KR20190024850A - Composition for preventing or treating neurodegenerative disease comprising inducer or activator of nSMase2 - Google Patents

Abstract

The present invention relates to a composition for preventing or treating a neurodegenerative disease containing nSMase2 expression promotors or activators as active ingredients. In the present invention, nSMase2 induces and mediates autophagy, and provides a cell protecting effect in a famine or toxic environment. The present invention identifies that nSMase2 expression is reduced in a striatum of an aged mouse which has reduced substantia nigra and autophagy of a patient with Parkinson′s disease, and nSMase2 is associated with neurodegenerative diseases. Accordingly, the nSmase2 proteins having the same effect, an expression promotor or activators of the proteins or genes coding the proteins may be usefully utilized in a pharmaceutical composition for preventing or treating neurodegenerative diseases, a biomarker composition for diagnosing neurodegenerative diseases, a kit for diagnosing neurodegenerative diseases, a sample composition for inducing autophagy and the like.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating degenerative neurological diseases comprising an nSMase2 expression promoter or an activator as an active ingredient.

The present invention relates to a composition for preventing or treating degenerative neurological diseases comprising an nSMase2 (Neutral sphingomyelinase 2, Sphingomyelin phosphodiesterase 3, SMPD3) protein, an expression promoter or activator of the protein, or a gene encoding the protein as an active ingredient .

Autophagy is an action that breaks down intracellular substances using lysosomes and is an essential process for normal function and survival of cells. In childhood, cells are surrounded by a membrane vacuole (autophagosome) composed of a lipid bilayer, such as starvation and ischemia, where cells lack nutrients in their cells and their unnecessary proteins, aged or damaged cellular organelles , And lysosomes to induce degradation of the enclosed internal material, thereby regenerating the intracellular nutrients, which plays an important role in maintaining the homeostasis of the cells.

Inhibition or activation of such a child action greatly affects not only simple cell activity but also in vivo physiological or pathological changes including the cells. Reduction of child action causes neurodegeneration by increasing the accumulation of misfolded protein. Upregulation of child action has been reported to reduce both levels of aggregated protein and symptoms of neurodegenerative diseases. Therefore, an agent that improves the cell function of a cell can act as a therapeutic agent for the prevention or treatment of a neurodegenerative disease.

However, only a few Adjuvants have been reported for the molecular mechanism of their use, and there are still many areas that need to be clarified. It is expected that the excavation of substances and mechanisms that regulate the child action will play a very important role in the development of therapeutic agents for degenerative neurological diseases, targeting a new point of action.

Korean Registered Patent No. 10-1130030 (registered on March 16, 2012)

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating degenerative neurological diseases.

Another object of the present invention is to provide a biomarker composition for diagnosing degenerative neurological diseases or a kit for diagnosing degenerative neurological diseases.

It is another object of the present invention to provide a method for providing information for diagnosing degenerative neurological diseases or a method for screening drugs for treating degenerative neurological diseases.

It is another object of the present invention to provide a reagent composition for inducing a child action or a method for inducing a child action in vitro.

It is still another object of the present invention to provide a biomarker composition for analyzing a child's action, a biomarker composition for identifying a dopaminergic neuron, or a biomarker composition for analyzing a dopaminergic neuron activity assay.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating degenerative neurological diseases, comprising an nSMase2 protein, an expression promoter or activator of the protein, or a gene encoding the protein as an active ingredient.

The present invention also provides a biomarker composition for diagnosing degenerative neurological diseases, which comprises an nSMase2 protein or a gene coding therefor as an active ingredient.

The present invention also provides a diagnostic kit for degenerative neurological disease, which comprises as an active ingredient an agent capable of detecting the expression level or activity level of nSMase2 protein or the expression level of a gene encoding the protein.

