KR20080051107A - Biological markers for diagnosing parkinson's disease and uses thereof - Google Patents

Abstract

Biological markers of which expression is reduced or increased by mutation of parkin gene causing Parkinson's disease are provided to diagnose and prognose Parkinson's disease, so that the markers are useful as target substances for alleviating symptoms of brain disease in a case of decreased expression or as treating agents of brain disease in a case of increased expression. The biological markers for diagnosis of Parkinson's disease include energy metabolism-related proteins containing glycogen phosphorylase, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, ubiquitin reductase, oxoglutarate dehydrogenase and hydrogen-transporting ATP synthase, and further include ribosomal protein S12, translation initiation factor 1(Trip 1), CG11474 and carbonic anhydrase 1.

Description

Biological markers for diagnosing Parkinson's disease and uses approximately}

The present invention relates to biological markers that can be used in the diagnosis of Parkinson's disease and uses of these markers, and more particularly to mutations in the parkin gene, which is the cause gene of Parkinson's disease. Protein markers whose expression changes quantitatively, and the use of these markers for diagnosis, treatment and prognosis of Parkinson's disease, etc. and the use of these markers as target substances for the prevention, alleviation and treatment of various brain diseases. It is about.

Parkinson's disease (Parkinson's disease) is a type of degenerative brain disease in which the neurons that secrete dopamine, a neurotransmitter that regulates muscle movement, degenerate as they age. The cause of Parkinson's disease is not yet fully understood, but studies on the related Parkin gene have been reported. The Parkin gene is a recessive gene, and when a genetic test is performed on patients with familial Parkinson's disease, a large number of mutations have been found on the Parkin gene.

Parkin protein, a protein product encoded by this Parkin gene, is an E3 ubiquitin ligase, which promotes the degradation of abnormal proteins present in the body. In addition, various substrates for these Parkin proteins have been reported. However, no detailed mechanism of Parkinson's disease is known at all if Parkin's protein is not normally produced by mutations on the Parkin gene.

To date, the following proteins have been reported associated with Parkinson's disease. First, tubulin is a component of the cytoskeleton, which is reported to promote ubiquitination by binding to Parkin protein (Ren et al ., 2003). In addition, glyceraldehyde-3-phosphate dehydrogenase is an enzyme related to glycolysis, which is well known in degenerative brain diseases such as Parkinson's disease and increases in the nucleus of Parkinson's disease patients. In addition, it has also been reported as a pro-apoptotic protein (Berry, 2004; Olanow, 2006). In addition, glutathione-S-transferase and NADH, which are related to oxidative stress, have also been associated with Parkinson's disease. Specifically, glutathione-S-transferase is an anti-oxidant protein that prevents the degeneration of neurons caused by oxidative stress, and modification of this protein has been reported to be associated with Parkinson's disease (Yi et. al ., 2003). Singer et al ., 1988) reported that Parkinson's disease-causing substance 1-methyl-4-phenylpyridinium (MPP +) inhibits ATP production by binding to NADH dehydrogenase, Burke. Mayer et Birkmayer et al ., 1989) reported that NADH could improve symptoms in Parkinson's disease patients.

In addition, a number of proteasomes and deubiquitinating enzymes have been observed in Parkinson's disease patients, which play an important role in protein degradation. Olano et al ., 2006) suggest that the ubiquitin-proteasome system (UPS), a proteolytic mechanism, plays a decisive role in the induction of Parkinson's disease, and that a UPS inhibitor is similar to that in Parkinson's patients in rats and monkeys. Research has been reported to show symptoms (Kordower et al ., 2006). In addition, mutations in the GTP cyclohydrolase I enzyme, a protein involved in the dopamine synthesis process, have been reported to reduce the amount of dopamine by reducing the activity of tyrosine hydroxylase (TH), a dopamine producing enzyme (Krishnakumar). et al ., 2000).

