Inhibition of miR-4763-3p expression in the brains of AD-MCI mice activates the PI3K/mTOR/Bcl2 autophagy signaling pathway to reverse neuronal loss and ameliorate cognitive decline

Inhibition of miR-4763-3p expression in the brains of AD-MCI mice activates the PI3K/mTOR/Bcl2 autophagy signaling pathway to reverse neuronal loss and ameliorate cognitive decline | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Inhibition of miR-4763-3p expression in the brains of AD-MCI mice activates the PI3K/mTOR/Bcl2 autophagy signaling pathway to reverse neuronal loss and ameliorate cognitive decline Wenxin Qi, Naijun Dong, Peiru WU, Wenjun Fu, Qian Liu, Xueqi Zhang, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4458094/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Cognitive decline and memory impairment are frequently observed in Alzheimer's disease (AD) patients and are closely associated with dysfunctional autophagy and neuroinflammation, which subsequently result in neuronal apoptosis and synaptic damage. Aberrant regulation of microRNAs (miRNAs) has been implicated in the pathogenesis of AD and may play a pivotal role in the early stages of the disease. Objectives To examine the role of a miR-4763-3p antagomir in ameliorating cognitive decline in mild cognitive impairment (MCI)-AD mice and to elucidate the underlying mechanisms involved. Methods Fluorescence in situ hybridization was used to demonstrate that miR-4763-3p is highly expressed in postmortem hippocampal tissue from AD patients and colocalizes with the Aβ and Tau proteins. Stereotactic injection of the miR-4763-3p antagomir and subsequent behavioral experiments revealed its ability to ameliorate cognitive decline in AD-MCI mice. RNA-seq, tissue staining, and SH-SY5Y cell experiments were used to explore specific molecular mechanisms and associated signaling pathways. Results The miR-4763-3p antagomir targeted ATP11A to enhance inward flipping of the "eat me" phosphatidylserine signal on the surface of neuronal cells, effectively alleviating brain inflammation and neuronal loss and improving synaptic morphology in AD-MCI mice. Furthermore, the miR-4763-3p antagomir increased autophagy in the early-stage AD-MCI brain, promoted the clearance of Aβ proteins, and reduced the deposition of lipofuscin. These findings confirm that miR-4763-3p targets ATP11A to regulate the PI3K/AKT/mTOR/Bcl2 signaling pathway, thereby promoting neuronal autophagy and reducing apoptotic crosstalk. Conclusions The miR-4763-3p antagomir has the potential to reverse neuronal apoptosis and enhance autophagy levels, improving the inflammatory microenvironment in brain tissue and thus improving learning and memory in early-stage AD-MCI mice to mitigate cognitive decline. Our data offer a promising strategy for the treatment of AD-MCI patients. AD-MCI ATP11A apoptosis autophagy phosphatidylserine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Cognitive decline and memory impairment are commonly observed in brain injury, aging, schizophrenia, and neurodegenerative diseases and have a significant impact on patients' quality of life. Alzheimer's disease (AD) is the most prevalent progressive neurodegenerative disease. According to an epidemiological data project, by 2050, the number of AD patients in the United States will double in comparison with that recorded in 2021 [ 1 ]; a similar trend in case statistics has been observed in China [ 2 ]. Alzheimer's disease is rapidly becoming one of the costliest, deadliest and most burdensome diseases of this century[ 3 ]. The AD brain exhibits a variety of pathological features, including the deposition of senile plaques and neuronal death [ 4 , 5 ]. The intracellular hyperphosphorylation of tau leads to its aggregation, and the formation of neurofibrillary tangles can serve as a diagnostic criterion for AD [ 6 , 7 ]. The downregulation of excitatory and inhibitory genes in the human frontal cortex is one of the most significant changes observed in AD [ 8 ]. Due to the presence of the blood‒brain barrier, there is currently no effective drug for the treatment of AD, and the irreversibility of this disease necessitates early intervention [ 9 ]. Accordingly, it is imperative to understand the pathological mechanisms underlying AD and develop targeted drugs. MicroRNAs (miRNAs) are a class of noncoding RNA molecules, typically consisting of approximately 22 nucleotides, that play a crucial role in post-transcriptional gene regulation and have been implicated in dysregulated gene expression. [ 10 , 11 ]. Recent studies have identified miR-31, miR-93, miR-143, miR-146a, miR-148a, and miR-191 as potential biomarkers for AD [ 10 , 12 ]. miR-331-3p and miR-9-5p have been shown to promote autophagy, improve cognitive ability in mice, and serve as diagnostic markers for early- and late-stage AD [ 13 ]. Overexpression of miR-455-3p can improve synaptic and cognitive function and extend lifespan in mouse models of AD [ 14 ]. In recent years, research on miRNAs as biomarkers of AD and the manner in which these molecules regulate related signaling pathways has become popular. The symptoms of AD are marked by an accumulation of protein aggregates (e.g., Aβ and tau) in the brain, which results in an inflammatory environment. This inflammatory environment contributes to the impairment of the autophagy-lysosome system. In turn, damage to this system leads to increased protein accumulation and inflammatory phenotypes, contributing to disease progression [ 15 ]. Autophagy plays a significant role in the maintenance of organismal homeostasis [ 16 ]. In neurons, autophagy has a protective effect and is essential for the maintenance and survival of neurons [ 17 ]. Damaged organelles and pathological proteins are encased in autophagolysosomes and subsequently degraded [ 18 ]. Dysautophagy is closely associated with the development of neurodegenerative diseases such as Alzheimer's disease [ 19 ]. Therefore, targeting and regulating the inflammatory microenvironment in the brain and the inflammatory environment of nerve cells, improving autophagy, and reducing nerve cell damage has become an effective strategy for the treatment of AD. In this study, we screened and found that miR-4763-3p is dysregulated in the early stages of AD and is positively correlated with age. Previous research [ 20 ] has shown that miR-4763-3p is closely related to cognition. Therefore, we further explored its ameliorative effect on cognitive decline in early-stage AD. In the present study, we used an early-stage AD mouse model that exhibits mild cognitive decline and memory impairment to evaluate treatment effects. Alleviation of symptoms in the early stages may prevent the occurrence of irreversible cognitive impairment in late-stage AD. Our study confirmed that miR-4763-3p was significantly upregulated in neurons in the AD brain and colocalized with the Aβ and Tau proteins. Stereotactic injection of the miR-4763-3p antagomir into the hippocampal CA1 region of early-stage AD-MCI mice (six-month-old 3×Tg-AD mice) improved learning and memory and alleviated cognitive decline. Treated mice exhibited a decrease in brain inflammation, an increase in the number of neurons, and changes in synaptic morphology. An investigation of the underlying mechanism of action revealed that miR-4763-3p targets ATP11A, thereby affecting the autophagy pathway in early-stage AD-MCI and promoting the clearance of the Aβ and Tau proteins. Furthermore, inhibiting miR-4763-3p can increase ATP11A-mediated inward flipping of phosphatidylserine on the surface of neuronal cells, which in turn reduces early-stage neuronal apoptosis. A combination of bioinformatics analysis and in vitro experiments revealed that miR-4763-3p participates in the PI3K/AKT/mTOR signaling pathway, thereby regulating the interplay between autophagy and apoptosis. Our data suggest that miR-4763-3p may serve as a potential therapeutic target for the clinical treatment of AD, providing novel strategies for the treatment of AD. Materials and Methods Animals C57BL/6 wild-type (WT) and 3xTg (a transgene with mutated human APP K670N/M671L and MAPT P301L and a knock-in mutation Psen1M146V) mice were obtained from Shanghai Model Organisms Center, Inc.[ 21 ]. Mice were raised at a constant temperature (22 ± 1°C) with a light/dark cycle of 12/12 h and were provided with free access to food and water. All animals were treated in accordance with international animal research guidelines. The study design was approved by the Animal Ethics Committee of Shanghai University. Identification of DEGs and functional annotation To explore the miRNAs affecting AD, we obtained raw RNA-Seq data downloaded from the GEO database (GEO accession number: GSE120584). The thresholds for the screening of differentially expressed genes (DEGs) were set to |log2FC|>1.5 and P < 0.05. To further explore the potential molecular mechanisms and downstream signaling pathways involved in miR-4763-3p regulation, we conducted sequencing analysis on the hippocampal region of AD-MCI model mice (each group n = 3) that received stereotactic injection of the miR-4763-3p antagomir or NC. To identify DEGs between AD patients and healthy controls, we obtained previously reported raw RNA-seq data from the human postmortem hippocampus (GEO accession number: GSE173955). The thresholds for the screening of DEGs were set to |log2FC| > 1.5 and P < 0.05. The P value was obtained using one-way ANOVA; P < 0.05 was considered to indicate a significant difference between values. DAVID 6.8 was used for functional enrichment analysis of AD-related genes [ 22 ]. Behavioral analysis Novel object recognition test The novel object recognition (commonly known as NOR) test was performed in an open-field box (40 cm × 40 cm). On the first day, the mice were habitually trained (5 min) and allowed to adapt to the test room for 48 h in an open area. On the third day, two identical objects were presented in opposite quadrants, and the mouse was allowed to freely explore for 5 min. Subsequently, the object was replaced with a new object, and a second 5 min of free exploration was conducted. Noldus software was used to analyze the total movement distance, residence time, number of entries, and average speed at which the mice entered the area around the two objects. Between tests, the instrument was cleaned with 75% ethanol. Morris water maze Following stereotactic injection, the mice were subjected to the Morris water maze (MWM) test to evaluate their cognitive abilities and memory performance. All mice underwent memory training four times a day, with an interval of 20 min, for four consecutive days. On the fifth day, the mice underwent a memory probe test to evaluate the retention of memory to find the hidden platform. The animals were video contemporaneously recorded via SMART 3.0 software (Panlab HARVARD, MA, USA), and behavioral metrics, including percentage of time in each quadrant, distance traveled, latency, and percentage of distance in each quadrant, were calculated. Y-maze On the testing day, the mice (6 months old) were allowed to freely explore two arms of the Y-maze for 5 min, while the other arms were blocked. After habituation training, the mice were allowed to freely explore the three arms for 5 min. During exploration, the duration and frequency of entry into the novel open arm were recorded. Spontaneous alternation was defined as consecutive entry into three different arms on the overlapping triplet. The apparatus was cleaned with 75% ethanol between tests. Barnes maze The Barnes maze test was used to evaluate the spatial memory ability of the mice. Briefly, the Barnes platform is circular, 92 cm in diameter, and contains 20 holes, 19 of which are blocked, leaving only one escape hole. Mice were acclimated to the experimental room one day in advance. During the training phase, each mouse was habituated to the escape hole for 4 min and then to the starting box for 30 s. The mice were then allowed to freely explore the platform, and spatial cues, bright light, and white noise were used to motivate the mice to find the escape hole within 4 min. Any mice that failed to find the escape hole were guided to it and allowed to habituate for 1 min. After three days of training, a 4-min platform probe test was performed, and the time taken and distance traveled by each mouse to enter the escape hole were recorded. Data analysis was performed using SMART software. RT-qPCR The mRNA levels were detected by RT-qPCR. The final reaction contained 2 µg of total RNA, 4 µL of 5× RT Master Mix (ABclonal; Wuhan, China), and RNase-free water (up to 20 µL). cDNA was reverse-transcribed using the following PCR parameters: 55°C for 15 min and 85°C for 5 min. Subsequently, qPCR SYBR Green Master Mix (ABclonal; Wuhan, China) was used for the qPCR amplification reactions. Each reaction contained 1 µL of cDNA sample (100–200 ng/µL), 10 µL of qPCR SYBR Green Master Mix, 0.8 µL (10 µM) of the designated primers, and RNase-free water (up to 20 µL). The qPCR conditions were as follows: 95°C for 3 min, 40 thermal cycles at 95°C for 5 s, and 60°C for 30 s. mRNA levels were normalized to those of GAPDH , and the relative gene expression was quantified using the ΔΔCt method. The primers used in this study are detailed in Table S1 -3. Western blotting The cells were washed with cold PBS and lysed on ice with tissue protein extraction reagent (Beyotime, China) for 30 min. Each homogenate was centrifuged at 12,000 rpm for 30 min at 4°C, the supernatant was collected, and the soluble protein concentration was determined using a BCA-100 protein detection kit. A total of 20 µg of medium-quality protein per sample was boiled for 10 min in 5× loading buffer, subjected to SDS-PAGE (12%), and then transferred to a nitrocellulose membrane. The membranes were blocked with 5% skim milk at room temperature for 2 h, incubated overnight at 4°C with primary antibodies (ABclonal, Wuhan, China), and then incubated with a secondary antibody at room temperature for 1 h. The protein bands were visualized using an Odyssey scanner and associated software (LI-COR Biosciences, USA). Relative protein levels were normalized to those of GAPDH, β-Tubulin, beta-actin or vinculin. Stereotactic injection C57BL/6 and 3xTg mice were obtained from Shanghai Model Organisms Center, Inc. The mice were divided into five groups (n = 10 per group). Targeted injection into the hippocampus was performed with the miR-4763-3p agomir, miR-4763-3p antagomir, and negative controls (NC: sequence-scrambled miRNA; Table S4, 5). The miR-4763-3p agomir and antagomir and the scrambled control were purchased from RiboBio (Guangzhou, China). The treatment groups were as follows: WT + PBS, AD-MCI + miR-4763-3p antagomir NC, AD-MCI + miR-4763-3p agomir NC, AD-MCI + miR-4763-3p antagomir, and AD-MCI + miR-4763-3p agomir. First, we made a longitudinal incision to expose the bregma and set this point to zero. From this point, we determined the hippocampal CA1 area (anterior and posterior: + 2.0 mm, inner and outer ± 0.3 mm, dorsoventral + 1.9 mm). Each mouse was injected with 2 µL of treatment solution within 10 min; the needle was then held for 10 min, after which the needle was slowly retracted. Seven days after stereotaxic injection, the mice were euthanized, and their hippocampi were isolated for total RNA extraction to test the effect of stereotaxic injection. After 7 days of free access to food and water, the behavioral test was conducted. Dual-luciferase reporter gene detection The relationship between hsa-miR-4763-3p and ATP11A was verified using a dual luciferase reporter assay kit (Vazyme, China). The binding sequences of miR-4763-3p and ATP11A were predicted by RNAhybrid software, and the free energy of their targeted binding sites was analyzed. We designed the mutated (MUT) binding site using the complementary sequence of wild-type (WT) ATP11A and constructed the pGL3 Basic reporter plasmid. HEK-293T cells were transfected with ATP11A-MUT (mutant 3'UTR) or ATP11A-WT (luciferase reporter plasmid with the correct sequence) and the miR-4763-3p mimic or NC (Shanghai Ribo, China, Table S4) for 48 h. After cell collection and lysis, the relative luciferase activity was calculated as the ratio of firefly luciferase to renilla luciferase. Three independent experiments were performed. Immunofluorescence imaging The NeuN, Iba1, Aβ, Tau, ATP11A, Annexin V, SQSMT1 and Caspase 3 levels were monitored by immunostaining (the relevant antibody information is provided in Table S6). Brain tissue slices (n = 3) on coverslips were washed three times with 1× PBS for 3 minutes each and fixed in 4% paraformaldehyde (PFA) for 15 minutes. After washing three times with 1× PBS for 3 minutes each, the slices were blocked and permeabilized in 1× PBS containing 1% goat serum and 0.25% Triton™ X-100 and then stained with a primary antibody overnight at 4°C. The following day, the slices were washed three times with 1× PBS for 3 minutes each, incubated with a secondary antibody at room temperature for 1 hour, and washed again. DAPI (Invitrogen, NBP2311561) was used to counterstain the nuclei, which were then incubated in the dark for 5 minutes. Finally, the slices were washed four times with PBST for 5 minutes each to remove excess DAPI and then mounted with mounting solution containing a fluorescence quencher. Images of immunostained brain tissue slices were obtained using confocal fluorescence microscopy (Zeiss). After adjusting the threshold, the fluorescence intensity was quantified using ImageJ® version 1.6.0 software (National Institute of Mental Health/NIH Research Services Branch, Bethesda, Maryland). Immunocytochemistry (IHC) Brain slices (10 µm thick) were obtained with a cryostat and rinsed with 1× PBS three times. Then, these slices were treated with 3% hydrogen peroxide for 30 min to block endogenous peroxidase activity. After incubation with 0.1% Triton X-100 for 20 min to permeabilize the membrane, the slices were blocked with 5% bovine serum albumin (BSA) for 30 min. The brain slices were incubated with the primary antibodies IL-6 and TNF-α overnight at 4°C. After washing with PBST, the slices were probed with a biotinylated secondary antibody and streptomycin-labeled peroxidase solution for 1 h at room temperature and then stained with 3,3'-diaminobenzidine (DAB) reagent for 1 to 10 min at 37°C. After washing, the brain slices were dehydrated with different concentrations of alcohol (75, 80, 95, and 100%), rendered transparent in xylene, and sealed on glass slides. The digital images of all slices were captured using a Pannoramic MIDl. Fluorescence in situ hybridization (FISH) A fluorescently labeled miR-4763-3p FISH probe was designed and synthesized by Servicebio (Wuhan, China). Fluorescence-labeled single-strand probes were hybridized. FISH was carried out according to the manufacturer’s instructions for SweAMI-FISH (Servicebio). All fluorescence images were captured using a confocal laser microscope (Zeiss, Germany). The miR-4763-3p probe sequence used was CCCGCCCAGCACCAGCCCCTGCCT. Flow cytometry An apoptosis detection kit (Yeasen, Shanghai, China) was used to measure the levels of apoptosis in the transfected cells. The cells were suspended in 50 µL of 1× binding buffer, to which 5 µL of Annexin V-FITC or 5 µL of anti-PI-PE was added, and the mixture was incubated for 15 minutes at room temperature. Next, 400 µL of 1× binding buffer was added to each sample. Flow cytometry (Beckman Coulter Cytoflex) was used to collect the data. ELISA Tissue: Frozen hippocampal tissue was rapidly homogenized using a homogenizer in PBS containing a protease inhibitor and then centrifuged at 5,000 × g for 10 min to collect the supernatant. The levels of the cytokines IL-6 and TNF-α in the serum and tissue were measured by ELISA kits (JiangLai, China). SH-SY5Y cells were plated in 6-well plates at 1 × 10 6 /well and transfected with NC (5 µM), siATP11A (5 µM), OEATP11A (4 µg of pcDNA3.0-ATP11A-overexpressing plasmid) or vector (4 µg) for 48 h. Supernatants were collected for detection of TNF-α and IL-6 by ELISA. Statistical analysis The data were analyzed and are presented as the mean ± standard error of the mean (SEM) of at least three independent experiments. The analytical methods used were GraphPad Prism version 8 (GraphPad Software, Boston, MA). For comparisons between two groups, a two-tailed unpaired Student’s t test was used for normally distributed data. For comparisons involving multiple groups, one-way ANOVA and two-way ANOVA followed by Tukey’s multiple comparison test were used for normally distributed datasets. P < 0.05 was considered to indicate statistical significance. Results MiR-4763-3p antagomir ameliorates cognitive decline