The present invention also provides a method for diagnosing a degenerative neurological disease of a subject, comprising the step of comparing the expression level or the activity level of the nSMase2 protein or the expression level of the gene encoding the protein from the sample separated from the subject and the normal control group The method comprising the steps of:

The present invention also provides a method for treating a candidate drug, comprising: a first step of treating a candidate drug from a sample suspected of being a degenerative neurological disease; A second step of measuring the expression level or activity level of the nSMase2 protein before or after the administration of the candidate drug, or the expression level of the gene encoding the protein; And a third step of selecting a candidate drug that increases the expression level or the activity level of the nSMase2 protein or the expression level of the gene encoding the protein as compared with the normal control sample, and a third step of screening for a drug screening method for treating a degenerative neurological disease .

The present invention also provides a reagent composition for inducing a child action comprising an nSMase2 protein or a gene encoding the same as an active ingredient.

In addition, the present invention provides a method for inducing in vitro action of a child by treating an nSMase2 protein, an expression promoter or activator of the protein, or a gene encoding the protein.

The present invention also provides a biomarker composition for analyzing a child's action, a biomarker composition for identifying a dopaminergic neuron, or a biomarker composition for analyzing a dopaminergic neuron activity, which comprises an nSMase2 protein or a gene encoding the nSMase2 protein as an active ingredient.

In the present invention, the expression of nSMase2 is reduced in the striatum of aged mice in which nSMase2 induces and mediates the child action, exhibits cytoprotective effect in a starvation or toxic environment, and has a reduced blackness and childhood function in patients with Parkinson's disease. The gene encoding the nSMase2 protein, the expression promoter or activator of the protein, or the gene encoding the protein may be used as a pharmaceutical composition for preventing or treating degenerative neurological diseases, A diagnostic biomarker composition, a kit for diagnosing degenerative neurological diseases, a reagent composition for inducing a child's action, and the like.

1 (B) and 1 (C) show the ceramide-producing enzymes and the inhibitors of the enzymes used to identify the enzymes that regulate the child action among the enzymes that produce ceramide. ) Were confirmed by nSMase2 induction and mediator effect by LC3 turnover and LC spot formation using nSMase2 inhibitor.
FIG. 2 shows the results of LC3 turnover, LC spot formation, and p62 degradation amount using nSMase2 and nSMase2 overexpression of (A) nSMase2 and (B) decrease in expression (nSMase2 siRNA).
Fig. 3 (A) and Fig. 3 (B) show the cytoprotective effect of nSMase2 by LDH release and PI staining using gene modification (nSMase2 siRNA), respectively.
Fig. 4 (A) shows the relationship between the amount of nSMase2 expressed by mouse brain and the distribution of dopaminergic neurons. Fig. 4 (B) shows the effect of nSMase2 on cytoprotective effect of LDH It is confirmed.
FIG. 5 shows that the cytoprotective effect by nSMase2 is dependent on the action of the child.
FIG. 6 shows the expression levels of nSMase2 gene and child-related genes in blacks of normal and Parkinson's patients.
7 shows the positive correlation between the expression of nSMase2 gene and the expression of the ATG9B and ATG10 genes in blacks of normal and Parkinson's patients.
Fig. 8 shows the relationship between the expression of nSMase2 and the offspring in the striatum of aged and young mice.

The inventors of the present invention have identified nSMase2 as an enzyme that regulates the action of the child and found that the expression of nSMase2 is reduced in the striatum of aged mice in which the blackness and the child action of Parkinson's disease are reduced, The present invention has been accomplished by confirming that nSMase2 can be utilized as a therapeutic agent for degenerative neurological diseases.

As used herein, the term "nSMase2 (Sphingomyelin phosphodiesterase 3, SMPD3)" is one of hydrolytic enzymes involved in the sphingolipid metabolism, and sphingomyelin (SM) phosphocholine and ceramide.

The term "biomarker" used in the present invention is a substance that can diagnose a tissue or cell of a subject in which degenerative neuropathy occurs, by distinguishing it from a tissue or cell of a normal control group. Or an organic biomolecule such as nucleic acid, lipid, glycolipid, glycoprotein,

The term "sample" used in the present invention refers to a tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or cerebrospinal fluid which differs from the normal control group in the expression level or the activity level of the nSMase2 protein, It may be, but not limited to, a sample such as urine.

