Levetiracetam ameliorated amyloidosis and tauopathy in mice with dexamethasone-induced Alzheimer’s disease

Levetiracetam ameliorated amyloidosis and tauopathy in mice with dexamethasone-induced 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 Research Article Levetiracetam ameliorated amyloidosis and tauopathy in mice with dexamethasone-induced Alzheimer’s disease Xiang-yu Zheng, Hai-Chen Zhang, Zhi-Wei Wei, Yu-Dan Lv, Feng-Yan Jin, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3517043/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Stress induced by glucocorticoids (GC), such as dexamethasone (DEX), has the potential to detrimentally impact the structure and function of the hippocampus and is closely associated with the development and progression of Alzheimer’s disease (AD). However, it remains uncertain whether LEV and TPM can effectively alleviate neuropathological and cognitive deficits in patients with DEX-induced AD by preserving or restoring neuronal network activities. This study aimed to investigate the mechanisms underlying the effect of DEX on AD development and progression and identify the role of NRP1 inflammasome in APP23/MAPT P301S mice. APP23/MAPT P301S mice were treated with DEX in the absence and presence of levetiracetam (LEV). After treatment, the mice were subjected to various cognitive and behavioral tests. DEX accelerated neuronal impairment by promoting the accumulation of β-amyloid protein and phosphorylation of tau in senile plaques and neurofibrillary tangles in APP23/MAPT P301S mice. Moreover, DEX significantly upregulated BACE1 and promoted the phosphorylation of cyclin-dependent kinase-5 and glycogen synthase kinase 3α/β, resulting in synaptic dystrophy and apoptosis. NLRP3 siRNA transfection showed that NLRP1 inflammasome activation is pivotal to the observed DEX effects. To counteract the adverse effects of DEX, LEV was administered to APP23/MAPT P301S mice, and it ameliorated DEX-induced AD via NLRP1-dependent mechanisms. This study underscores the detrimental impact of chronic glucocorticoid exposure on AD pathogenesis and the potential therapeutic benefits of compounds such as LEV in counteracting these effects by regulating neuroinflammation and key pathological markers. dexamethasone levetiracetam NLRP1 APP23/MAPTP301S Alzheimer’s Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Alzheimer’s disease (AD) is a neurodegenerative disorder and the leading cause of dementia and presents an increasing challenging with the rising global life expectancy. Although the primary pathological hallmarks of AD are commonly associated with the formation of senile plaques (SPs) from β-amyloid protein (Aβ) and neurofibrillary tangles formed by the aggregation of highly phosphorylated tau ( 1 ), other factors may play a role in AD development. Epidemiological evidence suggests that chronic stress is a potential risk factor for AD ( 2 ). Furthermore, preclinical data showed that prolonged administration of glucocorticoid (GC) induces Aβ deposition and tau hyperphosphorylation, contributing to synaptic dysfunction and neuronal impairment in AD ( 3 ). Recent research revealed that corticosterone-related stress can enhance the production of hippocampal Aβ 1−40 and Aβ 1−42 as well as the expression of BACE1 in APP/PS1 transgenic (Tg) mice ( 4 ). Moreover, chronic exposure to GC-related stress can cause functional and structural damage; disrupt the normal physiological function of hippocampal neurons; and cause neuronal atrophy, loss, and dysfunction in the brain ( 5 – 7 ) important for cognitive functions, including learning and memory ( 8 , 9 ). The glucocorticoid receptor (GR), a target of endogenous (corticosterone) or synthetic (dexamethasone, DEX) GC, is a widely used clinical therapeutic target ( 10 ). Impaired GR signaling exacerbates Aβ oligomers-induced AD ( 11 ). Moreover, GR dysfunction could prove highly detrimental to limbic structures (12), contributing to cognitive decline in AD. GR antagonists or selective inhibitors can potentially mitigate the effects of Aβo25-35 on the hippocampus, thereby strongly modulating GR activity as a therapeutic strategy ( 13 ). As a GR agonist, DEX induces pyroptosis by upregulating pyroptosis-related proteins, such as the NLR family pyrin domain containing 3 (NLRP3), caspase-1, and gasdermin-D (GSDMD) ( 14 ). Furthermore, downregulation of NLRP3 or GSDMD attenuates DEX-induced myotube pyroptosis and atrophy ( 14 ). However, this hypothesis should be considered cautiously, as a previous study reported that DEX could inhibit NLRP3 inflammasome activity in coronavirus disease 2019 ( 15 ). Additionally, DEX alleviates allergic airway inflammation in mice by inhibiting NLRP3 inflammasome activation ( 16 ). Similarly, intranasal curcumin combined with DEX inhibits NLRP3 inflammasome activation in lipopolysaccharide-induced asthma exacerbations ( 17 ). Given the roles of NLRP3 inflammasome in AD pathogenesis ( 18 ), it is possible that NLRP3 inflammasome will not be critical for the effects of DEX and GR on AD pathogenesis. Compared with NLRP3, NLRP1 is highly expressed in the central nervous system (CNS) and is involved in regulating the pathogenesis of various neurological diseases ( 19 ). NLRP1 inflammasome primarily comprises caspase-1 and NLRs, including apoptosis-associated speck-like protein (ASC) ( 20 , 21 ). Caspase-1 is a critical regulatory molecule that converts pre-interleukin (IL)-1β and IL-6 into mature active forms, which are released to participate in inflammatory reactions ( 21 , 22 ). More specifically, DEX significantly increased the mRNA and protein expression of NLRP1, caspase-1, ASC, and IL-1β, reducing spontaneous motor activity and exploratory behavior of male institute of cancer research mice. Moreover, the mice exhibited neurodegeneration and decreased microtubule-associated protein 2 (MAP2) levels in the frontal cortex and hippocampus CA3 region ( 23 ). In APP/PS1 Tg mice, DEX markedly increased the accumulation of Aβ 1−42 , leading to neuronal injury in primary hippocampal neurons. Furthermore, chronic DEX exposure significantly elevated the levels of reactive oxygen species and IL-1β and upregulated NLRP1 in primary hippocampal neurons of APP/PS1 mice ( 24 ). NLRP1 inhibition suppressed the accumulation of Aβ 1−42 and expression of APP, BACE1, and nicastrin (NCSTN) in APP/PS1 Tg mice ( 24 ), highlighting the pivotal mediating role of NLRP1 in the effects of DEX on AD pathogenesis. It is imperative to explore ways of mitigating the adverse effects of DEX. Based on our previous study, levetiracetam (LEV) alleviated cognitive decline in AD by ameliorating neuronal network dysfunction ( 25 ). Moreover, the therapeutic potential of LEV and topiramate (TPM) for AD has been demonstrated ( 26 ). The therapeutic effects of the antiepileptic drugs were reportedly attributed to their inhibition of histone deacetylase. Furthermore, LEV and TPM offer additional benefits, such as neuroprotective and anti-inflammatory properties ( 27 , 28 ). However, whether LEV and TPM can reverse neuropathological and cognitive deficits by preserving or restoring neuronal network activities in patients with DEX-induced AD remains uncertain. Therefore, this study aimed to elucidate the mechanisms underlying the effect of DEX on AD development and progression and identify the role of NRP1 inflammasome in APP23/MAPT P301S mice. Materials and Methods Mice and treatment protocol APP23 [B6-Tg (Thy1APP) 23SdZ] and MAPT [B6; C3- Tg (Prnp-MAPT P301S ) PS19Vle/J] mice were obtained from the Jackson Laboratory (Bar Harbor, ME, United States; Stock #030504 and #008169). The APP23 and MAPT mice carry the Swedish and P301S mutated human APP transgenes, respectively. The mice were generated from the C57BL/6 genetic background, as previously described ( 29 , 30 ). For the experimental cohort, the APP23 mice were crossbred with the MAPT transgenic mice to obtain APP23/MAPT mice. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of The First Hospital of Jilin University and adhered to the Guidelines for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health. The mice were housed in groups of five per cage in a room maintained at a temperature of 22°C ± 2°C and with a 12-h light-dark cycle. Food and water were provided ad libitum. Based on a previous study, six 5-month-old male mice per group were concurrently administered 10 mg/kg/d of DEX (Sigma-Aldrich Corp., St. Louis, MO, United States) intraperitoneally alone or with 100 mg/kg/d of LEV (Aladdin, Shanghai, China) emulsified in 0.9% phosphate-buffered saline [PBS (–)]. Male mice in the control group (n = 6) received PBS injection (–). After 3 months of treatment, the mice were subjected to behavioral tests. Subsequently, the mice were euthanized, and their brain tissue was harvested for further analysis. Morris water maze (MWM) test To assess cognitive changes, we conducted the MWM test using a circular water tank (diameter, 140 cm; height, 40 cm) filled with water to a depth of 20 cm and maintained at a temperature of 21°C ± 1°C. The tank was divided into four equal quadrants, and a submerged square platform was positioned in the third quadrant with its upper surface 1 cm below the water surface. The mice were introduced into the pool from four possible start locations, facing the pool wall, while a camera recorded their movements. Each mouse had a maximum of 60 s to locate the platform. If a mouse failed to find the platform within 60 s, a researcher gently guided it to the platform, where it remained for 2–3 s. Each mouse underwent three training sessions daily for 2 days to acclimate to the pool environment (the visible training platform) and subsequently participated in the test thrice daily for 4 days to assess their ability to locate the hidden platform. Latency (time taken to locate the platform in the water), distance traveled, and swim speed were recorded using an automated video-tracking software package (EthoVision 2.3.19; Noldus, Wageningen, the Netherlands). On the 7th day, the platform was removed, and we tracked and recorded the number of times the mice crossed the platform's original location. Moving times assay The mice were transferred to the testing room 1 h before the test to allow them to acclimatize to the environment. The mice were tested in a clear plastic cage, and the automated video-tracking software package (EthoVision 2.3.19; Noldus) was used to track their total movements. The movements of the mice in the center and periphery of the open field were recorded for further data analysis. Tissue procession The mice's brains were harvested and divided into two halves. Half the brain was immobilized in 4% paraformaldehyde for 24 h, then submerged in 30% sucrose solution before sectioning. The other half of the brain was used for protein extraction using a protein extraction kit (Thermo Fisher