Dipeptidyl Peptidase 4 mediated Caspase-8 Affects Cognitive Impairment in Mice with Alzheimer's Disease

Dipeptidyl Peptidase 4 mediated Caspase-8 Affects Cognitive Impairment in Mice with Alzheimer's Disease | 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 Dipeptidyl Peptidase 4 mediated Caspase-8 Affects Cognitive Impairment in Mice with Alzheimer's Disease XinYi Wang, Li Chen, Ke Wang, Yue Chen, Zhi He, XianWu Cheng, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4244824/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract To investigate the effect of dipeptidyl peptidase 4 (DPP4) on cognitive impairment in Alzheimer's disease (AD), the present study used seven-week-old male C57BL/6J and DPP4 knockout mice. The AD model was induced by microinjection of Aβ 25−35 into the lateral ventricle. Morris water maze test showed that DPP4 knockout significantly improved the spatial learning and memory abilitoes of AD mice. Western blot results showed that DPP4 knockout increased the expression levels of BDNF, CREB and Bcl-2 in the hippocampus of AD mice while the expression levels of Caspase-8, pyroptosis-related proteins NLRP3, Caspase-1, GSDMD, 1L-18, 1L-1β and apoptosis-related proteins Caspase-3 and Bax were decreased. Similar results were observed after HT22 neurons were treated with Aβ 25−35 and DPP4 inhibitor sitagliptin (Sit). Moreover, the treatment with Caspase-8 inhibitor (Z-LETD-FMK) showed that the inhibition of Caspase-8 inhibited the expression of NLRP3 and Caspase-1 in the AD model cells, but had no further inhibitory effect under the treatment of Sit. Our results suggests that DPP4 knockout may ameliorate learning and memory dysfunction in AD model mice by regulating pyroptosis and apoptosis pathways through Caspase-8. Biological sciences/Neuroscience Biological sciences/Physiology Dipeptidyl peptidase 4 Alzheimer's disease Learning and memory Caspase-8 Pyroptosis Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Dipeptidyl peptidase 4 (DPP4), also known as CD26, is widely expressed on the surface of a variety of cells. Studies have shown that DPP4 is expressed on neurons in the foetal brain, and a small amount of soluble DPP4 is present in the cerebrospinal fluid[ 1 ]. It has also been reported that the DPP4 gene is significantly correlated with hippocampal volume[ 2 ]. Cross-sectional studies of a population of Chinese elderly nondiabetic individuals (over 60 years old) showed that increased plasma DPP4 activity was positively correlated with cognitive impairment and negatively correlated with MoCA scores[ 3 ]. Significant increases in DPP4 activity have also been found in the brains of sporadic AD patients[ 4 ], suggesting that DPP4 may be associated with cognitive dysfunction. Several basic and clinical studies have shown that DPP4 inhibitors can play a neuroprotective role in the central nervous system. However, the specific mechanism through which DPP4 affects cognitive function is still unclear. As a multifunctional enzyme, DPP4 cleaves a variety of substrates and participates in various physiological and pathological activities. Most studies suggest that DPP4 inhibitors play a neuroprotective role by inhibiting the degradation of the main substrate glucagon-like peptide-1 (GLP-1), but one study reported that DPP4 inhibitors can improve AD-like pathological changes, while the effect of GLP-1 analogues is not significant[ 5 ], suggesting that there may be a GLP-1-independent mechanism by which DPP4 inhibitors improve AD. Progressive neuronal death is a major feature of AD and a direct cause of cognitive impairment. Recently, Nature reported that Caspase-8 is the molecular switch for apoptosis, pyroptosis and necroptosis[ 6 ]. One study reported that DPP4 inhibitors can reduce the expression of Caspase-8 and inhibit cell apoptosis induced by insulin resistance[ 7 ]. Inhibition of DPP4 can ameliorate cognitive dysfunction in vascular dementia model rats through antiapoptotic effects[ 8 ]. DPP4 inhibitors can also significantly reduce TNF-α and IL-6 levels in the hippocampus of rats[ 9 ], thereby inhibiting the neuroinflammatory response. As a highly proinflammatory form of programmed cell death[ 10 ], most scholars believe that inhibiting the pyroptosis pathway may constitute a breakthrough in the molecular mechanism of AD treatment[ 11 ]. However, most of the current central studies on DPP4 have focused only on unilateral apoptotic or pyroptotic effects[ 12 ], and it is not clear whether DPP4 may affect learning and memory dysfunction in AD through the regulation of Caspase-8-mediated programmed cell death. In this study, DPP4 knockout (DPP4 −/− ) mice were used to establish an AD model, and the neuromolecular mechanism by which DPP4 inhibition improves learning and memory dysfunction in AD model mice at the global and cellular levels was explored. These findings could lead to improved drug guidance, such as the use of DPP4 inhibitors, for the treatment of AD combined with diabetes. 2. Results 2.1 Effects of DPP4 knockout on learning and memory function in AD model mice As shown in Fig. 1 A, the escape latency of the four groups of mice gradually decreased as the number of training days increased (Fig. 1 A), and there was no significant difference between the control group and the DPP4 −/− group. Beginning on the third day of training, the escape latency of the DPP4 −/− + AD group was significantly shorter than that of the AD group. On the fifth day, the escape latency of the DPP4 −/− + AD group was 13.47 ± 7.91 seconds, which was significantly different from that of the AD group (34.73 ± 7.07 seconds) ( P < 0.01). The spatial exploration experiment showed that the exploratory swimming paths of the control group, DPP4 −/− group and DPP4 −/− + AD group were concentrated in the target quadrant, while the paths of the AD group were scattered (Fig. 1 B). Figure 1 C shows that the number of original platform areas crossed and the time spent in the target quadrant in the AD group were significantly lower than those in the control group ( P < 0.01), while the number of platform crossings in the DPP4 −/− + AD group (3.50 ± 1.87) was significantly different from that in the AD group (1.0 ± 0.63) ( P < 0.05). Similarly, there was a significant difference in the time spent in the target quadrant between the DPP4 −/− + AD group and the AD group ( P < 0.05). To exclude the possibility of differences in learning and memory function caused by motor dysfunction, the swimming speed and total swimming distance of each group were observed (Fig. 1 D), and there was no significant difference among the groups ( P > 0.05). In addition, as shown in Fig. 1 E, the expression levels of the learning-memory-related proteins CREB and BDNF in the hippocampus of the AD group were significantly lower than those in the control group ( P < 0.05), while the expression levels of these two proteins in the DPP4 −/− + AD group were significantly greater than those in the AD group ( P < 0.05). These results suggest that DPP4 knockout can improve the spatial learning and memory ability of AD model mice. 2.2 Effects of DPP4 knockout on the expression levels of Caspase-8 and pyroptosis-related proteins in the hippocampus As shown in Fig. 2 , the hippocampal Caspase-8 protein level was significantly greater in the AD group than in the control group ( P < 0.05). There was no difference in the DPP4 −/− group compared with the control group, while the DPP4 −/− + AD group exhibited significantly lower levels of the Caspase-8 protein than did the AD group ( P < 0.05). In addition, the expression levels of NLRP3, Caspase-1, GSDMD, IL-18 and IL-1β in the AD group were significantly greater than those in the control group ( P < 0.05), while the expression levels of the above pyroptosis-related proteins were significantly lower in the DPP4 −/− + AD group than in the AD group ( P < 0.05). Moreover, the expression levels of pyroptosis-related proteins, such as NLRP3, Caspase-1, and GSDMD, did not differ between the DPP4 −/− group and the control group. 2.3 Effects of DPP4 knockout on the expression of apoptosis-related proteins in the hippocampus of mice Compared with those in the control group, the hippocampal expression levels of Caspase-3 and Bax in the AD group were significantly greater, while the expression level of Bcl-2 was significantly lower (Fig. 3 ; P < 0.05). Compared with those in the AD group, the expression levels of Caspase-3 and Bax in the DPP4 −/− + AD group were significantly lower, and the expression level of Bcl-2 was significantly greater ( P < 0.05). 