KR102252879B1 - Method for diagnosing parkinson's disease using aimp2 in blood plasma, composition therefor and kit containing the same - Google Patents

Abstract

The present application relates to a method of diagnosing Parkinson's diseases, predicting the effect of a therapeutic agent, or classifying a Parkinson's patient group from the expression level of Parkinson's disease-related proteins in plasma. The present application also relates to a composition for diagnosing Parkinson's diseases by measuring the expression level of Parkinson's disease-related proteins in plasma, and a kit comprising the same.

Description

A method for diagnosing Parkinson's disease using AIMP2 in plasma, a composition therefor, and a kit containing the same {METHOD FOR DIAGNOSING PARKINSON'S DISEASE USING AIMP2 IN BLOOD PLASMA, COMPOSITION THEREFOR AND KIT CONTAINING THE SAME}

The present application relates to a method of diagnosing Parkinson's disease from the expression level of Parkinson's disease-related protein in plasma, predicting the effect of a therapeutic agent, or classifying a Parkinson patient group, and also by measuring the expression level of Parkinson's disease-related protein in plasma. It relates to a composition for diagnosing and a kit comprising the same.

In the modern era, which is rapidly entering an aging society, the number of Parkinson patients, a representative degenerative movement disorder brain disease, is on a rapid increase. Patients with Parkinson's disease show movement disorders as well as non-motor disorders (odor disorders, sleep disorders, depression, mania, cognitive disorders). However, since Parkinson patients do not show uniform clinical symptoms, attempts have been made to classify them into subgroups according to the manifestation pattern and degree of clinical motor and non-motor symptoms, and the timing of onset of motor symptoms and the speed of symptom progression. However, no plasma markers capable of distinguishing Parkinson's disease into subgroups in molecular pathology have been reported.

The major lesions in Parkinson's patients are Lewy body (LB) consisting of denatured α-synuclein, but a significant number of Parkinson's patients have neurofibrillary tangle lesions and amyloid plaque lesions consisting of denatured tau aggregates. Also shown.

These facts show that for precise diagnosis and treatment of Parkinson's patients, the desired therapeutic effect may not be obtained simply by targeting only the Lewy body, which is an α-synuclein lesion. This is because the coexistence of other protein aggregate lesions additionally present in various Parkinson patients is also an important factor in disease progression and treatment.

As described above, although the clinical symptoms and brain lesions of Parkinson's patients are expressed in various forms for each patient, there is no plasma marker that can more accurately distinguish patients. As metabolites and disease proteins in biological samples, α-synuclein (cerebrospinal fluid, saliva), LRRK2 (cerebrospinal fluid, blood, urine), apolipoprotein A1 (blood), etc. are being studied as diagnostic markers for Parkinson's disease. There are currently no diagnostic markers that have been proven effective. In addition, most of the Parkinson's disease markers presented above have a disadvantage that they are not suitable for precise treatment of Parkinson's disease patients according to additional lesions, since they cannot distinguish various other protein aggregate lesions and subgroups of Parkinson's disease appearing in Parkinson patients. .

Republic of Korea Patent Publication No. 10-2018-004674 Korean Patent Registration No. 10-0762995

In this study, we attempted to diagnose Parkinson's lesions by measuring the level of AIMP2 in the plasma of Parkinson's patients, and furthermore, by distinguishing subgroups according to lesions, we tried to enable more precise Parkinson diagnosis, distinction of patient groups, and treatment accordingly. That is, in the case of a patient diagnosed as an AIMP2-positive Parkinson patient, we tried to provide information for diagnosis and patient group classification so that it is possible to obtain high therapeutic effect by paralleling a treatment strategy capable of controlling AIMP2 proteomic toxicity.

However, the problem to be solved by the present application is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

The first aspect of the present application relates to a composition for diagnosing Parkinson's disease, including an agent for measuring the expression level of a protein related to Parkinson's disease in isolated plasma.

A second aspect of the present application relates to a kit for diagnosing Parkinson's disease comprising the composition according to the first aspect.

In a third aspect of the present application, the expression level of a Parkinson's disease-related protein is measured from plasma isolated from a patient suspected of Parkinson's disease; Normalizing the expression level to the expression of a standard protein; It relates to a method of providing information for diagnosis of Parkinson's disease comprising comparing the normalized expression level with the normalized expression level measured for a normal person.

A fourth aspect of the present application relates to a method of providing information for predicting the effect of a therapeutic agent on Parkinson's disease patients, comprising the step of confirming the expression level of a Parkinson's disease-related protein from plasma extracted from the patient.