in AD-MCI mice Dysregulation of miRNAs has been strongly implicated in the pathogenesis of AD. To determine the differences in miRNAs between patients with AD and healthy people, we analyzed noncoding RNAs in different human serum samples. After analyzing the noncoding RNA profile data from the serum samples of 50 AD patients and 50 healthy individuals (GSE120584), we identified differentially expressed miRNAs between the AD group and the healthy group. A total of 424 DEGs were screened (Figure S1 A). The p value was further optimized to < 0.01, and all differentially expressed genes were investigated in PubMed. Only 12 genes were found to be associated with neurological diseases (excluding tumors). According to the study, only hsa-miR-4763-3p, hsa-miR-342-3p, hsa-miR-361-3p, hsa-miR-485-3p and hsa-miR-211-5p are thought to be cognitively relevant [ 20 , 23 – 26 ]. We examined miRNA expression in the hippocampal tissues of AD-MCI and WT mice at 6 and 12 months. miRNA-qPCR experiments revealed that miR-361-3p and miR-4763-3p exhibited increased expression levels during the early stages of AD and continued to accelerate the progression of the disease in the later stages (Fig. 1 A, B). Given that existing studies have revealed the mode of action of miR-361-3p on neurons, the aim of this study was to further explore the effects of miR-4763-3p on AD neurons and its impact on cognitive function. Sequencing data (GSE120584) revealed significant upregulation of miR-4763-3p in the serum of AD patients (Fig. 1 C). Using clinical samples from 30 AD patients and 30 controls, we verified that miR-4763-3p expression increased with age in AD patients (Fig. 1 D). This finding reminds us of the strongest genetic risk factor for AD, apolipoprotein E epsilon 4 (APOE4), which is strongly associated with the risk of age-related cognitive decline in individuals without dementia[ 27 , 28 ]. Correlation analysis between the levels of miR-4763-3p and patient age in APOE4 high-risk AD patients revealed a positive association between miR-4763-3p and age (Fig. 1 E), suggesting that both could be markers for AD detection. Next, fluorescence in situ hybridization (FISH) experiments revealed the expression of miR-4763-3p in human neuroblastoma SH-SY5Y cells (Fig. 1 F). In contrast to the control, higher expression of miR-4763-3p was observed in postmortem hippocampal tissue from AD patients; colocalization with neurons was also detected (Fig. 1 G). Moreover, there was a positive correlation between the expression of miR-4763-3p and the expression of the Aβ or Tau proteins, with some degree of colocalization (Fig. 1 H, I), suggesting that miR-4763-3p may affect intracellular molecular mechanisms in neurons during the pathogenesis of AD. To further explore the mechanism of miR-4763-3p in the pathogenesis of AD and its ability to rescue cognitive and memory impairment in early AD mice, we stereotactically injected PBS, agomir (miRNA agonist), agomir NC, antagomir (miRNA antagonist), or antagomir NC into the hippocampal CA1 region of WT or AD-MCI mice (Figure S1 B). The detection of miR-4763-3p expression in the hippocampal region of the mouse brain showed that the antagomir decreased miR-4763-3p in the brain, while the agomir increased it, demonstrating effectiveness seven days after administration (Fig. 1 J). Then, we assessed the learning and memory abilities of these mice. The results of the novel object recognition test (assessing spatial learning) showed that the AD-MCI-miR-4763-3p agomir group had a lower frequency and duration of exploring objects than did the agomir NC group (Fig. 1 K, Figure S1 C), indicating that spatial memory was impaired in the model group. The AD-MCI-miR-4763-3p antagomir group spent almost twice as much time exploring novel objects than familiar objects and discovered novel objects more frequently, suggesting that their spatial memory was significantly improved in comparison with that of the NC group (Fig. 1 K, Figure S1 C). The results of the Y-maze experiment showed that AD-MCI mice in the NC and miR-4763-3p agomir treatment groups tended to stay and shuttle in the familiar arm, while those in the miR-4763-3p antagomir treatment group and WT group had a greater frequency and duration of stay in the open arm than in the familiar arm, demonstrating their greater spatial learning ability in comparison with other mice (Fig. 1 L, Figure S1 D). The results of the Morris water maze experiment demonstrated that the AD-MCI-miR-4763-3p antagomir group had a similar duration of stay and number of platform crossings as the WT group did, and these durations were significantly greater than those of the other groups, including the NC-treated AD-MCI group and miR-4763-3p agomir group, which spent the shortest amount of time in the target quadrant (Fig. 1 M, Figure S1 E). These observations indicate that the miR-4763-3p antagomir significantly improved the spatial and directional senses of AD-MCI mice. In the Barnes maze experiment, AD-MCI mice in the miR-4763-3p antagomir treatment group displayed a significantly shorter latency to reach the target hole during the search stage than did those in the NC and agomir treatment groups, indicating a significant improvement in learning ability and spatial memory following miR-4763-3p antagomir treatment (Fig. 1 N, Figure S1 F). These findings suggest that miR-4763-3p antagomir treatment has good diagnostic and promising therapeutic effects on early-stage AD. The miR-4763-3p antagomir rescued neuronal loss and synaptic morphology To further investigate the specific mechanisms underlying the improvements in cognitive and memory abilities following miR-4763-3p antagomir treatment, immunofluorescence staining was performed in the hippocampus of AD-MCI mice after stereotactic injection. The results demonstrated significant neuronal loss and microglial cell proliferation in the miR-4763-3p agomir group compared with the WT and NC groups. Conversely, following treatment with the miR-4763-3p antagomir, a significant decrease in the number of neurons and no increase in microglial cell proliferation were observed (Fig. 2 A, C and D). Nissl bodies can serve as a marker of neuronal functional status[ 29 ] since these structures decrease or disappear following neuronal damage. Interestingly, the staining intensity of Nissl bodies in the CA1 and CA3 hippocampal regions was significantly greater in the miR-4763-3p antagomir-treated group than in the NC group, while the miR-4763-3p agomir-treated group displayed lighter-stained Nissl bodies (Fig. 2 B and E). These results suggest that miR-4763-3p antagomir treatment may rescue neuronal loss and damage in the hippocampal CA1 and CA3 regions of AD-MCI mice and ameliorate cognitive decline. In addition, since the normal structure of hippocampal synapses is fundamental to learning and memory, Golgi staining was used to evaluate the morphology of brain dendrites and synapses. The results showed that the dendritic spines in the miR-4763-3p agomir group were relatively disorganized, while miR-4763-3p antagomir treatment significantly increased the length and density of the dendritic spines. Although there were still some differences in the Golgi between the miR-4763-3p antagomir group and the WT group, there was a significant improvement in the dendritic spine length and density in the miR-4763-3p antagomir NC group (Fig. 2 F, Figure S2 A). Moreover, Sholl analysis revealed that the dendrites of the miR-4763-3p agomir group exhibited the lowest complexity, and miR-4763-3p antagomir treatment significantly increased the complexity of dendrites (Fig. 2 F, H) and the percentage of mushroom-type spines (Fig. 2 G, I). Finally, the analysis of synaptic proteins revealed that the miR-4763-3p agomir reduced GLUR1/2 expression, while the antagomir increased GLUR1/2 expression, which was consistent with the WT results (Figure S2 B, C). These data suggest that miR-4763-3p antagomir treatment significantly rescues neuronal loss and reshaped synaptic morphology in AD-MCI mice, which may be the underlying mechanism for the restoration of cognition and enhancement of learning and memory. Bioinformatics analysis of the differential expression of miRNAs and related biochemical pathways in AD-MCI To further explore the underlying molecular mechanisms and downstream signaling pathways involved in the regulation of miR-4763-3p, RNA-seq analysis of the hippocampal region of AD-MCI mice was performed following stereotactic injection of the miR-4763-3p antagomir or NC (Fig. 3 A). The results revealed 430 upregulated genes and 48 downregulated genes in comparison with those in the NC group (Fig. 3 B). Functional enrichment analysis of the DEGs revealed enrichment mainly in the immune system, the ER-phagosome pathway and the role of phospholipids in phagocytosis (Fig. 3 C). The results of KEGG and GO enrichment analysis revealed that the DEGs were mainly enriched in the phagosomes, the PI3K-AKT signaling pathway, apoptosis, and the mTOR signaling pathway (Fig. 3 D), in addition to the cell surface receptor signaling pathway and the regulation of phagocytosis and apoptotic cell clearance (Fig. 3 E). Furthermore, the upregulated DEGs were mainly enriched in phagosomes, apoptosis and lysosomes (Fig. 3 F). The upregulated DEGs were associated with phagocytosis, inflammation regulation, phospholipid translocation (Fig. 3 G), membrane microdomains, membrane rafts (Figure S3A) and immune receptor activity (Figure S3B). GO enrichment analysis indicated that the downregulated DEGs were involved mainly in synapse organization, cognition and learning or memory (Fig. 3 H); neuron–neuron synapses (Figure S3C); and cytokine activity (Figure S3D). To determine the relationship between the regulatory role of miR-4763-3p and the pathological mechanisms of AD, we further analyzed the DEGs between publicly available datasets for AD patients and healthy controls. Analysis of the raw RNA-seq data (GSE173955) using FDR/adj P 1.5 identified 1429 DEGs between the AD group and the control group, of which 585 were upregulated and 844 were downregulated in the AD group. (Figure S3E). The DEGs were mainly enriched in synapse organization, regulation of transporter activity, cognition (Figure S3F) and the mTOR signaling pathway (Figure S3G). Therefore, miR-4763-3p antagomir may influence the PI3K or mTOR signaling pathway, potentially improving the immune microenvironment in the brain, managing phagocytosis, and ameliorating the loss of synaptic function and neuron count in the brain. ATP11A is a target gene of miR-4763-3p in neurons To verify our aforementioned hypothesis, bioinformatics analysis was conducted using online databases to predict the target genes of miR-4763-3p. The results showed that 1002 genes were repeatedly predicted by the TargetScan7, miRDB, and miRWalk databases (Fig. 4 A). Functional enrichment analysis indicated that the predicted target genes were involved mainly in signal transduction, nervous system development, PI3K/AKT signaling (Fig. 4 B), protein binding, the nucleus, phagocytosis (Fig. 4 C) and autophagy (Fig. 4 D). Consistent findings from bioinformatics analysis and mice hippocampal sequencing analysis highlighted the significant involvement of autophagy and phagocytosis in the learning and memory impairments observed in neurodegenerative diseases such as AD. Further investigation revealed only ATP11A at the intersection of five datasets: mice hippocampal sequencing data DEGs, predicted miR-4763-3p target genes, mass spectrometry results (SY5Y differentially transfected with miR-4763-3p inhibitor and NC), and human disease-related genes (Fig. 4 E). ATP11A is closely related to the nervous system [ 30 ] and was significantly upregulated in the hippocampal region of mice treated with the miR-4763-3p antagomir (Fig. 4 F). The First database ( https://alzmap.org/tsne/gene ) of the brain diagram shows that the Atp11a expression level in the CA1 brain region of the hippocampus decreased in AD-MCI mice at 6 months of age (Fig. 4 G) but not in 3-month-old mice compared with that in WT (C57BL/6) mice (Figure S4A, B). Therefore, we hypothesized that Atp11a may be closely related to the decline in learning and memory abilities and cognitive impairment observed in early AD. We extracted RNA and protein from the hippocampus of 3-month-old and 6-month-old WT or AD-MCI mice for detection and found that the expression of ATP11A in 3-month-old WT and AD-MCI mice was similar, while the expression of ATP11A in 6-month-old AD-MCI mice was lower than that in WT mice (Fig. 4 H-J). FISH analysis of human brain tissue revealed that miR-4763-3p was expressed at low levels in the control group, while ATP11A was significantly highly expressed in the hippocampal region (Fig. 4 K). In contrast, significant downregulation of ATP11A and elevated miR-4763-3p expression were observed in the hippocampus of patients diagnosed with Alzheimer's disease and were found to be colocalized with ATP11A (Fig. 4 K). Furthermore, FISH analysis revealed colocalization between miR-4763-3p and ATP11A in SH-SY5Y cells, suggesting that there may be a close relationship between the two miRNAs (Fig. 4 L, M). Subsequently, RT‒qPCR and western blotting were used to evaluate the effects of miR-4763-3p on ATP11A expression in cells and tissues (Fig. 4 N, O; Figure S4C-E). The results demonstrated that miR-4763-3p inhibited the expression of ATP11A at both the transcriptional and translational levels and that ATP11A expression was restored following treatment with the miR-4763-3p inhibitor. In addition, this mode of action also affects ATP11A expression in vivo. To verify the targeted binding between miR-4763-3p and ATP11A, RNAhybrid software was used to predict the binding sequence between miRNA and ATP11A. The prediction results revealed that the GGGACGG sequence (highlighted in yellow) was present in three predicted sequences (Figure S4F). The free energy of the targeted binding site was subsequently analyzed (Figure S4G). To further evaluate the molecular mechanism of miR-4763-3p, a mutant of ATP11A with a disrupted miR-4763-3p binding sequence was constructed. The sequencing results of the ATP11A mutant are shown in Figure S4H and I, and a schematic depicting the targeted binding mode between miR-4763-3p and WT or mutant ATP11A is illustrated in Fig. 4 P. A dual luciferase reporter gene assay was then employed to validate the targeted binding relationship between miR-4763-3p and ATP11A. HEK293T cells were cotransfected with ATP11A-WT or ATP11A-MUT and the miRNA mimic or NC. Compared with cells transfected with ATP11A-MUT, the miRNA mimic significantly reduced the luciferase activity in cells transfected with ATP11A-WT, indicating that miR-4763-3p inhibited the activity of the reporter gene and thus targeted regulated ATP11A (Fig. 4 Q). After revealing the targeting relationship between ATP11A and miR-4763-3p, we further determined the cognition-related functions of ATP11A. ShATP11A or NC was stereotactically injected into the brains of 6-month-old AD-MCI mice, and behavioral tests were performed 4 weeks later. The results showed that shATP11A mice had shorter dwell times and shorter distances to the new open arm in the Y maze (Figure S5A-C), and the dwell times and distances explored around new objects were significantly lower than those of NC mice (Figure S5D-F). In the Morris water maze test, the swimming time in the target quadrant was significantly lower than that in the NC group (Figure S5G, H), demonstrating that ATP11A deficiency led to learning and memory deficits in mice and that ATP11A, a target gene of miR-4763-3p, may play an important role in the process of AD disease. In general, miRNAs act in mammals by not fully binding to target genes, thereby inhibiting their translation [ 31 – 33 ]. However, our research findings demonstrated that miR-4763-3p also influences the transcription of ATP11A, which provoked our curiosity. Consequently, we conducted further investigations to explore the regulatory mechanisms underlying the transcription of miR-4763-3p. Considering that transcription factors (TFs) often play crucial roles in the mode of action of miRNAs [ 34 – 36 ], we employed gene function screening to predict the upstream TFs of miR-4763-3p and identified YY1 as a potential candidate (Figure S5I). Research has indicated that YY1 plays a vital role in regulating the expression of TREM2, which stimulates phagocytosis and suppresses cytokine production and inflammation [ 37 ]. Specifically, microglial YY1 maintains TREM2 expression levels, providing a therapeutic target for the prevention and treatment of AD [ 37 ]. Our experimental results revealed significant downregulation of YY1 expression in the hippocampus of AD-MCI patients compared to control subjects (Figure S5J), suggesting that YY1 may play an important role in the hippocampus. Based on the potential regulatory mechanism of YY1, the ChIP assay confirmed that YY1 could bind to the miR-4763-3p promoter in SH-SY5Y cells (Fig. 4 R), suggesting that YY1 can transcriptionally regulate miR-4763-3p expression. Inhibition of YY1 via siRNA upregulated the miR-4763-3p level (Figure S5K, L), but miR-4763-3p expression had no significant effect on YY1, demonstrating that YY1 may act only as an upstream regulatory element of miR-4763-3p. ChIP-qPCR was used to confirm that YY1 binds to the ATP11A promoter, initiating transcription in SH-SY5Y cells (Fig. 4 S). ATP11A expression was significantly downregulated after transfection with siYY1 (Figure S5M). In summary, our data revealed a feedforward regulatory mechanism of YY1-miR-4763-3p-ATP11A, which may play an important role in the nervous system of AD patients. MiR-4763-3p antagomir targets ATP11A to reverse early apoptosis in neurons by regulating PS flipping Based on our results in mouse hippocampal slices, we hypothesized that the miR-4763-3p antagomir may rescue neuronal loss; however, the specific mechanism is unclear. Currently known forms of regulated cell death include apoptosis, necrosis and autophagy [ 38 ]. Based on RNA-seq data, it is reasonable to suggest that miR-4763-3p is closely related to cellular apoptosis. Flow cytometry was used to detect apoptosis and necrosis in SH-SY5Y cells transfected with the miR-4763-3p mimic, inhibitor, corresponding NC or control. The results showed that compared with the NC, the miR-4763-3p mimic increased the levels of early and late apoptosis, while the miR-4763-3p inhibitor significantly reduced the proportion of early apoptotic cells (Fig. 5 A–C). Immunofluorescence and western blotting data revealed that the miR-4763-3p agomir/mimic significantly upregulated the expression of cleaved caspase 3 apoptosis-associated proteins in the SH-SY5Y cell line (Fig. 5 D) and hippocampal tissue (Fig. 5 E). Conversely, treatment with the miR-4763-3p antagomir/inhibitor resulted in a lower level of cleaved caspase 3, and the cleaved caspase 3 expression level in the antagomir treatment group was similar to that in the WT group (Fig. 5 D, E; Figure S6A, B). We hypothesize that the mechanism by which miR-4763-3p affects apoptosis is related to brain inflammation; therefore, ELISA, IHC and qPCR were performed to evaluate the expression of the inflammatory factors IL-6 and TNF-α in the mouse brain. The results revealed that the miR-4763-3p agomir significantly increased the expression levels of IL-6 and TNF-α, while treatment with the miR-4763-3p antagomir reduced brain inflammation (Fig. 5 F, G, Figure S6C-E). In addition, similar results were obtained for the qPCR detection of the inflammatory factors IL-6 and TNF-α (Figure S6F, G). We also further explored the sequencing data of miR-antagomir and AD-MCI-NC and found that IL-34 is significantly affected by miR-4763-3p, a cytokine that is closely associated with the clearance of Aβ and stimulates the release of proinflammatory factors from macrophages. We detected the effect of miR-4763-3p on IL-34 in brain tissue. The qPCR results showed that compared with the control, the miR-4763-3p antagomir significantly reduced the expression of IL-34. Conversely, the miR-4763-3p antagomir significantly increased the expression of IL-34 in brain tissue. Therefore, we speculate that the miR-4763-3p antagomir improves the inflammatory environment in the brain by reducing the expression of IL-34 and reducing the release of inflammatory factors from macrophages (Figure S6 H-I). We hypothesized that the target gene of miR-4763-3p, ATP11A, might be related to inflammation in the brain, and ELISA analysis was subsequently used to detect the expression of inflammatory factors. ELISA data demonstrated that siATP11A significantly increased the expression of IL-6 and TNF-α, while ATP11A