AVE 0991 suppresses neuroinflammation of Alzheimer's disease by enhancing autophagy in astrocytes

doi:10.21203/rs.3.rs-1967115/v1

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AVE 0991 suppresses neuroinflammation of Alzheimer's disease by enhancing autophagy in astrocytes

https://doi.org/10.21203/rs.3.rs-1967115/v1

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In our previous study, we have shown that AVE 0991, a nonpeptide analogue of Ang-(1–7), ameliorates cognitive decline and inhibits NLRP3 inflammasome of astrocytes in Alzheimer's disease model mice. Additionally, several studies have suggested that activation of autophagy appears to effectively inhibit the progression of neuroinflammation. However, it is unclear whether AVE 0991 can modulate astrocyte autophagy to suppress neuroinflammation in Alzheimer's disease. In our study, we showed that 30 days of intraperitoneal administration of AVE 0991 improved cognitive deficits and neuronal death in APP/PS1 Alzheimer's disease model mice. Moreover, AVE 0991 treatment greatly suppressed astrocyte-mediated inflammation and up-regulated the expression of autophagy. Furthermore, the inhibitory effect of AVE 0991 on the expression of inflammatory factors was reversed by 3-MA, an autophagy inhibitor. These findings suggest that regulation of autophagy is critical for inhibiting astrocyte neuroinflammatory responses and demonstrate a potential neuroprotective mechanism by which AVE 0991 could suppress neuroinflammatory responses by enhancing autophagy.

AVE 0991

Neuroinflammation

Alzheimer

Autophagy

Astrocytes

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by progressive cognitive impairment, memory degeneration and decreased locomotor activities [1]. Previously, mainstream research focused on the pathogenic effects of the two main AD drivers, Aβ peptide (Aβ) and hp-Tau protein [2, 3]. Recently, more and more evidence indicated that neuroinflammation plays a central role in the pathogenesis of AD [4, 5]. Emerging studies have suggested that controlling neuroinflammation mediated by glial cell activation is an important therapeutic strategy for AD [6, 7] and previous reports have also found that the glial responses activated by Aβ can promote the release of pro-inflammatory cytokines to cause neuronal dysfunction and memory impairment [8–10]. Therefore, targeting neuroinflammation induced by glial activation may be an effective way to slow or prevent the progression of AD.

Astrocytes are the most abundant glial cells in the brain [11] and play a vital role in maintaining brain homeostasis [12–14]. When AD occurs, the deposition of Aβ will induce astrocytes to transform from a resting state to a reactive state, which is manifested by increased levels of glial fibrillary acidic protein (GFAP) [15], which in turn produce more pro-inflammatory cytokines and mediators, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin- 6 (IL-6), which exacerbates the progression of inflammation [16]. Additionally, recent data found a specific accumulation of astrocytes associated with pro-inflammatory diseases in AD [17]. Therefore, inhibiting astrocyte-mediated inflammation may be a potentially effective strategy for reducing cognitive impairment in AD patients.

Emerging data reveal that autophagy may play a critical role in the regulation of astrocyte-mediated inflammatory response during AD [18]. Autophagy is one of the effective ways for the body to digest and degrade damaged or dysfunctional proteins and organelles in the cytoplasm and it plays an important role in maintaining cell survival, differentiation, development and homeostasis [19]. Previous studies have found that autophagy dysfunction occurs in both AD animal models and clinical patients, leading to the pathological accumulation of tau aggregates and Aβ, which in turn aggravates the course of AD [20]. Furthermore, another study demonstrated that Aβ-induced inflammatory cytokines release are related to astrocyte autophagy dysfunction, and activation of autophagy can inhibit Aβ-induced inflammatory responses [21]. Therefore, we can speculate that in the inflammatory environment mediated by astrocytes, the activation of autophagy may delay the progression of AD.

