COMPOSITION FOR TREATING NEUROINFLAMMATORY DISEASE COMPRISING COMPLEMENT COMPONENT 8 GAMMA PROTEIN OR FRAGMENT THEREOF

Abstract

The present invention relates to a composition for treating neuroinflammatory disease comprising a complement component 8 gamma protein or a fragment thereof, and more particularly, to use for treating neuroinflammatory disease of a complement component 8 gamma protein or a fragment thereof which exhibits an effect of reducing the expression of inflammatory cytokines in microglia.

The composition of the present invention has effects of reducing Alzheimer's abnormal behavior patterns and reducing the secretion of neuroinflammatory cytokines in brain microglia and thus can be very usefully used for development of an agent for preventing or treating neuroinflammatory disease.

Claims

1-11. (canceled)

12. A method for preventing, improving or treating neuroinflammatory disease, the method comprising administering an effective dose of a composition comprising a complement component 8-gamma protein or a fragment thereof as an active ingredient to a subject in need thereof.

13. (canceled)

14. A method for preventing, improving or treating neuroinflammatory disease, the method comprising administering an effective dose of a composition comprising a vector containing a polynucleotide encoding a complement component 8-gamma protein or a fragment thereof or a cell containing the vector as an active ingredient to a subject in need thereof.

15. The method of claim 12, wherein the complement component 8-gamma protein comprises an amino acid sequence defined by SEQ ID NO: 1.

16. The method of claim 12, wherein the neuroinflammatory disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Niemann's disease, amyotrophic axonal sclerosis, multiple sclerosis, neuroblastoma, stroke, Lou Gehrig's disease, Huntington's disease, Creutzfeldt-Jakob disease, post-traumatic stress disorder, depression, schizophrenia, and spinal muscular atrophy.

17. The method of claim 12, wherein the composition inhibits neuroinflammation by inhibiting the expression of inflammatory cytokines in microglia.

18. The method of claim 12, wherein the composition is a pharmaceutical composition or a food composition.

19. The method of claim 14, wherein the vector is at least one selected from the group consisting of linear DNA, plasmid DNA and recombinant viral vectors.

20. The method of claim 19, wherein the virus is at least one selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, and lentivirus.

21. The method of claim 14, wherein the cell is at least one selected from the group consisting of hematopoietic stem cell, dendritic cell, autologous tumor cell, and established tumor cell.

22. The method of claim 14, wherein the complement component 8-gamma protein comprises an amino acid sequence defined by SEQ ID NO: 1.

23. The method of claim 14, wherein the neuroinflammatory disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Niemann's disease, amyotrophic axonal sclerosis, multiple sclerosis, neuroblastoma, stroke, Lou Gehrig's disease, Huntington's disease, Creutzfeldt-Jakob disease, post-traumatic stress disorder, depression, schizophrenia, and spinal muscular atrophy.

24. The method of claim 14, wherein the composition inhibits neuroinflammation by inhibiting the expression of inflammatory cytokines in microglia.

25. The method of claim 14, wherein the composition is a pharmaceutical composition or a food composition.

Description

DESCRIPTION OF DRAWINGS

[0073] FIG. 1A illustrates a result of measuring cytotoxicity by MTT assay after BV2 microglia were pre-treated with a recombinant C8G protein for each concentration (0.001 to 10 μg/ml) and then treated with LPS (100 ng/ml) for 24 hours, and thereafter, a production level of nitrogen oxide (NO) was measured according to Griess assay (*P<0.05 (compared to control), #P<0.05, ##P<0.01 (compared to LPS-treated group)).

[0074] FIG. 1B is a graph showing expression levels of inflammatory cytokines and NOS2 mRNA after performing conventional RT-PCR in order to confirm whether recombinant C8G changes expression levels of proinflammatory cytokines and NOS2 mRNA increased by LPS (**P<0.01, *P<0.001 (compared to control), #P<0.01, ##P<0.01 (compared to LPS-treated group)).

