KR102187922B1 - Novel therapeutic agent for treating Alzheimer's disease - Google Patents

Abstract

The present invention relates to an effective prevention and treatment method for diabetic Alzheimer's disease, and more specifically, provides a pharmaceutical composition for the treatment of diabetic Alzheimer's disease using an inhibitor of expression or activity of stress-associated endoplasmic reticulum protein 1 (SERP1) as an active ingredient. do.

Description

Novel therapeutic agent for treating Alzheimer's disease

The present invention relates to a novel Alzheimer's disease treatment, and more particularly, to a novel pharmaceutical composition for treating diabetic Alzheimer's disease and a screening method thereof based on the interaction of SERP1 and γ-secretase.

Alzheimer's disease (AD) or Alzheimer's dementia is a progressive neurodegenerative disorder and is the most common form of dementia (Querfurth and LaFerla, N. Engl. J. Med. 362(4): 329-344, 2010). Two major pathological features of Alzheimer's disease are amyloid plaques consisting of amyloid beta (Aβ) peptides (Hardy and Selkoe, Science 297(5580): 353-356, 2002) and phosphorylated tau proteins (Ballatore et al ., Nat. Rev. Neurosci. 8(9): 663-672, 2007) is the presence of neurofibrillary tangles. Neurotoxic Aβ peptides are derived from amyloid beta precursor protein (APP) through sequential cleavage by β-site amyloid precursor proteolytic protein (BACE1) and γ-secretase. γ-secretase is a rate-limiting enzyme, presenillin 1 or 2 (PS1 or PS2), nicastrin (NCT), anterior pharyngeal-defective type 1 (APH-1) and presenillin enhancer 2 (PEN2) consists of four subunits (Roberson and Mucke, Science 314(5800): 781-784, 2006; De Strooper et al ., Nature 398(6727): 518-522, 1999). Among them, NCT serves as a substrate receptor for APP and Notch, and APH-1 serves as a support linking PS and NCT (Periz and Fortini, J. Neurosci. Res . 77(3):309-322, 2004). To date, additional proteins such as TMP21, GPR3 ????, and β-arrestin have been identified as regulators of γ-secretase (Chen et al ., Nature 440(7088): 1208-1212, 2006; Thathiah et al. ., Science 323(5916): 946-951, 2009; Liu et al ., Cell Res. 23(3): 351-365, 2013), The in vivo role of these proteins in the pathogenesis of AD in vivo has not yet been elucidated. . In addition, the presence of several substrates of γ-secretase is a factor limiting the therapeutic development of γ-secretase inhibitors (De Strooper et al ., Nat. Rev. Neurol . 6(2):99-107, 2010 ). Therefore, the identification of novel APP processing-preferred modulators of γ-secretase is important for drug development in AD.

Meanwhile, it is known that the activity of γ-secretase is regulated by many physiological and pathological signals, including hypoxia, growth factor signaling, reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress (Li et al. ., Neurobiol.Aging 30(7): 1091-1098, 2009; Matrone et al ., Proc. Natl. Acad. Sci. USA 105(35): 13139-13144, 2008; Ohta et al ., Biochem. Biophys. Res. Commun. 416(3-4): 362-366, 2011; Shen et al ., J. Biol. Chem. 283(25): 17721-17730, 2008; Wang et al ., FASEB J. 20(8 ): 1275-1277, 2006). In particular, ER stress caused by aging, environmental factors or genetic mutations is associated with neurodegenerative disorders including Alzheimer's disease. Metabolic diseases such as obesity and diabetes are also important risk factors for the later onset of Alzheimer's disease (Reitz et al ., Nat. Rev. Neurol . 7(3): 137-152, 2011; Frisardi et al ., Aging Res. Rev. 9(4): 399-417, 2010; Sims-Robinson et al ., Nat. Rev. Neurol. , 6(10): 551-559, 2010; Takeda et al ., Proc. Natl. Acad. Sci. US A 107(15): 7036-7041, 2010). Epidemiologic studies show that people with diabetes have a 1.5 to 2.5 times higher risk of Alzheimer's disease than people without diabetes (Ninomiya, Curr. Diab. Rep . 14: 487, 2014). AD model mice with diabetes (Takeda et al ., Proc. Natl. Acad. Sci. US A 107(15): 7036-7041, 2010) or AD model mice in which insulin resistance was induced by diet (Ho et al. ., FASEB J. 18(7): 902-904, 2004), Aβ production by high blood glucose levels known as hyperglycemia, insulin resistance associated with ER stress (Nagai et al ., Invest. Ophthalmol. Vis. Sci . , 57(3): 1408-1417, 2016; Tharp et al ., Obesity (Silver Spring) 24(7): 1471-1479, 2016) and Aβ deposition was found to increase. However, the molecular mechanisms of AD etiology associated with ER stress and diabetes are still unknown.

However, although ALK has been applied as a target treatment for the prevention of cancer, its role in neurodegenerative and AD pathology has not been identified yet, and therefore, the development of more efficient Alzheimer's disease treatments is delayed.

Accordingly, the present invention is to solve various problems including the above problems, and the interaction between SERP1 and γ-secretase has a close correlation with the pathology of diabetic Alzheimer's disease, and the mechanism and effect of Alzheimer's It is an object of the invention to present the possibility of developing a therapeutic agent for disease. However, these problems are exemplary, and the scope of the present invention is not limited thereby.

According to one aspect of the present invention, there is provided a pharmaceutical composition for treating diabetic Alzheimer's disease, comprising an inhibitor of expression or activity of stress-associated endoplasmic reticulum protein 1 (SERP1) as an active ingredient.

According to another aspect of the present invention, there is provided a method for screening a therapeutic agent for diabetic Alzheimer's disease, comprising the step of searching for a compound and/or natural product that inhibits the expression or activity of SERP1 protein.

According to another aspect of the present invention, there is provided a method for preventing and treating diabetic Alzheimer's disease, comprising administering to an individual a SERP1 inhibitor that inhibits the expression or activity of the SERP1 protein in a therapeutically effective amount.

