KR20220047124A - Parkinson's disease pharmaceutical composition and its therapeutic agent containing or modulating TRDN - Google Patents

Abstract

The present specification discloses a pharmaceutical composition for Parkinson's disease that provides an excellent therapeutic effect for Parkinson's disease, a therapeutic agent thereof, and a diagnostic kit thereof when used for Parkinson's disease. One embodiment provides the pharmaceutical composition for Parkinson's disease comprising, as an active ingredient, a substance by TRDN gene expression that regulates dopaminergic cells and alpha-synuclein in degenerative brain disease.

Description

Parkinson's disease pharmaceutical composition and its therapeutic agent containing or modulating TRDN

The present specification provides a pharmacological composition containing or modulating TRDN that modulates dopaminergic cells and alpha synuclein in degenerative brain disease (Pharmaceutical composition and its therapeutic agent containing or modulating TRDN that modulates dopaminergic cells and alpha synuclein in degenerative brain disease) and its provide treatment.

Parkinson's disease (PD) is a chronic progressive neurodegenerative disease. The main pathological feature is the dramatic loss of dopaminergic neurons, mainly in the Substantia Nigra (SN), and the formation of intracytoplasmic Lewy bodies and dystrophic neurites.

However, the mechanism behind the loss of dopaminergic neurons is not yet clear.

The present Examples provide a pharmaceutical composition for Parkinson's disease and a therapeutic agent thereof, which is used for Parkinson's disease and provides an excellent therapeutic effect on Parkinson's disease.

One embodiment provides a pharmaceutical composition for Parkinson's disease comprising, as an active ingredient, a substance by TRDN gene expression that regulates dopaminergic cells and alpha-synuclein in degenerative brain disease.

In another embodiment, a pharmaceutical composition for Parkinson's disease comprising a substance by TRDN gene expression regulating dopaminergic cells and alpha-synuclein as an active ingredient in degenerative brain disease, and a therapeutic agent for Parkinson's disease comprising a carrier containing the pharmaceutical composition provides

Another embodiment provides a method of screening substances affecting the expression level of the TRDN gene that regulates dopaminergic cells and alpha-synuclein in degenerative brain diseases as candidates for the treatment of Parkinson's disease.

This method of screening as a candidate for the treatment of Parkinson's disease includes the following steps.

(a) administering at least one candidate substance to the experimental animal generated in the Parkinson's disease model; and

(b) measuring the expression level of the TRDN gene, when the expression level of the TRDN gene is regulated

The candidate substance is determined to be a substance for preventing or treating Parkinson's disease.

Another embodiment is Parkinson's disease comprising the steps of isolating a sample from the subject, measuring the expression level of the TRDN gene from the sample, and diagnosing the subject as Parkinson's disease when the expression level of the TRDN gene is regulated Provides diagnostic methods.

According to the present embodiments, a pharmaceutical composition for Parkinson's disease and a therapeutic agent thereof are used for Parkinson's disease, thereby providing an excellent therapeutic effect on Parkinson's disease.

According to the present embodiments, a pharmaceutical composition for Parkinson's disease and a therapeutic agent thereof are used for Parkinson's disease to provide an effect of alleviating symptoms related to muscle function of Parkinson's disease.

According to this embodiment, the Parkinson's disease diagnostic kit provides an excellent effect in the treatment and prevention of Parkinson's disease by diagnosing the Parkinson's disease at an early stage.

According to the present embodiment, the method of screening as a candidate substance for a therapeutic agent for Parkinson's disease has an effect of screening a candidate substance for a therapeutic agent for Parkinson's disease to treat and prevent Parkinson's disease.

1 shows the results of performing the rota-road test and the pole test.
Figure 2 shows the results of confirming the expression levels of TH and TRDN by performing western blot (WB) to extract brain tissue, particularly SN from each group.
Figure 3 shows the results of confirming the TRDN and TH levels after culturing SH-SY5Y and human-derived dopamine cells.
4 shows the evaluation results for the level of cytochrome C (Cyt.C), Bcl-2-related X (Bax), Bcl-2 and Bax/Bcl-2 ratio using WB.
5 shows the results of performing a cytotoxicity assay.
6 shows the results of immunofluorescence (IF) to observe the TRDN and TH levels in SH-SY5Y cells of the control group and the MPP+ induced-PD model group.
7 is a control and 1-methyl-4-phenyl--1,2,3,6-tetrahydropyridine (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)) induced Parkinson's disease The group's rotarod and pole tests are shown.
8 shows Western blot analysis of proteins linked to skeletal muscle contraction.
9 shows Western blot analysis of components linked to Ca ²⁺ channels of sarcoplasmic reticulum at various 1-methyl-4-phenylpyridinium (MPP+) concentrations in C2C12 cells.
FIG. 10 shows western blot analysis of components linked to sarcoplasmic reticulum Ca ²⁺ channels after treatment of C2C12 cells with 1-methyl-4-phenyl pyridinium (MPP+) and TRDN siRNA.

Hereinafter, some embodiments of the present invention will be described in detail with reference to exemplary drawings. In adding reference numerals to the components of each drawing, it should be noted that the same components are given the same reference numerals as much as possible even though they are indicated on different drawings. In addition, in describing the embodiments of the present invention, if it is determined that a detailed description of a related well-known configuration or function may obscure the gist of the present invention, the detailed description thereof will be omitted.

As used herein, the terms “treatment”, “suppression” and “attenuation” refer to the use of the composition of the present invention in an individual to reduce the differentiation of specific cells, the level of a specific substance, or the progression of a specific disease or disorder. Any action that delays.

As used herein, the term “individual” refers to all animals including humans.

Hereinafter, a pharmaceutical composition for Parkinson's disease that provides an excellent therapeutic effect on Parkinson's disease and a therapeutic agent thereof according to embodiments of the present invention will be described.

