COMPOSITION FOR DIAGNOSIS OF DEGENERATIVE NEUROLOGICAL DISEASES

Abstract

The present invention relates to a composition for diagnosis of degenerative neurological diseases and, more specifically, to a composition for diagnosis of degenerative neurological diseases, comprising an agent for measuring the level of acetylation of COX2.

According to the present invention, since acetylation of COX2 in degenerative neurological diseases is significantly reduced, whether COX2 is acetylated may be utilized as a diagnostic marker for degenerative neurological diseases, and it is possible to diagnose degenerative neurological diseases more rapidly and accurately by using same.

Claims

1. A composition for diagnosis of degenerative neurological diseases, comprising an agent for measuring the level of acetylation of cyclooxygenase-2 (COX2).

2. The composition of claim 1, wherein the acetylation is acetylation at serine 565 (S565) of COX2 represented by SEQ ID NO: 1.

3. The composition of claim 1, wherein the agent is an antibody, a fragment of the antibody, or an aptamer that specifically binds to the acetylated COX2.

4. The composition of claim 3, wherein the antibody is an antibody that specifically binds to an epitope comprising an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 80% or more sequence identity thereto.

5. The composition of claim 1, wherein the degenerative neurological disease is at least one selected from the group consisting of Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, multi-system atrophy, olivopontocerebellar atrophy (OPCA), Shire-Dragger syndrome, striatonigral degeneration, Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremor, corticobasal degeneration, diffuse Lewy body disease, Parkin's-ALS-dementia complex, Niemann-Pick disease, Pick disease, cerebral ischemia, and cerebral infarction.

6. A kit for diagnosis of degenerative neurological diseases, comprising an agent for measuring the level of acetylation of cyclooxygenase-2 (COX2).

7. A method for providing information for diagnosis of degenerative neurological diseases, comprising: (a) providing a biological sample from a patient suspected of degenerative neurological diseases; (b) measuring the level of acetylation of COX2 in the sample; and (c) diagnosing degenerative neurological diseases when the level of acetylation of the COX2 is lower than that of a normal control sample.

8. The method of claim 7, wherein the method for measuring the level of acetylation of COX2 is at least one selected from the group consisting of autoradiography, liquid scintillation counting, molecular weight assay, liquid chromatographic mass assay, Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunofluorescence staining, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip.

9. The method of claim 7, wherein the sample is selected from the group consisting of blood, blood cells, brain tissue, nerve cells, cerebrospinal fluid, saliva, nasal fluid, sputum, synovial fluid, amniotic fluid, ascites, cervical or vaginal secretions, and urine.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIGS. 1a to 1f illustrate results of showing that SphK1 acetylates S565 of COX2 as an acetyltransferase.

[0050] FIGS. 1a and 1b illustrate results of evaluating binding (a) and dissociation (b) of SphK1 and acetyl-CoA.

[0051] FIG. 1c illustrates a result of evaluating acetylation of COX2 in the presence of SphK1, acetyl-CoA, and sphingosine ([.sup.14C] aspirin-treated test group is set as a positive control).

[0052] FIG. 1d illustrates a result of confirming changes in molecular weight by acetylation of COX2 in the presence of SphK1, acetyl-CoA and/or sphingosine using LC-MS/MS.

[0053] FIG. 1e illustrates a result of confirming that acetylation of COX2 at a residue S565 occurs in the presence of SphK1, acetyl-CoA and/or sphingosine using LC-MS/MS.

[0054] FIG. 1f illustrates a result of confirming that the acetylation does not occur well when S565 of a COX2 protein is mutated.

[0055] FIGS. 2a to 2d illustrate results of showing that neuroinflammatory resolution factor are decreased by reduction of acetylation of COX2 when SphK1 has been inhibited.

[0056] FIG. 2a illustrates a result of evaluating the expression of SphK1 and SphK2 when SphK1 siRNA has been treated to wild-type (WT) nerve cells.

[0057] FIG. 2b illustrates a result of confirming the acetylation of a COX2 protein when SphK1 siRNA has been treated in nerve cells [.sup.14C] aspirin-treated wild-type nerve cells are set as a positive control).

[0058] FIG. 2c illustrates a result of confirming the secretion of neuroinflammatory resolution factor (SPMs) such as LxA4, RvE1, and RvD1 when SphK1 siRNA has been treated in nerve cells.

[0059] FIG. 2d illustrates a result of confirming the secretion of 15-R-LxA4 using LC-MS/MS when SphK1 siRNA has been treated in nerve cells.

[0060] FIGS. 3a to 3c illustrate results of confirming that in APP/PS1 mice, acetylation of COX2 and secretion of neuroinflammatory resolution factor (SPMs) have been reduced and this reduction has been improved by overexpression of SphK1.

[0061] FIG. 3a illustrates a result of performing an acetylation assay of a COX2 protein in nerve cells derived from wild-type, APP/PS1, APP/PS1/SphK1 tg, and SphK1 tg mice. [14c] asprine-treated nerve cells have been set as a positive control.

[0062] FIG. 3b illustrates a result of measuring protein amounts of LxA4 and RvE1 in CM of nerve cells derived from wild-type, APP/PS1, APP/PS1/SphK1 tg, and SphK1 tg mice.

[0063] FIG. 3c illustrates a result of specifying an amount of 15-R-LxA4 using LC-MS/MS in nerve cells derived from wild-type, APP/PS1, APP/PS1/SphK1 tg, and SphK1 tg mice.