The present inventors investigated the effects of the mutation of the Parkin gene on the protein composition of brain cells and conducted a study to select proteins that cause Parkinson's disease. Specifically, an animal model of Drosophila showing human Parkinson's disease-like traits was used to analyze the brains of individuals carrying the mutated Parkin genes and to compare the results with those of normal individuals ( Proc . Natl . Acad . Sci . USA, 2005 102: 10345-10350).

Accordingly, the present inventors have made efforts to develop a new therapeutic agent for Parkinson's disease. As a result, the inventors searched for 29 protein markers affected by the Parkin gene mutation and determined whether the expression of these genes was reduced or increased. The present invention was successfully completed by screening each of them and confirming that they can be used as target substances or therapeutic agents for alleviating symptoms of brain disease and useful for diagnosis, treatment, and prognosis of Parkinson's disease.

It is an object of the present invention to provide biological markers that can be used in the diagnosis and prognosis of Parkinson's disease and the use of these markers as therapeutic agents for target substances and brain diseases.

In order to achieve the above object, the present invention provides a biological marker that can be used for the diagnosis of Parkinson's disease.

The present invention also provides biological markers that can be used to determine the course and prognosis of Parkinson's disease.

The present invention also provides a target material for alleviating or treating symptoms of brain diseases including Parkinson's disease using the biological markers.

In addition, the present invention provides a therapeutic agent for brain diseases using the biological marker as an active ingredient.

As described above, the present invention provides protein markers in which gene expression is changed quantitatively due to mutation of the parkin gene, which is the cause gene of Parkinson's disease, and the use of these markers for diagnosis and prognosis of Parkinson's disease. The present invention relates to the use of these markers as target materials for the prevention, alleviation and treatment of various brain diseases. Protein markers of the present invention can be used as a target material to attenuate the symptoms by amplifying brain disease when expression is reduced by the mutation of the Parkin gene, defense of the brain such as stress relief when expression is increased It can be a mechanism to be developed as a treatment for brain diseases. In addition, the quantitative increase by the mutation among the marker proteins is likely to be a direct cause of Parkinson's symptoms as a substrate of Parkin, which may be an important clue to determine the cause of Parkinson's disease in the future.

Hereinafter, the present invention will be described in detail.

The present invention provides biological markers that can be used for the diagnosis, treatment and / or prognosis of Parkinson's disease.

Specifically, the present invention relates to cytoskeleton-related proteins, energy metabolism-related proteins, signal transduction-related proteins, stress / detoxification-related proteins, and protein folding-related proteins. It provides biological markers selected from among proteins, proteins related to protein degradation and proteins related to neurotransmitter metabolism.

In addition, the present invention is selected from ribosomal protein S12, translation initiation factor Trip 1, CG11474 (isotype B) and carbonic anhydrase 1 in addition to the above. To provide a biological marker.

The cytoskeleton-related protein is preferably at least one selected from α-tubulin, β-tubulin, and actin, and the energy metabolism-related protein is glycogen phosphorylase, glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase), triose phosphate isomerase, ubiquitin reductase, oxoglutarate dehydrogense and hydrogen-transporting ATP synthase synthase), preferably, the signaling-related protein comprises a product of the CG5023 gene (calponin-like protein having actin binding, calponin repeats), and the stress / Detoxification-related proteins include glutathione-S-transferase, superoxide Seumyu hydratase (superoxide dismutase), preferably at least one selected from the group consisting of oxidative stress protein and a protein toxin reaction.

In addition, the protein folding-related protein preferably comprises a Tcp 1-like protein (CG5372), and the proteolysis-related protein comprises a proteasome and / or a deubiquitinated protein. Preferably, the neurotransmitter metabolism-related protein is at least one selected from GTP cyclohydrolase, alcohol dehydrogenase and aldehyde reductase.

The present invention also provides a biological marker that can be used as a target material to alleviate or treat symptoms of brain diseases, including Parkinson's disease.