Example 1: Pharmaceutical composition and therapeutic agent therefor

Parkinson's disease (PD) is a chronic progressive neurodegenerative disease. Parkinson's disease (PD) is the second most common degenerative brain disease in the elderly.

However, the cause is still unknown. Although TRDN is a factor of Ca2+ regulators in the sarcoplasmic reticulum (SR) of the heart and general muscle, its brain function is unclear.

As can be seen from the experimental examples below, the present inventors identified TRDN in the sarcoplasmic reticulum (SN) in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD mouse model by genetic sequence. It was confirmed that the expression level decreased.

To investigate the relationship between TRDN and PD, the present inventors studied the expression level of PD-related proteins and TRDN in the SH-SY5Y cell line using western blotting (WB) and siRNA, respectively. To investigate the apoptosis effect on dopaminergic cells by reduced TRDN level, cytotoxicity assay and fluorescence activated cell sorting (FACS) analysis were performed when TRDN level decreased. was used to evaluate cell viability and apoptosis, and the level of apoptosis-related proteins was confirmed by WB or FACS analysis.

These results showed a relationship between TRDN expression and PD in that reduced TRDN levels induce PD-like properties. Reduced TRDN levels also induced increases in Cyt.C and PS levels, and decreased Bcl-2 levels, a characteristic of apoptosis. FACS dot plot results showed that apoptosis increased with increasing TRDN siRNA concentration. As a result of cytotoxicity analysis, cell viability was decreased under the same conditions as in FACS analysis.

Therefore, reduced TRDN levels may be associated with apoptotic death of dopaminergic cells in the SN of PD, and TRDN administration may have a positive effect on PD by reducing apoptotic cell death.

Based on these studies, the present specification, dopaminergic cells in degenerative brain disease and alpha-synuclein (alpha-synuclein) to control the TRDN that contains or modulates a pharmacological composition is proposed.

One embodiment provides a pharmaceutical composition for Parkinson's disease comprising, as an active ingredient, a substance by TRDN gene expression that regulates dopaminergic cells and alpha-synuclein in degenerative brain disease.

The TRDN gene or Triadin gene is a human gene associated with the release of calcium ions from the myocytoplasmic reticulum that causes muscle contraction through calcium-induced calcium release. TRDNs are a family of polyproteins that result from different processing of the TRDN gene on chromosomes.

As will be described later in the experimental example, several isoforms of TRDN have been identified, and the function of TRDN is to inhibit Ca2 + release from depolarization-induced sarcoplasmic reticulum (SR).

In the present specification, the substance by TRDN gene expression is a protein expressed by the TRDN gene sequence, but may refer to a protein expressed by giving some modifications to the TRDN gene so that the protein is well expressed.

Alpha-synuclein is a protein abundant in the human brain. Small amounts are also found in the heart, muscle, and other tissues. In the brain, alpha-synuclein is mainly found at the ends of nerve cells (neurons) with special structures called presynaptic terminals. Human alpha-synuclein protein consists of 140 amino acids and is encoded by the SNCA gene.

Another embodiment, Parkinson's disease pharmaceutical composition comprising a substance by TRDN gene expression regulating dopaminergic cells and alpha-synuclein as an active ingredient in degenerative brain disease, and Parkinson's disease comprising a carrier containing the pharmaceutical composition provide treatment.

The pharmaceutical composition may further include a pharmaceutically acceptable carrier, and may be formulated with the carrier and provided as a food, drug, feed additive, and drinking water additive. As used herein, the term “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not inhibit the biological activity and properties of an administered compound without irritating the organism.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound. Examples of acceptable pharmaceutical carriers for compositions formulated as liquid solutions include sterile and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.

The composition for treating Parkinson's disease of the present specification is applicable to any formulation containing it as an active ingredient, and may be prepared as an oral or parenteral formulation. The pharmaceutical formulation of the present specification is oral, rectal, nasal, topical (including buccal and sublingual), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous). and those suitable for administration (including intravenous) or forms suitable for administration by inhalation or insufflation.

Formulations for oral administration containing the composition of the present specification as an active ingredient include, for example, tablets, troches, lozenges, aqueous or oily suspensions, powders or granules, emulsions, hard or soft capsules, syrups or elixirs. can do. For formulation into dosage forms such as tablets and capsules, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, an excipient such as dicalcium phosphate, a disintegrant such as corn starch or sweet potato starch, Masene stearate Lubricating oil such as calcium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax may be included, and in the case of capsule formulation, a liquid carrier such as fatty oil may be further contained in addition to the above-mentioned substances.

Formulations for parenteral administration containing the composition of the present specification as an active ingredient include injections such as subcutaneous injection, intravenous injection or intramuscular injection, a suppository injection method, or an aerosol for inhalation through the respiratory tract, etc. can be formulated as In order to formulate an injectable formulation, the composition of the present invention may be mixed with a stabilizer or buffer in water to prepare a solution or suspension, and it may be formulated for unit administration in ampoules or vials. For injection into a suppository, it may be formulated as a composition for rectal administration such as a suppository or enema containing a conventional suppository base such as cocoa butter or other glycerides. When formulated for spraying, such as an aerosol, a propellant or the like may be blended with an additive so that the water-dispersed concentrate or wet powder is dispersed.

The method of administering the pharmaceutical composition of the present invention and its therapeutic agent is not particularly limited, but depending on the desired method, parenteral administration or oral administration such as intravenous, subcutaneous, intraperitoneal, inhalation, skin application, patch or topical application can do.

The dosage varies according to the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease. Daily dosage means an amount of a therapeutic agent of the present invention sufficient to treat a disease state ameliorated by administration to a subject in need thereof. An effective amount of a therapeutic agent depends on the particular compound, the disease state and its severity, and the individual in need of treatment, which can be routinely determined by one of ordinary skill in the art. As a non-limiting example, the dosage of the composition according to the present invention to the human body may vary depending on the patient's age, weight, sex, dosage form, health status, and disease degree.

