Use of GLP-1 receptor agonists in treatment of Alzheimer's disease

Abstract

The present invention provides a method of treating Alzheimer's disease by activation of AMPK with a GLP-1 receptor agonist, the method involving the use of the GLP-1 receptor agonist to activate the AMPK signaling pathway in brain cells, thereby reducing beta-secretase (BACE1) mediated APP hydrolysis and A beta protein production, and promoting phagocytosis of microglial cells, thereby reducing the AMPK signaling pathway in brain cells. Therefore, neuroinflammation is reduced and cognitive impairment is improved. The invention further provides a pharmaceutical composition containing the GLP-1 receptor agonist and a corresponding medicine preparation application.

Description

Technical Field

[0001] This application relates to the use of GLP-1 receptor agonists in the treatment of Alzheimer's disease by activating AMPK, belonging to the technical field of molecular biology. This application also relates to pharmaceutical compositions containing GLP-1 receptor agonists and corresponding treatment methods and uses for the preparation of drugs. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by β-amyloid (Aβ) deposition, abnormal phosphorylation of Tau protein, and neuroinflammation. Currently, clinical therapeutic drugs (such as cholinesterase inhibitors, etc.) can only relieve symptoms or partially remove Aβ, and cannot fundamentally block the disease progression of Alzheimer's disease.

[0003] It is known that glucagon-like peptide-1 (GLP-1) receptor agonists (GLP-1RAs) such as exenatide (Exendin-4), tirzepatide, etc., which are drugs for the treatment of type 2 diabetes mellitus (T2DM), can regulate peripheral glucose and lipid metabolism. GLP-1RA is a synthetic analogue of GLP-1, which is an intestine-derived peptide hormone and plays a crucial role in reducing blood glucose levels. This effect is achieved by stimulating insulin secretion in response to oral glucose intake, a process known as the incretin effect. Different from the native GLP-1 peptide, these synthetic analogues are designed to resist degradation by dipeptidyl peptidase 4 (DPP-4), thereby prolonging the half-life. Exenatide is a 39-amino acid agonist of the GLP-1 receptor derived from the saliva of the Gila monster (Heloderma suspectum) and has been clinically approved for the treatment of type 2 diabetes. Tirzepatide is a GIP / GLP-1 dual receptor agonist and has recently been approved by the US FDA for T2DM management.

[0004] As an anti-diabetic drug, GLP-1RA achieves blood glucose control through various metabolic and energy regulation mechanisms. A recent study has shown that GLP-1RA has a protective effect on renal tubules by enhancing AMPK signaling, further activating fatty acid oxidation, and inhibiting lipid synthesis and glycolysis. The effects of AMPK on metabolism can be divided into two main functions: inhibiting anabolic processes to reduce ATP consumption and stimulating catabolic processes to enhance ATP production. During energy stress, activated AMPK promotes glucose uptake and lipid oxidation to generate energy, while shutting down energy-consuming processes such as glucose and lipid synthesis, thereby restoring energy balance and maintaining homeostasis. However, the effects of GLP-1RA on AMPK activity and energy metabolism in brain cells are still largely unknown.

[0005] In addition to its hypoglycemic effect, GLP-1RA also regulates the release of various neurotransmitters, including serotonin, dopamine, GABA, and glutamate. Furthermore, studies have shown that GLP-1RA exhibits certain neuroprotective effects in animal models, but its specific mechanism has not been elucidated, and there is a lack of systematic research on its regulation of brain energy metabolism and its actual impact on Alzheimer's disease. In-depth study of the specific molecular mechanism by which GLP-1RA regulates central nervous system energy metabolism not only has important theoretical significance for clarifying the pathogenesis of AD, but also provides potential intervention targets and treatment strategies for the development of new AD therapeutic drugs. Summary of the Invention

[0006] The inventors of the present application have for the first time demonstrated that GLP-1RA can alleviate AD-related phenotypes by activating 5'-AMP-activated protein kinase (AMPK) signaling. In particular, the inventors found that the decrease in plasma GLP-1 levels in AD model mice was negatively correlated with the β-amyloid (Aβ) load in AD patients. Enhancing GLP-1 signaling with GLP-1RA increased CaMKK2-AMPK signaling, subsequently reducing BACE1-mediated APP hydrolysis and Aβ production. GLP-1RA also increased AMPK activity in microglia, inhibited neuroinflammation, and promoted Aβ phagocytosis. Therefore, GLP-1RA inhibited plaque formation and improved memory deficits in AD model mice. The results of the inventors' study indicate that AMPK activation mediates the effect of GLP-1RA on AD, highlighting the therapeutic potential of GLP-1RA for the treatment of AD.

[0007] Based on the above unexpected findings, the present application provides a method for treating Alzheimer's disease by activating AMPK with a GLP-1 receptor agonist, which involves using a GLP-1 receptor agonist to activate the AMPK signaling pathway in brain cells, thereby reducing β-secretase (BACE1)-mediated APP hydrolysis and the production of Aβ protein, and promoting the phagocytosis of microglia, thereby reducing neuroinflammation and improving cognitive impairment. The present application also provides a pharmaceutical composition containing a GLP-1 receptor agonist and the corresponding use of the drug preparation. Brief Description of the Drawings

[0008] Figure 1It is shown that plasma GLP-1 levels are associated with cerebral energy metabolism disorders and Aβ pathology. (A) Plasma samples were collected from wild-type (WT) and APP23 / PS45 AD transgenic mice to measure GLP-1 levels. There were 12 mice in each group (6 males and 6 females). Statistical analysis was performed using a two-tailed Student's t-test. Primary neurons were isolated from WT and APP23 AD mouse (E17) embryos and subjected to glucose uptake assay (B) and ATP production assay (C). n = 3 independent experiments. One-way ANOVA was performed, followed by Tukey's multiple comparison test for statistical analysis. (D) Immunocytochemistry was performed to measure GLUT3 expression. Images were acquired using a Zeiss Apotome. The area within the box was magnified and shown in the bottom line with intensity traces (represented by white offset lines). The scale bar represents 5 μm. Quantification of GLUT3 expression (E) and translocation (F) in neurons. n = 10 cells from 2 independent experiments. One-way ANOVA was performed, followed by Tukey's multiple comparison test for statistical analysis. Primary astrocytes isolated from WT and APP23 AD mouse (E17) embryos were used for Seahorse XF96 cell mitochondrial stress assay to measure oxygen consumption rate (G and H). n = 3 independent experiments. One-way ANOVA was performed, followed by Tukey's multiple comparison test for statistical analysis. Twelve AD patients (6 males and 6 females) underwent 18 F-AV45 PET / MR scans to evaluate cerebral amyloid burden, and their blood samples were collected to measure plasma GLP-1 levels. (I) The correlation between plasma GLP-1 levels and cerebral Aβ pathology in AD patients was analyzed. (J) 18 F-AV45 PET / MR images of the patient with the lowest plasma GLP-1 level (Patient 1: 4.22 pg / mL) and the patient with the highest plasma GLP-1 level (Patient 2: 19.23 pg / mL) are shown. All results are expressed as mean ± SEM. C.C: Compound C, Ex-4: Exendin-4, OCR: Oxygen consumption rate.

[0009] Figure 2 showed that GLP-1RA via Ca 2+CaMKK2 activation mediated by [substance] induces AMPK phosphorylation. Primary neurons isolated from wild-type (WT) mouse embryos were cultured and treated with different doses of Exendin-4 (A and B) and tildatide (C and D) for 24 h. Western blot analysis of cell lysates was performed to detect phosphorylated AMPK, phosphorylated ACC, total AMPK, and total ACC. β-Actin was detected as an internal control. n = 3 independent experiments; one-way ANOVA was performed followed by Dunnett's multiple comparison test for statistical analysis. (E and F) Primary neurons were transduced with lentiviral shRNA vectors to reduce the expression of the GLP-1 receptor and then treated with or without Exendin-4. Control shRNA lentivirus was used as a negative control. Phosphorylated AMPK, total AMPK, and GLP-1R were detected by immunoblot analysis. n = 3 independent experiments; one-way ANOVA was performed followed by Tukey's multiple comparison test for statistical analysis. (G and H) Primary WT mouse neurons were labeled with fluo-4AM to measure intracellular Ca 2+ . The green fluorescence signal was analyzed. Scale bar represents 100 μm. n = 3 independent experiments; two-tailed Student's t-test was used for statistical analysis. (I and J) Primary WT mouse neurons were treated with Exendin-4 and immunoblotted to detect CaMKK2 and LKB1 levels. n = 3 independent experiments; two-tailed Student's t-test was used for statistical analysis. (K and L) Primary WT mouse neurons were treated with Exendin-4 with or without STO609, a CaMKK2 inhibitor. Western blot analysis of cell lysates was performed to detect phosphorylated AMPK and total AMPK. β-Actin was detected using the anti-actin antibody AC-15 as an internal control. n = 3 independent experiments; one-way ANOVA was performed followed by Tukey's multiple comparison test for statistical analysis. All results are expressed as mean ± SEM. ACC: acetyl-CoA carboxylase, CaMKK2: calcium / calmodulin-dependent protein kinase kinase 2, EX-4: Exendin-4, LKB1: liver kinase B1, TZP: tildatide.

