COMPOSITION FOR PREVENTING OR TREATING NEUROMUSCULAR DISEASE, COMPRISING PRMT1 PROTEIN OR GENE ENCODING SAME

Abstract

The present invention relates to a use for, by using the protein arginine methyltransferase 1 (PRMT1) protein or a gene encoding the same, preventing or treating a neuromuscular disease induced by motor neurons, particularly, damage to motor neurons caused by oxidative stress in the neuromuscular junction; and a method for screening a candidate material for activating the expression of PRMT1. PRMT1 deficiency in the motor nerve or neuromuscular junction induces aggravated degenerative motor nerve damage caused by aging, and DNA damage caused by oxidative stress and inflammation, thereby enabling the induction of neuromuscular disease, and thus the disease may be treated through the overexpression and activity of the PRMT1 protein and a gene encoding the same.

Claims

1. A method for protecting motor neuron cells, the method comprising: administering a PRMT1 protein or a gene encoding the same to a subject in need thereof.

2. The method of claim 1, wherein the protection of motor neuron cells prevents damage to motor neurons due to aging, oxidative stress and inflammation.

3. The method of claim 1, wherein the PRMT1 protein or the gene encoding the same promotes the re-innervation of damaged motor neuron cells.

4. A method for ameliorating a neuromuscular disease, the method comprising: administering a PRMT1 protein or a gene encoding the same.

5. The method of claim 4, wherein the neuromuscular disease is selected from the group comprising dystrophy, a motor neuron disease, myopathy, and a neuromuscular junction disease.

6. The method of claim 5, wherein the motor neuron disease is selected from the group comprising amyotrophic lateral sclerosis (ALS), infantile progressive spinal muscular atrophy (SMA), progressive bulbar palsy, pseudobulbar palsy, progressive muscular atrophy (PMA), progressive lateral sclerosis (PLS), monomelic amyotrophy (MMA) and Huntington's disease.

7-9. (canceled)

10. A method for screening a material preventing, ameliorating or treating a neuromuscular disease, the method comprising: i) treating motor neuron cells or neuromuscular junctions with a candidate; ii) determining the expression or activity of PRMT1 in the motor neuron cells or neuromuscular junctions treated with the candidate; and iii) selecting candidates which enhance the expression or activity of PRMT1 compared to a non-treatment group.

Description

DESCRIPTION OF DRAWINGS

[0047] FIG. 1 illustrates the results of observing senescence-based survival rate in mice specifically lacking PRMT1 in motor neurons (PRMT1 mnKO).

[0048] FIG. 2 illustrates the results of confirming body weight loss due to aging in PRMT1 mnKO mice.

[0049] FIG. 3 shows the results of limp clasping in PRMT1 mnKO mice in order to observe changes in behavioral ability due to aging.

[0050] FIG. 4 shows the results of comparing the distance moved and speed of PRMT1 mnKO mice with those of the control in an open field test.

[0051] FIG. 5 shows the results of comparing the time that RMT1.sup.mnKO mice endured in a rotarod test with that of the control.

[0052] FIG. 6 illustrates the results in which the hind leg muscle weight is recovered after damage to motor neurons in PRMT1 mnKO mice.

[0053] FIG. 7 shows the results of confirming that the muscle fiber size is significantly reduced in the TA muscle of PRMT1 mnKO mice.

[0054] FIG. 8 illustrates the results of confirming the degree of muscle fibrosis of PRMT1 mnKO mice.

[0055] FIG. 9 illustrates the results of comparing the expression degrees of nerve regeneration and muscle atrophy markers in PRMT1 mnKO mice with those in the control.

[0056] FIG. 10 shows the results of confirming the re-innervation degree of the axons in PRMT1 mnKO mice in comparison with the control.

[0057] FIG. 11 shows the results of confirming whether the stress-related sensitivity of PRMT1 mnKO mice is increased through the expression of DNA damage markers.

[0058] FIG. 12 shows the results of confirming, through western blot analysis, increases in expression level of stress-related proteins in a motor neuron cell line (NSC34) in which PRMT1 protein expression was suppressed.

[0059] FIG. 13 illustrates the results of confirming, by DHR123 staining, that ROS detection was increased in a motor neuron cell line (NSC34) in which PRMT1 protein expression was suppressed.