1A is a graph showing the results of ELISA analysis showing that SERP1 stimulates the activity of γ-secretase in relation to the production of Aβ, and FIG. 1B is a graph of Aβ by treatment with shRNA that specifically inhibits the expression of SERP1. It is a graph showing the result of ELISA analysis indicating that production is reduced, and FIG. 1C is a pre-immune serum (Pre) or anti-Aβ antibody (4G8) for the cell culture solution and cell lysate of cells transfected with pSERP1-HA plasmid. It is a photograph showing the result of performing Western blot analysis after immunoprecipitation. FIG. 1D is a control or pSERP1-HA transfected HEK293T cell by Comp. E is a graph showing the result of analyzing γ-secretase activity using a fluorescent substrate when cultured in the presence or absence of E. FIG. 1e is a control shRNA (shCtrl) or SERP1-specific shRNA-transfected HEK293T cells from Comp. It is a graph showing the result of analyzing γ-secretase activity using a fluorescent substrate when cultured in the presence or absence of E.
Figure 2a is a wild-type (WT) transfected with the SC100 expression construct SC100-GFP or SERP1 knocked out (SERP1 KO) mouse embryonic fibroblasts (MEF) Comp. E (10 nM) is a fluorescence micrograph of a fluorescence signal taken by GFP when cultured for 24 hours in the presence or absence of E (10 nM), Figure 2b is a truncated Notch expression construct, NΔE-GFP transfected wild type (WT) or SERP1 is sufficient Out (SERP1 KO) mouse embryonic fibroblasts (MEF) were transferred to Comp. E (10 nM) is a fluorescence micrograph photographing a fluorescence signal by GFP when cultured for 24 hours in the presence or absence of E (10 nM), Figure 2c is the MEF cells of Figures 2a and 2b transfected with the SERP1 construct, Comp . A Western blot analysis result (left) measuring the degree of production of NICD when cells are cultured in the presence or absence of E for 24 hours, and a graph quantifying it (right), and FIG. 2D is a variety of genes ( Hes1 , Hey1 , Tcf4 , Actb and Serp1 ) is a limb (left) showing the result of analyzing the expression level at the mRNA level by RT-PCT, and a graph that quantified it (right).
3A is a photograph showing the results of analyzing the expression levels of subunits constituting γ-secretase in HEK293T cells overexpressed in control or SERP1 by Western blot, and FIG. 3B is a view of FIG. 3A. It is a graph showing the RT-PCR result of measuring the expression of the subunit constituting γ-secretase in cells at the mRNA level, and FIG. 3C is a graph showing the APH1A/NCT subcomplex and γ in HEK293T cells transfected with the pSERP1-HA construct. -After developing the amount of secretase complex by blue native (BN) PAGE (top) and SDS-PAGE (bottom), a photograph showing the result measured by Western blot analysis (left) and a graph that quantified it (right) 3D shows the amount of APH1A/NCT subcomplex and γ-secretase complex in HEK293T cells transfected with the psgSERP1 construct and knocked out SERP1.Blue native (BN) PAGE (top) and SDS-PAGE (bottom ), a photograph showing the result measured by Western blot analysis (left) and a graph (right) quantifying it, and FIG. 3E is a control (Control) or PS1/2 double knockout MEF transfected with pSERP1-HA Digitonin-soluble membrane fraction of cells was developed by BN-PAGE, and then the amount of APH1A/NCT subcomplex and secretase complex was analyzed by Western blot.FIG. 3F is a control (shCtrl) or specific to SERP1. This is a photograph showing the results of Western blot analysis of the expression level of subunits constituting γ-citritase in HeLa cells knocked down with an appropriate shRNA, and FIG. 3G is a control or psgSERP1 transfected with SERP1 The Digitonin-soluble membrane fraction of this knocked out PS1/2 double knockout (DKO) MEF cell was developed by BN-PAGE, and then the amount of APH1A/NCT subcomplex and secretase complex was analyzed by Western blot. It's a picture.
Figure 4a is a graph showing the result of Western blot analysis after immunoprecipitation using an anti-SERP1 antibody or IgG for lysate of mouse cortical tissue, and Figure 4b is a graph showing the result of performing a Western blot analysis of HEK293T cell lysate using anti-SERP1 antibody or IgG. It is a graph showing the results of Western blot analysis after immunoprecipitation using, and FIG. 4C is a γ-sheet after developing membrane fractions recovered from wild-type (WT) and PS1/2 double knockout MEF cells by blue native-PAGE. A photograph showing the results of Western blot analysis using an antibody specific to the subunit constituting the retainase, and FIG. 4D is a glycerol rate for the microsomal membrane fraction solubilized with CHAPS-lysis buffer for the cells of FIG. 4B. After performing the gradient fraction, a photograph showing the results of Western blot analysis on each obtained fraction, and FIG. 4E is γ-secretase components in which the SERP1 protein purified from E. coli is present in mouse brain lysates. In order to analyze whether it interacts with or not, a photograph showing the result of confirming it by Western blot analysis after performing a protein overlay assay, and FIG. 4F is 1 hour with 1% digitonin or 1% Triton X-100 The crude membrane fraction recovered from the PS1/2 double knockout MEF cells lysed during was developed by blue native-PAGE, and then Western blot analysis was performed using an anti-NCT antibody to determine the APH1/NCT subcomplex. It is a photograph showing the result of confirming the degree of production, FIG. 4G is a schematic diagram for schematically explaining the experimental result of FIG. 4F, and FIG. 4H is of HEK293T cells cotransfected with pSERP1-HA and/or pAPH-1A-FLAG. Figure 4i is a photograph showing the result of immunoprecipitation analysis of the lysate, and Figure 4i is a photograph showing the result of immunoprecipitation analysis of the lysate of HEK293T cells cotransfected with pSERP1-HA and/or pNCT-V5, Figure 4j PS1-m HEK293T cells transfected with yc or SERP1-HA are solubilized with 1% CHAPS or 1% Triton X-100 lysis buffer, and then immunoprecipitation analysis is performed.FIG. 4K is a photograph showing the results of pSERP1-RFP and pAPH1-GFP cons. This is a fluorescence microscope photograph of the intracellular locations of SERP1 and APH1-A of HeLa cells cotransfected with trucking with a confocal microscope (bar: 10 μm).
Figure 5a is a variety of SERP1 and SEC61G, which is a topological analogue thereof, to confirm the part involved in the interaction with SERP1 and γ-secretase and its subcomplex, APH1/NCT subcomplex, according to an embodiment of the present invention. It is a schematic diagram schematically showing the structure of the variant, and FIG. 5B is a photograph showing the results of immunoprecipitation analysis on HEK293T cells transfected with various gene constructs and pNCT-V5 shown in FIG. 5A, and FIG. 5C Is a fluorescence micrograph showing the BiFC analysis result showing the interaction between SERP1 and NCT in HeLa cells cotransfected with pSERP1-HA and pNCT-V5, and FIG. 5D is a PS1/2 DKO MEF overexpressing SERP1 wild type or SERP1ΔC. A photograph showing the result of Western blot analysis after developing the membrane fraction recovered from the cells by BN-PAGE.FIG. 5E is a HEK293T cell cotransfected with pC99-Gv and pCDNA3 (PCD), pSERP1 WT or SERP1ΔC construct It is a graph showing the result of measuring the γ-secretase activity by measuring the luciferase activity of, and FIG. 5F is a graph showing the results of measuring the γ-secretase activity of, and FIG. 5f is This is a fluorescence microscope photograph showing the result of taking a fluorescence signal under a fluorescence microscope after 12 hours elapsed.
FIG. 6A shows HEK293T cells cotransfected with pAPPswe-FLAG and pSuper-neo (shCtrl) as a control group or pshSERP1 vector as an experimental group were treated with 0.5 μM thapsigargin (Tg) for 24 hours, and then the concentration of Aβ _{40 in} the culture medium was ELISA. It is a graph showing the results of the analysis, and FIG. 6B is a graph showing the results of analyzing the concentration of Aβ ₄₂ in the cells of FIG. 5A by ELISA, and FIG. 6C is a graph showing the result of analyzing the HEK293T cells transfected with shSERP1 with pC99-GVP and pUSA-luciferase. It is a graph showing the result of measuring the luciferase reporter activity after 24 hours treatment with 1 μM thapsigargin (Tg) for 24 hours after cotransfection, and FIG. 6D is a graph showing HEK29rT-APP695 cells transfected with a control vector (shCtrl) or pshSERP1 vector. It is a photograph showing the result of performing AICD production analysis on the membrane fraction after treatment with 0.5 μM thapsigargin (Tg) or 0.5 μm tumicamycine (Tuni) for 24 hours after infection, and FIG. 6E is a control vector (shCtrl) or pshSERP1 of the HEK293T cells. This is a photograph showing the results of Western blot analysis using antibodies specific to each of the γ-secretase subunits for the membrane fractions that were treated with 0.5 μM thapsigargin (Tg) for 24 hours after transfection with the vector and recovered. , Figure 6f shows the results of Western blot analysis after separating the crude membrane fraction recovered from the control group (HeLa/shCtrl) or the experimental group (HeLa/shSERP1) treated with 0.5 μM thapsigargin (Tg) by blue native-PAGE. It's a picture.
7A shows the effect of streptozotocin administration on serum glucose concentration in mice, as carriers (Veh) or streptozotocin (STZ, 60 mg/kg) in C57BL/6 mice (4-5 heads per group) of 2 months old ) Under daily intraperitoneal administration for 19 days, and a graph showing the results of measuring serum glucose concentration on days 12 and 19 from the administration of streptozotocin, and FIG. 7b is a graph showing the results of SERP1 in the hippocampus and cerebral cortex of the diabetic model mouse of FIG. 7A. It is a photograph showing the result of measuring the expression level through Western blot analysis, and FIG. 7C is a graph showing the result of quantifying the result of FIG. 7B by using a densitometer analysis and normalization using a β-actin signal, and FIG. 7D is This is a photograph showing the results of confirming the degree of formation of the γ-secretase complex in the brain of the STZ-induced diabetes model mouse by Western blot analysis using antibodies specific for each component of the γ-secretase complex, FIG. 7E Is a graph showing the result of quantification of the expression level of presenilin 1 (PS1) in the brain of the experimental animal of FIG. 7D through normalization using a β-actin signal, and FIG. 7F is a graph showing the result of quantification of the glucose (Glc) concentration. This is a photograph showing the difference in the expression level of each component of the SERP1 and γ-secretase complex in cultured SH-SY5Y cells after developing the cell lysate by SDS-PAGE and analyzing the result by Western blot analysis. 7G is a photograph showing the result of measuring the amount of the γ-secretase complex by analyzing the cell lysate of FIG. 7F by BN-PAGE and then analyzing it by Western blot analysis.
8A is a high concentration lentivirus expressing shSERP1 or shCtrl (control) in the hippocampus of a 2-month-old 3x Tg-AD mouse was injected intraventrally, and a carrier (Veh.) or streptozotocin (SZT) was intraperitoneally administered daily for 19 days. This is a graph showing the result of measuring the blood glucose concentration of the animal causing diabetes, and FIG. 8B is a graph showing the result of analyzing the level of Aβ ₄₀ by ELISA in the hippocampus extract of the experimental animal of FIG. 8A, and FIG. 8C is 8A is a graph showing the results of analyzing the level of Aβ ₄₂ by ELISA in the hippocampus extract of the experimental animal of FIG. 8A, and FIG. 8D is a digitonin collected from diabetic Alzheimer's disease model mice (STZ) and control mice (Veh.)- A photograph showing the result of Western blot analysis after developing the soluble membrane fraction by BN-PAGE and SDS-PAGE. FIG. 8E is a Western blot analysis result confirming the expression of SERP1 measured in the hippocampal tissue of an AD patient (top) And a graph showing the quantification of the relative expression level of SERP1 from the Western blot analysis, and FIG. 8F is a photograph showing the result of confirming the expression level of SERP1 in the parietal lobe of an AD patient in the Braak VI stage by Western blot analysis, and FIG. 8G is 8F is a graph quantified with a concentration analyzer, and FIG. 8H is a fluorescence micrograph showing immunohistochemical detection of SERP1 and APH1A in the hippocampus of an AD patient.

