Activation Of LXR By T0901317 Causes An Increases In DBP And CYP46 Expression And A Decrease In Aβ Level In Mouse Brains

Objective: Alzheimer's Disease (AD) is characterizedby the profuse accumulation of insoluble βamyloid (Aβ)peptides. Recent studies indicate a possible link betweencholesterol metabolism and Aβlevel in AD. Suppression ofcholesterol synthesis lowered the formation of Aβand theincidence of AD. In contrast, hypercholesterolemia increased theAβimmunocompetence in brain. Therefore , the research onrelationship between Aβand cholesterol metabolism has beingbecome an attractive field.Liver X-activated receptor (LXR) also expresses in brainand is a key regulated point for cholesterol metabolism in brain.Hydroxysterol and TO901317 are ligands for LXR. Theligand-actived LXR enhanced the expression of its target geneslike ATP-binding Cassette transporter (ABC) ABCA1, ABCG1increasing cholesterol efflux. Activation of LXR by22R-hydoxycholesteral or TO901317 reduced Aβlevel alongwith the increased expression of ABCA1 in transgenic mice andneuron primary culture cell. However, whether such situationhappens in normal animals is unclear.It is important for cholesterol balance in brain thatcholesterol traverses the blood-brain barrier into circulation,then is converted to bile acid in liver. Most of cholesterol shouldbe conversed to 24S-hydroxycholesterol byChlesterol-24S-hydroxylase (CYP46) before out of brain. Howis the expression of CYP46 after activation of LXR is unknown.D-3-Hydroxyacyl-CoA dehydratase /D-3-Hydroxyacyl-CoA dehydrogenase (D-bifunctional protein, DBP)locates in mammalian peroxisomes and involves in peroxisomalbeta-oxidation. In liver it is a necessary enzyme for bile acidsynthesis converting bile acid precursor into24R-hydroxycholesterol. DBP is also found in brain and playsan important role in the brain development. But its biochemicalfunction is still obscure. It has been established that there shouldbe other pathways for cholesterol out of brain beside the CYP46pathway. Therefore we are interested in studying whether DBPcontributes to cholesterol efflux by converting cholesterol to24R-hydroxycholesterol in brain.In present research, we treated the normal C57BL/6animals with the potent LXR agonist, TO901317 to induceexpression of its target genes ABCA1 and ABCG1. Then thelevel of total cholesterol and Aβand the expression of CYP46and DBP in the brain were assayed. The function of CYP46 andDBP in cholesterol homeostasis of brain was discussed. Thepresent research may provide a new solution and target point tocure AD.Methods1 AnimalsMale C57BL/6 mice aged 8-10 weeks were dividedrandomly into control group(n=10)and agonist group(n=15).Control group (control) and agonist group (TO) wereadministered orally with vehicle solution and LXR agongistTO901317 at 20mg/kg/day, respectively for 7 days. All animalswere sacrified at day 8. Brain was immediately submerged inliquid Nitrogen then stored at -70℃for the extraction of totalRNA and measurement of DBP activity, total cholesterol and Aβ.2 Measure Methods①DBP activity assayThe activity of D-3-hydoxyacyl-CoA dehydrogenase ofDBP was assayed by following change in absorbance at 303nmas described by Jiang LL. The enzyme activities were assayed at30℃, and one enzyme unit was defined as the amount of theenzyme converting 1μmol of substrate per min under the assayconditions.②Brain total cholesterol assayBrain cholesterol was extracted with chloroform/methanol(2:1) and was determinated by enzymatic-colorimetric assay.The values were expreeed as mg of cholesterol/g wet weight oftissue (mg/g).③The expression of mRNAassayTotal RNA was isolated using the guanidiumthiocyanate-phenol-chloroform method. The relative mRNAcontent were measured by RT-PCR using β-actin as innerstandard.④BrainAβassayAβwas determined by radioimmunoassay method usingthe Aβassay kit and expressed as pg of Aβ/mg of protein(pg/mg).Results1 Total cholesterol in mice brainCompared with control group(11.6±0.994 mg/g), braintotal cholesterol of TO group was singnificantly decreased(10.4±0.991 mg/g)(p<0.05).It indicated that enhanced cholesterolefflux by LXR agonist could decrease the level of totalcholesterol in brain.2 Aβcontent in mice brain Compared with control group (6.53±0.708 pg/mg), Aβcontent of TO group was singnificantly reduced (5.79±0.556 pg/mg )( p < 0.05 ) .It showed that the decreasedcholesterol by LXR agonist could effectively decrease theproduction of Aβ.3 Target genes of LXR, ABCA1 and ABCG1 mRNA expressionin mice brainIn TO group, mRNA expression of ABCA1(0.245±0.0319)and ABCG1(0.280±0.0572)was significantly higherthan that(0.161±0.0236,0.175±0.0167)(p<0.01)in controlgroup. It indicated that cholesterol efflux out of the cell could beenhanced because of the increased expression of ABCA1 and...