Method for inhibiting macrophage neurotoxicity

Abstract

The present application relates to a method for inhibiting macrophage neurotoxicity, comprising a step of increasing the expression of Aurora kinase in macrophages.

Description

Methods for inhibiting macrophage neurotoxicity

[0001] The present invention relates to a method for inhibiting macrophage neurotoxicity.

[0002] The central nervous system of vertebrates is protected by the skull and spine, but once damaged, recovery is very limited. Central nervous system damage, such as stroke, occurs due to impaired blood flow caused by blood clots or other factors, which can severely damage neural tissue. Currently, there are very limited innovative treatments that can effectively suppress neural tissue damage and promote recovery of neural function in the early stages of stroke.

[0003] Damage caused by stroke is further aggravated by secondary degeneration that occurs over several days following the primary injury. In particular, in ischemic stroke, the penumbra region surrounding the infarct core is a critical area with potential for recovery with appropriate treatment. Recent studies have highlighted the inflammatory response following stroke, particularly macrophage activation, as a major cause of secondary damage.

[0004] Macrophages are a crucial component of the immune system, and under normal conditions, they play a role in removing damaged tissue and promoting healing. However, in acute trauma situations such as stroke, overactivated macrophages can secrete neurotoxic substances, causing further nerve damage. This macrophage neurotoxicity exacerbates the post-stroke inflammatory response and, as a result, serves as a major factor impeding nerve tissue recovery.

[0005] Therefore, there is an urgent need to develop a new treatment method that can effectively suppress the neurotoxicity of macrophages after stroke, regulate the inflammatory response, minimize damage to neural tissue, and promote long-term recovery of neural function.

[0006] The background technology of this application, Republic of Korea Patent Publication No. 10-2021-0151945, relates to a method for regulating macrophage activity.

[0007] The present invention is intended to solve the problems of the above-mentioned prior art and provides a method for inhibiting macrophage neurotoxicity.

[0008] In addition, a pharmaceutical composition for inhibiting nerve tissue degeneration is provided.

[0009] Additionally, it provides biomarkers for diagnosing neurodegenerative diseases.

[0010] In addition, a kit for diagnosing a neurodegenerative disease is provided, which includes the biomarker for diagnosing the neurodegenerative disease.

[0011] Additionally, a method for screening for macrophage neurotoxicity inhibitors is provided.

[0012] However, the technical tasks to be achieved by the embodiments of the present invention are not limited to the technical tasks described above, and other technical tasks may exist.

[0013] As a technical means for achieving the above-mentioned technical task, the first aspect of the present invention provides a method for inhibiting macrophage neurotoxicity, comprising a step of increasing the expression of Aurora Kinase in macrophages.

[0014] According to one embodiment of the present invention, the aurora kinase may include, but is not limited to, one selected from the group consisting of AURKA, AURKB, and combinations thereof.

[0015] According to one embodiment of the present invention, the step of increasing the expression may include, but is not limited to, co-culturing the macrophages with neural cells.

[0016] According to one embodiment of the present invention, the nerve cells may include, but are not limited to, those selected from the group consisting of dorsal root ganglion sensory neurons, hippocampal neurons, dopaminergic neurons, motor neurons, cerebellar granule cells, thalamic neurons, spinal cord neurons, and combinations thereof.

[0017] In addition, the second aspect of the present invention provides a pharmaceutical composition for inhibiting nerve tissue degeneration, comprising an active substance that increases the expression of aurora kinase.

[0018] According to one embodiment of the present invention, the aurora kinase may include, but is not limited to, one selected from the group consisting of AURKA, AURKB, and combinations thereof.

[0019] In addition, the third aspect of the present invention provides a biomarker for diagnosing neurodegenerative diseases, comprising aurora kinase as an active ingredient.

[0020] According to one embodiment of the present invention, the aurora kinase may include, but is not limited to, one selected from the group consisting of AURKA, AURKB, and combinations thereof.

[0021] According to one embodiment of the present invention, if a downregulation in the expression level of the aurora kinase is observed, it may be determined that a neurodegenerative disease is present, but is not limited thereto.

[0022] According to one embodiment of the present invention, the neurodegenerative disease may include, but is not limited to, a disease selected from the group consisting of stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease, and combinations thereof.

[0023] According to one embodiment of the present invention, the neurodegenerative disease may include, but is not limited to, stroke.

