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The autocrine motility factor receptor delays the pathological progression of Alzheimer’s disease via regulating the ubiquitination-mediated degradation of APP

Alzheimer's Research & Therapy volume 17, Article number: 95 (2025) Cite this article

Abstract

Background

The ubiquitin-proteasome system (UPS) is responsible for most protein degradation and its malfunction is normally observed in neurodegenerative diseases, including Alzheimer’s disease (AD). The autocrine motility factor receptor (AMFR) is an E3 ubiquitin ligase that resides on the endoplasmic reticulum membrane and is involved in various essential biological processes. However, the role of AMFR in AD is still unidentified.

Methods

Behavioral experiments, including open-field test (OFT), novel object recognition test (NORT) and morris water maze test (MWMT) were conducted after adeno-associated virus (AAV) microinjection into AD model mice. Western blot, co-immunoprecipitation (Co-IP), qPCR and ubiquitination assay were used to analyze AMFR mediated ubiquitination degradation of amyloid precursor protein (APP). ELISA was employed to evaluate changes in amyloidogenic cleavage products of APP following upregulation or downregulation of AMFR in neural cells and analyze AMFR levels in serum and cerebrospinal fluid (CSF) of AD patients.

Results

The progressive decline in AMFR levels was found not only in the hippocampus of APPswe/PSEN1dE9 (APP/PS1) mice but also in the CSF and serum of patients with AD. Moreover, the interaction of AMFR and APP was observed both in hippocampal tissues and brain neurons. In addition, AMFR promoted the K11-linked polyubiquitination of APP to speed up its proteasomal degradation, resulting in decreased Aβ production. Importantly, AMFR overexpression largely rescued the cognitive and synaptic deficits in APP/PS1 mice.

Conclusions

Taken together, our results demonstrated that AMFR reduced Aβ production and alleviated cognitive impairment by promoting the ubiquitination-mediated degradation of APP. This study indicated that AMFR could have the potential to be a therapeutic target of early-stage AD.

Background

Alzheimer’s disease (AD), characterized by progressive cognitive decline, is the most common type of senile dementia [1], accounting for approximately 60%–70% of dementia cases worldwide [2]. Approximately 78 million patients will suffer from Alzheimer’s dementia in 2030 [3]. This number could grow to 139 million by 2050 owing to population aging [3]. Despite efforts exerted on the development of potential drugs that can slow down AD progression, no known drugs or effective treatments are in existence because of the limited understanding of the mechanism of AD [4].

The histopathological hallmarks of AD are extraneuronal amyloid-β (Aβ) plaques and intraneuronal neurofibrillary tangles, including hyperphosphorylated tau protein, which contributes to neuronal degeneration, synapse loss, and subsequent cognitive deficits [5]. Despite the complicated mechanism of AD, the accumulation of amyloid-β (Aβ) plaques generated by amyloid precursor protein (APP) is considered a central feature and initial factor of AD [6]. APP synthesis is regulated by various factors, including transcriptional regulation and post-translational modification (PTM), among which ubiquitylation is an important regulator of APP synthesis [7,8,9,10,11,12]. Ubiquitination modification mainly depends on the ubiquitin-proteasome pathway (UPP), which is essential for preventing the aggregation of abnormal intracellular proteins [13,14,15]. The ubiquitination process involves three component enzymes containing ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3) [16,17,18]. The combination of E3 ubiquitin ligase and the substrate protein is regarded as the most pivotal step in the proteasomal degradation of the target protein [19,20,21]. Recent studies have indicated that E3 ubiquitin ligase murine double minute 2 (MDM2) mediated PSD-95 turnover and played a role in AD pathogenesis [22]. Moreover, CHIP, a brain-enriched E3 ubiquitin ligase, promoted the ubiquitination and UPP-mediated degradation of β-secretase (BACE1), leading to the stabilization of APP in reducing Aβ generation [23]. However, whether E3s regulate APP degradation remains limited.

The autocrine motility factor receptor (AMFR), also known as gp78, is a RING-dependent and endoplasmic reticulum (ER) membrane-anchored E3 ubiquitin ligase that recognizes dysfunctional proteins for subsequent degradation via the ubiquitin-proteasome system (UPS) in an ER-associated degradation (ERAD) manner [24, 25]. AMFR reportedly participated in various biological and pathological processes, such as cellular signal transduction and metabolism, tumor formation and maintenance, neurodegenerative disorders, and innate immune response [24,25,26,27,28]. A previous study showed that AMFR might be involved in learning and memory in the central nervous system. Moreover, the mRNA and protein expression of AMFR were significantly decreased in the hippocampus of AD model mice [29]. The findings above indicated that AMFR might play a key role in the AD process.

In this study, we found that AMFR promoted APP degradation by interacting with APP to mediate its K11-linked polyubiquitination. In addition, AMFR overexpression markedly decreased amyloid deposition and ameliorated cognitive deficits in APP/PS1 mice.

Materials and methods

Human samples

The cerebrospinal fluid (CSF) samples from 164 participants were enrolled for examination: 30 patients with mild cognitive impairment (MCI) of Alzheimer’s type (nmale = 11, nfemale = 19), 35 patients with AD (nmale = 16, nfemale = 19), 64 patients with non-AD dementias (vascular dementia [VaD], n = 16, nmale = 10, nfemale = 6; Parkinson’s disease with dementia [PDD], n = 12, nmale = 7, nfemale = 5; dementia with Lewy bodies [DLB], n = 14, nmale = 6, nfemale = 8; and frontotemporal dementia [FTD], n = 22, nmale = 12, nfemale = 10), and 35 individuals with normal cognition (motor neuron disease, n = 6; hemifacial spasm, n = 4; trigeminal neuralgia, n = 5; peripheral nerve disease, n = 5; myelopathy, n = 6; cerebrovascular malformation, n = 4; body weakness, n = 5; nmale = 14, nfemale = 21). All patients were diagnosed by doctors of the Neurology Department of Xuanwu Hospital according to the international consensus criteria for MCI [30], AD [31], DLB [32], FTD [33], PDD [34], and VaD [35]. Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) were used as a measure of global cognition. In addition, 279 participants were enrolled for serum collection, including 40 patients with MCI (nmale = 18, nfemale = 22), 40 with AD (nmale = 17, nfemale = 23), 159 with non-AD dementias (VaD, n = 42, nmale = 26, nfemale = 16; PDD, n = 39, nmale = 26, nfemale = 13; DLB, n = 40, nmale = 22, nfemale = 18; and FTD, n = 38, nmale = 20, nfemale = 18) and 40 age-matched healthy controls (HCs) (nmale = 18, nfemale = 22). The same diagnostic criteria were applied. Patient demographics in different CSF and serum groups are listed in Table S1.

Serum and CSF samples were collected from other 60 participants (AD, n = 26; motor neuron disease, n = 3; hemifacial spasm, n = 4; trigeminal neuralgia, n = 3; peripheral nerve disease, n = 8; myelopathy, n = 7; cerebrovascular malformation, n = 2; body weakness, n = 5; papilledema, n = 2), with each pair of these two samples from the same individual. The participants included 27 males and 33 females, with the mean age of 60.08 years old.

CSF samples from other 155 participants with normal cognition (motor neuron disease, n = 15; hemifacial spasm, n = 27; trigeminal neuralgia, n = 16; peripheral nerve disease, n = 24; myelopathy, n = 28; cerebrovascular malformation, n = 11; body weakness, n = 25; papilledema, n = 9) were divided into four groups: 40–49 years old (n = 40; nmale = 18, nfemale = 22); 50–59 years old (n = 45; nmale = 23, nfemale = 22); 60–69 years old (n = 40; nmale = 22, nfemale = 18), 70–79 years old ( n = 30; nmale = 15, nfemale = 15). Participants characterization in different age groups are listed in Table S2.

Approximately 500 μL of CSF and 1 mL of serum was obtained and stored at − 80 °C for subsequent analyses. All procedures were approved by the Ethics Committee of Xuanwu Hospital of Capital Medical University and were performed according to the guidelines of the Declaration of Helsinki.

