KR20210026882A - Animal model with damage of cognitive function by optogenetic or chemogenetic stimulation of hippocampal astrocytes and screening method of drug for improving cognitive function using the same - Google Patents

Abstract

The present invention relates to an animal model of cognitive impairment using optogenetic or chemogenetic stimulation of hippocampal astrocytes, and a screening method for cognitive function improvement drugs using the same. More specifically, in order to regulate astrocytes in vivo, an adenovirus including a light- or chemical-responsive gene regulated by a cell-specific (GFAP) promoter is injected into the brain of a mouse, and thus light or chemical reaction genes are expressed in astrocytes. According to the present invention, it has been confirmed that cognitive impairment can be induced through stimulation of light or chemical activation of astrocytes in a hippocampal region. Also, it has been confirmed that inhibition of astrocyte activities using an inhibitory chemical reaction can protect cognitive impairment observed in neuroinflammatory situations. Accordingly, the technology developed according to the present invention can be useful to study cognitive impairment and presents the possibility of developing astrocyte-centered prevention/therapeutic strategies for controlling cognitive functions and treating related diseases.

Description

Animal model with damage of cognitive function by optogenetic or chemogenetic stimulation of hippocampal astrocytes and screening method of drug for improving cognitive function using the same}

The present invention relates to an animal model for impairing cognitive function through optogenetic or chemogenetic stimulation of hippocampal astrocytes and a method for screening a drug for improving cognitive function using the same.

Cognitive function is the ability to efficiently manipulate knowledge and information, and encompasses memory, spatial perception, judgment, executive function, and language ability. As medical technology advances and economic growth enters an aging society, both material damage caused by neurodegenerative diseases that impair cognitive function and cognitive ability and mental damage related to these diseases are appearing together. Decreased cognitive function often means a decline in functions and abilities related to memory, but in recent years, it is not limited to a decrease in memory, but complex attention, perceptual ability, executive function, learning and memory, language according to the process of changes in the brain. It is believed that it can appear in various aspects in terms of degraded abilities and areas such as ability, perceptual-motor function, and social cognition.

Astrocytes, a type of neuron, are also known as astrocytes, which occupy the largest number of cells in the nervous system, and play a role in supporting neuron activity by appropriately removing neurotransmitters secreted by neurons or regulating the concentration of ions in the brain. It is known to perform. Recently, as it has been found that it plays a certain role in the formation of synapses of neurons, regulation of synaptic numbers, and synaptic plasticity, plays a certain role in the onset of degenerative nervous system diseases, and plays a certain role even when neural stem cells differentiate into neurons. Research on whether astrocytes can be differentiated and used for treatment or improvement in degenerative neurological diseases is being actively conducted.

Korean Patent Publication 10-2018-0022150 (published on March 6, 2018)

An object of the present invention is a method for manufacturing a cognitive impaired animal model comprising the step of stimulating astrocytes in the brain hippocampus photogenetically or chemogeneically, a cognitive impaired animal model manufactured by the above method, and a drug for improving cognitive function using the same It is to provide a screening method of.

In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating brain diseases, comprising an inhibitor of activating cerebral hippocampal astrocytes as an active ingredient, and a health functional food composition for improving cognitive function.

The present invention provides a method for manufacturing a cognitive impaired animal model comprising the step of stimulating astrocytes in the hippocampus of the brain of animals other than humans by photogenetic or chemogenetic stimulation.

In addition, the present invention provides an animal model with impaired cognitive function except for humans produced by stimulating astrocytes in the brain hippocampus by photogenetic or chemogenetic stimulation.

In addition, the present invention comprises the steps of administering a drug candidate for improving cognitive function to the cognitive impaired animal model; And it provides a method for screening a drug for improving cognitive function, comprising the step of selecting a drug candidate material showing an effect of improving cognitive function compared to the animal group administered with the control substance.

In addition, the present invention provides a pharmaceutical composition for preventing or treating brain diseases comprising an inhibitor of activating cerebral hippocampal astrocytes as an active ingredient.

In addition, the present invention provides a health functional food composition for improving cognitive function comprising a brain hippocampal astrocyte activation inhibitor as an active ingredient.