A fifth aspect of the present application relates to a method for providing information for classification of a Parkinson patient group comprising the step of confirming the expression level of a Parkinson disease-related protein from plasma extracted from the patient.

The above-described problem solving means are merely exemplary and should not be construed as limiting the present invention. In addition to the exemplary embodiments described above, there may be additional embodiments and embodiments described in the drawings and detailed description of the invention.

The present application found that AIMP2, which is known to accumulate in the brain tissue of patients as a Parkinson's disease factor and contribute to the death of dopamine cells, has the property of being secreted out of the cells, and by measuring the level of AIMP2 in the plasma of Parkinson's patients, Parkinson's Diagnosis of disease, prediction of therapeutic effect, and discrimination of patient groups were made possible.

In the case of Parkinson's patients, it can be seen that plasma AIMP2 level measurement can be usefully used in the diagnosis of Parkinson patients through the fact that plasma AIMP2 is positively tested at a significantly high rate, and the detection method of AIMP2 in plasma according to the present application (e.g. For example, it is possible to develop a small new AIMP2 diagnostic kit using a dot blot detection method), and diagnosis of AIMP2 lesions in the brain that differs for each Parkinson patient is possible through quantification of AIMP2 in plasma. In addition, the Parkinson patient group can be further divided into AIMP2 positive/negative, so that it can be used to separate subgroups in clinical trials or to precisely apply treatments.

1A and 1B are experimental data for observing the extracellular secretion of AIMP2 in SH-SY5Y, a human neuroblastoma-derived cell line.
2A to 2C are experimental data confirming that AIMP2 in the cerebral cortex is also distributed in vascular endothelial cells in AIMP2 overexpressing mice.
3A and 3B are experimental data showing the accumulation of AIMP2 in neurons in the temporal lobe brain tissue of Parkinson's patient.
4A to 4C are experimental data showing the accumulation of AIMP2 in cerebrovascular endothelial cells in the temporal lobe brain tissue of Parkinson's patients.
5A to 5D are experimental data confirming detection of AIMP2 in plasma of Parkinson's patients.
6 is experimental data that attempts to detect various other disease-related factors in plasma of Parkinson's patients.

Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art may easily implement the present application. However, the present application may be implemented in various different forms and is not limited to the embodiments described herein. And in the drawings, in order to clearly describe the present application, parts not related to the description are omitted.

In the entire specification of the present application, when a certain part "includes" a certain component, it means that other components may be further included, rather than excluding other components unless specifically stated to the contrary.

The terms "about", "substantially", etc. to the extent used throughout the present specification are used at or close to the numerical value when manufacturing and material tolerances specific to the stated meaning are presented, and the understanding of the present application To assist, accurate or absolute numerical values are used to prevent unreasonable use of the stated disclosure by unscrupulous infringers. As used throughout the specification of the present application, the term "step (to)" or "step of" does not mean "step for".

In the entire specification of the present application, the term "combination of these" included in the expression of the Makushi format refers to one or more mixtures or combinations selected from the group consisting of components described in the expression of the Makushi format, and the component It means to include one or more selected from the group consisting of.

Throughout this specification, the description of “A and/or B” means “A, B, or A and B”.

Throughout this specification, “diagnosis” refers to ascertaining the presence or character of a pathological condition. Among them, the present invention is useful for diagnosis of Parkinson's disease. The present invention includes predicting the onset and progression of Parkinson's disease.

Hereinafter, embodiments of the present disclosure for the composition for diagnosis of Parkinson's disease, a kit including the same, a method for providing information for diagnosing Parkinson's disease, a method for providing information for predicting the effect of a therapeutic agent for Parkinson disease patients, and a method for providing information for classification of Parkinson's patient group. And it will be described in detail with reference to embodiments and drawings. However, the present application is not limited to these embodiments and examples and drawings.

The first aspect of the present application relates to a composition for diagnosing Parkinson's disease, including an agent for measuring the expression level of a protein related to Parkinson's disease in isolated plasma.

According to one embodiment of the present application, the Parkinson's disease-related protein is composed of AIMP2, α-synuclein (SNCA), tau, LRRK2, apolipoprotein A1, aggregates thereof, phosphorylated forms thereof, and combinations thereof. It may be selected from the group, but may not be limited thereto.

The composition of the present invention is for diagnosing Parkinson's disease using plasma that is easier to collect than the cerebrospinal fluid used for diagnosis of Parkinson's disease, and by measuring the expression level of a protein related to Parkinson's disease in plasma, it is easy to detect Parkinson's disease. Can be diagnosed.

