J Alzheimers Dis. 2014;40(3):575-86.

Proteomic Analysis of Serum Proteins in Triple Transgenic Alzheimer’s Disease Mice: Implications for Identifying Biomarkers for Use to Screen Potential Candidate Therapeutic Drugs for Early AD

Xiaojing Suia, b, c, Xiaohu Renb, Peiwu Huangb, Shuiming Lid, Quan Mab, Ming Yingd, Jiazuan Nia, d, Jianjun Liub and Xifei Yangb,*

aState Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry; Changchun 130022, P. R. China

bKey Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, China

cUniversity of Chinese Academy of Sciences, Beijing 100049, China

dCollege of Life Sciences, Shenzhen University, Shenzhen 518060, China

*Correspondence should be addressed to: Dr. Xifei Yang, No 8 Longyuan Road, Nanshan District, Shenzhen 518055, Guangdong, China; Tel: +0086-755-25601914; Fax: +0086-755-25508584, E-mail: xifeiyang@gmail.com.

 

Abstract

Alzheimer’s disease (AD) is the most common fatal neurodegenerative disease affecting the elderly worldwide. There is an urgent need to identify novel biomarkers of early AD. This study aims to search for potential early protein biomarkers in serum from a triple transgenic (PS1M146V/APPSwe/TauP301L) mouse model. Proteomic analysis via two-dimensional fluorescence difference gel electrophoresis was performed on serum samples from wild-type (WT) and triple transgenic mice that were treated with or without coenzyme Q10 (CoQ10) (800 mg/kg body weight/day), a powerful endogenous antioxidant displaying therapeutic benefits against AD pathology and cognitive impairment in multiple AD mouse models, for a period of three months beginning at two months of age. A total of 15 differentially expressed serum proteins were identified between the WT and AD transgenic mice. The administration of CoQ10 was found to alter the changes in the differentially expressed serum proteins by up-regulating 10 proteins and down-regulating 10 proteins. Among the proteins modulated by CoQ10, clusterin and alpha-2-macroglobulin were validated via ELISA assay. These findings revealed significant changes in serum proteins in the AD mouse model at an early pathological stage and demonstrated that administration of CoQ10 could modulate these changes in serum proteins. Our study suggested that these differentially expressed serum proteins could serve as potential protein biomarkers of early AD and that screening for potential candidate AD therapeutic drugs and monitoring of therapeutic effects could be performed via measurement of the changes in these differentially expressed serum proteins.

PMID: 24496070

 

Supplement

Alzheimer’s disease (AD) is a fatal neurodegenerative disease. It is estimated that AD will affect 66 million people by 2030 and 115 million people by 2050 worldwide if no effective therapeutic strategies can be discovered. To date, the diagnosis of AD is a costly and complicated clinical procedure, especially for early AD. With the advent of modification therapies that might be most effective when initiated very early in the course of AD, there is an urgent need for novel biomarkers to monitor and evaluate the biochemical effects of these putative treatment strategies.

Coenzyme Q10 (CoQ10), a powerful endogenous antioxidant, could prevent the oxidative damage caused by free radicals that are produced during oxidative phosphorylation via the respiratory chain in the mitochondrial membrane. CoQ10 was shown to exert neuroprotective effects in animal models and human trials of Parkinson’s disease (PD) and Huntington’s disease (HD). Previously, we reported that CoQ10 reduced the Aβ plaque burden in an APP/PS1 transgenic mouse model of AD. In recent studies, CoQ10 was found to be able to improve behavioral deficits and survival, up-regulate some key enzymes of the electron transport chain, and reduce oxidative stress in P301S tau transgenic mice suffering from tauopathy. The study by Magali et al., also found that CoQ10 reduced oxidative stress and attenuated amyloid pathology, as well as improved behavioral function, in the Tg19959 mouse model of AD. CoQ10 has also been demonstrated to be neuroprotective against Aβ-induced neural stem cell death. These findings suggest that CoQ10 can potentially be used for the treatment of AD.

Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) was employed to perform differential protein expression analysis. The critical attributes of DIGE are the capability to resolve multiple samples on one gel and the use of an internal standard for cross-gel normalization. 2D-DIGE allows for more accurate and sensitive quantitative proteomic studies than traditional two-dimensional gel electrophoresis [16]. This study was undertaken to screen for potential biomarkers of early AD using a triple transgenic (PS1M146V/APPSwe/TauP301L) AD mouse model (3xTg-AD), a commonly used AD model, and to validate the potential biomarkers by measuring the changes in the differentially expressed proteins after administration of CoQ10. The mice (male, 4 per group) were provided with either drinking water or drinking water supplemented with CoQ10 (800 mg/body weight/d) beginning at the age of 2 months for a duration of 3 months.