[0060] FIG. 14 confirms the result that the expression of a DNA damage-associated gene (γ-H2AX) was increased in a motor neuron cell line (NSC34) in which PRMT1 protein expression was suppressed.

[0061] FIG. 15 shows the results of confirming, through reduction of oxidative stress markers and inflammatory markers, the protective effect of neuronal cell stress induced by TNFα treatment in a motor neuron cell line (NSC34) in which PRMT1 protein was overexpressed.

MODES OF THE INVENTION

[0062] Hereinafter, the present specification will be described in detail with reference to Examples in order to specifically explain the present specification. However, the Examples according to the present specification may be modified in various forms, and it is not interpreted that the scope of the present specification is limited to the Examples described below in detail. The Examples of the present specification are provided to more completely describe the present specification to a person with ordinary skill in the art.

Example 1. Materials and Experimental Methods

1-1) Construction of Experimental Models

[0063] Constant conditions (temperature 22±2° C., humidity 55±5%) were maintained in an experimental animal breeding room. A light cycle of 12 hours a day and a dark cycle of 12 hours a day were adapted.

[0064] Mice carrying a PRMT1-floxed allelic gene (PRMT1f/f purchased from EUCOMM) were crossed with transgenic mice expressing Cre under the control of an Hb9 promoter (Hb9-Cre provided by Jackson Laboratory). For sciatic nerve denervation surgery, the anesthetization of the animals was induced with 4% isoflurane and then maintained by continuous inhalation of 2% isoflurane on a surface heated to 37° C. The skin was incised posteriorly parallel to the left femur. The sciatic nerve was exposed to the mid-thigh and then damaged using tongs for 10 seconds. After denervation, the nerve was repositioned, the muscle was closed with surgical dissolvable sutures, and the skin incision was sutured with suture tape. Carprofen (5 mg/kg) was subcutaneously administered for postoperative pain control.

1-2) Behavior Analysis

[0065] To analyze behavioral differences between PRMT1 f/f and PRMT1 hb9cre mice, open field and rotarod tests were performed.

[0066] The open field test was performed in an open plastic container (30×30×30 cm). When each experiment was initiated, mice were placed in the corners of the plastic container and movements were recorded for 20 minutes. The distance traveled and speed were analyzed through Noldus EthoVision 13.0 tracking software. Motor coordination and balance were measured using a rotarod apparatus (model 7600, Ugo Basile). For a fixed speed rotarod test (17 rpm), mice were disposed in a rotating cylinder after receiving 3 training sessions for 3 days before an actual experiment. A cut-off of 200 seconds per session was used. For an accelerated rotarod test (for 300 seconds, 4 to 40 rpm), mice received 3 training sessions for 3 days before an actual experiment. In both of the above two experiments, the time to fall off the rotarod was recorded.

1-3) Cell Culture and Transfection

[0067] NSC34 cells were cultured in a composition of 10% FBS, 1% GlutaMAX, 10 units/mL penicillin, and 10 μg/mL streptomycin based on DMEM media. NSC34 cells were transfected with a pcDNA, PRMT1-HA plasmid using a Mirus transfection reagent.

[0068] To make the expression of PRMT1 deficient in NSC 34 cells, the cells were transfected with Adenoviral pLKO.1-puro, PRMT1-shRNA.

1-4) Protein and RNA Analysis

[0069] After cells were harvested, cells were lysed by adding a lysis buffer (20 mM Tris-HCl, pH8, 150 mM NaCl, 1% Triton X, proteinase inhibitor) for 30 minutes, then centrifuged at 13000 rpm for 30 minutes, and then a cell lysate sample was quantified, the same amount of protein was subjected to SDS-PAGE electrophoresis, and transferred to a PVDF membrane. After reaction with the corresponding primary and secondary antibodies, protein expression levels were confirmed by exposing the protein to X-ray film using an ECL reagent.

[0070] NSC34 cells and muscle tissue were homogenized with FastPrepR-24 (MP Biomedicals) and RNA was extracted using an easy-spin Total RNA Extraction Kit (iNtRON Biotechnology). Fold changes in gene expression were normalized compared to the expression of the ribosomal gene L32.

