 2), but the current situation is still insufficient. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Korean Patent No. 10-1674920 [Patent Document 2] Korean Patent No. 10-1576602 [Non-patent literature]

[0010] [Non-Patent Document 1] Mackenzie et al., 2010 [Non-Patent Document 2] Sun et al., 2011 [Non-Patent Document 3] Chen et al., 2016 [Non-Patent Document 4] Jackel et al., 2015 Summary of the Invention [Problem to be solved by the invention]

[0011] As a result, the inventors were searching for a new marker for diagnosing nervous system degenerative diseases, and discovered that FUS protein, which should be located mainly in the nucleus, is located in the cytoplasm in the form of glutathionylated FUS protein aggregates. They confirmed that glutathionylated FUS protein could be a new marker for diagnosing nervous system degenerative diseases, and thus completed the present invention.

[0012] In addition, the present invention has been devised to solve the above problems. As a result of various studies, the inventors have confirmed that the formation of protein aggregates due to glutathionylation of FUS protein, which is known to be an amyotrophic lateral sclerosis-inducing protein, and its deposition in the cytoplasm of brain neurons are the causes of amyotrophic lateral sclerosis. They also confirmed the activity of GSTO (omega class glutathione transferase) in inhibiting the glutathionylation of FUS protein, and completed the present invention based on this.

[0013] An object of the present invention is to provide a marker composition for diagnosing nervous system degenerative diseases, which comprises a glutathionylated FUS protein.

[0014] Another object of the present invention is to provide a composition for diagnosing a nervous system degenerative disease, comprising a preparation for measuring the glutathionylation level of FUS protein, and a kit for diagnosing a nervous system degenerative disease, comprising the composition.

[0015] Another object of the present invention is to provide a method for providing information for diagnosing neurodegenerative diseases, comprising measuring the glutathionylation level of FUS protein in a biological sample derived from a subject and comparing it with that of a normal human.

[0016] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating neurodegenerative diseases, comprising the GSTO1 (omega class glutathione transferase 1) or GstO2 (omega class glutathione transferase 2) gene or a protein encoded by the gene as an active ingredient.

[0017] However, the technical problems that the present invention aims to achieve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0018] In order to achieve the above object, the present invention provides A marker composition for diagnosing nervous system degenerative diseases, comprising a glutathionylated FUS protein, is provided.

[0019] In one embodiment of the present invention, the nervous system degenerative disease may be amyotrophic lateral sclerosis.

[0020] In another embodiment of the present invention, the FUS protein may be glutathionylated at the Cys-447 residue.

[0021] The present invention also provides a composition for diagnosing a neurodegenerative disease, comprising a preparation for measuring the glutathionylation level of FUS protein.

[0022] In one embodiment of the present invention, the FUS protein may consist of the amino acid sequence represented by SEQ ID NO:1.

[0023] In one embodiment of the present invention, the nervous system degenerative disease may be amyotrophic lateral sclerosis.

[0024] The present invention also provides a kit for diagnosing a nervous system degenerative disease, comprising the composition.

[0025] The present invention also provides a method for providing information for diagnosing neurodegenerative diseases, comprising the steps of: a) measuring the glutathionylation level of FUS protein from a biological sample derived from a subject; and b) comparing said level with the glutathionylation level of said protein in a normal control sample.

[0026] The present invention also provides a pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising, as an active ingredient, the GSTO1 (omega class glutathione transferase 1) or GstO2 (omega class glutathione transferase 2) gene or a protein encoded by the gene.

[0027] In one embodiment of the present invention, the GSTO1 gene may consist of the base sequence represented by SEQ ID NO:1.

[0028] In another embodiment of the present invention, the GSTO1 protein may consist of the amino acid sequence represented by SEQ ID NO:2.

[0029] In yet another embodiment of the present invention, the GstO2 gene may consist of the base sequence represented by SEQ ID NO:3.

[0030] In yet another embodiment of the present invention, the GstO2 protein may consist of the amino acid sequence represented by SEQ ID NO:4.

[0031] In yet another embodiment of the present invention, the neurodegenerative disease may be amyotrophic lateral sclerosis.

[0032] In yet another embodiment of the present invention, the composition may inhibit glutathionylation of FUS protein.

[0033] In still another embodiment of the present invention, the glutathionylation of the FUS protein may be at the Cys-447 residue of the FUS.

[0034] The present invention also provides a method for preventing or treating a nervous system degenerative disease, comprising the step of administering the composition to an individual.

[0035] The present invention also provides an application of the composition for the prevention or treatment of neurodegenerative diseases. Effect of the Invention

[0036] The present inventors have demonstrated that glutathionylated FUS protein functions as a diagnostic marker for nervous system degenerative diseases, and the composition for diagnosing nervous system degenerative diseases according to the present invention contributes to the early diagnosis of nervous system degenerative diseases.

[0037] Furthermore, the present inventors have revealed that glutathionylation of FUS, known to be an amyotrophic lateral sclerosis-inducing protein, is the pathogenic mechanism of amyotrophic lateral sclerosis, which increases intracytoplasmic aggregation and neurotoxicity. Accordingly, the present composition for preventing or treating neurodegenerative diseases contains GSTO1 or GstO2, which induces deglutathionylation of FUS protein, and has been confirmed to have an inhibitory effect on brain cytoplasmic aggregation and neurotoxicity. It is expected that the composition can be usefully used for preventing, treating or ameliorating neurodegenerative diseases. [Brief description of the drawings]

[0038] [Figure 1a] This was done to confirm whether human FUS protein was glutathionylated, and the results of separating human FUS protein that was glutathionylated depending on the concentration of oxidized glutathione (GSSG) using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are shown. [Figure 1b] The present invention is for quantitatively analyzing glutathionylation of human FUS protein, and the results of quantitative analysis of the separated protein based on the concentration of oxidized glutathione (GSSG) are shown. [Figure 2a] To confirm the intracellular location of human FUS protein, the image shows that overexpressed human FUS protein aggregates are located in the cytoplasm of neurons in the Drosophila brain. [Figure 2b] This image shows that the reduced form of glutathione (GSH) generated by glutathionylation of the FUS protein is located in the cytoplasm of neurons in the Drosophila brain. [Figure 2c] This is to confirm the intracellular location of human FUS protein aggregates and reduced glutathione (GSH). The image shows that human FUS protein aggregates and reduced glutathione (GSH) are located in the cytoplasm of neurons in the Drosophila brain. [Figure 3a] This is to confirm the intracellular location of human wild-type FUS protein aggregates, and the image shows that human wild-type FUS protein aggregates are located in the cytoplasm of N2a. [Figure 3b] The image shows that reduced glutathione (GSH), generated by glutathionylation of human wild-type FUS protein, is located in the cytoplasm of N2a. [Figure 3c] This is to confirm the intracellular location of human wild-type FUS protein aggregates and reduced glutathione (GSH). The image shows that human wild-type FUS protein aggregates and reduced glutathione (GSH) are located in the cytoplasm of N2a. [Figure 4a] This is to confirm the intracellular location of human mutant FUSP525L protein aggregates, and the image shows that human mutant FUSP525L protein aggregates are located in the cytoplasm of N2a. [Figure 4b]This is an image showing that reduced form of glutathione (GSH) generated by glutathionylation of human mutant FUSP525L protein is located in the cytoplasm of N2a. [Figure 4c] This is to confirm the intracellular location of aggregates of human mutant FUSP525L protein and reduced form of glutathione (GSH). The image shows that aggregates of human mutant FUSP525L protein and reduced form of glutathione (GSH) are located in the cytoplasm of N2a. [Diagram 5] The present invention is to confirm the specific site of glutathionylation of the FUS protein, and the results of MALDI-mass spectrometry are shown. [Figure 6] The purpose of this study was to confirm whether the cysteine ​​sequence of the RanBP2 zinc-finger domain in the FUS protein is conserved between species. The results of analyzing the sequences of the RanBP2 zinc-finger domain from Drosophila melanogaster, Xenopus laevis, zebrafish D. rerio, house mouse M. musculus, and human H. sapiens are shown. [Figure 7] 1 is a schematic diagram of an experiment for forming aggregates of FUS protein. [Figure 8] FIG. 1 is a diagram for comparing the solubility of aggregates of glutathionylated FUS protein with that of non-glutathionylated FUS protein, and the solubility of the proteins is shown by Western blot analysis. [Figure 9] 3D homology model of the RanBP2 zinc-finger domain of the FUS protein and the relative position of Cys-447. [Figure 10a] This shows the results of confirming whether or not the FUS protein is glutathionylated in the presence of GSSG. [Figure 10b] The results show that glutathionylated FUS protein is located in the cytoplasm of neurons in the Drosophila brain. [Figure 10c]This shows the results of confirming aggregates of FUS and human mutant FUSP525L protein in the cytoplasm of N2a. [Figure 11a] This shows the results of confirming the eye phenotype of flies expressing FUS by GstO2. [Figure 11b] This shows the results of confirming the larval crawling activity of FUS-expressing flies by induction of GstO2. [Figure 11c] These are the results of confirming the number of synaptic boutons at the NMJ (neuromuscular junction) of flies expressing FUS using GstO2. [Figure 11d] The results of confirming the lifespan of FUS-expressing flies by GstO2 are shown. [Figure 11e] This shows the results of confirming the lifespan of FUS-expressing flies by GstO2 knockdown. [Figure 11f] These results confirm the climbing activity of FUS-expressing flies induced by GstO2. [Figure 12a] This shows the results of confirming the size of mitochondria in flies expressing FUS using GstO2. [Figure 12b] This shows the results of confirming mitochondrial morphology in flies expressing FUS using GstO2. [Figure 12c] This shows the results of confirming Marf expression in FUS-expressing flies induced by GstO2. [Figure 12d] This shows the results of confirming the mitochondrial complex content in FUS-expressing flies using GstO2. [Figure 12e] This shows the results of BN-PAGE of FUS-expressing flies induced by GstO2. [Figure 12f] This shows the results of confirming ROS production by GstO2 in FUS-expressing flies. [Figure 12g] This shows the results of confirming ATP levels in FUS-expressing flies by GstO2. [Figure 12h]The results of measuring the amount of cytoplasmic oxidized protein by GstO2 in FUS-expressing flies are shown. [Figure 13a] This is the result of immunoblotting of brain extracts from flies expressing FUS using GstO2. [Figure 13b] Immunoblotting results of brain extracts from GstO2-knockdown FUS-expressing flies. [Figure 13c] The results of nuclear / cytoplasmic fraction analysis of FUS-expressing flies using GstO2 are shown. [Figure 13d] This shows the results of a solubility analysis to confirm FUS aggregates in GstO2-induced FUS-expressing flies. [Figure 13e] Solubility analysis results to confirm FUS aggregates due to knockdown of GSTO1 or GstO3 are shown. [Figure 13f] This shows the results of confirming the FUS level in mitochondria of flies expressing FUS by GstO2. [Figure 14a] This shows the results of double immunofluorescence analysis to confirm the regulation of glutathionylation of FUS by GstO2 in neurons of FUS-expressing flies. [Figure 14b] The results confirming glutathionylation by endogenous GstO2 are shown. [Figure 15a] The figure shows the results of confirming the larval crawling activity of flies expressing FUSP525L by GstO2. [Figure 15b] The figure shows the results of immunoblotting of brain extracts from flies expressing FUSP525L with GstO2. [Figure 15c] The results of a solubility analysis to confirm FUS aggregates in flies expressing FUSP525L induced by GstO2 are shown. [Figure 16a] To confirm the regulation of FUS-induced neurotoxicity by GSTO1, the human chromosome ortholog of GstO2, we investigated the expression of GSTO1 in a stable N2a cell line expressing a Myc-DDK-GSTO1 fusion protein. [Figure 16b]Solubility analysis to confirm FUS aggregation with GSTO1, the human chromosomal ortholog of GstO2, is shown. [Figure 16c] This shows the results of confirming the effect of GSTO1, the human homologue of GstO2, in reversing neuronal cell death. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The present invention will be described in detail below.