[0075] FIG. 1C illustrates a result of confirming a change in expression level of inflammation-inducing chemokine mRNA by the treatment of recombinant C8G in BV2 microglia by conventional RT-PCR (*P<0.05, ***P<0.001 (compared to control), #P<0.01, ##P<0.01 (compared to LPS-treated group).

[0076] FIG. 1D illustrates a result of confirming a TNF-α protein level included in a culture medium by ELISA, by collecting a cell culture medium for each time after pre-treating a recombinant C8G protein (1 μg/ml) and then treating LPS (100 ng/ml) after 2 hours in BV2 microglia (*P<0.05, **P<0.001 (compared to control), #P<0.05, ##P<0.01 (compared to LPS-treated group).

[0077] FIG. 1E illustrates a result of measuring cytotoxicity by MTT assay after astrocytes were pre-treated with a recombinant C8G protein (1 μg/ml) for 2 hours and then treated with LPS (1 μg/ml) and IFN-γ (50 units/ml) for 24 hours, and an expression level of nitrogen oxide (NO) was measured according to Griess assay.

[0078] FIG. 1F illustrates an experiment for confirming an effect of the C8G recombinant protein in a neuroinflammation model induced by injecting LPS into the ventricle. Cannulation was performed before 14 days based on the LPS injection day. The cannulation was performed by making a small hole in the skull at a position of 1.0 mm to the right and 0.3 mm to the rear based on Bregma and then to a depth of 1.5 mm. The recombinant C8G protein (1 μg/ml, 3 μl) was injected into the ventricle for 4 days from a day before LPS injection (2 μg, ventricle). A control was injected with PBS instead of LPS. After 3 days of the LPS injection, a mouse was sacrificed after a depression behavioral test (sucrose preference test).

[0079] FIG. 1G illustrates a result of staining astrocytes (GFAP) and microglia (Iba-1) using immunohistochemical staining by extracting the brain of a mouse after 72 hours of intracerebroventricular injection of LPS (Scale bar=50 μm).

[0080] FIG. 1H illustrates a result of analyzing the number of activated astrocytes (GFAP) and the number of activated microglia (Iba-1) in the hippocampus of a mouse by an ImageJ program as illustrated in FIG. 1G.

[0081] FIG. 1I is a graph showing the measurement of sucrose preference, which is a measure of a level of depression due to neuroinflammation, through a sucrose intake of a mouse after 48 hours of intracerebroventricular injection of LPS.

[0082] FIG. 2A illustrates an experiment for confirming an absence effect of C8G by intracerebroventricular injection of AAV-shRNA C8G virus and intraperitoneal injection of LPS in a mouse. The cannulation was performed by making a small hole in the skull at a position of 1.0 mm to the right and 0.3 mm to the rear based on Bregma and then to a depth of 1.5 mm. The AAV-shRNA C8G virus (1×10.sup.9 TU/ml, 1.5 μl) was injected before 7 days of LPS injection (5 mg/kg). A control was injected with AAV-shRNA Scr (Scramble) instead of the AAV-shRNA C8G virus. The mouse was sacrificed after 1 day of LPS injection.

[0083] FIG. 2B illustrates a result of confirming changes in astrocytes (GFAP) and microglia (Iba-1) activated by intracerebroventricular injection of AAV-shRNA C8G virus (1×10.sup.9 TU/ml, 1.5 μl) and intraperitoneal injection of LPS (5 mg/kg) in a mouse through immunohistochemistry in the hippocampus of the mouse (Scale bar=50 μm).

[0084] FIG. 2C is a graph showing quantification of results illustrated in FIG. 2B. The number of astrocytes (GFAP) and the number of microglia (Iba-1) activated in the hippocampus of the mouse were analyzed by an ImageJ program.

[0085] FIG. 2D illustrates a result of confirming an mRNA expression level of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) increased by LPS by extracting the hippocampus of a C8G knockdown mouse by conventional RT-PCR.