Animals and treatment

For this study, 2-, 5-, and 8-month-old APPswe/PS1dE9 (APP/PS1) mice (n = 6 for each age group) and corresponding-age C57BL/6 mice (n = 6 for each group) were purchased from Zhishan Institute of Health Medicine Co. Ltd (Beijing, China). After a week of balancing, the mice were euthanized, and the brain tissues were collected for further experiments. In another experiment, 6-month-old APP/PS1 mice (n = 12) and 6-month-old C57BL/6 mice (n = 6) were used for adeno-associated virus (AAV) microinjection. APP/PS1 mice were further divided into two groups: the AAV9-Ctrl-microinjection group (n = 6) and the AAV9-AMFR-microinjection group (n = 6). The mice were anesthetized using sterile Avertin (2,2,2-tribromoethanol, 400–500 mg/kg body weight; Acros Organics), and the surgical area was prepared by removing hair. They were then secured in a stereotaxic frame (Kopf Instruments), and bilateral 1-mm-diameter openings were created in the skull using a mounted drill (Kopf Instruments). Subsequently, meloxicam (2 mg/kg) was administered subcutaneously for pain relief, and bupivacaine (1 mg/kg) was applied topically. For hippocampal injections, stereotaxic coordinates relative to the bregma were as follows: anterior/posterior, − 2.7; medial/lateral, ± 1.8; and dorsoventral, − 2.0 (mice weighing < 23 g) or − 2.1 (mice weighing > 23 g). A blunt 32-gauge, 1.27 cm-long needle attached to a 5-μL Hamilton syringe was affixed to the stereotaxic frame to deliver 1 μL of PAAV-ITR-CMV-mcherry-3Xflag-WPRE-SV40_polyA-ITR (2.56 × 1013 particles/mL), or PAAV-ITR-CMV-Amfr (mouse NM011787)-mcherry-3Xflag-WPRE-SV40_polyA-ITR (1.18 × 1012 particles/mL) at a rate of 0.1 μL/min into the right and left injection sites. The needle remained in place for another 3 min after infusion. After needle withdrawal, the surgical site was sealed with Vetbond tissue adhesive (3 M). The mice were monitored under a heating lamp until they fully recovered and then returned to their home cage. After 40 days of treatment, behavioral tests were conducted. Finally, all animals were euthanized, and brain tissues were collected for further analysis. The mice were kept in cages under a constant temperature and humidity and were exposed to a 12/12 h day–night cycle with free access to tap water and food. All mice in our study were male to exclude the effects of sex differences on AD-related cognitive impairment [36,37,38]. The animal experiments were approved by the Bioethics Committee of Xuanwu Hospital of Capital Medical University and complied with the National Institute of Health Guide for the Care and Use of Laboratory Animals.

Behavioral testing

Open-field test (OFT)

Mice were placed in the center of a clear plastic chamber (40 cm × 40 cm × 40 cm) with dim light and allowed to explore freely in the open field for 10 min using the Smart 3.0 video tracking system. The total distance traveled in the arena, time exploring in the center zone, and center entry times of each animal were analyzed. In addition, 70% ethanol was used to entirely clean the apparatus before subsequent tests to avoid the scent clues left by the previous mouse.

Novel object recognition test (NORT)

This test comprised habituation, training, and testing phases performed in an open-field arena (40 cm × 40 cm × 40 cm, same apparatus in OFT) for three consecutive days. Before training, mice were allowed to explore freely in the arena (no objects; data used for open-field assays) for 10 min. During training, mice were exposed to two identical objects (A and B) placed in two corners of the open field arena and allowed to explore for 5 min. After 24 h, object B was replaced with a novel object (C), and the mice were allowed to freely explore the arena for 5 min. To minimize olfactory cues left by the previous mouse, the objects and apparatus were cleaned with 70% ethanol before subsequent tests. The exploratory behavior of the mice was recorded and analyzed with a Smart 3.0 video tracking system. The time that the mice spent next to each object and the exploration time were recorded. The recognition index was calculated as follows: [(exploration time to the novel object)/(exploration time to the novel object + exploration times to the familiar object)].

Morris water maze test (MWMT)

The maze consisted of a 90-cm-diameter tank full of water (22℃ ± 2℃) made opaque with nontoxic white tempera paint (colored powder tempera paint), and a platform was placed in a designated quadrant of the tank. The walls surrounding the tank contained different shapes that served as visual reference cues. A camera was mounted above the maze to record the swimming behavior in the water maze. For hidden platform training, the platform was submerged 1.5 cm below the surface, and the mice were placed into the maze at one of four points (N, S, E, and W) facing the wall of the tank. Mice were subjected to five consecutive days of training to locate a hidden platform, with each day consisting of four trials. During each trial, mice were gently placed into the water from different release points and given 60 s to find the platform. The time taken to locate the hidden platform (escape latency) was recorded for each trial. If the mouse failed to find the platform within 60 s, it was gently guided to the submerged platform and allowed to remain there for 15 s, with the escape latency recorded as 60 s. On day 6, the hidden platform was removed, and a probe test was conducted. During this test, the following parameters were automatically recorded for subsequent analysis: swimming paths, number of crossings into the target quadrant, time spent in the target quadrant, and swimming speed. These measures were used to assess spatial memory and learning retention in mice after the training period.

Cell culture and transfection

HEK293T, SH-SY5Y, and N2a cells were sourced from ATCC. SH-SY5Y human neuroblastoma cells and SH-SY5Y cells stably expressing wild-type full-length human APP (designated as SH-SY5Y-hAPP) were cultured in minimum essential medium (Biological Industries, Israel) supplemented with 10% fetal bovine serum (Biological Industries) and 1% penicillin/streptomycin (Invitrogen, CA, USA; 15140122). Similarly, HEK293T cells and HEK293T cells stably expressing wild-type full-length human APP (referred to as HEK293T-hAPP) were cultured in Dulbecco’s modified eagle’s medium (Biological Industries) supplemented with 10% fetal bovine serum (Biological Industries) and 1% penicillin/streptomycin. These cells were maintained in a 37 °C humidified atmosphere with 5% CO2. For transfection purposes, DNA constructs were transfected into cells using the jetOPTIMUS Transfection Reagent (Polyplus-transfection Inc., France; 101000051) and Lipofectamine™ LTX Reagent with PLUS™ Reagent (Thermo Fisher Scientific, MA, USA; 15338100), while siRNA was transfected using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, MA, USA; 13778100).

DNA constructs and RNA interference

His-AMFR was constructed using the pLV-CMV-MCS-6×His (SyngenTech, Beijing) construct as backbones. Flag-APP was constructed using the pLV-CMV-MCS-3×Flag (SyngenTech) construct as the backbone. Wild-type (WT) human ubiquitin and a series of its truncated mutants tagged with HA were constructed using the CMV enhancer-MCS-SV40 (Genechem, Shanghai) construct as backbones.

Human AMFR siRNA and control siRNA were obtained from SyngenTech, and the target sequences for AMFR siRNA were as follows: siAMFR-1, 5′-GGGAAGAACAUCAAGGAGATT-3′; siAMFR-2, 5′-GCAAACGUUUCUUGAACAATT-3′; siAMFR-3, 5′-GCAAGGAUCGAUUUGAAUATT-3′.