Scientific, Shanghai, China) according to the manufacturer’s instructions. Cell culture BV2 and Neuroblastoma (N) 2a cells were cultured in Dulbecco-modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) medium as microglial and neuronal cells, respectively. In a 5% CO 2 incubator, the cells were treated with DEX in the absence or presence of LEV for the indicated time. Western blotting Total protein was extracted using a protein extraction kit (Thermo Fisher Scientific, Shanghai, China) according to the manufacturer’s instructions. The extracted proteins were separated using 10–15% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane at 100 V for 1 h. The membrane, containing protein extracts, was blocked with 5% non-fat skim milk diluted in tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature and thereafter incubated overnight with primary antibodies (diluted with 2% bovine serum albumin in TBST) at 4℃. The following primary antibodies were used: anti-β-actin (1:5,000), anti-p-NF-κB (1:2,000), anti-NLRP1 (1:3,000), anti-IL-1β (1:1,000), anti-ADAM10 (1:1,000), anti-β site APP cleavage enzyme 1 (BACE1) (1:2,000), anti-presenilin 1 (PS1) (1:1,000), anti-p-glycogen synthase kinase 3α/β (GSK3α/β) (1:4,000), anti-GSK3α/β (1:4000), anti-p-cyclin-dependent kinase 5 (CDK5) (1:4,000), anti-CDK5 (1:4,000), anti-p-tau (1:2,000), anti-tau (1:2,000), anti-GFAP (1:3,000), anti-Iba1 (1:1,000), anti-SYP (1:500), anti-NeuN (1:2,000), anti-Bax (1:2,000), and anti-Bcl-2 (1:2,000) (Cell Signaling Technology). On day 2, the proteins were visualized using an enhanced chemiluminescence detection system (Thermo Fisher Scientific) after they had been incubated with their corresponding secondary antibodies (1:10,000, Cell Signaling Technology) and visualized using Bio-Rad ChemiDoc XRS devices (Bio-Rad Laboratories, Shanghai, China). For quantitative analysis of band intensity, we used ImageJ software (National Institute of Health, Bethesda, MD, USA). For each blot, we multiplied the subtracted background density of the target protein in each lane with the ratio of the density of the loading control (such as a housekeeping protein) from a control sample in all the study blots to the other lanes in the gel. This provided a normalized density with respect to the loading control (NDL). We calculated the fold difference for each replicate by dividing the NDL from each lane by the NDL from the control sample. Enzyme-linked immunosorbent assay (ELISA) For a sandwiched ELISA, a 96-well plate (MaxiSorp, Nunc, Denmark) was coated with 100 µL of Aβ 42 -specific antibody (5.0 µg IgG/mL each) overnight at 4℃ in 100 mM carbonate buffer (pH 9.6) containing 0.05% sodium azide. After overnight blocking with 1% Block Ace in PBS at 4℃, both the standards (human synthetic Aβ peptides 1–42) and samples were loaded and incubated overnight at 4℃. Horseradish peroxidase-conjugated detection antibody was incubated for 4 h at room temperature, and the reaction was visualized using a TMB substrate. Survival rate We cultured 1 × 10 4 N2a mouse neuroblastoma cell lines in 96-well plates in a humidified incubator with 5% CO 2 at 37℃. The DMEM (Gibco, Grand Island, NY, United States) with 10% FBS (Gibco) was replaced when it turned yellow. After treatment with DEX (10 µM) in the absence or presence of LEV (100 µM) for 24 h, the survival rate of the cells was determined using an MTT assay kit (Abcam, Shanghai, China). Briefly, 50 µL of serum-free medium and 50 µL of MTT reagent were added to each well after the treatment medium was discarded. The plate was incubated at 37℃ for 3 h. Then, the MTT reagent-supplement medium was removed, and 150 µL of MTT solvent was added to each well. After wrapping the plate in a foil and shaking it using an orbital shaker for 15 min, absorbance at 590 nm was measured. The survival rate was calculated according to the following equation. Survival rate (%) = ODtreatment/ODcontrol×100% Aβ uptake and degradation BV2 cells were exposed to human Aβ. After 2 h, the cells were harvested and lysed to assess Aβ uptake using ELISA. For degradation assay, the cells were incubated with Aβ for 2 h and then placed in a fresh medium. After 22 h, the cells were collected using ELISA to determine the remaining Aβ concentration. Aβ degradation was calculated using the following formula. Degradation (%) = (Aβuptake-Aβremaining)/Aβuptake×100% Statistical analysis Data are presented as mean ± standard deviation and were analyzed using the SPSS 10.0 statistical software (SPSS Inc., Chicago, IL, United States). One-way and two-way analysis of variance tests were used to assess significant differences between the groups ( P < 0.05, P < 0.01, and P < 0.001). Results LEV improves learning and memory impairment induced by DEX in APP23/microtubule-associated tau protein in mice Based on the potential therapeutic effects of LEV on AD ( 26 ), we administered LEV (100 mg/kg/d) to APP/MAPT mice for 3 months to alleviate DEX (10 mg/kg/d)-induced cognitive impairments. Following the treatment, the MWM test was conducted to assess the learning and memory abilities of the mice. During the first 2 days of testing, the mice exhibited no discernible defects, as indicated by similar escape latency and path length to the visible platform (Fig. 1 a and 1 b). In contrast, DEX (10 mg/kg/d) increased the mean escape latency of the mice and path length, which were mitigated by LEV (100 mg/kg/d) (Fig. 1 c and 1 d). On the 7th day, the probe trial was conducted by removing the platform. As expected, DEX-treated mice had a shorter crossing time that was partially restored by LEV (Fig. 1 e). Similar results were obtained in the open field tests (Fig. 1 f). Taken together, these results demonstrated that LEV ameliorated DEX-induced memory deficits in AD progression. LEV reversed Aβ production and aggregation induced by DEX-associated chronic stress LEV alleviated DEX-induced memory deficits in APP/MAPT mice. Consequently, we assessed its impact on Aβ production and deposition in the brains of APP23/MAPT mice by quantifying the expressions of α-, β- and γ-secretases in the mice. The level of ADAM10 was decreased and the levels of BACE1 and PS1 were increased in the DEX-treated groups (Fig. 2 a–c). However, following LEV administration, the levels of ADAM10 increased, and levels of BACE1 and PS1 decreased (Fig. 2 a–c). Moreover, DEX considerably increased the BACE1/ADAM10 ratio and LEV considerably decreased the PS1/ADAM10 ratio in DEX-treated APP23/MAPT mice (Fig. 2 d). These results suggest that LEV may regulate Aβ production and deposition. ELISA and immunohistochemistry assays were performed to verify this hypothesis. In the DEX-treated groups, Aβ production was considerably increased; however, after LEV treatment, it decreased considerably (Fig. 2 e). Consistent with these findings, Aβ aggregation in DEX-treated mice was considerably decreased after LEV treatment (Fig. 2 f). Based on these findings, it is worth noting that LEV inhibited Aβ deposition in DEX-treated mice. LEV mitigates DEX-induced tau phosphorylation via the GSK3α/β and CDK5 pathways Tau phosphorylation is another prominent pathological feature of AD. Therefore, we examined phosphorylated tau levels in different APP/MAPT mice groups. The levels of p-CDK5 and p-GSK3α/β, which are positively associated with increased tau phosphorylation, were significantly elevated in the DEX-treated mice compared to those in the control mice (Fig. 3 a). However, p-CDK5 and p-GSK3α/β phosphorylation was attenuated by LEV in DEX-treated mice (Fig. 3 a), indicating that LEV inhibited tau phosphorylation by deactivating p-CDK5 and p-GSK3α/β. Based on these observations, tau phosphorylation was further evaluated using western blotting, which revealed that LEV suppressed tau phosphorylation in DEX-treated APP23/MAPT mice (Fig. 3 b). DEX-induced neuronal impairment via glial cell activation, which was mitigated by LEV Given the contrasting effects of DEX and LEV on the regulation of Aβ production, deposition, and tau phosphorylation, we proceeded to assess the expression of GFAP (an astrocyte biomarker) and Iba1 (a microglia biomarker) in the LEV-treated groups compared with that in the DEX-treated groups. A significant increase in optical densities of GFAP and Iba1 was observed in the DEX-treated APP23/MAPT mice, which was effectively reversed by LEV (Fig. 4 a). These findings suggest that LEV alleviated DEX-induced inflammatory stress in APP23/MAPT mice. Moreover, the BV2 cells were treated with DEX in the absence or presence of LEV, and their survival rates were determined using MTT assay. DEX and LEV decreased and increased BV2 cell survival rates, respectively (Fig. 4 b), suggesting that LEV attenuated DEX-induced pyroptosis of microglial cells. Aβ accumulation and tau hyperphosphorylation result in dystrophy and neuronal loss, which prompted us to examine the effects of LEV on neurons. For this purpose, the expression of the presynaptic marker, SYP, and the postmitotic neuronal marker, NeuN, were determined using western blotting. SYP was markedly downregulated in the DEX-treated mice compared to that in the control group (Fig. 4 c), indicating impaired synaptic plasticity induced by DEX in the brains of APP23/MAPT mice. Similarly, the protein level of NeuN was considerably lower in the DEX-treated mice than in the control mice (Fig. 4 c), suggesting that DEX-related stress promotes synaptic dystrophy and neuronal loss in the brains of APP23/MAPT mice. However, LEV attenuated the effects of DEX on neuronal dystrophy and loss (Fig. 4 c). Similar results were obtained for N2a cells, showing that DEX impaired the survival of neuronal cells and was ameliorated by LEV (Fig. 4 d), suggesting that DEX impaired the neurons by activating glial cells, which was ameliorated by LEV. LEV protects neurons from DEX-induced apoptosis Considering the aforementioned findings, we investigated apoptotic mechanisms underlying the loss of synapses or neurons. As anticipated, DEX induced neuronal apoptosis by upregulating Bax and downregulating Bcl2, thereby increasing the Bax/Bcl2 ratio in the brains of APP23/MAPT mice (Fig. 5 a and 5 b). However, LEV reduced the Bax/Bcl2 ratio in DEX-treated mice (Fig. 5 c). To validate these in vivo results, N2a cells were used as in vitro neuronal models, which showed that DEX simultaneously upregulates Bax and downregulates Bcl2, leading to the loss of neurons (Fig. 5 d and 5 e). In contrast, LEV protects neurons from apoptosis by downregulating Bax and upregulating Bcl2 in DEX-treated N2a cells (Fig. 5 d and 5 e). Similar to the in vivo results, DEX increases the Bax/Bcl2 ratio, which was reversed by LEV in N2a cells (Fig. 5 f), suggesting the mechanism by which LEV alleviate synapse or neuronal loss induced by DEX-related stress in APP23/MAPT mice. NLRP1 inflammasome activation by DEX is dependent on GR mechanisms, which was attenuated