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. 2023 Nov 29;49(3):620–630. doi: 10.1038/s41386-023-01774-2

Inhibition of asparagine endopeptidase (AEP) effectively treats sporadic Alzheimer’s disease in mice

Zhengjiang Qian 1, Bowei Li 2, Xin Meng 1, Jianming Liao 3, Guangxing Wang 4, Yanjiao Li 1, Qian Luo 5,✉, Keqiang Ye 1,✉
PMCID: PMC10789813  PMID: 38030711

Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative disease with cognitive dysfunction as its major clinical symptom. However, there is no disease-modifying small molecular medicine to effectively slow down progression of the disease. Here, we show an optimized asparagine endopeptidase (AEP, also known as δ-secretase) inhibitor, #11 A, that displays an orderly in vivo pharmacokinetics/pharmacodynamics (PK/PD) relationship and robustly attenuates AD pathologies in a sporadic AD mouse model. #11 A is brain permeable with great oral bioavailability. It blocks AEP cleavage of APP and Tau dose-dependently, and significantly decreases Aβ40 and Aβ42 and p-Tau levels in APP/PS1 and Tau P301S mice after oral administration. Notably, #11 A strongly inhibits AEP and prevents mouse APP and Tau fragmentation by AEP, leading to reduction of mouse Aβ42 (mAβ42), mAβ40 and mouse p-Tau181 levels in Thy1-ApoE4/C/EBPβ transgenic mice in a dose-dependent manner. Repeated oral administration of #11 A substantially decreases mAβ aggregation as validated by Aβ PET assay, Tau pathology, neurodegeneration and brain volume reduction, resulting in alleviation of cognitive impairment. Therefore, our results support that #11 A is a disease-modifying preclinical candidate for pharmacologically treating AD.

Subject terms: Pharmacology, Alzheimer's disease

Introduction

Alzheimer’s disease (AD) is the most common dementia. Its prominent pathological hallmarks are extracellular senile plaques with aggregated β-amyloid (Aβ) as main component, and neurofibrillary tangles (NFT), consisting of truncated and hyperphosphorylated Tau. In addition to pronounced Aβ and Tau pathologies, AD is also associated with extensive neuroinflammation, synaptic degeneration and neural cell death, resulting in brain volume reduction and cognitive impairment [1–5]. AD is a rapidly growing health crisis, exerting a huge healthcare burden. Treatments are urgently needed to prevent, delay the onset, slow the progression, improve cognition, and reduce behavioral disturbances of AD [6]. The currently approved therapy for AD are cholinesterase inhibitors, NMDA-receptor antagonists and their combination, but these therapies provide only temporary symptomatic relief [7]. Most recently, the US Food and Drug Administration (FDA) approved Lecanemab and Donanemab, two monoclonal antibodies designed to clear the brain of amyloid plaques, and slow down clinical progression in early symptomatic AD patients, but these are both associated associate with adverse events [8–10]. Clearly, different disease-modifying drugs targeting distinct mechanisms are needed for treating AD.

Asparagine endopeptidase (AEP) is an acidosis-activated cysteine protease that selectively cleaves substrates after asparagine (N) residues. We have reported that it is activated under stroke and cuts SET, a nuclear DNase inhibitor, triggering neuronal cell death [11]. Interestingly, AEP is progressively escalated in the brain during aging and acts as δ-secretase that simultaneously cleaves both APP and Tau at N585 and N368 residues, respectively. This facilitates Aβ production and NFT pathologαies in mouse models of AD. Inactivation of AEP substantially diminishes both Aβ and Tau pathologies [12, 13].

Remarkably, AEP also shreds β-secretase (BACE1) at N294 and enhances its protease activity. The enzymatic domain containing the BACE1 (1-294) fragment robustly cleaves APP C586-695 (C110), a C-terminal fragment of APP from AEP cleavage, into C99 truncate, accelerating Aβ generation [14]. We have also shown that AEP-truncated Tau N368 is present in the cerebrospinal fluid (CSF) of AD patients and that it correlates with Tau PET signals [15, 16]. Noticeably, CSF Tau N368/t-Tau ratios reflect cognitive impairment and Tau pathologies better than p-Tau 181 and p-Tau 217 in AD patients [17], underscoring that Tau N368 is a promising biomarker for AD early diagnosis.

C/EBPβ, an Aβ- or inflammatory cytokines-activated transcription factor, dictates AEP mRNA expression in the brain in an age-dependent manner. Deletion of C/EBPβ from 3xTg mouse model substantially attenuates AD pathologies [18]. Notably, C/EBPβ also modulates ApoE4 expression in AD [19] and ApoE4 feeds back and stimulates C/EBPβ activation, exacerbating AD pathogenesis [20]. Under physiological conditions, ApoE is primarily expressed in glial cells, but its neuronal expression is highly elevated under pathological stresses including AD [21, 22]. Neuronal ApoE4 strongly activates C/EBPβ and the resulting increase in δ-secretase activity leads to increased mouse APP and Tau, promoting AD-like pathologies. Notably, Thy1-ApoE4/C/EBPβ mice develop amyloid deposits, Tau aggregates and neurodegeneration in an age-dependent manner, leading to synaptic dysfunction and cognitive disorders. Hence, this mouse transgenic line acts as a sporadic AD mouse model [23].