The term "primer " as used herein means a short nucleic acid sequence capable of forming a base pair with a complementary template with a short free 3-terminal hydroxyl group and functioning as a starting point for template strand replication . The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. In addition, primers may incorporate additional features that do not alter the primer properties of the primers that serve as a starting point for DNA synthesis, as sense and antisense nucleic acids with 7 to 50 nucleotide sequences.

As used herein, the term "probe" means a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or hundreds of nucleotides that can specifically bind to an mRNA. , And the amount of expression can be confirmed. The probe may be prepared in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, or an RNA probe. Selection of suitable probes and hybridization conditions can be appropriately selected according to techniques known in the art.

The term "antibody" as used herein refers to a specific immunoglobulin directed against an antigenic site, and an antibody in the present invention refers to an antibody that specifically binds to nSMase2, Or those which are commercially available can be used. In addition, the antibody includes a polyclonal antibody, a monoclonal antibody, and a fragment capable of binding to an epitope. The antibody refers to a complete form having two full-length light chains and two long-length heavy chains, and also includes special antibodies such as humanized antibodies.

In addition, the kit of the present invention comprises an antibody that specifically binds to a marker component, a secondary antibody conjugate conjugated with a labeling substance that is colored by the reaction with the substrate, a coloring substrate solution that reacts with the labeling substance, Enzyme reaction termination solutions, and the like, and can be made from a number of separate packaging or compartments, including the reagent components used.

As used herein, the term "peptide" has a high binding capacity to the target material and does not cause denaturation during thermal / chemical treatment. In addition, since the molecular size is small, it can be used as a fusion protein by attaching to other proteins. It can be used as a diagnostic kit and a drug delivery material because it can be specifically attached to a polymer protein chain.

As used herein, the term "aptamer" is a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) having a stable tertiary structure and capable of binding to a target molecule with high affinity and specificity . Aptamers are comparable to monoclonal antibodies due to their inherent high affinity (usually pM levels) and their ability to bind to target molecules with specificity, and there is a high likelihood of being an alternative antibody, especially as a "chemoantibody".

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating degenerative neurological diseases comprising an nSMase2 protein, an expression promoter or activator of the protein, or a gene encoding the protein as an active ingredient.

The degenerative neurological diseases are selected from the group consisting of Parkinson's disease, Alzheimer's disease, Lou Gehrig's disease, Huntington's disease, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, multiple sclerosis, immune system dysfunction, progressive neurodegenerative disease, , Dementia due to cerebral ischemia and cerebral hemorrhage, amnesia, stroke, and stroke, but is not limited thereto.

The nSMase2 protein, the expression promoter or activator of the protein, or the gene encoding the protein may induce and mediate the action of the child and protect the cell in a starvation or toxic environment.

The nSMase2 protein or the gene encoding the protein is characterized in that the level of expression or activity is reduced in the degenerative neurological disease.

The composition of the present invention may contain, for administration, a pharmaceutically acceptable carrier, excipient or diluent in addition to the components described above. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical compositions of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations, suppositories or sterilized injection solutions according to a conventional method have. In detail, when formulating, it can be prepared using diluents or excipients such as fillers, weights, binders, humectants, disintegrants, surfactants and the like which are usually used. Solid form preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules and the like. Such a solid preparation may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin and the like in addition to the active ingredient. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration, liquid paraffin, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and tasks. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. As a base for suppositories, it is possible to use witepsol, macrosole, tween 61, cacao paper, laurin, glycerogelatin and the like.

A suitable dose of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, and the time, but the daily dose of the composition is preferably 0.01 mg / kg to 50 mg / kg, and may be administered once to several times per day as needed.

The present invention also provides a biomarker composition for diagnosing degenerative neurological diseases, which comprises an nSMase2 protein or a gene coding therefor as an active ingredient.

The present invention also provides a diagnostic kit for degenerative neurological disease, which comprises as an active ingredient an agent capable of detecting the expression level or activity level of nSMase2 protein or the expression level of a gene encoding the protein.