Specifically, the present invention relates to actin, trios phosphate isomerase, ubiquitin reductase, superoxide dismutase, oxidative stress protein, toxin reactive protein, proteosome, deubiquitated protein, GTP cyclohydrolase, aldehyde reductase And it provides a biological marker that can be used as a target material selected from ribosomal protein S12.

The present invention also provides a therapeutic agent for brain diseases containing the biological markers as an active ingredient.

The biological marker is preferably an expression of the gene is increased by the mutation of the Parkin gene, it is more preferable that the expression of the gene can be a defense mechanism of the brain, such as stress relief.

The therapeutic agent for brain diseases of the present invention may be used alone or as a pharmaceutical additive together with a pharmaceutically acceptable carrier or excipient according to the application, or in any other form suitable for administration to humans. Carriers that may be contained in the therapeutic agent for brain diseases of the present invention may include extenders, high fiber additives, encapsulating agents, lipids, and the like, examples of which are well known in the art.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example  1: mutation Parkin  Selection of Mutant Drosophila with Genes

In the present invention, in order to select a homozygous Drosophila flies of mutant Parkin genes showing human Parkinson's disease-like traits, it first has a TM6B chromosome as a balancer, that is, a tubular body is used as a genetic marker. Parkin-defective heterozygotes Drosophila bearing their characteristics were self-bred (http://flybase.bio.indiana.edu/.bin/fbidq.html?FBgn0003668). Drosophila without tubby, a genetic marker, was selected under the dissecting microscope. Homozygotes were experimental groups that lost Parkin's function, and heterozygotes were used as controls. In addition, fruit flies were awakened from the pupa and selected for not more than 10 days of adulthood, and immediately placed in a 50 ml tube and stored frozen at -70 ℃.

Example  2. Preparation of Mutant Drosophila Head Samples

50 ml tube containing the mutant fruit flies lacking the Parkin gene stored in Example 1 was strongly shaken to cut the head and torso joints (necks) of the fruit flies, which are vulnerable in structure. Drosophila sample mixed with the separated head and torso part was scrubbed with dry ice in an aluminum foil container in a styrofoam box capable of maintaining a temperature below -20 ° C, followed by screening of only 1.5 ml microcentrifuge tube. ) Here, all the work was done quickly under the condition of -20 ° C to prevent protein denaturation as much as possible, and the temperature was maintained by being inserted into dry ice.

Example  3. Marker  Separation of proteins

In the present invention, two-dimensional gel electrophoresis was used to isolate and identify marker proteins whose gene expression is quantitatively changed by mutation of the Parkin gene. Two-dimensional gel electrophoresis is performed twice by electrophoresis, which is first classified according to the isoelectric point of each protein and then reclassified according to the size of each protein. As a result of the two-dimensional gel electrophoresis, marker proteins were separated horizontally by pH and vertically by protein size.

Specifically, the lyophilized adult head was ground in 5 μl buffer solution (5% SDS, 0.1% DTT) and then suspended at 60 ° C. for 10 minutes. Then add 125 μl rehydration buffer (8 M urea, 2% CHAPS, bromophenol blue, 0.4 mg DTT) and 6.5 μl immobilized pH gradients (IPG) buffer, then suspend at room temperature for 30 minutes. Centrifugation again removed the remaining precipitate. The remaining solution is placed in a row of rehydrated molds (3.5 to 10 NL, 18 cm; Pharmacia Biotech.) And allowed to flow with increasing voltage from 300 V to 3500 V sequentially for 3 hours and then 3500 V Additional voltage was flowed for 3 hours. After primary sorting, the cells were suspended in 100 ml buffer A (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 10 mg DTT) for 20 minutes and 100 ml buffer B (50 mM Tris-HCl). , pH 8.8, 6M urea, 30% glycerol, 10 mg DTT, 25 mg iodoacetaamide) for 20 minutes.

