Withaferin A Rescues Brain Network Dysfunction and Cognitive Deficits in a Mouse Model of Alzheimer's Disease

Abstract

Background: Alzheimer's disease (AD) is the most common dementia, characterized by significant cognitive impairments and neural network dysfunction. Currently, multiple therapeutic strategies are being developed to design effective anti-AD drugs. Among them, Withaferin A (WA), a natural steroidal lactone extracted from Withania somnifera leaves, has been shown to reduce amyloid-β (Aβ) peptide levels in vitro. However, its potential to improve cognitive function in AD remains unclear. Methods: In this study, 5xFAD mice were administered WA (2 mg/kg intraperitoneally every 2 days) for 14 days, and its neuroprotective effects were evaluated through behavioral tests, wide-field imaging, immunohistochemistry, and ELISA. Results: WA significantly improved short-term memory, as evidenced by enhanced performance in the Novel Object Recognition Test (NORT) (p < 0.001, n = 10), Novel Location Recognition Test (NLRT) (p < 0.01, n = 14), and Three-Chamber Social Test (TCST) (p < 0.001, n = 8). WA also ameliorated long-term memory deficits in the Morris Water Maze Test (MWMT) (p < 0.05, n = 7). Furthermore, cortical wide-field Ca²⁺ imaging revealed that WA treatment rescued slow-wave impairments by enhancing long-range coherence (0.8363 ± 0.0185, p < 0.01, n = 8) and reducing the frequency of slow-wave activity (0.6578 ± 0.0512 Hz, p < 0.01, n = 8). Additionally, WA treatment significantly reduced Aβ plaque deposition in both cortical and hippocampal regions. Conclusions: These findings suggest that WA may be a promising therapeutic agent for AD, exerting neuroprotective effects.

Keywords: Alzheimer’s; cognitive function; cortical slow-wave activity; neuroprotection; withaferin A.

Figure 1

WA improves recognition memory in 5xFAD mice. (A) Experimental protocol for the NORT and representative heatmaps showing time distribution around each object. (B) Object discrimination index at 2 weeks. 5xFAD mice were treated with WA (n = 10) or DMSO (n = 8), and WT mice were treated with DMSO (n = 8). N, novel; F, familiar. (C) Summary change curve of the object discrimination index across varying WA doses (0.1 mg/kg, n = 8; 1 mg/kg, n = 8; 2 mg/kg, n = 8; and 10 mg/kg, n = 8). (D) Object discrimination index before and after WA treatment during the testing phase. 5xFAD mice were treated with WA (n = 8) or DMSO (n = 8). (E) Experimental protocol for the NLRT and representative heatmaps showing time distribution around each object. N, novel; F, familiar. (F) Location discrimination index at 2 weeks. 5xFAD mice were treated with WA (n = 14) or DMSO (n = 8), and WT mice were treated with DMSO (n = 8). (G) Summary change curve of the location discrimination index across varying WA doses (0.1 mg/kg, n = 8; 1 mg/kg, n = 8; 2 mg/kg, n = 8; and 10 mg/kg, n = 7). (H) Location discrimination index before and after WA treatment during the test phase. 5xFAD mice were treated with WA (n = 8) or DMSO (n = 8). Each dot represents an individual animal. Data in (B,C,F,G) are presented as the mean ± SEM. Data in (B,F) are analyzed using one-way ANOVA. Data in (D,H) are analyzed using a paired t-test. n.s., p > 0.05; **, p < 0.01; and ***, p < 0.001.

Figure 2

WA improves social memory in 5xFAD mice. (A) Experimental protocol for the Three-Chamber Social Test (TCST). E, empty; M, mouse; N, novel; F, familiar. (B) Representative heatmaps showing time distribution in the TCST. (C,E) Social preference index during training (C) and testing (E) in the social novelty task. (D,F) Total investigation time during training (D) and testing (F) in the social novelty task. 5xFAD mice were treated with WA (n = 8) or DMSO (n = 8), and WT mice were treated with DMSO (n = 8). Each dot represents an individual animal. Data in C–F are presented as the mean ± SEM. Data in (C–F) are analyzed using one-way ANOVA. n.s., p > 0.05; *, p < 0.05; and ***, p < 0.001.

Figure 3

WA improves long-term memory in 5xFAD mice via the Morris Water Maze Test (MWMT). (A) Representative swim path tracings from the MWMT. The grey solid circles represent the platform, and the grey dashed circles represent the removal of the platform. (B) Escape latency during the training phase of the MWMT. (C) Time spent in the target quadrant during the probe test. 5xFAD mice were treated with WA (n = 7) or DMSO (n = 8), and WT mice were treated with DMSO (n = 8). Each dot represents an individual animal. Data in (B,C) are presented as the mean ± SEM. Data in (B) are analyzed using two-way ANOVA. Data in (C) are analyzed using one-way ANOVA. n.s., p > 0.05; *, p < 0.05; and ***, p < 0.001.

Figure 4

WA improves long-range coherence of slow-wave activity in 5xFAD mice. (A,C,E) Schematic diagrams of the injection sites and comparison of slow waves in the AD control group (A), the treatment group (C), and the WT control group (E). Black circles and lines indicate the frontal cortical regions and their associated slow-wave activity, whereas red circles and lines correspond to the occipital cortical regions and their respective slow-wave activity. (B,D,F) Cross-correlation matrices from three representative experiments created from the cortical domains in the AD control group (B), the treatment group (D), and the WT control group (F). (G) Summary plot displaying the average cross-correlation coefficients and standard errors plotted against cortical distance. (H) Summary plot of the mean frequencies of slow waves along the anteroposterior cortical axis. 5xFAD mice were treated with WA (n = 8) or DMSO (n = 8), and WT mice were treated with DMSO (n = 8). Data in G and H are presented as the mean ± SEM and analyzed using two-way ANOVA. **, p < 0.01; and ***, p < 0.001.

Figure 5

WA ameliorates Aβ deposition in 5xFAD mice. (A,D) Thioflavin S staining (A) and Congo Red staining (D) of the WA treatment group and AD control group. The inset shows a magnified view of a representative image in the cortex (Ctx) and the hippocampus (Hp). (B,C) Comparison of Thioflavin S -positive plaques in the cortex (Ctx) (B) and hippocampus (Hp) (C) of the WA treatment group (n = 8) and AD control group (n = 8). (E,F) Comparison of Congo Red-positive plaques in the cortex (Ctx) (E) and hippocampus (Hp) (F) of the WA treatment group (n = 8) and AD control group (n = 8). (G,H) Quantification of Aβ₄₀ using ELISA in TBS (G) and SDS (H) of brain homogenates of the WA treatment group (n = 6) and AD control group (n = 6). (I,J) Quantification of Aβ₄₂ using ELISA in TBS (I) and SDS (J) of brain homogenates of the WA treatment group (n = 6) and AD control group (n = 6). Each dot represents an individual animal. Data in (B,C,E–J) are presented as the mean ± SEM and analyzed using an unpaired t-test. *, p < 0.05; **, p < 0.01; and ***, p < 0.001.