Implications of cerebrovascular ATP-binding cassette transporter G1 (ABCG1) and apolipoprotein M in cholesterol transport at the blood-brain barrier.

Impaired cholesterol/lipoprotein metabolism is linked to neurodegenerative diseases such as Alzheimer's disease (AD). Cerebral cholesterol homeostasis is maintained by the highly efficient blood-brain barrier (BBB) and flux of the oxysterols 24(S)-hydroxycholesterol and 27-hydroxycholesterol, potent liver-X-receptor (LXR) activators. HDL and their apolipoproteins are crucial for cerebral lipid transfer, and loss of ATP binding cassette transporters (ABC)G1 and G4 results in toxic accumulation of oxysterols in the brain. The HDL-associated apolipoprotein (apo)M is positively correlated with pre-ß HDL formation in plasma; its presence and function in the brain was thus far unknown. Using an in vitro model of the BBB, we examined expression, regulation, and functions of ABCG1, ABCG4, and apoM in primary porcine brain capillary endothelial cells (pBCEC). RT Q-PCR analyses and immunoblotting revealed that in addition to ABCA1 and scavenger receptor, class B, type I (SR-BI), pBCEC express high levels of ABCG1, which was up-regulated by LXR activation. Immunofluorescent staining, site-specific biotinylation and immunoprecipitation revealed that ABCG1 is localized both to early and late endosomes and on apical and basolateral plasma membranes. Using siRNA interference to silence ABCG1 (by 50%) reduced HDL-mediated [3H]-cholesterol efflux (by 50%) but did not reduce [3H]-24(S)-hydroxycholesterol efflux. In addition to apoA-I, pBCEC express and secrete apoM mainly to the basolateral (brain) compartment. HDL enhanced expression and secretion of apoM by pBCEC, apoM-enriched HDL promoted cellular cholesterol efflux more efficiently than apoM-free HDL, while apoM-silencing diminished cellular cholesterol release. We suggest that ABCG1 and apoM are centrally involved in regulation of cholesterol metabolism/turnover at the BBB.

Regulation of the yeast triacylglycerol lipases Tgl4p and Tgl5p by the presence/absence of nonpolar lipids.

Tgl3p, Tgl4p, and Tgl5p are the major triacylglycerol lipases of the yeast Saccharomyces cerevisiae Recently we demonstrated that properties of Tgl3p are regulated by the formation of nonpolar lipids. The present study extends these investigations to the two other yeast triacylglycerol lipases, Tgl4p and Tgl5p. We show that Tgl4p and Tgl5p, which are localized to lipid droplets in wild type, are partially retained in the endoplasmic reticulum in cells lacking triacylglycerols and localize exclusively to the endoplasmic reticulum in a mutant devoid of lipid droplets. In cells lacking steryl esters, the subcellular distribution of Tgl4p and Tgl5p is unaffected, but Tgl5p becomes unstable, whereas the stability of Tgl4p increases. In cells lacking nonpolar lipids, Tgl4p and Tgl5p lose their lipolytic activity but retain their side activity as lysophospholipid acyltransferases. To investigate the regulatory network of yeast triacylglycerol lipases in more detail, we also examined properties of Tgl3p, Tgl4p, and Tgl5p, respectively, in the absence of the other lipases. Surprisingly, lack of two lipases did not affect expression, localization, and stability of the remaining Tgl protein. These results suggest that Tgl3p, Tgl4p, and Tgl5p, although they exhibit similar functions, act as independent entities.

Nephroblastomas show low expression of microR-204 and high expression of its target, the oncogenic transcription factor MEIS1.

By comparing several studies we identified a possible deregulation of the transcription factors PBX2 (pre-B-cell leukemia homeobox 2) and one of its binding partners, MEIS1 (Meis homeobox 1) in nephroblastomas. The regulation of MEIS1 is complex, and its expression is known to be influenced by changes of promoter methylation and binding of microRNA-204 (miR-204). Therefore, in our study, we assessed the expression of MEIS1 and PBX2 and the factors regulating expression of MEIS1 in nephroblastomas. MEIS1 and PBX2 messenger RNA (mRNA) and protein levels were investigated by quantitative real-time-polymerase chain reaction (qRT-PCR) and immunohistochemistry. Promoter methylation of MEIS1 was evaluated using a methylation-specific PCR assay. Expression levels of miR-204 were examined by qRT-PCR. Eighteen of 21 nephroblastomas showed a high level of MEIS1 mRNA, and 22 of 26 samples had a specific nuclear protein expression. MicroRNA-204 had a statistically significantly lower expression in all nephroblastomas investigated compared with renal parenchyma, but no change of MEIS1 promoter methylation status was noted. Eleven of 23 nephroblastomas had a high expression of PBX2 mRNA, and 15 of 23 samples had a specific nuclear protein expression was noted. In our study, we demonstrated an expression of MEIS1 and its binding partner PBX2 in most nephroblastomas. The statistically significantly lower expression of miR-204 in all nephroblastomas investigated might point to an involvement of miR-204 in the regulation of MEIS1 in nephroblastomas.

Phospholipid transfer protein is expressed in cerebrovascular endothelial cells and involved in high density lipoprotein biogenesis and remodeling at the blood-brain barrier.

Phospholipid transfer protein (PLTP) is a key protein involved in biogenesis and remodeling of plasma HDL. Several neuroprotective properties have been ascribed to HDL. We reported earlier that liver X receptor (LXR) activation promotes cellular cholesterol efflux and formation of HDL-like particles in an established in vitro model of the blood-brain barrier (BBB) consisting of primary porcine brain capillary endothelial cells (pBCEC). Here, we report PLTP synthesis, regulation, and its key role in HDL metabolism at the BBB. We demonstrate that PLTP is highly expressed and secreted by pBCEC. In a polarized in vitro model mimicking the BBB, pBCEC secreted phospholipid-transfer active PLTP preferentially to the basolateral ("brain parenchymal") compartment. PLTP expression levels and phospholipid transfer activity were enhanced (up to 2.5-fold) by LXR activation using 24(S)-hydroxycholesterol (a cerebral cholesterol metabolite) or TO901317 (a synthetic LXR agonist). TO901317 administration elevated PLTP activity in BCEC from C57/BL6 mice. Preincubation of HDL3 with human plasma-derived active PLTP resulted in the formation of smaller and larger HDL particles and enhanced the capacity of the generated HDL particles to remove cholesterol from pBCEC by up to 3-fold. Pre-ß-HDL, detected by two-dimensional crossed immunoelectrophoresis, was generated from HDL3 in pBCEC-derived supernatants, and their generation was markedly enhanced (1.9-fold) upon LXR activation. Furthermore, RNA interference-mediated PLTP silencing (up to 75%) reduced both apoA-I-dependent (67%) and HDL3-dependent (30%) cholesterol efflux from pBCEC. Based on these findings, we propose that PLTP is actively involved in lipid transfer, cholesterol efflux, HDL genesis, and remodeling at the BBB.