[0024] In addition, the fourth aspect of the present invention provides a kit for diagnosing a neurodegenerative disease, comprising a biomarker for diagnosing a neurodegenerative disease according to the third aspect of the present invention.

[0025] In addition, the fifth aspect of the present invention provides a method for screening for a macrophage neurotoxicity inhibitor, comprising the steps of treating a candidate substance to macrophages; measuring the expression level of aurora kinase in the macrophages; and selecting the candidate substance as a macrophage neurotoxicity inhibitor when the expression of the aurora kinase increases.

[0026] According to one embodiment of the present invention, the aurora kinase may include, but is not limited to, one selected from the group consisting of AURKA, AURKB, and combinations thereof.

[0027] The above-described problem-solving methods are merely exemplary and should not be construed as limiting the present invention. In addition to the exemplary embodiments described above, additional embodiments may be included in the drawings and detailed description of the invention.

[0028] The present invention presents new possibilities in the field of research and therapeutic drug development for suppressing secondary degeneration caused by macrophage inflammatory responses, a field currently lacking. Furthermore, it experimentally demonstrates that modulating aurora kinase expression in infiltrating macrophages after ischemic stroke can modulate neurotoxicity and reduce neuronal damage. This finding offers a novel approach for suppressing secondary degeneration after central nervous system injury.

[0029] Furthermore, the present invention has significant implications for clinical practice after stroke. Given the current lack of prognostic tools for monitoring neurological status and predicting final outcome, the results of this study can lay the foundation for developing new prognostic tools. Furthermore, given the lack of clinically applicable stroke-related blood biomarkers, this study suggests the potential for developing novel biomarkers capable of monitoring inflammatory responses.

[0030] In addition, it can suppress neurotoxicity in other forms of nerve damage and degenerative neurodegenerative diseases other than ischemic damage caused by the inflammatory response of macrophages, thereby suggesting a therapeutic platform that can be commonly used in various neurological diseases.

[0031] However, the effects that can be obtained from this center are not limited to the effects described above, and other effects may exist.

[0032] Figure 1 shows the results of confirming neurotoxicity-induced neuronal cell death when a macrophage culture solution according to one experimental example of the present invention is applied to neurons.

[0033] Figure 2 shows the results of confirming the reduction in neurotoxicity when the culture solution of neural cells and co-cultured macrophages according to one experimental example of the present invention is treated to neural cells.

[0034] Figure 3a shows the results of analyzing the increased genome in co-cultured macrophages using IPA (Ingenuity Pathway Analysis) and confirming the increase in cell cycle-related mechanisms.

[0035] Figure 3b shows the results of cytoscape analysis of the network of genes increased in co-cultured macrophages, confirming that cell cycle-related genes are included in the core proteins of the network.

[0036] Figure 3c shows the results of analyzing the increased genome in co-cultured macrophages using the GSEA database, confirming an increase in cell cycle-related mechanisms.

[0037] Figure 3d shows the results of the GSEA database analysis, which confirmed that many genes are related to the cell cycle mechanism.

[0038] Figure 3e shows the results of PCR analysis confirming that cell cycle genes are increased in co-cultured macrophages in transcriptome analysis.

[0039] Figure 4a shows the results of observing the expression of AURKA, AURKB, and AURKC in the transcriptome results.

[0040] Figure 4b shows the results of PCR observation showing that the expression of AURKA and AURKB increased in co-cultured macrophages.

[0041] Figure 4c is the result of quantitative analysis of the PCR results performed in Figure 4b using Image J, and this confirms the increase in expression of AURKA and AURKB in co-cultured macrophages.

[0042] Figure 4d shows the results of Western blot observation showing that the expression of AURKA and AURKB increased in co-cultured macrophages.

[0043] Figure 4e is the result of quantitative analysis of the PCR results performed in Figure 4d using Image J, and through this, it is confirmed that the expression of AURKA and AURKB increased in co-cultured macrophages.

[0044] Figure 4f shows the results of PCR observation showing that the expression of AURKA and AURKB, which was increased in co-cultured macrophages, decreased when treated with a cell cycle inhibitor.

[0045] Figure 4g is the result of quantifying the PCR results performed in Figure 4f using Image J.

[0046] Figure 4h is a Western blot result showing that the expression of AURKA and AURKB, which was increased in co-cultured macrophages, decreased when treated with a cell cycle inhibitor.

[0047] Figure 4i is the result of quantitative analysis of the Western blot results performed in Figure 4h using Image J.