[0064] FIGS. 4a to 4c are results of confirming that neuroinflammatory resolution factor secreted by increased SphK1 in APP/PS1 mice reduce neuroinflammation.

[0065] FIG. 4a illustrates immunofluorescent images (left) of microglia (Iba1) in the brain cortex of wild-type, APP/PS1, APP/PS1/SphK1 tg and SphK1 tg mice and results (right) of quantifying the immunofluorescent images.

[0066] FIG. 4b illustrates immunofluorescent images (left) of astrocytes (GFAP) in the brain cortex of mice and results (right) of quantifying the immunofluorescent images.

[0067] FIG. 4c illustrates a result of evaluating mRNA expression levels of inflammatory markers M1 and M2 in the brain of mice (Marker M1: TNF-a, IL-1b, IL-6 and iNOS, Immunomodulatory factor: IL10, Marker M2: IL-4, TGF-b and Arg1).

[0068] FIGS. 5a to 5f illustrate results of confirming that neuroinflammatory resolution factor secreted by increased SphK1 improve the phagocytosis of microglia.

[0069] FIG. 5a illustrates a result of confirming microglia around A plaques in the brain cortex of APP/PS1 and APP/PS1/SphK1 tg mice.

[0070] FIG. 5b illustrates a result of confirming the phagocytic ability of microglia in the brain cortex of wild-type, APP/PS1, APP/PS1/SphK1 tg and SphK1 tg mice.

[0071] FIG. 5c illustrates a result of confirming that A plaques are digested within lysosomes of microglia in the brain cortex of APP/PS1 and APP/PS1/SphK1 tg mice.

[0072] FIGS. 5d and 5e illustrate results of confirming the expression of A degrading enzymes NEP, MMP9 and IDE and a phagocytic marker CD36 in microglia of wild-type, APP/PS1, APP/PS1/SphK1 tg, and SphK1 tg mice.

[0073] FIG. 5f illustrates a result of confirming sizes of A plaques where phagocytosis occurs in the brain cortex of APP/PS1 and APP/PS1/SphK1 tg mice.

[0074] FIGS. 6a to 6i illustrate results of showing that neuroinflammatory resolution factor secreted by increased SphK1 in APP/PS1 mice reduce AD lesions.

[0075] FIG. 6a illustrates a diagram (left) showing immunofluorescence staining of thioflavin S (ThioS, A plaques) in the brain medulla and hippocampus of APP/PS1 and APP/PS1/SphK1 tg mice and a result (right) of quantifying areas occupied by A plaques (n=6/group).

[0076] FIGS. 6b and 6c illustrate results of analyzing the accumulation of A1340 and A1342 in the mouse's brain by immunofluorescence staining (b) or an ELISA kit (c).

[0077] FIG. 6d illustrates a result of quantifying vasculopathy.

[0078] FIG. 6e illustrates a result of quantifying a tau protein.

[0079] FIGS. 6f to 6i illustrate results of showing immunofluorescence staining and quantification of synaptophysin (f), MAP2 (g), synapsin 1 (h) or PSD95 (i) in the brain cortex of each animal group.

[0080] FIGS. 7a to 7h are diagrams illustrating that an increase in neuroinflammatory resolution factor secreted by SphK1 overexpression in APP/PS1 mice has recovered a cognitive function.

[0081] FIG. 7a illustrates a result of evaluating learning and memory through a Morris Water Maze test of wild-type (n=14), APP/PS1 (n=12), APP/PS1/SphK1 tg (n=12), and SphK1 tg (n=13) mice.

[0082] FIGS. 7b to 7d illustrate results of measuring time spent in a target platform on day 11 (b), measuring time spent in other quadrants (c), and measuring a path length, a swim speed, and the number of times when each animal enters a small target area for 60 seconds (d).

[0083] FIG. 7e illustrates a swim path on day 10 of the test.

[0084] FIG. 7f illustrates a result of contextual and tone tasks during a fear conditioning test.

[0085] FIG. 7g illustrates a result of showing time spent on a wall side and a center region during an open field test and a ratio of the center region.

[0086] FIG. 7h illustrates movement paths of mice during the open field test.

[0087] FIGS. 8a to 8d are diagrams illustrating that a COX2 acetylating agent produced by SphK1 promotes the secretion of neuroinflammatory resolution factor.

[0088] FIG. 8a is a diagram illustrating a chemical structure of a COX2 acetylating agent.

[0089] FIG. 8b illustrates a result of confirming the secretion of neuroinflammatory resolution factor (SPMs) by treating a COX2 acetylating agent.

[0090] FIG. 8c illustrates a result of confirming that N-acetyl sphingosine causes COX2 acetylation.

[0091] FIG. 8d illustrates a result of confirming that acetylation of COX2 at a residue S565 occurs in the presence of N-acetyl sphingosine using LC-MS/MS.

[0092] FIGS. 9a to 9d are diagrams illustrating that a COX2 acetylating agent promotes secretion of neuroinflammatory resolution factor to reduce AD lesions of an Alzheimer's animal models.

[0093] FIG. 9a illustrates immunofluorescence images (left) of microglia (Iba1) in the brain cortex of wild-type, APP/PS1, and APP/PS1 mice injected with N-acetyl sphingosine, FTY720 (sphingosine derivative) and S1P and results (right) of quantifying the immunofluorescence images.