Hereinafter, the present invention will be described in more detail by describing an experimental example. However, the following experimental examples are merely examples of the present invention, and the content of the present invention should not be construed as being limited thereto.

Experimental Example 1: Pharmaceutical composition

1. Overview

Parkinson's disease (PD) is a neurodegenerative disease characterized by major behavioral symptoms and includes tremor, movement disorders, bradykinesia, and stiffness (Jankovic, 2008).

Parkinson's disease (PD) is the second most common degenerative brain disease in the elderly. PD causes clinical symptoms such as tremor, bradykinesia (slow movement), muscle stiffness, and postural instability. Some patients have dementia, depression and anxiety (Dauer and Przedborski, 2003).

Pathological features in the brain show loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) of the midbrain. In addition, the level of tyrosine hydroxylase (TH), which catalyzes tyrosine to form L-DOPA, is decreased in the striatum (striatum, ST) and substantia nigra (SN) (Haavik and Toska, 1998).

Although the cause of PD is still unknown, the probable cause of PD is genetic factors such as oxidative stress, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, and familial types (Haavik and Toska, 1998; Walden and Muqit, 2017). About 95% of PD cases are sporadic PD. Genetic PD due to gene mutations in α-synuclein, Parkin, and Dj-1 accounts for 5% of all PD cases (Dauer and Przedborski, 2003). On the other hand, exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP) was not only in humans but also in mice. Causes selective dopaminergic neurodegeneration in PD. Therefore, MPTP is most commonly used to create animal models of PD for research (Dauer and Przedborski, 2003).

In this experimental example, the SN part of the mouse brain was used to create an MPTP mouse model and perform gene sequencing. As a result, the Triadin (TRDN) gene expression level showed a significant decrease. Therefore, this specification focuses on the relationship between TRDN and PD.

TRDN has been mainly studied in cardiac and skeletal muscle. Several isoforms of TRDN have been identified, and the function of TRDN is to inhibit Ca2+ release from the depolarization-induced sarcoplasmic reticulum (SR). In addition, TRDN-deficient mice had reduced Ca2+ storage size of SR (Wang et al., 2009). Therefore, this specification can be inferred that TRDN expression is closely related to Ca2+ regulation. When the muscle contracts, Ca2+ release from the SR is generated by plasma membrane depolarization. Thus, TRDN deletion has also been reported to cause muscle dysfunction (Oddoux et al., 2009).

However, the relationship between TRDN and PD has not been reported. Therefore, in this specification, we investigated TRDN expression in relation to the pathological features of PD and how TRDN downregulation affects neurons in SH-SY5Y cells using siRNA transfection. In addition, based on the reported literature, apoptosis is considered to be neuronal degradation in PD (Anglade et al., 1997; Tatton et al., 2003). Therefore, the relationship between TRDN and PD was studied in terms of apoptosis.

2. Method

2.1. MPTP Model of Parkinsonism

Six-week-old male resistant C57BL/6 mice (20-22 g; DBL, Korea) were divided into two groups: a control group (CTL) and an MPTP-treated group (MPTP). Mice in the control group were intraperitoneally injected with 0.9% saline (100 μL) once daily for 4 weeks, and mice in the MPTP group were injected with MPTP-HCl (20 mg/kg of free base) with 0.9% saline (100 μL) intraperitoneally. did). A continuous chronic Parkinson's disease model is generated at 24-hour intervals for 4 weeks. After final MPTP treatment, mice were anesthetized using 16.5% urethane and perfused intracardially with cold 0.05-M sodium-phosphate buffer to allow immunohistochemical evaluation. The MPTP model of Parkinsonism was confirmed by dopaminergic cell death (Choiand Lim, 2010, Yeo et al., 2013).

The Upper School Animal Experimentation Committee approved all animal protocols used in this specification. Reagents used but not mentioned were purchased from Sigma, USA.

2.2. Rota-rod and pole testing

This test was performed to evaluate motor performance in an MPTP-induced 4-week Parkinsonism model before the last MPTP injection during the light phase. Rota-rod training was performed for 15 minutes at a constant 30 rpm on 2 days for 2 weeks, and pole test training was also performed once a day.

The rota-rod treadmill diameter was 280 mm, and the rota-rod test was performed in accelerated mode for 4 min from 10 rpm to 50 rpm at a run time of 5 min. After 4 minutes in accelerated mode, a constant 50 rpm was maintained for 1 minute until completion. The time to the first falling or the first drop was measured and the test was performed twice. In the pole test, a pole with a length of 548 mm and a diameter of 8 mm was placed vertically and made of wood. Mice were moved from top to bottom and time was measured (n=6/group).

2.3. RNA extraction and microarray analysis

Total RNA was extracted from bilateral SN tissues in each group using the RNeasy Plus Mini kit (QIAGEN, USA). Isolated RNA quality was estimated and quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, USA).

Aliquots of total RNA (300 ng) were applied to an Affymetrix GeneChip Mouse Gene 1.0 ST Array (genome wide expression profiling chip; 28,853 genes with 35,557 probes; Affymetrix, USA). The GeneChip Whole Transcript (WT) Sense Target marker analysis manual was followed as previously reported (Choi et al., 2011; Lin et al., 2010, Yeo et al., 2018).

1.4. Microarray Data Analysis

Significant signal intensities of the scanned images were first assessed using visual inspection. Quality control of the scanned data was performed by checking the sequence of signal intensities of poly-A and hybridization controls using Expression Console software (Affymetrix, USA). Microarray data was analyzed using GenPlex ver.3.0 (ISTECH, Korea). Four CEL files (2 CEL files generated in each group x 2 experimental groups) were uploaded and normalized under the following conditions; Exact match (PM)-PM intensity adjustment only, (2) robust multi-chip analysis (RMA) quantitation method as probe set summarization algorithm for log transformation using base 2 (log2), and quality of preliminary data in preprocessing module A quantization normalization method to evaluate the quantization (which serves as a data quality control). The average signal intensities of 28,853 genes were obtained from two chips in each group. After normalization, the differentially expressed genes (DEGs) of the GeneChip that satisfy the conditions of the fold change cutoff (1.5) and Student's t-test significance criterion (p <0.05) were identified using the DEG-finding module. did.