[0048] Figure 5a shows the results of confirming that neurotoxicity increased when the culture medium was secured after suppressing the expression of AURKA or AURKB in macrophages using siRNA and then treating the culture medium with nerve cells.

[0049] Figure 5b shows the results of confirming that neurotoxicity was reduced when the culture medium was secured after injecting a plasmid containing the AURKA or AURKB sequence into macrophages that were not co-cultured (macrophages cultured alone) to overexpress AURKA and AURKB, respectively, and then treating the culture medium to nerve cells.

[0050] Figure 5c shows the result of calculating the DI (Degeneration Index) after quantification based on the image obtained in Figure 5a.

[0051] Figure 5d shows the result of calculating the DI (Degeneration Index) after quantification based on the image obtained in Figure 5b.

[0052] Figure 6a is a schematic diagram illustrating the process of inducing photothrombotic stroke in a rat. This implies that damage can be observed 7 days after stroke induction in rats.

[0053] Figure 6b shows the results of observing the brain of a rat 7 days later, confirming that damage had occurred (indicated by a red circle).

[0054] Figure 6c shows the results of confirming that a lesion was formed through CV (Cresyl Violet) staining after cutting the tissue.

[0055] Figure 6d shows the results of Western blot using tissues obtained from the Sham, Stroke Contra, and Stroke ipsi sites 7 days after injury, and observing that AURKA and AURKB expression was reduced at the injury site.

[0056] Below, with reference to the attached drawings, embodiments of the present invention are described in detail to facilitate easy implementation by those skilled in the art. However, the present invention can be implemented in various different forms and is not limited to the embodiments described herein. In the drawings, irrelevant parts have been omitted for clarity, and similar reference numerals have been used throughout the specification to indicate similar elements.

[0057] Throughout this specification, when a part is said to be "connected" to another part, this includes not only cases where it is "directly connected" but also cases where it is "electrically connected" with another element in between.

[0058] Throughout this specification, when it is said that a member is located “on,” “above,” “upper,” “lower,” “lower” or “lower” another member, this includes not only cases where the member is in contact with the other member, but also cases where another member exists between the two members.

[0059] Throughout this specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0060] The terms "about," "substantially," and the like, as used herein, are used to mean at or near the numerical value when manufacturing and material tolerances inherent to the meanings referred to are presented, and are used to prevent unscrupulous infringers from unfairly exploiting disclosures that contain precise or absolute numerical values ​​to aid understanding of the present disclosure. Furthermore, throughout the present disclosure, the terms "step of ~" or "step of ~" do not mean "step for ~."

[0061] Throughout this specification, the term "combination thereof" included in the expressions in the Makushi format means one or more mixtures or combinations selected from the group consisting of the components described in the expressions in the Makushi format, and means including one or more selected from the group consisting of said components.

[0062] Throughout this specification, references to “A and / or B” mean “A, B, or A and B.”

[0063] Hereinafter, the method for inhibiting macrophage neurotoxicity of the present invention will be described in detail with reference to embodiments, examples, and drawings. However, the present invention is not limited to these embodiments, examples, and drawings.

[0064]

[0065] As a technical means for achieving the above-mentioned technical task, the first aspect of the present invention provides a method for inhibiting macrophage neurotoxicity, comprising a step of increasing the expression of Aurora Kinase in macrophages.

[0066] The present invention presents new possibilities in the field of research and therapeutic drug development for suppressing secondary degeneration caused by macrophage inflammatory responses, a field currently lacking. Furthermore, it experimentally demonstrates that modulating aurora kinase expression in infiltrating macrophages after ischemic stroke can modulate neurotoxicity and reduce neuronal damage. This finding offers a novel approach for suppressing secondary degeneration after central nervous system injury.

[0067] Furthermore, this invention has significant implications for clinical practice after stroke. Given the current lack of prognostic tools for monitoring neurological status and predicting final outcome, these findings may lay the foundation for developing new prognostic tools. Furthermore, given the lack of clinically applicable stroke-related blood biomarkers, this study suggests the potential for developing novel biomarkers capable of monitoring inflammatory responses.

[0068] According to one embodiment of the present invention, the aurora kinase may include, but is not limited to, one selected from the group consisting of AURKA, AURKB, and combinations thereof.

[0069] According to one embodiment of the present invention, the step of increasing the expression may include, but is not limited to, co-culturing the macrophages with neural cells.

