A novel arctigenin derivative ameliorates memory impairment and pathologies by activating adiponectin receptor 1-mediated autophagy and regulating amyloid precursor protein processing of Alzheimer's disease

A novel arctigenin derivative ameliorates memory impairment and pathologies by activating adiponectin receptor 1-mediated autophagy and regulating amyloid precursor protein processing of Alzheimer's disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A novel arctigenin derivative ameliorates memory impairment and pathologies by activating adiponectin receptor 1-mediated autophagy and regulating amyloid precursor protein processing of Alzheimer's disease Shangming Li, Bocheng Xiong, Nan Xu, Lulin Nie, Kaiwu He, Guiliang Zhang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7035906/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Feb, 2026 Read the published version in Molecular Neurobiology → Version 1 posted 11 You are reading this latest preprint version Abstract Alzheimer's disease (AD), the most prevalent form of dementia, is characterized as a slowly progressing neurodegenerative condition marked by neurotic plaques and neurofibrillary tangles due to the buildup of amyloid-beta peptide (Aβ) in the brain's medial temporal lobe and neocortical structures. It is reported that arctigenin (ATG) has the effect to reduce the expression of the enzyme 1 that cleaves β-site amyloid precursor protein and increase Aβ clearance by enhancing autophagy. Compound ARC-18 is a derivative of ATG. The main objective of this study is to investigate whether ARC-18 could improve cognitive function and disease progression in Alzheimer's mice by promoting autophagy. 3-month-old 5×FAD mice were orally treated with drug for 3 consecutive months. Water maze and new object recognition were used to assess cognitive impairment in 5xFAD mice. In the hippocampus of the mouse brain, APP processing-related proteins (sAPP β , BACE1) and autophagy (LC3B, P62, LAMP1) related proteins were detected. Some experiments related to animal data were conducted on N2a/APPswe cells to further identify the effect and mechanisms of drug. ARC-18 improved behavioral performance in water maze and new object recognition in 5xFAD mice. ARC-18 alleviated the over-aggregation of Aβ in the hippocampus and cortex of 5xFAD mice. ARC-18 promotes autophagy and inhibits amyloidogenic processing of APP in 5xFAD mice and N2a/APPswe cells. ARC-18 improves AD-like pathologies and memory impairment by increased clearance of Aβ by activating adiponectin receptor 1-mediated autophagy and reduced Abeta production via regulating amyloid precursor protein (APP) processing. Alzheimer's disease adiponectin receptor 1 autophagy A novel arctigenin derivative 5xFAD mice N2a/APPswe cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Alzheimer's disease (AD) is a progressive neurodegenerative disease that ultimately leads to irreversible loss of neurons and intelligence, including cognition and memory. Currently, around 50 million people worldwide have AD, and this figure is expected to double every five years, reaching 152 million by 2050. AD imposes a significant burden on individuals, families, and the economy, with global annual costs of US $ 1 trillion (Livingston et al., 2020 ; Yiannopoulou & Papageorgiou, 2020 ). Two distinct pathogenic protein aggregates, amyloid beta peptide (Aβ) plaques and neurofibrillary tangles containing hyperphosphorylated tau protein, are what characterize AD (Serrano-Pozo, Das, & Hyman, 2021 ). Aging is the most significant risk factor for late-onset Alzheimer's disease, responsible for over 94% of AD cases (Liu, 2022 ). Given the inevitability of aging, the prevalence of AD remains substantial. The research of new drugs to treat AD is a difficult problem that humans must overcome. The process of cell self-digesting is called autophagy. It consumes its own cytoplasmic material, wraps it into vesicles, and fuses with lysosomes to create autolysosomes, which have a degradative function. Autophagy selectively degrades aging organelles, faulty proteins, and other materials to maintain cellular homeostasis. Initially, autophagy was thought to be a large-scale, non-selective degradation system (Abdrakhmanov, Gogvadze, & Zhivotovsky, 2020 ). Autophagy, as an important cellular metabolic activity, is involved in amyloid-beta (Aβ) metabolism by regulating Aβ production and clearance. Aβ originates from the processing of amyloid precursor protein (APP), which is sequentially cleaved by β-secretase and γ-secretase. Furthermore, the observation that four subunits of the APP and γ-secretase complexes reside in the autophagosome suggests that at least some of the Aβ peptides are produced via the autophagy pathway (Di Meco, Curtis, Lauretti, & Praticò, 2020 ; Selkoe & Hardy, 2016 ). Previous studies have shown that when AD model mice were treated with rapamycin, a specific inhibitor of mTOR, autophagy was consequently enhanced. Significant reductions in both intracellular Aβ and extracellular amyloid deposition in the brain were observed, and the animals' cognitive performance was significantly improved (Caccamo, Majumder, Richardson, Strong, & Oddo, 2010 ). Autophagy appears to affect Aβ clearance at multiple stages. Histone B is a key lysosomal protease required to degrade autophagic substrates. Studies have shown that genetic elimination of histone B worsens AD pathology in AD model mice, including increased Aβ 42 abundance and amyloid deposition. In contrast, when histone B was overexpressed by lentiviral transduction, amyloid plaques were reduced even in aged AD mice (Mueller-Steiner et al., 2006 ). In transgenic Alzheimer's disease (AD) mouse models, the induction of autophagy has been shown to decrease BACE1 levels (Wu et al., 2015 ). These results suggest that promoting autophagic flow at any stage is beneficial in attenuating AD progression Arctigenin (ATG) is a phenylpropanoid dibenzyl butyrolactone lignan compound that occurs naturally in Arctium lappa L. Previous studies have shown that ATG attenuates learning and memory deficits through PI3k/Akt/GSK-3β pathway reducing Tau hyperphosphorylation in Aβ-Induced AD mice (Qi et al., 2017 ). Previous studies have also identified a deficiency of adiponectin in 5xFAD mice (K. He et al., 2021 ). ATG, an agonist of adiponectin receptor 1 (AdipoR1), is known to regulate adiponectin expression, and ARC-18, a derivative of ATG, may target the same pathway (Sun et al., 2013 ). Adiponectin is reported to inhibit apoptosis of brain cells induced by cardiac arrest/cardiopulmonary resuscitation by enhancing autophagy via the AdipoR1-AMPK signaling pathway (Y. He et al., 2020 ). Dysregulated adiponectin signaling may mediate the detrimental effects of obesity on the central nervous system and increase the risk of cognitive decline and Alzheimer’s disease (AD) (Forny-Germano, De Felice, & Vieira, 2018 ). Based on preliminary findings, we hypothesize that ARC-18 could promote autophagy through the AdipoR1-AMPK signaling pathway, thereby improving memory impairment and pathologies in 5xFAD mice. 2. Material and methods 2.1 Regents and antibodies ARC-18 (stated purity ≥ 98.86%) was synthesized by West China College of Pharmacy, Sichuan University. Arctigenin (stated purity ≥ 99.69%, catalog number: 7770-78-7), and chloroquine (CQ) (stated purity ≥ 99.50%, catalog number: 54-05-7) were purchased from the company of MedChemExpress (MCE, Monmouth Junction, NJ, USA). Information about antibodies and used in this study has been shown in Table S1 . All primary antibodies are diluted in a ratio of 1:1000. At a dilution of 1:5000, the secondary antibodies utilized were goat anti-mouse IgG H&L (HRP) and goat anti-rabbit IgG H&L (HRP) from Proteintech, USA. 2.2 Animal experiment 5xFAD (B6. Cg-Tg (APPSwFlLon, PSEN1*M146L*L286V)6799V) male mice were purchased from Jakson laboratory and bred at Shenzhen Center for Disease Control and Prevention. All experimental operations complied with the ARRIVE guidelines and were approved by the ethics committee of the Shenzhen Center for Disease Control and Prevention (Project No. 2023036). In this experiment, 3-month-old male mice were used as experimental subjects to test the cognitive impairment of mice with Morri’s water maze and Novel object recognition. The 3-month-old mice were selected because the transgenic mice began to develop disease at the age of 1.5 months, and the 5-month-old mice could already show significant Aβ aggregation. ARC-18 was dissolved in saline, and ATG was prepared in a solution containing 5% DMSO, 40% PEG400, 5% Tween-80, and 50% saline. The specific groups are as follows: wild-type mice (Saline, gavage), model group (Saline, gavage), ARC-18 low dose group (11.0mg/kg, gavage), ARC-18 median dose group (33.0mg/kg, gavage) ARC-18 high dose group (99.0mg/kg, gavage) and ATG group (24.3mg/kg, gavage). At the conclusion of the experiment, the mice were anesthetized and then sacrificed using 4% sodium pentobarbital to obtain blood and tissue samples. The dosage of the drug was determined based on acute toxicity tests (Table .S2) and articles on ARC-18 for amyotrophic lateral sclerosis (Xiong et al., 2024 ). 2.3 Behavioral tests 2.3.1 Morris water maze In a pool with a diameter of 120 cm and a depth of 76 cm; a platform of 10 cm 2 ; adjust the pool and the camera position to ensure that the platform position is at an angle of 45° in the middle of the axis and remains in the same position during the experiment; milky white water in the pool is submerged in the platform for 0.5-1 cm and the temperature of the water is maintained at 22 ± 2°C; four markers of different shapes and colours are affixed to the wall of the pool in the direction of the axis and the markers cannot be changed during the experiment. Training period: (1) Mice were placed in the desired starting position in the maze, facing the wall of the pool. The mice were released to the water surface. Once released, the computer tracking programme was initiated. (2) The timer was stopped when the mice reached (touched) the platform, and the mice were allowed 60 s per trial.(3) Animals that did not find the platform within this time period were artificially placed or guided to the platform and allowed to remain on the platform for 15 s. (4) The mice were taken out of the water maze, dried off, and placed in a warm environment to prevent them from catching cold. (5) Place the mice in a new starting position in the water maze and repeat steps 1–4 until the animal has completed the required 4 training sessions that day. The experiment was trained for a total of 6 days. Test period: The purpose of the exploration test was to determine if the animal remembered the location of the platform. After 5 consecutive days of training, the station in the water maze was removed and the mouse was placed in a new starting position from the maze to ensure that its behaviour reflected a memory of the target location rather than a specific swimming path. For example, from the initial platform position facing the wall of the pool, the mouse was released to the water surface and its trajectory was observed. After 2 min, the machine automatically stopped the timer and the mouse was removed. The first day was the platform visibility period to determine whether the mice had visual defects. The rest of the day is a platform hiding period (Vorhees & Williams, 2006 ). 2.3.2 Novel object recognition On the first day, mice were individually placed in a 50 cm 2 square box and allowed to adapt to the environment for 5 minutes. After exploration, the mice were removed and returned to breeding cages. The behavioral box was cleaned by removing feces and urine, followed by wiping with 75% alcohol to eliminate odor. On the second training day, mice were put in a cage with two identical objects. On the third day, one object was swapped with a unfamiliar one. For more details, refer to the previous report (Chen et al., 2021 ). The results were expressed as a preference index percentage, calculated using the formula [exploration time of the new object / (exploration time of the new object + exploration time of the old object) ×100%]. 