Antibodies and reagents

The following antibodies were purchased: rabbit anti-AMFR (1:1000, Proteintech, 16675-1-AP, Wuhan, China), rabbit anti-Flag (1:10,000, Proteintech, 20543-1-AP), mouse anti-His (1:10,000, Proteintech, 66005-1-Ig), mouse anti-GAPDH (1:10,000, Proteintech, 60004-1-Ig), mouse anti-ubiquitin (1:1000; Cell Signaling Technology, MA, USA; 3936), rabbit anti-APP (1:10,000; Abcam, Cambridge, UK; ab32136) mouse anti-APP (1:100; Invitrogen, CA, USA; 14-9749-82), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H + L) secondary antibody (1:10,000; ZSGB-BIO, Beijing, China; ZB-2306), HRP-conjugated goat anti-mouse IgG (H + L) secondary antibody (1:10,000; ZSGB-BIO, Beijing, China; ZB-2305), Alexa Fluor 488 donkey anti-rabbit IgG (H + L) (1:200, Abcam, ab150073), and Alexa Fluor 594 donkey anti-mouse IgG (H + L) (1:200, Abcam, ab150108). The proteasome inhibitor MG132 and protein synthesis inhibitor cycloheximide (CHX) were obtained from MedChemExpress (NJ, USA). 4′, 6-diamidino-2-phenylindole (DAPI) was from Sigma-Aldrich (MO, USA). Protease and phosphatase inhibitor cocktails were purchased from Beyotime Biotech Inc. (China), and the BCA protein assay kit was from Thermo Fisher Scientific.

Quantitative real-time polymerase chain reaction (RT-qPCR)

RNA was collected from brain tissue samples or cultured cells with Trizol reagent (Invitrogen, 15596018CN) according to the product manual and then quantified with a spectrophotometer. A reverse-transcription reaction was conducted using 1.0 μg of total RNA and 5 × All-In-One RT MasterMix (ABM). Subsequently, RT-qPCR was performed using SYBR green reagents (Takara, Japan) on a Roche 480 machine. Primer pairs for RT-qPCR are listed in Table 1. Gene expression analysis utilized the 2 − ΔΔCt method, with relative mRNA expression levels normalized to GAPDH mRNA levels. The PCR products of mice gene identification were used by agarose gel electrophoresis.

Table 1 Primer sequences for quantitative real-time polymerase chain reaction
Full size table

Co-immunoprecipitation (Co-IP) and Western blot

Total proteins were harvested from cells and brain tissue samples with ice-cold lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, and 1% Triton X-100, 1 mM EDTA) containing PMSF (Solarbio, China; P0100) and protease inhibitor cocktail (Beyotime, P1005). The lysate was centrifuged at 13,000 × g for 15 min at 4 °C. The protein concentration of the lysate was determined using the BCA protein assay kit (Thermo Fisher Scientific, 23227) according to the manufacturer’s protocol. For immunoprecipitation, 500–800 μg of protein was incubated with 4–5 μg of specific antibodies of APP (Abcam, ab32136), Flag-Tag (Proteintech, 20543-1-AP), and IgG Isotype Control (Invitrogen, 10400C) for 12 h at 4ºC with constant rotation, followed by incubation with 40 μL (0.40 mg) of PureProteome™ Protein A/G Mix Magnetic Beads (Millipore, LSKMAGAG10) at 4 °C for an additional 4–6 h with rotation. The magnetic beads were then washed three times with ice-cold lysis buffer. The proteins bound to the beads were extracted by resuspending the beads in 1 × sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and boiling at 95 °C for 10 min. The resultant IP products or cell lysates were resolved using 8% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes (Millipore, IPVH00010). After blocking, the membranes were incubated overnight at 4 °C with appropriate antibodies. Ultimately, specific protein bands were visualized using a chemiluminescence Western HRP substrate kit (Yeasen, 36222ES60).

Proximity ligation assay (PLA)

The protein binding level of AMFR with APP in the hippocampal tissues of APP/PS1 mice of different ages was detected in situ using the Duolink detection kit (Sigma, 92101) according to the instructions of the manufacturer. The primary antibody incubation against AMFR (1:100, Proteintech, 16675-1-AP) and APP (1:100, Invitrogen, 14-9749-82) were applied using the same conditions as immunocytochemistry staining. Duolink secondary antibodies against the primary antibodies were then added. Coverslips were mounted using Duolink In situ Mounting Media with DAPI. The signals were detected through rolling circle amplification, during which DNA polymerase incorporated fluorescently labeled nucleotides into the amplified products. The red fluorescent spots could be visualized, and each dot represented the interaction of two proteins. Images were captured through fluorescence microscopy.

Immunofluorescence analysis

For brain collection, adult mice were anesthetized with an intraperitoneal injection of 10% sterile Avertin and then intracardiacally perfused with 0.9% NaCl followed by cold freshly prepared 4% paraformaldehyde (PFA, Solarbio, P1110). Thereafter, the perfused brains were harvested and fixed with 4% PFA for 24 h at 4 °C. Frozen sections of fixed brain tissues with 10-μm thickness were prepared. Immunofluorescence co-staining was performed on free-floating sections with anti-AMFR (1:100, Proteintech, 16675-1-AP) and anti-APP antibody (1:100, Invitrogen, 14-9749-82). The sections were blocked with 10% donkey serum (Solarbio, SL050) for 2 h, followed by incubation with the primary antibodies diluted in 1% donkey serum overnight at 4 °C. Then, the sections were incubated for 1 h with fluorescent secondary antibodies: Alexa Fluor 488 donkey anti-rabbit IgG (H + L) (1:200, Abcam, ab150073) and Alexa Fluor 594 donkey anti-mouse IgG (H + L) (1:200, Abcam, ab150108). Nuclei were stained with 4′,6-diamidino-2-phenylindole (1:1000; Merck, NJ, USA; D9542). After mounting, the samples were analyzed by fluorescence microscopy.

Cells were fixed in 4% PFA and permeabilized in 0.1% Triton X-100. Subsequently, cells were blocked with 10% donkey serum and incubated with primary antibodies overnight at 4 °C, followed by the secondary antibodies (Alexa Flour 488 donkey anti-rabbit and Alexa Flour 594 donkey anti-mouse). The cell nuclei were stained with DAPI. Images were acquired using a Zeiss LSM 800 confocal laser microscope equipped with an Airyscan module.

In vitro and in vivo ubiquitination assay

To detect the Ub ligase activity of AMFR, the Ubiquitin Conjugation kit (UBBiotech) was used. Briefly, recombinant UBE1 (E1; 0.75 μg), UBE2G2 (E2; 6 μg), GST-AMFR-His (E3), ubiquitin (10 μg), and purified His-APP were mixed in the reaction buffer (1 mM ATP, 60 μM DTT, 500 μM Tris, and 100 μM MgCl2). The reactions were conducted at 37 °C for 4 h, terminated by adding 5 × loading buffer, and boiled at 95 °C for 5 min. The ubiquitinated APP was detected in a Western blot with the anti-ubiquitin (1:1000; Cell Signaling Technology, MA, USA; 3936) antibodies.

For the in vivo ubiquitination assays, cells were transfected with the indicated plasmids and then lysed in ice-cold buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) supplemented with phenylmethylsulfonyl fluoride (PMSF, Solarbio, P0100) and protease inhibitor cocktail (Beyotime, P1005). The co-immunoprecipitation assays were performed as previously described. The immunoprecipitated protein or cell lysate protein was detected with AMFR (1:1000, Proteintech, 16675-1-AP), APP (1:10,000, Abcam, ab32136), GAPDH (1:10,000, Proteintech, 10494-1-AP) and ubiquitin (1:1000, Cell Signaling Technology, 3936) antibodies.

ELISA

For AMFR overexpression, SH-SY5Y cells stably expressing APP were transfected with a His-AMFR plasmid using jetOPTIMUS Transfection Reagent (Polyplus-transfection Inc, 101000051). For the knockdown experiments of AMFR, SH-SY5Y cells stably expressing APP were transfected with AMFR-siRNA using LipofectAMINE RNAiMAX reagent (Thermo Fisher Scientific, 13778100) and incubated for 72 h. The amounts of Aβ40, Aβ42, and sAPPβ in cells were measured by sandwich ELISA kits (MLbio, Shanghai, China). Aβ40, Aβ42, and sAPPβ levels in the mouse hippocampus extract and the serum and CSF AMFR concentrations in patients were measured using the corresponding sandwich ELISA kits (Aβ40, Aβ42, and sAPPβ, MLbio; AMFR, FineTest, Wuhan, China) according to the manufacturer’s instructions.