1.5. immunohistochemistry

The brains of 4-week MPTP Parkinsonism mice were removed and fixed in 0.05-M sodium-phosphate buffer containing 4% paraformaldehyde at 4° for 1 day, and then the brains were dehydrated with sucrose buffer for 2 days.

Control brain sections (40-μm thick) were sectioned using a cryomicrotome. Immunohistochemical analyzes were performed using the ABC kit and the Mouse on Mouse (M.O.M) immunodetection kit (Vector Laboratories, CA) and a modification of the avidin-biotin-peroxidase method. Briefly, sections containing entire ST and SN regions were incubated in phosphate buffered saline (PBS; pH 7.4) with 3% H2O2 followed by blocking buffer (3% bovine serum albumin (BSA) and 10% horse serum). (horse serum)). When a rabbit anti-TH antibody (1:200; Santa Cruz Biotechnology, USA) was used, the tissue was incubated with the primary antibody overnight at 4° for 1 h at room temperature with M.O.M. mice were treated with Ig-blocking reagent (Vector Laboratories).

Thereafter, the sections were treated with biotinylated anti-mouse IgG and avidin-biotin-peroxidase complexes, which were used to treat dopaminergic neurons in the triamino and SN regions. It can be reacted with diaminobenzidine-hydrogen peroxide solution to confirm. Neuronal cells were observed using a Nicon X-cite series 120 Q microscope (Nikon; Japan).

2.6. Cell Lines and Cultures

Cells from the human-derived SH-SY5Y cell line were cultured under standard culture conditions (5% CO2, 37°C). 10% fetal bovine serum (BioWest), 100-U/mL penicillin-streptomycin (Gibco; USA) and 0.1 mM nonessential amino acids (Gibco, USA) It was cultured in a minimum essential medium (MEM; Welgen, Namcheon-myeon, Korea) containing the

2.7. siRNA (small interfering RNA) knockdown

To down-regulate the expression of TRDN and investigate the effect, siRNA (5'-UCAUG UGG GUA GAC UCA GU-3') and negative control duplex (siRNA, 5'-UUC UCC GAACGU GUC ACG UTT-3') to TRDN ) was used. siRNA was obtained from Bioneer Inc. of Korea.

Transfection reagent (Promega, USA) was used in Opti-MEM (Gibco) medium at a 3.5:1 transfection reagent-to-duplex RNA ratio (3.5:1 transfection reagent-to-duplex RNA ratio) during transfection. .

2.8. Western blotting

SH-SY5Y cells were homogenized in 20 mM radioimmunoprecipitation assay buffer (RIPA) on ice for 20 min after PBS washing. When ST and SN tissues were homogenized in 20 mM RIPA using a sonicator (Qsonica Q55; USA) and incubated on ice for 20 min. After centrifugation at 12,000 rpm for 15 min at 4 °C, a sample of the supernatant of the same protein concentration was separated using 4-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) followed by polyvinylidene difluoro It was transferred to polyvinylidene difluoride (PVDF) membrane (Pall Life Science, USA). Membranes were blocked with 3% BSA in 0.1% Tris-buffered saline (TBS; 150mM NaCl containing 0.1% Tween-20, TBST) with 20-mM Tris-HCl [pH 7.5] and incubated for 1 h at room temperature. It was washed overnight with the primary antibody and then washed with 0.1% TBST. The membrane was incubated with the secondary antibody for 1 hour and washed with 0.1% TBST. Rabbit anti-TRDN (1:2000; MyBioSource), rabbit anti-TH (1:5000; Abcam), rabbit anti-β actin (1:5000; Cloud Clone), rabbit anti-cytochrome C (1:600; Abcam) Rabbit anti-Bax (1:1000; Abcam), rabbit anti-Bcl-2 (1:5000; Abcam) and mouse anti-β actin (1:5000; Santa Cruz Biotechnology) were used as primary antibodies. Goat anti-mouse IgG (HRP; suitable ratio; Abcam) and goat anti-rabbit IgG H & L (HRP; suitable ratio: Santa Cruz Biotechnology) were used as secondary antibodies. Antigen-antibody complexes were visualized by Amersham Imager 680 (GE Healthcare) using ECL detection reagent (GE Healthcare; USA).

2.9. Cytotoxicity assay

A cytotoxicity assay was performed to determine the effect of reduced TRDN levels on cells. Cell viability and cytotoxicity were investigated using EZ-CYTOX (DoGenBio, Korea). Following the recommended protocol, cell viability was measured after TRDN siRNA (siTRDN) treatment for 24 hours. SH-SY5Y cells were treated with various concentrations of siTRDN (5, 10, 25, 50 and 100 nM) and 100-nM negative control siRNA.

2.10. Fluorescence-activated cell sorting assay

Following siTRDN treatment, fluorescence-activated cell sorting (FACS) analysis was performed to analyze apoptosis in cells. siTRDN treatment was performed in the same manner as the cytotoxicity assay. Controls were added for compensation and none of the control cells were treated. Cells were collected with a cell scraper and washed twice with PBS at 2500 rpm, 4° C. for 5 minutes. Cells were resuspended in binding buffer at 106 cells/mL and 100 μL (105 cells) was aliquoted. Fluorescein (FITC)-annexin V) and propidium iodide (PI) were treated according to the manufacturer's suggested protocol (BD Parmingen™). Samples were diluted 5 times with binding buffer and detection was performed with cytoFLEX (Beckman Coulter Life Sciences; US).