AVE 0991 (5-Formyl-4-methoxy-2-phenyl-1-[[4-[2-(ethylaminocarbonylsulfonamido) 5-isobutyl-3-thienyl]-phenyl]-meth-yl]-imidazole) is a nonpeptide analogue of Ang-(1–7), which can simulate the multiple functions of Ang-(1–7) in the nervous system. Our previous study proofed that intracerebroventricular injection of angiotensin-(1–7) can inhibit the excessive activation of autophagy in the brain of spontaneously hypertensive rats [22]. In Parkinson's disease, Ang-(1–7) reduced the aggregation of α-synuclein in rotenone-induced cell models by enhancing autophagy activity [23]. In fact, our previous studies have directly confirmed that AVE 0991 inhibits the inflammatory response mediated by microglia in a MAS receptor-dependent manner, thereby reducing the aging-related neuroinflammation [24]. Notably, AVE 0991 also enhances the clearance of NLRP3 inflammasomes by promoting the process of autophagy, thereby protecting the brain from LPS-induced excessive inflammation [25]. However, it is not clear whether AVE 0991 regulates autophagy and its neuroprotective mechanisms in AD.

Therefore, we hypothesized that AVE 0991 exerts its neuroprotective effect by promoting autophagy of astrocytes and inhibiting neuroinflammation in AD. In the present study, we investigated the effects of AVE 0991 on behavior, inflammation and autophagy in APP/PS1 AD model mice. Meanwhile, we treated primary astrocytes with AVE 0991 to investigate whether AVE 0991 inhibited the neuroinflammatory response induced by Aβ1–42 in astrocytes by promoting autophagy. Our data indicated that AVE 0991 protects against astrocyte-mediated inflammation by promoting autophagy.

Animals and treatments

Eight-month-old male APPswe/PSEN1dE9 (APP/PS1) double-transgenic mice and age-matched male wild-type C57BL/6J mice (WT) were purchased from the Beijing Zhishan Co., Ltd. All experiments were performed in accordance with the guidelines for the care and use of laboratory animals, and were approved by the Animal Research and Ethics Committee of Nanjing First Hospital (protocol#: IACUC-1911032).

WT and APP/PS1 mice were randomly divided into six groups: WT group, APP/PS1 group, APP/PS1 + 1 mg/kg AVE 0991 group, APP/PS1 + 3 mg/kg AVE 0991 group, APP/PS1 + 10 mg/kg AVE 0991 group and APP/PS1 + 10 mg/kg AVE 0991 + 30 mg/kg 3-MA group. All mice were given intraperitoneal injection once daily for 30 consecutive days. Mice in WT group and APP/PS1 group were given equal volumes of normal saline. The Morris Water Maze (MWM) experiment was started on the 25th day. After the behavioral experiment was completed, all mice were quickly sacrificed. The brain was promptly removed on ice, and then stored at -80°C for molecular detections such as gene and protein analysis.

Morris water maze (MWM) test

The Morris Water Maze (MWM) was widely used to evaluate learning and memory of mice [26]. In short, a hidden round platform was placed 1.00 cm below the water surface. A video tracking system (Ethovision 3.0, Noldus Information Technology B.V.) was used to automatically track mice. Data of learning and memory were collected for 5 days. The animals were randomly put into the water from four points every day until the platform was found and stayed for 10 s within 60 s. If the animals cannot find the platform within 60 s, they then were guided to the platform. One day after the last trial, the platform was removed and the mice were placed into the water from the opposite quadrant of the platform. The path length to the merged platform was recorded, and the average value of four trials was calculated.

Nissl staining

Nissl staining was performed as described [27]. In brief, paraffin-embedded sections were deparaffinized and rehydrated according to standard protocols. The sections then were stained with tar violet at 56℃ for 1 h. Next, the sections were dehydrated with gradient concentrations of ethanol, rinsed with xylene, and sealed with neutral gum after removing the dye by washing. Then a CX23 microscope (Olympus) was used to randomly select six fields of cortex on each coronal slice for quantitative analysis. The cell bodies of injured neurons were more shrunken and the nuclei was darker than normal neurons. Nissl positive neurons % = positive neurons/total neurons ×100%.

Oligomeric Aβ₁₋₄₂ preparation

The preparation of Aβ1–42 oligomer was performed as previously described [28]. In short, Aβ1–42 monomer was prepared by evaporating 2 mg of Aβ1–42 dissolved in 1, 1, 1, 3, 3, 3, hexafluoro-2-propanol for 30 minutes under N2 gas at room temperature. Then, the monomer was diluted to 10 µM. Subsequently, the diluted solution was incubated to form oligomers. Next, The Aβ1–42 oligomer preparations were centrifuged to remove any insoluble fibrils.