[0094] FIG. 9b illustrates immunofluorescence images (left) of astrocytes (GFAP) in the brain cortex of mice and results (right) of quantifying the immunofluorescence images.

[0095] FIG. 9c is a diagram (top) illustrating immunofluorescence staining of Thioflavin S (ThioS, A plaques) in the brain medulla and hippocampus of APP/PS1 and APP/PS1 mice injected with N-acetyl sphingosine, FTY720 (sphingosine derivative) and S1P and illustrates a result (bottom) of quantifying areas occupied by A.

[0096] FIG. 9d illustrates a result of evaluating learning and memory of wide-type, APP/PS1, and APP/PS1 mice injected with N-acetyl sphingosine, FTY720 (sphingosine derivative) and S1P through a Morris Water Maze test.

[0097] FIG. 10 illustrates a result of confirming that an ac-S565 antibody specifically detects the acetylation of a S565 residue of COX2 (N-AS: N-acetyl sphingosine).

[0098] FIG. 11a illustrates a result of confirming the levels of acetylation of a COX2 protein and S565 of COX2 expressed in microglia derived from wild-type (WT) and APP/PS1 mice through fluorescence immunostaining, and FIG. 11b illustrates a result of quantifying the levels (n=6/group).

[0099] FIG. 12a illustrates a result of confirming the levels of acetylation of a COX2 protein and S565 of COX2 expressed in microglia derived from wild-type and APP/PS1 mice through Western blotting, and FIG. 12b illustrates a result of quantifying the levels (n=6/group).

[0100] FIG. 13a illustrates a result of confirming the levels of acetylation of a COX2 protein and S565 of COX2 expressed in microglia derived from human treated with amyloid beta through fluorescence immunostaining, and FIG. 13b illustrates a result of quantifying the levels (n=4/group).

[0101] FIG. 14a illustrates a result of confirming the levels of acetylation of a COX2 protein and S565 of COX2 expressed in microglia derived from human treated with amyloid beta through Western blotting, and FIG. 14b illustrates a result of quantifying the levels (n=4/group).

[0102] FIG. 15a illustrates a result of confirming the levels of acetylation of a COX2 protein and S565 of COX2 expressed in blood cells derived from wild-type and APP/PS1 mice through Western blotting, and FIG. 15b illustrates a result of quantifying the levels (n=3/group).

MODE FOR CARRYING OUT INVENTION

[0103] Hereinafter, the present invention will be described in detail.

[0104] However, the following Examples are just illustrative of the present invention, and the contents of the present invention are not limited to the following Examples.

[0105] Experiment Method

[0106] 1. Mouse

[0107] A mouse test was approved by the Kyungpook National University Institutional Animal Care and Use Committee (IACUC). Based on C57BL/6 mice (Charles River, UK), a transgenic mouse line overexpressing APPswe (hAPP695swe) or PS1 (presenilin-1M146V) was used [hereinafter, APP mouse: mouse overexpressing APPswe, PS1 mouse: mouse overexpressing presenilin-1M146V; GlaxoSmithKline]

[0108] SphK1 tg (SphK1 gene overexpressing mice) was crossed with APP mice and APP/PS1 mice to prepare APP/PS1/SphK1 tg mice.

[0109] 2. Isolation of Monocytes into Mouse Blood

[0110] After the blood from a mouse was collected through heart blood collection, ammonium chloride was added in an amount equal to 10 times of the amount of the collected blood to dissolve red blood cells in the blood. The blood from which the red blood cells were dissolved was centrifuged to remove a supernatant, and then monocytes were isolated.

[0111] 3. Preparation of ac-S565 Antibody

[0112] In order to prepare an antibody capable of detecting whether serine 565 (S565) of a COX2 protein of SEQ ID NO: 1 was acetylated, a peptide of a N-GCPFTS.sup.acFSVPD-C sequence represented by SEQ ID NO: 2 was prepared and conjugated with a carrier protein (keyhole limpet hemocyanin), and then the prepared peptide was immunized to rabbits according to an immune progression table shown in Table 1 below to prepare a rabbit polyclonal antibody.

TABLE-US-00002 TABLE 1 Day 1 Pre-immune serum collection Day 0 Primary immunization Day 14 1.sup.st boost Day 21 Test beleed and ELISA test Day 35 2.sup.nd boost Day 42 Product on bleed Day 56 3.sup.rd boost Day 63 Production teed Day 77 ELISA [0113] Time [0114] Step

[0115] 4. Treatment of SphK siRNA

[0116] SphK1 siRNA (Dharmacon SMART pool) and a siRNA control (Dharmacon) were treated on nerve cells of E18 C57BL/6 mice for 48 hours. The nerve cells were collected and the acetylation and neuroinflammatory resolution factor were analyzed.

[0117] 5. Immunofluorescence

[0118] After the cerebrum and the hippocampus of a mouse were immobilized, anti-20G10 against amyloid- (A) 42 (mouse, 1:1000) and anti-G30 against A 40 (rabbit, 1:1000), anti-MAP2 (chicken, 1:2000), anti-Synaptophysin (mouse, 1:100), anti-Synapsin1 (rabbit, 1:500), anti-PSD95 (mouse, 1:100), anti-Iba-1 (rabbit, 1:500), and anti-GFAP (rabbit, 1:500) were incubated together. The sites were analyzed using a confocal laser scanning microscope or an Olympus BX51 microscope equipped with Fluoview SV1000 imaging software (Olympus FV1000, Japan). Percentages of areas of the stained sites to an area of total tissues were quantified using Metamorph software (Molecular Devices).