[0086] FIG. 3A is a diagram illustrating a result of measuring cytotoxicity by MTT assay after HT-22 hippocampal neurons were pre-treated with a recombinant C8G protein (1 μg/ml) for 2 hours and then treated with ADDL (2 μM) for 24 hours.

[0087] FIG. 3B illustrates an indirect toxicity experiment design. BV2 microglia were pre-treated with a recombinant C8G protein (1 μg/ml) for 2 hours, and then treated with ADDL (2 μM) and LPS (100 ng/ml) for 6 hours. Thereafter, the BV2 microglia were washed with PBS and cultured in a new serum-deprivation medium. After the treatment, conditioned media were produced for 18 hours.

[0088] FIG. 3C is a diagram illustrating a result of measuring cytotoxicity by MTT assay by treating the conditioned media produced in FIG. 3B to HT-22 hippocampal neurons for 24 hours.

[0089] FIG. 3D illustrates an experimental design for confirming an effect of C8G using an acute Alzheimer's disease animal model in which ADDL was injected into the ventricle of a mouse. Cannulation was performed before 14 days based on the ADDL injection day. The cannulation was performed by making a small hole in the skull at a position of 1.0 mm to the right and 0.3 mm to the rear based on Bregma and then to a depth of 1.5 mm A recombinant C8G protein (1 μg/ml, 3 μl) was injected into the ventricle, and ADDL (1 μM, 7.5 μl) was injected into the ventricle after one day. At this time, a control was injected with PBS instead of ADDL. The mouse was sacrificed after a behavioral experiment at 72 hours after ADDL injection.

[0090] FIG. 3E is a graph showing a result of confirming changes in activated astrocytes (GFAP) and microglia (Iba-1) after 72 hours of the intracerebroventricular injection of ADDL in a mouse through immunohistochemistry in the hippocampus of the mouse and a result of quantifying the changes.

[0091] FIG. 3F illustrates a result of confirming an mRNA expression level of pro-inflammatory cytokines (TNF-α and, IL-1β) increased by ADDL by extracting the hippocampus by Conventional RT-PCR.

[0092] FIG. 3G illustrates a result of performing a passive avoidance test (conditioned fear test) of a mouse. After a learning session (electric shock, 0.5 mA), a movement time when the mouse entered a dark room at 24 hours (1 d), 48 hours (2 d), and 72 hours (3 d) after ADDL injection was measured.

[0093] FIG. 3H illustrates a result of confirming an effect of C8G injection on a damaged spatial working memory by using an AD animal model with intracerebroventricular injection of ADDL by a Y-maze test. The spatial working memory was measured by a behavioral alternation in the Y-maze. The basic activity of the mouse was confirmed as the total number of times of passing through the Y-maze (Number of arm entries).

[0094] FIG. 3I illustrates a forced swim test performed to confirm an effect of C8G on depression-like symptoms shown in an AD animal model made by intracerebroventricular injection of ADDL.

[0095] FIG. 4A illustrates an experimental design for confirming an effect of C8G using a sporadic Alzheimer's disease animal model in which STZ was injected into the ventricle in a mouse. Cannulation was performed before 14 days based on the STZ (3 mg/kg) injection day. The cannulation was performed by making a small hole in the skull at a position of 1.0 mm to the right and 0.3 mm to the rear based on Bregma and then to a depth of 1.5 mm After 60 days after the STZ injection, a recombinant C8G protein (1 μg/ml, 3 μl) was injected into the ventricle. A control was injected with saline instead of STZ. The mouse was sacrificed after a behavioral experiment at 72 hours after first C8G injection.

[0096] FIG. 4B illustrates a result of confirming changes in activated astrocytes (GFAP) and microglia (Iba-1) after 72 hours of the intracerebroventricular injection of C8G in a STZ-induced sporadic AD mouse through immunohistochemistry in the hippocampus of the mouse and a result of quantifying the changes.

[0097] FIG. 4C illustrates results of confirming and quantifying an mRNA expression level of pro-inflammatory cytokines (TNF-α and, IL-1β) increased by STZ by extracting the hippocampus of the STZ-induced sporadic AD mouse by Conventional RT-PCR.