[0010] Figure 3Reduced AMPK phosphorylation decreases BACE1-mediated APP processing. (A and B) Primary WT mouse neurons were treated with different doses of Exendin-4, with or without compound C, an AMPK inhibitor. Western blot analysis was performed to detect endogenous BACE1, phosphorylated AMPK, and total AMPK levels. β-Actin was detected using the anti-actin antibody AC-15 as an internal control. n = 3 independent experiments; one-way ANOVA followed by Tukey's multiple comparison test was performed for statistical analysis. (C and D) The Swedish mutant APP stable cell line 20E2 was cultured and treated with Exendin-4, with or without compound C to inhibit AMPK activity. Full-length APP and APP CTF were detected using the C20 antibody. Endogenous BACE1 was detected using an anti-BACE1 antibody. β-Actin was detected using the anti-actin antibody AC-15 as an internal control. n = 3 independent experiments; one-way ANOVA followed by Tukey's multiple comparison test was performed for statistical analysis. Aβ ELISA was performed to detect Aβ40 (E) and Aβ42 (F) in the conditioned medium of 20E2 cells. n = 3 independent experiments; one-way ANOVA followed by Tukey's multiple comparison test was performed for statistical analysis. All results are presented as mean ± SEM. APP: amyloid-β precursor protein, Aβ: amyloid-β, BACE1: β-site APP cleaving enzyme 1, C.C: compound C, EX-4: Exendin-4.

[0011] Figure 4The AMPK regulation of BACE1 transcription depends on NF-κB p65 activity. (A) The human BACE1 promoter was transfected into N2a cells and treated with different doses of compound C or AICAR. AMPK activity regulated luciferase activity. n = 3 independent experiments; one-way ANOVA was performed, followed by Dunnett's multiple comparison test for statistical analysis. Primary mouse neurons were treated with compound C or AICAR. (B) RNA was extracted and qPCR was performed to measure the endogenous mouse Bace1 mRNA level. One-way ANOVA was performed, followed by Dunnett's multiple comparison test for statistical analysis. (C and D) Western blot analysis was performed to measure the level of endogenous BACE1 protein. n = 3 independent experiments; one-way ANOVA was performed, followed by Dunnett's multiple comparison test for statistical analysis. (E) Primary APP23 mouse neurons were treated with compound C or AICAR for 24 h and then subjected to cell fractionation. The cytoplasmic and nuclear fractions were detected by SDS-PAGE. AMPK activity regulated the level of NF-κB p65 in the nuclear and cytoplasmic fractions (F). n = 3 independent experiments; one-way ANOVA was performed, followed by Dunnett's multiple comparison test for statistical analysis. N2a cells were infected with lentiviruses carrying scrambled sequences or short hairpin RNA (shRNA) sequences targeting AMPK, with or without the NF-κB inhibitor helenalin. (G) ChIP assay was performed to detect the binding of NF-κB to the Bace1 promoter. n = 3 independent experiments; one-way ANOVA was performed, followed by Tukey's multiple comparison test for statistical analysis. All results are expressed as mean ± SEM. AICAR: 5-aminoimidazole-4-carboxamide ribonucleoside, C.C: compound C, NF-κB: nuclear factor κB.

[0012] Figure 5GLP-1RA was shown to promote microglial phagocytosis and inhibit neuroinflammation. BV2 cells were treated with Exendin-4 or tizepatide for 24 h. (A and B) Western blot analysis was performed to detect the levels of phosphorylated AMPK and total AMPK. β-actin was detected using the anti-actin antibody AC-15 as an internal control. n = 3 independent experiments; one-way ANOVA was performed followed by Dunnett's multiple comparison test for statistical analysis. BV2 cells were cultured with Aβ oligomers or pretreated with Exendin-4 and then cultured with Aβ oligomers. After RNA extraction, the samples were used for RNA-seq analysis. (C) Differentially expressed genes (DEGs) were measured using DESeq2 (version 1.40.2) with default parameters. (D–F) Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to explore biological processes. Total sample number = 6. A one-sided hypergeometric test was used, and the false discovery rate for multiple comparisons was controlled using the Benjamini–Hochberg (BH) method. BV2 cells were treated with Exendin-4 or tizepatide for 24 h. (G–I) Immunocytochemistry was performed. Images were acquired using a Zeiss microscope. Scale bars represent 20 μm. n = 6 images from 3 independent quantitative experiments. One-way ANOVA was performed followed by Dunnett's multiple comparison test for statistical analysis. (J and K) The uptake of carboxyfluorescein (FAM)-labeled Aβ42 (FAM-Aβ42) by BV2 cells was measured. Scale bars represent 20 μm. n = 8 images from 3 independent quantitative experiments. One-way ANOVA was performed followed by Dunnett's multiple comparison test for statistical analysis. All results are expressed as mean ± SEM. CD68: cluster of differentiation 68, EX-4: Exendin-4, LAMP1: lysosome-associated membrane protein 1, TZP: tizepatide.

[0013] Figure 6GLP-1RA was shown to inhibit BACE1 cleavage of APP and Aβ production in vivo. Hemibrains from Exendin-4-treated and control APP23 / PS45 mice (n = 36 in total) were homogenized in RIPA-Doc lysis buffer and separated by 8% Tris-glycine or 16% Tris-tricine SDS-PAGE. Phosphorylated AMPK and total AMPK levels (A and B) as well as full-length APP, APP CTF (C99 and C89), and endogenous BACE1 levels (C and D) were detected. β-actin was detected using anti-actin antibody AC-15 as an internal control. Statistical analysis was performed using a two-tailed Student's t-test. (E) ELISA was performed to measure Aβ40 and Aβ42 levels in the brain tissues of APP23 / PS45 mice injected or not injected with Exendin-4. n = 10 per group; statistical analysis was performed using a two-tailed Student's t-test. Total RNA was isolated from the cortex of APP23 / PS45 mice. (F) The App and Bace1 genes were amplified using a set of gene-specific primers. qPCR was performed. β-actin was used as an internal reference. n = 4 per group; statistical analysis was performed using a two-tailed Student's t-test. All results are expressed as mean ± SEM. APP: amyloid-β precursor protein, Aβ: amyloid-β, BACE1: β-site APP cleaving enzyme 1, EX-4: Exendin-4.

[0014] Figure 7Exendin-4 treatment significantly reduced neuritic plaque formation and improved memory deficits in AD transgenic mice. Neutral plaques were detected using 4G8 antibody and Thioflavin S fluorescence staining. (A) Representative brain sections of control and Exendin-4 injected APP23 / PS45 mice. White arrows point to plaques. Scale bar represents 100 μm. (B) Quantification of neuritic plaques, numbers represent mean ± SEM, n = 30 mice in total, two-tailed Student's t-test. Morris water maze test was performed (n = 36, 18 Exendin-4 treated and 18 sham treated). On the first day of the visible platform test, Exendin-4 treated and control APP23 / PS45 mice showed similar latencies (C) and swimming distances (D) to escape onto the visible platform. P>0.05, Student's t-test. In the hidden platform test, mice were trained 5 times a day for 4 days. APP23 / PS45 mice treated with Exendin-4 had shorter latencies to escape onto the hidden platform on the 3rd and 4th days (E), and shorter swim lengths before escaping onto the hidden platform on the 4th day (F). Tukey post hoc analysis was performed for statistical analysis. (G) In the probe test on the sixth day, APP23 / PS45 mice treated with Exendin-4 entered the third quadrant where the hidden platform was previously placed, significantly more than the control group. Student's t-test was performed for statistical analysis. (H) Summary graph. All results are expressed as mean ± SEM. AMPK: 5'AMP-activated protein kinase, APP: β-amyloid precursor protein, BACE1: β-site APP cleaving enzyme 1, CaMKK2: calcium / calmodulin-dependent protein kinase kinase 2, CD68: cluster of differentiation 68, EX-4: Exendin-4, FAO: fatty acid oxidation, GLP-1RA: glucagon-like peptide-1 receptor agonist, GLUT3: glucose transporter 3, IL-1β: interleukin-1β, IL-6: interleukin 6, LAMP1: lysosome-associated membrane protein 1, NF-κB: nuclear factor κB, PGC-1α: peroxisome proliferator-activated receptor γ coactivator 1α, TNF-α: tumor necrosis factor-α.

[0015] Figure 8Exendin-4 treatment was shown to restore the decreased expression of neuronal GLUT3 and PGC-1α induced by AD. Primary neurons were isolated from embryos of WT mice and APP23 AD mice (E17), and then treated with Exendin-4, with or without the AMPK inhibitor Compound C. Cell lysates were analyzed by Western blotting to detect the levels of GLUT3 (A and B) and PGC-1α (C and D). β-Actin was detected as an internal control. n = 3 independent experiments; statistical analysis was performed using one-way ANOVA. All results are expressed as mean ± SEM. C.C: Compound C. EX-4: Exendin-4, GLUT3: Glucose transporter 3, PGC-1α: Peroxisome proliferator-activated receptor γ coactivator 1α.

[0016] Figure 9 Show the effects of GLP-IRA on fatty acid oxidation in astrocytes. (A) Mitochondrial respiration of astrocytes isolated from WT mice and APP23 AD mice was measured. Astrocytes were treated with Exendin-4 and / or Compound C. Untreated cells were used as controls. The fatty acid oxidation inhibitor Etomoxir was injected before oligomycin injection for evaluation. The oxygen consumption rate (OCR) was quantitatively analyzed, including basal respiration (B), maximal respiration (C), and spare respiratory capacity (D). (E) ATP production analysis. n = 3 independent experiments; *P < 0.01, one-way ANOVA. (F and G) Primary APP23 mouse astrocytes were isolated, treated with Etomoxir to inhibit fatty acid oxidation, and then co-cultured with primary APP23 mouse neurons, with or without Exendin-4. APP CTF was detected using a C20 antibody. Endogenous BACE1 was detected using an anti-BACE1 antibody. β-Actin was detected using an anti-actin antibody AC-15 as an internal control. n = 3 independent experiments; statistical analysis was performed using one-way ANOVA. (H) Aβ40 and Aβ42 in the conditioned medium of primary neurons were detected using Aβ ELISA. n = 3 independent experiments; statistical analysis was performed using one-way ANOVA. All results are expressed as mean ± SEM. C.C: Compound C. EX-4: Exendin-4, OCR: Oxygen consumption rate.