overexpression significantly reduced the expression levels of IL-6 and TNF-α, indicating that ATP11A overexpression may alleviate brain inflammation (Fig. 5 H–K). ATP11A is generally present in plasma membranes, and studies have shown that it has phospholipid flipping activity [ 39 ]; therefore, this study further explored whether ATP11A may play a role in phospholipid flipping in neuronal cell membranes. Immunofluorescence staining of brain tissue was used to evaluate the expression of ATP11A in the brain. ATP11A was significantly upregulated in the CA1 and CA3 regions of the hippocampus in the miR-4763-3p antagomir-treated group, while its expression was relatively low in the miR-4763-3p agomir-treated group (Fig. 5 P). Notably, ATP11A was colocalized with neurons in the hippocampus, and its abundance and density were greater in the brains of mice treated with the miR-4763-3p antagomir. This may be more beneficial for its function: flipping the membrane of phosphatidylserine (PS). Flow cytometry analysis was performed in SH-SY5Y cells stimulated with LPS using Annexin V-FITC staining to measure phosphatidylserine expression levels, and the shift in the X-axis represents the effect of ATP11A on extracellular PS. The results demonstrated that overexpression of ATP11A and treatment with the miR-4763-3p inhibitor significantly decreased the level of extracellular PS (Fig. 5 L), while siATP11A treatment significantly increased PS in the outer leaflet of the plasma membrane of neurons. Moreover, compared with siATP11A treatment alone, cotransfection of cells with the miR-4763-3p inhibitor resulted in a significant decrease in the outer member PS level, especially following LPS treatment (Fig. 5 L; Figure S7A-C). Studies have shown that intracellular PS is an important component of cells that can regulate the activity of neuroreceptors, enzymes, and signaling molecules and improve neuronal signaling. Therefore, it can be inferred that ATP11A enhances PS levels within the neuronal cell membrane, thereby improving neuronal function [ 40 – 44 ]. These results demonstrate that miR-4763-3p plays an important role in stabilizing the phospholipid balance inside and outside the cell membrane under inflammatory conditions. Immunofluorescence staining of brain tissue revealed much stronger PS staining in the NC group than in the miR-4763-3p antagomir-treated group (Fig. 5 Q), indicating that the miR-4763-3p antagomir stimulates ATP11A to inwardly flip PS into the cytoplasm, reducing the early apoptotic "eat me" signal on the cell surface. Flow cytometry analysis confirmed that siATP11A significantly increased apoptosis, particularly in the early stages. Conversely, treatment with an inhibitor of miR-4763-3p effectively mitigated both the early and late stages of neuronal apoptosis induced by ATP11A deficiency (Fig. 5 O, N; Figure S7D-H). Additionally, this regulatory mechanism of apoptosis did not affect PS synthase expression levels, which demonstrated that the miR-4763-3p inhibitor affected only PS flipping activity rather than synthesis (Fig. 5 M; Figure S7I, J). These results revealed that the miR-4763-3p inhibitor can stimulate PS flipping into cells by increasing ATP11A levels. This flip can greatly reduce the recognition of "eat me" signals on the surface of glial cells under inflammatory conditions, reshaping the inflammatory environment in the brain; consequently, this mechanism reduces the phagocytic impact of glial cells on neuronal synapses and cell bodies, thus achieving the desired outcome of reducing early apoptosis of neurons. MiR-4763-3p antagomir/ATP11A increases autophagy levels in neurons Since the miR-4763-3p antagomir improves PS flipping under inflammatory conditions and reverses the occurrence of apoptosis, whether cellular homeostasis within neurons is also improved is unclear. Revisiting RNA-seq data and functional enrichment data for miR-4763-3p target genes points to autophagy. The gradual deposition of amyloid plaques in the brains of AD patients has been identified as the primary cause of cognitive decline in the early stages of the disease [ 45 ]. To investigate the involvement of miR-4763-3p in the regulation of autophagy and its potential regulatory role in early Aβ deposition, we conducted IF staining of hippocampal tissue sections from the stereotactic injections of miR-4763-3p antagomir, agomir, antagomir NC, and agomir NC in AD-MCI mice. The results showed that the NC group, particularly the miR-4763-3p agomir-treated group, exhibited significant Aβ deposition, whereas the miR-4763-3p antagomir-treated group exhibited almost no Aβ deposition; these results were most similar to those of the WT group (Fig. 6 A, B). We speculated that the miR-4763-3p antagomir may reduce Aβ deposition during the early stages of the disease by facilitating autophagy or phagocytosis. TEM was used to evaluate changes in intracellular structures in the hippocampal region of AD-MCI mice following treatment with NC or the miR-4763-3p antagomir, revealing greater amounts of lipofuscin in the neurons of mice in the NC and agomir-treated groups (Fig. 6 C). Excess lipofuscin indicates incomplete elimination of lipids and/or misfolded proteins in the cell body, suggesting insufficient lysosomal activity and excessive accumulation of metabolic waste, which may further impair neuronal function or cause neuronal death [ 46 , 47 ]. However, the amount and size of lipofuscin were significantly reduced in the miR-4763-3p antagomir-treated group, which led us to believe that the miR-4763-3p antagomir may improve the intracellular environment of neurons by facilitating autophagy or lysosomal phagocytosis. A growing body of evidence supports the involvement of autophagy in the pathogenesis of neurodegenerative diseases, immune disorders, and various human tumors [ 48 – 50 ]; therefore, we aimed to explore the role of miR-4763-3p in the regulation of autophagy. During the early stages of autophagy, SQSTM1 functions as a selective autophagy receptor and serves as an important protein marker. Studies have demonstrated that SQSTM1 binds to arginine-modified substrates and induces autophagy. The depletion of SQSTM1 inhibited the recruitment of LC3 to autophagosomes, hindered the formation of autophagosomes within cells, and may impair autophagy [ 51 – 53 ]. The basal levels of autophagy and lysosomal biogenesis can be reflected by the expression levels of LC3B II/I and SQSTM1. Compared with those in the control group, the miR-4763-3p inhibitor significantly increased the mRNA expression levels of LC3B and SQSTM1, as well as the protein expression levels of LC3B II/I and SQSTM1, whereas the mimic decreased the expression of these genes. The same trend was observed in the hippocampal CA1 and CA3 regions of mice following injection (Fig. 6 D-F; Figure S8A–C). Transmission electron microscopy revealed a significant increase in the number of autophagosomes in hippocampal neurons after miR-4763-3p antagomir treatment compared with those in the NC- and miR-4763-3p agomir-treated groups (Fig. 6 G). In addition, SH-SY5Y cells were transfected with LC3-GFP-RFP to determine the effect of miR-4763-3p on autophagic flux. GFP is an acid-sensitive protein that emits yellow fluorescence in autophagosomes and red fluorescence in autolysosomes. After miR-4763-3p inhibitor treatment, the number of red and yellow fluorescent intracellular structures increased significantly in comparison with that in the control group, indicating an increase in autophagic flux. In contrast, only green fluorescent structures were observed following miR-4763-3p mimic treatment, suggesting that miR-4763-3p may block the fusion of autophagosomes with lysosomes (Fig. 6 H). To further elucidate the underlying mechanism by which miR-4763-3p regulates autophagic flux, we hypothesized that its target gene ATP11A may play a role in modulating the expression of SQSTM1; therefore, we investigated the impact of ATP11A on SQSTM1. The results demonstrated that the levels of LC3B and SQSTM1 decreased after treatment with siATP11A compared with those in the control and NC groups (Fig. 6 I–K; Figure S8D, E). Interestingly, cotransfection of miR-4763-3p inhibitor and siATP11A restored autophagic flux (Fig. 6 L–N; Figure S8F, G). These data suggested that miR-4763-3p can target ATP11A to inhibit the fusion of autophagosomes with lysosomes, thereby reducing autophagy levels. The miR-4763-3p antagomir can increase autophagic flux in the brain to some extent and clear the excessive deposition of Aβ and lipofuscin in a timely manner, improving the brain environment and restoring spatial memory and cognitive abilities in AD-MCI mice. Inhibition of the PI3K/AKT/mTOR/Bcl2 axis results in crosstalk between autophagy and apoptosis Autophagy and apoptosis exhibit a dynamic balance in the brain, which is mediated by signaling pathways. To further investigate the regulatory mechanism of the miR-4763-3p antagomir/ATP11A, we hypothesized that miR-4763-3p is likely involved in modulating the PI3K/AKT/mTOR signaling pathway. This hypothesis is based on combined KEGG analysis of sequencing data from the hippocampal region of AD-MCI mice treated with miR-4763-3p antagomir, reactome analysis of target genes regulated by miR-4763-3p, and KEGG analysis comparing DEGs between AD patients and normal controls. To verify this hypothesis, we examined the protein expression of p-mTOR, mTOR, p-PI3K, PI3K, p-AKT, and AKT (Fig. 7 ). The immunoblotting results showed that p-mTOR/mTOR, p-PI3K/PI3K, and p-AKT/AKT were significantly upregulated in the siATP11A- and miR-4763-3p mimic-treated cells (Fig. 7 A–D, H–K; Figure S9A–C, F–H). In addition, Beclin1 and Bcl2 were downregulated following siATP11A and mimic treatment (Fig. 7 E–G, L–N; Figure S9D, E, I, J), indicating that activation of the PI3K/AKT/mTOR signaling pathway inhibited autophagy and activated the apoptotic pathway. The levels of p-mTOR/mTOR, p-PI3K/PI3K, and p-AKT/AKT were significantly decreased in SH-SY5Y cells treated with the miR-4763-3p inhibitor, indicating that the PI3K/AKT/mTOR signaling pathway was inhibited (Fig. 7 H–K, Figure S9F-H). Bcl2 and Beclin1 were also significantly increased in these cells, indicating that the antiapoptotic pathway was activated (Fig. 7 L-N, Figure S9I, J). Ultimately, cotransfection of siATP11A and the miR-4763-3p inhibitor had an appreciable ameliorating effect (Fig. 7 O–U; Figure S9K-O). Specifically, the PI3K/AKT/mTOR signaling pathway was activated following the administration of siATP11A compared with that in the control group. In addition, Bcl2 and Beclin1 expression was downregulated, which could be reversed by the introduction of the miR-4763-3p inhibitor. In conclusion, these findings confirmed that miR-4763-3p can target ATP11A to modulate the PI3K/AKT/mTOR/Bcl2 signaling pathway, thereby stabilizing the levels of autophagy and apoptosis in the brain and establishing specific cross-talk relationships. Discussion Here, bioinformatics analysis revealed significant upregulation of miR-4763-3p in the serum of individuals with AD, and this upregulation was positively correlated with age. Subsequently, we determined that miR-4763-3p was highly expressed in the brains of AD patients and colocalized with the Aβ and tau proteins. Intracerebral stereotactic injection of a miR-4763-3p antagomir significantly improved learning and memory impairment and cognitive decline in an AD-MCI mouse model. We found that miR-4763-3p antagomir treatment improved the progressive loss of hippocampal neurons in AD-MCI mice, which was accompanied by an increase in the number of Nissl bodies, dendritic number and synaptic complexity. RNA-seq analysis of hippocampal tissue from control and miR-4763-3p antagomir-treated AD-MCI mice demonstrated potential involvement in regulating the immune system, phagosomes, and apoptosis. Subsequently, we identified the YY1-miR-4763-3p-ATP11A feedforward regulatory mechanism, which reduces the inflammatory environment in the brain and enhances the ability of ATP11A to inwardly flip PS in the neuronal cell membrane under inflammatory conditions. This reduces the level of early apoptosis and inflammation in neurons and regulates autophagy through the PI3K/AKT/mTOR/Bcl2 signaling pathway. AD is the most common neurodegenerative disease and causes a heavy burden on patients, their caregivers, and society as a whole [ 54 , 55 ]. Late-stage AD is an irreversible progressive brain disorder characterized by memory loss and cognitive decline, which is accompanied by severe neuronal loss and widespread inflammation, and there is currently no effective cure [ 56 – 58 ]. Therefore, early detection, prevention, and intervention strategies for the early stages of the disease are increasingly considered the keys to more effective management and treatment [ 59 , 60 ]. MCI is a transitional state between normal aging and dementia, during which cognitive impairment does not yet affect daily life; nevertheless, reports suggest that 15–20% of these patients subsequently develop AD [ 61 – 63 ]. Therefore, MCI is considered a possible early manifestation of AD pathology, other pathological entities such as cerebrovascular disease and Lewy body disease, or mixed pathology [ 61 , 64 ]. Studies have shown that MCI can be stratified by biomarkers [β-amyloid (A+/A-), tau (T+/T-), and neurodegeneration (N+/N-]; A + T-(N+) MCI is defined as "mismatch MCI", and A-T-(N+) MCI is defined as "neurodegeneration-only MCI". An examination of clinical samples revealed that mismatch MCI reflects early prodromal AD symptoms, which are characterized by rapid cognitive decline over time and susceptibility to non-AD pathology, such as amyloid angiopathy [ 65 ]. The mismatch MCI group largely overlapped with the neurodegeneration-only MCI group [ 66 ]. In addition, tau pathology plays a key role in the cognitive and neurodegenerative disease phenotype of AD, with the deposition of tau aggregates being a pathological marker [ 67 ]. Some studies suggest that the failure of experimental disease treatments to date is due to their being conducted in patients who already meet the AD criteria, which may represent too late a time point [ 68 , 69 ]. During the preclinical stage of AD, a series of events occur after Aβ deposition, including tauopathy and abnormalities in markers associated with synaptic dysfunction and neuronal death [ 70 – 73 ]. AD is a disease closely related to age [ 74 ]. An increasing number of studies have shown that APOE4 is an extremely important genetic risk factor for AD, which is related to the risk of age-related cognitive decline in individuals without dementia [ 75 ]. It has the potential to become a detection clinical treatment process. Age factors and APOE risk genes have good reference value in the study of AD pathogenesis and early detection markers. In our study, we found that miR-4763-3p expression appeared to increase with age in patients of different age groups, similar to APOE expression patterns, so we hypothesized that miR-4763-3p may be related to age. We used APOE4 as one of the screening factors during the screening process to explore the correlation between miR-4763-3p and age in high-risk AD patients with APOE4. The results showed that miR-4763-3p was positively correlated with age (Fig. 1 E), suggesting that miR-4763-3p and APOE may jointly serve as age-related markers of AD and early warning signs of AD. Surprisingly, miR-4763-3p was highly expressed in the hippocampus of AD patients and appeared to colocalize with neurons, Aβ, and Tau, while its expression level in healthy controls was low. This finding suggested that miR-4763-3p may play an important role in the course of AD. Six-month-old 3×Tg mice exhibit early AD symptoms but no tau pathology, but Aβ deposition and synaptic dysfunction are already present, which manifests as early cognitive deficits, and these mice can be used as an AD-MCI model. Here, AD-MCI mice were subjected to stereotactic injection of a miR-4763-3p agomir or antagomir to explore their effects. Multiple behavioral experiments related to memory demonstrated that the miR-4763-3p antagomir effectively rescued the cognitive deficits of AD-MCI mice. Moreover, examination of hippocampal tissue revealed that the miR-4763-3p antagomir significantly restored the number and synaptic density of neurons and Nissl bodies. Therefore, the specific mechanism by which the miR-4763-3p antagomir rescued AD-MCI at an early stage was investigated. The mode of action of miRNAs is generally considered to involve targeted inhibition of gene expression [ 76 ]; thus, RNA-seq is undoubtedly the most direct method for revealing the mechanism of action of miR-4763-3p. The results showed that the target genes of miR-4763-3p are mainly involved in the immune system, phagocytosis, synapse organization, regulation of nervous system processes, cognition, and learning or memory in biological processes. We identified a direct target of miR-4763-3p, ATP11A , which encodes a P4-type ATPase that functions redundantly as a phospholipid flippase in the plasma membrane. Previous studies have shown that mutations in ATP11A cause developmental delays and neurological deterioration [ 30 ], and ATP11A deficiency leads to mouse embryonic death at E14.5, demonstrating its important role in the formation of the syncytiotrophoblast layer during placental development [ 39 ]. These observations suggest that ATP11A plays an important role in the development of the nervous system and may be closely related to the development or death of neurons. A common feature of neurodegenerative diseases is neuronal loss caused by apoptosis. The brains of AD patients are considered to have an inflammatory microenvironment, and the present study proposes an unknown regulatory mechanism between autophagy and apoptosis. The miR-4763-3p antagomir targets ATP11A to reduce the expression of inflammatory factors in the brain and enhances the ability of ATP11A to inwardly flip PS inside the neuronal cell membrane. This reduces the exposure of the "eat me" signal on the neuronal surface, thereby decreasing the recognition and phagocytosis of neurons by glial cells and reversing their early apoptosis. Moreover, homeostasis within neurons is improved, autophagy levels are increased to clear metabolic waste, brain lipofuscin levels and Aβ deposition are reduced, and a healthy microenvironment is restored within brain neurons. Autophagy defects often occur in early AD-MCI, where abnormal protein aggregates and Aβ + autophagic vacuoles (AVs) containing incompletely digested autophagic substrates accumulate in neurons. These effects are associated with an age-induced reduction in autophagy-related gene expression and late-onset AD [ 77 – 79 ]. Therefore, autophagy dysfunction may act as an upstream event of AD amyloid pathology, rendering it an attractive target for therapeutic intervention [ 80 ] since maintaining the crosstalk balance between autophagic flux and apoptosis is particularly important for the treatment of nervous system diseases. The balance between autophagy and apoptosis is intricately regulated in brain tissues. Autophagy can reduce cellular apoptosis by eliminating damaged fragments or degraded subcellular components [ 81 , 82 ]. Studies have shown that activation of the TNF-α/TNFR1 signaling pathway in AD leads to the recruitment of RIPK1 by accumulated p62, which induces its oligomerization and results in necroptotic death of neurons. Ectopic accumulation of p62 is caused by impaired autophagic flux mediated by TNF-α-induced downregulation of UVRAG during the necroptotic process [ 83 ]. Moreover, in a mouse model of rotenone-induced Parkinson's disease (PD), PLG-induced autophagy inhibited cell apoptosis through Ser70 phosphorylation, stabilizing the balance between autophagy and apoptosis [ 84 , 85 ]. Autophagy also limits endoplasmic reticulum (ER) stress by degrading unfolded protein aggregates. Simultaneously, autophagy can recycle protein aggregates and misfolded proteins to maintain ER function, thus attenuating the ER stress response and subsequent cell apoptosis [ 86 ]. However, aberrant autophagy can promote cell apoptosis. For example, it has been proposed that autophagy induces cell apoptosis by modulating the levels of interferon-beta (IFN-β) induced by Toll-like receptor (TLR)/interleukin-1 receptor (TIR) domain-containing adaptor-inducing interferon-β (TRIF) [ 87 ]. In a rat model of cerebral ischemia, conventional protein kinase C gamma (cPKCγ) alleviated stroke damage, possibly by downregulating ubiquitin C-terminal hydrolase L1 (UCHL1), which upregulated the ERK-mTOR pathway, alleviated autophagy and cell apoptosis, and ultimately exerted a neuroprotective effect [ 