[0070] According to one embodiment of the present invention, the nerve cells may include, but are not limited to, those selected from the group consisting of dorsal root ganglion sensory neurons, hippocampal neurons, dopaminergic neurons, motor neurons, cerebellar granule cells, thalamic neurons, spinal cord neurons, and combinations thereof.

[0071] In addition, the second aspect of the present invention provides a pharmaceutical composition for inhibiting nerve tissue degeneration, comprising an active substance that increases the expression of aurora kinase.

[0072] Regarding the pharmaceutical composition for inhibiting nerve tissue degeneration according to the second aspect of the present invention, detailed descriptions of parts overlapping with the first aspect of the present invention have been omitted, but even if the descriptions have been omitted, the contents described in the first aspect of the present invention can be equally applied to the second aspect of the present invention.

[0073] According to one embodiment of the present invention, the aurora kinase may include, but is not limited to, one selected from the group consisting of AURKA, AURKB, and combinations thereof.

[0074] In addition, the third aspect of the present invention provides a biomarker for diagnosing neurodegenerative diseases, comprising aurora kinase as an active ingredient.

[0075] Regarding the biomarker for diagnosing neurodegenerative diseases according to the third aspect of the present invention, detailed descriptions of parts overlapping with the first aspect of the present invention have been omitted. However, even if the descriptions have been omitted, the contents described in the first aspect of the present invention can be equally applied to the third aspect of the present invention.

[0076] According to one embodiment of the present invention, the aurora kinase may include, but is not limited to, one selected from the group consisting of AURKA, AURKB, and combinations thereof.

[0077] According to one embodiment of the present invention, if a downregulation in the expression level of the aurora kinase is observed, it may be determined that a neurodegenerative disease is present, but is not limited thereto.

[0078] According to one embodiment of the present invention, the neurodegenerative disease may include, but is not limited to, a disease selected from the group consisting of stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease, and combinations thereof.

[0079] According to one embodiment of the present invention, the neurodegenerative disease may include, but is not limited to, stroke.

[0080] In addition, the fourth aspect of the present invention provides a kit for diagnosing a neurodegenerative disease, comprising a biomarker for diagnosing a neurodegenerative disease according to the third aspect of the present invention.

[0081] Regarding the neurodegenerative disease diagnostic kit according to the fourth aspect of the present invention, detailed descriptions of parts overlapping with the third aspect of the present invention have been omitted, but even if the descriptions have been omitted, the contents described in the third aspect of the present invention can be equally applied to the fourth aspect of the present invention.

[0082] In addition, the fifth aspect of the present invention provides a method for screening for a macrophage neurotoxicity inhibitor, comprising the steps of treating a candidate substance to macrophages; measuring the expression level of aurora kinase in the macrophages; and selecting the candidate substance as a macrophage neurotoxicity inhibitor when the expression of the aurora kinase increases.

[0083] Regarding the method for screening for a macrophage neurotoxicity inhibitor according to the fifth aspect of the present invention, detailed descriptions of parts overlapping with the first aspect of the present invention have been omitted. However, even if the descriptions have been omitted, the contents described in the first aspect of the present invention can be equally applied to the fifth aspect of the present invention.

[0084] According to one embodiment of the present invention, the aurora kinase may include, but is not limited to, one selected from the group consisting of AURKA, AURKB, and combinations thereof.

[0085] The present invention will be described in more detail through the following examples; however, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0086]

[0087] [Experimental Method]

[0088] laboratory animals

[0089] All animals used in this experiment were C57BL / 6 wild-type mice purchased from Koatech Co. For cortical neuron culture, pregnant mice at the E17 stage were purchased from Koatech Co. All animal experimental procedures were approved by the Animal Ethics Committee of Ajou University College of Medicine.

[0090] Macrophage (BMDM, bone marrow-derived macrophage) culture

[0091] The experiment was conducted after euthanizing the rats by CO2 gas inhalation. The skin covering both hind legs was incised, and the bones with attached muscles were removed and placed in a sterile petri dish (100 mm) containing cold PBS. 2 ) was placed in. After removing the muscles, the bone marrow was extracted from the bone by cutting between the femur and the knee joint with 10 ml of PBS. The bone marrow was centrifuged at 1,200 rpm for 5 minutes, the supernatant was discarded, and the precipitated cells were diluted in DMEM medium containing 10% FBS and 1% Penicillin and then plated on a 150 mm plate. 2 Cultured in petri dishes.