by LEV Considering the potential role of inflammasomes in AD ( 24 ), we investigated the relationship between DEX and NLRP1 inflammasomes. Western blotting revealed a significant increase in NF-κB phosphorylation and NLRP1 and IL-1β expressions in the brains of DEX-treated APP23/MAPT mice (Fig. 6 a). Furthermore, western blotting revealed that the optical densities of phosphorylated NF-κB, NLRP1, and IL-1β were considerably reduced in the LEV-treated groups compared to those in the DEX-treated group (Fig. 6 a). These results suggest that LEV inhibited inflammatory stress by deactivating inflammasomes in DEX-activated mice. To verify the role of GR in the activation of NLRP1 inflammasome, mifepristone (MIF), a GR antagonist, was used to treat APP/MAPT mice. Western blotting showed decreased optical densities of phosphorylated NF-κB, NLRP1, and IL-1β in the MIF-treated groups compared to those in the DEX-treated group (Fig. 6 b). Therefore, DEX activated NLRP1 inflammasome via GR. NLRP1 inflammasome activation is critical for Aβ accumulation and tau phosphorylation Based on the above results, it was necessary to determine whether NLRP1 is pivotal for the observed Aβ accumulation and tau phosphorylation in DEX-treated APP/MAPT mice. Therefore, siRNA interference experiments were performed to knock down NLRP1 in BV2 cells. Following incubation with human (h) Aβ, BV2 cells' ability to uptake and degrade hAβ was impaired by DEX (Fig. 7 a and 7 b). Conversely, knocking down the NLRP1 in BV2 cells partially restored the hAβ uptake and degradation ability of the BV2 cells (Fig. 7 a and 7 b). Furthermore, tau phosphorylation was attenuated in the NLRP1 knockdown group compared with the control group (Fig. 7 c). Altogether, NLRP1 is critical for the effects of DEX on Aβ accumulation and tau phosphorylation. Discussion As a synthetic GC, DEX impairs the structure and function of the hippocampus, ultimately contributing to AD progression ( 31 ). In this study, we expanded upon our previous research by investigating the effects of DEX on memory impairment and neuropathology in APP23/MAPT mice. Our findings revealed that DEX exacerbated behavioral deficits by reducing SPs burden and tau phosphorylation. In addition, DEX reduced Aβ degradation and clearance in APP23/MAPT mice. Moreover, we found that NLRP1 inflammasome played key roles in AD regulation by DEX. Interestingly, LEV suppressed neuroinflammation by deactivating inflammasomes and protected neurons from dystrophy and loss by suppressing DEX-induced apoptosis. DEX was suspected to impair learning and memory abilities. As anticipated, DEX increased the time taken and distance traveled by mice to find the platform and decreased crossing times (Fig. 1 ). Similar to our findings, DEX reportedly exacerbated Aβ-induced learning and memory impairment in rats in a previous study ( 32 ). In another study, DEX was confirmed to induce memory and learning impairment in senescent mice ( 33 ). In addition, melatonin could reportedly attenuate DEX-induced spatial memory impairment and DEX-induced reduction of synaptic protein levels in the mouse brain ( 34 ). In line with these observations, our results demonstrated that LEV could ameliorate AD progression by inhibiting Aβ production and deposition and tau phosphorylation in the brains of DEX-treated APP23/MAPT mice (Figs. 1 – 3 ). Although no related associated study exists, LEV reportedly normalizes hippocampal CA3/DG activity and improves memory performance in patients with amnestic mild cognitive impairment based on the results of high-resolution functional magnetic resonance imaging techniques ( 35 ). In addition, hippocampal spatial memory decline in aged rats was rescued by chronic infusion or a single injection of LEV and sodium valproate before training, as reflected in the MWM test performance ( 36 ). In a previous study including APP/PS1 mice, LEV effectively alleviated behavioral deficits in AD ( 37 ). Consistent with these findings, LEV administered intraperitoneally improves neuronal functions in APP/PS1 Tg mice, suggesting its ability to penetrate the blood-brain barrier ( 37 ). Likewise, LEV reversed the abnormal expression of neuronal activity-related proteins that reflect hippocampal remodeling and cognitive deficits in hAPPJ20 mice ( 38 ). Therefore, these results suggest the roles of DEX in exacerbating the pathological features of AD, which were ameliorated by LEV in the brains of APP23/MAPT mice. Considering that Aβ production and deposition are widely accepted as key pathogenic factors of AD ( 39 ), we initially investigated the effect of DEX on Aβ production and deposition. The results indicated that DEX induced Aβ production and deposition in the brains of APP23/MAPT mice (Fig. 2 ). In agreement with our finding, chronic DEX treatment accelerates Aβ production in the neurons of APP/PS1 mice ( 24 ). DEX further induces Aβ production with cholinergic dysfunction similar to AD ( 40 ). Aβ results from β-site APP-cleaving enzyme 1 (BACE1) and γ-secretase-mediated cleavage of APP. Consistently, we found that DEX elevated BACE1 and PS1 levels responsible for Aβ production in the brains of APP/MAPT mice (Fig. 2 ). Similarly, GC exposure elevated Aβ production by increasing BACE1 and APP gene expression in primary astrocytes ( 41 ). In addition to inducing Aβ production and deposition, DEX induces tau hyperphosphorylation ( 40 ), which is consistent with our results (Fig. 3 ). GC exposure induces tau hyperphosphorylation and neurostructural deficits in hippocampal neurons in vitro and in vivo ( 42 ). To counteract the side effects of DEX, LEV was used to treat APP23/MAPT mice. The results demonstrated that LEV suppressed Aβ production and deposition and tau phosphorylation (Figs. 2 and 3 ). Although no relevant research exists, LEV and TPM reportedly reduce Aβ production by inhibiting the activity of γ-secretase ( 37 ). In addition, the underlying mechanisms may involve Aβ uptake and degradation ( 43 ). In line with our findings, the lack of Aβ uptake by glial cells is a major cause of sporadic AD ( 44 ), which might be caused by impaired Aβ clearance in AD ( 45 ). Regarding the underlying mechanism, LEV increases Aβ uptake (Fig. 7 a). Aβ degradation is regarded as a new therapeutic target for AD treatment ( 46 , 47 ). Our study contributed to previous studies showing that LEV enhanced Aβ degradation (Fig. 7 b). Moreover, tau phosphorylation can be induced by Aβ production and deposition ( 48 ). We found that LEV decreased tau phosphorylation via CDK5- and GSK3α/β-dependent mechanisms (Fig. 3 ). Similarly, tau phosphorylation is reportedly mediated by CDK5 and GSK3α/β ( 49 , 50 ). Based on these findings, we can infer that LEV counteracts the effects of DEX on Aβ production and deposition and tau hyperphosphorylation. Excessive Aβ loading and tau hyperphosphorylation result in neuroinflammation and neuronal loss in AD ( 51 ). Inflammasomes contribute to various disorders in the CNS by causing neuroinflammation. Extracellular accumulation of Aβ in SPs in brains with AD is a principal event in AD ( 52 ). Deposition of Aβ peptide initiates inflammasome activity in the microglia ( 53 ). Moreover, inflammasome activation causes AD impairment through Aβ deposition and loss of spatial memory via harmful chronic inflammatory response. It is important to note that NLRP1 activation in the brain is restricted to plaque-associated microglia, suggesting that microglial activation of the NLRP1 inflammasome is a pivotal event in AD pathogenesis ( 54 ). Furthermore, the correlation between AD and local neuroinflammation has been established. As an important inflammasome component, IL-1β can induce tau phosphorylation ( 55 ), leading to compromised learning and memory in animals with AD ( 56 ). By blocking IL-1β, AD was ameliorated in animals with AD ( 57 ). Consistent with other studies, our study further shows that LEV deactivates DEX-induced inflammasomes, thus suppressing glial cell activity (Fig. 6 a). The glial cells are responsible for Aβ clearance, whereas the microglial cells are responsible for degrading Aβ degradation via autophagy ( 58 ). Accumulating evidence has indicated that elevated levels of GC cause chronic environmental stress, which is a risk factor for AD ( 59 ). Evidence also suggests that high but not low levels of DEX exposure impair hippocampal neurons in rats ( 60 ). Using SH-SY5Y cells, high levels of DEX induce neuronal loss and neurotoxicity by impairing the mitochondria ( 61 ). Consistent with previous studies, we further found that DEX inhibited the expression of both NeuN and SYP, leading to the loss of neurons in the synapse (Fig. 4 c). Moreover, the apoptotic mechanisms underlying the above process were identified (Fig. 5 ). LEV attenuated DEX-induced apoptosis, ameliorating neuronal and synaptic loss (Figs. 4 c and 5 ). Our previous study had shown the protective effects of LEV on neurons via apoptosis inhibition in kainic acid (KA)-activated APP23/MAPT mice ( 25 ). In support of our findings, LEV administration after hypoxia reduces neuronal apoptosis in a neonatal rat model of hypoxic-ischemic brain injury ( 62 ). Additionally, a study showed that LEV conferred neuroprotective effects against focal cerebral ischemia-reperfusion injury in mice ( 63 ). In conclusion, this study builds on previous studies by showing that LEV ameliorated amyloidosis and tauopathy by inhibiting the activity of NLRP1 inflammasome in mice with DEX-induced AD. Declarations Disclosure of potential conflicts of interest: The authors have no relevant financial or non-financial interests to disclose. Research involving Human Participants and/or Animals: All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of The First Hospital of Jilin University and adhered to the Guidelines for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health. Informed consent: Not applicable Funding This study was supported by the National Natural Science Foundation of China (No. 81873812), the Fundamental Research Funds for the Central Universities, the Jilin Province Department of Finance (No. JLSWSRCZX2023-36), and the Science and Technology Development Program of the Jilin Province (No. 20220203122SF). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions Xiang-Yu and Yang Ruan conceived the project. Hai-Chen, Zhi-Wei, Yu-Dan, Feng-Yan, and Jie Zhu conducted the research or assisted the research, discussed the project, and assisted the manuscript preparation. Xiang-Yu and Yang Ruan wrote the manuscript. Jie Zhu supervised the project. All authors are accountable for all aspects of the work and all persons designated as authors qualify for the authorship, and all those who qualify for authorship are listed. All authors read and approved the final version of the manuscript submitted for publication. Data Availability The data presented in this study are included in the article/supplementary material. Further inquiries should be directed to the corresponding author(s). The experimental animal procedures performed in this study were reviewed and approved by the Animal Care and Use Committee of The First Hospital of Jilin University, Changchun, China. Ethics approval All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of The First Hospital of Jilin University and adhered to the Guidelines for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health. Consent to participate Not applicable Consent to publish Not applicable References Zhang Y, Zhao Y, Zhang L, Yu W, Wang Y, Chang W (2019) Cellular prion protein as a receptor of toxic amyloid-β42 oligomers is important for Alzheimer’s disease. Front Cell Neurosci 13:339. https://doi.org/10.3389/fncel.2019.00339 Bisht K, Sharma K, Tremblay MÈ (2018) Chronic stress as a risk factor for Alzheimer’s disease: Roles of microglia-mediated synaptic remodeling, inflammation, and oxidative stress. 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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-3517043","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":245807133,"identity":"9b3e346e-6350-4b3b-b17e-54df54ee6950","order_by":0,"name":"Xiang-yu Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYDACZihtACI+8FjA2cRpYZzBI0GEFhgAKWPmYSBCi8Fx5mcPv7Ydtjdn7z382kZGIrGBvXmbBEPNHZxaJJvZzI1l2w4zW/acS7PO4QFq4TlWJsFw7BlOLfzMDGbSkm2H2Qxu5JgZg7VI5JhJMDYcxqmFjZn9G0gLj8H9N2bGFiAt8m/wa+Fn5jGT/Nh2WMLgBo/xYwawLTz4tUg285RJM5xLNzA4k2PG2MMjYdzGk1ZskXAMtxaD88e3Sf4os7Y3OH7G+MPPHhvZfvbDG298qMGtBQSYedkg/pJg7AGSIGYCXg3ASP/xB6L1A8MPAkpHwSgYBaNgRAIAuq5LwpZvDN0AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-9898-1748","institution":"the First Hospital of Jilin University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xiang-yu","middleName":"","lastName":"Zheng","suffix":""},{"id":245807134,"identity":"d98d46c2-97d0-4aa2-96fb-5d51362bced3","order_by":1,"name":"Hai-Chen Zhang","email":"","orcid":"","institution":"The First Hospital of Jilin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hai-Chen","middleName":"","lastName":"Zhang","suffix":""},{"id":245807135,"identity":"41f0fcef-f22d-4bd5-9852-c1ba4c80a48c","order_by":2,"name":"Zhi-Wei Wei","email":"","orcid":"","institution":"The First Hospital of Jilin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhi-Wei","middleName":"","lastName":"Wei","suffix":""},{"id":245807136,"identity":"bb0dd819-976c-4b8b-8df4-5adf30b7a868","order_by":3,"name":"Yu-Dan Lv","email":"","orcid":"","institution":"The First Hospital of Jilin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu-Dan","middleName":"","lastName":"Lv","suffix":""},{"id":245807137,"identity":"dba7634d-c062-495d-894d-d03c9fdc14e8","order_by":4,"name":"Feng-Yan Jin","email":"","orcid":"","institution":"The First Hospital of Jilin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Feng-Yan","middleName":"","lastName":"Jin","suffix":""},{"id":245807138,"identity":"3aa01450-84cf-4337-9af9-35bf9137905e","order_by":5,"name":"Jie Zhu","email":"","orcid":"","institution":"The First Hospital of Jilin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Zhu","suffix":""},{"id":245807139,"identity":"cc922b52-714e-47ff-8b44-77bc8b0e076d","order_by":6,"name":"Yang Ruan","email":"","orcid":"","institution":"key laboratory of pathobiology, ministry of education, colleage of Basic Medical Science, Jilin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Ruan","suffix":""}],"badges":[],"createdAt":"2023-10-30 22:55:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3517043/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3517043/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":46050344,"identity":"67f0bdfc-8bc7-491d-8058-ef39e43ce99e","added_by":"auto","created_at":"2023-11-07 23:50:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":150466,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLEV mitigates DEX-induced behavioral abnormalities in APP23/MAPT mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a, b) During the first 2 days of the visible platform tests, the control, LEV-treated, and DEX-treated APP23/MAPT mice exhibit similar latencies in escape time (P \u0026gt; 0.05, ANOVA); (c, d) In the hidden platform tests, DEX-treated APP23/MAPT mice showed a longer latency period and distance traveled to escape on days 3 and 4, which were ameliorated by low concentrations of LEV on day 4 (*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. the control group; ##\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ###\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 vs. the DEX-treated group by ANOVA); (e) During the probe trial on day 7, DEX-treated APP23/MAPT mice travel into the third quadrant (where the hidden platform was previously placed) in significantly shorter times than the controls after treatment with LEV (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. the control group; #\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. the DEX-treated group by ANOVA); (f) Total mice movements in the different groups tested in the open field experiments (∗\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 vs. the control group; #P \u0026lt; 0.05 vs. the DEX-treated group by ANOVA). DEX, dexamethasone; LEV, levetiracetam; ANOVA, analysis of variance.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3517043/v1/4e6ea20ee77ba174876c24dc.png"},{"id":46049562,"identity":"e496c789-f619-4705-8290-8ed80b4145a1","added_by":"auto","created_at":"2023-11-07 23:34:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":149045,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLEV suppress Aβ production and deposition in DEX-treated APP23/MAPT mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a–c) Protein levels of ADAM10, BACE1, and PS1 in the brains of DEX- and LEV-treated APP23/MAPT mice; (d) BACE1/ADAM10 and PS1/ADAM10 ratio in the brains of DEX- and LEV-treated APP23/MAPT mice; (e) Aβ production was determined using enzyme-linked immunosorbent assay; (f) The number of Aps was determined by immunohistochemical staining (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 vs. controls; #\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ##\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01 vs. the DEX group; one-way ANOVA). DEX, dexamethasone; LEV, levetiracetam; SPs, senile plaques; Aβ, amyloid β-peptide; ANOVA, analysis of variance.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3517043/v1/76473a90c03ba4181c425a5b.png"},{"id":46049773,"identity":"64086738-7d89-4d8c-9d3c-7f26073e77d6","added_by":"auto","created_at":"2023-11-07 23:42:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":107298,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLEV suppresses DEX-induced tau phosphorylation in the brains of APP23/MAPT mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Phosphorylated and total protein levels of GSK3α/β and CDK5 in the brains of DEX- and LEV-treated APP23/MAPT mice were determined using western blotting; (b) Phosphorylated and total protein levels of tau in the brains of DEX- and LEV-treated APP23/MAPT mice were determined using western blotting; Optical densities of bands in the western blots were evaluated using the ImageJ software (∗\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; ∗∗\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01 vs. the control group; #\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ##\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. the DEX-only group, one-way ANOVA). DEX, dexamethasone; LEV, levetiracetam; Aβ, amyloid β-peptide; ANOVA, analysis of variance.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3517043/v1/1d0a861a8cb08f761a2561a5.png"},{"id":46049559,"identity":"0f4eda54-b8f5-44a3-9a69-c403ffa07285","added_by":"auto","created_at":"2023-11-07 23:34:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":105662,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLEV mitigates DEX activation of microglia, thereby protecting synapse loss of neurons in the brains of APP23/MAPT mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) The total protein levels of GFAP and Iba1 in the brains of DEX- and LEV-treated APP23/MAPT mice determined using western blots; (b) The survival rates of BV2 cells in DEX- and LEV-treated cells; (c) The protein levels of SYP and NeuN in the brains of DEX- and LEV-treated APP23/MAPT mice were determined using western blotting; (c) The survival rates of N2a cells in DEX- and LEV-treated cells. Optical densities of bands in the western blots were evaluated using the ImageJ software (∗∗\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ∗∗∗\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001 vs. the control group; #\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ##\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. the DEX-only group). DEX, dexamethasone; LEV, levetiracetam; Aβ, amyloid β-peptide.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3517043/v1/5dde28f7be08d093f0f55ca6.png"},{"id":46049565,"identity":"97752134-86e6-4da0-bc0a-4d2c3ee87fbb","added_by":"auto","created_at":"2023-11-07 23:34:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":138583,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLEV protects neurons from neuronal loss in DEX-treated APP23/MAPT mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a, b) The protein levels of Bax and Bcl2 in the brains of DEX- and LEV-treated APP23/MAPT mice were determined using western blotting. Optical densities of bands in the western blots were evaluated using ImageJ software. (c) Bax/Bcl2 ratio; (d, e) The protein levels of Bax and Bcl2 in the brains of DEX- and LEV-treated N2a cells were determined using western blotting. The optical densities of bands in the western blots were evaluated using ImageJ software; (f) The ratio of Bax and Bcl2 was calculated; (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 vs. the control group; #\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; ##\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; and ###\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 vs. the DEX-only group). DEX, dexamethasone; LEV, levetiracetam.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3517043/v1/cf8b7fd8b55ea8eab7ba3696.png"},{"id":46049560,"identity":"2521be5c-85c5-4cb2-9489-66822aedf5e4","added_by":"auto","created_at":"2023-11-07 23:34:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":180236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLEV mitigates DEX activation of inflammasomes via GR-dependent mechanisms in the brains of APP23/MAPT mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) The phosphorylation level of NF-κB and total protein levels of NLRP3 and IL-1β in the brains of DEX- and LEV-treated APP23/MAPT mice were determined using western blotting; (b) The phosphorylation level of NF-κB and total protein levels of NLRP3 and IL-1β in the brains of DEX- and MIF-treated APP23/MAPT mice were determined using western blotting. Optical densities of bands in the western blots were evaluated using the ImageJ software (∗P \u0026lt; 0.05; ∗∗\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ∗∗∗\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01 vs. the control group; #\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ##\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. the DEX-only group). DEX, dexamethasone; LEV, levetiracetam; Aβ, amyloid β-peptide; MIF, mifepristone.