[0119] In addition, cerebral tissues of wild-type and APP/PS1 9-month-old mice and human-derived microglia were incubated together with anti-ac-S565 (rabbit, 1:100), anti-COX2 (mouse, 1:500, Thermo Fisher Scientific), and anti-Iba1 (goat, 1:500, Abcam). In the cerebral tissues and the human-derived microglia, percentages of cells stained with anti-ac-S565, anti-COX2 and anti-Iba1 among cells stained with anti-COX2 and anti-Iba1 were quantified and analyzed using MetaMorph (Molecular Devices, USA).

[0120] 6. Quantitative Real-Time PCR

[0121] RNA was extracted according to a manufacturer's manual using a commercially available RNeasy kit (QIAGEN). cDNA was synthesized from 5 g of total RNA using a commercially available cDNA kit (Takara Bio Inc.). Quantitative real-time PCR was performed using a Corbett research RG-6000 real-time PCR instrument.

[0122] 7. Western Blot

[0123] The expression of the proteins was analyzed using Western blotting. First, antibodies against CD36 (Novus biolobicals) and -actin (Santa Cruz) were used, and densitometric quantification was performed using ImageJ software (US National Institutes of Health).

[0124] In addition, a Western blot sample was prepared by reacting a recombinant COX2 protein with aspirin and N-acetyl sphingosine (Sigma, 01912, N-AS). In addition, microglia were isolated from the cerebra of wild-type and APP/PS1 9-month-old mice to prepare a Western blot sample, and microglia (Applied Biologics Materials, USA) were incubated with amyloid beta, and then a Western blot sample was prepared. The prepared Western blot sample was subjected to electrophoresis on a polyacrylamide gel to isolate proteins, transferred to a nitrocellulose filter, and then bound with anti-ac-S565 (rabbit, 1:500), anti-COX2 (rabbit, 1:1,000, Abcam), and anti-actin (mouse, 1:1,000, Santa Cruz) antibodies, and then bands were shaped using secondary antibodies, and quantified and analyzed using ImageJ (National Institutes of Health, USA).

[0125] 8. Immunoenzyme Assay

[0126] A commercially available ELISA kit (Biosource) was used, and the hemispheres of mice were homogenized and added in a buffer containing 0.02 M of guanidine to prepare a sample for A ELISA. In order to measure neuroinflammatory resolution factor, nerve cells from the mouse cerebrum were incubated to prepare conditioned media (CM). Thereafter, according to the manufacturer's manual, ELISA for A and SPM was performed.

[0127] 9. Behavioral Test

[0128] In order to confirm potential effects on learning and memory, Morris water maze (MWM) and fear conditioning tests were performed. In the MWM, the mouse learned a task 4 times a day for 10 days, a platform was removed on day 11, and a probe trial was performed. In the fear conditioning, on day 1, the mouse was added in a conditioning chamber, and sound stimulation (10 kHz, 70 dB) and electrical stimulation (0.3 mA, 1 s) were applied. On day 2, the memory on a space was confirmed without stimulation in the same conditioning chamber as day 1, and on day 3, a memory test for fear was performed when only the sound stimulation was applied in another conditioning chamber. An open field test was performed to evaluate motor ability and immediate activity. In the open field test, the mouse was placed in a quadrangular box for 10 minutes and then overall motor ability and time and distances were measured.

[0129] 10. Method for Measuring Level of COX2 Acetylation

[0130] A COX2 protein of nerve cells reacted for 1 hour at 37 C. in the presence of [.sup.14C] acetyl-CoA was isolated by immunoprecipitation, and then liquid scintillation counting was performed on [.sup.14C].

[0131] 11. Enzymatic Analysis of Acetyltransferase

[0132] The acetyl-CoA binding activity of SphK1 was analyzed by filter binding assay in the presence of 10 mM sphingosine. The binding rate (Vbinding) of [.sup.3H] acetyl-CoA for SphK1 was expressed as an acetyl-CoA concentration. Nonlinear regression analysis of a saturation plot showed acetyl-CoA and SphK1 binding activities using K.sub.cat (catalyst constant) and K.sub.M (Michaelis-Menten constant).

[0133] 12. LC-MS/MS

[0134] In order to confirm the relationship between the secretion of SphK1 and the secretion of neuroinflammatory resolution factor in nerve cells, the nerve cells were isolated from 9-month-old WT, APP/PS1, APP/PS1/SphK1 tg, and SphK1 tg mice. The nerve cells were sonicated and incubated with 2.5 mM acetyl-CoA (Sigma) (24 hours, 37 C.). In addition, CM was harvested from nerve cells treated with SphK1 siRNA or control siRNA. 200 l aliquots of each cell lysate or CM were mixed with 100 g/ml 100 l of a 15-S-LxA4-d5 (internal standard, Cayman chemical) solution, 100 l of a 1% formic acid solution, and 600 l of water, and then added with 4 ml of ethyl acetate. After vortexing and centrifuging (13,200 rpm), the mixture was frozen in a deep freezer for each 10 minutes and 2 hours. An organic supernatant was separated and dried under a nitrogen stream. The remaining solution was reconstituted with a 60% acetonitrile solution injected into an LC-MS/MS system. This sample was subjected to 15-R-LxA4 concentration analysis using an Agilent 6470 Triple Quad LC-MS/MS system (Agilent, Wilmington, Del., USA) connected to an Agilent 1290 HPLC system.

