WO2013036031A2

WO2013036031A2 - Pharmaceutical composition including a microrna486 expression inhibitor as an active ingredient for preventing and treating neurological disorders

Info

Publication number: WO2013036031A2
Application number: PCT/KR2012/007112
Authority: WO
Inventors: 강수경
Original assignee: 서울대학교 산학협력단
Priority date: 2011-09-06
Filing date: 2012-09-05
Publication date: 2013-03-14
Also published as: KR20130026950A; WO2013036031A3

Abstract

The present invention relates to a pharmaceutical composition including a miRNA486 expression inhibitor as an active ingredient for preventing and treating neurological disorders. More particularly, since it has been verified that interference with the expression of miR486 using an miR486 inhibitor overcomes the inhibition of NeuroD6, thereby inducing improvements in spinal cord injuries (SCI), the miR486 inhibitor can be used as an active ingredient in a pharmaceutical composition for preventing and treating neurological disorders, and further, the mechanism by which miR486 controls the expression of NeuroD6 can be applied in screening for candidate materials for preventing and treating neurological disorders.

Description

【Specification】

[Name of invention]

Pharmaceutical composition for preventing and treating neurological diseases, including microRNA486 expression inhibitors as an active ingredient

Technical Field

The present invention relates to a pharmaceutical composition for preventing and treating neurological diseases, including miRNA486 expression inhibitors as an active ingredient.

BACKGROUND OF THE INVENTION Self-proliferation and differentiation of stem cells and somatic cells are regulated by intracellular and intercellular mechanisms. Intercellular mechanisms include differential gene expression that is regulated at epigenetic, transcriptional, translational and posttranslational levels. Small, unencoded RNAs (microRNAs, miRNAs), a novel class of regulatory molecules as factors involved in gene expression, are known to be involved in tissue regeneration. miRNAs are emerging as important factors in translational regulation, involved in regulating the function of cell fate, and alterations in functional gene expression have been shown to play an important role in the secondary damage process in some disease models. Since miRNAs can regulate the expression of a specific set of functional genes, they are useful candidates as regulators upstream of the secondary spinal cord injury process among disease model miRNAs signaling mediators. Many miRNAs play an important role in neurodevelopment and are believed to be important mediators of cell differentiation. Based on the specific interaction of miRNAs with their target genes, RNA-based techniques have potential as therapeutic strategies. miRNAs expression will be a novel therapeutic target for the treatment of various diseases, including cancer, cardiovascular disease and wounded invasion. As one of the diseases, neurodestruction is due to various factors that depend on the inherent properties of the underlying neurodestructive disorder. This indicates the homeostasis of unstable cells along with the cytotoxicity of by-products mediated by overloaded immune cells. In particular, previous studies have explored the potential for miRNAs in neurodestruction. It has been shown that altered expression of miRNAs contributes to secondary damage following trauma to the central nervous system.

This study highlights the fact that all gene regulation requires proper regulation of both positive and negative regulators for tissue regeneration. New findings on the specific role of miRNAs indicate that miRNAs are important contributors to the diversity of nerve function. During the pathogenesis of neurodestructive disease, the miRNA paradigm is closely related to the regulation of gene expression and influences signaling pathways.

NeuroD6 is a factor associated with nerve regeneration. NeuroD6 is known to have a neuroprotective role in the central nervous system against R0S-mediated secondary injury. The NeuroD6 protein promotes neuronal differentiation and survival in the pathological microenvironment and stimulates the biomass of the mitochondria to initiate anti-apoptosis and chaperone responses. The NeuroD6 expression interference is associated with the induction of motor neuron apoptosis initiated by actively proliferating macrophages and the secretion of pro-inflammatory factors mediating apoptosis and myelin destruction of motor neurons. These successive pathological events result in secondary damage for a long time after traumatic primary injury to the spinal cord. In addition to initiation of neuronal differentiation, NeuroD6 promotes neuronal survival through expression of anti-apoptotic regulators that maintain mitochondrial preservation. Previously, the function of miR miR-133b, a miRNA during spinal cord regeneration of zebrafish, has been studied, and an increase in miR-133b expression in brain regeneration has been identified. MiR-486 has been reported to be ubiquitous in rat brain, spinal cord, liver and heart. And identifying miR-486 or miR-422a, which regulates cell survival in the heart, and treating or preventing cardiac hypertrophy, heart failure, or myocardial infarction following increased expression of miR-486 and / or miR-422a in heart tissue. It has been reported to include a method of inhibiting smooth muscle cell proliferation through contact of a smooth muscle cell with a miR-486 inhibitor such as antisense oligonucleotide. In addition, a method of treating or preventing heart failure by interfering with miR-486 expression by administering an inhibitor of miRNAs such as antisense -miR486 has been known. However, in the spinal cord injury model, the antisense miR-486 was suppressed by interfering with the expression of miR-486. Neuroprotective function and function in recovery of disease have not been studied. Therefore, the present inventors have studied to identify the relationship between miR As and the therapeutic target using spinal cord injury. As a result, motor neurons related to pathophysiology mediated by reactive oxygen species (ROS) in sustained nerve injury Since miR486 was specifically overexpressed in the eye, miR486 inhibitor expression interference was found to induce spinal cord injury (SCI) improvement by restoring the inhibition of NeuroD6. The present invention was completed by confirming that miR486 can be used as an active ingredient for prevention and treatment, and miR486 can be used for screening candidates for preventing and treating neurological diseases.

[Detailed Description of the Invention] [Technical Issues]

An object of the present invention is a pharmaceutical composition for preventing and treating neurological diseases, a method for treating neurological diseases, a method for screening candidate substances for preventing and treating neurological diseases, a kit for diagnosing neurological diseases, and a diagnostic for neurological diseases. It provides a gene detection method and a neurological disease diagnosis method for providing information.

Technical Solution In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing and treating neurological diseases, comprising miR486 expression inhibitor as an active ingredient. The present invention also provides a method for preventing and treating cancer, comprising administering a pharmaceutically effective amount of a miR486 expression inhibitor to an individual suffering from a neurological disease. In addition, the present invention

1) treating the test substance to miR486 expressing cell line;

2) measuring the expression level of miR486 of the cell line; and 3) provides a method for screening a candidate substance for preventing and treating neurological diseases, including selecting a test substance whose expression level is reduced compared to a control group that does not process the test substance.

In addition, the present invention

1) treating the test substance to miR486 and NeuroD6 expressing cell line;

2) measuring the amount of NeuroD6 expression in said cell line; and

3) It provides a method for screening a candidate substance for preventing and treating neurological diseases, comprising selecting a test substance whose NeuroD6 expression amount is increased compared to a control group not treated with the test substance.

The present invention also provides a kit for diagnosing a neurological disease, comprising a nucleotide of the miR486 gene, a nucleotide having a sequence complementary to the nucleotide, or a fragment thereof.

In addition, the present invention

1) measuring the expression level of miR486 in a sample derived from the subject as an experimental group;

2) comparing the expression level of miR486 of step 1) with the expression level of miR486 of a sample derived from a normal individual as a control; And

3) Provides a gene detection method for providing information for diagnosing neurological disease, comprising determining that the miR486 expression level of step 2) is higher than that of the control group.

In addition, the present invention

1) measuring the expression level of miR486 in the subject-derived sample as an experimental group;

2) comparing the expression level of miR486 of step 1) and the expression level of miR486 of a sample derived from a normal individual as a control; And

3) When the miR486 expression level of step 2) is increased compared to the control group provides a method for diagnosing a neurological disease comprising the step of determining that the subject has a neurological disease. The present invention also provides a pharmaceutical composition for the prevention and treatment of neurological diseases containing NeuroD6 as an active ingredient. Advantageous Effects In the present invention, expression interference of miR486 using miR486 expression inhibitor

Since it was confirmed that induction of spinal cord injury (SCI) is improved by restoring the inhibition of NeuroD6, the niiR486 inhibitor can be used as an active ingredient of a pharmaceutical composition for preventing and treating neurological diseases, and miR486 expresses NeuroD6. Mechanism to control the can be used to screen for candidates for the prevention and treatment of neurological diseases, can be used as a marker for diagnosing neurological diseases. [Brief Description of Drawings]

1 shows overexpression of miR486 in motor neurons after traumatic injury and inhibition of target genes in spinal cord tissue:

FIG. 1A shows the expression of NeuroD6, a miR486 target gene, before spinal cord injury and after 7 days of injection of miR486 or scrambled RNA;

(B) shows in situ hybridization analysis of miR486 expression in spinal cord injury lesions before and after 7 days of miR486 or scrambled RNA injection; FIG. 1C shows miR486 region ί “in motor neurons in injured lesions in spinal cord tissue;

FIG. 1D shows immunohistochemical images of NeuroD6 expression of NF160 + motor neurons in spinal cord injury lesions (T10), and arrows represent NeuroD6 expressing in NF160 + motor neurons; and

FIG. 1E shows a schematic flow diagram of an experimental procedure for treating and evaluating spinal cord injury.

