KR20150135867A - Composition comprising AEG-1 for preventing or treating Parkinson's disease - Google Patents

Abstract

The present invention relates to a composition comprising astrocyte elevated gene-1 (AEG-1) protein or a nucleic acid molecule for coding AEG-1 protein as an active ingredient for preventing or treating Parkinson′s disease. According to the present invention, AEG-1 with excellent protective effects of dopamine neuron in an animal model with Parkinson′s disease can be usefully used for preventing, alleviating, and treating Parkinson′s disease.

Description

AEG-1 for preventing or treating Parkinson's disease comprising AEG-1 as an active ingredient.

The present invention relates to a composition for preventing or treating Parkinson's disease comprising, as an active ingredient, a nucleic acid molecule encoding an AEG-1 (astrocyte elevated gene-1) protein or an AEG-1 protein.

Parkinson's disease is a chronic progressive degenerative disease of the nervous system characterized by stable tremor, stiffness, gait, and postural instability, which results in gradual loss of dopaminergic neurons in the subcortical nigra pars compacta (SNc) of the brain (Calne et al., 1983, Heikkila 1984). Patients with Parkinson's disease are estimated to be approximately 1% of the population at approximately 60 years of age or older.

Although the cause of Parkinson's disease is not clear, the 'multinomial hypothesis' that genetic and environmental factors interact with each other is the most commonly accepted. Most patients with Parkinson's disease develop without familial involvement, but about 10% are familial Parkinson's.

L-Dopa is currently commonly used as a remedy for the increased amount of dopamine in Parkinson's disease. Although L-Dopa slows the progression of Parkinson's disease and alleviates clinical symptoms after use, long-term adverse effects include involuntary exercise and vomiting (Clarke and Deane, 2001). In addition, therapeutic agents used to treat Parkinson's disease include FDA-approved dopamine agonists, catechol-omethyltransferase inhibitors (COMT inhibitors), monoamine oxidase B B, MAO-B inhibitors, and anti-cholinergics.

As a result of further studies to develop a novel therapeutic agent for Parkinson's disease, the present inventors have confirmed that the expression of AEG-1 is significantly reduced in the cerebral brain of Parkinson's disease patients and the AEG-1 gene is introduced using AAV The present inventors have completed the present invention by confirming that AEG-1 can be used for the treatment of Parkinson's disease.

It is an object of the present invention to provide a composition for preventing or treating Parkinson's disease comprising, as an active ingredient, a nucleic acid molecule encoding an AEG-1 (astrocyte elevated gene-1) protein or an AEG-1 protein.

It is another object of the present invention to provide a method for screening candidate substances for treating Parkinson's disease using AEG-1.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating Parkinson's disease comprising, as an active ingredient, a nucleic acid molecule encoding an AEG-1 (astrocyte elevated gene-1) protein or an AEG- to provide.

The present invention also provides a food composition for preventing or ameliorating Parkinson's disease comprising an AEG-1 protein or a nucleic acid molecule encoding an AEG-1 protein as an active ingredient.

Also, the present invention provides a method for producing an AEG-1 (astrocyte elevated gene-1) expression cell line comprising the steps of: 1)

2) measuring the level of expression or activity of AEG-1 in the cell line of step 1); And

3) screening the candidate substance for Parkinson's disease treatment, wherein the level of expression or activity of AEG-1 in step 2) is increased compared to the control not treated with the test substance.

AEG-1 according to the present invention is excellent in the protective effect of dopamine neurons in an animal model of Parkinson's disease, and can be usefully used for prevention, improvement and treatment of Parkinson's disease.

