Increased Plasma Homosysteine Levels Precede Early Intraneuronal β-amyloid Pathology and Cognitive Impairment in a Triple Transgenic Mouse Model of Alzheimer’s Disease
Ming Ying1, a, Zhijun Huang1, c, Zhonghao Zhanga, Quan Mab, Junjun Wanga, Jiazuan Nia,*, Xifei Yang1, b,*
Alzheimer’s disease (AD) is the most common form of neurodegenerative disease. Homocysteine (Hcy) has been implicated as a risk factor for the onset of AD; however, there are also some conflicting results. Here report that the levels of Hcy are significantly increased in plasma of a triple transgenic mouse model of AD (3 x Tg-AD) harboring PS1M146V, APPSwe, and tau p301L transgenes at an early age of one month, occurring before early intracellular β-amyloid pathology and spatial memory impartment. These data provide evidence demonstrating that plasma Hcy could serve as an early biomarker for AD.
Key words: Homocysteine (Hcy); β-amyloid; Biomarker; Alzheimer’s disease (AD)
Alzheimer’s disease (AD), characterized pathologically by neuritic plaques composed predominantly of amyloid-Beta (Aβ) peptides and neurofibrillary tangles formed by hyperphosphorylated forms of tau proteins in the brain (Small and Cappai 2006), is one of the most common neurodegenerative diseases. Aβs are produced from the amyloid precursor protein (APP) by the sequential cleavage of the β-secretase-1 (β-amyloid cleaving enzyme, BACE-1) and γ-secretase (Sinha and Lieberburg 1999). The etiology of AD remains largely unclear. Epidemiological evidence indicates that elevated blood levels of homosystemine (Hcy), called hyperhomocysteinemia, are a risk factor of AD (Morris 2003). A number of studies have demonstrated that Hcy could induce Alzheimer-like cognitive impairment and pathological changes (Ataie et al. 2010, Ho et al. 2010, Wei et al. 2011, Zhang et al. 2009, Zhang et al. 2008). However, there are also some findings which do not support a causal relationship between high Hcy levels and AD (Aisen et al. 2008, Malouf et al. 2003, Zhang et al. 2008), and high levels of Hcy are only indicative of being a biomarker for AD (Seshadri 2006).
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Hcy is a sulfur-containing, non-protein amino acid produced in the methionine cycle. Hcy is biosynthesized from methionine and can be recycled into methionine or converted into cysteine with the aid of B-vitamins. High Hcy levels have been implicated in many diseases such as cardiovascular diseases, diabetes, high blood pressure, neurodegenerative diseases. It is unclear that Hcy of high levels only act as a biomarker of AD or only a risk factor of AD. It is also not clear whether there is a correlation of Hcy levels with the progression of the disease.
In this study, we examined the changes of Hcy in plasma at different stages of AD in a triple transgenic model of AD harboring PS1M146V, APPSwe, and tau p301L transgenes and determined the temporal relationship with the occurrence of early Aβ deposit and cognitive impartment.
Materials and Methods
Antibodies and chemicals
The polyclonal antibody against Aβ1-42 was purchased from Chemicon International/Millipore (Temecula, CA, USA). The mousehomocysteine (Hcy)ElisakitwaspurchasedfromBmassay (Beijing,China).
The triple AD transgenic mice (B6; 129-Psen1tm1Mpm Tg(APPSwe,tauP301L)1Lfa/Mmjax) and the control mice (B6129SF2/J) were purchased form Jackson Laboratory. The animal experiments were approved by the Ethics Committee of Shenzhen University. The animals were housed for 12h light and 12 night in circle. All the mice were housed with free access to food and water, and were maintained on 12 h light to 12 h dark cycle (lights on at 5:00 p.m., off at 5:00 a.m.) at stable temperature (23–25â„ƒ).
The blood samples (about 500-1000 µl) were drawn from the eye.Total plasma Hcy was measured using an enzymatic assay according to instructions provided by the kit.
Morris Water maze
The memory acquisition test was lasted for 5 days and each training session consisted of four trials altogether (one trial per quadrant). Before each experiment, the rats were brought to the
site to allow them to be acclimatized. The temperature of the room and water was kept at 26±2 °C. The water in the pool is made opaque with milk to hide the escape platform. The Plexiglas platform is AAA cm high, AAA cm in diameter, and its surface was scarred to help the rats climb on it. The water surface is AAA cm from the rim of the pool, and the inner wall is always carefully wiped to eliminate any local cues. A camera is fixed to the ceiling of the room, AAA m from the water surface. The camera is connected to a digital tracking device. The mice were not allowed to search for the platfrom more than 60 s, after which they were guided to the platform. On the fifth day the mice were trained as usual and their escape latency and swimming path were recorded as primary protocols. The memory retention test was performed 5 days after the last training day, and all the mice were tested and their escape latency and swimming pathways were recorded in the same way.
The control mice and transgenic mice brain sections embedded in paraffin were prepared for immunohistochemical analysis through xylene treatment and gradual rehydration with 100
–70% ethanol. Sections were blocked and then incubated with primary antibody overnight at 4°C in 0.3% Triton X-100 phosphate buffered saline (PBS). Secondary antibody was incubated for 1 h at room temperature in the dark. The immunoreactivity of Aβ1-42 (1:500) was probed using secondary antibodies Alexa Fluor® 488 goat anti-rabbit IgG (H+L) . The images were taken using a general light microscope. The number of Abeta1-42-positive cells was counted in a double blind way.