Through a high-throughput screening and co-crystallization, we identified a small molecular inhibitor #11 that binds to both the active site and allosteric site of AEP, robustly inhibiting active AEP [24]. AEP is upregulated in human cancers and mediates their metastasis. We have demonstrated that blockade of AEP with its optimized inhibitor #11 A blunts cancer cells from spreading [25]. Furthermore, we showed that AEP cleaves α-Synuclein at N103 in Parkinson’s disease (PD), accelerating α-Synuclein aggregation and Lewy body formation [26]. Accordingly, antagonizing AEP with #11 A ameliorates PD pathogenesis in mouse models [27]. In the current report, we show that #11 A possesses acceptable in vivo pharmacokinetics (PK) profiles and it exhibits orderly in vivo pharmacokinetics/pharmacodynamics (PK/PD) relationships in AD mouse models upon acute oral administration. It dose-dependently blocks AEP-truncated APP N585, C586, and Tau N368 fragments in CSF and plasma, decreasing Aβ and p-Tau 181 levels. Repeated oral administration of #11 A in Thy1-ApoE4/C/EBPβ transgenic mice significantly lessens AD pathologies and alleviates cognitive dysfunctions. Because #11 A is safe for repeated treatment [25, 27, 28], it represents a preclinic candidate for the innovative disease-modifying pharmacological treatment of AD.

Material and methods

Mice and reagents

The Tau P301S (line PS19) and APP/PS1 mice were originally purchased from the Jackson Laboratory. The P301S mouse expresses a mutant form of human Tau and that the APP/PS1 mouse is engineered to express mutant of both human APP and presenilin. The Thy1-ApoE4/C/EBPβ transgenic mice with C57BL/6 J background were generated and maintained as previously described [23]. The CD1 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All the mice were bred in specific pathogen-free facilities, with 12-h light/12-h dark cycle and free access to water and food, at Shenzhen Institute of Advanced Technology (SIAT), Chinese Academy of Sciences. Both male and female mice were interchangeably used for experiments. Anti-phospho TrkB Y816, Anti-TrkB N486, anti-APP Y687 were generated as described previously [29]. The detailed information of antibodies and ELISA kits were provided in Supplementary Table 1.

#11 A treatment

The AEP inhibitor, #11 A, was ultrasonically dissolved in DMSO and diluted with 0.1% methyl cellulose solution. For analysis of in vivo PK parameters, the CD1 mice at the age of 2 months were treated with #11 A by intravenous injection (I.V., 2 mg/kg) or oral administration (P.O., 10 mg/kg) for respective different time points. At each indicated time point, three mice were sacrificed for the collection of blood and brain tissue, which were then used for LC-MS/MS analysis. For acute treatment, mice, including APP/PS1 (age at 10 months), Tau P301S (age at 8 months) and Thy1-ApoE4/C/EBPβ Tg mice (age at 6 months), were treated orally with #11 A by P.O. for 2 h, the blood and brain tissue were then collected for subsequent experiments. Thy1-ApoE4/C/EBPβ Tg mice were reported to develop AD-like pathological features, including intraneuronal Aβ and cognitive disorders starting from 3 months of age [23]. Therefore, for repeated administration, these transgenic mice were treated daily with #11 A by P.O. for 3 months at the age of 3-month, and then subjected to pathological analysis. All the experimental procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee at SIAT.

Single molecular array (SiMoA) assay of CSF and blood samples

Collection of CSF and serum samples were performed as previously described [30]. All the SiMoA assays were performed on the Quanterix SR-X analyzer (Quanterix). The human Aβ 40 and Aβ 42 levels were measured using the Simoa® Neurology 3-Plex A Advantage Kit (catalog #101995), and human pTau-181 was measured using the Simoa® pTau-181 Advantage V2 Kit (catalog #103714) according to the manufacturer’s instructions. CSF and serum samples were diluted 1:100 and 1:6, respectively. The measurement of APP N585, APP C586 and Tau N368 levels were performed according to the Quanterix homebrew protocol. In brief, homebrew monoclonal antibodies of APP N585, APP C586 and Tau N368 were first conjugated with beads, followed by the biotinylation of detection antibody. Then the SiMoA testing was optimized and performed on the Quanterix SR-X analyzer. CSF and serum samples were diluted 1:400 and 1:20, respectively. All measurements were carried out in one round of experiment using the same batch of reagents.

ELISA assay

Mouse brain samples were homogenized in lysis buffer and centrifuged at 16,000 g for 20 min at 4 ◦C. The supernatant was analyzed by ELISA kits according to the manufacturer’s instructions. The insoluble protein including Aβ and Tau aggregates were extracted following the protocol as described in previous work [31, 32] and then were analyzed by ELISA kits.

Small animal PET

Mice were anesthetized following a standardized protocol for Radiochemistry, acquisition, and post processing. Inhalation anesthesia with isoflurane was used throughout the experiments, and PET images were recorded on a high-resolution small animal PET scanning device (microPET) with a spatial resolution of 1.0 mm. Brain emission scans were acquired in volumetric mode for 20 min after an intravenous injection of 10–15 MBq of 18F-florbetapir (AV45) in approximately 100 μL of saline into the tail. The PET images were reconstructed by using ordered-subset expectation maximization (OSEM) with 16 subsets and 5 iterations. Target-to-reference tissue (the cerebellum) standard uptake value ratios (SUVR) were calculated for 18F-AV45. The statistical results of SUVR in the region of interesting (ROI) were calculated and analyzed by Amide 1.0.4-1 (San Diego, CA 92101).