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3517043/v1/3512ad5dd18f3fc2e9a61871.png"},{"id":46049774,"identity":"38c32e37-1b05-4ccb-a84a-44d748b5565c","added_by":"auto","created_at":"2023-11-07 23:42:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":87152,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLEV mitigates DEX activation of inflammasomes via GR-dependent mechanisms in the brains of APP23/MAPT mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) The phosphorylation level of NF-κB and total protein levels of NLRP3 and IL-1β in the brains of DEX- and LEV-treated APP23/MAPT mice were determined using western blotting; (b) The phosphorylation level of NF-κB and total protein levels of NLRP3 and IL-1β in the brains of DEX- and MIF-treated APP23/MAPT mice were determined using western blotting. Optical densities of bands in the western blots were evaluated using the ImageJ software (∗P \u0026lt; 0.05; ∗∗\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ∗∗∗\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01 vs. the control group; #\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ##\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. the DEX-only group). DEX, dexamethasone; LEV, levetiracetam; Aβ, amyloid β-peptide; MIF, mifepristone.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-3517043/v1/71d0d6ff391f7cf3382fcee8.png"},{"id":46051195,"identity":"dd299609-c2df-4e0f-a638-d7e33fed8703","added_by":"auto","created_at":"2023-11-07 23:58:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2177302,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3517043/v1/aaaa4744-e372-4a71-9ebb-b55858b5e337.pdf"}],"financialInterests":"","formattedTitle":"Levetiracetam ameliorated amyloidosis and tauopathy in mice with dexamethasone-induced Alzheimer’s disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlzheimer\u0026rsquo;s disease (AD) is a neurodegenerative disorder and the leading cause of dementia and presents an increasing challenging with the rising global life expectancy. Although the primary pathological hallmarks of AD are commonly associated with the formation of senile plaques (SPs) from β-amyloid protein (Aβ) and neurofibrillary tangles formed by the aggregation of highly phosphorylated tau (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), other factors may play a role in AD development. Epidemiological evidence suggests that chronic stress is a potential risk factor for AD (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Furthermore, preclinical data showed that prolonged administration of glucocorticoid (GC) induces Aβ deposition and tau hyperphosphorylation, contributing to synaptic dysfunction and neuronal impairment in AD (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Recent research revealed that corticosterone-related stress can enhance the production of hippocampal Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e and Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e as well as the expression of BACE1 in APP/PS1 transgenic (Tg) mice (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Moreover, chronic exposure to GC-related stress can cause functional and structural damage; disrupt the normal physiological function of hippocampal neurons; and cause neuronal atrophy, loss, and dysfunction in the brain (\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) important for cognitive functions, including learning and memory (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe glucocorticoid receptor (GR), a target of endogenous (corticosterone) or synthetic (dexamethasone, DEX) GC, is a widely used clinical therapeutic target (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Impaired GR signaling exacerbates Aβ oligomers-induced AD (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Moreover, GR dysfunction could prove highly detrimental to limbic structures (12), contributing to cognitive decline in AD. GR antagonists or selective inhibitors can potentially mitigate the effects of Aβo25-35 on the hippocampus, thereby strongly modulating GR activity as a therapeutic strategy (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs a GR agonist, DEX induces pyroptosis by upregulating pyroptosis-related proteins, such as the NLR family pyrin domain containing 3 (NLRP3), caspase-1, and gasdermin-D (GSDMD) (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Furthermore, downregulation of NLRP3 or GSDMD attenuates DEX-induced myotube pyroptosis and atrophy (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). However, this hypothesis should be considered cautiously, as a previous study reported that DEX could inhibit NLRP3 inflammasome activity in coronavirus disease 2019 (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Additionally, DEX alleviates allergic airway inflammation in mice by inhibiting NLRP3 inflammasome activation (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Similarly, intranasal curcumin combined with DEX inhibits NLRP3 inflammasome activation in lipopolysaccharide-induced asthma exacerbations (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Given the roles of NLRP3 inflammasome in AD pathogenesis (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), it is possible that NLRP3 inflammasome will not be critical for the effects of DEX and GR on AD pathogenesis.\u003c/p\u003e \u003cp\u003eCompared with NLRP3, NLRP1 is highly expressed in the central nervous system (CNS) and is involved in regulating the pathogenesis of various neurological diseases (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). NLRP1 inflammasome primarily comprises caspase-1 and NLRs, including apoptosis-associated speck-like protein (ASC) (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Caspase-1 is a critical regulatory molecule that converts pre-interleukin (IL)-1β and IL-6 into mature active forms, which are released to participate in inflammatory reactions (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). More specifically, DEX significantly increased the mRNA and protein expression of NLRP1, caspase-1, ASC, and IL-1β, reducing spontaneous motor activity and exploratory behavior of male institute of cancer research mice. Moreover, the mice exhibited neurodegeneration and decreased microtubule-associated protein 2 (MAP2) levels in the frontal cortex and hippocampus CA3 region (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). In APP/PS1 Tg mice, DEX markedly increased the accumulation of Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e, leading to neuronal injury in primary hippocampal neurons. Furthermore, chronic DEX exposure significantly elevated the levels of reactive oxygen species and IL-1β and upregulated NLRP1 in primary hippocampal neurons of APP/PS1 mice (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). NLRP1 inhibition suppressed the accumulation of Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e and expression of APP, BACE1, and nicastrin (NCSTN) in APP/PS1 Tg mice (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), highlighting the pivotal mediating role of NLRP1 in the effects of DEX on AD pathogenesis. It is imperative to explore ways of mitigating the adverse effects of DEX. Based on our previous study, levetiracetam (LEV) alleviated cognitive decline in AD by ameliorating neuronal network dysfunction (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Moreover, the therapeutic potential of LEV and topiramate (TPM) for AD has been demonstrated (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). The therapeutic effects of the antiepileptic drugs were reportedly attributed to their inhibition of histone deacetylase. Furthermore, LEV and TPM offer additional benefits, such as neuroprotective and anti-inflammatory properties (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). However, whether LEV and TPM can reverse neuropathological and cognitive deficits by preserving or restoring neuronal network activities in patients with DEX-induced AD remains uncertain.\u003c/p\u003e \u003cp\u003eTherefore, this study aimed to elucidate the mechanisms underlying the effect of DEX on AD development and progression and identify the role of NRP1 inflammasome in APP23/MAPT\u003csup\u003eP301S\u003c/sup\u003e mice.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice and treatment protocol\u003c/h2\u003e \u003cp\u003eAPP23 [B6-Tg (Thy1APP) 23SdZ] and MAPT [B6; C3- Tg (Prnp-MAPT\u003csup\u003eP301S\u003c/sup\u003e) PS19Vle/J] mice were obtained from the Jackson Laboratory (Bar Harbor, ME, United States; Stock #030504 and #008169). The APP23 and MAPT mice carry the Swedish and P301S mutated human APP transgenes, respectively. The mice were generated from the C57BL/6 genetic background, as previously described (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). For the experimental cohort, the APP23 mice were crossbred with the MAPT transgenic mice to obtain APP23/MAPT mice. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of The First Hospital of Jilin University and adhered to the Guidelines for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health. The mice were housed in groups of five per cage in a room maintained at a temperature of 22\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and with a 12-h light-dark cycle. Food and water were provided ad libitum.