1 is a diagram showing a vector map of pBL-AEG-1.
FIG. 2 shows the result of double fluorescent staining of mouse brain tissue with HA-tag and dopaminergic neuron (TH) after 3 weeks of AAV-AEG-1 (HA-tag) administration (Scale bar, 10 μm) .
FIG. 3 is a graph showing the result of staining mouse brain tissue with AEG-1 after 3 weeks of administration of AAV-AEG-1 (HA-tag).
FIG. 4 is a graph showing the neuroprotective effect of AAV-AEG-1 (HA-tag) by injecting 6-OHDA after 3 weeks of AAV-AEG-1 (HA-tag) administration and TH staining A, a: control, B, b: 6-OHDA only, C, c: 6-OHDA after 3 weeks of AAV-AEG-1 (HA-tag) administration).
FIG. 5 is a graph showing the expression of AEG-1 (Blue) in dopamine neurons (Neuromelanin positive cell, Brown) by staining cytoplasmic blackening of PD patients (Scale bar, 25 μm).
6 is a graph showing AEG-1 protein expression in midbrain substantia nigra of PD patients.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for preventing or treating Parkinson's disease comprising an astrocytic elevated gene-1 (AEG-1) protein or a nucleic acid molecule encoding an AEG-1 protein as an active ingredient.

The composition comprises a pharmaceutical composition and a food composition.

The AST-1 (astrocyte elevated gene-1) protein preferably comprises a protein having an amino acid sequence represented by SEQ ID NO: 1 or a protein encoded by a nucleotide sequence represented by SEQ ID NO: 2 and a functional equivalent of the protein . Is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70%, more preferably at least 90%, more preferably at least 90% Quot; refers to a protein having substantially the same physiological activity as a protein having the amino acid sequence represented by SEQ ID NO: 1, having a sequence homology of 95% or more.

In the AEG-1 protein of the present invention, not only a protein having a naturally occurring amino acid sequence but also an amino acid sequence variant thereof are also included in the scope of the present invention. A variant of an AEG-1 protein refers to a protein having a sequence that differs by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, with the AEG-1 native amino acid sequence and one or more amino acid residues. Amino acid exchanges in proteins and peptides that do not globally alter the activity of the molecule are known in the art. The AEG-1 protein or its variants can be prepared by natural extraction or by synthesis (Merrifleld, J. Amer. Chem. Soc. 85: 2149-2156, 1963) or by DNA recombination methods Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, Second Edition, 1989).

The nucleic acid molecule encoding the AEG-1 protein may preferably consist of the nucleotide sequence shown in SEQ ID NO: 2, and variants of the nucleotide sequence are included within the scope of the present invention. Specifically, the nucleic acid molecule has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 . ≪ / RTI > "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

The nucleic acid molecule encoding the AEG-1 protein of the present invention may be in a form contained in a vector. The vector comprising the nucleic acid molecule encoding the AEG-1 protein may be a vector comprising a linear DNA, a plasmid vector, a vector comprising a viral expression vector, or a recombinant retrovirus vector, recombinant adenovirus ( a recombinant viral vector comprising an adenovirus vector, an adeno-associated virus (AAV) vector, a recombinant herpes simplex virus vector or a recombinant lentivirus vector is preferable, More preferably, it is an adeno-associated virus (AAV) vector, but is not limited thereto.

AEG-1 according to the present invention is excellent in the protective effect of dopamine neurons in an animal model of Parkinson's disease, and can be usefully used for prevention, improvement and treatment of Parkinson's disease.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. The pharmaceutical composition according to the present invention may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or other oral formulations, external preparation, suppository and sterilized injection solution, . Suitable formulations known in the art are preferably those disclosed in Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Easton PA.

Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention 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. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations are prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, It is prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and suspension include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. For a desired effect, the composition of the present invention can be administered at a daily dose of 0.001 to 1000 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way. The dosage may vary depending on factors such as the type of disease of the individual, the severity, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, Can be determined.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. However, at the time of oral administration, since the protein is digested, it is preferable to formulate the oral composition so as to coat the active agent or protect it from decomposition at the top. Preferably in the form of an injection.

In the present invention, the term "health functional food" refers to a food group imparted with added value to function or express the function of the food by physical, biochemical or biotechnological techniques, Means a food which has been designed and manufactured so that the body's control function regarding disease prevention and recovery is sufficiently expressed to the living body.

The health functional food may include food-acceptable food supplementary additives, and may further include suitable carriers, excipients and diluents conventionally used in the production of health functional foods.