[0040] As a result of conducting various studies related to neurodegenerative diseases, the inventors discovered that FUS protein, which is known to be one of the causes of amyotrophic lateral sclerosis, is glutathionylated to form FUS protein aggregates. Through research into this, the inventors have completed the present invention as a diagnostic marker for amyotrophic lateral sclerosis.

[0041] Thus, the present invention provides a marker composition for diagnosing nervous system degenerative diseases comprising glutathionylated FUS protein, a composition for diagnosing nervous system degenerative diseases comprising a preparation for measuring the glutathionylation level of FUS protein, and a kit for diagnosing nervous system degenerative diseases comprising the composition.

[0042] The gene encoding the FUS protein according to the present invention may consist of the nucleotide sequence shown in SEQ ID NO: 1, or may consist of the amino acid sequence shown in SEQ ID NO: 2. In this case, it may also include a nucleotide sequence having a sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more with the nucleotide sequence shown in SEQ ID NO: 1.

[0043] The term "neurodegenerative diseases" as the target disease of the present invention collectively refers to diseases in which the death of nerve cells progresses in a part or various parts of the nervous system. The form of nerve cell death includes cell necrosis and cell apoptosis. Preferably, the disease may include Alzheimer's disease, mild cognitive impairment, stroke, vascular dementia, frontotemporal dementia, dementia with Lewy bodies, Creutzfeldt-Jakob disease, traumatic head injury, syphilis, acquired immune deficiency syndrome, other viral infections, brain abscess, brain tumor, multiple sclerosis, Parkinson's disease, Huntington's disease, Pick's disease, amyotrophic lateral sclerosis, epilepsy, ischemia, and stroke, and more preferably, amyotrophic lateral sclerosis, but is not limited thereto.

[0044] In the present invention, the "FUS (Fused in Sarcoma) protein," which is known to be one of the causes of the onset of neurodegenerative diseases, is an RNA and DNA binding protein, and the N-terminus of FUS is known to be associated with transcriptional activity, while the C-terminus is involved in protein and RNA binding.

[0045] The term "glutathionylation" as used herein refers to the formation of a disulfide bond between cysteine ​​and the reduced form of glutathione (GSH). Glutathionylation of proteins induces changes in the structure and function of proteins.

[0046] As described above, the formation of FUS protein aggregates due to glutathionylation of FUS protein, which is known to be an amyotrophic lateral sclerosis-inducing protein, and the deposition of the aggregates in the cytoplasm are the causes of amyotrophic lateral sclerosis. In one embodiment of the present invention, a Western blot analysis experiment using an anti-GSH antibody and an anti-myc antibody was performed to confirm that glutathionylation of FUS protein occurs in vitro depending on the concentration of oxidized glutathione (Glutathione disulfide, GSSH) (see Example 2), and an experiment was performed to confirm whether glutathionylation of FUS protein occurs in vivo, and glutathionylated FUS protein aggregates were confirmed in the cytoplasm of neurons (see Example 3).

[0047] In another embodiment of the present invention, the human FUS protein and the mutant FUS protein are expressed in Neuron2a (N2a). P525L Protein expression experiments were performed to express human wild-type FUS protein and mutant FUS P525L It was confirmed that glutathionylation of proteins occurs identically in mammalian systems (see Example 4).

[0048] In yet another embodiment of the present invention, MALDI-mass spectrometry and sequence analysis of the RanBP2 zinc-finger domain were performed to confirm that glutathioneylation by oxidized glutathione (Glutathione disulfide, GSSH) occurs at Cys-447 of the RanBP2 zinc-finger domain in the FUS protein (see Example 5).

[0049] In yet another embodiment of the present invention, a solubility analysis of glutathionylated FUS protein and a 3D homology model analysis experiment of the RanBP2 zinc-finger domain of FUS protein were performed to confirm that glutathionylation of FUS protein induces aggregate formation of the protein (see Example 6).

[0050] The above results indicate that glutathionylation occurs at Cys-447 in the RanBP2 zinc-finger domain in the FUS protein, resulting in the formation of glutathionylated FUS protein aggregates. It was confirmed from Figures 2 to 4 that the above aggregates are poorly soluble and are deposited in the cytoplasm.

[0051] The term "diagnosis" as used in the present invention means, in a broad sense, the determination of all aspects of the actual condition of a patient's illness. The contents of the determination include the name of the illness, the cause of the illness, the type of illness, the severity, detailed conditions of the hospital bed, and the presence or absence of complications. In the present invention, diagnosis refers to determining the presence or absence of the onset of a nervous system degenerative disease and the level of progression.

[0052] In another aspect of the present invention, there is provided a method for providing information for diagnosing neurodegenerative diseases, comprising the steps of determining the glutathionylation level of FUS protein from a biological sample derived from a subject and comparing said level with the glutathionylation level of said protein in a normal control sample.

[0053] The term "method of providing information for diagnosing a nervous system degenerative disease" used in the present invention refers to providing objective basic information necessary for diagnosing a nervous system degenerative disease as a preliminary step for diagnosis or prognosis prediction, excluding a doctor's clinical judgment or findings. The biological sample derived from the subject may be, for example, a tissue, a cell, etc., but is not limited thereto.

[0054] In addition, the inventors have conducted various studies related to the treatment of neurodegenerative diseases and have confirmed that the formation of protein aggregates due to glutathionylation of FUS protein, which is known to be an amyotrophic lateral sclerosis-inducing protein, and its deposition in the cytoplasm of brain neurons are the causes of amyotrophic lateral sclerosis. They have also confirmed the glutathionylation inhibitory activity of FUS protein by GSTO (omega class glutathione transferase), thereby completing the present invention.

[0055] Accordingly, the present invention provides a pharmaceutical composition for preventing or treating nervous system degenerative diseases, comprising the GSTO1 (omega class glutathione transferase 1) or GstO2 (omega class glutathione transferase 2) gene or a protein encoded by the gene as an active ingredient.

