Sporadic Alzheimer’s disease is a late-onset dementia characterized by the presence of senile plaques made of Aβ, neurofibrillary tangles (NFT), and cognitive decline. So far, epidemiological and clinical studies have suggested a link between cholesterol metabolism and sporadic AD pathogenesis (Kivipelto et al., 2001
;Kivipelto et al., 2005
). Despite the therapeutic potential of this link, mechanisms by which cholesterol metabolism influence AD pathogenesis remain uncertain(Puglielli et al., 2003
). The LXRs, LXRα and LXRβ, are transcription factors that control the expression of genes involved in cholesterol metabolism(Kalaany and Mangelsdorf, 2005
). In the brain, in addition to normal neuronal function, cholesterol metabolism is of utmost importance for secretase activities, APP proteolytic cleavage, Aβ aggregation and its clearance from the brain. Most importantly, APOE
, the only proven risk factor for late onset AD (Strittmatter et al., 1993
), is an LXR target gene (Laffitte et al., 2001
). This is the first study to demonstrate that a treatment can ameliorate both memory deficits and amyloid pathology caused by high fat/high cholesterol diet in AD mouse model.
In the present study we have investigated the effect of LXR agonist treatment on AD phenotype in APP expressing mice fed HF diet for 4 months. The design of the study considered a therapeutic application of an LXR ligand and therefore treatment was started at an age when APP23 mice already exhibit deposited amyloid plaques. Our results show that HF diet significantly worsens memory deficits in APP23 mice such that they were completely unable to learn the MWM paradigm task when compared to ND fed mice. In contrast, our experiments clearly demonstrate a disease-modifying effect of chronic LXR ligand treatment in APP23 mice fed HF diet as exemplified by improved learning curves during the acquisition phase. Furthermore, during the probe trial LXR agonist treated mice performed remarkably better and did not differ from APP23 mice on ND. The performance of T0 and vehicle treated mice on HF diet evaluated by multiple comparison procedures at the end of each of the trial blocks, as well as during the probe trial, confirmed the internal validity of the study.
To gain further insight into the effect of extended T0 treatment on AD phenotype in APP23 mice fed HF diet, we evaluated a number of parameters related to amyloid load and deposition of soluble and fibrillar Aβ peptides. We did not find an effect of HF diet or T0 treatment on APP processing. Few previous studies reported that high fat/high cholesterol diet affects APP processing in APP transgenic mice (George et al., 2004
;Refolo et al., 2000
;Thirumangalakudi et al., 2008
). The reason for this discrepancy is unknown but it is possible that it is caused by the different composition of the diets (our diet does not contain cholate); the duration of the diet which continued 4 months in this study as opposite to 6–12 weeks, or the mouse models used in other studies. However, HF diet in vehicle treated mice led to a significant increase in deposited plaques and fibrillar Aβ, which are well known determinants of memory deficits in APP expressing mice. On the other hand, treatment with the LXR ligand T0 led to a significant decrease in amyloid load and soluble and insoluble Aβ levels. In contrast to insoluble Aβ, HF diet did not increase the amount of Aβ oligomers but their level was significantly decreased following T0 treatment. Remarkably, the amount of A11 positive oligomers correlated significantly to the behavioral phenotype.
Our in vitro
data suggests that Aβ clearance by microglia is affected by ApoE-containing lipoproteins in agreement with a recent study by Jiang et al. (Jiang et al., 2008
). demonstrates that the amount of fully lipidated ApoE is important for Aβ degradation by glia because: 1) Lipid-poor ApoE produced by Abca1ko
astrocytes is less efficient at promoting Aβ clearance and 2) T0 facilitates Aβ degradation by increasing the secretion of lipidated ApoE from WT but not from Abca1ko
astrocytes. The results from microdialysis experiments confirm the in vitro
data and point to the conclusion that Aβ clearance is increased following T0 treatment. The level of ISF Aβ in the brain is mainly the result of an equilibrium between its production, degradation by intracellular and extracellular proteases, efflux from the brain and deposition in plaques. Due to Aβ deposition in senile plaques, ISF Aβ levels decrease in older mice. For this reason we chose to examine the effect of T0 on ISF Aβ levels in mice younger than our original cohort. Collectively the in vitro
and microdialysis data presented in this report, together with the lack of effect on APP processing suggests that T0 facilitates and increases Aβ clearance.