Morris water maze (MWM)

Mice were trained in a round tub filled with water in an environment surrounded by extra maze cues. Each mouse was trained 3 trails/day for 5 consecutive days with a 15-min intertrial interval. The maximum trial time was 60 s, and if the mouse did not reach the platform in the allotted time, they were manually guided to do it. Following the 5 days task training, a probe trial was given, during which time the platform was removed. All trials were analyzed for latency using EthoVision XT-Video tracking software (Noldus Information Technology Co., Ltd).

Novel object recognition test (NORT)

Mice subjected to NORT underwent three phases: habituation, familiarization and discrimination. In the habituation phase, each mouse was placed in a square open field (40 × 40 × 40 cm) individually for 10 min per day for 3 consecutive days. Then, the animals entered into the familiarization phase. In this phase, each mouse was allowed to explore in the open field with two identical objects located in opposite and equidistant positions for 10 min. Exploration was defined as sniffing or touching the objects with the nose and/or forepaws when the nose was in contact with or directed at the object at a distance of ≤1.5 cm. After a three-hour retention interval, the mouse returned to the open field, with one of the familiar objects replaced by a novel object. For the discrimination phase, each mouse was allowed to explore for 10 min and the time for exploring each object was recorded. Mice touching an object or facing an object within 2 cm around the object were taken as measure of object exploration behavior. To eliminate olfactory cues, the objects and field were cleaned with 75% ethanol between each trial. The exploring index was determined as: (exploration time for the novel—exploration time for the familiar object) / (total exploration time during the test session) × 100%.

Statistical analysis

Statistical analyses were performed with the SPSS software (SPSS, Chicago, IL, USA) for Windows. All data are presented as means ± SD unless otherwise stated. When only two groups were compared, the statistical differences were assessed with the double-sided Student’s t test. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) with Tukey’s post hoc test. Two-way ANOVA was used for analysis of multiple groups with Tukey’s multiple comparison post hoc test. For all experiments, *P ≤ 0.05 was considered a significant difference.

Results

In vivo Pharmacokinetic profiling of #11 A

To explore the druggability of #11 A, we analyzed its in vivo PK profiles by orally dosing 10 mg/kg (P.O.) and 2 mg/kg via intravenous injection (I.V.) into 2 months old CD1 mice (Fig. 1A). The concentrations of #11 A in the brain and plasma from three mice of each route of administration were measured using LC-MS/MS analysis at different time points. The detailed levels were summarized in Supplementary Table 2. The kinetic curves of each route and #11 A distribution in the plasma and the brain are presented in Fig. 1B–D. The in vivo PK parameters were shown in Fig. 1E. The t1/2 values of #11 A in the plasma and the brain were 1.31 h and 1.63 h via I.V. injection. Notably, t1/2 values were enhanced to 2.75 h and 2.19 h via P.O., respectively. Through oral administration of #11 A, the tmax appeared at 1.33 h with Cmax of 102.78 ng/ml and 1.00 h with Cmax of 27.01 ng/ml in the plasma and the brain, respectively. The volume of distribution (Vz) parameter was 78.88 L/kg via P.O., suggesting that it is lipophilic. The plasma clearance rates by both routes were 262 and 328 ml/min/kg, which are relatively high, accountable for the medium half-life in the plasma. The oral bioavailability (F) was 80.96%. Hence, #11 A is highly oral bioactive and brain permeable with acceptable in vivo PK profiles.

Fig. 1. In vivo pharmacokinetics (PK) profiles of #11 A by the treatment of intravenous (I.V.) injection and oral administration (P.O.) in CD1 mouse.

Fig. 1

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A Diagram showing experimental schedule for in vivo PK profile analysis of #11 A. Total of 42 CD1 mice were equally divided into two groups, which were subjected to #11 A treatment by I.V. (2 mg/kg) and P.O. (10 mg/kg) treatment, respectively. Blood and brain tissue were collected from three mice at each indicated time point, and the concentration of #11 A was determined by LC-MS/MS for PK parameters analysis. B Concentrations of #11 A in plasma (ng/mL) after I.V. and P.O. treatment with #11 A in CD1 mice. C Concentrations of #11 A in plasma (ng/mL) and brain (ng/g) after I.V. treatment with #11 A in CD1 mice. D Concentrations of #11 A in plasma (ng/mL) and brain (ng/g) after P.O. treatment with #11 A in CD1 mice. E In vivo plasma and brain PK parameters of #11 A in CD1 mice by I.V. and P.O. treatment.

Acute oral administration of #11 A displays dose-dependent inhibition of AEP in Tau P301S mice

To assess #11 A’s in vivo PK/PD relationship, we employed 8 months old Tau P301S mice and orally administrated 3.5, 7.5, and 15 mg/kg doses and collected the brain, CSF and plasma samples 2 h after drug treatment (Fig. 2A). #11 A was dose-dependently elevated in the serum and brain (Fig. 2B). Correspondingly, AEP enzymatic activities were repressed in a dose-dependent manner (Fig. 2C). Immunoblotting (IB) showed that active AEP levels were progressively decreased as #11 A concentrations were gradually escalated in the brain. In consequence, AEP-cleaved Tau N368 and p-Tau (AT8 and AT100) levels were consequently reduced. The ratios of aggregated Tau, monitored by its specific antibody T22, versus Tau 5 for total Tau were significantly blunted (Fig. 2D, E). A similar dose-dependent reduction of human p-Tau 181 levels in the brain was found, whereas total human Tau remained unaltered (Fig. 2F). The concentrations of p-Tau 181 and Tau N368 in the CSF and plasma were further analyzed by Single Molecular Array (SiMoA). Both of them were robustly inhibited by #11 A in a gradient way (Fig. 2G, H). Thus, #11 A displays an orderly PK/PD relationship. It inhibits AEP in the brain of Tau P301S mice, repressing both p-Tau 181 and Tau N368 levels in the body fluids.