The present invention relates to an animal model for cognitive function impairment through optogenetic or chemogenetic stimulation of hippocampal astrocytes, and a method for screening a drug for improving cognitive function using the same, and more specifically, to a cell for controlling astrocytes in vivo. An adenovirus containing a gene that responds to light or chemicals regulated by a specific (GFAP) promoter was injected into the brain of a mouse to express a light or chemically reactive gene in astrocytes. Through the present invention, it was confirmed that cognitive impairment can be induced through light or chemical activation stimulation of astrocytes in the hippocampus region, and inhibition of astrocyte activity using an inhibitory chemical reaction can reduce cognitive impairment seen in neuroinflammation situations. It was confirmed that it can be protected. Therefore, the technology developed through the present invention not only can be usefully used to study cognitive impairment, but also suggests the possibility of developing a stellate cell-centered prevention/treatment strategy to control cognitive function and treat related diseases.

1 shows the results of induction of astrocyte-specific ChR2 expression of hippocampal CA1 by virus. A, virus configuration diagram is shown. B, Astrocytes and neurons in the hippocampus of mice 2 weeks after injection of the control and ChR2 virus were visualized by fluorescence immunostaining using antibodies (GFAP and NeuN). C, astrocytes (GFAP) and ChR2 were confirmed to overlap (yellow arrows), and neurons did not overlap.
2 shows the result of impairment of spatial cognitive function according to the duration of astrocyte stimulation. A, A schematic diagram of the optical stimulation stimulation method of astrocytes and the experimental design for the Y-maze test is shown. B, the animals were given photostimulation of 5 minutes, 10 minutes, and 20 minutes per day. At the 3rd day of 20-minute stimulation, spatial cognitive function was impaired in the ChR2 group compared to the control virus group (eYFP). There was no difference between the group not treated with virus and the control group.
3 shows the results of spatial search and memory impairment according to astrocyte-specific activation duration. A, A schematic diagram of the experimental design for the astrocyte photostimulation method and passive avoidance test is shown. B, during the training period, 5 minutes, 10 minutes, and 20 minutes of photostimulation were repeated for 3 days. There was a significant difference in the speed of finding an exit by training on the 3rd day of 20-minute stimulation compared to other groups. The memory test by the probe test showed impairment on the 4th day, but there was no difference compared to the other groups on the 6th day.
4 shows the result of increasing the expression of pro-inflammatory substances in the hippocampus by astrocyte-specific stimulation. The mRNA expressions of Il-1b, Lcn2, and Tnf were increased in the ChR2 group in the hippocampal tissues of animals that were stimulated for a total of 3 days for a total of 3 days in the group without virus injection, the control virus injection group, and the ChR2 virus injection group. In the absence of photostimulation, there was no change.
5 shows the results of DREADD expression in the CA1 region of the hippocampus. Expression of hM3D(Gq)-mCherry (A) and hM4Di-mCherry (B) in the hippocampal CA1 region was at the same location as the astrocyte marker GFAP (white) (yellow arrow). However, it did not appear in the neuronal marker NeuN (red).
6 shows the results of impairment of hippocampal-dependent cognitive function by activation of astrocytes by hM3Dq. A, A schematic diagram of the experimental design for hM3Dq stimulation and Y-maze test for astrocyte activation is shown. The cross-visit behavior was significantly decreased at 90 and 120 minutes after intraperitoneal injection of B and CNO (3 mg/kg). There was no difference in the total number of visits used as an indicator of activity. C, a schematic diagram of the experimental design for hM3Dq stimulation for astrocyte activation and passive avoidance test is shown. There was no difference between the eYFP and hM3Dq groups during training 30 minutes after D, CNO (3 mg/kg) intraperitoneal injection. There was a significant difference in the memory related to fear learning performed 24 hours later in the hM3Dq group.
7 shows the results of impairment of hippocampal-dependent cognitive function by activation of astrocytes by hM4Di. A, A schematic diagram of the experimental design for hM4Di stimulation for astrocyte activation and Y-maze test is shown. B, CNO (3 mg/kg) 30 minutes before LPS treatment, and CNO intraperitoneally injected 3 times at 6 hour intervals after LPS treatment. After 24 hours of LPS treatment, the cross-visit behavior was significantly reduced in the hM4Di CNO+LPS group. There was no difference in the total number of visits used as an indicator of activity. C, a schematic diagram of the experimental design for hM4Di stimulation for astrocyte activation and passive avoidance test is shown. D, CNO (3 mg/kg) 30 minutes before LPS treatment, and CNO intraperitoneally three times at 6 hour intervals after LPS treatment. During the training period, there was a significant decrease compared to the hM4Di LPS group control group. There was a significant difference in the memory related to fear learning performed after 24 hours in the hM4Di group.