2.4 DPP4 inhibition reduced HT22 cell damage in an AD model The results of 2.6Hoechst 33342 and PI staining (Fig. 4 ) showed that the number of cells with membrane damage in the AD group was significantly greater than that in the control group ( P < 0.05). The number of damaged cells in the AD + Sit group was significantly lower than that in the AD group ( P < 0.05), indicating that the DPP4 inhibitor could improve the cell death induced by Aβ 25−35 . 2.5 DPP4 inhibition affects the expression of Caspase-8 Figure 5 Immunofluorescence staining results showed that the average fluorescence intensity of Caspase-8 in the AD group was significantly greater than that in the other groups ( P < 0.01), while the intensity in the AD + Sit group was significantly lower than that in the AD group ( P < 0.01). These results indicated that the DPP4 inhibitor could reduce the expression of Caspase-8. 2.6 Effect of DPP4 inhibition on the expression of Caspase-8 and pyroptosis-related proteins in HT22 cells As shown in Fig. 6 , the expression level of Caspase-8 in the AD group was significantly greater than that in the control group, and there was no difference in the expression level of Caspase-8 between the control + Sit group and the control group. However, Sit (a DPP4 inhibitor) significantly reversed the increase in the expression level of the Caspase-8 protein in the AD group. Similarly, the expression levels of NLRP3, Caspase-1, GSDMD, IL-18, and IL-1β in the AD group were significantly greater than those in the control group ( P < 0.05). Compared with those in the AD group, the expression levels of NLRP3, Caspase-1, GSDMD, IL-18, and IL-1β in the AD + Sit group were significantly lower ( P < 0.05). 2.7 Effect of DPP4 inhibition on the expression of apoptosis-related proteins in HT22 cells As shown in Fig. 7 , the expression levels of Caspase-3 and Bax in the AD group were significantly greater than those in the control group. Compared with those in the AD group, the expression levels of Caspase-3 and Bax in the AD + Sit group were significantly lower ( P < 0.05), and the AD + Sit treatment significantly reversed the decrease in the expression of the antiapoptotic protein Bcl-2 in the AD group ( P < 0.05). 2.8 DPP4 inhibition affects pyroptosis through Caspase-8 As shown in Fig. 8 , compared with those in the control group, the AD group had significantly greater NLRP3 and Caspase-1 levels ( P < 0.01), and C8i did not change the expression levels of NLRP3 or Caspase-1 in normal cells. Compared with those in the AD group, the expression levels of NLRP3 and Caspase-1 in the AD + Sit group were significantly lower ( P < 0.01, P < 0.05), while the AD + Sit + C8i group had no significant changes in the expression levels of these two proteins compared with those in the AD + Sit group. These results further suggested that the DPP4 inhibitor inhibited pyroptosis induced by the AD model through regulating Caspase-8. 3. Discussion The aim of this study was to investigate the effect of central DPP4 on spatial learning and memory function and the underlying mechanism in the AD mouse model. The main findings of this study are as follows: (1) DPP4 knockout can ameliorate spatial learning and memory dysfunction in AD model mice; (2) DPP4 knockout or inhibition significantly reduces the expression of Caspase-8 in hippocampal tissues and HT22 cells in the AD model; (3) DPP4 knockout or inhibition inhibit pyroptosis and apoptosis in hippocampal tissues and HT22 cells in the AD model. (4) DPP4 knockout inhibited neuronal death by regulating Caspase-8 in AD model mice. Dipeptidyl peptidase-4 (DPP4), a serine protease, is a membrane-bound extracellular peptidase. It plays an important role by cleaving a large number of biomolecules (such as growth factors, cytokines, and glucagon-like peptides) under pathophysiological conditions. DPP4 is widely expressed on the cell surface of tissues such as the intestine, liver, bone marrow and brain, and soluble DPP4 without a transmembrane domain can be detected in plasma and cerebrospinal fluid[ 13 ]. In recent years, it has been reported that the increased activity of DPP4 is related to the cognitive dysfunction caused by neurodegenerative diseases such as AD. A significant increase in DPP4 activity has also been found in the brains of sporadic AD patients[ 4 ], and the use of DPP4 inhibitor therapy can ameliorate the cognitive impairment of subjects[ 14 ], decreasing the risk of AD[ 15 ]. In the present study, DPP4 knockout was used to confirm that DPP4 was involved in cognitive impairment in AD mice. DPP4 knockout significantly improved spatial learning and memory dysfunction in AD mice and was accompanied by significantly increased expression levels of the learning and memory-related proteins BDNF and CREB. DPP4 can cleave GLP-1, stromal-derived factor-1 (SDF-1), neuropeptide Y and other substrates and participate in related signalling pathways to regulate the central nervous system in vivo. At present, studies on the central effect of DPP4 have focused mostly on GLP-1, which is believed to act on the NLRP3 inflammasome through the P-AMPK/SIRT-1 and p38 MAPK/NF-κB pathways[ 16 , 17 ]. However, it has also been reported that GLP-1 receptor agonists do not improve AD-like pathological changes[ 5 ]. This finding suggested that there may be a non-GLP-1-dependent pathway involved. Programmed cell death is an active extinction process that occurs to maintain the homeostasis of the internal environment when cells receive internal or external stimuli. The process mainly includes apoptosis, pyroptosis, autophagy and other processes. In neurodegenerative diseases such as AD, stimulation, such as mitochondrial damage in the central nervous system caused by abnormal protein aggregation, will overactivate cell death, causing neuronal degeneration and cognitive decline. Recently, Nature reported that Caspase-8 may be the molecular switch for pyroptosis and apoptosis[ 6 ]. In the present study, Western blot and immunofluorescence analyses revealed that DPP4 knockout or inhibition inhibited Caspase-8 expression and promoted pyroptosis and apoptosis in neurons in the AD model. However, the use of the Caspase-8 inhibitor did not further decrease the expression of NLRP3 or Caspase-1 in the AD model cells treated with the DPP4 inhibitor, suggesting that DPP4 knockout improves cognitive dysfunction in AD model mice, which may be related to DPP4 inhibiting pyroptosis and apoptosis in AD model cells by regulating Caspase-8. It has been suggested that Caspase-8 promotes the posttranslational activation of the NLRP3 inflammasome and that Caspase-8 is required for Caspase-1 activation and production of IL-1β and IL-18[ 18 ]. Recent studies have suggested that pyroptosis is closely related to the pathogenesis of AD[ 19 ], and recently, Cell reported[ 12 ] that inhibiting the pyroptosis pathway may constitute a breakthrough in the molecular mechanism of AD treatment. Moreover, the expression level of Caspase-1 in the brains of APP/PS1 transgenic mice increased. Aβ induces the expression of the NLRP3/Caspase-1 signalling pathway in cortical neurons in vitro, leading to pyroptosis[ 20 ]. In the present study, the pyroptosis-related proteins NLRP3, Caspase-1 and GSDMD were significantly increased in the hippocampus and neurons in the AD model, leading to the production of IL-18 and IL-1β. However, DPP4 gene knockout or drug inhibition in AD models significantly reduced the expression of Caspase-1, GSDMD and other related proteins and inhibited classical pyroptosis. Pyroptosis is a highly proinflammatory programmed death process, and the persistent excessive inflammatory response plays an important role in the occurrence and development of AD. Studies have shown that DPP4 induces the differentiation of macrophages to the M1 phenotype, which is involved in affecting the inflammatory response[ 21 ], and that DPP4 inhibition significantly reduces inflammatory marker levels in the hippocampus of ischaemic rats[ 9 ]. The downregulation of the antiapoptotic protein Bcl-2 and upregulation of the proapoptotic proteins Bax and Caspase-3 were also observed in the hippocampi of AD model mice and in vitro-cultured neurons. This finding is consistent with the results of previous studies[ 22 , 23 ], which showed that a large number of neurons undergo apoptosis in the brain in AD, especially in the cortex and hippocampus, and impair cognitive function over time[ 24 ]. In the present study, DPP4 knockout or inhibition significantly reduced the expression of the Bax and Caspase-3 proteins and increased the expression of Bcl-2 in the mouse hippocampus and in vitro neuronal cells from the AD model, suggesting that DPP4 knockout or inhibition may inhibit the occurrence of apoptosis caused by Caspase-8 in the AD model. Ma et al. also reported that the DPP4 inhibitor vildagliptin could reduce hippocampal neuron apoptosis and ameliorate memory deficits in AD model rats[ 25 ]. The present study suggested that DPP4 is involved in the pathological process of AD and affects cognitive impairment. DPP4 gene knockout can ameliorate spatial learning and memory impairments in AD model mice via a mechanism involving the regulation of neuronal apoptosis and classical pyroptosis via the inhibition of Caspase-8 (Fig. 9 ). However, the target and effect of DPP4 on Caspase-8 were not explored in this study, and further studies are needed. 