Thioflavin S staining

Thioflavin S staining was performed to label Aβ plaques. Frozen sections of brain tissues were stained with 0.3% Thioflavin-S (Invitrogen) in the dark for 8 min in 50% ethanol and washed with 50% ethanol thrice and with PBS three times. Afterward, brain sections were covered with a glass cover using an antifade mounting medium and observed under a fluorescence microscope.

Electrophysiology

Acute hippocampal transversal slices were prepared from WT mice and APP/PS1 mice injected with the AAV. Briefly, mice were anesthetized with isoflurane and then decapitated, and their brains were dropped in a chilled high-sucrose cutting solution consisting of the following: 5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 212.7 mM sucrose, 10 mM glucose, 1 mM CaCl2, and 3 mM MgCl2. Hippocampi were dissected and cut into 300-μm thick transverse slices with the Leica VT1200 Vibratome. The slices were then incubated for 30 min at 32 °C in artificial CSF (ACSF) containing the following: 5 mM KCl, 124 mM NaCl, 1.25 mM NaH2PO4, 10 mM glucose, 26 mM NaHCO3, 2.4 mM CaCl2, and 1.2 mM MgCl2. Meanwhile, the ACSF was bubbled with 95% O2 and 5% CO2 to maintain pH, and the slices were allowed to recover at room temperature for 30 min before recording. Whole-cell patch-clamp recordings were performed on pyramidal neurons in the CA1 stratum, using a BX51WI microscope and an infrared-sensitive charge-coupled device camera for identification. All neurons selected had pyramidal-shaped cell bodies and single apical dendrites, and experiments were limited to the densely populated CA1 region. The Axon Multiclamp 700B amplifier was used for recording, and data were digitized using pClamp version 10.8.

Statistical analysis

The statistical analyses of data were conducted using GraphPad Prism 9 software. All data were presented as mean ± standard error of the mean (SEM). One- or two-way analysis of variance (ANOVA) was performed for multiple comparisons. Student’s t-test was used for the statistical analysis of two independent treatments. The relationship between serum and CSF AMFR level was assessed with simple linear regression analysis. The operating characteristic curves (ROC) for analytes were generated to evaluate the clinical efficiency of serum or CSF AMFR level in AD and other types of dementia. Analysis of covariance (ANCOVA) was used to compare variables among different groups after adjusting for age and sex. P < 0.05 indicated statistical significance.

Results

AMFR levels decrease in AD

To clarify the involvement of AMFR in AD pathology, the AMFR mRNA and protein levels in the hippocampal tissues of WT and APP/PS1 mice at different ages were initially investigated. Our data showed that the AMFR mRNA levels in the 8-month-old hippocampal tissues of APP/PS1 mice notably decreased compared to those of age-matched WT mice and 2- and 5-month-old APP/PS1 mice (Fig. 1A). Likewise, AMFR protein level showed an obvious trend of reduction during aging, and the 8-month-old APP/PS1 mice exhibited a significant decrease compared to the age-matched WT mice, which indicated a potential role of AMFR in AD progression (Fig. 1B). A similar result which AMFR level reduced during aging was also observed in human CSF (Fig. S1). To further support the role of AMFR in AD and explore the clinical significance of this E3 ubiquitin ligase, we examined the levels of AMFR in both serum and paired CSF of patients and analyzed their correlation. The data showed that there were a certain positive correlation between the levels of serum and CSF AMFR (Fig. 1C). This indicated that changes in level of serum AMFR might be influenced by AD-related pathology as CSF AMFR level. Moreover, after standardizing the results on subjects’ ages and sex, we found that serum AMFR level slightly decreased at the MCI stage; however, it was lower in the AD group than that in the HC and MCI groups (Fig. 1D). In the CSF samples, the AMFR levels evidently decreased at the MCI stage (Fig. 1E). The detailed data adjusted for age and sex were displayed in Table S3 and Table S4. The alteration of AMFR level in the serum and CSF of patients at different stages of AD further demonstrated the involving of AMFR in the development of AD. In addition, we also investigated AMFR levels in the serum and CSF of different types of dementia and found differences among these diseases, suggesting that AMFR might be meaningful in their pathogenesis (Fig. S2). These results indicated that the level of AMFR decreased with AD progression and might be crucially involved in AD.

Fig. 1
figure 1

AMFR levels in APP/PS1 mice and in the serum and CSF of AD patients. A AMFR mRNA levels in the hippocampus of WT (n = 6) and APP/PS1 (n = 6) mice in different age periods. Data are shown as mean ± SEM from three independent experiments (*P < 0.05). B AMFR protein levels in the hippocampus of WT (n = 6) and APP/PS1 (n = 6) mice in different age periods. Data are shown as mean ± SEM from three independent experiments (*P < 0.05, ****P < 0.0001). C Relationship between the serum AMFR concentration (X-axis) and CSF AMFR concentration (Y-axis) of participants (n = 60). D Serum AMFR levels of patients with different stages of AD by ELISA (HC, n = 40; MCI, n = 40; AD, n = 40). Data are shown as mean ± SEM (**P < 0.01, ***P < 0.001). E CSF AMFR levels of patients with different stages of AD by ELISA (NC, n = 35; MCI, n = 30; AD, n = 35). Data are shown as mean ± SEM (*P < 0.05)

Full size image

AMFR interacts with APP

Considering that AMFR is an ER-resident E3 ubiquitin ligase, AMFR deficiency might counteract the APP ubiquitin system. To test this, whether AMFR interacted with APP was first examined. Co-IP results showed that endogenous AMFR interacted with APP in hippocampal tissues of 8-month-old APP/PS1 mice (Fig. 2A). In addition, endogenous and exogenous AMFR interacted with APP in SH-SY5Y (Fig. 2B, C) and HEK293T cells (Fig. 2D, E). Then, the subcellular localization of AMFR and APP was determined by confocal microscopy assay. As shown in Fig. 2F, AMFR and APP were localized together in primary neurons of APP/PS1 embryonic mice, SH-SY5Y, HT22, and N2a cells. To further confirm the interaction between these two proteins, the Duolink PLA was conducted using primary neurons of APP/PS1 embryonic mice (Fig. 2G). PLA confirmed the direct interaction between the two proteins, in which AMFR and APP with primary antibodies were recognized by anti-mouse and anti-rabbit PLA DNA probes. If the probes were within 40 nm of each other, the oligos were ligated and amplified to produce red fluorescent spots. Taken together, these data indicated that AMFR interacted with APP, resulting in the generation of an AMFR-APP complex.

Fig. 2
figure 2

AMFR associates with APP. A Endogenous AMFR and APP interaction in the hippocampus of 8-month APP/PS1 mice by Co-IP assays. The protein expression levels of AMFR and APP in the tissue lysates were confirmed. Data presented are representative of three separate experiments. B Endogenous AMFR and APP interaction in SH-SY5Y cells by Co-IP assays. The protein expression levels of AMFR and APP in the cell lysates were confirmed. Data presented are representative of three separate experiments. C Exogenous AMFR and APP interaction in SH-SY5Y cells transfected with empty vector or His-AMFR and empty vector or Flag-APP by Co-IP assays. The protein expression levels of AMFR and APP in the cell lysates were confirmed. Data presented are representative of three separate experiments. D Endogenous AMFR and APP interaction in HEK293T cells by Co-IP assays. The protein expression levels of AMFR and APP in the cell lysates were confirmed. Data presented are representative of three separate experiments. E Exogenous AMFR and APP interaction in HEK293T cells transfected with empty vector or His-AMFR and empty vector or Flag-APP by Co-IP assays. The protein expression levels of AMFR and APP in the cell lysates were confirmed. Data presented are representative of three separate experiments. F AMFR and APP co-localization in primary neurons of APP/PS1 embryonic mice, SH-SY5Y, HT22, and neuro-2a cells by confocal microscopy assays. AMFR (green), APP (red), cell nucleus (DAPI, blue), merge (AMFR + APP + DAPI); scale bars represent 50 μm. Data presented are representative of three separate experiments. G Endogenous AMFR and APP interaction in primary neurons of APP/PS1 embryonic mice by proximity ligation assay (PLA). The PLA signal is shown in red. Scale bars represent 1000 μm. Data presented are representative of three separate experiments.