The primary fraction obtained as described above was subjected to SDS-polyacrylamide gel electrophoresis again as follows to perform secondary classification. Primary sorting by pouring SDS-polyacrylamide gel (0.5% w / v agarose, 198 mM to 25 mM, 0.1% w / v tris-glycine-SDS pH 8.3) into a vertical gradient slab Water was loaded to apply a 100 V voltage and electrophoresis was performed for 2 hours for 2 hours. The secondary fraction obtained as above was suspended in a dye solution (0.2% Coomassie blue, 40% methanol, 7% acetic acid) for 2 hours by Coomassie staining and then decolorized solution (45% methanol, 7%). % Stained with acetic acid).

Example  4. Marker  Analysis and Identification of Proteins

MALDI- to analyze the mass by the difference in the separation time of the ion generated by using a laser ionization sample for the object of difference compared to the classification of wild-type fruit flies compared to the classification of wild-type Drosophila The amino acid sequence of each protein was determined using a matrix-assisted laser desorption ionization-time of flight (TOF) method.

Specifically, the selected subjects were cut into pieces (1 mm × 1 mm size) in a gel and placed in a 1.5 ml tube and then with 100 μl wash solution C (400 mM ammonium bicarbonate: 100% acetonitrile = 1: 1). The wash was repeated twice for 20 minutes. Then washed again for 15 minutes using 100 μl acetonitrile, dried for 10 minutes in air, 5 μl trypsin solution (including 50 ng of 10 mM ammonium bicarbonate) and incubated at 37 ° C. for 3 hours. Then, add 5 µl of 5% formic acid, shake, and stir for 10 minutes. The solution is cyano-4-hydroxycinnamic acid (CCA), acetone, isopropanol and nitro. It was placed in a matrix composed of cellulose (nitrocellulose). This matrix was placed in a Reflex III MALDI-ToF mass spectrometer (Bruker UK Ltd., Coventry) and the mass of each object was analyzed using a nitrogen laser.

The mass information of the object obtained by the above method is obtained by requesting mass information from Matrix Science (http://www.matrixscience.com) having an amino acid database and using the MASCOT search engine. The information was accurately identified.

Two-dimensional gel electrophoresis analysis using heterozygous and homozygotes of the Parkin gene and the head of a revertant Drosophila that eliminated the P transfer factor that caused the Parkin mutation revealed that the homozygous of the Parkin gene was heterozygous. Compared to the case of the conjugate and the reduced type, as shown in FIG. 4, protein spots (arrows) showing expression changes in quantity were distinguished and selected. Specifically, 34 protein spots were isolated and identified by the MALDI-TOF method were identified as 29 different proteins. The CG numbers are displayed according to the notation of flybase (), a database for fruit flies. As shown in Table 1, proteins suspected of changing the expression of the gene were classified by labeling by the number of spots.

Aspect Number of spots Protein classification Protein Name Increased expression 1, 2 CG9277 β-tubulin at 56D 3 CG1913 α-tubulin at 84B 4, 5 CG18290 Actin 87E 6, 7 CG7254 Glycogen phosphorylase 8 CG12055 Glyceraldehyde-3-phosphate dehydrogenase 1 9 CG4181 Glutathione S transferase D2 10 CG3481 Alcohol dehydrogenase 16 CG8882 Trip1 17 CG11474 CG11474 20 CG5023 CG5023 21 CG3835 CG3835 30 CG1544 CG1544 34 CG3612 Bellwether Reduced expression 11, 12 CG18290 Actin 87E 13 CG6084 CG6084 14 CG9327 Proteasome 29kD subunit 15 CG11271 Ribosomal protein S12 18 CG12082 CG12082 19 CG5867 CG5867 22 CG11793 Superoxide dismutase 23 CG17331 CG17331 24 CG2171 Triose phosphate isomerase 25 CG7820 Carbonic anhydrase 1 26 CG14207 CG14207 27 CG1341 Rpt1 28 CG5374 Tcp1-like 29 CG2286 NADH: ubiquinone reductase 75kD subunit precursor 31 CG6673 CG6673 32 CG4904 Proteasome 35kD subunit 33 CG9441 Punch

Twelve of the marker proteins selected as above increased expression in Parkin mutants and 16 decreased expression. In addition, protein actin was found to increase some modified forms and decrease other modified forms.

