HIF-1α exerts a double-edged sword regulatory role in hippocampal neuronal PANoptosis of Alzheimer's disease through HK2/VDAC1/NLRP3 axis and RIPK3 signal

HIF-1α exerts a double-edged sword regulatory role in hippocampal neuronal PANoptosis of Alzheimer's disease through HK2/VDAC1/NLRP3 axis and RIPK3 signal | 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 Article HIF-1α exerts a double-edged sword regulatory role in hippocampal neuronal PANoptosis of Alzheimer's disease through HK2/VDAC1/NLRP3 axis and RIPK3 signal Yang Li Chao, Siqi Wang, Laixi Luo, Xilin Jiang, Tong Ren, Weilin Liu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7239681/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Extensive neuronal loss in brain regions critical for learning and memory is a hallmark of Alzheimer's disease (AD). PANoptosis, a newly characterized form of programmed cell death, integrates the key features of pyroptosis, apoptosis and necroptosis, and explains the molecular crosstalk among these pathways. However, whether PANoptosis is a new manner for hippocampal neuron death in AD, and the involved regulatory mechanisms remains largely unknown. Here, we demonstrate that PANoptosis is a crucial mechanism driving hippocampal neuronal loss in an AD mouse model. Moreover, we uncovered that the HIF-1α signaling pathway exerts adouble-edged sword effect on hippocampal neuronal PANoptosis by activating the HK2/VDAC1/NLRP3 axis while concurrently suppressing RIPK3signal. This observation may offer a partial explanation for the double-edged sword role of HIF-1α as both a neuroprotective and neurotoxic factor in AD. Finally, we uncovered that semaglutide, a glucagon-like peptide-1 receptor agonist (GLP-1RA) currently undergoing phase 3 clinical trials for AD, mitigates hippocampal neuronal PANoptosis by upregulating HIF-1α expression while suppressing its downstream HK2/VDAC1/NLRP3 axis and RIPK3 signal, highlighting its potential as a therapeutic avenue for AD. These findings uncover a previously unrecognized role of PANoptosis in AD and provide new insights into the HIF-1α-mediated regulatory mechanisms, offering a promising target for therapeutic intervention. Biological sciences/Neuroscience Health sciences/Diseases Alzheimer’s disease Hippocampal neuronal PANoptosis HIF-1α signaling pathway Double-edged sword effect Semagrutide Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Alzheimer’s disease (AD), the most prevalent neurodegenerative disorder, is pathologically characterized by the extracellular accumulation of β-amyloid (Aβ) plaques and the intracellular formation of neurofibrillary tangles composed of hyperphosphorylated Tau protein 1 . A hallmark of AD is the extensive loss of neurons, particularly in regions such as the hippocampus, which are crucial for memory and higher cognitive functions 2 . The intricate interaction among various programmed cell death (PCD) pathways is thought to be the primary cause of neuronal death in AD. Numerous molecules involved in neuronal death under AD pathology have been identified across distinct PCD pathways, including Gasdermin D (GSDMD) and NOD-like receptor family pyrin domain containing 3 (NLRP3) in pyroptosis 3 , Caspase-3/-8/-9 and the Bcl-2 family in apoptosis 4 , and receptor-interacting protein kinase 1 (RIPK1) along with the necrosome complex in necroptosis 5 . Therefore, a comprehensive elucidation of molecular mechanisms and regulatory network underlying neuronal death in AD, along with the exploration of effective therapeutic strategies to mitigate neuronal loss, is critical for advancing precise therapy in AD. Recent evidences have highlighted a strong association between AD and neuronal PANoptosis 6 , 7 , which is a novel form of proinflammatory PCD that intricately integrates the pyroptosis, apoptosis, and necroptosis pathways 8 . This process is orchestrated by a multiprotein complex known as the PANoptosome, which facilitates the crosstalk and regulation among these distinct cell death mechanisms through key components such as GSDMD, NLRP3, Caspase-1, Caspase-8, RIPK1, and RIPK3 9 . This conceptualized complex is thought to provide a flexible framework, allowing the recruitment of core components from various cell death pathways to execute a unified cell death response 10 . Together, these insights provide a novel perspective on the complexity and diversity of neuronal cell death pathways in AD and emphasize the importance of elucidating the molecular crosstalk of neuronal PANoptosome in understanding the pathogenesis and finding new treatment strategies for AD. Nevertheless, the occurrence and underlying regulatory mechanisms of neuronal PANoptosis in the AD hippocampus remain largely unexplored. Here, we identified the activation of neuronal PANoptosis in the hippocampus in AD. We also revealed a significant correlation between the hypoxia-inducible factor 1-alpha (HIF-1α) signalling pathway and PANoptosis in hippocampal neurons during both AD and the aging process. HIF-1α has been recognized as a pivotal regulator of Aβ plaque deposition, tau hyperphosphorylation, angiogenesis, glucose metabolism, and neuronal survival in AD pathology 11 . Notably, HIF-1α exhibits a complex dual role in AD, characterized by both neuroprotective and neurotoxic effects, with the mechanisms underlying this duality largely unexplored. As a result, although HIF-1 presents significant potential as a therapeutic target for AD, its dual effects pose substantial challenges to its further application in AD treatment. In this work, we further elucidated that the HIF-1α signalling pathway plays a dual role in regulating hippocampal neuronal PANoptosis in AD. HIF-1α facilitates neuronal PANoptosis by activating the downstream hexokinase 2 (HK2)/voltage-dependent anion channel 1 (VDAC1)/NLRP3 axis, while concurrently inhibiting PANoptosis through the transcriptional suppression of RIPK3. It indicates that the modulation of HIF-1α on PANoptosis in hippocampal neurons could potentially be one of the mechanisms through which it exerts a dual function in the pathological process of AD. Additionally, we demonstrated that semaglutide, a novel glucagon-like peptide-1 receptor agonist (GLP-1RA) currently undergoing two phase 3 clinical trials in AD patients 12 , exerts therapeutic effects on AD treatment by activating the HIF-1α signalling pathway while inhibiting its downstream HK2/VDAC1/NLRP3 axis. Collectively, our study uncovers that PANoptosis drives hippocampal neuronal loss in AD, and highlights the dual regulatory role of HIF-1α in this process. These findings deepen our understanding of hippocampal neurodegeneration in AD, while the therapeutic effect of semaglutide on neuronal PANoptosis through targeting HIF-1α signalling pathway underscores the new AD treatment strategies in future. 2. Materials and methods 2.1 Animals The male C57BL/6 mice aged 9 to 11 months were purchased from Xiamen university laboratory animal centre (Xiamen, China). The male 4×FAD mice aged 9 to 11 months (FAD 4T [B6/JGpt-Tg(Thy-APP/Thy-PSEN1)5/Gpt] mice) were provided by the Gempharmatech Co., Ltd (Jiangsu, China). All mice were housed under specific pathogen-free (SPF) conditions, with a 12-hour light/dark cycle and ad libitum access to food and water. All mice in the study were not previously involved in other experimental procedures and following “3Rs” principles (Replacement, Reduction and Refinement) in all experimental procedures. The study protocol was approved by the Xiamen University Animal Ethics Committee (XMULAC20230130). All animals were randomly grouped using a random number table method, and the acquisition and analysis of relevant data were conducted in a double-blind experiment. 2.2 Isolation of primary hippocampal neurons and cell culturing Mouse hippocampus tissues were dissected from pups at postnatal day 1, and dissociated using enzymatic digestion. The isolated primary neurons were plated on poly-D-lysine coated dishes, and cultured in Neurobasal medium supplemented with B27 (GIBCO), 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Invitrogen). Mouse hippocampal neuronal cell lines HT22 were maintained in DMEM (High glucose) containing 10% FBS. All cells were cultured in an incubator at 37 ℃ under humidified air with 5% CO 2 . For the AD model, the primary hippocampal neurons and HT22 cells were treated with 5 µM Aβ 25−35 (CSN21486, Neural Signalling) and 20 nM Okadaic acid (S30686, Shanghai yuanye Bio-Technology Co., Ltd). For activation and inhibition of HIF-1α, HT22 cells were treated with 500 µM DMOG (A160647, Ambeed) and 5 nM LW6 (A2667376, Ambeed), respectively. For targeted degradation of HK2 protein, HT22 cells were treated with 0.5 µM HK2 degrader-1, a proteolysis-targeting chimera (PROTAC) (HY-155008, MCE). For inhibition of VDAC1, HT22 cells were treated with 10 µM VBIT-4 (A1364932, Ambeed). 2.3 Drug treatment The 4×FAD mice received intraperitoneal injections of either 10 mL/kg/day saline or 10 nmol/kg/day semaglutide (SJ20210015, Novo Nordisk) for 35 consecutive days, starting 28 days prior to the water maze test. 2.4 Morris water maze Morris water maze is a very popular tool for assessing spatial learning and memory. In this study, the maze comprised a circular tank with a diameter of 122 cm, containing a platform submerged in tap water maintained at a temperature of 22 ± 2 ℃. Distinctive shapes were placed along the tank walls to serve as spatial reference cues. A camera mounted above the maze recorded the swimming traces of the mice. During the acquisition trials, the platform was submerged 1 cm below the water surface. Mice were introduced into the maze at one of four designated entry points (N, S, E, W), facing the tank wall. They were given 60 seconds to locate the platform. If a mouse failed to find the platform within this time, it was guided to the platform and allowed to remain there for 10 seconds. Each day, two trials were conducted with a 1-hour interval between them over a period of 5 days. And the escape latency, an indicator of spatial learning and memory acquisition, was recorded for each trial. On the 6th day, a probe test was conducted with the platform removed. Metrics recorded during this test included the latency to the first entry into the platform’s target location, the number of crossings over the platform’s target area, and the time spent in the target quadrant. 2.5 Novel object recognition The Novel object recognition test, used to evaluate learning and memory in mice, was conducted following the Morris water maze test. Two similar objects differing in shape and color were selected to serve as experimental tools, with one designated as the “familiar object” and the other as the “novel object”. The mice were placed in an open field (40 cm × 40 cm × 40 cm) for unrestricted exploration, allowing them to acclimate to the environment for 10 minutes. In the familiarization phase, two identical “familiar objects” were placed in the open field and the mice were allowed to explore freely for 5 minutes. One hour after the familiarization phase ends, one of “familiar objects” was replaced with the “novel object”, and the mouse was returned to the open field for another 5 minutes of unrestricted exploration. The exploration time of the mice for the “new object” (T new ) and the “familiar object” (T familiar ) was recorded. The “Novel Object Recognition Index” (Cognition Index) was then caculated as follows: Cognition Index (%) = [T new / (T new + T familiar )] × 100%. 2.6 CCK-8 assay The CCK-8 assay was used to assess the effects of DMOG, LW6, or semaglutide on neuronal viability. Once the cells were in optimal condition and fully adherent, they were treated with different concentrations of DMOG, LW6, or semaglutide. After replacing the medium, 10% CCK-8 solution was added to each well, and the plates were incubated in the dark for 1 hour. Absorbance at 450 nm was measured, ensuring that the maximum value exceeded 0.9. The absorbance for each well was recorded, and the cell survival rate was calculated using the formula: Survival rate (%) = [(Experimental group – Medium control group) / (Control group – Medium control group)​] × 100%.​ 2.7 Tissue and cell lysate preparation, and antibodies used for immunoblotting Mouse hippocampal tissues were collected from at least three mice each group. Proteins extracted from HT22 cells, primary hippocampal neurons or hippocampal tissue lysates subjected to western blot analysis. The samples were separated using SDS-polyacrylamide gel electrophoresis and probed with specific antibodies listed in Table 1 . Goat-anti-mouse secondary antibodies and goat-anti-rabbit secondary antibodies were purchased from the Millipore (#AP132P, #AP124P). For quantification, band intensities were measured using Image J (a public domain software from the National Institutes of Health), normalized to β-actin, and averaged from at least four independent experiments. Table 1 Antibodies used in the western blotting, immunofluorescence and Tyramide signal amplification. Antibodies Source Identifier β-actin Invitrogen MA1-140 Anti-β-actin Proteintech 66009-1-Ig Anti-APP/β-amyloid Proteintech 25524-1-AP Anti-VEGFA Proteintech 19003-1-AP Anti-HK2 Proteintech 22029-1-AP Anti-HMOX1/HO-1 Proteintech 10701-1-AP Anti-MEFV/Pyrin Proteintech 24280-1-AP Anti-ZBP1 Proteintech 13285-1-AP Anti-AIM2 Proteintech 20590-1-AP Anti- MLKL Proteintech 66675-1-Ig Anti-p-MLKL CST #37333 Anti-Casp1 Proteintech 22915-1-AP Anti-C-Casp3 CST #9661 Anti-Casp3 Proteintech 19677-1-AP Anti-RIPK1 Proteintech 29932-1-AP Anti-p-RIPK1 Immunoway YP1467 Anti-RIPK3 Proteintech 17563-1-AP Anti- p-RIPK3 Immunoway YP1468 Anti-TUBA1B Proteintech 66031-1-Ig Anti-TOM20 Proteintech 11802-1-AP Anti-dsDNA Abcam ab27156 Anti-NLRP3 Proteintech 30109-1-AP Anti-ASC Proteintech 67494-1-Ig Anti-GSDMD Proteintech 66387-1-Ig Anti-N-GSDMD Immunoway YT7991 Anti-P63 Proteintech 12143-1-AP Anti-PIK3CB Proteintech 20584-1-AP Anti-HIF-1α Proteintech 20960-1-AP Anti-VDAC1 Proteintech 10866-1-AP Goat-anti-mouse secondary antibody Proteintech SA00001-1 Goat-anti-rabbit secondary antibody Proteintech SA00001-2 350-labeled Tyramide Immunoway YS0006 Multiplex fluorescent staining kit AiFang Bioligical AFIHC024 2.8 Frozen section After anesthesia, the mouse is rapidly euthanized by cervical dislocation. The brain is carefully removed and washed with physiological saline to eliminate blood and impurities. The brain tissue is then fixed overnight at 4℃ in 4% paraformaldehyde. After fixation, the tissue is fully immersed in optimal cutting temperature and placed in a -80℃ freezer to ensure complete freezing. The frozen brain tissue is placed in a pre-cooled cryostat (Leica CM 1950, German), then quickly sectioned with 20 µm thicken using the cryostat. The sections are immediately placed on pre-cooled glass slides. The prepared tissue sections are stored at -80℃ until further processing. 2.9 Toluidine blue staining Brain tissue sections were placed in a 37℃ oven for 20 minutes. After washing with PBS, submerge the sections sequentially in 75%, 95%, 100%, 95%, and 75% ethanol for 3 minutes each. After another PBS wash, submerge the sections in a 0.1% toluidine blue solution for 20 minutes. Then, thoroughly wash off the excess stain with distilled water. Next, submerge the sections sequentially in 75%, 95%, and 100% ethanol for 3 minutes each, followed by clearing with xylene for 5 minutes. Finally, mount the sections with neutral resin and observe the staining results under a microscope. 2.10 Immunostaining Tyramide signal amplification system was used for chromogenic immunostaining. For double-staining with primary antibodies, HT22 cells, primary hippocampal neurons or hippocampal tissue sections were incubated in 3% H 2 O 2 for 10 minutes at room temperature to block the endogenous peroxidase, then rinsed in PBS and subsequently incubated with 3% BSA at room temperature for 1 hour, following by incubating with the first primary antibody and with HRP-linked secondary antibody. After the second round of heating and washing as described above, the sections were incubated with another primary antibody and with HRP-linked secondary antibody. For multiplex with primary antibodies, the primary hippocampal neurons on slides were submerged in citrate buffer and microwave-heated for 15 minutes to release the antigens. To block the endogenous peroxidase, the sections were incubated in 3% H 2 O 2 for 10 minutes at room temperature. They were then rinsed in PBS and subsequently incubated with 3% BSA at room temperature for an hour. After that, they were incubated with primary antibody ASC, following by an HRP-linked secondary antibody and 350-labeled tyramide. After the second round of heating and washing as described above, the sections were incubated with primary antibody MEFV or ZBP1, following by an HRP-linked secondary antibody and 520-labeled tyramide. After the third round of heating and washing as described above, the sections were incubated with primary antibody NLRP3 or RIPK1, following by an HRP-linked secondary antibody and 570-labeled tyramide. After the fourth round of heating and washing as described above, the sections were incubated with primary antibody AIM2 or RIPK3, following by an HRP-linked secondary antibody and 690-labeled tyramide. Images were acquired using a Nikon confocal microscope. Antibodies used for immunostaining were listed in Table 1 . 