[0135] To confirm an acetylation site of COX2, a COX2 enzyme was precipitated with trichroloacetic acid (Merck) and dried. The dried extract was resuspended in 10 L of a 5 M urea solution, and a 0.1 M ammonium bicarbonate buffer was incubated at 37 C. with 1 g trypsin (Promega) for 16 hours. Thereafter, the sample was treated with 1 M DTT (GE Healthcare) at room temperature for 1 hour and then alkylated with 1M iodoacetamide (Sigma) for 1 hour. A protein sample was loaded onto a ZORBAX 300SB-C18 column for sequencing. Peptides were identified with BioTools 3.2 SR5 (Bruker Daltonics).

[0136] 13. Treatment of COX2 Acetylating Agent

[0137] In order to measure neuroinflammatory resolution factor, nerve cells from the mouse's cerebrum were incubated and then treated with 10 nM N-acetyl sphingosine 1 phsosphate (Toronto Research chemicals, C262710) and N-acetyl sphingosine (Sigma, 01912) to prepare CM. For COX2 acetylation analysis, nerve cells were incubated from the mouse's cerebrum and treated with 2uCi [.sup.14C] N-acetyl sphingosine (ARC, ARC1024) and then prepared. In addition, for an in vivo experiment, 5 mg/kg N-acetyl sphingosine (Sigma, 01912), 1 mg/kg FTY720, and 3 uM S1P were injected into 7-month-old APP/PS1 mice daily for 4 weeks through intraperitoneal injection.

[0138] 14. Statistical Analysis

[0139] For comparison of two groups, a T-test of students was performed, while for comparison of multiple groups, repeated measurement analysis of a Tukey's HSD test and a variance test was performed according to an SAS statistical package (release 9.1; SAS Institute Inc., Cary, N.C.). *p<0.05 and **p<0.01 were considered to be significant.

[0140] Experiment Results

[0141] 1. SphK1 Induces Acetylation at an S565 Residue of COX2 as an Acetyltransferase.

[0142] In order to confirm the acetyltransferase activity of SphK1, analysis of binding and dissociation of an acetyl groups from an enzyme was performed. The binding of the acetyl group to SphK1 was saturated as the concentration of acetyl-CoA was increased, and K.sub.M and K.sub.cat values were 58.2 m and 0.0185 min.sup.1, respectively (FIG. 1a). After an equilibrium dialysis test, the bound acetyl group was also dissociated from an acetyl-CoA:SphK1 complex in the presence of concentration-dependently competitive free acetyl-CoA. This dissociation of acetyl-CoA and SphK1 was saturated at a high inhibitor concentration and showed a K.sub.D value of 6.8 m (FIG. 1b). A K.sub.D (i.e., dissociation constant) value lower than a K.sub.M value (i.e., binding affinity) represented acetyltransferase properties of SphK1.

[0143] Next, in order to confirm the acetyltransferase activity of SphK1 in relation to COX2, purified SphK1 was incubated with COX2 and [.sup.14C] acetyl-CoA in the presence or absence of sphingosine to measure the acetylation. In addition, aspirin, known to cause acetylation at a COX2 S516 residue, was used as a positive control to confirm the level of acetylation. Referring to the results of FIG. 1c, it can be seen that SphK1 induces a higher level of acetylation for COX2 than aspirin in the presence of Sphingosine, which indicates that SphK1 exhibits acetyltransferase activity to acetylate COX2 through a sphingosine or sphingosine intermediate (FIG. 1c).

[0144] Finally, SphK1, acetyl-CoA and sphingosine were treated to COX2 in order to confirm an acetylation position of COX2 acetylated by SphK1.

[0145] As such, COX2 treated with SphK1, acetyl-CoA and sphingosine had an acetyl group, and COX2 treated without sphingosine had no acetyl group. In addition, it was confirmed that serine 565 (S565) for a peptide 560-GCPFTSFSVPDPELIK-575 of COX2 was acetylated in the presence of SphK1 (FIGS. 1d and 1e). In order to establish its causal relationship, the present inventors mutated S565 of COX2 to an Ala 565 residue (S565A) and then performed acetylation analysis. Wild-type COX2 was acetylated by SphK1 and sphingosine, but S565A-mutated COX2 had reduced acetylation in the presence of SphK1, and these results indicate that S565 of COX2 is a major target site for SphK1-mediated COX2 acetylation (FIG. 1e. In particular, it was confirmed that the position of the acetylation of COX2 S565 by SphK1 was different from the position (S516) acetylated with aspirin.

[0146] 2. When SphK1 has been Inhibited in Nerve Cells, a Decrease in Secretion of Neuroinflammatory Resolution Factor is Caused by Reduction of COX2 Acetylation.

[0147] In order to more directly confirm the correlation between SphK1 and COX2 acetylation in nerve cells, wild-type nerve cells were treated with SphK1 siRNA and COX2 acetylation was confirmed. It was confirmed that the COX2 acetylation was reduced in nerve cells treated with SphK1 siRNA (FIG. 2b).

[0148] Next, changes in neuroinflammatory resolution factor by COX2 acetylation were observed. It was confirmed that LxA4 and RvE1, neuroinflammatory resolution factor, were reduced in CM derived from nerve cells treated with SphK1 siRNA (FIG. 2c).