2.11. immunofluorescence

SH-SY5Y cells were used for immunofluorescence, and 1 mM MPP+ was treated in the MPP+ group for 18 hours. Cells were fixed using 4% paraformaldehyde and methanol. After fixation, cells were incubated in blocking buffer (1% BSA, 5% goat serum in PBS) for 30 min. Cells from each group were incubated with primary antibodies, mouse anti-TH and rabbit anti-TRDN, followed by secondary antibodies, goat anti-mouse IgG Flamma 488 and goat anti-rabbit IgG (H + L) tetramethylrhodamine (TRITC). ) join . Finally, DAPI (1 μg/ml) was used to stain the nuclei of the cells.

Photographic documentation was performed using a Nicon X-cite series 120 Q microscope (Nikon; Japan), and exposure parameters were the same for the control and MPP+ groups.

2.12. statistical analysis

Statistical analysis was performed using Student's t-test and the analysis of variance (ANOVA) of SPSS 25 (Release 2017, PASW Statistics for Windows, version 25.0, Chicago: SPSS Inc.). .

3. Results

To create an MPTP-induced PD mouse model, mice were injected with MPTP-HCL (20 mg/kg) every 24 hours for 4 weeks. Rota-rod test and pole test were performed to confirm the condition of the mice, and the results are shown in FIG. 1 . In the rota-rod test, the mice in the MPTP group fell earlier than the mice in the control group (t(14)=3.17, P=0.007). In the pole test, mice in the MPTP group reached the bottom earlier than mice in the control group ((t (12) = 2.52, P = 0.027)).

Mice in the MPTP group appeared to crawl faster because they did not have the strength to grip the poles with their hind legs. These behavioral tests showed decreased mobility of mice in the MPTP group and clear differences between the control and MPTP groups.

The result of the gene arrangement confirmed that the TRDN gene expression level was decreased in FIG. 2C (t (2) = 59.77, P = 0.00028). In addition, the expression levels of TH and TRDN were confirmed by performing western blotting (WB) to extract brain tissue, particularly SN, from each group. The results are shown in Fig. 2 (Fig. 2A and B). Similar to the gene sequence results, the WB results showed decreased expression levels of TRDN and TH (TRDN; t (4) = 11.67, P = 0.0003, TH; t (2) = 53.3, P = 0.00035).

To reconfirm the reduction in THB, immunohistochemistry (IHC) with 3,3-diamino-benzidine (DAB) was performed, and the reduction in TH levels in SN and ST is shown in Fig. 2D. These results confirmed the reduction of TRDN levels in the MPTP-induced PD mouse model.

Meanwhile, in order to observe the effect in human cells, SH-SY5Y and human-derived dopaminergic cells were cultured and treated with TRDN siRNA (siTRDN) at concentrations of 10 and 100 nM for 24 hours. After 24 h, TRDN and TH levels decreased as shown in Figure 3 (TRDN; F (2, 3) = 25.34, P = 0.013, TH; F (2, 3) = 82.0, P = 0.002). These results showed that reduced TRDN induced a decrease in TH levels.

In addition to the expression levels of PD-associated proteins, to determine the effect of TRDN reduction on cells, WB was used to determine the levels of cytochrome C (Cyt.C), Bcl-2-associated X (Bax), Bcl-2 and Bax/ The Bcl-2 ratio was evaluated. As a result, Cyt.C levels increased (t (4) = -2.79, P = 0.049), Bax levels did not change significantly, but Bcl-2 levels decreased (F (2, 3) = 12.97, P = 0.033), The Bax/Bcl-2 ratio increased as shown in FIG. 4 (t (2) = -4.60, P = 0.044). From this result, it can be expected that TRDN reduction will cause apoptosis.

This is important because it implies that a decrease in TRDN levels induces apoptosis in dopaminergic cells of PD. To confirm apoptosis, a cytotoxicity assay was performed, and the results showed cell viability shown in Figure 5C. We found no significant change in cell viability at 10-nM siTRDN, but cell viability decreased at siTRDN concentrations >25 nM (Fig. 5C). At 100-nM siTRDN, cell viability was reduced by 71.50%.

To more accurately confirm that cell death was due to apoptosis, cells were analyzed using flow cytometry. Phosphatidylserine (PSs), known as apoptosis signaling factor in cells, was stained with annexin V-FITC, and the high FITC signal means that the cells are undergoing apoptosis. On the other hand, the nuclei were stained with PI, and the low PI signal means that the cells are still alive. Therefore, it was confirmed that the H1-LR zone, high FITC signal and low PI signal were useful for detecting apoptosis in negative control (100-nM NC siRNA treatment) and 10-, 25-, 50-, 100-, and nM siTRDN treatment. was effective against SH-SY5Y cells (Fig. 5A).

The percentage of the result is expressed as a bar graph in FIG. 5B . The increase in apoptosis was significant at 25-nM siTRDN and continued to increase up to 100-nM siTRDN (F(4, 5) = 5.23, P = 0.049). These results are semantically consistent with the results of the cytotoxicity assay in which cell viability significantly affected apoptosis from 25 nM siTRDN (F(4, 25) = 9.11, P = 0.00011).

Meanwhile, to observe the TRDN and TH levels in SH-SY5Y cells of the control group and the MPP+ induced-PD model group, immunofluorescence (IF) was performed. TH was stained with FITC and TRDN was stained with TRITC. The results showed that TH and TRDN were successfully incorporated into the control group (WT group), especially in the nucleus (Fig. 6D). On the other hand, TH and TRDN were not merged in the MPP+ group (FIG. 6H). Thus, under WT conditions, TRDN and TH interact with each other, but when PD occurs, TRDN levels decrease and the interaction between TRDN and TH also decreases.