The present invention provides a method for manufacturing a cognitive impaired animal model comprising the step of stimulating astrocytes in the hippocampus of the brain of animals other than humans by photogenetic or chemogenetic stimulation. Preferably, the animal may be a mouse, but is not limited thereto.

Preferably, the step of optogeneically stimulating astrocytes in the hippocampus of the brain is 1) transforming astrocytes in the hippocampus of the brain with an expression vector containing a light-activated ion channel gene, the Expressing a photoactivated ion channel; And 2) photostimulating astrocytes in the hippocampus of the brain, but is not limited thereto.

In the present invention, optogenetics is one of the biotechnology fields that have been in the spotlight recently, and it means controlling something with light by manipulating genes. Early research in optogenetics was applied to control the activity of nerve cells using light, which refers to a technology that activates nerve cells when light is irradiated to them. By controlling the activity of nerve cells using light in this way, you can control the activity of nerve cells more precisely than controlling the nerve cells using conventional electrical stimulation. This is because, while electrical stimulation stimulates a large number of unspecified nerve cells, optogenetic technology can accurately stimulate specific cells of interest. Accordingly, an object of the present invention is to transform a photoactivated ion channel capable of activating astrocytes by the photostimulation under the control of an astrocyte-specific promoter.

In the present invention, the light-activated ion channel is a group of membrane-permeable proteins that open and close pores by light stimulation. In the present invention, the photoactivated ion channel can be used without limitation as long as it is a protein capable of opening an ion channel of a cell membrane in response to exposure to light stimulation. Proteins such as (archaerhodopsin) can be used. Preferably, in one embodiment of the present invention, channelrhodopsin-2 (ChR2) was used. On the other hand, channelrhodopsin-2 used in the present invention is a mutant in which the 401th base of genbank number EF47017 (ChR2) is point mutated to guanine, and is also indicated as ChR2 (H134R).

The channel of the channel rhodopsin means that something passes, and the word rhodopsin is related to light. This has the characteristic that when it receives light, the channel of channel rhodopsin opens and something enters the cell, and without light, the channel does not open. Therefore, the channel rhodopsin-2 protein was injected into astrocytes to control the opening of the channel in the astrocyte cell membrane with light, and when the channel of the astrocyte is opened, cations such as potassium (K) enter the cell into the cell. As a result, a potential difference between the inside and outside of the cell is generated, and the astrocyte is activated.

In the present invention, the method of transforming astrocytes with a photoactivated ion channel may use a transformation method known in the art without limitation, and non-limiting examples thereof include transformation, transfection, and It may be selected from the group consisting of electrophoresis, transduction, microinjection, and balliatic introduction.

In the present invention, the optical stimulation is preferably irradiated with blue light having a wavelength of 450 to 480 nm, and the time for irradiating the optical stimulation is 5 to 30 minutes, preferably 1 to 3 times. On the other hand, the method of irradiating the optical stimulation is not particularly limited, and the animal may be immobilized and the optical stimulation may be irradiated from the outside, and an optical fiber may be implanted into the hippocampus to irradiate the direct optical stimulation to the hippocampus. The wavelength of the light may vary depending on the type of the photoactivated ion channel to be transformed, and the optimal light wavelength can be easily selected by a person skilled in the art through repeated tests.

Preferably, the step of stimulating astrocytes in the brain hippocampus chemogeneically comprises: 1) transforming astrocytes in the brain hippocampus with an expression vector containing an hM3Dq DREADD (designer receptors exclusively activated by designer drugs) receptor, the expressing the hM3Dq DREADD receptor; And 2) reacting the expressed hM3Dq DREADD receptor with Clozapine N-oxide (CNO) to activate astrocytes in the hippocampus of the brain, but is not limited thereto.

In the present invention, chemogenetics, also called chemical biology or chemogenomics, is an applied technology that helps understanding physiological functions by controlling the activity of cells by applying a protein that reacts to an unknown low-molecular synthetic material. Proteins such as kinase, non-kinase enzyme, GPCR (G protein-coupled receptors), and ligand-gated ion channels have been made chemically. DREADDs (designer) lowered reactivity with acetylcholine (Ach) and increased reactivity to new synthetic substances (e.g., clozapine-N-oxide) by partially mutating wild type muscarinic receptors among various chemogeneically designed proteins. receptors exclusively activated by designer drugs) are most commonly used.