[0056] The GSTO1 (omega class glutathione transferase 1) gene according to the present invention may comprise the nucleotide sequence represented by SEQ ID NO: 2 (Human GSTO1 NCBI Accession: NM_004832.2) or the amino acid sequence represented by SEQ ID NO: 3 (Human GSTO1 NCBI Accession: NP_004823.1). In this case, it may comprise a nucleotide sequence having sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more to the nucleotide sequence represented by SEQ ID NO: 2.

[0057] Furthermore, the GstO2 (omega class glutathione transferase 2) gene according to the present invention may consist of the nucleotide sequence shown in SEQ ID NO: 4 (Drosophila GstO2 NCBI Accession: NM_168277.2) or the amino acid sequence shown in SEQ ID NO: 5 (Drosophila GstO2 NCBI Accession: NP_729388.1). In this case, it may include a nucleotide sequence having sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more to the nucleotide sequence shown in SEQ ID NO: 4.

[0058] The term "glutathionylation" as used in the present invention is the result of disulfide bonds between cysteine ​​and reduced glutathione (GSH), which is known to be capable of leading to changes in protein structure and function.

[0059] As described above, the present invention confirmed that the formation of protein aggregates due to glutathionylation of FUS protein, known as an amyotrophic lateral sclerosis-inducing protein, and its deposition in the cytoplasm of brain neurons are the causes of amyotrophic lateral sclerosis. Accordingly, in one embodiment of the present invention, FUS was cultured in the presence of GSSG, and it was confirmed that the amount of glutathionylated FUS increased as the amount of GSH increased. It was also confirmed that FUS was glutathionylated at Cys-447 (see Example 7), and the formation of FUS aggregates due to glutathionylation of FUS was specifically confirmed (see Example 8).

[0060] In another embodiment of the present invention, we identified GstO as a protein that can regulate the pathology caused by glutathionylated FUS, and confirmed the alleviating effect of GstO2 on the phenotypic defects and mitochondrial fission and dysfunction caused by FUS overexpression in Drosophila (see Examples 9 and 10).

[0061] In yet another embodiment of the present invention, we not only confirmed the inhibitory effect of GstO2 on the formation of FUS aggregates in Drosophila neurons, but also specifically confirmed whether GstO2 regulates the glutathionylation of FUS in Drosophila neurons (see Examples 11 and 12). We also confirmed that GstO2 inhibits the formation of FUS aggregates in Drosophila neurons (see Examples 12 and 13). P525L It was also confirmed that overexpression of GstO2 had the same effect as overexpression of FUS on flies that exhibited an ALS phenotype due to expression of FUS (see Example 13). Finally, to confirm whether the recovery effect of GstO2 against FUS-induced neuronal toxicity is also applicable to mammalian systems, the inventors confirmed the neuronal cell toxicity suppression effect on GSTO1, the human homologue of GstO2, and as a result, specifically confirmed the effect of improving FUS insolubility and FUS-induced cell death in a mammalian neuronal cell model of ALS (see Example 14).

[0062] The above process suggests that GSTO1 (omega class glutathione transferase 1) or GstO2 (omega class glutathione transferase 2) induces the deglutathionylation of FUS and suppresses FUS cytoplasmic aggregation and neurotoxicity, and may be useful as a therapeutic agent for neurodegenerative diseases.

[0063] The pharmaceutical composition according to the present invention includes the GSTO1 (omega class glutathione transferase 1) or GstO2 (omega class glutathione transferase 2) gene or a protein encoded by the gene as an active ingredient, and may also include a pharma- ceutical acceptable carrier. The pharma-ceutical acceptable carrier is one that is commonly used in formulation, and includes, but is not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, etc., and may further include other common additives such as antioxidants and buffers, if necessary. In addition, the composition may be formulated into an injectable dosage form such as an aqueous solution, suspension, emulsion, pills, capsules, granules, or tablets by additionally adding a diluent, dispersant, surfactant, binder, lubricant, etc. With regard to suitable pharma-ceutical acceptable carriers and formulation, each component may be suitably formulated using the method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in its dosage form, and can be formulated into an injection, inhalant, topical agent for skin application, or oral ingestible agent.

[0064] The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, or by application to the skin, nasal cavity, or airway) depending on the intended method, and the dosage varies depending on the condition and weight of the patient, the degree of disease, drug form, administration route, and time, but can be appropriately selected by those skilled in the art.

[0065] The composition according to the present invention is administered in a pharma- tically effective amount. In the present invention, the term "pharma- tically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level can be determined by factors including the type and severity of the patient's disease, the activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, duration of treatment, co-administered drugs, and other factors well known in the medical field. The composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or multiple times. It is important to administer an amount that can obtain the maximum effect at the minimum amount without side effects, taking into account all of the above factors, and this can be easily determined by one skilled in the art.

[0066] Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex, and weight of the patient, and may generally be administered at 0.001-150 mg, preferably 0.01-100 mg per kg of body weight every day or every other day, or may be administered in one to three divided doses per day. However, the dosage may increase or decrease depending on the route of administration, the severity of the neurodegenerative disease, sex, weight, age, etc., and therefore the above dosage does not limit the scope of the present invention in any way.

[0067] In another aspect, the present invention provides a method for preventing, regulating, or treating a nervous system degenerative disease, comprising administering the pharmaceutical composition to an individual.

[0068] The term "prevention" as used in the present invention means any action to suppress or delay the onset of a neurodegenerative disease by administering the pharmaceutical composition according to the present invention.

[0069] The term "treatment" as used in the present invention refers to any action that improves or favorably alters the symptoms of a neurodegenerative disease by administering a pharmaceutical composition according to the present invention.

[0070] In the present invention, the term "individual" refers to a subject in need of a method for preventing, regulating or treating a disease, and more specifically refers to mammals such as human or non-human primates, mice, rats, dogs, cats, horses and cows.

[0071] In the following, preferred examples are presented to aid in understanding the present invention. However, the following examples are merely provided to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples.

[0072] [Example] Example 1. Experimental preparation and method 1-1. Preparation of Drosophila melanogaster All Drosophila stocks were kept under standard food conditions, normal temperature (25°C) and normal humidity conditions (60%). Drosophila crossings were performed according to standard procedures and all progeny were reared at 25°C.

[0073] 1-2.Cell culture Mouse neuroblastoma Neuro2a cells were cultured in DMEM (Life Technologies) containing 10% fetal bovine serum (FBS) (Gibco) and penicillin-streptomycin (50 mg / ml) (Gibco) solution at 37°C in 5% CO2 / 95% air.

[0074] 1-3.In vitro glutathionylation assay Recombinant full-length myc-tagged human FUS protein (0.51 μg) purified from HEK293 (OriGene) cells was incubated in 50 mM Tris-HCl (pH 7.5) in the presence of various concentrations of oxidized glutathione (GSSG) at 25°C. After 1 hour of incubation, the samples were placed on ice and 3× non-reducing LDS sample buffer (Invitrogen) was added. The samples were then separated by 12% SDS-PAGE and subjected to Western blot analysis using mouse anti-GSH (1:1,000; ViroGen Corp.) and mouse anti-myc (1:1,000; Millipore) antibodies.

[0075] 1-4. Transgenic flies UAS-FUS, UAS-FUS P525L Cell lines were obtained from Nancy M. Bonini (University of Pennsylvania), and the UAS-GstO2 cell line was obtained as previously described ( Kim et al., 2012 ; Kim and Yim, 2013 ). In addition, UAS-GSTO1 RNAi (BL34727, v26711), UAS-GstO2 RNAi (v109255), and UAS-GstO3 RNAi (v105274) cell lines were obtained from the Bloomington Drosophila stock center and the Vienna Drosophila RNAi center, UAS-mitoGFP cell lines were obtained from HJ Bellen (Baylor College of Medicine), and pan-neuronal driver, elav-Gal4, muscle-specific driver, mhc-Gal4, motor neuron-specific driver, and D42-Gal4 cell lines were also obtained from the Bloomington Drosophila stock center and the Vienna Drosophila RNAi center. In this case, W1118 flies were used as a control group due to genetic background technology.

[0076] 1-5.Transfection N2a cells were plated in 6-well plates, and 4 μg of pCMV6-FUS-GFP (green fluorescent protein)-labeled human wild-type FUS and pCMV6-FUS were added to each well. P525L -GFP-tagged mutant FUS P525L were transfected using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer's instructions, with blank pCMV6-AC-GFP plasmid used as a negative control.

[0077] 1-6.Stable cell line generation N2a cells in a 24-well plate were transfected with 1 μg of human GSTO1 cDNA using Lipofectamine 3000 reagent (Invitrogen), and stable transformants were selected in the presence of G418 (600 μg / ml) for 2 days after transfection. Overexpression of GSTO1 protein in the stable transformants was confirmed by immunoblot analysis.

