COMPOSITION, COMPRISING TMEM176B OR AN EXPRESSION OR ACTIVITY REGULATOR THEREOF AS AN ACTIVE INGREDIENT, FOR PREVENTION OR TREATMENT OF DEGENERATIVE BRAIN DISEASE

Abstract

This application relates to a composition for prophylaxis or treatment of a neurodegenerative brain disease, the composition including TMEM176B as an active ingredient. In one aspect, the composition not only promotes phagocytosis of amyloid-beta in astrocytes and reduces amyloid plaques, but also exhibits an effect of ameliorating behavior and cognitive functions in an animal model having Alzheimer's disease. Therefore, the composition can be usefully utilized for the prophylaxis and/or treatment of various neurodegenerative brain diseases.

Claims

1. A method of preventing, ameliorating, or treating a neurodegenerative brain disease, the method comprising: increasing the expression of transmembrane protein 176B (TMEM176B) in a subject having a neurodegenerative brain disease.

2. The method of claim 1, wherein the increasing comprises administering to the subject a therapeutically effective amount of TMEM176B, a polynucleotide encoding the TMEM176B, or an agent for enhancing activity or expression of the TMEM176B or the polynucleotide.

3. The method of claim 2, wherein the TMEM176B comprises an amino acid sequence of SEQ ID NO: 1.

4. The method of claim 1, wherein the subject is an individual in which a level of amyloid-beta or an aggregation level of amyloid-beta is higher than normal or at risk of being higher than normal, the normal not having neurodegenerative brain disease.

5. The method of claim 1, wherein the subject is an individual in which phagocytosis activity of glial cells more dysfunctional than normal or at risk of being lower than normal, the normal not having a neurodegenerative brain disease.

6. The method of claim 1, wherein the neurodegenerative brain disease is selected from the group consisting of preclinical Alzheimer's disease, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, Down syndrome, amyloid stroke, systemic amyloidosis, Dutch type amyloidosis, Niemann-Pick disease, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette's syndrome, Friedrich's ataxia, Machado-Joseph's disease, Lewy body dementia, dystonia, progressive supranuclear palsy, and frontotemporal dementia.

7. The method of claim 2, wherein the TMEM176B, the polynucleotide, or the agent increases phagocytosis of amyloid-beta by a glial cell.

8. The method of claim 7, wherein the glial cell is an ependymal cell, an oligodendrocyte, an astrocyte, or a microglial cell.

9. A method of preventing, ameliorating, or treating a neurodegenerative brain disease, the method comprising: administering to a subject in need thereof a therapeutically effective amount of a polynucleotide encoding transmembrane protein 176B (TMEM176B) or a vector comprising the polynucleotide.

10. The method of claim 9, wherein the vector is any one selected from the group consisting of a plasmid vector, a cosmid vector, a bacteriophage vector, an adenoviral vector, a retroviral vector, and an adeno-associated viral vector.

11. The method of claim 10, wherein the adeno-associated viral vector is any one selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11.

12. A method of preventing, ameliorating, or treating a neurodegenerative brain disease, the method comprising: administering to a subject in need thereof a therapeutically effective amount of a cell transformed with a vector comprising a polynucleotide encoding transmembrane protein 176B (TMEM176B).

13. The method of claim 12, wherein the cell is any one selected from the group consisting of a stem cell, a progenitor cell, and an animal cell.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0080] FIG. 1 is a graph showing results of measuring phagocytosis of astrocytes treated with TMEM176B inhibitor on FAM-labeled amyloid-beta.

[0081] FIG. 2 shows Western blot results and a graph confirming increased expression of TMEM176B in astrocytes into which a TMEM176B gene is introduced using an adeno-associated virus.

[0082] FIG. 3 is a graph confirming increased phagocytosis of astrocytes into which a TMEM176B gene is introduced using an adeno-associated virus.

[0083] FIG. 4 is a schematic diagram describing administration and evaluation methods for confirming a therapeutic effect of the TMEM176B-adeno-associated virus in a mouse model having Alzheimer's disease.

