INFORMATION PROVIDING METHOD FOR DIAGNOSING PARKINSONS DISEASE

Abstract

Provided is an information providing method for diagnosing Parkinson's disease by measuring the amount of any one target selected from the group consisting of a Proteus mirabilis strain, a metabolite produced by the Proteus mirabilis strain, and -synuclein in a biological sample of a subject; a composition comprising a Proteus mirabilis strain as an active ingredient for fabricating a Parkinson's disease animal model; a method for fabricating a Parkinson's disease animal model, the method comprising a step for administering a Proteus mirabilis strain to an animal excluding human; and a method for screening a Parkinson's disease medicine, the method comprising a step for administering a candidate drug of the Parkinson's disease medicine to the Parkinson's disease animal model and a step for observing the degree of mitigation of Parkinson's disease symptoms to determine the treatment effect of the candidate drug on Parkinson's disease.

Claims

1. An information providing method for diagnosing Parkinson's disease, the method comprising: a) measuring the amount of any one target selected from the group consisting of a Proteus mirabilis strain, a metabolite produced by the Proteus mirabilis strain, and -synuclein in a biological sample of a subject; and b) comparing the amount of the target measured in the above step a) with the amount of the target measured from a biological sample of a normal control group which is not suffering from Parkinson's disease.

2. The information providing method of claim 1, further comprising: c) classifying, i) when the amount of the target measured in the above step a) is greater than the amount of the target measured from a biological sample of a normal control group which is not suffering from Parkinson's disease, that the subject develops or has a high risk of developing Parkinson's disease, and classifying, ii) when the amount of the target measured in the above step a) is similar or equal to the amount of the target measured from a biological sample of a normal control group which is not suffering from Parkinson's disease, that the subject does not develop Parkinson's disease.

3. The information providing method of claim 1, wherein the biological sample is tissue, blood, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, urine, colonic tissue or feces.

4. The information providing method of claim 1, wherein the metabolite produced by the Proteus mirabilis strain is lipopolysaccharide (LPS).

5. A composition for fabricating a Parkinson's disease animal model, comprising a Proteus mirabilis strain as an active ingredient.

6. The composition of claim 5, wherein the composition comprises the Proteus mirabilis strain in a concentration of 1 to 11020 CFU/ml (per animal).

7. The composition of claim 6, wherein the composition comprises the Proteus mirabilis strain in a concentration of 2109 CFU/ml (per animal).

8. The composition of claim 5, wherein the composition is orally administered once a day for 3 to 7 days.

9. The composition of claim 5, wherein the composition is a feed composition.

10. A method for fabricating a Parkinson's disease animal model, the method comprising: administering a Proteus mirabilis strain to an animal excluding humans.

11. The method of claim 10, the method comprising: further administering a neurotoxin causing Parkinson's disease, selected from the group consisting of 6-OHDA (6-hydroxydopamine), MPTP (1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine), MPTP/probenecid, rotenone and paraquat.

12. The method of claim 11, wherein the Proteus mirabilis strain and the neurotoxin causing Parkinson's disease are administered to the animal sequentially, simultaneously or intermittently.

13. The method of claim 11, wherein the neurotoxin causing Parkinson's disease is MPTP.

14. The method of claim 13, wherein the MPTP is administered in an amount of 1 to 1000 mg/kg.

15. The method of claim 14, wherein the MPTP is administered in an amount of 15 mg/kg when administered sequentially with the Proteus mirabilis strain.

16. A Parkinson's disease animal model fabricated by the method of claim 10.

17. A method for screening an agent for treating Parkinson's disease, the method comprising: administering a candidate drug of the agent for treating Parkinson's disease to the Parkinson's disease animal model of claim 16; and observing the degree of mitigation of Parkinson's disease symptoms to determine the treatment effect of the candidate drug on Parkinson's disease.

18. The Parkinson's disease animal model of claim 16, wherein the Parkinson's disease animal model further comprises a neurotoxin causing Parkinson's disease, selected from the group consisting of 6-OHDA (6-hydroxydopamine), MPTP (1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine), MPTP/probenecid, rotenone and paraquat.

19. The method of claim 17, wherein the Parkinson's disease animal model further comprises a neurotoxin causing Parkinson's disease, selected from the group consisting of 6-OHDA (6-hydroxydopamine), MPTP (1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine), MPTP/probenecid, rotenone and paraquat.