One of the DREADDs, hM3Dq, is a receptor for promoting neuronal activity. A combination of hM3Dq using Gq protein and clozapine N-oxide (CNO) as a ligand is generally used. Gq-DREADDs are activated at low concentrations of CNO (nM) to increase intracellular calcium secretion and promote neuronal activity.

One of the DREADDs, hM4Di, is a receptor for inhibiting neuronal activity and was developed using Gi protein. hM4Di is activated by CNO, perlapine, and compound 21.

In the present invention, the expression vector may be selected from the group consisting of a linear DNA vector, a plasmid DNA vector, and a recombinant viral vector, but is not limited thereto, and all conventional vectors used for transformation in the art are the methods of the present invention. Can be used for

Preferably, the expression vector used in an embodiment of the present invention may be an adenovirus vector or an adeno-associated viral vector, but is not limited thereto.

In addition, the present invention provides an animal model with impaired cognitive function except for humans produced by stimulating astrocytes in the brain hippocampus by photogenetic or chemogenetic stimulation. Preferably, the animal may be a mouse, but is not limited thereto.

Preferably, the optogenetic stimulation transforms astrocytes in the brain hippocampus with an expression vector containing a photoactivated ion channel gene, expressing the photoactivated ion channel, and photostimulating astrocytes in the brain hippocampus. It may be, but is not limited thereto.

Preferably, the chemogenetic stimulation transforms astrocytes in the brain hippocampus with an expression vector containing the hM3Dq DREADD receptor to express the hM3Dq DREADD receptor, and the expressed hM3Dq DREADD receptor and clozapine N-oxide (Clozapine N-oxide; CNO) may be reacted to activate astrocytes in the hippocampus of the brain, but is not limited thereto.

In addition, the present invention comprises the steps of administering a drug candidate for improving cognitive function to the cognitive impaired animal model; And it provides a method for screening a drug for improving cognitive function, comprising the step of selecting a drug candidate material showing an effect of improving cognitive function compared to the animal group administered with the control substance.

In the present invention, the type of the candidate drug for improving cognitive function is not particularly limited, and preferably natural products, synthetic compounds, RNA, DNA, polypeptides, enzymes, proteins, ligands, antibodies, metabolites and bioactivity of bacteria or fungi It may be selected from the group consisting of molecules.

The method of administering the cognitive improvement drug candidate to an animal can be used without limitation in the art of administering the drug to the animal, for example, intraperitoneal administration, oral administration, intravenous administration, negative administration, intrathecal administration. Etc. can be used. The timing of administration of the candidate substance may be within 1 day immediately after optogenetic or chemogenetic stimulation, but is not limited thereto. Depending on the type of candidate substance, the drug may be administered before optogenetic or chemogenetic stimulation. The effect of improving cognitive function can also be evaluated. The timing of administration, route of administration, administration interval, and dosage of the drug can be applied by a person skilled in the art by easily selecting and applying optimal conditions through preliminary experiments according to the type, nature, and mechanism of action of the candidate substance.

In addition, the present invention provides a pharmaceutical composition for preventing or treating brain diseases comprising an inhibitor of activating cerebral hippocampal astrocytes as an active ingredient.

The pharmaceutical composition of the present invention may include chemical substances, nucleotides, antisense, siRNA oligonucleotides, and natural product extracts as active ingredients. The pharmaceutical composition or complex formulation of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant may be an excipient, a disintegrant, a sweetening agent, a binder, a coating agent, an expanding agent, and a lubricant. , A solubilizing agent such as a lubricant or a flavoring agent may be used. The pharmaceutical composition of the present invention may be preferably formulated as a pharmaceutical composition, including at least one pharmaceutically acceptable carrier in addition to the active ingredient for administration. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions are sterilized and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injection formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

The pharmaceutical formulation form of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, and sustained-release formulations of active compounds. I can. The pharmaceutical composition of the present invention is in a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. It can be administered as. The effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for prevention or treatment of a disease. Therefore, the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, weight, general health condition, sex and diet, administration time, administration route and composition It can be adjusted according to various factors, including the rate of secretion, duration of treatment, and drugs used concurrently. Although not limited thereto, for example, in the case of an adult, when administered once to several times a day, the composition of the present invention is administered once to several times a day, in the case of a compound, 0.1 ng/kg to 10 g/kg, a polypeptide, In the case of protein or antibody, 0.1 ng/kg to 10 g/kg, for antisense nucleotides, siRNA, shRNAi, and miRNA, it can be administered at a dose of 0.01 ng/kg to 10 g/kg.

