25-Hydroxycholesterol in health and diseases.

Cholesterol is an essential structural component of all membranes of mammalian cells where it plays a fundamental role not only in cellular architecture, but also, for example, in signaling pathway transduction, endocytosis process, receptor functioning and recycling, or cytoskeleton remodeling. Consequently, intracellular cholesterol concentrations are tightly regulated by complex processes, including cholesterol synthesis, uptake from circulating lipoproteins, lipid transfer to these lipoproteins, esterification, and metabolization into oxysterols that are intermediates for bile acids. Oxysterols have been considered for long time as sterol waste products, but a large body of evidence has clearly demonstrated that they play key roles in central nervous system functioning, immune cell response, cell death, or migration and are involved in age-related diseases, cancers, autoimmunity, or neurological disorders. Among all the existing oxysterols, this review summarizes basic as well as recent knowledge on 25-hydroxycholesterol which is mainly produced during inflammatory or infectious situations and that in turn contributes to immune response, central nervous system disorders, atherosclerosis, macular degeneration, or cancer development. Effects of its metabolite 7α,25-dihydroxycholesterol are also presented and discussed.

The Tetraspanin Tspan8 Associates with Endothelin Converting Enzyme ECE1 and Regulates Its Activity.

Tspan8 is a member of the tetraspanins family of cell surface molecules. The ability of tetraspanins to organize membrane microdomains with other membrane molecules and interfere with their function suggests that they could act as surface integrators of external or internal signals. Among the first identified tetraspanins, Tspan8 promotes tumor progression and metastasis, presumably by stimulating angiogenesis and cell motility. In patients, its expression on digestive tract tumors seems to be associated with a bad prognosis. We showed previously that Tspan8 associates with E-cadherin and EGFR and modulates their effects on cell motility. Using Mass spectrometry and western blot, we found a new partner, the endothelin converting enzyme ECE1, and showed that Tspan8 amplifies its activity of conversion of the endothelin-1 precursor bigET1 to endothelin. This was observed by transduction of the colon carcinoma cell line Isreco1, which does not express Tspan8, and on ileum tissue fragments of tspan8ko mice versus wild type mice. Given these results, Tspan8 appears to be a modulator of the endothelin axis, which could possibly be targeted in case of over-activity of endothelins in biological processes of tissues expressing Tspan8.

TNFα Activates the Liver X Receptor Signaling Pathway and Promotes Cholesterol Efflux from Human Brain Pericytes Independently of ABCA1.

Neuroinflammation and brain lipid imbalances are observed in Alzheimer's disease (AD). Tumor necrosis factor-α (TNFα) and the liver X receptor (LXR) signaling pathways are involved in both processes. However, limited information is currently available regarding their relationships in human brain pericytes (HBP) of the neurovascular unit. In cultivated HBP, TNFα activates the LXR pathway and increases the expression of one of its target genes, the transporter ATP-binding cassette family A member 1 (ABCA1), while ABCG1 is not expressed. Apolipoprotein E (APOE) synthesis and release are diminished. The cholesterol efflux is promoted, but is not inhibited, when ABCA1 or LXR are blocked. Moreover, as for TNFα, direct LXR activation by the agonist (T0901317) increases ABCA1 expression and the associated cholesterol efflux. However, this process is abolished when LXR/ABCA1 are both inhibited. Neither the other ABC transporters nor the SR-BI are involved in this TNFα-mediated lipid efflux regulation. We also report that inflammation increases ABCB1 expression and function. In conclusion, our data suggest that inflammation increases HBP protection against xenobiotics and triggers an LXR/ABCA1 independent cholesterol release. Understanding the molecular mechanisms regulating this efflux at the level of the neurovascular unit remains fundamental to the characterization of links between neuroinflammation, cholesterol and HBP function in neurodegenerative disorders.

Regulation of the trafficking and the function of the metalloprotease ADAM10 by tetraspanins.

By interacting directly with partner proteins and with one another, tetraspanins organize a network of interactions referred to as the tetraspanin web. ADAM10 (A Disintegrin And Metalloprotease 10), an essential membrane-anchored metalloprotease that cleaves off the ectodomain of a large variety of cell surface proteins including cytokines, adhesion molecules, the precursor of the ß-amyloid peptide APP or Notch, has emerged as a major component of the tetraspanin web. Recent studies have shown that ADAM10 associates directly with all members (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33) of a subgroup of tetraspanins having eight cysteines in the large extracellular domain and referred to as TspanC8. All TspanC8 regulate ADAM10 exit from the endoplasmic reticulum, but differentially regulate its subsequent trafficking and its function, and have notably a different impact on Notch signaling. TspanC8 orthologs in invertebrates also regulate ADAM10 trafficking and Notch signaling. It may be possible to target TspanC8 tetraspanins to modulate in a tissue- or substrate-restricted manner ADAM10 function in pathologies such as cardiovascular diseases, cancer or Alzheimer's disease.

New insights into the tetraspanin Tspan5 using novel monoclonal antibodies.