[0092] Plasmids were transduced to increase AURKA or AURKB expression. After 10 days of macrophage culture, plasmids containing the AURKA or AURKB genes were mixed with jetplus, a reagent, and incubated at room temperature for 10 minutes before treatment with macrophages. After 4 hours of treatment, the medium was replaced with fresh medium and left for 72 hours to collect the culture medium.

[0093] Dorsal root ganglion sensory neuron (DRG) culture

[0094] The experiment was conducted after euthanizing the mice by CO2 gas inhalation. After resection of the L3, L4, and L5 ganglia, single cells were dissociated with type XI collagenase (Sigma) at 37°C for 90 minutes, and centrifuged at 1,200 rpm for 5 minutes. After centrifugation, the sedimented cells were resuspended in culture medium supplemented with 2% B27, and then cultured in a 24-well plate for 4 hours.

[0095] For co-culture of dorsal root ganglion sensory neurons and macrophages, macrophages were cultured in a 1:5 ratio in a Trans-well 4 hours after neuron culture. The culture medium was collected after 72 hours of culture.

[0096] Primary cortical neuron (CN) culture

[0097] Neuronal cell culture was performed using 17-day-old pregnant female mice. Embryos were separated from the placenta, the fetal heads were decapitated, and transferred to 60-mm petri dishes. The brains were isolated, the meninges removed, and the cerebral cortex was excised. The collected cerebral cortex was transferred to a 15-ml tube filled with papain enzyme (Worthington, final concentration 14 U / ml) diluted in HBSS (Hank's Balanced Salt Solution) and digested at 37°C for 15 minutes. After 15 minutes, the cells were washed three times with 5 ml of HBSS, and 1 ml of media (CNB: Neurobasal media, 2% B27, 1% Sodium pyruvate, 1% Glutamax, 1% antibiotics, 0.5% Fetal Bovine Serum) was added. The cells were dissociated into single cells 40 times with a pipette. The cells were then placed in a 24-well plate and cultured. After 1 day of culture, the medium was replaced with fresh CNB medium. To observe neurotoxicity, the CNB medium was replaced with macrophage culture medium on the 7th day of culture. After 1 day, the medium was removed, fixed with 4% PFA, and immunostained.

[0098] To quantify neurotoxicity, images were acquired at 200x magnification. Images were analyzed using Image J software. The entire stained area in the images was quantified, and the brightness of the corresponding area was measured by setting the particle area (pixels, 10-10,000) for damaged neurons. The degeneration index (DI) was then calculated by dividing the particle area by the brightness of the entire stained area.

[0099] RNA isolation and PCR

[0100] Total RNA was isolated from cultured macrophages using Trizol (Gibco) according to the manufacturer's protocol. Reverse transcription was performed using Accupower PCR Premix (Bioneer). Quantitative real-time PCR was performed on an Applied Biosystems 7500 Real-Time PCR System using sybrpremix ex taq (Takara), and relative expression of 18S rRNA was quantified using the comparative Ct method.

[0101] Protein isolation and Western blot

[0102] Lysis buffer was added to the isolated tissues and macrophages to lyse them. The tissue extracts from the lysed tissues were centrifuged at 13,000 rpm for 15 minutes at 4°C, and the supernatants were used to measure protein concentration. Protein samples were separated on a 4–20% gel and transferred to PVDF membranes. The membrane-transferred proteins were incubated with AURKA (abcam, ab13824, 1:1000) and AURKB (abcam, ab2254, 1:1000) antibodies, followed by incubation with the appropriate HRP-conjugated secondary antibody. The immunoreactive bands were detected using a chemiluminescent substrate (ECL solution).

[0103] Photothrombosis model

[0104] A photothrombotic ischemic stroke model was created in adult rats anesthetized with ketamine. The anesthetized animals were secured in a stereotaxic frame, and two skin incisions were made. After the incision, Rose Bengal (1010 μg / ml, Sigma, St. Louis, USA) was administered at a concentration of 100 μl / g, followed by a 5-minute wait for absorption into the bloodstream. After 5 minutes, a light source was irradiated onto the skull for 15 minutes, using a KL 1500 HAL (laser). The light source was irradiated onto the skull 1.8 mm lateral to the left of bregma.