4. Materials and methods 4.1 Experimental animals and grouping C57BL/6J wild-type mice were purchased from Weitong Lihua Laboratory Animal Technology Co., Ltd. (Beijing, China). DPP4 −/− mice were provided by the Laboratory Animal Research Center of Yanbian University; all the mice were 7-week-old males weighing 25–30 g. Mice were randomly divided into the C57BL/6J sham operation group (control group), DPP4 −/− sham operation group (DPP4 −/− group), model group (AD group) and DPP4 −/− model group (DPP4 −/− +AD group), with 10 mice in each group. The feeding environment was a sterile environment at room temperature (26 ℃), humidity (60%), light-dark cycle (12 h), and free access to food and water. This study was approved by the Committee of Animal Care of Jiaxing University (Permit Number: JUMC2023-142). All methods were performed in accordance with the relevant guidelines and regulations, as well as in accordance with the ARRIVE guidelines. 4.2 Establishment of an animal model of AD AD model was established in mice by brain localization injection. The right lateral ventricle was located (AP: -0.5 mm; ML: 1.1 mm; DV: -2 mm) using a mouse brain stereotaxic instrument (RWD Life Science, Shenzhen, China). The needle was slowly inserted, and 5 µl of 1 mM/mL Aβ 25−35 solution was microinjected (MedChemExpress, Shanghai, China), then the needle was slowly withdrawn, and the skin was sutured. The sham operation group was operated on in the same way and microinjected with the same amount of artificial cerebrospinal fluid. The postoperative recovery time was one week. 4.3 Morris water maze test The Morris water maze (MWM) test was carried out in a circular pool with a diameter of 120 cm and a height of 40 cm. The water temperature of the pool was controlled at 22 ± 2°C, and an escape platform with a diameter of 9 cm was placed inside the platform. The MWM test was divided into a navigation localization test and a space exploration experiment. In the positioning navigation experiment, the mice were trained to find the platform within a limited time of 60 s. If the mice successfully found the platform and stayed for 5 s or more, the time to find the platform was recorded as the escape latency. If the mice did not find the platform, they were guided to find the platform and stayed there for at least 15 s, and the escape latency was recorded as 60 s. Each mouse was trained 3 times, each time starting from a different quadrant of the pool. In the spatial exploration experiment, the platform was removed. The mice were placed from the quadrant opposite the platform to record their swimming path, swimming speed and distance, number of original platform area crossings and time ratio in the target quadrant within 120 s. 4.4 Western blot Hippocampal tissue and cells were lysed with RIPA lysate, and protein concentrations were determined using a BCA kit (Beyotime, Shanghai, China). The samples were subjected to electrophoresis at 65 V for 30 min, 85 V for 90 min, and 100 V for 1-1.5 h. Rabbit primary antibodies against BDNF, CREB, NLRP3, Caspase-1, GSDMD, IL-18, IL-1β, Caspase-3, Bcl-2, Bax (1:1500; ABclonal, Wuhan, China), Caspase-8 (1:1500; Cell Signaling Technology, MA, USA) and GAPDH (1:1500; Proteintech, Chicago, USA) were incubated overnight at 4°C after blocking with 5% skim milk for 2 h. Goat anti-rabbit IgG (H + L)-HRP secondary antibody (1:3000; Proteintech) was incubated for 2 h at room temperature. Finally, an enhanced chemiluminescence (ECL) kit (Beyotime) and ImageJ software were used to quantify the protein expression levels. The experiment was repeated three times, and GAPDH was used as the internal reference protein. 4.5 Cell culture and treatment Mouse hippocampal HT22 cells were cultured in DMEM supplemented with 10% foetal bovine serum (Biochannel, Nanjing, China) at 37°C in a 5% CO 2 incubator. Cells were treated with 40 µM Aβ 25−35 (MedChemExpress) for 24 h to establish an AD cell model. The DPP4 inhibitor sitagliptin (Sit) (MedChemExpress) was also used for 24 h, and the optimal concentration was determined by the MTT assay to be 0.25 mM. The cells were divided into a normal cell group (control group), a DPP4 inhibitor treatment group (control + Sit group), an AD model group (AD group), and an AD model with the DPP4 inhibitor treatment group (AD + Sit group). 4.6 Hoechst 33342 and PI staining After treatment with Aβ 25−35 and Sit, 200 µL of Hoechst 33342 staining solution (Beyotime) was added to each well. After 20 min of incubation in the dark, 100 µL of 15 µM PI staining solution (Biosharp, Anhui, China) was added to each well for another 10 min of incubation. After washing with PBS, the cells were observed under an inverted fluorescence microscope. Three different fields were taken, and the cell death rate in each field was calculated with ImageJ software (cell death rate = number of PI-positive cells/number of Hoechst 33342-positive cells×100%). The experiment was repeated three times, after which the average cell death rate was calculated. 4.7 Immunofluorescence staining The cells were treated with 4% paraformaldehyde (Biosharp) for 20 min and 0.3% Triton X-100 (Solarbio, Beijing, China) for 10 min, blocked with 5% goat serum for 30 min and incubated with the primary antibody against Caspase-8 (1:200; ABclonal) overnight at 4°C. IgG (H + L) Fluor488 (1:500; Beyotime) was incubated at room temperature for 2 hours, after which the antifade mounting medium containing DAPI (Solarbio) was added. An inverted fluorescence microscope was used to observe and take pictures, and the average fluorescence intensity of Caspase-8 was calculated by ImageJ software. The experiment was repeated three times, and the mean value was calculated. 4.8 Inhibition of Caspase-8 and detection of pyroptosis proteins The cells in the corresponding experimental groups were pretreated with the 20 µM Caspase-8 inhibitor (C8i) Z-LETD-FMK (MedChemExpress) for 24 hours and then treated with Aβ 25−35 and/or Sit for 24 hours. The expression levels of the pyroptosis-related proteins NLRP3 and Caspase-1 were detected via Western blot. 4.9 Statistical analysis All experiments were completely randomized design, all data were expressed as the mean ± standard deviation (x ± s), SPSS 25.0 software was used for statistical analysis, GraphPad Prism 8.0 software was used to draw statistical maps, P < 0.05 was considered statistically significant. Declarations Conflicts of interest The authors declare that they have no conflicts of interest to disclose with respect to this manuscript. Funding This work was funded by the National Natural Science Foundation of China (No. 82204837 and 81760207) and Natural Science Foundation of Zhejiang Province (No. LQ23H290004). Author Contribution XYW performed experiments and data collection, analyzed and discussed the data, and contributed to the manuscript's composition;LC, KW and YC performed experiments and collected data and analyzed and discussed the data; XWC and ZH managed and designed the study, HYJ handled the funding and wrote the manuscript, which was revised and approved by all authors. Data Availability All data used to support the findings of this study are included within the article. All data used to support the findings of this study are available from the corresponding author upon request. References Lambeir, A.M.; Durinx, C.; Scharpe, S.; De Meester, I. Dipeptidyl-peptidase IV from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme DPP IV. 