[0084] FIG. 5 is a photograph confirming reduction of plaques in the TMEM176B-adeno-associated virus administration group through amyloid-beta staining.

[0085] FIG. 6 shows graphs confirming reduction of plaques in the TMEM176B-adeno-associated virus administration group through amyloid-beta staining.

[0086] FIG. 7 is a graph confirming that the time to reach the platform is shortened in the TMEM176B-adeno-associated virus administration group through the water maze test.

[0087] FIG. 8 shows graphs confirming that the cognitive function of the TMEM176B-adeno-associated virus administration group is improved in the behavior test after removal of the platform.

DETAILED DESCRIPTION

[0088] Meanwhile, an important pathological feature of Alzheimer's, which is a type of neurodegenerative brain disease, is formation of peptide aggregates called senile plaques, wherein the senile plaques cause synaptic dysfunction and apoptosis of neurons. One of components of the senile plaques is amyloid-beta, which is 40 to 42 amino acids long. Amyloid-beta monomers are easy to self-assemble into oligomers, protofibrils, fibers rich in beta-sheets, and are related to the pathogenesis of neurotoxicity.

[0089] Although the correlation between amyloid-beta plaques and neurotoxicity has not yet been clearly defined, the self-assembly of amyloid-beta into intermediate oligomers or aggregates is considered to be related to the development of cranial nerve diseases such as Alzheimer's disease.

[0090] Meanwhile, it has been recently reported that glial cells and microglial cells have phagocytosis activity on can phagocytize amyloid-beta, which is a peptide as a major cause of Alzheimer's disease (Chung, W S, et al., Do glia drive synaptic and cognitive impairment in disease?, Nat Neurosci, 2015.18(11): p.1539-45.)

[0091] Therefore, inhibiting aggregation of amyloid-beta or disaggregating aggregates of amyloid-beta, reducing tau proteins that are abnormally hyperphosphorylated, or increasing phagocytosis activity of glial cells is suggested as a method of treating neurodegenerative brain disease such as Alzheimer's disease and Parkinson's disease.

[0092] Hereinafter, the present disclosure will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the scope of the present disclosure is not intended to be limited by these Examples. It is apparent to those skilled in the art that the these Examples below may be modified without departing from the essential gist of the disclosure.

EXAMPLE 1

Confirmation of Phagocytic Activity of Astrocyte by TMEM176B Inhibition

[0093] It was confirmed how inhibition of TMEM176B affected phagocytic activity of an astrocyte.

[0094] In detail, BAYK8644 (by Merck) was used as a TMEM176B inhibitor, a method of confirming phagocytic activity of an astrocyte (by Abm) after treatment with the inhibitor was performed as follows. The astrocyte was cultured in a 96-well black cell culture plate (by SPL) using a PriGrow IV medium containing 10% FBS (by GIBCO), penicillin/streptomycin (by Thermo), human EGF 10 ng/mL (by Abm), and L-glutamine (by Abm), and incubation thereof was started at a density of 50,000 cells per well. Here, the TMEM176B inhibitor was treated at concentrations of 0 μM, 3.703 μM, 11.11 μM, 33.33 μM, and 100 μM, and the then the cells were stabilized by incubation at 37° C. in a 5% CO.sub.2 incubator for 18 to 24 hours. Fluorescent FAM-labeled amyloid-beta was dissolved in cold PBS (by Anaspec) at a concentration of 5 μM, blocked from light, and stored in a refrigerator at 4° C. for 24 hours to prepare amyloid-beta in oligomers. After the stabilization was completed, the cells were treated with oligomeric amyloid-beta that was stored for 24 hours, and then incubated at 37° C. in a 5% CO.sub.2 incubator for 18 to 24 hours. After the incubation was completed, the culture solution was removed by suction, and 0.4% trypan blue (by GIBCO) was added thereto and incubated for 1 minute at 37° C. in a 5% CO.sub.2 incubator. After 1 minute, trypan blue was removed by suction, and the phagocytized oligomeric amyloid-beta was measured at an excitation wavelength of 485 nm/emission wavelength of 535 nm using a microplate reader capable of measuring fluorescence. After the measurement was completed, a washing process was performed thereon once with PBS, and 50 μg/ml of Hoechst Dye 33342 (by abcam) was added and incubated at 37° C. in a 5% CO.sub.2 incubator for 30 minutes to stain the nuclei. After the incubation was completed, a washing process was performed thereon once with PBS, and the stained nuclei were measured at an excitation wavelength of 360 nm/emission wavelength of 465 nm. Results thereof are shown in FIG. 1.