[0017] Figure 10Shows the effect of GLP-IRA on fatty acid oxidation in astrocytes treated with palmitic acid. (A) Measurement of mitochondrial respiration in astrocytes isolated from wild-type (WT) mice and APP23 AD mice. Palmitic acid was applied to cultured astrocytes treated with Exendin-4 and / or compound C. (F) Evaluation was performed by injecting the fatty acid oxidation inhibitor Etomoxir before oligomycin. The oxygen consumption rate (OCR) was quantified, including basal respiration (B and G), maximal respiration (C and H), and spare respiratory capacity (D and I). (E and J) ATP production analysis. n = 3 independent experiments; statistical analysis was performed using one-way ANOVA. All results are expressed as mean ± SEM. C.C: Compound C. EX-4: Exendin-4, OCR: Oxygen consumption rate.

[0018] Figure 11 Shows the effect of GLP-IRA on glycolysis in AD astrocytes. (A) Determination of glycolysis in astrocytes isolated from wild-type (WT) mice and APP23 AD mice. Astrocytes were treated with Exendin-4 and / or compound C. Untreated cells were used as controls. The extracellular acidification rate (ECAR) was quantitatively analyzed, including glycolysis (B), glycolytic capacity (C), and non-glycolytic acidification (D). n = 3 independent experiments; statistical analysis was performed using one-way ANOVA. All results are expressed as mean ± SEM. C.C: Compound C. EX-4: Exendin-4, ECAR: Extracellular acidification rate.

[0019] Figure 12Shows the effects of GLP-IRA on oxidative phosphorylation and glycolysis in AD neurons. (A) Determination of mitochondrial respiration in neurons isolated from WT mice and APP23 AD mice. Neurons were treated with Exendin-4 and / or Compound C. Untreated cells served as controls. Quantitative oxygen consumption rate (OCR), including basal respiration (B), maximal respiration (C), and spare respiratory capacity (D). (E) ATP production analysis. n = 4 independent experiments; statistical analysis was performed using one-way ANOVA. (F) Determination of glycolysis in neurons isolated from WT mice and APP23 AD mice. Neurons were treated with Exendin-4 and / or Compound C. Untreated cells served as controls. Quantitative extracellular acidification rate (ECAR), including glycolysis (G), glycolytic capacity (H), and non-glycolytic acidification (I). n = 3 independent experiments; statistical analysis of the role of AMPK-mediated GLP-1RA in AD was performed using one-way ANOVA. The expression of pyruvate (J) and reactive oxygen species (K and L) was measured. Scale bar represents 20 μm. n = 9 cells from 3 independent experiments. Statistical analysis was performed using one-way ANOVA. All results are expressed as mean ± SEM. C.C: Compound C. EX-4: Exendin-4, ECAR: extracellular acidification rate, OCR: oxygen consumption rate, ROS: reactive oxygen species.

[0020] Figure 13 Shows that reduced AMPK expression alleviates the effect of Exendin-4 on BACE1-mediated APP processing. Primary APP23 mouse neurons were infected with lentiviruses carrying scrambled sequences or short hairpin RNA (shRNA) sequences targeting AMPK and treated with or without Exendin-4. (A and B) Western blot analysis was used to detect the levels of full-length APP, APP CTF, endogenous BACE1, and total AMPK. β-Actin was detected as an internal control. n = 3 independent experiments: statistical analysis was performed using one-way ANOVA. (C) Aβ40 and Aβ42 in the conditioned medium of APP23 neurons were detected by Aβ ELISA. n = 3 independent experiments; statistical analysis was performed using one-way ANOVA. All results are expressed as mean ± SEM. APP: amyloid-β precursor protein, Aβ: amyloid-β, BACE1: β-site APP cleaving enzyme 1, EX-4: Exendin-4.

[0021] Figure 14Show the effect of GLP-IRA on lysosomal protein expression. BV2 cells were treated with GLP-1RA (including Exendin-4 and tildatide). Western blot analysis of cell lysates was performed to detect the levels of CD68 (A and B) or LAMP1 (A and C). β-actin was detected with the anti-actin antibody AC-15 as an internal control. n = 3 independent experiments; one-way ANOVA was used for statistical analysis. All results are expressed as mean ± SEM. CD68: cluster of differentiation 68, EX-4: Exendin-4, LAMP1: lysosome-associated membrane protein 1, TZP: tildatide.

[0022] Figure 15 Show the effect of GLP-IRA on inflammation. BV2 cells were pretreated with Exendin-4 or tildatide and then co-cultured with Aβ oligomers for 24 hours, with or without the presence of compound C. The levels of IL-1β (A), IL-6 (B), TNF-α (C), and TGF-β (D) were measured by ELISA. n = 4 independent experiments; two-tailed Student's t-test was performed for statistical analysis. All results are expressed as mean ± SEM. C.C: compound C, EX-4: Exendin-4, IL-1β: interleukin-1β. IL-6: interleukin 6, TGF-β: transforming growth factor-β, TNF-α: tumor necrosis factor-α, TZP: tildatide.

[0023] Figure 16It is shown that AMPK inhibition can alleviate the effect of Exendin-4 on APP processing in vivo. AD mice were treated with Exendin-4 with or without compound C to inhibit AMPK activity. Mouse hemibrains were homogenized in RIPA-Doc lysis buffer and separated by 8% Tris-glycine or 16% Tris-tricine SDS-PAGE electrophoresis. The levels of full-length APP, APP CTF (C99), and endogenous BACE1 in the cortex (A and B) and hippocampus (E and F) were detected. β-actin was detected with anti-actin antibody AC-15 as an internal control. n = 3 independent experiments; statistical analysis was performed by one-way ANOVA. The levels of Aβ40 and Aβ42 in the cortex (C) and hippocampus (G) were determined by ELISA. n = 3 independent experiments; statistical analysis was performed by one-way ANOVA. Total RNA was extracted from mouse cortex and hippocampus. The App and Bacel genes were amplified using gene-specific primers. The mRNA levels of APP and BACE1 in the cortex (D) and hippocampus (H) were detected by qPCR. β-actin was used as an internal control. n = 4 per group; statistical analysis was performed by one-way ANOVA. All results are expressed as mean ± SEM. APP: amyloid-β precursor protein, Aβ: amyloid-β protein, BACE1: β-site APP cleaving enzyme 1, C.C: compound C, EX-4: Exendin-4.

[0024] Figure 17 It is shown that GLP-IRA can alleviate neuroinflammation and enhance the phagocytic function of microglia in APP23 / PS45 mice. Six-week-old APP23 / PS45 double transgenic mice were treated with Exendin-4 for 8 weeks, while age-matched control APP23 / PS45 mice were treated with vehicle solution. Mice were sacrificed after Morris water maze behavioral test, and the brain tissues were homogenized in lysis buffer or dissected, fixed, and sectioned. The levels of IL-1β (A), IL-6 (B), TNF-α (C), and TGF-β (D) were detected by ELISA. n = 4 independent experiments; statistical analysis was performed by two-tailed Student's t-test. (E) Representative brain sections of control group and APP23 / PS45 mice injected with Exendin-4 sacrificed immediately after behavioral analysis. Scale bar represents 100 μm. Squares (white dotted lines) correspond to the magnified areas in each image. (F) Quantitative analysis of CD68 expression in the brain tissues of APP23 / PS45 mice, values represent mean ± SEM, n = 30 mice, statistical analysis was performed by two-tailed Student's t-test. All results are expressed as mean ± SEM. EX-4: Exendin-4, IL-1β: interleukin-1β, IL-6: interleukin-6. TGF-β: transforming growth factor-β, TNF-α: tumor necrosis factor-α. Detailed implementation manners

[0025] The present application provides a method for treating Alzheimer's disease by activating AMPK with a GLP-1 receptor agonist. The method involves using a GLP-1 receptor agonist to activate the AMPK signaling pathway in brain cells, thereby reducing β-secretase (BACE1)-mediated hydrolysis of APP and the production of Aβ protein, and promoting phagocytosis of microglia, thereby reducing neuroinflammation and improving cognitive impairment. The present application also provides a pharmaceutical composition containing a GLP-1 receptor agonist and the corresponding pharmaceutical preparation use.

[0026] In one aspect, there is provided the use of a GLP-1 receptor agonist in the preparation of a medicament for treating Alzheimer's disease (AD).

[0027] In some embodiments, the term "GLP-1 receptor agonist" as used herein refers to a compound that fully or partially activates the human GLP-1 receptor.

[0028] In some embodiments, the term "Alzheimer's disease" as used herein is an irreversible neurodegenerative disease with Aβ and Tau pathologies at its core. The main features include: progressive decline in cognitive function (such as impaired memory, language, and executive function); pathological changes in the brain: including deposition of β-amyloid protein (Aβ) to form senile plaques; abnormal phosphorylation of Tau protein leading to neurofibrillary tangles (NFTs); neuronal loss (especially in the hippocampus and cerebral cortex); and accompanied by neuroinflammation, synaptic damage, and brain atrophy.

