Yuan-Hu Zhi Tong Prescription Mitigates Tau Pathology and Alleviates Memory Deficiency in the Preclinical Models of Alzheimer's Disease

Abstract

Alzheimer's disease (AD) is characterized by memory dysfunction, Aβ plaques together with phosphorylated tau-associated neurofibrillary tangles. Unfortunately, the present existing drugs for AD only offer mild symptomatic cure and have more side effects. As such, developments of effective, nontoxic drugs are immediately required for AD therapy. Present study demonstrates a novel role of Chinese medicine prescription Yuan-Hu Zhi Tong (YZT) in treating AD, and it has substantiated the in vivo effectiveness of YZT in two different transgenic mice models of AD, namely P301S tau and 3XTg-AD mice. Oral treatment of YZT significantly ameliorates motor dysfunction as well as promotes the clearance of aggregated tau in P301S tau mice. YZT improves the cognitive function and reduces the insoluble tau aggregates in 3XTg-AD mice model. Furthermore, YZT decreases the insoluble AT8 positive neuron load in both P301S tau and 3XTg-AD mice. Using microarray and the "Connectivity Map" analysis, we determined the YZT-induced changes in expression of signaling molecules and revealed the potential mechanism of action of YZT. YZT might regulate ubiquitin proteasomal system for the degradation of tau aggregates. The research results show that YZT is a potential drug candidate for the therapy of tau pathogenesis and memory decline in AD.

Keywords: Alzheimer’s disease; Chinese medicine; P301S tau mice; connectivity map; microarray; neurofibrillary tangles; yuan-hu zhi tong.

FIGURE 1

Qualitative analysis and herbal preparation of YZT. (A) The herbal materials of YZT were powdered boiled in water; the extract was concentrated and lyophilized to prepare YZT extract powder. LC-ESI-Q/TOF chromatograms (TIC) of (B) YZT, (C) CY, and (D) ADH. (E) Peaks: 1. Protopine, 2. Tetrahydropalmatine, 3. α-Allocryptopine, 4. Byakangelicin, 5. Coptisine, 6. Tetrahydroberberine, 7. Corydaline, 8. Berberine, 9. Palmatine, 10. Coptisine, 11. Dehydrocorydaline, 12. Psolaren, 13. Imperatorin, and 14. Isoimperatorin.

FIGURE 2

YZT treatment improved motor function and enhances learning and memory function in P301S and 3XTg-AD mice models. (A) The timeline schedule of drug administration and behavior tests in P301S tau mice. (B) In tail hanging test, YZT improved the motor function of 4 months P301S tau mice when compared to vehicle. (C) In rotarod test, regular treatment of 2-month old P301S tau mice with YZT (2 or 4 g/kg) via food admixture for 2 months significantly ameliorated motor function. The average latency of mice (N = 14) fall was calculated. ####p < 0.01 (WT/Vehicle), ***p < 0.001(YZT 2 g/kg/d), and ****p < 0.0001 (YZT 4 g/kg/d) vs Tg-vehicle. (D) The timeline schedule of drug administration and behavior tests in 3XTg-AD mice. (E) After the visible platform training in Morris water maze test, the experimental mice were trained for 6 days with four trials per day to learn the place and location of hidden platform in the tank. The learning potential of the YZT treatment group improved with the time and days of learning when compared to the Tg-vehicle. Each point represents the mean length values of four trials per day, (N = 14). ^^^^p < 0.001 (WT-vehicle vs. Tg-vehicle); ###p < 0.001 (YZT -1 g/kg vs. Tg-vehicle), ∼∼∼∼ p < 0.0001 (YZT-2 g/kg vs. Tg-vehicle), **p < 0.01 (YZT-4 g/kg vs. Tg-vehicle). (F) on the seventh Day, the probe trial demonstrated that YZT treatment showed improved memory function in probing the platform in target quadrant when compared to the Tg-vehicle. (G) The displayed pictures illustrate YZT improved the memory retention in animal’s behavior during the probe trial in the animal tracking camera videos.