Primary astrocytes culture and treatment

Primary astrocytes were isolated from the brain of newborn WT mice (< 24 h). Briefly, the minced brain was digested, the digestion solution was filtered and centrifuged, and then the cells were seeded in DMEM/F12 complete medium, and incubated in a 5% CO₂ incubator at 37°C. After 10 days of incubation, the cells were shaken to remove microglial contamination from adherent astrocytes. Then, the purified astrocytes were cultured in DMEM/F12 complete medium and placed in a humidified incubator at 37°C and 5% CO₂. The third generation of cells were used for subsequent experiments. The purity of astrocytes was evaluated by immunofluorescence staining with anti-GFAP antibody.

Astrocytes were treated for 24 h with 5 µM Aβ₁₋₄₂ with or without 24 h incubation with AVE 0991 (1×10^− 8, 1×10^− 7 or 1×10^− 6 M) in the presence or absence of A-779 (1×10^− 6 M). Besides, 3-MA was added 1 h before Aβ₁₋₄₂ treatment, while AVE 0991 was added for 24 h co-treatment upon Aβ₁₋₄₂ exposure. DMEM (10% FBS) was used as a control.

Immunofluorescence staining

Immunofluorescence was used to identify primary astrocytes. Astrocytes were fixed, penetrated and blocked. Afterwards, astrocytes were incubated with GFAP antibody (1:300, #3670, Cell Signaling Technology) overnight at 4°C. After washing with PBS, the cells were incubated with Alexa Fluor 488-labeled goat anti-mouse secondary antibody (1:500, ab150113, Abcam). The nuclei were stained with DAPI, and a confocal microscope (LSM780, Zeiss, Germany) was used to observed the samples.

Cell viability assay

Cell viability was assessed using Cell Counting Kit-8(CCK-8, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. Astrocytes were seeded in 96-well plates at a density of 5×10⁴ cells/well 24 h. The cells were then treated for 24 h with 5 µM Aβ₁₋₄₂ with or without 24 h incubation with AVE 0991 (1×10^− 8, 1×10^− 7 or 1×10^− 6 M). Then, 10 µl CCK-8 was added to the cells and incubated at 37°C for 2 h. The absorbance at 450 nm was measured using a microplate reader (Thermo Scientific, San Jose, CA, USA).

Enzyme-Linked Immunosorbent Assay (ELISA)

The concentrations of Ang-(1–7) and inflammatory cytokines including IL-1β, TNF-α and IL-6 in mouse brain and the conditioned medium of each group were measured by commercial ELISA kits (R&D Systems, Inc.). The absorbance was detected and quantified with a microplate reader (Molecular Device Corporation, Sunnyvale, CA, USA).

Real-time quantitative reverse-transcription PCR (qRT-PCR)

Total RNA from mice brain or astrocytes were isolated and extracted using Trizol reagent (Invitrogen, USA) according to the manufacturer's protocol. Reverse transcription was performed using Exscript RT Reagent Kit (Takara Bio Inc., China). qRT-PCR was performed on a fluorescent thermocycler iQ5 (Bio-Rad) using SYBR® Premix Ex Taq™ II. β-actin was used as an endogenous control. Relative mRNA levels were analyzed using the 2^−ΔΔCt method. The primers used in this study were listed in Supplementary Table S1.

Western blot analysis

Western blot was performed as previously described [29]. The brain or astrocytes were lysed by RIPA lysis buffer to extract protein. A BCA protein assay kit (Thermo, Hercules, CA) was used to determine the protein concentrations. Equal amounts of protein samples were separated by SDS-PAGE and transferred to PVDF membrane (Merck Group, Darmstadt, Germany). The membrane was then blocked with 5% non-fat milk for 1 h at room temperature, and incubated with primary antibodies against Mas1 (1:200, PA5-77282, Thermo Fisher Scientific), Synaptophysin (1:1500, #SAB4502906, Sigma-Aldrich), GFAP (1:1000, #3670, Cell Signaling Technology), LC3B (1:1000, #43566, Cell Signaling Technology), Beclin-1 (1:1000, #3495, Cell Signaling Technology), p62 (1:1000, #23214, Cell Signaling Technology) and β-actin (1:1000, #4970, Cell Signaling Technology) overnight at 4°C. After washing with TBST buffer, the membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibody. Next, the membranes were visualized with a chemiluminescent HRP substrate (#32132, Thermo Fisher Scientific, Waltham, MA, USA). Protein bands were quantified by Image J software.