\u003c/p\u003e \u003cp\u003eBased on a previous study, six 5-month-old male mice per group were concurrently administered 10 mg/kg/d of DEX (Sigma-Aldrich Corp., St. Louis, MO, United States) intraperitoneally alone or with 100 mg/kg/d of LEV (Aladdin, Shanghai, China) emulsified in 0.9% phosphate-buffered saline [PBS (\u0026ndash;)]. Male mice in the control group (n\u0026thinsp;=\u0026thinsp;6) received PBS injection (\u0026ndash;). After 3 months of treatment, the mice were subjected to behavioral tests. Subsequently, the mice were euthanized, and their brain tissue was harvested for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMorris water maze (MWM) test\u003c/h2\u003e \u003cp\u003eTo assess cognitive changes, we conducted the MWM test using a circular water tank (diameter, 140 cm; height, 40 cm) filled with water to a depth of 20 cm and maintained at a temperature of 21\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. The tank was divided into four equal quadrants, and a submerged square platform was positioned in the third quadrant with its upper surface 1 cm below the water surface. The mice were introduced into the pool from four possible start locations, facing the pool wall, while a camera recorded their movements. Each mouse had a maximum of 60 s to locate the platform. If a mouse failed to find the platform within 60 s, a researcher gently guided it to the platform, where it remained for 2\u0026ndash;3 s. Each mouse underwent three training sessions daily for 2 days to acclimate to the pool environment (the visible training platform) and subsequently participated in the test thrice daily for 4 days to assess their ability to locate the hidden platform. Latency (time taken to locate the platform in the water), distance traveled, and swim speed were recorded using an automated video-tracking software package (EthoVision 2.3.19; Noldus, Wageningen, the Netherlands). On the 7th day, the platform was removed, and we tracked and recorded the number of times the mice crossed the platform's original location.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMoving times assay\u003c/h2\u003e \u003cp\u003eThe mice were transferred to the testing room 1 h before the test to allow them to acclimatize to the environment. The mice were tested in a clear plastic cage, and the automated video-tracking software package (EthoVision 2.3.19; Noldus) was used to track their total movements. The movements of the mice in the center and periphery of the open field were recorded for further data analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTissue procession\u003c/h2\u003e \u003cp\u003eThe mice's brains were harvested and divided into two halves. Half the brain was immobilized in 4% paraformaldehyde for 24 h, then submerged in 30% sucrose solution before sectioning. The other half of the brain was used for protein extraction using a protein extraction kit (Thermo Fisher Scientific, Shanghai, China) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eBV2 and Neuroblastoma (N) 2a cells were cultured in Dulbecco-modified Eagle\u0026rsquo;s medium (DMEM) with 10% fetal bovine serum (FBS) medium as microglial and neuronal cells, respectively. In a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator, the cells were treated with DEX in the absence or presence of LEV for the indicated time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003e Total protein was extracted using a protein extraction kit (Thermo Fisher Scientific, Shanghai, China) according to the manufacturer\u0026rsquo;s instructions. The extracted proteins were separated using 10\u0026ndash;15% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane at 100 V for 1 h. The membrane, containing protein extracts, was blocked with 5% non-fat skim milk diluted in tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature and thereafter incubated overnight with primary antibodies (diluted with 2% bovine serum albumin in TBST) at 4℃. The following primary antibodies were used: anti-β-actin (1:5,000), anti-p-NF-κB (1:2,000), anti-NLRP1 (1:3,000), anti-IL-1β (1:1,000), anti-ADAM10 (1:1,000), anti-β site APP cleavage enzyme 1 (BACE1) (1:2,000), anti-presenilin 1 (PS1) (1:1,000), anti-p-glycogen synthase kinase 3α/β (GSK3α/β) (1:4,000), anti-GSK3α/β (1:4000), anti-p-cyclin-dependent kinase 5 (CDK5) (1:4,000), anti-CDK5 (1:4,000), anti-p-tau (1:2,000), anti-tau (1:2,000), anti-GFAP (1:3,000), anti-Iba1 (1:1,000), anti-SYP (1:500), anti-NeuN (1:2,000), anti-Bax (1:2,000), and anti-Bcl-2 (1:2,000) (Cell Signaling Technology). On day 2, the proteins were visualized using an enhanced chemiluminescence detection system (Thermo Fisher Scientific) after they had been incubated with their corresponding secondary antibodies (1:10,000, Cell Signaling Technology) and visualized using Bio-Rad ChemiDoc XRS devices (Bio-Rad Laboratories, Shanghai, China). For quantitative analysis of band intensity, we used ImageJ software (National Institute of Health, Bethesda, MD, USA). For each blot, we multiplied the subtracted background density of the target protein in each lane with the ratio of the density of the loading control (such as a housekeeping protein) from a control sample in all the study blots to the other lanes in the gel. This provided a normalized density with respect to the loading control (NDL). We calculated the fold difference for each replicate by dividing the NDL from each lane by the NDL from the control sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eFor a sandwiched ELISA, a 96-well plate (MaxiSorp, Nunc, Denmark) was coated with 100 \u0026micro;L of Aβ\u003csub\u003e42\u003c/sub\u003e-specific antibody (5.0 \u0026micro;g IgG/mL each) overnight at 4℃ in 100 mM carbonate buffer (pH 9.6) containing 0.05% sodium azide. After overnight blocking with 1% Block Ace in PBS at 4℃, both the standards (human synthetic Aβ peptides 1\u0026ndash;42) and samples were loaded and incubated overnight at 4℃. Horseradish peroxidase-conjugated detection antibody was incubated for 4 h at room temperature, and the reaction was visualized using a TMB substrate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSurvival rate\u003c/h2\u003e \u003cp\u003eWe cultured 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e N2a mouse neuroblastoma cell lines in 96-well plates in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃. The DMEM (Gibco, Grand Island, NY, United States) with 10% FBS (Gibco) was replaced when it turned yellow. After treatment with DEX (10 \u0026micro;M) in the absence or presence of LEV (100 \u0026micro;M) for 24 h, the survival rate of the cells was determined using an MTT assay kit (Abcam, Shanghai, China). Briefly, 50 \u0026micro;L of serum-free medium and 50 \u0026micro;L of MTT reagent were added to each well after the treatment medium was discarded. The plate was incubated at 37℃ for 3 h. Then, the MTT reagent-supplement medium was removed, and 150 \u0026micro;L of MTT solvent was added to each well. After wrapping the plate in a foil and shaking it using an orbital shaker for 15 min, absorbance at 590 nm was measured. The survival rate was calculated according to the following equation.\u003c/p\u003e \u003cp\u003eSurvival rate (%)\u0026thinsp;=\u0026thinsp;ODtreatment/ODcontrol\u0026times;100%\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAβ uptake and degradation\u003c/h2\u003e \u003cp\u003eBV2 cells were exposed to human Aβ. After 2 h, the cells were harvested and lysed to assess Aβ uptake using ELISA. For degradation assay, the cells were incubated with Aβ for 2 h and then placed in a fresh medium. After 22 h, the cells were collected using ELISA to determine the remaining Aβ concentration. Aβ degradation was calculated using the following formula.\u003c/p\u003e \u003cp\u003eDegradation (%) = (Aβuptake-Aβremaining)/Aβuptake\u0026times;100%\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation and were analyzed using the SPSS 10.0 statistical software (SPSS Inc., Chicago, IL, United States). One-way and two-way analysis of variance tests were used to assess significant differences between the groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLEV improves learning and memory impairment induced by DEX in APP23/microtubule-associated tau protein in mice\u003c/h2\u003e \u003cp\u003eBased on the potential therapeutic effects of LEV on AD (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), we administered LEV (100 mg/kg/d) to APP/MAPT mice for 3 months to alleviate DEX (10 mg/kg/d)-induced cognitive impairments. Following the treatment, the MWM test was conducted to assess the learning and memory abilities of the mice. During the first 2 days of testing, the mice exhibited no discernible defects, as indicated by similar escape latency and path length to the visible platform (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In contrast, DEX (10 mg/kg/d) increased the mean escape latency of the mice and path length, which were mitigated by LEV (100 mg/kg/d) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). On the 7th day, the probe trial was conducted by removing the platform. As expected, DEX-treated mice had a shorter crossing time that was partially restored by LEV (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Similar results were obtained in the open field tests (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Taken together, these results demonstrated that LEV ameliorated DEX-induced memory deficits in AD progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLEV reversed Aβ production and aggregation induced by DEX-associated chronic stress\u003c/h2\u003e \u003cp\u003eLEV alleviated DEX-induced memory deficits in APP/MAPT mice. Consequently, we assessed its impact on Aβ production and deposition in the brains of APP23/MAPT mice by quantifying the expressions of α-, β- and γ-secretases in the mice. The level of ADAM10 was decreased and the levels of BACE1 and PS1 were increased in the DEX-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026ndash;c). However, following LEV administration, the levels of ADAM10 increased, and levels of BACE1 and PS1 decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026ndash;c). Moreover, DEX considerably increased the BACE1/ADAM10 ratio and LEV considerably decreased the PS1/ADAM10 ratio in DEX-treated APP23/MAPT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). These results suggest that LEV may regulate Aβ production and deposition. ELISA and immunohistochemistry assays were performed to verify this hypothesis. In the DEX-treated groups, Aβ production was considerably increased; however, after LEV treatment, it decreased considerably (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Consistent with these findings, Aβ aggregation in DEX-treated mice was considerably decreased after LEV treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Based on these findings, it is worth noting that LEV inhibited Aβ deposition in DEX-treated mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eLEV mitigates DEX-induced tau phosphorylation via the GSK3α/β and CDK5 pathways\u003c/h2\u003e \u003cp\u003eTau phosphorylation is another prominent pathological feature of AD. Therefore, we examined phosphorylated tau levels in different APP/MAPT mice groups. The levels of p-CDK5 and p-GSK3α/β, which are positively associated with increased tau phosphorylation, were significantly elevated in the DEX-treated mice compared to those in the control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). However, p-CDK5 and p-GSK3α/β phosphorylation was attenuated by LEV in DEX-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), indicating that LEV inhibited tau phosphorylation by deactivating p-CDK5 and p-GSK3α/β. Based on these observations, tau phosphorylation