To determine the effects of CoQ10 on brain protein expression in 3xTg-AD mice, we performed quantitative proteomic analysis on brain samples from 3xTg-AD mice. Typical 2D-DIGE gel images of the brain proteins isolated from WT and 3xTg-AD mice treated with or without CoQ10 are presented in Supplementary Figs. 1 and 2. Among these differentially expressed proteins, 40 proteins were up-regulated and 3 were down-regulated.

Comparative proteomic analysis via depletion of IgG and albumin, 2D-DIGE, image analysis, in-gel digestion, MS identification and database searching were performed on the serum proteins collected from WT and 3xTg-AD mice treated with or without CoQ10. To screen for potential biomarkers of early AD and to further evaluate the potential effects of CoQ10 treatment on the differentially expressed serum proteins in 3xTg-AD mice, two separate comparisons were performed as follows: (1) WT mice versus untreated 3xTg-AD mice; (2) untreated 3xTg-AD mice versus 3xTg-AD mice treated with CoQ10.

fig1

Fig. 1.

fig2 Fig. 2.

Representative 2D-DIGE gel images of serum proteins isolated from WT and 3xTg-AD mice treated with or without CoQ10 are presented in Fig. 1 and Fig. 2. Several serum proteins were found to be differentially expressed between the untreated WT and untreated 3xTg-AD mice (Table 1). Among these proteins, eighteen protein spots were up-regulated in untreated 3xTg-AD mice relative to untreated WT mice, and fourteen protein spots were down-regulated.

 

tab1

Table 1. Differentially expressed serum protein spots between WT and 3xTg-AD mice identified via 2D-DIGE/MALDI-TOF-MS/MS.

 

A total of 33 protein spots were found to be differentially expressed between the 3xTg-AD mice treated with or without CoQ10 (Table 2). Among these differentially expressed proteins, nineteen protein spots were up-regulated in the 3xTg-AD mice treated with CoQ10 compared to the untreated 3xTg-AD mice, and fourteen protein spots were down-regulated in the 3xTg-AD mice treated with CoQ10 compared to the untreated 3xTg-AD mice.

 tab2

Table 2. Differentially expressed serum protein spots between 3xTg-AD mice treated with and without CoQ10 identified via 2D-DIGE/MALDI-TOF-MS/MS.

 

Among the differentially expressed serum proteins between the WT and 3xTg-AD mice, the expression of twelve proteins was modulated by CoQ10 treatment. These proteins included clusterin, α2M, TTR, apoA-I, apoA-II, hemopexin, haptoglobin, MUP1, A1BG, kininogen-1, MBP-C, and IGKC.

To confirm the 2D-DIGE results, ELISA assays were performed. Two CoQ10-modulated serum proteins, clusterin and α2M, were selected for validation. In accordance with the 2D-DIGE results, a significant decrease in the serum levels of clusterin in the 3xTg-AD mice and a modulatory effect of CoQ10 treatment on the serum levels of clusterin were detected (Fig. 3A). Similarly, a significant increase in the serum levels of α2M in the 3xTg-AD mice and a modulatory effect of CoQ10 treatment on the serum levels of α2M were found (Fig. 3B).

 fig3

Fig. 3.

 

In summary, our study revealed that significant changes in the serum levels of proteins occurred in AD mice at an early pathological stage and that administration of CoQ10 could modulate the changes in the serum proteins, such as clusterin, α2M, TTR, apoA-I, apoA-II, MBP-C, hemopexin, haptoglobin, MUP1, A1BG, kininogen-1, and IGKC. These data suggested that the differentially expressed serum proteins could serve as potential protein biomarkers of early AD and that screening for potential candidate therapeutic drugs for AD and monitoring of therapeutic effects could be performed by measuring the changes in these serum proteins. However, further studies are still necessary to validate the above results using serum samples from healthy age-matched controls and patients with mild cognitive impairment (MCI), early AD, and moderate/severe AD cases. It will also be useful to analyze the relationship between the expression of these differentially expressed serum proteins and the AD state to evaluate the efficiency of these potential biomarkers.

 

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