KR101485163B1 - Composition of Regulating Differentiation into Dopaminergic Neurons by ADAM17 - Google Patents

Abstract

The present invention relates to a method for controlling the differentiation of neural stem cells or neural progenitor cells into dopaminergic neurons, comprising the step of increasing or inhibiting the activity of ADAM17 in neural stem cells or neural precursor cells, and a composition for treating cerebral neurological diseases The methods and compositions of the present invention modulate the activity of ADAM17 on neural stem cells or neural progenitor cells to increase dopaminergic neurons in the midbrain region and induce dopaminergic neuronal cell death such as Parkinson's disease And by inhibiting the activity, it is possible to improve the therapeutic effect of a disease such as a tumor.

Description

METHODS FOR REGULATING DERIVATIVES IN DOPAMINERGIC NEURONES BY ADAM 17 AND METABOLO ROTATE ADAM17

The present invention relates to a method for regulating the differentiation of neural stem cells or neural progenitor cells into ADAM17 to regulate dopaminergic neuron differentiation and a composition containing an activator or inhibitor of ADAM17 as an active ingredient.

Dopamine is produced in various areas of the brain, including substantia nigra and spinal cord, and is secreted by the hypothalamus. It is a catecholamine-based neurohormone that is known to be involved in various neurological diseases, Schizophrenia, drug addiction, and depression are caused by deficits in the dopaminergic system. Dopamine is synthesized by the midbrain neurons present in the substantia nigra and intermediate cortical areas of the brain. The dopaminergic neurons transfer neurites from the black area and midcortical area to the striatum, cerebral cortex, limbic system, etc., Compensatory circuit response, regulation of hormone secretion from the hypothalamus to the pituitary gland, and other physiological functions.

Dopamine can be used as an intravenous drug that acts on the sympathetic nervous system by increasing heart rate and blood pressure, but since dopamine can not pass through the blood-brain barrier under normal conditions, not a precursor, it is directly influenced by the central nervous system Can not give.

If an abnormality occurs in the regulation of dopamine secretion, for example, if the secretion of dopamine is excessive or active, it causes bupropathy or schizophrenia, and when the secretion of dopamine is reduced, it causes a clinical depression. In addition, when neurons that produce dopamine are damaged, they cause movement disorders and cause Parkinson's disease. Nicotine, which is absorbed by smoking, activates dopamine and makes you feel pleasure. Hallucinations and pleasures through drugs are also obtained by stimulating and activating the secretion of dopamine. Therefore, controlling the action of dopamine, suppressing damage to dopaminergic neurons, and maintaining growth can be an important therapeutic method for treating diseases.

The function of the dopamine binds to a dopamine receptor (Dopamin Receptor), which interacts with G-proteins (GTP-binding proteins) to activate a secondary signal transducer or to activate a specific signal transduction system Is known to lead to physiological responses. There are five subtypes of dopamine receptors known to date. They include D1 and D5 due to their structure and pharmacological properties. D1-like Recepter (D1R), which activates adenylate cyclase and promotes intracellular cAMP formation, ) Group, D3, D4 and the like, and in contrast to the D1R, it can be classified into a D2-like receptor (D2R) group inhibiting cAMP formation, and since the functional group of D2R is not clear yet, And a number of molecular or cellular biologic methods have been used to investigate the mechanism of action.

Activation of D2R phosphorylates ERK (extracellular signal-regulated kinases), in which EGF (epidermal growth factor) binds to EGFR (epidermal growth factor receptor) . However, the mechanism by which D2R activates EGF has not yet been elucidated.

As a result, the inventors of the present invention have found that ADAM17 is involved in promoting the differentiation of dopaminergic neurons in the midbrain region as a result of intense efforts to clarify the D2R mechanism of dopamine, thereby completing the present invention.

1. Berridge KC et al. , BMC Biology 3 : 4, 2005
2. Brun P et al. , ≪ / RTI > J Neurochem 94 (10): 63, 2005

It is an object of the present invention to provide a method of regulating the activity of ADAM17 in neural stem cells or neural precursor cells to regulate differentiation into dopaminergic neurons.

It is another object of the present invention to provide a composition for promoting the differentiation of dopaminergic neurons containing an activator of ADAM17 as an active ingredient.

It is still another object of the present invention to provide a composition for treating cerebral neurological diseases containing an activator or an inhibitor of AADAM17 as an active ingredient.