Description

DESCRIPTION OF DRAWINGS

[0054] FIG. 1 illustrates the immunohistochemical stain results and graphs showing the decrease of dopaminergic neurons in the corpus striatum and the substantia nigra of the brain in a Proteus mirabilis strain administration group as compared with a normal control group;

[0055] FIG. 2 illustrates the immunohistochemical stain results and graphs showing the decrease of dopaminergic neurons in the corpus striatum and the substantia nigra of the brain in an MPTP 15 mg/kg administration group, an MPTP 30 mg/kg administration group, and a Proteus mirabilis strain administration group;

[0056] FIG. 3 illustrates the drawing and graph showing that in a Proteus mirabilis strain administration group, astrocyte (GFAP-positive cells) and microglia (CD11b-positive cells) are activated in the corpus striatum and the substantia nigra of the brain so that cranial nerve inflammation occurs;

[0057] FIG. 4 illustrates the graphs showing that LPS increases in the feces and plasma over time after administering the Proteus mirabilis strain;

[0058] FIG. 5 illustrates the graphs showing that -synuclein increases in the large intestine and the substantia nigra of the brain over time after administering the Proteus mirabilis strain;

[0059] FIG. 6 illustrates the graphs showing the pole test result and the rotarod test result performed on an MPTP 15 mg/kg administration group, an MPTP 30 mg/kg administration group, and a Proteus mirabilis strain administration group;

[0060] FIG. 7 illustrates the graphs showing the open field test result and the rotarod test result performed on a Proteus mirabilis strain administration group;

[0061] FIG. 8 illustrates the graphs showing the decrease in dopamine content and DOPAC content in the corpus striatum of the brain in a Proteus mirabilis strain administration group; and

[0062] FIG. 9 illustrates the graphs showing the measurement of the number of enterobacteria and the number of bacteria of the Proteus mirabilis strain in an MPTP/Proteus mirabilis strain administration group and an MPTP or 6-OHDA administration group.

MODES OF THE INVENTION

[0063] Hereinafter, the present invention will be explained in more detail with reference to the examples. These examples are to explain the present invention more specifically, and do not intend to limit the scope of the present invention.

EXAMPLES

Example 1: Fabrication of Parkinson's Disease Animal Model

[0064] 1-1. Preparation of Test Animal

[0065] A C57 black 6 (C57BL/6 test animal, Daehanbiolink, Republic of Korea) male test animal at 7 weeks of age with a body weight of 25-28 g was purchased, and supplied with sufficient feed and water to adapt to the test environment. The animal test was performed after an adjustment period of about one week.

[0066] 1-2. Separation of Proteus mirabilis Strain

[0067] Fresh feces (about 0.3 g) was obtained from a Parkinson's disease animal model fabricated by intraperitoneally administering 30 mg/kg of MPTP hydrochloride to the test animal once a day for 5 days, and diluted to be cultured in a BL agar medium (for 3 days) or a DHL agar medium (for 2 days) at 37 C. under anaerobic conditions (Nissui Pharmaceutical Co., Japan).

[0068] The cultured strain was identified by using gram staining, sugar utility and 16S rRNA sequencing. As a result of confirming the base sequence (SEQ ID NO: 1) of the 16S rRNA gene of the cultured strain, it could be confirmed that the cultured strain is Proteus mirabilis.

[0069] 1-3. Proteus mirabilis Strain Induced Parkinson's Disease Animal Model

[0070] A Parkinson's disease animal model was fabricated by orally administering the Proteus mirabilis strain cultured in 1-2 above to the test animal in a concentration of 210.sup.9 CFU/ml (per animal) once a day for 5 days (hereinafter, Proteus mirabilis strain administration group).

[0071] 1-4. Fabrication of MPTP Induced Parkinson's Disease Animal Model

[0072] A Parkinson's disease animal model was fabricated by intraperitoneally administering to the test animal 15 mg/kg of MPTP hydrochloride (hereinafter, MPTP 15 mg/kg single administration group) or 30 mg/kg of MPTP hydrochloride (hereinafter, MPTP 30 mg/kg single administration group) once a day for 5 days. The control group was administered with the same amount of sterilized physiological saline by the same method.

[0073] 1-5. Fabrication of MPTP/Proteus Mirabilis Strain Induced Parkinson's Disease Animal Model

[0074] In order to find out the effect of the Proteus mirabilis strain in increasing toxicity of MPTP, a Parkinson's disease animal model was fabricated by intraperitoneally administering 15 mg/kg of MPTP hydrochloride to the test animal once a day for 5 days simultaneously with orally administering the Proteus mirabilis strain in a concentration of 210.sup.9 CFU/ml (per animal) (hereinafter, MPTP/Proteus mirabilis strain administration group).