In particular, it has not been known that AIMP2, known as a protein accumulated in dopamine neurons related to Parkinson's disease, can be quantitatively measured in plasma, but in the present application, it is known that AIMP2 is secreted extracellularly and thus can be measured in plasma. It was first demonstrated and tried to be used for purposes such as diagnosis of Parkinson's disease.

According to the embodiment of the present application, a quantitative increase of AIMP2 in vascular endothelial cells was observed in the brain tissue of a mouse overexpressing AIMP2 in neurons, which can be detected by secreting AIMP2 from neurons into vascular endothelial cells and further into blood. It is to show that it has a property. In fact, a quantitative increase in AIMP2 neurons and endothelial vascular cells was observed in the brain tissues of Parkinson's patients. Since the amount of AIMP2 in neurons in the brain tissue of Parkinson's patients and the amount of AIMP2 in vascular endothelial cells show a correlation, the accumulation of AIMP2 in the brain tissue can be derived from the high level of AIMP2 in plasma.

In particular, when analyzing plasma proteins collected from Parkinson's patient group, plasma AIMP2 was detected as positive in a significantly higher proportion of patients compared to the control group, so Parkinson's patients were divided into AIMP2 positive and negative AIMP2 positive and negative groups through simple quantification of plasma AIMP2 for the patient. It is also possible to segment and refine treatment strategies accordingly.

For example, it is possible to establish a precise Parkinson treatment strategy, such as using a therapeutic drug capable of controlling AIMP2 toxicity for additional treatment by discriminating among Parkinson patients who are positive for plasma AIMP2.

According to one embodiment of the present application, the formulation for measuring the expression level may include an antibody or aptamer that specifically binds to a protein related to Parkinson's disease, but may not be limited thereto, and in particular, expression using an antibody When measuring the level, the expression level of the protein is, for example, Dot blot, Western blot, ELISA (enzyme linked immunosorbent assay), Immunoprecipitation Assay, and Complement Fixation. Assay), flow cytometry (Fluorecence Activated Cell Sorter, FACS), mass spectrometry, protein chip (protein chip) or antibody chip (antibody chip) can be selected and measured by a person skilled in the art without particular limitation among known analytical methods. In this case, it is possible to measure the amount of protein in plasma using the recombinant protein as a standard.

A second aspect of the present application relates to a kit for diagnosing Parkinson's disease comprising the composition according to the first aspect.

Any kit for diagnosing Parkinson's disease according to the present invention may be used without limitation as long as it is for measuring the presence of a protein related to Parkinson's disease in a sample. That is, the detection of the protein related to Parkinson's disease present in the 　 kit 　 or the sample can be performed using conventional techniques or methods known to those of ordinary skill in the art. Such methods include, for example, Dot blot, Western blot, ELISA (enzyme linked immunosorbent assay), immunoprecipitation assay (Immunoprecipitation Assay) using antibodies that specifically bind to proteins related to Parkinson's disease. , Complement Fixation Assay, mass spectrometry, protein chip or antibody chip, or a modification method thereof.

For example, the kit may contain materials suitable for carrying out various immunochemical reactions using an antibody, for example, a preservative and/or a buffer solution for the antibody. In addition, an additional reagent may be included, and for example, a secondary antibody may be included as a conjugate labeled with a color developing enzyme, a fluorescent substance, a radioactive isotope or a colloid. The coloring enzyme may be peroxidase, alkaline phosphatase, or acid phosphatase. In the case of a fluorescent substance, fluorescein carboxylic acid (FCA), fluorescein isothiocyanate (FITC ), fluorescein thiourea (FTH), 7-acetoxycoumarin-3-yl, fluorescein-5-yl, fluorescein-6-yl, 2',7'-dichlorofluorescein-5- Yl, 2',7'-dichlorofluorescein-6-yl, dihydrotetramethylrosamin-4-yl, tetramethylrhodamine-5-yl, tetramethylrhodamine-6-yl, 4,4- Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-ethyl or 4,4-difluoro-5,7-diphenyl-4-bora-3a ,4a-diaza-s-indacene-3-ethyl. In addition, the kit may include a washing solution or an eluent capable of retaining only the bound complex, removing a substrate for color development reaction with an enzyme and an unbound protein.

In a third aspect of the present application, the expression level of a Parkinson's disease-related protein is measured from plasma isolated from a patient suspected of Parkinson's disease; Normalizing the expression level to the expression of a standard protein; It relates to a method of providing information for diagnosis of Parkinson's disease comprising comparing the normalized expression level with the normalized expression level measured for a normal person.