It is still another object of the present invention to provide siRNA of ADAM17 having the nucleotide sequence shown in SEQ ID NO: 1.

In order to achieve the above object, the present invention provides a method for regulating differentiation of neural stem cells or neural progenitor cells into dopaminergic neurons, comprising the step of increasing or inhibiting the activity of ADAM17 in neural stem cells or neural progenitor cells do.

The present invention provides a composition for promoting the differentiation of dopaminergic neurons containing an activator of ADAM17 as an active ingredient.

The present invention provides a composition for treating a cerebral neurological disease comprising an activator or an inhibitor of ADAM17 as an active ingredient.

The present invention also provides siRNA of ADAM17 having the nucleotide sequence shown in SEQ ID NO: 1.

According to the present invention, by modulating the activity of ADAM17 on neural stem cells or neural progenitor cells, dopamine neurons in the midbrain region are increased, and the therapeutic effect of diseases induced by the death of dopaminergic neurons such as Parkinson's disease And by inhibiting the activity, it is possible to improve the therapeutic effect of a disease such as a tumor.

FIG. 1 shows that the development of dopaminergic neurons (TH-positive cells) induced by EGF was induced in normal rat and D2R - / - mice cerebral neurons (A: immunohistochemistry of the cerebral nerve cells and the control group; B: TH-positive cells, C: neurite length, D: number of neurites, 100 μm (*, p <0.05; **, p <0.01; ***, p <0.001) ††, p <0.05; ††, p <0.01 normal mice vs. D2R - / - mice)).
FIG. 2 shows that the development of dopaminergic neurons (TH-positive cells) induced by D2R agonists was induced in normal rat and D2R - / - mice cerebral neurons (A: Immunostaining of cerebral nerve cells and control , P <0.05, **, p <0.01), B: TH-positive cells, C: neurite length, D: ; ††, p <0.01; †††, p <0.001 normal mice vs. D2R - / - mice)
Figure 3 shows that ERK phosphorylation is induced by EGF on normal rat and D2R - / - mice derived mesenchymal neurons (A: EGF, quinpirole and haloperidol treatment; B: combination treatment of EGF, quinpirole and AG1478; *, p <0.05; **, p <0.01; ***, p <0.001 control vs drug treatment comparison group; †, p <0.05; ††, p <0.001 normal rat vs. D2R - / - rat).
Figure 4 shows that D2R and metalloprotease are involved in ERK phosphorylation in normal rat and D2R - / - rat cerebellum neurons (A: combination of EGF and GM6001; B: combination of quinpirole and GM6001; ***, p <0.001 Control group vs drug treatment group; †, p <0.05; †††, p <0.001 normal rat vs. D2R - / - rat).
FIG. 5 shows that D2R and metalloprotease-mediated development of dopaminergic neurons (TH-positive cells) were induced in normal rat and D2R - / - mice cerebral neurons (A: D: Neurite number, Scale bar: 100um (*, p <0.05; **, p <0.01; ***, p <0.001) ††, p <0.01; †††, p <0.001 normal mice vs. D2R - / - mice).
FIG. 6 is a photograph showing the expression patterns of ADAM10 and ADAM17 in the TH positive cells at fetal SN and VTA (A: ADAM10; B: ADAM17).
FIG. 7 shows that the development of D2R ERK phosphorylation and resulting dopaminergic neurons (TH-positive cells) in normal rat and D2R - / - mice induced cerebral neurons were attributed to ADAM17 (A: C: Analysis of the inhibitory effect of siADAM17 on D: siADAM17 and D2R antagonist-induced ERK phosphorylation change; scale bar ††, p <0.01, ††, p <0.001), p <0.05, p <0.05; Normal mice vs. D2R - / - mice).
FIG. 8 is a schematic diagram of a dopaminergic neuron cell division mechanism by ERK phosphorylation via D2R and ADAM17 (D2R: dopamine D2 receptor; ADAM: a disintegrin and metalloprotease; heparin-binding EGF-like growth factor; EGF: epidermal growth factor (EGFR); extracellular signal-regulated kinase (ERK).

In one aspect, the present invention relates to a method for regulating the differentiation of neural stem cells or neural progenitor cells into dopaminergic neurons, comprising increasing or inhibiting the activity of ADAM17 in neural stem cells or neural progenitor cells.

In the present invention, it was confirmed that phosphorylation is a major mechanism of extracellular signal-regulated kinases (ERKs) induced by dopamine D2 receptor and epidermal growth factor (EGF) in the differentiation mechanism into dopaminergic neurons. .