[0105] Tissue immunostaining and imaging

[0106] Tissue samples for tissue immunostaining were prepared by fixing experimental animals anesthetized with ketamine and perfusing them with PBS and 4% PFA. After perfusion, the tissues were collected, placed in a 30% sucrose solution, and kept at 4°C for one day. After sucrose treatment, the tissues were sectioned at 30 μm thickness using a frozen microtome. The sectioned tissues were then attached to tissue slides and subjected to tissue immunostaining. After attaching the tissues to the slides, they were placed on a slide warmer for 1 hour to dry. The dried tissue slides were immersed in dH2O for 2 minutes and then dehydrated in alcohol concentrations of 70% (2 minutes), 90% (2 minutes), 100% (4 minutes), 90% (2 minutes), and 70% (2 minutes). After dehydration, the slides were immersed in dH2O for 30 seconds and then stained in Cresyl Violet solution for 2 minutes. After staining, the sections were rinsed 3–4 times in glacial acetic acid and then dehydrated 10 times in 95%, 100%, and 100% alcohol concentrations. After placing a drop of Permount on one end of the dehydrated, stained tissue, the sections were covered with a coverslip and dried at room temperature for 1 day before being imaged. The stained tissue slides were photographed using a Zeiss LSM880 (Jena, Germany) microscope. Each tissue slide was photographed at 0.5x magnification with a 100x lens.

[0107]

[0108] [Experimental Example 1]

[0109] After extracting hematopoietic cells from the bone marrow of mice, they were cultured for 10 days with M-CSF (Macrophage colony stimulating factor) to differentiate them into macrophages. After one day, the medium was replaced with fresh medium and cultured for 72 hours. After 72 hours, the macrophage culture medium was collected, treated with primary cultured cerebral cortical neurons, and after 48 hours, images of neurons were obtained through Tuj-1 antibody staining, which labels neurons. The degree of neurotoxicity in the acquired images was quantified using the Degeneration Index (DI) to observe the neurotoxicity of neurons caused by the macrophage culture medium. For comparison, a condition in which the culture medium was not treated was used as a control.

[0110] Figure 1 shows the results of confirming neurotoxicity-induced neuronal cell death when a macrophage culture solution according to one experimental example of the present invention is applied to neurons.

[0111] Referring to Figure 1, it was observed that neurotoxicity increased when macrophage culture was treated.

[0112] [Experimental Example 2]

[0113] In a study in which the inventors of this institute participated, it was reported that when macrophages co-cultured with dorsal root ganglion sensory neurons were treated with cyclic AMP (cAMP), and the culture medium obtained was treated to neurons, the culture medium enhanced the regenerative capacity of the neurons. In order to confirm the degree of neurotoxicity of macrophages with regenerative capacity, dorsal root ganglion sensory neurons were first cultured, and macrophages differentiated for 4 hours were cultured for 24 hours. After that, the culture medium was replaced and the cells were cultured again for 72 hours. The culture medium was then treated to cerebral cortex cells, and after 48 hours, images of the neurons were obtained through antibody staining using the same method as in Figure 1, and the degree of neurotoxicity was quantified.

[0114] Figure 2 shows the results of confirming the reduction in neurotoxicity when the culture solution of neural cells and co-cultured macrophages according to one experimental example of the present invention is treated to neural cells.

[0115] Referring to Figure 2, it was observed that the neurotoxicity of the culture medium of macrophages co-cultured with neurons was significantly reduced compared to that of macrophages cultured alone.

[0116] [Experimental Example 3]

[0117] To determine differences in gene expression in macrophages cultured alone or co-cultured with neurons, RNA-sequencing capable of analyzing the entire cellular genome was performed. Macrophage culture conditions were the same as in Experimental Examples 1 and 2.

[0118] Cultured macrophages were lysed with Qiazol (Qiagen, Dusseldorf, Germany), and then centrifuged after mixing 20% ​​of the Qizaol volume in chloroform. The supernatant was separated, and 1 ml of isopropanol and yeast tRNA were treated and incubated at low temperature for 1 hour. After incubation, centrifugation was performed for 30 minutes. The precipitated RNA was washed with a 70% ethanol solution and dried at room temperature for 5 minutes. The dried RNA was diluted in RNase-free water and subjected to RNA-sequencing. The fastaq files obtained after RNA-sequencing were used to isolate differentially expressed genes (DEGs) using the DESeq2 program in the R program. Differentially expressed genes were analyzed using Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Assay (GSEA) to identify genes that were increased in macrophages co-cultured with neurons and their associated signaling mechanisms.

[0119] Figure 3a shows the results of analyzing the increased genome in co-cultured macrophages using IPA (Ingenuity Pathway Analysis) and confirming the increase in cell cycle-related mechanisms.