For example, the comparison of the normalized expression level may be a comparison with a predetermined cutoff value, but may not be limited thereto. In this case, the cutoff value may be determined according to the sensitivity of the checked value and the ROC curve (Receiver Operation Characterstic Curve), which is a curve drawn with 1-specificity. In other words, after normalizing the expression level of Parkinson's disease-related protein in the plasma of a Parkinson patient prepared in advance and the expression level of Parkinson's disease-related protein in the plasma of a normal person, and determining the cutoff value in advance from the ROC curve result using this, plasma of the target to be diagnosed Information for diagnosis of Parkinson's disease may be provided by comparing the normalized value of the gene expression level in the inner and the predetermined cutoff value, but the present disclosure may not be limited thereto.

In the method for providing information for diagnosis of Parkinson's disease according to the present application, the Parkinson's disease-related protein expression level (normalized expression level) measurement result may be compared with the measurement result of a normal group to diagnose Parkinson's disease, or a predetermined cutoff You can also diagnose against the value.

According to one embodiment of the present application, the Parkinson's disease-related protein is composed of AIMP2, α-synuclein (SNCA), tau, LRRK2, apolipoprotein A1, aggregates thereof, phosphorylated forms thereof, and combinations thereof. It may be selected from the group, but may not be limited thereto.

According to the exemplary embodiment of the present disclosure, the normalizing of the expression of the standard protein may include normalizing the result of quantification of Ponceau staining of the entire plasma protein, but may not be limited thereto.

Alternatively, the standard protein may be CD31, but may not be limited thereto.

According to one embodiment of the present application, the expression level of the protein is Dot blot, Western blot, ELISA (enzyme linked immunosorbent assay), immunoprecipitation assay, complement fixation assay (Complement Fixation). Assay), flow cytometry (Fluorecence Activated Cell Sorter, FACS), mass spectrometry, protein chips, or antibody chips may be used, but may not be limited thereto.

A fourth aspect of the present application relates to a method of providing information for predicting the effect of a therapeutic agent on Parkinson's disease patients, comprising the step of confirming the expression level of a Parkinson's disease-related protein from plasma extracted from the patient.

According to the exemplary embodiment of the present disclosure, when the protein related to Parkinson's disease is expressed, it may be determined that the effect of the therapeutic agent will appear, but the present disclosure may not be limited thereto.

A fifth aspect of the present application relates to a method for providing information for classification of a Parkinson patient group comprising the step of confirming the expression level of a Parkinson disease-related protein from plasma extracted from the patient.

The present invention is to be described in more detail through the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[ Example ]

1. Experimental method

1.1. Cell culture and Transfection ( Transfection )

1.1.1. Subculture (Cell subculture)

1) The culture medium of human neuroblastoma SH-SY5Y cells was removed (media conditions: DMEM containing 10% FBS, 1% P/S).

2) Washed with PBS.

3) After adding 2 ml of trypsin, it _{was reacted in a 37°C CO 2} incubator for 1 to 2 minutes.

4) After inactivating trypsin by adding 2 ml medium, the cells were suspended using a pipette.

5) 10 ml of the medium was added to a new dish, and 1 ml of the suspended cell solution was added and cultured.

1.1.2. Transfection

1) 2 µg of DNA was added to 200 µl of Opti-MEM. (Based on 6-well scale)

2) After adding 2 μl transfection reagent (X-tremeGENE HP), tapping was performed lightly.

3) It was reacted for about 15 minutes at room temperature.

4) The DNA-X-treme gene mixture was evenly sprayed on the cell medium by dropping.

1.2. Western Blot analysis

1.2.1. Running & transfer

※ TBS-T preparation (1L)

Using 900 ml D.W + 100 ml 10X TBS + [1 ml Tween-20] (cut off the tip and use), the required% gel was prepared in advance. The gel was placed in the tank, a new running buffer was filled inside, and the existing running buffer (used running buffer) was filled up to the 2 gel and 4 gel lines outside.

※ Assemble the gel plate by fitting it well into the groove, and put it in the tank with the lower glass plate facing each other.

※ When the number of gels was odd, a plastic plate was hung.

※ Running buffer (1X, 4℃) = 10X Running 100 + D.W 900

1) 20 µl of the incubated protein was mixed with 20 µl of 2X sample buffer + 100 µl of 1X sample buffer, and then boiled at 95° C. for 5 minutes.

2) Samples along with the markers were loaded into the comb of the gel by 15 µl each.

3) Transfer buffer (0.5X, 4℃) = D.W 800 + MeOH 150 + 10X transfer buffer 50 → Pre-made and cooled.

4) After the run, ice was placed in a styrofoam box, the tank was completely buried, and the ice pack and rack were assembled.