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Studies on the Neuroprotection of Osthole on Glutamate-Induced Apoptotic Cells and an Alzheimer's Disease Mouse Model via Modulation Oxidative Stress. Appl Biochem Biotechnol 2020, 190 , 634–644, doi: 10.1007/s12010-019-03101-2 . Morley, J.E.; Farr, S.A.; Nguyen, A.D. Alzheimer Disease. Clin Geriatr Med 2018, 34 , 591–601, doi: 10.1016/j.cger.2018.06.006 . Ma, Q.H.; Jiang, L.F.; Mao, J.L.; Xu, W.X.; Huang, M. Vildagliptin prevents cognitive deficits and neuronal apoptosis in a rat model of Alzheimer's disease. Mol Med Rep 2018, 17 , 4113–4119, doi: 10.3892/mmr.2017.8289 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-4244824","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":294295557,"identity":"ea8a4c28-ff7f-47b5-a667-95d0d5793f7b","order_by":0,"name":"XinYi Wang","email":"","orcid":"","institution":"Jiaxing University Medical College","correspondingAuthor":false,"prefix":"","firstName":"XinYi","middleName":"","lastName":"Wang","suffix":""},{"id":294295558,"identity":"61c16e87-2e0c-48e2-9bae-eb6b8cbc5343","order_by":1,"name":"Li Chen","email":"","orcid":"","institution":"Northeast Yunnan Regional Central Hospital, Yunnan PR","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Chen","suffix":""},{"id":294295559,"identity":"6cb38870-ba47-457d-a3f6-b5b1c0ef632e","order_by":2,"name":"Ke Wang","email":"","orcid":"","institution":"Jiaxing University Medical College","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Wang","suffix":""},{"id":294295560,"identity":"50ca7a50-b825-4664-9e52-1208a2502e44","order_by":3,"name":"Yue Chen","email":"","orcid":"","institution":"Jiaxing University Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Chen","suffix":""},{"id":294295561,"identity":"a500adaa-9402-414c-a2e5-57d1adac98e9","order_by":4,"name":"Zhi He","email":"","orcid":"","institution":"Jiaxing University Medical College","correspondingAuthor":false,"prefix":"","firstName":"Zhi","middleName":"","lastName":"He","suffix":""},{"id":294295562,"identity":"f00125e7-db48-4ab5-939c-e12d3fde162d","order_by":5,"name":"XianWu Cheng","email":"","orcid":"","institution":"Yanbian University Hospital, Jilin PR","correspondingAuthor":false,"prefix":"","firstName":"XianWu","middleName":"","lastName":"Cheng","suffix":""},{"id":294295563,"identity":"cd6516c1-0731-43d4-8f0f-179be72109ef","order_by":6,"name":"Haiying Jiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYBACAwaGhANAur6fvfngAwYemCARWhhn9hxLNiBWCxgwbpiRYyaBJogdmEskPDzwc0ctswHPGbPKHzKHExvYm7dJMNTcwanFckZCwsHeM8fZzNnbym5I8KQlNvAcK5NgOPYMt8NuJCQc4G07xmPZc3jbDQMem8QGCaALGRsO49Vy8G/bMQkgw6wggUcisUH+DWEth3nbagwMbqSYMRwA28JDQMuZBwmHZdsOJEgCA1mygSfNuI0nrdgi4RgeLcdzkj++batL4AdG5cefPYdl+9kPb7zxoQa3FgaBnAQgCVXA2MPAwAZiJODWwMDAf/wAkKyD8n7gUzoKRsEoGAUjFQAAETZgDQ6PYIoAAAAASUVORK5CYII=","orcid":"","institution":"Jiaxing University Medical College","correspondingAuthor":true,"prefix":"","firstName":"Haiying","middleName":"","lastName":"Jiang","suffix":""}],"badges":[],"createdAt":"2024-04-10 03:29:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4244824/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4244824/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55331993,"identity":"12cf71d5-582f-45d1-9f2a-17955efe8987","added_by":"auto","created_at":"2024-04-25 20:06:51","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":73881,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of DPP4 knockout on learning and memory function in AD model mice.A: the escape latency changes of each group in the localization navigation experiment; B: Typical images of swimming path in each group during the spatial exploration experiment. C: the number of the original platform area crossing and the time ratio in the target quadrant in the space exploration experiment; D: swimming speed and total distance of each group in the space exploration experiment; E: Typical plots and corresponding statistical plots of CREB and BDNF protein expression in the hippocampus. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, AD vs. Control; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05,\u003csup\u003e ##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, DPP4\u003csup\u003e-/-\u003c/sup\u003e + AD vs. AD。\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4244824/v1/252937b28d93b9691072a8f9.jpg"},{"id":55331992,"identity":"53ad1c30-8ef3-49ab-bc9a-7477a9cf14f5","added_by":"auto","created_at":"2024-04-25 20:06:51","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":64300,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of DPP4 knockout on the expression levels of Caspase-8 and pyroptosis-related proteins in the hippocampus. A: Typical pattern of Caspase-8 and pyroptosis protein bands; B: Statistical plot of Caspase-8 and pyroptosis protein levels. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, AD vs. Control; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05,\u003csup\u003e ##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01,\u003csup\u003e \u003c/sup\u003e\u0026nbsp;DPP4\u003csup\u003e-/-\u003c/sup\u003e + AD vs. AD 。\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4244824/v1/e78b6b3d1ac3232a1c066c2a.jpg"},{"id":55331994,"identity":"a89d1e04-7ad4-4e3b-9757-fc160ee849b9","added_by":"auto","created_at":"2024-04-25 20:06:51","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":39010,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of DPP4 knockout on expression levels of apoptosis-related proteins within the hippocampus. A: Typical pattern of apoptotic protein bands; B: Statistical plot of apoptotic protein levels.\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, AD vs. Control; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, DPP4\u003csup\u003e-/-\u003c/sup\u003e + AD vs. AD。\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4244824/v1/2702af0fc90c5a536cb7c5cd.jpg"},{"id":55331995,"identity":"ca7c62ce-8151-4bee-ba55-21b4a0a00496","added_by":"auto","created_at":"2024-04-25 20:06:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":34498,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of DPP4 inhibitor on cell death rate. Scale bar = 50 µm, the magnification of merge is 200×. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, AD vs. Control; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, AD + Sit vs. AD.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4244824/v1/8d99644200bae552e04c746f.jpg"},{"id":55332104,"identity":"057f55f1-80ab-40a9-afaf-0b8b084deecf","added_by":"auto","created_at":"2024-04-25 20:14:51","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":46572,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of DPP4 inhibitor on Caspase-8 expression in cells. Scale bar = 50 µm, the magnification of merge is 200×. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, AD vs. Control; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, AD + Sit vs. AD。\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4244824/v1/0725633f9ba01263b30dfa1b.jpg"},{"id":55332219,"identity":"f48468f2-27f1-4bfd-bcd8-bbb75a78cb1a","added_by":"auto","created_at":"2024-04-25 20:22:51","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":63085,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of DPP4 inhibitor on the expression levels of Caspase-8 and pyroptosis-related proteins. A: Typical pattern of Caspase-8 and pyroptosis protein bands; B: Statistical plot of Caspase-8 and pyroptosis protein levels.\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, AD vs. Control; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05,\u003csup\u003e ##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, AD + Sit vs.\u003csup\u003e \u003c/sup\u003eAD.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4244824/v1/77017795371a8256c9bf9130.jpg"},{"id":55331999,"identity":"8690612e-2792-4c89-84d3-04486024ac60","added_by":"auto","created_at":"2024-04-25 20:06:51","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":37560,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of DPP4 inhibitor on the expression levels of apoptosis-related proteins in HT22 cells. A: Typical pattern of apoptotic protein bands; B: Statistical plot of apoptotic protein levels. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, AD vs. Control; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, AD + Sit vs.