4. Conclusion

To date, the function and role of TRDN in neurons is unknown. However, the results of this study show that TRDN and PD are related. TRDN levels were decreased in MPTP-induced Parkinsonism model mice, and SH-SY5Y cells showed the characteristics of PD when TRDN was knocked down. That is, the reduced TRDN level decreased the TH level as a characteristic of PD. Through this, the relationship between TRDN level and PD was confirmed.

In addition, significant changes in apoptosis-related factors in cells with decreased TRDN levels were also associated with apoptosis. The results showed that when the TRDN level decreased, Cyt.C increased and Bcl-2 increased. Bcl-2 is known to block the release of Cyt.C from mitochondria and prevent cell apoptosis (Yang et al., 1997).

Indeed, we deduced that reduced Bcl-2 levels increased the efflux of Cyt.C from mitochondria and increased the release of Cyt.C, leading to apoptosis via the caspase 9 pathway (Li et al., 1997). Decreased TRDN levels may allow the apoptotic process to proceed in this way. This means that it can be inferred that TRDN is deeply associated with dopaminergic cell death in PD. In general, TRDN is already known to be a factor associated with inhibition of Ca ²⁺ release from SR in heart and general muscle (Chen et al., 2013, Guo and Campbell, 1995, Marty, 2015). Reduced TRDN levels can be considered to induce dysfunction of Ca ²⁺ regulation in the ER of neurons in the SN, and the apoptotic process associated with Ca ²⁺ is activated. However, the mechanisms of neuronal Ca ²⁺ regulation changes and TRDN-induced apoptosis have not been elucidated.

In conclusion, TRDN expression is associated with PD in that TRDN levels were reduced in the MPTP-induced PD mouse model and the pathological features of PD were induced by reduced TRDN levels. In addition, cells started to die at concentrations of siTRDN >25 nM. Because changes in the levels of apoptosis-related factors such as Cyt.C, Bcl-2, Bax/Bcl-2 ratio and PS indicate that apoptosis has occurred, cell death by reduced TRDN expression levels is is cell death.

Moreover, as the concentration of siTRDN increased, the level of apoptosis also increased. Thus, TRDN is associated with neuronal apoptosis and apoptotic cell death in PD. Thus, in dopaminergic cell death in PD, TRDN administration may be a potential candidate for PD treatment.

Through this Experimental Example 1, Parkinson's disease pharmaceutical composition and its therapeutic agent containing a substance by TRDN gene expression that regulates dopaminergic cells and alpha-synuclein in degenerative brain disease as an active ingredient were used for Parkinson's disease. It can be seen that an excellent therapeutic effect is provided.

Experimental Example 2 : Pharmaceutical composition and its therapeutic agent

1. Overview

Muscle contraction is regulated by cytoplasmic Ca ²⁺ levels, which in turn are associated with Ca ²⁺ channels. Ryanodine receptor (RYR), calsequestrin 1 (CSQ1), triadin (TRDN), junctin, and dihydropyridine receptors are involved in Ca ²⁺ homeostasis. It is a protein involved (Oddoux et al., 2009).

In this Experimental Example 2, focusing on the role of TRDN in muscle, proteins such as RYR and CSQ1 related to TRDN in the sarcoplasmic reticulum membrane Ca ^{2 +} channel were studied.

TRDN has mostly been studied in cardiac and skeletal muscle. TRDN regulates Ca ²⁺ release from the SR. In cardiac muscle, TRDN mutations cause leakage of Ca ²⁺ ions from the sarcoplasmic reticulum lumen because CSQ1 is unable to inhibit the release of Ca ²⁺ ions by RYR. Reduction of TRDN impairs muscle function. Furthermore, while CSQ1 serves as a luminal calcium sensor for RYR, TRDN may also mediate the interaction between RYR and CSQ1 to detect Ca ²⁺ levels in the SR lumen.

In this Experimental Example 2, the TRDN-related factor is 1-methyl-4-phenyl--1,2,3,6-tetrahydropyridine (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) )) was studied in an induced PD mouse model. The quadriceps (QF) were used in the study because the quadriceps (QF) are the most important muscles for walking and jumping. The mouse model was a semi-chronic model induced by 4 weeks of MPTP administration. The C2C12 cell line, an immortalized mouse myoblast cell line, was used to identify changes in TRDN-related factors after treatment with 1-methyl-4-phenylpyridinium (MPP+) and TRDN siRNA.

2. Method

2.1. MPTP-induced PD mouse model and Rota-rod and pole tests

MPTP-induced PD mouse models were generated by injecting MPTP-HCL (20 mg/kg) every 24 h for 4 weeks. Rotarod and pole tests were performed to determine the condition and motor ability of mice in the control and MPTP groups.

7 is a control and 1-methyl-4-phenyl--1,2,3,6-tetrahydropyridine (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)) induced Parkinson's disease The group's rotarod and pole tests are shown.

In Figure 7 (A) rotarod (diameter 28mm) test was performed in Accel Forward mode for 4 minutes at 10-50 rpm for 5 minutes of running time. The final 1 min was performed at a constant 50 rpm. (n = 5, **P < 0.005) (B) In the pole test, the length and diameter of the column were 548 mm and 8 mm, respectively. The mouse was moved from the top to the bottom of a vertical wooden pole. (n = 5, *P < 0.05) Statistical analysis was performed using Student's t-test.

As can be seen from Figure 7, in the rotarod test, the mice in the MPTP group fell faster than the mice in the control group, and the average difference was 44 seconds. In the pole test, mice in the MPTP group reached the floor faster, on average, than the control mice. Mice in the MPTP group were found to lack strength by sliding down without gripping the pole with their hind legs. This indicated that MPTP mice showed reduced motor capacity compared to control mice.