Statistical analysis

All experiments were performed at least three times. Prism 8.0 software (GraphPad, San Diego, CA, USA) was used for all analysis data. The results of the escape latency in MWM were analyzed by two-way analysis of variance with Tukey's multiple comparison test. All other data were analyzed using one-way analysis of variance (ANOVA) and Tukey's test for post hoc analysis. Data were presented as the mean ± standard error of the mean (SEM). A value of P < 0.05 was considered to be statistically significant.

AVE 0991 activates the Ang-(1–7)/Mas1 axis in the brain of APP/PS1 mice

As a non-peptide analogue of Ang-(1–7), AVE 0991 acts on the Ang-(1–7)/Mas1 axis and exerts various functions in neurological diseases. As shown in Fig. 1A, Ang (1–7) levels were significantly decreased in APP/PS1 mice compared with WT group, while AVE 0991 treatment (1, 3 and 10 mg/kg) have no influence on this level. Furthermore, AVE 0991 injections at 3 and 10 mg/kg dramatically increased Mas1 mRNA expression (Fig. 1B). In coincide with the qRT-PCR results, Western blot analysis indicated that AVE 0991 administration (3 and 10 mg/kg) remarkly increased the protein expression level of Mas1 (Fig. 1C, D).

AVE 0991 attenuates cognitive deficits and neuron death in AD mice

To study the effect of AVE 0991 treatment on AD model mice, 8-month-old APP/PS1 mice were intraperitoneally injected with AVE 0991 daily for 30 days. The MWM test was conducted for 5 days to assess their spatial learning and memory abilities before execution. As shown in Fig. 2A, there was no difference in swimming speed between APP/PS1 mice and their age-matched WT controls, whereas injection of different doses of AVE 0991 did not significantly affect the swimming speed of APP/PS1 mice. Meanwhile, there was no significant difference in cognitive function among the five groups on 1–2 days during the MWM test. On day 4 and 5, APP/PS1 mice exhibit significant spatial cognitive impairment, while AVE 0991 (3 and 10 mg/kg) rescues this impairment (Fig. 2B). Nissl staining was used to observe the injury of mice neurons. The results indicated that there was significant neuronal death in the cortex of APP/PS1 mice, which was notably rescued by AVE 0991 treatment (3 and 10 mg/kg) (Fig. 2C, D). As displayed in Fig. 2E and F, compared with the WT group, APP/PS1 mice had significant synapse loss, while AVE 0991 treatment (3 and 10 mg/kg) strikingly increased synaptophysin protein levels. Taken together, these results demonstrated that AVE 0991 can alleviate memory and cognitive impairment in the development of AD. In this part, we selected three doses of AVE 0991 (1, 3, and 10 mg/kg) for in vivo assay, and the 1 mg/kg AVE 0991 group did not obtain a positive effect.

AVE 0991 restrains astrocyte activation and inflammation in AD mice

In light of the fact that the deposition of Aβ in AD lesions can promote the proliferation of glial cells and trigger inflammatory response, and astrocytes are one of the most intensively studied cell types in neuroinflammation and AD [30]. Therefore, we further tested the effects of AVE 0991 treatment on the activation of astrocytes and related pro-inflammatory factors in APP/PS1 mice, including IL-1β, TNF-α and IL-6. Western blot results suggested that the GFAP expression in the brain of APP/PS1 mice was higher than that of the WT group, while was, however, inhibited by AVE 0991 treatment in a dose-dependent manner (Fig. 3A, B). Next, to evaluate whether AVE 0991 can decrease the pro-inflammatory cytokines levels in the brain of APP/PS1 mice, we measured the levels of IL-1β, IL-6 and TNF-α in mice brains. As shown in Fig. 3C-E, the mRNA expression of IL-1β, IL-6 and TNF-α was remarkably increased in the brain of the APP/PS1 mice compared with the WT mice. Intriguingly, AVE 0991 treatment (3 and 10 mg/kg) markably decreased the expression of these cytokines. In addition, in order to further verify the above results, we then tested the effect of AVE 0991 on IL-1β, IL-6 and TNF-α by ELISA. Results indicated that the concentrations of IL-1β, IL-6 and TNF-α in the brain was in line with qRT-PCR results (Fig. 3F-H). Taken together, these results showed that AVE 0991 could effectively inhibit the activation of astrocytes and pro-inflammatory cytokines transcription and secretion in APP/PS1 mice.