In addition, the present invention provides a health functional food composition for improving cognitive function comprising a brain hippocampal astrocyte activation inhibitor as an active ingredient.

The health functional food composition of the present invention may be provided in the form of powder, granule, tablet, capsule, syrup or beverage, and the health functional food composition is used with other foods or food additives in addition to the active ingredient, according to a conventional method. Can be used appropriately. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use, for example, prevention, health or therapeutic treatment.

The effective dose of the active ingredient contained in the health functional food composition may be used in accordance with the effective dose of the pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control, it should be less than the above range. It is clear that the active ingredient can be used in an amount beyond the above range because there is no problem in terms of safety.

There are no particular restrictions on the types of health functional foods, examples of which include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea. , Drinks, alcoholic beverages, and vitamin complexes.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art.

< Experimental example >

The following experimental examples are intended to provide experimental examples commonly applied to each of the examples according to the present invention.

1. Experimental animals

As the experimental animal, 18-20g male C57BL/6 mice purchased from Coretech were used. The breeding and treatment of experimental animals followed the policy of the Kyungpook National University Laboratory Animal Ethics Committee and the regulations related to animal testing. The mice used in the experiment were subjected to temperature (21-23℃), humidity (40-60%) and lighting (12 hours). / Cancer) was raised in a breeding room where drinking water and feed were supplied freely, and was used after acclimating to the laboratory environment for at least 1 week.

2. Virus delivery and optical fiber implantation into animal hippocampal CA1 tissue

Mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (20 mg/kg) and placed in a Stereotaxic Brain Surgery Instrument (Harvard Apparatus). The skull was exposed by a small incision in the scalp and a small hole was drilled for virus injection and cannulation. Insert the injection needle into the brain and AAV5/GFAP-ChR2-eYFP virus (titer: 2.0 x 10 ¹² particles per ml); AAV5/GFAP-hM3D(Gq)-mCherry-WPRE (titer: 1.0×10 ¹² particles per ml); AAV5/GFAP-hM4D(Gi)-mCherry-WPRE (titer: 4.0 × 10 ¹² particles per ml) virus and control AAV5/GFAP-eYFP virus (titer: 2.5 × 10 ¹² particles per ml) virus to hippocampus CA1 (coordinates , bregma: -2 mm; midline: ±1.8 mm; skull surface: -2.5 mm) A total of 0.5 μl was injected at a rate of 0.1 μl per minute, and the needle was taken out after 10 minutes. After virus injection, a 26 gauge stainless steel cannula was inserted in the same position. The guide cannula is fixed to the skull using dental cement, and a single optical fiber (Doric Lenses Inc, Quebec, Canada) is implanted for optogenetic stimulation, and the tip is positioned 0.5 mm above the hippocampus CA1. It was fixed with cement. After the surgical operation of the mice, the mice were recovered from the cages alone for at least 2 weeks for viral expression and recovery from surgery.

3. Photostimulation

After virus injection and surgical recovery, photostimulation was applied unilaterally using a blue laser (473 nm; SDL-473-100MFL, Shanghai Dream Laser Technology Co., Shanghai, China) driven at a frequency of 20 Hz by a stimulator. (Model S-88, Grass medical Inc., Quincy, MA). The light intensity at the tip of the fiber optic vein was 1.7mW/mm ² (PM204, Tholabs Co., Ann Arbor, MI).

4. Behavioral experiment

The mice used in the present invention were kept in individual cages individually for at least a week to acclimatize to the new environment before starting behavioral experiments. Each behavioral experiment was outlined graphically in the relevant drawing and measured at different time intervals according to different experimental requirements.

(1) Y-maze test

Spatial cognitive function was investigated through voluntary shift work in Y-maze. Y-maze is a maze with three arms horizontally (40 cm long and 3 cm wide with 12 cm high walls). Mice were initially placed in the center, the sequence (ABCCAB) and the number of visits to the horizontal arm were measured and recorded manually for 7 minutes for each individual. Spontaneous selection is defined as sequential visits for all 3 arms at consecutive visits (ie ABC, CAB or BCA, excluding BAB). After the end of the experiment for continuous measurement of animals, the arm of the maze was removed with 70% ethanol to remove residual odor from the animals. The shift ratio was defined according to the following equation. % alternation = [(number of alternations) / (total arm entries)] × 100. The total number of maze arms in and out is used as an indicator of motor activity.