2.4 Immunohistochemistry The Abcam ABC HRP kit (ab64264) was used, and all procedures were performed according to the manufacturer’s instructions. Specifically, (1) paraffin-embedded brain sections were first deparaffinized in xylene and then rehydrated in a gradient of ethanol-water. (2) The brain sections were placed in citrate buffer, heated in a microwave for 10 minutes, and then cooled at room temperature for 30 minutes to retrieve antigens. (3) 3% H2O2 was added, covering the sections for 10 minutes, followed by 3 washes in PBS, each for 5 minutes. 3% bovine serum albumin was applied onto the brain sections and incubated at room temperature for 1 hour. (4) The samples were incubated with Aβ antibody (#803015, Biolegend) overnight at 4°C, followed by incubation with biotinylated goat secondary antibody at room temperature for 1 hour, and then with avidin-peroxidase at room temperature for 30 minutes. After each antibody incubation, the sections were washed three times with PBST for 10 minutes each. (5) The brain sections were stained with diaminobenzidine (DAB) for 2 minutes. After dehydration in a gradient of ethanol, the sections were cleared in xylene and sealed with neutral resin. (6) The brain sections were imaged using an optical microscope (Aperio GT 450, Leica Biosystems, USA). 2.5 Cell experiment Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (P/S), 0.25% trypsin-EDTA were purchased from Gibco/Invitrogen (USA). N2a/APPswe cells (kindly gifted by Professor Jian-zhi Wang, Tongji Medical School, HUST, China) were cultured in dishes with DMEM supplemented with 10% FBS in the presence of 200µg/mL geneticin(G418) and 1% P/S. The concentrations of ARC-18 on cells were 50, 100 and 200 µM, which were determined based on the IC50 of the ARC-18 assay and the concentration of arctigenin administered on cells (Song, Li, Liu, Hu, & Yang, 2019 ). Chloroquine was administered at a concentration of 50 micromoles. 2.6 Concentration tests for Aβ 1−40 and Aβ 1−42 Concentrations of Aβ 1−40 and Aβ 1−42 were tested on brain tissue and cells. The contents of Aβ 1−40 and Aβ 1−42 were determined using an ELISA assay, following the protocol provided by the manufacturer (Elabscience Biotechnology Co., Ltd, China). The levels of amyloid-beta (Aβ) were subsequently normalized to the total protein concentrations. 2.7 Interruption of AdipoR1 siRNA is chemically synthesized and Lipofectamine 3000 (Thermo Fisher Scientific, USA) is transfected into N2a/APPswe cells. The sequence of AdipoR1 (mouse) targeting siRNA is as follows: CAGCTTTCGTCC ACTTCTA. Unrelated siRNA sequences were used as negative controls. AdipoR1 targeted siRNA and unrelated siRNA sequences were synthesized by SANTA CRUZ BIOTECHNOLOGY (El-Andaloussi et al., 2012 ). The final concentration of siRNA is 50nM. 2.8 Statistical analysis Data were presented as means ± standard error of the mean (SEM). Statistical analyses were conducted using GraphPad Prism version 8.0 software. Comparisons between two groups were performed using an unpaired t-test. A one-way analysis of variance (ANOVA) was employed to assess the statistical significance of differences among multiple groups, followed by Dunnett's multiple comparison test. A p -value of less than 0.05 was considered statistically significant. 3. Result 3.1ARC-18 improved the performance of Morris water maze and Novel object recognition in 5xFAD mice To assess the cognitive differences between wild-type mice and 5xFAD mice, as well as the therapeutic effects of ARC-18, we conducted the Morris water maze test (MWM) and Novel object recognition (NOR) test. As shown in Fig .1a, there were no significant differences in swimming speed among the groups, indicating comparable locomotor abilities that would not affect cognitive function assessment. The wild-type mice exhibited a significantly higher number of platform crossings compared to the untreated 5xFAD group, suggesting that the untreated 5xFAD mice demonstrated impaired cognitive function in locating the platform. Following administration of a median dose of ARC-18 (33.0 mg/kg), the 5xFAD mice showed a marked increase in the number of platform crossings, surpassing the effects observed with equimolar doses of ATG (Fig .1b). Compared to the untreated 5xFAD group, the median-dose ARC-18 (33.0mg/kg) group improved the target quadrant cumulative time percentage in 5xFAD mice, and the effect was superior to ATG (Fig .1c). This is a water maze movement track of mice (Fig .1d). As illustrated in Fig .1e, the graph delineates the time required by mice to locate the platform during the initial trial, segmented into three distinct phases. On the first day of training, the platform is conspicuously positioned above the water surface to evaluate potential disparities in visual acuity among the groups, thereby mitigating experimental errors attributable to visual impairments. This phase is designated as the “visible platform period”. Subsequently, from the second to the fifth day, the platform is submerged, and the latency for the mice to identify the platform during their initial trial is systematically recorded. This phase is known as the “hidden platform period”. On the sixth day of testing, untreated 5xFAD mice took significantly longer than wild-type mice to find the platform. Median-dose ARC-18 (33.0 mg/kg) treatment reduced this latency in 5xFAD mice more effectively than equimolar doses of ATG. In Fig .1f, compared to wild-type mice, the untreated 5xFAD mice exhibited a significant decrease in the percentage of time spent exploring the novel object. Notably, after median-dose ARC-18 treatment, the percentage of time spent exploring the novel object in 5xFAD mice significantly increased. Based on the above experimental results, it could be concluded that ARC-18 improves cognitive dysfunction in 5xFAD mice. 3.2 ARC-18 alleviated Aβ deposition and reduced the concentrations of total Aβ in the hippocampus and cortex of 5xFAD mice Overdeposition of Aβ is an important factor in the development of AD. To assess the pathogenesis of 5xFAD mice, we performed immunohistochemical staining of Aβ and tested the concentrations of Aβ 1−42 and Aβ 1−40 in the hippocampus and prefrontal cortex by Elisa. Aβ aggregation was markedly elevated in 5xFAD mice relative to wild-type counterparts. Administration of a median-dose ARC-18 (33.0 mg/kg) resulted in a significant reduction in Aβ deposition, demonstrating a more pronounced effect than equimolar doses of ATG (Fig. 2 a and Fig. 2 b). ARC-18 did not affect the levels of Aβ 1−40 in the prefrontal cortex; however, it significantly decreased the levels of Aβ 1−42 (Fig .2c). In comparison to untreated 5xFAD mice, ARC-18 treatment led to a significant reduction in the concentrations of both Aβ 1−40 and Aβ 1−42 in the hippocampus (Fig .2d). These results suggest that ARC-18 mitigates Aβ deposition and lowers the overall levels of Aβ in 5xFAD mice. 3.3 ARC-18 promoted autophagy and regulated the processing of APP in 5xFAD mice To investigate how ARC-18 reduces Aβ deposition, we examined the expression of proteins involved in the APP processing pathway through Western blot analysis. Untreated 5xFAD mice had significantly higher APP expression than wild-type mice, which was reduced by ARC-18 treatment. Median-dose ARC-18 (33.0 mg/kg) significantly increased sAPPα expression and decreased sAPP β expression, outperforming equimolar doses of ATG (Fig .3b). Compared to wild-type mice, the expression of BACE1 and Presenilin 1(PS1) was significantly increased in untreated 5xFAD mice. Moreover, after ARC-18 treatment, the expression of BACE1 and PS1 was significantly decreased (Fig .3c). These results suggest that ARC-18 reduced Aβ production by modulating APP processing. It has been reported that excessive deposition of Aβ could lead to dysfunction in autophagy. Arctigenin (ATG) has been shown to increase Aβ clearance by promoting autophagy in AD mouse model (Zhu et al., 2013 ). ARC-18, a derivative of ATG may have similar effects. To investigate whether ARC-18 could enhance Aβ clearance by promoting autophagy, we conducted Western blot validation in the hippocampal area of 5xFAD mice. Compared to the 5xFAD mice treated with saline, the high-dose ARC-18 group (33.0 mg/kg) significantly decreased the expression of p-mTOR/mTOR and P62, while increasing the expression of p-ULK1/ULK1, ATG5, Beclin1, and LC3B/LC3A (Fig .3d and Fig .3e). LAMP1 and Cathepsin D (CTSD) are proteins associated with autophagic lysosomes. Compared to the 5xFAD mice treated with saline, ARC-18 promoted the degradation of LAMP1 but did not affect the expression of CTSD (Fig .3f). These results demonstrate that ARC-18 ameliorates autophagic dysfunction in 5xFAD mice, and the effect is superior to equimolar doses of ATG. 3.4 CQ reversed the effect of ARC-18 in amyloidogenic processing of APP in vitro To investigate the effects of ARC-18 on Aβ production, the levels of Aβ 1−40 and Aβ 1−42 were tested in the cell lysate of N2a/APPswe cells with or without ARC-18 treatment. At the same time, we verified the proteins associated with the APP processing pathway. The IC50 of ARC-18 was approximately 471.0 µM in N2a/APPswe cells (Fig .4b). Based on the IC50 data, the drug demonstrated effective activity at concentrations of 50, 100, and 200 µM without adversely affecting normal cell growth. At a concentration of 100 µM, ARC-18 significantly reduced the levels of Aβ 1−40 and Aβ 1−42 in N2a/APPswe cells, which was reversed by the autophagy inhibitor CQ (Fig .4c). ARC-18 was also found to upregulate the expression of sAPP α , while exhibiting a certain trend on APP and sAPP β levels. Administration of ARC-18 in combination with a CQ inhibitor upregulated the expression of APP and sAPP β , compared to administration of ARC-18 (100µM) alone. (Fig .4d). Additionally, ARC-18 decreased the expression of Presenilin 1(PS1), but did not affect BACE1 levels with a certain trend. The expressions of BACE1 and PS1 were changed after CQ administration (Figure .4e). These findings indicate that ARC-18 may influence Aβ production through its involvement in APP processing, potentially in connection with autophagy. 3.5 ARC-18 improved autophagic lysosomal disorders, while CQ reversed this effect in N2a/APPswe cells According to the results of previous Western blot experiments, ARC-18 significantly upregulated the expression of LC3B and downregulated the expression of LAMP1 in 5xFAD mice, improving autophagic lysosomal disorders. Therefore, we performed detection of the co-localization of LC3B and LAMP1 in N2a/APP cells. Based on the co-localization results and statistical analysis, ARC-18 upregulated fluorescence intensity of LC3B and downregulated fluorescence intensity of LAMP1, promoting the fusion and degradation of autophagolysosomes (Fig .5c and Fig .5d). Previous studies have shown that ARC-18 promotes autophagy in 5xFAD mice. To investigate the effects of ARC-18 on autophagy, we conducted a series of experiments using N2a/APPswe cells, incorporating the autophagy inhibitor chloroquine (CQ) for validation purposes. Western blot analysis revealed that ARC-18 did not significantly alter the expression levels of p-mTOR/mTOR, although a slight decreasing trend was noted. However, ARC-18 at concentrations of 100 µM and 200 µM was observed to upregulate the expression of p-ULK1/ULK1, ATG5, Beclin1, and LC3B/LC3A, while simultaneously downregulating P62 protein expression. ARC-18(100 µM) also down-regulates the expression of LAMP1. CQ reversed the autophagy promotion of ARC-18 (Fig .5e and Fig .5f). These findings suggest that ARC-18 facilitates autophagy in N2a/APPswe cells. 3.6 ARC-18 promoted autophagy through the AdipoR1-AMPK signaling pathway We found that ARC-18 was able to up-regulate p-AMPK/AMPK, APN, and AdipoR1 levels in 5xFAD mice (Fig .6e and Fig .6f). To verify whether ARC-18 affects autophagy through AdipoR1-AMPK signaling pathway, we silenced AdipoR1 in N2a/APPswe cells. ARC-18 increased the levels of p-AMPK/AMPK, APN, and AdipoR1 in N2a/APPswe cells. Additionally, ARC-18 upregulated autophagy-related proteins p-ULK1/ULK1 and LC3B/A, and reduced the expression of p62 (Fig .6c and Fig .6d). In addition, silencing AdipoR1 reversed the ARC-18-mediated autophagy promotion through AMPK. These results suggest that ARC-18 promotes autophagy via the AdipoR1-AMPK signaling pathway. 