AVE 0991 alleviates inflammation and promotes autophagy in Aβ-treated astrocytes

In order to explore the effect of AVE 0991 on astrocyte-mediated inflammation, astrocytes were induced by Aβ₁₋₄₂ in vitro experiments. First, immunofluorescence identification with GFAP proved that the primary astrocytes isolated in this study met the requirements (Fig. 4A). Then we incubated astrocytes with different doses of AVE 0991 (1×10^− 8, 1×10^− 7和1×10^− 6 M) and Aβ₁₋₄₂ for 24 h. CCK-8 results indicated that Aβ₁₋₄₂ stimulation resulted in a prominent decrease in astrocyte viability, whereas AVE 0991 increased cell viability in a dose-dependent manner (Fig. 4B). Meanwhile, Western blot analysis showed that the expression of GFAP in astrocytes stimulated by Aβ₁₋₄₂ was strongly increased compared with the normal group, while was inhibited by AVE 0991 in a dose-dependent manner (Fig. 4C, D). Besides, compared with the normal group, Aβ₁₋₄₂ considerably increased the mRNA expression of IL-1β, IL-6 and TNF-α in astrocytes. However, these increases were remarkably cancelled by AVE 0991 (Fig. 4E-G). In addition, the protein levels of IL-1β, IL-6 and TNF-α in the culture medium also showed a similar trend by ELISA (Fig. 4H-J). These data sugggest that the neuroprotective effect of AVE 0991 on Aβ ₁₋₄₂-induced neurotoxicity is associated with reduced release of inflammatory cytokines in astrocytes.

To further verify whether AVE 0991 can promote autophagy in Aβ₁₋₄₂-induced astrocytes, the expression of autophagy-related proteins LC3, Beclin-1 and P62 in astrocytes was determined by Western blot. Aβ₁₋₄₂ stimulation significantly reduced LC3 processing and Beclin-1 expression, while increased the protein abundance of P62 compared with the normal group, indicating that the autophagy function of astrocytes induced by Aβ₁₋₄₂ was impaired (Fig. 4K-N). Different concentrations of AVE 0991 upregulated autophagy activity in Aβ₁₋₄₂-induced astrocytes via increasing LC3 processing and Beclin-1 expression, whereas decreasing P62 level (Fig. 4K-N). These results indicate that AVE 0991 triggers autophagy in astrocytes.

AVE 0991 promotes Aβ-induced astrocytic autophagy and suppresses inflammation through a Mas1 dependent manner

Previous studies have identified that Mas1 is the functional receptor of Ang-(1–7) and is responsible for the beneficial physiological effects of Ang-(1–7) [31]. In order to determine whether the effect of AVE 0991, a selective agonist of Mas1, in regulating inflammation and autophagy was associated with the Mas1, astrocytes treated with AVE 0991 were co-incubated with Mas1 antagonist A-779 for 24 h concurrently. As expected, qRT-PCR results showed that AVE 0991 (1×10^− 6 M) substantially decreased the mRNA expression of cytokine in astrocytes, while A-779 abolished this inhibitory effect of AVE 0991 (Fig. 5A-C). Similarly, ELISA results indicated that A-779 partially reversed the inhibitory effect of AVE 0991 on proinflammatory cytokines IL-1β, IL-6 and TNF-α (Fig. 5D-F). In addition, Western blot analysis showed that AVE 0991 (1×10^− 6 M) notably increased the expression of LC3 processing and Beclin-1, while decreased the expression of P62. However, the effects of AVE 0991 on protein levels of LC3, Beclin-1 and P62 were partially eliminated by A-779 (Fig. 5G-J). This observation indicated that AVE 0991 promoted astrocytic autophagy and inhibit astrocytic inflammation via a Mas1-dependent manner.