[0075] 1-6. Fabrication of MPTP/Probenecid Induced Parkinson's Disease Animal Model

[0076] 25 mg/kg of MPTP hydrochloride was intraperitoneally administered to the test animal 30 minutes after dissolving probenecid in 5% NaHCO.sub.3 and intraperitoneally administering the same in an amount of 100 mg/kg. A Parkinson's disease animal model was induced by administering the same a total of 10 times at an interval of 3.5 days (hereinafter. MPTP/p administration group). The control group was administered with the same amount of sterilized physiological saline by the same method.

[0077] 1-7. Fabrication of 6-OHDA (6-Hydroxydopamine) Induced Parkinson's Disease Animal Model

[0078] A Parkinson's disease animal model was fabricated by diluting 16 g of 6-OHDA in 2 l of 0.1% ascorbic acid, and injecting the same to the test animal once through stereotaxic injection at the corpus striatum site (AP +0.5, ML +2.0, DV 3.0 according to The Mouse Brain in Stereotaxic Coordinates second edition) at a rate of 0.5 l/minute (hereinafter, 6-OHDA administration group:). The control group was injected once with the same amount of sterilized physiological saline by the same method.

[0079] 1-8. Fabrication of Rotenone Induced Parkinson's Disease Animal Model

[0080] A Parkinson's disease animal model was fabricated by intravenously administering to the test animal a solution in which 2 mg/kg of rotenone was dissolved in a solution diluting dimethylsulfoxide (DMSO) and polyethylene glycol (PEG) in a ratio of 1:1 using an osmotic mini pump once a day for 35 days. The control group was injected with the same amount of a 1:1 diluted solution of DMSO and PEG by the same method (Nat Neurosci. 2000 December; 3(12):1301-6).

[0081] 1-9. Fabrication of Paraquat Induced Parkinson's Disease Animal Model

[0082] A Parkinson's disease animal model was fabricated by intraperitoneally administering to the test animal a solution in which 10 mg/kg of paraquat was dissolved in sterilized physiological saline a total of three times for three weeks at an interval of once a week. The control group was injected with the same amount of sterilized physiological saline by the same method (Neurobiol Dis. 2002 July; 10(2):119-27).

Example 2: Confirmation of Likelihood of Occurrence of Parkinson's Disease by Proteus mirabilis Strain

[0083] In order to confirm the likelihood of the occurrence of Parkinson's disease by Proteus mirabilis strain, tests were performed to evaluate the damage to dopaminergic neurons, the degree of expression of inflammation, and the degree of expression of -synuclein in the Parkinson's disease animal models fabricated in Examples 1-3 to 1-5 above.

[0084] 2-1. Confirmation of Damage to Dopaminergic Neuron

[0085] In order to evaluate the degree of damage to dopaminergic neurons in the Parkinson's disease animal model, an immunohistochemical staining test was performed.

[0086] After anesthetizing each Parkinson's disease animal model fabricated according to the methods in Examples 1-3 to 1-5 above and conducting perfusion, the brain was extracted and the brain tissue was fixed with 4% PFA. Thereafter, the brain tissue subjected to a postfixation process was sliced with a thickness of 30 m using a frozen slicer and fixed to a slide. Each tissue of the corpus striatum and the substantia nigra was immunostained using a tyrosine hydroxylase (TH) antibody, and colored using diaminobenzidine. The optical density (OD) of TH positive cells in the corpus striatum and the number of TH positive cells in the substantia nigra were counted to quantify the degree of damage to dopaminergic neurons. The results are shown in FIGS. 1 and 2.

[0087] As illustrated in FIG. 1, it has been confirmed that the dopaminergic neurons decreased in the corpus striatum and the substantia nigra of the Proteus mirabilis strain administration group as compared with the normal control group. Also, as illustrated in FIG. 2, the MPTP/Proteus mirabilis strain administration group showed a higher degree of damage to dopaminergic neurons in the corpus striatum and the substantia nigra of the brain as compared with the MPTP 15 mg/kg single administration group. It was confirmed that this is a level similar to the MPTP 30 mg/kg single administration group.

[0088] 2-2. Confirmation of Expression of Cranial Nerve Inflammation

[0089] In order to confirm the expression of cranial nerve inflammation in the Proteus mirabilis strain administration group, an immunohistochemical staining test was performed.

[0090] The tissues of the corpus striatum and the substantia nigra obtained from the Proteus mirabilis strain administration group were immunostained using a glial fibrillary acidic protein (GFAP) and a CD11b antibody, respectively, and colored using diaminobenzidine. The results are shown in FIG. 3.