[0098] FIG. 4D illustrates a result of performing a passive avoidance test (conditioned fear test) of a mouse. After a learning session (electric shock, 0.5 mA), a movement time when the mouse entered a dark room at 24 hours (1 d), 48 hours (2 d), and 72 hours (3 d) after C8G injection was measured.

[0099] FIG. 4E illustrates a result of confirming an effect of C8G injection on a damaged spatial working memory by using a sporadic AD animal model with intracerebroventricular injection of STZ by a Y-maze test. The spatial working memory was measured by a behavioral alternation in the Y-maze.

[0100] FIG. 4F illustrates a result of performing a forced swim test to confirm an effect of C8G on depression-like symptoms shown in a sporadic AD animal model made by intracerebroventricular injection of STZ.

MODE FOR CARRYING OUT INVENTION

[0101] Hereinafter, the present invention will be described in detail.

[0102] However, the following Examples are just illustrative of the present invention, and the contents of the present invention are not limited to the following Examples.

[0103] Experimental Method

[0104] 1. Cell Culture

[0105] Cells used in an experiment were BV2 cells in a microglia cell line, and HT-22 cells in a hippocampal nerve cell line, and were maintained in a Dulbecco's modified Eagle medium (DMEM) supplemented with 5% bovine serum (FBS) and antibiotics. As primary cultured astrocytes, mixed neuroglia were obtained by extracting the brain from a 3-day-old mouse. After 2 weeks of culture, non-astrocytes were removed by shaking at 250 rpm.

[0106] 2. Griess Assay

[0107] The level of nitric oxide, an indicator of the inflammatory response of microglia, was measured indirectly using the amount of nitrogen dioxide (NO.sub.2—) due to the instability of nitric oxide. After 24 hours after inflammatory stimulation of microglia, 50 μl of a culture medium was transferred to a 96-well plate, and 50 μl of a Griess reagent (1% sulfanylamide/0.1% naphthylethylenediamine dihydrochloride/2% phosphoric acid) was mixed with the culture medium in the 96-well plate. The absorbance at 550 nm was measured using a plate reader. Using NaNO.sub.2 as a standard, a NO.sub.2 concentration in the culture medium was calculated.

[0108] 3. MTT Assay

[0109] In order to check the viability of the cells, the cells were treated with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and then cultured at 37° C. for 2 hours. An intracellular insoluble formazan crystal was completely dissolved in dimethyl sulfur monoxide (DMSO) and the absorbance was measured at 570 nm.

[0110] 4. Conventional RT-PCR

[0111] RNA was isolated from cells and brain tissues according to a manufacturer's protocol using a QIAzol reagent (QIAGEN, Valencia, Calif.). PCR amplification and reverse transcription were performed using a thermal cycler (Bio-Rad, Hercules, Calif.). The primers used were as follows.

TABLE-US-00002 IL-1β[NM_008361,Forward, 5′-GCA ACT GTT CCT GAA CTC-3′ (SEQ ID NO: 2) Reverse, 5′-CTC GGA GCC TGT AGT GCA-3′ (SEQ ID NO: 3)], NO52 [NM_010927, Forward, 5′-CCC TTC CGA AGT TTC TGG CAG CAG C-3′ (SEQ ID NO: 4), Reverse, 5′-GGC TGT CAG AGC CTC GTG GCT TTG G-3′ (SEQ ID NO: 5)], TNF-cc [NM_013693, Forward, 5′-CAT CTT CTC AAA ATT CGA GTG ACA A-3′ (SEQ ID NO: 6), Reverse, 5′-ACT TGG GCA GAT TGA CCT CAG-3′ (SEQ ID NO: 7)], IL-6 [NM_031168, Forward, 5′-CGG CCT TCC CTA CTT CAC AA-3′ (SEQ ID NO: 8), Reverse, 5′-TAA CGC ACT AGG TTT GCC GA-3′ (SEQ ID NO: 9)], GAPDH [NM_008084, Forward, 5′-ACC ACA GTC CAT GCC ATC AC-3′ (SEQ ID NO: 10), Reverse, 5′-TCC ACC ACC CTG TTG CTG TA-3′ (SEQ ID NO: 11)]. Cxcl10 [NM_021274, Forward, 5′-GAG AGA CAT CCC GAG CCA AC-3′ (SEQ ID NO: 12), Reverse, 5′-GAG GCT CTC TGC TGT CCA TC-3′ (SEQ ID NO: 13)], Cc12 [NM_011333, Forward, 5′-ATG CAG TTA ACG CCC CAC TC-3′ (SEQ ID NO: 14), Reverse, 5′-TAA GGC ATC ACA GTC CGA GTC-3′ (SEQ ID NO: 15)]