Data were expressed as the mean ± SD and analyzed using SPSS 13.0 statistical software (SPSS Inc., Chicago, Illinois, USA). One-way ANOVA, followed by Student-Newman Keuls (SNK), or a Student’s t-test was used to determine the different means among the groups. The level of significance was set at p<0.05.
1. Plasma Hcy Levels are significantly increased in an age-dependent manner in a triple transgenic mouse model of AD.
In order to determine the effects of the genotype on plasma Hcy levels in the triple AD transgenic mice, we measured the levels of Hcy using Hcy assay kit. We find that plasma Hcy levels are significantly increased (approximately 1.5 fold) as early as one month of age compared to the non-transgenic control mice (Fig. 1). As the mice ages, plasma Hcy levels are gradually increased. The levels of Hcy are increased by about 4 fold and 8 fold at the age of 2 and 4 months, respectively. Hcy levels seem to become stable when the mice are at the age of 4 moths or above (Fig. 1).
2. Early intracellular Aβ1-42 deposit occurs at the age of 2 months in the AD mice.
Intracellular Aβ1-42 deposit is an early pathological sign in the pathogenesis of AD. We measured the distribution and the levels of Aβ1-42 by immunohistochemistry in cortex and hippocampus of the AD mice at the age of one and two month. We find that two-month-old but not one-month-old AD mice display increased Aβ1-42 deposit in both cortex and hippocampal subfields (Fig. 2A, 2B, 2C, 2D). These data indicate that early intracellular Aβ1-42 deposit occurs after elevated plasma Hcy.
3. Spatial memory impairment is revealed at the age of 4 months in the AD mice.
In order to determine the temporal relationship of plasma Hcy change with the occurrence of cognitive impartment, we assess the changes of spatial learning and memory of the AD mice at the age of one and two months using Morris water maze test. We do not find obvious impairment in spatial learning for the two or four-month-old AD mice (data not shown), but find obvious deficit in spatial memory in the AD mice at the age of 4 months (Fig. 3). These data indicate that cognitive impairment occurs after early intracellular Aβ1-42 deposit and plasma Hcy elevation in the AD mice.
In this study, we find that plasma Hcy levels are significantly elevated in a triple transgenic AD mouse model early at the age of one month when no obvious AD-like pathology and cognitive deficit occur. With the age increased, the mice display higher plasma Hcy levels in an age-dependent manner.
Hcy is suggested to be a potential risk factor for AD, and high levels of Hcy were shown to be able to contribute to Aβ accumulation, hyperphosphorylation of tau and spatial learning and memory impairment. The studies using in vitro and in vivo models have revealed diverse possible mechanisms involved in the pathogenic effects of Hcy on AD developement. These mechanisms include oxidative stress, demethylation, excitatory damage, endoplasmic reticulum stress (Zhuo et al. 2011). Our present findings for the first time demonstrate abnormally elevated Hcy levels occur early at the age of one month while early intracellular β-amyloid pathology can be revealed at the age of 2 months and spatial memory impartment at the age of 4 moths in the AD mice. Furthermore, plasma Hcy levels are increased in an age-dependent manner. These data indicate that Hcy metabolism is disturbed by the genotype at a very early stage in the AD mice. Consistent with our data, a previous study reported that 16-18-month-old Tg 2576 AD mice have higher Hcy levels than wild-type mice in the brains (Bernardo et al. 2007). In contrast, a previous study reported that in the aging brain of APP/PS1 mice, the factors of Hcy metabolism S-adenosyl-methionine (SAM) and S-adenosyl-homocysteine (SAH) levels are significantly changed in the aging brains of APP/PS1 mice, and that the SAM/SAH ratio (methylation index) was significantly increased (Hooijmans et al. 2009). This piece of data also indicates that Hcy could be over-produced due to the disturbed Hcy metabolism resulting from AD pathology. However, the mechanisms underlying the increase of plasma Hcy levels remain to be elucidated in future studies.
Given the contribution of high levels of Hcy to the development of Aβ and tau pathology, and cognitive deficit, we speculate that elevated Hcy levels and AD pathology could form a vicious cycle in the development of AD.
In summary, our study for the first time demonstrates that plasma Hcy is elevated as early as one month of age, occurring before early intracellular β-amyloid pathology and spatial memory impartment. These data indicate that plasma Hcy could serve as an early biomarker for AD along with as a risk factor for AD, although further studies on early AD subjects are still warranted to validate the conclusions drawn in this study.
This work was supported by NSFC (the National Natural Science Foundation of China) (81102154,21271131,31470804), Medical Scientific Research Foundation of Guangdong Province (A2013598), Shenzhen Scheme of Science and Technology (Medicine and Health) (201302148), Shenzhen Special Fund Project on Strategic Emerging Industry Development (JCYJ20130329103949650, JCYJ20120616144140857) and the Upgrade Scheme of Shenzhen Municipal Key Laboratory (JCYJ20130408172946974).
Declaration of interest
None of the authors have any potential conflicts of interest or financial interests to disclose.
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Elevated plasma Hcy levels in a triple transgenic mouse model of AD.
Increased plasma Hcy levels are revealed at an early age of one month in the triple transgenic AD mice. Plasma Hcy levels are dramatically elevated with aging in the AD mice (n=8-12?).
Early intracellular Aβ1-42 deposit occurs at the age of 2 months in the AD mice.
Immunohistochemistry reveals that early intracellular Aβ1-42 deposit occurs at the age of two months but not one month in cortex and hippocampus of the AD mice (2A, 2B, 2C, 2D). n=4?.
Spatial memory impairment occurs until the age of 4 months in the AD mice.
Morris water maze test reveals spatial memory is significantly impaired at the age of 4 months in the AD mice (n=6-10?).
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