Effect of liver X receptor agonist TO901317 on cognitive function in APP/PS1 double transgenic mice with Alzheimer's disease and the underlying mechanism. / 肝脏X受体激动剂TO901317对APP/PS1åŒè½¬åŸºå› é˜¿å°”èŒ¨æµ·é»˜ç— å°é¼ è®¤çŸ¥åŠŸèƒ½çš„å½±å“åŠå ¶æœºåˆ¶.

OBJECTIVES: The liver X receptors (LXRs) are members of the nuclear hormone receptor superfamily, and LXR-ß is an important receptor for cholesterol content in brain cells. LXR-ß/retinoic X receptor (RXR-α)/ATP binding cassette transporter A1 (ABCA1) cholesterol transmembrane transport system is closely related to the occurrence and development of Alzheimer's disease (AD). LXR agonist TO901317 can affect the accumulation of ß- amyloid protein in the brain tissue of APP/PS1 double transgenic AD mice. However, the molecular mechanism is not clarified in detail. This study aims to evaluate the effects of LXR agonist TO901317 on the cognitive function of AD mice fed with high cholesterol diet, and to explore its possible mechanism from the perspective of cholesterol metabolism. METHODS: Twenty four male 6-month-old APP/PS1 double transgenic AD mice were randomly divided into 4 groups, 6 mice in each group: a control group (fed with normal diet), a cholesterol rich diet (CRD) group, a TO901317 group (fed with CRD combined with TO901317), and a GSK2033 group (fed with CRD combined with TO901317 and LXR antagonist GSK2033). The mice were fed with pellet feed made of high cholesterol feed, mixed with lard, egg yolk powder, and cod liver oil twice a day. TO901317 and GSK2033 were dissolved and diluted to a final concentration at 0.03%. The drugs were given to the mice daily through gastric tube according to their body weight. Meanwhile, the mice in the drug group were fed with high cholesterol diet . After feeding for 3 months, Morris water maze was used to observe the changes of spatial exploration and memory ability of AD mice in each group. The contents of TC, LDL, and HDL in serum of mice in each group were detected by cholesterol enzyme colorimetry, and the differences among the groups were compared. The expression of Aß42 in the brain of AD mice was detected by ELISA. Western blotting was used to detect the protein levels of LXR-ß, RXR-α, ABCA1, and Caveolin-1 in the brain of each group. RESULTS: Morris water maze results showed that the times, distance and the duration of mice crossing the platform in the CRD group were significantly decreased compared with the control group (all P<0.05), while these three figures in TO901317 group were significantly increased compared with the CRD group (all P<0.05). Compared with the TO901317 group, there was a decrease of these figures in the GSK2033 group (all P<0.05). The serum TC and LDL levels in the CRD group were significantly higher than those in the control group, while HDL levels were significantly lower (all P<0.001). The figures of the TC and LDL contents level in the TO901317 group were lower than those in the CRD group, while HDL levels were higher (all P<0.001). Compared with TO901317 group, the contents of the TC and LDL in GSK2033 group were significantly increased, while HDL content was significantly decreased (all P<0.001). ELISA results showed that the production of Aß42 peptides in the brain of CRD group was the highest while the content in the TO901317 group was significantly decreased (P<0.001), which was the lowest among the groups. The figure in the control group was close to the GSK2033 group. Western blotting results showed that the protein levels of LXR-ß, RXR-α, and ABCA1 in the CRD group were significantly decreased compared with the control group, but the protein level of Caveolin-1 was increased (all P<0.01). After TO901317 treatment, the protein levels of LXR-ß, RXR-α and ABCA1 were significantly increased, while the protein level of Caveolin-1 was decreased partially (all P<0.001). In the GSK2033 group, the effect of TO901317 on AD mice was partially reversed by GSK2033. Compared to TO901317 group, the protein levels of LXR-ß, RXR-α, and ABCA1 showed a decrease trend, while the protein level of Caveolin-1 showed an increase state (all P<0.05). CONCLUSIONS: High cholesterol diet leads to severer spatial exploration, learning and memory impairment in transgenic AD mice, while the LXR agonist TO901317 attenuates this effect. The mechanism may be that TO901317 promotes cholesterol efflux by activating LXR-ß/RXR-α/ABCA1 transmembrane transport system, reduces the expression of Caveolin-1, improves the composition of lipid raft, and ultimately reduces the production of Aß42 in the brain.