AVE 0991 suppresses Aβ-induced inflammatory responses by activating astrocyte autophagy

Recent studies have shown that autophagy can limit harmful and uncontrolled inflammation by removing pro-inflammatory cytokines. Considering the aforementioned neuroprotective ability of AVE 0991 in regulating autophagy and inflammation, we speculate that AVE 0991 may reduce neuroinflammation by promoting autophagy in Aβ₁₋₄₂-induced astrocytes. 3-MA, the autophagy inhibitor, was used to block autophagy activation of astrocytes. 3-MA (5 µM) was added at 1 h before Aβ₁₋₄₂ treatment for 24 h, and AVE 0991 was added at the same time as Aβ₁₋₄₂ exposure. The protein abundance of autophagy markers in astrocytes was determined by Western blot. As expected, compared with Aβ₁₋₄₂ + AVE 0991 group, 3-MA treatment robustly decreased the expression of LC3 processing and Beclin-1, whereas the expression of P62 was enhanced by 3-MA (Fig. 6A-D). This indicated that 3-MA prevents the promoting effect of AVE 0991 on astrocytes autophagy. Besides, we further evaluated the relationship between the inhibition of neuroinflammation and autophagy activation by AVE 0991 in Aβ₁₋₄₂ treated astrocytes. ELISA results showed that AVE 0991 greatly reduced the increase of cytokines levels in astrocytes induced by Aβ₁₋₄₂, while 3-MA eliminated the inhibitory effect of AVE 0991 on the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α (Fig. 6E-G). Consistent with ELISA results, qRT-PCR results proved that 3-MA significantly reduced the inhibitory effect of AVE 0991 on pro-inflammatory cytokines mRNA expression (Fig. 6H-J). These findings suggested that AVE 0991 inhibits the inflammatory responses induced by Aβ₁₋₄₂ by promoting autophagy in astrocytes.

AVE 0991 has been studied in different central nervous system diseases, including subarachnoid hemorrhage (SAH), chronic cerebral hypoperfusion (CCH) and ischemic stroke. For example, the expression of Ang-(1–7) and Mas1 observed was markedly increased in during cerebral ischemia, and AVE 0991 can protect neurons from ischemic damage when applied early during the ischemic period [32]. Moreover, Mas expression was obviously decreased after SAH, while AVE 0991 reduced neuronal apoptosis and oxidative stress, and improved short-term and long-term neurological function [33]. Furthermore, our previous study found that AVE 0991 significantly reduced hippocampal synaptic degeneration in CCH rats by promoting CBF recovery, reducing hippocampal Aβ levels and inhibiting neuroinflammatory responses [34]. However, whether AVE 0991 has a neuroprotective effect in AD and its potential mechanisms are still largely unknown. In the current study, we demonstrated AVE 0991 plays a protective role in APP/PS1 mice, and illustrated its anti-inflammatory and pro-autophagy mechanisms in vivo and in vitro. In APP/PS1 mice, AVE 0991 treatment improved memory and cognitive impairment, neuronal loss and synaptic damage. Subsequently, we aimed to clarify whether the expression of pro-inflammatory cytokines and autophagy was regulated by AVE 0991. Our results indicated that AVE 0991 suppressed the neuroinflammation of Alzheimer's disease by enhancing autophagy of astrocytes.

Neuroinflammation is currently considered to be widely involved in the occurrence and development of AD pathology [35]. This inflammatory response can often promote tissue repair and remove cellular debris to produce beneficial effects. However, sustained inflammation is detrimental [36, 37]. In fact, elevated inflammatory markers such as cytokines IL-1β, IL-6 and TNF-α have been detected in AD patients or in different animal models with AD-like pathology [38]. Therefore, maintaining the balance of inflammation in AD may be very important. Recently, the relationship between AVE 0991 and inflammation has been reported, suggesting that AVE 0991 may inhibit inflammation in response to stressful conditions. For instance, AVE 0991 alleviated pyrolysis and liver damage by inhibiting the ROS-NLRP3 inflammatory signaling pathway after heatstroke [39]. In a mouse model of AKI induced by bilateral I/R injury, AVE 0991 treatment attenuated local and systemic inflammatory responses and reduce renal functional impairment [40]. Wang et al. observed that AVE 0991 alleviated inflammation-induced arthritis by attenuating the NF-κB and MAPK pathways [41]. In addition, our previous study confirmed that AVE 0991 inhibits microglia-mediated inflammatory response in a MAS receptor-dependent manner, thereby attenuating aging-related neuroinflammation [24]. However, whether AVE 0991 plays a protective role in AD by improving inflammation has not been confirmed. In the present study, our data found that AVE 0991 improved cognitive and memory impairment in mice. Furthermore, the inflammation levels of APP/PS1 mice were up-regulated, whereas AVE 0991 could down-regulated the expression of IL-1β, IL-6 and TNF-α in the brain by a dose-dependent manner, indicating that AVE 0991 may play a neuroprotective effect in AD by suppressing inflammation.