88 ]. In the treatment of hepatocellular carcinoma, the activation of autophagy through the JNK/beclin-1 pathway can induce cancer cell apoptosis, achieving partial therapeutic efficacy [ 89 ]. Autophagy is regulated by various signaling pathways, among which mTOR, a protein kinase, senses the availability of cellular energy and regulates cell proliferation. Reports have shown excessive activation of mTOR signaling in specific brain regions of AD patients [ 90 – 92 ]. The mTOR pathway is critical for the regulation of autophagy and is controlled by the upstream PI3K/AKT pathway. Overactivation of mTOR leads to the accumulation of Aβ and exerts a negative feedback effect on autophagy [ 93 ]. Beclin-1, a well-known inducer of autophagy, regulates the initiation of autophagosome formation, and its activation promotes autophagy [ 94 ]. Therefore, inhibition of the PI3K/AKT/mTOR pathway can activate autophagy, which is consistent with our functional enrichment and immunoblotting data. Inhibition of miR-4763-3p resulted in a significant increase in the expression levels of LC3B II/I and SQSTM1, as well as a notable reduction in the accumulation of Aβ and lipofuscin in the hippocampal region. Additionally, an increase in the number of autophagosomes was observed. Moreover, the expression levels of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR were significantly decreased, while those of Beclin1 and Bcl2 were upregulated. Taken together, these data indicated that the miR-4763-3p antagomir promoted autophagy by inhibiting the PI3K/AKT/mTOR/Bcl2 signaling pathway and reducing cell apoptosis, which suggests that the PI3K/AKT/mTOR/Bcl2 pathway participates in the balance between brain autophagy and early neuronal apoptosis in AD-MCI mice. Finally, there are some limitations in this paper. First, although a feedback loop mechanism involving YY1-miR-4763-3p-ATP11A was found in this study, the interaction between these two factors and the YY1 transcription of these two genes was not fully explored in this study, which may be a shortcoming of this study. In addition, ATP11A, the key target gene of this protein, is a phospholipid flipping enzyme. The effect of PS in this study was mainly to reduce the inflammatory microenvironment in the brain and flip PS into the inner membrane of nerve cells, thereby improving the level of inflammation in nerve cells and improving autophagy. Does PS play a role in autophagy in nerve cells? How PS, as a lipid structure, may affect autophagosome assembly may be worth further exploration. In addition, while we have contributed to understanding the role of miR-4763-3p in relevant therapeutic outcomes in mouse models, its preclinical application needs to be further explored. In future studies, it will be necessary to optimize the biologics of miR-4763-3p antagomirs to improve their stability and therapeutic efficacy in vivo. Conclusions We demonstrated that miR-4763-3p was highly expressed in the serum of AD patients and highly colocalized with Aβ and tau in the brain. The miR-4763-3p antagomir targeted ATP11A inwardly flipped PS in the neuronal cell membrane to reduce early apoptosis. In addition, we showed that miR-4763-3p/ATP11A regulated autophagy through the PI3K/AKT/mTOR/Bcl2 signaling pathway, improved the internal microenvironment in neuronal cells and repaired damaged synaptic morphology, thus improving learning and memory in AD-MCI mice to mitigate cognitive decline. This finding points to a promising strategy for the treatment of AD-MCI patients. Abbreviations AD-MCI: Alzheimer’s disease-mild cognitive impairment; FISH: fluorescence in situ hybridization; AVs: autophagic vacuoles; PS: phosphatidylserine; ER: endoplasmic reticulum; IFN-β: interferon-beta; TLR: Toll-like receptor; TIR: Toll/interleukin-1 receptor; cPKCγ: conventional protein kinase C gamma; UCHL1: ubiquitin C-terminal hydrolase L1; MWM: Morris water maze; PFA: paraformaldehyde Declarations Ethics approval and consent to participate Sample collection was approved by Shanghai University (Shanghai, China). Informed consent was obtained from patients or their guardians, as appropriate. All animal experiments were carried out following the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Care Committee of Shanghai University (ECSHU 2023-004). Data availability All data required for evaluation of the conclusions are presented in the paper and/or Supplementary Materials. The data that support the findings of this study are available from the corresponding author upon reasonable request. Conflict of interest statement The authors declare no potential conflicts of interest. Consent for publication Not applicable. Acknowledgments We thank the Shanghai Jiao Tong University School of Medicine for its technical support. Funding This work was sponsored by the National Key Research and Development Program of China (2020YFA0113000, 2018YFA0109800), Basic Research Program of Shanghai (20JC1412200), National Natural Science Foundation of China (81971324), and CAMS Innovation Fund for Medical Sciences (2022-I2M-1-012). Author contributions W.Q. and N.D. were involved in the conception and design of the study and performed the experiments. X.Z., Q.L., W.F., P.W., and H.W. were involved in the data analysis and interpretation. X.H., L.W., and N.W. were involved in manuscript editing. X.D., Y.L., R.Z. and J.W. were involved in the design of the study, review, editing, funding acquisition, resources, supervision, and project administration. All authors meet authorship requirements. All the authors have read and approved the final manuscript. 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Supplementary Files Graphicalabstract.tif Supplementarymaterial.docx WBgels.pdf FigureS1.pdf FigureS2.pdf FigureS3.pdf FigureS4.pdf FigureS5.pdf FigureS6.pdf FigureS7.pdf FigureS8.pdf FigureS9.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4458094","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":309596928,"identity":"7ba76d81-f409-400e-9334-b51ff1528866","order_by":0,"name":"Wenxin Qi","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Wenxin","middleName":"","lastName":"Qi","suffix":""},{"id":309596929,"identity":"a4591da0-4ad1-4106-8351-82cb347422e8","order_by":1,"name":"Naijun Dong","email":"","orcid":"","institution":"Shanghai 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Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yihao","middleName":"","lastName":"Liu","suffix":""},{"id":309596940,"identity":"f6f50248-3db2-4cad-a560-a262e0c203c1","order_by":12,"name":"Robert Chunhua Zhao","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"Chunhua","lastName":"Zhao","suffix":""},{"id":309596941,"identity":"d9a481f4-867d-4f38-a889-b47647520ded","order_by":13,"name":"Jiao Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYBACxgb+D4f/VDAkgDgSRGphMDzAc4YULUBgfIC3jRQtzO0NCQck59XlGRxgPnibh8Euj7DDeg4cOGC47XCxwQG2ZGsehuRiwlpmJDYcSNx2IHHDAR4zaR6GA4kNhLUkMxw4OKcOqIX/G7Fa0hgONjYwg2xhI1JLzxmGwwzHDifOPMxmbDnHIJmwFsP2HubPDDV1iX3Hmx/eeFNhR4QWuApmEGFASD0QyBOhZhSMglEwCkY6AAAQ30CE19BFfAAAAABJRU5ErkJggg==","orcid":"","institution":"Shanghai University","correspondingAuthor":true,"prefix":"","firstName":"Jiao","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-05-22 04:16:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4458094/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4458094/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57954543,"identity":"34edf8a5-965d-459f-a6d4-2c013e56f0c5","added_by":"auto","created_at":"2024-06-07 23:16:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":607701,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMiR-4763-3p was highly expressed in the serum and hippocampal tissue of AD patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, B: Expression levels of miRNAs in hippocampal tissues of AD-MCI and WT mice at 6 and 12 months.\u003c/p\u003e\n\u003cp\u003eC: Standardized relative expression of miR-4763-3p in serum samples from AD patients and healthy controls in the GSE120584 dataset.\u003c/p\u003e\n\u003cp\u003eD: miRNA-qPCR assay of clinical serum samples from AD patients (AD) and normal controls (control). (AD: n=30, Control: n=30)\u003c/p\u003e\n\u003cp\u003eE: Correlation analysis of miR-4763-3p levels in APOE4 high-risk AD patients according to patient age. Correlation analysis was performed using the RStudio (version 4.3) cor function and Pearson correlation analysis method.\u003c/p\u003e\n\u003cp\u003eF: Fluorescence in situ hybridization (FISH) of miR-4763-3p (red) in SH-SY5Y cells. Nuclei were stained with DAPI (blue). Scale bar, 10 μm.\u003c/p\u003e\n\u003cp\u003eG-I: FISH and immunofluorescence(IF) staining of NeuN, Aβ, tau (green), and miR-4763-3p (red) in control (normal) or AD (Alzheimer's disease patient) brain hippocampal tissue. Nuclei were stained with DAPI (blue). Scale bar, 50 μm.\u003c/p\u003e\n\u003cp\u003eJ: Expression levels of miR-4763-3p in mouse hippocampal tissue seven days after injection of the miR-4763-3p agomir, agomir NC, antagomir or antagomir NC.\u003c/p\u003e\n\u003cp\u003eK: The object recognition test was conducted to assess spatial memory capacity. The duration of exploration of novel objects wasrecorded (n = 10 per group).\u003c/p\u003e\n\u003cp\u003eL: The Y maze test was conducted to evaluate spatial memory capacity. The duration of exploration of the novel and familiar arms was recorded (n = 10 per group).\u003c/p\u003e\n\u003cp\u003eM: The Morriswater maze test was performed to analyze long-term memory. The duration of occupancy of the platform area wasmeasured (n =10 per group).\u003c/p\u003e\n\u003cp\u003eN: The Barnes maze test was performed to assess changes in cognitive ability. The latency to reach the target hole was recorded (n = 10 per group).\u003c/p\u003e\n\u003cp\u003eThe data are expressed as the mean ± SEM of three independent experiments. \u003cem\u003e*P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4458094/v1/44e7ef43ae506704c3f6c4a0.png"},{"id":57954894,"identity":"59d6d0ef-ea27-46d4-a7cc-0948a91bf990","added_by":"auto","created_at":"2024-06-07 23:24:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":209690,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeuronal deficits and changes in synaptic morphology in the brains of AD mice were restored after miR-4763-3p antagomir treatment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: IF staining of NeuN (green) and Iba1 (red) in mouse brain tissues from the NC antagomir, NC antagomir, NC agomir, and agomir groups. Scale bar, 200 or 50 μm.\u003c/p\u003e\n\u003cp\u003eB: Nissl staining of the CA1 and CA3 regions of the hippocampus from different groups. Scale bar, 50 μm.\u003c/p\u003e\n\u003cp\u003eC-E: Statistical analysis of Iba1\u003csup\u003e+\u003c/sup\u003e (C) and NeuN\u003csup\u003e+\u003c/sup\u003e cell numbers (D) and Nissl bodies (E).\u003c/p\u003e\n\u003cp\u003eF: Golgi staining showing dendritic spine morphology in mice injected with the NC antagomir or antagomir. Scale bar, 25 μm.\u003c/p\u003e\n\u003cp\u003eG: An example of Golgi staining examining the dendritic spine morphology of tertiary neurons from mice injected with the antagomir NC or antagomir. Scale bar, 5 μm.\u003c/p\u003e\n\u003cp\u003eH: Sholl analysis was performed to evaluate the dendritic complexity of the WT-, agomir-, agomir NC-, antagomir-, and antagomir NC-injected mice.\u003c/p\u003e\n\u003cp\u003eI: Quantitative analysis of spine density (per 5 μm)\u003c/p\u003e\n\u003cp\u003eThe data are expressed as the mean ± SEM of three independent experiments. \u003cem\u003e*P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4458094/v1/11843f54961b6d474bb139aa.png"},{"id":57954547,"identity":"b490d830-d793-4e41-9c1a-964c0a0bb5a3","added_by":"auto","created_at":"2024-06-07 23:16:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":132980,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBioinformatics analysis of the differential miRNA expression and associated biochemical pathways in AD.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: The differential gene expression heatmap identified by RNA-seq for mice injected with the miR-4763-3p antagomir was significantly different from that for mice injected with the antagomir NC (AD-MCI-NC) (n = 3 per group).\u003c/p\u003e\n\u003cp\u003eB: The volcano plot shows a log2-fold change on the x-axis and statistical significance on the y-axis, with genes significantly different in abundance between mice injected with AD-MCI-NC and those injected with the miR-4763-3p antagomir. The names of some differentially expressed genes are shown.\u003c/p\u003e\n\u003cp\u003eC: Reactome enrichment analysis of the DEGs.\u003c/p\u003e\n\u003cp\u003eD, E: Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis (D) and Gene Ontology (GO) enrichment analysis (E) of all DEGs.\u003c/p\u003e\n\u003cp\u003eF, G: KEGG enrichment analysis (F) and GO biological process (BP) enrichment analysis (G) of all upregulated genes.\u003c/p\u003e\n\u003cp\u003eH: GO-BP enrichment analysis of all downregulated genes.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4458094/v1/88cd38f2a3b937f718fc2c5e.png"},{"id":57954895,"identity":"946a86d8-6d97-4f00-8e8c-ba5c0f9dd680","added_by":"auto","created_at":"2024-06-07 23:24:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":393403,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMiR-4763-3p targets ATP11A,and the transcription initiation site is regulated by the transcription factor YY1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Venn diagrams were generated using the target genes to predict miR-4763-3p. The data were obtained from the TargetScan7, miRDB, and miRWalk databases.\u003c/p\u003e\n\u003cp\u003eB: Reactome enrichment analysis of the target genes.\u003c/p\u003e\n\u003cp\u003eC: GO enrichment analysis of the target genes.\u003c/p\u003e\n\u003cp\u003eD: KEGG enrichment analysis of the target genes.\u003c/p\u003e\n\u003cp\u003eE: Venn diagrams were generated using RNA-seq-determined differentially expressed genes, miR-4763-3p predictedtarget genes, mass spectrometry results (SY5Y differentially transfected with miR-4763-3p inhibitor and NC), and human disease-related genes.\u003c/p\u003e\n\u003cp\u003eF: The histogram shows the level of ATP11A expression in the hippocampus.\u003c/p\u003e\n\u003cp\u003eG: Differential expression of ATP11A in different brain regions according to the AlzMap database.\u003c/p\u003e\n\u003cp\u003eH-J: Protein and mRNA expression of ATP11A in the hippocampus of WT and 3×Tg mice at 3 and 6 months of age and a diagram of the statistical analysis.\u003c/p\u003e\n\u003cp\u003eK: FISH and IF imagesof ATP11A (green) and miR-4763-3p (red) in the hippocampus of AD patients and normal controls were obtained. Nuclei are stained with DAPI (blue). Scale bar, 40 μm.\u003c/p\u003e\n\u003cp\u003eL: FISH and IF staining of ATP11A (green) and miR-4763-3p (red) in SH-SY5Y cells. Nuclei are stained with DAPI (blue). Scale bar, 10 μm.\u003c/p\u003e\n\u003cp\u003eM: Colocalization of miR-4763-3p and ATP11A in SH-SY5Y cells.\u003c/p\u003e\n\u003cp\u003eN, O: Immunoblotting was used to evaluatethe expression level of ATP11A in SH-SY5Y cells transfected with the miR-4763-3p mimic or inhibitor and NC.\u003c/p\u003e\n\u003cp\u003eP: The predicted binding site between miR-4763-3p and wild-type ATP11A (ATP11A-WT) or between miR-4763-3p and mutant ATP11A (ATP11A-MUT).\u003c/p\u003e\n\u003cp\u003eQ: Dual-luciferase activity in HEK-293T cells cotransfectedwith ATP11A-WT or ATP11A-MUT and the miR-4763-3p mimic or miR-4763-3p mimic NC.\u003c/p\u003e\n\u003cp\u003eR: ChIP assays were carried out in SH-SY5Y cells using an antibody against YY1, and IgG was used as a negative control. The enrichment of YY1 binding to the miR-4763-3p promoter was quantified using qPCR.\u003c/p\u003e\n\u003cp\u003eS: ChIP‒qPCR analysisof the association between YY1 and the promoter region of ATP11A in SH-SY5Y cells.\u003c/p\u003e\n\u003cp\u003eThe data are expressed as the mean ± SEM of three independent experiments. \u003cem\u003e*P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4458094/v1/75721902f734056b67cdccc2.png"},{"id":57954898,"identity":"5665bfd4-43c6-4ec0-be5c-031a27651b70","added_by":"auto","created_at":"2024-06-07 23:24:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":990443,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMiR-4763-3p antagomir targets ATP11A to rescue early apoptosis in neurons by regulating PS flipping\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Flow cytometry analysis of the apoptotic rate of SH-SY5Y cells transfected with the miR-4763-3p mimic, mimic NC, inhibitor, and inhibitor NC and control cells.\u003c/p\u003e\n\u003cp\u003eB, C: The histograms show the proportions of early, late and all apoptotic cells.\u003c/p\u003e\n\u003cp\u003eD, E: Immunoblotting of cleaved caspase 3.\u003c/p\u003e\n\u003cp\u003eF, G: The histograms of IL-6 and TNF-α in the hippocampus of mice injected with the miR-4763-3p agomir, agomir NC, antagomir, antagomir NC and WT were analyzed by ELISA (n=3).\u003c/p\u003e\n\u003cp\u003eH-K: ELISA measurement of plasma IL-6 and TNF-α levels in SH-SY5Y cells transfected with NC or overexpressingATP11A (OE ATP11A) or siATP11A.\u003c/p\u003e\n\u003cp\u003eL: Flow cytometry analysis of PS exposure in SH-SY5Y cells transfected with the miR-4763-3p inhibitor, siATP11A, or OE ATP11A and subsequently stimulated with LPS.\u003c/p\u003e\n\u003cp\u003eO, N: Flow cytometry analysis of the apoptotic rate of SH-SY5Y cells transfected with the miR-4763-3p inhibitor or siATP11A and subsequently stimulated with LPS. The histogram shows the percentage of early and late apoptotic cells.\u003c/p\u003e\n\u003cp\u003eM: Immunoblotting of PTDSS1 and cleaved caspase 3. Vinculin served as an internal reference protein.\u003c/p\u003e\n\u003cp\u003eP: IF staining of NeuN (green) and ATP11A (red) in mouse brains from the WT, antagomir NC, antagomir, agomir NC, and agomir groups. Nuclei were stained with DAPI (blue). Scale bar, 10 μm.\u003c/p\u003e\n\u003cp\u003eQ: IF staining of NeuN (green) and PS (red) in mouse brainsfrom the WT, antagomir NC and antagomir groups. Nuclei were stained with DAPI (blue). Scale bar, 20 μm.\u003c/p\u003e\n\u003cp\u003eThe data are expressed as the mean ± SEM of three independent experiments. \u003cem\u003e*P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4458094/v1/e82a8e0a72e11ff60c70c5e8.png"},{"id":57954556,"identity":"4f4eff69-6064-4266-83b4-bc773d9b2b69","added_by":"auto","created_at":"2024-06-07 23:16:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1219987,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMiR-4763-3p antagomir/ATP11A\u003c/strong\u003e \u003cstrong\u003eincreases autophagic flux in neurons.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, B: IF staining of Aβ in the mouse hippocampus. The graph displays the results of quantitative Aβ staining (n=3). Scale bar, 20 μm.\u003c/p\u003e\n\u003cp\u003eC: Transmission electron microscopy (TEM) of the ultrastructure of the mouse hippocampus (the red arrow indicates lipofuscin). Scale bar, 1 μm, 5 μm.\u003c/p\u003e\n\u003cp\u003eD-F: RT‒qPCR and immunoblotting of autophagic flux markersin SH-SY5Y cells transfected with the miR-4763-3p mimic, mimic NC, inhibitor, inhibitor NC orcontrol.\u003c/p\u003e\n\u003cp\u003eG: TEM of neuronal autophagosomes in the mouse hippocampus (red arrows indicate autophagosomes). Scale bar, 1 μm, 5 μm.\u003c/p\u003e\n\u003cp\u003eH: IF staining of LC3B-GFP (green) and RFP (red) in SH-SY5Y cells from the control, mimic NC, mimic, inhibitor NC, and inhibitor groups. Scale bar, 10 μm.\u003c/p\u003e\n\u003cp\u003eI–K: RT‒qPCR and immunoblotting of autophagic flux markersin SH-SY5Y cells transfected with control, NC or siATP11A.\u003c/p\u003e\n\u003cp\u003eL‒N: RT‒qPCR and immunoblotting of autophagic flux markersin SH-SY5Y cells transfected with control, inhibitor and inhibitor cotransfected with siATP11A.\u003c/p\u003e\n\u003cp\u003eThe data are expressed as the mean ± SEM of three independent experiments. \u003cem\u003e*P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4458094/v1/cfb47228617f6924cbbfe389.png"},{"id":57955373,"identity":"3c0ddc2f-bca8-4442-b430-79bd81da5824","added_by":"auto","created_at":"2024-06-07 23:32:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":314853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe miR-4763-3p antagomir/ATP11A inhibits the PI3K/AKT/mTOR signaling pathway to mediate crosstalk between the autophagic protein Beclin1 and the apoptotic protein Bcl2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA:Immunoblotting of p-mTOR, mTOR, p-PI3K, PI3K, p-AKT and AKT in SH-SY5Y cells transfected with control, NC and siATP11A. β-Tubulin served as an internal reference protein.