[0029] In some embodiments, the term "treatment" as used herein includes preventive, delaying, reducing the risk of occurrence, improving, or curative treatment for the mentioned medical indication. Treatment can be symptomatic treatment or treatment for improving the disease. In some embodiments, the term treatment as used herein refers to preventing the mentioned medical indication. In some embodiments, the term treatment as used herein refers to delaying (e.g., delaying the onset) of the mentioned medical indication. In some embodiments, the term treatment as used herein refers to reducing the risk of occurrence of the mentioned medical indication. In some embodiments, the term treatment as used herein refers to improving the mentioned medical indication. In some embodiments, the term treatment as used herein refers to curatively treating the mentioned medical indication.

[0030] In some embodiments, the GLP-1 receptor agonist treats Alzheimer's disease by activating AMPK.

[0031] In some embodiments, the term "AMPK" as used herein refers to AMP-activated protein kinase, which is a highly conserved metabolic regulatory enzyme that acts as an "energy sensor" in eukaryotic cells and is responsible for maintaining cellular energy homeostasis. It is activated under low energy conditions (such as reduced ATP, elevated AMP / ADP) and restores energy balance by regulating multiple metabolic pathways.

[0032] In some embodiments, the GLP-1 receptor agonist activates the AMPK signaling pathway in brain cells.

[0033] In some embodiments, the GLP-1 receptor agonist reduces β-secretase (BACE1)-mediated hydrolysis of amyloid precursor protein (APP) and the production of amyloid-β (Aβ) protein.

[0034] In some embodiments, the term "BACE1" as used herein, also known as β-secretase 1, is a transmembrane aspartic protease. It is a key enzyme in the pathogenesis of Alzheimer's disease and is responsible for catalyzing the cleavage of amyloid precursor protein (APP) to generate β-amyloid (Aβ), and the abnormal aggregation of Aβ is one of the main pathological features of AD.

[0035] In some embodiments, the GLP-1 receptor agonist promotes phagocytosis of microglia.

[0036] In some embodiments, the GLP-1 receptor agonist reduces neuroinflammation and / or improves cognitive impairment.

[0037] In some embodiments, the GLP-1 receptor agonist is selected from exenatide (Exendin-4), tirzepatide, lixisenatide, liraglutide, albiglutide, dulaglutide, and semaglutide.

[0038] In some embodiments, the GLP-1 receptor agonist is selected from exenatide and tirzepatide.

[0039] In one aspect, there is provided a pharmaceutical composition for the treatment of Alzheimer's disease (AD), which comprises the GLP-1 receptor agonist described herein and a pharmaceutically acceptable carrier.

[0040] In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents.

[0041] In some embodiments, the additional therapeutic agent is selected from cholinesterase inhibitors, NMDA receptor antagonists, anti-Aβ monoclonal antibodies, and Tau protein-targeting drugs.

[0042] In some embodiments, the cholinesterase inhibitor includes donepezil, rivastigmine, and galantamine.

[0043] In some embodiments, the NMDA receptor antagonist includes memantine.

[0044] In some embodiments, the anti-Aβ monoclonal antibody includes aducanumab, lecanemab, and donanemab.

[0045] In some embodiments, the Tau protein-targeting drug includes anti-Tau antibodies (such as semorinemab) and Tau aggregation inhibitors (such as TRx0237).

[0046] In some embodiments, the additional therapeutic agent is selected from AMPK activators.

[0047] In some embodiments, the AMPK activator is selected from AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) or metformin.

[0048] In one aspect, a method for treating Alzheimer's disease (AD) is provided, the method comprising administering a therapeutically effective amount of the GLP-1 receptor agonist described herein or a pharmaceutical composition comprising the GLP-1 receptor agonist described herein.

[0049] In one aspect, the GLP-1 receptor agonist described herein is provided for treating Alzheimer's disease (AD), for example, by the mechanism discovered herein.

[0050] In one aspect, the use of the GLP-1 receptor agonist described herein in the preparation of a reagent for activating the AMPK signaling pathway in brain cells is provided.

[0051] In one aspect, the use of the GLP-1 receptor agonist described herein in the preparation of a reagent for regulating BACE1 expression and / or Aβ production in vivo is provided.

[0052] Embodiment

[0053] Summary of experimental methods:

[0054] The following summarizes the experimental methods and experimental conditions / information used in the various examples of the present invention.

[0055] 1. Research Approval:

[0056] For human research, written informed consent was obtained from all subjects. This study was approved by the Ethics Committee of the First Affiliated Hospital of Dalian Medical University. Animals were cared for according to the "Guide for the Care and Use of Laboratory Animals" and the standard procedures established by the Ethics Committee of Wenzhou Medical University. All animal experimental protocols were conducted in accordance with the regulations of the Institutional Animal Care and Use Committee of Wenzhou Medical University.

[0057] 2. AD patients and 18 F-AV45 positron emission tomography:

[0058] Alzheimer's disease was diagnosed by at least two experts according to the NINCDs-ADRDA criteria. All participants underwent a comprehensive medical history and clinical neurological examination. Patients were excluded according to the following criteria: 1) clinical conditions such as diabetes, epilepsy, stroke, or long-term use of drugs known to cause progressive cognitive impairment; 2) Hachinski Ischemia Scale (HIS) > 4 points, Hamilton Anxiety Scale (HAMA) score ≥ 7 points, Hamilton Depression Scale (HAMD) score ≥ 7 points; 3) presence of severe heart, liver, lung, kidney, or other organ diseases. A total of 12 AD patients (6 males and 6 females) underwent 18 F-AV45 PET / MR scan (United Imaging, China). 18 The F-AV45 imaging agent was synthesized by the AllinOne module of Trasis, and the 18 F labeling was completed by the Nuclear Medicine Department of the First Affiliated Hospital of Dalian Medical University. The NeuroQ software (version 3.7) was used to quantitatively analyze the changes in Aβ load in each brain region.

[0059] 3. ELISA quantification of human plasma GLP-1 concentration:

[0060] 20 μL of dipeptidyl peptidase IV (DPP IV) inhibitor (Millipore sigma, Cat# DPP4-010) was added to the EDTA tube to inhibit the degradation of GLP-1. Blood samples were collected from each patient under fasting conditions and then centrifuged at 1000 g for 10 minutes. The plasma was aliquoted, immediately frozen, and stored at -80 °C. The plasma concentration of GLP-1 was measured by a human GLP-1 ELISA kit (Thermo Fisher Scientific, Cat# EH221RB).

[0061] 4. Cell culture, transfection, and treatment:

[0062] Cells were cultured at 37 °C in 5% CO 2Cultured in an incubator. HEK293 (human embryonic kidney), N2a (mouse neuroblastoma), and BV2 (mouse microglia) were maintained in high-glucose Dulbecco's modified Eagle's medium (Cytiva, Cat#SH30243.01) supplemented with 10% fetal bovine serum (Gibco, Cat#12483020) and 100 U / mL penicillin-streptomycin (Gibco, Cat#15140122). HEK cells were transfected with polyethylenimine (MedChemExpress, Cat#HY-K2014), and N2a cells were transfected with Lipofectamine 2000 reagent (Invitrogen, Cat#11668019). The 20E2 cell line is a Swedish mutant APP695-stable HEK293 cell line cultured in complete DMEM containing 50 μg / mL geneticin. For treatment, HEK cells and N2a cells were transfected in 6 cm main plates and then split into 4 x 35 mm plates 4 hours after transfection and maintained overnight (16 hours) before treatment. Primary neurons were derived from WT and APP23 mice at embryonic day 17 and cultured in Neurobasal medium (Gibco, Cat#21103049) supplemented with B27 (Gibco, Cat#17504044) and GlutaMAX (Gibco, Cat#35050061). Primary astrocytes were also derived from WT and APP23 mice at embryonic day 17 and maintained in high-glucose DMEM. The medium was enriched with 10% fetal bovine serum (FBS; Gibco, Cat#10270106). Primary neurons were infected with lentiviruses encoding sequences targeting the GLP-1 receptor (GLP-1R) or AMPK or short hairpin RNA (shRNA) sequences. Cells were treated with Aβ42 peptide (Millipore Sigma, Cat#PP69), Exendin 4 (MilliporeSigma, Cat#141758-74-9), Teprotide (Selleck, Cat#LY3298176), Compound C (Sigma-Aldrich, Cat#171260), AICAR (Sigma-Aldrich, Cat#A9978), Helenalin (MedChemExpress, Cat#HY-119970), or STO609 (MedChemExpress, Cat#HY-19805).

[0063] 5. Preparation of Aβ oligomers

[0064] First, the lyophilized Aβ42 peptide was dissolved in 100% hexafluoroisopropanol (HFIP) to a final concentration of 1 mM to monomerize the lyophilized Aβ42 peptide. Subsequently, HFIP was evaporated under vacuum using a SpeedVac, and the resulting peptide film was stored at -20 °C. For oligomeric Aβ, the peptide film was resuspended in dimethyl sulfoxide (DMSO) and then sonicated in a water bath for 10 minutes. Then the solution was diluted to a final concentration of 100 μM in cold F-12 cell medium without phenol and incubated at 4 °C for 24 hours.

[0065] 6. Luciferase assay

[0066] The BACE1 promoter construct pB1-luc (-1942 to +292) was transfected into N2a cells together with the Renilla luciferase vector pCMV-Rluc to normalize transfection efficiency. Luciferase assays were performed 48 hours after transfection using the Dual-Luciferase Reporter Assay System (Promega).

[0067] 7. Chromatin immunoprecipitation

[0068] Chromatin immunoprecipitation (ChIP) assays were performed using the Pierce™ Agarose ChIP Kit (Thermo Fisher Scientific, Cat# 26156). N2a cells were crosslinked in 1% formaldehyde (Sigma-Aldrich, Cat# 252549) and the DNA was fragmented to a length of 200 - 600 bp by enzymatic digestion. Immunoprecipitation of every 500 μL of sheared chromatin was performed overnight at 4 °C using 5 μL of monoclonal anti-NF-κB p65 (Cell Signaling Technology, Cat# 8242S) or IgG control. Input and immunoprecipitated samples were digested with proteinase K to reverse crosslinking at 65 °C for 90 minutes. DNA was purified and further analyzed by qPCR using primers specific for the BACE1 promoter.