Tspan5 is a member of a subgroup of tetraspanins referred to as TspanC8. These tetraspanins directly interact with the metalloprotease ADAM10, regulate its exit from the endoplasmic reticulum and subsequent trafficking, and differentially regulate its ability to cleave various substrates and activate Notch signaling. The study of Tspan5 has been limited by the lack of good antibodies. This study provides new insights into Tspan5 using new monoclonal antibodies (mAbs), including two mAbs recognizing both Tspan5 and the highly similar tetraspanin Tspan17. Using these mAbs, we show that endogenous Tspan5 associates with ADAM10 in human cell lines and in mouse tissues where it is the most abundant, such as the brain, the lung, the kidney, or the intestine. We also uncover two TspanC8-specific motifs in the large extracellular domain of Tspan5 that are important for ADAM10 interaction and exit from the endoplasmic reticulum. One of the anti-Tspan5 mAbs does not recognize Tspan5 associated with ADAM10, providing a convenient way to measure the fraction of Tspan5 not associated with ADAM10. This fraction is minor in the cell lines tested, and it increases upon transfection of cells with TspanC8 tetraspanins such as Tspan15 or Tspan33 that inhibit Notch signaling. Finally, two antibodies inhibit ligand-induced Notch signaling, and this effect is stronger in cells depleted of the TspanC8 tetraspanin Tspan14, further indicating that Tspan5 and Tspan14 can compensate for each other in Notch signaling.

TspanC8 tetraspanins differentially regulate the cleavage of ADAM10 substrates, Notch activation and ADAM10 membrane compartmentalization.

The metalloprotease ADAM10 mediates the shedding of the ectodomain of various cell membrane proteins, including APP, the precursor of the amyloid peptide Aß, and Notch receptors following ligand binding. ADAM10 associates with the members of an evolutionary conserved subgroup of tetraspanins, referred to as TspanC8, which regulate its exit from the endoplasmic reticulum. Here we show that 4 of these TspanC8 (Tspan5, Tspan14, Tspan15 and Tspan33) which positively regulate ADAM10 surface expression levels differentially impact ADAM10-dependent Notch activation and the cleavage of several ADAM10 substrates, including APP, N-cadherin and CD44. Sucrose gradient fractionation, single molecule tracking and quantitative mass-spectrometry analysis of the repertoire of molecules co-immunoprecipitated with Tspan5, Tspan15 and ADAM10 show that these two tetraspanins differentially regulate ADAM10 membrane compartmentalization. These data represent a unique example where several tetraspanins differentially regulate the function of a common partner protein through a distinct membrane compartmentalization.

Bexarotene Promotes Cholesterol Efflux and Restricts Apical-to-Basolateral Transport of Amyloid-ß Peptides in an In Vitro Model of the Human Blood-Brain Barrier.

One of the prime features of Alzheimer's disease (AD) is the excessive accumulation of amyloid-ß (Aß) peptides in the brain. Several recent studies suggest that this phenomenon results from the dysregulation of cholesterol homeostasis in the brain and impaired bidirectional Aß exchange between blood and brain. These mechanisms appear to be closely related and are controlled by the blood-brain barrier (BBB) at the brain microvessel level. In animal models of AD, the anticancer drug bexarotene (a retinoid X receptor agonist) has been found to restore cognitive functions and decrease the brain amyloid burden by regulating cholesterol homeostasis. However, the drug's therapeutic effect is subject to debate and the exact mechanism of action has not been characterized. Therefore, the objective of this present study was to determine bexarotene's effects on the BBB. Using an in vitro model of the human BBB, we investigated the drug's effects on cholesterol exchange between abluminal and luminal compartments and the apical-to-basolateral transport of Aß peptides across the BBB. Our results demonstrated that bexarotene induces the expression of ABCA1 but not ApoE. This upregulation correlates with an increase in ApoE2-, ApoE4-, ApoA-I-, and HDL-mediated cholesterol efflux. Regarding the transport of Aß peptides, bexarotene increases the expression of ABCB1, which in turn decreases Aß apical-to-basolateral transport. Our results showed that bexarotene not only promotes the cholesterol exchange between the brain and the blood but also decreases the influx of Aß peptides across BBB, suggesting that bexarotene is a promising drug candidate for the treatment of AD.

Apical-to-basolateral transport of amyloid-ß peptides through blood-brain barrier cells is mediated by the receptor for advanced glycation end-products and is restricted by P-glycoprotein.

Several studies have highlighted the close relationship between Alzheimer's disease (AD) and alterations in the bidirectional transport of amyloid-ß (Aß) peptides across the blood-brain barrier (BBB). The brain capillary endothelial cells (BCECs) that compose the BBB express the receptors and transporters that enable this transport process. There is significant in vivo evidence to suggest that P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) restrict Aß peptides entry into the brain, whereas the receptor for advanced glycation end-products (RAGE) seems to mediate apical-to-basolateral passage across the BBB. However, deciphering the molecular mechanisms underlying these in vivo processes requires further in vitro characterization. Using an in vitro BBB model and specific competition experiments against RAGE, we have observed a significant decrease in apical-to-basolateral (but not basolateral-to-apical) transport of Aß1-40 and Aß1-42 peptides through BCECs. This transport is a caveolae-dependent process and fits with the apical location of RAGE observed in confocal microscopy experiments. Inhibition of P-gp and BCRP using different inhibitors increases transport of Aß peptides suggesting that these efflux pumps are involved in Aß peptide transport at the BCECs level. Taken as a whole, these results demonstrate the involvement of the caveolae-dependent transcytosis of Aß peptides through the BBB in a RAGE-mediated transport process, reinforcing the hypothesis whereby this receptor is a potential drug target in AD.