[0149] In addition, when the neuroinflammatory resolution factor were measured using LC-MS/MS, 15-R-LxA4 produced by COX2 acetylation was reduced in the nerve cells treated with SphK1 siRNA (FIG. 2d). That is, when SphK1 was inhibited, it could be confirmed that the neuroinflammatory resolution factor (especially, 15-R-LxA4) was reduced due to the reduction in COX2 acetylation.

[0150] 3. In an Alzheimer's Animal Model, COX2 Acetylation and the Secretion of Neuroinflammatory Resolution Factor have been Reduced, which was Improved by SphK1 Overexpression.

[0151] The present inventors treated [.sup.14C] acetyl-CoA to nerve cells isolated from 9-month-old mice and analyzed the level of acetylation by purifying COX2 in order to confirm whether the results shown after treatment of SphK1 siRNA also occurred in an Alzheimer's animal model. Compared with wild-type mice, a low level of COX2 acetylation was shown in the nerve cells of APP/PS1 mice, and the acetylation of COX2 was increased in the nerve cells of APP/PS1/SphK1 tg mice (FIG. 3a).

[0152] Expression levels of LxA4 and RvE1 were significantly decreased in CM derived from APP/PS1 nerve cells as compared to those in CM derived from wild-type nerve cells, and recovered in CM derived from APP/PS1/SphK1 tg cells (FIG. 3b). In addition, when the neuroinflammatory resolution factor were measured using LC-MS/MS, 15-R-LxA4 produced by COX2 acetylation was reduced in the Alzheimer's animal model and recovered when SphK1 was overexpressed (FIG. 3c). That is, the COX2 acetylation and the secretion of neuroinflammatory resolution factor were reduced in the Alzheimer's animal model, and it is meant that the reduction can be improved by overexpression of SphK1.

[0153] 4. Increased SphK1 Regulates Neuroinflammation by Secreting Neuroinflammatory Resolution Factor in APP/PS1 Mice.

[0154] The present inventors observed changes in microglia and astrocytes in order to determine an effect of increased SphK1 on neuroinflammatory response by secreting neuroinflammatory resolution factor. The APP/PS1/SphK1 tg mice showed a remarkable decrease in microglia and astrocytes compared to the APP/PS1 mice (FIGS. 4a and 4b). In addition, APP/PS1/SphK1 tg mice showed a decrease in pro-inflammatory M1 markers and immune regulatory cytokines compared to APP/PS1 mice, and the expression of anti-inflammatory M2 markers was increased (FIG. 4c).

[0155] Collectively, these results indicate that SphK1 overexpression can improve the inflammatory response in the AD brain by promoting the secretion of neuroinflammatory resolution factor by inducing the acetylation of COX2.

[0156] 5. Neuroinflammatory Resolution Factor Secreted by SphK1 Overexpression Regulate Ali Phagocytosis of Microglia.

[0157] To determine whether the neuroinflammatory resolution factor secreted by increased SphK1 recover the recruitment of microglia with A, the number of microglia around plaques was quantified. As a result, the recruitment of microglia was increased in APP/PS1/SphK1 tg mice compared to APP/PS1 mice (FIG. 5a).

[0158] Next, a phagocytosis assay was performed using brain pieces. Compared to APP/PS1 mice, the number of microglia exhibiting phagocytosis was increased in APP/PS1/SphK1 tg mice (FIG. 5b). To further determine this effect, the A phagocytosis of microglia was evaluated in vivo. The APP/PS1/SphK1 tg brain had an increased number of microglia stained with lysosomes and A. Importantly, phagolysosomes in microglia were increased in the cortex of APP/PS1/SphK1 tg mice compared to APP/PS1 mice. As a result of analyzing plaque-related microglia, it was found that a ratio of cells containing A incorporated in the phagolysosomes was increased in APP/PS1 mice overexpressing SphK1. The amount of A contained in the phagolysosomes was increased in the brain of APP/PS1/SphK1 tg mice (FIG. 5c).

[0159] Next, the expression of A degrading enzymes such as neprilysin (NEP), matrix metallopeptidase 9 (MMP9), and insulin degrading enzyme (IDE) was analyzed. Although the expression levels of these enzymes were not changed, CD36, which was known to increase when the phagocytosis of microglia occurred, was recovered in APP/PS1/SphK1 tg mice (FIGS. 5d and 5e).

[0160] On the other hand, it is known that the phagocytosis of microglia induces a decrease in an outer part of A compared to a core of the A plaque. In the analysis of the morphology of A plaques, in APP/PS1/SphK1 tg mice, it was confirmed that small (<25 m) plaques were significantly increased, and medium (25 to 50 m) and large (>50 m) plaques were significantly decreased, and thus, the outer part was phagocytosed by microglia (FIG. 5f).

[0161] Through the above results, it was found that the increased SphK1 of the nerve cells increased the acetylation of COX2 to increase the secretion of the neuroinflammatory resolution factor, and as a result, it could be seen that the A phagocytosis of microglia has increased in APP/PS1 mice.

[0162] 6. Neuroinflammatory Resolution Factor Secreted by SphK1 Overexpression Alleviate AD Lesions in Mice.