[0095] Consequently, as shown in FIG. 1, it was confirmed that the treatment of astrocyte with the TMEM176B inhibitor decreased the phagocytosis function on the amyloid-beta oligomer. These results indicate that, if the TMEM176B were overexpressed or the activity thereof were enhanced, there would be a therapeutic effect on neurodegenerative brain diseases including Alzheimer's disease.

EXAMPLE 2

Confirmation of Effect on Astrocyte by Enhancement of Expression or Activity of TMEM176B

[0096] By using a recombinant adeno-associated virus prepared to express a TMEM176B gene, overexpression of TMEM176B was induced in an astrocyte, and an effect of the overexpression was confirmed.

[0097] <2-1> Preparation of TMEM176B-Adeno-Associated Virus

[0098] By requesting Vigene Biosciences, a TMEM176B-adeno-associated virus was produced as a virus to which a CMV promoter and mouse-derived TMEM176B sequence were introduced using adeno-associated virus serotype 9.

[0099] <2-2> Confirmation of Expression of TMEM176B in Astrocyte Inoculated with TMEM176B-Adeno-Associated Virus

[0100] An astrocyte was inoculated on a 12-well cell culture plate (by Corning) using a PriGrow IV medium (by Abm) containing 10% FBS (by GIBCO), penicillin/streptomycin (by Thermo), and L-glutamin (by Abm), and L-glutamine (Abm) at a density of 100,000 cells per well. After 24 hours, to find out an appropriate inoculation concentration, an adeno-associated virus was inoculated thereon at concentrations of 0.5 genome copies (GC)/cell, 1 CG/cell, and 2×10.sup.6 GC/cell. 48 hours later, the infection of the cells was determined by a western blot. The western blot was performed using an anti-TMEM176B antibody (by Abclonal) and an HRP-conjugated anti-rabbit immunoglobulin G antibody (by Thermo), and results are shown in FIG. 2.

[0101] Consequently, as shown in FIG. 2, it was confirmed that the expression of the TMEM176B was approximately doubled at the inoculation concentration of 1×10.sup.6 GC/cell.

[0102] <2-3> Confirmation of Phagocytic Activity of Astrocyte with Overexpressed TMEM176B

[0103] A TMEM176B-infected astrocyte and a normal astrocyte were separately cultured into wells of a 96-well black cell culture plate (by SPL) at a density of 50,000 cells per well, the plate using PriGrow IV medium containing 10% FBS (by GIBCO), penicillin/streptomycin (by Thermo), 10 ng/mL of human EGF (by Abm), and L-glutamine (by Abm). Here, only the astrocytes inoculated with the TMEM176B-adeno-associated virus were treated with the TMEM176B inhibitor at concentrations of 0 μM, 10 μM, and 50 μM, and then stabilized by incubation at 37° C. in a 5% CO.sub.2 incubator for 18 to 24 hours. FAM-labeled amyloid-beta (by Anaspec) was dissolved in cold PBS at a concentration of 5 μM, blocked from light, and stored in a refrigerator at 4° C. for 24 hours to prepare an oligomer. After the stabilization was completed, the cells were treated with oligomeric amyloid-beta stored for 24 hours, and then incubated at 37° C. in a 5% CO.sub.2 incubator for 18 to 24 hours. After the incubation was completed, the culture solution was removed, and 0.4% trypan blue (by GIBCO) was added thereto and incubated for 1 minute at 37° C. in a 5% CO.sub.2 incubator. After 1 minute, trypan blue was removed, and the phagocytized oligomeric amyloid-beta was measured at an excitation wavelength of 485 nm/emission wavelength of 535 nm using a microplate reader capable of measuring fluorescence. After the measurement was completed, a washing process was performed thereon once with PBS, 50 μg/ml of Hoechst Dye 33342 (abcam) was added and incubated at 37° C. in a 5% CO.sub.2 incubator for 30 minutes to stain the nuclei. After the incubation was completed, a washing process was performed thereon once with PBS, and the stained nuclei were measured at an excitation wavelength of 360 nm/emission wavelength of 465 nm using a microplate reader capable of measuring fluorescence. Results thereof are shown in FIG. 3.