[0078] 1-7. Whole brain immunostaining The adult brains of 7-day-old male flies were fixed with 4% formaldehyde in a fixing buffer (100 mM PIPES, 1 mM EGTA, 1% Triton X-100, 2 mM MgSO4, pH 6.9), blocked with a washing buffer solution (50 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100 and 0.5 mg / ml BSA, pH 6.8) containing 10 mg / ml BSA, and then incubated with a primary antibody diluted in a blocking buffer at 4°C for 12 hours. The antibodies used were as follows: rabbit anti-FUS (1:100, Bethyl Laboratories), mouse anti-FUS (1:50, Santa Cruz Biotechnology), rabbit anti-GstO2 (1:20), and mouse anti-GSH.

[0079] Afterwards, the samples were incubated with Alexa 488-conjugated secondary antibodies (1:200, Invitrogen), Cy3-conjugated secondary antibodies (1:200, Jackson Immuno Research Laboratories), and DAPI (1:500, Sigma-Aldrich), and then the brains were washed three times for 10 min each with washing buffer and then subjected to SlowFade. TM The sections were fixed with Gold antifade reagent (Invitrogen), and all images were captured using a Carl Zeiss confocal microscope (LSM710).

[0080] 1-8.Immunohistochemistry The cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 minutes, and then washed three times for 10 minutes with PBS containing 0.3% Triton X-100 (PBST). The cells were then incubated with blocking buffer (PBST containing 5% NGS (normal goat serum)) at 25°C for 1 hour, and then incubated with primary antibodies diluted in blocking buffer at 4°C for 12 hours. The antibodies used were as follows: mouse anti-GSH (1:100; ViroGen Corp.), rabbit anti-FUS (1:100; Bethyl Laboratories), mouse anti-GFP (1:200; Roche), and rabbit anti-cleaved caspase 3 (1:500, Cell Signaling).

[0081] After incubation with the primary antibody, the cells were washed three times with PBST for 10 minutes each, and then incubated with secondary antibodies diluted 1:2000 in PBST at 25°C for 1 hour. The secondary antibodies used were as follows: Alexa-594 conjugated goat anti-rabbit IgG, Alexa-488 conjugated goat anti-rabbit IgG, Alexa 594 conjugated goat anti-mouse IgM and Alexa-488 conjugated goat anti-mouse IgG (Jackson Immuno Research laboratory).

[0082] The samples were then observed using a Leica confocal microscope.

[0083] Next, for NMJ (neuromuscular junction) analysis in Drosophila, 3rd instar larvae were dissected and fixed in 4% formaldehyde in PBS for 15 min. Samples were then washed three times for 10 min each with PBS containing 0.1% Triton X-100, blocked in PBST with 5% BSA, and incubated with primary antibodies for 12 h at 4°C. FITC-conjugated anti-HRP (Jackson Immuno Research Laboratories) was used at 1:150 and SlowFade staining was performed. TM The sections were fixed with Gold antifade reagent (Invitrogen), and all images were acquired with a Leica TCS SP5 AOBS confocal microscope.

[0084] 1-9.Homology modeling The 3D structure of the FUS ZnF domain (32 amino acid residues, 422-RAGDWKCPNPTCENMNFSWRNECNQCKAPKPD-453) was predicted and generated for protein structure and function prediction using the I-TASSER server based on Molegro Molecular Viewer 2.5.0 (Molegro ApS, Aarhus C, Denmark).

[0085] 1-10.In vitro protein aggregation assay After in vitro glutathionylation, samples were heat blocked at 37°C and then centrifuged at 20,000 × g for 30 min at 4°C to separate the supernatant and pellet fractions. The pellets were washed five times for 10 min each with 50 mM Tris-HCl (pH 7.5) and then dissolved in LDS sample buffer (2% SDS) (Invitrogen) with a reducing agent. FUS was detected in both fractions by Western blot analysis using rabbit anti-FUS (1:1000; Bethyl Laboratories) antibody.

[0086] 1-11.External eye microscopy For imaging of adult Drosophila eyes, the heads were fixed on a slide and images were taken with a digital camera. Five-day-old male Drosophila were used in the experiment. Images were taken with a Leica MZ10 F stereo microscope and a Leica DFC450 camera system.

[0087] 1-12.Locomotive activity and lifespan assays For larval crawling analysis, 3rd instar larvae were washed with PBS to remove any remaining food debris, dried on clean filter paper, and placed on a 2% grape juice-agar petri dish and allowed to crawl for 90 seconds. To quantify the crawling behavior of the larvae, Image J software was used to track the larvae and measure the distance, and the results for at least 10 larvae were averaged for each transgenic line.

[0088] Next, for the climbing analysis, 10 male Drosophila of each age group were anesthetized with carbon dioxide and placed in a column vial, then the Drosophila were transferred to an empty vial and cultured at room temperature for 1 hour to acclimate to the environment, and the number of Drosophila that climbed to the top of the vial within 10 seconds was counted. At this time, the experiment was repeated four times independently for each transgenic line at 5-minute intervals, and all climbing experiments were performed at 25°C.

[0089] Finally, for lifespan analysis, 20 male Drosophila of each genotype (>150 Drosophila) were placed in different vials and maintained at 25°C, and the next day, all groups were transferred to new vials, after which the number of dead Drosophila was recorded.

[0090] 1-13.Immunoblot analysis Protein extracts for Western blot analysis were prepared by homogenizing ten 14-day-old male Drosophila heads in LDS sample buffer (Invitrogen), and total protein extracts were separated using 4%-12% gradient SDS-PAGE gels and transferred to PVDF membranes (Millipore). The membranes were then blocked for 1 h with Tris-buffered saline (TBS) containing 4% nonfat dry milk or 4% bovine serum albumin (BSA) and incubated with primary antibodies for 12 h at 4°C.

[0091] The primary antibodies used were as follows: Rabbit anti-FUS (1:1000, Bethyl Laboratories), rabbit anti-Drosophila Marf (1:1000, a gift from Leo Pallanck, University of Washington), mouse anti-Opa1 (1:1000; mouse anti-UQCRC2 (1:1000, Abcam), mouse anti-ATP5A (1:10000, Abcam), mouse anti-Drosophila GstO2 (1:1000), rabbit anti-lamin C (Developmental Studies Hybrodoma Bank, DSHB), rabbit anti-α-tubulin (1:2000, Sigma) and rabbit anti-β-actin (1:4000, Cell Signaling).

[0092] Blots were washed with TBS containing 0.1% Tween-20 (TBST) and incubated with secondary antibodies, and the primary antibodies were detected with HRP-conjugated secondary antibodies using goat anti-rabbit IgG HRP conjugate and goat anti-mouse IgG HRP conjugate (1:2000, Millipore) using the ECL-Plus kit (Amersham).

[0093] Protein extracts were then homogenized in RIPA buffer (Cell Signaling) containing a protease-phosphatase inhibitor cocktail (Roche) and mixed with LDS sample buffer (Invitrogen) with a reducing agent. Protein samples were then separated on 4%-12% Bis-Tris gels (Novex) and transferred to PVDF membranes (Novex) for Western blot analysis using rabbit anti-TurboGFP (1:2000, OriGene), mouse anti-GSTO1 (1:1000, Proteintech), rabbit anti-DDK (1:1000, OriGene), and rabbit anti-α-tubulin (1:2000, Sigma) primary antibodies.

[0094] 1-14. Mitochondrial image and morphology To image mitochondria in thoracic muscle tissue, 7-day-old adult male Drosophila were dissected in PBS and fixed with 3% formaldehyde in fixation buffer (100 mM PIPES, 1 mM EGTA, 1% Triton X-100, and 2 mM MgSO4, pH 6.9) for 25 min, then washed with wash buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100, and 0.5 mg / ml BSA, pH 6.8). TM The sections were fixed with Gold antifade reagent (Invitrogen) and images were acquired using a Carl Zeiss confocal microscope (LSM710).

[0095] Next, to confirm the image of mitochondria in leg motor neurons, 7-day-old adult male Drosophila were dissected in PBS, and the forelegs were fixed in 4% formaldehyde in fixation buffer (100 mM PIPES, 1 mM EGTA, 1% Triton X-100 and 2 mM MgSO4, pH 6.9) for 25 min, then washed with PBST and SlowFade. TM The sections were fixed with Gold antifade reagent (Invitrogen) and images were acquired using a Carl Zeiss confocal microscope (LSM710).

[0096] 1-15.BN-PAGE(Blue native polyacrylamide gel electrophoresis) Mitochondria were isolated from 14-day-old male adult Drosophila using a mitochondrial isolation kit (Pierce) according to the manufacturer's protocol, and the purified mitochondrial extract was resuspended in 60 μL of 1× Native PAGE sample buffer (Invitrogen) containing 2% n-dodecyl-β-D-maltoside (DDM), 1% Digitonin, and a protease inhibitor (Halt). The sample was then incubated on ice for 15 min and centrifuged at 12,000×g, after which the concentration of mitochondrial protein was measured in the supernatant. The supernatant (20 μg) was mixed with 0.5% G-250 sample additive and then subjected to BN-PAGE (Blue native polyacrylamide gel electrophoresis) using a 3%-12% Native PAGE Bis-Tris gel (Invitrogen) at 4°C. In this case, anode running buffer was used outside the gel and cathode running buffer was used inside the gel. After PAGE, Western blot analysis was performed using mouse anti-NDUFS3 (1:5000, Abcam), mouse anti-UQCRC2 (1:1000, Abcam), and mouse anti-ATP5A (1:10000, Abcam) antibodies.