Detailed description of the invention:

Hereinafter, the present invention will be described in more detail.

According to one aspect of the present invention, there is provided a pharmaceutical composition for treating diabetic Alzheimer's disease, comprising an inhibitor of expression or activity of stress-associated endoplasmic reticulum protein 1 (SERP1) as an active ingredient.

In the composition, the SERP1 expression or activity inhibitor may be an anti-SERP1 antisense oligonucleotide, siRNA, shRNA or miRNA that specifically inhibits the expression of SERP1, and optionally, the SERP1 expression or activity inhibitor is specific for SERP1. It may be an antagonistic antibody, an aptamer, or an inactive SERP1 analog that binds to, and the inactive SERP1 analog may be a peptide fragment containing a 4 amino acid residue at the C-terminal of SERP1.

The composition can be administered orally or parenterally during clinical administration, and when administered parenterally, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or intrathoracic injection It can be administered by, and can be used in the form of a general pharmaceutical formulation.

The therapeutic agent according to an embodiment of the present invention further includes an inactive ingredient including a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to a composition, specifically, a component of a pharmaceutical composition other than an active substance. Examples of pharmaceutically acceptable carriers include binders, disintegrants, diluents, fillers, lubricants, solubilizers or emulsifiers and salts.

In practical use, the composition according to an embodiment of the present invention may be combined with a pharmaceutical carrier according to conventional pharmaceutical formulation techniques. The carrier can take a wide variety of forms, depending on the preparation desired, for example for oral or parenteral administration (including intravenous administration).

In addition, the composition according to an embodiment of the present invention may be administered at a dose of 0.1 mg/kg to 1 g/kg, more preferably 0.1 mg/kg to 500 mg/kg. Meanwhile, the dosage may be appropriately adjusted according to the patient's age, sex, and condition.

According to another aspect of the present invention, there is provided a method for screening a therapeutic agent for diabetic Alzheimer's disease, comprising the step of searching for a compound and/or natural product that inhibits the expression or activity of SERP1 protein.

In the screening method, the compound and/or natural product that inhibits the activity of the SERP1 protein is a candidate compound and/or a natural product in a sample containing a SERP1 protein and a SERP1 interactor consisting of APH1A, NCT and APH1A/NCT subcomplexes. Processing; And it can be selected through the step of selecting a candidate compound and / or natural product that significantly inhibits the interaction between the SERP1 protein and the SERP1 interactor compared to the control group not treated with the candidate compound and / or natural product.