[0091] As illustrated in FIG. 3, it has been confirmed from the activation of the astrocyte (GFAP-positive cell) and the microglia (CD11b-positive cell) in the corpus striatum and the substantia nigra of the Proteus mirabilis strain administration group that cranial nerve inflammation occurred.

[0092] Also, in order to evaluate the degree of expression of inflammation in the Proteus mirabilis strain administration group, the amount of LPS was measured.

[0093] After administering the Proteus mirabilis strain to the test animal, feces were collected on the 1.sup.st, 8.sup.th, 16.sup.th and 32.sup.nd day after administration. Blood was collected 24 hours after completion of the motor ability test. From the feces and plasma, LPS was quantified according to the user manual of the LAL quantifying kit (Cape Cod Inc., Falmouth, U.S.A.). The results are shown in FIG. 4.

[0094] As illustrated in FIG. 4, it has been confirmed that the amount of LPS increased statistically significantly on the 1.sup.st, 8.sup.th and 16.sup.th day after administering the Proteus mirabilis strain and on the 16.sup.th and 32.sup.nd day after administering the Proteus mirabilis strain in the feces and the plasma, respectively, as compared with the normal control group.

[0095] 2-3. Confirmation of Expression of -Synuclein

[0096] In order to evaluate the degree of expression of -synuclein in the substantia nigra of the brain and the intestinal tissue in the Proteus mirabilis strain administration group, western blot was performed.

[0097] After treating the tissue of the large intestine and the substantia nigra of the brain extracted from the Proteus mirabilis strain administration group with an -synuclein primary antibody (BD Biosciences. USA) 1:1000), the degree of expression was confirmed with LAS-4000 mini system (Fujifilm Corp., Japan), and quantified using Image J software (National Institute of Health. USA). The results are shown in FIG. 5.

[0098] As illustrated in FIG. 5, it has been confirmed that in the Proteus mirabilis strain group as compared with the normal group, the amount of -synuclein has a tendency of increasing on the 8.sup.th, 16.sup.th and 32.sup.nd day after administering the Proteus mirabilis strain and on the 1.sup.th, 8.sup.th, 16.sup.th and 32.sup.nd day after administering the Proteus mirabilis strain in the substantia nigra of the brain and the large intestine, respectively, as compared with the normal control group. In particular, it has been confirmed that the level increased statistically significantly on the 16.sup.th day after administering the Proteus mirabilis strain and on the 32.sup.nd day after administering the Proteus mirabilis strain in the substantia nigra of the brain and the large intestine, respectively, as compared with the normal control group.

[0099] 2-4. Motor Ability Behavior Evaluation

[0100] In order to evaluate behavior disorder, a pole test, an open field test and a rotarod test were performed on the Parkinson's disease animal models fabricated in Examples 1-3, 1-4 and 1-5.

[0101] Specifically, for the pole test, on the 16.sup.th day after single administration of 15 mg/kg of MPTP, administration of 15 mg/kg of MPTP and Proteus mirabilis strain, and single administration of 30 mg/kg of MPTP, the test animal was placed on a pole having a width of 0.8 cm at a height of 55 cm facing the sky, and then the total time required (T-LA time) for the test animal to get to the floor after being rotated was measured.

[0102] Further, in order to evaluate the motor deficit and balance maintenance of the test animal, a rotarod test was performed. The rotarod device used for the test is a rotatable cylindrical rod with five partitions having a diameter of 7 cm and a height of 60 cm at an interval of 15 cm. The latency time (sec) from the time of placing the test animal on a rod rotating at a rate of 20 rpm until the test animal falls off was measured by a rotarod. All test animals were subjected to the test after sufficient training, and the average value was set to be the latency time. The maximum measurement time was limited to 300 seconds. The results are shown in FIGS. 6 and 7.

[0103] In addition, in order to evaluate the walking activity level of the test animal, an open field test was performed. The open field test device used for the test is an acryl box with a white bottom, which has a width of 40 cm, a length of 25 cm, and a depth of 18 cm. In order to evaluate the walking activity level according to the instinctive nocturnal habit of a mouse, the test was performed between 9:00 pm and 2:00 am. The test animal was placed in the middle of the acryl box, and the total moving distance (cm) of the test animal was calculated using a viewer system (Viewer. Biobserve) for 30 minutes. The results are shown in FIG. 7.

[0104] As illustrated in FIG. 6, in the case of administering 15 mg/kg of neurotoxic substance MPTP together with the Proteus mirabilis strain, it has been confirmed from the pole test and the rotarod test that motor ability was statistically significantly damaged as compared with the MPTP 15 mg/kg single administration group, and that motor ability was damaged to a level similar to the MPTP 30 mg/kg single administration group.