4. Discussion In this investigation, we discovered that a novel derivative of arctigenin(ATG), designated as ARC-18, effectively reduces amyloid-beta (Aβ) deposition and production in both cellular and animal models of AD. Most importantly, ARC-18 rescued cognitive impairments in 5xFAD mice, demonstrating superior efficacy compared to equimolar concentrations of ATG. To elucidate the underlying mechanisms by which ARC-18 mitigates Aβ deposition, we conducted comprehensive mechanistic studies. Our findings reveal that ARC-18 facilitates autophagy and modulates the processing of APP, thereby diminishing Aβ deposition and production. Based on previous studies, we know that 5xFAD mice exhibit disease onset at 1.5 months of age, with evident Aβ deposition and cognitive impairments in the brain by five to six months of age (Chen et al., 2018 ; T. Li et al., 2022 ). In in vivo experiments utilizing the water maze and novel object recognition tests, we identified cognitive impairments in 5xFAD mice at 6 months of age. Notably, treatment with ARC-18 ameliorated these cognitive deficits. Furthermore, pathological analysis and ELISA experiments demonstrated that ARC-18 reduces Aβ deposition in both 5xFAD mice and N2a/APPswe cells, and its effectiveness was superior to equimolar doses of ATG. Amyloid-beta (Aβ) is produced through the proteolytic processing of amyloid precursor protein (APP), a process in which the enzymes β-secretase(sAPPβ) and γ-secretase are integral. Initially, APP undergoes cleavage by β-secretase, resulting in the formation of a C-terminal fragment (CTF) comprising 99 amino acids, referred to as C99. This fragment is subsequently cleaved by γ-secretase, leading to the generation of Aβ peptides. This sequence of enzymatic events is termed the amyloidogenic pathway, culminating in the production of the neurotoxic peptides Aβ 1−40 and Aβ 1−42 (Y. Li, Zhang, Wan, Liu, & Sun, 2020 ; Wang et al., 2017 ). APP can be processed non-amyloidogenically by α-secretase, producing soluble APP-α (sAPPα) and an 83-amino-acid C-terminal fragment (C83). Encouraging this pathway over amyloidogenic processing can reduce Aβ production and delay Alzheimer's disease progression (Jiao et al., 2015 ; Y. Li et al., 2020 ). Although ARC-18 does not significantly affect APP protein levels, it surprisingly reduces sAPPβ and increases sAPPα expression. BACE1, a 501-amino-acid transmembrane protease, acts as β-secretase and is linked to amyloid formation in early-stage AD brain tissue. γ-secretase is a complex involving presenilin 1 (PS1) or presenilin 2 (PS2). PS1 and PS2 contain two aspartate residues crucial for the intramembrane proteolysis of various transmembrane proteins by γ-secretase (Bergmans & De Strooper, 2010 ; O'Brien & Wong, 2011 ). ARC-18 also inhibited BACE1 activity, reduced the expression of PS1. The findings suggest that ARC-18 has the potential to augment α-secretase activity and promote non-amyloidogenic processing, while concurrently inhibiting β-secretase activity and diminishing amyloidogenic processing. A large number of recent studies have shown that defects in Aβ- induced autophagy and mitochondrial autophagy are important events in the pathogenesis of AD (Reddy & Oliver, 2019 ). Electron microscopy has revealed the presence of immature autophagosomes in the brains of AD patients (Nixon et al., 2005 ). These abnormal autophagosomes accumulate due to defects in axonal transport from the distal axon terminals to the cell body, which is caused by nutrient deprivation in neuronal processes. Immature autophagosomes are typically retrogradely transported to the cell body for lysosomal degradation (Z. Zhang, Yang, Song, & Tu, 2021 ). Furthermore, several crucial autophagy-related proteins are downregulated in AD patients, indicating that impaired autophagy exacerbates the progression of AD (K. He et al., 2021 ). In this study, we observed upregulation of LC3B/LC3A expression in 5xFAD mice compared to wild-type mice, which is consistent with previous reports of abnormal accumulation of immature autophagosomes in AD patients (Nixon et al., 2005 ). The important selective autophagy receptor p62 is involved in the clearance of ubiquitinated proteins. Since P62 bound to substrates is degraded by proteases in the lysosomal degradation process, increased levels of P62 are generally considered a marker of suppressed autophagic activity (Dikic, 2017 ; D. Zhang et al., 2023 ). Our study demonstrated that 5xFAD mice exhibited an upregulation of LC3B and P62, indicative of late-stage autophagic dysfunction. Treatment with ARC-18 resulted in increased expression levels of p-ULK1/ULK1, ATG5, Beclin1, and the LC3B/LC3A ratio, while concurrently decreasing the expression of p-mTOR/mTOR and P62. These findings suggest that ARC-18 has the potential to ameliorate late-stage autophagic dysfunction in 5xFAD mice. N2a/APPswe cells are a type of mouse neuroblastoma N2A cells that can stably transfect the human Swedish mutant APP695. It is a classic in vitro model of AD (Yi, Luo, Wang, Dong, & Du, 2023 ; Zou et al., 2024 ). We chose to use an autophagy inhibitor for the reversal experiment because we found that ATG improves cognitive impairments in APP/PS1 mice by promoting autophagy (Zhu et al., 2013 ). ARC-18, as a derivative of ATG, may possess similar effects. Chloroquine (CQ) impedes autophagy by obstructing the fusion of autophagosomes with lysosomes, a process mediated through lysosomal acidification (Ferreira, Sousa, Ferreira, Militão, & Bezerra, 2021 ). It was reported that 5xFAD mice had autophagic lysosomal disorders accompanied by abnormal elevations of P62,LAMP1and CTSD (Chen et al., 2021 ). To investigate ARC-18's impact on autolysosomes, CQ was used to inhibit autophagy in N2a/APPswe cells. The expression of LC3B was positively correlated with the number of autophagosomes. We found that ARC-18 increased the number of autophagosomes and downregulated the expression of p62 protein. Additionally, ARC-18 reduced the expression of the lysosomal marker LAMP1, improving autolysosomal dysfunction, which is consistent with the downregulation of P62 and LAMP1 in ARC-18-treated animals (33..0mg/kg). After conducting a reversal experiment with CQ, we found that the number of autophagosome (LC3B) remained higher than N2a/APPswe cells without ARC-18 treatment group, but the expression of lysosomal marker proteins (LAMP1) was still reduced. This phenomenon may be attributed to ARC-18's capacity to enhance the proliferation of autophagosomes, whereas CQ does not directly influence autophagosomes formation but rather affects the fusion of autolysosomes with lysosomes. Through a combination of in vivo and in vitro experiments utilizing ARC-18, we provide evidence that ARC-18 facilitates the comprehensive autophagic process, encompassing the formation of autophagosomes as well as the fusion and degradation of autolysosomes. According to the current results, ARC-18 promotes autophagy and inhibits the production of Aβ in the APP processing pathway. Understanding the connection between APP processing pathways and autophagy is crucial. Autophagy stress associated with Alzheimer's disease and alterations in autophagosome transport contribute to the accumulation of BACE1 in distal axons. This accumulation exacerbates the amyloidogenic process in amyloid precursor protein (APP), ultimately resulting in increased production of amyloid-beta (Aβ). Subsequently, BACE1 is transported to the autophagolysosome for degradation (Feng, Tammineni, Agrawal, Jeong, & Cai, 2017 ). Our experimental findings demonstrated a significant upregulation of LC3B/LC3A in 5xFAD mice, which facilitated the accumulation of BACE1. Concurrently, there was a notable increase in the levels of P62 and LAMP1 in 5xFAD mice, indicating the presence of autophagic lysosomal dysfunctions that impede the successful degradation of BACE1. The excessive accumulation of BACE1 further exacerbates the production of amyloid-beta (Aβ) within the amyloid precursor protein (APP) processing pathway. ARC-18 not only (33.0mg/kg) up-regulated LC3B/LC3A, but also down-regulated the expression of P62 and LAMP1, which improved autophagic lysosomal disorders, so BACE1 did not accumulate excessively. In order to further substantiate the effect of autophagic lysosomal disorder on BACE1 expression. We selected the autophagic lysosome inhibitor CQ to perform the experiment in N2a/APPswe cells. CQ reversed the autophagy promoting effect of ARC-18 and up-regulated the expression of BACE1, which was consistent with the results of literature reports and animal experiments in this study. These observations imply that BACE1 is excessively recruited and aggregated under conditions of autophagolysosomal dysfunction in Alzheimer's disease models, thereby augmenting the production of Aβ within the APP processing pathway. Adiponectin (APN) is a plasma protein with multipotent effects such as anti-diabetic effects, increased insulin sensitivity in target organs, anti-inflammatory and anti-atherosclerosis (Achari & Jain, 2017 ; Nigro et al., 2014 ). Adiponectin receptors, including AdipoR1 and AdipoR2, are expressed in various regions of the brain, with AdipoR1 performing various roles by activating the AMP-activated protein kinase (AMPK) pathway. Maria Rosaria Rizzo et al. found that serum APN levels may serve as a marker of cognitive decline, highlighting the importance of prevention. In addition, serum APN levels affect cognitive performance and are not associated with obesity (Rizzo, Fasano, & Paolisso, 2020 ). ARC-18 has been shown to improve adiponectin deficiency in 5xFAD mice, potentially functioning through the AdipoR1-AMPK signaling pathway. Previous studies demonstrated that ARC-18 promotes autophagy and reduces the formation of amyloid plaques; however, the relationship between AdipoR1 and autophagy requires further investigation. Autophagy-mediated lipotoxicity is crucial in the progression of diabetic nephropathy. Impaired autophagy is linked to reduced expression of AdipoR1 and phosphorylated AMPK, as well as heightened renal injury (Han et al., 2021 ). We silenced AdipoR1 to examine changes in autophagy-related proteins. The results revealed that silencing AdipoR1 abolished the autophagy-promoting effects of ARC-18, accompanied by downregulation of both p-AMPK and adiponectin expression. These findings suggest that ARC-18 mediates autophagy through the AdipoR1-AMPK signaling pathway. Based on the results of HE staining of individual organs, ARC-18 did not exhibit significant toxic effects at the dose administered. In addition, ARC-18 was able to reduce the expression of GFAP (Glial Fibrillary Acidic Protein) and IBA1(Ionized Calcium Binding Adapter Molecule 1) and alleviate neuroinflammation in 5xFAD mice (Figure S1 - S3 ). ARC-18 has the potential to enhance the limitations associated with the poor water solubility and low bioavailability of ATG in vivo (Table S3 and Table S4 ). This improvement may account for the superior performance of ARC-18 compared to equimolar concentrations of ATG. 5. Conclusions In summary, The pathogenesis of Alzheimer's disease is complex, and there is still a lack of effective drugs to cure the disease. Aβ deposition and autophagy dysfunction are important pathological manifestations of AD. ARC-18 is a derivative of ATG. We have done some studies on autophagy and APP processing and compared it with equimolar ATG. Fig .7 is the mechanism diagram of ARC-18. ARC-18 ameliorates memory impairment and pathologies by activating adiponectin receptor 1-mediated autophagy and regulating the process of APP (Fig .7) . The effectiveness of ARC-18 is superior to equimolar doses of ATG. our study demonstrates that ARC-18 holds promising therapeutic potential for the treatment of Alzheimer’s disease. Declarations CRediT authorship contribution statement Shangming Li: Writing – original draft, Methodology, Investigation, Data curation. Nan Xu, Bocheng Xiong: Methodology, Investigation. Lulin Nie, Kaiwu He, Guiliang Zhang, Chongyang Chen: Methodology, Data curation. Zaijun Zhang: Project administration, Maggie Pui Man Hoi, Xifei Yang: Supervision, Writing – review & editing, Project administration, Investigation, Funding acquisition. Declaration of Competing Interest The authors confirm that they do not have any financial interests or personal ties that could have affected the findings of this paper. Funding declaration This work was funded by the Science and Technology Development Fund of the Macao SAR (FDCT/0023/2020/AFJ, FDCT/0035/2020/AGJ, SKL-QRCM(UM)-2023-2025), the University of Macau Research Funding (MYRG-CRG2022-00010-ICMS, MYRG2022-00248-ICMS), the Key Basic Research Program of Shenzhen Science and Technology Innovation Commission (JCYJ20200109150717745 XY) and Sanming Project of Medicine in Shenzhen (SZSM202211010). Ethical approval Ethics approval for animal work was provided by the Shenzhen Center for Disease Control and Prevention (Project No. 2023036). Data availability The dataset used to support the findings of this study are available from the corresponding author upon request. References Abdrakhmanov, A., Gogvadze, V., & Zhivotovsky, B. (2020). To Eat or to Die: Deciphering Selective Forms of Autophagy. Trends Biochem Sci, 45 (4), 347-364. doi:10.1016/j.tibs.2019.11.006 Achari, A. E., & Jain, S. K. (2017). 