Fig. 2. Acute treatment with #11 A inhibits activity of AEP and its downstream targets in Tau P301S mouse.

Fig. 2

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A Diagram showing experiment of acute #11 A treatment in Tau P301S mice. The mice were treated with #11 A at different concentration (i.e., 3.5, 7.5 and 15 mg/kg) by P.O. for 2 h. Blood, cerebrospinal fluid (CSF) and brain tissue were collected from three mice treated with each indicated concentration of #11 A, and the #11 A level, AEP activity and the downstream targets of AEP were analysed. B Concentration of #11 A in plasma (ng/mL) and brain (ng/g) of Tau P301S mice with acute treatment of #11 A. C AEP enzymatic activity in brain tissue of Tau P301S mice with acute treatment of #11 A at different concentration. (n = 3, **p < 0.01, ***p < 0.001, compared with vehicle). D Representative immunoblot images showing the effects of #11 A treatment on the expression of AEP and its downstream targets in Tau P301S mice. E Relative quantification of protein levels in (D). (n = 3; *p < 0.05, **p < 0.01, compared with vehicle). F ELISA analysis of total human Tau (h-Tau, left panel) and pTau 181 (h-pTau181, right panel) levels in brain tissue of Tau P301S mice with acute treatment of #11 A. (n = 3, *p < 0.05, compared with vehicle). G, H SiMoA analysis of pTau 181 and AEP downstream target Tau N368 levels in CSF (G) and plasma (H) of Tau P301S mice with acute treatment of #11 A. (n = 3, *p < 0.05, compared with vehicle).

Acute oral administration of #11 A exhibits dose-dependent blockade of AEP in APP/PS1 mice

Next, we further examined the in vivo PK/PD relationship of #11 A’s in the APP/PS1 mouse model. As described above for other mouse strains, #11 A concentrations in both the serum and the brain of APP/PS1 mice were gradually elevated 2 h after oral administration as the doses augmented (Supplementary Fig. 1A, B). As expected, AEP activities in the brain tissues in these mice were steadily antagonized by increased levels of #11 A. Using IB analysis, we found that the reduced active AEP levels were coupled with decreased APP N585 and APP C586 fragmentation (Supplementary Fig. 1C–E). As a result, human Aβ40 and Aβ42 levels in the brain tissues were dose-dependently attenuated by #11 A (Supplementary Fig. 1F). SiMoA analysis showed that human Aβ42 but not Aβ40 concentrations in the CSF and plasma were progressively reduced by #11 A. We made the similar observations with APP N585 and C586 truncates in these tissues, correlating with their lessened fragmentation in the brain tissues (Supplementary Fig. 1G, H). Therefore, #11 A reveals a strong in vivo PK/PD relationship and blocks AEP activity in the brains of APP/PS1 mice, diminishing human Aβ42 and APP N585 and C586 levels.

Acute oral administration of #11 A demonstrates dose-dependent suppression of AEP in sporadic Thy1-ApoE4/C/EBPβ transgenic mice

We have recently developed a sporadic AD mouse employing neuronal ApoE4 driving the activation of C/EBPβ, which subsequently dictates APP and MAPT and BACE1 expression. In this mouse model (Thy1-ApoE4/C/EBPβ transgenic mouse) aggregated mouse Aβ and NFT-like mouse Tau inclusions are found, as is the expression of age-dependent cognitive defects [27]. To investigate whether #11 A also applies to this sporadic AD mouse model, we conducted the in vivo PK/PD profiling assay. Remarkably, #11 A concentrations in the serum and brain tissues were dose-dependently augmented, correlating with significant reduction of AEP activities in the brains from Thy1-ApoE4/C/EBPβ transgenic mice (Fig. 3A–C). IB analysis validated that active AEP levels were dose-dependently reduced, associated with gradually decreased APP N585, C586 and Tau N368 fragmentation (Fig. 3D, E). Though total Tau levels were not altered by #11 A, mouse p-Tau 181 quantities in the brain were progressively repressed (Fig. 3F). Interestingly, both mouse Aβ40 and Aβ42 concentrations were dose-dependently suppressed (Fig. 3G). In alignment with these effects in the brain, concentrations of APP N585, C586, and Tau N368 fragments in the CSF and plasma were reduced by #11 A treatment in a similar manner (Fig. 3H, I). Hence, #11 A also displays a close in vivo PK/PD relationship in the brain and body fluids in Thy1-ApoE4/C/EBPβ transgenic mice, attenuating mouse Aβ and p-Tau 181 levels, which echo AEP-truncated APP and Tau fragments’ concentrations.

Fig. 3. Acute treatment with #11 A inhibits activity of AEP and its downstream targets in Thy1-ApoE4/C/EBPβ Tg mouse.