When the composition of the present invention is used as a food additive, the composition can be added as it is or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). In general, the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may comprise flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Also, the present invention provides a method for producing an AEG-1 (astrocyte elevated gene-1) expression cell line comprising the steps of: 1)

2) measuring the level of expression or activity of AEG-1 in the cell line of step 1); And

3) screening the candidate substance for Parkinson's disease treatment, wherein the level of expression or activity of AEG-1 in step 2) is increased compared to the control not treated with the test substance.

The term "candidate substance" means a substance to be tested for a Parkinson's therapeutic activity, and may include any molecule such as an extract, protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide and a wide variety of compounds. Such candidate materials also include synthetic materials as well as natural materials.

Methods for measuring the expression level include RT-PCR, competitive RT-PCR, real-time quantitative RT-PCR, RNase protection assay RNase protection method, Northern blotting, and DNA chip technology. The present invention is not limited to these methods.

Methods for measuring the activity level include western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, Immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, immunohistochemical staining, immunohistochemical staining, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, A fluorescence activated cell sorter analysis, a protein chip technology, and the like, but the present invention is not limited thereto.

In one embodiment of the present invention, the expression of AEG-1 in the cerebral blood of Parkinson's patients is decreased and the protective effect of dopamine neurons is excellent when administered to an animal model of Parkinson's disease, which can be used to treat Parkinson's disease Respectively. Therefore, AEG-1 can be useful for screening candidates for the treatment of Parkinson's disease.

Hereinafter, the present invention will be described in more detail by way of examples. It should be noted, however, that the following examples are provided to further illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1. Preparation of AAV-AEG-1

1-1. Preparation of pBL-AEG-1 Recombinant Vector

A recombinant vector containing the AEG-1 gene was prepared by introducing the astrocyte elevated gene-1 (AEG-1) gene into the pBL plasmid (HA-tag). The vector map is shown in Fig.

1-2. Preparation of AAV-AEG-1

Adeno-associated virus (AAV) -AEG-1 was constructed with the request of the University of North Carolina Vector Core and the genetic potency was 9.4 × 10 ¹² viralgenomes / ml. Activated GFP (greenfluorescent protein) was subcloned into the same viral backbone and used as a control.

Example 2. Analysis results using an animal model of Parkinson's disease

2-1. Experimental animal

Male 8 week old C57BL / 6 mice were purchased from Daehan Biolink (Eumseong, Korea). Each mouse was anesthetized by ip (intraperitoneal) administration of chloral hydrate (360 mg / kg; Sigma, St. Louis, MO.) Followed by stereotaxic frame (David Kopf Instrument, Tujunga, CA, USA). Using a 10 μl Hamilton syringe (30S needle) attached to a pneumatic pump (KD Scientific, New Hope, Pa.) Containing AAV-AEG-1 in the midbrain black part of each mouse according to the conventional method, the right SN (AP: -0.35 (Paxinos and Watson, 1998) were injected intraperitoneally to the mouse (Fig. After injection, the needle was held for an additional 5 minutes before slowly withdrawing. At this time, 2.0 μl of viral vector suspension was injected at a rate of 0.1 μl / min for 20 minutes.

2-2. Immunohistochemical staining

Immunohistochemical staining was performed by modifying a known experimental method (Kim et al., 2005; Kim et al., 2011; Kim et al., 2012). Each of the mice of Example 2-1 was perfused with perfusion, the tissue was fixed, and brain tissue was sectioned to a thickness of 30 mu m. Tissue sections were washed with PBS and incubated with primary antibody for 48 hours at room temperature. The primary antibodies were rabbit anti-tyrosine hydroxylase (TH, 1: 2000; Pel-Freez, Brown Beer, WI) and mouse anti-HA (1: 100; Cell-signaling, Beverly, MA). After washing with PBS, the cells were incubated with secondary antibody and treated with avidin-biotin complex kit (Vector Laboratories, Burlingame, CA). The tissue sections were incubated with 0.5 mg / ml 3,3'-diaminobenzidine (Sigma, St. Louis, MO) in 0.1 M PB containing 0.003% H ₂ O ₂ , Respectively. The dyed samples were analyzed using a bright-field microscope (Carl Zeiss, Gottingen, Germany).