[0163] In order to find how the neuroinflammatory resolution factor secreted by the increased SphK1 activity in APP/PS1/SphK1 tg mice had affected the lesions of AD, an A profile was firstly identified. As experiment results of thioflavin S (ThioS) staining, immunofluorescence staining, and ELISA of A1340 and A1342, it was shown that A was significantly lowered in APP/PS1/SphK1 tg mice compared to APP/PS1 mice (FIGS. 6a to 6c). In APP/PS1/SphK1 tg mice, amyloid angiopathy of the brain was also reduced (FIG. 6d). Compared to wild-type mice, label densities of synaptophysin, MAP2, synapsin1 and PSD95 were reduced in APP/PS1 mice. However, in APP/PS1/SphK1 tg mice, the label densities were recovered to levels similar to those of the wild-type mice (FIGS. 6f to 6i).

[0164] 7. Neuroinflammatory Resolution Factor Secreted by SphK1 Overexpression Recover Cognitive Function in an Alzheimer's Animal Model.

[0165] The present inventors also performed a Morris Water Maze test and a fear conditioning test to evaluate changes in learning and memory. It was confirmed that the old APP/PS1 mice exhibited serious problems in memory formation, while the APP/PS1/SphK1 tg mice alleviated these problems to some extent (FIGS. 7a to 7f).

[0166] An open field test was performed to evaluate motor ability and immediate activity. The APP/PS1/SphK1 tg mice showed improved motor ability and immediate activity compared to the APP/PS1 mice (FIGS. 7g to 7h).

[0167] Overall, these results indicate that compared to APP/PS1 mice, in APP/PS1/SphK1 tg mice, the expression of SphK1 is increased in nerve cells, resulting in increased acetylation of COX2, and as a result, the accumulation of A has been reduced and learning and learning have been improved.

[0168] 8. A COX2 Acetylating Agent Produced by SphK1 Promotes the Secretion of Neuroinflammatory Resolution Factor.

[0169] Based on the experimental results, the present inventors conducted a series of experiments to directly confirm whether a compound capable of inducing the acetylation of COX2 exhibits an effect of preventing or treating degenerative neurological diseases.

[0170] Specifically, the present inventors predicted that N-acetyl sphingosine 1 phsosphate and N-acetyl sphingosine could induce the acetylation of COX2, and the following experiments were conducted using this (FIG. 8a).

[0171] First, in order to confirm whether the selected compounds promote the secretion of neuroinflammatory resolution factor in nerve cells, APP/PS1 nerve cells were treated with N-acetyl sphingosine 1 phsosphate or N-acetyl sphingosine, and then the expression levels of the neuroinflammatory resolution factor were confirmed. As a result, it was confirmed that the expression levels of LxA4 and RvE1 in nerve cells of APP/PS1 mice were recovered when treated with N-acetyl sphingosine 1 phsosphate or N-acetyl sphingosine (FIG. 8b).

[0172] Next, in order to confirm whether the secretion of the neuroinflammatory resolution factor was caused by the increase in acetylation of COX2, it was confirmed by using N-acetyl sphingosine labeled to C.sup.14, and it was confirmed that C.sup.14 N-acetyl sphingosine caused more acetylation than a sample mixed with SphK1, acetyl-CoA, and sphingosine confirmed above (FIG. 8c). In addition, it was confirmed that serine 565 (S565) for a peptide 560-GCPFTSFSVPDPELIK-575 of COX2 was acetylated in the presence of N-acetyl sphingosine (FIG. 8d).

[0173] That is, it was confirmed that the compounds induced the COX2 acetylation to promote the secretion of the neuroinflammatory resolution factor, and in particular, by directly confirming that this acetylation occurred at S565 of COX2, in the treatment of degenerative neurological diseases, it was confirmed once again that S565 acetylation of COX2 may be a very critical therapeutic target.

[0174] 9. The COX2 Acetylating Agent Reduces AD Lesions in an Alzheimer's Animal Model by Promoting the Secretion of Neuroinflammatory Resolution Factor.

[0175] The present inventors confirmed AD lesions by injecting N-acetyl sphingosine, one of COX2 acetylating agents identified through the above experiment, into an APP/PS1 animal model. First, in order to determine an effect of N-acetyl sphingosine on neuroinflammatory response by secreting the neuroinflammatory resolution factor, changes in microglia and astrocytes were observed. The APP/PS1 mice injected with N-acetyl sphingosine showed a remarkable decrease in microglia and astrocytes compared to the APP/PS1 mice (FIGS. 9a and 9b). In addition, it was found that the amount of A was significantly lowered in APP/PS1 mice injected with N-acetyl sphingosine compared to APP/PS1 mice, and it was confirmed that memory and cognition were improved (FIGS. 9c and 9d). However, when sphingosine derivatives FTY720 and S1P were injected, there was no difference in the activity of microglia and astrocytes compared to the Alzheimer's animal model, and there was no effect of reducing A deposition and improving memory (FIGS. 9a and 9b).

[0176] Through the above results, it was confirmed that unlike sphingosine derivatives such as FTY720 and S1P, the COX2 acetylating agent promoted the secretion of neuroinflammatory resolution factor to exhibit an effect of reducing AD lesions, such as reduction in neuroinflammation, a decrease in A deposition, and improvement in memory in APP/PS1 mice.

[0177] 10. Preparation and Efficacy Verification of Antibody (ac-S565) that Specifically Recognizes Acetylated S565 Residue in COX2 Protein

[0178] Based on the results of the above Examples, the present inventors have prepared an antibody that specifically recognized S565 acetylated in a COX2 protein represented by SEQ ID NO: 1 and intended to use the antibody in the following experiments.