\u003csup\u003e \u003c/sup\u003eAD.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4244824/v1/11cb618d0d920bea5250e6b2.jpg"},{"id":55331996,"identity":"58b1d0a0-b1f6-4562-bfe2-14d1b5ce4c73","added_by":"auto","created_at":"2024-04-25 20:06:51","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":42593,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of DPP4 inhibitor on pyroptotic protein expression content through Caspase-8. A: Typical pattern of pyroptotic protein bands; B: Statistical plot of pyroptotic protein content. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01，AD vs. Control or AD vs. AD + Sit and AD+C8i.\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4244824/v1/ffe06e694d9800e58a57bdda.jpg"},{"id":55332001,"identity":"0b3f7459-7efc-451f-aea2-1a1acf8c6e45","added_by":"auto","created_at":"2024-04-25 20:06:51","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":68492,"visible":true,"origin":"","legend":"\u003cp\u003eDPP4 affects the learning and memory ability of mice by regulating apoptosis and pyroptosis of neurons through Caspase-8.\u003c/p\u003e","description":"","filename":"Picture9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4244824/v1/7fdc890fb7bd0807e4eea811.jpg"},{"id":60874158,"identity":"8a346d4f-41c6-46e9-9df1-408061e2677b","added_by":"auto","created_at":"2024-07-23 05:27:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1089462,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4244824/v1/6f5935d7-219a-4534-ba48-699c6005a081.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dipeptidyl Peptidase 4 mediated Caspase-8 Affects Cognitive Impairment in Mice with Alzheimer's Disease","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDipeptidyl peptidase 4 (DPP4), also known as CD26, is widely expressed on the surface of a variety of cells. Studies have shown that DPP4 is expressed on neurons in the foetal brain, and a small amount of soluble DPP4 is present in the cerebrospinal fluid[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It has also been reported that the DPP4 gene is significantly correlated with hippocampal volume[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Cross-sectional studies of a population of Chinese elderly nondiabetic individuals (over 60 years old) showed that increased plasma DPP4 activity was positively correlated with cognitive impairment and negatively correlated with MoCA scores[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Significant increases in DPP4 activity have also been found in the brains of sporadic AD patients[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], suggesting that DPP4 may be associated with cognitive dysfunction.\u003c/p\u003e \u003cp\u003eSeveral basic and clinical studies have shown that DPP4 inhibitors can play a neuroprotective role in the central nervous system. However, the specific mechanism through which DPP4 affects cognitive function is still unclear. As a multifunctional enzyme, DPP4 cleaves a variety of substrates and participates in various physiological and pathological activities. Most studies suggest that DPP4 inhibitors play a neuroprotective role by inhibiting the degradation of the main substrate glucagon-like peptide-1 (GLP-1), but one study reported that DPP4 inhibitors can improve AD-like pathological changes, while the effect of GLP-1 analogues is not significant[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], suggesting that there may be a GLP-1-independent mechanism by which DPP4 inhibitors improve AD.\u003c/p\u003e \u003cp\u003eProgressive neuronal death is a major feature of AD and a direct cause of cognitive impairment. Recently, Nature reported that Caspase-8 is the molecular switch for apoptosis, pyroptosis and necroptosis[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. One study reported that DPP4 inhibitors can reduce the expression of Caspase-8 and inhibit cell apoptosis induced by insulin resistance[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Inhibition of DPP4 can ameliorate cognitive dysfunction in vascular dementia model rats through antiapoptotic effects[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. DPP4 inhibitors can also significantly reduce TNF-α and IL-6 levels in the hippocampus of rats[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], thereby inhibiting the neuroinflammatory response. As a highly proinflammatory form of programmed cell death[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], most scholars believe that inhibiting the pyroptosis pathway may constitute a breakthrough in the molecular mechanism of AD treatment[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, most of the current central studies on DPP4 have focused only on unilateral apoptotic or pyroptotic effects[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and it is not clear whether DPP4 may affect learning and memory dysfunction in AD through the regulation of Caspase-8-mediated programmed cell death.\u003c/p\u003e \u003cp\u003eIn this study, DPP4 knockout (DPP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) mice were used to establish an AD model, and the neuromolecular mechanism by which DPP4 inhibition improves learning and memory dysfunction in AD model mice at the global and cellular levels was explored. These findings could lead to improved drug guidance, such as the use of DPP4 inhibitors, for the treatment of AD combined with diabetes.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Effects of DPP4 knockout on learning and memory function in AD model mice\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, the escape latency of the four groups of mice gradually decreased as the number of training days increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), and there was no significant difference between the control group and the DPP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e group. Beginning on the third day of training, the escape latency of the DPP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e + AD group was significantly shorter than that of the AD group. On the fifth day, the escape latency of the DPP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e + AD group was 13.47\u0026thinsp;\u0026plusmn;\u0026thinsp;7.91 seconds, which was significantly different from that of the AD group (34.73\u0026thinsp;\u0026plusmn;\u0026thinsp;7.07 seconds) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eThe spatial exploration experiment showed that the exploratory swimming paths of the control group, DPP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e group and DPP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e + AD group were concentrated in the target quadrant, while the paths of the AD group were scattered (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC shows that the number of original platform areas crossed and the time spent in the target quadrant in the AD group were significantly lower than those in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while the number of platform crossings in the DPP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e + AD group (3.