Effect of activation of liver X receptor alpha on cardiac & hepatic ABCC10 and SLC17A5 drug transporters in hypercholesterolemic rat model.

BACKGROUND: Liver x receptor α (LXRα) is a ligand-activated transcription factor belonging to the nuclear receptor superfamily. Oxysterols (endogenous oxidized cholesterol derivatives) are the most potent endogenous LXRα-agonist. LXRα has a direct impact on several members of drug transporter superfamilies; ATP-binding cassette (ABC) and solute linked carrier (SLC). OBJECTIVE: The current study aimed to investigate the effect of LXRα-activation by either endogenous oxysterols or a synthetic LXRα-agonist (LXRa) such as TO901317 on hepatic and cardiac gene expression of ABCC10 and SLC17A5 drug transporters in an experimentally hypercholesterolemic rat model. METHODS: 48 male rats were divided randomly into four groups (n = 12); control group rats received vehicle; hypercholesterolemic group (HCH group) rats received diet contain 2.5% cholesterol &deoxycholic acid for 8 weeks; (LXRa group) rats were fed standard pellet chow for 8 weeks, then a single dose of LXRa was administered (IP) at a dose of 10 mg/kg; (HCH + LXRa group) rats received diet contain 2.5% cholesterol &deoxycholic acid for 8 weeks, then a single dose of LXRa was administered (IP) at a dose of 10 mg/kg. RESULTS: Our findings revealed that hypercholesterolemia and LXRa significantly activated LXRα to varying degrees in both hepatic and cardiac tissues with subsequent alteration of LXRα and ABCC10 gene expression. Whereas, SLC17A5 gene expression was primarily affected by elevated serum cholesterol level and unmediated via LXRα-activation. CONCLUSIONS: Accordingly, it was concluded that ABCC10 is a specific LXRα-target gene and that LXRα autoregulates its own expression in rats.

TO901317 activation of LXR-dependent pathways mitigate amyloid-beta peptide-induced neurotoxicity in 3D human neural stem cell culture scaffolds and AD mice.

Alzheimer's disease (AD) is the major cause of neurodegeneration worldwide and is characterized by the accumulation of amyloid beta (Aß) in the brain, which is associated with neuronal loss and cognitive impairment. Liver X receptor (LXR), a critical nuclear receptor, and major regulator in lipid metabolism and inflammation, is suggested to play a protective role against the mitochondrial dysfunction noted in AD. In our study, our established 3D gelatin scaffold model and a well characterized in vivo (APP/PS1) murine model of AD were used to directly investigate the molecular, biochemical and behavioral effects of neuronal stem cell exposure to Aß to improve understanding of the in vivo etiology of AD. Herein, human neural stem cells (hNSCs) in our 3D model were exposed to Aß, and had significantly decreased cell viability, which correlated with decreased mRNA and protein expression of LXR, Bcl-2, CREB, PGC1α, NRF-1, and Tfam, and increased caspase 3 and 9 activities. Cotreatment with a synthetic agonist of LXR (TO901317) significantly abrogated these Aß-mediated effects in hNSCs. Moreover, TO901317 cotreatment both significantly rescues hNSCs from Aß-mediated decreases in ATP levels and mitochondrial mass, and significantly restores Aß-induced fragmented mitochondria to almost normal morphology. TO901317 cotreatment also decreases tau aggregates in Aß-treated hNSCs. Importantly, TO901317 treatment significantly alleviates the impairment of memory, decreases Aß aggregates and increases proteasome activity in APP/PS1 mice; whereas, these effects were blocked by cotreatment with an LXR antagonist (GSK2033). Together, these novel results improve our mechanistic understanding of the central role of LXR in Aß-mediated hNSC dysfunction. We also provide preclinical data unveiling the protective effects of using an LXR-dependent agonist, TO901317, to block the toxicity observed in Aß-exposed hNSCs, which may guide future treatment strategies to slow or prevent neurodegeneration in some AD patients.