[0120] Figure 3b shows the results of cytoscape analysis of the network of genes increased in co-cultured macrophages, confirming that cell cycle-related genes are included in the core proteins of the network.

[0121] Figure 3c shows the results of analyzing the increased genome in co-cultured macrophages using the GSEA database, confirming an increase in cell cycle-related mechanisms.

[0122] Figure 3d shows the results of the GSEA database analysis, which confirmed that many genes are related to the cell cycle mechanism.

[0123] Figure 3e shows the results of PCR analysis confirming that cell cycle genes are increased in co-cultured macrophages in transcriptome analysis.

[0124] Referring to Figures 3a to 3e, the analysis results confirmed that cell cycle-related genes and mechanisms increased in neural cells and co-cultured macrophages.

[0125] [Experimental Example 4]

[0126] Among cell cycle genes that regulate neurotoxicity, research has reported that AURK (Aurora Kinase) gene expression is suppressed by cell cycle inhibitors, and that inhibition of the AURK gene alters the phenotype of macrophages. Therefore, RNA-seq analysis was performed based on Experimental Example 3.

[0127] Figure 4a shows the results of observing the expression of AURKA, AURKB, and AURKC in the transcriptome results.

[0128] Figure 4b shows the results of PCR observation showing that the expression of AURKA and AURKB increased in co-cultured macrophages.

[0129] Figure 4c is the result of quantitative analysis of the PCR results performed in Figure 4b using Image J, and this confirms the increase in expression of AURKA and AURKB in co-cultured macrophages.

[0130] Figure 4d shows the results of Western blot observation showing that the expression of AURKA and AURKB increased in co-cultured macrophages.

[0131] Figure 4e is the result of quantitative analysis of the PCR results performed in Figure 4d using Image J, and through this, it is confirmed that the expression of AURKA and AURKB increased in co-cultured macrophages.

[0132] Figure 4f shows the results of PCR observation showing that the expression of AURKA and AURKB, which was increased in co-cultured macrophages, decreased when treated with a cell cycle inhibitor.

[0133] Figure 4g is the result of quantifying the PCR results performed in Figure 4f using Image J.

[0134]

[0135] Figure 4h is a Western blot result showing that the expression of AURKA and AURKB, which was increased in co-cultured macrophages, decreased when treated with a cell cycle inhibitor.

[0136] Figure 4i is the result of quantitative analysis of the Western blot results performed in Figure 4h using Image J.

[0137] Referring to Figures 4a to 4i, it was confirmed that the expression of AURKA (Aurora Kinase A) and AURKB (Aurora Kinase B) among the AURK family members increased in macrophages when co-cultured with neurons and macrophages through PCR (Polymerase chain reaction) to confirm gene expression of cells and Western blot to confirm protein expression. In addition, it was observed that the expression of AURKA and AURKB decreased when treated with a cell cycle inhibitor.

[0138] [Experimental Example 5]

[0139] To determine whether inhibition of the functions of the cell cycle regulatory genes AURKA and AURKB results in changes in the neurotoxicity of macrophages, we treated cells with inhibitors specific for AURKA and AURKB, respectively, and then examined changes in neurotoxicity.

[0140] After co-culture of neurons and macrophages, inhibitors were treated and cultured for 24 hours. After replacing with fresh medium, the culture was continued for 72 hours. The culture medium obtained after culture was treated with cerebral cortical neurons to quantify the degree of neuronal damage, and it was confirmed that the neurotoxicity of macrophages increased when treated with an AURK inhibitor. Conversely, to observe whether neurotoxicity changed when AURK expression was increased, a plasmid containing the AURK gene was injected into macrophages and changes in neurotoxicity were observed. First, the plasmid containing the AURKA or AURKB gene and the injection solution (reagent) interferin were mixed and left at room temperature for 10 minutes, and then treated with macrophages. After 4 hours of treatment, the medium was replaced with fresh medium and cultured for 72 hours.

[0141] Figure 5a shows the results of confirming that neurotoxicity increased when the culture medium was secured after suppressing the expression of AURKA or AURKB in macrophages using siRNA and then treating the culture medium with nerve cells.

[0142] Figure 5b shows the results of confirming that neurotoxicity was reduced when the culture medium was secured after injecting a plasmid containing the AURKA or AURKB sequence into macrophages that were not co-cultured (macrophages cultured alone) to overexpress AURKA and AURKB, respectively, and then treating the culture medium to nerve cells.