1-5) Histology and Immunofluorescence Analysis

[0071] The tibialis anterior muscle and extensor digitorum longus muscle were used as tissues used in the experiments, and the tibialis anterior muscle was frozen and fixed with OCT compound using liquefied nitrogen immediately after isolation, and then stored in an ultra-low temperature freezer at −80° C. Tissue sections were cut into a thickness of 7 μm using a cryomicrotome maintained at −20° C., and the degree of fibrosis was observed after Sirius Red staining.

[0072] Immunohistochemical staining was performed using the tibialis anterior muscle isolated from the animal tissues. The frozen sections stored in an ultra-low temperature freezer at −80° C. were washed twice with 1×PBS. The frozen sections were fixed at ordinary temperature for 15 minutes using 4% paraformaldehyde and washed twice with 1×PBS. The tissue was reacted with a permeabilization buffer (0.5% Triton-X/PBS) at ordinary temperature for 5 minutes, and washed twice with 1×PBS. The tissue was reacted with a TE buffer at 100° C. for 10 minutes for antigen retrieval, and washed twice with 1 ×PBS. After blocking with a blocking buffer (5% goat serum, 0.1% Triton-X) for 1 hour, a primary antibody (laminin) was diluted 1:500 in the blocking buffer and reacted at 4° C. for 12 hours, and then washed twice with 1×PBS. After reacting with a secondary antibody, the tissue was washed twice with 1×PBS, encapsulated, and analyzed under a fluorescence microscope.

[0073] Further, neuromuscular junction staining (NMJ staining) was performed using the extensor digitorum longus muscle isolated from the animal tissue. The extensor digitorum longus muscle was isolated, immediately fixed using 4% paraformaldehyde at ordinary temperature for 15 minutes, and washed three times with 1×PBS. After blocking with a blocking buffer (3% BSA, 0.5% Triton-X) for 2 hours, a primary antibody (neurofilament, synaptophysin) was diluted 1:300 in the blocking buffer and reacted at 4° C. for 24 hours, and then washed three times with 1×PBS. A secondary antibody and BTX antibody conjugated with the secondary antibody were reacted at room temperature for 2 hours, then washed twice with 1×PBS, and then encapsulated for analysis under a fluorescence microscope.

1-6) Statistical Analysis

[0074] All values were expressed as mean±SEM or SD. Statistical significance was calculated by a paired or unpaired two-tailed Student's t-test or analysis of variance (ANOVA) test followed by Tukey's test. Differences were considered significant at P<0.05.

Experimental Example 1. Effect of Motor Neuron-Specific PRMT1 Deficiency

1-1) Effect of Aging-Based Survival Rate and Weight Loss in PRMT1-Deficient Mice

[0075] The survival rate and body weight of mice specifically lacking PRMT1 in motor neurons(PRMT1 mnKO) were measured at each month of age. As a result, it was confirmed that the survival rate and body weight of mice decreased sharply as aging progressed compared to wild-type mice (FIGS. 1 and 2). That is, it was confirmed that PRMT1, which is expressed in motor neurons, plays an important role in the maintenance of survival rate and body weight of aged mice.

1-2) Changes in Behavioral Ability Due to Aging in PRMT1-Deficient Mice

[0076] In the case of PRMT1 mnKO mice specifically lacking PRMT1 in motor neuron, as shown in FIG. 3, it could be confirmed that motor nerves were degenerated because a phenomenon of limp clasping was observed. In addition, as a result of confirming by performing an open field test as a behavioral evaluation, as shown in FIG. 4, it was confirmed that the moving distance and walking speed in the PRMT1 mnKO mice were decreased compared to normal mice (PRMT1 f/f).

[0077] Furthermore, as a result of performing a rotarod test to evaluate the ability to regulate exercise capacity, as shown in FIG. 5, it was confirmed that PRMT1 mnKO mice had a shorter endurance time than PRMT1 f/f. From the experimental results described above, it can be seen that PRMT1 plays an important role in maintaining behavioral ability in aged mice in motor neurons.

1-3) Delayed Nerve Recovery in PRMT1-Deficient Mice

[0078] After sciatic nerve injury was induced in PRMT1 mnKO mice and normal mice (PRMT1 f/f), respectively, and re-innervation was induced for 28 days, the mice were sacrificed after behavioral analysis.