5) Pour half of the cold transfer buffer into the tank, and pour the rest into another bucket (1 L).

6) A black sponge was immersed in the buffer, and two used 3M papers were stacked in the order of the membrane. (Sponge → 2 used 3M sheets → membrane → gel → 2 new 3M sheets → sponge)

7) Take out the gel, remove the small plate, and attach it to the large plate.

8) Cut the lower dye and cut out the comb part.

9) The upper left was cut out slightly, marked, turned over and dropped onto the membrane.

10) Arranged so that air bubbles or dust do not enter.

11) Put two new 3M papers in the 3M on the back, pick them up and put them on a white board.

12) Wet the transfer buffer in a black sponge, put it on 3M, and then assemble.

13) The remaining buffer was poured into the tank and transferred at 100 V for 1.5 hours.

1.2.2. Blocking & Primary Antibody (Blocking & 1 ^st Ab )

1) The transferred membrane was immersed in a bucket poured with poncean S so that the band was visible.

2) Washed in TBS.

3) Cut it in a straight line with scissors according to the required size.

4) Each of them was put in a labeled container.

5) 5% skim milk was soaked and blocked for 45 minutes to 1 hour.

6) When blocking was over, the skim milk was discarded and washed with TBS-T.

7) The primary antibody was poured and left on a rocker at room temperature for 1 hour (primary antibody-TBS-T: Ab = 5000: 1 + 10% sodium azide 10 µl (0.01%)).

8) The membrane to which the primary antibody was attached was taken out, and the primary antibody was poured into a 15 ml tube and stored at 4°C.

9) Washed three times with TBS-T for 5 minutes each (speed 42 at RT Rocking).

10) In 10 ml of 5% skim milk, 2 µl of the secondary antibody was added at 5000:1 and mixed well.

11) Pour enough to submerge the membrane and let it stand for 30 minutes at RT (speed 32 at RT Rocking).

12) After 1.5 hours, it was washed 3 times with TBS-T for 5 minutes each.

13) ECL was added 1 ml each to a 15 ml tube, vortexed well, and then poured into a bucket.

14) Put the membrane in the ECL container and shake it well.

15) Take it out, slightly dry it, and put it on the developer board.

16) A little about 500 μl to 1 ml of ECL was picked up and placed on the membrane and covered with vinyl.

17) I picked up the developer plate and film and moved to the dark room to print.

18) Information such as date, experiment content, antibody name, size, and cell line were written on the film.

1.3. Immunofluorescence staining

1.3.1. Day 1

1) Buffer preparation: (i) 10 ml 0.1% Triton X-100 (100 μl 10% Triton X-100 + 1 ml 10X PBS + 8.9 ml DW), (ii) 5 ml 5% blocking solution (4.75 ml 0.1% Triton X-100 buffer + 250 µl 100% Goat serum)

2) Among the brain slices sectioned to a thickness of 35-40 μm, sections to be used for the experiment were selected, transferred to a 12 well plate containing 1X PBS, and washed once for 5 minutes.

3) 1 ml of 0.1% Triton X-100 was added to the wells, and then the sections were transferred to the wells and permeabilized by shaking for 10 minutes at room temperature.

4) 1 ml of 5% blocking solution was added to the wells, and then the sections were transferred to the wells and blocked by shaking for 1 hour at room temperature.

5) After 1 hour, AIMP2 antibody (Proteintech #10424-1-AP) and CD31 antibody (BD bioscience #550274) were added at a ratio of 1:1000 to 1:500 and shaken overnight at 4°C.

1.3.2. Day 2

1) The plate incubated overnight was taken out and the sections were washed 3 times for 5 minutes each with 1X PBS.

2) Secondary antibody mCy3 (Alexa568 #A10037) + pCy2 (Aleca488 #A11008) was diluted in 1X PBS at a ratio of 1:500, and then the fragments were transferred thereto and shaken at room temperature for 1 hour.

3) After 1 hour, it was washed 3 times with 1X PBS.

4) DAPI dye (Stock conc. 0.2mg/ml) was diluted in 1X PBS at a ratio of 1:1000, and then the sections were transferred and incubated for 5 minutes.

5) After 5 minutes, it was washed 3 times with 1X PBS.

6) It was placed on a microscope slide (Thermo #J1800AMNZ), and before drying, a cover glass (22x50 mm) was covered with an Immu-mount solution (thermos #9990402).

7) It was photographed using a fluorescence microscope.

1.4. Dot Blot (Dot blot) experiment

1) The NC membrane was prepared by immersing it in TBS buffer.