In the screening method, the compound and/or the natural product that inhibits the expression of the SERP1 protein is treated with a candidate compound and/or a natural product to cells expressing the SERP1 protein; And it may be selected through the step of selecting a candidate compound and / or natural products that significantly inhibited the expression of the SERP1 protein in the cell compared to the control.

According to another aspect of the present invention, there is provided a method for preventing and treating diabetic Alzheimer's disease, comprising administering to an individual a SERP1 inhibitor that inhibits the expression or activity of the SERP1 protein in a therapeutically effective amount.

In order to identify important genetic factors regulating γ-secretase activity under stressful and pathological conditions, the present inventors have developed a γ-secretase activity-based cell assay and cDNA expression library encoding thousands of membrane proteins. Was used to perform genome-scale functional screening. In the function acquisition screen, we isolated a single transmembrane protein, stress-related endoplasmic reticulum protein 1 (SERP1) as a modulator of γ-secretase activity. The function of SERP1 is not well known except for some roles in ER stress and protein modification in the liver. The present inventors confirmed that SERP1 interacts with the γ-secretase subcomplex to promote γ-secretase assembly and stimulate the processing of APP, but not the Notch. In particular, SERP1 was upregulated in the brain of AD patients and was essential for γ-secretase activation and Aβ production in diabetic AD model mice. Therefore, the present inventors confirmed the present invention by confirming that SERP1 is a γ-secretase modulator that confers substrate selectivity and is an important target molecule for the treatment of diabetic Alzheimer's disease, as a deficient ring linking ER stress with γ-secretase. Completed.

Hereinafter, the present invention will be described in more detail through examples.

General method

ELISA analysis

According to the manufacturer's instructions, Aβ in the condition culture medium or brain extract of HEK/APPsw and SH-SY5Y/APPsw cells was measured using a sandwich ELISA kit using anti-Aβ antibodies (Invitrogen and IBL).

Antibody and Western Blot Analysis

In the present invention, the following antibodies were used:

Anti-Tubulin antibody (Sigma-Aldrich, T6074), anti-Actin antibody (Sigma-Aldrich, A1978), anti-HA antibody (HA hybridoma), anti-FLAG antibody (Sigma-Aldrich, S7425), anti-GFP antibody ( Santa Cruz Biotechnology, sc-8334), anti-V5 antibody (Sigma-Aldrich, S2540), anti-Aβ antibody (4G8, GTX109283, Genetax) anti-BIP antibody (Santa Cruz Biotechnology, sc-33757), anti-CAV1 antibody (Abcam, ab2910), anti-PS1-NTF antibody (Santa Cruz Biotechnology, sc-8760), anti-APP-CTF antibody (Sigma-Aldrich, A8717), anti-NCT antibody (Sigma-Aldrich, N1660), anti- NICD antibody (Novus, NB200-251), anti-Tim23 antibody (BD bioscience, 611222), anti-CANX antibody (Santa Cruz Biotechnology, sc-11397), anti-APH-1A antibody and anti-PEN2 antibody (kindly Japanese Donated by Dr. A. Takashima of RIKEN BSI). Anti-SERP1 antibody (produced by the present inventors) was prepared by performing standard immunization procedures using the SERP1 protein purified from E. coli. Western blot analysis was performed using standard techniques. Cells were lysed with sample buffer (10% glycerol, 2% SDS, 62.5 mM Tris-HCl and 2% β-mercaptoethanol, pH 6.8). For the preparation of membrane proteins, cells were lysed in 1% CHAPS buffer containing proteinase inhibitor cocktail and centrifuged at 10,000 xg for 10 minutes. After separating the aqueous supernatant using SDS-PAGE, the separated protein was transferred with a Bio-Rad semi-dry transfer unit (Bio-Rad), and then Western blot analysis was performed on the transferred membrane. And visualized with an improved chemiluminescence method.

Cell culture and DNA transfection

SY5Y-APPswe cells and HEK-APP695 cells are as previously reported (Gwon et al ., Aging cell 11(4): 559-568, 2012; Mitsuishi et al ., J. Biol. Chem . 285(20) : 14920-14931, 2010). HEK293T, HeLa, PS 1/2 DKO MEF, CHO-7PA2 cells and stable transfected cells were added to Dulbecco's with 10% fetal bovine serum (HyClone, SH30919.03) and 50 μg/ml Gentamicin (GIBCO, 15750-060) Modified Eagle Medium (DMEM, HyClone, SH30243.01) and 37 ℃, 5% CO ₂ (v / v) was cultured in conditions. HeLa/shCtrl and HeLa/shSERP1 stable transfected cells were selected with 2 mg/ml G418 (Gold Bio, G418-5) and maintained at 1 mg/ml G418 for the experiment. SERP1 KO MEF cells were selected with 1 μg/ml puromycin. According to the manufacturer's instructions, transfection was performed using Polyfect (Qiagen, 1015586) or PEI (Sigma, 764647).

Luciferase reporter γ-secretase activity assay

Luciferase reporter γ-secretase activity assay was performed as previously reported (Han et al ., Cell. Mol. Life Sci ., 71(13): 2561-2576, 2014). HEK293T cells were cotransfected with experimental control genes with or without pC99-GVP, pUAS-luciferase, pβ-galactosidase, and 1 μM Compound E. After 24 hours, the cell extract was analyzed by Luciferase Reporter according to the manufacturer's instructions (Promega, USA). Luciferase activity was normalized to β-galactosidase activity for transfection efficiency.

Glycerol velocity gradient fractionation

HEK293T cells were homogenized with homogenization buffer (5 mM HEPES pH 7.4, 1 mM EDTA, 250 mM sucrose and proteinase inhibitor cocktail), and the supernatant was prepared by centrifugation at 800 xg. After centrifugation at 4° C. for 1 hour at a rate of 100,000 xg, the pellet was solubilized with CHAPS buffer (2% CHAPS, 50 mM Tris pH 7.5, 2 mM EDTA and 150 mM NaCl). The soluble lysate was centrifuged again at 100,000 xg for 30 minutes and the supernatant was subjected to glycerol gradient centrifugation. The same amount of protein extract was loaded on top of a 10-40% (w/v) straight-chain glycerol gradient and centrifuged at 4° C. for 15 hours at a rate of 100,000 xg using a Beckman SW32.1Ti rotor. Each fraction was collected from the top to the bottom of the gradient, and the same volume of each fraction was analyzed by Western blot analysis.

Protein overlap analysis

Protein overay analysis was performed as previously reported (Nah et al ., Autophagy 9(12): 2009-2021, 2013). SERP1 protein (10 μg) expressed and purified in E. coli was analyzed by SDS-PAGE and transferred to a nitrocellulose membrane. After blocking, the membrane was incubated at 4° C. for 12 hours with total brain lysate of 3 month old C57BL/6 mice. Then, Western blot analysis was performed on the membrane.