For immunofluorescence labeling, sections of brain tissue were rinsed and incubated with rabbit anti-tyrosine hydroxylase (TH, 1: 2000; Pel-Freez, Brown Beer, WI) ; Cell-signaling, Beverly, MA). The next day, tissue sections were washed and incubated for 1 hour with Cy3 conjugated anti-rabbit IgG (1: 200; Millipore, Bedford, Mass.) And FITC-conjugated anti- rabbit IgG (1: 200; Millipore) Lt; / RTI > This was mounted with a Vectashield mounting medium (Vector). Dyed samples were observed using a confocal microscope (LSM700, Carl Zeiss, Germany).

The above experimental results are shown in FIG. 2 and FIG.

As shown in FIG. 2, it was confirmed by HA-tag staining that AAV-AEG-1 was expressed in the black part (SNpc) into which AAV-AEG-1 was injected (upper part of FIG. 2) 1 gene (HA-tag, green) was expressed in Tyrosine hydroxylase (TH) -positive cell, red (lower part of FIG. 2).

Further, as shown in FIG. 3, it was confirmed that AEG-1 can be more expressed by the introduction of AAV-AEG-1 in black dopamine neuron cells.

2-3. 6-OHDA lesion

As in Example 2-1, AAV-AEG-1 was injected into each mouse and after 3 weeks, desipramine was pretreated to prevent absorption of 6-OHDA. Each mouse was anesthetized by intraperitoneal administration of chloral hydrate (360 mg / kg; Sigma, St. Louis, MO.) And then placed in a stereotaxic frame. Thereafter, a solution of 6-OHDA (5.0 μg / μl in 0.9% NaCl / 0.02% ascorbate) at a total concentration of 15.0 μg / 3 μl was injected at a rate of 0.5 μl / min using a microliter syringe. The injection was on the left striatum (AP: +0.09 cm; ML: +0.22 cm; DV: -0.25 cm relative to bregma). After waiting for 5 minutes, the needle was slowly removed. After brain tissue was isolated from each mouse, TH staining was performed in the same manner as in Example 2-2. Also, for Nissl staining, SN tissue samples were mounted on gelatin coated slides and stained with 0.5% cresyl violet (Sigma). Thereafter, it was analyzed using a bright field microscope. The results are shown in Fig.

As shown in FIG. 4, in mice (C, c) injected with 6-OHDA after 3 weeks of AEG-1 injection compared with mice injected with only 6-OHDA, which is a neurotoxic substance, It was confirmed that brown color remained and the size of the remaining dopamine neurons was also preserved.

Example 3 Analysis Result Using Human Brain Cells

3-1. Immunohistochemical analysis

Brain (midbrain and black) tissue of Parkinson's patients was obtained from VBBN (Victoria Brain Bank Network). The human brain tissue was deparaffinized in xylene and antigen retrieval was performed prior to immunohistochemistry. Tissue sections were washed with cold PBS for 15 minutes and then blocked with universal blocking solution (0.3% Triton X-100, 1% bovine serum albumin, 0.05% Tween 20, 0.1% cold fish gelatin and 0.05% sodium azide in PBS ) For 1 hour at room temperature. The tissue sections were diluted with antibody Diluent (Dako; S3022) and the sections were incubated with streptavidin-conjugated HRP (horseradish peroxidase) (Vectastain ABC (Invitrogen) kit, Vector Lavoratories) followed by biotin-conjugated secondary antibody. Each tissue sample was visualized by incubation with 0.1 M PB containing 0.05% DAB (diaminobenzidine-HCl), 0.05% nickel ammonium sulfate, 0.05% cobalt chloride hexahydrate and 0.003% hydrogen peroxide. The dyed samples were analyzed using a bright field microscope. The results are shown in Fig.

As shown in FIG. 5, expression of AEG-1 (Blue) was confirmed in dopamine neurons (Neuromelanin positive cell, Brown) in patients with Parkinson's disease and AEG-1 expression was decreased in Parkinson's disease Respectively.