(2) Passive avoidance test

For training of the passive avoidance test, the mice were first placed in a bright chamber. When the mice passed into the dark chamber, a light impact (0.25 mA/1 sec) was applied to the paws. The waiting time to enter the dark chamber was used as the reference measurement. During the probe test 24 hours after training, the mice were put back into the bright chamber and the waiting time until entering the dark chamber was measured as a passive fear avoidance index.

(3) Barnes maze test

Barnes maze is a circular platform (90 cm high) (100 cm in diameter) with an escape hole (5 cm direct), connected to a small chamber under the platform. To make it difficult to find the real hole, 19 different fake holes were made on the platform around the maze. The fake hole looks like a way out, but it doesn't lead to the way out. The area around the maze was lit with a 100 W bulb. On the first day, the mice were placed in an opaque box in the center of the maze. After 10 seconds had elapsed, the opaque box was removed and the mouse was guided to the escape hole by hand. The first escape attempt began 15 minutes later. In the first attempt, the mice were placed in an opaque box for 10 seconds. The mouse, released from the opaque box, took three minutes to find a way out, and the light went out as soon as it found the hole. The mice stayed in the chamber at the exit for 1 minute and then returned to their original cage. This procedure was recorded with a video camera and tracking software, and the waiting time and distance traveled to find the way out were recorded. The mice were trained four times a day at 15-minute intervals. After learning for 3 days, the mice were placed in a maze on the 4th and 6th days, and a probe test was performed. This time the escape was covered so that it looked like all the other holes. The mouse's search behavior was recorded and the time spent around the exit was recorded. Also, the amount of search for fake holes was recorded. 90 seconds after the test was over, it was returned to the original cage.

5. Drugs and Administration

Clozapine N-oxide (CNO) used in the present invention was purchased from Enzo Life Sciences (Postfach, Lausen, Switzerland) and 3 mg/kg was dissolved in physiological saline and administered intraperitoneally, and lipopolysaccharide; LPS) was purchased from Sigma-Aldrich (St. Louis, MO, USA), and 2 μg per individual was dissolved in physiological saline and injected into the ventricle.

6. Treatment of brain tissue

While the experimental animals were anesthetized with tiletamine and zolazepam, the RT-PCR mice were sacrificed with a single head, the brain was removed, and the hippocampal tissue was isolated. Mice for tissue staining were open thoracic, and sufficiently perfused with 0.05 M phosphate buffered saline (PBS) and 4% paraformaldehyde through the heart. Then, the brain was excised, post-fixed for about 24 hours, immersed in sucrose solution and precipitated, and then the brain tissue was frozen with a dry ice-isopentane solution at -40°C. The frozen tissue was prepared as a 30 μm thick section with a cryo-cut (Leica, 2800N, Germany) and used for immunohistochemistry.

7. Immune tissue staining

Mouse anti-GFAP antibody (1:500 dilution; BD Biosciences), rabbit anti-GFAP (1:1000 dilution; Dako), rabbit anti-Iba-1 (1:1000 dilution; Wako), and mouse anti-NeuN (1:500 dilution; Millipore) antibodies were reacted and for visualization Cy3-, Cy5-, and FITC-conjugated anti-mouse, rabbit, or goat IgG antibody ( The Jackson Laboratory, Bar Harbor, ME, Cy3-mouse, Cy3-rabbit, Cy-goat, Cy5-rabbit, FITC-mouse, FITC-rabbit, FITC-goat) were used. Fluorescence or confocal microscopy was used for image acquisition or analysis.

8. Inflammatory cytokines mRNA RT- PCR Measure

Total RNA was extracted from the hippocampal tissue isolated from the mouse brain using Trizol (Quagen, Germany). Afterwards, 1 μg of total RNA and Script cDNA synthesis kit (Bio-Rad, USA) were used to reverse transcription into DNA. For PCR amplification, a specific primer set was used with DNA Engine Tetrad Peltier Thermal Cycler (MJ Research, Waltham, MA). Il- 1b (forward, 5'-AGT TGC CTT CTT GGG ACT GA-3'; reverse, 5'-TCC ACG ATT TCC CAG AGA AC-3'); Lcn2 (forward, 5'-ATG TCA ACC TCC ACC TGG TC-3'; reverse, 5'-CAC ACT CAC CAC CCA TTC AG-3'); Tnf (forward, 5'-CAT CTT CTC AAA ATT CGA GTG ACA A-3'; reverse, 5'-ACT TGG GCA GAT TGA CCT CAG-3'); Gapdh (forward, 5'-TGG GCT ACA CTG AGC ACC AG-3'; reverse, 5'-GGG TGT CGC TGT TGA AGT CA-3').