Mouse brain extraction

Cortex and hippocampus from C57BL/6J mice and 3 xTg-AD mice were sonicated in ice-cold Tris-buffered saline containing 20 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1% NP40, and proteinase inhibitor. . The homogenate was centrifuged at 4°C to make it transparent, dispensed, and stored at -70°C. For analysis, 20 μg of the supernatant was used for quantification of Aβ by ELISA Kit (IBL), and SDS-PAGE and Western blot analysis were performed. For BN-PAGE analysis, mouse brains were sonicated in ice-cold PBS, and then the crude membrane fraction was centrifuged at 4° C. to obtain a pellet, which was analyzed by blue native (BN)-PAGE.

Human brain sample preparation

At Harvard Brain Tissue Resource Center (McLean Hospital, Boston), AD (Braak V-VI) patients (71-93 years old, 2 to 16 hours post-mortem) and hippocampal tissues of age-matched controls were provided. Hippocampal tissues of AD patients were homogenized in ice-cold Tris buffered saline containing 20 mM Tris-Cl (pH 7.4), 150 mM NaCl, and proteinase inhibitor. The homogenate was centrifuged at 4°C to make it transparent, aliquoted and stored at -70°C, and the supernatant was analyzed by SDS-PAGE.

Immunohistochemistry

Immunohistochemical analysis of brain sections of patients with Alzheimer's disease (AD) has been previously reported (Park et al ., Hum. Mol. Genet ., 21(12): 2725-2737, 2012). The hippocampus of the AD patient was fixed for 48 hours before slicing to the continuous coronal plane under paraformaldehyde and sliced using a frozen microtome. The flake was placed on the slide and refilled with 4% paraformaldehyde. Samples were incubated for 20 minutes in 0.1% Triton X-100 and 1 hour in 1% BSA before the primary antibody was applied. Blocked slices were incubated with anti-SERP1 (1:200) and anti-APH1A (1:300) antibodies, washed, and then reacted with FITC-conjugated and TRITC-conjugated secondary antibodies (Jackson Laboratory, Inc.) Made it. The sample was washed and observed under a Zeiss LSM700 microscope.

Analysis of AICD production in vitro

In vitro AICD (amyloid precursor protein intracellular domain) production analysis was performed with minor modifications as previously reported (Tesco et al ., J. Nneurochem. 95(2): 446-456, 2005). Specifically, harvested cells were lysed by sonication in buffer A (50 mM HEPES pH 7.4, 150 mM NaCl, 5 mM 1,10-phenathroline monohydrate, 2 mM EDTA, and proteinase inhibitor cocktail). The homogenate was centrifuged at 1,000 xg for 10 minutes, and the supernatant was centrifuged at 10,000 xg for 30 minutes. The membrane fraction of the pellet was washed once with buffer A. The final membrane pellet was resuspended in buffer A and the protein level of the sample was measured using Bradford assay (Biorad). The same amount of protein was reacted with fluorescent C99 substrate (Invitrogen) or purified C99-FLAG protein at 37° C. for 12 hours.

BN-PAGE

BN-PAGE was performed as previously reported (Thathiah et al ., Science 323(5916): 946-951, 2009). Membrane proteins were solubilized in BN-PAGE buffer (1% digitonin, 20% glycerol and 25 mM Bis-Tris pH 7.0) on ice for 60 minutes and centrifuged at 100,000 xg for 30 minutes. The remaining supernatant was used for BN-PAGE. The same volume of protein was separated by BN-PAGE at 4° C. and transferred to a PVDF membrane. The transferred blot was washed in a destaining solution (distilled water:methanol:acetic acid=6:3:1) for 15 minutes, and Western blot analysis was performed.

Immunoprecipitation assay

A typical immunoprecipitation (IP) assay is 1% Triton X-100 buffer (50 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate and proteinase inhibitor cocktail). This was done using. Immunoprecipitation of the γ-secretase protein complex was performed in 1% CHAPS buffer (25 mM HEPES pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% CHAPS and protease inhibitor cocktail) or 1% digitonin buffer (25 mM HEPES pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% digitonin and protease inhibitor cocktail). Cell lysates were prepared by sonication in Triton X-100, digitonin or CHAPS buffer. After short centrifugation, the supernatant was reacted overnight at 4° C. with anti-HA, anti-GFP, anti-SEP1, anti-PS1 or anti-APH1A antibodies, and then pulled down into Protein-G Sepharose beads (Ge Healthcare). .

STZ induction in diabetes

Diabetes was controlled by daily administration of Streptozotocin (STZ, 0.1M citrate buffer, 60 mg/kg body weight, Sigma-Aldrich) to male C57BL/6J mice and 3x Tg-AD mice (8-12 weeks old) by intraperitoneal injection for 5 days. Induced. Control mice were injected with buffer only. The glucose concentration was measured with a glucose meter (Accu-Check Instant, Boehringer Mannheim Corporation), and the blood glucose value was measured 7 days after STZ administration. Mice with blood glucose values of 300 mg/dl or more were considered to have diabetes. After the blood glucose measurement was performed again at the time of brain excision, which is the 19th day after the first STZ administration, the mice were sacrificed and the brain was excised and analyzed. Animal experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Seoul National University (SNU IACUC).

Statistical analysis

All experiments were performed in 3 parallel runs and repeated at least 3 times. Statistical analysis was performed using Microsoft Office 2016 Excel software package (Microsoft Corporation). Mean values were compared using an independent sample t-test.

Example 1: Genome-scale gene screening

In order to identify important genetic factors regulating γ-secretase activity under stress conditions and pathological conditions, the present inventors developed γ-secretase activity-based cellular assays and cDNA expression libraries encoding thousands of membrane proteins. Genome-scale functional screening was performed as previously reported using (Han et al ., Cell. Mol. Life Sci . 71(13): 2561-2576, 2014). Specifically, HEK293T cells were cotransfected with pC99-TetOn, pTRE-GFP, and monomeric RFP vector and control vector (pCtrl) or each cDNA for 18 hours. Then, it was treated with 100 ng/ml deoxycycline (Sigma-Aldrich, USA) for 12 hours. AICD-rtTA is produced by endogenous γ-secretase and then transported to the nucleus to induce GFP expression. Presumed positive cDNA clones that significantly increased green fluorescence were isolated under a fluorescence microscope (Olympus). The cDNA encoding the membrane protein was generated using RT-PCR and subcloned into a mammalian expression vector or commercially available one was purchased (ORIGENE, Inc). After the first screening, the second screening was performed using pC99-GVP and pUAS-luciferase reporter assays. The cDNA library was prepared as previously reported (Shim et al ., Cell Rep . 2(3): 603-615, 2012; Lee et al ., Hum. Mol. Genet . 21(1): 101-114 , 2012).

Through the gain-of-function screening, the present inventors isolated stress-assosiated endoplasmic reticulum protein 1 (SERP1), a single transmembrane protein, as a modulator of γ-secretase activity. .