[0104] Consequently, as shown in FIG. 3, it was confirmed that phagocytosis was increased in the astrocytes inoculated with theTMEM176B-adeno-associated virus while phagocytosis was decreased in a concentration-dependent manner by the TMEM176B inhibitor. As the phagocytosis of amyloid-beta by the astrocytes was increased when the TMEM176B was overexpressed or the activity thereof was enhanced, the results above indicate that there is a therapeutic effect on neurodegenerative brain disease including Alzheimer's disease.

EXAMPLE 3

Confirmation of Therapeutic Effect by Administration of TMEM176B-Adeno-Associated Virus to Mice with Alzheimer's Disease

[0105] By introducing TMEM176B into an adeno-associated virus, the TMEM176B was set to be overexpressed upon in vivo infection. Mice with Alzheimer's disease were infected with the TMEM176B-adeno-associated virus, and it was confirmed whether aggregates of amyloid-beta were formed in the brain. Then, through behavior test using a water maze test, a therapeutic effect was confirmed.

[0106] <3-1> Experiment to Confirm TMEM176B Efficacy Using Alzheimer's Disease Mouse Model

[0107] APP/PS1 Alzheimer's disease mouse models purchased from Jackson Lab were used, and the breeding and experiments were conducted at KPC. The received mice were acclimatized and aged for 6 months, and then administration of a TMEM176B-adeno-associated virus was started. During the acclimatization period, the mice were each bred one per cage and marked with ear-tacks. Labels were additionally attached to the cages to distinguish each mouse. When separating groups, the unique number for each mouse was used. After the group separation and initiation of the test, an individual identification card (group information, mouse number, sex, administration period, etc.) was attached to each breeding box. Each group consisted of 5 wild-type (WT) control groups, 8 APP/PS1 control groups, and 8 APP/PS1 TMEM176B administration groups. Animals were anesthetized using isoflurane inhalation anesthetic, and the head was fixed on a stereotaxic instrument. A Hamilton syringe was inserted into the brain location (AP: −0.58, ML: −1.25, DV: −2.1) for intracerebroventricular (ICV) administration, and 5 μl of a test substance was injected at a rate of 1 μl/min. After waiting for 5 minutes to 10 minutes to allow the test substance to diffuse to the surroundings, the syringe was withdrawn. After administered once at 32nd to 34th week, the behavior test was performed 8 weeks later. As shown in FIG. 4, after administered once at the 8th month, the behavior test was performed 2 months later to confirm the efficacy of the TMEM176B.

[0108] FIG. 4 shows an experiment schedule for confirming the therapeutic effect of the TMEM176B-adeno-associated virus by using the Alzheimer's disease mouse models.