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AMFR is involved in APP degradation via the proteasome-dependent pathway

The results above demonstrated that AMFR physically associated with APP (Fig. 2). Then, we explored whether AMFR regulated APP expression. The Western blot analysis showed that AMFR upregulation decreased APP protein level in SH-SY5Y cells stably overexpressing human APP (SH-SY5Y-hAPP) (Fig. 3A) and HEK293T-hAPP cells (Fig. 3B). In contrast, AMFR silencing significantly increased APP protein level (Fig. 3C). Meanwhile, the changes of AMFR had no effects on APP mRNA levels in SH-SY5Y-hAPP cells (Fig. 3D) and HEK293T-hAPP cells (Fig. 3E), which indicated that AMFR negatively regulated APP at protein level. To further test whether AMFR affected APP protein stability, SH-SY5Y-hAPP cells transfected with control or His-AMFR plasmid were treated with 50 µM CHX, a protein synthesis inhibitor, and harvested at the indicated time points. The Western blot assay showed that AMFR overexpression obviously shortened the half-life of APP protein (Fig. 3F), demonstrating that AMFR could regulate APP degradation. Considering there are two mainly pathways of protein degradation–the autophagy lysosomal pathway and the ubiquitin–proteasome pathway, we investigated the autophagy lysosomal pathway and found no alteration with increased or decreased levels of AMFR (Fig. S3). Thus, whether AMFR could regulate APP degradation through the proteasome pathway was explored. The results showed when AMFR expression was upregulated or downregulated in the presence of MG132, a protease inhibitor, which can accurately inhibit the ubiquitin-mediated proteasome pathway, the AMFR-induced decrease in APP level was inhibited in SH-SY5Y-hAPP cells (Fig. 3G, H). Similarly, the increase in APP level was observed after MG132 administration in HEK293T-hAPP cells transfected with His-AMFR plasmid (Fig. 3I). Collectively, these data indicated the involvement of AMFR in the proteasome-mediated degradation of APP.

Fig. 3
figure 3

AMFR mediates proteasome-associated APP degradation. A AMFR and APP protein levels in SH-SY5Y-hAPP cells transfected with empty vector or His-AMFR. Data are shown as mean ± SEM from three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001). B AMFR and APP protein levels in HEK293T-hAPP cells transfected with empty vector or His-AMFR. Data are shown as mean ± SEM from three independent experiments (**P < 0.01, ****P < 0.0001). C AMFR and APP protein levels in SH-SY5Y-hAPP cells transfected with siRNA-control or siRNA-AMFR. Data are shown as mean ± SEM from three independent experiments (*P < 0.05). D mRNA levels of AMFR and APP in SH-SY5Y-hAPP cells transfected with empty vector or His-AMFR by RT-qPCR assays. Data are shown as mean ± SEM from three independent experiments (ns, not significant, ****P < 0.0001). E AMFR mRNA levels in HEK293T-hAPP cells transfected with empty vector or His-AMFR by quantitative real-time PCR assays. Data are shown as mean ± SEM from three independent experiments (ns, not significant, **P < 0.01, ****P < 0.0001). F AMFR and APP protein levels in SH-SY5Y-hAPP cells transfected with empty vector or His-AMFR at different durations of cycloheximide administration. Data are shown as mean ± SEM from three independent experiments (*P < 0.05). G AMFR and APP protein levels in SH-SY5Y-hAPP cells transfected with empty vector or His-AMFR at DMSO or MG132 administration for 6 h. Data are shown as mean ± SEM from three independent experiments (*P < 0.05, ***P < 0.001, ****P < 0.0001). H AMFR and APP protein levels in SH-SY5Y-hAPP cells transfected with siRNA-control or siRNA-AMFR at DMSO or MG132 administration for 6 h. Data are shown as mean ± SEM from three independent experiments (*P < 0.05, **P < 0.01). I APP protein levels in HEK293T-hAPP cells transfected with His-AMFR at DMSO or MG132 administration for 6 h. Data are shown as mean ± SEM from three independent experiments (*P < 0.05, ***P < 0.001)

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AMFR reduces APP amyloidogenic cleavage products

Given that AMFR regulated APP degradation (Fig. 3), the role of AMFR in the regulation of amyloidogenic cleavage products was then examined. The protein level of APP was reduced in SH-SY5Y-hAPP cells with human plasmid-mediated AMFR overexpression (Fig. 4A). The amounts of amyloidogenic cleavage products of APP, including sAPPβ, Aβ40, and Aβ42, in cells transfected with His-AMFR plasmid, significantly decreased (Fig. 4B–D). Meanwhile, we also explored the effect of mouse AMFR overexpression on the level of Aβ in primary neurons of APP/PS1 embryonic mice. Similarly, mouse AMFR upregulation promoted the decline in APP and its amyloidogenic cleavage products (Fig. 4E–H). In contrast, the levels of APP, sAPPβ, Aβ40, and Aβ42 increased in SH-SY5Y-hAPP cells when the siRNA-AMFR was transfected compared with control cells (Fig. 4I–L). Furthermore, we found that in SH-SY5Y-hAPP cells treated with MG132, the levels of Aβ40 and Aβ42 which initially decreased due to AMFR overexpression, were restored (Fig. S4A, B). These results indicated that AMFR-mediated APP degradation led to a decrease in Aβ production.

Fig. 4
figure 4

Effects of AMFR on APP amyloidogenic cleavage products. A AMFR and APP protein levels in SH-SY5Y-hAPP cells transfected with empty vector or His-AMFR. Data are shown as mean ± SEM from three independent experiments (*P < 0.05, ****P < 0.0001). B-D sAPPβ, Aβ40, and Aβ42 levels in SH-SY5Y-hAPP cells transfected with empty vector or His-AMFR by ELISA assays. Data are shown as mean ± SEM from three independent experiments (*P < 0.05, ****P < 0.0001). E AMFR and APP protein levels in SH-SY5Y-hAPP cells transfected with siRNA-control or siRNA-AMFR. Data are shown as mean ± SEM from three independent experiments (*P < 0.05, ***P < 0.001). F–H sAPPβ, Aβ40, and Aβ42 levels in SH-SY5Y-hAPP cells transfected with siRNA-control or siRNA-AMFR by ELISA assays. Data are shown as mean ± SEM from three independent experiments (*P < 0.05). I AMFR and APP protein levels in primary neurons of APP/PS1 embryonic mice transfected with empty vector or His-AMFR. Data are shown as mean ± SEM from three independent experiments (*P < 0.05). J-L sAPPβ, Aβ40, and Aβ42 levels in primary neurons of APP/PS1 embryonic mice transfected with empty vector or His-AMFR by ELISA assays. Data are shown as mean ± SEM from three independent experiments (*P < 0.05)

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AMFR promotes K11-linked polyubiquitination of APP

E3 ubiquitin ligase AMFR was found to promote APP degradation via the proteasome-dependent pathway (Fig. 3); accordingly, the mechanism of whether AMFR mediates APP ubiquitination was then investigated. As shown in Fig. 5A, the inclusion of his-APP protein, GST-AMFR-his protein, E1, UBE2G2 (E2), and ubiquitin induced APP polyubiquitination in vitro. Furthermore, AMFR upregulation significantly induced APP ubiquitination in HEK293T-hAPP (Fig. 5B) and SH-SY5Y-hAPP cells cotransfected with HA-Ub and His-AMFR plasmids (Fig. 5C). The biological functions of ubiquitination are influenced by the type of ubiquitin chain: K48, K11 and part of K29 chains typically target substrates for the proteasomal degradation, while chains linked via K6, K27, K33, K63 and linear residues are primarily involved in non-degradative processes, such as signal transduction, DNA damage repair and intracellular trafficking [39,40,41]. In order to dissect which polyubiquitin linkage on APP is mediated by AMFR, a representative group of ubiquitin mutants were used, including those with all lysines replaced with arginines, except for the indicated one (K11, K29, K33, K48 and K63) and the one with a point mutation at position 11 lysine (K11R) (Fig. 5D). By co-transfecting his-AMFR plasmid with K11, K29, K33, K48, and K63 ubiquitin mutants into HEK293T-hAPP (Fig. 5E) and SH-SY5Y-hAPP cells (Fig. 5F), AMFR was found to notably promote K11-related polyubiquitination of APP. Consistently, the ubiquitination level decreased when the K11R ubiquitin mutant and his-AMFR plasmid were transfected into HEK293T-hAPP (Fig. 5G) and SH-SY5Y-hAPP cells (Fig. 5H). These results demonstrated that AMFR positively promoted K11-linked polyubiquitination of APP to speed up its degradation.