9. Statistical processing

Results obtained from various experiments were expressed as mean ± standard error. The statistical significance of each data was tested with a significance level of p <0.05 or higher using the student's t-test.

< Example 1> Optogenetic For access ChR2 Astrocyte Induction of specific expression

In order to investigate the potential role of astrocytes in the hippocampus, which is known as an important brain region for the regulation of cognitive function, cell-specific expression of ChR2 in astrocytes was induced using a virus using an optogenetic approach (FIG. 1). The virus used induces specific expression of ChR2 in astrocytes using the GFAP promoter, and it was confirmed by immunofluorescence staining that the expression of ChR2 did not appear in neurons. In the subsequent experiment, an experiment was conducted to specifically activate astrocytes using the same virus.

< Example 2> Astrocyte continuous To photostimulation Cognitive impairment due to

In order to confirm the effect on cognitive function when the activation of astrocytes is continuously induced, the animals expressing ChR2 are subjected to photostimulation for 5 minutes, 10 minutes, and 20 minutes a day, and the Y-maze test is performed to determine spatial perception. Function was tested. This was repeated for 3 days in all groups and stimulation conditions. On the first and second days of photostimulation, there was no significant difference in all stimulation times, and it was confirmed that spatial cognitive function decreased on the third day (Fig. 2). These results indicate that continuous activation of astrocytes can impair spatial cognitive function. It was verified once more that cognitive impairment was induced using the Barnes maze test, which can test spatial search ability and memory (FIG. 3).

< Example 3> By photostimulation Induced Astrocyte In the hippocampus by specific activation Induction of neuroinflammation

Astrocytes play a role in maintaining homeostasis under normal circumstances, but are known to mediate inflammatory responses by disease or injury. In the results of the present invention, in order to confirm the possibility of neuroinflammation in cognitive function impairment due to specific activation of astrocytes, a ChR2-expressing animal was treated with photostimulation for 20 minutes a day and repeated for 3 days, followed by proinflammatory cytokines. The expression of was confirmed. As a result, the expression of Il- 1b , Lcn2 , and Tnf was significantly increased in the hippocampal tissues of ChR2-expressing animals, which were repeated 20 minutes a day photostimulation for 3 days (FIG. 4). These results indicate that there is a correlation between cognitive impairment and neuroinflammation caused by 20-minute photostimulation.

< Example 4> Using chemogenetic methods Astrocyte Cognitive function control by selective activation

To investigate the potential role of astrocytes in the hippocampal region in regulating cognitive function, a chemogenetic DREADD approach was used in conjunction with cognitive function analysis. DREADD uses an engineered G-protein-coupled receptor (GPCR) that is activated by a ligand that selectively binds to the GPCR. For example, engineered human muscarinic type 3 GPCRs (hM3D(Gq) and hM4D(Gi) do not respond to endogenous ligands, but are instead activated by pharmacologically inactive clozapine-N-oxide (CNO)). The muscarinic receptor variants hM3D(Gq) and hM4D(Gi) fused to the red fluorescent protein mCherry were encoded in astrocytes of the hippocampal CA1 region using a virus containing the GFAP promoter. In order to characterize the expressing cell types, we immunostained GFAP in brain sections of animals injected with virus and confirmed that cells expressing hM3Dq- and hM4Di-mCherry express GFAP (Fig. 5). Y-maze and passive avoidance test were performed to investigate the effect of CNO (3 mg) 30 minutes before the start to induce activation of astrocytes by hM3Dq to proceed with the Y-maze test. /kg) was injected intraperitoneally, and then significantly decreased compared to the control group at 90 and 120 minutes (Fig. 6B) Passive avoidance test to test memory related to fear learning by applying electrical stimulation to animals In the first training phase, the animals were first placed in a bright chamber to measure the speed of moving to the dark chamber, and there was no difference between the two groups. Fear was learned, and in the memory test of electrical stimulation after 24 hours, the rate of movement from the light chamber to the dark chamber was significantly reduced in the hM3Dq group. This means that you don't remember well that when you move to the chamber, you are given a harmful stimulus (Fig. 6D). The following experiment confirmed the effect of astrocytes activated by hM4Di on cognitive function. CNO was administered intraperitoneally, and there was no effect on cognitive function in Y-maze and passive avoidance test (Fig. 7B and 7D).