FIGURE 3

YZT treatment mitigates tau pathology in P301S and 3XTg-AD mice. (A) Chronic treatment of YZT reduced AT8, AT100 and MC1 in the cortico-hippocampal brain region of P301S tau mice. The displayed pictures are fluorescent images of brain slice taken in fluorescent detecting microscope. (B) The quantified results of AT8, AT100 and MC1 positive neurons in the cortico-hippocampal brain region of P301S tau mice was performed using Image J software N = 14, **p < 0.01; ***p < 0.001. (C) Chronic treatment of YZT reduced AT8-, PHF- and HT7-positive neurons in the CA2, CA3 hippocampal region of 3XTg-AD mice. (D) Quantification of AT8-, HT7- and PHF1 positive neurons in the CA2, CA3 hippocampal brain region of 3XTg-AD mice. The number of phospho and total tau-positive neurons were quantified using ImageJ software N = 4.

FIGURE 4

YZT reduces the pathological phospho tau in P301S and 3XTg-AD mice models. (A) Regular YZT treatment decreased the sarcosyl insoluble tau protein levels in the brain homogenates of P301S tau mice. There was no change in the soluble tau in the sarcosyl soluble brain homogenate fraction. (B) The quantified results of different tau epitopes protein levels in brain lysates of P301S tau mice was performed using Image J software. (C) Long term YZT treatment lowered the sarcosyl insoluble phospho tau protein levels in the brain homogenates of 3XTg-AD mice model. There was no change in the soluble tau in the sarcosyl soluble brain homogenate fraction. (D) The densitometric analysis of the various tau epitopes protein levels in the brain lysates of 3XTg-AD mice models was performed using Image J software. **p < 0.01; ***p < 0.001.

FIGURE 5

YZT-mediated network analysis reveals UPS as potential target. (A) Potential network involved in YZT-treated SHSY5Y-P301L cells. The gene expression profile of YZT-treated SHSY5Y-P301L cells were analyzed by using Ingenuity Pathway Analysis (IPA and STITCH (http://stitch.embl.de/). Red: up-regulation. Green: down-regulation.

FIGURE 6

Involvement of UPS in YZT-mediated degradation of tau. (A) Viability test of YZT on SH-SY5Y-P301L cells. Cell viability was determined using the MTT assay. (B) Effect of YZT on the level of total phospho tau (tau 5) in SH-SY5Y-P301L cells. (C) YZT significantly decreased the levels of polyubiquitin in SH-SY5Y-P301L treated cells. (D) YZT significantly decreased the levels of ubiquitin K48 in SH-SY5Y-P301L treated cells. (E,F) YZT reduces the level of insoluble phosphorylated and misfolded tau via UPS mediated degradation pathway. YZT significantly reduced the levels of polyubiquitin in SH-SY5Y-P301L treated cells. The reduction of polyubiquitin was restored when the cells were pretreated with MG132 (proteasome inhibitor), thus clearly illustrating that involvement of UPS in YZT-mediated degradation of tau. (G,H) YZT- treated SH-SY5Y-P301L cells in the presence of autophagy inhibitors (CQ and wortmannin) the tau reducing effect of YZT was not blocked but in the presence of MG132 blocked the YZT’s tau reducing effect. All the results were represented as mean ± SEM of three independent experiments. N = 3, **p < 0.01; ***p < 0.001.

FIGURE 7

Schematic representation of YZT-mediated UPS pathway in vivo. (A) YZT treatment significantly decreases the poly ubiquitin levels in P301S mice brain homogenate compared to the Tg-vehicle and its quantification. (B) Long term YZT treatment in 3XTg-AD mice significantly reduced the ploy ubiquitin protein levels when compared to Tg-vehicle. (C) Schematic diagram illustrating that involvement of ubiquitin proteasomal pathway in YZT-mediated degradation of tau.

FIGURE 8

Schematic illustration of YZT's effect on tau pathology and its mechanism of action.