Huperzine A Regulates the Physiological Homeostasis of Amyloid Precursor Protein Proteolysis and Tau Protein Conformation-A Computational and Experimental Investigation

Abstract

The beneficial actions of the natural compound Huperzine A (Hup A) against age-associated learning and memory deficits promote this compound as a nootropic agent. Alzheimer's disease (AD) pathophysiology is characterized by the accumulation of amyloid beta (Aβ). Toxic Aβ oligomers account for the cognitive dysfunctions much before the pathological lesions are manifested in the brain. In the present study, we investigated the effects of Hup A on amyloid precursor protein (APP) proteolysis in SH-SY5Y neuroblastoma cells. Hup A downregulated the expression of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and presenilin 1 (PS1) levels but augmented the levels of A disintegrin and metalloproteinase 10 (ADAM10) with significant decrement in the Aβ levels. We herein report for the first time an in silico molecular docking analysis that revealed that Hup A binds to the functionally active site of BACE1. We further analyzed the effect of Hup A on glycogen synthase kinase-3 β (GSK3β) and phosphorylation status of tau. In this scenario, based on the current observations, we propose that Hup A is a potent regulator of APP processing and capable of modulating tau homeostasis under physiological conditions holding immense potential in preventing and treating AD like disorders.

Keywords: A disintegrin and metalloproteinase 10; Alzheimer’s disease; Huperzine A; amyloid beta; amyloid precursor protein; presenilin 1; tau; β-site amyloid precursor protein cleaving enzyme 1.

Figure 1

Effect of huperzine A (Hup A) on the amyloidogenic pathway in SH-SY5Y cells. Cells were treated with various concentrations of Hup A (0, 0.1, 1, and 10 µM) for 24 h. The levels of β-site amyloid precursor protein cleaving enzyme 1 (BACE1), a 99 amino acid C-terminal fragment of amyloid beta precursor protein (APP-C99), presenilin-1 (PS1), and amyloid beta 42 (Aβ42) protein were assessed using Western blot analysis. (A) Effect of Hup A on the levels of BACE1 protein. (B) Effect of Hup A on the levels of APP-C99 protein. (C) Effect of Hup A on the levels of the carboxy-terminal fragment of PS1 (PS1-CTF) protein. (D) Effect of Hup A on the levels of Aβ42 protein. (E) Confocal image of BACE1 immunostaining was captured after incubation in culture medium for 24 h. (a) Basal expression in the control cells. (b) Hup A-treated cells stained with anti-BACE1. Scale bar = 20 µm (20× magnification). Data are expressed as a percentage of the control and represented as the mean ± standard error of the mean (n = 4). Statistical significance was calculated using one-way ANOVA analysis (*, **, and *** denote the statistical significance at p < 0.05, p < 0.01, and p < 0.001, respectively, compared to the control group). (white bar = control and black bar = Hup A).

Figure 2

Effect of Huperzine A (Hup A) on the nonamyloidogenic pathway in SH-SY5Y cells. Cells were treated with various concentrations of Hup A (0, 0.1, 1, and 10 µM) for 24 h. The levels of a disintegrin and metalloproteinase 10 (ADAM10) and an 83 amino acid C-terminal fragment of amyloid beta precursor protein (APP-C83) were assessed using Western blot analysis. (A) Effect of Hup A on the levels of ADAM10 protein. (B) Effect of Hup A on the levels of APP-C83 protein. Data are expressed as a percentage of the control and represented as the mean ± standard error of the mean (n = 4). Statistical significance was calculated using one-way ANOVA analysis *, **, and *** denote the statistical significance at p < 0.05, p < 0.01, and p < 0.001, respectively, compared to the control group). (white bar = control and black bar = Hup A).

Figure 3

Effect of huperzine A (Hup A) on β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and a disintegrin and metalloproteinase 10 (ADAM10) activity. The activity was assayed for α- or β-secretase activity as described in the section of materials and methods. (A) BACE1 catalytic activity. (B) ADAM10 catalytic activity. The results are expressed as a percentage of the control (white bar represents no drug treatment; dark bar represents different concentration of Hup A treatment; grey bar represents 1 µM LY2886721, the BACE1-specific inhibitor, or 10 µM GM6001, the ADAM10-specific inhibitor). The values in the bar graph represent the mean ± standard error of the mean of the specific fluorescence recorded during independent experiment (n = 4). Statistical analysis was performed using one-way ANOVA analysis (*** denotes statistical significance at p < 0.001 compared with untreated control). (white bar = control, black bar = Hup A, and grey bar = positive control).

Figure 4

Molecular docking study of huperzine A (Hup A) and β-site amyloid precursor protein cleaving enzyme 1 (BACE1)/a disintegrin and metalloproteinase 10 (ADAM10) interactions. (A) The active site and protein structure of BACE1 are shown. The active site of BACE1 constitutes amino acids residues 90–101 (pink) and two aspartic residues (Asp93 and Asp and amino acids 289). (B) The best binding position of Hup A on BACE1 was predicted to be at the active site. (C) The 2D diagram shows amino acid residues lining the active site pocket interacting with Hup A. (D) The most favorable binding position of Hup A on ADAM10 was predicted at disintegrin domain (pink ribbons). (E) The interacting residues involved in Hup A interaction are illustrated using the 2D plot. (C,D) Van der Waals, attractive charges, and pi–pi interaction are indicated by green, orange, and pink dashed lines, respectively. The molecular structure of Hup A is shown as sticks with carbon, hydrogen, oxygen, and nitrogen atoms labelled with grey, white, red, and blue, respectively.

Figure 5

Effect of huperzine A (Hup A) on the glycogen synthase kinase-3 beta (GSK3β and tau pathways in SH-SY5Y cells. Cells were treated with various concentrations of Hup A (0, 0.1, 1, and 10 µM) for 24 h. The levels of phosphorylation of glycogen synthase kinase-3 beta (p-GSK3β), GSK3β, phosphorylation of tau (p-TAU), and total tau (TAU) protein were assessed using Western blot analysis. (A) Effect of Hup A on the ratio of the phosphorylated GSK3β to the total GSK3β (p-GSK3β/GSK3β). (B) Effect of Hup A on the ratio of phosphorylated tau to total tau protein (p-TAU/TAU). Data are expressed as a percentage of the control and represented as the mean ± standard error of the mean (n = 4). Statistical significance was calculated using one-way ANOVA analysis (** and *** denote the statistical significance at p < 0.01 and p < 0.001, respectively, compared to the control group). (white bar = control, black bar = Hup A).

Figure 6

Schematic summary of the neuroprotective properties of huperzine A (Hup A). Hup A enhanced the expression of proteins in the nonamyloidogenic pathway by increasing the protein levels of a disintegrin and metalloproteinase 10 (ADAM10) and an 83 amino acid C-terminal fragment of amyloid beta precursor protein (APP-C83), which might help to improve neuronal loss and normal brain function. On the other hand, Hup A diminished the expression of proteins in the amyloidogenic pathway by lowering the levels of two major secretases, β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and presenilin-1 (PS1), subsequently resulting in decreases in the production of both a 99 amino acid C-terminal fragment of amyloid beta precursor protein (APP-C99) and amyloid beta (Aβ). Moreover, Hup A increased the levels of phosphorylation of glycogen synthase kinase-3 beta (Serine9) (GSK3β (Ser9)), an inactive form of GSK3β protein, whereas it decreased the levels of phosphorylation of tau. Therefore, Hup A might help to reduce pathological events, including Aβ plaque and neurofibrillary tangle (NFT) formation, and relieve the progression of Alzheimer’s disease (AD). ( = increase, = decrease).