Example  6. Marker  Examination of functional classification of proteins

As a result of comparing the protein expression levels in the Parkin-deficient fruit flies and the control fruit flies as examined in Example 5, it was confirmed that the proteins whose expression amount was significantly reduced or increased could be divided into seven functional categories (Table 2).

First of all, the most common category is the cytoskeletal protein, and Parkin's gene forms the cytoskeleton, as proteins such as actin and tubulin, which support the inside of the cell, are noticeably reduced or increased. It was found to play an important role. In addition, as a second category, proteins related to energy metabolism include proteins associated with glycolysis, ATP synthesis, flavo protein, hydrogen ion transfer, FAD, NAD / NADP, mitochondria. Mitochondria use intracellular nutrients to provide energy for many metabolisms, so it appears that energy metabolism can occur when Parkin genes are missing. In addition, proteolytic categories include proteins associated with ubiquitin and proteasomes that remove proteins that no longer require function or overproduced proteins that have a lethal effect on cells. The Parkin gene has been reported to function to remove the protein by attaching ubiquitin to the obsolete protein, indicating that the deletion of the Parkin gene causes abnormalities in this elimination function. In addition, the proteins in the stress / detoxification category was quantitatively changed compared to the control group, and specifically, the amount of GTP cyclohydrolase (GTP cyclohydrolase I), a dopamine regulatory protein belonging to the neurotransmitter category, was confirmed. Since dopamine itself acts as a neurotransmitter to neurons in the central nervous system and when Parkinson's disease is known, the amount of dopamine in the brain is known to be reduced. In addition, quantitative changes have been observed in signaling proteins essential for many intracellular mass transfer processes (calponin-related), proteins associated with protein folding and alcoholases, and proteins whose function has not yet been identified.

Functional Classification of Proteins function Protein Name Number of spots Cytoskeleton β-tubulin at 56D 1, 2 α-tubulin at 84B 3 Actin 87E 4, 5, 11, 12 Energy metabolism Glycogen phosphorylase 6, 7 Glyceraldehyde-3-phosphate dehydrogenase 1 8 Triose phosphate isomerase 24 NADH: ubiquinone reductase 75kD subunit precursor 29 CG1544 (Oxoglutarate dehydrogenase, mitochondrial protein) 30 Bellwether (Hydrogen-transporting ATP synthase activity) 34 Signal transduction CG5023 (Actin-binding, calponin repeat; calponin like) 20 Stress / detoxification Glutathione S transferase D2 9 Superoxide dismutase 22 CG14207 (Oxidative stress gene) 26 CG6673 (Glutathione transferase activity, response to toxin) 31 Protein folding Tcp1-like (ATP binding, unfolded protein binding) 28 Protein degradation Proteasome 29kD subunit 14 CG12082 (Protein deubiquitination) 13 CG17331 (Endopeptidase activity, proteasome core complex) 23 Rpt1 (ATP binding, proteasome regulatory particle) 27 proteasome 35KD subunit 32 Neurotransmitter metabolism Punch (GTP cyclohydrolase I) 33 Other Alcohol dehydrogenase 10 CG6084 (Aldehyde reductase) 13 Ribosomal protein S12 15 Trip1 (Translation initiation factor activity) 16 CG11474 (Isoform B) 17 Carbonic anhydrase 1 25

Example  7. Marker  Comparative Analysis of Proteins

In the present invention, marker proteins found in the Drosophila model having the mutant Parkin gene and the conventional mouse model were compared and analyzed as follows. As shown in Table 3, the names of the marker proteins observed in Drosophila are listed in comparison to the results in a mouse model with the mutated Parkin gene already reported (Periquet et al ., 2005).