2 shows the therapeutic application of miR486 interference and evaluation of traumatically injured spinal cord:

2 (a) shows the functional outcome of antisense _−m iR486 (antisense ™ miR486) treatment in the injured spinal cord assessed with Beattie and Bresnahan (BBB);

FIG. 2 (b) shows antisense-injured spinal cord in the damaged spinal cord assessed on the Basso mouse scale (BMS). miR486 treatment results are shown;

Figure 2 (c) shows the induction of traumatic spinal cord injury symptoms when injecting miR486;

Figure 2 (d) shows histological analysis and apoptotic cell death evaluation of neuronal destruction when miR486 or scrambled RNA treated normal and damaged spinal cord;

2 (e) shows normal spinal cord (Normal SC), spinal cord treated with scrambled RNA (scramRNA SO, damaged spinal cord (SCI), damaged spinal cord treated with scrambled RNA (SCI / scramRNA), normal spinal cord treated with miR486 ( comparison of TUNEL-positive populations in injured spinal cords treated with itiiR486 / SC) and antisense_1 1¾86 (3 (: 1 / ^^ ᅳ 111? 486);

FIG. 2 (f) shows caspase-3 (caspase) in the normal spinal cord, spinal cord treated with scrambled RNA, damaged spinal cord, damaged spinal cord treated with scrambled RNA, normal spinal cord treated with miR486, and damaged spinal cord treated with antisense miR486. -3) express expression;

Figure 2 (g) shows ATP production in normal spinal cord, spinal cord treated with scrambled RNA, damaged spinal cord, damaged spinal cord treated with scrambled RNA, normal spinal cord treated with miR486 and damaged spinal cord treated with antisense-miR486;

Figure 2 (h) shows immunohistochemical analysis of neurodestructive phenotypes in spinal cord injury and miR486 interference in spinal cord injury mice 5 and 10 days after antisense miR486 or scrambled RNA treatment;

2 (i) shows the evaluation of myelin destruction and neurodestruction by Luxal Fast Blue (LFB) staining of damaged spinal cord tissue; And

FIG. 2 (j) shows the expression of TuJ, MBP and GFAP of neuronal markers NeuroD6 and NF160 after post-treatment on days 0, 2 and 7 of miR486 expression interference in injured spinal cord tissue. 3 shows that miR486 expression interference markedly inhibited pro-inflammatory factor secretion and also induced R0SVIII gene expression in spinal cord injury:

3 (a) and 3 (b) show real-time RT-PCR analysis and histochemical analysis of other pro-inflammatory cytokine expression before interference with the damaged spinal cord and after 7 days of miR486 expression interference or scramble A treatment. Represents;

3 (c) shows real-time RT-PCR of MPO, iNOS and eNOS expression in spinal cord injury lesions. Is a diagram showing the analysis;

FIG. 3D shows real-time RT-PCR analysis of SEPN1, TXNL1, GPxl and GPx3, genes involved in the R0S-elimination system 7 days after interference or expression of miR486 on medullary spinal cord lesions;

3 (e) shows the expression of P13K, p-Akt, Bax, Caspase-3 and cytokine CCcytochrome C, Cytoch C) when treated with antisense _miR486 and scrambled RNA in injured spinal cord tissues;

3 (f) shows an immunohistochemical analysis of the expression of the redox removal protein TXNL1 in motor neurons of injured spinal cord tissue (T10);

Figure 3 (g) shows an immunohistochemical analysis of the expression of the redox protein GPx3 in motor neurons of injured spinal cord tissue (T10); and

Figure 3 (h) shows an immunohistochemical analysis of the expression of SEPN1, a redox removal protein in motor neurons of injured spinal cord tissue (T10).

4 shows the potential role of miR486 in the secondary injury process in the injured spinal cord:

4 (a) shows a schematic flow chart of an experimental procedure for i R486 and miR486 expression interference and functional analysis;

Figure 4 (b) shows the expression of Tuj, NF160, MBP and GFAP when injected miR486 and scrambled RNA into normal spinal cord, damaged spinal cord, normal spinal cord;

4 (c) shows miR486, NeuroD6, Cox2, IL-Ιβ, IL-6 and TNFci expression when scrambled R A, miR486 and antisense-miR486 were injected separately or together with normal and injured spinal cord;

4 (d) shows NeuroD6, EDI, MPO, iNOS and eNOS expression in the group injected with miR486 in the normal spinal cord, the damaged spinal cord and the normal spinal cord;

4 (e) shows R0S expression levels in spinal cord lesions, the group injected with scrambled R A to the normal spinal cord, the group injected with miR486 to the normal spinal cord, and the group injected with miR486 and antisense -miR486 to the normal spinal cord;

4 (f) shows SEPNl, TX L1, Gpxl and Gpx3 expression when scrambled RNA, miR486 and antisense—miR486 were injected separately or together with normal and injured spinal cord. Represents;

Figure 4 (g) shows the expression of P-P13K, p-Akt, p-p38, p-JNK, Bax, caspase-3 and cytochrome C when treated with scrambled RNA and miR486 in the normal and damaged spinal cord ;

Figure 4 (h) shows the flow cytometry results and TUNEL analysis results when the scrambled RNA and miR486 injected into the cultured NPC;

4 (i) shows C0X2, MPO, iNOS, eNOS, p-p38, p-JNK, Bax caspase 3, in the group treated with scrambled RNA, mi 486 or miR486 and antisense _m iR486 in cultured NPCs. Expression of cytokine C; and

4 (j) shows NeuroD6 expression when treated with scrambled R A, antisense -miR486 or miR486 in cultured NPC.

FIG. 5 shows knockdown of NeuroD6 expression leading to traumatic spinal cord injury-like phenotype:

Figure 5 (a) shows the miR486-binding site of 3UTR of the 醒 u-NeuroD6 gene of chromosome 6 and the base sequence complementary between NeuroD6 and the miR486 gene;

5 (b) shows the chip (CHIP) -PCR results for miR486 expression interference inducing NeuroD6 binding to regulatory sites of GPx3 and TXNL1 genes;

Figure 5 (c) shows the result of traumatic spinal cord injury-like symptoms such as hind limb paralysis in mice 1 day after injection of NeuroD6 into the normal spinal cord;

FIG. 5D shows in situ analysis (day 7) of NeuroD6 expression in spinal cord injury, miR486-implanted normal spinal cord and iniR486 / simiR486-implanted normal spinal cord;

FIG. 5 (e) shows Reactive Oxygen Species (ROS) expression in 7-day spinal cord tissue when NeuroD6 is knocked down in normal spinal cord tissue; And

5 (f) shows NF160, Tuj and MBP expression 1 day after NeuroD6 injection when NeuroD6 knocked down in normal spinal cord;

Figure 5 (g) shows the expression of inflammatory factors EDI, Cox2, iNOS and eNOS when NeuroD6 knocked down in normal spinal cord, 1 day after NeuroD6 injection;

Figure 5 (h) is the day of NeuroD6 injection when knocked down NeuroD6 in normal spinal cord Later IL-Iβ IL-6 and TNFa expression;

FIG. 5 (i) shows that NeuroD6 expression interference in normal spinal cord expresses SEPN1, TXNL1, TXNL2, Gpxl and Gpx3, factors related to the R0SVIII deletion system, compared to control (scrambled RNA in normal spinal cord);

5 (j) shows ATP activity levels and caspase-3 activity for knockdown and miR486 injection of NeuroD6;

5 (k) and (1) show miR486 and NeuroD6 expression levels when hydrogen peroxide was treated to cultured NPCs;

5 (m) to (p) show the expression levels of SEPN1, TXNL1, GPx3 and NF160 when treated with hydrogen peroxide in cultured NPCs; And

FIG. 5 (q) shows a schematic flow chart of R0S / miR486-mediated neurodestruction of motor neurons after traumatic injury in the spinal cord.

Figure 6 shows the expression of R0S-removing enzymes and neurodegeneration of motor neurons when restoring NeuroD6 expression in spinal cord injury:

FIG. 6A is a flow chart illustrating an experimental procedure for processing miR486, antisense -miR486, siNeuroD6 and NeuroD6 and functional judgment; FIG.

6 (b) to (f) show the expression of NeuroD6, SEPNl, TXNL1, GPx3 and NF160 when treated with scrambled RNA, miR486, siNeuroD6 or NeuroD6 to normal or damaged spinal cord, respectively;

Figure 6 (g) shows co-expression of NF160 and Cox2, co-expression of ED1 and GFAP, co-expression of NF160 and NeuroD6 when treated with scrambled RNA, NeuroD6, miR486, mir486 and NeuroD6, siNeuroD6 or siNeuroD6 and NeuroD6 in the injured spinal cord Expression; and

6 (h) and (i) show the results of BBS and BMS for the normal spinal cord, the damaged spinal cord, and the damaged spinal cord treated with scrambled RNA and the damaged spinal cord treated with antisense _miR486.

[Best form for implementation of the invention]

The present invention prevents neurological diseases comprising miR486 expression inhibitor as an active ingredient And pharmaceutical compositions for treatment.

The present invention also provides a method of treating a neurological disorder comprising administering a pharmaceutically effective amount of a miR486 expression inhibitor to a subject suffering from the neurological disorder.