Fig. 3

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A Diagram showing experiment of acute #11 A treatment in Thy1-ApoE4/C/EBPβ Tg mice. The mice were treated with #11 A at different concentration (i.e., 3.5, 7.5, and 15 mg/kg) by P.O. for 2 h. Blood, CSF and brain tissue were collected from three mice treated with each indicated concentration of #11 A, and the #11 A level, AEP activity and the downstream targets of AEP were analysed. B Concentration of #11 A in plasma (ng/mL) and brain (ng/g) of Thy1-ApoE4/ C/EBPβ Tg mice with acute treatment of #11 A. C AEP enzymatic activity in brain tissue of Thy1-ApoE4/C/EBPβ Tg mice with acute treatment of #11 A at different concentration (n = 3, *p < 0.05, **p < 0.01, compared with vehicle). D Representative immunoblot images showing the effects of #11 A treatment on the expression of AEP and its downstream targets in Thy1-ApoE4/C/EBPβ Tg mice. E Relative quantification of protein levels in (D). (n = 3; *p < 0.05, **p < 0.01, compared with vehicle). F, G ELISA analysis of mouse total Tau (left panel of F), pTau181 (right panel of F), Aβ42 (top panel of G) and Aβ40 (bottom panel of G) in brain tissue of Thy1-ApoE4/C/EBPβ Tg mice with acute treatment of #11 A at different concentration (n = 3 mice, *p < 0.05, compared with vehicle). H, I SiMoA analysis of AEP downstream targets APP N585, APP C586 and Tau N368 levels in CSF (H) and plasma (I) of Thy1-ApoE4/C/EBPβ Tg mice with acute treatment of #11 A at different concentration (n = 3 mice, *p < 0.05, **p < 0.01, compared with vehicle).

Repeated treatment of Thy1-ApoE4/C/EBPβ transgenic mice with #11 A diminishes AD pathologies

Our PK/PD relationship studies support that oral treatment with #11 A at a dose of 7.5 mg/kg is sufficient to interfere with AEP-mediated Aβ and Tau pathologies. To examine whether repeated treatment of Thy1-ApoE4/C/EBPβ transgenic mice with #11 A will abrogate the AD pathologies, we orally treated the mice with 7.5 mg/kg daily for 3 months. The experimental outlines are presented in Supplementary Fig. 2A. As expected, AEP activities in the brains were significantly repressed after repeated drug treatment. IB showed that active AEP levels were also pronouncedly blunted. As a consequence, the downstream APP N585 and Tau N368 fragmentation was substantially lessened by #11 A treatments (Supplementary Fig. 2B–D). Immunofluorescent (IF) staining of the brain sections showed that active AEP and APP C586 signals were strongly suppressed by #11 A (Supplementary Fig. 2E, F). Quantification of levels of these fragments in the CSF and plasma tissues demonstrated that the APP N585, C586, and Tau N368 truncates were significantly dampened by #11 A (Supplementary Fig. 2G, H).

To explore whether mouse Aβs form senile plaque-like structures in the Thy1-ApoE4/C/EBPβ transgenic mouse brain, we performed an Aβ PET study with 18F-AV45. Notably, #11 A treatments significantly abrogated Aβ aggregates in the brains (Fig. 4A, B). The effect on Aβ pathology was further confirmed by IF co-staining on the brain sections. We found that anti-Aβ (4G8) activities and ThS fluorescent signals were robustly abolished by #11 A (Fig. 4C, D). Results of an Aβ ELISA assay also supported that both mouse Aβ40 and Aβ42 concentrations were considerably decreased by treatments with #11 A (Fig. 4E). Silver staining of Tau inclusions in the hippocampal regions revealed its aggregation was attenuated by #11 A, which was validated by immunohistochemical (IHC) staining with anti-AT100 (Fig. 4F). Quantification of soluble and insoluble Tau from the brain tissues showed that both of them were substantially diminished by #11 A (Fig. 4G). Moreover, we conducted IB with anti-AT8 and AT100 antibodies and found that immunoreactivity to both was decreased, correlating with IF co-staining with AT8/Tau N368 on the brain sections (Fig. 4H–K).

Fig. 4. Repeated treatment with #11 A inhibits Aβ deposition and Tau pathology in Thy1-ApoE4/C/EBPβ Tg mouse.

Fig. 4

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A Representative Aβ PET images showing the effects of #11 A treatment on the Aβ plaque deposition in brain of Thy1-ApoE4/C/EBPβ Tg mice. B Relative quantification of Aβ PET standardized uptake value ratio (SUVR) in (A) (n = 3 mice, **p < 0.01, compared with vehicle). C Immunofluorescence staining of Aβ and Thioflavin S (Ths) in hippocampus of Thy1-ApoE4/C/EBPβ Tg mice with repeated #11 A treatment. Scale bar, 20 μm. D Relative quantification of Aβ and Ths fluorescence intensity in (C) (n = 3 mice, *p < 0.05, **p < 0.01, compared with vehicle). E ELISA analysis of mouse Aβ42 (left panel) and Aβ40 (right panel) levels in brain tissue of Thy1-ApoE4/C/EBPβ Tg mice with repeated #11 A treatment. (n = 3 mice, *p < 0.05, compared with vehicle). F Silver staining (Top panels) and immunohistochemistry (IHC) staining of AT100 in hippocampus of Thy1-ApoE4/C/EBPβ Tg mice with repeated #11 A treatment. Scale bar, 50 μm. G ELISA analysis of soluble Tau (left panel) and insoluble Tau (right panel) in brain tissue of Thy1-ApoE4/C/EBPβ Tg mice with repeated #11 A treatment. (n = 3 mice, *p < 0.05, compared with vehicle). H Representative immunoblot images showing the effects of #11 A treatment on the expression of AT8 and AT100. I Relative quantification of AT8 and AT100 levels in (H). (n = 3, *p < 0.05, **p < 0.01, compared with vehicle). J Immunofluorescence staining of AT8 and Tau N368 in hippocampus of Thy1-ApoE4/C/EBPβ Tg mice with repeated #11 A treatment. K Relative quantification of AT8 and Tau N368 fluorescence intensity in (J). Scale bar, 20 μm. (n = 3, *p < 0.05, compared with vehicle).