3-2. Western blot

Western blotting was performed according to a conventionally known method using the brain tissue of the Parkinson's disease patient of Example 3-2. The primary antibodies were mouse anti-β-actin (1: 5000; Abcam, Cambridge, UK) and rabbit anti-AEG-1 (1: 500; Invitrogen). The results are shown in Fig.

As shown in FIG. 6, it was confirmed that the amount of AEG-1 protein was decreased in the cerebral blood of Parkinson's disease patients compared with the control group.

These results suggest that the AEG-1 protein levels in the cerebral blood of patients with Parkinson's disease are significantly decreased and that the AEG-1 gene introduced by AAV has a protective effect against dopaminergic neurons and Parkinson's disease .

Hereinafter, formulation examples of the pharmaceutical composition and the food composition of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described .

Formulation Example 1. Preparation of a pharmaceutical composition

1-1. Manufacture of Powder

2 g of AEG-1 protein

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

1-2. Manufacture of tablets

100 mg of AEG-1 protein

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

1-3. Preparation of capsules

100 mg of AEG-1 protein

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

Formulation Example 2. Preparation of Food Composition

2-1. Manufacture of health food

100 mg of AEG-1 protein

Vitamin mixture quantity

70 g of vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

0.2 g of vitamin B12

Vitamin C 10 mg

Biotin 10 g

Nicotinic acid amide 1.7 mg

Folate 50 g

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

<110> Kyungpook National University Industry-Academic Cooperation Foundation          University-Industry Cooperation Group of Kyung Hee University <120> Composition comprising AEG-1 for or treating          Parkinson's disease <130> 196 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 582 <212> PRT <213> Homo sapiens <400> 1 Met Ala Ala Arg Ser Trp Gln Asp Glu Leu Ala Gln Gln Ala Glu Glu   1 5 10 15 Gly Ser Ala Arg Leu Arg Glu Met Leu Ser Val Gly Leu Gly Phe Leu              20 25 30 Arg Thr Glu Leu Gly Leu Asp Leu Gly Leu Glu Pro Lys Arg Tyr Pro          35 40 45 Gly Trp Val Ile Leu Val Gly Thr Gly Ala Leu Gly Leu Leu Leu Leu      50 55 60 Phe Leu Leu Gly Tyr Gly Trp Ala Ala Ala Cys Ala Gly Ala Arg Lys  65 70 75 80 Lys Arg Arg Ser Pro Pro Arg Lys Arg Glu Glu Ala Ala Ala Val Pro                  85 90 95 Ala Ala Ala Pro Asp Asp Leu Ala Leu Leu Lys Asn Leu Arg Ser Glu             100 105 110 Glu Gln Lys Lys Lys Asn Arg Lys Lys Leu Ser Glu Lys Pro Lys Pro         115 120 125 Asn Gly Arg Thr Val Glu Val Ala Glu Gly Glu Ala Val Arg Thr Pro     130 135 140 Gln Ser Val Thr Ala Lys Gln Pro Pro Glu Ile Asp Lys Lys Asn Glu 145 150 155 160 Lys Ser Lys Lys Asn Lys Lys Lys Ser Lys Ser Asp Ala Lys Ala Val                 165 170 175 Gln Asn Ser Ser Arg His Asp Gly Lys Glu Val Asp Glu Gly Ala Trp             180 185 190 Glu Thr Lys Ile Ser His Arg Glu Lys Arg Gln Gln Arg Lys Arg Asp         195 200 205 Lys Val Leu Thr Asp Ser Gly Ser Leu Asp Ser Thr Ile Pro Gly Ile     210 215 220 Glu Asn Thr Ile Thr Val Thr Thr Glu Gln Leu Thr Thr Ala Ser Phe 225 230 235 240 Pro Val Gly Ser Lys Lys Asn Lys Gly Asp Ser His Leu Asn Val Gln                 245 250 255 Val Ser Asn Phe Lys Ser Gly Lys Gly Asp Ser Thr Leu Gln Val Ser             260 265 270 Ser Gly Leu Asn Glu Asn Leu Thr Val Asn Gly Gly Gly Trp Asn Glu         275 280 285 Lys Ser Val Lys Leu Ser Ser Gln Ile Ser Ala Gly Glu Glu Lys Trp     290 295 300 Asn Ser