2.11 Differently expressed genes analysis of transcriptome sequencing Micro-array dataset GSE48350 used in this study was obtained from the Gene Expression Omnibus (GEO) database. This dataset comprises the gene expression matrix of the hippocampus of the AD patients (n = 19), the Aging (n = 25) and the Adult (n = 18). Using R software’s “limma” packages 13 , differently expressed genes (DEGs) in hippocampus between the groups were analyzed, with the following criterion: adjust p < 0.05. 2.12 Differently expressed genes analysis of single-nucleus transcriptomic sequencing Single-nucleus transcriptomic sequencing (snRNA-seq) datasets GSE199243, GSE198323 and GSE185553 were obtained from the GEO database. These datasets comprise gene expression matrix of the hippocampus of the AD patients (n = 8), aging individuals (n = 7), and adults (n = 6). Raw expression matrix of each individual specimen were loaded into the R (v4.3.2) package “Seurat”, following by the data analysis was carried out based on the published methods 14 . The 11 principal components (PCs) were then used to generate a shared nearest neighbor graph which was then clustered under the Louvain algorithm with a resolution of 0.4. The t-distributed Stochastic Neighbor Embedding (t-SNE) was then performed using the first 11 PCs. Then, using human cell markers in the CellMarker 2.0 ( http://117.50.127.228/CellMarker/CellMarkerBrowse.jsp ) as the standard of cell annotation, 7 kinds of cell types including Glutamatergic neuron (Glu_N, cell makers: “SLC17A7”), GABAergic neuron (GABA_N, cell makers: “GAD2”), Dopaminergic neurons (Dopa_N, cell makers: “FOLR1”), Oligodendrocytes (Oligo, cell makers: “MBP”, “MOBP”) and Oligodendrocytes precursor cells (OPCs, cell makers: “LHFPL3”), Astrocytes (Astro, cell makers: “AQP4”, “GFAP”) and Microglia (Micro, cell makers: “APBB1IP”) were identified. To investigate DEGs in each cell type between the groups, analysis was performed on the integrated dataset using the ‘RNA slot’ of the Seurat object and a two-sided Wilcoxon rank sum test (implemented in “FindMarkers”). Finally, the DEGs in each hippocampal cell type between the groups were analyzed, with the following criteria: p 0.6. 2.13 Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis The clusterProfiler package in R was used for functional enrichment analyses of DEGs based on KEGG enrichment analysis, a statistical significance threshold of p < 0.05 was set to determine significant results in the analysis 15 . 2.14 Single-gene gene set enrichment analysis (sgGSEA) The clusterProfiler package in R was employed for sgGSEA 16 . The read count values of gene were used as input for the sgGSEA package to explore the related pathways associated with the TP63 and TUBA1B. The correlation between the TP63 and TUBA1B and all other genes in the transcriptome data of the GSE48350 dataset was calculated. The genes were then sorted in descending order based on their correlations. This sorted gene list was considered as the gene set to be tested for pathway enrichment analysis. The analysis further involved the utilization of the predefined KEGG signaling pathway set to evaluate the enrichment of the sorted genes in the KEGG pathways. This step aimed to identify the specific KEGG pathways that demonstrated significant enrichment among the genes associated with TP63 and TUBA1B. 2.15 Statistical analysis All statistical analyses related to Micro-array and snRNA-seq data in the present study were implemented using R software (version 4.3.2). Unless otherwise stated, P < 0.05 was deemed as statistically significant, and all P values were two tailed. Statistical analyses of experimental data were performed using GraphPad Prism version 9.0 (GraphPad Inc., San Diego, CA, USA). For experiments with two groups, unpaired two-tailed Student's t-test was used. For experiments with three or more groups, one-way or two-way ANOVA with Tukey’s adjustment for multiple comparisons was applied. Data are presented as mean ± SEM (standard error of the mean). Values were considered statistically significant at P < 0.05. 3. Results 3.1 PANoptosis was observed in the hippocampus of 4×FAD mice and in neurons exposed to Aβ 25-35 and OA PANoptosis, a novel proinflammatory PCD pathway characterized by the interplay among apoptosis, necroptosis, and pyroptosis, has been reported to be linked to AD 6 , 7 . However, the direct experimental evidence of neuronal PANoptosis in AD has yet to be reported. To investigate the occurrence of neuronal PANoptosis in AD, the 4×FAD mouse model was employed. Compared to age-matched control mice, the 4×FAD mice exhibited elevated gene and protein expression levels of amyloid precursor protein (APP) and more pronounced neuronal loss in the hippocampal CA1 region (Fig. 1 A-F). The immunofluorescence staining revealed significantly increased levels of cleaved Caspase-3 (C-Caspase-3), phosphorylated mixed lineage kinase domain-like protein (p-MLKL), and N-terminal GSDMD (N-GSDMD) in the hippocampus of 4×FAD mice relative to aging controls (Fig. 1 G-I). These findings suggest a strong association between hippocampal neuronal loss and PANoptosis in AD. Okadaic acid (OA) has been shown to be more potent than Aβ in inducing cell death, primarily by promoting Tau hyperphosphorylation 17 , 18 . To determine whether PANoptosis is a new manner for hippocampal neuron death, primary hippocampal neurons for WT mice were simultaneously exposed to 5 mM Aβ 25−35 and 20 nM OA for 24 hours to establish an AD hippocampal neuron model (Fig. 1 J). Immunoblotting results revealed that neurons treated with Aβ 25−35 and OA exhibited significantly elevated protein expression in PANoptosis, including C-Caspase-3, C-Caspase-1, p-MLKL, N-GSDMD, ASC, AIM2, ZBP1, Pyrin, p-RIPK1, p-RIPK3, and NLRP3, compared to untreated control neurons (Fig. 1 K-N). Moreover, PANoptosis is mediated by the PANoptosome complex, which is assembled through the integration of key components from the apoptosis, necroptosis, and pyroptosis 9 . Multiplex immunofluorescence (mIF) staining demonstrated that Pyrin, NLRP3, AIM2, ZBP1, RIPK1, and RIPK3 were highly co-localized with ASC specks in primary hippocampal neurons treated with Aβ 25−35 and OA, compared to untreated control neurons (Fig. 1 O and P ), indicating that Aβ 25−35 and OA induced the assembly of the PANoptosome complex. Together, these findings suggest that PANoptosis is a novel form of PCD that leads to hippocampal neuronal loss in AD. 3.2 HIF-1α exerted the key role in regulating hippocampal neuronal PANoptosis in AD We have observed the hippocampal neurons PANoptosis in AD (Fig. 1 ). Neuronal degeneration and death are key pathological features of AD 2 . As an integrated form of PCD that encompasses apoptosis, pyroptosis, and necroptosis, PANoptosis is a new pathogenic way in AD neuronal death. Therefore, elucidating the molecular and signaling pathways that regulate PANoptosis in hippocampal neurons is crucial for developing new targets or strategies for Alzheimer's disease. A total of 902 PANoptosis-related genes (PRGs) were retrieved from the GeneCards database ( https://www.genecards.org/ ) and the PubMed 19 (Supplementary Table 1). Aging (senescence) is recognized as a major risk factor for AD, and targeting senescent cells has grown into a promising therapeutic approach to mitigating the onset and progression of AD 20 , 21 . To identify the differentially expressed PRGs (DE-PRGs) involved in AD pathology and aging process, the bioinformatics analyses were performed on the hippocampal gene expression matrix from AD patients, aging individuals and healthy adults. A total of 239 DE-PRGs related to AD pathology and 267 DE-PRGs related to aging process were identified ( Supplementary Fig. 1A, B ). KEGG enrichment analysis revealed that both sets of DE-PRGs were significantly enriched in PI3K-Akt, apoptosis, NOD-like receptor, necroptosis and HIF-1 signalling pathways ( Supplementary Fig. 1C, D ). To obtain the DE-PRGs in hippocampal neurons associated with both AD pathology and aging process, the snRNA-seq clinical datasets comprising hippocampal gene expression matrix in the AD patients, aging individuals and adult was analyzed. Information on datasets before and after quality control was presented in Supplementary Fig. 2 and Supplementary Fig. 3 , respectively. Based on established markers from the CellMarker 2.0, seven distinct cell clusters were identified: glutamatergic neurons (Glu_N), GABAergic neurons (GABA_N), dopaminergic neurons (Dopa_N), oligodendrocytes (Oligo), astrocytes (Astro), oligodendrocyte progenitor cells (OPCs), and microglia (Micro) (Fig. 2 A). Results showed that the proportions of Glu_N and GABA_N in AD group were significantly higher than that in aging group (Fig. 2 B). By collectively classifying the DEGs identified in Glu_N and GABA_N as neuronal DEGs, 35 neuronal DE-PRGs associated with AD pathology and 47 neuronal DE-PRGs linked to the aging process were identified (Fig. 2 C-H). Furthermore, by intersecting the neuronal DE-PRGs with those derived from hippocampal tissues, TP63 and TUBA1B were identified as the key DE-PRGs (Fig. 2 I). Notably, the immunoblotting results revealed that the expression levels of both TP63 and TUBA1B in the hippocampus of 4×FAD and adult mice were significantly higher than those of aging mice (Fig. 2 J-L). These findings indicate that TP63 and TUBA1B are the central DE-PRGs in the hippocampus and hippocampal neurons involving the AD pathology and the aging process. Previous studies have demonstrated that TP63 deficiency accelerates the aging process, with its expression in hippocampal neurons diminishing progressively with age 22 , 23 . However, the molecular mechanisms through which TP63 and TUBA1B influence AD and neuronal PANoptosis remain inadequately elucidated. To investigate this, single-gene gene set enrichment analysis (sgGSEA) of TP63 and TUBA1B was performed in hippocampal expression matrix from AD patients and aging individuals. A total of 93 signaling pathways were obtained from the intersection of those significantly enriched for TP63 and TUBA1B ( Supplementary Fig. 4 and Supplementary Table 2 ). Following the exclusion of functional and disease pathways, several key pathways emerged prominently, including HIF-1, IL-17, Hippo, ErbB, cGMP-PKG, and Wnt signaling pathways (Fig. 2 M, N, Supplementary Fig. 5A-J ). Among these the HIF-1 signaling pathway was prioritized for further investigation based on integrated analysis. By intersecting the 239 DE-PRGs linked to AD with genes involved in the HIF-1 signaling pathway enriched from TP63 and TUBA1B, four potential key genes in HIF-1 signaling pathway significantly associated with hippocampal neuronal PANoptosis in AD pathology were identified: PIK3CB, VEGFA, HO-1, and HK2 (Fig. 2 O). Subsequent validation through immunoblotting demonstrated a significant decrease in the protein expressions of PIK3CB, HIF-1α, and VEGFA, whereas the protein expressions of HO-1 and HK2 were significantly increased in the hippocampus of 4×FAD mice (Fig. 2 P-U). These findings underscore a significant association between hippocampal neuronal PANoptosis and dysregulation of the HIF-1α signaling pathway in AD. 3.3 HIF-1α activation played the dual regulatory effects on the hippocampal neuronal PANoptosis As previously noted, our bioinformatic analyses indicate that dysregulation of the HIF-1α signaling pathway is strongly associated with hippocampal neuronal PANoptosis in AD (Fig. 2 ). To investigate the effect of HIF-1α on regulating neuronal PANoptosis in AD, the hippocampal neuronal cell line HT22 was exposed to Aβ 25−35 and OA, followed by treatment with either the HIF-1α agonist DMOG or the inhibitor LW6. CCK-8 assays revealed that pharmacological activation of HIF-1α significantly enhanced the viability of HT22 cells under Aβ 25−35 and OA- induced stress, whereas its inhibition markedly decreased cell survival under the same conditions (Fig. 3 A, B). These findings suggest that pharmacological activation of HIF-1α may represent a promising therapeutic strategy for promoting neuronal survival in AD pathology. To further explore whether the neuroprotective effect of HIF-1α activation is mediated by inhibiting neuronal PANoptosis, we examined the expression of key proteins involved in the PANoptosis in HT22 cells treated with DMOG or LW6, with or without exposure to Aβ 25−35 and OA. Interestingly, the results do not fully match our speculation. Immunoblotting results revealed that HIF-1α activation significantly reduced the expression of ZBP1, AIM2, Pyrin, p-RIPK3, p-RIPK1, p-MLKL, C-GSDMD, C-Caspase1, and C-Caspase3 in HT22 cells exposed to Aβ 25−35 and OA, but did not decrease the expression of NLRP3. Similarly, HIF-1α inhibition markedly increased the expression of ZBP1, AIM2, p-RIPK3, p-RIPK1, p-MLKL, GSDMD, C-Caspase1, and C-Caspase3, while decreasing the expression level of NLRP3 and Pyrin under the same conditions (Fig. 3 C, D). These findings suggest that although activation of the HIF-1α pathway exerts neuroprotective effects at the cellular level, it may play a dual role in regulating hippocampal neuronal PANoptosis, potentially through the activation of NLRP3. This underscores the complex and multifaceted regulatory function of HIF-1α activation within the PANoptosis pathway in neurons affected by AD. Notably, HIF-1α has previously been reported to exerts both protective and detrimental effects in AD treatment 11 , and our results suggest that this duality may, in part, be attributed to its regulation of neuronal PANoptosis. 3.4 HIF-1α activation promoted neuronal PANoptosis by upregulating HK2/VDAC1/NLRP3 axis NLRP3 is a critical component of the PANoptosome, and its activation during the inflammatory response is mediated through multiple mechanisms 24 . Previous studies have demonstrated that the dissociation of HK2 from mitochondria facilitates the oligomerization of VDAC1, the release of oxidized mitochondrial DNA (mtDNA) fragments, and the subsequent binding of NLRP3 to VDAC1, thereby promoting the assembly and activation of NLRP3 25, 26 . HK2, a pivotal enzyme in glycolysis, is upregulated by HIF-1α through its interaction with hypoxia response elements in the HK2 promoter 27 . Studies have demonstrated that HK2 protein expression is significantly elevated in the cerebral cortex and hippocampus of AD patients and 5×FAD mice, while the expression levels of other hexokinase isoforms, including HK1—commonly recognized as the “brain hexokinase”—remain largely unaltered 28 , 29 . In this study, HK2 was identified as a pivotal molecule within the HIF-1α signaling pathway involved in regulating neuronal PANoptosis (Fig. 2 ). Immunoblotting analysis revealed that co-treatment with Aβ 25−35 and OA markedly increased HK2 expression in HT22 cells, an effect was further potentiated by HIF-1α activation (Fig. 4 A-C). Consequently, we hypothesize that HIF-1α activation upregulates the expression and cytoplasmic translocation of HK2, thereby promoting NLRP3 activation. To test this, immunofluorescence staining was performed to assess HK2 dissociation from mitochondria, the release of mtDNA fragments, and the binding of NLRP3 to VDAC1. Results showed that treatment with Aβ 25−35 and OA facilitated HK2 dissociation from mitochondria (Fig. 4 D), increased the release of mtDNA fragments (Fig. 4 E), and enhanced co-localization of VDAC1 and NLRP3 in HT22 cells (Fig. 4 F). These effects were further amplified in HT22 cells by co-treatment with DMOG, resulting in even greater HK2 dissociation from mitochondria, an increased release of mtDNA fragments, and more pronounced co-localization of VDAC1 and NLRP3. Conversely, treatment with LW6 markedly reduced HK2 dissociation from mitochondria, decreased the release of mtDNA fragments, and disrupted the co-localization of VDAC1 and NLRP3 in HT22 cells treated with Aβ 25−35 and OA (Fig. 4 D-F). These findings suggest that the HIF-1α activation promotes AD-related neuronal PANoptosis by inducing HK2-dependent NLRP3 activation. To further validate the mediating role of HK2 in the relationship between HIF-1α activation and NLRP3 activation, HT22 cells exposed to Aβ 25−35 and OA were treated with DMOG either alone or in combination with PROTAC-HK2-Degrade-1, a compound specifically designed to degrade the HK2 protein (Fig. 4 G). Immunoblotting and immunostaining results indicated that the combined treatment of DMOG and PROTAC-HK2-Degrade-1 counteracted the effects induced by DMOG alone in HT22 cells exposed to Aβ 25−35 and OA. This was evidenced by reduced HK2 dissociation from mitochondria, decreased release of mtDNA fragments, and significantly diminished co-localization of VDAC1 and NLRP3 (Fig. 4 H-J). These findings suggest HIF-1α activation mediates HK2 dissociates from mitochondria and triggers