[0097] 1-16. Mitochondrial superoxide measurement Mitochondrial ROS generation in Drosophila was measured using the mitochondrial oxygen free radical indicator mitoSOX-Red (Invitrogen) according to the manufacturer's protocol. More specifically, muscle tissues dissected from the thorax of 11-day-old Drosophila in cold PBS were incubated with 5 μM MitoSOX-Red in DMSO at 25°C for 20 min. After washing three times with cold PBS, the muscle tissue samples were rapidly subjected to SlowFade. TMAfter treatment with Gold antifade (Invitrogen) reagent and fixation, the cells were observed within 15 minutes using a Carl Zeiss confocal microscope (LSM710), and the fluorescence intensity was quantified using Image J software.

[0098] 1-17.ATP analysis (ATP assay) The thorax of 28-day-old Drosophila was homogenized in 100 μl of extraction buffer (6 M Guanidine-HCl, 100 mM Tris, 4 mM EDTA, pH 7.8) to inhibit ATPase enzyme activity, and the extract was immediately frozen in liquid nitrogen and heated to denature ATP synthase. The sample was then centrifuged at 20,000 × g for 15 minutes, the supernatant was transferred to a new tube, and diluted with extraction buffer (1 / 100). The sample was then placed in each well of a 96-well plate, mixed with the luminescence solution of the Enliten ATP Assay Kit (Promega), and luminescence was measured at 10-second intervals using a Glomax microplate reader (Promega). The supernatant was then diluted with extraction buffer (1 / 2) and protein concentration was measured using a BCA protein assay kit (Pierce). The relative ATP level compared to the standard was then calculated by dividing by the total protein concentration.

[0099] 1-18. Protein oxidation assay Protein oxidation was detected using an OxyBlot protein oxidation detection kit (Millipore) according to the manufacturer's protocol. Specifically, the thorax of 28-day-old Drosophila was homogenized in a dissolution buffer containing 6% SDS and 2% β-mercaptoethanol, and the homogenate was centrifuged at 20,000×g for 30 minutes to discard the pellet debris. The denatured protein was derivatized with DNPH (2,4-dinitrophenylhydrazine) at 25°C and neutralized using a neutralization solution. The DNPH-labeled protein was then applied to SDS-PAGE and transferred to a PVDF membrane (Millipore), and the membrane was analyzed with an anti-DNP antibody (1:150, Millipore), and the signal was detected using a goat anti-rabbit IgG HRP-conjugated secondary antibody (1:300; Millipore). Detection was performed using an ECL-Plus kit (Amersham), and band density was measured using Image J software.

[0100] 1-19.Nuclear / cytoplasmic fractionation assay Twenty male Drosophila heads were lysed with nuclear extract kit (Active Motif) reagent according to the manufacturer's protocol, and the protein extracts from different cell fractions were mixed with SDS loading buffer, heated, and applied to SDS-PAGE for Western blot analysis.

[0101] 1-20. Protein solubility assay Total protein was fractionated by solubility using a modified version of a previously described protocol (Woo et al., 2017). Twenty heads from 7- or 14-day-old Drosophila were homogenized in lysis buffer without SDS (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 0.1% NP-40, and 10% glycerol, pH 7.5). The homogenized samples were then centrifuged at 100,000 × g for 30 min at 4 °C, and the supernatant was collected as the soluble fraction. The remaining pellet was further extracted with 50 μl 2 × buffer containing 2% SDS, sonicated, and heated at 95 °C for 10 min. The supernatant from the above process was collected as the insoluble fraction.

[0102] 1-21.GSH / GSSG content The method for measuring oxidized (glutathione sulfide, GSSG) and reduced glutathione (GSH) was measured using a glutathione analysis kit (Cayman Chemical) according to the manufacturer's protocol. More specifically, the total glutathione from 10 heads of 10-day-old Drosophila melanogaster was homogenized in 50 μl of 50 mM MES buffer, the sample was centrifuged at 10,000 × g for 15 minutes, and the supernatant was transferred to a new tube. The same amount of MPA reagent was then added to the sample and left at room temperature for 5 minutes, the mixture was centrifuged at 2,000 × g for 2 minutes, and 2.5 μl of TEAM reagent was added and the sample was vortexed immediately. Next, the sample was placed in each well of a 96-well plate, and a mixture of MES buffer, cofactor mixture, enzyme mixture, and DTNB was added. Then, the absorbance at 405 nm was measured using a Glomax microplate reader (Promega), and 2.5 μl of TEAM reagent was added, and oxidized glutathione (GSSG) was measured using the same method as the total glutathione assay. 2-vinylpyridine was also added, and the cells were incubated at 25°C for 1 hour. The same process as the total glutathione assay was carried out, and the supernatant was diluted with extraction buffer (1 / 10) and the protein concentration was measured using a BCA protein assay kit (Pierce). The calculated levels of oxidized glutathione (GSSG) and reduced glutathione (GSH) were divided by the total protein concentration to obtain the calculated values ​​for the levels of oxidized glutathione (GSSG) and reduced glutathione (GSH) compared to the standard levels.

[0103] Example 2. Confirmation of in vitro glutathionylation of FUS protein In this example, to confirm whether glutathionylation of FUS protein occurs in vitro, myc-tagged human FUS recombinant full-length protein was incubated with oxidized glutathione (Glutathione disulfide, GSSH), separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and Western blot analysis was performed using mouse anti-GSH antibody and mouse anti-myc antibody.

[0104] As a result, as shown in Figure 1a and Figure 1b, it was confirmed that the greater the concentration of oxidized glutathione (Glutathione disulfide, GSSH) (0mM, 0.25mM, 0.05mM, 1mM), the greater the amount of glutathionylated FUS protein detected. Inferred from the above results, it was confirmed that glutathionylation of FUS protein occurs in vitro in a manner dependent on the concentration of oxidized glutathione (Glutathione disulfide, GSSH).

[0105] Example 3. Confirmation of in vivo glutathionylation of FUS protein In this example, to confirm whether glutathionylation of FUS protein occurs in vivo, human FUS protein, which is specifically expressed in neurons, was expressed in Drosophila neurons using the elav-Gal4 driver. Specifically, Drosophila was transfected with human wild-type FUS protein tagged with pCMV6-FUS-GFP. The area stained with DAPI indicates the location of the nucleus.

[0106] As a result, human wild-type FUS protein was mainly located in the nucleus in Drosophila brain neurons, but when human wild-type FUS protein was overexpressed, human wild-type FUS protein aggregates were observed in large amounts in the cytoplasm (green) as shown by the arrow in Figure 2a. In addition, reduced glutathione (GSH) was observed in the cytoplasm (red) as shown by the arrow in Figure 2b. In addition, FUS protein aggregates in the cytoplasm of brain neurons were observed in the same location as reduced glutathione (GSH) as shown by the arrow in Figure 2d, which is a merger of Figures 2a and 2b. The above results indicate that human wild-type FUS protein glutathionylated by oxidized glutathione (Glutathione disulfide, GSSH) and reduced glutathione (GSH) generated by the glutathionylation exist together in the cytoplasm in vivo.

[0107] Example 4. In vitro glutathionylation of FUS protein in mammalian systems In this example, human wild-type FUS protein and human mutant FUS P525L To determine whether protein glutathionylation also occurs in mammalian systems, we cultured human wild-type and human mutant FUS proteins in the mouse neuroblastoma cell line Neuron2a (N2a). P525L Proteins were expressed (transfected). The DAPI stained sections indicate the location of the nuclei.

[0108] As a result, as shown by the arrow in Figure 3a, it was confirmed that human wild-type FUS protein aggregates were observed in large amounts in the cytoplasm (green). In addition, as shown by the arrow in Figure 3b, it was confirmed that reduced glutathione (GSH) was observed in the cytoplasm (red). As shown by the arrow in Figure 3d, which is a merger of Figures 3a and 3b, it was confirmed that the positive signal for reduced glutathione (GSH) was mainly detected in the cytoplasm where human wild-type FUS protein aggregates existed. In addition, as shown by the arrow in Figure 4a, it was confirmed that human mutant FUS P525LIt was confirmed that protein aggregates were observed in large amounts in the cytoplasm (green). In addition, as shown by the arrow in Figure 4b, reduced glutathione (GSH) was observed in the cytoplasm (red). As shown by the arrow in Figure 4d, which is a merger of Figures 4a and 4b, the positive signal for reduced glutathione (GSH) was observed in the human mutant FUS P525L The glutathionylation of human FUS protein in Drosophila was confirmed to occur in mammalian systems.

[0109] Example 5. Localization of glutathionylation of FUS protein 5-1. Confirmation of glutathionylation site of FUS protein using mass spectrometry In this example, to confirm the site of glutathionylation of human FUS protein I, glutathionylation of myc-tagged human FUS protein was induced in vitro, and glutathionylated human FUS protein was detected using a Coomassie stained gel. The human FUS protein band was then excised from the gel, digested with trypsin, and analyzed by MALDI (Matrix Assisted Laser Desorption / Ionization)-mass spectrometry.