2.2. Western blotting

8 shows Western blot analysis of proteins linked to skeletal muscle contraction. In FIG. 8 , the quadriceps muscle tissue important for walking and jumping was used for B. Western blot analysis like ScN (sciatic nerve). (B) Western blot analysis of TRDN, RYR and CSQ1 are factors of Ca ²⁺ channels in the sarcoplasmic reticulum membrane of control and MPTP-treated groups. (C) Protein levels for Western blot results are shown as bar graphs (N = 3, *P <0.05, **P <0.005, # P <0.0005).

Western blot analysis of the muscle tissue of the quadriceps muscle (see (A) in FIG. 8) showed that the expression levels of TRDN, RYR and CSQ1 were decreased in the MPTP group (see (B) and (C) in FIG. 8). RYR and CSQ1 levels were at least 50% lower compared to controls. Considering that TRDN, RYR and CSQ1 are components of Ca ²⁺ channels in the SR membrane of T-tubules, this may indicate a reduced level of Ca ²⁺ channels in the rcoplasmic reticulum (SR) membrane.

FIG. 9 shows Western blot analysis of components linked to Ca ²⁺ channels of sarcoplasmic reticulum at various 1-methyl-4-phenylpyridinium (MPP+) concentrations in C2C12 cells. In Fig. 9 (A) at various MPP+ treatment concentrations, 1 mM, 2.5 mM, 5 mM MPP+ was applied for 18 hours in Western blot analysis of TRDN, RYR and CSQ1, which are components of Ca ²⁺ channels of the sarcoplasmic reticulum membrane. . In Figure 9 (A) shows the Western blot result, (B) histogram shows the quantified protein level. (N = 3, *P <0.05, **P <0.005)

C2C12 (mouse myoblast) cells were used to examine the results at the cellular level. After induction of the PD model with the neurotoxin MPP+, the effect of MPP+ on C2C12 cells was investigated when the MPP+ concentration was increased. High MPP+ concentration induced a decrease in the expression levels of large TRDN, RYR, and CSQ1 of Ca ²⁺ channels in C2C12 cells (see FIG. 9 ).

FIG. 10 shows western blot analysis of components linked to sarcoplasmic reticulum Ca ²⁺ channels after treatment of C2C12 cells with 1-methyl-4-phenyl pyridinium (MPP+) and TRDN siRNA.

In FIG. 10 (A), Western blot analysis of TRDN, RYR and CSQ1, which are proteins of the sarcoplasmic reticulum membrane, is shown after application of MPP+ and TRDN siRNA. Negative control cells were treated with negative control siRNA (100 nM) under the same conditions as TRDN siRNA treatment. MPP+ was applied at a concentration of 1 mM for 18 hours. The incubation time of TRDN and negative control siRNA was 48 hours. In FIG. 10 (B), the histogram shows quantified protein levels from Western blot results.

NC, negative control siRNA treatment (100 nM for 2 days); TRDN siRNA 10 nM, TRDN siRNA treatment (10 nM for 2 days); TRDN siRNA 100 nM, TRDN siRNA treatment (100 nM for 2 days). Stealth siRNA for TRDN (5'-UC AUG UGG GUA GAC UCA GU-3') and negative control siRNA (5'-UUC UCC GAA CGU GUC ACG UTT-3') were purchased from Bioneer Korea. (n = 4, *P<0.05, **P<0.005, #P<0.0005) Statistical analysis was performed with ANOVA of SPSS 25.

In addition, when the TRDN level was decreased through TRDN siRNA, changes in the composition of Ca ²⁺ channels were analyzed in MPP+-treated C2C12 cells (see FIG. 10 ). Since the protein levels of TRDN, RYR, and CSQ1 were decreased in C2C12 cells treated with 1 mM MPP+ (see FIG. 9 ), MPP+ treatment was performed identically with 1 mM in FIG. 10 . In C2C12 cells treated with 10 nM or 100 nM TRDN siRNA, the level of TRDN expression was also reduced. In addition, the expression levels of RYR and CSQ1 were also decreased (see FIG. 10 ). This indicated that decreasing TRDN levels in this PD model also decreased RYR and CSQ1 expression.

In Experimental Example 2, we developed an MPTP-induced PD mouse model and confirmed whether the expression level of Ca ²⁺ channel components was decreased in the quadriceps muscle of the leg using the MPTP-induced PD mouse model. In addition, the expression levels of TRDN, RYR and CSQ1 were found to be decreased by MPP+ treatment in C2C12 cells at the cellular level.

These results indicated that the expression of Ca2 + channel components TRDN, RYR and CSQ1 was reduced in the MPTP-induced PD mouse model. It was also shown that Ca ²⁺ channel expression was decreased in the SR membrane of skeletal muscle. RYR-associated Ca ²⁺ channels in T-tubules suggest that they may affect muscle function in relation to dyskinesia in Parkinson's disease. Since Ca ²⁺ release from the rcoplasmic reticulum lumen of muscle is important for contraction, insufficient Ca ²⁺ release due to decreased Ca ²⁺ channel expression may impair muscle contraction regulation. This may be related to acromancy (slow movement), which is one of the symptoms seen in people with Parkinson's disease. However, further investigation into factors involved in the regulation of Ca ²⁺ homeostasis is needed.

In conclusion, the expression levels of RYR, TRDN, and CSQ1, which are components of Ca ²⁺ channels, were decreased in the quadriceps muscle of MPTP-induced PD mice and in C2C12 cells treated with MPP+. Reducing TRDN levels decreased RYR and CSQ1 levels, which may lead to Ca ²⁺ leakage through RYR and decreased Ca ²⁺ release channel levels. Furthermore, it delays Ca ²⁺ uptake and muscle contraction from the sarcoplasmic reticulum lumen until the required Ca ²⁺ concentration in the cytoplasm is reached, potentially implicating this mechanism in motor symptoms of PD. Restoring TRDN, which is a component of the Ca ²⁺ channel irradiated in Experimental Example 2, can alleviate symptoms related to muscle function of Parkinson's disease.