[0179] Specifically, after preparing a peptide of SEQ ID NO: 2, a rabbit was immunized by injecting the peptide, and then a polyclonal antibody (hereinafter referred to as ac-S565 antibody) was isolated and purified from the rabbit serum.

[0180] Meanwhile, referring to the above experimental results, the COX2 protein was acetylated at a residue S516 by aspirin, and was not acetylated at S565. However, the acetylation occurred specifically at the residue S565 of COX2 by N-acetyl sphigosine (N-AS).

[0181] Based on these results, the present inventors confirmed whether to acetylate S565 of COX2 by performing Western blotting using an ac-S565 antibody without treating an acetyl transfer to a recombinant COX2 protein, or after treating aspirin or N-AS.

[0182] As a result, it was confirmed that a band was hardly detected in the Western blot experiment using the ac-S565 antibody in the COX2 that was not treated with acetyl transfer or treated with aspirin, but the band was strongly shown in the COX2 treated with N-AS (FIG. 10). Through these results, it was confirmed that the prepared ac-S565 antibody could specifically detect whether to acetylate S565 of COX2, and furthermore, it was confirmed again that N-AS was a substance that acetylated S565 of COX2.

[0183] 11. Confirmation of Reduction of Acetylation of S565 of COX2 in Brain Tissue of Alzheimer's Animal Model

[0184] The present inventors confirmed the level of COX2 S565 acetylation in brain tissues of wild-type mice (WT) and an Alzheimer's animal model (APP/PS1) by using the prepared ac-S565 antibody through fluorescence immunostaining.

[0185] As a result, it was confirmed that the expression of COX2 was increased and the S565 acetylation of COX2 was decreased in microglia of the brain tissue of an Alzheimer's animal compared to wild-type mice. That is, these results indicate that the S565 acetylation of COX2 is reduced in microglia of the Alzheimer's animal model (FIGS. 11a and 11b).

[0186] The present inventors reconfirmed the level of COX2 S565 acetylation in microglia of the Alzheimer's animal model through Western blot.

[0187] As a result, like the fluorescence immunostaining result, it was confirmed that the expression of COX2 was increased and the S565 acetylation of COX2 was decreased in the microglia of the Alzheimer's animal compared to wild-type mice. That is, these results indicate that the S565 acetylation of COX2 is reduced in the microglia of the Alzheimer's animal model (FIGS. 12a and 12b).

[0188] Through this result, it can be confirmed that degenerative neurological diseases can be diagnosed by analyzing the level of S565 acetylation of COX2.

[0189] 12. Confirmation of Reduction in S565 Acetylation of COX2 in Microglia Treated with Amyloid Beta

[0190] The present inventors created a neuroinflammatory environment by treating human-derived microglia with amyloid beta, and then confirmed the level of S565 acetylation of COX2 through fluorescence immunostaining.

[0191] As a result, it was confirmed that when the amyloid beta was treated, the expression of COX2 was increased and the S565 acetylation of COX2 was decreased in the microglia. These results indicate that the level of S565 acetylation of COX2 is reduced in a neuroinflammatory environment due to the accumulation of amyloid beta (FIGS. 13a and 13b).

[0192] The present inventors created a neuroinflammatory environment by treating human-derived microglia with amyloid beta, and then reconfirmed the level of S565 acetylation of COX2 through Western blotting.

[0193] As a result, like the fluorescence immunostaining result, it was confirmed that when the amyloid beta was treated, the expression of COX2 was increased and the S565 acetylation of COX2 was decreased in the microglia. These results indicate that the level of S565 acetylation of COX2 is reduced in a neuroinflammatory environment due to the accumulation of amyloid beta (FIGS. 14a and 14b).

[0194] Through this result, it can be confirmed that degenerative neurological diseases can be diagnosed by analyzing the level of S565 acetylation of COX2.

[0195] 13. Confirmation of Reduction in COX2 S565 Acetylation Through Western Blot in Blood Cells (Monocytes) of Alzheimer's Animal Model

[0196] In order to confirm the possibility of diagnosing degenerative neurological diseases by analyzing the level of COX2 S565 acetylation in blood cells (monocytes) of the blood as well as microglia, the present inventors confirmed the level of S565 acetylation of COX2 in blood cells (monocytes) of an Alzheimer's animal model (APP/PS1) through Western blot.

[0197] As a result, like the result, it was confirmed that the expression of COX2 was increased and the S565 acetylation of COX2 was decreased in the blood cells (monocytes) of the Alzheimer's animal compared to wild-type mice. These results indicate that the S565 acetylation of COX2 is reduced even in the blood cells (monocytes, monocytes) as well as the microglia of the Alzheimer's animal model, and it can be determined that degenerative neurological diseases can be easily diagnosed by analyzing the level of COX2 S565 acetylation in the blood cells (monocytes) of the blood (FIGS. 15a and 15b).

INDUSTRIAL APPLICABILITY

[0198] According to the present invention, since the acetylation of COX2 in degenerative neurological diseases is significantly reduced, whether COX2 is acetylated may be utilized as a diagnostic marker for degenerative neurological diseases, and it is possible to diagnose degenerative neurological diseases more rapidly and accurately by using same. Therefore, the present invention has industrial applicability.