Epidemiological, clinical and experimental studies have suggested a link between cholesterol metabolism and AD pathogenesis [35–41]. Despite the therapeutic potential of this link, the mechanisms by which cholesterol metabolism influences AD pathogenesis are still unclear. LXR target genes control the removal of excess cholesterol through efflux, catabolism or decreased absorption, lipid metabolism and lipoprotein remodelling . Because of their regulatory effect on different aspects of lipid metabolism it has been speculated that LXR ligands might impact metabolic disease and atherosclerosis particularly. We have reported that a short treatment, 6 days, of young APP23 transgenic mice with the synthetic LXR ligand T0 significantly decreased the level of both Aβ40 and Aβ42 . Using another APP transgenic model (Tg2576) or WT mice and applying T0 for up to 14 days, two other groups found a decrease in Aβ42 but not in Aβ40 levels [19, 42]. At the age of treatment, the mice used in the second study were pre-deposing, i.e. lacking insoluble Aβ, and therefore the observed effect was a result of a decreased level of soluble Aβ peptides.
In the present study we wanted to determine which genes are affected on the expression level by extended T0 treatment and if the administration of T0 to APP23 mice decreases amyloid deposition at the age when plaque formation begins. Our results show that prolonged administration of T0 at 50 mg/kg/day in APP23 mice up-regulated a number of LXR target genes related to lipid metabolism. A previous study exploring transcriptional profiling of T0 treated non-APP transgenic mice identified some of these genes as differentially up-regulated in the brain of LXRwt but not in LXRdko mice . In terms of AD phenotype, we observed that T0 mediated activation of LXR increased protein level of soluble apoE and apoA-I in the brain which correlated negatively to the level of insoluble Aβ. This is in agreement with our previous studies in APP/ABCA1-/- mice where the elevation of amyloid load was accompanied by considerable decrease of soluble brain apoE . At the same time the magnitude of the increase of apoE protein did not correspond to the rather small increase of apoE mRNA as identified in gene arrays, and by RT-QPCR. One reason for this is that apoE is selectively up-regulated in different brain cell types – in glia but not in neurons. Similarly, in peripheral cells apoE is transcriptionally regulated by LXR in macrophages and adipocytes but not in liver cells . We speculate that the increased protein level of apoE following T0 treatment is a combination of: (1) LXR mediated transcriptional up-regulation of apoE and (2) transcriptional up-regulation of ABCA1 in glia that leads to an increased cholesterol efflux. The net effect is an increased level of properly lipidated brain apoE.
In brain the lipidation of apoA-I, which comes primarily from the circulation, depends on ABCA1, as it does in the periphery and similarly to the mechanism for apoE lipidation, which has been extensively discussed in relation to Aβ deposition and clearance [2, 12, 14, 16]. There are no published data that LXRα/β exert a regulatory role on apoA-I expression at the transcriptional level in mouse or human cells and cell lines, although, such a regulatory mechanism has been demonstrated in chick embryo hondrocytes . Nevertheless, T0 administered to APP23 mice causes an increase in apoA-I transcript (albeit not statistically significant) and protein. The results from our study allow an explanation, based on the so-called antagonizing effect of T0 on NF-κB, which results in down-regulation of NF-κB responsive genes . It has been shown that cytokines exert a strong inhibitory effect on apoA-I expression at the mRNA and protein levels in both human and mouse primary cells, established cell lines, and in vivo [45, 46]. Our Affymetrix gene array and RT-QPCR assays clearly demonstrate that a prolonged application of T0 to APP23 mice ultimately causes a decrease in TNFα and IL-6 expression levels. While ABCA1 upregulation and ABCA1-facilitated cholesterol efflux are the primary mechanisms for apoA-I lipidation and its increased stability, we consider the sustained inhibition of cytokines as one additional mechanism for elevated expression of apoA-I and thus an increased amount of soluble apoA-I in brain.
Properly lipidated and therefore stable apoE and apoA-I, are an indispensable part of functional HDL-like lipoprotein complexes in brain. The legitimacy of this hypothesis is confirmed by the studies demonstrating that LXR agonist treatment of mice increases plasma levels of HDL, HDL particle size and apoA-I protein  – effects attributed to the increased ABCA1 expression and cholesterol efflux. In brain the increased expression of ABCA1 could lead to: (1) an increase in the amount of Aβ bound to lipid-rich apoE, which may have a significant role in maintaining the level of soluble Aβ and preventing it from aggregation, and (2) an increased delivery of Aβ to astrocytes for degradation, or Aβ clearance through the blood brain barrier. Support for this hypothesis is provided by a recent study , which revealed an increased plaque load in APP transgenic mice with global deletion of LXRα or LXRβ, and further reinforced the idea that LXRs and their responsive genes are important determinants in AD pathogenesis [2, 14, 20].
Our results from gene array assays demonstrated that a number of genes related to immune response and inflammation were down-regulated in the brain of APP23 mice. This group of genes was not identified previously  as differentially affected by T0 in LXRwt or LXRdko probably because the animals in that study were not APP expressing transgenic mice. In the course of AD, CNS inflammation is an invariant finding and is considered a result of a localized activation of microglia by fibrillar or oligomeric Aβ. AD brain with active chronic neurodegeneration is primed to elicit faster and more pronounced proinflammatory responses to central and systemic inflammatory challenges. The up-regulation of inflammatory genes identified by microarray in incipient AD supports such a notion . Moreover, a recent study demonstrated that with aging, without any neurodegenerative pathology, there was an up-regulation in immune/inflammatory pathways . Different protein markers including MHC class I and class II proteins, IgG Fc-gamma receptors, pro-inflammatory cytokines and tumor necrosis factor alpha (TNF-α) were found upregulated in AD and some of those were down-regulated by LXR ligand in our gene array assays. Similar data have been recently published by Zelcer et al . Using primary glia, the authors demonstrated up-regulation of proinflammatory genes in response to fibrillar Aβ and a strong inhibitory effect of GW. In their study expression profiling of GW treated BV2 cells revealed up- or down-regulation of genes clustered in two groups primarily related to lipid metabolism and transport, while LPS treatment of the cells strongly induced a battery of inflammatory genes, which were potently repressed if GW was added to the culture.
In our study, Serpina3n is a gene that was down-regulated (more than 2-fold, Fig. 1C) after extended T0 treatment of APP23 mice and may have an impact on amyloid deposition. Serpina3n is a murine homolog of human α-Antichymotrypsin and is one of 13 closely related inhibitors within the murine serpina3 cluster . In brain, α-Antichymotrypsin is a component of the acute inflammatory response. Human α-Antichymotrypsin has been identified in amyloid plaques and was shown to promote Aβ aggregation as well as tau phosphorylation [51–53]. In this study we also demonstrate that LXR ligands have anti-inflammatory effect in primary microglia and astrocytes activated by LPS or fibrillar Aβ. Most importantly we found that T0 inhibits the expression of pro-inflammatory cytokines in the brain of APP23 mice after extended but not short-term (24 hours) application.