The present invention also provides a method for preventing a nervous system disease comprising administering to a subject a pharmaceutically effective amount of a miR486 expression inhibitor.

The present invention also provides a use of miR486 expression inhibitor in the manufacture of a pharmaceutical composition for preventing and treating neurological diseases.

The miR486 expression inhibitor may be selected from the group consisting of antisense oligonucleotides complementary to the miR486 gene, small interfering RNA (siRNA), short hairpin A and ribozyme, but are antisense nucleotides But may not be limited thereto.

The antisense nucleotides, as defined in the Watson-click base pair, bind (combine) to the complementary sequencing of DNA, immature -mR A or mature mR A to disrupt the flow of genetic information as a protein in DNA. The nature of antisense nucleotides specific to the target sequence makes them exceptionally multifunctional. Since antisense nucleotides are long chains of monomeric units they can be easily synthesized for the target RNA sequence. Many recent studies have demonstrated the utility of antisense nucleotides as biochemical means for studying target proteins (Rothenberg et al., J. Natl. Cancer Inst., 81: 1539-1544, 1999). The use of antisense nucleotides can be considered as a novel form of inhibitor because of recent advances in nucleotide synthesis and in the field of nucleotide synthesis that exhibit improved cell adsorption, target binding affinity and nuclease resistance.

The siRNA is composed of a 15-30 mer sense sequence selected from the base sequence of the miR486 gene and an antisense sequence complementarily binding to the sense sequence, wherein the sense sequence is not particularly limited thereto. It may be composed of 25 bases, but is not limited thereto.

The miR486 expression inhibitor may be to increase the expression of NeuroD6, but is not limited thereto. The neurological diseases include spinal cord injury, Parkinson's disease, stroke, muscular dystrophy, scoliosis, motor nerve injury, peripheral nerve injury due to trauma, ischemic brain injury, neonatal hypoxic ischemic brain injury, cerebral palsy, epilepsy, refractory epilepsy, Al Sheimer's disease, congenital metabolic neurological disease and traumatic brain injury may be any one selected from the group 5, but may be more specifically spinal cord injury, but not limited to this, may occur due to nerve damage Applicable to all neurological diseases present.

The present inventors have identified the effect of miR486 overexpression on neuronal destruction of R0S mediated spinal cord injury in motor neurons. It was found that most target genes of miR486 were downregulated, including overexpression of miR486 10 and NeuroD6 in spinal cord injury tissues (see FIGS. La and lb). In spinal cord injury lesions, only miR486 was expressed in NF160 + motor neurons, and 2 ', 7'-dichlorodihydrofluorescein diacetate (2 ᅳ, 7'-dichlorodihydrof kiorescein diacetate, DCFDA) was produced in the cytoplasm and reactive oxygen species (Reactive) oxygen species (R0S) was confirmed to accumulate (see FIG. 15). NeuroD6 expression was detected in the nucleus and cytoplasm of lesions of spinal cord injury and motor neurons of normal spinal cord tissue. In addition, only ED1 + macrophages produced R0S, but did not express miR486 (see FIG. ID).

. In addition, as a result of confirming the recovery of motor function according to the interference of miR486 expression, miR486

. Expression interfered spinal cord injury animals showed improved motor function. Three days after spinal cord injury, ^• 20 miR486 expression interference spinal cord injury animals despite the ^'most there was a part that rotates during their feet walking, and parallel to the walking continued their were weighed maintained during the arrest phenotype was gradually restored to . In contrast, injured animals injected only with scrambled siRNA had limited joint movement (see FIGS. 2A and 2B).

When only miR486 was injected into 25 normal spinal cords, it was confirmed to show a spinal cord-like phenotype (see FIG. 2D), which included specific dysfunction, motor neuron death (TU EL-positive), and cell survival inhibition ( 2e). Interference with miR486 expression in spinal cord injured animals markedly improved motor function and protected TUNEL-positive apoptotic cell death through caspase-3 activation and ATP production dysfunction (See FIGS. 2F and 2G). Well-developed motor neurons (NF 160+) of spinal cord tissue lesions were prominent in spinal cord injured tissues treated with antisense -miR486 (see FIG. 2H) and survival of myelin in motor neurons in miR486 expressing interfering spinal cord injury animal models And significantly increased preservation (see FIG. 2J). When scrambled RNA or antisense -miR486 was treated to the normal and damaged spinal cord, strong LFB staining was observed in the antisense -miR486 treated group (see FIG. 2I).

In addition, as a result of neuroD6-mediated neuroprotective induction through the GPx3, TXNLl and SEPNl upregulation of miR486 expression interference, miR486 interference effectively expressed the expression of i R486 and Cox2, EDI, ΙΙβ IL6 and TNFCL. Inhibition (see FIG. 3a), the expression of redox-causing factors MP0 and eNOS was reduced, and the R0S-removing enzymes SEPNl, TXNLl, GPxl and GPx3 were significantly increased (FIGS. 3c, 3d, 3f and 3h). Increased miR486 expression induced downregulated expression of NeuroD6 ᅳ GPx3, TXNLl and SEPNl in NF160-positive motor neurons. In addition, the GPx3, TXNLl and SEPNl expression was detected in NF160 + motor neurons in normal spinal cord tissue (see FIG. 3E). After interference with miR486 expression in spinal cord injury, the number of ED1-positive inflammatory cells, macrophages, microglial cells was significantly reduced (see FIG. 3B). In cultured neural progenitor cells (NPC), knockdown of miR486 expression effectively induced p-PI3K / p-Akt expression, improved NPC survival, and allowed cells to escape neuronal cell death. It was. Interference with miR486 expression after antisense -miR486 injection in spinal cord injury mice resulted in increased target gene expression. This resulted in elevated levels of lethal neuronal destruction and myelin destruction (see FIG. 4B). High levels of expression of EDI, MP0, iNOS and eNOS, which cause redox factors, were seen in miR486-injected animals (see FIG. 4D). Spinal cord injury and high R0S production in miR486-injected normal spinal cord were confirmed (see FIG. 4E).

After introducing miR486 and antisense-miR486 into the normal spinal cord, it was confirmed that expression levels of proinflammatory factors such as Cox2, IL-Ιβ, IL-6, and TNFc were lowered (see FIG. 4C). In contrast, _m iR486 After injection of redox-removing SEPNl, TXNL1 and GPx3 expression were significantly downregulated and its expression after miR486 expression interference Confirmed recovery (see FIG. 4F). The DNA-binding frequency of the regulatory site of NeuroD6 was increased due to the interference of i R486 expression, but miR486-overexpressing NPCs were markedly reduced (see FIG. 4I). Induction of miR486 expression in NPCs effectively inhibited NeuroD6 expression and R0S and proinflammatory factor production, resulting in a markedly decreased cell survival, while an apoptotic cell signal was increased (see FIG. 4H). When cultured NPCs were treated with scrambled RNA, antisense -miR486 and miR486, respectively, it was confirmed that the expression of NeuroD6 of the group injected with antisense ᅳ miR486 was increased (see FIG. 4J). In addition, the present inventors confirmed that miR486 expression in the injured spinal cord and the initiation of anti-oxidative reaction through GPx3 and TXNL1 ol by insufficient NeuroD6 resulted in interference of miR486 expression induced NeuroD6 binding to the regulatory sites of GPx3 and TXNL1 (see FIG. 5B). ). It was confirmed that R0S accumulated significantly in spinal cord injury and miR486-injected spinal cord tissue and siNeuroD6-injected spinal cord tissue (see FIG. 5E). It was confirmed that in vivo expression of the motor neuron markers NF160, Tuj and MBP were downregulated in spinal cord tissue after siNeuroD6 injection (see FIG. 5F). NeuroD6 expression knockdown in the intact spinal cord has cytotoxic effects on motor neurons, increased expression of TNF α, ΙΙΛβ, IL6, C0X2, iNOS and eNOS, and expression of the R0S clearance factors SEPNl, TXNL2, GPxl and GPx3 Was down-regulated (see FIGS. 5G-5I).

It was confirmed whether NeuroD6 or R0S regulated miR486 expression. As a result of confirming ATP expression, NeuroD6-mediated neuronal protection in spinal cord or cultured NPCs was also associated with caspase-3 downregulation and normalized ATP synthesis ability (see FIG. 5J). In particular, miR486 expression increased more than 3-fold (320%) in hydrogen peroxide treatment and NeuroD6 or ascorbic acid treatment significantly reduced hydrogen peroxide-mediated miR486 increase (194% and 132%, respectively) (see FIG. 5K). Hydrogen peroxide-induced R0S production was significantly inhibited by the expression of NeuroD6 (53%), SEPNl (37%), TXNL1 (23%) and GPx3 (40%). Prior to hydrogen peroxide exposure to NPC, NeuroD6 or ascorbic acid (5 / zg / ml) treatment significantly upregulated SEPN1, TXNL1 and GPx3 compared to the exposure effect with hydrogen peroxide alone (see FIGS. 51-5O). In addition, exogenous NeuroD6 protected neurons from ROS-mediated neurotoxicity (see Figure 5p).