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Supplementary Files supplementalmaterial.docx Highlights.docx WBoriginalforFigure3.docx WBoriginalforFigure4.docx WBoriginalforFigure5.docx WBoriginalforFigure6.docx Cite Share Download PDF Status: Published Journal Publication published 14 Feb, 2026 Read the published version in Molecular Neurobiology → Version 1 posted Editorial decision: Revision requested 06 Sep, 2025 Reviews received at journal 05 Sep, 2025 Reviewers agreed at journal 19 Aug, 2025 Reviewers agreed at journal 17 Aug, 2025 Reviewers agreed at journal 16 Aug, 2025 Reviews received at journal 15 Aug, 2025 Reviewers agreed at journal 14 Aug, 2025 Reviewers invited by journal 14 Aug, 2025 Editor assigned by journal 17 Jul, 2025 Submission checks completed at journal 17 Jul, 2025 First submitted to journal 03 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7035906","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":502804371,"identity":"c9b9626b-bd0a-4635-9163-3c1e73cca078","order_by":0,"name":"Shangming Li","email":"","orcid":"","institution":"Shenzhen Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Shangming","middleName":"","lastName":"Li","suffix":""},{"id":502804372,"identity":"a26044cf-d1bf-42d1-bd28-13e91df07f24","order_by":1,"name":"Bocheng Xiong","email":"","orcid":"","institution":"Shenzhen Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Bocheng","middleName":"","lastName":"Xiong","suffix":""},{"id":502804373,"identity":"1101e512-d2e7-4bab-8603-9d3e8f41dafb","order_by":2,"name":"Nan Xu","email":"","orcid":"","institution":"Institute of Chinese Medical Sciences, University of Macau, SAR of China","correspondingAuthor":false,"prefix":"","firstName":"Nan","middleName":"","lastName":"Xu","suffix":""},{"id":502804374,"identity":"333a145b-81d5-4116-a724-caa8614d39b6","order_by":3,"name":"Lulin Nie","email":"","orcid":"","institution":"Shenzhen Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Lulin","middleName":"","lastName":"Nie","suffix":""},{"id":502804375,"identity":"48388ef3-0eb5-4317-8977-c261c838467e","order_by":4,"name":"Kaiwu He","email":"","orcid":"","institution":"Shenzhen Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Kaiwu","middleName":"","lastName":"He","suffix":""},{"id":502804436,"identity":"3b22efa0-f6ad-43de-88a7-ff209c3ee10d","order_by":5,"name":"Guiliang Zhang","email":"","orcid":"","institution":"Institute of Chinese Medical Sciences, University of Macau, SAR of China","correspondingAuthor":false,"prefix":"","firstName":"Guiliang","middleName":"","lastName":"Zhang","suffix":""},{"id":502804437,"identity":"4b0455f7-61a8-45fa-9a84-44a4fdabe143","order_by":6,"name":"Chongyang Chen","email":"","orcid":"","institution":"Shenzhen Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Chongyang","middleName":"","lastName":"Chen","suffix":""},{"id":502804438,"identity":"4204120a-f77b-4b7e-ad07-c17f0837f69f","order_by":7,"name":"Zaijun Zhang","email":"","orcid":"","institution":"Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Zaijun","middleName":"","lastName":"Zhang","suffix":""},{"id":502804439,"identity":"2844925b-98f9-4543-911d-0a60f9a85bd5","order_by":8,"name":"Maggie Pui Man Hoi","email":"","orcid":"","institution":"Institute of Chinese Medical Sciences, University of Macau, SAR of China","correspondingAuthor":false,"prefix":"","firstName":"Maggie","middleName":"Pui Man","lastName":"Hoi","suffix":""},{"id":502804441,"identity":"02d2a590-2d12-4b4b-8965-ea77e413fc71","order_by":9,"name":"Xifei Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYDACZsYHDAkgBnsDkDCwIEYLswFEC88BkBYJoqwxgNASYI1EaDE4DnTZwza7PPnI51c3/CiQYOBv707Aq0WyGeiyxLbkYsPbOWU3e4AOkzhzdgNeLfzM/MckEtuYEzfOzkm7wQPUYiCRi18LGzMz+4/EtvrEjTPPpN38Q4wWfmZmNobEtsOJ8yXYj90myhaQXyQSzh1P3MCTw3ZbxkCCh6BfDM4fZvz4o6w6cX778Wc33/yxkeNv78WvBQwY2YB6D/CAI4iHsHIw+MPAIN/A/oBI1aNgFIyCUTDSAACCpUVwmWsSZgAAAABJRU5ErkJggg==","orcid":"","institution":"Shenzhen Center for Disease Control and Prevention","correspondingAuthor":true,"prefix":"","firstName":"Xifei","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-07-03 08:38:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7035906/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7035906/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12035-026-05731-0","type":"published","date":"2026-02-14T15:59:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89646366,"identity":"893768ee-3c0c-41cf-a6de-f288b90ab73d","added_by":"auto","created_at":"2025-08-22 09:01:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":684365,"visible":true,"origin":"","legend":"\u003cp\u003eARC-18 improved behavioural performance in water maze and novel object recognition in 5xFAD mice. (\u003cstrong\u003ea\u003c/strong\u003e) Mouse movement speed in water maze test. (\u003cstrong\u003eb\u003c/strong\u003e) Number of times for mice traversed the platform in the water maze test. (\u003cstrong\u003ec\u003c/strong\u003e) Percentage of cumulative time in the target area of mice in the water maze test. (\u003cstrong\u003ed\u003c/strong\u003e) Water maze movement track of mice. (\u003cstrong\u003ee\u003c/strong\u003e) Time to first latency of mice in the water maze test. (\u003cstrong\u003ef\u003c/strong\u003e) Preference index in the novel object recognition test. Data are expressed as mean ± SEM. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01 and ****\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs. Wild type mice; #\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, ##\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01 and ###\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001 vs. 5xFAD mice.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7035906/v1/ce640b5482fd59fd91cccb3d.png"},{"id":89646368,"identity":"4265254b-4fb0-430b-a3a9-fc498b2e58ff","added_by":"auto","created_at":"2025-08-22 09:01:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":729018,"visible":true,"origin":"","legend":"\u003cp\u003eARC-18 alleviated Aβ deposition and reduces the levels of total Aβ in 5xFAD mice (\u003cstrong\u003ea\u003c/strong\u003e) Immunohistochemical staining of 6E10 in the hippocampus and cortex of 5 × FAD mice. (\u003cstrong\u003eb\u003c/strong\u003e) amyloid plaque quantification in the hippocampus and frontal cortex. (\u003cstrong\u003ec\u003c/strong\u003e) Levels of Aβ1-40 and Aβ1-42 in frontal cortex. (\u003cstrong\u003ed\u003c/strong\u003e) Levels of Aβ1-40 and Aβ1-42 in the hippocampus. Data are expressed as mean ± SEM. *\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 and ****\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.0001 vs. Wild type mice; #\u003cem\u003e p\u003c/em\u003e\u0026lt; 0.05, ##\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01 and ###\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.001 vs. 5xFAD mice. Multiple comparisons were analyzed by One-Way ANOVA followed by Dunnett test or t-test.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7035906/v1/f921fe277900e86896f0185b.png"},{"id":89646369,"identity":"cdf22186-642e-4a29-bb4a-399f0a485cf2","added_by":"auto","created_at":"2025-08-22 09:01:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":896062,"visible":true,"origin":"","legend":"\u003cp\u003eARC-18 had the effects in promoting autophagy and regulating APP processing pathways in 5xFAD mice. (\u003cstrong\u003ea\u003c/strong\u003e) Western blots of proteins in autophagy pathways and APP processing pathways. (\u003cstrong\u003eb\u003c/strong\u003e) The quantitative analysis of the expression of APP, sAPPα and sAPP\u003csub\u003eβ\u003c/sub\u003e. (\u003cstrong\u003ec\u003c/strong\u003e) The quantitative analysis of the expression of BACE1 and PS1. (\u003cstrong\u003ed\u003c/strong\u003e) The quantitative analysis of the expression of p-mTOR / mTOR, p-ULK1 / ULK1 and P62. (\u003cstrong\u003ee\u003c/strong\u003e) The quantitative analysis of the expression of ATG5, LC3B/LC3A and Beclin1. (\u003cstrong\u003ef\u003c/strong\u003e) The quantitative analysis of the expression of LAMP1 and CTSD. Data are expressed as mean ± SEM. *\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05 and **\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01 vs. Wild type mice; #\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, ##\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01 and ###\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.001 vs. 5xFAD mice.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7035906/v1/47c006311115b9323dd90176.png"},{"id":89646373,"identity":"5fdb588f-f618-40b4-bdaa-b1dc91cdc82f","added_by":"auto","created_at":"2025-08-22 09:01:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":570072,"visible":true,"origin":"","legend":"\u003cp\u003eThe treatment of ARC-18 reduced Aβ production in N2a/APPswe cells by regulating the APP processing pathways. (\u003cstrong\u003ea\u003c/strong\u003e) Western blots of proteins in APP processing pathways. (\u003cstrong\u003eb\u003c/strong\u003e) the IC\u003csub\u003e50\u003c/sub\u003e of ARC-18 in N2a/APPswe cells. (\u003cstrong\u003ec\u003c/strong\u003e) Levels of Aβ1-40 and Aβ1-42 in N2a/APPswe cells. (\u003cstrong\u003ed\u003c/strong\u003e) The quantitative analysis of the expression of APP, sAPPα and sAPP\u003csub\u003eβ. \u003c/sub\u003e(\u003cstrong\u003ee\u003c/strong\u003e) The quantitative analysis of the expression of BACE1 and PS1. Data are expressed as mean ± SEM. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01 vs. N2a/APPswe cells without ARC-18 treatment. #\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, ##\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01, ###\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.001 and ####\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.0001 vs. N2a/APPswe cells with ARC-18 treatment (100μM).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7035906/v1/335f94bba3dc7fb84f4ce49f.png"},{"id":89646378,"identity":"9c73e8b9-f6fa-4aff-b50b-689d77124cc7","added_by":"auto","created_at":"2025-08-22 09:01:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":834569,"visible":true,"origin":"","legend":"\u003cp\u003eARC-18 improved autophagic dysfunction in N2a/APPswe cells. (\u003cstrong\u003ea\u003c/strong\u003e) Western blots of proteins in autophagy pathways. (\u003cstrong\u003eb\u003c/strong\u003e) Immunofluorescence co-localization staining results of LC3B and LAMP1. (\u003cstrong\u003ec\u003c/strong\u003e) Statistical results of LAMP1 fluorescence intensity. (\u003cstrong\u003ed\u003c/strong\u003e) Statistical results of LC3B fluorescence intensity. (\u003cstrong\u003ee\u003c/strong\u003e) The quantitative analysis of the expression of p-mTOR / mTOR, p-ULK1 / ULK1,P62 and Beclin1. (\u003cstrong\u003ef\u003c/strong\u003e) The quantitative analysis of the expression of ATG5, LC3B / LC3A and LAMP1. Data are expressed as mean ± SEM. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01 and ****\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.0001 vs. N2a/APPswe cells without ARC-18 treatment. #\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05 and ##\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01vs. N2a/APPswe cells with ARC-18 treatment (100μM).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7035906/v1/3701ea7337d9a784d5bc672a.png"},{"id":89646391,"identity":"6a05f92d-5112-490b-8bcf-624ddff8e41f","added_by":"auto","created_at":"2025-08-22 09:01:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":658569,"visible":true,"origin":"","legend":"\u003cp\u003eSilencing of AdipoR1 abrogates autophagy promotion by ARC-18\u003cstrong\u003e.