VDAC1-dependent NLRP3 translocation and activation. Moreover, while DMOG treatment promoted NLRP3 translocation without altering its protein expression in HT22 cells exposed to Aβ 25−35 and OA (Fig. 3 C, D and Fig. 4 L, M), combination treatment with DMOG and PROTAC-HK2-Degrade-1 or VDAC1 inhibitor VBIT-4 significantly reduced the NLRP3 protein expression and the co-localization of VDAC1 and NLRP3 in HT22 cells exposed to Aβ 25−35 and OA (Fig. 4 K-O). These findings highlight that HK2 and VDAC1 as critical regulators capable of reversing the heterogeneous expression of key protein in PANoptosis pathway induced by HIF-1α activation. Collectively, our results suggest that HIF-1α activation promotes neuronal PANoptosis in AD by upregulating HK2/VDAC1/NLRP3 axis. 3.5 HIF-1α activation suppressed neuronal PANoptosis by transcriptionally downregulating RIPK3 We have demonstrated that HIF-1α activation exerts a double-edged sword effect on hippocampal neuronal PANoptosis in AD. However, the molecular mechanisms by which HIF-1α activation inhibits PANoptosis remain largely undefined. Resent study has shown that HIF-1α expression in intestinal epithelium restricts arthritis inflammation by transcriptionally inhibiting RIPK3-induced cell death machinery 30 . Our results showed that HIF-1α activation significantly downregulated the gene and protein expression levels of RIPK3 in HT22 cells exposed to Aβ 25−35 and OA. Conversely, HIF-1α inhibition markedly upregulated the protein expression of RIPK3 and showed a trend toward increasing its mRNA expression in HT22 cells under the same conditions (Fig. 5 A-C), suggesting that HIF-1α activation also transcriptionally inhibits RIPK3 expression in AD hippocampal neuron. RIPK3 knockdown has been reported to effectively suppress PANoptosis in bone-marrow-derived macrophages (BMDMs) 31 . To further explore the effect of RIPK3 inhibition on neuronal PANoptosis in AD, HT22 cells exposed to Aβ 25−35 and OA were treated with the GSK-872, a selective RIPK3 inhibitor. Immunoblotting revealed that GSK-872 treatment significantly reduced the expression levels of RIPK3, p-MLKL, NLRP3, GSDMD, C-Caspase-1, and C-Caspase-3 in HT22 cells exposed to Aβ 25−35 and OA (Fig. 5 D-J). These results suggest that HIF-1α activation suppresses neuronal PANoptosis by transcriptional downregulation of RIPK3. Consequently, HIF-1α activation exerts a dualistic role in regulating neuronal PANoptosis: it exacerbates PANoptosis through upregulation of the HK2/VDAC1/NLRP3 axis, while concurrently suppressing PANoptosis via the downregulation of RIPK3. This dual effect poses a challenge to pharmacological strategies for targeting HIF-1α activation. Thus, our subsequent research aims to elucidate methods to preserve the inhibitory effect of HIF-1α signaling on neuronal PANoptosis, while attenuating its pro-PANoptosis activity. 3.6 Semaglutide suppressed hippocampal neuronal PANoptosis by enhacing HIF-1α signaling pathway in AD Given the double-edged sword role of HIF-1α in neuroprotection and neurotoxicity, it is crucial to harness its beneficial effects while minimizing its detrimental outcomes. HIF-1α may serve as a co-linker between AD and T2DM, highlighting its potential as a therapeutic target for AD 32 . The GLP-1RAs, which are widely used for T2DM in clinical practice, have also shown promise in alleviating AD pathology 12 , 33 , 34 . Moreover, GLP-1RAs have been reported to enhance HIF-1α expression in the brain and maintain glucose homeostasis, thereby exhibiting therapeutic potential across a range of diseases, including neurodegenerative disorders 35 , 36 . In our study, we further identified a significant upregulation of HK2—a critical downstream effector of the HIF-1α signaling pathway—in the hippocampus of AD models. Based on this, we propose that GLP-1RAs may serve as promising candidates to eliminate the double-edged sword effect of HIF-1α signaling in AD. Semaglutide, a novel GLP-1RA and an approved clinical medication for TM2D, is currently undergoing two phase 3 clinical trials in AD patients 12 . It has been shown to improve learning and memory in APP/PS1 and 3×Tg mouse models, as well as human brain organoid models 37 , 38 . Similarly, we validated this conclusion in 4×FAD mice received semaglutide injections for 35 days, with a 6-day Morris water maze test initiated on day 28 of treatment, followed by a novel object recognition test, before being humanely euthanized for subsequent analysis (Fig. 6 A). The results showed that the semaglutide-treated mice exhibited shorter latency to reach the platform, spent more time in the target zone, and crossed the platform location more frequently compared to the untreated AD mice (Fig. 6 B-E). And, the semaglutide group demonstrated a significant higher cognitive index than the AD group (Fig. 6 F). Next, we performed CCK-8 assays to determine the protective effects of semaglutide on Aβ 25−35 and OA induced HT22 cells. HT22 cells were treated with different concentrations of semaglutide (5-100 nM) for 24 hours in the presence of Aβ 25−35 and OA. The results showed that the protective effects of semaglutide were dose-dependent, with the greatest effect seen at 50 nM ( Supplementary Fig. 6 ). Thus, 50 nM doses were chosen to determine the modulatory role of semaglutide in the double-edged effects of HIF-1α in the HT22 cells exposed to Aβ 25−35 and OA. We found that semaglutide significantly upregulated the gene and protein expression of HIF-1α, while without markedly affecting the gene and protein levels of HK2 in HT22 cells exposed to Aβ 25−35 and OA (Fig. 6 G-K). This indicates that, unlike HIF-1α agonists, semaglutide activates HIF-1α expression while suppressing the expression of its downstream target HK2. Moreover, double immunostaining revealed that semaglutide significantly decreased HK2 dissociation from mitochondria, reduced the release of fragmented mtDNA, and diminished co-localization of VDAC1 and NLRP3 in HT22 cells exposed to Aβ 25−35 and OA (Fig. 6 L). The assembly and activation of NLRP3 inflammasome is the classical indication of PANoptosis 24 . Therefore, our results indicate that semaglutide inhibits the NLRP3 inflammasome activation-induced neuronal PANoptosis through activating HIF-1α/HK2/VDAC1 axis. Additionally, to clarify the more all-sided mechanisms by which semaglutide suppresses hippocampal neuronal PANoptosis, the RIPK3 branch of HIF-1α signaling pathway was examined in the HT22 cells exposed to Aβ 25−35 and OA. Immunoblotting results showed that semaglutide significantly reduced the key proteins’ expression in PANoptosis, including RIPK1, p-RIPK1, RIPK3, p-RIPK3, ZBP1, GSDMD, AIM2, NLRP3, C-Caspase3, C-Caspase1 and ASC in HT22 cells exposed to Aβ 25−35 and OA (Fig. 6 M, N). These findings suggest that semaglutide also exerts the inhibition on neuronal PANoptosis through suppressing RIPK3 by activating HIF-1α signal. Moreover, toluidine blue staining demonstrated that semaglutide significantly mitigated the neuronal loss in the hippocampus of 4×FAD mice (Fig. 6 O, P). Immunostaining exhibited that semaglutide markedly reduced the expression levels of C-Caspase-3, p-MLKL, and N-GSDMD in the hippocampus of 4×AD mice (Fig. 6 Q). These results suggest that semaglutide effectively suppresses hippocampal neuronal PANoptosis in AD. Therefore, semaglutide suppresses the neuronal PANoptosis by activating HIF-1α-induced both HK2/VDAC1 axis and RIPK3 signal, thereby salvaging hippocampus neuronal loss and exerting therapeutic effects of AD. 4. Discussion PANoptosis integrates the key feature of pyroptosis, apoptosis, and necroptosis, and which is a critical focus in future research on PCD 8 . However, definitive evidence supporting the occurrence of PANoptosis in AD neurons remains lacking. Therefore, exploring the occurrence and molecular mechanisms of hippocampal neuronal PANoptosis in AD may offer valuable insights for the treatment of AD. Previous reports have suggested a strong association between PANoptosis and AD 6 , 7 ; however, specific experimental evidence characterizing this relationship in vivo and in vitro is still lacking. Here, we confirmed the hippocampal neuronal PANoptosis in AD by identifying the expression of PANoptosis-related proteins and the formation of PANoptosome in the 4×FAD mouse and primary hippocampal neurons exposed to Aβ 25−35 and OA. Subsequently, through comprehensive bioinformatic analysis of clinical data combined with experimental validation, we found that the HIF-1α signal pathway is a crucial regulator of hippocampal neuronal PANoptosis in AD. HIF-1α, a central transcription factor mediating cellular adaptation to hypoxic conditions, exhibits a complex bidirectional regulatory role in AD 11 . HIF-1α has been shown to exert beneficial effects in AD, including the regulation of energy metabolism, promotion of neuroprotection and neural repair, enhancement of neurogenesis, and attenuation of oxidative stress. However, HIF-1α also elicits detrimental effects in AD, such as upregulating β-site APP cleaving enzyme 1 (BACE1), enhancing β-secretase activity to promote Aβ production, impairing cerebral microvascular function, and triggering aberrant cell cycle re-entry-induced neuronal apoptosis. This dual role becomes particularly evident in pharmacological interventions: several AD-related therapeutic agents, including deferoxamine, obalt chloride, lactoferrin and simvastatin, facilitate Aβ clearance by increasing HIF-1α protein levels. Meanwhile, neuroprotective agent neurotropin alleviate AD neuroinflammation by inhibiting the NF-κB/HIF-1α signaling axis 32 . However, the precise underlying mechanism through which HIF-1α exerts a dual function in AD has remained elusive for a long time, and effective strategies to intervene in the neurotoxic effects mediated by the HIF-1α pathway are still unknown, significantly hindering the further development and application of related pharmaceuticals. In the present study, pharmacological activation of HIF-1α using DMOG significantly improved the viability of HT22 cells exposed to Aβ 25–35 and OA, and exerted a dual regulatory effect on the PANoptosis pathway in these neurons. Therefore, to investigate whether HIF-1α exerts the dual effects on AD pathology by regulating hippocampal neuronal PANoptosis, which will hold a significant promise for identifying HIF-1α as a therapeutic target for AD. As the first rate-limiting enzyme in the glycolytic pathway, HK2 is the only one among the four hexokinase isoenzymes that is aberrantly expressed in the cerebral cortex and hippocampus of both AD patients and the 5×FAD mice 28 , 29 . However, the role of HK2 in hippocampal neuronal death in AD remains unknown. In the present study, HK2 was identified as one of the key genes involved in hippocampal neuronal PANoptosis in AD, and exhibited significantly higher expression in AD mouse hippocampal tissue and cell cultures. Furthermore, studies have shown that HIF-1α can transcriptionally upregulate HK2 expression by binding to hypoxia response elements of the HK2 promoter, and inflammation can induce the HK2 dissociation from the mitochondria, ultimately leading to the activation and assembly of NLRP3 through VDAC1 oligomerization 25 , 26 , 27 . Our study has demonstrated that NLRP3, a key component of PANoptosome 24 , did not exhibit reduced expression but rather showed prominent mitochondria translocation when treatment with HIF-1α agonist, in HT22 cells co-treated with Aβ 25−35 and OA. To this, we hypothesize that HIF-1α activation may enhance NLRP3 activation by upregulating HK2, thereby driving hippocampal neuronal PANoptosis in AD. We found that the HIF-1α activation significantly promotes HK2 expression and its dissociation from mitochondria, while decreasing the expression of key components of PANoptosome, with the exception of NLRP3. In addition, our results showed that both HK2-targeted degradation and suppression of VDAC1 oligomerization significantly reduced the expression and mitochondria translocation of NLRP3 in the hippocampal neurons in AD when treated with HIF-1α agonist, suggesting that the inhibition of HK2 or VDAC1 significantly reversed the heterogeneity caused by HIF-1α activation. Therefore, HIF-1α activation promotes the HK2 expression and its dissociation from mitochondria, ultimately leading to the activation of NLRP3 through VDAC1 oligomerization, in HT22 cells exposed to Aβ 25−35 and OA. Together, our findings suggest that HIF-1α activation promotes the hippocampal neuronal PANoptosis by upregulating HK2/VDAC1/NLRP3 axis in AD. RIPK3 is the crucial component of PANoptosome 31 . Study has showed that HIF-1α alleviates arthritis inflammation by inhibiting RIPK3 expression in intestinal epithelial cells 30 . We further found that HIF-1α activation remarkedly downregulated RIPK3 expression in the hippocampal neurons under AD pathology. However, the effect of HIF-1α on RIPK3-mediated hippocampal neuronal PANoptosis in AD has not been reported. Our results showed that pharmacological inhibition of RIPK3 significantly suppressed hippocampal neuronal PANoptosis in HT22 cells exposed to Aβ 25−35 and OA, suggesting that HIF-1α activation suppresses the hippocampal neuronal PANoptosis by transcriptionally downregulating RIPK3 in AD. Altogether, our finding revealed that HIF-1α may exert neuroprotective or neurotoxic effects in AD pathology through dual regulation of neuronal PANoptosis, and effectively activating the HIF-1α pathway and blocking its downstream HK2/VDAC1/NLRP3 axis is key to leveraging the HIF-1α signaling pathway to suppress hippocampal neuronal PANoptosis in AD. Recent studies have shown that GLP-1RAs reduce the risk of neurodegenerative diseases such as AD 39 . Multiple studies have demonstrated that GLP-1RAs—including liraglutide, exenatide, lixisenatide, and semaglutide—reduce Aβ accumulation, tau hyperphosphorylation, and neuroinflammation 33 , 34 , 37 , 38 . Moreover, AMPK activation has been implicated in mediating these beneficial effects by suppressing BACE1 and Aβ production, alleviating neuroinflammation, and enhancing Aβ clearance. While AMPK has been identified as one mechanism by which the GLP-1RAs mitigate AD-related phenotypes 40 , our study uncovers an alternative pathway whereby the semaglutide—currently in two phase 3 clinical trials for AD 12 —activates HIF-1α signaling and blocks the downstream HK2/VDAC1/NLRP3 axis, thereby suppressing hippocampal neuronal PANoptosis and improving cognitive function in AD mice. Our findings provide a novel mechanistic insight into how GLP-1RAs regulate HIF-1α signaling to mitigate PANoptosis and cognitive dysfunction, offering a compelling rationale for further clinical development of GLP-1RAs in AD. 5. Conclusion In summary, our study demonstrated the occurrence of hippocampal neuronal PANoptosis in AD. Additionally, we revealed that HIF-1α activation has a dual effect on hippocampal neuronal PANoptosis: it suppresses neuronal PANoptosis by transcriptional downregulation of RIPK3, while simultaneously promoting neuronal PANoptosis via upregulation of HK2/VDAC1/NLRP3 axis, deepening the understanding of the double-sword effect of HIF-1α in AD and providing new research directions for investigation in the development of AD medications. Finally, we discovered that semaglutide alleviates cognition deficits by suppressing hippocampal neuronal PANoptosis mediated by activating HIF-1α signaling pathway and inhibiting its downstream HK2/VDAC1/NLRP3 axis, which provides a new theoretical basis for the treatment of Alzheimer's disease with semaglutide. Declarations Acknowledgements This study was supported by grants from the National Science Foundation of China (Nos.: 82273910, 81973306, 81872866 and 82002162), Major Scientific Research Program for Young and Middle-aged Health Professionals of Fujian Province, China (No. 2023ZQNZD019), Fujian Province Science and Technology Innovation Joint Fund Project (Nos. 2024Y9722, 2024Y9700), Fujian Province Science and Technology Plant Project (2022J05325), the Natural Science Foundation of Xinjiang Autonomous Region (No. 2022D01D15), the Xinjiang Tianshan Talent Cultivation Program (2023TSYCLJ0036), the Guiding Medical and Health Projects of Xiamen (Nos.: 35O2Z20214ZD1257, 35O2Z20214ZD1257, 35O2Z20214ZD1326 and 35O2Z20209117), and the Science and Technology Plan of Xiamen Medical College (No.: K2021-03[3]). Conflict of interest None of the authors have any conflict of interest with respect to the study. Author Contributions SQW: data collection, statistical analyses and the initial draft of the manuscript; LXL and XHZ data collection, statistical analyses and the initial draft of the manuscript; XLJ, Y.L and R.T data collection and statistical analyses; LCY: conceptualized and designed the study, analyses and interpretation of the results, statistical analyses, final writing and approval of the manuscript for publication. Data availability The data that support the findings of this study are available from corresponding author upon reasonable request. Description of sample size (n-number) For the behavioural tests described in this study, the number of mice (n = 8-13) analysed in each group is provided in the corresponding figure legends (Figures 6A-F). For other experiments—including western blotting, immunofluorescence, Golgi staining, and toluidine blue staining—the definition of “n” in the context of independent experimental replicates is also detailed in the respective figure legends. All statistical information, including the exact value of n, what n represents, and the statistical tests used, is clearly described in the figure legends. References Bostanciklioglu M. An update on the interactions between Alzheimer's disease, autophagy and inflammation. Gene 2019, 705: 157-166. Goel P, Chakrabarti S, Goel K, Bhutani K, Chopra T, Bali S. Neuronal cell death mechanisms in Alzheimer's disease: An insight. Front Mol Neurosci 2022, 15: 937133. Moonen S, Koper MJ, Van Schoor E, Schaeverbeke JM, Vandenberghe R, von Arnim CAF , et al. Correction: Pyroptosis in Alzheimer's disease: cell type‑specific activation in microglia, astrocytes and neurons. Acta Neuropathol 2024, 149 (1) : 3. Kumari S, Dhapola R, Reddy DH. 