AD is associated with extensive neuroinflammation in the brain accompanied with abundant gliosis and microglia activation, a prominent pathology in neurodegenerative diseases. To test whether #11 A also ameliorates this detrimental effect, we measured activity of the inflammatory cytokines and found that IL-6 and TNFα were potently dampened, at both the mRNA and protein levels, in the brains of mice treated with #11 A (Supplementary Fig. 3A, B). Similarly, mRNA levels of both GFAP and Iba-1 were diminished by #11 A, fitting with reduced IF staining signals on the brain sections (Supplementary Fig. 3C–G). Therefore, #11 A treatment significantly alleviates these AD pathologies in Thy1-ApoE4/C/EBPβ transgenic mouse brains.

Repeated treatment of Thy1-ApoE4/C/EBPβ transgenic mice with #11 A alleviates synaptic degeneration and rescues cognitive functions

AD is associated with wide-spread synaptic degeneration with impaired synaptic plasticity, which is frequently accompanied with massive neuronal cell loss. Noticeably, IB analysis demonstrated that repeated treatment with #11 A significantly increased synaptic protein levels including Glut2, PSD95, Spinophilin and Synapsin 1 in the Thy1-ApoE4/C/EBPβ transgenic mouse brain (Fig. 5A, B). Neuronal cell death was prominently blocked by #11 A, which was analyzed by IF co-staining with NeuN and TUNEL on the brain sections (Fig. 5C, D). Based on the results of the Morris Water Maze (MWM) assay, #11 A repeated treatment significantly increased the learning and memory in both the training and probe trials (Fig. 5E, F). The results of the Novel Object Recognition test also supported that #11 A treatment appreciably enhanced the memory capability (Fig. 5G, H). Together, these findings manifest that #11 A repeated treatment ameliorates synaptic degeneration and neuronal cell death, resulting in alleviation of cognitive dysfunctions of Thy1-ApoE4/C/EBPβ transgenic mice.

Fig. 5. Repeated treatment with #11 A inhibits neuronal loss and improve cognitive function in Thy1-ApoE4/C/EBPβ Tg mouse.

Fig. 5

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A Representative immunoblot images showing the effects of #11 A treatment on the expression of synaptic markers. B Relative quantification of protein level in (A). (n = 3, **p < 0.01, ***p < 0.001, compared with vehicle). C Immunofluorescence staining of NeuN and TUNEL in hippocampus of Thy1-ApoE4/C/EBPβ Tg mice with repeated #11 A treatment. Scale bar, 20 μm. D Relative quantification of NeuN and TUNEL fluorescence intensity in (C). (n = 6, *p < 0.05, compared with vehicle). E Diagram showing experiment of Morris Water Maze (MWM). F Latency to mount the submerged platform, percentage of time in platform quadrant, swim speed and total travel distance in MWM assay (n = 6, *p < 0.05, compared with vehicle). G Diagram showing experiment of Novel Object Recognition Test (NORT). H Exploring index of Thy1-ApoE4/C/EBPβ Tg mice with repeated #11 A treatment. (n = 6, *p < 0.05, compared with vehicle).

Repeated treatment of Thy1-ApoE4/C/EBPβ transgenic mice with #11 A inhibits brain volume reduction

Magnetic Resonance Imaging (MRI) analysis of brain volume changes in AD patient is one of the “4 clinical diagnosis golden standards” for the clinical diagnosis. Usually, the extensive neuron loss results in the brain volume shrinkage. To further interrogate the therapeutic efficacy of #11 A, we performed MRI analysis on Thy1-ApoE4/C/EBPβ transgenic mice. MRI revealed that Thy1-ApoE4/C/EBPβ transgenic mouse brain volumes in both the hippocampus and the cortex were significantly increased by #11 A as compared to vehicle control (Supplementary Fig. 4A–C). Nissl staining also confirmed that #11 A augmented the brain volumes in the hippocampus by decreasing the ventricle zone’s size (Supplementary Fig. 4D, E).

Mounting evidence shows that BDNF/TrkB neurotrophic signaling is abrogated in AD brains [33–35]. We have previously reported that Tau N368 physically binds the tyrosine kinase domain on the TrkB receptor and blocks its neurotrophic activities [36]. Moreover, activated TrkB phosphorylates APP on Y687, inhibiting its cleavage by AEP and reducing Aβ production [29]. On the other hand, we also show that BDNF/TrkB signaling inhibits AEP activation via Akt phosphorylation of T322 residue on AEP [37]. Accordingly, we explored these biochemical events in the brain tissues after #11 A repeated treatment and found that the trophic factors including netrin-1 and BDNF were both elevated, accordingly, p-APP Y687 activities were augmented. These effects were inversely correlated with TrkB N486 fragmentation by AEP (Supplementary Fig. 5A, B), consistent with significantly AEP inhibition by #11 A. BDNF upregulation and p-TrkB Y816 activation in the hippocampus was corroborated by IF co-staining. In alignment with activated p-TrkB signals, APP Y687 phosphorylation was consequently increased (Supplementary Fig. 5C, D). Together, our data support that #11 A increases BDNF levels and prevents TrkB cleavage by AEP, resulting in reduced neuronal degeneration and increased brain volumes in Thy1-ApoE4/C/EBPβ transgenic mice.