Astrocytes and microglia have important activities in homeostasis and brain function, and are the major factors involved in the inflammatory process of AD [42]. When inflammation occurs, both activated astrocytes and microglia produce and secrete several pro-inflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), all of which could result in neurotoxicity [43]. In fact, astrocytes are the most widely distributed cell type in brain and the time scale of pro-inflammatory signaling of astrocytes lasts longer than that of microglia [44, 45]. Besides, it is reported that GFAP expression increased in reactive astrocytes during AD, and this increase was associated with the progression of the disease [46]. Moreover, among the different GFAP subtypes, GFAPα and GFAPδ immunoreactive astrocytes are often abundantly distributed near Aβ plaques [47]. Thus, some authors proposed that Aβ-induced reactive astrocytes were the main driver of AD′s neuroinflammation [48]. A previous study demonstrated that safflower leaf powder improved inflammatory responses and cognitive function in APP/PS1 mice by suppressing excessive astrocyte activation [49]. In addition, Silibinin-loaded exosomes prevented astrocyte activation and attenuated astrocyte inflammation-mediated neuronal damage [50]. Similar to these findings, our data proved that the elevation of GFAP in the brain of APP/PS1 mice can be effectively inhibited by AVE 0991. Meanwhile, AVE 0991 treatment suppresses Aβ-stimulated astrocyte activation and decreases the production and secretion of pro-inflammatory cytokines in vitro, which was keeping with the results in vivo. These results indicated that the neuroprotective effect of AVE 0991 appears to be related to the reduced release of inflammatory cytokines after astrocyte activation.

Autophagy is one of the more effective ways by which the body removes misfolded proteins and damaged organelles, which is responsible for maintaining cell survival and normal function [19]. It is reported that dysfunction in the process of autophagy is closely related to the pathology of AD [51]. When AD occurs, it will lead to the accumulation of misfolded proteins (mainly composed of Aβ). Under physiological conditions, autophagy usually maintains Aβ homeostasis in healthy brains by clearing Aβ [52]. Autophagy stimulation, moreover, can inhibit tau hyperphosphorylation [53]. However, when autophagy is impaired, the clearance of these harmful proteins is delayed [54]. Therefore, activating autophagy to enhance the elimination of harmful proteins was considered to be a promising target for discovery of anti-AD drugs. Recently, the association between AVE 0991 and autophagy has been studied. AVE 0991 treatment aggravated palmitic acid-induced autophagy and ER stress in human proximal renal tubular HK2 cells [55]. In LPS-stimulated microglia and mouse brain tissues, activation of Mas receptor by AVE 0991 can significantly trigger FOXO1 signaling and promote autophagy [25]. Here, we investigated the potential ability of AVE 0991 to activate autophagy in AD. Our results showed that Aβ1–42 stimulation reduced the autophagy of astrocytes, as assessed by down-regulating LC3 processing, Beclin-1 expression and up-regulating p62 expression, while AVE 0991 treatment promoted the autophagy activity of astrocytes. This effect, nevertheless, can be partially eliminated by the Mas receptor antagonist A-779. This observation indicated that AVE 0991 promoted astrocyte autophagy through a Mas1 dependent manner. Current evidence suggested that astrocyte autophagy could limit harmful and uncontrolled inflammation, thereby acting as a central fulcrum to balance inflammatory responses [56]. A recent study has confirmed that AVE 0991 enhanced the clearance of NLRP3 inflammasomes by promoting autophagy, thereby protecting the brain from excessive inflammation caused by LPS exposure [25]. Therefore, we used 3-MA to study whether the anti-inflammatory effect of AVE 0991 was mediated by autophagy. Our results indicated that blockage of autophagy activation in astrocyte with 3-MA remarkably increased the release of pro-inflammatory cytokines induced by Aβ1–42 following AVE 0991 treatment. Similarly, the anti-inflammatory effect of AVE 0991 in vivo was remarkably eliminated by 3-MA. These data strongly indicated that AVE 0991 inhibited neuroinflammatory injury and brain damage in AD by promoting autophagy activation.