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) The result of western blots for adiponectin receptor-associated proteins in 5xFAD mice. (\u003cstrong\u003eb\u003c/strong\u003e) The result of western blots of p-AMPK/AMPK, p-ULK1/ULK1, P62, LC3B/ LC3A, APN and AdipoR1 in N2a/APPswe cells. (\u003cstrong\u003ec\u003c/strong\u003e) The quantitative analysis of the expression of AdipoR1, APN and p-AMPK/AMPK in N2a/APPswe. (\u003cstrong\u003ed\u003c/strong\u003e) The quantitative analysis of the expression of LC3B/ LC3A, p-ULK1/ULK1 and P62 in N2a/APPswe. (\u003cstrong\u003ee\u003c/strong\u003e) The quantitative analysis of the expression of APN, AdipoR1 and AdipoR2 in 5xFAD mice. (\u003cstrong\u003ef\u003c/strong\u003e) The quantitative analysis of the expression of p-AMPK/AMPK in 5xFAD mice. Data are expressed as mean ± SEM. #\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, ##\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01, ###\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.001 and ####\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.0001 vs. si-NC with ARC-18 treatment. *\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05 and **\u003cem\u003e p\u003c/em\u003e\u0026lt; 0.01 vs. Wild type mice; #\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, ##\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01 , ###\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 and ####\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.0001vs. 5xFAD mice.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7035906/v1/1eaf21accdef6bb5191f17c0.png"},{"id":89646380,"identity":"b6333656-3bad-43de-99d3-4680aa7b463f","added_by":"auto","created_at":"2025-08-22 09:01:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":292467,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism diagram:\u003cstrong\u003e \u003c/strong\u003eARC-18 improves memory and Alzheimer's symptoms by activating autophagy through adiponectin receptor 1 and modulating amyloid precursor protein processing.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7035906/v1/e625a57c3abe4d710043a9e5.png"},{"id":102785450,"identity":"ead4af88-7fd8-456f-9d34-bc09817cb24e","added_by":"auto","created_at":"2026-02-16 16:06:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5738669,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7035906/v1/9a60b892-3f36-4100-a4af-f68733fb5126.pdf"},{"id":89649314,"identity":"bfd8b175-af1d-4d89-b054-8da045629a77","added_by":"auto","created_at":"2025-08-22 09:25:23","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2320751,"visible":true,"origin":"","legend":"","description":"","filename":"supplementalmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7035906/v1/ef7bba424c747471b4adc612.docx"},{"id":89646365,"identity":"d3af19d4-d0ab-414f-af3a-ca14981320e6","added_by":"auto","created_at":"2025-08-22 09:01:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15259,"visible":true,"origin":"","legend":"","description":"","filename":"Highlights.docx","url":"https://assets-eu.researchsquare.com/files/rs-7035906/v1/b598d3409b9a6eda5d71e1a9.docx"},{"id":89646384,"identity":"1eb18cf9-92e0-4861-aabe-6b7d07acf50e","added_by":"auto","created_at":"2025-08-22 09:01:23","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6720922,"visible":true,"origin":"","legend":"","description":"","filename":"WBoriginalforFigure3.docx","url":"https://assets-eu.researchsquare.com/files/rs-7035906/v1/8a369f692ebfd3ba17ebf378.docx"},{"id":89647553,"identity":"3146c7fd-512a-4289-bfe8-08d42357ce1f","added_by":"auto","created_at":"2025-08-22 09:09:23","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1979707,"visible":true,"origin":"","legend":"","description":"","filename":"WBoriginalforFigure4.docx","url":"https://assets-eu.researchsquare.com/files/rs-7035906/v1/fc45ed3d3e3be6dfcc1a393a.docx"},{"id":89646392,"identity":"8b928b38-9fb2-4209-bf4c-d6e892d38829","added_by":"auto","created_at":"2025-08-22 09:01:23","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":4126160,"visible":true,"origin":"","legend":"","description":"","filename":"WBoriginalforFigure5.docx","url":"https://assets-eu.researchsquare.com/files/rs-7035906/v1/62ebd4d288aa763d45c4c6df.docx"},{"id":89646394,"identity":"fb58f8c7-6dac-407d-9cfc-4f9913edf082","added_by":"auto","created_at":"2025-08-22 09:01:24","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2718157,"visible":true,"origin":"","legend":"","description":"","filename":"WBoriginalforFigure6.docx","url":"https://assets-eu.researchsquare.com/files/rs-7035906/v1/aec75ddfdd2d8a413e44d285.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A novel arctigenin derivative ameliorates memory impairment and pathologies by activating adiponectin receptor 1-mediated autophagy and regulating amyloid precursor protein processing of Alzheimer's disease","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAlzheimer's disease (AD) is a progressive neurodegenerative disease that ultimately leads to irreversible loss of neurons and intelligence, including cognition and memory. Currently, around 50\u0026nbsp;million people worldwide have AD, and this figure is expected to double every five years, reaching 152\u0026nbsp;million by 2050. AD imposes a significant burden on individuals, families, and the economy, with global annual costs of US\u003cspan\u003e$\u003c/span\u003e1 trillion (Livingston et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yiannopoulou \u0026amp; Papageorgiou, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Two distinct pathogenic protein aggregates, amyloid beta peptide (Aβ) plaques and neurofibrillary tangles containing hyperphosphorylated tau protein, are what characterize AD (Serrano-Pozo, Das, \u0026amp; Hyman, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Aging is the most significant risk factor for late-onset Alzheimer's disease, responsible for over 94% of AD cases (Liu, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Given the inevitability of aging, the prevalence of AD remains substantial. The research of new drugs to treat AD is a difficult problem that humans must overcome.\u003c/p\u003e\u003cp\u003eThe process of cell self-digesting is called autophagy. It consumes its own cytoplasmic material, wraps it into vesicles, and fuses with lysosomes to create autolysosomes, which have a degradative function. Autophagy selectively degrades aging organelles, faulty proteins, and other materials to maintain cellular homeostasis. Initially, autophagy was thought to be a large-scale, non-selective degradation system (Abdrakhmanov, Gogvadze, \u0026amp; Zhivotovsky, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAutophagy, as an important cellular metabolic activity, is involved in amyloid-beta (Aβ) metabolism by regulating Aβ production and clearance. Aβ originates from the processing of amyloid precursor protein (APP), which is sequentially cleaved by β-secretase and γ-secretase. Furthermore, the observation that four subunits of the APP and γ-secretase complexes reside in the autophagosome suggests that at least some of the Aβ peptides are produced via the autophagy pathway (Di Meco, Curtis, Lauretti, \u0026amp; Pratic\u0026ograve;, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Selkoe \u0026amp; Hardy, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Previous studies have shown that when AD model mice were treated with rapamycin, a specific inhibitor of mTOR, autophagy was consequently enhanced. Significant reductions in both intracellular Aβ and extracellular amyloid deposition in the brain were observed, and the animals' cognitive performance was significantly improved (Caccamo, Majumder, Richardson, Strong, \u0026amp; Oddo, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Autophagy appears to affect Aβ clearance at multiple stages. Histone B is a key lysosomal protease required to degrade autophagic substrates. Studies have shown that genetic elimination of histone B worsens AD pathology in AD model mice, including increased Aβ\u003csub\u003e42\u003c/sub\u003e abundance and amyloid deposition. In contrast, when histone B was overexpressed by lentiviral transduction, amyloid plaques were reduced even in aged AD mice (Mueller-Steiner et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In transgenic Alzheimer's disease (AD) mouse models, the induction of autophagy has been shown to decrease BACE1 levels (Wu et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These results suggest that promoting autophagic flow at any stage is beneficial in attenuating AD progression\u003c/p\u003e\u003cp\u003eArctigenin (ATG) is a phenylpropanoid dibenzyl butyrolactone lignan compound that occurs naturally in Arctium lappa L. Previous studies have shown that ATG attenuates learning and memory deficits through PI3k/Akt/GSK-3β pathway reducing Tau hyperphosphorylation in Aβ-Induced AD mice (Qi et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Previous studies have also identified a deficiency of adiponectin in 5xFAD mice (K. He et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). ATG, an agonist of adiponectin receptor 1 (AdipoR1), is known to regulate adiponectin expression, and ARC-18, a derivative of ATG, may target the same pathway (Sun et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Adiponectin is reported to inhibit apoptosis of brain cells induced by cardiac arrest/cardiopulmonary resuscitation by enhancing autophagy via the AdipoR1-AMPK signaling pathway (Y. He et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Dysregulated adiponectin signaling may mediate the detrimental effects of obesity on the central nervous system and increase the risk of cognitive decline and Alzheimer\u0026rsquo;s disease (AD) (Forny-Germano, De Felice, \u0026amp; Vieira, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Based on preliminary findings, we hypothesize that ARC-18 could promote autophagy through the AdipoR1-AMPK signaling pathway, thereby improving memory impairment and pathologies in 5xFAD mice.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Regents and antibodies\u003c/h2\u003e\u003cp\u003eARC-18 (stated purity\u0026thinsp;\u0026ge;\u0026thinsp;98.86%) was synthesized by West China College of Pharmacy, Sichuan University. Arctigenin (stated purity\u0026thinsp;\u0026ge;\u0026thinsp;99.69%, catalog number: 7770-78-7), and chloroquine (CQ) (stated purity\u0026thinsp;\u0026ge;\u0026thinsp;99.50%, catalog number: 54-05-7) were purchased from the company of MedChemExpress (MCE, Monmouth Junction, NJ, USA). Information about antibodies and used in this study has been shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. All primary antibodies are diluted in a ratio of 1:1000. At a dilution of 1:5000, the secondary antibodies utilized were goat anti-mouse IgG H\u0026amp;L (HRP) and goat anti-rabbit IgG H\u0026amp;L (HRP) from Proteintech, USA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Animal experiment\u003c/h2\u003e\u003cp\u003e5xFAD (B6. Cg-Tg (APPSwFlLon, PSEN1*M146L*L286V)6799V) male mice were purchased from Jakson laboratory and bred at Shenzhen Center for Disease Control and Prevention. All experimental operations complied with the ARRIVE guidelines and were approved by the ethics committee of the Shenzhen Center for Disease Control and Prevention (Project No. 2023036). In this experiment, 3-month-old male mice were used as experimental subjects to test the cognitive impairment of mice with Morri\u0026rsquo;s water maze and Novel object recognition. The 3-month-old mice were selected because the transgenic mice began to develop disease at the age of 1.5 months, and the 5-month-old mice could already show significant Aβ aggregation. ARC-18 was dissolved in saline, and ATG was prepared in a solution containing 5% DMSO, 40% PEG400, 5% Tween-80, and 50% saline. The specific groups are as follows: wild-type mice (Saline, gavage), model group (Saline, gavage), ARC-18 low dose group (11.0mg/kg, gavage), ARC-18 median dose group (33.0mg/kg, gavage) ARC-18 high dose group (99.0mg/kg, gavage) and ATG group (24.3mg/kg, gavage). At the conclusion of the experiment, the mice were anesthetized and then sacrificed using 4% sodium pentobarbital to obtain blood and tissue samples. The dosage of the drug was determined based on acute toxicity tests (Table .S2) and articles on ARC-18 for amyotrophic lateral sclerosis (Xiong et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Behavioral tests\u003c/h2\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1 Morris water maze\u003c/h2\u003e\u003cp\u003eIn a pool with a diameter of 120 cm and a depth of 76 cm; a platform of 10 cm\u003csup\u003e2\u003c/sup\u003e; adjust the pool and the camera position to ensure that the platform position is at an angle of 45\u0026deg; in the middle of the axis and remains in the same position during the experiment; milky white water in the pool is submerged in the platform for 0.5-1 cm and the temperature of the water is maintained at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C; four markers of different shapes and colours are affixed to the wall of the pool in the direction of the axis and the markers cannot be changed during the experiment. Training period: (1) Mice were placed in the desired starting position in the maze, facing the wall of the pool. The mice were released to the water surface. Once released, the computer tracking programme was initiated. (2) The timer was stopped when the mice reached (touched) the platform, and the mice were allowed 60 s per trial.