In addition, the neuroprotective effect of exogenous NeuroD6 in the injured spinal cord NeuroD6 overexpression significantly increased SEPNl, TXNL1 and GPx3 expression (see Figures 6B-6E). After 4 weeks of spinal cord injury, NeuroD6-injected animals recovered from paralysis but damaged with scrambled _s iRNA. Animals had limited joint mobility (see FIGS. 6H and 6I).

Therefore, it was confirmed that the expression interference of miR486 using miR486 expression inhibitors induces improvement of spinal cord injury (SCI) by restoring the inhibition of NeuroD6. Thus, the miR486 inhibitors are effective in preventing and treating neurological diseases. It can be usefully used as a component. The composition containing the miR486 expression inhibitor of the present invention as an active ingredient may include, but is not limited to, the active ingredient in an amount of 0.0001 to 50 weight ¾> based on the total weight of the composition.

The composition of the present invention may contain one or more active ingredients exhibiting the same or similar function in addition to the miR486 expression inhibitor.

The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, saturated saline, textose solution, maltodextrin solution, glycerol, ethanol, liposomes, and one or more of these components. And other conventional additives such as buffers and bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets, and can act specifically on target organs. Target organ specific antibodies or other ligands may be used in combination with the carriers so as to be used. Furthermore, it can be formulated according to each disease or component using an appropriate method in the art or using the method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). ·

Nucleotides or nucleic acids used in the present invention can be prepared for oral, topical, parenteral, nasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal and the like. More specifically, nucleic acids or vectors are used in injectable forms. Thus, in particular the area to be treated may be combined with any pharmaceutically acceptable vehicle for injectable compositions for direct infusion. The compositions of the present invention may in particular comprise isotonic sterile solutions or lyophilized compositions which allow the composition of injectable solutions upon the addition of dry, in particular sterile water or appropriate physiological saline. Direct injection of nucleic acid into a patient's tumor is advantageous because it allows the treatment efficiency to be focused on the infected tissue. The dosage of nucleic acid used can be adjusted by various parameters, in particular by gene, vector, mode of administration used, disease in question or alternatively desired duration of treatment. In addition, the range varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate and the severity of the disease. The daily dosage is specifically about 0.0001 to 100 mg / kg, more specifically 0.001 to 10 mg / kg, and may be administered once to several times a day. The present invention also provides a method for preventing or treating a neurological disease, comprising administering to a subject a pharmaceutically effective amount of the composition.

The neurological disorders specifically include spinal cord injury, Parkinson's disease, stroke, muscular dystrophy, scoliosis, motor neuron injury, peripheral nerve injury due to trauma, ischemic brain injury, neonatal hypoxic ischemic brain injury, cerebral palsy, epilepsy, refractory epilepsy, Al-Hymer's disease, congenital metabolic nervous system disease and traumatic brain injury may be any one selected from the group, and more specifically, spinal cord injury, but not limited thereto, but may be caused by nerve injury. Applicable to all possible neurological diseases.

The pharmaceutically effective amount is 0.0001 to 100 mg / kg, 0.001 to 10 mg / kg, but is not limited thereto. Dosage may vary depending on the weight, age, sex, health status, diet, duration of administration, rate of administration, elimination rate, and severity of the particular patient.

The composition can be administered orally or parenterally during clinical administration and intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or intrathoracic injection during parenteral administration. Can be administered by the general It can be used in the form of a pharmaceutical system. ^'

The subject is a spinal cord animal, specifically a mammal, more specifically an experimental object such as a rat, a rabbit, a guinea pig, a hamster, a dog or a cat, and more specifically, an ape-like animal such as a chimpanzee or a gorilla. Can be.

Interference of expression of miR486 with miR486 expression inhibitors was demonstrated in NeuroD6. Since it was confirmed to induce improvement in spinal cord injury by restoring inhibition, the pharmaceutical composition comprising the _m iR486 inhibitor as an active ingredient can be usefully used for preventing or treating neurological diseases. In addition, the present invention

1) treating the test substance to miR486 expressing cell line;

2) measuring the expression level of miR486 of the cell line; and

3) provides a method for screening a candidate substance for preventing and treating neurological diseases, including selecting a test substance whose expression level is reduced compared to a control group that does not process the test substance.

As another way,

1) treating the test substance to miR486 and NeuroD6 expressing cell line;

2) measuring the amount of NeuroD6 expression in said cell line; and

Expression measurement of step 2) is RT-PCR, Quantitative or semi-Quentitative RT-PCR, Quantitative or semi-Quentitative real-time RT-PCR -PCR), Northern blot, and DNA or RNA chip may be measured using any one method selected from the group consisting of, but is not limited thereto.

Since miR486 gene is overexpressed in the spinal cord injury and miR486 is overexpressed to inhibit the expression of NeuroD6, the miR486 and NeuroD6 prevent neurological diseases and 1 can be used for screening therapeutic candidates.

The present invention also provides a kit for diagnosing a neurological disease comprising a nucleotide of the miR486 gene, a nucleotide having a sequence complementary to the nucleotide, or a fragment thereof.

In addition, the present invention

1) measuring the expression level of miR486 in a sample separated from the subject as an experimental group;

2) comparing the expression level of miR486 of step 1) with the expression level of n] iR486 in a sample isolated from normal individuals as a control; And

In addition, the present invention

2) comparing the expression level of miR486 of step 1) with the expression level of miR486 of a sample isolated from a normal individual as a control; And

3) When the miR486 expression level of step 2) is increased compared to the control group provides a method for diagnosing a neurological disease comprising the step of determining that the subject has a neurological disease. The nervous system diseases include spinal cord injury, Parkinson's disease, stroke, muscular dystrophy, scoliosis, motor nerve injury, peripheral nerve injury due to trauma, ischemic brain injury, neonatal hypoxic ischemic brain injury, cerebral palsy, epilepsy, refractory epilepsy, Alzheimer's disease. The disease may be one selected from the group consisting of congenital metabolic neurological disease and traumatic brain injury, but is not limited thereto.

Since itiiR486 gene is overexpressed in the spinal cord injury and miR486 is overexpressed to inhibit NeuroD6 expression, the miR486 and NeuroD6 can be used in kits for providing information for diagnosing neurological disease, gene detection and diagnosing neurological disease. The present invention also provides a pharmaceutical composition for preventing and treating neurological diseases, including NeuroD6 as an active ingredient.

The present invention also provides a method of treating a neurological disease, comprising administering a pharmaceutically effective amount of NeuroD6 to a subject having a neurological disease.

The present invention also provides a method for preventing neurological disease, comprising administering to a subject a pharmaceutically effective amount of NeuroD6.

The present invention also provides the use of NeuroD6 in the manufacture of a pharmaceutical composition for preventing and treating neurological diseases. The nervous system diseases include spinal cord injury, Parkinson's disease, stroke, muscular dystrophy, scoliosis, motor nerve injury, peripheral nerve injury due to trauma, ischemic brain injury, neonatal hypoxic ischemic brain injury, cerebral palsy, epilepsy, intractable epilepsy, Alzheimer's disease. The disease may be one selected from the group consisting of congenital metabolic neurological disease and traumatic brain injury, but is not limited thereto.

In addition, the present invention provides a vector comprising a polynucleotide encoding NeuroD6, or a pharmaceutical composition for preventing and treating neurological diseases containing the cells containing the vector as an active ingredient.

The nervous system diseases include spinal cord injury, Parkinson's disease, stroke, muscular dystrophy, scoliosis, motor neuron injury, peripheral nerve injury due to trauma, ischemic brain injury, neonatal hypoxic ischemic brain injury, cerebral palsy, epilepsy, intractable epilepsy, Alpheimer's disease. The disease may be one selected from the group consisting of congenital metabolic. Nervous system disease and traumatic brain injury, but is not limited thereto.

In addition, the present invention

1) treating the test substance to the NeuroD6 expressing cell line;

2) measuring the amount of NeuroD6 expression in said cell line; and

3) provides a method for screening a candidate substance for preventing and treating neurological diseases, the method comprising selecting a test substance in which the amount of NeuroD6 expression is increased compared to a control group not treated with the test substance. The nervous system diseases include spinal cord injury, Parkinson's disease, stroke, muscular dystrophy, scoliosis, motor neuron injury, peripheral nerve injury due to trauma, ischemic brain injury, neonatal hypoxic ischemic brain injury, cerebral palsy, epileptic refractory epilepsy, Alpheimer's disease. It may be any one selected from the group consisting of congenital metabolic neurological disease and traumatic brain injury, but is not limited thereto.

In addition, the present invention

1) treating the test compound or composition to NeuroD6 protein;

2) measuring the activity of the NeuroD6 protein of step 1); And

3) A method for screening a candidate substance for preventing and treating neurological diseases, comprising selecting a test compound or composition in which the activity of the NeuroD6 protein of step 2) is increased compared to the activity of the NeuroD6 protein untreated with the test compound or composition. The neurological disorders include spinal cord injury, Parkinson's disease, stroke, muscular dystrophy, scoliosis, motor neuron injury, peripheral nerve injury due to trauma, ischemic brain injury, neonatal hypoxic ischemic brain injury, cerebral palsy, epilepsy, refractory epilepsy, egg ^ Hemer's disease, congenital metabolic nervous system disease and traumatic brain injury may be any one selected from the group consisting of, but is not limited thereto.