Effect of TO901317 on GF to promote the differentiation of human bone marrow mesenchymal stem cells into dopamine neurons on Parkinson's disease.

BACKGROUND: Human bone marrow mesenchymal stem cells (hBMSCs) could differentiate into dopamine-producing cells and ameliorate behavioral deficits in Parkinson's disease (PD) models. Liver X receptors (LXRs) are involved in the maintenance of the normal function of central nervous system myelin. Therefore, the previous work of our team has found the induction of cocktail-induced to dopaminergic (DA) phenotypes from adult rat BMSCs by using sonic hedgehog (SHH), fibroblast growth factor 8 (FGF8), basic fibroblast growth factor (bFGF), and TO901317 (an agonist of LXRs) with 87.42% of efficiency in a 6-day induction period. But we did not verify whether the induced cells had the corresponding neural function. METHODS: Expressions of LXRα, LXRß, and tyrosine hydroxylase (TH) were detected by immunofluorescence and western blot. Adenosine triphosphate-binding cassette transporter A1 (ABCA1) was detected by quantitative real-time PCR. The induced cells were transplanted into PD rats to study whether the induced cells are working. RESULTS: The induced cells can release the dopamine transmitter; the maximum induction efficiency of differentiation of hBMSCs into DA neurons was 91.67% under conditions of combined use with TO901317 and growth factors (GF). When the induced-cells were transplanted into PD rats, the expression of TH in the striatum increased significantly, and the behavior of PD rats induced by apomorphine was significantly improved. CONCLUSION: The induced cells have the function of DA neurons and have the potential to treat PD. TO901317 promoted differentiation of hBMSCs into DA neurons, which may be related to activation of the LXR-ABCA1 signaling pathway.

The Liver X Receptor Agonist TO901317 Ameliorates Behavioral Deficits in Two Mouse Models of Autism.

Autism spectrum disorder (ASD) is a developmental disability characterized by social deficits and repetitive stereotyped behaviors. There are currently no drugs available for the treatment of the core symptoms of ASD, suggesting an urgent need for new therapeutic strategies. The neurobiology of autism is complex, but emerging research indicates that defects in hippocampal neurogenesis are associated with ASD in both humans and mouse models of ASD, leading to the suggestion that restoring neurogenesis may be a novel therapeutic approach for ASD. Here, we found that postnatal treatment with TO901317 (TO), a potent liver X receptor (LXR) agonist, typically activated LXRß and its target genes in the hippocampus, and alleviated the social deficits and stereotypical behaviors in BTBR T+ tf/J (BTBR) and valproic acid (VPA)-induced mouse models. In addition, we further confirmed that TO postnatal treatment also rescued the inhibition of adult hippocampal neurogenesis in these two models. In summary, our study suggests that LXR agonist targeting hippocampal neurogenesis may represent a novel potential therapy for ASD.

TO901317 inhibits the development of hepatocellular carcinoma by LXRα/Glut1 decreasing glycometabolism.