was further evaluated using western blotting, which revealed that LEV suppressed tau phosphorylation in DEX-treated APP23/MAPT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDEX-induced neuronal impairment via glial cell activation, which was mitigated by LEV\u003c/h2\u003e \u003cp\u003eGiven the contrasting effects of DEX and LEV on the regulation of Aβ production, deposition, and tau phosphorylation, we proceeded to assess the expression of GFAP (an astrocyte biomarker) and Iba1 (a microglia biomarker) in the LEV-treated groups compared with that in the DEX-treated groups. A significant increase in optical densities of GFAP and Iba1 was observed in the DEX-treated APP23/MAPT mice, which was effectively reversed by LEV (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). These findings suggest that LEV alleviated DEX-induced inflammatory stress in APP23/MAPT mice. Moreover, the BV2 cells were treated with DEX in the absence or presence of LEV, and their survival rates were determined using MTT assay. DEX and LEV decreased and increased BV2 cell survival rates, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), suggesting that LEV attenuated DEX-induced pyroptosis of microglial cells. Aβ accumulation and tau hyperphosphorylation result in dystrophy and neuronal loss, which prompted us to examine the effects of LEV on neurons. For this purpose, the expression of the presynaptic marker, SYP, and the postmitotic neuronal marker, NeuN, were determined using western blotting. SYP was markedly downregulated in the DEX-treated mice compared to that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), indicating impaired synaptic plasticity induced by DEX in the brains of APP23/MAPT mice. Similarly, the protein level of NeuN was considerably lower in the DEX-treated mice than in the control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), suggesting that DEX-related stress promotes synaptic dystrophy and neuronal loss in the brains of APP23/MAPT mice. However, LEV attenuated the effects of DEX on neuronal dystrophy and loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Similar results were obtained for N2a cells, showing that DEX impaired the survival of neuronal cells and was ameliorated by LEV (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), suggesting that DEX impaired the neurons by activating glial cells, which was ameliorated by LEV.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eLEV protects neurons from DEX-induced apoptosis\u003c/h2\u003e \u003cp\u003eConsidering the aforementioned findings, we investigated apoptotic mechanisms underlying the loss of synapses or neurons. As anticipated, DEX induced neuronal apoptosis by upregulating Bax and downregulating Bcl2, thereby increasing the Bax/Bcl2 ratio in the brains of APP23/MAPT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). However, LEV reduced the Bax/Bcl2 ratio in DEX-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). To validate these \u003cem\u003ein vivo\u003c/em\u003e results, N2a cells were used as \u003cem\u003ein vitro\u003c/em\u003e neuronal models, which showed that DEX simultaneously upregulates Bax and downregulates Bcl2, leading to the loss of neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). In contrast, LEV protects neurons from apoptosis by downregulating Bax and upregulating Bcl2 in DEX-treated N2a cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Similar to the \u003cem\u003ein vivo\u003c/em\u003e results, DEX increases the Bax/Bcl2 ratio, which was reversed by LEV in N2a cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef), suggesting the mechanism by which LEV alleviate synapse or neuronal loss induced by DEX-related stress in APP23/MAPT mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eNLRP1 inflammasome activation by DEX is dependent on GR mechanisms, which was attenuated by LEV\u003c/h2\u003e \u003cp\u003eConsidering the potential role of inflammasomes in AD (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), we investigated the relationship between DEX and NLRP1 inflammasomes. Western blotting revealed a significant increase in NF-κB phosphorylation and NLRP1 and IL-1β expressions in the brains of DEX-treated APP23/MAPT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Furthermore, western blotting revealed that the optical densities of phosphorylated NF-κB, NLRP1, and IL-1β were considerably reduced in the LEV-treated groups compared to those in the DEX-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). These results suggest that LEV inhibited inflammatory stress by deactivating inflammasomes in DEX-activated mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo verify the role of GR in the activation of NLRP1 inflammasome, mifepristone (MIF), a GR antagonist, was used to treat APP/MAPT mice. Western blotting showed decreased optical densities of phosphorylated NF-κB, NLRP1, and IL-1β in the MIF-treated groups compared to those in the DEX-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Therefore, DEX activated NLRP1 inflammasome via GR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eNLRP1 inflammasome activation is critical for Aβ accumulation and tau phosphorylation\u003c/h2\u003e \u003cp\u003eBased on the above results, it was necessary to determine whether NLRP1 is pivotal for the observed Aβ accumulation and tau phosphorylation in DEX-treated APP/MAPT mice. Therefore, siRNA interference experiments were performed to knock down NLRP1 in BV2 cells. Following incubation with human (h) Aβ, BV2 cells' ability to uptake and degrade hAβ was impaired by DEX (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Conversely, knocking down the NLRP1 in BV2 cells partially restored the hAβ uptake and degradation ability of the BV2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Furthermore, tau phosphorylation was attenuated in the NLRP1 knockdown group compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). Altogether, NLRP1 is critical for the effects of DEX on Aβ accumulation and tau phosphorylation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs a synthetic GC, DEX impairs the structure and function of the hippocampus, ultimately contributing to AD progression (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). In this study, we expanded upon our previous research by investigating the effects of DEX on memory impairment and neuropathology in APP23/MAPT mice. Our findings revealed that DEX exacerbated behavioral deficits by reducing SPs burden and tau phosphorylation. In addition, DEX reduced Aβ degradation and clearance in APP23/MAPT mice. Moreover, we found that NLRP1 inflammasome played key roles in AD regulation by DEX. Interestingly, LEV suppressed neuroinflammation by deactivating inflammasomes and protected neurons from dystrophy and loss by suppressing DEX-induced apoptosis.\u003c/p\u003e \u003cp\u003eDEX was suspected to impair learning and memory abilities. As anticipated, DEX increased the time taken and distance traveled by mice to find the platform and decreased crossing times (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Similar to our findings, DEX reportedly exacerbated Aβ-induced learning and memory impairment in rats in a previous study (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). In another study, DEX was confirmed to induce memory and learning impairment in senescent mice (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). In addition, melatonin could reportedly attenuate DEX-induced spatial memory impairment and DEX-induced reduction of synaptic protein levels in the mouse brain (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). In line with these observations, our results demonstrated that LEV could ameliorate AD progression by inhibiting Aβ production and deposition and tau phosphorylation in the brains of DEX-treated APP23/MAPT mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Although no related associated study exists, LEV reportedly normalizes hippocampal CA3/DG activity and improves memory performance in patients with amnestic mild cognitive impairment based on the results of high-resolution functional magnetic resonance imaging techniques (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). In addition, hippocampal spatial memory decline in aged rats was rescued by chronic infusion or a single injection of LEV and sodium valproate before training, as reflected in the MWM test performance (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). In a previous study including APP/PS1 mice, LEV effectively alleviated behavioral deficits in AD (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Consistent with these findings, LEV administered intraperitoneally improves neuronal functions in APP/PS1 Tg mice, suggesting its ability to penetrate the blood-brain barrier (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Likewise, LEV reversed the abnormal expression of neuronal activity-related proteins that reflect hippocampal remodeling and cognitive deficits in hAPPJ20 mice (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Therefore, these results suggest the roles of DEX in exacerbating the pathological features of AD, which were ameliorated by LEV in the brains of APP23/MAPT mice.\u003c/p\u003e \u003cp\u003eConsidering that Aβ production and deposition are widely accepted as key pathogenic factors of AD (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), we initially investigated the effect of DEX on Aβ production and deposition. The results indicated that DEX induced Aβ production and deposition in the brains of APP23/MAPT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In agreement with our finding, chronic DEX treatment accelerates Aβ production in the neurons of APP/PS1 mice (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). DEX further induces Aβ production with cholinergic dysfunction similar to AD (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Aβ results from β-site APP-cleaving enzyme 1 (BACE1) and γ-secretase-mediated cleavage of APP. Consistently, we found that DEX elevated BACE1 and PS1 levels responsible for Aβ production in the brains of APP/MAPT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Similarly, GC exposure elevated Aβ production by increasing BACE1 and APP gene expression in primary astrocytes (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). In addition to inducing Aβ production and deposition, DEX induces tau hyperphosphorylation (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), which is consistent with our results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). GC exposure induces tau hyperphosphorylation and neurostructural deficits in hippocampal neurons \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo counteract the side effects of DEX, LEV was used to treat APP23/MAPT mice. The results demonstrated that LEV suppressed Aβ production and deposition and tau phosphorylation (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Although no relevant research exists, LEV and TPM reportedly reduce Aβ production by inhibiting the activity of γ-secretase (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In addition, the underlying mechanisms may involve Aβ uptake and degradation (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). In line with our findings, the lack of Aβ uptake by glial cells is a major cause of sporadic AD (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e), which might be caused by impaired Aβ clearance in AD (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Regarding the underlying mechanism, LEV increases Aβ uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Aβ degradation is regarded as a new therapeutic target for AD treatment (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Our study contributed to previous studies showing that LEV enhanced Aβ degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Moreover, tau phosphorylation can be induced by Aβ production and deposition (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). We found that LEV decreased tau phosphorylation via CDK5- and GSK3α/β-dependent mechanisms (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Similarly, tau phosphorylation is reportedly mediated by CDK5 and GSK3α/β (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Based on these findings, we can infer that LEV counteracts the effects of DEX on Aβ production and deposition and tau hyperphosphorylation.