[0069] 8. qPCR analysis

[0070] RNA was isolated using the FastPure Cell / Tissue Total RNA Isolation Kit v2 (Vazyme, Cat# RC112-01). First-strand cDNA was synthesized using the HiScript III First Strand cDNA Synthesis Kit (Vazyme, Cat# R312-01) according to the manufacturer's instructions. The cDNA was added to a reaction mixture (final volume 20 μL) containing gene-specific primers and Taq Pro Universal SYBR qPCR Premix (Vazyme, Cat# Q712). All samples were run in duplicate and using Analysis was performed on the 480 system. Relative gene expression was normalized to the actin control. Primer sequences and information are as follows:

[0071] BACE1 forward 5′-GGAACCCATCTCGGCATCC and reverse 5′-TCCGATTCCTCGTCGGTCTC;

[0072] APP forward 5′-TCCGAGAGGTGTGCTCTGAA and reverse 5′-CCACATCCGCCGTAAAAGAATG.

[0073] ACTIN forward 5'-CATTGCTGACAGGATGCAGAAGG and reverse 5'-TGCTGGAAGGTGGACAGTGAGG.

[0074] 9. Immunoblot analysis

[0075] Homogenize brain tissues or cells in RIPA lysis buffer (1% Triton X-100, 1% sodium deoxycholate, 4% SDS, 0.15 M NaCl, 0.05 M Tris-HCl, pH 7.2), and the RIPA lysis buffer is supplemented with sodium orthovanadate (200 mM), β-glycerophosphate (25 mM), sodium pyrophosphate (20 mM), sodium fluoride (30 mM), phenylmethylsulfonyl fluoride (PMSF, 1 mM), and a complete mini protease inhibitor cocktail tablet (Roche Diagnostics, catalog number #04693159001). Dilute the samples in 4× SDS sample buffer, boil, and separate on 10% or 12% Tris-glycine SDS-PAGE or 16% Tris-tricine SDS-PAGE, and then transfer to a nitrocellulose membrane. For immunoblot analysis, block the membrane in PBS containing 5% non-fat milk for 1 hour, and then incubate overnight with shaking in the primary antibody at 4 °C. Rabbit anti-APP C-terminal polyclonal antibody C20 detects APP and its CTF products. BACE1 is detected with an anti-BACE1 antibody 208 that recognizes the C-terminus (Abcam, Cat# ab183612, 1:1000). Total AMPK is determined using a rabbit anti-AMPKα antibody (Cell Signaling Technology, Cat# 2532S, 1:1000). Phosphorylated AMPK is determined using a rabbit anti-phosphorylated AMPKα (Thr172) antibody (Cell Signaling Technology, Cat# 2535S, 1:1000). NF-κB p65 subunit is determined using a mouse anti-p65 (Sigma-Aldrich, Cat# 8242S, 1:1000). GLP-1 receptor is determined using a mouse monoclonal anti-GLP-1R antibody (Santa Cruz Biotechnology, Cat# sc-390774, 1:500). CaMKK2 and LKB1 expressions are determined using a rabbit anti-CaMKK2 antibody (Cell Signaling Technology, Cat# 16810S, 1:1000) and a rabbit anti-LKB1 antibody (Thermo Fisher Scientific, Cat# PA5-PA96062, 1:500), respectively. CD68 is determined by a rabbit anti-CD68 antibody (Proteintech, Cat# 28058-1-AP, 1:1000). LAMP1 is determined by a rabbit anti-LAMP1 antibody (Cell Signaling Technology, Cat# 99437S, 1:1000).GLUT3 and PGC-1-α were determined using rabbit polyclonal anti-GLUT3 antibody (Proteintech, Cat#20403-1-AP, 1:1000) and mouse monoclonal anti-PGC-1-α antibody (Proteintech, Cat#66369-1-Ig, 1:1000). The internal control β-actin was analyzed using monoclonal antibody AC-15 (Sigma-Aldrich, Cat#A5441, 1:8000). The next day, the membranes were incubated with IRDye TM 680-labeled or IRDye TM 800CW-labeled secondary antibodies (LI-COR Biosciences) for 1 hour at room temperature. Then the membranes were visualized using a LI-COR imaging system.

[0076] 10. ELISA detection of Aβ, mouse GLP-1, and inflammatory cytokines

[0077] HEK293 cells stably expressing the human APP gene with the Swedish mutation were cultured in medium supplemented with 1% FBS. After 24 hours of Exendin-4 treatment, with or without the AMPK inhibitor compound C, the conditioned medium was collected and protease inhibitors and AEBSF (Roche Diagnostics, Cat#101500) were added to prevent Aβ peptide degradation. Before performing the ELISA protocol, cortical tissues from APP23 / PS45 double transgenic mice were prepared according to the manufacturer's protocol. The concentrations of Aβ40 and Aβ42 were measured using an Aβ1-40 or Aβ1-42 colorimetric ELISA kit (Invitrogen, Cat#KHB3544; KHB3481). The plasma concentration of GLP-1 in 12-week-old APP23 / PS45 and WT mice was measured using a mouse GLP-1 ELISA kit (Elabscience, Cat#E-EL-M3012). The conditioned medium of BV-2 microglia was collected and centrifuged to remove particles. Cortical tissues from APP23 / PS45 double transgenic mice were prepared according to the manufacturer's instructions. The levels of TNF-α (Elabscience, Cat#E-EL-M3063), IL-6 (Elabscience, Cat#E-EL-M0044), IL-1β (Elabscience, Cat#E-EL-M0037), and TGF-β (Multi Sciences, Cat#70-EK981-96) in the medium or mouse cortical tissues were quantified using commercially available ELISA kits.

[0078] 11. Evaluation of mitochondrial oxidative phosphorylation

[0079] Oxygen consumption rate (OCR) was evaluated using a Seahorse XF96 Flux Analyzer (Agilent Technologies) and Seahorse XF Cell Mitochondrial Stress Test Kit (Agilent, Cat#103015-100). Primary astrocytes and neurons isolated from APP23 mice were seeded and sequentially injected with oligomycin (2 μM), FCCP (2 μM), and rotenone / antimycin A (R / A) (1 μM) to measure OCR. To evaluate fatty acid oxidation (FAO), cells were incubated with Seahorse assay medium supplemented with palmitate (167 μM) and etomoxir (40 μM, HY-50202), an irreversible inhibitor of FAO).

[0080] 12. Evaluation of extracellular acidification rate (ECAR)

[0081] In a Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies), ECAR was measured as for OCR, except that the neuronal medium was replaced with XF DMEM (pH 7.4) medium without glucose, glutamine, and pyruvate. After incubation in a 37 °C non-CO 2 incubator for 1 h, ECAR was measured by sequentially injecting glucose, oligomycin, and 2-deoxyglucose. Basal glycolysis and glycolytic capacity were calculated and normalized to the protein concentration in each corresponding microplate well measured by the BCA protein assay (Thermo Fisher Scientific, Cat#A55864).

[0082] 13. Glucose uptake assay

[0083] To evaluate 2-deoxyglucose (2DG) uptake, a Glucose Uptake-Glo assay (Promega, J1341) was performed. Primary neurons were cultured on coverslips pre-coated with 10% poly-D-lysine (Solarbio, Cat#P2100). After treatment, cells were washed twice with PBS and then incubated with 1 mM 2DG for 10 min at room temperature, followed by sequential addition of stop buffer, neutralization buffer, and detection buffer. Luminescence was measured using a microplate reader ( system).

[0084] 14. ATP production assay

[0085] The ATP levels in neurons were measured using an ATP detection kit (Abcam, Cat# ab83355). After washing the cells twice with PBS, 100 μL of lysis buffer was added to each tube and then kept on ice. The lysate was centrifuged at 13,000 g for 5 minutes at 4 °C. The supernatant was transferred to a new 1.5 mL tube for ATP determination using the detection kit. The relative ATP level was calculated according to the following formula: relative ATP level = ATP value / protein value (nmol / mg).

[0086] 15. ROS measurement

[0087] Changes in the levels of reactive oxygen species (ROS) were measured using a Cellular ROS Detection Kit (Abcam, Cat# ab186027). Briefly, primary neurons were seeded onto 24-well confocal culture dishes pre-coated with poly-D-lysine. After treatment with 1 μM Exendin-4 for 24 hours, the cells were washed once with PBS. Subsequently, 200 μL of ROS RedStain working solution was added to each well, and the dishes were incubated at 37 °C in the dark for 30 minutes. After incubation, the staining results were observed under a fluorescence microscope (Ex / Em = 520 / 605 nm).

[0088] 16. Intracellular pyruvate quantification

[0089] Pyruvate was measured using a Pyruvate Detection Kit (Abcam, Cat# ab65342). Briefly, primary neurons were collected in detection buffer and exposed to a premix containing pyruvate oxidase to generate a fluorescence signal that directly reflects the pyruvate level (excitation / emission = 535 / 587 nm). To inhibit the conversion of pyruvate to lactate, the samples were deproteinized using a 10 kDa cutoff centrifugal filter (Pierce 88 513). Then the pyruvate level was determined using a standard curve

[0090] 17. Phagocytosis assay

[0091] BV-2 cells were treated with 1 μM Exendin-4 or 0.5 μM telotristat for 24 hours, then exposed to 10 μM cytochalasin D (MedChemExpress, Cat# HY-N6682) or left untreated for 30 minutes, and then incubated with 500 nM FAM-Aβ1-42 (AS-23525-05) oligomers for 1 hour. After treatment, the cells were washed and fixed with 4% paraformaldehyde (PFA). Phagocytosis of FAM-labeled oAβ1-42 in BV-2 cells was quantified by a Leica confocal microscope. The phagocytic ability was quantified by calculating the ratio of the cumulative fluorescence signal area to the total number of phagocytic cells.