Fig. 5
figure 5

AMFR promotes K11-related polyubiquitination of APP. A Ubiquitination alteration of APP incubated in vitro with AMFR recombinant protein and E1, E2, and Ub at 37 °C for 1 h. Data presented are representative of three separate experiments. B APP ubiquitination in HEK293T-hAPP cells transfected with empty vector or His-AMFR along with HA-Ub by Co-IP assays. The protein expression levels of AMFR and APP in the cell lysates were confirmed. Data presented are representative of three separate experiments. C APP ubiquitination in SH-SY5Y-hAPP cells transfected with empty vector or His-AMFR along with HA-Ub by Co-IP assays. The protein expression levels of AMFR and APP in the cell lysates were confirmed. Data presented are representative of three separate experiments. D Schematic presentation of Ub and its mutants. E APP ubiquitination in HEK293T-hAPP cells transfected with empty vector or His-AMFR along with HA-Ub or its mutants (K11, K29, K33, K48 and K63) by Co-IP assays. Data presented are representative of three separate experiments. F APP ubiquitination in SH-SY5Y-hAPP cells transfected with empty vector or His-AMFR along with HA-Ub or its mutants (K11, K29, K33, K48 and K63) by Co-IP assays. Data presented are representative of three separate experiments. G APP ubiquitination in HEK293T-hAPP cells transfected with empty vector or His-AMFR along with HA-Ub or its mutant (K11R) by Co-IP assays. Data presented are representative of three separate experiments. H APP ubiquitination in SH-SY5Y-hAPP cells transfected with empty vector or His-AMFR along with HA-Ub or its mutant (K11R) by Co-IP assays. Data presented are representative of three separate experiments

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AMFR upregulation alleviates the cognitive impairment of APP/PS1 mice

Known that AMFR regulated APP turnover by K11-linked polyubiquitination and thus reduced amyloid β production, whether E3 ubiquitin ligase AMFR could be therapeutic in AD mouse models was further studied. AAV preparations carrying either Flag-labeled control-mcherry (Flag-AAV9-control) or AMFR-mcherry (Flag-AAV9-AMFR) were microinjected into the hippocampus of APP/PS1 mice to investigate the effect of AMFR on AD pathology (Fig. 6A). At the end of the time point, an MWMT was performed to detect cognitive function. The results of the latency to the hidden platform showed that the control mice with AD spent longer time than the WT control mice on day 5 (Fig. 6B). This result validated the significant impairment of learning and memory in AD control mice. However, after training, the Flag-AAV9-AMFR-microinjected mice displayed evident shorter escape latency in finding the hidden platform than the AD control mice (Fig. 6B). During the probe trial in the hidden platform task, WT and Flag-AAV9-AMFR-microinjected mice spent more time swimming in the target quadrant than the AD control mice (Fig. 6C). Besides, both WT and Flag-AAV9-AMFR-microinjected mice crossed more times to reach the target platform than the AD mice (Fig. 6D, F). No significant differences in the swimming speed were noted among all groups (Fig. 6E). These experimental results verified that AMFR rescued the impaired learning and memory of APP/PS1 mice.

Fig. 6
figure 6

Effects of AMFR on learning and memory behaviors of APP/PS1 mice. A Experimental design and treatment schedule to assess the effect of Flag-AAV9-AMFR in the behavior and memory of APP/PS1 mice. 6-month-old APP/PS1 mice were injected bilaterally in the hippocampus with 1 µg of Flag-AAV9-AMFR or Flag-AAV9-Ctrl. B Mean escape latency of mice in the Morris water maze test within a 5-day training period. (WT, n = 7; APP/PS1-Flag-AAV9-Ctrl, n = 7; APP/PS1-Flag-AAV9-AMFR, n = 7). Data are shown as mean ± SEM from three independent experiments (*P < 0.05, ***P < 0.001). C Swimming time in the target quadrant of mice within a probe-test period. (WT, n = 7; APP/PS1-Flag-AAV9-Ctrl, n = 7; APP/PS1-Flag-AAV9-AMFR, n = 7). Data are shown as mean ± SEM from three independent experiments (*P < 0.05). D Number of crossings over the original platform site within a probe-test period. (WT, n = 7; APP/PS1-Flag-AAV9-Ctrl, n = 7; APP/PS1-Flag-AAV9-AMFR, n = 7). Data are shown as mean ± SEM from three independent experiments (*P < 0.05, **P < 0.01). E Swimming speed of mice within a probe-test period. (WT, n = 7; APP/PS1-Flag-AAV9-Ctrl, n = 6; APP/PS1-Flag-AAV9-AMFR, n = 7). Data are shown as mean ± SEM from three independent experiments (ns, not significant). F Representative swimming trajectories of mice in the Morris water maze probe test. G Discrimination index of mice in relation to the novel and familiar objects during the testing phase (WT, n = 7; APP/PS1-Flag-AAV9-Ctrl, n = 7; APP/PS1-Flag-AAV9-AMFR, n = 7). Data are shown as mean ± SEM from three independent experiments (*P < 0.05). H Exploration time of mice in relation to novel and familiar objects during the testing phase of the novel object test. (WT, n = 7; APP/PS1-Flag-AAV9-Ctrl, n = 7; APP/PS1-Flag-AAV9-AMFR, n = 7). Data are shown as mean ± SEM from three independent experiments (ns, not significant, *P < 0.05). I Representative motion traces of mice during the testing phases in the novel object test. J Representative heatmap images of mice during the testing phases in the novel object test

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The NORT was used to assess recognition memory in mice. This test was based on the spontaneous tendency of mice to spend more time exploring a novel object than a familiar one. As shown in Fig. 6G, the control mice with AD had a markedly lower recognition index than the WT group, suggesting that the AD control mice had an impaired ability to discriminate between familiar and novel objects. The microinjection of AMFR effectively increased the recognition index in the AD mice when compared with that in AD control mice (Fig. 6G). In addition, the exploration time with novel objects of the AMFR-microinjected mice was longer than that with familiar objects, whereas the AD control mice exhibited similar exploration time for two different objects during the training phase (Fig. 6H–J).

The OFT was used in studies on the neurobiological basis of anxiety. When compared with the AD control mice, the AMFR-microinjected APP/PS1 mice exhibited a higher frequency of exploration in the center zone (Fig. S5A, C, D). In addition, the AD control mice featured slightly lower locomotor activity than the Flag-AAV9-AMFR-microinjected mice (Fig. S5B–D), indicating that AMFR could alleviate the anxiety-related behavior deficits in APP/PS1 mice. Together, all these experimental findings suggested that AMFR obviously ameliorated cognitive decline in APP/PS1 mice.