Val Ser Pro Ala Ser Ala Gly Lys Arg Lys Thr Glu Pro Ser 305 310 315 320 Ala Trp Ser Gln Asp Thr Gly Asp Ala Asn Thr Asn Gly Lys Asp Trp                 325 330 335 Gly Arg Ser Serp Ser Asp Arg Ser Ile Phe Ser Gly Ile Gly Ser Thr             340 345 350 Ala Glu Pro Val Ser Gln Ser Thr Thr Ser Asp Tyr Gln Trp Asp Val         355 360 365 Ser Arg Asn Gln Pro Tyr Ile Asp Asp Glu Trp Ser Gly Leu Asn Gly     370 375 380 Leu Ser Ser Ala Asp Pro Asn Ser Asp Trp Asn Ala Pro Ala Glu Glu 385 390 395 400 Trp Gly Asn Trp Val Asp Glu Glu Arg Ala Ser Leu Leu Lys Ser Gln                 405 410 415 Glu Pro Ile Pro Asp Asp Gln Lys Val Ser Asp Asp Asp Lys Glu Lys             420 425 430 Gly Glu Gly Ala Leu Pro Thr Gly Lys Ser Lys Lys Lys Lys Lys Lys Lys         435 440 445 Lys Lys Lys Gln Gly Glu Asp Asn Ser Thr Ala Gln Asp Thr Glu Glu     450 455 460 Leu Glu Lys Glu Ile Arg Glu Asp Leu Pro Val Asn Thr Ser Lys Thr 465 470 475 480 Arg Pro Lys Gln Glu Lys Ala Phe Ser Leu Lys Thr Ile Ser Thr Ser                 485 490 495 Asp Pro Ala Glu Val Leu Val Lys Asn Ser Gln Pro Ile Lys Thr Leu             500 505 510 Pro Pro Ala Thr Ser Thr Glu Pro Ser Val Ile Leu Ser Lys Ser Asp         515 520 525 Ser Asp Lys Ser Ser Ser Gln Val Pro Pro Ile Leu Gln Glu Thr Asp     530 535 540 Lys Ser Lys Ser Asn Thr Lys Gln Asn Ser Val Pro Pro Ser Gln Thr 545 550 555 560 Lys Ser Glu Thr Ser Trp Glu Ser Pro Lys Gln Ile Lys Lys Lys Lys                 565 570 575 Lys Ala Arg Arg Glu Thr             580 <210> 2 <211> 1746 <212> DNA <213> Homo sapiens <400> 2 atggctgcac ggagctggca ggacgagctg gcccagcagg ccgaggaggg ctcggcccgg 60 ctgcgggaaa tgctctcggt cggcctaggc tttctgcgca ccgagctggg cctcgacctg 120 gggctggagc cgaaacggta ccccggctgg gtgatcctgg tgggcactgg cgcgctcggg 180 ctgctgctgc tgtttctgct gggctacggc tgggccgcgg cttgcgccgg cgcccgcaaa 240 aagcggagga gcccgccccg caagcgggag gaggcggcgg ccgtgccggc cgcggccccc 300 gacgacctgg ccttgctgaa gaatctccgg agcgaggaac agaagaagaa gaaccggaag 360 aaactgtccg agaagcccaa accaaatggg cggactgttg aagtggctga gggtgaagct 420 gttcgaacac ctcaaagtgt aacagcaaag cagccaccag agattgacaa gaaaaatgaa 480 aagtcaaaga aaaataagaa gaaatcaaag tcagatgcta aagcagtgca aaacagttca 540 cgccatgatg gaaaggaagt tgatgaagga gcctgggaaa ctaaaattag tcacagagag 600 aaacgacagc agcgtaaacg tgataaggtg ctgactgatt ctggttcatt ggattcaact 660 atccctggga tagaaaatac catcacagtt accaccgagc aacttacaac cgcatcattt 720 cctgttggtt ccaagaagaa taaaggtgat tctcatctaa atgttcaagt tagcaacttt 780 aaatctggaa aaggagattc tacacttcag gtttcttcag gattgaatga aaacctcact 840 gtcaatggag gaggctggaa tgaaaagtct gtaaaactct cctcacagat cagtgcaggt 900 gaggagaagt ggaactccgt ttcacctgct tctgcaggaa agaggaaaac tgagccatct 960 gcctggagtc aagacactgg agatgctaat acaaatggaa aagactgggg aaggagttgg 1020 agtgaccgtt caatattttc tggcattggg tctactgctg agccagtttc tcagtctacc 1080 acttctgatt atcagtggga tgttagccgt aatcaaccct atatcgatga tgaatggtct 1140 gggttaaatg gtctgtcttc tgctgatccc aactctgatt ggaatgcacc agcagaagag 1200 tggggcaatt gggtagacga agaaagagct tcacttctaa agtcccagga accaattcct 1260 gatgatcaaa aggtctcaga tgatgataaa gaaaagggag agggagctct tccaactggg 1320 aaatccaaaa agaaaaaaaa gaaaaagaag aagcaaggtg aagataactc tactgcacag 1380 gacacagaag aattagaaaa agagattaga gaagaccttc cagtgaatac ctctaaaacc 1440 cgtccaaaac aggaaaaagc tttttccttg aagaccataa gcactagtga tccagccgaa 1500 gtactcgtca aaaatagcca gcctatcaag actcttccac ctgctacttc taccgagcca 1560 tctgtaatct tatcaaaaag tgattctgac aagagctctt cccaagtgcc gccaatacta 1620 caagagacag ataaatccaa gtcaaatacc aagcaaaata gtgtgcctcc ttcacagacc 1680 aagtctgaaa ctagctggga atctcccaaa caaataaaaa agaagaaaaa agccagacga 1740 gaaacg 1746