\u003c/p\u003e\n\u003cp\u003eB-D: Quantitative analysis of p-mTOR/mTOR, p-PI3K/PI3K and p-AKT/AKT protein expression.\u003c/p\u003e\n\u003cp\u003eE-G: Immunoblotting of Beclin1 and Bcl2 in SH-SY5Y cells transfected with control, NC orsiATP11A. GAPDH served as an internal reference protein. Quantitative analysis was performed on the obtained data.\u003c/p\u003e\n\u003cp\u003eH-K: Immunoblotting of p-mTOR, mTOR, p-PI3K, PI3K, p-AKT and AKT in SH-SY5Y cells transfected with control, mimic, mimic NC, inhibitor or inhibitor NC. β-Tubulin served as an internal reference protein (H). Quantitative analysis was performed on the obtained data (I-K).\u003c/p\u003e\n\u003cp\u003eL-N: Immunoblotting of Beclin1 and Bcl2 in SH-SY5Y cells transfected with control, mimic, mimic NC, inhibitor orinhibitor NC. β-Tubulin served as an internal reference protein. Quantitative analysis was performed on theobtained data.\u003c/p\u003e\n\u003cp\u003eO-U: Immunoblotting of SH-SY5Y cells cotransfected with siATP11A and control, inhibitor or inhibitor. β-Tubulin served as an internal reference protein. Quantitative analysis was performed on the obtained data.\u003c/p\u003e\n\u003cp\u003eThe data are expressed as the mean ± SEM of three independent experiments. \u003cem\u003e*P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure72.png","url":"https://assets-eu.researchsquare.com/files/rs-4458094/v1/73f35906195f48c692bb4ca1.png"},{"id":57994122,"identity":"29356281-ecff-4d4c-a3c3-93eceeb404a6","added_by":"auto","created_at":"2024-06-09 07:46:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5166569,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4458094/v1/e73105a7-3297-4949-b01c-2f674317d13c.pdf"},{"id":57954546,"identity":"26570722-9d48-4cc6-85c4-e3d8d70773c0","added_by":"auto","created_at":"2024-06-07 23:16:17","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3344810,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-4458094/v1/9d08c2b090fdb70d0a123ce7.tif"},{"id":57954545,"identity":"2480b2c1-f2de-4262-ab43-c4dc77427490","added_by":"auto","created_at":"2024-06-07 23:16:17","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16834,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4458094/v1/553b549f0949686b7e019064.docx"},{"id":57954557,"identity":"f7a2fff8-1254-4cd5-afde-a4d8417bbf4a","added_by":"auto","created_at":"2024-06-07 23:16:17","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1767018,"visible":true,"origin":"","legend":"","description":"","filename":"WBgels.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4458094/v1/c7f9cbbdc9fd5dd181be34fa.pdf"},{"id":57954896,"identity":"6e4f69fc-ac89-4e11-b2ce-ba3819b5abd2","added_by":"auto","created_at":"2024-06-07 23:24:17","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":5679294,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4458094/v1/931f8ec2a3723a8b458033c6.pdf"},{"id":57954554,"identity":"def7342d-c926-472c-ae19-bb424a8086eb","added_by":"auto","created_at":"2024-06-07 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23:16:17","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":2120667,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4458094/v1/68008747ae99b1886deafd33.pdf"},{"id":57954559,"identity":"33a3b644-d01e-4e59-992c-db23b89e2c07","added_by":"auto","created_at":"2024-06-07 23:16:18","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":4147043,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4458094/v1/768e1cb91f8a416aa501279b.pdf"},{"id":57954899,"identity":"adba6926-1c54-4327-b252-20f7a9f84f11","added_by":"auto","created_at":"2024-06-07 23:24:18","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":1631076,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS8.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4458094/v1/e654d1f03fc89bfd99f3eee7.pdf"},{"id":57954551,"identity":"501a5a88-be6c-480f-a374-4cdf1ac5da97","added_by":"auto","created_at":"2024-06-07 23:16:17","extension":"pdf","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":579884,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS9.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4458094/v1/a96f02b17aa096ea208d1ea5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Inhibition of miR-4763-3p expression in the brains of AD-MCI mice activates the PI3K/mTOR/Bcl2 autophagy signaling pathway to reverse neuronal loss and ameliorate cognitive decline","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCognitive decline and memory impairment are commonly observed in brain injury, aging, schizophrenia, and neurodegenerative diseases and have a significant impact on patients' quality of life. Alzheimer's disease (AD) is the most prevalent progressive neurodegenerative disease. According to an epidemiological data project, by 2050, the number of AD patients in the United States will double in comparison with that recorded in 2021 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]; a similar trend in case statistics has been observed in China [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Alzheimer's disease is rapidly becoming one of the costliest, deadliest and most burdensome diseases of this century[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe AD brain exhibits a variety of pathological features, including the deposition of senile plaques and neuronal death [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The intracellular hyperphosphorylation of tau leads to its aggregation, and the formation of neurofibrillary tangles can serve as a diagnostic criterion for AD [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The downregulation of excitatory and inhibitory genes in the human frontal cortex is one of the most significant changes observed in AD [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Due to the presence of the blood‒brain barrier, there is currently no effective drug for the treatment of AD, and the irreversibility of this disease necessitates early intervention [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Accordingly, it is imperative to understand the pathological mechanisms underlying AD and develop targeted drugs.\u003c/p\u003e \u003cp\u003eMicroRNAs (miRNAs) are a class of noncoding RNA molecules, typically consisting of approximately 22 nucleotides, that play a crucial role in post-transcriptional gene regulation and have been implicated in dysregulated gene expression. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Recent studies have identified miR-31, miR-93, miR-143, miR-146a, miR-148a, and miR-191 as potential biomarkers for AD [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. miR-331-3p and miR-9-5p have been shown to promote autophagy, improve cognitive ability in mice, and serve as diagnostic markers for early- and late-stage AD [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Overexpression of miR-455-3p can improve synaptic and cognitive function and extend lifespan in mouse models of AD [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In recent years, research on miRNAs as biomarkers of AD and the manner in which these molecules regulate related signaling pathways has become popular.\u003c/p\u003e \u003cp\u003eThe symptoms of AD are marked by an accumulation of protein aggregates (e.g., Aβ and tau) in the brain, which results in an inflammatory environment. This inflammatory environment contributes to the impairment of the autophagy-lysosome system. In turn, damage to this system leads to increased protein accumulation and inflammatory phenotypes, contributing to disease progression [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Autophagy plays a significant role in the maintenance of organismal homeostasis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In neurons, autophagy has a protective effect and is essential for the maintenance and survival of neurons [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Damaged organelles and pathological proteins are encased in autophagolysosomes and subsequently degraded [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Dysautophagy is closely associated with the development of neurodegenerative diseases such as Alzheimer's disease [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Therefore, targeting and regulating the inflammatory microenvironment in the brain and the inflammatory environment of nerve cells, improving autophagy, and reducing nerve cell damage has become an effective strategy for the treatment of AD.\u003c/p\u003e \u003cp\u003eIn this study, we screened and found that miR-4763-3p is dysregulated in the early stages of AD and is positively correlated with age. Previous research [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] has shown that miR-4763-3p is closely related to cognition. Therefore, we further explored its ameliorative effect on cognitive decline in early-stage AD. In the present study, we used an early-stage AD mouse model that exhibits mild cognitive decline and memory impairment to evaluate treatment effects. Alleviation of symptoms in the early stages may prevent the occurrence of irreversible cognitive impairment in late-stage AD. Our study confirmed that miR-4763-3p was significantly upregulated in neurons in the AD brain and colocalized with the Aβ and Tau proteins. Stereotactic injection of the miR-4763-3p antagomir into the hippocampal CA1 region of early-stage AD-MCI mice (six-month-old 3\u0026times;Tg-AD mice) improved learning and memory and alleviated cognitive decline. Treated mice exhibited a decrease in brain inflammation, an increase in the number of neurons, and changes in synaptic morphology. An investigation of the underlying mechanism of action revealed that miR-4763-3p targets ATP11A, thereby affecting the autophagy pathway in early-stage AD-MCI and promoting the clearance of the Aβ and Tau proteins. Furthermore, inhibiting miR-4763-3p can increase ATP11A-mediated inward flipping of phosphatidylserine on the surface of neuronal cells, which in turn reduces early-stage neuronal apoptosis. A combination of bioinformatics analysis and in vitro experiments revealed that miR-4763-3p participates in the PI3K/AKT/mTOR signaling pathway, thereby regulating the interplay between autophagy and apoptosis. Our data suggest that miR-4763-3p may serve as a potential therapeutic target for the clinical treatment of AD, providing novel strategies for the treatment of AD.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eC57BL/6 wild-type (WT) and 3xTg (a transgene with mutated human APP K670N/M671L and MAPT P301L and a knock-in mutation Psen1M146V) mice were obtained from Shanghai Model Organisms Center, Inc.[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Mice were raised at a constant temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) with a light/dark cycle of 12/12 h and were provided with free access to food and water. All animals were treated in accordance with international animal research guidelines. The study design was approved by the Animal Ethics Committee of Shanghai University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of DEGs and functional annotation\u003c/h2\u003e \u003cp\u003eTo explore the miRNAs affecting AD, we obtained raw RNA-Seq data downloaded from the GEO database (GEO accession number: GSE120584). The thresholds for the screening of differentially expressed genes (DEGs) were set to |log2FC|\u0026gt;1.5 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. To further explore the potential molecular mechanisms and downstream signaling pathways involved in miR-4763-3p regulation, we conducted sequencing analysis on the hippocampal region of AD-MCI model mice (each group n\u0026thinsp;=\u0026thinsp;3) that received stereotactic injection of the miR-4763-3p antagomir or NC. To identify DEGs between AD patients and healthy controls, we obtained previously reported raw RNA-seq data from the human postmortem hippocampus (GEO accession number: GSE173955). The thresholds for the screening of DEGs were set to |log2FC| \u0026gt; 1.5 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The P value was obtained using one-way ANOVA; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate a significant difference between values. DAVID 6.8 was used for functional enrichment analysis of AD-related genes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral analysis\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eNovel object recognition test\u003c/h2\u003e \u003cp\u003eThe novel object recognition (commonly known as NOR) test was performed in an open-field box (40 cm \u0026times; 40 cm). On the first day, the mice were habitually trained (5 min) and allowed to adapt to the test room for 48 h in an open area. On the third day, two identical objects were presented in opposite quadrants, and the mouse was allowed to freely explore for 5 min. Subsequently, the object was replaced with a new object, and a second 5 min of free exploration was conducted. Noldus software was used to analyze the total movement distance, residence time, number of entries, and average speed at which the mice entered the area around the two objects. Between tests, the instrument was cleaned with 75% ethanol.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMorris water maze\u003c/h2\u003e \u003cp\u003eFollowing stereotactic injection, the mice were subjected to the Morris water maze (MWM) test to evaluate their cognitive abilities and memory performance. All mice underwent memory training four times a day, with an interval of 20 min, for four consecutive days. On the fifth day, the mice underwent a memory probe test to evaluate the retention of memory to find the hidden platform. The animals were video contemporaneously recorded via SMART 3.0 software (Panlab HARVARD, MA, USA), and behavioral metrics, including percentage of time in each quadrant, distance traveled, latency, and percentage of distance in each quadrant, were calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eY-maze\u003c/h2\u003e \u003cp\u003eOn the testing day, the mice (6 months old) were allowed to freely explore two arms of the Y-maze for 5 min, while the other arms were blocked. After habituation training, the mice were allowed to freely explore the three arms for 5 min. During exploration, the duration and frequency of entry into the novel open arm were recorded. Spontaneous alternation was defined as consecutive entry into three different arms on the overlapping triplet. The apparatus was cleaned with 75% ethanol between tests.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eBarnes maze\u003c/h2\u003e \u003cp\u003eThe Barnes maze test was used to evaluate the spatial memory ability of the mice. Briefly, the Barnes platform is circular, 92 cm in diameter, and contains 20 holes, 19 of which are blocked, leaving only one escape hole. Mice were acclimated to the experimental room one day in advance. During the training phase, each mouse was habituated to the escape hole for 4 min and then to the starting box for 30 s. The mice were then allowed to freely explore the platform, and spatial cues, bright light, and white noise were used to motivate the mice to find the escape hole within 4 min. Any mice that failed to find the escape hole were guided to it and allowed to habituate for 1 min. After three days of training, a 4-min platform probe test was performed, and the time taken and distance traveled by each mouse to enter the escape hole were recorded. Data analysis was performed using SMART software.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eRT-qPCR\u003c/h2\u003e \u003cp\u003eThe mRNA levels were detected by RT-qPCR. The final reaction contained 2 \u0026micro;g of total RNA, 4 \u0026micro;L of 5\u0026times; RT Master Mix (ABclonal; Wuhan, China), and RNase-free water (up to 20 \u0026micro;L). cDNA was reverse-transcribed using the following PCR parameters: 55\u0026deg;C for 15 min and 85\u0026deg;C for 5 min. Subsequently, qPCR SYBR Green Master Mix (ABclonal; Wuhan, China) was used for the qPCR amplification reactions. Each reaction contained 1 \u0026micro;L of cDNA sample (100\u0026ndash;200 ng/\u0026micro;L), 10 \u0026micro;L of qPCR SYBR Green Master Mix, 0.8 \u0026micro;L (10 \u0026micro;M) of the designated primers, and RNase-free water (up to 20 \u0026micro;L). The qPCR conditions were as follows: 95\u0026deg;C for 3 min, 40 thermal cycles at 95\u0026deg;C for 5 s, and 60\u0026deg;C for 30 s. mRNA levels were normalized to those of \u003cem\u003eGAPDH\u003c/em\u003e, and the relative gene expression was quantified using the ΔΔCt method. The primers used in this study are detailed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-3.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eThe cells were washed with cold PBS and lysed on ice with tissue protein extraction reagent (Beyotime, China) for 30 min. Each homogenate was centrifuged at 12,000 rpm for 30 min at 4\u0026deg;C, the supernatant was collected, and the soluble protein concentration was determined using a BCA-100 protein detection kit. A total of 20 \u0026micro;g of medium-quality protein per sample was boiled for 10 min in 5\u0026times; loading buffer, subjected to SDS-PAGE (12%), and then transferred to a nitrocellulose membrane. The membranes were blocked with 5% skim milk at room temperature for 2 h, incubated overnight at 4\u0026deg;C with primary antibodies (ABclonal, Wuhan, China), and then incubated with a secondary antibody at room temperature for 1 h. The protein bands were visualized using an Odyssey scanner and associated software (LI-COR Biosciences, USA). Relative protein levels were normalized to those of GAPDH, β-Tubulin, beta-actin or vinculin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStereotactic injection\u003c/h2\u003e \u003cp\u003eC57BL/6 and 3xTg mice were obtained from Shanghai Model Organisms Center, Inc. The mice were divided into five groups (n\u0026thinsp;=\u0026thinsp;10 per group). Targeted injection into the hippocampus was performed with the miR-4763-3p agomir, miR-4763-3p antagomir, and negative controls (NC: sequence-scrambled miRNA; Table S4, 5). The miR-4763-3p agomir and antagomir and the scrambled control were purchased from RiboBio (Guangzhou, China). The treatment groups were as follows: WT\u0026thinsp;+\u0026thinsp;PBS, AD-MCI\u0026thinsp;+\u0026thinsp;miR-4763-3p antagomir NC, AD-MCI\u0026thinsp;+\u0026thinsp;miR-4763-3p agomir NC, AD-MCI\u0026thinsp;+\u0026thinsp;miR-4763-3p antagomir, and AD-MCI\u0026thinsp;+\u0026thinsp;miR-4763-3p agomir. First, we made a longitudinal incision to expose the bregma and set this point to zero. From this point, we determined the hippocampal CA1 area (anterior and posterior: + 2.0 mm, inner and outer\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 mm, dorsoventral\u0026thinsp;+\u0026thinsp;1.9 mm). Each mouse was injected with 2 \u0026micro;L of treatment solution within 10 min; the needle was then held for 10 min, after which the needle was slowly retracted. Seven days after stereotaxic injection, the mice were euthanized, and their hippocampi were isolated for total RNA extraction to test the effect of stereotaxic injection. After 7 days of free access to food and water, the behavioral test was conducted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDual-luciferase reporter gene detection\u003c/h2\u003e \u003cp\u003eThe relationship between hsa-miR-4763-3p and \u003cem\u003eATP11A\u003c/em\u003e was verified using a dual luciferase reporter assay kit (Vazyme, China). The binding sequences of miR-4763-3p and \u003cem\u003eATP11A\u003c/em\u003e were predicted by RNAhybrid software, and the free energy of their targeted binding sites was analyzed. We designed the mutated (MUT) binding site using the complementary sequence of wild-type (WT) \u003cem\u003eATP11A\u003c/em\u003e and constructed the pGL3 Basic reporter plasmid. HEK-293T cells were transfected with ATP11A-MUT (mutant 3'UTR) or ATP11A-WT (luciferase reporter plasmid with the correct sequence) and the miR-4763-3p mimic or NC (Shanghai Ribo, China, Table S4) for 48 h. After cell collection and lysis, the relative luciferase activity was calculated as the ratio of firefly luciferase to renilla luciferase. Three independent experiments were performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence imaging\u003c/h2\u003e \u003cp\u003eThe NeuN, Iba1, Aβ, Tau, ATP11A, Annexin V, SQSMT1 and Caspase 3 levels were monitored by immunostaining (the relevant antibody information is provided in Table S6). Brain tissue slices (n\u0026thinsp;=\u0026thinsp;3) on coverslips were washed three times with 1\u0026times; PBS for 3 minutes each and fixed in 4% paraformaldehyde (PFA) for 15 minutes. After washing three times with 1\u0026times; PBS for 3 minutes each, the slices were blocked and permeabilized in 1\u0026times; PBS containing 1% goat serum and 0.25% Triton\u0026trade; X-100 and then stained with a primary antibody overnight at 4\u0026deg;C. The following day, the slices were washed three times with 1\u0026times; PBS for 3 minutes each, incubated with a secondary antibody at room temperature for 1 hour, and washed again. DAPI (Invitrogen, NBP2311561) was used to counterstain the nuclei, which were then incubated in the dark for 5 minutes. Finally, the slices were washed four times with PBST for 5 minutes each to remove excess DAPI and then mounted with mounting solution containing a fluorescence quencher. Images of immunostained brain tissue slices were obtained using confocal fluorescence microscopy (Zeiss). After adjusting the threshold, the fluorescence intensity was quantified using ImageJ\u0026reg; version 1.6.0 software (National Institute of Mental Health/NIH Research Services Branch, Bethesda, Maryland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunocytochemistry (IHC)\u003c/h2\u003e \u003cp\u003eBrain slices (10 \u0026micro;m thick) were obtained with a cryostat and rinsed with 1\u0026times; PBS three times. Then, these slices were treated with 3% hydrogen peroxide for 30 min to block endogenous peroxidase activity. After incubation with 0.1% Triton X-100 for 20 min to permeabilize the membrane, the slices were blocked with 5% bovine serum albumin (BSA) for 30 min. The brain slices were incubated with the primary antibodies IL-6 and TNF-α overnight at 4\u0026deg;C. After washing with PBST, the slices were probed with a biotinylated secondary antibody and streptomycin-labeled peroxidase solution for 1 h at room temperature and then stained with 3,3'-diaminobenzidine (DAB) reagent for 1 to 10 min at 37\u0026deg;C. After washing, the brain slices were dehydrated with different concentrations of alcohol (75, 80, 95, and 100%), rendered transparent in xylene, and sealed on glass slides. The digital images of all slices were captured using a Pannoramic MIDl.