This study was conducted to observe the effect and possible mechanism of TO901317 in vivo and in vitro to provide a new basis for the targeted therapy of hepatocellular carcinoma (HCC). The expressions of liver X receptor (LXR)-α, glucose transporter (Glut)-1, proliferating cell nuclear antigen (PCNA), and matrix metalloproteinase (MMP)-9 were analyzed from HCC public database (NCBI PubMed database). The result showed that LXRα was downregulated, whereas Glut1, PCNA, and MMP9 were upregulated in human HCC compared with normal liver. Furthermore, LXRα mRNA was negatively correlated with Glut1 mRNA. At the same time, HCC cells were cultivated in vitro and axillary injected in nude mice to establish the xenograft model. The xenograft in the TO901317-treated group was slower and smaller than the control group. The protein expression of LXRα, Glut1, and MMP9 could be detected by Western blot and glucose level. As a result, TO901317 could inhibit the cell proliferation of HCC in a dose-dependent manner by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. With the increase of TO901317 concentration, the cellular glucose concentration and ATP level were gradually decreased. Western blot results showed TO901317 could upregulate LXRα expression but downregulate MMP9 and Glut1 expression. Transwell and wound-healing analysis confirmed that, by increasing the concentration of TO901317, the cell invasion and migration were both decreased. LXRα small-interfering RNA (siRNA) could relieve the suppression effect of TO901317 on the cell invasion and migration and the expression of LXRα, Glut1, and MMP9. The glucose concentration was also raised. TO901317 could repress the progress of HCC cells by reducing the glucose concentration, upregulating LXRα expression, but downregulating the expression of Glut1 and MMP9. NEW & NOTEWORTHY This subject confirmed that TO901317, a specific liver X receptor agonist, could inhibit the progression of liver cancer through upregulating liver X receptor-α, downregulating the expression of glucose transporter-1 and matrix metalloproteinase-9, and decreasing the glucose content in SMMC-7721 and HepG2 cells.

Treatment with TO901317, a synthetic liver X receptor agonist, reduces brain damage and attenuates neuroinflammation in experimental intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) induces a series of inflammatory processes that contribute to neuronal damage and neurological deterioration. Liver X receptors (LXRs) are nuclear receptors that negatively regulate transcriptional processes involved in inflammatory responses, but their role in the pathology following ICH remains unclear. The present study investigated the neuroprotective effects and anti-inflammatory actions of TO901317, a synthetic LXR agonist, in a model of collagenase-induced ICH and in microglial cultures. METHODS: Mice subjected to collagenase-induced ICH injury were injected with either TO901317 (30 mg/kg) or vehicle 10 min after ICH and subsequently daily for 2 days. Behavioral studies, histology analysis, and assessments of hematoma volumes, brain water content, and blood-brain barrier (BBB) permeability were performed. The protein expression of LXR-α, LXR-ß, ATP binding cassette transporter-1 (ABCA-1), and inflammatory molecules was analyzed. The anti-inflammatory mechanism of TO901317 was investigated in cultured microglia that were stimulated with either lipopolysaccharide (LPS) or thrombin. RESULTS: ICH induced an increase in LXR-α protein levels in the hemorrhagic hemisphere at 6 h whereas LXR-ß expression remained unaffected. Both LXR-α and LXR-ß were expressed in neurons and microglia in the peri-ICH region and but rarely in astrocytes. TO901317 significantly attenuated functional deficits and brain damage up to 28 days post-ICH. TO901317 also reduced neuronal death, BBB disruption, and brain edema at day 4 post-ICH. These changes were associated with marked reductions in microglial activation, neutrophil infiltration, and expression levels of inflammatory mediators at 4 and 7 days. However, TO901317 had no effect on matrix metalloproteinase-9 activity. In BV2 microglial cultures, TO901317 attenuated LPS- and thrombin-stimulated nitric oxide production and reduced LPS-induced p38, JNK, MAPK, and nuclear factor-kappa B (NF-κB) signaling. Moreover, delaying administration of TO901317 to 3 h post-ICH reduced brain tissue damage and neuronal death. CONCLUSIONS: Our results suggest that enhancing LXR activation may provide a potential therapy for ICH by modulating the cytotoxic functions of microglia.

ABCA1 contributes to macrophage deposition of extracellular cholesterol.