Example 2: Analysis of the relationship between SERP1 and γ-secretase activity

In order to investigate whether the expression of SERP1 affects Aβ production, the present inventors measured the levels of Aβ ₄₀ and Aβ ₄₂ secreted from SH-SY5Y-APP _swe cells expressing the Swedish mutant form of APP (APP _swe ). I did. As a result, corresponding to the results of the functional screening of Example 1, the ectopic expression of SERP1 significantly increased the levels of Aβ ₄₀ and Aβ ₄₂ by about 30 to 50% compared to the control (condition culture medium) (Fig. 1a). Conversely, when SERP1 expression was knocked down, the levels of Aβ ₄₀ and Aβ ₄₂ were reduced (FIG. 1B). For SERP1 expression knockdown, the target sequence was cloned into a pSuper-Neo vector (pshRNA, OligoEngine) to prepare a SERP1-specific shRNA expression vector pSuper-neo-shSERP1 (hereinafter, abbreviated as'shSERP1'or'pshSERP1'). The sequence of preparation of pSuper-neo-shSERP1 is as follows: Forward primer (5'-GAT CCC CGC ACT TAG CTT AAA TTA CAT TCA AGA GAT ATT TAA GCT AAG TGC TTT TTA-3', SEQ ID NO: 1) and reverse oligo After synthesizing a nucleotide (5'-AGC TTA AAA AGC ACT TAG CTT AAA TTA CAT CTC TTG AAT GTA ATT TAA GCT AAG TGC GGG-3, SEQ ID NO: 2), it was mixed and then ligated to pSuper-Neo vector. I did. The genetic knock-out of SERP1 in cells was performed using the CRISPR system. Specifically, for SERP1 sgRNA, the target sequence (forward: 5'-CAC CGC TTG GCG ACG TTG CCG CGC T-3' (SEQ ID NO: 3), reverse: 5'-AAA CAG CGC GGC AAC GTC GCC AAG C-3' (SEQ ID NO: 4) was cloned into lentiCRISPR v2 vector (Addgene, # 52961).

When a similar assay was _{carried out} in CHO-7PA2 cells expressing APP _swe (Walsh et al ., Nature 416(6880): 535-539, 2002), Western blot results showed that SERP1 expression was determined by the total Aβ level in the medium. It increased by 70%, but the expression of full-length APP, α-secretase-cleavage products of APP, sAPPα and C38 (α-APP-CTF) was not affected (FIG. 1C). These results suggest that SERP1 stimulates the production of Aβ in cultured cells.

Next, the present inventors performed an in vitro AICD (amyloid precursor protein intracellular domain) generation analysis to investigate whether SERP1 affects the enzyme activity of γ-secretase. As a result, as a result of performing an in vitro enzyme analysis using a fluorescent substrate, it was found that the enzyme activity of γ-secretase increased by 50% upon overexpression of SERP1 (FIG. 1D). As a result of investigating the enzyme activity in the microsome membrane fraction using the purified C99 substrate, a rapid increase in AICD production was confirmed by treatment with Compound E, a γ-secretase inhibitor, when SERP1 was overexpressed (FIG. 1A). Conversely, when SERP1 expression was knocked down, γ-secretase activity was reduced by 30% (Fig. 1e). Consistently, as a result of an in vitro AICD production assay, it was shown that AICD production was decreased upon SERP1 knockdown in HEK-APP695 cells (FIG. 1B ). Overall, these results indicate that SERP1 promotes APP processing through an increase in γ-secretase activity.

Example 3: Analysis of the effect of SERP1 on the formation of NICD by γ-secretase

Since SERP1 increases γ-secretase activity to stimulate Aβ production, the present inventors investigated whether SERP1 influences the cleavage of other substrates of γ-secretase. In particular, the present inventors investigated the effect of γ-secretase on the Notch cleavage. Cytoplasmic pattern analysis of GFP-tagged SC100 (SC100-GFP) under a fluorescence microscope, as previously reported (He et al ., Nature 467(7311): 95-98, 2010), AICD-GFP was wild-type (WT ) It was confirmed that the mouse embryonic fibroblasts (MEFs) were distributed in a diffusion pattern (FIG. 2A ). On the other hand, SC100-GFP was observed in a number of small vesicles in the Serp1 knockout MEF, and the same pattern was observed even after suppressing γ-secretase with Compound E (FIG. 2A). Compared with the pattern of SC100-GFP, NICD-GFP generated from NotchΔE-GFP was accumulated in the nucleus of wild-type MEF, as previously observed (Liu et al ., Cell Res. 23(3): 351-365, 2013) This pattern did not change in Serp1 knockout MEF (Fig. 2b). This means that Notch is processed by γ-secretase regardless of the SERP1 level. In fact, as a result of Western blot analysis, it was confirmed that SERP1 overexpression reduced the production of NICD (notch intracellular domain) by about 30% (Figure 2c). In addition, the present inventors confirmed that the mRNA level of the NICD downstream genes such as Hes1, Hey1, and Tcf4 was also reduced by SERP1 expression (FIG. 2D). Thus, the possibility remains that SERP1 negatively regulates Notch's γ-secretase processing.

Example 4: Effect of SERP1 on the stability of APH1A/NCT subcomplexes

To investigate the molecular mechanisms underlying SERP1-mediated stimulation of γ-secretase activity, the expression level of each subunit in the γ-secretase complex was investigated by Western blot analysis. Interestingly, the inventors have found that the levels of APH1A and NCT increase after SERP1 overexpression (Figure 3a). Subsequently, RT-PCR analysis was performed to confirm whether the increase in the γ-secretase subunit by SERP1 is due to transcriptional regulation. As a result, it was found that SERP1 expression did not significantly affect the mRNA level of the γ-secretase subunit (Fig. 3b). In addition, as a result of measuring the level of the γ-secretase complex using blue-native (BN)-PAGE analysis, the level of mature γ-secretase complex of about 440 kDa was significantly up to about 50% by SERP1 expression. It was found to increase (Fig. 3c). Conversely, when SERP1 was genetically knocked out, the amount of mature γ-secretase complex (FIG. 3D) and γ-secretase constituent (FIG. 3e) was significantly reduced. These results indicate that SERP1 regulates the level of the γ-secretase complex.

From the above results, the present inventors confirmed the need to further elucidate the effect of SERP1 on the formation of a γ-secretase subcomplex in PS1/2 double knockout (PS1/2 DKO) MEF. Interestingly, as a result of BN gel analysis, the intracellular level of the APH1A/NCT subcomplex of about 240 kDa rapidly increased due to SERP1 overexpression (Fig. 3F), but decreased due to SERP1 knockout in PS1/2 DKO MEF (Fig. 3G). . When these results are considered comprehensively, it can be seen that SERP1 increases the level of the γ-secretase complex by controlling the amount of the APH1A/NCT subcomplex.