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence in situ hybridization (FISH)\u003c/h2\u003e \u003cp\u003eA fluorescently labeled miR-4763-3p FISH probe was designed and synthesized by Servicebio (Wuhan, China). Fluorescence-labeled single-strand probes were hybridized. FISH was carried out according to the manufacturer\u0026rsquo;s instructions for SweAMI-FISH (Servicebio). All fluorescence images were captured using a confocal laser microscope (Zeiss, Germany). The miR-4763-3p probe sequence used was CCCGCCCAGCACCAGCCCCTGCCT.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eAn apoptosis detection kit (Yeasen, Shanghai, China) was used to measure the levels of apoptosis in the transfected cells. The cells were suspended in 50 \u0026micro;L of 1\u0026times; binding buffer, to which 5 \u0026micro;L of Annexin V-FITC or 5 \u0026micro;L of anti-PI-PE was added, and the mixture was incubated for 15 minutes at room temperature. Next, 400 \u0026micro;L of 1\u0026times; binding buffer was added to each sample. Flow cytometry (Beckman Coulter Cytoflex) was used to collect the data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eTissue: Frozen hippocampal tissue was rapidly homogenized using a homogenizer in PBS containing a protease inhibitor and then centrifuged at 5,000 \u0026times; g for 10 min to collect the supernatant. The levels of the cytokines IL-6 and TNF-α in the serum and tissue were measured by ELISA kits (JiangLai, China).\u003c/p\u003e \u003cp\u003eSH-SY5Y cells were plated in 6-well plates at 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e/well and transfected with NC (5 \u0026micro;M), siATP11A (5 \u0026micro;M), OEATP11A (4 \u0026micro;g of pcDNA3.0-ATP11A-overexpressing plasmid) or vector (4 \u0026micro;g) for 48 h. Supernatants were collected for detection of TNF-α and IL-6 by ELISA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data were analyzed and are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) of at least three independent experiments. The analytical methods used were GraphPad Prism version 8 (GraphPad Software, Boston, MA). For comparisons between two groups, a two-tailed unpaired Student\u0026rsquo;s t test was used for normally distributed data. For comparisons involving multiple groups, one-way ANOVA and two-way ANOVA followed by Tukey\u0026rsquo;s multiple comparison test were used for normally distributed datasets. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMiR-4763-3p antagomir ameliorates cognitive decline in AD-MCI mice\u003c/h2\u003e \u003cp\u003eDysregulation of miRNAs has been strongly implicated in the pathogenesis of AD. To determine the differences in miRNAs between patients with AD and healthy people, we analyzed noncoding RNAs in different human serum samples. After analyzing the noncoding RNA profile data from the serum samples of 50 AD patients and 50 healthy individuals (GSE120584), we identified differentially expressed miRNAs between the AD group and the healthy group. A total of 424 DEGs were screened (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). The p value was further optimized to \u0026lt;\u0026thinsp;0.01, and all differentially expressed genes were investigated in PubMed. Only 12 genes were found to be associated with neurological diseases (excluding tumors). According to the study, only hsa-miR-4763-3p, hsa-miR-342-3p, hsa-miR-361-3p, hsa-miR-485-3p and hsa-miR-211-5p are thought to be cognitively relevant [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. We examined miRNA expression in the hippocampal tissues of AD-MCI and WT mice at 6 and 12 months. miRNA-qPCR experiments revealed that miR-361-3p and miR-4763-3p exhibited increased expression levels during the early stages of AD and continued to accelerate the progression of the disease in the later stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Given that existing studies have revealed the mode of action of miR-361-3p on neurons, the aim of this study was to further explore the effects of miR-4763-3p on AD neurons and its impact on cognitive function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSequencing data (GSE120584) revealed significant upregulation of miR-4763-3p in the serum of AD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Using clinical samples from 30 AD patients and 30 controls, we verified that miR-4763-3p expression increased with age in AD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). This finding reminds us of the strongest genetic risk factor for AD, apolipoprotein E epsilon 4 (APOE4), which is strongly associated with the risk of age-related cognitive decline in individuals without dementia[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Correlation analysis between the levels of miR-4763-3p and patient age in APOE4 high-risk AD patients revealed a positive association between miR-4763-3p and age (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), suggesting that both could be markers for AD detection. Next, fluorescence in situ hybridization (FISH) experiments revealed the expression of miR-4763-3p in human neuroblastoma SH-SY5Y cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). In contrast to the control, higher expression of miR-4763-3p was observed in postmortem hippocampal tissue from AD patients; colocalization with neurons was also detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Moreover, there was a positive correlation between the expression of miR-4763-3p and the expression of the Aβ or Tau proteins, with some degree of colocalization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, I), suggesting that miR-4763-3p may affect intracellular molecular mechanisms in neurons during the pathogenesis of AD. To further explore the mechanism of miR-4763-3p in the pathogenesis of AD and its ability to rescue cognitive and memory impairment in early AD mice, we stereotactically injected PBS, agomir (miRNA agonist), agomir NC, antagomir (miRNA antagonist), or antagomir NC into the hippocampal CA1 region of WT or AD-MCI mice (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). The detection of miR-4763-3p expression in the hippocampal region of the mouse brain showed that the antagomir decreased miR-4763-3p in the brain, while the agomir increased it, demonstrating effectiveness seven days after administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003eThen, we assessed the learning and memory abilities of these mice. The results of the novel object recognition test (assessing spatial learning) showed that the AD-MCI-miR-4763-3p agomir group had a lower frequency and duration of exploring objects than did the agomir NC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC), indicating that spatial memory was impaired in the model group. The AD-MCI-miR-4763-3p antagomir group spent almost twice as much time exploring novel objects than familiar objects and discovered novel objects more frequently, suggesting that their spatial memory was significantly improved in comparison with that of the NC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). The results of the Y-maze experiment showed that AD-MCI mice in the NC and miR-4763-3p agomir treatment groups tended to stay and shuttle in the familiar arm, while those in the miR-4763-3p antagomir treatment group and WT group had a greater frequency and duration of stay in the open arm than in the familiar arm, demonstrating their greater spatial learning ability in comparison with other mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD). The results of the Morris water maze experiment demonstrated that the AD-MCI-miR-4763-3p antagomir group had a similar duration of stay and number of platform crossings as the WT group did, and these durations were significantly greater than those of the other groups, including the NC-treated AD-MCI group and miR-4763-3p agomir group, which spent the shortest amount of time in the target quadrant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE). These observations indicate that the miR-4763-3p antagomir significantly improved the spatial and directional senses of AD-MCI mice. In the Barnes maze experiment, AD-MCI mice in the miR-4763-3p antagomir treatment group displayed a significantly shorter latency to reach the target hole during the search stage than did those in the NC and agomir treatment groups, indicating a significant improvement in learning ability and spatial memory following miR-4763-3p antagomir treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF). These findings suggest that miR-4763-3p antagomir treatment has good diagnostic and promising therapeutic effects on early-stage AD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eThe miR-4763-3p antagomir rescued neuronal loss and synaptic morphology\u003c/h2\u003e \u003cp\u003eTo further investigate the specific mechanisms underlying the improvements in cognitive and memory abilities following miR-4763-3p antagomir treatment, immunofluorescence staining was performed in the hippocampus of AD-MCI mice after stereotactic injection. The results demonstrated significant neuronal loss and microglial cell proliferation in the miR-4763-3p agomir group compared with the WT and NC groups. Conversely, following treatment with the miR-4763-3p antagomir, a significant decrease in the number of neurons and no increase in microglial cell proliferation were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, C and D). Nissl bodies can serve as a marker of neuronal functional status[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] since these structures decrease or disappear following neuronal damage. Interestingly, the staining intensity of Nissl bodies in the CA1 and CA3 hippocampal regions was significantly greater in the miR-4763-3p antagomir-treated group than in the NC group, while the miR-4763-3p agomir-treated group displayed lighter-stained Nissl bodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and E). These results suggest that miR-4763-3p antagomir treatment may rescue neuronal loss and damage in the hippocampal CA1 and CA3 regions of AD-MCI mice and ameliorate cognitive decline. In addition, since the normal structure of hippocampal synapses is fundamental to learning and memory, Golgi staining was used to evaluate the morphology of brain dendrites and synapses. The results showed that the dendritic spines in the miR-4763-3p agomir group were relatively disorganized, while miR-4763-3p antagomir treatment significantly increased the length and density of the dendritic spines. Although there were still some differences in the Golgi between the miR-4763-3p antagomir group and the WT group, there was a significant improvement in the dendritic spine length and density in the miR-4763-3p antagomir NC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). Moreover, Sholl analysis revealed that the dendrites of the miR-4763-3p agomir group exhibited the lowest complexity, and miR-4763-3p antagomir treatment significantly increased the complexity of dendrites (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, H) and the percentage of mushroom-type spines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, I). Finally, the analysis of synaptic proteins revealed that the miR-4763-3p agomir reduced GLUR1/2 expression, while the antagomir increased GLUR1/2 expression, which was consistent with the WT results (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB, C). These data suggest that miR-4763-3p antagomir treatment significantly rescues neuronal loss and reshaped synaptic morphology in AD-MCI mice, which may be the underlying mechanism for the restoration of cognition and enhancement of learning and memory.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eBioinformatics analysis of the differential expression of miRNAs and related biochemical pathways in AD-MCI\u003c/h2\u003e \u003cp\u003eTo further explore the underlying molecular mechanisms and downstream signaling pathways involved in the regulation of miR-4763-3p, RNA-seq analysis of the hippocampal region of AD-MCI mice was performed following stereotactic injection of the miR-4763-3p antagomir or NC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The results revealed 430 upregulated genes and 48 downregulated genes in comparison with those in the NC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Functional enrichment analysis of the DEGs revealed enrichment mainly in the immune system, the ER-phagosome pathway and the role of phospholipids in phagocytosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The results of KEGG and GO enrichment analysis revealed that the DEGs were mainly enriched in the phagosomes, the PI3K-AKT signaling pathway, apoptosis, and the mTOR signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), in addition to the cell surface receptor signaling pathway and the regulation of phagocytosis and apoptotic cell clearance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Furthermore, the upregulated DEGs were mainly enriched in phagosomes, apoptosis and lysosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). The upregulated DEGs were associated with phagocytosis, inflammation regulation, phospholipid translocation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), membrane microdomains, membrane rafts (Figure S3A) and immune receptor activity (Figure S3B). GO enrichment analysis indicated that the downregulated DEGs were involved mainly in synapse organization, cognition and learning or memory (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH); neuron\u0026ndash;neuron synapses (Figure S3C); and cytokine activity (Figure S3D). To determine the relationship between the regulatory role of miR-4763-3p and the pathological mechanisms of AD, we further analyzed the DEGs between publicly available datasets for AD patients and healthy controls. Analysis of the raw RNA-seq data (GSE173955) using FDR/adj P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log2FC|\u0026gt;1.5 identified 1429 DEGs between the AD group and the control group, of which 585 were upregulated and 844 were downregulated in the AD group. (Figure S3E). The DEGs were mainly enriched in synapse organization, regulation of transporter activity, cognition (Figure S3F) and the mTOR signaling pathway (Figure S3G). Therefore, miR-4763-3p antagomir may influence the PI3K or mTOR signaling pathway, potentially improving the immune microenvironment in the brain, managing phagocytosis, and ameliorating the loss of synaptic function and neuron count in the brain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eATP11A\u003c/b\u003e \u003cb\u003eis a target gene of miR-4763-3p in neurons\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo verify our aforementioned hypothesis, bioinformatics analysis was conducted using online databases to predict the target genes of miR-4763-3p. The results showed that 1002 genes were repeatedly predicted by the TargetScan7, miRDB, and miRWalk databases (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Functional enrichment analysis indicated that the predicted target genes were involved mainly in signal transduction, nervous system development, PI3K/AKT signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), protein binding, the nucleus, phagocytosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) and autophagy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Consistent findings from bioinformatics analysis and mice hippocampal sequencing analysis highlighted the significant involvement of autophagy and phagocytosis in the learning and memory impairments observed in neurodegenerative diseases such as AD. Further investigation revealed only ATP11A at the intersection of five datasets: mice hippocampal sequencing data DEGs, predicted miR-4763-3p target genes, mass spectrometry results (SY5Y differentially transfected with miR-4763-3p inhibitor and NC), and human disease-related genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). ATP11A is closely related to the nervous system [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and was significantly upregulated in the hippocampal region of mice treated with the miR-4763-3p antagomir (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). The First database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alzmap.org/tsne/gene\u003c/span\u003e\u003cspan address=\"https://alzmap.org/tsne/gene\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) of the brain diagram shows that the \u003cem\u003eAtp11a\u003c/em\u003e expression level in the CA1 brain region of the hippocampus decreased in AD-MCI mice at 6 months of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG) but not in 3-month-old mice compared with that in WT (C57BL/6) mice (Figure S4A, B). Therefore, we hypothesized that Atp11a may be closely related to the decline in learning and memory abilities and cognitive impairment observed in early AD. We extracted RNA and protein from the hippocampus of 3-month-old and 6-month-old WT or AD-MCI mice for detection and found that the expression of ATP11A in 3-month-old WT and AD-MCI mice was similar, while the expression of ATP11A in 6-month-old AD-MCI mice was lower than that in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-J). FISH analysis of human brain tissue revealed that miR-4763-3p was expressed at low levels in the control group, while ATP11A was significantly highly expressed in the hippocampal region (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK). In contrast, significant downregulation of ATP11A and elevated miR-4763-3p expression were observed in the hippocampus of patients diagnosed with Alzheimer's disease and were found to be colocalized with ATP11A (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK). Furthermore, FISH analysis revealed colocalization between miR-4763-3p and ATP11A in SH-SY5Y cells, suggesting that there may be a close relationship between the two miRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL, M). Subsequently, RT‒qPCR and western blotting were used to evaluate the effects of miR-4763-3p on ATP11A expression in cells and tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eN, O; Figure S4C-E). The results demonstrated that miR-4763-3p inhibited the expression of ATP11A at both the transcriptional and translational levels and that ATP11A expression was restored following treatment with the miR-4763-3p inhibitor. In addition, this mode of action also affects ATP11A expression in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo verify the targeted binding between miR-4763-3p and ATP11A, RNAhybrid software was used to predict the binding sequence between miRNA and ATP11A. The prediction results revealed that the GGGACGG sequence (highlighted in yellow) was present in three predicted sequences (Figure S4F). The free energy of the targeted binding site was subsequently analyzed (Figure S4G). To further evaluate the molecular mechanism of miR-4763-3p, a mutant of ATP11A with a disrupted miR-4763-3p binding sequence was constructed. The sequencing results of the ATP11A mutant are shown in Figure S4H and I, and a schematic depicting the targeted binding mode between miR-4763-3p and WT or mutant ATP11A is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eP. A dual luciferase reporter gene assay was then employed to validate the targeted binding relationship between miR-4763-3p and ATP11A. HEK293T cells were cotransfected with ATP11A-WT or ATP11A-MUT and the miRNA mimic or NC. Compared with cells transfected with ATP11A-MUT, the miRNA mimic significantly reduced the luciferase activity in cells transfected with ATP11A-WT, indicating that miR-4763-3p inhibited the activity of the reporter gene and thus targeted regulated ATP11A (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eQ). After revealing the targeting relationship between ATP11A and miR-4763-3p, we further determined the cognition-related functions of ATP11A. ShATP11A or NC was stereotactically injected into the brains of 6-month-old AD-MCI mice, and behavioral tests were performed 4 weeks later. The results showed that shATP11A mice had shorter dwell times and shorter distances to the new open arm in the Y maze (Figure S5A-C), and the dwell times and distances explored around new objects were significantly lower than those of NC mice (Figure S5D-F). In the Morris water maze test, the swimming time in the target quadrant was significantly lower than that in the NC group (Figure S5G, H), demonstrating that ATP11A deficiency led to learning and memory deficits in mice and that ATP11A, a target gene of miR-4763-3p, may play an important role in the process of AD disease.