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87) was significantly different from that in the AD group (1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, there was a significant difference in the time spent in the target quadrant between the DPP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e + AD group and the AD group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). To exclude the possibility of differences in learning and memory function caused by motor dysfunction, the swimming speed and total swimming distance of each group were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), and there was no significant difference among the groups (\u003cem\u003eP\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05). In addition, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, the expression levels of the learning-memory-related proteins CREB and BDNF in the hippocampus of the AD group were significantly lower than those in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the expression levels of these two proteins in the DPP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e + AD group were significantly greater than those in the AD group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results suggest that DPP4 knockout can improve the spatial learning and memory ability of AD model mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cp\u003e \u003cb\u003e2.2 Effects of DPP4 knockout on the expression levels of Caspase-8 and pyroptosis-related proteins in the hippocampus\u003c/b\u003e \u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the hippocampal Caspase-8 protein level was significantly greater in the AD group than in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was no difference in the DPP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e group compared with the control group, while the DPP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e + AD group exhibited significantly lower levels of the Caspase-8 protein than did the AD group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, the expression levels of NLRP3, Caspase-1, GSDMD, IL-18 and IL-1β in the AD group were significantly greater than those in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the expression levels of the above pyroptosis-related proteins were significantly lower in the DPP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e + AD group than in the AD group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, the expression levels of pyroptosis-related proteins, such as NLRP3, Caspase-1, and GSDMD, did not differ between the DPP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e group and the control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Effects of DPP4 knockout on the expression of apoptosis-related proteins in the hippocampus of mice\u003c/h2\u003e \u003cp\u003eCompared with those in the control group, the hippocampal expression levels of Caspase-3 and Bax in the AD group were significantly greater, while the expression level of Bcl-2 was significantly lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared with those in the AD group, the expression levels of Caspase-3 and Bax in the DPP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e + AD group were significantly lower, and the expression level of Bcl-2 was significantly greater (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4 DPP4 inhibition reduced HT22 cell damage in an AD model\u003c/h2\u003e \u003cp\u003eThe results of 2.6Hoechst 33342 and PI staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) showed that the number of cells with membrane damage in the AD group was significantly greater than that in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The number of damaged cells in the AD\u0026thinsp;+\u0026thinsp;Sit group was significantly lower than that in the AD group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that the DPP4 inhibitor could improve the cell death induced by Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5 DPP4 inhibition affects the expression of Caspase-8\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e Immunofluorescence staining results showed that the average fluorescence intensity of Caspase-8 in the AD group was significantly greater than that in the other groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while the intensity in the AD\u0026thinsp;+\u0026thinsp;Sit group was significantly lower than that in the AD group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results indicated that the DPP4 inhibitor could reduce the expression of Caspase-8.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Effect of DPP4 inhibition on the expression of Caspase-8 and pyroptosis-related proteins in HT22 cells\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the expression level of Caspase-8 in the AD group was significantly greater than that in the control group, and there was no difference in the expression level of Caspase-8 between the control\u0026thinsp;+\u0026thinsp;Sit group and the control group. However, Sit (a DPP4 inhibitor) significantly reversed the increase in the expression level of the Caspase-8 protein in the AD group. Similarly, the expression levels of NLRP3, Caspase-1, GSDMD, IL-18, and IL-1β in the AD group were significantly greater than those in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared with those in the AD group, the expression levels of NLRP3, Caspase-1, GSDMD, IL-18, and IL-1β in the AD\u0026thinsp;+\u0026thinsp;Sit group were significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Effect of DPP4 inhibition on the expression of apoptosis-related proteins in HT22 cells\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the expression levels of Caspase-3 and Bax in the AD group were significantly greater than those in the control group. Compared with those in the AD group, the expression levels of Caspase-3 and Bax in the AD\u0026thinsp;+\u0026thinsp;Sit group were significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the AD\u0026thinsp;+\u0026thinsp;Sit treatment significantly reversed the decrease in the expression of the antiapoptotic protein Bcl-2 in the AD group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.8 DPP4 inhibition affects pyroptosis through Caspase-8\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, compared with those in the control group, the AD group had significantly greater NLRP3 and Caspase-1 levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and C8i did not change the expression levels of NLRP3 or Caspase-1 in normal cells. Compared with those in the AD group, the expression levels of NLRP3 and Caspase-1 in the AD\u0026thinsp;+\u0026thinsp;Sit group were significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the AD\u0026thinsp;+\u0026thinsp;Sit\u0026thinsp;+\u0026thinsp;C8i group had no significant changes in the expression levels of these two proteins compared with those in the AD\u0026thinsp;+\u0026thinsp;Sit group. These results further suggested that the DPP4 inhibitor inhibited pyroptosis induced by the AD model through regulating Caspase-8.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThe aim of this study was to investigate the effect of central DPP4 on spatial learning and memory function and the underlying mechanism in the AD mouse model. The main findings of this study are as follows: (1) DPP4 knockout can ameliorate spatial learning and memory dysfunction in AD model mice; (2) DPP4 knockout or inhibition significantly reduces the expression of Caspase-8 in hippocampal tissues and HT22 cells in the AD model; (3) DPP4 knockout or inhibition inhibit pyroptosis and apoptosis in hippocampal tissues and HT22 cells in the AD model. (4) DPP4 knockout inhibited neuronal death by regulating Caspase-8 in AD model mice.\u003c/p\u003e \u003cp\u003eDipeptidyl peptidase-4 (DPP4), a serine protease, is a membrane-bound extracellular peptidase. It plays an important role by cleaving a large number of biomolecules (such as growth factors, cytokines, and glucagon-like peptides) under pathophysiological conditions. DPP4 is widely expressed on the cell surface of tissues such as the intestine, liver, bone marrow and brain, and soluble DPP4 without a transmembrane domain can be detected in plasma and cerebrospinal fluid[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In recent years, it has been reported that the increased activity of DPP4 is related to the cognitive dysfunction caused by neurodegenerative diseases such as AD. A significant increase in DPP4 activity has also been found in the brains of sporadic AD patients[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and the use of DPP4 inhibitor therapy can ameliorate the cognitive impairment of subjects[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], decreasing the risk of AD[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In the present study, DPP4 knockout was used to confirm that DPP4 was involved in cognitive impairment in AD mice. DPP4 knockout significantly improved spatial learning and memory dysfunction in AD mice and was accompanied by significantly increased expression levels of the learning and memory-related proteins BDNF and CREB. DPP4 can cleave GLP-1, stromal-derived factor-1 (SDF-1), neuropeptide Y and other substrates and participate in related signalling pathways to regulate the central nervous system in vivo. At present, studies on the central effect of DPP4 have focused mostly on GLP-1, which is believed to act on the NLRP3 inflammasome through the P-AMPK/SIRT-1 and p38 MAPK/NF-κB pathways[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, it has also been reported that GLP-1 receptor agonists do not improve AD-like pathological changes[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This finding suggested that there may be a non-GLP-1-dependent pathway involved.