[0092] 18. Real-time Fluo-4 AM calcium imaging

[0093] The Fluo-4 Calcium Assay Kit (Beyotime, Cat#S1061S) facilitated the measurement of intracellular calcium levels in neurons. Primary neurons were seeded onto 24-well confocal culture dishes pre-coated with poly-D-lysine. After treatment with 1 μM Exendin-4 for 24 h, the cells were washed once with PBS. Subsequently, 250 μL of 1X Fluo-4 staining solution was added to each well, and the dishes were incubated at 37 °C in the dark for 30 min. After incubation, the staining results were observed under a fluorescence microscope (Fluo-4 AM showed green fluorescence, Ex / Em = 490 / 525 nm).

[0094] 19. RNA sequencing and data processing

[0095] RNA sequencing was performed on 6 independent RNA samples of BV2 cells: BV2 cells cultured with Aβ oligomers (3 samples) and BV2 cells pretreated with Exendin-4 before culturing with Aβ oligomers (3 samples). After RNA extraction, the samples were subjected to quality control using Qubit 2.0 and 2100 BioAnalyzer. Libraries were prepared using the TIANGEN Biotech kit and sequenced using illumina novaseq / Xplus in PE150 mode. To measure differentially expressed genes (DEGs), DESeq2 (version 1.40.2) with default parameters was used. Based on the results of biological process analysis using the DESeq2 package and GO database, gene set enrichment analysis (GSEA) of biological processes related to inflammation and glucose metabolism was performed using the R package clusterProfiler (version 4.8.3). An absolute NES value greater than 1 and a P value less than 0.05 were considered statistically significant. Based on the KEGG database, functional enrichment analysis was performed using the R package clusterProfiler (version 4.8.3), and an adjusted P value less than 0.05 was considered statistically significant.

[0096] 20. Transgenic mice and drug treatment

[0097] APP23 mice overexpressing the Swedish APP751 (KM→NL) mutant transgene under the control of the murine Thy1.2 promoter were originally developed at Novartis Pharma. APP23 mice were crossed with mice overexpressing human G384A mutant PS1 under the regulation of the murine Thy1 promoter (B6, D2-TgN(Thy1-PS1G384A)45) to generate APP23 / PS45 double transgenic mice. APP23 and PS45 mice had been backcrossed to C57BL / 6 mice for more than 7 generations before breeding the double transgenic mice. The genotype of the mice was confirmed by PCR using DNA from tail tissue. Although this mouse model does not fully capture the complexity of human AD, it provides valuable insights into amyloid-related mechanisms. The treatment group consisted of 18 animals (9 females, 9 males), and the sham injection group consisted of 18 animals (9 females, 9 males). No statistical method was used to predetermine the sample size, but the sample size in this article was similar to that reported in previous publications. Mice in the treatment group were intraperitoneally injected with 25 nmol / kg Exendin-4 at the same time every day, with or without 20 mg / kg Compound C, diluted with 0.9% saline. Control group mice were injected with a vehicle solution containing DMSO diluted only with 0.9% saline.

[0098] 21. Immunohistochemical staining

[0099] After the behavioral tests, the mice were euthanized and one hemisphere of the brain was homogenized rapidly for protein, RNA, or DNA extraction. The other hemisphere was fixed in 4% paraformaldehyde and sectioned at 30 μm thickness using a Leica cryostat. Every 12th section was mounted on a glass slide maintaining the same reference position for subsequent staining. Immunocytochemistry staining was performed on the floating sections. Plaques in the sections were detected with a 1:500 diluted biotinylated monoclonal 4G8 antibody (Biolegend, Cat#800708), visualized by the ABC and DAB methods, and counted under a ×40 objective. The plaques were quantified and the mean plaque count per section per mouse was recorded. Thioflavin-S staining of the plaques was performed with 1% Thioflavin-S and visualized using a fluorescence microscope (Olympus). The brain sections were also stained with anti-CD68 (1:200, Proteintech, Cat#28058-1-AP). BV2 cells were fixed with 4% paraformaldehyde and stained with anti-CD68 (1:200) or anti-LAMP1 (1:200, Cell Signaling Technology, Cat#99437S), followed by secondary antibody staining. Primary neurons isolated from WT and APP23 mice were labeled with the DiO cell membrane staining kit (Byotime, Cat#C1038) for 15 minutes, then fixed with 4% paraformaldehyde and stained with anti-GLUT3 (1:400, Proteintech, Cat#20403010AP), followed by secondary antibody staining. The staining results were observed under a 63× oil immersion objective of a Zeiss fluorescence microscope.

[0100] 22. Morris water maze test

[0101] The Morris water maze test was performed as previously reported in the literature. APP23 / PS45 mice treated with Exendin-4 or saline (control group) underwent the Morris water maze test 1 day after the last injection. The test was conducted in a pool with a platform diameter of 10 cm located in the southeast (SE) quadrant. In the visible platform test on the first day, the mice completed 5 consecutive trials with an inter-trial interval of 60 minutes. For the hidden platform test, the mice were subjected to 5 trials per day with a 60-minute rest between each trial. Each mouse had 60 seconds to locate the platform. If the platform was not found within this time, the mouse was guided to the platform and allowed to rest for 15 seconds. On the last day, a probe test was performed in which the platform was removed and each mouse had 60 seconds to locate the original position of the platform. Mouse behavior, including distance traveled, escape latency, and number of platform crossings, was recorded using automated video tracking (ANY-maze, Stoelting).

[0102] 23. Statistics and reproducibility

[0103] All statistical analyses were performed using GraphPad Prism 7 software. All data are expressed as mean ± SEM. For comparison between two groups, two-tailed Student's t-test was used for statistical analysis. For comparison among multiple groups, the results were analyzed by one-way analysis of variance (ANOVA) or two-way ANOVA, and then post hoc tests were performed when appropriate. Normality test and homogeneity of variance test were performed before data analysis to ensure the premise assumptions of statistical methods were met. The data collection and analysis processes were not blinded. A P value < 0.05 was set as statistically significant difference. Specific embodiment

[0105] Example 1: Study on the relationship between plasma GLP-1 level and the pathological process of Alzheimer's disease

[0106] To explore the role of GLP-1 in the pathogenesis of AD, in this example, comparative analysis of plasma GLP-1 levels was performed between wild-type (WT) mice and AD transgenic mice APP23 / PS45. The present invention found that the plasma GLP-1 level in APP23 / PS45 was significantly reduced to nearly half of that in WT mice (P = 0.0288, Figure 1 A), and at the same time, glucose uptake ( Figure 1 B) and ATP production in neurons of AD mice decreased ( Figure 1 C). In addition, the expression ( Figure 1 D and 1E; Figure 8 A and 8B) and membrane translocation ( Figure 1 D and 1F) of glucose transporter 3 (GLUT3) were decreased. Glucose transporter 3 (GLUT3) is the main glucose transporter in neurons. Considering that approximately 20% of the total brain energy consumption comes from fatty acid oxidation (FAO), in this example, Seahorse XF96 cell mitochondrial stress test was performed to measure the oxygen consumption rate (OCR) and ATP production of astrocytes ( Figure 1 G and 1H; Figure 9 and 10 ). Compared with WT mice, AD mice showed decreased basal respiration, maximal respiration and reserve respiratory capacity, ultimately resulting in reduced OXPHOS-dependent ATP synthesis ( Figure 1 H). Using 18 F-AV45 PET imaging to evaluate the brain amyloid burden in Alzheimer's disease patients, in this example, a strong negative correlation was found between plasma GLP-1 level and hippocampal Aβ plaque accumulation (R = -0.825, p = 0.0017, Figure 1I). Among the participants, individuals with the lowest plasma GLP-1 levels (4.22 pg / mL) showed significantly higher hippocampal amyloid burden (SUVR = 1.44), compared to those with the highest plasma GLP-1 levels (19.23 pg / mL), whose SUVR was 0.87( Figure 1 J). These findings suggest that lower plasma GLP-1 levels are associated with increased amyloid burden in the hippocampus.

[0107] Next, in this example, GLP-1RA Exendin-4 was administered to enhance GLP-1 signaling, and successfully restored the glucose uptake ability in AD neurons( Figure 1 B) and ATP production( Figure 1 C), as well as fatty acid oxidation in AD astrocytes( Figure 1 G and 1H; Figure 9 and 10 ). However, Exendin-4 treatment had no effect on glycolysis in AD astrocytes( Figure 11 ). Exendin-4 also alleviated the AD-induced decrease in the levels of GLUT3 and the mitochondrial regulator PGC-1α( Figure 1 D-F; Figure 8 C and 8D). Then, in this example, the effects of Exendin-4 on neuronal glycolysis and oxidative phosphorylation were further explored by performing OCR and extracellular acidification rate (ECAR) assays. The results showed that after the improvement of glucose uptake, Exendin-4 treatment significantly enhanced oxidative phosphorylation( Figure 12 A-E) and glycolytic capacity( Figure 12 F-I) in AD neurons. In addition, in this example, cellular metabolites such as pyruvate and reactive oxygen species (ROS) were evaluated. Exendin-4 did not change the pyruvate level( Figure 12 J), but significantly reduced the AD-induced increase in ROS, indicating its role in preventing excessive oxidative phosphorylation( Figure 12 K and 12L). In addition, in this example, it was observed that the administration of compound C (also known as Dorsomorphin, a widely used AMPK inhibitor) counteracted the beneficial effects of Exendin-4, indicating that the action of Exendin-4 is closely related to AMPK activity( Figure 1 B-H; Figures 8 - 10 and 12).