In conclusion, our data demonstrated that AVE 0991 alleviated the activation of astrocytes, attenuated neuroinflammation and improved memory and cognitive function in AD. This effect may be regulated by astrocyte autophagy following AVE 0991 administration. Therefore, promoting autophagy to resist astrocyte-mediated inflammation may be a neuroprotective mechanism of AVE 0991 in AD. Our study proved that astrocyte autophagy was a potential therapeutic target for AD, and provided a new insight into the neuroprotective mechanism of AVE 0991.

AUTHOR CONTRIBUTION

Yingdong Zhang and Rui Duan revised the manuscript and advised experimental design; Rui Duan wrote the manuscript; Yang Deng, Siyu Wang and Qingguang Wang performed the research; Qiang Peng and Lin Zhu conducted and helped with the experiment methods; Zhaohan Xu analyzed the data and participated in statistical analyses; Shuaiyu Chen was responsible for treatments. All of the authors read and approved the final manuscript.

FUNDING

This work was supported by the National Science and Technology Innovation 2030 - Major program of "Brain Science and Brain-Inspired Intelligence Research" (2021ZD0201807), the Natural Science Foundation of Jiangsu Province (BK20201117), the International Joint Research and Development Project of Nanjing (202201030), the Natural Science Foundation of Jiangsu Province (BK20220196), China Postdoctoral Science Foundation (2022M711666) and Xinghuo Talent Program of Nanjing First Hospital.

DATA AVAILABILITY All data present in the paper is available for reasonable request to the corresponding author.

Ethics approval and consent to participate. All animal studies were approved by the Animal Research and Ethics Committee of Nanjing First Hospital.

Consent for Publication All authors read and approved the final manuscript.

Conflict of Interest The authors declare that there is no conflict of interest.

Acknowledgements Not Applicable.

Authors' information

Yang Deng, Email: [email protected];

Siyu Wang, Email: [email protected];

Qingguang Wang, Email: [email protected];

Zhaohan Xu, Email: [email protected];

Qiang Peng, Email: [email protected];

Shuaiyu Chen, Email: [email protected];

Lin Zhu, Email: [email protected];

Yingdong Zhang, Email: [email protected];

Rui Duan, Email: [email protected].

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No competing interests reported.

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AVE 0991 suppresses neuroinflammation of Alzheimer's disease by enhancing autophagy in astrocytes

Status:

Version 1

Abstract

Figures

Introduction

Matetial And Methods

Animals and treatments

Morris water maze (MWM) test

Nissl staining

Oligomeric Aβ₁₋₄₂ preparation

Primary astrocytes culture and treatment

Immunofluorescence staining

Cell viability assay

Enzyme-Linked Immunosorbent Assay (ELISA)

Real-time quantitative reverse-transcription PCR (qRT-PCR)

Western blot analysis

Statistical analysis

Result

AVE 0991 activates the Ang-(1–7)/Mas1 axis in the brain of APP/PS1 mice

AVE 0991 attenuates cognitive deficits and neuron death in AD mice

AVE 0991 restrains astrocyte activation and inflammation in AD mice

AVE 0991 alleviates inflammation and promotes autophagy in Aβ-treated astrocytes

AVE 0991 promotes Aβ-induced astrocytic autophagy and suppresses inflammation through a Mas1 dependent manner

AVE 0991 suppresses Aβ-induced inflammatory responses by activating astrocyte autophagy

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

AVE 0991 suppresses neuroinflammation of Alzheimer's disease by enhancing autophagy in astrocytes

Status:

Version 1

Abstract

Figures

Introduction

Matetial And Methods

Animals and treatments

Morris water maze (MWM) test

Nissl staining

Oligomeric Aβ1−42 preparation

Primary astrocytes culture and treatment

Immunofluorescence staining

Cell viability assay

Enzyme-Linked Immunosorbent Assay (ELISA)

Real-time quantitative reverse-transcription PCR (qRT-PCR)

Western blot analysis

Statistical analysis

Result

AVE 0991 activates the Ang-(1–7)/Mas1 axis in the brain of APP/PS1 mice

AVE 0991 attenuates cognitive deficits and neuron death in AD mice

AVE 0991 restrains astrocyte activation and inflammation in AD mice

AVE 0991 alleviates inflammation and promotes autophagy in Aβ-treated astrocytes

AVE 0991 promotes Aβ-induced astrocytic autophagy and suppresses inflammation through a Mas1 dependent manner

AVE 0991 suppresses Aβ-induced inflammatory responses by activating astrocyte autophagy

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Oligomeric Aβ₁₋₄₂ preparation