\u003c/p\u003e \u003cp\u003eProgrammed cell death is an active extinction process that occurs to maintain the homeostasis of the internal environment when cells receive internal or external stimuli. The process mainly includes apoptosis, pyroptosis, autophagy and other processes. In neurodegenerative diseases such as AD, stimulation, such as mitochondrial damage in the central nervous system caused by abnormal protein aggregation, will overactivate cell death, causing neuronal degeneration and cognitive decline. Recently, Nature reported that Caspase-8 may be the molecular switch for pyroptosis and apoptosis[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In the present study, Western blot and immunofluorescence analyses revealed that DPP4 knockout or inhibition inhibited Caspase-8 expression and promoted pyroptosis and apoptosis in neurons in the AD model. However, the use of the Caspase-8 inhibitor did not further decrease the expression of NLRP3 or Caspase-1 in the AD model cells treated with the DPP4 inhibitor, suggesting that DPP4 knockout improves cognitive dysfunction in AD model mice, which may be related to DPP4 inhibiting pyroptosis and apoptosis in AD model cells by regulating Caspase-8. It has been suggested that Caspase-8 promotes the posttranslational activation of the NLRP3 inflammasome and that Caspase-8 is required for Caspase-1 activation and production of IL-1β and IL-18[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent studies have suggested that pyroptosis is closely related to the pathogenesis of AD[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and recently, Cell reported[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] that inhibiting the pyroptosis pathway may constitute a breakthrough in the molecular mechanism of AD treatment. Moreover, the expression level of Caspase-1 in the brains of APP/PS1 transgenic mice increased. Aβ induces the expression of the NLRP3/Caspase-1 signalling pathway in cortical neurons in vitro, leading to pyroptosis[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In the present study, the pyroptosis-related proteins NLRP3, Caspase-1 and GSDMD were significantly increased in the hippocampus and neurons in the AD model, leading to the production of IL-18 and IL-1β. However, DPP4 gene knockout or drug inhibition in AD models significantly reduced the expression of Caspase-1, GSDMD and other related proteins and inhibited classical pyroptosis. Pyroptosis is a highly proinflammatory programmed death process, and the persistent excessive inflammatory response plays an important role in the occurrence and development of AD. Studies have shown that DPP4 induces the differentiation of macrophages to the M1 phenotype, which is involved in affecting the inflammatory response[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and that DPP4 inhibition significantly reduces inflammatory marker levels in the hippocampus of ischaemic rats[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe downregulation of the antiapoptotic protein Bcl-2 and upregulation of the proapoptotic proteins Bax and Caspase-3 were also observed in the hippocampi of AD model mice and in vitro-cultured neurons. This finding is consistent with the results of previous studies[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], which showed that a large number of neurons undergo apoptosis in the brain in AD, especially in the cortex and hippocampus, and impair cognitive function over time[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In the present study, DPP4 knockout or inhibition significantly reduced the expression of the Bax and Caspase-3 proteins and increased the expression of Bcl-2 in the mouse hippocampus and in vitro neuronal cells from the AD model, suggesting that DPP4 knockout or inhibition may inhibit the occurrence of apoptosis caused by Caspase-8 in the AD model. Ma et al. also reported that the DPP4 inhibitor vildagliptin could reduce hippocampal neuron apoptosis and ameliorate memory deficits in AD model rats[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe present study suggested that DPP4 is involved in the pathological process of AD and affects cognitive impairment. DPP4 gene knockout can ameliorate spatial learning and memory impairments in AD model mice via a mechanism involving the regulation of neuronal apoptosis and classical pyroptosis via the inhibition of Caspase-8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). However, the target and effect of DPP4 on Caspase-8 were not explored in this study, and further studies are needed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4. Materials and methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Experimental animals and grouping\u003c/h2\u003e \u003cp\u003eC57BL/6J wild-type mice were purchased from Weitong Lihua Laboratory Animal Technology Co., Ltd. (Beijing, China). DPP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were provided by the Laboratory Animal Research Center of Yanbian University; all the mice were 7-week-old males weighing 25\u0026ndash;30 g. Mice were randomly divided into the C57BL/6J sham operation group (control group), DPP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e sham operation group (DPP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e group), model group (AD group) and DPP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e model group (DPP4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e+AD group), with 10 mice in each group. The feeding environment was a sterile environment at room temperature (26 ℃), humidity (60%), light-dark cycle (12 h), and free access to food and water. This study was approved by the Committee of Animal Care of Jiaxing University (Permit Number: JUMC2023-142). All methods were performed in accordance with the relevant guidelines and regulations, as well as in accordance with the ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Establishment of an animal model of AD\u003c/h2\u003e \u003cp\u003eAD model was established in mice by brain localization injection. The right lateral ventricle was located (AP: -0.5 mm; ML: 1.1 mm; DV: -2 mm) using a mouse brain stereotaxic instrument (RWD Life Science, Shenzhen, China). The needle was slowly inserted, and 5 \u0026micro;l of 1 mM/mL Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e solution was microinjected (MedChemExpress, Shanghai, China), then the needle was slowly withdrawn, and the skin was sutured. The sham operation group was operated on in the same way and microinjected with the same amount of artificial cerebrospinal fluid. The postoperative recovery time was one week.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Morris water maze test\u003c/h2\u003e \u003cp\u003eThe Morris water maze (MWM) test was carried out in a circular pool with a diameter of 120 cm and a height of 40 cm. The water temperature of the pool was controlled at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, and an escape platform with a diameter of 9 cm was placed inside the platform. The MWM test was divided into a navigation localization test and a space exploration experiment. In the positioning navigation experiment, the mice were trained to find the platform within a limited time of 60 s. If the mice successfully found the platform and stayed for 5 s or more, the time to find the platform was recorded as the escape latency. If the mice did not find the platform, they were guided to find the platform and stayed there for at least 15 s, and the escape latency was recorded as 60 s. Each mouse was trained 3 times, each time starting from a different quadrant of the pool. In the spatial exploration experiment, the platform was removed. The mice were placed from the quadrant opposite the platform to record their swimming path, swimming speed and distance, number of original platform area crossings and time ratio in the target quadrant within 120 s.