The formulation may be selected from the group consisting of primers, probes, antibodies, peptides, and aptamers, but is not limited thereto.

The present invention also provides a method for diagnosing a degenerative neurological disease of a subject, comprising the step of comparing the expression level or the activity level of the nSMase2 protein or the expression level of the gene encoding the protein from the sample separated from the subject and the normal control group The method comprising the steps of:

The present invention also provides a method for treating a candidate drug, comprising: a first step of treating a candidate drug from a sample suspected of being a degenerative neurological disease; A second step of measuring the expression level or activity level of the nSMase2 protein before or after the administration of the candidate drug, or the expression level of the gene encoding the protein; And a third step of selecting a candidate drug that increases the expression level or the activity level of the nSMase2 protein or the expression level of the gene encoding the protein as compared with the normal control sample, and a third step of screening for a drug screening method for treating a degenerative neurological disease .

The level of expression or activity of the nSMase2 protein in the second step or the expression level of the gene encoding the protein may be determined by reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay assay, ELISA), radioimmunoassay, immunohistochemistry, microarray, western blotting and flow cytometry (FACS). However, , But not limited to,

The present invention also provides a reagent composition for inducing a child action comprising an nSMase2 protein or a gene encoding the same as an active ingredient.

In addition, the present invention provides a method for inducing in vitro action of a child by treating an nSMase2 protein, an expression promoter or activator of the protein, or a gene encoding the protein.

The present invention also provides a biomarker composition for analyzing a child's action, a biomarker composition for identifying a dopaminergic neuron, or a biomarker composition for analyzing a dopaminergic neuron activity, which comprises an nSMase2 protein or a gene encoding the nSMase2 protein as an active ingredient.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  One : nSMase2  Child action induction and mediating effect

As shown in Fig. 1 (A), PC12 cells (ATCC) were treated with an inhibitor of each enzyme for 1 hour and the culture solution was incubated with amino acids The degree of starvation induction was evaluated by exchanging with growth factor-deficient HBSS (Hank's balanced salt solution) and culturing for 2 hours in the presence or absence of chloroquine (CQ) (50 μM). Inhibitors of nSMase2 include GW4869 (5 μM), FB1 (10 μM) as an inhibitor of ceramide synthase and myriocin (5 μM) as an inhibitor of serine-palmitoyltransferase , and desipramine (10 μM) was used as an inhibitor of acidic sphingomyelinase.

The degree of autophagic flux was measured by using chloroquine, a lysosomal inhibitor, to determine the degree of degradation of LC3 II, which is specifically formed during childhood. Comparing the degree of degradation of LC3 II with or without chloroquine under the same conditions, the degree of decomposition of LC3 II by the child action, that is, LC3 turnover, can be measured. The amount of LC3 rotation was measured by Western blot. As a result, it was confirmed that LC3 rotation amount was inhibited when GW4869, an inhibitor of nSMase2, was treated as shown in Fig. 1 (B).

As a result of measuring the autophagosome or autolysosome, which is specifically formed during child action, with LC3 spot (puncta), as shown in Fig. 1 (C), the LC3 spot induced by starvation GW4869 treatment. Therefore, it was confirmed that nSMase2 induces the action of the enzyme in the ceramide generation pathway.

The results were confirmed not only by nSMase2 inhibitors but also by gene modulation. PC12 cells were transfected with 1 [mu] g of plasmid-encoded V5-tagged nSMase2 or empty vector. In addition, PC12 cells were transfected with 50 nM of nSMase2 siRNA (siSmpd3) (Mixture of RSS331830 and RSS331831, Stealth siRNA, Thermo Fisher Scientific) or untargeted negative control siRNA (siControl) (# 12935-300).

As a result, referring to FIG. 2 (A), when overexpression of nSMase2, LC3 spots were observed similar to the starvation state, and it was confirmed that the child action was induced. In addition, induction of nociception by nSMase2 was also confirmed by LC3 rotation amount and p62 decomposition amount. p62 is a substrate which is degraded by the action of a child, and the degree of the child action can be analyzed by using the amount of decomposition of the p62. Analysis of LC3 turnover and p62 degradation revealed that nMase2 overexpression induces a child action.