In the present invention, the neural stem cells or neural progenitor cells are neural stem cells or neural progenitor cells of the midbrain.

The term &quot; stem cell &quot; or &quot; precursor cell &quot; of the present invention is collectively referred to as undifferentiated cells at a stage before differentiation into each cell constituting the tissue. Stem cells have the ability to divide into cells. When the differentiation stimulus is applied, the stem cells have the ability to differentiate into specific cells. The stem cells have plasticity that changes the characteristics of the final cells differentiated according to the environment or stimulation. Cells can be classified into embryonic stem cells, adult stem cells, depending on their origin.

The term 'neuron' of the present invention is a cell of the nervous system and can be used interchangeably with the term 'neuron' or 'neuron cell'.

The term &quot; differentiation &quot; of the present invention refers to a phenomenon in which cells and tissues specialize in structure or function while cells grow and proliferate, thereby changing behaviors or functions in order to perform tasks assigned to each of them. As a final step leading to cell specificization during development, cell differentiation is a phenomenon in which the gene activities in each cell are different from each other and genes are expressed in different ways. As a result, each cell has a completely differentiated structure and function I have.

The term &quot; treatment &quot; of the present invention means an approach for obtaining a beneficial or desired clinical result. For purposes of the present invention, a beneficial or desired clinical result indicates, but is not limited to, relief of symptoms, reduction of lesions, inhibition of deterioration, delayed progression of disease, amelioration or temporary alleviation or alleviation of disease states, Also, 'treatment' may mean increasing the survival rate compared to the expected survival rate when untreated. &Quot; Treatment &quot; refers to both therapeutic treatment and prophylactic or preventative measures. Such treatment includes not only the disorder to be prevented, but also the treatment required for the disorder already occurring.

The term 'dopaminergic neuron' of the present invention is a nerve cell that expresses tyrosine hydrozylase (TH), and is specifically located in the middle cerebral substantia nigra. It stimulates striatum, limbic system and cortex in vivo, Motion, and compensation related behavior. In fact, in order to function as a dopaminergic neuron in the body, it must exhibit mesencephalic characteristics.

In another aspect, the present invention relates to a composition for promoting the differentiation of dopaminergic neurons containing an activator of ADAM17 as an active ingredient.

 In the composition for promoting differentiation according to the present invention and the composition for promoting differentiation into dopaminergic neurons, not only ADAM17 but also an activator or an inhibitor for increasing or inhibiting the activity of ADAM10 may be additionally used.

ADAM17, one of ADAM (a disintegrin and a metalloproteinase) -family, is known as CD156b, cSVP, MGC71942, or TACE (tumor necrosis factor converting enzyme) and has α-secretase activity. ADAM10 has 30% There is a feature that has the same sex.

ADAM17 and ADAM10 can be regulated by TAPI-1, TAPI-2, GI254023, and GW280264, but are not limited thereto.

In another aspect, the present invention relates to a composition for treating a cerebral neurological disease containing an activator or an inhibitor of ADAM17 as an active ingredient.

In the present invention, the term 'cerebral neurological disease' refers to a disorder of the nervous system such as Parkinson's disease, neuralgia, arthritis, headache, schizophrenia, epilepsy, cerebral palsy, dementia, depression, dyskinesia, Alzheimer's disease, , Anxiety, learning and memory impairment, neurodegenerative diseases such as degenerative neurological diseases, and the like.

The 'activator' in the present invention enhances the activity of ADAM 17 or ADMA10 using an agonist, and is preferably applicable to the treatment of brain diseases caused by the death of dopaminergic neurons.

The 'inhibitor' in the present invention inhibits the activity of ADAM 17 or ADMA10 using an antagonist, and is preferably applicable to diseases caused by the activity of ADAM17 such as tumor.

The inhibitor is also preferably an siRNA or antisense RNA that recognizes the sequence of ADAM17 or ADAM10.

The present invention relates to siRNA of ADAM17 having the nucleotide sequence shown in SEQ ID NO: 1 from another viewpoint.