(3) Animals that did not find the platform within this time period were artificially placed or guided to the platform and allowed to remain on the platform for 15 s. (4) The mice were taken out of the water maze, dried off, and placed in a warm environment to prevent them from catching cold. (5) Place the mice in a new starting position in the water maze and repeat steps 1\u0026ndash;4 until the animal has completed the required 4 training sessions that day. The experiment was trained for a total of 6 days. Test period: The purpose of the exploration test was to determine if the animal remembered the location of the platform. After 5 consecutive days of training, the station in the water maze was removed and the mouse was placed in a new starting position from the maze to ensure that its behaviour reflected a memory of the target location rather than a specific swimming path. For example, from the initial platform position facing the wall of the pool, the mouse was released to the water surface and its trajectory was observed. After 2 min, the machine automatically stopped the timer and the mouse was removed. The first day was the platform visibility period to determine whether the mice had visual defects. The rest of the day is a platform hiding period (Vorhees \u0026amp; Williams, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2 Novel object recognition\u003c/h2\u003e\u003cp\u003eOn the first day, mice were individually placed in a 50 cm\u003csup\u003e2\u003c/sup\u003e square box and allowed to adapt to the environment for 5 minutes. After exploration, the mice were removed and returned to breeding cages. The behavioral box was cleaned by removing feces and urine, followed by wiping with 75% alcohol to eliminate odor. On the second training day, mice were put in a cage with two identical objects. On the third day, one object was swapped with a unfamiliar one. For more details, refer to the previous report (Chen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The results were expressed as a preference index percentage, calculated using the formula [exploration time of the new object / (exploration time of the new object\u0026thinsp;+\u0026thinsp;exploration time of the old object) \u0026times;100%].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Immunohistochemistry\u003c/h2\u003e\u003cp\u003eThe Abcam ABC HRP kit (ab64264) was used, and all procedures were performed according to the manufacturer\u0026rsquo;s instructions. Specifically, (1) paraffin-embedded brain sections were first deparaffinized in xylene and then rehydrated in a gradient of ethanol-water. (2) The brain sections were placed in citrate buffer, heated in a microwave for 10 minutes, and then cooled at room temperature for 30 minutes to retrieve antigens. (3) 3% H2O2 was added, covering the sections for 10 minutes, followed by 3 washes in PBS, each for 5 minutes. 3% bovine serum albumin was applied onto the brain sections and incubated at room temperature for 1 hour. (4) The samples were incubated with Aβ antibody (#803015, Biolegend) overnight at 4\u0026deg;C, followed by incubation with biotinylated goat secondary antibody at room temperature for 1 hour, and then with avidin-peroxidase at room temperature for 30 minutes. After each antibody incubation, the sections were washed three times with PBST for 10 minutes each. (5) The brain sections were stained with diaminobenzidine (DAB) for 2 minutes. After dehydration in a gradient of ethanol, the sections were cleared in xylene and sealed with neutral resin. (6) The brain sections were imaged using an optical microscope (Aperio GT 450, Leica Biosystems, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Cell experiment\u003c/h2\u003e\u003cp\u003eDulbecco\u0026rsquo;s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (P/S), 0.25% trypsin-EDTA were purchased from Gibco/Invitrogen (USA). N2a/APPswe cells (kindly gifted by Professor Jian-zhi Wang, Tongji Medical School, HUST, China) were cultured in dishes with DMEM supplemented with 10% FBS in the presence of 200\u0026micro;g/mL geneticin(G418) and 1% P/S. The concentrations of ARC-18 on cells were 50, 100 and 200 \u0026micro;M, which were determined based on the IC50 of the ARC-18 assay and the concentration of arctigenin administered on cells (Song, Li, Liu, Hu, \u0026amp; Yang, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Chloroquine was administered at a concentration of 50 micromoles.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Concentration tests for Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e and Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e\u003c/h2\u003e\u003cp\u003eConcentrations of Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e and Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e were tested on brain tissue and cells. The contents of Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e and Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e were determined using an ELISA assay, following the protocol provided by the manufacturer (Elabscience Biotechnology Co., Ltd, China). The levels of amyloid-beta (Aβ) were subsequently normalized to the total protein concentrations.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Interruption of AdipoR1\u003c/h2\u003e\u003cp\u003esiRNA is chemically synthesized and Lipofectamine 3000 (Thermo Fisher Scientific, USA) is transfected into N2a/APPswe cells. The sequence of AdipoR1 (mouse) targeting siRNA is as follows: CAGCTTTCGTCC ACTTCTA. Unrelated siRNA sequences were used as negative controls. AdipoR1 targeted siRNA and unrelated siRNA sequences were synthesized by SANTA CRUZ BIOTECHNOLOGY (El-Andaloussi et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The final concentration of siRNA is 50nM.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e\u003cp\u003eData were presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical analyses were conducted using GraphPad Prism version 8.0 software. Comparisons between two groups were performed using an unpaired t-test. A one-way analysis of variance (ANOVA) was employed to assess the statistical significance of differences among multiple groups, followed by Dunnett's multiple comparison test. A \u003cem\u003ep\u003c/em\u003e-value of less than 0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Result","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1ARC-18 improved the performance of Morris water maze and Novel object recognition in 5xFAD mice\u003c/h2\u003e\u003cp\u003eTo assess the cognitive differences between wild-type mice and 5xFAD mice, as well as the therapeutic effects of ARC-18, we conducted the Morris water maze test (MWM) and Novel object recognition (NOR) test. As shown in Fig .1a, there were no significant differences in swimming speed among the groups, indicating comparable locomotor abilities that would not affect cognitive function assessment. The wild-type mice exhibited a significantly higher number of platform crossings compared to the untreated 5xFAD group, suggesting that the untreated 5xFAD mice demonstrated impaired cognitive function in locating the platform. Following administration of a median dose of ARC-18 (33.0 mg/kg), the 5xFAD mice showed a marked increase in the number of platform crossings, surpassing the effects observed with equimolar doses of ATG (Fig .1b). Compared to the untreated 5xFAD group, the median-dose ARC-18 (33.0mg/kg) group improved the target quadrant cumulative time percentage in 5xFAD mice, and the effect was superior to ATG (Fig .1c). This is a water maze movement track of mice (Fig .1d). As illustrated in Fig .1e, the graph delineates the time required by mice to locate the platform during the initial trial, segmented into three distinct phases. On the first day of training, the platform is conspicuously positioned above the water surface to evaluate potential disparities in visual acuity among the groups, thereby mitigating experimental errors attributable to visual impairments. This phase is designated as the \u0026ldquo;visible platform period\u0026rdquo;. Subsequently, from the second to the fifth day, the platform is submerged, and the latency for the mice to identify the platform during their initial trial is systematically recorded. This phase is known as the \u0026ldquo;hidden platform period\u0026rdquo;. On the sixth day of testing, untreated 5xFAD mice took significantly longer than wild-type mice to find the platform. Median-dose ARC-18 (33.0 mg/kg) treatment reduced this latency in 5xFAD mice more effectively than equimolar doses of ATG. In Fig .1f, compared to wild-type mice, the untreated 5xFAD mice exhibited a significant decrease in the percentage of time spent exploring the novel object. Notably, after median-dose ARC-18 treatment, the percentage of time spent exploring the novel object in 5xFAD mice significantly increased. Based on the above experimental results, it could be concluded that ARC-18 improves cognitive dysfunction in 5xFAD mice.\u003c/p\u003e\u003ch2\u003e3.2 ARC-18 alleviated Aβ deposition and reduced the concentrations of total Aβ in the hippocampus and cortex of 5xFAD mice\u003c/h2\u003e\u003cp\u003eOverdeposition of Aβ is an important factor in the development of AD. To assess the pathogenesis of 5xFAD mice, we performed immunohistochemical staining of Aβ and tested the concentrations of Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e and Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e in the hippocampus and prefrontal cortex by Elisa. Aβ aggregation was markedly elevated in 5xFAD mice relative to wild-type counterparts. Administration of a median-dose ARC-18 (33.0 mg/kg) resulted in a significant reduction in Aβ deposition, demonstrating a more pronounced effect than equimolar doses of ATG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). ARC-18 did not affect the levels of Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e in the prefrontal cortex; however, it significantly decreased the levels of Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e (Fig .2c). In comparison to untreated 5xFAD mice, ARC-18 treatment led to a significant reduction in the concentrations of both Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e and Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e in the hippocampus (Fig .2d). These results suggest that ARC-18 mitigates Aβ deposition and lowers the overall levels of Aβ in 5xFAD mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3 ARC-18 promoted autophagy and regulated the processing of APP in 5xFAD mice\u003c/h2\u003e\u003cp\u003eTo investigate how ARC-18 reduces Aβ deposition, we examined the expression of proteins involved in the APP processing pathway through Western blot analysis. Untreated 5xFAD mice had significantly higher APP expression than wild-type mice, which was reduced by ARC-18 treatment. Median-dose ARC-18 (33.0 mg/kg) significantly increased sAPPα expression and decreased sAPP\u003csub\u003eβ\u003c/sub\u003e expression, outperforming equimolar doses of ATG (Fig .3b). Compared to wild-type mice, the expression of BACE1 and Presenilin 1(PS1) was significantly increased in untreated 5xFAD mice. Moreover, after ARC-18 treatment, the expression of BACE1 and PS1 was significantly decreased (Fig .3c). These results suggest that ARC-18 reduced Aβ production by modulating APP processing.