 Conversely, as shown in FIG. 2 (B), knockdown of nSMase2 using nSMase2 siRNA resulted in inhibition of starvation induced by LC3 spot formation, LC3 turnover, and p62 degradation amount.

Therefore, it was confirmed from the above results that nSMase2 induces and mediates the action of the child.

Example  2 : nSMase2  Cytoprotective effect

In order to confirm that nSMase2, which induces and mediates the action of a child, plays a protective role in cells, the degree of starvation induced cell toxicity was analyzed when nSMase2 expression was decreased using nSMase2 siRNA.

PC12 cells were transfected with 50 nM of nSMase2 siRNA (siSmpd3) or untargeted negative control siRNA (siCON). Forty-eight hours after transfection, cells were incubated with HBSS for 7 or 10 hours. The levels of lactate dehydrogenase (LDH) and total LDH released into the medium were quantitated to determine cytotoxicity. In addition, PC12 cells transfected with siRNA were double-stained with PI (propidium iodide) and Hoechst 3334. Apoptotic cells are positive for PI, and total cells are labeled Hoechst 33342.

As a result, as shown in FIG. 3 (A), cytotoxicity was confirmed through LDH release. As a result, when knockdown (si-Smpd3) expression of nSMase2 was observed in starvation, cell death was more increased than control si- Respectively.

As shown in FIG. 3 (B), when cell staining was performed using PI, it was confirmed that the cell death was increased when knockdown of nSMase2 expression (si-Smpd3) was performed compared to the control (si-Control) .

nSMase2 is known to be expressed mainly in brain tissue and bone tissue. In order to determine which part of the brain is mainly expressed, C57BL / 6 mouse (6 weeks old) brain was dissected by each site to express nSMase2 Were analyzed by immunoblotting. As a result, as shown in Fig. 4 (A), it was confirmed that the striatum and the midbrain, in which the dopaminergic neurons are widely distributed, are particularly expressed. Tyrosine hydroxylase (TH) is an enzyme expressed in dopaminergic neurons. Through TH expression, the distribution of actual dopaminergic neurons in the brain region can be confirmed.

Therefore, in order to confirm whether nSMase2 exhibits cytoprotective effect even under dopaminergic toxic stimulation, PC12 cells were transfected with siRNA of nSMase2, and a high concentration of dopamine or mitochondrial uncoupler, carbonyl cyanide After treating m-chlorophenylhydrazone (CCCP) with m-chlorophenyl hydrazone, the degree of cytotoxicity was analyzed by LDH release. CCCP treatment is a commonly used method in in vitro disease models of Parkinson's disease, a neurodegenerative disease characterized by loss of dopaminergic neurons.

 As a result, referring to Fig. 4 (B), it was confirmed that the cell death was increased compared to the control (si-Control) when knockdown (si-Smpd3) of nSMase2 expression was observed in the toxicity by dopamine or CCCP Respectively. This means that nSMase2 has a cytoprotective effect on the toxins. In addition, the cells exposed to the toxic stimuli were more likely to die in the presence of chloroquine, indicating that the child acts cytoprotective against the toxic stimulants.

To confirm whether the cytoprotective effect of this nSMase2 appears dependent on the child's behavior, the experiment was carried out in the presence of the child's inhibitor chloroquine. Cells transfected with siRNA were treated with starvation in the absence or presence of 50 [mu] M chloroquine for 7 hours or 20 [mu] M CCCP for 24 hours. Cytotoxicity was analyzed by LDH release.

As a result, as shown in Fig. 5, it was confirmed that there was no effect of increasing the cell death by nSMase2 knockdown (si-Smpd3) in the toxicity by starvation or CCCP. This reflects the fact that the cytoprotective effect of nSMase2 on these toxic stimuli is child-dependent.

Therefore, it was confirmed from the above results that nSMase2 exhibited cytoprotective effect from toxic stimuli through child action.