In addition, the present invention

1) measuring NeuroD6 protein expression in a sample derived from the subject as an experimental group;

2) NeuroD6 protein expression level of step 1) and control samples

Comparing NeuroD6 protein expression levels; And

3) Provides a protein detection method for providing information of neurological disease diagnosis, comprising the step of determining that the risk of neurological disease is high when the expression level of NeuroD6 protein is reduced compared to the control group.

In addition, the present invention

2) comparing the NeuroD6 protein expression level of step 1) with the NeuroD6 protein expression level of a normal subject-derived sample as a control; And 3) When the expression level of NeuroD6 protein is reduced compared to the control group, it provides a method for diagnosing a neurological disease, comprising the step of determining that the subject has a neurological disease. The nervous system diseases include spinal cord injury, Parkinson's disease, stroke, muscular dystrophy, scoliosis, motor neuron injury, peripheral nerve injury due to trauma, ischemic brain injury, neonatal hypoxic ischemic brain injury, cerebral palsy, epilepsy, refractory epilepsy, Alzheimer's disease. It may be one selected from the group consisting of a disease, congenital metabolic nervous system disease and traumatic brain injury, but is not limited thereto.

Since interference of miR486 expression with miR486 expression inhibitors has been shown to induce improvement in spinal cord injury by restoring inhibition of NeuroD6, NeuroD6 can be usefully used as an active ingredient in pharmaceutical compositions for preventing and treating neurological diseases. Candidate screening methods for preventing and treating neurological diseases, protein detection methods for providing information on neurological disease diagnosis, and neurological disease diagnostic methods. Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples illustrate the present invention in detail, and the content of the present invention is not limited to the examples.

Example 1 miR486 Expression Interference on Spinal Cord Injury Model

<1-1> Induction of Spinal Cord Injury (SCI)

The mice were intraperitoneally administered in an amount of 25 ^ / g using 1.25% avertin and then anesthetized, and the back skin of the mouse was incised. ), The spinal cord at the T10 region was damaged to a size of about 1 mm deep in the dorsal direction and about 2 mm wide, and then sutured to receive antibiotics (amicin). Antibiotics were administered intramuscularly every two days.

<1-2> miR486 expression interference

For in vivo miR486 expression interference studies, mice were fixed with stereotaxic instruments under anesthesia. DharmaFECT (Dharma Inc., Chicago Antisense miR486 (anti _miR486 or anti miR; 20 μΜ stock solution, Dhamacon) diluted with IL) was injected once 2 days after SCI with a 25 μί Hamilton syringe with 26 gauge needles. The antisense -miR486 injection dose finally used 10 μΜ. The rate of microinjector in the stereosuspension instrument was 1 / min. The syringe was secured at an angle of about 90 degrees above the spinal cord of the stereosuspension instrument. The needle was injected into the site of injury, the syringe was left at the site of injury for 3 minutes after the end of the injection, and slowly removed to prevent the outflow of antisense -miR486. <1-3> Induction of NeuroD6 Expression

NeuroD6 (0pen Biosystems) was injected once 2 days after spinal cord injury. NeuroD6 infusion doses were 100, 200 and 400 ng. NeuroD6 was diluted with lipofectamin (Klinvitrogen, Carlsbad, Calif.) And NeuroD6 Vector DNA constructs were obtained from Open Biosystems (Thermo Scientific Co.). NeuroD6 injection method was carried out according to the same method as the antisense -miR486 injection method of Example <1-2>. Under anesthesia by intravenous anesthesia (Avert in), the rat spinal cord was opened using a surgical knife, and the spinal cord bone was removed using surgical scissors and bone tongs. <1-4> Infusion of miR486

miR486 (Dharmcon) was injected only once into the Hamilton syringe with a 26-gauge needle with a microinjector into the T11-L1 site. The concentration of miR486 stock solution was 20 μΜ, which was diluted with DharmaFECTCDharmacon Inc., Chicago, IL). The miR486 injection dose was finally 10 μΜ and the total injection volume of miR486 was 5 per rat. The miR486 injection rate of the microinjector was 1 ^ / tnin. The syringe was fixed to a stereosuspension instrument 90 degrees above the spinal cord. The needle was injected into the T11-L1 site of the spinal cord and the syringe was left in the site for 3 minutes to prevent leakage after miR486 injection.

<1-5> Inhibition of NeuroD6 Expression In order to perform injection of NeuroD6 siRNAs (siNeuroD6; Dharmcon) into the normal spinal cord, it was prepared in the same manner as the miR486 injection method of Example <1-2>. Mice were fixed in stereosuspension instruments under anesthesia via intravenous anesthesia injection. NeuroD6 siRNAs were administered once by injection dose of NeuroD6 siRNAs (10 μΜ), and the 20 μΜ stock solution of NeuroD6 siRNAs was also diluted with the same volume of DharmaFECT (Dharaacon Inc., Chicago, IL). After NeuroD6 siRNAs (10 μΜ) injection into the normal spinal cord, NeuroD6 (100 ng) was injected once a day at the same site of NeuroD6 siRNAs injection. Example 2 Spinal Cord-derived Neuronal Progenitor Cell Culture and Neurogenesis Induction

Neural progenitor cells (PC) were cultured as follows. Spinal cord-derived neural progenitor cells (NPCs) were isolated from 5-6 week old adult ICR mice. To separate the nerve cells from the adult rat spinal cord, and washed with a strong Ca ₂ ⁺ or Mg ₂ ⁺ volume of 4 ^° C HBSS as no. The tissue was crushed into small pieces (~ 1 1 丽³ ) and digested with 0.025% trypsin at 371: for 30 minutes. The enzyme activity of the trypsin was neutralized with DMEM containing 10% FBS, and the sample was centrifuged at 1500 rpm for 5 minutes to obtain a high concentration of cell pellet. The NPC pellets were incubated overnight at 37 ^° C. and 5% CO ₂ conditions in DMEM containing 10% FBS. The medium was changed 48 hours and then every 4 days. After 2 or 3 passages, cells were used to characterize their differentiation capacity. The cells were placed in differentiation conditions to confirm the neural differentiation capacity of the cultured cells. The cultured NPCs formed globular clumps of cells and floating neurospheres in the culture medium. Transfer the cells to a petri dish and add Neurobasal neuron-specific medium (B27 (Invitrogen, Gaithersburg, MD), 20 ng / ml bFGF and 20 ng / ml BDNF (Sigma, St Louis, M0)) NB, Invitrogen, Gaithersburg, MD) was incubated for 3 days. The culture concentration of the spheres was maintained at 1 20 cells / cm ² to prevent self-counseling. For neuronal differentiation, neuron-derived neurospheres were placed in a cover-slip double-coated with PDL-laminin. During differentiation, 70% of the medium every 4 days Replaced. All data presented were run at least three times.

Example 3 Identification of miR486 overexpression on neurodestruction of R0S-mediated spinal cord injury in motor neurons

<3-1> Confirmation of expression of miR486 and its target genes following spinal cord injury Extract RNA of whole cells with TrizoKLife Technologies, Frederick, Mass., USA), and PCR by using 20 pni specific oligo—dT primers. Amplified by cycle (95 ^° C. 1 minute; 55 ^° C. 1 minute; 72 ^° C. 1 minute) and reverse transcribed into first strand cDNA. The PCR reaction was performed using ABI 7700 Prism Sequence Detection System and SYBER green detection kit (Applied Biosystems, Foster, CA, USA). The primer sequence was designed with Primer Express software (PE-Ai Bio Bios, Warrington, UK) using the gene sequence obtained from the GeneBank database (Table 1). For PCR product labeling, the SYBER green detection kit (Applied Biosystems) was used.

Table 1

In addition, whole tissue specimen sites (/ 2 situ) were analyzed to confirm miR486 and NeuroD6 expression in spinal cord tissue. In situ rocking of miR486 and NeuroD6 was performed in spinal cord injury tissue or normal spinal cord tissue. Several types of gene oligonucleotide probes were synthesized (Dharmacon RNA Technologies, USA). Tissue slides were dried in air and prepared for 15 minutes in saline citrate (pH 7.0) containing 0.1 mg / ml proteinase K (Sigma Aldrich) at room temperature. Sections were washed twice for 5 minutes in IX SSC. The labeled oligonucleotides were diluted in a shake buffer containing 50% ion free formamide, 4X SSC and sodium pyrophosphate and covered with parafilm. The reaction was carried out in a humidified bath ^at 45 ^° C for 12 hours. After reaction, the sections were washed with IX SSC for 10 minutes at room temperature, washed with IX SSC for 20 minutes at 60 t, and distilled water for 5 minutes at room temperature. It was washed, dehydrated with 70%, 95% and 100% isopropanol and finally dried in air. Controls missing specific probes did not have detectable staining. The samples were evaluated by Leica fluorescence microscopy (Leica Microsystems, Exon, PA, USA). This test was repeated at least three times.