Discussion

AD is an age-dependent neurodegenerative disease and the most common type of dementia, leading to progressive memory loss along with neuropsychiatric symptoms and a decline in the activities of daily life. In the current study, we manifest that #11 A robustly inhibits AEP in the mouse brains of various AD models including the genetic APP/PS1 and Tau P301S mice and sporadic Thy1-ApoE4/C/EBPβ transgenic mice. #11 A is highly orally bioavailable and brain permeable, exhibiting acceptable in vivo PK profiles (Fig. 1). Moreover, it displays an orderly in vivo PK/PD relationship after oral administration in a variety of AD mouse models, blocking AEP in the brain, associated with subsided human Aβ42 and mouse Aβs and p-Tau 181 signals. These effects are closely coupled with reduced AEP-truncated APP N585, C586 and Tau N368 levels in the CSF and plasma (Figs. 2, 3 and Supplementary Fig. 1). These strong PK/PD relationships in the body fluids lay a solid foundation for closely monitoring #11 A’s drug engagement in future clinical trials. Using biochemical assays, IHC and IF staining and neuroimaging, we have provided abundant evidence that AD pathologies including amyloid plaques, Tau aggregation and neuroinflammation and neurodegeneration are all significantly blunted by repeated treatment with #11 A in sporadic Thy1-ApoE4/C/EBPβ transgenic mice (Figs. 4, 5, and Supplementary Fig. 3).

In Tau P301S mice, the brain/plasma (B/P) ratios for #11 A at 2 h change from 0.14 to 0.29 to 0.45, while in APP/PS1 mice, the B/P ratios alter from 0.32 to 0.29 to 0.48, as the doses are gradually increased from 3.5 to 7.5 to 15 mg/kg. Hence, in genetic AD mouse models, #11 A B/P ratios are augmented as the doses elevate. By contrast, in the sporadic Thy1-ApoE4/C/EBPβ transgenic mice, the B/P ratios remain around 0.25 on average regardless of the dose escalation (Fig. 3). The main reason for the moderate B/P ratio may be due to numerous O and N atoms in #11 A. Presumably, reduction of the H-bond acceptors or donors and increase of hydrophobicity will improve the brain penetration of #11 A.

Interestingly, AEP is highly upregulated in several cancer types such as colon, prostate and breast cancer [38]. AEP has been found to promote cancer cell invasiveness both in vitro and in vivo [39]. Recently, we showed that #11 A blocks breast cancer cells to lung metastasis via blocking C/EBPβ/AEP signaling [25]. When combined with TrkB agonist CF3CN, #11 A inhibits AEP activities in the 3xTg [28] and α-SNCA PD mice [27], attenuates the pathologies and restores cognitive functions and motor movements. Fitting with these findings, we show that #11 A treatment elevates BDNF levels in the brain and activates TrkB signaling in addition to upregulation of netrin-1, a secreted hormone implicated in AD pathologies [40], resulting in escalation of p-APP Y687 signals and repression of Aβ production (Supplementary Fig. 5). In addition to the genetic AD mouse models including Tau P301S, APP/PS1 and 3xTg, we also demonstrate that #11 A exerts profound therapeutic efficacy in the sporadic Thy1-ApoE4/C/EBPβ transgenic mice that exhibit AD pathologies with mouse endogenous machinery without any patient-derived mutations. These findings indicate that this compound may act as an idea clinical candidate for treating most of AD patients, because more than 95% of AD patients are sporadic.

ApoE4 is the biggest genetic risk factor for AD. We have reported that neuronal C/EBPβ is escalated during aging and stimulated by various AD risk factors, dictating mRNA transcription of all of the major AD-related players, including APP, MAPT, ApoE4, BACE1, and AEP [23]. Physiologically, ApoE4 is mainly expressed in astrocytes, it can be upregulated in neurons under stress or inflammation, hence, we overexpress neuronal ApoE4 under the Thy1 promoter for both ApoE4 and C/EBPβ. As we showed, this mouse model recapitulates most of AD pathologies temporo-spatially in the absence of any human APP, PS1/2 mutation. This mouse model displays cognitive defects and acts as a sporadic mouse model [23]. To our knowledge, this is the best sporadic mouse model at present that reconstitutes most of AD pathologies. Nevertheless, it should be noted that the sporadic mouse model may not represent the development of AD disease in humans. However, compared to the commonly used genetic AD mouse models, it provides another experimental evidence that #11 A could serve as a preclinical candidate for pharmacologically treating AD.