We previously reported that cholesterol-enriched macrophages excrete cholesterol into the extracellular matrix. A monoclonal antibody that detects cholesterol microdomains labels the deposited extracellular particles. Macro-phage deposition of extracellular cholesterol depends, in part, on ABCG1, and this cholesterol can be mobilized by HDL components of the reverse cholesterol transport process. The objective of the current study was to determine whether ABCA1 also contributes to macrophage deposition of extracellular cholesterol. ABCA1 functioned in extracellular cholesterol deposition. The liver X receptor agonist, TO901317 (TO9), an ABCA1-inducing factor, restored cholesterol deposition that was absent in cholesterol-enriched ABCG1(-/-) mouse macrophages. In addition, the ABCA1 inhibitor, probucol, blocked the increment in cholesterol deposited by TO9-treated wild-type macrophages, and completely inhibited deposition from TO9-treated ABCG1(-/-) macrophages. Lastly, ABCA1(-/-) macrophages deposited much less extracellular cholesterol than wild-type macrophages. These findings demonstrate a novel function of ABCA1 in contributing to macrophage export of cholesterol into the extracellular matrix.

Differential regulation of ABCA1 and ABCG1 gene expressions in the remodeling mouse hippocampus after entorhinal cortex lesion and liver-X receptor agonist treatment.

Entorhinal cortex lesioning (ECL) causes an extensive deafferentation of the hippocampus that is classically followed by a compensatory reinnervation, where apolipoprotein E, the main extracellular lipid-carrier in the CNS, has been shown to play a crucial role by shuttling cholesterol to reconstructing neurons terminals. Hence, we investigated whether the ATP-binding cassette (ABC) transporters -A1 and -G1, known to regulate cellular cholesterol efflux and lipidation of the apolipoprotein E-containing lipoprotein complex are actively involved in this context of brain׳s plastic response to neurodegeneration and deafferentation. We assessed ABCA1 and ABCG1 mRNA and protein levels throughout the degenerative phase and the reinnervation process and evaluated the associated cholinergic sprouting following ECL in the adult mouse brain. We subsequently tested the effect of the pharmacological activation of the nuclear receptor LXR, prior to versus after ECL, on hippocampal ABCA1 and G1 expression and on reinnervation. ECL induced a time-dependent up-regulation of ABCA1, but not G1, that coincided with a significant increase in acetylcholine esterase (AChE) activity in the ipsilateral hippocampus. Pre-ECL, but not post-ECL i.p. treatment with the LXR agonist TO901317 also led to a significant increase solely in hippocampal ABCA1 expression, paralleled by increases in both AchE and synaptophysin protein levels in the deafferented hippocampus. Thus, ABCA1 and -G1 are differentially regulated in the lesioned brain and upon treatment with an LXR agonist. Further, TO901317-induced up-regulation of ABCA1 appears to be more beneficial in a prevention (pre-lesion) than rescue (post-lesion) treatment; both findings support a central role for ABC transporters in brain plasticity.

Blockage of progesterone receptor effectively protects pancreatic islet beta cell viability.

The progesterone receptor (PR), a member of nuclear receptor superfamily, is closely associated with gestational, type 1 and type 2 diabetes. However, the underlying mechanisms remain obscure. Here we found that PR activation increased the pro-inflammatory cytokines (PIC)-induced injury in Min6 cells, and PR blockage with siRNA interference protected the cells from damage. Moreover, the new discovered PR antagonist SC51089 effectively improved cell survival by reducing the PIC-stimulated cell apoptosis in Min6 cells. Immunoblotting assays indicated that either PR agonist progesterone (P4) or PR-B over-expression promoted the PIC-induced reinforces of extracellular-signal-regulated kinase 1/2 phosphorylation (p-Erk) and protein 53 (p53), and the attenuations of protein kinase B phosphorylation (p-AKT) and tumor necrosis factor receptor-associated factor 2 (TRAF2). SC51089 could reverse all the P4- or PR-B over-expression induced effects. In addition, PR siRNA inference based assay further supported that SC51089 protected pancreatic islet beta cells from the PR activation or PIC-induced injury by targeting PR and this protective action was mediated by AKT signaling pathway. To our knowledge, this current work might be the first report on the regulation of PR in pancreatic islet beta cell survival. It is expected that SC51089, as a non-steroid PR antagonist, might also find its potential in anti-diabetic research.