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Additional Declarations There is a duality of interest Supplementary Files SupplementaryTable1.xlsx Supplementary table1 SupplementaryTable2.xlsx Supplementary table2 SupplementaryFigure.docx Supplementary figures Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: revise 29 Sep, 2025 Review # 2 received at journal 26 Aug, 2025 Review # 1 received at journal 19 Aug, 2025 Reviewer # 3 agreed at journal 15 Aug, 2025 Reviewer # 2 agreed at journal 15 Aug, 2025 Reviewer # 1 agreed at journal 29 Jul, 2025 Reviewers invited by journal 29 Jul, 2025 Submission checks completed at journal 29 Jul, 2025 Editor assigned by journal 29 Jul, 2025 First submitted to journal 29 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7239681","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":492610528,"identity":"e39c4040-bd11-4e53-a958-c02b766e802b","order_by":0,"name":"Yang Li Chao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYLCCBwwSPPzsDWA2YwNRWhIYLGQkew6QpqXCxmBGApFaDI6fPfwioUaCx0DyjelmHgYb2Q0HmJ89wKvlTF6aRcIxCR5z6bS02zwMacYbDrCZG+DVciDHzCCxQYLHcnbyMaCWw4kbDvCwSeDVcv4NRIvBzYNtQC3/idByI8f4AVjLDWaQLQcIa5G88caMAeQXyZ60tJtzDJKNZx5mM8Orhe98jvGHDzV19vzsZ8xuvKmwk+073vwMrxaFAwzIzgAFFTM+9UAg38DA/IGAmlEwCkbBKBjpAAAHJErv68ediwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-9945-2338","institution":"Medicine School","correspondingAuthor":true,"prefix":"","firstName":"Yang","middleName":"Li","lastName":"Chao","suffix":""},{"id":492610529,"identity":"9e284030-ed54-435d-9287-7f02e26b64de","order_by":1,"name":"Siqi Wang","email":"","orcid":"","institution":"[email protected]","correspondingAuthor":false,"prefix":"","firstName":"Siqi","middleName":"","lastName":"Wang","suffix":""},{"id":492610530,"identity":"e22f68f9-d3cc-489c-9d46-e7ff4c6c55fe","order_by":2,"name":"Laixi Luo","email":"","orcid":"","institution":"[email protected]","correspondingAuthor":false,"prefix":"","firstName":"Laixi","middleName":"","lastName":"Luo","suffix":""},{"id":492610531,"identity":"b1722728-b2cb-492b-bd7c-cbecc57a5206","order_by":3,"name":"Xilin Jiang","email":"","orcid":"","institution":"Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Xilin","middleName":"","lastName":"Jiang","suffix":""},{"id":492610532,"identity":"8c8c4c59-0283-4d5d-9cfb-0e68910b99a4","order_by":4,"name":"Tong Ren","email":"","orcid":"","institution":"[email protected]","correspondingAuthor":false,"prefix":"","firstName":"Tong","middleName":"","lastName":"Ren","suffix":""},{"id":492610533,"identity":"c839af77-9c1e-4f9a-ab7f-73a99f512d81","order_by":5,"name":"Weilin Liu","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Weilin","middleName":"","lastName":"Liu","suffix":""},{"id":492610534,"identity":"935547b5-73ea-4239-805b-dc1833dedbd1","order_by":6,"name":"Jun Hai","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Hai","suffix":""},{"id":492610535,"identity":"7fd55f13-8576-4cb1-9ade-cf5828309f74","order_by":7,"name":"Xianhui Zhou","email":"","orcid":"","institution":"The First Affiliated Hospital of Xinjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xianhui","middleName":"","lastName":"Zhou","suffix":""},{"id":492610536,"identity":"bb3919dc-245f-4fe4-8896-e8f114cafb33","order_by":8,"name":"Ying Li","email":"","orcid":"","institution":"Xiamen Medical College","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-07-29 06:30:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7239681/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7239681/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88030586,"identity":"86881f17-a519-484c-a7fb-7068a1bd2e82","added_by":"auto","created_at":"2025-07-31 15:24:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":484891,"visible":true,"origin":"","legend":"\u003cp\u003eActivated neuronal PANoptosis in the hippocampus under AD pathology. (A) Representative images of immunofluorescent staining for APP (green) in the hippocampal tissues from 4×FAD mice or age-matched mice (aging mice). Nuclei stained with DAPI were shown in blue. n = 3. Scale bar = 100 μm. (B) The RT-PCR analysis of APP in hippocampus tissues from 4×FAD mice or aging mice. n = 3. (C, D) Representative images and quantification of immunoblotting for APP in the hippocampal tissues from 4×FAD mice or aging mice. β-actin was used as a loading control. n = 3. (E, F) Representative images of toludine blue staining for hippocampal tissues from 4×FAD mice or aging mice, and the number of cells in the CA1 region was quantified and is shown as a bar graph. Scale bars: 600 μm in hippocampus and 1.5 μm in CA1. n = 6-8. (G-I) Representative images of immunofluorescent staining for NeuN (green), C-Caspase3 (red), p-MLKL (yellow) and N-GSDMD (far-red/turquoise) in the hippocampal tissues from 4×FAD mice or age-matched mice (aging mice). Nuclei stained with DAPI were shown in blue. n = 3. Scale bar = 50 μm. (J) Schematic diagram for extraction and treatment of primary mouse hippocampal neurons. (K-N) Representative images and quantification of immunoblotting for p-MLKL, N-GSDMD, C-Caspase1, C-Caspase3, ZBP1, ASC, p-RIPK1, p-RIPK3, NLRP3, AIM2 and Pyrin in the mouse primary hippocampal neurons expose or unexposed to Aβ\u003csub\u003e25-35\u003c/sub\u003e and OA. β-actin was used as a loading control. n = 3. (O) Representative immunofluorescence staining images showing the co-localization of ASC (cyan), Pyrin (green), NLRP3 (red), and AIM2 (far-red/turquoise), as well as the co-localization of ASC (cyan), ZBP1 (green), RIPK1 (red), and RIPK3 (far-red/turquoise), in mouse primary hippocampal neurons exposed or unexposed to Aβ\u003csub\u003e25-35\u003c/sub\u003e and OA. n = 3. Scale bar = 25 μm. The \u003cem\u003eP\u003c/em\u003e-values were determined via unpaired two-tailed Student’s t-test (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7239681/v1/930fb96e21859e19200ffab3.png"},{"id":88030588,"identity":"3903f433-fc6d-437d-925e-dadcbc78e692","added_by":"auto","created_at":"2025-07-31 15:24:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":342496,"visible":true,"origin":"","legend":"\u003cp\u003eAbnormally expression of TP63 and TUBA1B, the hub DE-PRGs in the hippocampus and hippocampal neurons involved in AD pathology and aging process, was associated with HIF-1α signalling pathway. (A) Cell markers for cell clusters, including astrocyte (Astro), dopaminergic neuron (Dopa_N), GABAergic neuron (GABA_N), Glutaminergic neuron (Glu_N), microglia (Micro), oligodendrocyte (Oligo) and oligodendrocyte precursor cells (OPCs), in the hippocampus were shown in the violin diagram. (B) Proportion of each cell type in the hippocampus of AD patients, aging individuals and healthy adults. (C-F) DEGs in the Glu_N and GABA_N of hippocampus in AD pathology and aging process. (G) Venn diagram of PRGs and the DEGs in hippocampal neurons (the intergroup DEGs of the Glu_N and GABA_N were collectively classified as the intergroup DEGs of neurons) of AD patients and aging individuals. (H) Venn diagram of PRGs and the DEGs in hippocampal neuron of aging individuals and healthy adults. (I) Venn diagram of DE-PRGs in the hippocampus and hippocampal neurons of AD patients versus aging individuals, and in the hippocampus and hippocampal neurons of aging individuals versus healthy adults. (J-L) Images and analysis of western blot for TP63 and TUBA1B protein in the hippocampus of 4×FAD mice, aging mice and adult mice. The \u003cem\u003eP-\u003c/em\u003evalues were determined via one-way ANOVA with Tukey’s adjustment for multiple comparisons (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). (M, N) HIF-1α signaling pathway obtained from sgGSEA analysis for TP63 and TUBA1B. (O) Venn diagram showing the overlap between DE-PRGs in the hippocampus of AD patients and aging individuals, and genes involved in the HIF-1α signaling pathway identified through TP63 and TUBA1B sgGSEA analysis. (P-U) Images and analysis of western blot for PIK3CB, HIF-1α, VEGFA, HO-1 and HK2 proteins in the hippocampus of 4×FAD and Aging mice. The \u003cem\u003eP\u003c/em\u003e-values were determined via unpaired two-tailed Student’s t-test (*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7239681/v1/fec437b774d2834280ab487e.png"},{"id":88030591,"identity":"ae7246a2-edd8-4347-abce-81100e5f7929","added_by":"auto","created_at":"2025-07-31 15:24:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":237530,"visible":true,"origin":"","legend":"\u003cp\u003eHIF-1α activation exerts a dual regulatory effect in regulating PANoptosis of HT22 cells exposed to Aβ\u003csub\u003e25-35\u003c/sub\u003e and OA. (A, B) Cell viability of HT22 cells exposed to Aβ\u003csub\u003e25-35\u003c/sub\u003e and OA was assessed following treatment with different concentrations of DMOG or LW6 for 1 hour. n = 3. (C, D) Representative images and quantification of immunoblotting for p-MLKL, GSDMD, C-Caspase3, C-Caspase1, NLRP3, p-RIPK3, Pyrin, p-RIPK1, ZBP1, and AIM2 in HT22 cells exposed or unexposed to Aβ\u003csub\u003e25-35\u003c/sub\u003e and OA, with or without DMOG or LW6 treatment. β-actin was used as a loading control. n = 3. The \u003cem\u003eP\u003c/em\u003e-values were determined via one-way ANOVA with Tukey’s adjustment for multiple comparisons (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7239681/v1/4dd08e946fceef819336eaf9.png"},{"id":88030590,"identity":"d79db426-0638-4cd7-bf8b-baaa6d7fcf5b","added_by":"auto","created_at":"2025-07-31 15:24:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":421809,"visible":true,"origin":"","legend":"\u003cp\u003eHIF-1α activation promotes hippocampal neuronal PANoptosis by upregulating HK2/VDAC1/NLRP3 axis under AD pathology. (A-C) Representative images and quantification of immunoblotting for HIF-1α and HK2 in HT22 cells exposed or unexposed to Aβ\u003csub\u003e25-35\u003c/sub\u003e and OA, with or without DMOG or LW6 treatment. β-actin was used as a loading control. n = 3. (D-F) Representative images of double immunofluorescent staining for HK2 (cyan) and MitoTracker (far-red/turquoise), for TOM20 (green) and dsNDA (red), and for VDAC1 (yellow) and NLRP3 (grey) in HT22 cells exposed or unexposed to Aβ\u003csub\u003e25-35\u003c/sub\u003e and OA, with or without DMOG or LW6 treatment. Nuclei stained with DAPI were shown in blue. n = 3. Scale bar = 10 μm. (G) Schematic diagram of the mechanism by which PROTAC HK2 Degrader-1 targets and degrades HK2 protein. (H-J) Representative images of double immunofluorescent staining for HK2 (cyan) and MitoTracker (far-red/turquoise), for TOM20 (green) and dsNDA (red), and for VDAC1 (yellow) and NLRP3 (grey) in HT22 cells exposed to Aβ\u003csub\u003e25-35\u003c/sub\u003e and OA, treated with either DMOG or a combination of DMOG and PROTAC HK2 Degrader-1. Nuclei stained with DAPI were shown in blue. n = 3. Scale bar = 10 μm. (K) Representative images of double immunofluorescent staining for VDAC1 (yellow) and NLRP3 (grey) in HT22 cells exposed to Aβ\u003csub\u003e25-35\u003c/sub\u003e and OA, treated with either DMOG or a combination of DMOG and VBIT4. Nuclei stained with DAPI were shown in blue. n = 3. Scale bar = 10 μm. (L-N) Representative images and quantification of immunoblotting for NLRP3 in HT22 cells exposed or unexposed to Aβ\u003csub\u003e25-35\u003c/sub\u003e and OA, treated with either DMOG or a combination of DMOG and PROTAC HK2 Degrader-1 or VBIT4. β-actin was used as a loading control. n = 3. The \u003cem\u003eP\u003c/em\u003e-values were determined via one-way ANOVA with Tukey’s adjustment for multiple comparisons (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7239681/v1/0afb91b0a5635812ce5a2f18.png"},{"id":88030592,"identity":"d189b5bd-2ebd-414a-94d7-132c8a714fd4","added_by":"auto","created_at":"2025-07-31 15:24:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":152366,"visible":true,"origin":"","legend":"\u003cp\u003eHIF-1α activation suppresses PANoptosis of HT22 cells by transcriptionally downregulating RIPK3 under AD pathology. (A) Quantification of immunoblotting (representative images shown in Figure 4C) for RIPK3 in HT22 cells exposed or unexposed to Aβ\u003csub\u003e25-35\u003c/sub\u003e and OA, with or without DMOG or LW6 treatment. β-actin was used as a loading control. n = 3. The \u003cem\u003eP\u003c/em\u003e-values were determined via one-way ANOVA with Tukey’s adjustment for multiple comparisons (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001). (B) Schematic diagram of the mechanism by which HIF-1α transcriptionally downregulates RIPK3. (C) The RT-PCR analysis of RIPK3 in HT22 cells exposed or unexposed to Aβ\u003csub\u003e25-35\u003c/sub\u003e and OA, DMOG or LW6 treatment. n = 6. The \u003cem\u003eP\u003c/em\u003e-values were determined via one-way ANOVA with Tukey’s adjustment for multiple comparisons (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). (D-J) Representative images and quantification of immunoblotting for RIPK3, p-MLKL, NLRP3, GSDMD, C-Caspase3 and C-Caspase1 in HT22 cells exposed or unexposed to Aβ\u003csub\u003e25-35 \u003c/sub\u003eand OA, with or without GSK-872 treatment. β-actin was used as a loading control. n = 3. The \u003cem\u003eP\u003c/em\u003e-values were determined via unpaired two-tailed Student’s t-test (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7239681/v1/23b8ffacd8380cd06e0a52d6.png"},{"id":88030596,"identity":"91240214-dc65-4e7e-9382-f32b56432b57","added_by":"auto","created_at":"2025-07-31 15:24:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":621550,"visible":true,"origin":"","legend":"\u003cp\u003eSemaglutide suppresses hippocampal neuronal PANoptosis by modulating HIF-1α signaling pathway under AD pathology. (A) Schematic diagram of 4×FAD mice injected with vehicle or semaglutide, following by behavioural tests conducted 28 days post-injection. (B-E) Morris water maze tests were performed in WT mice or 4×FAD mice after injection of either vehicle or semaglutide, and data was analysed for latency first entrance to target during a 5-day training period, as well as for time spent in the target zone, target crossing, and latency first entrance to target during memory tests. n = 8-11. The \u003cem\u003eP\u003c/em\u003e-values were determined via one-way ANOVA with Tukey’s adjustment for multiple comparisons (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001). (F) Novel object recognition tests were performed in WT mice or 4×FAD mice after injection of either vehicle or semaglutide, and data was analysed for cognition index. n = 8-13. The \u003cem\u003eP\u003c/em\u003e-values were determined via unpaired two-tailed Student’s t-test (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). (G-I) Representative images and quantification of immunoblotting for HIF-1α and HK2 in HT22 cells exposed to Aβ\u003csub\u003e25-35\u003c/sub\u003e and OA, treated with either vehicle or semaglutide (50 nM) treatment. β-actin was used as a loading control. n = 3. The \u003cem\u003eP\u003c/em\u003e-values were determined via unpaired two-tailed Student’s t-test (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). (J, K) The RT-PCR analysis of HIF-1α and HK2 in HT22 cells exposed to Aβ\u003csub\u003e25-35\u003c/sub\u003e and OA, treated with either vehicle or semaglutide treatment. n = 3. The \u003cem\u003eP\u003c/em\u003e-values were determined via one-way ANOVA with Tukey’s adjustment for multiple comparisons (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u0026nbsp; (L) Representative images of double immunofluorescent staining for HK2 (cyan) and MitoTracker (far-red/turquoise), for TOM20 (green) and dsNDA (red), and for VDAC1 (yellow) and NLRP3 (grey) in HT22 cells exposed to Aβ\u003csub\u003e25-35\u003c/sub\u003e and OA, treated with either vehicle or semaglutide. Nuclei stained with DAPI were shown in blue. n = 3. Scale bar = 10 μm. (M, N) Representative images and quantification of immunoblotting for p-MLKL, MLKL, NLRP3, p-RIPK1, RIPK1, Pyrin, RIPK3, p-RIPK3, AIM2, GSDMD, ZBP1, C-Caspase3, C-Caspase1 and ASC in HT22 cells exposed or unexposed to Aβ\u003csub\u003e25-35\u003c/sub\u003e and OA, treated with either vehicle or semaglutide. β-actin was used as a loading control. n = 3. The \u003cem\u003eP\u003c/em\u003e-values were determined via unpaired two-tailed Student’s t-test (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).. (O, P) Representative images of immunofluorescent staining for NeuN (green), C-Caspase3 (red), p-MLKL (yellow) and N-GSDMD (far-red/turquoise) in the hippocampal tissues from 4×FAD mice after injection of either vehicle or semaglutide. Nuclei stained with DAPI were shown in blue. n = 3. Scale bar = 50 μm. (Q) Representative images of toludine blue staining for hippocampal tissues from 4×FAD mice after injection of either vehicle or semaglutide, and the number of cells in the CA1 region was quantified and is shown as a bar graph. Scale bars: 600 μm in hippocampus and 1.5 μm in CA1. n = 6-8.