[0108] In summary, this example confirmed that GLP-1 signaling system dysfunction is closely related to the pathological process of Alzheimer's disease, and GLP-1RA restores glucose metabolism and mitochondrial function in AD neurons through an AMPK-dependent mechanism and enhances fatty acid oxidation in astrocytes.

[0109] Example 2: GLP-1RA enhances CaMKK2-mediated phosphorylation of AMPK

[0110] To examine the effect of GLP-1 signaling on AMPK activity, primary neurons isolated from wild-type (WT) mouse embryos (E17) were treated with Exendin-4 or tirzepatide. Treatment with Exendin-4 at concentrations of 0.25, 0.5, and 1 μM significantly increased the level of phosphorylated AMPK to 251.2% ± 7.9% (P = 0.0006), 258.9% ± 23.7% (P = 0.0005), 275.0% ± 12.5% (P = 0.0002) ( Figure 2 A and 2B). Activation of AMPK led to a further increase in the level of phosphorylated acetyl-CoA carboxylase (ACC) to 174.5% ± 13.3% (P = 0.0169), 163.5% ± 10.8% (P = 0.0378), and 194.1% ± 17.9% (P = 0.0044) ( Figure 2 A and 2B). Similarly, 0.1, 0.25, and 0.5 μM of tirzepatide also significantly increased the level of phosphorylated AMPK to 150.9% ± 13.4% (P = 0.0151), 155.1% ± 15.1% (P = 0.0103), and 162.2% ± 0.5 (P = 0.0055) ( Figure 2 C and 2D). To confirm that these effects of GLP-1RA depend on the binding of GLP-1 to its receptor GLP-1R, primary neurons were transduced with a lentiviral shRNA vector to knockdown GLP-1R expression. Inhibition of GLP-1R expression led to a reduced response to GLP-1RA ( Figure 2 E and 2F). It is well known that an increase in intracellular calcium levels triggers AMPK phosphorylation, which is a process mediated by calcium / calmodulin-dependent protein kinase kinase 2 (CaMKK2). Treatment with Exendin-4 significantly increased calcium influx into neurons ( Figure 2 G and 2H), which further increased the level of CaMKK2 to 137.1% ± 7.5% (P = 0.0331) ( Figure 2 I and 2J). In addition, the application of the CaMKK inhibitor STO609 significantly attenuated the effect of Exendin-4 on AMPK phosphorylation ( Figure 2 K and 2L). Liver kinase B1 (LKB1) is recognized as directly phosphorylating threonine 172 (Thr 172 ) of AMPKα. The results of this example study showed that GLP-1RA had no significant effect on LKB1 expression (P = 0.5729) ( Figure 2 I and 2J). These results suggest that GLP-1RA enhances AMPK activity through a CaMKK2-mediated mechanism.

[0111] Example 3: AMPK phosphorylation by GLP-1RA reduces the production of BACE1 and Aβ

[0112] In this example, it was found that Exendin-4 at a concentration of 1 μM significantly reduced the levels of BACE1 in primary mouse neurons and the human Swedish mutant APP stable cell line 20E2 to 53.6% ± 6.6% (P = 0.0349) ( Figure 3 A and 3B) and 39.8% ± 10.5% (P = 0.035) ( Figure 3 C and 3D), respectively. The treatment significantly reduced the level of the β-secretase cleavage product APP C99 to 65.2% ± 5.6% (P = 0.0143) ( Figure 3 C and D). However, Exendin-4 treatment had no significant effect on APP expression (P = 0.4649) ( Figure 3 C and D). To evaluate the levels of Aβ40 and Aβ42 in the conditioned medium of 20E2 cells, an Aβ ELISA was performed. Exendin-4 significantly reduced the levels of Aβ40 and Aβ42 to 65.1% ± 8.7% (P = 0.043) ( Figure 3 E) and 52.8% ± 4.6% (P = 0.0031) ( Figure 3 F), respectively. In contrast, the addition of compound C to inhibit AMPK activity significantly reduced the effects of Exendin-4 on BACE1 expression ( Figure 3 A-D) as well as the production of C99 and Aβ ( Figure 3 C-F). To confirm the results, this example further infected primary APP23 mouse neurons with AMPK-targeted shRNA and found that the reduction in AMPK expression significantly alleviated the effects of Exendin-4 on BACE1-mediated APP processing ( Figure 13 A and 13B) and Aβ production ( Figure 13 C). In addition, by inhibiting fatty acid oxidation in AD astrocytes, the effects of Exendin-4 on BACE1 expression ( Figure 9 F and 9G) and the amyloidogenic process of APP to Aβ production ( Figure 9 H) were significantly altered. In addition, this example also demonstrated the effects of Exendin-4 on AKT and PKA signaling, which together with AMPK signaling regulate BACE1 expression ( Figure 9 I-K). These findings clearly show that Exendin-4 inhibits BACE1-mediated APP processing to produce Aβ, and this effect is at least partially mediated by AMPK activity.

[0113] Example 4: NF-κB-mediated AMPK-induced BACE1 transcription

[0114] To determine how AMPK regulates BACE1 expression, the transcriptional regulation of the BACE1 gene promoter was first assayed. A BACE1 promoter construct pB1-luc was constructed by cloning the human BACE1 promoter region spanning -1942 to +292 bp upstream of the firefly luciferase reporter gene into the promoterless vector pGL3-basic( Figure 4 A). N2a cells were transfected with the promoter construct and then treated with an AMPK inhibitor (compound C) or an AMPK activator (AICAR). Compound C inhibited AMPK signaling at 50 or 100 μM, increasing the promoter activity to 343.5% ± 28.0% (P = 0.0011) and 499.8% ± 33.7% (P = 0.0003( Figure 4 A), respectively. In contrast, treatment with 1 or 2 mM AICAR, which increased AMPK activity, significantly decreased the promoter activity to 60.7% ± 2.1% (P = 0.0061) and 50.7% ± 1.8% (P = 0.0026)( Figure 4 A), respectively. Consistently, inhibition of AMPK by compound C also increased the mRNA level of BACE1 in primary mouse neurons( Figure 4 B) and the protein level( Figure 4 C and 4D), while stimulation of AMPK activity by AICAR significantly decreased the mRNA level of BACE1( Figure 4 B) and the protein level( Figure 4 C and 4D). Previous studies by the inventors have shown that NF-κB significantly enhances BACE1 promoter activity. To investigate whether the effect of AMPK on BACE1 expression is mediated by NF-κB, primary APP23 mouse neurons were treated with compound C or AICAR and then subjected to subcellular fractionation( Figure 4 E). Compared with the control group, AICAR treatment significantly decreased the nuclear NF-κB p65 level to 65.0% ± 9.7% (P = 0.0343)( Figure 4 F). In addition, compared with the control group, AICAR treatment also decreased the cytoplasmic NF-κB p65 level to 65.1% ± 8.9% (P = 0.0262)( Figure 4 F). These findings indicate that AMPK activation achieved by AICAR leads to a decrease in NF-κB activity by reducing the NF-κB p65 level. In addition, a ChIP assay was also performed in this example, and the results showed that inhibition of AMPK significantly enhanced the binding of NF-κB p65 to the BACE1 promoter, resulting in an increase in BACE1 expression( Figure 4G). Overall, the results indicate that NF-κB signal transduction plays a role in mediating the regulatory effect of GLP-1RA on BACE1 gene expression.

[0115] Example 5: GLP-1RA-AMP activation promotes microglial phagocytosis

[0116] In this example, it was observed that GLP-1RAs, including Exendin-4 and tirzepatide, also enhanced AMPK phosphorylation in the microglial cell line BV to 124.6% ± 6.1% (P = 0.0132) and 129.8% ± 4.0% (P = 0.0056), respectively ( Figure 5 A and 5B). To determine the biological functions affected by GLP-1RA in microglia, RNA-seq analysis was performed to detect differentially expressed genes (DEGs) between cells cultured with Aβ oligomers and cells pretreated with Exendin-4 before culturing with Aβ oligomers. The results showed that Exendin-4 treatment increased the expression of genes related to phagocytosis, while decreasing the expression of disease-associated microglia (DAM) genes and inflammation-related genes ( Figure 5 C). In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were also performed to further explore the biological processes involved. Treatment with Exendin-4 enhanced the expression of genes related to the AMPK signaling pathway ( Figure 5 D) and glucose metabolism, including genes involved in glucose catabolism, gluconeogenesis, and glucose transmembrane transport ( Figure 5 E). In addition, inflammation-related biological processes were also downregulated after Exendin-4 treatment ( Figure 5 F). To further verify the results of the RNA-seq analysis, immunocytochemistry ( Figure 5 G-I) and Western blot analysis ( Figure 14 ) were performed. Exendin-4 or tirzepatide significantly increased the expression of CD68 ( Figure 5 G and 5H; Figure 14 A and 14B) and LAMP1 ( Figure 5 G and 5I; Figure 14 A and 14C) in BV2 cells, which are two well-recognized markers of microglial phagocytosis and lysosomal degradation.