As a result, it was confirmed that the damaged spinal cord tissues overexpressed miR486 significantly compared to normal spinal cord tissues 7 days after spinal cord injury or 6 days after miR486 interference with the damaged spinal cord. In spinal cord injury lesions, the target genes of miR486, including downregulated NeuroD6, Dicer, RxR (Retinoic Acid Receptor) y, EphrinAl and Enoxl were shown to be low expression (FIGs. La and lb).

<3-2> Confirmation of R0S-related neurodestruction induced by immunohistochemical analysis

The sections were fixed for 30 minutes in 4¾ paraformaldehyde for immunohistochemical analysis of spinal cord tissue. The sections were then washed three times in PBS and the nonspecific binding was blocked with normal horse serum. The cut sections were reacted with the following antibodies at 4 ^° C. for 12 hours; Anti-GFAP (1: 2000; Dako), anti-EDI (1: 1000; Cell Signal), anti-Tuj (1: 250; Sigma) and anti-NF160 (1: 250; Sigma). After washing off the primary antibody, the cut sections were incubated for 1 hour. After strong washing in PBS, the cells were reacted with FITC or Texas-red conjugated secondary antibody (1: 250; Molecular Probe; and 1: 250; Jackson Laboratory, respectively) for 30 minutes. Controls using IgG with or without irrelevant primary antibody were not stained. The samples were evaluated using a Leica fluorescence microscope (Leica Microsystems, Exon, PA, USA). Immunocytochemical analysis was repeated at least three times. Double-labeled cells were identified through 1 iffli cutting plane collection through the sample.

As a result, miR486 was expressed only in NF160 + motor nerve, and cytoplasm was accumulated in R0S by producing 2 ', 7'-dichlorodihydrofluorescein diacetate (2', 7'-dichlorodihydrof luorescein diacetate, DCFDA). It was confirmed (Fig. Lc). NeuroD6 expression was also detected at high levels in the nucleus and cytoplasm of motor neurons in lesions of spinal cord injury and in normal spinal cord tissue, and R0S in ED1 + macrophages. Produced, but did not express miR486 (FIG. Id).

Example 4 Confirmation of recovery of motor function due to miR486 expression interference

<4-1> Confirmation of motor function recovery by interference with miR486 expression

modified Basso, to confirm the recovery of spinal cord injury by interference with miR486 expression

Motor function was measured for 30 days through Beat tie and Bresnahan (BBB) locomotor rating scale and Basso mouse scale (BMS). BBB and BMS are shown in the table below, respectively.

It measured according to 2 and Table 3.

Table 2

As a result, interference with miR486 expression in spinal cord injury animals has been shown to improve motor function as compared to spinal cord injury. Compared to uninjured animals, spinal cord injured animals severely degraded motor function after induction of injury. After spinal cord injury, the paralyzed phenotypes on flat walking continued to recover, even though they were mostly in the rotating area while their feet were walking. Stayed constant. In contrast, injured animals injected with only scrambled siRNA had limited joint movement (FIGS. 2A and 2B).

<4-2> Apoptosis by miR486 injection

To identify apoptosis-positive cells after injection of miR486, spinal cord tissue and culture

The effect of traumatic injury on induction of apoptosis of NPCs was confirmed using the TdT in situ apoptosis detector kit (Roche, USA) according to the manufacturer's instructions. After the damaged spinal cord tissue is fixed, the damaged tissue. Sections were placed in 4% paraformaldehyde, 90% at 37 ^° C with TUNEL reaction mixture containing deoxynucleot idyl transferase (TdT) buffer and biotinylated dUTP. Reaction in humid environment for minutes. Then, the secondary antibody bound to the fluorescent label After reaction, the results were analyzed using a fluorescence microscope (Leica Microsystem, PA). TUNEL-positive apoptotic cells and cells at spinal cord lesion sites were quantified by counting positively stained cells. Three 100-fold microscopic images were randomly captured in the range where the positive cells were abundant around the lesion site for each cut. The number of positively stained cells of the three images was averaged. The results are expressed as relative cell ratios per field of view under the microscope.

As a result, when only miR486 was injected into the normal spinal cord, it was confirmed that the spinal cord showed the same phenotype (FIG. 2D), which showed specific dysfunction, motor neuron death (TUNEL-positive), and cell survival inhibition (FIG. 2e).

<4-3> Caspase-3 and ATP Analysis after miR486 Interference

We measured caspase 3 activity in traumatic spinal cord tissue 1 week after antisense-miR486 treatment. For caspase-3 activity analysis, 10 proteins in a total 50 ^ volume were mixed with the equilibrated caspase-3 reagent (Promega). After reacting at room temperature for 1 hour, luminescence was measured using a TD 20/20 luminometer (LuminoiTieter) (Turner Designs, Sunnyvale, CA). Blank values were excluded and fold increase in activity was calculated based on activity measured from untreated cells. Each sample was measured three times.

In addition, the amount of protein was confirmed using the Protein Assay Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. Cells were 150 mmol / 1 KC1, 25 mmol / 1 Tris-HCl; pH 7.6, 2 mmol / 1 EDTA pH 7.4, 10 mmol / 1 KP0 ₄ pH 7.4, 0.1 醒 ol / l MgCl ₂ and 1 mg protein concentration per ml of 0.1% (w / v) buffer Bovine Serum Albumin (BSA) Suspended in the containing buffer. ATP synthesis was carried out in substrate buffer 750 βί {10 mmol / 1 malate, 10 mmol / 1 pyruvate, 1 mmol / 1 ADP, 40 zg / ml digitonin and 0.15 mmol / 1 adenosine pentaphosphate. The cell suspension was added to add 250 ^. Cells were reacted at 37 ^° C for 10 minutes. 50 μί of the reaction mixture was removed at 0 and 10 minutes, and 100 mmol / 1 Tris-HCl, 4 mmol / 1 EDTA (pH 7.75) boiling for 2 minutes was removed. Digestion and the digested buffer were diluted to 1/10. The amount of ATP was measured with an ATP Bioluminescence Assay Kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions on a luminometer (Berthold, Detection Systems, Pforzheim, Germany).

As a result, miR486 expression interference from spinal cord injury animals sikyeotgo significantly improved motor function, through the cascaded L-rise eu 3 ^activation, and ATP production dysfunction showed that protect the TU EL- positive cell death (Fig. 2f and 2g) .

<4-4> Confirmation of motor nerve

According to the method described above, changes in NF160 protein expression were confirmed.

As a result, immunochemical analysis of spinal cord tissue lesions also showed that well-developed motor neurons (NF 160+) were prominent in spinal cord injury tissues treated with antisense—miR486 (FIG. 2H). Interference with miR486 expression in spinal cord injury has been shown to significantly increase the survival and preservation of myelin in motor neurons compared to control animals (FIG. 2J).

In addition, LFB staining was performed as follows to confirm morphological changes. The frozen section was first placed overnight in 1: 1 alcohol / chloroform and dehydrated using 9¾ ethyl alcohol. The dehydrated sections were reacted overnight at 56 ^° C. with 0.1% luxal fast blue solution (up to 16 hours). Next, excess dye was washed off with 95% ethyl alcohol and then washed with distilled water. The slide was reacted with 0.05% Lithium carbonate solution for 30 seconds and then reacted with 70% ethyl alcohol for 30 seconds to distinguish between tissues. After washing with distilled water, it was dyed with cresyl violet solution for counterstaining. After rinsing with distilled water again, in order to facilitate differentiation between the dyes, it was left for 5 minutes in 95% ethyl alcohol, treated twice with 5% of 100% alcohol, twice with 5 minutes of xylene, and then mounted. mounting). Through this staining, the myelin is colored blue and the nerve is colored in red or purple.

As a result, scrambled RNA or antisense ᅳ in normal and damaged spinal cord When treated with miR486, strong LFB staining was observed in the group treated with antisense -miR486 (FIG. 2I).

Example 5 Confirmation of Induction of NeuroD6 Mediated Neuroprotection by GPx3, TXNLl and SEPNl Upregulation of miR486 Expression Interference

<5-1> Confirmation of Cox2, EDI, ΙΙβ IL6 and TNFa, ROS-removing enzymes SEPNl, TXNLl, GPxl and GPx3

In order to confirm the effect of miR486 expression interference, the expression of proinflammatory factors Cox2, EDI, ΙΙβ IL6 and TNF a, and ROS scavenging enzymes SEPNl, TXNLl, GPxl and GPx3 were confirmed after miR486 expression interference.