[0079] As a result of measuring and comparing the hindlimb muscle weights of PRMT1 mnKO mice and PRMT1 f/f, it was confirmed that the muscle recovery was delayed in PRMT1 mnKO mice (FIG. 6), and as a result of confirming the tibialis anterior (TA) muscle by laminin staining, it could be confirmed that the size of the myofiber of the PRMT1 mnKO mice was significantly decreased (FIG. 7).

[0080] Furthermore, as a result of performing sirus red staining on muscle cells to confirm the degree of fibrosis, which is known to increase during muscle atrophy, it could be confirmed that the degree of fibrosis was increased in PRMT1 mnKO mice (FIG. 8), and in qPCR results for TA muscle, markers indicating denervation and muscle atrophy were increased compared to PRMT1 f/f mice (FIG. 9).

[0081] That is, from the above results, it can be seen that PRMT1 present in motor neurons plays an important role in the re-innervation of damaged motor neurons.

1-4) Delayed Re-Innervation of Axons in PRMT1-Deficient Mice

[0082] In neuromuscular junction staining (NMJ staining), it could be confirmed that in the flexor digitorum brevis (FDB) muscle and extensor digitorum longus (EDL) muscle, the thickness of motor neuron axons and the size of alpha_bungarotoxin (α-BTX), which is a neuromuscular junction marker, were thinner and smaller than those in PRMT1 f/f mice (FIG. 10). From this, it can be confirmed that re-innervation was delayed in the axons in the case of PRMT1 mnKO mice.

1-5) Increase in DNA Damage and Stress Sensitivity in PRMT1-Deficient Mice

[0083] In order to confirm DNA damage and stress-related sensitivity in PRMT1 mnKO mice, the lumbar spinal cord, which is responsible for the movement of the hind leg muscles of mice, was serially sectioned and then observed after co-staining with choline acetyltransferase (ChAT), which is a motor neuron marker, and gamma H2AX. As a result, it could be confirmed that the expression of gamma H2AX, which is a DNA damage marker, was increased compared to PRMT1 f/f (FIG. 11). From the above results, it can be seen that PRMT1-deficient motor neurons have increased stress-related sensitivity.

Experimental Example 2. Effect of PRMT1 Expression in Motor Neuron Cells

2-1) Effect of Increase in Cellular Stress when PRMT1 Expression is Suppressed

[0084] By inhibiting PRMT1 expression using an shPRMT1 adenovirus in an NSC34 cell line, which is a motor neuron, changes in the expression levels of cellular stress markers expressed in the cell line were confirmed. As a result, as shown in FIG. 12, it was confirmed, by western blotting analysis, that the expression of stress-related proteins p-eIF2a and c-caspase3 was significantly increased compared to the control, and the expression of gamma H2AX, which is a DNA damage protein, was also increased compared to the control. Further, through dihydrorhodamine123 (DHR123) staining, which is ROS staining, it was confirmed that the amount of ROS detected in NSC34 cells in which the expression of PRMT1 was inhibited was increased compared to the control (FIG. 13).

[0085] In addition, it was confirmed that when PRMT1 expression was inhibited using an shPRMT1 adenovirus in an NSC34 cell line, which is a motor neuron cell, and the cell line was stained with γ-H2AX, which is a DNA damage-related gene, γ-H2AX expression was significantly increased compared to the control (FIG. 14).

2-2) Effect of Protecting Neuron Cells from Cellular Stress During Overexpression of PRMT1

[0086] PRMT1 was overexpressed in an NSC34 cell line, which is a motor neuron cell, and mRNA expression of stress genes was observed by qPCR after TNFα treatment, which induces cell stress. It was confirmed that in TNFα-treated motor neuron cells, the expression levels of ROS stress markers (Gpx1, Sod1, and Sod2) and inflammatory stress markers (Ccl2, Cxcl1, and Cxcl10) were all increased, but all the markers were significantly decreased when PRMT1 was overexpressed (FIG. 15). From these results, it can be confirmed that overexpression of PRMT1 in motor neuron cells has an effect of being able to protect cell stress.

[0087] In the foregoing, the present invention has been examined mainly based on the preferred examples thereof. A person with ordinary skill in the art to which the present invention pertains will be able to understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed examples should be considered not from a restrictive viewpoint, but from an explanatory viewpoint. The scope of the present invention is defined not in the above-described explanation, but in the claims, and it should be interpreted that all the differences within a range equivalent thereto are included in the present invention.