2) The prepared NC membrane was set in a Bio-Dot microfiltration device (Bio-Rad) and a vacuum tube was connected.

3) Samples were loaded into each well.

4) The Bio-Dot microfiltration device was separated, the NC membrane was taken out, and washed 3 times for 5 minutes each with a TBS-T buffer (TBS buffer containing 5% tween 20).

5) The NC membrane was blocked for 1 hour using 2.5% skim milk (based on TBS-T).

6) The NC membrane was incubated using the primary antibody (O/N).

7) Washing was performed 3 times for 10 minutes each using TBS-T buffer.

8) HRP-conjugated secondary antibody suitable for the primary antibody was used and incubated for 1 hour and 30 minutes.

9) Step 7 was repeated.

10) The results were confirmed in the dark using the ECL solution.

2. Experiment result

2.1. AIMP2 Overexpression and Extracellular secretion

AIMP2 is a matrix protein of Parkin, a recessive gene for Parkinson's disease, and has been reported to be accumulated in dopamine neurons in the midbrain black matter of patients with Parkinson's disease. Is known. However, it has not been reported that AIMP2 can be secreted out of cells when accumulated in cells like other disease proteins (α-synuclein, tau). To confirm the possibility of extracellular secretion of AIMP2, SH-SY5Y cells were transfected with GFP-tagged AIMP2 (GFP-AIMP2) and HA-tagged α-synuclein plasmid (HA-α-synuclein) and then α- We verified whether synuclein or AIMP2 was detected through Western blot and dot blot.

First, it was confirmed by Western blot using specific antibodies indicated for expression of each protein in SH-SY5Y cell lysates transfected with GFP-AIMP2, HA-a-synuclein, or both (Fig. 1A). . It was confirmed that α-synuclein and AIMP2 were successfully overexpressed by each DNA transfection into SH-SY5Y cells. In addition, the proteins secreted into the culture medium in the cell culture of FIG. 1A were bound to the nitrocellulose membrane using the dot blot technique, and then each protein was detected and confirmed using appropriate specific antibodies (Fonso (as a loading control for the dot blot)). ponceau) staining) (Fig. 1B). As a result, it was confirmed that AIMP2, like α-synuclein, is secreted into the cell culture fluid upon overexpression. Curiously, it was observed that when AIMP2 and α-synuclein were simultaneously overexpressed in SH-SY5Y cells, the secretion of α-synuclein was suppressed while the secretion of AIMP2 was still observed.

2.2. AIMP2 Transition from nerve cells to vascular cells

AIMP2 Tg , which overexpresses AIMP2 in neurons to confirm the mechanism of the extracellular secretion of AIMP2 Expression distribution of AIMP2 in brain tissue sections of mice ( CamKIIα - tTA; TetP - AIMP2) was analyzed.

Specifically, transformed AIMP2 at 6 months of age Tg Immunofluorescence staining was performed using an AIMP2-specific antibody for cerebral cortical brain tissue sections of mice and control mice (nuclei were counter-stained with blue DAPI) (Fig. 2a), AIMP2 Tg Double immunofluorescence staining was performed using AIMP2 and a specific antibody against CD31, which is a vascular marker, in cortex brain tissue sections of mice and control mice (Fig. 2b), and the right panel of Fig. 2b is shown in the middle panel of Fig. 2b. An enlarged image of the area indicated by the square of the Tg fluorescence image is shown. Also, AIMP2 Tg The intensity of expression of AIMP2 in vascular endothelial cells labeled with CD31 was quantified for fluorescence images of brain sections of mice and control mice, and is shown in FIG. 2C (12 endothelial cells were analyzed in 3 Tg mice, and 1 control mouse Analyzed 12 endothelial cells). Quantitative data were expressed as mean +/- standard error, and the significance test was confirmed by unpaired two tailed student t- test. *** P <0.001

Experimental results, AIMP2 that over-expression compared with the control group in the cortex AIMP2 Tg In mice, it was confirmed that a quantitative increase in AIMP2 expression appeared even on vascular endothelial cells stained with CD31. This suggests the possibility that the accumulation of AIMP2 in neurons was caused by the secretion of AIMP2 and accumulation in vascular endothelial cells, and the accumulation of AIMP2 in vascular endothelial cells suggests the possibility of secretion of AIMP2 into blood and detection of AIMP2 in plasma. .

2.3. Parkinson's brain tissue neurons and Blood vessel cell AIMP2 Manifestation correlation

AIMP2 Tg In order to verify the clinical linkage of the facts confirmed in mice, a confirmation experiment was performed on autopsy brain tissue sections of Parkinson patients.

