Effects Of Zinc On β-amyloid Deposition In APP/PS1 Transgenic Mouse Brain

PrefaceAlzheimer's disease (AD) is a disease cinically characterized by progressive intellectual deterioration. AD is pathologically characterized by senile plaques (SP) formed by pathological deposition ofβ-amyloid (Aβ). Aβpeptide is generated by a proteolytic process of amyloid precursor protein (APP). The ectodomain of APP is first cleaved byβ-secretase and produces an APP C-terminal fragment. The latter is further cleaved within the transmembrane domain by theγ-secretase complex, and hence leads to the generation of Aβpeptides. Interestingly, both APP and its proteolytic product Aβare metalloproteinases containing metal binding sites, which are believed to be associated with metal regulation in AD brain. Recently, a marked accumulation of zinc has found in the amyloid plaques in the brains of AD patients and AD transgenic mouse models and APP expression can be promote via its 5'-untranslational region (5'-UTR) in mRNA by zinc. The more important is that zinc ions can trigger a deposition of Aβby connecting the 13th amino acides between the neighboring Aβmolecules. Furthermore, zinc chelating agents, such as clioquinol (CQ) and DP-109. that reduce zinc levels in brain can inhibit the formation of amyloid plaques in APP transgenic mouse brain and improve the cognitive function in AD patients. Taken altogether, abnormal zinc homeostasis may be an important factor leading to the AP accumulation in AD brain, and amelioration of zinc homeostasis is a potential therapeutic strategy for AD. However, there is no detailed study of zinc ions on the process of AD pathogenesis and related mechanisms, in vivo. Therefore, in the present study, APP/PS1 transgenic mice were treated with zinc ions and zinc chelator Clioquinol (CQ) separately. The cognitive abilities of transgenic mice were tested with Morris water maze. The levels of the brain senile plaques were detected with Aβimmunohistochemistry. Furthermore, the expression levels of proteins involving APP processing, including ADAM10, BACE1 and PS1, were examined with Western blot analysis. The present study indicates that chronic high intake of dietary zinc plays a role in the neuropathphysiology of AD, through enhancement of the amyloidogenesis.Materials and MethodsThree-month-old male C57BL/6 mice were randomly and equally divided into 3 groups (n=14 in each group). (1) Normal control group. (2) Dimethyl sulfoxide (DMSO) control group. (3) CQ group. The CQ group and the DMSO group mice were injected intraperitoneally with CQ and DMSO respectively. Two hours later, brains of the three groups'mice were removed and cryostat sections were prepared. TSQ fluorescence probe and immersion autometallography (AMG) were performed to analyze the distribution of free zinc ions in mouse brain.Double transgenic APPswe/PS1dE9 (APP/PS1) mice were used in the present study. Mice at the age of 3 months were randomly assigned to one of four groups (n= 12 in each group). (1) Control group:mice were given standard diet and deionized water ad libitum. (2) Zn group:mice were given standard diet and deionized water containing ZnSO4(20 mg/mL). (3) Zn+CQ group:mice were given standard diet and deionized water containing ZnSO4 (20 mg/mL). At the age of 7 months, they were gavaged once a day with CQ (30 mg/kg/day) dissolved in 0.05% carboxymethylcellulose. (4) CQ group:mice were gavaged once a day with CQ (30 mg/kg/day) dissolved in carboxymethylcellulose started from the age of 7 months. The animal body weights were monitored and general health was observed on a daily basis. Just prior to decapitation, the blood samples were drawn from the heart for serum zinc measurement. Morris water maze tests were performed to evaluate the mice learning and memory. Immersion autometallography (AMG) and immunostainning were used to detect the senile plaques (SP) generation in each group mice. Finally, ELISA and Western blot were used to detect the Aβsecretion and expression levels of proteins, including APP, ADAM10, BACE1, PS1, sAPPα, sAPPβ,.Results1. The chelation effect of CQ on the mouse brain zinc.Both TSQ fluorescence and AMG results indicated that CQ group had a dramatic reduction in free zinc ions in the brain compared with the normal control group and the DMSO control group, but there were no statistical differences between two control groups.2. Chronic high intake of dietary zinc on the impact of the transgenic mice body weight, serum and brain zinc levelCompared with the control group, the transgenic mice in zinc group showed a significantly decreased body weight, and the coat color turn to brown yellow. The serum and brain zinc content were significantly increased that that in control mice. Furthermore, CQ could reverse the toxic effects of the zinc to the transgenic mice, but its own had no effect to the body weight and coat color.3. Chronic high intake of dietary zinc on the impact of the transgenic mice learning and memory ability.Compared with the control group, the learning and memory ability in space exploration significantly reduced in the transgenic mice of the zinc group. CQ can reverse the zinc toxic effects and itself also can significantly improve the cognitive abilities of the transgenic mice.4. Chronic high intake of dietary zinc on the formation of senile plaques in transgenic mice brain.Compared with the control group, the senile plaques had significantly increased in the zinc group mouse brain. CQ could slow down the formation of zinc on the promotion of the senile plaques, and its own could significantly reduce APP/PS1 transgenic mice brain levels of senile plaques.5. Chronic high intake of dietary zinc on the APP protein processing.Compared with the control group, the protein level of the APP, BACE1, PS1, sAPPβand the Aβlevel were significantly increased and the ADAM10 and sAPPαwere significantly decreased in the zinc groups transgenic mouse brain. CQ could reverse the zinc ions this effects. Conclusion1. CQ has a significantly chelation effect on free zinc ions.2. Chronic high intake of dietary zinc induces body weight loss, serum and brain zinc content increased in APP/PS1 transgenic mice.3. Chronic high intake of dietary zinc significantly decreases the cognitive abilities of transgenic mice.4. Chronic high intake of dietary zinc promotes the senile plaques generation.5. Chronic high intake of dietary zinc enhances APP protein and AP generation in transgenic mice brain.