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7239681/v1/63832ccae7371b3061a243a1.png"},{"id":105724863,"identity":"79bb732b-b1ea-4549-9391-0bd45e072b54","added_by":"auto","created_at":"2026-03-30 10:14:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3338522,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7239681/v1/3b1a3c7f-3e63-44d6-a660-a902dfb0f567.pdf"},{"id":88030589,"identity":"63b34278-7839-4a07-97be-5c8b2d543d3b","added_by":"auto","created_at":"2025-07-31 15:24:38","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":54563,"visible":true,"origin":"","legend":"Supplementary table1","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7239681/v1/ad7da3a09d4e77d8f2b2b741.xlsx"},{"id":88031028,"identity":"15684d3c-666f-4736-9c12-a2b7cc1369ce","added_by":"auto","created_at":"2025-07-31 15:32:38","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11791,"visible":true,"origin":"","legend":"Supplementary table2","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7239681/v1/48c0013bdf6bdd9ebb8b97ad.xlsx"},{"id":88030604,"identity":"f8d6c935-0f7b-47bf-bb2f-e5f7ee242487","added_by":"auto","created_at":"2025-07-31 15:24:38","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":6168684,"visible":true,"origin":"","legend":"Supplementary figures","description":"","filename":"SupplementaryFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-7239681/v1/1a8fd9e47cb3ef8f5c101f9e.docx"}],"financialInterests":"There is a duality of interest","formattedTitle":"HIF-1α exerts a double-edged sword regulatory role in hippocampal neuronal PANoptosis of Alzheimer's disease through HK2/VDAC1/NLRP3 axis and RIPK3 signal","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAlzheimer\u0026rsquo;s disease (AD), the most prevalent neurodegenerative disorder, is pathologically characterized by the extracellular accumulation of β-amyloid (Aβ) plaques and the intracellular formation of neurofibrillary tangles composed of hyperphosphorylated Tau protein\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. A hallmark of AD is the extensive loss of neurons, particularly in regions such as the hippocampus, which are crucial for memory and higher cognitive functions\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The intricate interaction among various programmed cell death (PCD) pathways is thought to be the primary cause of neuronal death in AD. Numerous molecules involved in neuronal death under AD pathology have been identified across distinct PCD pathways, including Gasdermin D (GSDMD) and NOD-like receptor family pyrin domain containing 3 (NLRP3) in pyroptosis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, Caspase-3/-8/-9 and the Bcl-2 family in apoptosis\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, and receptor-interacting protein kinase 1 (RIPK1) along with the necrosome complex in necroptosis\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Therefore, a comprehensive elucidation of molecular mechanisms and regulatory network underlying neuronal death in AD, along with the exploration of effective therapeutic strategies to mitigate neuronal loss, is critical for advancing precise therapy in AD.\u003c/p\u003e\u003cp\u003eRecent evidences have highlighted a strong association between AD and neuronal PANoptosis\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, which is a novel form of proinflammatory PCD that intricately integrates the pyroptosis, apoptosis, and necroptosis pathways\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. This process is orchestrated by a multiprotein complex known as the PANoptosome, which facilitates the crosstalk and regulation among these distinct cell death mechanisms through key components such as GSDMD, NLRP3, Caspase-1, Caspase-8, RIPK1, and RIPK3\u003csup\u003e9\u003c/sup\u003e. This conceptualized complex is thought to provide a flexible framework, allowing the recruitment of core components from various cell death pathways to execute a unified cell death response\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Together, these insights provide a novel perspective on the complexity and diversity of neuronal cell death pathways in AD and emphasize the importance of elucidating the molecular crosstalk of neuronal PANoptosome in understanding the pathogenesis and finding new treatment strategies for AD. Nevertheless, the occurrence and underlying regulatory mechanisms of neuronal PANoptosis in the AD hippocampus remain largely unexplored.\u003c/p\u003e\u003cp\u003eHere, we identified the activation of neuronal PANoptosis in the hippocampus in AD. We also revealed a significant correlation between the hypoxia-inducible factor 1-alpha (HIF-1α) signalling pathway and PANoptosis in hippocampal neurons during both AD and the aging process. HIF-1α has been recognized as a pivotal regulator of Aβ plaque deposition, tau hyperphosphorylation, angiogenesis, glucose metabolism, and neuronal survival in AD pathology\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Notably, HIF-1α exhibits a complex dual role in AD, characterized by both neuroprotective and neurotoxic effects, with the mechanisms underlying this duality largely unexplored. As a result, although HIF-1 presents significant potential as a therapeutic target for AD, its dual effects pose substantial challenges to its further application in AD treatment. In this work, we further elucidated that the HIF-1α signalling pathway plays a dual role in regulating hippocampal neuronal PANoptosis in AD. HIF-1α facilitates neuronal PANoptosis by activating the downstream hexokinase 2 (HK2)/voltage-dependent anion channel 1 (VDAC1)/NLRP3 axis, while concurrently inhibiting PANoptosis through the transcriptional suppression of RIPK3. It indicates that the modulation of HIF-1α on PANoptosis in hippocampal neurons could potentially be one of the mechanisms through which it exerts a dual function in the pathological process of AD. Additionally, we demonstrated that semaglutide, a novel glucagon-like peptide-1 receptor agonist (GLP-1RA) currently undergoing two phase 3 clinical trials in AD patients\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, exerts therapeutic effects on AD treatment by activating the HIF-1α signalling pathway while inhibiting its downstream HK2/VDAC1/NLRP3 axis. Collectively, our study uncovers that PANoptosis drives hippocampal neuronal loss in AD, and highlights the dual regulatory role of HIF-1α in this process. These findings deepen our understanding of hippocampal neurodegeneration in AD, while the therapeutic effect of semaglutide on neuronal PANoptosis through targeting HIF-1α signalling pathway underscores the new AD treatment strategies in future.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Animals\u003c/h2\u003e\u003cp\u003eThe male C57BL/6 mice aged 9 to 11 months were purchased from Xiamen university laboratory animal centre (Xiamen, China). The male 4\u0026times;FAD mice aged 9 to 11 months (FAD\u003csup\u003e4T\u003c/sup\u003e [B6/JGpt-Tg(Thy-APP/Thy-PSEN1)5/Gpt] mice) were provided by the Gempharmatech Co., Ltd (Jiangsu, China). All mice were housed under specific pathogen-free (SPF) conditions, with a 12-hour light/dark cycle and ad libitum access to food and water. All mice in the study were not previously involved in other experimental procedures and following \u0026ldquo;3Rs\u0026rdquo; principles (Replacement, Reduction and Refinement) in all experimental procedures. The study protocol was approved by the Xiamen University Animal Ethics Committee (XMULAC20230130). All animals were randomly grouped using a random number table method, and the acquisition and analysis of relevant data were conducted in a double-blind experiment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Isolation of primary hippocampal neurons and cell culturing\u003c/h2\u003e\u003cp\u003eMouse hippocampus tissues were dissected from pups at postnatal day 1, and dissociated using enzymatic digestion. The isolated primary neurons were plated on poly-D-lysine coated dishes, and cultured in Neurobasal medium supplemented with B27 (GIBCO), 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Invitrogen). Mouse hippocampal neuronal cell lines HT22 were maintained in DMEM (High glucose) containing 10% FBS. All cells were cultured in an incubator at 37 ℃ under humidified air with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eFor the AD model, the primary hippocampal neurons and HT22 cells were treated with 5 \u0026micro;M Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e (CSN21486, Neural Signalling) and 20 nM Okadaic acid (S30686, Shanghai yuanye Bio-Technology Co., Ltd). For activation and inhibition of HIF-1α, HT22 cells were treated with 500 \u0026micro;M DMOG (A160647, Ambeed) and 5 nM LW6 (A2667376, Ambeed), respectively. For targeted degradation of HK2 protein, HT22 cells were treated with 0.5 \u0026micro;M HK2 degrader-1, a proteolysis-targeting chimera (PROTAC) (HY-155008, MCE). For inhibition of VDAC1, HT22 cells were treated with 10 \u0026micro;M VBIT-4 (A1364932, Ambeed).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Drug treatment\u003c/h2\u003e\u003cp\u003eThe 4\u0026times;FAD mice received intraperitoneal injections of either 10 mL/kg/day saline or 10 nmol/kg/day semaglutide (SJ20210015, Novo Nordisk) for 35 consecutive days, starting 28 days prior to the water maze test.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Morris water maze\u003c/h2\u003e\u003cp\u003eMorris water maze is a very popular tool for assessing spatial learning and memory. In this study, the maze comprised a circular tank with a diameter of 122 cm, containing a platform submerged in tap water maintained at a temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2 ℃. Distinctive shapes were placed along the tank walls to serve as spatial reference cues. A camera mounted above the maze recorded the swimming traces of the mice.\u003c/p\u003e\u003cp\u003eDuring the acquisition trials, the platform was submerged 1 cm below the water surface. Mice were introduced into the maze at one of four designated entry points (N, S, E, W), facing the tank wall. They were given 60 seconds to locate the platform. If a mouse failed to find the platform within this time, it was guided to the platform and allowed to remain there for 10 seconds. Each day, two trials were conducted with a 1-hour interval between them over a period of 5 days. And the escape latency, an indicator of spatial learning and memory acquisition, was recorded for each trial. On the 6th day, a probe test was conducted with the platform removed. Metrics recorded during this test included the latency to the first entry into the platform\u0026rsquo;s target location, the number of crossings over the platform\u0026rsquo;s target area, and the time spent in the target quadrant.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Novel object recognition\u003c/h2\u003e\u003cp\u003eThe Novel object recognition test, used to evaluate learning and memory in mice, was conducted following the Morris water maze test. Two similar objects differing in shape and color were selected to serve as experimental tools, with one designated as the \u0026ldquo;familiar object\u0026rdquo; and the other as the \u0026ldquo;novel object\u0026rdquo;. The mice were placed in an open field (40 cm \u0026times; 40 cm \u0026times; 40 cm) for unrestricted exploration, allowing them to acclimate to the environment for 10 minutes. In the familiarization phase, two identical \u0026ldquo;familiar objects\u0026rdquo; were placed in the open field and the mice were allowed to explore freely for 5 minutes. One hour after the familiarization phase ends, one of \u0026ldquo;familiar objects\u0026rdquo; was replaced with the \u0026ldquo;novel object\u0026rdquo;, and the mouse was returned to the open field for another 5 minutes of unrestricted exploration. The exploration time of the mice for the \u0026ldquo;new object\u0026rdquo; (T\u003csub\u003enew\u003c/sub\u003e) and the \u0026ldquo;familiar object\u0026rdquo; (T\u003csub\u003efamiliar\u003c/sub\u003e) was recorded. The \u0026ldquo;Novel Object Recognition Index\u0026rdquo; (Cognition Index) was then caculated as follows: Cognition Index (%) = [T\u003csub\u003enew\u003c/sub\u003e / (T\u003csub\u003enew\u003c/sub\u003e + T\u003csub\u003efamiliar\u003c/sub\u003e)] \u0026times; 100%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 CCK-8 assay\u003c/h2\u003e\u003cp\u003eThe CCK-8 assay was used to assess the effects of DMOG, LW6, or semaglutide on neuronal viability. Once the cells were in optimal condition and fully adherent, they were treated with different concentrations of DMOG, LW6, or semaglutide. After replacing the medium, 10% CCK-8 solution was added to each well, and the plates were incubated in the dark for 1 hour. Absorbance at 450 nm was measured, ensuring that the maximum value exceeded 0.9. The absorbance for each well was recorded, and the cell survival rate was calculated using the formula: Survival rate (%) = [(Experimental group \u0026ndash; Medium control group) / (Control group \u0026ndash; Medium control group)​] \u0026times; 100%.​\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Tissue and cell lysate preparation, and antibodies used for immunoblotting\u003c/h2\u003e\u003cp\u003eMouse hippocampal tissues were collected from at least three mice each group. Proteins extracted from HT22 cells, primary hippocampal neurons or hippocampal tissue lysates subjected to western blot analysis. The samples were separated using SDS-polyacrylamide gel electrophoresis and probed with specific antibodies listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Goat-anti-mouse secondary antibodies and goat-anti-rabbit secondary antibodies were purchased from the Millipore (#AP132P, #AP124P). For quantification, band intensities were measured using Image J (a public domain software from the National Institutes of Health), normalized to β-actin, and averaged from at least four independent experiments.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAntibodies used in the western blotting, immunofluorescence and Tyramide signal amplification.