[0112] 5. Enzyme-Linked Immunosorbent Assay (ELISA)

[0113] In order to measure the concentration of TNF-α in the cultured cells, the concentration of TNF-α in the cells was measured according to a manufacturer's protocol using a sandwich ELISA Kit (R&D Systems). Specifically, each cell culture medium was placed in a 96-well plate coated with an antibody of anti-TNF-α (rat monoclonal anti-mouse TNF-a, 1:180) and left at 4° C. for 18 hours. Then, the cell culture medium reacted with a detection antibody (Goat biotinyl-ated polyclonal antimouse TNF-a, 1:180). Color development was performed using streptavidin-horseradish peroxidase (HRP, 1:120) and 3,3′,5,5′-tetramethylbenzidine (TMB). Finally, 2N H.sub.2SO.sub.4 was added to terminate the reaction. The absorbance at 450 nm was measured using a micro plate reader.

[0114] 6. Intracerebroventricular Drug Injection Animal Model

[0115] The mouse was anesthetized with isoflurane and then fixed on a stereoscopic instrument. The drug injection was performed by making a small hole in the skull at a position of 1.0 mm to the right and 0.3 mm to the rear based on Bregma and then to a depth of 1.5 mm LPS was injected into the ventricle in a dose of 2 μg, and an oligomeric amyloid beta mixture was injected in a dose of 7.5 μl at a concentration of 1 μM. Streptozotocin (STZ) was injected in a dose of 3.0 mg/kg. The following control mouse was treated in the same manner as those described above, except for injecting PBS.

[0116] 7 Immunohistochemistry

[0117] A C57BL/6 mouse was perfused with saline to remove the blood and then isolate the brain. The isolated brain was immersed in 4% paraformaldehyde (PFA) for 72 hours. To protect the frozen tissue, the brain was left for 72 hours with 30% sucrose diluted in 0.1M PBS, and then embedded in an optimal cutting temperature (OCT) compound and cut to a thickness of 20 μm. Thereafter, brain slices were left at room temperature for 1 hour with 0.1% Triton X-100, 1% BSA, and 5% normal donkey serum. The brain slices were cultured overnight at 4° C. with a primary antibody [anti-GFAP (1:200 dilution; DakoCytomation, Glostrup), anti-Iba-1 (1:200 dilution; Wako, Osaka) antibody], and cultured at room temperature for 2 hours with a secondary antibody (FITC-conjugated Donkey anti-rabbit IgG antibody; Jackson ImmunoResearch Laboratories, West Grove, Pa.). Thereafter, counter staining was performed using gelatin containing DAPI, and images were obtained using a microscope (Leica, DM2500). Iba-1 was used as a marker for microglia, GFAP was used as a marker for astrocytes, and DAPI was used as a marker for nuclei. The images were analyzed using an Image J program.

[0118] 8. Sucrose Preference Test

[0119] The sucrose intake of a C57BL/6 mouse was measured from 48 hours after intracerebral injection of LPS, and one bottle of water filled with a 1% sucrose solution and one bottle filled with water were replaced every 24 hours for 3 days. Sucrose preference was measured as follows: (A Sucrose weight)/(A Sucrose weight+A water weight)×100.