Claims (10)

A pharmaceutical composition for preventing or treating Parkinson's disease comprising an AEG-1 (astrocyte elevated gene-1) protein or a nucleic acid molecule encoding an AEG-1 protein as an active ingredient.
The pharmaceutical composition for preventing or treating Parkinson's disease according to claim 1, wherein the AEG-1 protein is an amino acid sequence represented by SEQ ID NO: 1.
2. The pharmaceutical composition for preventing or treating Parkinson's disease according to claim 1, wherein the nucleic acid molecule encoding the AEG-1 protein comprises the nucleotide sequence of SEQ ID NO: 2.
The pharmaceutical composition for preventing or treating Parkinson's disease according to claim 1, wherein the nucleic acid molecule encoding the AEG-1 protein is contained in a vector.
5. The pharmaceutical composition according to claim 4, wherein the vector is at least one selected from the group consisting of linear DNA, plasmid DNA, and recombinant viral vectors.
6. The pharmaceutical composition according to claim 5, wherein the recombinant viral vector is Adeno-associated virus (AAV).
A food composition for preventing or ameliorating Parkinson's disease comprising an AEG-1 (astrocyte elevated gene-1) protein or a nucleic acid molecule encoding an AEG-1 protein as an active ingredient.
1) treating the test substance with an AEG-1 (astrocyte elevated gene-1) expressing cell line;
2) measuring the level of expression or activity of AEG-1 in the cell line of step 1); And
3) screening the candidate substance for Parkinson's disease, wherein the level of expression or activity of AEG-1 in step 2) is increased relative to the control not treated with the test substance.
9. The method according to claim 8, wherein the expression level is measured by RT-PCR, competitive RT-PCR, real-time quantitative RT- PCR method, RNase protection method, Northern blotting, and DNA chip technology. 2. The method according to claim 1, wherein the candidate compound is selected from the group consisting of: Screening method.
9. The method of claim 8, wherein the activity level is measured by Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemical staining, Wherein the method is one or more selected from the group consisting of immunochromatography, fluorescence activated cell sorter analysis, and protein chip technology.

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