Example 5: Analysis of correlation with γ-secretase through APH1A/NCT subcomplex of SERP1

The present inventors investigated whether SERP1 physically interacts with γ-secretase in order to study how SERP1 modulates the formation of γ-secretase complexes. To this end, the present inventors specifically performed an immunoprecipitation analysis, and as a result, the γ-secretase subunit was combined with SERP1 in HEK293T cells transfected with a cerebral cortex extract of mouse brain (Fig. 4a) and SERP1 construct (Fig. 4b). It was confirmed that immunoprecipitation was performed. In addition, SERP1 is BN- with a γ-secretase preenzyme complex of about 440 kDa in wild-type (WT) MEF and APH1A/NCT subcomplex of about 240 kDa in PS1/2 DKO (double knockout) MEF (Fig. 4c). It was co-moved onto the gel. Through the glycerol rate gradient fraction analysis of the cell extract, the present inventors also found that SERP1 was also detected in the fraction overlapping the APH1A/NCT subcomplex (5-7) and the fraction containing the γ-secretase complex (8-9). It was confirmed (Fig. 4d). In addition, it was confirmed that the purified His-tagged SERP1 protein interacts with the γ-secretase component in mouse brain lysates through protein overlap analysis (Fig. 4e). These results indicate that SERP1 physically interacts with γ-secretase through the APH1/NCT subcomplex.

Next, the present inventors analyzed the APH1/NCT subunit that interacts with SERP1 using a detergent. As a result of BN-PAGE analysis, the APH1A/NCT subcomplex could be detected in the 1% CHAPS/digitonin soluble fraction, but not detected in the 1% Trition X-100 soluble fraction (Fig. 4f), which was found in the APH1A/NCT subcomplex by detergent. It indicates dissociation (FIG. 4G). As a result of immunoprecipitation analysis performed under the conditions of using 1% Trition X-100, it was confirmed that SERP1-HA interacts with APH1A-FLAG or NCT-V5 (Figs. 4h and 4i), which means that SERP1 interacts with APH1A and NCT. It shows that it works. As expected, SERP1-HA also interacted with PS1-NTF of the γ-secretase complex in the 1% CHAPS-soluble membrane fraction (Fig. 4j). However, this interaction was not observed in the 1% Triton-X100 soluble fraction. In addition, the present inventors observed colocalization of SERP1-RFP and APH1A-GFP in HeLa cells (FIG. 4K). Therefore, the present inventors confirmed for the first time that SERP1 interacts with the APH1A/NCT subcomplex of γ-secretase.

Although the functionally conserved domain of SERP1 has not yet been identified, SERP1 has an N-terminal (residues 1-35), transmembrane region (residues 35-61) and a C-terminal lumenal domain (residues 62-66). (Favaloro et al ., J. Cell Sci . 121(11): 1832-1840, 2008. The present inventors created a deletion (△) structure of SERP1 to identify the SERP1 region involved in the interaction with NCT. Since SERP1 is too small to generate a deletion structure, a chimeric structure was generated using SEC61G having a structure similar to SERP1 30. Surprisingly, a SERP1ΔC mutant lacking C-terminal 4 residues Did not interact well with NCT-V5 in the immunoprecipitation assay (Fig. 5b) In addition, it was confirmed that the SEC61G chimera having four residues of SERP1 (SERP1ΔNTM) at the C-terminus strongly binds to the NCT. In order to do so, the present inventors have proposed bimolecular fluorescence complementation, which enables fluorescence detection of the protein-protein interaction between VN-labeled NCT (NCT-VN) and VC-tagged SERP1 (VC-SERP1) in living cells. , BiFC) analysis (Wong et al ., J. Vis. Exp . (50): 2643, 2011).As a result, when both NCT-VN and VC-SERP1 are simultaneously expressed in cells, fluorescence Fluorescence complementarity was observed under the microscope (Fig. 5c) Conversely, fluorescence was not observed in cells expressing NCT-VN and VC-SERP1ΔC. In addition, compared to SERP1 WT, the SERP1ΔC mutant strain was PS1/2 DKO MEFs (FIG. 5D ). There was little effect on the formation of γ-secretase subcomplexes and the activity of γ-secretase in HEK293 cells (Figs. 5e and 5f).These results show that the C-terminus of SERP1 interacts with NCT and γ -This suggests that it is important for the regulation of secretase activity.

Example 6: Correlation between ER stress-dependent γ-secretase activation and Aβ formation

Conventionally, SERP1 is upregulated in ER stress conditions (Yamaguchi et al ., J. Cell Biol . 147(6): 1195-1204, 1999), and it has been reported that ER stress increases γ-secretase activity. Because (Ohta et al ., Biochem. Biophys. Res. Commun. 416(3-4): 362-366, 2011), the present inventors investigated the role of SERP1 in regulating γ-secretase activity under ER stress conditions. I did. As a result of monitoring the effect of ER stress on Aβ production, it has been previously reported that when HEK-APP695 cells were treated with thapsigargin, an inhibitor of ER Ca ²⁺ channel, SERP1 expression increased and Aβ ₄₀ and Aβ ₄₂ production increased. It was confirmed that it is promoted (Fig. 6A). On the other hand, in the case of SERP1 knockdown cells, Aβ ₄₀ and Aβ ₄₂ production were not increased even under ER stress conditions (FIG. 6B). In addition, treatment of control cells with thapsigargin increased C99-GVP/UAS luciferase reporter activity, which reflects γ-secretase activity (Fig. 6c) and AICD production (Fig. 6d), showing similar results to Aβ production. , Not in SERP1 knockdown cells. Therefore, it can be seen that SERP1 is a factor necessary for Aβ production through γ-secretase activation under ER stress conditions.

In addition, as a result of Western blot analysis to confirm the level of the γ-secretase component, the level of APH1A increased when thapsigargin (Tg) was treated in the control cells, but the expression of APH1A was rather increased in the SERP1 knockdown cells (Fig. 6e). It was confirmed that it was alleviated. Under these conditions, the level of the ER protector, BIP, was not affected by the SERP1 trough. Corresponding to the increase in APH1A by ER stress, the formation of the active γ-secretase complex was also simultaneously regulated by ER stress. ER stress increased the amount of γ-secretase preenzyme (holoenzyme) in wild-type cells, but did not increase in SERP1 knockdown cells (FIG. 6F). These results show that the increase in the γ-secretase activity under ER stress conditions is due to the increase in the formation of the γ-secretase complex by SERP1.

Example 7: Effect of SERP1 on Aβ in Diabetic AD Model Mice

ER stress is an important etiology in diabetes, and it is well known that diabetes is an important risk factor in the later onset of Alzheimer's disease (Ninomiya, Curr. Diab. Rep . 14: 487, 2014; Takeda et al ., Proc. Natl. Acad. Sci. USA 107(15): 7036-7041, 2010). Accordingly, the present inventors hypothesized that SERP1 may be an important risk factor linking diabetes and AD. In order to verify this hypothesis, the expression level of SERP1 in the brain of diabetes model mice was monitored. Because hyperglycemia, a hallmark of diabetes, increases ER stress (Lakshmanan et al ., I nt. J. Biochem. Cell Biol . 45(2): 438-447, 2013; Sheikh-Ali et al ., Nutrition 26(11) -12): 1146-1150, 2010), glucose levels were measured, and the effect of SERP1 on γ-secretase in a diabetic mouse model was investigated. When streptozotocin (STZ), which exhibits specific toxicity to pancreatic β-cells, was administered to mice for 12 and 19 days, the blood glucose level of STZ-treated mice increased by about 2 times compared to the control group (Fig. 7a). As expected, SERP1 levels were very high in both the cortex and hippocampus of the brain of STZ-induced diabetic mice (FIGS. 7B and 7C ). Moreover, as a result of BN-PAGE analysis, it was confirmed that the level of the active γ-secretase complex was significantly increased in diabetic model mice (FIGS. 7D and 7E ). In addition, when cultured in high glucose medium, the level of SERP1 and γ-secretase complex increased in SH-SY5Y cells (FIGS. 7F and 7G ).