[0143] Figure 5c shows the result of calculating the DI (Degeneration Index) after quantification based on the image obtained in Figure 5a.

[0144] Figure 5d shows the result of calculating the DI (Degeneration Index) after quantification based on the image obtained in Figure 5b.

[0145] Referring to Figures 5a to 5d, when the culture medium of cultured macrophages was treated with cerebral cortical neurons and the degree of neuronal damage was quantified, it was observed that when the expression of AURKA or AURKB was increased, neurotoxicity was reduced compared to macrophages that did not increase gene expression.

[0146] [Experimental Example 6]

[0147] After inducing a photothrombotic stroke model, tissue immunostaining was performed on day 7, when macrophage-induced degeneration increased, to confirm damage. To confirm the expression of AURKA and AURKB after stroke induction, tissues were collected from rats that had not been induced with photothrombotic stroke and rats that had been induced with photothrombotic stroke. In particular, in the induced rats, tissues were collected from the injured area (Ipsi) and the contralateral area (Contra).

[0148] Figure 6a is a schematic diagram illustrating the process of inducing photothrombotic stroke in a rat. This implies that damage can be observed 7 days after stroke induction in rats.

[0149] Figure 6b shows the results of observing the brain of a mouse 7 days later, confirming that damage had occurred (indicated by a red circle).

[0150] Figure 6c shows the results of confirming that a lesion was formed through CV (Cresyl Violet) staining after cutting the tissue.

[0151] Figure 6d shows the results of Western blot using tissues obtained from the Sham, Stroke Contra, and Stroke ipsi sites 7 days after injury, and observing that AURKA and AURKB expression was reduced at the injury site.

[0152] Referring to Figures 6a to 6d, tissues were collected and the amount of protein was analyzed, and it was confirmed that the expression of AURKA and AURKB was reduced in the damaged area.

[0153]

[0154] The above description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0155] The scope of the present invention is indicated by the claims described below rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. A method for inhibiting macrophage neurotoxicity, comprising a step of increasing the expression of Aurora Kinase in macrophages.

2. In paragraph 1, The above Aurora kinase comprises a selected from the group consisting of AURKA, AURKB and combinations thereof. Methods for inhibiting macrophage neurotoxicity.

3. In paragraph 1, The step of increasing the above expression is: Including co-culturing the above macrophages with neural cells, Methods for inhibiting macrophage neurotoxicity.

4. In paragraph 3, The above neurons include those selected from the group consisting of dorsal root ganglion sensory neurons, hippocampal neurons, dopaminergic neurons, motor neurons, cerebellar granule cells, thalamic neurons, spinal neurons, and combinations thereof. Methods for inhibiting macrophage neurotoxicity.

5. Containing an active substance that increases the expression of aurora kinase, A pharmaceutical composition for inhibiting nerve tissue degeneration.

6. In paragraph 5, The above Aurora kinase comprises a selected from the group consisting of AURKA, AURKB and combinations thereof. A pharmaceutical composition for inhibiting nerve tissue degeneration.

7. Containing Aurora phosphorylation enzyme as an active ingredient, Biomarkers for diagnosing neurodegenerative diseases.

8. In paragraph 7, The above Aurora kinase comprises a selected from the group consisting of AURKA, AURKB and combinations thereof. Biomarkers for diagnosing neurodegenerative diseases.

9. In paragraph 7, If a downregulation of the expression level of the above aurora kinase is observed, it is judged to be a neurodegenerative disease. Biomarkers for diagnosing neurodegenerative diseases.

10. In paragraph 7, The above neurodegenerative disease includes a disease selected from the group consisting of stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease and combinations thereof. Biomarkers for diagnosing neurodegenerative diseases.

11. In paragraph 10, The above neurodegenerative disease includes stroke, Biomarkers for diagnosing neurodegenerative diseases.

12. A kit for diagnosing a neurodegenerative disease, comprising a biomarker for diagnosing a neurodegenerative disease according to any one of claims 7 to 11.

13. Step of treating candidate substances to macrophages; A step of measuring the expression level of aurora kinase of the above macrophages; and A step of selecting the candidate substance as a macrophage neurotoxicity inhibitor when the expression of the above aurora kinase is increased; including, A method for screening inhibitors of macrophage neurotoxicity.

14. In paragraph 13, The above Aurora kinase comprises a selected from the group consisting of AURKA, AURKB and combinations thereof. A method for screening inhibitors of macrophage neurotoxicity.

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