3A is an immunofluorescence image using specific antibodies against MAP2 and AIMP2, which are neuronal dendrites markers, in autopsy brain tissue obtained from a healthy control group of a similar age to Parkinson's patients. Each image shows representative immunofluorescence images from temporal lobe brain tissue samples from 4 controls and 4 Parkinson patients. Figure 3b shows a quantitative graph of the amount of AIMP2 expression in MAP2-labeled neurons in the temporal lobe brain tissue of Parkinson's patient and control group ( n = 60 brain sections from 6 Con, 60 brain sections from 6 PD). 4A is a result of immunofluorescence imaging using specific antibodies against CD31 and AIMP2, which are vascular endothelial cell markers, for autopsy brain tissue obtained from a healthy control group of a similar age to Parkinson's patient. Each image shows representative immunofluorescence images of CD31 (red channel) and AIMP2 (green channel) in temporal lobe brain tissue samples from 4 controls and 4 Parkinson patients. Figure 4b shows a quantitative graph of AIMP2 expression in CD31-labeled vascular endothelial cells in the temporal lobe brain tissue of Parkinson patients and controls ( n = 60 brain sections from 6 Con, 60 brain sections from 6 PD). Figure 4c is a graph showing the correlation analysis of the signal intensity of AIMP2 in the CD31-labeled cells and the MAP2-labeled cells quantified in the temporal lobe brain tissue of Parkinson's patients and controls ( n = con 5, PD 6). Pearson correlation analysis showed a correlation with a significance P <0.01.

AIMP2 has previously been reported to accumulate in the midbrain black matter of Parkinson's patients with dopaminergic neurons, but according to FIGS. 3A and 3B, it was confirmed that it also accumulates in neurons present in the temporal lobe of Parkinson's patients. There was a difference in degree for neurons stained with MAP2, but AIMP2 was significantly accumulated in Parkinson patients compared to the control group. In addition, according to FIGS. 4A and 4B, even in the case of vascular endothelial cells labeled with CD31 for the same brain tissue sample, the accumulation of AIMP2 was hardly observed in the normal group, whereas AIMP2 in vascular endothelial cells in the temporal lobe brain tissue of Parkinson's patients. 4C showed a positive correlation between the amount of AIMP2 in neurons (MAP2 staining) and the amount of AIMP2 in vascular endothelial cells (CD31 staining) in all samples. This shows that the accumulation of AIMP2 in neurons is linked to the accumulation of AIMP2 in blood vessels, and through this, the secretion of AIMP2 in the blood can represent the accumulation of AIMP2 in the brain.

2.4. In Parkinson's plasma AIMP2 detection

The pattern of AIMP2 accumulation in cerebrovascular endothelial cells in Parkinson's patients suggests the possibility of detection of AIMP2 in the patient's blood. To reconfirm this, AIMP2 detection experiments were performed from plasma obtained from Parkinson's patients and plasmas of other controls (musculoskeletal disorder patients in their 50s, dementia patients in their 70s, and normal groups in their 30s).

Specifically, plasma from Parkinson patients (30 patients), dementia patients (24 patients), and musculoskeletal disease patients (24 patients) of a similar age group as shown in Tables 1 to 3 below were obtained from the Human Resource Bank. Tables 1 and 2 show the Parkinson patient and normal group brain tissue information, Table 3 shows the Parkinson patient and disease control / normal group plasma donor information.

Experimental group Clinical pathology age gender race PMI (h) PD1 LB/dementia 78 F White 3.5 PD2 LB/dementia 79 M White 2.5 PD3 LB/dementia 87 F White 3 PD4 LB/dementia 82 M White 4.25 PD5 LB/dementia 72 F White 3.58 PD6 LB 77 M White 4 Ctrl1 NA 90 F White 3 Ctrl2 NA 87 M White 3.5 Ctrl3 NA 86 M White 4.75 Ctrl4 NA 79 M White 2 Ctrl5 NA 73 F White 1.5 Ctrl6 NA 70 F White 2

Note: PMI, postmortem interval hours

Parkinson patient Normal note N 6 6 age 76.17 ± 5.04 80.83 ± 8.13 P = 0.67 Gender (M:F) 3:3 3:3

Age expressed as mean ± standard deviation. Statistical processing was analyzed by unpaired two tailed student t -test. Brain tissues from Parkinson patients and normal groups are donated from the Brain Body Donation Program.