AMFR alleviates AD-related pathological damage in APP/PS1 mice

The protein levels of AMFR and APP, APP polyubiquitination, amyloidogenic cleavage products of APP, Aβ plaques, and synaptic plasticity were examined among the groups of mice. After stereotactic injection, the presence of a red mcherry fluorescence indicated that the AAV had transducted into the hippocampus of APP/PS1 mice (Fig. 7A). The protein levels of APP were upregulated in the hippocampal tissues of the AD control mice compared with that in the WT mice but dramatically downregulated in the Flag-AAV9-AMFR-injected AD mice (Fig. 7B). Compared with the control group, AMFR overexpression significantly reduced the sAPPβ, Aβ40, and Aβ42 levels and Aβ plaques in the hippocampus of APP/PS1 mice (Fig. 7C–E, G). And we also observed an obviously increase of APP polyubiquitination in the hippocampus of Flag-AAV9-AMFR-injected APP/PS1 mice (Fig. 7F). Then, synaptic plasticity alteration was examined by monitoring long-term potentiation (LTP) in CA1 pyramidal neurons of these three mouse groups (Fig. 7H, I). The potentiation of excitatory postsynaptic currents (EPSCs) was significantly impaired in neurons of the AD control group, whereas AMFR upregulation could reverse the LTP impairment. However, the paired-pulse ratio was not significantly changed among these mouse groups (Fig. S6). Therefore, these results revealed that AMFR could reduce Aβ deposition and improve synaptic functions in APP/PS1 mice.

Fig. 7
figure 7

Effects of AMFR on AD-like pathologies and synaptic plasticity in APP/PS1 mice. A Viral diffusion in the hippocampus of the APP/PS1-Flag-AAV9-Ctrl and APP/PS1-Flag-AAV9-AMFR groups by immunofluorescence analysis. mCherry (red), nuclei (DAPI, blue), merge (mCherry + DAPI); scale bars represent 2000 μm. Data presented are representative of three separate experiments. B AMFR and APP protein levels in the hippocampus of WT (n = 7), APP/PS1-Flag-AAV9-Ctrl (n = 7), and APP/PS1-Flag-AAV9-AMFR (n = 7) groups. Data are shown as mean ± SEM from three independent experiments (*P < 0.05, **P < 0.01). C-E sAPPβ, Aβ40, and Aβ42 levels in the hippocampus of the three mouse groups by ELISA (n = 7). Data are shown as mean ± SEM from three independent experiments (ns, not significant, *P < 0.05, **P < 0.01). F APP ubiquitination in the hippocampus of the three mouse groups by Co-IP assays (n = 7). The protein levels of APP and AMFR in the tissue lysates were confirmed. Data presented are representative of three separate experiments. G Amyloid plaque deposition (green) in the hippocampus of different groups of mice by immunofluorescence analysis (n = 3). Scale bars represent 500 μm. Data are shown as mean ± SEM from three independent experiments (*P < 0.05, ***P < 0.001). H Representative fEPSP recording traces and normalized slopes of the fEPSP of the three mouse groups before and 60 min after high-frequency stimulation with electrophysiology assays. (n = 5 slices from 5 mice per group). Data are shown as mean ± SEM from three independent experiments. I Quantifications of the average percentage changes in the fEPSP slope 0–60 min after high-frequency stimulation. (n = 5 slices from five mice per group). Data are shown as mean ± SEM from three independent experiments (*P < 0.05)

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Discussion

It was reported that the roles of AMFR have been investigated in various neurodegenerative disorders [42]. Cyclin-dependent kinase 5 (CDK5) directly phosphorylated AMFR at Ser516, which caused AMFR ubiquitination and degradation and resulted in the increased rate of neuronal death in cellular and animal PD models [43]. However, the influence of AMFR on neuronal death in AD remains to be confirmed, and this pathology is observed only in aged APP/PS1 mice and localizes in brain of these animals [44, 45], suggesting more suitable mouse model might be needed for further research, such as mouse with xenografted human neurons in brain [46]. Another study demonstrated that mutant huntingtin protein interacted with the CUE domain of AMFR, which competitively interfered with the interaction of AMFR and p97/VCP, a molecular chaperone that was important for ERAD. However, AMFR was still able to promote the ubiquitination and degradation of huntingtin proteins with expanded polyglutamine repeats, which confirmed that AMFR played an essential role in Huntington’s disease (HD) [47]. Likewise, aging studies have indicated that the mRNA and protein expression of AMFR significantly decreased in the hippocampus of senescence-accelerated mouse strain (SAM) prone/8 (SAMP8), a mouse model of AD [29]. In this study, we examined AMFR mRNA and protein levels in the hippocampus of AD mouse model–the APP/PS1 mice, and further investigated the levels of AMFR in serum and CSF from patients with different stages of AD (Fig. 1). The AMFR protein, being ubiquitously expressed, could be altered in serum levels due to various pathologies, such as decreased level of plasma AMFR in osteoporosis [48], as well as changes observed in some muscle diseases and tumors [27, 49, 50]. Considering the correlation between the levels of serum and CSF AMFR, the change in serum AMFR might be linked to AD pathology to some extent as CSF AMFR level (Fig. 1). Our study showed that the serum and CSF AMFR level of the AD group was lower than that of the HC group (Fig. 1). Based on this, we further evaluated their clinical efficiency in AD (Fig. S7, Table S5). These findings revealed that AMFR might play an important role in the development of AD.

Aβ plaques, as the major pathological component of AD, mainly consisted of Aβ40 and the more fibrillogenic Aβ42, which originated from APP cleaved by β- and γ-secretases [51]. Therefore, the regulation of APP processing is a basic focus in AD. A previous study demonstrated that E3 ubiquitin ligase FBL2-mediated ubiquitination of APP inhibited its endocytosis and reduced Aβ generation [52]. The other E3 ubiquitin ligase, HRD1, promoted APP degradation and aggresome formation through ERAD to prevent Aβ generation [53]. APP maturation was also influenced by E3 ubiquitin ligase Mahogunin (MGRN1) by delaying the proteolytic processing of APP, which led to reduced Aβ40 and Aβ42 release [54]. To explore whether AMFR could influence the processing of APP like other E3 ubiquitin ligases, we first investigated the association between these two protein. The results demonstrated that AMFR interacted with APP in neuronal cells and mouse brain tissues (Fig. 2). Thus, we speculated that AMFR might be involved in AD pathogenesis via regulating the biological process of APP (Fig. 3). In the study research, we demonstrated in a time-course experiment with CHX that AMFR could promote the degradation of APP. Since we found that AMFR changes may not affect autophagy lysosomal pathway, an alternative pathway for APP degradation, the ubiquitin-proteasome pathway, was further investigated. The result showed that the protein level of APP reduced by AMFR upregulation was reverted to normal when cells were treated with a proteasome inhibitor–MG132, suggesting that AMFR degraded APP via proteasomal system (Fig. 3). To further explore whether it has an impact of AMFR-mediated APP degradation on the generation of amyloidogenic cleavage products, we examined levels of them and observed that AMFR could reduce the production of Aβ by regulating APP stabilization (Fig. 4). In summary, we identified that AMFR could accelerate the proteasomal degradation of APP and affect its amyloid metabolism.

As an E3 ubiquitin ligase, AMFR mainly functions as a regulator of ubiquitination of substrate proteins. Ubiquitination is an extremely important part of PTMs and has a critical function in AD [55,56,57,58]. The transcription factor CCAAT/enhancer binding protein beta (c/EBPβ) orchestrated microglial proinflammatory genes and was upregulated in AD. The ubiquitin ligase COP1 mediated the ubiquitination of c/EBPβ to regulate its expression in microglia [59]. RPS23RG1, a newly identified protein associated with AD, interacted with PSD-93/PSD-95 and sequestered it from MDM2-mediated ubiquitination and degradation, consequently maintaining synaptic integrity [22]. Recently, ubiquilin-1, the protein product of UBQLN1 with single nucleotide polymorphisms, was implicated in late-onset AD, and ubiquilin-1 mediated K63-linked polyubiquitination of full-length APP at lysine 688 [60]. To investigate the mechanism of how AMFR promoted APP degradation, our in vitro and in vivo ubiquitination assays revealed significantly increased ubiquitination levels with AMFR overexpression. In addition, AMFR conjugated the K11-linked polyubiquitin chain to APP through its E3 ligase activity (Fig. 5). The result was consistent with current findings that K48-linked and K11-linked polyubiquitin chains accelerated target protein degradation [41, 61,62,63] and K63-linked polyubiquitin chains normally mediated the repair of DNA damage and cell signal transduction [64,65,66].