\u003c/p\u003e\u003cp\u003eIt has been reported that excessive deposition of Aβ could lead to dysfunction in autophagy. Arctigenin (ATG) has been shown to increase Aβ clearance by promoting autophagy in AD mouse model (Zhu et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). ARC-18, a derivative of ATG may have similar effects. To investigate whether ARC-18 could enhance Aβ clearance by promoting autophagy, we conducted Western blot validation in the hippocampal area of 5xFAD mice. Compared to the 5xFAD mice treated with saline, the high-dose ARC-18 group (33.0 mg/kg) significantly decreased the expression of p-mTOR/mTOR and P62, while increasing the expression of p-ULK1/ULK1, ATG5, Beclin1, and LC3B/LC3A (Fig .3d and Fig .3e). LAMP1 and Cathepsin D (CTSD) are proteins associated with autophagic lysosomes. Compared to the 5xFAD mice treated with saline, ARC-18 promoted the degradation of LAMP1 but did not affect the expression of CTSD (Fig .3f). These results demonstrate that ARC-18 ameliorates autophagic dysfunction in 5xFAD mice, and the effect is superior to equimolar doses of ATG.\u003c/p\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.4 CQ reversed the effect of ARC-18 in amyloidogenic processing of APP in \u003cem\u003evitro\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTo investigate the effects of ARC-18 on Aβ production, the levels of Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e and Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e were tested in the cell lysate of N2a/APPswe cells with or without ARC-18 treatment. At the same time, we verified the proteins associated with the APP processing pathway. The IC50 of ARC-18 was approximately 471.0 \u0026micro;M in N2a/APPswe cells (Fig .4b). Based on the IC50 data, the drug demonstrated effective activity at concentrations of 50, 100, and 200 \u0026micro;M without adversely affecting normal cell growth. At a concentration of 100 \u0026micro;M, ARC-18 significantly reduced the levels of Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e and Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e in N2a/APPswe cells, which was reversed by the autophagy inhibitor CQ (Fig .4c). ARC-18 was also found to upregulate the expression of sAPP\u003csub\u003eα\u003c/sub\u003e, while exhibiting a certain trend on APP and sAPP\u003csub\u003eβ\u003c/sub\u003e levels. Administration of ARC-18 in combination with a CQ inhibitor upregulated the expression of APP and sAPP\u003csub\u003eβ\u003c/sub\u003e, compared to administration of ARC-18 (100\u0026micro;M) alone. (Fig .4d). Additionally, ARC-18 decreased the expression of Presenilin 1(PS1), but did not affect BACE1 levels with a certain trend. The expressions of BACE1 and PS1 were changed after CQ administration (Figure .4e). These findings indicate that ARC-18 may influence Aβ production through its involvement in APP processing, potentially in connection with autophagy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.5 ARC-18 improved autophagic lysosomal disorders, while CQ reversed this effect in N2a/APPswe cells\u003c/h2\u003e\u003cp\u003eAccording to the results of previous Western blot experiments, ARC-18 significantly upregulated the expression of LC3B and downregulated the expression of LAMP1 in 5xFAD mice, improving autophagic lysosomal disorders. Therefore, we performed detection of the co-localization of LC3B and LAMP1 in N2a/APP cells. Based on the co-localization results and statistical analysis, ARC-18 upregulated fluorescence intensity of LC3B and downregulated fluorescence intensity of LAMP1, promoting the fusion and degradation of autophagolysosomes (Fig .5c and Fig .5d).\u003c/p\u003e\u003cp\u003ePrevious studies have shown that ARC-18 promotes autophagy in 5xFAD mice. To investigate the effects of ARC-18 on autophagy, we conducted a series of experiments using N2a/APPswe cells, incorporating the autophagy inhibitor chloroquine (CQ) for validation purposes. Western blot analysis revealed that ARC-18 did not significantly alter the expression levels of p-mTOR/mTOR, although a slight decreasing trend was noted. However, ARC-18 at concentrations of 100 \u0026micro;M and 200 \u0026micro;M was observed to upregulate the expression of p-ULK1/ULK1, ATG5, Beclin1, and LC3B/LC3A, while simultaneously downregulating P62 protein expression. ARC-18(100 \u0026micro;M) also down-regulates the expression of LAMP1. CQ reversed the autophagy promotion of ARC-18 (Fig .5e and Fig .5f). These findings suggest that ARC-18 facilitates autophagy in N2a/APPswe cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.6 ARC-18 promoted autophagy through the AdipoR1-AMPK signaling pathway\u003c/h2\u003e\u003cp\u003eWe found that ARC-18 was able to up-regulate p-AMPK/AMPK, APN, and AdipoR1 levels in 5xFAD mice (Fig .6e and Fig .6f). To verify whether ARC-18 affects autophagy through AdipoR1-AMPK signaling pathway, we silenced AdipoR1 in N2a/APPswe cells. ARC-18 increased the levels of p-AMPK/AMPK, APN, and AdipoR1 in N2a/APPswe cells. Additionally, ARC-18 upregulated autophagy-related proteins p-ULK1/ULK1 and LC3B/A, and reduced the expression of p62 (Fig .6c and Fig .6d). In addition, silencing AdipoR1 reversed the ARC-18-mediated autophagy promotion through AMPK. These results suggest that ARC-18 promotes autophagy via the AdipoR1-AMPK signaling pathway.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this investigation, we discovered that a novel derivative of arctigenin(ATG), designated as ARC-18, effectively reduces amyloid-beta (Aβ) deposition and production in both cellular and animal models of AD. Most importantly, ARC-18 rescued cognitive impairments in 5xFAD mice, demonstrating superior efficacy compared to equimolar concentrations of ATG. To elucidate the underlying mechanisms by which ARC-18 mitigates Aβ deposition, we conducted comprehensive mechanistic studies. Our findings reveal that ARC-18 facilitates autophagy and modulates the processing of APP, thereby diminishing Aβ deposition and production.\u003c/p\u003e\u003cp\u003eBased on previous studies, we know that 5xFAD mice exhibit disease onset at 1.5 months of age, with evident Aβ deposition and cognitive impairments in the brain by five to six months of age (Chen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; T. Li et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In in vivo experiments utilizing the water maze and novel object recognition tests, we identified cognitive impairments in 5xFAD mice at 6 months of age. Notably, treatment with ARC-18 ameliorated these cognitive deficits. Furthermore, pathological analysis and ELISA experiments demonstrated that ARC-18 reduces Aβ deposition in both 5xFAD mice and N2a/APPswe cells, and its effectiveness was superior to equimolar doses of ATG.\u003c/p\u003e\u003cp\u003eAmyloid-beta (Aβ) is produced through the proteolytic processing of amyloid precursor protein (APP), a process in which the enzymes β-secretase(sAPPβ) and γ-secretase are integral. Initially, APP undergoes cleavage by β-secretase, resulting in the formation of a C-terminal fragment (CTF) comprising 99 amino acids, referred to as C99. This fragment is subsequently cleaved by γ-secretase, leading to the generation of Aβ peptides. This sequence of enzymatic events is termed the amyloidogenic pathway, culminating in the production of the neurotoxic peptides Aβ \u003csub\u003e1\u0026minus;40\u003c/sub\u003e and Aβ \u003csub\u003e1\u0026minus;42\u003c/sub\u003e (Y. Li, Zhang, Wan, Liu, \u0026amp; Sun, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). APP can be processed non-amyloidogenically by α-secretase, producing soluble APP-α (sAPPα) and an 83-amino-acid C-terminal fragment (C83). Encouraging this pathway over amyloidogenic processing can reduce Aβ production and delay Alzheimer's disease progression (Jiao et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Y. Li et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Although ARC-18 does not significantly affect APP protein levels, it surprisingly reduces sAPPβ and increases sAPPα expression. BACE1, a 501-amino-acid transmembrane protease, acts as β-secretase and is linked to amyloid formation in early-stage AD brain tissue. γ-secretase is a complex involving presenilin 1 (PS1) or presenilin 2 (PS2). PS1 and PS2 contain two aspartate residues crucial for the intramembrane proteolysis of various transmembrane proteins by γ-secretase (Bergmans \u0026amp; De Strooper, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; O'Brien \u0026amp; Wong, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). ARC-18 also inhibited BACE1 activity, reduced the expression of PS1. The findings suggest that ARC-18 has the potential to augment α-secretase activity and promote non-amyloidogenic processing, while concurrently inhibiting β-secretase activity and diminishing amyloidogenic processing.\u003c/p\u003e\u003cp\u003eA large number of recent studies have shown that defects in Aβ- induced autophagy and mitochondrial autophagy are important events in the pathogenesis of AD (Reddy \u0026amp; Oliver, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Electron microscopy has revealed the presence of immature autophagosomes in the brains of AD patients (Nixon et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). These abnormal autophagosomes accumulate due to defects in axonal transport from the distal axon terminals to the cell body, which is caused by nutrient deprivation in neuronal processes. Immature autophagosomes are typically retrogradely transported to the cell body for lysosomal degradation (Z. Zhang, Yang, Song, \u0026amp; Tu, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, several crucial autophagy-related proteins are downregulated in AD patients, indicating that impaired autophagy exacerbates the progression of AD (K. He et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this study, we observed upregulation of LC3B/LC3A expression in 5xFAD mice compared to wild-type mice, which is consistent with previous reports of abnormal accumulation of immature autophagosomes in AD patients (Nixon et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The important selective autophagy receptor p62 is involved in the clearance of ubiquitinated proteins. Since P62 bound to substrates is degraded by proteases in the lysosomal degradation process, increased levels of P62 are generally considered a marker of suppressed autophagic activity (Dikic, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; D. Zhang et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Our study demonstrated that 5xFAD mice exhibited an upregulation of LC3B and P62, indicative of late-stage autophagic dysfunction. Treatment with ARC-18 resulted in increased expression levels of p-ULK1/ULK1, ATG5, Beclin1, and the LC3B/LC3A ratio, while concurrently decreasing the expression of p-mTOR/mTOR and P62. These findings suggest that ARC-18 has the potential to ameliorate late-stage autophagic dysfunction in 5xFAD mice.\u003c/p\u003e\u003cp\u003eN2a/APPswe cells are a type of mouse neuroblastoma N2A cells that can stably transfect the human Swedish mutant APP695. It is a classic in vitro model of AD (Yi, Luo, Wang, Dong, \u0026amp; Du, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zou et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). We chose to use an autophagy inhibitor for the reversal experiment because we found that ATG improves cognitive impairments in APP/PS1 mice by promoting autophagy (Zhu et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). ARC-18, as a derivative of ATG, may possess similar effects. Chloroquine (CQ) impedes autophagy by obstructing the fusion of autophagosomes with lysosomes, a process mediated through lysosomal acidification (Ferreira, Sousa, Ferreira, Milit\u0026atilde;o, \u0026amp; Bezerra, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It was reported that 5xFAD mice had autophagic lysosomal disorders accompanied by abnormal elevations of P62,LAMP1and CTSD (Chen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To investigate ARC-18's impact on autolysosomes, CQ was used to inhibit autophagy in N2a/APPswe cells. The expression of LC3B was positively correlated with the number of autophagosomes. We found that ARC-18 increased the number of autophagosomes and downregulated the expression of p62 protein. Additionally, ARC-18 reduced the expression of the lysosomal marker LAMP1, improving autolysosomal dysfunction, which is consistent with the downregulation of P62 and LAMP1 in ARC-18-treated animals (33..0mg/kg). After conducting a reversal experiment with CQ, we found that the number of autophagosome (LC3B) remained higher than N2a/APPswe cells without ARC-18 treatment group, but the expression of lysosomal marker proteins (LAMP1) was still reduced. This phenomenon may be attributed to ARC-18's capacity to enhance the proliferation of autophagosomes, whereas CQ does not directly influence autophagosomes formation but rather affects the fusion of autolysosomes with lysosomes. Through a combination of in vivo and in vitro experiments utilizing ARC-18, we provide evidence that ARC-18 facilitates the comprehensive autophagic process, encompassing the formation of autophagosomes as well as the fusion and degradation of autolysosomes.