Musculoskeletal disorders Parkinson's disease dementia Jeong Jeong-gun note N 24 30 24 48 age 53±8.87 70.59±9.69 70.5±6.63 34.77±9.7 F = 132.312 P <0.001 Gender (M:F) 16:8 16:14 8:16 12:36

Age expressed as mean ± standard deviation. Statistical treatment was analyzed by ANOVA Tukey's post-test. Plasma samples from patients and normal groups are donated from the Korea Human Resource Bank (musculoskeletal system samples, Keimyung University Hospital; Parkinson patient samples, Gyeongsang National University Hospital and Inje University Hospital; dementia samples, Chungbuk National University Hospital; Jeongsang-gun, Chonbuk National University Hospital. Research using derived plasma samples was approved by the Institutional Bioethics Committee of Sungkyunkwan University (SKKU 2018-07-018-001).

First, 20 μl of plasma samples of each experimental group were diluted in PBS and bound to a nitrocellulose membrane using a dot blot device. In the left column, 50, 100, and 200 pg of each of the recombinant AIMP2 proteins were bound. Figure 5a is an image of plasma proteins that are printed and bound to the nitrocellulose membrane through fonso staining, and Figure 5b shows the expression level of AIMP2 in plasma using an AIMP2 specific antibody for plasma samples from AIMP2 standard and each group. The result confirmed by dot blot, and FIG. 5C is the result of performing dot blot using Fonso staining and AIMP2-specific antibody in the same manner for 48 plasmas obtained from healthy young people, and FIG. 5D shows the results of FIG. 5B and 5C. The concentration of AIMP2 in plasma of patients and normal groups was quantified and expressed as a plot graph. When there is no AIMP2 dot blot signal, it is indicated by a gray circle. For the quantification of AIMP2, the amount of standard recombinant AIMP2 and the dot blot signal intensity were obtained through a Lineweaver-Burk plot, and the final concentration was obtained by inputting the AIMP2 dot blot signal to this function formula. In addition, phosphorylated Tau (pTau), LRRK2 and α-synuclein for plasma samples from Parkinson's (30), dementia (24), and similar age groups of musculoskeletal disorders (24) bound to a nitrocellulose membrane. A dot blot experiment was performed using the antibody (FIG. 6).

Plasma proteins from the experimental groups were bound to the nitrocellulose membrane (Fig. 5a), and analysis was performed using specific antibodies through dot blot. As a result, in patients with musculoskeletal diseases or relatively young normal groups, 6.2 to 8.3% of patient plasma The detection of AIMP2 was confirmed in, but in the case of dementia patients, plasma detection of AIMP2 was confirmed in about twice as many patients as in musculoskeletal patients (FIGS. 5b, 5c and 5d). On the other hand, in the case of Parkinson's disease patients, plasma AIMP2 was confirmed in a significantly greater proportion of patients than other diseases and normal groups, specifically about 30% (FIGS. 5b, 5c and 5d). In addition, it was confirmed that the amount of AIMP2 detected in plasma showed a significant variation for each Parkinson patient. This suggests the possibility of dividing patients into subgroups based on the detection of plasma AIMP2 within the Parkinson patient group. On the other hand, in the case of LRRK2 or α-synuclein, which were previously reported to be used as diagnostic markers for Parkinson patients, detection in plasma could not be confirmed in the dot blot experiment of this example. In some cases, phosphorylated Tau in plasma was detected (FIG. 6).

The foregoing description of the present application is for illustrative purposes only, and those of ordinary skill in the art to which the present application pertains will be able to understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present application. Therefore, the embodiments described above are illustrative in all respects, and should be understood as non-limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as being distributed may also be implemented in a combined form.

The scope of the present application is indicated by the claims to be described later rather than the detailed description, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present application.

Claims (11)

As a method for providing information for classification of a group of Parkinson patients in which AIMP2 is expressed in plasma in Parkinson patients, comprising: checking the expression level of Parkinson's disease-related proteins from plasma isolated from Parkinson patients,
The Parkinson's disease-related protein is characterized in that the AIMP2 protein, the method. delete The method of claim 1,
The expression level is measured by a formulation containing an antibody or aptamer that specifically binds to Parkinson's disease-related AIMP2 protein, a method for providing information for classification of Parkinson's patient group. delete delete delete delete The method of claim 1,
Confirmation of the expression level of the protein was performed by Dot blot, Western blot, ELISA (enzyme linked immunosorbent assay), immunoprecipitation assay (Immunoprecipitation Assay), complement fixation assay (Complement Fixation Assay), flow cytometry ( Fluorecence Activated Cell Sorter (FACS), mass spectrometry, protein chip (protein chip) or antibody chip (antibody chip) is measured using the information providing method for classification of Parkinson's patient group. delete delete delete

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