br ABCA modulates intracellular sphingolipid metabolism in

ABCA2 modulates intracellular sphingolipid metabolism in the LE/LY Recycling of plasma membrane lipids that occurs by constitutive endocytosis and vesicularization of complex glycolipids, complex sphingolipids and cholesterol as well as free cholesterol liberated from LDL after receptor-mediated endocytosis of the LDLR. As depicted in Fig. 2, following endocytosis of plasma membrane lipids into vesicles, further invagination of endosomal vesicles into intraluminal vesicles and the progressive acidification of the lumen by proton pumps establish distinct compartments, (i.e., early endosome to late endosome/lysosome maturation) favorable to the enzymatic activities (glucocerebrosidase, Dioscin sphingomyelinase, acid ceramidase). Their catalytic activities are restricted to defined acidic pH optima. The results of the enzymatic activities recycle membrane lipid components (salvage pathway) to other intracellular compartments for structural and signaling functions dependent on lipid metabolism [84] (Fig. 2). At the acidic pH of LE/LY (∼5.0 to 5.5), the action of sphingolipid recycling enzymes (salvage pathway), (e.g. glucocerebrosidase, acid sphingomyelinase and acid ceramidase) modulates the catabolism of the membrane sphingolipids derived from the plasma membrane [85]. The central molecule of sphingolipid metabolism is ceramide, which consists of the sphingoid base sphingosine covalently linked to a fatty acid [86]. Ceramide is produced through the action of de novo synthetic pathways as well as through catabolism of more complex sphingolipids in the salvage/recycling pathway in the LE/LY [85]. Ceramide is the precursor for the synthesis of more complex lipids, including sphingomyelin, cerebrosides and complex glycosphingolipids. In the umbrella model both ceramide and free cholesterol, which have small polar head groups and relatively large nonpolar bodies, compete for coverage of their headgroups by phospholipids in the membrane bilayer, to prevent exposure of their nonpolar bodies to aqueous environments [87]. Elevated ceramide levels contained within intraluminal vesicles in the LE/LY displace free cholesterol onto the aqueous free cholesterol acceptor NPC2 protein. NPC2 loaded with free cholesterol then interacts with NPC1 present at the limiting membrane of the LE/LY and transfers free-cholesterol to the limiting membrane of the LE/LY [88]. We hypothesize that the ceramide/sphingosine ratio is a key regulator of the movement of LE/LY cholesterol onto NPC2. Since sphingosine is less effective than ceramide in displacing cholesterol onto NPC2 [89], elevation of sphingosine in the salvage pathway at the expense of ceramide may reduce free cholesterol loading onto aqueous NPC2, resulting in cholesterol sequestration in the LE/LY. Sphingosine is produced by catabolism of membrane sphingolipids to ceramide (i.e., glucocerebrosidase and sphingomyelinase to ceramide) and by the deacylation of the resulting ceramide by acid ceramidase in the LE/LY [90], [91]. Agents that decrease ceramide levels or increase sphingosine in the LE/LY would be expected to decrease free cholesterol loading onto NPC2 for transfer to NPC1 and cholesterol exit, resulting in cholesterol sequestration in the LE/LY. In fact, Niemann-Pick Type C disease has been characterized as a sphingosine storage disease since elevation of sphingosine levels precedes the sequestration of other lipids associated with the disease [92]. We have reported that modulation of ABCA2 expression level alters the cellular sphingolipid profile by electrospray ionization mass spectrometry (ESI/MS/MS). Lipidomics analysis was performed in control N2a cells, in N2a cells that overexpress human ABCA2, in control D6P2T cells that express high levels of endogenous ABCA2 and in RNAi-treated D6P2T [61], [64]. ABCA2 overexpression in mouse N2a neuroblastoma cells exhibited reduced levels of key ceramide metabolites and a greater than twofold increase in sphingosine levels. Conversely, depletion of endogenous ABCA2 expression in D6P2T cells exhibited elevated the levels of key ceramide metabolites. In addition, we measured an increase in in vitro acid ceramidase activity in microsomes isolated from ABCA2 overexpressing N2a cells. These results suggest that ABCA2 expression may modulate the ceramide: sphingosine ratio necessary for cholesterol loading onto NPC2. In a broader sense, our studies have suggested a convergence between ABCA2 expression, sphingolipid salvage pathway catabolism and cholesterol exit from the LE/LY.