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAntibodies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSource\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIdentifier\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eβ-actin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eInvitrogen\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMA1-140\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-β-actin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e66009-1-Ig\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-APP/β-amyloid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25524-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-VEGFA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19003-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-HK2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e22029-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-HMOX1/HO-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10701-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-MEFV/Pyrin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24280-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-ZBP1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13285-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-AIM2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20590-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti- MLKL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e66675-1-Ig\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-p-MLKL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCST\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e#37333\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-Casp1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e22915-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-C-Casp3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCST\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e#9661\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-Casp3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19677-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-RIPK1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29932-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-p-RIPK1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eImmunoway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYP1467\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-RIPK3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17563-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti- p-RIPK3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eImmunoway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYP1468\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-TUBA1B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e66031-1-Ig\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-TOM20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11802-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-dsDNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAbcam\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eab27156\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-NLRP3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30109-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-ASC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e67494-1-Ig\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-GSDMD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e66387-1-Ig\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-N-GSDMD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eImmunoway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYT7991\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-P63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12143-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-PIK3CB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20584-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-HIF-1α\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20960-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-VDAC1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10866-1-AP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGoat-anti-mouse secondary antibody\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSA00001-1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGoat-anti-rabbit secondary antibody\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteintech\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSA00001-2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e350-labeled Tyramide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eImmunoway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYS0006\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMultiplex fluorescent staining kit\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAiFang Bioligical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAFIHC024\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Frozen section\u003c/h2\u003e\u003cp\u003eAfter anesthesia, the mouse is rapidly euthanized by cervical dislocation. The brain is carefully removed and washed with physiological saline to eliminate blood and impurities. The brain tissue is then fixed overnight at 4℃ in 4% paraformaldehyde. After fixation, the tissue is fully immersed in optimal cutting temperature and placed in a -80℃ freezer to ensure complete freezing. The frozen brain tissue is placed in a pre-cooled cryostat (Leica CM 1950, German), then quickly sectioned with 20 \u0026micro;m thicken using the cryostat. The sections are immediately placed on pre-cooled glass slides. The prepared tissue sections are stored at -80℃ until further processing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Toluidine blue staining\u003c/h2\u003e\u003cp\u003eBrain tissue sections were placed in a 37℃ oven for 20 minutes. After washing with PBS, submerge the sections sequentially in 75%, 95%, 100%, 95%, and 75% ethanol for 3 minutes each. After another PBS wash, submerge the sections in a 0.1% toluidine blue solution for 20 minutes. Then, thoroughly wash off the excess stain with distilled water. Next, submerge the sections sequentially in 75%, 95%, and 100% ethanol for 3 minutes each, followed by clearing with xylene for 5 minutes. Finally, mount the sections with neutral resin and observe the staining results under a microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Immunostaining\u003c/h2\u003e\u003cp\u003eTyramide signal amplification system was used for chromogenic immunostaining. For double-staining with primary antibodies, HT22 cells, primary hippocampal neurons or hippocampal tissue sections were incubated in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 10 minutes at room temperature to block the endogenous peroxidase, then rinsed in PBS and subsequently incubated with 3% BSA at room temperature for 1 hour, following by incubating with the first primary antibody and with HRP-linked secondary antibody. After the second round of heating and washing as described above, the sections were incubated with another primary antibody and with HRP-linked secondary antibody.\u003c/p\u003e\u003cp\u003eFor multiplex with primary antibodies, the primary hippocampal neurons on slides were submerged in citrate buffer and microwave-heated for 15 minutes to release the antigens. To block the endogenous peroxidase, the sections were incubated in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 10 minutes at room temperature. They were then rinsed in PBS and subsequently incubated with 3% BSA at room temperature for an hour. After that, they were incubated with primary antibody ASC, following by an HRP-linked secondary antibody and 350-labeled tyramide. After the second round of heating and washing as described above, the sections were incubated with primary antibody MEFV or ZBP1, following by an HRP-linked secondary antibody and 520-labeled tyramide. After the third round of heating and washing as described above, the sections were incubated with primary antibody NLRP3 or RIPK1, following by an HRP-linked secondary antibody and 570-labeled tyramide. After the fourth round of heating and washing as described above, the sections were incubated with primary antibody AIM2 or RIPK3, following by an HRP-linked secondary antibody and 690-labeled tyramide. Images were acquired using a Nikon confocal microscope. Antibodies used for immunostaining were listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Differently expressed genes analysis of transcriptome sequencing\u003c/h2\u003e\u003cp\u003eMicro-array dataset GSE48350 used in this study was obtained from the Gene Expression Omnibus (GEO) database. This dataset comprises the gene expression matrix of the hippocampus of the AD patients (n\u0026thinsp;=\u0026thinsp;19), the Aging (n\u0026thinsp;=\u0026thinsp;25) and the Adult (n\u0026thinsp;=\u0026thinsp;18). Using R software\u0026rsquo;s \u0026ldquo;limma\u0026rdquo; packages\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, differently expressed genes (DEGs) in hippocampus between the groups were analyzed, with the following criterion: adjust \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12 Differently expressed genes analysis of single-nucleus transcriptomic sequencing\u003c/h2\u003e\u003cp\u003eSingle-nucleus transcriptomic sequencing (snRNA-seq) datasets GSE199243, GSE198323 and GSE185553 were obtained from the GEO database. These datasets comprise gene expression matrix of the hippocampus of the AD patients (n\u0026thinsp;=\u0026thinsp;8), aging individuals (n\u0026thinsp;=\u0026thinsp;7), and adults (n\u0026thinsp;=\u0026thinsp;6). Raw expression matrix of each individual specimen were loaded into the R (v4.3.2) package \u0026ldquo;Seurat\u0026rdquo;, following by the data analysis was carried out based on the published methods\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The 11 principal components (PCs) were then used to generate a shared nearest neighbor graph which was then clustered under the Louvain algorithm with a resolution of 0.4. The t-distributed Stochastic Neighbor Embedding (t-SNE) was then performed using the first 11 PCs. Then, using human cell markers in the CellMarker 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://117.50.127.228/CellMarker/CellMarkerBrowse.jsp\u003c/span\u003e\u003cspan address=\"http://117.50.127.228/CellMarker/CellMarkerBrowse.jsp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) as the standard of cell annotation, 7 kinds of cell types including Glutamatergic neuron (Glu_N, cell makers: \u0026ldquo;SLC17A7\u0026rdquo;), GABAergic neuron (GABA_N, cell makers: \u0026ldquo;GAD2\u0026rdquo;), Dopaminergic neurons (Dopa_N, cell makers: \u0026ldquo;FOLR1\u0026rdquo;), Oligodendrocytes (Oligo, cell makers: \u0026ldquo;MBP\u0026rdquo;, \u0026ldquo;MOBP\u0026rdquo;) and Oligodendrocytes precursor cells (OPCs, cell makers: \u0026ldquo;LHFPL3\u0026rdquo;), Astrocytes (Astro, cell makers: \u0026ldquo;AQP4\u0026rdquo;, \u0026ldquo;GFAP\u0026rdquo;) and Microglia (Micro, cell makers: \u0026ldquo;APBB1IP\u0026rdquo;) were identified. To investigate DEGs in each cell type between the groups, analysis was performed on the integrated dataset using the \u0026lsquo;RNA slot\u0026rsquo; of the Seurat object and a two-sided Wilcoxon rank sum test (implemented in \u0026ldquo;FindMarkers\u0026rdquo;). Finally, the DEGs in each hippocampal cell type between the groups were analyzed, with the following criteria: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log\u003csub\u003e2\u003c/sub\u003e(fold change)| \u0026gt;0.6.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13 Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis\u003c/h2\u003e\u003cp\u003eThe clusterProfiler package in R was used for functional enrichment analyses of DEGs based on KEGG enrichment analysis, a statistical significance threshold of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was set to determine significant results in the analysis\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14 Single-gene gene set enrichment analysis (sgGSEA)\u003c/h2\u003e\u003cp\u003eThe clusterProfiler package in R was employed for sgGSEA\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The read count values of gene were used as input for the sgGSEA package to explore the related pathways associated with the TP63 and TUBA1B. The correlation between the TP63 and TUBA1B and all other genes in the transcriptome data of the GSE48350 dataset was calculated. The genes were then sorted in descending order based on their correlations. This sorted gene list was considered as the gene set to be tested for pathway enrichment analysis. The analysis further involved the utilization of the predefined KEGG signaling pathway set to evaluate the enrichment of the sorted genes in the KEGG pathways. This step aimed to identify the specific KEGG pathways that demonstrated significant enrichment among the genes associated with TP63 and TUBA1B.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.15 Statistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses related to Micro-array and snRNA-seq data in the present study were implemented using R software (version 4.3.2). Unless otherwise stated, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was deemed as statistically significant, and all \u003cem\u003eP\u003c/em\u003e values were two tailed.\u003c/p\u003e\u003cp\u003eStatistical analyses of experimental data were performed using GraphPad Prism version 9.0 (GraphPad Inc., San Diego, CA, USA). For experiments with two groups, unpaired two-tailed Student's t-test was used. For experiments with three or more groups, one-way or two-way ANOVA with Tukey\u0026rsquo;s adjustment for multiple comparisons was applied. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (standard error of the mean). Values were considered statistically significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cb\u003e3.1 PANoptosis was observed in the hippocampus of 4\u0026times;FAD mice and in neurons exposed to Aβ\u003c/b\u003e\u003csub\u003e\u003cb\u003e25-35\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eand OA\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePANoptosis, a novel proinflammatory PCD pathway characterized by the interplay among apoptosis, necroptosis, and pyroptosis, has been reported to be linked to AD\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, the direct experimental evidence of neuronal PANoptosis in AD has yet to be reported. To investigate the occurrence of neuronal PANoptosis in AD, the 4\u0026times;FAD mouse model was employed. Compared to age-matched control mice, the 4\u0026times;FAD mice exhibited elevated gene and protein expression levels of amyloid precursor protein (APP) and more pronounced neuronal loss in the hippocampal CA1 region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-F). The immunofluorescence staining revealed significantly increased levels of cleaved Caspase-3 (C-Caspase-3), phosphorylated mixed lineage kinase domain-like protein (p-MLKL), and N-terminal GSDMD (N-GSDMD) in the hippocampus of 4\u0026times;FAD mice relative to aging controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-I). These findings suggest a strong association between hippocampal neuronal loss and PANoptosis in AD.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOkadaic acid (OA) has been shown to be more potent than Aβ in inducing cell death, primarily by promoting Tau hyperphosphorylation\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. To determine whether PANoptosis is a new manner for hippocampal neuron death, primary hippocampal neurons for WT mice were simultaneously exposed to 5 mM Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and 20 nM OA for 24 hours to establish an AD hippocampal neuron model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). Immunoblotting results revealed that neurons treated with Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA exhibited significantly elevated protein expression in PANoptosis, including C-Caspase-3, C-Caspase-1, p-MLKL, N-GSDMD, ASC, AIM2, ZBP1, Pyrin, p-RIPK1, p-RIPK3, and NLRP3, compared to untreated control neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK-N). Moreover, PANoptosis is mediated by the PANoptosome complex, which is assembled through the integration of key components from the apoptosis, necroptosis, and pyroptosis\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Multiplex immunofluorescence (mIF) staining demonstrated that Pyrin, NLRP3, AIM2, ZBP1, RIPK1, and RIPK3 were highly co-localized with ASC specks in primary hippocampal neurons treated with Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA, compared to untreated control neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO and \u003cb\u003eP\u003c/b\u003e), indicating that Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA induced the assembly of the PANoptosome complex. Together, these findings suggest that PANoptosis is a novel form of PCD that leads to hippocampal neuronal loss in AD.\u003c/p\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.2 HIF-1α exerted the key role in regulating hippocampal neuronal PANoptosis in AD\u003c/h2\u003e\u003cp\u003eWe have observed the hippocampal neurons PANoptosis in AD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Neuronal degeneration and death are key pathological features of AD\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. As an integrated form of PCD that encompasses apoptosis, pyroptosis, and necroptosis, PANoptosis is a new pathogenic way in AD neuronal death. Therefore, elucidating the molecular and signaling pathways that regulate PANoptosis in hippocampal neurons is crucial for developing new targets or strategies for Alzheimer's disease.\u003c/p\u003e\u003cp\u003eA total of 902 PANoptosis-related genes (PRGs) were retrieved from the GeneCards database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the PubMed\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e (Supplementary Table\u0026nbsp;1). Aging (senescence) is recognized as a major risk factor for AD, and targeting senescent cells has grown into a promising therapeutic approach to mitigating the onset and progression of AD\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. To identify the differentially expressed PRGs (DE-PRGs) involved in AD pathology and aging process, the bioinformatics analyses were performed on the hippocampal gene expression matrix from AD patients, aging individuals and healthy adults. A total of 239 DE-PRGs related to AD pathology and 267 DE-PRGs related to aging process were identified (\u003cb\u003eSupplementary Fig.\u0026nbsp;1A, B\u003c/b\u003e). KEGG enrichment analysis revealed that both sets of DE-PRGs were significantly enriched in PI3K-Akt, apoptosis, NOD-like receptor, necroptosis and HIF-1 signalling pathways (\u003cb\u003eSupplementary Fig.\u0026nbsp;1C, D\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eTo obtain the DE-PRGs in hippocampal neurons associated with both AD pathology and aging process, the snRNA-seq clinical datasets comprising hippocampal gene expression matrix in the AD patients, aging individuals and adult was analyzed. Information on datasets before and after quality control was presented in \u003cb\u003eSupplementary Fig.