\u003c/p\u003e \u003cp\u003eExcessive Aβ loading and tau hyperphosphorylation result in neuroinflammation and neuronal loss in AD (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Inflammasomes contribute to various disorders in the CNS by causing neuroinflammation. Extracellular accumulation of Aβ in SPs in brains with AD is a principal event in AD (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Deposition of Aβ peptide initiates inflammasome activity in the microglia (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Moreover, inflammasome activation causes AD impairment through Aβ deposition and loss of spatial memory via harmful chronic inflammatory response. It is important to note that NLRP1 activation in the brain is restricted to plaque-associated microglia, suggesting that microglial activation of the NLRP1 inflammasome is a pivotal event in AD pathogenesis (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Furthermore, the correlation between AD and local neuroinflammation has been established. As an important inflammasome component, IL-1β can induce tau phosphorylation (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e), leading to compromised learning and memory in animals with AD (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). By blocking IL-1β, AD was ameliorated in animals with AD (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Consistent with other studies, our study further shows that LEV deactivates DEX-induced inflammasomes, thus suppressing glial cell activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The glial cells are responsible for Aβ clearance, whereas the microglial cells are responsible for degrading Aβ degradation via autophagy (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccumulating evidence has indicated that elevated levels of GC cause chronic environmental stress, which is a risk factor for AD (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). Evidence also suggests that high but not low levels of DEX exposure impair hippocampal neurons in rats (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). Using SH-SY5Y cells, high levels of DEX induce neuronal loss and neurotoxicity by impairing the mitochondria (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). Consistent with previous studies, we further found that DEX inhibited the expression of both NeuN and SYP, leading to the loss of neurons in the synapse (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Moreover, the apoptotic mechanisms underlying the above process were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). LEV attenuated DEX-induced apoptosis, ameliorating neuronal and synaptic loss (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Our previous study had shown the protective effects of LEV on neurons via apoptosis inhibition in kainic acid (KA)-activated APP23/MAPT mice (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In support of our findings, LEV administration after hypoxia reduces neuronal apoptosis in a neonatal rat model of hypoxic-ischemic brain injury (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). Additionally, a study showed that LEV conferred neuroprotective effects against focal cerebral ischemia-reperfusion injury in mice (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn conclusion, this study builds on previous studies by showing that LEV ameliorated amyloidosis and tauopathy by inhibiting the activity of NLRP1 inflammasome in mice with DEX-induced AD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eDisclosure of potential conflicts of interest:\u003c/strong\u003e The authors have no relevant financial or non-financial interests to disclose.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eResearch involving Human Participants and/or Animals:\u0026nbsp;\u003c/strong\u003eAll animal experimental procedures were approved by the Institutional Animal Care and Use Committee of The First Hospital of Jilin University and adhered to the Guidelines for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eInformed consent:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/li\u003e\n\u003c/ul\u003e\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (No. 81873812), the Fundamental Research Funds for the Central Universities, the Jilin Province Department of Finance (No. JLSWSRCZX2023-36), and the Science and Technology Development Program of the Jilin Province (No. 20220203122SF).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiang-Yu and Yang Ruan conceived the project. Hai-Chen, Zhi-Wei, Yu-Dan, Feng-Yan, and Jie Zhu conducted the research or assisted the research, discussed the project, and assisted the manuscript preparation. Xiang-Yu and Yang Ruan wrote the manuscript. Jie Zhu supervised the project. All authors are accountable for all aspects of the work and all persons designated as authors qualify for the authorship, and all those who qualify for authorship are listed. All authors read and approved the final version of the manuscript submitted for publication.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data presented in this study are included in the article/supplementary material. Further inquiries should be directed to the corresponding author(s). The experimental animal procedures performed in this study were reviewed and approved by the Animal Care and Use Committee of The First Hospital of Jilin University, Changchun, China.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experimental procedures were approved by the Institutional Animal Care and Use Committee of The First Hospital of Jilin University and adhered to the Guidelines for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhang Y, Zhao Y, Zhang L, Yu W, Wang Y, Chang W (2019) Cellular prion protein as a receptor of toxic amyloid-β42 oligomers is important for Alzheimer\u0026rsquo;s disease. 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Histopathology 5:549\u0026ndash;564. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-2559.1981.tb01818.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2559.1981.tb01818.x\" 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":false,"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":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"dexamethasone, levetiracetam, NLRP1, APP23/MAPTP301S, Alzheimer’s","lastPublishedDoi":"10.21203/rs.3.rs-3517043/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3517043/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eStress induced by glucocorticoids (GC), such as dexamethasone (DEX), has the potential to detrimentally impact the structure and function of the hippocampus and is closely associated with the development and progression of Alzheimer\u0026rsquo;s disease (AD). However, it remains uncertain whether LEV and TPM can effectively alleviate neuropathological and cognitive deficits in patients with DEX-induced AD by preserving or restoring neuronal network activities. This study aimed to investigate the mechanisms underlying the effect of DEX on AD development and progression and identify the role of NRP1 inflammasome in APP23/MAPT\u003csup\u003eP301S\u003c/sup\u003e mice. APP23/MAPT\u003csup\u003eP301S\u003c/sup\u003e mice were treated with DEX in the absence and presence of levetiracetam (LEV). After treatment, the mice were subjected to various cognitive and behavioral tests. DEX accelerated neuronal impairment by promoting the accumulation of β-amyloid protein and phosphorylation of tau in senile plaques and neurofibrillary tangles in APP23/MAPT\u003csup\u003eP301S\u003c/sup\u003e mice. Moreover, DEX significantly upregulated BACE1 and promoted the phosphorylation of cyclin-dependent kinase-5 and glycogen synthase kinase 3α/β, resulting in synaptic dystrophy and apoptosis. NLRP3 siRNA transfection showed that NLRP1 inflammasome activation is pivotal to the observed DEX effects. To counteract the adverse effects of DEX, LEV was administered to APP23/MAPT\u003csup\u003eP301S\u003c/sup\u003e mice, and it ameliorated DEX-induced AD via NLRP1-dependent mechanisms. This study underscores the detrimental impact of chronic glucocorticoid exposure on AD pathogenesis and the potential therapeutic benefits of compounds such as LEV in counteracting these effects by regulating neuroinflammation and key pathological markers.\u003c/p\u003e","manuscriptTitle":"Levetiracetam ameliorated amyloidosis and tauopathy in mice with dexamethasone-induced Alzheimer’s disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-07 23:34:48","doi":"10.21203/rs.3.rs-3517043/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Reject and Resubmit","date":"2023-11-22T14:20:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-11-03T02:45:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurobiology","date":"2023-10-30T05:09:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e83052df-beff-4b89-92b0-74dc8e23dd8d","owner":[],"postedDate":"November 7th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-27T13:51:09+00:00","versionOfRecord":[],"versionCreatedAt":"2023-11-07 23:34:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3517043","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3517043","identity":"rs-3517043","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}