As a result, interference with miR486 expression in spinal cord injury rats resulted in increased target gene expression following direct injection of antisense -miR486 and lipofectamine combinations. Mainly, miR486 interference effectively inhibited the expression of miR486 (88% of miR486 expression was downregulated) and the expression of the proinflammatory factors Cox2, EDI, ΙΙΙβ IL6 and TNF α (FIG. 3A). After 7 days of miR486 expression interference, myeloperoxidase (MP0) and nitric oxide synthase (eNOS) expressions were markedly reduced, while 3 and 7 days after miR486 expression interference in spinal cord injury. SEPNl, TXNLl, GPxl and GPx3, which are the enzymes depleting R0S, were significantly increased (FIGS. 3C and 3D). In addition, as a result of confirming the protein expression, the number of ED1-positive inflammatory cells, macrophages, microglyal cells significantly decreased after 7 days of miR486 expression interference in spinal cord injury (Fig. 3b). Seven days after simiR486 injection in spinal cord injured animals, the GPx3, TXNLl and SEPNl expression was detected in NF160 + motor neurons in normal spinal cord tissue (FIGS. 3 f and 3h). <5-2> Confirmation of protein expression through western blot

Western blots were performed to identify proteins expressed in spinal cord tissue or cultured NPCs. NPCs or tissues were dissolved in lysis buffer 500 ^ containing 1 mM phenylmethanesulfonylfluoride (PMSF). The lysate at 15,000 g for 10 minutes After centrifugation, the total protein content was confirmed by Rad (Millan, Italy) protein assay kit. For Western blotting, the same amount of protein extract (40) in lysis buffer was analyzed by 10% SDS-PAGE and transferred to nitrocel lulose membrane. Then—p ᅳ PI3K (1: 1000, Cell Signaling), anti-p-Rac (1: 1000, Cell Signaling), anti-pc-Raf (1: 1000, Cell Signaling), anti-p-MEK ( 1: 1000, Cell Signaling), anti-p-ERK (1: 1000, Cell Signaling), anti-Akt (1: 1000, Cell Signaling), anti-p-SAPK / JNK (1: 1000, Cell Signaling), Anti-JAK2 (1: 1000, Cell signaling), anti-p21 (1: 200, Santa Cruz Biotechnology), anti-p53 (1: 200, Santa Cruz Biotechnology), anti— p— c 一 Myc (1: 200, Santa Cruz Biotechnology), Anti-MAP2ab (1: 500, Sigma), Anti-NF160 (1:40, Sigma), Anti-Nestin (1: 500, BD Biosciences), Anti-GFGF (1: 3000, Dakocytomat ion) , Anti-NeuroD6 (1: 3000), anti-Tuj (1: 400, Sigma) or anti-pActin (1: 500, Sigma) antibodies were reacted with the membrane. Relative band intensities were confirmed with Quality-one 1-D analysis software (Bio-Rad, USA).

As a result, increased miR486 expression was found in NeuroD6, GPx3, NF160-positive motor neurons.

Downregulated expression of TXNL1 and selenoprotein (Selenoprotein Nl, SEPN1) was induced. In addition, P13k and p-Akt expression was induced when interference with ^ !? 486 expression in the injured spinal cord, but the expression of caspase-3 and cytochrome-C was reduced ( 3e).

<5-3> Effect of miR486 expression interference on NPC

In the NPC prepared in Example 2, as a result of confirming the effect of miR486 expression knockdown, effectively induced p-PI3K / p-Akt expression, improved the survival of cultured NPCs, Freed from cell death. In addition, direct injection of antisense-miR486 and lipofectamine combinations into spinal cord injury mice followed by miR486 expression interference resulted in increased target gene expression. This resulted in elevated levels of lethal neuronal and myelin destruction (FIG. 4B). Compared to spinal cord injured animals, miR486—injected animals showed higher levels of expression of redox-causing factors, EDI, MP0, iNOS and eNOS (FIG. 4D). In vivo, spinal cord injury and miR486-injected normal spinal cord effectively induced high R0S production (FIG. 4E).

Induction of miR486 and antisense-miR486 into the normal spinal cord revealed that expression levels of proinflammatory factors such as Cox2, IL-β, IL-6, and TNFa were reduced (FIG. 4C). In contrast, injection of miR486 Later, mechanism expression such as redox-removing SEPN1, TXNL1 and GPx3 was significantly downregulated and its expression recovered after miR486 expression interference (FIG. 4F). The DNA-binding frequency of the regulatory site of NeuroD6 was increased due to interference of miR486 expression, but miR486-overexpressing NPCs were significantly reduced (FIG. 4I).

Cell viability was assessed by visible cell counts associated with trypan blue staining. Mitochondrial activity is formazan stained with 3, 4, 5—dimethyl thi azo 1-2-y 1 -2, 5-di pheny 1 tetrazolium bromide (MTT; Sigma, St. Louis, MO, USA). Evaluation was made using a plate reader by evaluating the ability of the cortical culture to decrease. In all viability assays, three wells were established under each experimental condition and each experiment was repeated at least three times. The original data of each experiment was analyzed through change analysis of Fisher's or t_tests. In addition, for flow cytometry, the cells were incubated at concentrations that ensured exponential growth when harvested in 100-mm dishes. Harvest and test protocols were used with propidium iodide to detect DNA via flow cytometry. The cells were analyzed with a BD Biosciences FACScan system (San Jose, CA, USA). The proportion of these cells at the G0 / G1, S and G2 / M stages of the cell cycle was confirmed using a DNA histogram fitting program (MODFIT; Verity Software, Topsham, ME, USA). As a result, induction of miR486 expression in NPC cultures isolated from spinal cord tissues effectively inhibited NeuroD6 expression and production of R0S and proinflammatory factors, resulting in a markedly decreased cell survival, while increased apoptotic cell signaling. (FIG. 4H).

In addition, chip (CHIP) analysis was performed to confirm NeuroD6 expression after miR486 interference. The monoclonal antibody, anti-NeuroD6, was purchased from Santa Cruze and rabbit IgG (PP64B) antibody was purchased from Upstate. Cultured NPCs were harvested and chemically crosslinked with formaldehyde for 20 hours at 4 ^° C. Fixation was restrained by adding 1/20 volume 2.5 M glycine at room temperature for 5 minutes. Cells were centrifuged at 500 g at 4 ^° C., washed with cold PBS and twice with lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl ₂ , 0.5% GEPAL, 1 mM PMSF). After washing, the cells were centrifuged to make cell pellets and cryopreserved in liquid nitrogen. The pellet was pre-IP lime buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl ₂ , 1 mM CaCl _2> 4% GEPAL, 1 mM PMSF), 60 mL PMSF and additional components (100 mM PMSF, 256 Protease inhibitor, 20% SDS, 5 M NaCl, ¾0 ₂ ). Cells were sonicated with a 1261 minute wave at 50% amplitude using a Branson Sonifier 450D and placed in ice water after 1 minute. Sonicated fractions ranged in size from 200-1000 bp. After sonication, the samples were centrifuged at 14,000 rpm for 10 minutes at 4 ^° C, aliquoted and rapidly frozen in liquid nitrogen. Ultrasonicated cell extracts equivalent to 2 × 10 ⁶ cells were used for the next immunoprecipitation. Samples were prepared with protein G Dynabeads (Dynal) and 1000 mL dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1), 167 mM NaCl, 5 mL Upstate protease inhibitor cocktail Π First).

As a result, when NPCs were treated with scrambled RNA, antisense -miR486 and miR486, it was confirmed that the expression of NeuroD6 of the group injected with antisense _miR486 was increased (FIG. 4J).

Example 6 Confirmation of Initiation of Antioxidant Response Induced by GPx3 and TXNL1 by MiR486 Expression and Deficient NeuroD6 in the Injured Spinal Cord

Chip analysis after interference with miR486 expression confirmed the induction of NeuroD6 binding to regulatory sites of GPx3 and TXNL1 (FIG. 5B). React ive oxygen species (ROS) accumulated significantly in spinal cord injury and miR486—injected spinal cord tissue and siNeuroD6-injected spinal cord tissue (FIG. 5E). After 48 hours of siNeuroD6 injection, protein expression levels were confirmed and in vivo expression of the motor neuron markers NF160, Tuj and MBP in spinal cord tissues was downregulated (FIG. 50. NeuroD6 expression knockdown in intact spinal cord cells in motor neurons It has a toxic effect, increased the expression of TNFa, ΙΙΙβ, IL6, C0X2, iNOS and eNOS (FIG.) And confirmed gene expression.Knockdown of NeuroD6 expression in the intact spinal cord has a cytotoxic effect on motor neurons. To Increased the expression of TNFa, Ιίΐβ, IL6, C0X2, iNOS and eNOS, and down-regulated expression of SEPNl, TXNL2, GPxl and GPx3, which are factors that eliminate R0S, and the composition of pathological microenvironment according to tissue R0S accumulation. (Fig. 5g-i). NeuroD6-mediated neuronal protection in spinal cord or cultured NPCs was also associated with caspase-3 downregulation and normalized ATP synthesis ability (FIG. 5J).

In order to determine whether NeuroD6 or R0S regulated miR486 expression, we determined that miD486 expression levels in neuroD6, hydrogen peroxide (¾), hydrogen peroxide / NeuroD6 and hydrogen peroxide / ascorbic acid in cultured spinal cord-derived differentiated neurons. (5 yg / ml) before and after treatment.

As a result, _m iR486 expression increased more than 3-fold (320%) in hydrogen peroxide treatment and NeuroD6 or ascorbic acid markedly decreased hydrogen peroxide-mediated miR486 increase (194% and 13¾ respectively) (FIG. 5K). . Hydrogen peroxide-induced R0S production was markedly inhibited by the expression of NeuroD6 (53%), SEPNl (37%), TXNL1 (23%) and GPx3 (40%). Prior to hydrogen peroxide exposure to NPC, NeuroD6 or ascorbic acid (5 ug / ml) treatment significantly upregulated SEPN1, TXNL1 and GPx3 compared to the exposure effect with hydrogen peroxide alone (FIGS. 51-5O). In addition, exogenous NeuroD6 protected neurons from R0S-mediated neurotoxicity (FIG. 5P).