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Western blot\u003c/h2\u003e \u003cp\u003eHippocampal tissue and cells were lysed with RIPA lysate, and protein concentrations were determined using a BCA kit (Beyotime, Shanghai, China). The samples were subjected to electrophoresis at 65 V for 30 min, 85 V for 90 min, and 100 V for 1-1.5 h. Rabbit primary antibodies against BDNF, CREB, NLRP3, Caspase-1, GSDMD, IL-18, IL-1β, Caspase-3, Bcl-2, Bax (1:1500; ABclonal, Wuhan, China), Caspase-8 (1:1500; Cell Signaling Technology, MA, USA) and GAPDH (1:1500; Proteintech, Chicago, USA) were incubated overnight at 4\u0026deg;C after blocking with 5% skim milk for 2 h. Goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L)-HRP secondary antibody (1:3000; Proteintech) was incubated for 2 h at room temperature. Finally, an enhanced chemiluminescence (ECL) kit (Beyotime) and ImageJ software were used to quantify the protein expression levels. The experiment was repeated three times, and GAPDH was used as the internal reference protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Cell culture and treatment\u003c/h2\u003e \u003cp\u003eMouse hippocampal HT22 cells were cultured in DMEM supplemented with 10% foetal bovine serum (Biochannel, Nanjing, China) at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. Cells were treated with 40 \u0026micro;M Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e (MedChemExpress) for 24 h to establish an AD cell model. The DPP4 inhibitor sitagliptin (Sit) (MedChemExpress) was also used for 24 h, and the optimal concentration was determined by the MTT assay to be 0.25 mM. The cells were divided into a normal cell group (control group), a DPP4 inhibitor treatment group (control\u0026thinsp;+\u0026thinsp;Sit group), an AD model group (AD group), and an AD model with the DPP4 inhibitor treatment group (AD\u0026thinsp;+\u0026thinsp;Sit group).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Hoechst 33342 and PI staining\u003c/h2\u003e \u003cp\u003eAfter treatment with Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and Sit, 200 \u0026micro;L of Hoechst 33342 staining solution (Beyotime) was added to each well. After 20 min of incubation in the dark, 100 \u0026micro;L of 15 \u0026micro;M PI staining solution (Biosharp, Anhui, China) was added to each well for another 10 min of incubation. After washing with PBS, the cells were observed under an inverted fluorescence microscope. Three different fields were taken, and the cell death rate in each field was calculated with ImageJ software (cell death rate\u0026thinsp;=\u0026thinsp;number of PI-positive cells/number of Hoechst 33342-positive cells\u0026times;100%). The experiment was repeated three times, after which the average cell death rate was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.7 Immunofluorescence staining\u003c/h2\u003e \u003cp\u003eThe cells were treated with 4% paraformaldehyde (Biosharp) for 20 min and 0.3% Triton X-100 (Solarbio, Beijing, China) for 10 min, blocked with 5% goat serum for 30 min and incubated with the primary antibody against Caspase-8 (1:200; ABclonal) overnight at 4\u0026deg;C. IgG (H\u0026thinsp;+\u0026thinsp;L) Fluor488 (1:500; Beyotime) was incubated at room temperature for 2 hours, after which the antifade mounting medium containing DAPI (Solarbio) was added. An inverted fluorescence microscope was used to observe and take pictures, and the average fluorescence intensity of Caspase-8 was calculated by ImageJ software. The experiment was repeated three times, and the mean value was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.8 Inhibition of Caspase-8 and detection of pyroptosis proteins\u003c/h2\u003e \u003cp\u003eThe cells in the corresponding experimental groups were pretreated with the 20 \u0026micro;M Caspase-8 inhibitor (C8i) Z-LETD-FMK (MedChemExpress) for 24 hours and then treated with Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and/or Sit for 24 hours. The expression levels of the pyroptosis-related proteins NLRP3 and Caspase-1 were detected via Western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were completely randomized design, all data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (x\u0026thinsp;\u0026plusmn;\u0026thinsp;s), SPSS 25.0 software was used for statistical analysis, GraphPad Prism 8.0 software was used to draw statistical maps, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflicts of interest to disclose with respect to this manuscript.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was funded by the National Natural Science Foundation of China (No. 82204837 and 81760207) and Natural Science Foundation of Zhejiang Province (No. LQ23H290004).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXYW performed experiments and data collection, analyzed and discussed the data, and contributed to the manuscript's composition;LC, KW and YC performed experiments and collected data and analyzed and discussed the data; XWC and ZH managed and designed the study, HYJ handled the funding and wrote the manuscript, which was revised and approved by all authors.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data used to support the findings of this study are included within the article. All data used to support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLambeir, A.M.; Durinx, C.; Scharpe, S.; De Meester, I. Dipeptidyl-peptidase IV from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme DPP IV. Crit Rev Clin Lab Sci 2003, \u003cem\u003e40\u003c/em\u003e, 209\u0026ndash;294, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/713609354\u003c/span\u003e\u003cspan address=\"10.1080/713609354\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHorgusluoglu-Moloch, E.; Risacher, S.L.; Crane, P.K.; Hibar, D.; Thompson, P.M.; Saykin, A.J.; Nho, K. Genome-wide association analysis of hippocampal volume identifies enrichment of neurogenesis-related pathways. 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Vildagliptin prevents cognitive deficits and neuronal apoptosis in a rat model of Alzheimer's disease. Mol Med Rep 2018, \u003cem\u003e17\u003c/em\u003e, 4113\u0026ndash;4119, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3892/mmr.2017.8289\u003c/span\u003e\u003cspan address=\"10.3892/mmr.2017.8289\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Dipeptidyl peptidase 4, Alzheimer's disease, Learning and memory, Caspase-8, Pyroptosis, Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-4244824/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4244824/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo investigate the effect of dipeptidyl peptidase 4 (DPP4) on cognitive impairment in Alzheimer's disease (AD), the present study used seven-week-old male C57BL/6J and DPP4 knockout mice. The AD model was induced by microinjection of Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e into the lateral ventricle. Morris water maze test showed that DPP4 knockout significantly improved the spatial learning and memory abilitoes of AD mice. Western blot results showed that DPP4 knockout increased the expression levels of BDNF, CREB and Bcl-2 in the hippocampus of AD mice while the expression levels of Caspase-8, pyroptosis-related proteins NLRP3, Caspase-1, GSDMD, 1L-18, 1L-1β and apoptosis-related proteins Caspase-3 and Bax were decreased. Similar results were observed after HT22 neurons were treated with Aβ\u003csub\u003e25\u0026minus;35\u003c/sub\u003e and DPP4 inhibitor sitagliptin (Sit). Moreover, the treatment with Caspase-8 inhibitor (Z-LETD-FMK) showed that the inhibition of Caspase-8 inhibited the expression of NLRP3 and Caspase-1 in the AD model cells, but had no further inhibitory effect under the treatment of Sit. Our results suggests that DPP4 knockout may ameliorate learning and memory dysfunction in AD model mice by regulating pyroptosis and apoptosis pathways through Caspase-8.\u003c/p\u003e","manuscriptTitle":"Dipeptidyl Peptidase 4 mediated Caspase-8 Affects Cognitive Impairment in Mice with Alzheimer's Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-25 20:06:46","doi":"10.21203/rs.3.rs-4244824/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"86ca69b0-4c75-4d2c-98c5-89c278307a65","owner":[],"postedDate":"April 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":31022133,"name":"Biological sciences/Neuroscience"},{"id":31022134,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2024-07-23T05:11:42+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-25 20:06:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4244824","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4244824","identity":"rs-4244824","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}