Example  3: With nSMase2  Correlation with Degenerative Neurological Disorders

The expression levels of nSMase2 in Parkinson's disease (PD) patients and normal blacks were measured by GEO database in order to confirm the association of nSMase2, which induces and mediates child action, And analyzed using GSE 7621.

As a result, referring to FIG. 6, it was confirmed that the expression level of nSMase2 was decreased in the black blood of patients with Parkinson's disease compared with that of normal persons. In addition, it was confirmed that the expression of the genes related to the child action was also decreased.

As shown in FIG. 7, the expression level of nSMase2 and the expression level of ATG9B or ATG10 were found to be positively correlated with each other, Respectively.

In addition, the most important risk factor of degenerative brain disease is aging. Using aging C57BL / 6 mice (20 months old) and young C57BL / 6 mice (6 weeks old) The expression level of nSMase2 was analyzed. As a result, as shown in Fig. 8, it was confirmed that the expression amount of nSMase2 in aged mice decreased and the accumulation of p62, a substrate degraded by child action, was increased. In other words, it was confirmed that nSMase2 mediating the child action was reduced in the aged mice in which the child action was decreased, which means that nSMase2 is associated with degenerative neurological diseases.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims (15)

A pharmaceutical composition for preventing or treating degenerative neurological diseases comprising an nSMase2 protein, an expression promoter or activator of the protein, or a gene encoding the protein as an active ingredient. The method of claim 1, wherein the degenerative neurological disease is selected from the group consisting of Parkinson's disease, Alzheimer's disease, Lou Gehrig's disease, Huntington's disease, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, multiple sclerosis, immune system dysfunction, Wherein the disease is selected from the group consisting of dementia caused by cerebral ischemia and cerebral hemorrhage, amnesia, stroke, and stroke. 2. The method according to claim 1, wherein the nSMase2 protein, an expression promoter or activator of the protein, or a gene encoding the protein induces and mediates the action of a child and protects cells in a starvation or toxic environment. A pharmaceutical composition for preventing or treating diseases. The pharmaceutical composition for preventing or treating neurodegenerative diseases according to claim 1, wherein the nSMase2 protein or the gene encoding the protein has decreased expression or activity level in a neurodegenerative disease. A biomarker composition for diagnosing degenerative neurological disease comprising an nSMase2 protein or a gene encoding the same as an active ingredient. A kit for diagnosing degenerative neurological disease, comprising an agent capable of detecting the expression level or activity level of nSMase2 protein or the expression level of a gene encoding said protein as an active ingredient. 7. The kit for diagnosing a neurodegenerative disease according to claim 6, wherein the agent is selected from the group consisting of primers, probes, antibodies, peptides and aptamers. Comparing the expression or activity level of the nSMase2 protein or the expression level of the gene encoding the protein from the sample isolated from the subject and the normal control group. A first step of treating the candidate drug with a sample separated from a suspected neurodegenerative disease subject;
A second step of measuring the expression level or activity level of the nSMase2 protein before or after the administration of the candidate drug, or the expression level of the gene encoding the protein; And
A third step of selecting a candidate drug that increases the expression level or the activity level of the nSMase2 protein or the expression level of the gene encoding the protein as compared with the normal control sample;
≪ / RTI > wherein the method comprises the steps of: 10. The method according to claim 9, wherein the expression level or activity level of the nSMase2 protein in the second step or the expression level of the gene encoding the protein is determined by reverse transcription-polymerase chain reaction (RT-PCR) Or any one selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunohistochemistry, microarray, western blotting and flow cytometry (FACS) Of the total amount of the drug. A reagent composition for inducing a child action comprising an nSMase2 protein or a gene encoding the same as an active ingredient. A method for inducing a child's action in vitro by treating an nSMase2 protein, an expression promoter or activator of the protein, or a gene encoding the protein. and a nSMase2 protein or a gene coding therefor as an active ingredient. A biomarker composition for identifying a dopaminergic neuron comprising an nSMase2 protein or a gene encoding the same as an active ingredient. A biomarker composition for analyzing dopaminergic neuron activity comprising an nSMase2 protein or a gene encoding the same as an active ingredient.

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