\u003c/p\u003e\u003cp\u003eAccording to the current results, ARC-18 promotes autophagy and inhibits the production of Aβ in the APP processing pathway. Understanding the connection between APP processing pathways and autophagy is crucial. Autophagy stress associated with Alzheimer's disease and alterations in autophagosome transport contribute to the accumulation of BACE1 in distal axons. This accumulation exacerbates the amyloidogenic process in amyloid precursor protein (APP), ultimately resulting in increased production of amyloid-beta (Aβ). Subsequently, BACE1 is transported to the autophagolysosome for degradation (Feng, Tammineni, Agrawal, Jeong, \u0026amp; Cai, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our experimental findings demonstrated a significant upregulation of LC3B/LC3A in 5xFAD mice, which facilitated the accumulation of BACE1. Concurrently, there was a notable increase in the levels of P62 and LAMP1 in 5xFAD mice, indicating the presence of autophagic lysosomal dysfunctions that impede the successful degradation of BACE1. The excessive accumulation of BACE1 further exacerbates the production of amyloid-beta (Aβ) within the amyloid precursor protein (APP) processing pathway. ARC-18 not only (33.0mg/kg) up-regulated LC3B/LC3A, but also down-regulated the expression of P62 and LAMP1, which improved autophagic lysosomal disorders, so BACE1 did not accumulate excessively. In order to further substantiate the effect of autophagic lysosomal disorder on BACE1 expression. We selected the autophagic lysosome inhibitor CQ to perform the experiment in N2a/APPswe cells. CQ reversed the autophagy promoting effect of ARC-18 and up-regulated the expression of BACE1, which was consistent with the results of literature reports and animal experiments in this study. These observations imply that BACE1 is excessively recruited and aggregated under conditions of autophagolysosomal dysfunction in Alzheimer's disease models, thereby augmenting the production of Aβ within the APP processing pathway.\u003c/p\u003e\u003cp\u003eAdiponectin (APN) is a plasma protein with multipotent effects such as anti-diabetic effects, increased insulin sensitivity in target organs, anti-inflammatory and anti-atherosclerosis (Achari \u0026amp; Jain, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Nigro et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Adiponectin receptors, including AdipoR1 and AdipoR2, are expressed in various regions of the brain, with AdipoR1 performing various roles by activating the AMP-activated protein kinase (AMPK) pathway. Maria Rosaria Rizzo et al. found that serum APN levels may serve as a marker of cognitive decline, highlighting the importance of prevention. In addition, serum APN levels affect cognitive performance and are not associated with obesity (Rizzo, Fasano, \u0026amp; Paolisso, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). ARC-18 has been shown to improve adiponectin deficiency in 5xFAD mice, potentially functioning through the AdipoR1-AMPK signaling pathway. Previous studies demonstrated that ARC-18 promotes autophagy and reduces the formation of amyloid plaques; however, the relationship between AdipoR1 and autophagy requires further investigation. Autophagy-mediated lipotoxicity is crucial in the progression of diabetic nephropathy. Impaired autophagy is linked to reduced expression of AdipoR1 and phosphorylated AMPK, as well as heightened renal injury (Han et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We silenced AdipoR1 to examine changes in autophagy-related proteins. The results revealed that silencing AdipoR1 abolished the autophagy-promoting effects of ARC-18, accompanied by downregulation of both p-AMPK and adiponectin expression. These findings suggest that ARC-18 mediates autophagy through the AdipoR1-AMPK signaling pathway.\u003c/p\u003e\u003cp\u003eBased on the results of HE staining of individual organs, ARC-18 did not exhibit significant toxic effects at the dose administered. In addition, ARC-18 was able to reduce the expression of GFAP (Glial Fibrillary Acidic Protein) and IBA1(Ionized Calcium Binding Adapter Molecule 1) and alleviate neuroinflammation in 5xFAD mice (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-\u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). ARC-18 has the potential to enhance the limitations associated with the poor water solubility and low bioavailability of ATG in \u003cem\u003evivo\u003c/em\u003e (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e and Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). This improvement may account for the superior performance of ARC-18 compared to equimolar concentrations of ATG.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn summary, The pathogenesis of Alzheimer's disease is complex, and there is still a lack of effective drugs to cure the disease. Aβ deposition and autophagy dysfunction are important pathological manifestations of AD. ARC-18 is a derivative of ATG. We have done some studies on autophagy and APP processing and compared it with equimolar ATG. \u003cb\u003eFig .7\u003c/b\u003e is the mechanism diagram of ARC-18. ARC-18 ameliorates memory impairment and pathologies by activating adiponectin receptor 1-mediated autophagy and regulating the process of APP \u003cb\u003e(Fig .7)\u003c/b\u003e. The effectiveness of ARC-18 is superior to equimolar doses of ATG. our study demonstrates that ARC-18 holds promising therapeutic potential for the treatment of Alzheimer\u0026rsquo;s disease.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShangming Li:\u0026nbsp;\u003c/strong\u003eWriting – original draft, Methodology, Investigation, Data curation. \u003cstrong\u003eNan Xu, Bocheng Xiong:\u0026nbsp;\u003c/strong\u003eMethodology, Investigation. \u003cstrong\u003eLulin Nie, Kaiwu He,\u003c/strong\u003e \u003cstrong\u003eGuiliang Zhang, Chongyang Chen:\u003c/strong\u003e Methodology, Data curation.\u003cstrong\u003e\u0026nbsp;Zaijun Zhang:\u0026nbsp;\u003c/strong\u003eProject administration, \u003cstrong\u003eMaggie Pui Man Hoi, Xifei Yang:\u003c/strong\u003eSupervision, Writing – review \u0026amp; editing, Project administration, Investigation, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that they do not have any financial interests or personal ties that could have affected the findings of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Science and Technology Development Fund of the Macao SAR (FDCT/0023/2020/AFJ, FDCT/0035/2020/AGJ, SKL-QRCM(UM)-2023-2025), the University of Macau Research Funding (MYRG-CRG2022-00010-ICMS, MYRG2022-00248-ICMS), the Key Basic Research Program of Shenzhen Science and Technology Innovation Commission (JCYJ20200109150717745 XY) and Sanming Project of Medicine in Shenzhen (SZSM202211010).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval for animal work was provided by the Shenzhen Center for Disease Control and Prevention (Project No.\u0026nbsp;2023036).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset used to support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdrakhmanov, A., Gogvadze, V., \u0026amp; Zhivotovsky, B. 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Inhibition of autophagosome-lysosome fusion contributes to TDCIPP-induced A\u0026beta;1-42 production in N2a-APPswe cells. \u003cem\u003eHeliyon, 10\u003c/em\u003e(8), e26832. doi:10.1016/j.heliyon.2024.e26832\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Alzheimer's disease, adiponectin receptor 1, autophagy, A novel arctigenin derivative, 5xFAD mice, N2a/APPswe cells","lastPublishedDoi":"10.21203/rs.3.rs-7035906/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7035906/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlzheimer's disease (AD), the most prevalent form of dementia, is characterized as a slowly progressing neurodegenerative condition marked by neurotic plaques and neurofibrillary tangles due to the buildup of amyloid-beta peptide (Aβ) in the brain's medial temporal lobe and neocortical structures. It is reported that arctigenin (ATG) has the effect to reduce the expression of the enzyme 1 that cleaves β-site amyloid precursor protein and increase Aβ clearance by enhancing autophagy. Compound ARC-18 is a derivative of ATG. The main objective of this study is to investigate whether ARC-18 could improve cognitive function and disease progression in Alzheimer's mice by promoting autophagy. 3-month-old 5\u0026times;FAD mice were orally treated with drug for 3 consecutive months. Water maze and new object recognition were used to assess cognitive impairment in 5xFAD mice. In the hippocampus of the mouse brain, APP processing-related proteins (sAPP\u003csub\u003eβ\u003c/sub\u003e, BACE1) and autophagy (LC3B, P62, LAMP1) related proteins were detected. Some experiments related to animal data were conducted on N2a/APPswe cells to further identify the effect and mechanisms of drug. ARC-18 improved behavioral performance in water maze and new object recognition in 5xFAD mice. ARC-18 alleviated the over-aggregation of Aβ in the hippocampus and cortex of 5xFAD mice. ARC-18 promotes autophagy and inhibits amyloidogenic processing of APP in 5xFAD mice and N2a/APPswe cells. ARC-18 improves AD-like pathologies and memory impairment by increased clearance of Aβ by activating adiponectin receptor 1-mediated autophagy and reduced Abeta production via regulating amyloid precursor protein (APP) processing.\u003c/p\u003e","manuscriptTitle":"A novel arctigenin derivative ameliorates memory impairment and pathologies by activating adiponectin receptor 1-mediated autophagy and regulating amyloid precursor protein processing of Alzheimer's disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 09:01:18","doi":"10.21203/rs.3.rs-7035906/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-06T22:36:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-05T17:18:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62007924717729750241676804866271962100","date":"2025-08-20T00:22:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"97278701636526385992772303058753683029","date":"2025-08-18T01:08:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"65373914409709596476507288799793109130","date":"2025-08-16T14:19:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-15T08:12:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"243601311808042578675067503071537921258","date":"2025-08-15T00:40:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-14T15:15:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-17T08:44:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-17T08:39:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurobiology","date":"2025-07-03T08:29:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6bbb602b-b362-4c7b-a607-0b49b9f10fdc","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T16:03:34+00:00","versionOfRecord":{"articleIdentity":"rs-7035906","link":"https://doi.org/10.1007/s12035-026-05731-0","journal":{"identity":"molecular-neurobiology","isVorOnly":false,"title":"Molecular Neurobiology"},"publishedOn":"2026-02-14 15:59:01","publishedOnDateReadable":"February 14th, 2026"},"versionCreatedAt":"2025-08-22 09:01:18","video":"","vorDoi":"10.1007/s12035-026-05731-0","vorDoiUrl":"https://doi.org/10.1007/s12035-026-05731-0","workflowStages":[]},"version":"v1","identity":"rs-7035906","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7035906","identity":"rs-7035906","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}