\u003c/p\u003e \u003cp\u003eIn general, miRNAs act in mammals by not fully binding to target genes, thereby inhibiting their translation [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, our research findings demonstrated that miR-4763-3p also influences the transcription of ATP11A, which provoked our curiosity. Consequently, we conducted further investigations to explore the regulatory mechanisms underlying the transcription of miR-4763-3p. Considering that transcription factors (TFs) often play crucial roles in the mode of action of miRNAs [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], we employed gene function screening to predict the upstream TFs of miR-4763-3p and identified YY1 as a potential candidate (Figure S5I). Research has indicated that YY1 plays a vital role in regulating the expression of TREM2, which stimulates phagocytosis and suppresses cytokine production and inflammation [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Specifically, microglial YY1 maintains TREM2 expression levels, providing a therapeutic target for the prevention and treatment of AD [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Our experimental results revealed significant downregulation of YY1 expression in the hippocampus of AD-MCI patients compared to control subjects (Figure S5J), suggesting that YY1 may play an important role in the hippocampus. Based on the potential regulatory mechanism of YY1, the ChIP assay confirmed that YY1 could bind to the miR-4763-3p promoter in SH-SY5Y cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eR), suggesting that YY1 can transcriptionally regulate miR-4763-3p expression. Inhibition of YY1 via siRNA upregulated the miR-4763-3p level (Figure S5K, L), but miR-4763-3p expression had no significant effect on YY1, demonstrating that YY1 may act only as an upstream regulatory element of miR-4763-3p. ChIP-qPCR was used to confirm that YY1 binds to the ATP11A promoter, initiating transcription in SH-SY5Y cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eS). ATP11A expression was significantly downregulated after transfection with siYY1 (Figure S5M). In summary, our data revealed a feedforward regulatory mechanism of YY1-miR-4763-3p-ATP11A, which may play an important role in the nervous system of AD patients.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eMiR-4763-3p antagomir targets ATP11A to reverse early apoptosis in neurons by regulating PS flipping\u003c/h2\u003e \u003cp\u003eBased on our results in mouse hippocampal slices, we hypothesized that the miR-4763-3p antagomir may rescue neuronal loss; however, the specific mechanism is unclear. Currently known forms of regulated cell death include apoptosis, necrosis and autophagy [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Based on RNA-seq data, it is reasonable to suggest that miR-4763-3p is closely related to cellular apoptosis. Flow cytometry was used to detect apoptosis and necrosis in SH-SY5Y cells transfected with the miR-4763-3p mimic, inhibitor, corresponding NC or control. The results showed that compared with the NC, the miR-4763-3p mimic increased the levels of early and late apoptosis, while the miR-4763-3p inhibitor significantly reduced the proportion of early apoptotic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;C). Immunofluorescence and western blotting data revealed that the miR-4763-3p agomir/mimic significantly upregulated the expression of cleaved caspase 3 apoptosis-associated proteins in the SH-SY5Y cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) and hippocampal tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Conversely, treatment with the miR-4763-3p antagomir/inhibitor resulted in a lower level of cleaved caspase 3, and the cleaved caspase 3 expression level in the antagomir treatment group was similar to that in the WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E; Figure S6A, B). We hypothesize that the mechanism by which miR-4763-3p affects apoptosis is related to brain inflammation; therefore, ELISA, IHC and qPCR were performed to evaluate the expression of the inflammatory factors IL-6 and TNF-α in the mouse brain. The results revealed that the miR-4763-3p agomir significantly increased the expression levels of IL-6 and TNF-α, while treatment with the miR-4763-3p antagomir reduced brain inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, G, Figure S6C-E). In addition, similar results were obtained for the qPCR detection of the inflammatory factors IL-6 and TNF-α (Figure S6F, G). We also further explored the sequencing data of miR-antagomir and AD-MCI-NC and found that IL-34 is significantly affected by miR-4763-3p, a cytokine that is closely associated with the clearance of Aβ and stimulates the release of proinflammatory factors from macrophages. We detected the effect of miR-4763-3p on IL-34 in brain tissue. The qPCR results showed that compared with the control, the miR-4763-3p antagomir significantly reduced the expression of IL-34. Conversely, the miR-4763-3p antagomir significantly increased the expression of IL-34 in brain tissue. Therefore, we speculate that the miR-4763-3p antagomir improves the inflammatory environment in the brain by reducing the expression of IL-34 and reducing the release of inflammatory factors from macrophages (Figure S6 H-I).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe hypothesized that the target gene of miR-4763-3p, ATP11A, might be related to inflammation in the brain, and ELISA analysis was subsequently used to detect the expression of inflammatory factors. ELISA data demonstrated that siATP11A significantly increased the expression of IL-6 and TNF-α, while ATP11A overexpression significantly reduced the expression levels of IL-6 and TNF-α, indicating that ATP11A overexpression may alleviate brain inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH\u0026ndash;K). ATP11A is generally present in plasma membranes, and studies have shown that it has phospholipid flipping activity [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]; therefore, this study further explored whether ATP11A may play a role in phospholipid flipping in neuronal cell membranes. Immunofluorescence staining of brain tissue was used to evaluate the expression of ATP11A in the brain. ATP11A was significantly upregulated in the CA1 and CA3 regions of the hippocampus in the miR-4763-3p antagomir-treated group, while its expression was relatively low in the miR-4763-3p agomir-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eP). Notably, ATP11A was colocalized with neurons in the hippocampus, and its abundance and density were greater in the brains of mice treated with the miR-4763-3p antagomir. This may be more beneficial for its function: flipping the membrane of phosphatidylserine (PS). Flow cytometry analysis was performed in SH-SY5Y cells stimulated with LPS using Annexin V-FITC staining to measure phosphatidylserine expression levels, and the shift in the X-axis represents the effect of ATP11A on extracellular PS. The results demonstrated that overexpression of ATP11A and treatment with the miR-4763-3p inhibitor significantly decreased the level of extracellular PS (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL), while siATP11A treatment significantly increased PS in the outer leaflet of the plasma membrane of neurons. Moreover, compared with siATP11A treatment alone, cotransfection of cells with the miR-4763-3p inhibitor resulted in a significant decrease in the outer member PS level, especially following LPS treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL; Figure S7A-C). Studies have shown that intracellular PS is an important component of cells that can regulate the activity of neuroreceptors, enzymes, and signaling molecules and improve neuronal signaling. Therefore, it can be inferred that ATP11A enhances PS levels within the neuronal cell membrane, thereby improving neuronal function [\u003cspan additionalcitationids=\"CR41 CR42 CR43\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. These results demonstrate that miR-4763-3p plays an important role in stabilizing the phospholipid balance inside and outside the cell membrane under inflammatory conditions. Immunofluorescence staining of brain tissue revealed much stronger PS staining in the NC group than in the miR-4763-3p antagomir-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eQ), indicating that the miR-4763-3p antagomir stimulates ATP11A to inwardly flip PS into the cytoplasm, reducing the early apoptotic \"eat me\" signal on the cell surface. Flow cytometry analysis confirmed that siATP11A significantly increased apoptosis, particularly in the early stages. Conversely, treatment with an inhibitor of miR-4763-3p effectively mitigated both the early and late stages of neuronal apoptosis induced by ATP11A deficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eO, N; Figure S7D-H). Additionally, this regulatory mechanism of apoptosis did not affect PS synthase expression levels, which demonstrated that the miR-4763-3p inhibitor affected only PS flipping activity rather than synthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM; Figure S7I, J). These results revealed that the miR-4763-3p inhibitor can stimulate PS flipping into cells by increasing ATP11A levels. This flip can greatly reduce the recognition of \"eat me\" signals on the surface of glial cells under inflammatory conditions, reshaping the inflammatory environment in the brain; consequently, this mechanism reduces the phagocytic impact of glial cells on neuronal synapses and cell bodies, thus achieving the desired outcome of reducing early apoptosis of neurons.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eMiR-4763-3p antagomir/ATP11A increases autophagy levels in neurons\u003c/h2\u003e \u003cp\u003eSince the miR-4763-3p antagomir improves PS flipping under inflammatory conditions and reverses the occurrence of apoptosis, whether cellular homeostasis within neurons is also improved is unclear. Revisiting RNA-seq data and functional enrichment data for miR-4763-3p target genes points to autophagy. The gradual deposition of amyloid plaques in the brains of AD patients has been identified as the primary cause of cognitive decline in the early stages of the disease [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. To investigate the involvement of miR-4763-3p in the regulation of autophagy and its potential regulatory role in early Aβ deposition, we conducted IF staining of hippocampal tissue sections from the stereotactic injections of miR-4763-3p antagomir, agomir, antagomir NC, and agomir NC in AD-MCI mice. The results showed that the NC group, particularly the miR-4763-3p agomir-treated group, exhibited significant Aβ deposition, whereas the miR-4763-3p antagomir-treated group exhibited almost no Aβ deposition; these results were most similar to those of the WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). We speculated that the miR-4763-3p antagomir may reduce Aβ deposition during the early stages of the disease by facilitating autophagy or phagocytosis. TEM was used to evaluate changes in intracellular structures in the hippocampal region of AD-MCI mice following treatment with NC or the miR-4763-3p antagomir, revealing greater amounts of lipofuscin in the neurons of mice in the NC and agomir-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Excess lipofuscin indicates incomplete elimination of lipids and/or misfolded proteins in the cell body, suggesting insufficient lysosomal activity and excessive accumulation of metabolic waste, which may further impair neuronal function or cause neuronal death [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. However, the amount and size of lipofuscin were significantly reduced in the miR-4763-3p antagomir-treated group, which led us to believe that the miR-4763-3p antagomir may improve the intracellular environment of neurons by facilitating autophagy or lysosomal phagocytosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA growing body of evidence supports the involvement of autophagy in the pathogenesis of neurodegenerative diseases, immune disorders, and various human tumors [\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]; therefore, we aimed to explore the role of miR-4763-3p in the regulation of autophagy. During the early stages of autophagy, SQSTM1 functions as a selective autophagy receptor and serves as an important protein marker. Studies have demonstrated that SQSTM1 binds to arginine-modified substrates and induces autophagy. The depletion of SQSTM1 inhibited the recruitment of LC3 to autophagosomes, hindered the formation of autophagosomes within cells, and may impair autophagy [\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe basal levels of autophagy and lysosomal biogenesis can be reflected by the expression levels of LC3B II/I and SQSTM1. Compared with those in the control group, the miR-4763-3p inhibitor significantly increased the mRNA expression levels of LC3B and SQSTM1, as well as the protein expression levels of LC3B II/I and SQSTM1, whereas the mimic decreased the expression of these genes. The same trend was observed in the hippocampal CA1 and CA3 regions of mice following injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-F; Figure S8A\u0026ndash;C). Transmission electron microscopy revealed a significant increase in the number of autophagosomes in hippocampal neurons after miR-4763-3p antagomir treatment compared with those in the NC- and miR-4763-3p agomir-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). In addition, SH-SY5Y cells were transfected with LC3-GFP-RFP to determine the effect of miR-4763-3p on autophagic flux. GFP is an acid-sensitive protein that emits yellow fluorescence in autophagosomes and red fluorescence in autolysosomes. After miR-4763-3p inhibitor treatment, the number of red and yellow fluorescent intracellular structures increased significantly in comparison with that in the control group, indicating an increase in autophagic flux. In contrast, only green fluorescent structures were observed following miR-4763-3p mimic treatment, suggesting that miR-4763-3p may block the fusion of autophagosomes with lysosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). To further elucidate the underlying mechanism by which miR-4763-3p regulates autophagic flux, we hypothesized that its target gene ATP11A may play a role in modulating the expression of SQSTM1; therefore, we investigated the impact of ATP11A on SQSTM1. The results demonstrated that the levels of LC3B and SQSTM1 decreased after treatment with siATP11A compared with those in the control and NC groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI\u0026ndash;K; Figure S8D, E). Interestingly, cotransfection of miR-4763-3p inhibitor and siATP11A restored autophagic flux (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL\u0026ndash;N; Figure S8F, G). These data suggested that miR-4763-3p can target ATP11A to inhibit the fusion of autophagosomes with lysosomes, thereby reducing autophagy levels. The miR-4763-3p antagomir can increase autophagic flux in the brain to some extent and clear the excessive deposition of Aβ and lipofuscin in a timely manner, improving the brain environment and restoring spatial memory and cognitive abilities in AD-MCI mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eInhibition of the PI3K/AKT/mTOR/Bcl2 axis results in crosstalk between autophagy and apoptosis\u003c/h2\u003e \u003cp\u003eAutophagy and apoptosis exhibit a dynamic balance in the brain, which is mediated by signaling pathways. To further investigate the regulatory mechanism of the miR-4763-3p antagomir/ATP11A, we hypothesized that miR-4763-3p is likely involved in modulating the PI3K/AKT/mTOR signaling pathway. This hypothesis is based on combined KEGG analysis of sequencing data from the hippocampal region of AD-MCI mice treated with miR-4763-3p antagomir, reactome analysis of target genes regulated by miR-4763-3p, and KEGG analysis comparing DEGs between AD patients and normal controls. To verify this hypothesis, we examined the protein expression of p-mTOR, mTOR, p-PI3K, PI3K, p-AKT, and AKT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The immunoblotting results showed that p-mTOR/mTOR, p-PI3K/PI3K, and p-AKT/AKT were significantly upregulated in the siATP11A- and miR-4763-3p mimic-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;D, H\u0026ndash;K; Figure S9A\u0026ndash;C, F\u0026ndash;H). In addition, Beclin1 and Bcl2 were downregulated following siATP11A and mimic treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE\u0026ndash;G, L\u0026ndash;N; Figure S9D, E, I, J), indicating that activation of the PI3K/AKT/mTOR signaling pathway inhibited autophagy and activated the apoptotic pathway. The levels of p-mTOR/mTOR, p-PI3K/PI3K, and p-AKT/AKT were significantly decreased in SH-SY5Y cells treated with the miR-4763-3p inhibitor, indicating that the PI3K/AKT/mTOR signaling pathway was inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH\u0026ndash;K, Figure S9F-H). Bcl2 and Beclin1 were also significantly increased in these cells, indicating that the antiapoptotic pathway was activated (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eL-N, Figure S9I, J). Ultimately, cotransfection of siATP11A and the miR-4763-3p inhibitor had an appreciable ameliorating effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eO\u0026ndash;U; Figure S9K-O). Specifically, the PI3K/AKT/mTOR signaling pathway was activated following the administration of siATP11A compared with that in the control group. In addition, Bcl2 and Beclin1 expression was downregulated, which could be reversed by the introduction of the miR-4763-3p inhibitor. In conclusion, these findings confirmed that miR-4763-3p can target ATP11A to modulate the PI3K/AKT/mTOR/Bcl2 signaling pathway, thereby stabilizing the levels of autophagy and apoptosis in the brain and establishing specific cross-talk relationships.