Withania somnifera reverses Alzheimer's disease pathology by enhancing low-density lipoprotein receptor-related protein in liver

Abstract

A 30-d course of oral administration of a semipurified extract of the root of Withania somnifera consisting predominantly of withanolides and withanosides reversed behavioral deficits, plaque pathology, accumulation of β-amyloid peptides (Aβ) and oligomers in the brains of middle-aged and old APP/PS1 Alzheimer's disease transgenic mice. It was similarly effective in reversing behavioral deficits and plaque load in APPSwInd mice (line J20). The temporal sequence involved an increase in plasma Aβ and a decrease in brain Aβ monomer after 7 d, indicating increased transport of Aβ from the brain to the periphery. Enhanced expression of low-density lipoprotein receptor-related protein (LRP) in brain microvessels and the Aβ-degrading protease neprilysin (NEP) occurred 14-21 d after a substantial decrease in brain Aβ levels. However, significant increase in liver LRP and NEP occurred much earlier, at 7 d, and were accompanied by a rise in plasma sLRP, a peripheral sink for brain Aβ. In WT mice, the extract induced liver, but not brain, LRP and NEP and decreased plasma and brain Aβ, indicating that increase in liver LRP and sLRP occurring independent of Aβ concentration could result in clearance of Aβ. Selective down-regulation of liver LRP, but not NEP, abrogated the therapeutic effects of the extract. The remarkable therapeutic effect of W. somnifera mediated through up-regulation of liver LRP indicates that targeting the periphery offers a unique mechanism for Aβ clearance and reverses the behavioral deficits and pathology seen in Alzheimer's disease models.

Fig. 1.

Behavioral recovery and reduction of amyloid pathology in middle-aged and old APP/PS1 mice after 30 d of WS treatment. (A and B) Reversal of behavioral deficits in middle-aged (A) and old (B) APP/PS1 (Tg) mice measured as a significant decrease in reference memory errors (RME), correct working memory errors (CWME), and incorrect working memory errors (ICWME) on an eight-arm radial maze (RAM) task. (C) The plaque area was decreased in the CT and HP of middle-aged and old animals. Sections stained by normal IgG were used as negative controls. (D and E) Aβ42 levels were significantly reduced in the CT, HP, and plasma of middle-aged and old animals treated with WS. Aβ42 levels in WT and middle-aged and old mice were as follows: CT, 0.63 pmol/mg protein; HP, 0.30 pmol/mg protein; plasma, 0.52 pmol/100 mL and CT, 0.78 pmol/mg protein; HP, 0.33 pmol/mg protein; plasma, 0.61 pmol/100 mL, respectively. (F) A significant reduction of Aβ42 monomer and lower oligomers was seen in CT. Values are mean ± SEM (A and B) or mean ± SD (C–F), n = 10 mice. *P ≤ 0.05.

Fig. 2.

Time course of clearance of AD pathology by WS in APP/PS1 mice. (A) A significant decrease in silver-stained plaques was seen in CT after 14–30 d of treatment. (B) Aβ42 monomer and oligomers were decreased significantly after 7 and 14 d, respectively. (C and D) Cortical Aβ42/40 decreased progressively from day 14 to day 30; however, corresponding plasma levels were significantly elevated from day 7 to day 14 and decreased thereafter up to day 30. Values are mean ± SD (n = 8 mice). *P ≤ 0.05.

Fig. 3.

WS induces the expression of LRP, NEP, and sLRP in APP/PS1 mice. (A and B) mRNA and protein levels of LRP (A) increased progressively in CT from day 14 onward, whereas NEP mRNA, protein, and activity (B) increased after day 21. (C and D) LRP expression increased in endothelial cells as detected by colocalization with CD31, but not in cortical neurons (arrowheads). (Inset) Negative control stained by normal IgG. (E and F) In contrast, hepatic LRP, NEP (E), and plasma sLRP (F) increased significantly from day 7 onward. (G) Pearson correlation analysis indicates significant negative correlations among brain Aβ42 and liver LRP, NEP, and sLRP. Values are mean ± SD (n = 8 mice). *P ≤ 0.05.

Fig. 4.

Down-regulation of hepatic LRP abolishes the therapeutic efficacy of WS. Young adult WT mice treated with WS for 30 d showed significant increases in hepatic LRP, NEP (A), and plasma sLRP (B), but no change in brain levels. (C) Aβ42/40 was decreased in brain and plasma. APP/PS1 mice given WS for 7 d along with antisense oligos to LRP (AS) showed a significant decrease in mRNA and protein of liver LRP (D) and plasma sLRP (E), leading to an increase in brain Aβ42/40 (G) and a decrease in plasma (H) Aβ42/40 compared with mice treated with WS and random (R) oligos. (F) Antisense oligos to LRP had no effect on liver NEP. Values are mean ± SD (n = 9 mice). *P ≤ 0.05.