[0120] 9. Knockdown of C8G Gene

[0121] In order to knock down a C8G gene, a gene of a mouse shRNA C8G sequence was inserted into a pSicoR vector using a HpaI/XhoI region. After cloning, in order to prepare AAV shRNA C8G, the gene was inserted into a pAAV-MCS vector using a MluI/BglII region.

[0122] A high-concentration recombinant AAV vector was obtained from HEK293TN cells using a helper virus-free system. An Amicon ultra-15 centrifugal filter was used to concentrate the virus.

[0123] 10. Passive Avoidance Test

[0124] For a passive avoidance test, a chamber was divided into two zones (17 cm×12 cm×10 cm) of a bright chamber with an illumination and a dark chamber, and an electric grid was installed on the floor to give an electric shock. As a learning test, the mouse was adapted for 30 seconds in an illuminated chamber while the illumination was turned off, and then the illumination was turned on. When the mouse moved to the dark chamber, an electric shock was applied at 0.5 mA for 3 seconds. Thereafter, each mouse was subjected to a memory test (teat trial). The time taken to move from the illuminated chamber to the dark chamber was performed with the step-through latency limited to a maximum of 300 seconds.

[0125] 11. Y Maze Test

[0126] A Y-maze test apparatus consisted of a Y-shaped maze made of an acrylic plate (40 cm width, 3 cm length, and 12 cm height), and each maze was disposed at an angle of 120° to each other. After each maze was designated as regions A, B, and C, the mouse was placed in the middle and moved freely for 7 minutes, and then the regions into which the experimental animal entered were recorded (e.g., ABCCAB . . . ). The number and order of entering each maze were recorded to evaluate alternation behavior (%). When entering three different regions sequentially, one point (actual change, that is, in the order of ABC, BCA, CAB, etc.) was recorded. No score was not recorded if the mouse did not enter consecutively. When a next animal was tested, after removing a residual odor, the maze was thoroughly cleaned with water. The alternation behavior was calculated according to the following equation. Alternation behavior %=[(Actual alternation count)/(Total alternation count)]×100. The total alternation count was used as an indicator of exercise activity.

[0127] 12. Forced Swim Test

[0128] A vertical acrylic cylinder (height: 60 cm, diameter: 20 cm) was filled with 26° C. tap water and a C57BL/6 mouse fell into the water and then the test was performed. After observing the behavior for 6 minutes, the animal was removed from the water, dried, and returned to a mouse cage. Behavioral differences were classified as follows. (1) Not moving—It was judged that the mouse did not move when passively wetted with water, and it was meant that there was only a small movement to lift its nose above the surface. (2) Climbing—it was meant that the forefoot moved upward in and out of the water along the side of a swimming chamber. (3) Swimming—Referred to necessary active movements rather than keeping its head on the water. A non-moving time was measured.

[0129] Experimental Results

[0130] 1. C8G Protein Regulated the Microglial Activation

[0131] According to examination of the present inventors, there was no literature of reporting that C8G functioned as immunocalin in the brain. In order or the present inventors to confirm a potential effect of C8G on neuroinflammation, BV2 cells, microglia, were treated with a C8G recombinant protein for each dose, and then treated with LPS (100 ng/ml) after 2 hours. After 24 hours, nitric oxide (NO) and cell viability were measured using Greiss assay and MTT assay. As a result, C8G exhibited a remarkable inhibitory effect on the production of nitric oxide, which exhibited the microglial activation (inhibition concentration IC50=1 μg/ml, FIG. 1A).

[0132] Next, the present inventors have confirmed whether proinflammatory cytokines produced by LPS stimulation in microglia may be regulated by C8G. The BV2 cells were pre-treated with C8G (1 μg/ml) for 2 hours in the same manner as the method, and then treated with LPS (100 ng/ml). As a result of a RT-PCR test, changes in pro-inflammatory cytokines and chemokines were observed in a time-dependent manner. That is, LPS stimulation increased the production of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and NOS2) and chemokines (CCL2 and CXCL10) (FIGS. 1B and 1C), and the production of cytokines and chemokines increased by the LPS-stimulation was inhibited by C8G treatment (FIGS. 1B to 1D). However, there was no effect on the activation of astrocytes (FIG. 1E).

