| It is well known that as a progressive neurodegenerative disorder the Alzheimer’s disease is characterized by the presence of senile plaques and neurofibrillary tangles. Senile plaques are composed ofβ-amyloid peptides (Aβ) generated from a two-step proteolytic processing of the amyloid precursor protein (APP) byβ-secretase and y-secretase.β-secretase also known asβ-site amyloid precursor protein cleaving enzyme 1 (BACE1), is recognized as an initiator of the generation of Aβ.A number of studies suggest a potential link between abnormal cholesterol levels and AD pathogenesis.Clinical studies indicate that risk factors linked to elevated cholesterol levels in the elderly also increase the risk of developing AD. A recent report indicates that altered cholesterol metabolism does exist in the brain of AD patients. Animal studies have demonstrated that diet-induced hypercholesterolemia could enhance Aβaccumulation. Conversely, cholesterol-lowering drugs administration can reduce serum AP peptides in a transgenic mouse model of AD.Liver X receptor (LXR) is a ligand-activated transcription factor that plays a central role in the regulation of cholesterol transport and metabolism. Some LXR-responsive target genes such as ATP binding cassette proteins Al(ABCAl) and apolipoprotein E (ApoE) regulate the expression of various molecules associated with cholesterol homeostasis. The LXR family comprises two isoforms, LXRa and LXRβ, both of which are expressed in various tissues, including the brain, which makes it possible to be therapeutic agents for AD. Physiologic activators for the LXR include 24(S)-hydroxycholesterol,27-hydroxycholesterol and the synthetized LXR agonists such as T0901317 and GW3965. Moreover, studies have demonstrated that synthetic LXR agonists, such as T0901317 and GW3695, reduce the levels of Aβin AD models, but the mechanism is not fully understood.Is the cholesterol homeostasis affected in the brain of APP/PS1 transgenic mice after T0901317 treatment? Is the secretases activity affected? And what is the mechanism of T0901317 in reducing the levels of Aβin AD models? The answer to these questions remains unknown. In this study, we focused on the relationship of the cholesterol content, secretases activity, AP productions in APP/PS1 transgenic mice and in primary neuron culture after T0901317 treatment. We hope to explore the possible pathomechanism of AD. The study is divided into 2 parts:In the first part, we tested the modifications of the cholesterol homeostasis, APP processing, the cognitive performance of APP/PS1 mice in response to T0901317 administration. Realtime qRT-PCR showed that the increased mRNA expression of ABCA1 and ApoE was significant in the group of T0901317 administration when compared with the vehicle-treated control group. Statistical quantitative analysis of Western blot demonstrated that the protein levels of ABCA1 and ApoE were higher in the T0901317-treated group than in the control.Membrane cholesterol levels decreased significantly with T0901317 treatment. However, the total cholesterol contents were of no significant differences between T0901317-treated and vehicle-treated control mice. There was a significant difference in the percentage of the area occupied by plaque load in HC between the T0901317-treated and vehicle-treated group determined by immunohistochemical staining for Aβ. ELISA measurements found that treatment with T0901317 induced significant changes in Aβ40 and Aβ42 levels compared to levels from vehicle-treated APP/PS1 mice. There were significantly decreased BACE1 protein levels andβ-secretase activity after T0901317 administration. We all found that T0901317 has beneficial effects on the treatment of cognitive impairment in the APP/PS1 mice. All these results indicate that T0901317 treatment can reduce theβ-secretase activity and Aβlevels.In the second part, in order to test the mechanism of theβ-secretase activity and Aβlevels in response to T0901317 administration, we performed a series of experiments to measure the expression of ABCA1 mRNA, membrane cholesterol content,β-secretase activity and Aβlevels in primary neuron from the APP/PS1 transgenic mice. Cholesterol assay showed that the membrane cholesterol levels were significantly increased in exogenous cholesterol-treated cells as compared to the controls. We also revealed thatβ-secretase activity and the secretion of Aβlevels in cholesterol-treated cells were significantly higher than those in control cells. Cholesterol depletion induced byβ-methyl cyclodextrin reduced membrane cholesterol levels,β-secretase activity and Aβproduction in primary neuron cells. These two results indicate that membrane cholesterol content may influenceβ-secretase activity. T0901317 increased ABCA1 expression, attenuated membrane cholesterol levels, decreasedβ-secretase activity and Aβproduction in primary neuron cells.To further investigate the role of ABCA1 in the cholesterol-lowering effect of T0901317, primary neuron cells were pretreated for 1 h with glyburide, which is an effective inhibitor of ABCA1. Pre-incubation with glyburide abolished the T0901317-mediated decrease in membrane cholesterol levels,β-secretase activity and Aβproduction, which implied that induction of ABCA1 was required for T0901317-mediated decrease in membrane cholesterol levels andβ-secretase activity. To determine whether the T0901317-mediated increase in ABCA1 expression and the reduction in membrane cholesterol levels were dependent on the activation of LXR, we used the LXR antagonist, geranylgeranyl pyrophosphate (GGPP) and T0901317 for co-incubation for 24 h. GGPP completely blocked the T0901317-induced increase in ABCA1 mRNA expression and reduction in membrane cholesterol levels,β-secretase activity and Aβproduction. These results imply an essential role of LXR activation in T0901317-regulated expression of ABCA1, which may contribute to the suppressive effect of T0901317 on membrane cholesterol levels in vitro.In conclusion, we demonstrated that LXR agonist, T0901317, can affect the cholesterol homeostasis, decrease the BACE1 protein expression and Aβlevels in APP/PS1 transgenic mice. Our results suggest that activation of LXR may decrease BACE1 expression,β-secretase activity and Aβlevels through a pathway associated with ABCA1-mediated reduction in membrane cholesterol levels. |
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