[0105] Also, as illustrated in FIG. 7, in the case of the Proteus mirabilis single administration group, it has been confirmed from the open field test and the rotarod test that motor ability was statistically significantly damaged as compared with the normal group.

[0106] 2-5. Measurement of the Contents of Dopamine (DA) and Dopamine Metabolite (DOPAC) in the Corpus Striatum

[0107] In order to confirm the content of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC), which is one of dopamine metabolites, in the corpus striatum of the brain, a high performance liquid chromatography (HPLC) was performed on the Parkinson's disease animal model fabricated in Example 1-3.

[0108] Specifically, after homogenizing the tissue of the corpus striatum of the brain obtained by sacrificing the test animal together with 0.2 M perchloric acid, a supernatant obtained by centrifugation (0 C. 14000g) for 20 minutes was used as a sample, and Dionex HPLC was used as a THERMO Hypersil GOLD column (2502.1 mm, 5 m). As a mobile phase, 150 mM of ammonium acetate of pH 4.0, 140 M of ethylene diamine tetra acetic acid, 15% methanol and 5% acetonitrile were used at a rate of 0.2 ml/minute. For data analysis, Chromeleon software (Version 6.40) was used. The contents of dopamine and dopamine metabolite were converted in comparison with the standard and quantified, and the proteins were quantified using the Bradford test method.

[0109] As illustrated in FIG. 8, it has been confirmed that the Proteus mirabilis strain administration group presented the reduced contents of dopamine and DOPAC in the corpus striatum, as compared with the normal control group.

[0110] Through the above tests, it could be confirmed that the Parkinson's disease animal models were fabricated by the Proteus mirabilis strain.

Example 3: Measurement of the Number of Bacteria in Parkinson's Disease Animal Model

[0111] After collecting fresh feces from each test animal, the feces were diluted by 10 times with sterilized PBS and spread on DHL and BL agar plates. Enterobacteriaceae. E. coli. Klebsiella sp. and Proteus sp. strains grew in the DHL agar plate, and the number of bacteria was confirmed through the color and shape of colonies one day after aerobic respirational culturing. The E. coli and Klebsiella sp. strains form red colonies, and the Proteus mirabilis strain forms black colonies. The number of bacteria was measured by selecting colonies having black spots or black colonies from the colonies.

[0112] 3-1. Measurement of the Number of Enterobacteria

[0113] The number of enterobacteria was measured in each of the MPTP 15 mg/kg single administration group, MPTP/p administration group and 6-OHDA administration group. The results are shown in the following table 1 and FIG. 9.

TABLE-US-00001 TABLE 1 MPTP/ Normal Proteus Normal Normal control mirabilis control control group strain group MPTP group 6-OHDA 1.95 10.sup.5 7.4 10.sup.5 2.18 10.sup.3 26.28 10.sup.3 1.6 10.sup.3 7.56 10.sup.3

[0114] As illustrated in FIG. 9, it has been confirmed that the number of bacteria of Enterobacteria strains in the three types of Parkinson's disease animal models of the MPTP/Proteus mirabilis strain administration group, MPTP 30 mg/kg single administration group and 6-OHDA administration group was remarkably greater than that of the normal control group.

[0115] 3-2. Measurement of the Number of Bacteria of Proteus mirabilis Strain

[0116] The number of bacteria of E. coli, Klebsiella sp. and Proteus mirabilis strains in the MPTP/Proteus mirabilis strain administration group was measured. The results are shown in the following table 2 and FIG. 9.

TABLE-US-00002 TABLE 2 Number of bacteria in the Number of MPTP/Proteus mirabilis bacteria in control Strain administration group (CFU (10.sup.4) group (CFU (10.sup.4) E. coli 1.0 13.5 Klebsiella sp. 1.66 2 P. mirabilis 62.33 4

[0117] Also, the number of bacteria of the Proteus mirabilis strains in the normal control group and the MPTP 30 mg/kg administration group was measured. The results are shown in the following table 3 and FIG. 9.

TABLE-US-00003 TABLE 3 Number of bacteria of Proteus mirabilis strain Strain (CFU (10.sup.2) Normal control group 7.3 MPTP administration 49.8 group

[0118] As illustrated in FIG. 9, as a result of measuring the number of bacteria of the Proteus mirabilis strain and the number of bacteria of other strains of the Enterobacteriaceae family to which the Proteus mirabilis strain belongs, i.e., E. coli and Klebsiella sp. strains, in the Parkinson's disease animal model, and comparing the same with the normal control group, it has been confirmed that the number of bacteria of the Proteus mirabilis strain was remarkably greater than that of the normal control group, as compared with the two strains.