SiRNA or antisense RNA molecules that inhibit the expression of ADAM17 and ADAM10 according to the present invention have a form in which a short nucleotide sequence (e.g., about 5-15 nt) is inserted between self-complementary sense and antisense strands In particular, siRNA molecules formed by the expression of nucleotide sequence form a hairpin structure by intramolecular hybridization and form a stem-and-loop structure as a whole. Such a stem-and-loop structure can be processed in vitro or in vivo to produce siRNA molecules that are capable of mediating RNAi. Introducing the siRNA into cells decreases the mRNA level of ADAM17 and thus decreases the activity of ADAM17.

siRNAs can be introduced into cells using shRNA molecules, and shRNA constructs encode stem-loop RNA. After introduction into the cell, the stem-loop RNA is processed into double-stranded RNA whose sequence corresponds to the stem of the original RNA molecule, and the double-stranded RNA can be prepared according to any method known in the art .

For in vivo administration of siRNA or antisense RNA, shRNA or antisense RNA may be inserted into plasmids and used in vivo administration by preparation of AAV vectors, retroviral vectors, particularly lentiviral vectors, adenoviral vectors, For example, by direct injection into the target tissue selected according to the route, subcutaneous tissue or conventional application.

The route of administration of siRNA or antisense RNA varies from direct local delivery to systemic intravenous administration. The advantage of local delivery is that the dose of siRNA required for efficacy is substantially lower because the molecule is injected into or near the target tissue. Also, topical administration allows intensive delivery of siRNA. For such direct delivery, naked siRNA can be used. "Naked siRNA" refers to the delivery of siRNA (unmodified or modified) in saline or other simple excipients, for example, 5% dextrose. Such molecules are an attractive treatment because they are easy to formulate and administer. In addition, naked DNA can be formulated into lipids, particularly liposomes.

Systemic application of siRNA or antisense RNA is often less invasive, and more importantly not limited to tissues accessible from the outside. For systemic delivery, siRNA can be formulated with cholesterol conjugates, liposomes, or polymer-based nanoparticles. Liposomes have traditionally been used to provide increased pharmacokinetic properties and / or reduced toxicity profiles. These allow for successful and repeatable in vivo delivery. Presently, the use of lipid-based formulations for systemic delivery of siRNAs to hepatocytes is likely to present one of the most promising near future opportunities for the development of RNAi therapeutics. Formulations using polymers, such as dynamic poly-conjugates (e. G., Coupled to N-acetylglucosamine for hepatocyte targeting) and cyclodextrin-based nanoparticles, provide both targeted delivery and endosome escape mechanisms Allow. Other polymers, such as atelocollagen and chitosan, allow therapeutic effects on subcutaneous tumor xenografts and bone metastases.

siRNAs can also be directly conjugated to molecular entities designed to facilitate targeted delivery. In view of the nature of the siRNA duplex, the presence of an inactive or sense strand is directed to an ideal site for conjugation. An example of a conjugate is a lipophilic conjugate, such as a cholesterol, or an aptamer-based conjugate.

Cationic peptides and proteins can also be used to complex with the negatively charged phosphate backbone of siRNA duplexes.

The therapeutic composition according to the present invention is an activator, inhibitor of the therapeutically active components ADAM17 and ADAM10. In addition to its siRNA and antisense RNA, it may further comprise suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions.

Examples of carriers, excipients or diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method .

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, Lactose, gelatin, etc. are mixed to prepare.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

Activator and inhibitor of ADAM17 and ADAM10 used in the present invention. The dosage of siRNA and antisense RNA thereof may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, nasal, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be apparent to those skilled in the art that these embodiments are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples

Example 1 : EGF of Dopamine  Neuron development Induction ability  Confirm

(1) Confirming the ability to induce dopaminergic neuron development by EGF treatment

After the treatment of EGF, EGF + haloperidol, EGF + AG1478, and EGF + PD98059 on normal brain and D2R - / - mice, TH was stained by immunocytochemistry and TH nerve The developmental potential of dopaminergic neurons was examined by comparing cell numbers, length of neurites and number of neurites. As a result, the development of dopaminergic neurons by EGF in both normal and D2R - / - And this effect was completely blocked by the EGFR inhibitor AG1478 and the MAPK inhibitor PD98059, but not by the D2R antagonist haloperidol (Fig. 1).