[0109] <3-2> Confirmation of Reduction in Plaques Through Amyloid-Beta Staining

[0110] After completion of the treatment and behave test, the mice were sacrificed. Then, amyloid-beta plaques were detected from the wild-type (WT) control group (G1), the APP/PS1 control group (G2), and the APP/PS1 TMEM176 administration group (G3) for analysis. In detail, after fixing the brain in a 4% PFA solution and preparing a paraffin block, the paraffin block was cut using a microtome (HM340E, Thermo, USA) to a thickness of 4 μm and then attached on a slide. The slide was deparaffinized with xylene and rehydrated with ethanol (100%-95%-80%-70%) to completely remove paraffin. After treatment with 0.03% H.sub.2O.sub.2 for blocking of endogenous peroxidase, heat-mediated antigen retrieval was performed with a Tris/EDTA pH 9.0 buffer solution. The slide was incubated with a primary antibody (6E10, 1:2000) for 1 hour and a secondary antibody (Envision anti-rabbit HRP) for 30 minutes, developed with diaminobenzidine (DAB), and then counter-stained with Mayer's Hematoxylin. The analysis was performed using a confocal microscope, and the number and area of plaques were measured. Results are shown in FIGS. 5 and 6.

[0111] As a result, as shown in FIGS. 5 and 6, in the APP/PS1 TMEM176B administration group (G3), the number of plaques decreased in the whole brain, hippocampus, and cortex.

[0112] <3-3> Confirmation of Cognitive Function Improvement Through Morris Water Maze Test

[0113] Based on the method devised by Morris, the water maze experiment was conducted. In detail, a stainless pool (a diameter of 90 cm and a height of 50 cm) was filled with water (having a temperature of 22±1° C.) to the level of 30 cm. A hidden platform (a diameter of 9 cm) was placed 1 cm below the water level. On the first day of evaluation, 3 to 4 training sessions were performed. The total swimming time per mouse was set to 60 seconds, and the mice that found the platform within 60 seconds were allowed to stay on the platform for 10 seconds to induce memory. The mice that failed to find the platform after 60 seconds were artificially guided to the platform and stayed on the platform for 10 seconds. Here, the escape time was set to 60 seconds. The acquisition test (AQ test) was conducted for 5 days, and the time to reach the platform was compared by date to verify the effectiveness of the test substance in relation to the cognitive function. On the first day, without recording the time to reach the platform, the training of reaching the platform using visual cues in four directions, i.e., east, west, north, and south directions, was conducted. For 4 days from the second day, the time to reach the platform was recorded (indicated by AQ1). Starting from each of the east, west, south, and north directions, the time to reach the platform was recorded. 4 data were recorded per mouse, and results are shown in FIG. 7.

[0114] Consequently, as shown in FIG. 7, in comparison with the G2 group by date, the G1 group showed a significant decrease in the time to reach the platform from AQ2 to AQ4 (last day) (AQ2, p=0.025; Aq3, p=0.018; Aq4, p<0.001). In addition, when comparing the G2 group with the G3 group which is the TMEM176B administration group, a significant difference was observed on the day of AQ4 (p=0.042).

[0115] Next, a probe test performed after removing the platform on the 6th day of the water maze test to measure the space perception ability (e.g., latency to target, target crossing numbers, percentage of time in SW area (%), etc.). The analysis was performed using a SMART VIDEO TRACKING Software (by Panlab, USA). A percentage of the time spent in the quadrant where the platform was located to the total swimming time (Percentage of time in SW area, %), the time required to reach a place where the platform was located (Latency to target, sec), and the number of times that the mice passed by the platform (Target Crossing, numbers) were compared. Results were calculated using a total of these three indicators, and the calculated results are shown in FIG. 8.

[0116] All data obtained in the above experiment were expressed as mean±standard error mean (SEM), and all results were analyzed by one-way ANOVA using SPSS (by IBM SPSS Statistics, Version 22). The post-hoc test was performed by the least significant difference (LSD) test.

[0117] Referring to FIG. 8, as a result of comparing the G3 group with the G2 group, the G3 group showed a significant difference in the latency to target index (p=0.012), as well as in the target crossing (p=0.040).

[0118] The results above indicate that the TMEM176B according to an embodiment promotes phagocytosis of amyloid-beta by astrocytes, reduces amyloid plaques, and exhibits an effect of improving behavioral and cognitive functions in an animal model with Alzheimer's disease, thereby showing availability for remarkable use in the treatment of neurodegenerative brain disease.