\u0026nbsp;2 and Supplementary Fig.\u0026nbsp;3\u003c/b\u003e, respectively. Based on established markers from the CellMarker 2.0, seven distinct cell clusters were identified: glutamatergic neurons (Glu_N), GABAergic neurons (GABA_N), dopaminergic neurons (Dopa_N), oligodendrocytes (Oligo), astrocytes (Astro), oligodendrocyte progenitor cells (OPCs), and microglia (Micro) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Results showed that the proportions of Glu_N and GABA_N in AD group were significantly higher than that in aging group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). By collectively classifying the DEGs identified in Glu_N and GABA_N as neuronal DEGs, 35 neuronal DE-PRGs associated with AD pathology and 47 neuronal DE-PRGs linked to the aging process were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-H). Furthermore, by intersecting the neuronal DE-PRGs with those derived from hippocampal tissues, TP63 and TUBA1B were identified as the key DE-PRGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Notably, the immunoblotting results revealed that the expression levels of both TP63 and TUBA1B in the hippocampus of 4\u0026times;FAD and adult mice were significantly higher than those of aging mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ-L). These findings indicate that TP63 and TUBA1B are the central DE-PRGs in the hippocampus and hippocampal neurons involving the AD pathology and the aging process.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePrevious studies have demonstrated that TP63 deficiency accelerates the aging process, with its expression in hippocampal neurons diminishing progressively with age\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. However, the molecular mechanisms through which TP63 and TUBA1B influence AD and neuronal PANoptosis remain inadequately elucidated. To investigate this, single-gene gene set enrichment analysis (sgGSEA) of TP63 and TUBA1B was performed in hippocampal expression matrix from AD patients and aging individuals. A total of 93 signaling pathways were obtained from the intersection of those significantly enriched for TP63 and TUBA1B (\u003cb\u003eSupplementary Fig.\u0026nbsp;4 and Supplementary Table\u0026nbsp;2\u003c/b\u003e). Following the exclusion of functional and disease pathways, several key pathways emerged prominently, including HIF-1, IL-17, Hippo, ErbB, cGMP-PKG, and Wnt signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM, N, \u003cb\u003eSupplementary Fig.\u0026nbsp;5A-J\u003c/b\u003e). Among these the HIF-1 signaling pathway was prioritized for further investigation based on integrated analysis. By intersecting the 239 DE-PRGs linked to AD with genes involved in the HIF-1 signaling pathway enriched from TP63 and TUBA1B, four potential key genes in HIF-1 signaling pathway significantly associated with hippocampal neuronal PANoptosis in AD pathology were identified: PIK3CB, VEGFA, HO-1, and HK2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eO). Subsequent validation through immunoblotting demonstrated a significant decrease in the protein expressions of PIK3CB, HIF-1α, and VEGFA, whereas the protein expressions of HO-1 and HK2 were significantly increased in the hippocampus of 4\u0026times;FAD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eP-U). These findings underscore a significant association between hippocampal neuronal PANoptosis and dysregulation of the HIF-1α signaling pathway in AD.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.3 HIF-1α activation played the dual regulatory effects on the hippocampal neuronal PANoptosis\u003c/h2\u003e\u003cp\u003eAs previously noted, our bioinformatic analyses indicate that dysregulation of the HIF-1α signaling pathway is strongly associated with hippocampal neuronal PANoptosis in AD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). To investigate the effect of HIF-1α on regulating neuronal PANoptosis in AD, the hippocampal neuronal cell line HT22 was exposed to Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA, followed by treatment with either the HIF-1α agonist DMOG or the inhibitor LW6. CCK-8 assays revealed that pharmacological activation of HIF-1α significantly enhanced the viability of HT22 cells under Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA- induced stress, whereas its inhibition markedly decreased cell survival under the same conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). These findings suggest that pharmacological activation of HIF-1α may represent a promising therapeutic strategy for promoting neuronal survival in AD pathology.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further explore whether the neuroprotective effect of HIF-1α activation is mediated by inhibiting neuronal PANoptosis, we examined the expression of key proteins involved in the PANoptosis in HT22 cells treated with DMOG or LW6, with or without exposure to Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA. Interestingly, the results do not fully match our speculation. Immunoblotting results revealed that HIF-1α activation significantly reduced the expression of ZBP1, AIM2, Pyrin, p-RIPK3, p-RIPK1, p-MLKL, C-GSDMD, C-Caspase1, and C-Caspase3 in HT22 cells exposed to Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA, but did not decrease the expression of NLRP3. Similarly, HIF-1α inhibition markedly increased the expression of ZBP1, AIM2, p-RIPK3, p-RIPK1, p-MLKL, GSDMD, C-Caspase1, and C-Caspase3, while decreasing the expression level of NLRP3 and Pyrin under the same conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). These findings suggest that although activation of the HIF-1α pathway exerts neuroprotective effects at the cellular level, it may play a dual role in regulating hippocampal neuronal PANoptosis, potentially through the activation of NLRP3. This underscores the complex and multifaceted regulatory function of HIF-1α activation within the PANoptosis pathway in neurons affected by AD. Notably, HIF-1α has previously been reported to exerts both protective and detrimental effects in AD treatment\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, and our results suggest that this duality may, in part, be attributed to its regulation of neuronal PANoptosis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.4 HIF-1α activation promoted neuronal PANoptosis by upregulating HK2/VDAC1/NLRP3 axis\u003c/h2\u003e\u003cp\u003eNLRP3 is a critical component of the PANoptosome, and its activation during the inflammatory response is mediated through multiple mechanisms\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Previous studies have demonstrated that the dissociation of HK2 from mitochondria facilitates the oligomerization of VDAC1, the release of oxidized mitochondrial DNA (mtDNA) fragments, and the subsequent binding of NLRP3 to VDAC1, thereby promoting the assembly and activation of NLRP3\u003csup\u003e25, 26\u003c/sup\u003e. HK2, a pivotal enzyme in glycolysis, is upregulated by HIF-1α through its interaction with hypoxia response elements in the HK2 promoter\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Studies have demonstrated that HK2 protein expression is significantly elevated in the cerebral cortex and hippocampus of AD patients and 5\u0026times;FAD mice, while the expression levels of other hexokinase isoforms, including HK1\u0026mdash;commonly recognized as the \u0026ldquo;brain hexokinase\u0026rdquo;\u0026mdash;remain largely unaltered\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In this study, HK2 was identified as a pivotal molecule within the HIF-1α signaling pathway involved in regulating neuronal PANoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Immunoblotting analysis revealed that co-treatment with Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA markedly increased HK2 expression in HT22 cells, an effect was further potentiated by HIF-1α activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). Consequently, we hypothesize that HIF-1α activation upregulates the expression and cytoplasmic translocation of HK2, thereby promoting NLRP3 activation. To test this, immunofluorescence staining was performed to assess HK2 dissociation from mitochondria, the release of mtDNA fragments, and the binding of NLRP3 to VDAC1. Results showed that treatment with Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA facilitated HK2 dissociation from mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), increased the release of mtDNA fragments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), and enhanced co-localization of VDAC1 and NLRP3 in HT22 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These effects were further amplified in HT22 cells by co-treatment with DMOG, resulting in even greater HK2 dissociation from mitochondria, an increased release of mtDNA fragments, and more pronounced co-localization of VDAC1 and NLRP3. Conversely, treatment with LW6 markedly reduced HK2 dissociation from mitochondria, decreased the release of mtDNA fragments, and disrupted the co-localization of VDAC1 and NLRP3 in HT22 cells treated with Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F). These findings suggest that the HIF-1α activation promotes AD-related neuronal PANoptosis by inducing HK2-dependent NLRP3 activation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further validate the mediating role of HK2 in the relationship between HIF-1α activation and NLRP3 activation, HT22 cells exposed to Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA were treated with DMOG either alone or in combination with PROTAC-HK2-Degrade-1, a compound specifically designed to degrade the HK2 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Immunoblotting and immunostaining results indicated that the combined treatment of DMOG and PROTAC-HK2-Degrade-1 counteracted the effects induced by DMOG alone in HT22 cells exposed to Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA. This was evidenced by reduced HK2 dissociation from mitochondria, decreased release of mtDNA fragments, and significantly diminished co-localization of VDAC1 and NLRP3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-J). These findings suggest HIF-1α activation mediates HK2 dissociates from mitochondria and triggers VDAC1-dependent NLRP3 translocation and activation. Moreover, while DMOG treatment promoted NLRP3 translocation without altering its protein expression in HT22 cells exposed to Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL, M), combination treatment with DMOG and PROTAC-HK2-Degrade-1 or VDAC1 inhibitor VBIT-4 significantly reduced the NLRP3 protein expression and the co-localization of VDAC1 and NLRP3 in HT22 cells exposed to Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK-O). These findings highlight that HK2 and VDAC1 as critical regulators capable of reversing the heterogeneous expression of key protein in PANoptosis pathway induced by HIF-1α activation. Collectively, our results suggest that HIF-1α activation promotes neuronal PANoptosis in AD by upregulating HK2/VDAC1/NLRP3 axis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.5 HIF-1α activation suppressed neuronal PANoptosis by transcriptionally downregulating RIPK3\u003c/h2\u003e\u003cp\u003eWe have demonstrated that HIF-1α activation exerts a double-edged sword effect on hippocampal neuronal PANoptosis in AD. However, the molecular mechanisms by which HIF-1α activation inhibits PANoptosis remain largely undefined. Resent study has shown that HIF-1α expression in intestinal epithelium restricts arthritis inflammation by transcriptionally inhibiting RIPK3-induced cell death machinery\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Our results showed that HIF-1α activation significantly downregulated the gene and protein expression levels of RIPK3 in HT22 cells exposed to Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA. Conversely, HIF-1α inhibition markedly upregulated the protein expression of RIPK3 and showed a trend toward increasing its mRNA expression in HT22 cells under the same conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C), suggesting that HIF-1α activation also transcriptionally inhibits RIPK3 expression in AD hippocampal neuron.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRIPK3 knockdown has been reported to effectively suppress PANoptosis in bone-marrow-derived macrophages (BMDMs)\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. To further explore the effect of RIPK3 inhibition on neuronal PANoptosis in AD, HT22 cells exposed to Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA were treated with the GSK-872, a selective RIPK3 inhibitor. Immunoblotting revealed that GSK-872 treatment significantly reduced the expression levels of RIPK3, p-MLKL, NLRP3, GSDMD, C-Caspase-1, and C-Caspase-3 in HT22 cells exposed to Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-J). These results suggest that HIF-1α activation suppresses neuronal PANoptosis by transcriptional downregulation of RIPK3.\u003c/p\u003e\u003cp\u003eConsequently, HIF-1α activation exerts a dualistic role in regulating neuronal PANoptosis: it exacerbates PANoptosis through upregulation of the HK2/VDAC1/NLRP3 axis, while concurrently suppressing PANoptosis via the downregulation of RIPK3. This dual effect poses a challenge to pharmacological strategies for targeting HIF-1α activation. Thus, our subsequent research aims to elucidate methods to preserve the inhibitory effect of HIF-1α signaling on neuronal PANoptosis, while attenuating its pro-PANoptosis activity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Semaglutide suppressed hippocampal neuronal PANoptosis by enhacing HIF-1α signaling pathway in AD\u003c/h2\u003e\u003cp\u003eGiven the double-edged sword role of HIF-1α in neuroprotection and neurotoxicity, it is crucial to harness its beneficial effects while minimizing its detrimental outcomes. HIF-1α may serve as a co-linker between AD and T2DM, highlighting its potential as a therapeutic target for AD\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The GLP-1RAs, which are widely used for T2DM in clinical practice, have also shown promise in alleviating AD pathology\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Moreover, GLP-1RAs have been reported to enhance HIF-1α expression in the brain and maintain glucose homeostasis, thereby exhibiting therapeutic potential across a range of diseases, including neurodegenerative disorders\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In our study, we further identified a significant upregulation of HK2\u0026mdash;a critical downstream effector of the HIF-1α signaling pathway\u0026mdash;in the hippocampus of AD models. Based on this, we propose that GLP-1RAs may serve as promising candidates to eliminate the double-edged sword effect of HIF-1α signaling in AD.\u003c/p\u003e\u003cp\u003eSemaglutide, a novel GLP-1RA and an approved clinical medication for TM2D, is currently undergoing two phase 3 clinical trials in AD patients\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. It has been shown to improve learning and memory in APP/PS1 and 3\u0026times;Tg mouse models, as well as human brain organoid models\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Similarly, we validated this conclusion in 4\u0026times;FAD mice received semaglutide injections for 35 days, with a 6-day Morris water maze test initiated on day 28 of treatment, followed by a novel object recognition test, before being humanely euthanized for subsequent analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The results showed that the semaglutide-treated mice exhibited shorter latency to reach the platform, spent more time in the target zone, and crossed the platform location more frequently compared to the untreated AD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-E). And, the semaglutide group demonstrated a significant higher cognitive index than the AD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, we performed CCK-8 assays to determine the protective effects of semaglutide on Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA induced HT22 cells. HT22 cells were treated with different concentrations of semaglutide (5-100 nM) for 24 hours in the presence of Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA. The results showed that the protective effects of semaglutide were dose-dependent, with the greatest effect seen at 50 nM (\u003cb\u003eSupplementary Fig.