[0117] To further evaluate whether GLP-1RA promotes microglial phagocytosis, the uptake of carboxyfluorescein (FAM)-labeled Aβ42 (FAM-Aβ42) by BV2 cells was measured. Both Exendin-4 and tirzepatide significantly enhanced the phagocytic activity of BV2 cellsFigure 5 J and 5K). Previous studies have shown that GLP-1RA has anti-inflammatory properties that may be beneficial for the treatment of lung injury. The study in this example found that GLP-1RA also significantly reduced Aβ-induced inflammatory cytokines produced by BV2 cells, including a decrease in IL-1β, IL-6, and TNF-α ( Figure 15 A-C), while increasing the production of the anti-inflammatory cytokine TGF-β ( Figure 15 D). In addition, application of compound C to inhibit AMPK activity attenuated the effect of GLP-1RA on neuroinflammation ( Figure 15 ). These results indicate that the anti-inflammatory effect of GLP-1RA is mediated through AMPK signaling.

[0118] Example 6: Regulation of BACE1 expression and Aβ production by GLP-1R in vivo

[0119] To investigate the effect of GLP-1RA on AMPK activity and APP processing in vivo, AMPK phosphorylation, APP CTF, and Aβ production in the brains of APP23 / PS45 mice were first evaluated. APP23 / PS45 mice express the familial AD-related human Swedish mutant APP751 transgene and the G384A mutant presenilin-1 transgene (PS1). These double transgenic mice develop neuritic plaques and cognitive impairment in the neocortex and hippocampus. Mice were treated with Exendin-4 (25 nmol / kg) daily from 6 weeks of age for 8 weeks, while age-matched control mice received the vehicle solution. Compared with the control group ( Figure 6 C and 6D), treatment with Exendin-4 significantly increased the level of phosphorylated AMPK protein in the brain to 174.9% ± 20.6% (P = 0.0186) ( Figure 6 A and 6B), which was associated with a decrease in the level of C99 produced by β-secretase in the cortex (56.4% ± 3.5%, P = 0.001) and hippocampus (42.7% ± 3.4%, P = 0.0006). Compared with the control group, the cortical levels of Aβ40 and Aβ42 in Exendin-4-treated mice were reduced to (77.7% ± 5.7%, P = 0.0055) and (81.5% ± 4.3%, P = 0.0034) ( Figure 6 E). The hippocampal levels of Aβ40 and Aβ42 in Exendin-4-treated mice were also reduced to (75.1% ± 4.9%, P = 0.0009) and (78.1% ± 4.4%, P = 0.0021) ( Figure 6 E). These results indicate that enhanced AMPK activity induced by GLP-1RA treatment reduces β-secretase cleavage of APP and Aβ production in vivo.

[0120] In addition, this example investigated whether the expression of BACE1 is affected by in vivo GLP-1RA treatment. Western blot analysis showed that compared with control mice, Exendin-4 treatment significantly reduced the protein levels of BACE1 in the cortex (58.7% ± 2.0%, P = 0.0003) and hippocampus (62.3% ± 7.1%, P = 0.0102), and had no significant effect on the protein level of APP (p > 0.05)( Figure 6 C and 6D). In vitro studies showed that GLP-1RA regulates the transcription of the BACE1 gene. To confirm that the decrease in BACE1 protein levels in the brains of Exendin-4-treated mice was due to reduced BACE1 gene transcription, the endogenous Bace1 mRNA levels were measured( Figure 6 F). Exendin-4 treatment significantly reduced the Bace1 mRNA levels to 0.36 ± 0.14 (P = 0.0052), while the App mRNA levels did not change significantly( Figure 6 F). In addition, AD mice were injected with Exendin-4 and compound C to inhibit AMPK activity. The results showed that the inhibition of AMPK activity significantly alleviated the effect of Exendin-4 on the regulation of BACE1 expression, and the amyloidogenic processing of APP mediated by BACE1 in the cortex and hippocampus( Figure 16 ). These findings indicate that, consistent with the in vitro results, GLP-1RA inhibits BACE1 gene expression and its β-secretase activity in vivo by regulating AMPK activity.

[0121] Example 7: GLP-1R reduces AD pathology and cognitive deficits in mice

[0122] To investigate the specific effects of GLP-1RA on the pathogenesis of AD, 4G8 immunostaining and Thioflavin-S staining were used to detect Aβ-containing neuritic plaques in the brains of APP23 / PS45 mice( Figure 7 A). Compared with the vehicle-treated group, treatment with Exendin-4 significantly reduced the number of plaques in APP23 / PS45 mice to approximately half (18.9 ± 1.4 vs. 35.2 ± 2.0 per section, P < 0.0001, Figure 7 B). This example further found that Exendin-4 treatment significantly reduced the levels of pro-inflammatory cytokines in the brains of mice, and the levels of IL-1β, IL-6, and TNF-α decreased to 83.3% ± 1.8% (P = 0.0008), 70.0% ± 2.5% (P < 0.0001), and 48.1% ± 2.4 (P < 0.0001), respectively( Figure 17 A-D). In addition, the expression of CD68 increased after Exendin-4 treatment( Figure 17E and 17F). To evaluate the effect of Exendin-4 treatment on cognitive deficits, the Morris water maze test was performed in APP23 / PS45 mice after 8 weeks of Exendin-4 treatment. In the visible platform test, both Exendin-4-treated and control APP23 / PS45 mice showed similar escape latencies (29.2 ± 1.5 vs. 28.8 ± 1.8 s, P = 0.8691)( Figure 7 C) and path lengths (4.5 ± 0.2 m vs. 3.9 ± 0.4 m, P = 0.2525)( Figure 7 D), indicating that Exendin-4 treatment did not affect mouse activity or vision. During the hidden platform test, mice treated with Exendin-4 showed significant improvement compared to vehicle-treated controls. Exendin-4-treated mice had shorter escape latencies (30.2 ± 2.9 and 29.9 ± 2.8 s) on days 4 and 5 of the hidden platform test than vehicle-treated mice (40.0 ± 2.8 and 42.3 ± 2.8 s)( Figure 7 E). In addition, Exendin-4-treated mice swam significantly shorter distances on day 4 compared to control mice (2.8 ± 0.3 m vs. 5.3 ± 0.6 m, P = 0.0007)( Figure 7 F). In the probe test on the last test day, when the platform was removed, Exendin-4 treatment significantly improved the spatial memory of APP23 / PS45 mice, as demonstrated by an increased number of entries into the third quadrant where the hidden platform had previously been located (3.7 ± 0.7 vs. 1.6 ± 0.4 fold, P = 0.0143)( Figure 7 G). These results indicate that GLP-1RA treatment significantly improves cognitive impairment in AD model mice.

[0123] In summary, the present invention demonstrates that GLP-1RA is a promising therapeutic strategy for AD. More importantly, the present invention surprisingly demonstrates that GLP-1RA is an activator of AMPK and can be used to regulate energy metabolism and the function of brain cells to inhibit BACE1-mediated amyloidosis and cognitive impairment. The said regulation is the key mechanism for its therapeutic effect on AD. The present invention provides valuable insights into the potential of GLP-1RA as a treatment for AD and provides a clear mechanistic basis to support further clinical trials.

[0124] Although the present invention has been specifically shown and described with reference to its preferred embodiments, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the scope of the invention as covered by the appended claims.

Claims

1. Use of a GLP-1 receptor agonist in the preparation of a medicament for treating Alzheimer's disease (AD).

2. The use of claim 1, wherein the drug treats Alzheimer's disease by activating AMPK.

3. The use of claim 2, wherein the drug activates the AMPK signaling pathway in brain cells.

4. The use of any one of claims 1 to 3, wherein the drug reduces β-secretase (BACE1)-mediated APP hydrolysis and the generation of amyloid-β (Aβ) protein.

5. The use of any one of claims 1-4, wherein the drug promotes phagocytosis of microglia.

6. The use according to any one of claims 1 to 5, wherein the drug reduces neuroinflammation and / or improves cognitive impairment.

7. The use of any one of claims 1-6, wherein the GLP-1 receptor agonist is selected from Exendin-4, Tirzepatide, Lixisenatide, Liraglutide, Albiglutide, Dulaglutide and Semaglutide.

8. The use according to any one of claims 1 to 7, wherein the GLP-1 receptor agonist is selected from exenatide and tilpotide.

9. A pharmaceutical composition for treating Alzheimer's disease (AD), comprising a GLP-1 receptor agonist and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition of claim 9, wherein the GLP-1 receptor agonist is selected from Exendin-4, Tirzepatide, Lixisenatide, Liraglutide, Albiglutide, Dulaglutide and Semaglutide.

11. The use according to any one of claims 9 to 10, wherein the GLP-1 receptor agonist is selected from exenatide and tirpotide.

12. The pharmaceutical composition of any one of claims 9-11, further comprising one or more additional therapeutic agents.

13. The pharmaceutical composition of claim 12, wherein the additional therapeutic agent is selected from the group consisting of a cholinesterase inhibitor, an NMDA receptor antagonist, an anti-Aβ monoclonal antibody, and a Tau protein targeting drug.

14. The pharmaceutical composition of claim 12, wherein the additional therapeutic agent is selected from an AMPK activator.

15. The pharmaceutical composition of claim 14, wherein the AMPK activator is selected from AICAR and metformin.

16. A method for treating Alzheimer's disease (AD), the method comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of claims 9 to 15 to a patient in need of treatment.

17. A GLP-1 receptor agonist for use in the treatment of Alzheimer's disease (AD).

18. Use of a GLP-1 receptor agonist in the preparation of an agent for activating the AMPK signaling pathway in brain cells.

19. Use of a GLP-1 receptor agonist in the preparation of an agent for regulating BACE1 expression and / or Aβ production in vivo.

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