It is known that as a side effect, treatments with LXR agonists increase serum triglyceride levels, which depends on the mouse strain and duration of the treatment. For example, in WT type C57BL6 mice short treatment (less than a week) causes an increase in serum triglycerides (Grefhorst et al., 2002
) whereas long-term (more than a month) reduced or did not change triglycerides in serum (Peng et al., 2010
). In accordance with these studies we did not find an increase in liver weight (not shown) and serum triglycerides, but there was an increase in liver triglycerides (Supplementary Figure 2C and D
The behavioral and morphological phenotype of APP23 mice on HF diet raises two important questions. First, what are the consequences of HF diet at the cellular and molecular levels in the brain that cause the observed memory deficits and the increased Aβ deposition? Based on previous studies, done primarily in vitro
(Simons et al., 1998
) it is believed that increased intracellular levels or abnormal distribution of cholesterol in the brain can increase β-and γ-secretase activities and thus Aβ production. However, there is no animal model demonstrating that the relatively autonomous brain cholesterol metabolism in the adult mouse can be influenced by systemic hypercholesterolemia resulting from prolonged dietary intake. The results presented here show that HF diet treatment for 4 months does not change the amount of total cholesterol in the brain of vehicle treated APP23 mice regardless of its increased levels in plasma (Supplementary Figure 2
). Although not addressed by this study, HF diet may dysregulate two functionally unrelated physiological systems that independently can exacerbate AD phenotype: insulin sensitivity in the brain and the intactness of the blood brain barrier (BBB). Insulin resistance in the brain has been demonstrated in rats fed HF diet, and was implicated as a reason for behavioral deficits (Parrott and Greenwood, 2007
). On the other hand, an impairment of the BBB can allow leakage of circulating lipoprotein-Aβ complexes into the brain as evidenced by co-localization of plasma lipoproteins (ApoB) and Aβ in close proximity to cerebral vessels, which ultimately exacerbates an AD-like morphological phenotype (Takechi et al., 2010
The second question is related to the therapeutic effects of synthetic LXR ligands applied to APP23 mice on HF diet. We consider transcriptional upregulation of LXR responsive genes and functional changes in the brain lipoproteins relevant to deposition and clearance of Aβ as the most likely mediators of T0 effects on AD phenotype. Previous reports demonstrated that functional impairment of the critical LXR target Abca1 have a negative effect on AD phenotype presumably caused by decreased lipidation of ApoE and ApoA-I and thus an increased aggregation of Aβ (Kim et al., 2009
;Koldamova and Lefterov, 2007
;Lefterov et al., 2007
;Lefterov et al., 2009
). Activation of LXR by extended application of synthetic ligands has been proven an efficient approach leading to sustainable increases in expression levels of Abca1, Abcg1 and brain lipoproteins – primarily ApoE, but also ApoD and ApoC1. Presence of functional cholesterol transporters and properly lipidated ApoE and ApoA-I can have a dual effect on the development and progression of pathological phenotypes related to increased Aβ level: first by decreasing Aβ aggregation and second by increasing Aβ clearance. The effect on Aβ aggregation could directly or indirectly increase Aβ clearance: directly because properly lipidated ApoE increases Aβ degradation, and indirectly because it would be easier for soluble Aβ to be cleared through the BBB. Our experiments show that LXR agonist treatment effectively increases Aβ degradation only when functional Abca1 and LXR are present. The results from the in vitro
experiments of this study as well as previously published studies by other groups (Jiang et al., 2008
) suggest that properly lipidated brain lipoproteins mediated by functional Abca1 are important for Aβ degradation.
The overall implications of this study are that high fat cholesterol-containing diets, common in developed countries, can exacerbate AD pathology, although there may be therapeutic potential for reversing such effects by pharmacological activation of LXR in the brain. This study conclusively shows for the first time, that LXR agonist treatment can lessen the detrimental effects of metabolic perturbations on both pathology and cognition due to Aβ accumulation.