[0110] As a result, an amino acid polymer (peptide) with a mass difference of 305 Da was detected by mass spectrometry as shown in Figure 5. This corresponds to a reduced form of glutathione (GSH moiety), and by combining the MALDI-mass spectrometry graph in Figure 5, it was confirmed that glutathionylation of human FUS protein occurs at the Cys-447 site of the zinc-finger domain of RanBP2, as shown by the arrow in Figure 6.

[0111] 5-2. Confirmation of the conservation of the RanBP2 zinc-finger domain sequence in FUS proteins among species The RanBP2 zinc-finger domain of the FUS protein contains four cysteines. To confirm whether the sequence of the cysteines in the RanBP2 zinc-finger domain is conserved in eukaryotes, the sequences of the RanBP2 zinc-finger domains from D. melanogaster, X. laevis, D. rerio, M. musculus, and H. sapiens were analyzed.

[0112] As a result, as shown in Figure 12, it was confirmed that all four cysteines in the RanBP2 zinc-finger domain are conserved in Drosophila melanogaster, Xenopus laevis, D. rerio, M. musculus, and humans.

[0113] Example 6. Confirmation of aggregate formation by glutathionylation of FUS protein 6-1. Solubility analysis of glutathionylated FUS As confirmed in Examples 1 to 3, glutathionylated human FUS protein aggregates were observed in the cytoplasm of Drosophila brain neurons, and as confirmed in Example 5, glutathionylation occurred at Cys-447, the RanBP2 zinc-finger domain in the FUS protein. Based on the above examples, an experiment was carried out to measure the solubility of glutathionylated FUS protein to confirm whether glutathionylation of FUS protein affects the formation of FUS protein aggregates. Specifically, as shown in FIG. 7, oxidized glutathione (Glutathione disulfide, GSSH) was added to myc-tagged human FUS protein purified from HEK293 cells to induce glutathionylation of human FUS protein, and then the cells were exposed to heat-stress, which is a proteotoxic stress. The solubility of glutathionylated human FUS protein was measured at different time points through Western blot analysis by quantifying the human FUS protein in the supernatant and pellet fractions of each sample.

[0114] As a result, as shown in FIG. 8, it was confirmed that the soluble fraction of human FUS protein without the addition of oxidized glutathione (Glutathione disulfide, GSSH) maintained a high level of solubility for 2 hours at 37°C.

[0115] In addition, when oxidized glutathione (Glutathione disulfide, GSSH) was added and the solubility of the soluble fraction of glutathionylated human FUS protein was measured under the above conditions, it was found that the degree of solubility rapidly decreased from 90% to 20%.

[0116] The above results together with the results of Example 5 confirmed that glutathionylation of FUS protein induces the formation of FUS protein aggregates.

[0117] 6-2. 3D homology model analysis of the RanBP2 zinc-finger domain of FUS To obtain a structural basis for the relative position of the Cys-447 residue, a three-dimensional homology model of the RanBP2 zinc-finger domain in human FUS protein was generated using the I-TASSER server, and the RanBP2 zinc-finger domain model was used to search for proteins with structural homology regions in the protein database bank (PDB).

[0118] As a result, we confirmed that the Cys-447 residue in the RanBP2 zinc-finger domain is exposed to the surface due to oxidative transformation, as shown in Figure 9. Based on the structure of the ectogene 3D homology model, we demonstrated the high susceptibility of Cys-447 in the glutathionylation of human FUS protein, and demonstrated the possibility of structural changes in the RanBP2 zinc-finger domain.

[0119] Example 7. Confirmation of glutathionylation of FUS It is known that glutathionylation is the result of a disulfide bond between cysteine ​​and reduced glutathione (GSH), which can lead to changes in protein structure and function.

[0120] In order to confirm whether glutathionylation of FUS occurs, we performed an in vitro glutathionylation assay, and as shown in Figure 10a, we confirmed that wild-type FUS protein was glutathionylated in the presence of various concentrations of oxidized glutathione (GSSG). In addition, we performed Western blot analysis of glutathionylation of FUS using anti-GSH and anti-myc antibodies, and confirmed that glutathionylated FUS was reduced to FUS when treated with reducing agents such as 2-mercaptoethanol or dithiothreitol, which cleave mixed disulfide bonds. In other words, we were able to confirm that glutathionylation of FUS protein occurs depending on the concentration of GSSG.

[0121] To confirm glutathionylation of FUS in vivo, human FUS, which is specifically expressed in neurons, was expressed in Drosophila neurons using the elav-Gal4 driver.

[0122] As a result, we were able to confirm that FUS protein was mainly restricted to the nuclei of neurons, but that cytoplasmic FUS aggregates were abundantly observed in neurons of FUS-overexpressing fly brains, as indicated by the white arrow in Figure 10b. Interestingly, we were also able to confirm that cytoplasmic and mislocalized FUS was co-localized with GSH in brain tissue, similar to the results shown in the in vitro study.

[0123] Next, FUS and its mutant form, FUS P252L To confirm whether glutathionylation of FUS would produce the same results in a mammalian system, we used mouse neuroblastoma cell line Neuron2a (N2a) to express human FUS and FUS P525L As a result, as shown in Fig. 10c, GSH-positive signals were observed in the presence of FUS or FUS P525L It was confirmed that the expression was restricted to aggregates.

[0124] Therefore, FUS and FUS P525L It was confirmed that glutathionylation occurred both in vitro and in vivo.

[0125] Finally, to identify the glutathionylation site, myc-tagged human FUS was induced to undergo glutathionylation in vitro and the bands were detected on a Coomassie stained gel. The FUS protein bands were then excised from the gel, digested with trypsin, and analyzed by MALDI-mass spectrometry.

[0126] As a result, a peptide with a mass difference of 305 Da, which indicates one GSH moiety, was detected by mass spectrometry, as shown in Figure 5. The RanBP2 zinc-finger (ZnF) domain of FUS has four cysteines and may be sensitive to oxidative stress in vivo.

[0127] In addition, we investigated the FUS sequence for cysteine ​​conservation in eukaryotes and confirmed that all four cysteines are well conserved from flies to humans. Among these, the glutathionylation site was identified in the RanBP2-type ZnF domain, Cys-447 (Figure 7, arrow).

[0128] Therefore, it was confirmed that human FUS is specifically glutathionylated at the Cys-447 residue.

[0129] Example 8. Confirmation of aggregate formation by glutathionylation of FUS Post-translational modification has been known to be an important mediator of pathogenic protein aggregation in various neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). We were able to confirm that cytoplasmic or mislocalized FUS protein is colocalized with GSH in Drosophila brain, as shown in Figure 1b. The above experimental background indicates that glutathionylation of Cys-477 in the ZnF domain of FUS may lead to the generation of FUS aggregates. Therefore, we predicted that cytoplasmic FUS aggregation may be regulated by FUS glutathionylation.

[0130] Thus, a solubility study was performed on FUS to determine whether FUS glutathionylation at the Cys-477 residue modulates FUS protein aggregation.

[0131] More specifically, purified myc-tagged human FUS protein was incubated with GSSG in HEK293 cells to induce glutathionylation, and then exposed to heat shock to impart proteotoxic stress (see FIG. 7).

[0132] FUS solubility was assessed by quantifying FUS protein in the supernatant and pellet fractions of each sample at different time points by Western blot analysis.

[0133] As a result, as shown in Figure 8, FUS protein without GSSG treatment remained highly soluble for 2 hours at 37°C, but after GSSG treatment to induce glutathionylation, the solubility of FUS protein in the soluble fraction rapidly decreased from 90% to 20% after 2 hours compared to FUS protein without GSSG treatment.

[0134] This indicates that glutathionylation of FUS significantly induces the formation of aggregates, proving the hypothesis that FUS glutathionylation at Cys-447 residue induces protein aggregation and supporting cysteine ​​glutathionylation as a critical determinant of FUS aggregate formation.

[0135] To further evaluate the structural basis for the relative position of the Cys-447 residue, a 3D homology model of the human FUS ZnF domain was generated using the I-TASSER server, and the FUS ZnF domain model was used to search for proteins with structural homology regions in the protein database bank (PDB).

[0136] As a result, as shown in Figure 9, it was confirmed that the Cys-447 residue in the ZnF domain was exposed to the surface for oxidative transformation, which demonstrated the high sensitivity of Cys-447 to glutathionylation and indicated the possibility of a structural change in the ZnF domain.

[0137] Example 9. Confirmation of the inhibitory effect of GstO2 on neuronal toxicity caused by FUS To reduce the pathology caused by glutathionylated FUS, we sought to identify the exact molecular mechanisms and proteins that could regulate the glutathionylation process and selected omega class glutathione transferase (GSTO) as a potential candidate.

[0138] Human GSTO1 has been previously reported to be capable of acting as a deglutathionylation enzyme, and we have previously reported that GstO2, the Drosophila homologue of human GSTO1, suppresses neurotoxicity by regulating glutathionylation of ATP synthase β subunit in a Drosophila PD model (Kim et al., 2012).

