Withania somnifera (Ashwagandha) Improves Spatial Memory, Anxiety and Depressive-like Behavior in the 5xFAD Mouse Model of Alzheimer's Disease

Abstract

Withania somnifera (WS), also known as ashwagandha, is a popular botanical supplement used to treat various conditions including memory loss, anxiety and depression. Previous studies from our group showed an aqueous extract of WS root (WSAq) enhances cognition and alleviates markers for depression in Drosophila. Here, we sought to confirm these effects in the 5xFAD mouse model of β-amyloid (Aβ) accumulation. Six- to seven-month-old male and female 5xFAD mice were treated with WSAq in their drinking water at 0 mg/mL, 0.5 mg/mL or 2.5 mg/mL for four weeks. In the fourth week of treatment, spatial memory, anxiety and depressive-like symptoms were evaluated. At the conclusion of behavioral testing, brain tissue was harvested, immunohistochemistry was performed, and the cortical expression of antioxidant response genes was evaluated. Both concentrations of WSAq improved spatial memory and reduced depressive and anxiety-related behavior. These improvements were accompanied by a reduction in Aβ plaque burden in the hippocampus and cortex and an attenuation of activation of microglia and astrocytes. Antioxidant response genes were upregulated in the cortex of WSAq-treated mice. Oral WSAq treatment could be beneficial as a therapeutic option in AD for improving disease pathology and behavioral symptoms. Future studies focused on dose optimization of WSAq administration and further assessment of the mechanisms by which WSAq elicits its beneficial effects will help inform the clinical potential of this promising botanical therapy.

Keywords: 5xFAD mice; Alzheimer’s disease; anxiety; ashwagandha; beta-amyloid; depression; memory; neuroinflammation; oxidative stress.

Figure 1

Treatment timeline for experiments.

Figure 2

WSAq improves OLM performance in 5xFAD mice. WSAq treatment attenuated deficits in OLM performance at both 2 hours (A) and 24 hours (B). n = 9–12 per treatment group * p < 0.05.

Figure 2

WSAq improves OLM performance in 5xFAD mice. WSAq treatment attenuated deficits in OLM performance at both 2 hours (A) and 24 hours (B). n = 9–12 per treatment group * p < 0.05.

Figure 3

WSAq improves performance on the OF in 5xFAD mice. WSAq improved performance on the OF at the 0.5 g/L dose and approached significant improvement at the 2.5 g/L dose. * p < 0.05, ** p < 0.01. n = 8–12 per treatment group.

Figure 4

WSAq improves performance on the FST in 5xFAD mice. WSAq improved performance on the FST at both the 0.5 g/L and 2.5 g/L doses. WSAq treated groups were not significantly different from the WT group. * p < 0.05, ** p < 0.01, *** p < 0.001. n = 9–12 per treatment group.

Figure 5

Aβ accumulation is attenuated following WSAq treatment. (A) Representative images from each 5xFAD treatment condition. In both the (B) cortex and (C) hippocampus a significant reduction of pan-Aβ staining at the 2.5 g/L dose was observed, * p < 0.05, ** p < 0.01. n = 13–15 per treatment group.

Figure 6

Astrocytic activation is reduced following WSAq treatment. Representative images of GFAP staining given by (A). A significant reduction in GFAP staining was observed at the 0.5 g/L dose but was not observed in the 2.5 g/L group in both the cortex (B) and hippocampus (C). **** p < 0.0001. n = 7–15 per treatment group.

Figure 7

WSAq reduces microglial activation in 5xFAD mice. Representative images of Iba1 staining given by (A). Treatment with 2.5 g/L WSAq reduced activated microglia in the hippocampus (C) and approached significant reduction of IBA1 in the cortex (B). Activation following WSAq 0.5 g/L was not significantly different from the 5xFAD controls. * p < 0.05, **** p < 0.0001. n = 11–15 per treatment group.

Figure 8

WSAq upregulates antioxidant response in the cortex of 5xFAD mice. (A) Gene expression of antioxidant response genes is increased in the cortex of WSAq treated 5xFAD mice. (B) NRF2 protein expression is likewise increased in the cortex of 5xFAD mice treated with 2.5 g/L WSAq. * p < 0.05. n = 9–12 per treatment condition.

Figure 8

WSAq upregulates antioxidant response in the cortex of 5xFAD mice. (A) Gene expression of antioxidant response genes is increased in the cortex of WSAq treated 5xFAD mice. (B) NRF2 protein expression is likewise increased in the cortex of 5xFAD mice treated with 2.5 g/L WSAq. * p < 0.05. n = 9–12 per treatment condition.