Next, the present inventors investigated the effect of SERP1 on Aβ production in STZ-induced diabetic 3x Tg-AD mice, an AD mouse model expressing APP, presenilin and tau genes. In order to knock down SERP1 expression in the mouse brain, the hippocampus of 3x Tg-AD mice was administered a non-target shRNA (shCtrl) or a lentiviral vector (shSERP1) encoding shSERP1 in both directions. When compared with the control group, the blood glucose level of the STZ-administered mice was increased by about 2 times regardless of the SERP1 level (FIG. 8A). As previously reported (Devi et al ., PLoS One 7(3): e32792, 2012; Wang et al ., Int. J. Neurosci. 124(8): 601-608, 2014; Zhou et al ., Behav.Brain Funct. 11: 24, 2015), Aβ ₄₀ and Aβ ₄₂ levels were significantly increased in STZ-administered mice to which non-target shRNA was administered (FIGS. 8B and 8C ). On the other hand, Aβ levels in the group of shSERP1-administered mice did not increase even after STZ administration. Accordingly, as a result of BN-PAGE analysis, it was found that the level of the γ-secretase complex in STZ-administered mice was also regulated depending on SERP1 (FIG. 8D). These results suggest that SERP1 contributes to the increase of γ-secretase levels and Aβ production in diabetic AD model mice.

In addition, the present inventors were able to confirm that this phenomenon also occurs in the human body through an experiment using human autopsy tissue. Specifically, it was confirmed that the expression of SERP1 in the autopsy hippocampal tissue obtained from AD patients increased by 9.6 times compared to that of non-AD patients of the same age (FIG. 8E). In addition, a 5.5-fold upregulation pattern of SERP1 was observed in the parietal lobe of AD patients at Braak VI stage (FIGS. 8F and 8G ). As a result of immunohistochemical analysis at the cellular level, APH1A levels were higher in the hippocampal tissue of AD patients compared to the control group of non-AD patients of the same age, and this increased level of APH1A is consistent with the higher level of SERP1 immune response (Fig. 8h). The above results show the pathological importance of showing the close relationship of the level of SERP1 to the onset of AD, and support the potential role of SERP1 in the onset of AD.

As described above, according to epidemiological studies, diabetes is an important risk factor for the later onset of Alzheimer's disease, and insulin plays an important role in maintaining normal brain function (Gray et al ., Diabetes 63(12): 3992-3997, 2014. ) Because insulin degrading enzyme (IDE) can degrade Aβ (Crane et al ., N. Engl. J. Med. 369(19): 1863-1864, 2013), most of the in vitro and in vivo studies The focus has been on demonstrating the correlation of insulin, IDE, and Aβ degradation with Alzheimer's disease. According to a recent study, hyperglycemia increases the risk of dementia (Qiu et al ., J. Biol. Chem . 273(49): 32730-32738, 1998), and was found to increase Aβ levels by promoting Aβ production in vitro and in vivo (Tharp et al ., Obesity (Silver Spring) 24(7): 1471-1479, 206; Macauley et al ., J. Clin. Invest . 125(6): 2463-2467, 2016). In spite of increasing evidence, the molecular mechanisms underlying the relationship between hyperglycemia and AD pathogenesis have not been identified. The present inventors believe that SERP1 induced by hyperglycemia/ER stress can be an important factor that can explain the risk of Alzheimer's disease high-risk group in diabetic patients, and therefore, inhibitors that can selectively inhibit the expression or function of SERP1, such as , By administering a SERP1-specific siRNA or shRNA or a SERP1 inactive analog containing the C-terminal 4 amino acids of SERP1, it was confirmed that Alzheimer's disease, which may be caused by hyperglycemia or ER stress, can be prevented or treated, thereby completing the present invention. I did.

The present invention has been described with reference to the above-described embodiments, but these are merely exemplary, and those of ordinary skill in the art will appreciate that various modifications and equivalent other embodiments are possible therefrom. Therefore, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims.

<110> Seoul National University R&DB Foundation <120> Novel therapeutic agent for treating Alzheimer's disease <130> PD19-5786 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> sense oliguncleotide for shSERP1 <400> 1 gatccccgca cttagcttaa attacattca agagatattt aagctaagtg cttttta 57 <210> 2 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> antisense oliguncleotide for shSERP1 <400> 2 gcttaaaaag cacttagctt aaattacatc tcttgaatgt aatttaagct aagtgcggg 59 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> target sense sequence for SERP1 sgRNA <400> 3 caccgcttgg cgacgttgcc gcgct 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> target antisense sequence for SERP1 sgRNA <400> 4 aaacagcgcg gcaacgtcgc caagc 25

Claims (7)

Anti-SERP1 antisense oligonucleotide that specifically inhibits the expression of SERP1, a stress-associated endoplasmic reticulum protein 1 (SERP1) expression inhibitor selected from the group consisting of siRNA, shRNA, and miRNA, or an antagonistic antibody that specifically binds to SERP1 , A pharmaceutical composition for the treatment of diabetic Alzheimer's disease, comprising a SERP1 activity inhibitor selected from the group consisting of an aptamer and a peptide fragment containing 4 amino acid residues at the C-terminus of SERP1 as an active ingredient. delete delete delete A method for screening a therapeutic agent for diabetic Alzheimer's disease, comprising the step of searching for a compound and/or a natural product that inhibits the expression or activity of the SERP1 protein. The method of claim 5,
The compound and/or natural product that inhibits the activity of the SERP1 protein is the SERP1 protein or a peptide fragment containing the C-terminal 4 amino acid residue of the SERP1 protein. And treating a candidate compound and/or a natural product to a sample containing a SERP1 interactor consisting of APH1A, NCT and APH1A/NCT subcomplexes. And the candidate compound and/or the peptide fragment containing the C-terminal 4 amino acid residue of the SERP1 protein or the SERP1 protein compared to the control not treated with the natural product, and a candidate that significantly inhibits the interaction between the SERP1 interactor. A method for screening a therapeutic agent for diabetic Alzheimer's disease, which is selected through the step of selecting a compound and/or a natural product. The method of claim 5,
The compound and/or natural product that inhibits the expression of the SERP1 protein is treated with a candidate compound and/or a natural product on the cell expressing the SERP1 protein; And selecting a candidate compound and/or a natural product that significantly inhibited the expression of SERP1 protein in the cell compared to the control group.

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