[0139] Based on the above background knowledge, we used Drosophila as a genetic tool to identify novel roles and regulators of glutathionylation in the pathogenesis of FUS-induced ALS. First, we sought to determine whether increasing GstO2 could alleviate the phenotype caused by overexpression of human FUS in Drosophila.

[0140] More specifically, to assess the functional relationship between GstO2 and FUS in Drosophila, we generated transgenic Drosophila that overexpressed GstO2 and FUS inducible by the eye-specific Gal4, GMR-Gal4.

[0141] In addition, a recent study using Drosophila genetic approaches has shown that FUS expression in the adult eye is known to result in a rough eye phenotype. Similar to previous reports, we confirmed that FUS expression results in a rough eye phenotype and ruptures ommatidial tissues.

[0142] However, co-expression of FUS and GstO2 significantly rescued the rough eye phenotype, and no changes in the burst eyes were found in the single GstO2-expressing flies (Fig. 11a).The above results indicate that GstO2 genetically interacts with FUS in Drosophila.

[0143] Next, to confirm the mechanism of the motility defect caused by FUS expression, we performed larval crawling experiments to investigate the motility of larvae expressing FUS in neurons using pan-neuronal Gal4 and elav-Gla4.

[0144] As a result, as shown in Figure 11b, Drosophila expressing FUS in neurons showed a significant decrease in motility compared to the control group, but when co-expressed with GstO2, it was confirmed that the crawling activity of the larvae was improved. On the other hand, it was confirmed that larvae expressing GstO2-knockdown FUS lost their crawling activity. It was also confirmed that overexpression or knockdown of GstO2 alone had no effect on the crawling activity of the larvae.

[0145] Based on the above results, the present inventors investigated the number of synaptic boutons at the neuron muscular junction (NMJ) to determine whether the larval motility defects were caused by defects at the NMJ.

[0146] As a result, as shown in Figure 11c, we confirmed that the total number of boutons was significantly reduced at the NMJ in flies expressing FUS under the regulation of elav-Gal4, and this phenotype was significantly restored by co-expression of GstO2. However, GstO2 alone had no effect in suppressing the reduction in bouton number, whereas flies expressing GstO2-knockdown FUS did not show a reduction in the number of boutons at the NMJ.

[0147] We also confirmed that expression of FUS in neurons significantly shortened lifespan, but co-expression of GstO2 restored lifespan, as shown in Fig. 11d, whereas GstO2-knockdown in FUS-expressing flies not only partially shortened lifespan, but was unaffected by GstO2 RNAi, as shown in Fig. 11e.

[0148] In addition, the negative geotaxis assay is an assay to evaluate dysfunction of the nervous system in various neurodegenerative disease research including ALS. When applied to the Drosophila model system according to the present invention, as shown in FIG 11f, it was confirmed that FUS-expressing flies showed significantly reduced climbing activity compared to age-matched controls, consistent with previous research, but that the climbing defect was significantly suppressed in the case of FUS-expressing flies co-expressing GstO2 in neurons. It was confirmed that overexpression of GstO2 alone did not affect climbing ability.

[0149] Therefore, the above data confirmed that FUS-induced neurotoxicity could be significantly suppressed or enhanced through regulation of GstO2 expression.

[0150] Example 10. Confirmation of suppression of FUS-induced mitochondrial dynamics and OXPHOS dysfunction by GstO2 It is known that abnormal mitochondria are consistently observed in animal models of ALS (Dal Canto and Gurney, 1994; Magrane et al., 2014). In addition, overexpression of mutant FUS in motor neurons caused mitochondrial fission (Tradewell et al., 2012), and expression of wild-type and mutant forms of FUS in NSC34 motor neuron cells reduced mitochondrial ATP production (Stoica et al., 2016). Even though mitochondrial dysfunction remains a common feature of ALS in various previous studies, it has not yet been demonstrated that dysfunction of mitochondrial dynamics and OXPHOS (oxidative phosphorylation) system is a major cause of ALS pathogenesis in FUS-induced proteinopathy. Accordingly, the inventors confirmed in previous studies that mitochondrial fission is enhanced in muscle or motor neurons of FUS-expressing flies, and that Marf instability caused by mitochondrial fusion protein causes imbalance of mitochondrial dynamics (Altanbyek et al., 2016).

[0151] Based on the above findings, we sought to determine whether GstO2 expression suppresses mitochondrial division in FUS-expressing flies and to this end, we characterized the effects of GstO2 in flies expressing FUS in the thoracic muscle.

[0152] To characterize the effects of GstO2, a FUS-expressing Drosophila line was crossed with a muscle-specific Gal4, Mhc-Gal4, and mitochondria were visualized by expressing mitochondria-targeted GFP (mitoGFP).

[0153] As a result, as shown in Figure 12a, the size of mitochondria in FUS-expressing flies was significantly smaller and fragmented compared to the control group, and no detectable changes in mitochondrial morphology were found in the GstO2 overexpression group alone, but the size of mitochondria was dramatically restored when GstO2 was co-expressed in FUS-expressing flies.

[0154] Next, to determine whether GstO2 restores mitochondrial morphology in motor neurons, we overexpressed GstO2 using the motor neuron-specific Gal4, D42-Gal4, together with mitoGFP.

[0155] As shown in Figure 12b, we confirmed that expression of FUS in leg motor neurons of adult flies increased mitochondrial fission, consistent with the restorative effect of GstO2 on fission mitochondria in muscle of FUS-expressing flies, but this phenotype was also suppressed by co-expression of GstO2.

[0156] We also investigated whether mitochondrial fission was due to the dysfunction of proteins that regulate mitochondrial dynamics. Having previously confirmed that reduced Marf expression in FUS-expressing flies induces excessive mitochondrial fission (Altanbyek et al., 2016), we assessed whether Marf expression was altered by GstO2.

[0157] As a result, as shown in FIG. 12c, it was confirmed that Marf expression was abundantly increased in FUS flies co-expressing GstO2, while Opa1 levels remained unchanged.

[0158] Next, we assessed the functional relevance of imbalanced mitochondrial dynamics in FUS-induced ALS to determine whether mitochondrial morphological changes upon FUS overexpression affect the OXPHOS system. More specifically, we analyzed the levels of various complex subunits in FUS-expressing flies, with the content of mitochondrial complexes in the ETC indirectly measured.

[0159] As a result, as shown in Figure 12d, FUS-expressing flies significantly reduced the levels of complex I subunit NDUFS3 and complex III subunit UQCRC2, but the α-subunit of complex V, ATP5A, showed almost no change. In addition, we were unable to measure the levels of Drosophila complexes II and IV subunits because we were unable to find an antibody that cross-reacted with either of these. Here, we were able to confirm that overexpression of GstO2 using FUS restored the levels of NUDFS3 and UQCRC2 to similar levels to the control group.

[0160] To further study complex assembly, we performed blue native gel electrophoresis (BN-PAGE) on mitochondria purified from adult fly thorax tissue.

[0161] As a result, as shown in Figure 12e, we were able to confirm that the levels of assembled complexes I and III were reduced in FUS-expressing flies by BN-PAGE, which was consistent with the effect of reducing the expression of complexes I and III subunits in FUS-expressing flies. Notably, we confirmed that FUS-induced disassembly of complexes I and III could also be alleviated by overexpression of GstO2, whereas the state of complex V assembly was unchanged in all mutant lines.

[0162] Next, to determine whether GstO2 could restore mitochondrial functionality, we measured mitochondrial production of reactive oxygen species (ROS) in FUS-expressing flies using mitoSOX.

[0163] As a result, as shown in FIG. 12f, it was confirmed that ROS production was increased in FUS-expressing flies and was restored by GstO2 expression.

[0164] In addition, mitochondria produce cellular energy in the form of ATP, and it was confirmed that ATP levels were significantly reduced in FUS-expressing flies compared to controls (FIG. 12g).

[0165] To determine whether GstO2 could attenuate the increase in oxidative stress in the cytoplasm, the amount of oxidized protein in the cytoplasm was measured.

[0166] As a result, as shown in FIG. 12h, we observed that oxidized proteins increased in FUS-expressing flies and confirmed that this was restored by GstO2 expression.

[0167] Therefore, it was confirmed that GstO2 could significantly prevent the intracytoplasmic oxidative stress and mitochondrial dynamics imbalance and dysfunction induced by FUS.

[0168] Example 11. Confirmation of the inhibitory effect of GstO2 on FUS aggregate formation in Drosophila neurons The above results confirm that GstO2 overexpression suppresses all defective phenotypes caused by FUS expression in Drosophila, suggesting that GstO2 can promote the reduction of FUS, a pathogenic factor, in neuronal cells.

[0169] Therefore, to confirm the inhibitory effect of GstO2 on FUS aggregate formation in Drosophila neurons, we performed immunoblotting of brain extracts from FUS-expressing adult flies. We found that co-expression of GstO2 did not affect FUS levels (see Figure 13a), whereas knockdown of GstO2 in FUS-expressing flies increased FUS protein, as shown in Figure 13b.