Cognitive impairment was confirmed by optogenetic stellate cell activation and chemogenetic method using hM3Dq. However, there was no cognitive impairment response to the activation of astrocytes using hM4Di. These results basically show that activation by ChR2 and activation by hM3Dq may increase neuroinflammation and impair cognitive function, and activation by hM4Di did not show this reaction. The present inventors conducted an experiment to confirm the effect of activation by hM4Di on the regulation of cognitive function by neuroinflammation. The concentration of LPS (2 μg/mouse) used in the experiment was low enough to not affect behavior and was found to cause weak cognitive impairment. In the Y-maze and passive avoidance test, CNO was pretreated to suppress the effect of LPS, and LPS was injected into the ventricle 30 minutes later, and three additional times at 6 hour intervals to induce continuous hM4Di activity. Was injected (Fig. 7A and 7C). As a result, in the Y-maze test, when LPS was treated in the control virus group, cognitive function was impaired, which showed that the cognitive function impairment was alleviated in the hM4Di virus group (Fig. 7B). Similarly in the passive avoidance test, it was confirmed that cognitive impairment significantly decreased cognitive impairment in the hM4Di virus group (Fig. 7D). These results indicate that the activation of astrocytes through hM4Di can alleviate cognitive function caused by neuroinflammation.

Claims (14)

Cognitive function impaired animal model manufacturing method comprising the step of stimulating astrocytes in the hippocampus of the brain of animals other than humans photogenetically or chemogeneically. The method of claim 1, wherein the step of optogeneically stimulating astrocytes in the cerebral hippocampus comprises: 1) transforming astrocytes in the cerebral hippocampus with an expression vector containing a light-activated ion channel gene. , Expressing the photo-activated ion channel; And 2) photostimulating astrocytes in the brain hippocampus. The method of claim 2, wherein the light-activated ion channel gene is channelrhodopsin-2 (ChR2), which is a light-sensitive cation channel. The method of claim 2, wherein the step of photostimulating astrocytes in the hippocampus of the brain comprises photostimulating a light source of 450 to 480 nm for 5 to 30 minutes, 1 to 3 times. . The method of claim 1, wherein the step of stimulating astrocytes in the cerebral hippocampus chemogeneically comprises: 1) transforming astrocytes in the cerebral hippocampus with an expression vector containing an hM3Dq DREADD (designer receptors exclusively activated by designer drugs) receptor , Expressing the hM3Dq DREADD receptor; And 2) reacting the expressed hM3Dq DREADD receptor with Clozapine N-oxide (CNO) to activate astrocytes in the hippocampus of the brain. The method of claim 2 or 5, wherein the expression vector is an adenovirus vector or an adeno-associated viral vector. The method of claim 1, wherein the animal is a mouse. An animal model with impaired cognitive function excluding humans produced by stimulating astrocytes in the hippocampus of the brain through optogenetic or chemogenetic stimulation. The method of claim 8, wherein the optogenetic stimulation transforms astrocytes in the hippocampus of the brain with an expression vector containing a photoactivated ion channel gene, thereby expressing the photoactivated ion channel, and lightening the astrocytes in the hippocampus. An animal model of cognitive impairment except for humans, characterized in that stimulation. The method of claim 8, wherein the chemogenetic stimulation transforms astrocytes in the brain hippocampus with an expression vector containing the hM3Dq DREADD receptor to express the hM3Dq DREADD receptor, and the expressed hM3Dq DREADD receptor and clozapine N-oxide (Clozapine N-oxide; CNO), a cognitive impairment animal model except for humans, characterized in that astrocytes in the brain hippocampus are activated. 11. The animal model for impaired cognitive function excluding humans according to any one of claims 8 to 10, wherein the animal is a mouse. Administering a drug candidate substance for improving cognitive function to the animal model for cognitive impairment according to any one of claims 8 to 10; And
A method for screening a drug for improving cognitive function, comprising the step of selecting a drug candidate that exhibits an effect of improving cognitive function compared to the animal group administered with a control substance. A pharmaceutical composition for preventing or treating cerebral diseases comprising a cerebral hippocampal astrocyte activation inhibitor as an active ingredient. A health functional food composition for improving cognitive function comprising a brain hippocampal astrocyte activation inhibitor as an active ingredient.

Images