[0133] In order to confirm a neuroinflammation regulating effect of C8G in vivo, glial activation was measured (FIGS. 1F to 1H). The activity was measured by immunohistochemical staining. Recombinant C8G (1 μg/ml, 3 μl) was administered to a mouse injected with LPS according to a schedule shown in FIG. 1F. The morphological activation of astrocytes and microglia was remarkably increased, but the glial activation was decreased by injection of C8G (FIGS. 1G and 1H). Moreover, administration of icv-C8G alleviated behavioral patterns such as depression (sucrose preference test) (FIG. 1I).

[0134] 2. In Vivo C8G Downregulation Deteriorated Systemic Neuroinflammation Induced by LPS

[0135] To confirm the anti-inflammatory effect of the C8G protein confirmed in Experimental Result 1 again, the present inventors performed an experiment to confirm a change in neuroinflammation due to decreased expression of the C8G protein in the brain of a neuroinflammatory animal induced by intraperitoneal injection of LPS.

[0136] Before 7 days of LPS injection, icy AAV-shRNA C8G virus was injected (1×10.sup.9 TU/ml, 1.5 μl) to induce knockdown of C8G (FIG. 2A). C8G AAV-shRNA scramble (1×10.sup.9 TU/ml, 1.5 μl) was used as a control. As a result of the experiment, the knockdown of C8G further deteriorated microgliosis induced by LPS, but did not affect astrogliosis (FIGS. 2B and 2C), and the production of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 induced by LPS was further increased (FIG. 2D).

[0137] 3. C8G Inhibited Microglial Activation Induced by Oligomeric-Amyloid Beta Mixture (ADDL) and Exhibited a Neuroprotective Effect.

[0138] The present inventors then tried to confirm whether the regulation of microglia by C8G in neuroinflammation may act as a positive effect in Alzheimer's disease (AD).

[0139] Although the AD was typically a progressive neurodegenerative disease characterized by the presence of amyloid plaques and neurofibrillary tangles, there have been many studies demonstrating that neuroinflammation was also a major cause of AD. The microglial activation played an important role in maintaining homeostasis, such as cleaning up cell debris and abnormally misfolded proteins in the early stages of AD. However, when pathological stimuli persisted chronically, the microglia converted physiological and beneficial functions. Uncontrolled microglia may directly cause synaptic loss and neurotoxicity. Thus, the regulation of microglia activation in AD could be a new treatment.

[0140] In connection with this concept, the present inventors confirmed the effect of C8G on acute neurocytotoxicity of the oligomeric-Aβ mixture (ADDL) (FIG. 3A). However, the viability of HT-22 cells as a hippocampal neuronal cell line was significantly reduced by ADDL, but C8G did not protect neurons. Accordingly, the present inventors attempted to confirm whether C8G may affect indirect toxicity to hippocampal cells. In this study, a non-contact co-culture system was used. The present inventors obtained a conditioned medium (CM) from BV2 cells cultured with ADDL or LPS for 24 hours with or without C8G pretreatment. Thereafter, the neurons were cultured with the conditioned medium for 24 hours (FIGS. 3B and 3C). As a result, when HT-22 cells were cultured in a conditioned medium without containing C8G, the cell viability significantly decreased. However, the viability of cells cultured with the conditioned medium containing C8G was similar to that of the cells of the control. The result means that C8G protects neurons from indirect toxicity induced by ADDL (FIG. 3C).

[0141] Next, the present inventors examined whether C8G may prevent hippocampal neuroinflammation and cognitive impairment in an ADDL-injected mouse. C8G was applied every 4 days from the day before icv-ADDL injection through a pre-installed icy injection cannula (FIG. 3D). In the ADDL-injected mouse, reactive astrogliosis and microgliosis of the hippocampus were clearly observed, but significantly reduced by C8G injection (FIG. 3E). In addition, the level of pro-inflammatory cytokines induced by ADDL was significantly reduced in a C8G/ADDL-injected mouse (FIG. 3F).