\u0026nbsp;6\u003c/b\u003e). Thus, 50 nM doses were chosen to determine the modulatory role of semaglutide in the double-edged effects of HIF-1α in the HT22 cells exposed to Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA. We found that semaglutide significantly upregulated the gene and protein expression of HIF-1α, while without markedly affecting the gene and protein levels of HK2 in HT22 cells exposed to Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-K). This indicates that, unlike HIF-1α agonists, semaglutide activates HIF-1α expression while suppressing the expression of its downstream target HK2. Moreover, double immunostaining revealed that semaglutide significantly decreased HK2 dissociation from mitochondria, reduced the release of fragmented mtDNA, and diminished co-localization of VDAC1 and NLRP3 in HT22 cells exposed to Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL). The assembly and activation of NLRP3 inflammasome is the classical indication of PANoptosis\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Therefore, our results indicate that semaglutide inhibits the NLRP3 inflammasome activation-induced neuronal PANoptosis through activating HIF-1α/HK2/VDAC1 axis. Additionally, to clarify the more all-sided mechanisms by which semaglutide suppresses hippocampal neuronal PANoptosis, the RIPK3 branch of HIF-1α signaling pathway was examined in the HT22 cells exposed to Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA. Immunoblotting results showed that semaglutide significantly reduced the key proteins\u0026rsquo; expression in PANoptosis, including RIPK1, p-RIPK1, RIPK3, p-RIPK3, ZBP1, GSDMD, AIM2, NLRP3, C-Caspase3, C-Caspase1 and ASC in HT22 cells exposed to Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM, N). These findings suggest that semaglutide also exerts the inhibition on neuronal PANoptosis through suppressing RIPK3 by activating HIF-1α signal.\u003c/p\u003e\u003cp\u003eMoreover, toluidine blue staining demonstrated that semaglutide significantly mitigated the neuronal loss in the hippocampus of 4\u0026times;FAD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eO, P). Immunostaining exhibited that semaglutide markedly reduced the expression levels of C-Caspase-3, p-MLKL, and N-GSDMD in the hippocampus of 4\u0026times;AD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eQ). These results suggest that semaglutide effectively suppresses hippocampal neuronal PANoptosis in AD. Therefore, semaglutide suppresses the neuronal PANoptosis by activating HIF-1α-induced both HK2/VDAC1 axis and RIPK3 signal, thereby salvaging hippocampus neuronal loss and exerting therapeutic effects of AD.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePANoptosis integrates the key feature of pyroptosis, apoptosis, and necroptosis, and which is a critical focus in future research on PCD\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. However, definitive evidence supporting the occurrence of PANoptosis in AD neurons remains lacking. Therefore, exploring the occurrence and molecular mechanisms of hippocampal neuronal PANoptosis in AD may offer valuable insights for the treatment of AD. Previous reports have suggested a strong association between PANoptosis and AD\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e; however, specific experimental evidence characterizing this relationship \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e is still lacking. Here, we confirmed the hippocampal neuronal PANoptosis in AD by identifying the expression of PANoptosis-related proteins and the formation of PANoptosome in the 4\u0026times;FAD mouse and primary hippocampal neurons exposed to Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA. Subsequently, through comprehensive bioinformatic analysis of clinical data combined with experimental validation, we found that the HIF-1α signal pathway is a crucial regulator of hippocampal neuronal PANoptosis in AD.\u003c/p\u003e\u003cp\u003eHIF-1α, a central transcription factor mediating cellular adaptation to hypoxic conditions, exhibits a complex bidirectional regulatory role in AD\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. HIF-1α has been shown to exert beneficial effects in AD, including the regulation of energy metabolism, promotion of neuroprotection and neural repair, enhancement of neurogenesis, and attenuation of oxidative stress. However, HIF-1α also elicits detrimental effects in AD, such as upregulating β-site APP cleaving enzyme 1 (BACE1), enhancing β-secretase activity to promote Aβ production, impairing cerebral microvascular function, and triggering aberrant cell cycle re-entry-induced neuronal apoptosis. This dual role becomes particularly evident in pharmacological interventions: several AD-related therapeutic agents, including deferoxamine, obalt chloride, lactoferrin and simvastatin, facilitate Aβ clearance by increasing HIF-1α protein levels. Meanwhile, neuroprotective agent neurotropin alleviate AD neuroinflammation by inhibiting the NF-κB/HIF-1α signaling axis\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. However, the precise underlying mechanism through which HIF-1α exerts a dual function in AD has remained elusive for a long time, and effective strategies to intervene in the neurotoxic effects mediated by the HIF-1α pathway are still unknown, significantly hindering the further development and application of related pharmaceuticals. In the present study, pharmacological activation of HIF-1α using DMOG significantly improved the viability of HT22 cells exposed to Aβ\u003csub\u003e25\u0026ndash;35\u003c/sub\u003e and OA, and exerted a dual regulatory effect on the PANoptosis pathway in these neurons. Therefore, to investigate whether HIF-1α exerts the dual effects on AD pathology by regulating hippocampal neuronal PANoptosis, which will hold a significant promise for identifying HIF-1α as a therapeutic target for AD.\u003c/p\u003e\u003cp\u003eAs the first rate-limiting enzyme in the glycolytic pathway, HK2 is the only one among the four hexokinase isoenzymes that is aberrantly expressed in the cerebral cortex and hippocampus of both AD patients and the 5\u0026times;FAD mice\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. However, the role of HK2 in hippocampal neuronal death in AD remains unknown. In the present study, HK2 was identified as one of the key genes involved in hippocampal neuronal PANoptosis in AD, and exhibited significantly higher expression in AD mouse hippocampal tissue and cell cultures. Furthermore, studies have shown that HIF-1α can transcriptionally upregulate HK2 expression by binding to hypoxia response elements of the HK2 promoter, and inflammation can induce the HK2 dissociation from the mitochondria, ultimately leading to the activation and assembly of NLRP3 through VDAC1 oligomerization\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Our study has demonstrated that NLRP3, a key component of PANoptosome\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, did not exhibit reduced expression but rather showed prominent mitochondria translocation when treatment with HIF-1α agonist, in HT22 cells co-treated with Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA. To this, we hypothesize that HIF-1α activation may enhance NLRP3 activation by upregulating HK2, thereby driving hippocampal neuronal PANoptosis in AD. We found that the HIF-1α activation significantly promotes HK2 expression and its dissociation from mitochondria, while decreasing the expression of key components of PANoptosome, with the exception of NLRP3. In addition, our results showed that both HK2-targeted degradation and suppression of VDAC1 oligomerization significantly reduced the expression and mitochondria translocation of NLRP3 in the hippocampal neurons in AD when treated with HIF-1α agonist, suggesting that the inhibition of HK2 or VDAC1 significantly reversed the heterogeneity caused by HIF-1α activation. Therefore, HIF-1α activation promotes the HK2 expression and its dissociation from mitochondria, ultimately leading to the activation of NLRP3 through VDAC1 oligomerization, in HT22 cells exposed to Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA. Together, our findings suggest that HIF-1α activation promotes the hippocampal neuronal PANoptosis by upregulating HK2/VDAC1/NLRP3 axis in AD.\u003c/p\u003e\u003cp\u003eRIPK3 is the crucial component of PANoptosome\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Study has showed that HIF-1α alleviates arthritis inflammation by inhibiting RIPK3 expression in intestinal epithelial cells\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. We further found that HIF-1α activation remarkedly downregulated RIPK3 expression in the hippocampal neurons under AD pathology. However, the effect of HIF-1α on RIPK3-mediated hippocampal neuronal PANoptosis in AD has not been reported. Our results showed that pharmacological inhibition of RIPK3 significantly suppressed hippocampal neuronal PANoptosis in HT22 cells exposed to Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and OA, suggesting that HIF-1α activation suppresses the hippocampal neuronal PANoptosis by transcriptionally downregulating RIPK3 in AD. Altogether, our finding revealed that HIF-1α may exert neuroprotective or neurotoxic effects in AD pathology through dual regulation of neuronal PANoptosis, and effectively activating the HIF-1α pathway and blocking its downstream HK2/VDAC1/NLRP3 axis is key to leveraging the HIF-1α signaling pathway to suppress hippocampal neuronal PANoptosis in AD.\u003c/p\u003e\u003cp\u003eRecent studies have shown that GLP-1RAs reduce the risk of neurodegenerative diseases such as AD\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Multiple studies have demonstrated that GLP-1RAs\u0026mdash;including liraglutide, exenatide, lixisenatide, and semaglutide\u0026mdash;reduce Aβ accumulation, tau hyperphosphorylation, and neuroinflammation\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Moreover, AMPK activation has been implicated in mediating these beneficial effects by suppressing BACE1 and Aβ production, alleviating neuroinflammation, and enhancing Aβ clearance. While AMPK has been identified as one mechanism by which the GLP-1RAs mitigate AD-related phenotypes\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, our study uncovers an alternative pathway whereby the semaglutide\u0026mdash;currently in two phase 3 clinical trials for AD\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e\u0026mdash;activates HIF-1α signaling and blocks the downstream HK2/VDAC1/NLRP3 axis, thereby suppressing hippocampal neuronal PANoptosis and improving cognitive function in AD mice. Our findings provide a novel mechanistic insight into how GLP-1RAs regulate HIF-1α signaling to mitigate PANoptosis and cognitive dysfunction, offering a compelling rationale for further clinical development of GLP-1RAs in AD.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, our study demonstrated the occurrence of hippocampal neuronal PANoptosis in AD. Additionally, we revealed that HIF-1α activation has a dual effect on hippocampal neuronal PANoptosis: it suppresses neuronal PANoptosis by transcriptional downregulation of RIPK3, while simultaneously promoting neuronal PANoptosis via upregulation of HK2/VDAC1/NLRP3 axis, deepening the understanding of the double-sword effect of HIF-1α in AD and providing new research directions for investigation in the development of AD medications. Finally, we discovered that semaglutide alleviates cognition deficits by suppressing hippocampal neuronal PANoptosis mediated by activating HIF-1α signaling pathway and inhibiting its downstream HK2/VDAC1/NLRP3 axis, which provides a new theoretical basis for the treatment of Alzheimer's disease with semaglutide.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the National Science Foundation of China (Nos.: 82273910, 81973306, 81872866 and 82002162), Major Scientific Research Program for Young and Middle-aged Health Professionals of Fujian Province, China (No. 2023ZQNZD019), Fujian Province Science and Technology Innovation Joint Fund Project (Nos. 2024Y9722, 2024Y9700), Fujian Province Science and Technology Plant Project (2022J05325), the Natural Science Foundation of Xinjiang Autonomous Region (No. 2022D01D15), the Xinjiang Tianshan Talent Cultivation Program (2023TSYCLJ0036),\u0026nbsp;the Guiding Medical and Health Projects of Xiamen (Nos.: 35O2Z20214ZD1257, 35O2Z20214ZD1257, 35O2Z20214ZD1326 and 35O2Z20209117), and the Science and Technology Plan of Xiamen Medical College (No.: K2021-03[3]).\u003c/p\u003e\n\u003cp\u003eConflict of interest\u003c/p\u003e\n\u003cp\u003eNone of the authors have any conflict of interest with respect to the study.\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eSQW: data collection, statistical analyses and the initial draft of the manuscript; LXL\u0026nbsp;and XHZ\u0026nbsp;data collection, statistical analyses and the initial draft of the manuscript; XLJ, Y.L and R.T\u0026nbsp;data collection and statistical analyses; LCY: conceptualized and designed the study, analyses and interpretation of the results, statistical analyses, final writing and approval of the manuscript for publication.\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003eDescription of sample size (n-number)\u003c/p\u003e\n\u003cp\u003eFor the behavioural tests described in this study, the number of mice (n = 8-13) analysed in each group is provided in the corresponding figure legends (Figures 6A-F). For other experiments—including western blotting, immunofluorescence, Golgi staining, and toluidine blue staining—the definition of “n” in the context of independent experimental replicates is also detailed in the respective figure legends. All statistical information, including the exact value of n, what n represents, and the statistical tests used, is clearly described in the figure legends.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBostanciklioglu M. An update on the interactions between Alzheimer\u0026apos;s disease, autophagy and inflammation. \u003cem\u003eGene\u003c/em\u003e 2019, \u003cstrong\u003e705:\u003c/strong\u003e 157-166.\u003c/li\u003e\n\u003cli\u003eGoel P, Chakrabarti S, Goel K, Bhutani K, Chopra T, Bali S. 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PANoptosis, a newly characterized form of programmed cell death, integrates the key features of pyroptosis, apoptosis and necroptosis, and explains the molecular crosstalk among these pathways. However, whether PANoptosis is a new manner for hippocampal neuron death in AD, and the involved regulatory mechanisms remains largely unknown. Here, we demonstrate that PANoptosis is a crucial mechanism driving hippocampal neuronal loss in an AD mouse model. Moreover, we uncovered that the HIF-1α signaling pathway exerts adouble-edged sword effect on hippocampal neuronal PANoptosis by activating the HK2/VDAC1/NLRP3 axis while concurrently suppressing RIPK3signal. This observation may offer a partial explanation for the double-edged sword role of HIF-1α as both a neuroprotective and neurotoxic factor in AD. Finally, we uncovered that semaglutide, a glucagon-like peptide-1 receptor agonist (GLP-1RA) currently undergoing phase 3 clinical trials for AD, mitigates hippocampal neuronal PANoptosis by upregulating HIF-1α expression while suppressing its downstream HK2/VDAC1/NLRP3 axis and RIPK3 signal, highlighting its potential as a therapeutic avenue for AD. These findings uncover a previously unrecognized role of PANoptosis in AD and provide new insights into the HIF-1α-mediated regulatory mechanisms, offering a promising target for therapeutic intervention.\u003c/p\u003e","manuscriptTitle":"HIF-1α exerts a double-edged sword regulatory role in hippocampal neuronal PANoptosis of Alzheimer's disease through HK2/VDAC1/NLRP3 axis and RIPK3 signal","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-31 15:24:33","doi":"10.21203/rs.3.rs-7239681/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-09-29T14:17:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-08-26T14:04:41+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-08-19T07:46:09+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-08-15T13:57:14+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-08-15T11:41:58+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-07-29T14:40:39+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-07-29T12:54:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-29T10:09:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-29T06:27:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Differentiation","date":"2025-07-29T06:27:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-differentiation","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cdd","sideBox":"Learn more about [Cell Death \u0026 Differentiation](http://www.nature.com/cdd/)","snPcode":"41418","submissionUrl":"https://mts-cdd.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Differentiation","twitterHandle":"@cddpress","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a0d60cb9-a628-41ea-9b45-e2afaeb37ed6","owner":[],"postedDate":"July 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":52306856,"name":"Biological sciences/Neuroscience"},{"id":52306857,"name":"Health sciences/Diseases"}],"tags":[],"updatedAt":"2026-04-28T05:51:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-31 15:24:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7239681","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7239681","identity":"rs-7239681","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}