Treatments whose purpose is cognitive enhancement or control of neuropsychiatric symptoms without claiming to impact the underlying biological causes of AD are classified as “symptomatic”, including ACHE inhibitors. Treatments intended to change the biology of AD and slow the course of the disease are listed as “disease modifying.” [41]. Most recently, Lecanemab, a humanized IgG1 monoclonal antibody that binds with high affinity to Aβ soluble protofibrils. It reduces markers of senile plaques in early AD and results in moderately less decline on measures of cognition and function than placebo at 18 months but it is also associated with adverse events [8]. In addition, Donanemab, an antibody designed to clear brain amyloid plaques, significantly slows clinical progression at 76 weeks among participants with early symptomatic AD and Aβ and tau pathology [10]. Hence, these two monoclonal anti-Aβ antibodies are disease-modifying therapies, directly targeting the amyloid pathologies in AD patient brains. Nevertheless, these immunotherapies are associated with adverse effects. For instance, Lecanemab results in infusion-related reactions in 26.4% of the participants and amyloid-related imaging abnormalities with edema or effusions in 12.6%. Donanemab also has similar adverse effects. Moreover, three deaths in the donanemab group and 1 in the placebo group are considered treatment related [8, 10]. Clearly, safer and more effective drugs for treating this devastating disease are urgently demanded to meet the clinical needs.

AEP is an emerging innovative drug target for halting AD onset and progression. Accumulative evidence supports that it acts as a δ-secretase that simultaneously cleaves both APP and Tau, facilitating both Aβ and Tau pathologies [12, 13, 42]. Previously, we showed that AEP is activated by acidosis after ischemia or excitotoxicity. Activated AEP cleaves SET, the nuclear protein inhibiting DNase, at the N175 residue and leads to neuronal cell death [11]. Therefore, AEP drives neurodegeneration not only by triggering DNase activation but also elevating cytotoxic Aβ and Tau N368. Of note, the AEP-generated Tau N368 is abundant in the brain of AD patients but barely detectable in the control brain. Moreover, Tau N368 is more prone to aggregate and is highly neurotoxic. Knockout of AEP from Tau P301S mice partially alleviates Tau deposition and memory loss [12]. We further found that AEP binds to BACE1 and cleaves it at the N294 residue in an age-dependent manner. Interestingly, the truncated BACE1 enzymatic domain (1-294) exhibits increased secretase activity and accelerates Aβ production, promoting AD pathogenesis and cognitive dysfunctions in an APP/PS1 AD mouse model [14].

Physiologically, AEP is bound and inhibited by cystatin C in the lysosomes. However, the cystatin C levels in the CSF and brain are lower in AD patients than in control individuals, resulting in AEP activation [43]. AEP is highly expressed in the proximal tubular cells (PTCs) of kidney [44]. The PTCs is responsible for the uptake of proteins from the crude urine. AEP knockout mice show accumulated proteins in their PTC endosomes and lysosomes, indicating AEP is required for the normal processing of these proteins. As a result, AEP knockout mice develop hyperplasia of PTCs, interstitial fibrosis, glomerular cysts, proteinuria and decreased glomerular filtration [44]. We have reported that AEP knockout mice also show anemia and extramedullary hematopoiesis. Some plasma membrane components are altered in red blood cells from AEP-null mice. The activity of natural killer cells is also affected in AEP knockout mice. These symptoms are similar to hemophagocytic syndrome/hemophagocytic lymphohistiocytosis (HLH) [45]. Additionally, AEP plays an important role in immunity. For instance, AEP is implicated in class II MHC maturation through proteolysis of the invariant chain (li) [46]. Employing AEP null mice, Ploegh et al. demonstrated that AEP is essential for the processing of cathespsin L but not for class II MHC-restricted antigen presentation in mice [47]. Although AEP homozygous knockout mice exhibit the above adverse effects, heterozygous knockout mice are completely normal without any demonstrable defects. These are consistent with our observation of repeated treatment with AEP inhibitor #11 [24] or its derivative #11 A that exhibit no any noticeable toxicity or side effects [27, 28]. At 7.5 mg/kg dose, only 30–40% of AEP activity is blocked by the brain penetrated #11 A in various AD mouse models upon acute treatment (Figs. 2, 3 and Supplementary Fig. 1). In repeated treatment with the same dose, we observed approximately 50–60% reduction of brain AEP activities (Supplementary Fig. 2) and significant therapeutic efficacy in alleviating AD pathologies and restoring cognitive functions (Figs. 4, 5 and Supplementary Fig. 3, 4). Hence, partial suppression of active AEP in the brain is sufficient to demonstrate the prominent therapeutic efficacy, indicating that it might be safe for repeated drug treatment for AD patients in the near future.

Supplementary information

Supplementary files (1.1MB, pdf)

Author contributions

KY conceived the project, designed the experiments, analyzed the data, and wrote the manuscript. ZQ designed and performed most of the experiments, analyzed the data and wrote the manuscript. BL performed the SiMoA experiments. XM and GW performed the LC-MS/MS analysis. JL performed part of the immunofluorescence staining experiments. YL and QL assisted with data analysis and interpretation. All the authors approved the manuscript.

Funding

This work was supported by Start-up fund from SIAT and the National Natural Science Foundation of China (32330040) to KY, the Guangdong Basic and Applied Basic Research Foundation (2023A1515030296) and the Shenzhen Government Basic Research Program (JCYJ20220531100802005) to ZQ, and the National Natural Science Foundation of China (32200928) to YL, and the “Hundred, Thousand and Ten Thousand” Science and Technology Major Special Project of Heilongjiang Province (No. 2020ZX07B01) to QL.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Qian Luo, Email: qian.luo@siat.ac.cn.

Keqiang Ye, Email: kq.ye@siat.ac.cn.

Supplementary information

The online version contains supplementary material available at 10.1038/s41386-023-01774-2.

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