<Example 7> Confirmation of neuroprotective effect of exogenous NeuroD6 in the damaged spinal cord In order to determine the neuroprotective effect of NeuroD6 in spinal cord injury, the expression of SEPNl, TX L1 and GPx3 and BBS and BMS were confirmed.

As a result, NeuroD6 overexpression significantly increased SEPN1, TXNL1 and GPx3 expression (FIGS. 6B-E). Four weeks after spinal cord injury, NeuroD6-injected animals recovered from paralysis, maintained their weight consistently during parallel gait, and exhibited a mainly rotated foot position while walking. In contrast, injured animals injected with scrambled siRNA only had limited joint mobility (FIGS. 6H and 6I).

Claims

[Range of request]

[Claim 1]

Pharmaceutical composition for preventing and treating neurological diseases, including miR486 expression inhibitors as an active ingredient.

[Claim 2]

The method of claim 1, wherein the miR486 expression inhibitor is selected from the group consisting of antisense oligonucleotides that bind complementarily to the miR486 gene, small interfering RNA (siRNA), short hairpin RNA, and ribozyme (r ibozyme) Pharmaceutical composition for the prevention and treatment of diseases of the nervous system, characterized in that any one.

[Claim 3]

The pharmaceutical composition for preventing and treating neurological diseases according to claim 1, wherein the miR486 expression inhibitor increases the expression of NeuroD6.

[Claim 4]

According to claim 1, wherein the neurological disease is spinal cord injury, Parkinson's disease, stroke, muscular dystrophy myocardial sclerosis, motor nerve injury, peripheral nerve injury due to trauma, ischemic brain injury, neonatal hypoxic ischemic brain injury, cerebral palsy, epilepsy ， The pharmaceutical composition for preventing and treating neurological diseases, characterized in that any one selected from the group consisting of refractory epilepsy, egg ≥heimer's disease, congenital metabolic nervous system disease and traumatic brain injury.

[Claim 5]

1) treating the test substance to miR486-expressing cell line;

2) measuring the expression level of miR486 of the cell line; and

3) selecting the test material, the expression of the miR486 expression is reduced compared to the control group does not process the test substance, for preventing and treating neurological diseases Candidate Screening Methods

[Claim 6]

1) treating the test substance to miR486 and NeuroD6 expressing cell line;

2) measuring the amount of NeuroD6 expression in said cell line; and

3) A method for screening candidate substances for preventing and treating neurological diseases, comprising selecting a test substance whose NeuroD6 expression amount is increased compared to a control group not treated with the test substance. .

[Claim 7]

The method of any one of claims 5 and 6, wherein the measurement of miR486 expression in step 2) is RT-PCR, quantitative or semi-quantitative RT-PCR, quantitative or semi-quantitative. What is measured using any one method selected from the group consisting of real-time or semi-Quentitative real-time RT-PCR, Northern blot, and DNA or RNA chip Screening method characterized by.

[Claim 8]

A kit for diagnosing a nervous system disease comprising a nucleotide of a miR486 gene, a nucleotide having a sequence complementary to the nucleotide, or a fragment thereof.

[Claim 9]

2) comparing the expression level of miR486 in step 1) with the expression level of miR486 in a sample isolated from normal individuals as a control; And

3) A gene detection method for providing information for diagnosing a neurological disease, comprising determining that the miR486 expression level of step 2) is higher than that of the control group, and thus, the risk of developing the neurological disease is high.

[Claim 10]

10. The neurological disorders of claim 9, wherein the neurological disorders include spinal cord injury, Parkinson's disease, stroke, amyotrophic lateral sclerosis, motor neuron injury, peripheral nerve injury by trauma, ischemic brain injury, neonatal hypoxic ischemic brain injury, cerebral palsy, epilepsy, A gene detection method for providing information for diagnosing a neurological disease, wherein the disease is any one selected from the group consisting of refractory epilepsy, Alzheimer's disease, congenital metabolic nervous system disease, and traumatic brain injury.

[Claim 11]

Pharmaceutical composition for preventing and treating neurological diseases, including NeuroD6 as an active ingredient. -

[Claim 12]

The method of claim 11, wherein the neurological disorders include spinal cord injury, Parkinson's disease, stroke, amyotrophic lateral sclerosis, motor neuron injury, peripheral nerve injury due to trauma, ischemic brain injury, hypoxic ischemic brain injury, cerebral palsy, Epilepsy, refractory epilepsy, Alpheimer's disease, congenital metabolic neurological disease and traumatic brain injury (traumatic brain injury), characterized in that any one selected from the group consisting of a pharmaceutical composition for preventing and treating neurological diseases.

[Claim 13]

A vector comprising a polynucleotide encoding NeuroD6 according to claim 11, or a pharmaceutical composition for preventing and treating neurological diseases containing the cell containing the vector as an active ingredient.

[Claim 14]

According to claim 13, wherein the neurological disease is spinal cord injury, Parkinson's disease, stroke, muscular dystrophy myocardial sclerosis, motor neuron injury, peripheral nerve injury due to trauma, ischemic Brain injury, neonatal hypoxic ischemic brain injury, cerebral palsy, epileptic refractory epilepsy, Alheimer's disease, congenital metabolic neurological disease and traumatic brain injury

Pharmaceutical composition for the prevention and treatment of diseases of the nervous system, characterized in that any one selected from the group.

[Claim 15]

1) treating the test substance to the NeuroD6 expressing cell line;

2) measuring the amount of NeuroD6 expression in said cell line; and

3) A method for screening candidate substances for preventing and treating neurological diseases, comprising selecting a test substance whose NeuroD6 expression amount is increased compared to a control group not treated with the test substance.

[Claim 16]

16. The neurological disorder of claim 15, wherein the neurological disorder includes spinal cord injury, Parkinson's disease, stroke, muscular dystrophy, lateral sclerosis, motor neuron injury, peripheral nerve injury due to trauma, ischemic brain injury, neonatal hypoxic ischemic brain injury, cerebral palsy, epilepsy. ^' Candidate screening method for the prevention and treatment of neurological diseases, characterized in that any one selected from the group consisting of refractory epilepsy, Alzheimer's disease, congenital metabolic neurological disease and traumatic brain injury.

[Claim 17]

1) treating the test compound or composition to the NeuroD6 protein;

2) measuring the activity of the NeuroD6 protein of step 1); And

3) A method for screening a candidate substance for preventing and treating neurological diseases, comprising selecting a test compound or composition in which the activity of the NeuroD6 protein of step 2) is increased compared to the activity of the NeuroD6 protein untreated with the test compound or composition.

[Claim 18]

18. The method of claim 17, wherein the neurological disease is spinal cord injury, Parkinson's disease, stroke Amyotrophic spinal lateral sclerosis, motor nerve injury, peripheral nerve injury due to trauma, ischemic brain injury, neonatal hypoxic ischemic brain injury, cerebral palsy, epilepsy, refractory epilepsy

Alzheimer's disease, congenital metabolic nervous system disease and traumatic brain injury

Candidate screening method for preventing and treating neurological diseases, characterized in that any one selected from the group.

[Claim 19]

2) NeuroD6 protein expression level of step 1) and control samples

Comparing NeuroD6 protein expression levels; And

3) A method for detecting a protein for providing information for diagnosing a neurological disease, comprising determining that the risk of developing the nervous system disease is high when the expression level of NeuroD6 protein is decreased compared to the control group.

[Claim 20]

20. The method of claim 19, wherein the neurological disorders include spinal cord injury, Parkinson's disease, stroke atrophic spinal sclerosis, motor neuron injury, peripheral nerve injury due to trauma, ischemic brain injury, neonatal hypoxic ischemic brain injury, cerebral palsy, epilepsy, A protein detection method for providing information for diagnosing a neurological disease, characterized in that it is any one selected from the group consisting of refractory epilepsy, Alzheimer's disease, congenital metabolic neurological disease and traumatic brain injury. ^'

[Claim 21]

A method of preventing and treating cancer comprising administering a pharmaceutically effective amount of a miR486 expression inhibitor to an individual suffering from a neurological disease.

[Claim 22]

miR486 expression inhibitors for the preparation of pharmaceutical compositions for the prevention and treatment of diseases of the nervous system Use to use.

[Claim 23]

3) A method for diagnosing a neurological disease, comprising determining that the miR486 expression level of step 2) is higher than that of a control group.

[Claim 24]

A method of preventing and treating cancer comprising administering a pharmaceutically effective amount of NeuroD6 to an individual suffering from a neurological disease.

[Claim 25]

Use of NeuroD6 in the manufacture of a pharmaceutical composition for the prevention and treatment of neurological diseases.

[Claim 26]

2) comparing NeuroD6 protein expression level of step 1) and NeuroD6 protein expression level of a sample derived from a normal individual as a control; And

3) A method for diagnosing a neurological disease, comprising determining that the individual has a neurological disease when the expression level of NeuroD6 protein is decreased compared to the control group.