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, bioinformatics analysis revealed significant upregulation of miR-4763-3p in the serum of individuals with AD, and this upregulation was positively correlated with age. Subsequently, we determined that miR-4763-3p was highly expressed in the brains of AD patients and colocalized with the Aβ and tau proteins. Intracerebral stereotactic injection of a miR-4763-3p antagomir significantly improved learning and memory impairment and cognitive decline in an AD-MCI mouse model. We found that miR-4763-3p antagomir treatment improved the progressive loss of hippocampal neurons in AD-MCI mice, which was accompanied by an increase in the number of Nissl bodies, dendritic number and synaptic complexity. RNA-seq analysis of hippocampal tissue from control and miR-4763-3p antagomir-treated AD-MCI mice demonstrated potential involvement in regulating the immune system, phagosomes, and apoptosis. Subsequently, we identified the YY1-miR-4763-3p-ATP11A feedforward regulatory mechanism, which reduces the inflammatory environment in the brain and enhances the ability of ATP11A to inwardly flip PS in the neuronal cell membrane under inflammatory conditions. This reduces the level of early apoptosis and inflammation in neurons and regulates autophagy through the PI3K/AKT/mTOR/Bcl2 signaling pathway.\u003c/p\u003e \u003cp\u003eAD is the most common neurodegenerative disease and causes a heavy burden on patients, their caregivers, and society as a whole [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Late-stage AD is an irreversible progressive brain disorder characterized by memory loss and cognitive decline, which is accompanied by severe neuronal loss and widespread inflammation, and there is currently no effective cure [\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Therefore, early detection, prevention, and intervention strategies for the early stages of the disease are increasingly considered the keys to more effective management and treatment [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. MCI is a transitional state between normal aging and dementia, during which cognitive impairment does not yet affect daily life; nevertheless, reports suggest that 15\u0026ndash;20% of these patients subsequently develop AD [\u003cspan additionalcitationids=\"CR62\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Therefore, MCI is considered a possible early manifestation of AD pathology, other pathological entities such as cerebrovascular disease and Lewy body disease, or mixed pathology [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Studies have shown that MCI can be stratified by biomarkers [β-amyloid (A+/A-), tau (T+/T-), and neurodegeneration (N+/N-]; A\u0026thinsp;+\u0026thinsp;T-(N+) MCI is defined as \"mismatch MCI\", and A-T-(N+) MCI is defined as \"neurodegeneration-only MCI\". An examination of clinical samples revealed that mismatch MCI reflects early prodromal AD symptoms, which are characterized by rapid cognitive decline over time and susceptibility to non-AD pathology, such as amyloid angiopathy [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. The mismatch MCI group largely overlapped with the neurodegeneration-only MCI group [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. In addition, tau pathology plays a key role in the cognitive and neurodegenerative disease phenotype of AD, with the deposition of tau aggregates being a pathological marker [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSome studies suggest that the failure of experimental disease treatments to date is due to their being conducted in patients who already meet the AD criteria, which may represent too late a time point [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. During the preclinical stage of AD, a series of events occur after Aβ deposition, including tauopathy and abnormalities in markers associated with synaptic dysfunction and neuronal death [\u003cspan additionalcitationids=\"CR71 CR72\" citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. AD is a disease closely related to age [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. An increasing number of studies have shown that APOE4 is an extremely important genetic risk factor for AD, which is related to the risk of age-related cognitive decline in individuals without dementia [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. It has the potential to become a detection clinical treatment process. Age factors and APOE risk genes have good reference value in the study of AD pathogenesis and early detection markers. In our study, we found that miR-4763-3p expression appeared to increase with age in patients of different age groups, similar to APOE expression patterns, so we hypothesized that miR-4763-3p may be related to age. We used APOE4 as one of the screening factors during the screening process to explore the correlation between miR-4763-3p and age in high-risk AD patients with APOE4. The results showed that miR-4763-3p was positively correlated with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), suggesting that miR-4763-3p and APOE may jointly serve as age-related markers of AD and early warning signs of AD. Surprisingly, miR-4763-3p was highly expressed in the hippocampus of AD patients and appeared to colocalize with neurons, Aβ, and Tau, while its expression level in healthy controls was low. This finding suggested that miR-4763-3p may play an important role in the course of AD. Six-month-old 3\u0026times;Tg mice exhibit early AD symptoms but no tau pathology, but Aβ deposition and synaptic dysfunction are already present, which manifests as early cognitive deficits, and these mice can be used as an AD-MCI model. Here, AD-MCI mice were subjected to stereotactic injection of a miR-4763-3p agomir or antagomir to explore their effects. Multiple behavioral experiments related to memory demonstrated that the miR-4763-3p antagomir effectively rescued the cognitive deficits of AD-MCI mice. Moreover, examination of hippocampal tissue revealed that the miR-4763-3p antagomir significantly restored the number and synaptic density of neurons and Nissl bodies. Therefore, the specific mechanism by which the miR-4763-3p antagomir rescued AD-MCI at an early stage was investigated.\u003c/p\u003e \u003cp\u003eThe mode of action of miRNAs is generally considered to involve targeted inhibition of gene expression [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]; thus, RNA-seq is undoubtedly the most direct method for revealing the mechanism of action of miR-4763-3p. The results showed that the target genes of miR-4763-3p are mainly involved in the immune system, phagocytosis, synapse organization, regulation of nervous system processes, cognition, and learning or memory in biological processes. We identified a direct target of miR-4763-3p, \u003cem\u003eATP11A\u003c/em\u003e, which encodes a P4-type ATPase that functions redundantly as a phospholipid flippase in the plasma membrane. Previous studies have shown that mutations in ATP11A cause developmental delays and neurological deterioration [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], and ATP11A deficiency leads to mouse embryonic death at E14.5, demonstrating its important role in the formation of the syncytiotrophoblast layer during placental development [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. These observations suggest that ATP11A plays an important role in the development of the nervous system and may be closely related to the development or death of neurons. A common feature of neurodegenerative diseases is neuronal loss caused by apoptosis. The brains of AD patients are considered to have an inflammatory microenvironment, and the present study proposes an unknown regulatory mechanism between autophagy and apoptosis. The miR-4763-3p antagomir targets \u003cem\u003eATP11A\u003c/em\u003e to reduce the expression of inflammatory factors in the brain and enhances the ability of ATP11A to inwardly flip PS inside the neuronal cell membrane. This reduces the exposure of the \"eat me\" signal on the neuronal surface, thereby decreasing the recognition and phagocytosis of neurons by glial cells and reversing their early apoptosis. Moreover, homeostasis within neurons is improved, autophagy levels are increased to clear metabolic waste, brain lipofuscin levels and Aβ deposition are reduced, and a healthy microenvironment is restored within brain neurons.\u003c/p\u003e \u003cp\u003eAutophagy defects often occur in early AD-MCI, where abnormal protein aggregates and Aβ\u0026thinsp;+\u0026thinsp;autophagic vacuoles (AVs) containing incompletely digested autophagic substrates accumulate in neurons. These effects are associated with an age-induced reduction in autophagy-related gene expression and late-onset AD [\u003cspan additionalcitationids=\"CR78\" citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. Therefore, autophagy dysfunction may act as an upstream event of AD amyloid pathology, rendering it an attractive target for therapeutic intervention [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e] since maintaining the crosstalk balance between autophagic flux and apoptosis is particularly important for the treatment of nervous system diseases.\u003c/p\u003e \u003cp\u003eThe balance between autophagy and apoptosis is intricately regulated in brain tissues. Autophagy can reduce cellular apoptosis by eliminating damaged fragments or degraded subcellular components [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. Studies have shown that activation of the TNF-α/TNFR1 signaling pathway in AD leads to the recruitment of RIPK1 by accumulated p62, which induces its oligomerization and results in necroptotic death of neurons. Ectopic accumulation of p62 is caused by impaired autophagic flux mediated by TNF-α-induced downregulation of UVRAG during the necroptotic process [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. Moreover, in a mouse model of rotenone-induced Parkinson's disease (PD), PLG-induced autophagy inhibited cell apoptosis through Ser70 phosphorylation, stabilizing the balance between autophagy and apoptosis [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. Autophagy also limits endoplasmic reticulum (ER) stress by degrading unfolded protein aggregates. Simultaneously, autophagy can recycle protein aggregates and misfolded proteins to maintain ER function, thus attenuating the ER stress response and subsequent cell apoptosis [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. However, aberrant autophagy can promote cell apoptosis. For example, it has been proposed that autophagy induces cell apoptosis by modulating the levels of interferon-beta (IFN-β) induced by Toll-like receptor (TLR)/interleukin-1 receptor (TIR) domain-containing adaptor-inducing interferon-β (TRIF) [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. In a rat model of cerebral ischemia, conventional protein kinase C gamma (cPKCγ) alleviated stroke damage, possibly by downregulating ubiquitin C-terminal hydrolase L1 (UCHL1), which upregulated the ERK-mTOR pathway, alleviated autophagy and cell apoptosis, and ultimately exerted a neuroprotective effect [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. In the treatment of hepatocellular carcinoma, the activation of autophagy through the JNK/beclin-1 pathway can induce cancer cell apoptosis, achieving partial therapeutic efficacy [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAutophagy is regulated by various signaling pathways, among which mTOR, a protein kinase, senses the availability of cellular energy and regulates cell proliferation. Reports have shown excessive activation of mTOR signaling in specific brain regions of AD patients [\u003cspan additionalcitationids=\"CR91\" citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. The mTOR pathway is critical for the regulation of autophagy and is controlled by the upstream PI3K/AKT pathway. Overactivation of mTOR leads to the accumulation of Aβ and exerts a negative feedback effect on autophagy [\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e]. Beclin-1, a well-known inducer of autophagy, regulates the initiation of autophagosome formation, and its activation promotes autophagy [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e]. Therefore, inhibition of the PI3K/AKT/mTOR pathway can activate autophagy, which is consistent with our functional enrichment and immunoblotting data. Inhibition of miR-4763-3p resulted in a significant increase in the expression levels of LC3B II/I and SQSTM1, as well as a notable reduction in the accumulation of Aβ and lipofuscin in the hippocampal region. Additionally, an increase in the number of autophagosomes was observed. Moreover, the expression levels of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR were significantly decreased, while those of Beclin1 and Bcl2 were upregulated. Taken together, these data indicated that the miR-4763-3p antagomir promoted autophagy by inhibiting the PI3K/AKT/mTOR/Bcl2 signaling pathway and reducing cell apoptosis, which suggests that the PI3K/AKT/mTOR/Bcl2 pathway participates in the balance between brain autophagy and early neuronal apoptosis in AD-MCI mice.\u003c/p\u003e \u003cp\u003eFinally, there are some limitations in this paper. First, although a feedback loop mechanism involving YY1-miR-4763-3p-ATP11A was found in this study, the interaction between these two factors and the YY1 transcription of these two genes was not fully explored in this study, which may be a shortcoming of this study. In addition, ATP11A, the key target gene of this protein, is a phospholipid flipping enzyme. The effect of PS in this study was mainly to reduce the inflammatory microenvironment in the brain and flip PS into the inner membrane of nerve cells, thereby improving the level of inflammation in nerve cells and improving autophagy. Does PS play a role in autophagy in nerve cells? How PS, as a lipid structure, may affect autophagosome assembly may be worth further exploration. In addition, while we have contributed to understanding the role of miR-4763-3p in relevant therapeutic outcomes in mouse models, its preclinical application needs to be further explored. In future studies, it will be necessary to optimize the biologics of miR-4763-3p antagomirs to improve their stability and therapeutic efficacy in vivo.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe demonstrated that miR-4763-3p was highly expressed in the serum of AD patients and highly colocalized with Aβ and tau in the brain. The miR-4763-3p antagomir targeted ATP11A inwardly flipped PS in the neuronal cell membrane to reduce early apoptosis. In addition, we showed that miR-4763-3p/ATP11A regulated autophagy through the PI3K/AKT/mTOR/Bcl2 signaling pathway, improved the internal microenvironment in neuronal cells and repaired damaged synaptic morphology, thus improving learning and memory in AD-MCI mice to mitigate cognitive decline. This finding points to a promising strategy for the treatment of AD-MCI patients.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAD-MCI:\u0026nbsp;Alzheimer\u0026rsquo;s disease-mild cognitive impairment;\u003c/p\u003e\n\u003cp\u003eFISH: fluorescence in situ hybridization;\u003c/p\u003e\n\u003cp\u003eAVs: autophagic vacuoles;\u003c/p\u003e\n\u003cp\u003ePS: phosphatidylserine;\u003c/p\u003e\n\u003cp\u003eER: endoplasmic reticulum;\u003c/p\u003e\n\u003cp\u003eIFN-\u0026beta;: interferon-beta;\u003c/p\u003e\n\u003cp\u003eTLR: Toll-like receptor;\u003c/p\u003e\n\u003cp\u003eTIR: Toll/interleukin-1 receptor;\u003c/p\u003e\n\u003cp\u003ecPKC\u0026gamma;: conventional protein kinase C gamma;\u003c/p\u003e\n\u003cp\u003eUCHL1: ubiquitin C-terminal hydrolase L1;\u003c/p\u003e\n\u003cp\u003eMWM: Morris water maze;\u003c/p\u003e\n\u003cp\u003ePFA: paraformaldehyde\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSample collection was approved by Shanghai University (Shanghai, China). Informed consent was obtained from patients or their guardians, as appropriate. All animal experiments were carried out following\u0026nbsp;the National Institutes of Health (NIH)\u0026nbsp;Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Care Committee of Shanghai University (ECSHU 2023-004).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data required for evaluation of the conclusions are\u0026nbsp;presented\u0026nbsp;in the paper and/or Supplementary Materials. The data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Shanghai Jiao Tong University School of Medicine for its technical support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was sponsored by the National Key Research and Development Program of China (2020YFA0113000, 2018YFA0109800), Basic Research Program of Shanghai (20JC1412200), National Natural Science Foundation of China (81971324), and CAMS Innovation Fund for Medical Sciences (2022-I2M-1-012).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;W.Q. and N.D. were involved in the conception and design of the study and performed the experiments. X.Z., Q.L., W.F., P.W., and H.W. were involved in the data analysis and interpretation. X.H., L.W., and N.W. were involved in manuscript editing. X.D., Y.L., R.Z. and J.W. were involved in the design of the study, review, editing, funding acquisition, resources, supervision, and project administration. All authors meet authorship requirements. All the authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRights and permissions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen Access\u0026nbsp;\u003c/strong\u003eThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article\u0026apos;s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article\u0026apos;s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eR. Kaur, A. Sood, D.K. Lang, S. Bhatia, A. Al-Harrasi, L. Aleya, T. 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Rubinsztein, VCP/p97 regulates Beclin-1-dependent autophagy initiation, Nat Chem Biol 17(4) (2021) 448-455 https://doi.org/10.1038/s41589-020-00726-x.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"AD-MCI, ATP11A, apoptosis, autophagy, phosphatidylserine","lastPublishedDoi":"10.21203/rs.3.rs-4458094/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4458094/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCognitive decline and memory impairment are frequently observed in Alzheimer's disease (AD) patients and are closely associated with dysfunctional autophagy and neuroinflammation, which subsequently result in neuronal apoptosis and synaptic damage. Aberrant regulation of microRNAs (miRNAs) has been implicated in the pathogenesis of AD and may play a pivotal role in the early stages of the disease.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjectives\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo examine the role of a miR-4763-3p antagomir in ameliorating cognitive decline in mild cognitive impairment (MCI)-AD mice and to elucidate the underlying mechanisms involved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFluorescence in situ hybridization was used to demonstrate that miR-4763-3p is highly expressed in postmortem hippocampal tissue from AD patients and colocalizes with the Aβ and Tau proteins. Stereotactic injection of the miR-4763-3p antagomir and subsequent behavioral experiments revealed its ability to ameliorate cognitive decline in AD-MCI mice. RNA-seq, tissue staining, and SH-SY5Y cell experiments were used to explore specific molecular mechanisms and associated signaling pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe miR-4763-3p antagomir targeted ATP11A to enhance inward flipping of the \"eat me\" phosphatidylserine signal on the surface of neuronal cells, effectively alleviating brain inflammation and neuronal loss and improving synaptic morphology in AD-MCI mice. Furthermore, the miR-4763-3p antagomir increased autophagy in the early-stage AD-MCI brain, promoted the clearance of Aβ proteins, and reduced the deposition of lipofuscin. These findings confirm that miR-4763-3p targets ATP11A to regulate the PI3K/AKT/mTOR/Bcl2 signaling pathway, thereby promoting neuronal autophagy and reducing apoptotic crosstalk.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe miR-4763-3p antagomir has the potential to reverse neuronal apoptosis and enhance autophagy levels, improving the inflammatory microenvironment in brain tissue and thus improving learning and memory in early-stage AD-MCI mice to mitigate cognitive decline. Our data offer a promising strategy for the treatment of AD-MCI patients.\u003c/p\u003e","manuscriptTitle":"Inhibition of miR-4763-3p expression in the brains of AD-MCI mice activates the PI3K/mTOR/Bcl2 autophagy signaling pathway to reverse neuronal loss and ameliorate cognitive decline","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-07 23:16:12","doi":"10.21203/rs.3.rs-4458094/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2be66143-a4df-4666-bcf7-c869e2b4a97d","owner":[],"postedDate":"June 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-09T07:38:31+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-07 23:16:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4458094","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4458094","identity":"rs-4458094","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}