As a result, up to 41.4% of the marker proteins found were found in mouse model experiments, which could analyze the cause of Parkinson's disease common between the two animals. First, tubulin (CG9277) and actin (CG18290) are major constituents of the cytoskeleton and have been reported to bind Parkin protein to promote ubiquitination of Parkin and to degrade tubulin ( Ren et al , 2003). In addition, it was found that glyceraldehyde-3-phosphate dehydrogenase (CG12055), which is involved in glycolysis, was also quantitatively increased in both experimental animal models. In addition, glutathione-S-transferase (CG4181) and NADH (CG2286) related to oxidative stress were also found in both models. CG 12082) were also observed in large quantities in both animal models. In addition, a t-complex protein (CG5374) has been found that is associated with the folding of actin and tubulin (Periquet, 2005). In addition, mitochondrial-associated proteins, NADH and mitochondrial ATP synthase (CG3612), which are thought to be one of the major mechanisms of Parkinson's disease, were observed in both animal models. Therefore, in the development of Parkinson's disease, changes in cytoskeletal structure, proteolysis, oxidative stress and energy generation were found to be important.

Protein Name function Drosophila mouse β-Tubulin at 56D Tubulin α1 chain GTPbinding / structural constituent of cytoskeleton / tubulin binding / GTPase activity Actin 87E γ-actin structural constituent of cytoskeleton / motor activity Glyceraldehyde3 phosphate dehydrogenase 1 Glyceraldehyde3 phosphate dehydrogenase glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) activity Glutathione S transferase D2 Glutathione S transferase glutathione transferase activity / glutathione peroxidase activity Proteasome 29kD subunit Proteasome subunit, beta type 5 endopeptidase activity CG12082 Deubiquitinating enzyme OTUB1 ubiquitin-specific protease activity / cysteine-type endopeptidase activity / ubiquitin-specific protease 5 activity CG17331 Proteasome subunit, beta type 5 endopeptidase activity Tcp1-like t-complex protein A TP binding / hydrogen-exporting ATPase activity, phosphorylative mechanism / ATPase activity, coupled / unfolded protein binding NADH: ubiquinone reductase 75kD subunit precursor NADH ubiquinone oxidoreductase NADH dehydrogenase activity / electron carrier activity NADH dehydrogenase (ubiquinone) activity / transferase activity / iron ion binding CG6673 Glutathione S transferase glutathione transferase activity Proteasome 35kD subunit Proteasome subunit, beta type 5 endopeptidase activity Bellwether ATP synthase α chain, mitochondrial ATP binding / hydrogen-exporting ATPase activity, phosphorylative mechanism / hydrogen-transporting ATP synthase activity, rotational mechanism /

Figure 1 shows the results of analysis of two-dimensional gel electrophoresis of the head proteins of the heterozygous Drosophila mutated Parkin gene.

Figure 2 shows the results of analysis of two-dimensional gel electrophoresis of the head proteins of the homozygous Drosophila mutated Parkin gene.

Figure 3 shows the results of the analysis of the two-dimensional gel electrophoresis of the reduced Drosophila head proteins mutated Parkin gene.

Figure 4 shows the isolation and identification of Drosophila head proteins mutated by Parkin gene quantitatively increased protein in homozygotes in red, reduced protein in blue is shown.

Claims (2)

Glycogen phosphorylase, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, ubiquitin reductase, oxoggle Biological markers can be used to diagnose Parkinson's disease, which consists of one or more energy metabolism-related proteins selected from oxoglutarate dehydrogense and hydrogen-transporting ATP synthase. . The method of claim 1, In addition to the above, a biological marker, characterized in that it is used in the treatment process and prognosis of Parkinson's disease.

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