[0170] Next, to determine whether expression of GstO2 suppresses FUS mislocalization in brain tissue, we performed nuclear / cytoplasmic fraction analysis to measure FUS levels in the cytoplasm and nucleus.

[0171] As a result, as shown in Figure 13c, it was confirmed that co-expression of GstO2-FUS reduced FUS protein levels in the cytoplasmic fraction but not in the nuclear fraction, confirming that GstO2 may act as a suppressor of FUS toxicity by reducing FUS protein levels in the cytoplasm.

[0172] To further examine the effect of GstO2 on FUS aggregation in transgenic flies, fly heads of diverse genotypes were collected and lysed in modified lysis buffer, followed by separation into detergent-soluble and -insoluble fractions.

[0173] As a result, as shown in Figure 13d, we confirmed that GstO2 and FUS co-expressing flies significantly increased the amount of FUS in the soluble fraction and decreased the amount of FUS in the insoluble fraction. On the other hand, we confirmed that neuronal-specific knockdown of GstO2 induced FUS conversion from the soluble fraction to the insoluble fraction. In addition, we could see through quantification of soluble and insoluble FUS that the ratio of insoluble / soluble FUS was reduced to >40% in GstO2 co-expressing flies compared to FUS-expressing flies, whereas the ratio of insoluble / soluble FUS was greatly increased in GstO2 knockdown FUS-expressing flies.

[0174] On the other hand, as shown in FIG. 13e, knockdown of either GSTO1 or GstO3 alone was not sufficient to enhance FUS aggregation by immunoblotting.

[0175] Thus, we demonstrate that GstO2, a member of the GstO family, has a protective function in FUS-induced ALS by regulating FUS aggregate formation in the cytoplasm.

[0176] Increased cytoplasmic FUS expression is known to promote the binding of FUS to mitochondria and induce mitochondrial dysfunction ( Deng et al., 2015 ). To confirm whether GstO2 regulates mitochondrial FUS levels, we coexpressed FUS and GstO2 in fly muscle tissue and then examined FUS levels from purified mitochondria by Western blotting.

[0177] As a result, as shown in FIG. 13f, it was confirmed that mitochondrial FUS levels were significantly reduced in flies co-expressing GstO2.

[0178] Example 12. Confirmation of the regulation of glutathionylation of FUS in Drosophila neurons by GstO2 The phenotypic rescue of FUS-expressing flies by GstO2 may be associated with glutathionylation of FUS, and thus we hypothesized that GstO2 could regulate FUS glutathionylation and aggregation in the Drosophila brain. To confirm this, we performed the following experiment.

[0179] First, double immunofluorescence analysis of FUS-expressing adult fly brain tissue confirmed that FUS aggregates were colocalized with GSH in the cytoplasm, as shown in Figure 5a, and that coexpression of GstO2 suppressed the increase in FUS aggregate formation and reduced the glutathione-saturation of FUS. Next, we analyzed the glutathione-saturation-induced aggregation of cytoplasmic FUS with down-regulated GstO2 using RNAi. As a result, we confirmed that knockdown of GstO2 increased cytoplasmic FUS aggregates and glutathione-saturation.

[0180] From the above, it is predicted that deglutathionylation of FUS by GstO2 may be useful in suppressing FUS aggregate formation in the cytoplasm of neurons and delaying FUS-induced neurotoxicity.

[0181] Next, we investigated whether endogenous GstO2 interacts with FUS in neurons. As a result, as shown in Figure 5b, endogenous GstO2 localization was dispersed in the cytoplasm of Drosophila neurons, and endogenous GstO2 was colocalized with cytoplasmic FUS aggregates in FUS-expressing flies, confirming that GstO2 and FUS interact with each other, and inferring that GstO2 may play a role in FUS-induced ALS.

[0182] Example 13. GstO2-induced FUS in Drosophila P525L Confirmation of induced neurotoxicity and insoluble inhibition Previous studies have confirmed that FUS mutations are present in the majority of ALS patients (Mackenzie et al., 2010). P525L The FUS mutant showed cytoplasmic mislocalization and was associated with acute FUS-induced ALS (Sun et al., 2011), and both the FUS wild type and its variant, FUS P525L is known to exhibit the same phenotype in Drosophila, including rough eyes and reduced motility ( Chen et al., 2016 ; Jackel et al., 2015 ).

[0183] FUS in FIG. 10c according to one embodiment of the present invention P525L Based on the results of confirming glutathionylation in the cytoplasm of neuro2a, GstO2 is a FUS-dependent agonist in the Drosophila brain. P525L We anticipated that the expression of this gene could contribute to the phenotype, and therefore carried out the following experiment.

[0184] More specifically, GstO2 expression was associated with FUS P525L To determine whether elav-Gal4 could suppress the induced motor deficits, we transfected neurons with a mutant form of FUS, FUS P525L We then expressed FUS and performed an experiment to confirm larval motility. P525L The mutation was less toxic than wild-type FUS.

[0185] As a result, as shown in Figure 15a, crawling activity in larvae was reduced by about 40%, similar to that in FUS-expressing flies, and co-expression of GstO2 was confirmed to activate crawling in the opposite way. As shown in Figure 15b, GstO2 expression was significantly reduced by FUS in whole brain extracts of adult flies. P525L It had no effect on the level.

[0186] Next, FUS P525L We attempted to confirm the effect of GstO2 on the solubility of FUS, as shown in Fig. 15c. P525L -Insoluble / soluble FUS in GstO2-coexpressing flies compared to FUS-single expressing flies P525L The ratio of soluble and insoluble FUS was reduced by more than 50%. P525L This was proven through quantification.

[0187] Overall, our results suggest that GstO2 inhibits FUS through protein aggregate formation. P525L It was confirmed that this compound regulates the toxicity of

[0188] Example 14. Confirmation of regulation of FUS-induced neurotoxicity in mammals To determine whether the restorative effect of GstO2 against FUS-induced neuronal cell toxicity also applies to mammalian systems, we developed a stable N2a cell line expressing a Myc-DDK-GSTO1 fusion protein against GSTO1, the human homologue of GstO2 (see Figure 16a), and used it to transfect the GSTO1 stable cell line with GFP-tagged FUS and then investigated the effect of GSTO1 on FUS.

[0189] As a result, as shown in FIG. 16b, quantification of soluble and insoluble FUS confirmed that the ratio of insoluble / soluble FUS was reduced by more than 50% in GSTO1-coexpressing flies compared to FUS-expressing flies.

[0190] Next, based on previous findings that increased expression of FUS promotes cell death in mammalian neural cells ( Deng et al., 2015 ), we stained N2a cells with anti-cleaved caspase 3 antibody to investigate the effect of GSTO1 on FUS-induced cell death.

[0191] As a result, as shown in Figure 16c, we confirmed that cleaved caspase 3 signals were significantly increased in N2a cells expressing FUS-GFP compared to control cells, but when FUS was co-expressed with GSTO1, cleaved caspase 3 signals were reduced and the neuronal death phenotype was restored.

[0192] Thus, we could confirm that expression of GSTO1, the human homologue of GstO2, also ameliorates FUS insolubility and FUS-induced cell death in a mammalian neuronal model of ALS.

[0193] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified in other specific forms without changing the technical concept or essential features of the present invention. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not restrictive. [Industrial Applicability]

[0194] The marker composition for diagnosing neurodegenerative diseases, which contains the glutathionylated FUS protein of the present invention, enables early diagnosis of amyotrophic lateral sclerosis, and a pharmaceutical composition for preventing or treating neurodegenerative diseases, which contains the GSTO gene or a protein encoded by the gene as an active ingredient, can be used to treat amyotrophic lateral sclerosis.

Claims

1. A pharmaceutical composition for preventing or treating amyotrophic lateral sclerosis, comprising as an active ingredient a GSTO1 (omega class glutathione transferase 1) or GstO2 (omega class glutathione transferase 2) gene or a protein encoded by said gene, The GSTO1 gene comprises a base sequence represented by SEQ ID NO: 2 or a base sequence having 90% or more sequence homology to the base sequence represented by SEQ ID NO: 2; A pharmaceutical composition for therapeutic use, wherein the GstO2 gene comprises a base sequence represented by SEQ ID NO:4 or a base sequence having 90% or more sequence homology with the base sequence represented by SEQ ID NO:

4.

2. The pharmaceutical composition according to claim 1 , wherein the GSTO1 gene consists of the base sequence represented by SEQ ID NO:

2.

3. The pharmaceutical composition according to claim 1, characterized in that the GSTO1 protein consists of the amino acid sequence represented by SEQ ID NO:

3.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the GstO2 gene consists of the base sequence represented by SEQ ID NO:

4.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the GstO2 protein consists of the amino acid sequence represented by SEQ ID NO:

5.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the composition inhibits glutathionylation of FUS protein.

7. The pharmaceutical composition according to claim 6, wherein the glutathionylation of the FUS protein is at the Cys-447 residue of the FUS.

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