Memory loss and cognitive impairment are major symptoms of AD [67]. RNF220, depending on its E3 ubiquitin ligase activity, could inhibit postsynaptic AMPA receptor (AMPAR)-mediated excitatory synaptic transmission and its cellular surface expression. In addition, learning and memory abilities changed in the forebrain of RNF220-deficient mice, indicating that the E3 ubiquitin ligase for AMPARs played a substantial role in excitatory synaptic activity and brain physiological function [68]. Of note, a previous study demonstrated that E3 ubiquitin ligase AMFR could also strengthen learning and establish memory abilities by conducting several experiments on rats and mice [29]. Thus, we microinjected AAV preparations carrying either Flag-AAV9-control or Flag-AAV9-AMFR into the hippocampus of APP/PS1 mice to further determine and confirm the biological role of AMFR on AD pathology. Behavioral and electrophysiological tests showed that AMFR overexpression significantly mitigated cognitive and synaptic plasticity deficits of APP/PS1 mice (Figs. 6 and 7). Additionally, AMFR overexpression reduced APP protein level and Aβ accumulation in the hippocampus of APP/PS1 mice (Fig. 7). Consequently, our results further supported the evidence suggesting the key role of AMFR in the clearance of amyloid deposition and cognitive improvement.

So far, although compounds targeting AMFR have been investigated in cancer, such as tumor-suppressing AMFR monoclonal antibody [69] and EGFR antibody inhibiting AMF/AMFR overexpression in breast cancer [70], the study of drugs targeting AMFR in AD are still limited. Further investigation in this field might be required in the future.

Conclusions

In conclusion, we found that AMFR levels were reduced in AD model mice. AMFR interacted with APP and degraded APP through the UPS system, resulting in decreased Aβ production. Furthermore, AMFR was verified to alleviate cognitive impairment and AD-related pathological damage in AD model mice (Fig. 8). However, we have not conclusively evaluated the role of AMFR in patients with AD and other neurodegenerative diseases through relevant clinical trials. Collectively, our findings revealed a novel pathway for APP/Aβ metabolism whereby AMFR regulated APP ubiquitination and might bring a new direction in exploring therapeutic strategies for AD and related neurodegenerative disorders.

Fig. 8
figure 8

A schematic diagram depicting that AMFR functions as an E3 ubiquitin ligase that promotes the proteasomal degradation of APP, thereby ameliorating Alzheimer’s pathology

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Data availability

No datasets were generated or analysed during the current study.

Abbreviations

AMFR:

Autocrine Motility Factor Receptor

UPS:

Ubiquitin-Proteasome System

AD:

Alzheimer’s Disease

APP/PS1:

APPswe/PSEN1dE9

CSF:

Cerebrospinal Fluid

APP:

Amyloid Precursor Protein

PTM:

Post-Translational Modification

UPP:

Ubiquitin-proteasome pathway

MDM2:

Murine Double Minute 2

BACE1:

 β-site APP Cleaving Enzyme 1

ER:

Endoplasmic Reticulum

ERAD:

ER-Associated Degradation

MCI:

Mild Cognitive Impairment

VaD:

Vascular Dementia

PDD:

Parkinson’s Disease with Dementia

DLB:

Dementia with Lewy Bodies

FTD:

Frontotemporal Dementia

HC:

Healthy Control

AAV:

Adeno-Associated Virus

OFT:

Open-Field Test

NORT:

Novel Object Recognition Test

MWMT:

Morris Water Maze Test

WT:

Wild-Type

LTP:

Long-Term Potentiation

EPSCs:

Excitatory Postsynaptic Currents

NC:

Normal Cognition

AUC:

Area Under the Curve

CDK5:

Cyclin-Dependent Kinase 5

HD:

Huntington’s Disease

SAMP8:

Senescence-Accelerated Mouse strain Prone/8

c/EBPβ:

CCAAT/Enhancer Binding Protein beta

AMPAR:

AMPA Receptor

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Acknowledgements

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Funding

This research was financially supported by Beijing Natural Science Foundation (grant number 7252060); the State Key Program of National Natural Science Foundation of China (grant number 82030064); Beijing Hospital Authority Youth Program (grant number QML20230812); National Natural Science Foundation of China (grant number 82102487 and 81871714); Research and Development Foundation of Capital Medical University (grant number PYZ23052).

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  1. Peichang Wang and Yaqi Wang contributed equally to this work.

Authors and Affiliations

  1. Department and Institute of Clinical Laboratory, Xuanwu Hospital, National Clinical Research Center for Geriatric Diseases, Capital Medical University, Beijing, 100053, China

    Jingjing Zhang, Congcong Liu, Jing Liu, Yuting Cui, Yuli Hou, Qiao Song, Xiaomin Zhang, Xiaoling Wang, Qian Zhang, Wenchao Wang, Peichang Wang & Yaqi Wang

  2. Department of Clinical Laboratory, Beijing Huairou Hospital, Beijing, 101400, China

    Min Cao

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Contributions

PW and YW conceived the design of the study and provided funding. JZ performed the experiments, analyzed the data and drafted substantial part of the manuscript and figures. CL, JL, YC, YH, QS, XZ, XW, QZ, MC and WW assisted with the experiments. All authors reviewed and approved the final manuscript.

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Correspondence to Peichang Wang or Yaqi Wang.

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All procedures were approved by the Ethics Committee of Xuanwu Hospital of Capital Medical University and were performed according to the guidelines of the Declaration of Helsinki. All animal experiments were approved by the Bioethics Committee of Xuanwu Hospital of Capital Medical University and complied with the National Institute of Health Guide for the Care and Use of Laboratory Animals.

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Supplementary Information

13195_2025_1741_MOESM1_ESM.docx

Supplementary Material 1: Figure S1. The levels of AMFR in the CSF from normal cognitive participants of different age groups. Figure S2. AMFR levels in the serum and CSF of patients with different types of dementia. Figure S3. The protein levels of markers in autophagy lysosomal pathway in cells with AMFR alteration. Figure S4. Effects of AMFR on APP amyloidogenic cleavage products in proteasomal degradation-inhibited system. Figure S5. Behavioral characterization of APP/PS1-Flag-AAV9-Ctrl and APP/PS1-Flag-AAV9-AMFR groups of mice. Figure S6. Whole-cell recordings from the hippocampal neurons of WT, APP/PS1-Flag-AAV9-Ctrl and APP/PS1-Flag-AAV9-AMFR groups of mice by paired-pulse ratio assays. Figure S7. The ROC analysis of AMFR in AD and other dementia.

Supplementary Material 2: Table S1. Characterization of patients in different groups.

Supplementary Material 3: Table S2. Characterization of participants in different age groups.

Supplementary Material 4: Table S3. Age- and sex-adjusted effects of AMFR levels in serum of participants.

Supplementary Material 5: Table S4. Age- and sex-adjusted effects of AMFR levels in CSF of participants.

13195_2025_1741_MOESM6_ESM.xlsx

Supplementary Material 6: Table S5. The clinical efficiency of serum and CSF AMFR levels in AD and other types of dementia.

Supplementary Material 7.

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Zhang, J., Liu, C., Liu, J. et al. The autocrine motility factor receptor delays the pathological progression of Alzheimer’s disease via regulating the ubiquitination-mediated degradation of APP. Alz Res Therapy 17, 95 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13195-025-01741-7

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Keywords

Alzheimer's Research & Therapy

ISSN: 1758-9193