br Theoretical mechanism of ABCA

Theoretical mechanism of ABCA2 activity in LE/LY in modulation of cholesterol trafficking It is important to confirm that ABCA2 is a lipid transporter and identify the substrates that are mechanistic in its cholesterol sequestering activity. We hypothesize that ABCA2 modulates cholesterol sequestration in the LE/LY by altering the intraluminal membrane lipid bilayer organization that results in increased Nitecapone ceramidase activity in the LE/LY, facilitating ceramide deacylation to sphingosine (Fig. 3). At the acidic pH of the LE/LY, enzymes in the sphingolipid salvage pathway are positively charged. In addition, Saposin Activator Proteins (e.g. SAP-C, SAP-D) that facilitate salvage pathway enzymatic activity are also positively charged [90]. Electrostatic interactions between the positively charged SAP proteins and anionic phospholipids [e.g. bis-monoacylglycerol phosphate (BMP), PS, phosphatidylinositol (PI), phosphatidic acid (PA) phosphatidylglycerol (PG)] mediate salvage pathway enzymatic activity (e.g., glucocerebrosidase, acid sphingomyelinase [91], [93], [94] (Fig. 4). In addition, acid ceramidase activity also requires the anionic phospholipid interaction with salvage pathway enzymes and SAP proteins. We hypothesize that ABCA2 may function as an anionic phospholipid transporter, facilitating acid ceramidase activity and elevation of LE/LY sphingosine levels. Interestingly, elevated sphingosine levels in the LE/LY are associated with depleted calcium levels in the LE/LY by modulating calcium channel activity [95], [96]. Elevated calcium levels in the LE/LY are critical for vesicular budding in the non-vesicular pathway of cholesterol trafficking [92]. Depleted calcium levels in the LE/LY may indirectly result in cholesterol sequestration.
ABCA2 and Alzheimer’s disease ABCA2 has been most strongly linked to Alzheimer’s disease (AD). Three independent groups have identified a genetic link to both early-onset and sporadic late-onset AD. The first group identified a synonymous single nucleotide polymorphism (C to T transition in the third nucleotide position of the aspartate codon, rs908832) (C679T) in exon 14 of ABCA2 [97]. A second independent group confirmed the first finding that rs908832 was linked to AD, although in an ethnicity-dependent manner [98]. A more recent third epidemiological study, employing a meta-analysis of eight polymorphisms associated with AD, identified the same rs908832 SNP as a significant risk factor for AD in certain ethnic populations [99]. How the rs908832 SNP mutation modulates ABCA2 function and AD etiology is unknown. Our laboratory demonstrated that overexpression of ABCA2 in human embryonic kidney cells was associated with increased expression of genes associated with AD, including the amyloid precursor protein (APP) [24]. We also determined that overexpression of ABCA2 in N2a mouse neuroblastoma cells increased transcription from the human APP promoter and resulted in a shift in the subcellular trafficking and processing of the APP holoprotein to early endosomal compartments that promoted Aβ production [100]. In contrast, Michaki et al. found that knockdown of endogenous ABCA2 in mammalian cells in vitro, in a Drosophila melanogaster model system and in ABCA2 knockout mice reduced Aβ production by altering nicastrin maturation, a key component of γ-secretase [101]. As described above, ABCA2 overexpression in N2a cells increases sphingosine levels. We determined that elevated sphingosine directly modulated a protein kinase C signaling pathway that regulated APP transcription at the AP-1 and E-box control sites in the basal APP promoter [102]. These results indicate that ABCA2 is a viable target for continuing studies into its role in Alzheimer’s disease pathology.
ABCA2 and cardiovascular disease A principal mechanism for initiation and progression of cardiovascular disease involves the cellular response to lipoprotein-derived cholesterol, i.e., low-density lipoprotein (LDL) [103], [104]. The effects of ABCA2 on LDLR expression in N2a cells provides a viable experimental model to study these effects. Steady-state levels of both LDLR mRNA and LDLR proteins are decreased when ABCA2 is overexpressed. Immunoprecipitation of biotinylated cell surface proteins indicated that ABCA2 overexpression reduced surface LDLR levels [61]. Measurement of LDL uptake by the LDLR using a fluorescently labeled LDL (DiI-LDL) was also lower in ABCA2 overexpressing N2a cells. Kinetic experiments by metabolic radiolabeling with [35S]methionine and immunoprecipitation of the LDLR indicated that ABCA2 overexpression decreased LDLR stability (i.e., increased metabolic turnover). These results in neuroblastoma cells are in contrast to those described for stable ABCA2 overexpression in CHO cells and suggest that mechanisms of modulation of LDLR by ABCA2 may be cell type/tissue type dependent. However, such results could be significant given the various pharmacological efforts employed to increase plasma LDL clearance (e.g., antibodies to PCSK9, inhibition of internalization of dietary cholesterol through NPC1L1) [105], [106], [107]. Although only modest reductions in plasma cholesterol levels were measured in ABCA2 knockout mice [19], these experiments were performed on mice fed standard rodent chow and not chow enriched in cholesterol, which may more accurately assess the effects of ABCA2 on modulation of LDL and plasma cholesterol levels. Importantly, ABCA2 overexpression was sufficient to reduce LDLR and therefore may be an attractive target for LDLR therapies (e.g., selective reduction in endogenous ABCA2 to increase LDLR levels and increase plasma LDL clearance).