[0142] Next, the present inventors examined whether C8G may improve severe damage to memory caused by ADDL. In this study, the present inventors performed a passive avoidance test and a Y-maze test as a hippocampal dependent behavioral test.

[0143] In the passive avoidance test (FIG. 3G), the time taken for the mouse to move to a darker part preferred to a bright illumination of a shuttle box before exposed to an electric shock, that is, the step-through latency was not significantly different in all groups. After electric shock, the step-through latency was clearly reduced in the ADDL-injected mouse group compared to a control mouse group. This means that a response in which the electric shocked mouse remembered an unpleasant experience and avoided entering the dark room was reduced to toxicity by ADDL. In contrast, the step-through latency significantly increased from 48 hours after electric shock in a C8G/ADDL-injected mouse group compared to the ADDL-injected mouse group.

[0144] A Y-maze test of measuring a spatial working memory was performed after 1 hour of the last session of the passive avoidance test (FIG. 3H). As a result, a change in decreased impaired spatial short-term memory in the ADDL-injected mouse was significantly recovered by C8G injection.

[0145] In addition, an effect of C8G on depression-like behavior induced by ADDL injection was evaluated (FIG. 3I). Compared with the control mouse, the immobility of the ADDL-injected mouse significantly increased in a forced swim test (FST). However, the increased immobility was reduced by C8G to a level similar to that of the control.

[0146] 4. C8G Alleviated Neuroinflammation and Behavioral Impairment in a Sporadic Alzheimer's Model Induced by Intracerebroventricular Injection of Streptozotocin (STZ).

[0147] In the past 20 years, transgenic mouse models produced by overexpression of genetically modified human PS1, APP and/or tau proteins have been the most used in an Alzheimer's disease study. However, these animal models have limitations without showing all the anomalies observed in human AD and showing sporadic forms of AD, which account for 99% of AD patients. Moreover, since conventional animal models exhibited acute neuropathology, there was a limitation in examining a correlation between neuroinflammation and a cause of AD.

[0148] Therefore, the present inventors used an experimental method of making a sporadic AD model by intracerebroventricular injection (icy) of streptozotocin (STZ), and tried to confirm whether injection of C8G could alleviate chronic neuroinflammation and neural death (FIG. 4A).

[0149] According to the experimental results, GFAP-positive astrocytes and Iba-1-positive microglia were significantly increased by icv-STZ and significantly decreased by C8G injection in microglia (FIG. 4B). In addition, the level of pro-inflammatory cytokines induced by STZ was significantly reduced in a STZ/C8G-injected mouse (FIG. 4C).

[0150] As a result of the passive avoidance test, the step-through latency was significantly reduced in the STZ-injected mouse group after electric shock compared to the control mouse group (FIG. 4D). However, the step-through latency increased significantly from 24 hours in the STZ+C8G-injected mouse group compared to the STZ-injected mouse group. As a result of the Y-maze test (spatial working memory), it was also confirmed that the impaired behavior shown in the STZ-injected mouse was remarkably recovered by C8G (FIG. 4E).

[0151] In addition, an effect of C8G on depression-like behavior induced by STZ injection was evaluated. Compared with the control mouse, the STZ-injected mouse significantly increased in immobility in the forced swim test (FST), while the immobility was significantly reduced by C8G (FIG. 4F).

INDUSTRIAL APPLICABILITY

[0152] According to the present invention, the composition of the present invention containing the complement component 8-gamma protein or the fragment thereof as the active ingredient has effects of reducing Alzheimer's abnormal behavior patterns and reducing the secretion of neuroinflammatory cytokines in brain microglia, and can be very usefully used for development of agents for preventing or treating neuroinflammatory disease. Therefore, the industrial applicability of the present invention is very excellent.