Through this Experimental Example 2, Parkinson's disease muscle function by using the Parkinson's disease pharmaceutical composition and the therapeutic agent for Parkinson's disease containing the substance by TRDN gene expression described in Example 1 as an active ingredient in degenerative brain diseases such as Parkinson's disease It can be seen that it provides an effect that can relieve symptoms related to

Hereinafter, with reference to the above-described experimental examples, a method for screening as a candidate material for a therapeutic agent for Parkinson's disease, a diagnostic method for diagnosing Parkinson's disease, and a diagnostic kit thereof according to other embodiments will be described in detail.

Example 2: Method of screening

Another embodiment provides a method of screening substances affecting the expression level of the TRDN gene that regulates dopaminergic cells and alpha-synuclein in degenerative brain diseases as candidates for the treatment of Parkinson's disease.

In the above-described experimental example, it can be seen that TRDN expression is related to PD in that the TRDN level is decreased in the MPTP-induced PD mouse model and the pathological features of PD are induced by the decreased TRDN level. In addition, as the concentration of siTRDN increased, the level of apoptosis also increased, confirming that TRDN was associated with neuronal apoptosis and apoptotic cell death in PD.

Another embodiment provides a screening method for a substance for preventing or treating Parkinson's disease, comprising the following steps.

(a) administering at least one candidate substance to the experimental animal generated in the Parkinson's disease model; and

(b) measuring the expression level of the TRDN gene, when the expression level of the TRDN gene is regulated

The candidate substance is determined to be a substance for preventing or treating Parkinson's disease.

The Parkinson's disease model may be the MPTP model of Parkinsonism described in Experimental Examples, but the present invention is not limited thereto. does not In addition, the experimental animal may be the mouse described in the experimental example, but the present invention is not limited thereto.

The expression level may be measured as described in Experimental Examples, but may also be performed by other general methods.

Example 3: Diagnostic method

Another embodiment provides a diagnostic method for diagnosing Parkinson's disease with the degree of expression of the TRDN gene that regulates dopaminergic cells and alpha-synuclein in degenerative brain disease.

Parkinson's disease diagnosis method according to another embodiment may diagnose Parkinson's disease by measuring the expression level of TRDN in a subject, for example, a material isolated from a human or animal, for example, blood or cells.

Specifically, the method for diagnosing Parkinson's disease according to another embodiment includes the steps of isolating a sample from a subject, measuring the expression level of the TRDN gene from the sample, and when the expression level of the TRDN gene is regulated, the subject is diagnosed with Parkinson's disease including the steps of

As described above, the sample may be any material isolated from the subject, for example, blood or cells. The subject may be an animal, including a human.

In general, the step of separating the specimen may include methods of naturally separating the specimen from the subject or artificially separating the specimen using a syringe or the like.

Measuring the expression level of the TRDN gene in the step of measuring the expression level of the TRDN gene from the sample may be the general methods described in Experimental Examples.

In the step of diagnosing Parkinson's disease for the subject, if the expression level of the TRDN gene is statistically significantly regulated, the subject may be diagnosed with Parkinson's disease.

Another embodiment provides a diagnostic kit for diagnosing Parkinson's disease with the degree of expression of the TRDN gene that regulates dopaminergic cells and alpha-synuclein in degenerative brain disease. This diagnostic kit is a kit for performing the aforementioned diagnostic method, and may have a configuration of a general diagnostic kit.

The present invention provides a therapeutic agent for PD by specifying TRDN that affects dopaminergic cells and alpha-synuclein in Parkinson's disease. It is possible to provide a therapeutic agent for Parkinson's disease by specifying substances that prevent or increase the decrease.

In addition, the present invention can provide a method for screening substances affecting the expression level of a specific gene as candidate substances for treatment, a method or diagnostic kit for diagnosing Parkinson's disease based on the gene expression level of TRDN.

Although the embodiments have been described with reference to the drawings, the present invention is not limited thereto, and the above description is merely illustrative of the technical idea of the present invention, and those of ordinary skill in the art to which the present invention pertains. Various modifications and variations will be possible without departing from the essential characteristics of the invention. Accordingly, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, but to explain, and the scope of the technical spirit of the present invention is not limited by these embodiments. The protection scope of the present invention should be construed by the following claims, and all technical ideas within the scope equivalent thereto should be construed as being included in the scope of the present invention.

Claims (6)

A pharmaceutical composition for Parkinson's disease comprising, as an active ingredient, a substance by TRDN gene expression that regulates dopaminergic cells and alpha-synuclein in degenerative brain disease. The method of claim 1,
The substance by the TRDN gene expression is a protein expressed by the TRDN gene sequence, or a pharmaceutical composition for Parkinson's disease, which is a protein expressed by giving some modifications to the TRDN gene so that the protein expression is good. Parkinson's disease pharmaceutical composition comprising, as an active ingredient, a substance by TRDN gene expression that regulates dopaminergic cells and alpha-synuclein in degenerative brain disease; and
A therapeutic agent for Parkinson's disease comprising a carrier containing the pharmaceutical composition. 4. The method of claim 3,
The substance caused by the TRDN gene expression is a protein expressed by the TRDN gene sequence, or a therapeutic agent for Parkinson's disease, which is a protein expressed by partially modifying the TRDN gene so that the protein expression is good. There is provided a method of screening as a candidate for a therapeutic agent for Parkinson's disease, comprising the steps of:
(a) administering at least one candidate substance to the experimental animal generated in the Parkinson's disease model; and
(b) measuring the expression level of the TRDN gene, when the expression level of the TRDN gene is regulated
The candidate substance is determined to be a substance for preventing or treating Parkinson's disease. isolating the sample from the subject:
measuring the expression level of the TRDN gene from the sample; and
A method for diagnosing Parkinson's disease comprising the step of diagnosing the subject as Parkinson's disease when the expression level of the TRDN gene is regulated.

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