(2) Confirming the ability of D2R agonists to induce dopaminergic neuronal development

After treatment of quinpirole, EGF and quinpirole, quinpirole and AG1478, EGF and quinpirole, AG1478, EGF, quinpirole and haloperidol on midbrain neurons derived from normal rat and D2R - / - mice, immunohistochemistry stained TH And the number of TH neurons, the length of neurites, and the number of neurites in the middle cerebral neurons were examined. As a result, the effects of EGF and quinpirole, an agent of D2R, on the development of dopaminergic neurons, There was no synergistic effect on the increase of gonadal cell number, and the development of dopaminergic neurons by EGF + Quinpirole was not inhibited by haloperidol in both normal and D2R - / - mice, but by AG1478 (Fig. 2). These results indicate that D2R is located in a higher signal system than EGF and induces the development of dopaminergic neurons through EGFR.

Example 2 : EGF On by ERK  Phosphorylation

(1) Identification of ERK phosphorylation by D2R and EGF

To determine whether the developmental process of dopaminergic neurons via D2R and EGFR is mediated through the ERK signaling pathway, EGF and quinpirole were treated with haloperidol and AG1478 to observe ERK phosphorylation. As a result, ERK phosphorylation by EGF was increased by 417% compared with the control, and this effect was not inhibited by haloperido. In contrast, it was confirmed that ERK phosphorylation (194%) by quinpirole was inhibited by AG1478 (FIG. 3). It was confirmed that the dopamine D2 receptor phosphorylates ERK in an EGFR-dependent manner, thereby promoting the development of dopaminergic neurons.

(2) Confirmation of metalloprotease action on EGFR activation of D2R

To confirm whether the developmental process of D2R-mediated dopaminergic neurons is mediated through the ERK signaling pathway, we investigated whether metalloprotease inhibitor, GM6001, is involved in the activation of EGFR through D2R by EGF or quinpirole And phosphorylation of ERK was observed. ERK phosphorylation by EGF was not affected by GM6001, but phosphorylation of ERK by quinpirole was inhibited to basal levels (FIG. 4). In addition, in the development of dopaminergic neurons, the effect of EGF was not inhibited by GM6001, but the effect of quinpirole was inhibited (FIG. 5).

Example 3 : D2R  And EGFR On by ERK  In phosphorylation ADAM17 Confirmation of action of

We performed immunohistochemical staining for ADAM10 and ADAM17, the most active in the brain in relation to EGFR, and found that the dopaminergic neuron specific protein Tyrosine hydroxylase (TH).

As a result, it was confirmed that both ADAM10 and ADAM17 were expressed in some TH positive cells (Fig. 6).

In addition, ADAM17 transfected ADAM17 siRNA (SEQ ID NO: 1: siADAM17), which was prepared to confirm that D2R activates EGFR and induces the development of dopaminergic neurons and phosphorylates ERK, into primary cultured midbrain neurons We also observed the development of dopaminergic neurons by quinpirole and EGF and the phosphorylation of ERK by quinpirole. As a result, when glycosylated ADAM17 (100 kDa) was knocked down, the development of dopaminergic neurons by EGF was not affected, but it was confirmed that the effect of quinpirole was partially reduced (FIGS. 7A and 7B) Phosphorylation of ERK was also inhibited (Fig. 7D). These results indicate that ADAM17 is involved in the phosphorylation of ERK by D2R activation.

siRNA 17 (antisense): 5'-GGCAGACUUUAGAUGCUUCUUTT-3 '(SEQ ID NO: 1)

The mechanism by which ADAM17 acts on the signaling of D2R and EGFR is that it acts to dissociate the EGF precursor and regulates the phosphorylation of ERK depending on the activation of EGFR, thereby regulating the differentiation of dopaminergic neurons (Fig. 8).

Having thus described the present invention in detail, it will be apparent to those skilled in the art that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereby . It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

AG1478 is an inhibitor of EGFR.
PD98059 is an MAPK inhibitor.
GM6001 is a metalloprotease inhibitor.
Quinpirole is a D2R agonist.
Haloperidol is a D2R antagonist.

<110> Korea University <120> Method for Regulating Differentiation into Dopaminergic Neurons          by ADAM17 <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ADAM17 siRNA antisense <220> <223> siRNA <400> 1 ggcagacuuu agaugcuucu utt 23

Claims (7)

A method of inhibiting the gene expression of ADAM17 in the cerebellum neurons to inhibit the development of neurons from the middle cerebral neurons to the dopaminergic neurons (except for humans).
The method according to claim 1, further comprising inhibiting the enzyme activity of ADAM10 or inhibiting the expression of ADAM10 gene.
delete delete delete delete SiRNA of ADAM17 having the nucleotide sequence shown in SEQ ID NO: 1.

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