Statement of the Problem: ATP-binding-cassette (ABC) transporters utilize the energy of ATP hydrolysis to pump substrates across cell membranes. ABC transporters have been identified that are specific for a variety of substrates including amino acids, sugars, inorganic ions, polysaccharides, lipids, peptides and proteins. ABC transporters have been implicated in human diseases as well as contributing to the multi-drug resistance phenotype in response to cancer chemotherapy. Diseases with a demonstrated etiology to defects in ABC transporter function include: cystic fibrosis and the cystic fibrosis transmembrane receptor protein (CTFR or ABCC7), X-linked sideroblastic anemia and ataxia and the mitochondrial half-transporter (ABCB7), Stargardt’s disease and other retinal disorders and the rod photoreceptor ABC transporter (ABCA4), Tangier disease, affecting reverse cholesterol transport and susceptibility to atherosclerosis and the ABCA1 transporter. A central role for ABCA1 in cholesterol homeostasis has been demonstrated by its facilitation of the removal of excess cholesterol from macrophages. This removal is critical for the formation of high-density lipoprotein (HDL). The cholesterol content of most cellular membranes is regulated by a feedback system consisting of de novo synthesis from acetyl CoA (synthetic pathway) and through uptake of cholesterol-containing low-density lipoprotein (LDL) by receptor-mediated endocytosis (salvage pathway). The ABCA2 transporter is a member of the sub-family A of ABC transporters, including ABCA1 described above, with which it shares the highest structural homology. The human gene was identified by this laboratory in investigating mechanisms of acquired resistance of ovarian carcinoma cells to the anticancer drug estramustine. Presumably, the over-expression of the transporter by gene amplification in drug-resistant cells inhibits the accumulation of the drug with a consequent reduction of toxicity and cell death. The human ABCA2 gene comprises 48 exons, which are localized within a genomic region of 21 kb. Studies examining the expression of the gene have detected the greatest abundance in the brain, and greatest immunoreactivity is detected in oligodendrocytes. Sub-cellular localization experiments have detected ABCA2 within late-endosomal/lysosomal and trans-golgi network organelles [14, 15]. A recent study has demonstrated that ABCA2 is expressed in peripheral nerves and in spinal cord oligodendrocyte progenitors of the rat and continues to be expressed into adulthood in mature oligodendrocytes during postnatal development. In spite of the original identification of the ABCA2 gene almost a decade ago, a clear demonstration of its functional role in cells has not been established. Its structural similarity to family member ABCA1 is suggestive of a role in cholesterol homeostasis. We undertook experiments to evaluate the function of ABCA2 in cholesterol homeostasis and report that ABCA2 may have an important role in the delivery of low-density lipoprotein-derived free cholesterol to the endoplasmic reticulum for esterification. Chinese hamster ovary cells engineered to constitutively express the ABCA2 protein display increased expression of the LDL receptor (LDLR) and 3-hydroxy-3-methylglutaryl CoA synthase (HMGCoA S) genes and reduced esterification of LDL-derived free cholesterol. We found that treatment of CHO cells with the steroid hormone, progesterone or the hydrophobic amine, U18666A, which cause accumulation of LDL-derived cholesterol in lysosomes, and also inhibit cholesterol synthesis, both elevate ABCA2 expression. We also measured an increase in ABCA2 expression in Niemann-Pick and Familial Hypercholesterolemia fibroblasts. In preliminary studies, we have also observed that selective reduction of ABCA2 expression by siRNA results in a corresponding decrease in LDLR expression in CHO and HepG2 cells (Warren Davis, unpublished observations). Having established a correlation between expression of ABCA2 and the LDLR we next wish to examine if ABCA2 is a physiological regulator of LDLR function in inflammatory processes associated with neurodegeneration and Alzheimer's disease. Numerous reports have documented the effects of inflammatory cytokines on elevation of the expression of the LDLR and increased binding and uptake of LDL lipoproteins. These cytokines include: tumor necrosis factor-??(TNF), and interleukin-1?, interleukin-6 (IL-6), transforming growth factor-?1 (TGF-????[24] and evidence suggests a role for the LDLR in modulation of the inflammatory response mediated by these cytokines. In the brain, microglia and astrocytes produce these same inflammatory cytokines and they have been implicated in an inflammatory model of cell death associated with Alzheimer's disease. A goal of this project is to determine the role of the ABCA2 transporter in the regulation of cholesterol metabolism and signaling that promotes the cytokine-driven inflammatory response in glial cells. A second goal is to evaluate the effects of modulation of ABCA2 expression levels on the stimulus-induced elaboration of inflammatory cytokines by glial cells. A third goal is to investigate ABCA2 as a potential anti-inflammatory therapeutic target through inhibition of its putative PDZ ligand-binding domain. PDZ domains are structural modules for protein complex assembly and ligand binding to PDZ domains have been identified in the c-terminus of several ABC transporters, including ABCA1 and ABCC2. These PDZ ligand-binding domains and their interacting proteins have been demonstrated to be required for correct intracellular sorting of these transporters. The ABCA2 c-terminus has a "XXC" putative PDZ ligand motif. We will seek to target ABCA2 function by introducing peptides into cells that correspond to the putative PDZ ligand-binding domain. We expect that by competitive peptide inhibition of its binding to its PDZ domain-containing protein partners, ABCA2 function may be abrogated. We will also seek to identify in vivo protein-protein interaction binding partners that may interact through the putative PDZ ligand-binding domain.

Methods: For Specific Aim 1, the BV-2 murine microglial and the CCF-STTG1 human astrocytic cell lines will be selected as model systems. Both lines have been well characterized in the literature. We will generate stable cell lines that express the ABCA2 transporter under tetracycline-inducible control and examine the effect on cholesterol uptake and metabolism. We will measure the expression of the LDLR and perform LDL binding and uptake studies with radiolabeled ligands. We will measure esterification of LDL-derived cholesterol in these cells following thin layer chromatography of radiolabeled metabolic end products. We will measure the trafficking of LDL-derived free cholesterol (LDL-FC) to the plasma membrane by cholesterol oxidase assays. We will measure the effect of ABCA2 expression on cholesterol efflux as well as on the secretion of apolipoprotein E by ELISA and Western blot. To examine the role of ABCA2 expression on modulation of the inflammatory response, we will measure the effects of administration of inflammatory cytokines, IL-1?, IL-1?, TNF?, TGF-1?, and IL-6 on ABCA2 mRNA and protein expression by real-time PCR and Western blot. In order to determine if ABCA2 expression is responsive to exposure to inflammatory stimuli, we will treat glial cells with the amyloid protein A? fragment or the bacterial endotoxin lipopolysaccharide (LPS). To determine if expression of ABCA2 modulates the production of inflammatory cytokines produced in response to A? or LPS we will measure the production and secretion of IL-1?, IL-1?, TNF?, TGF-1?, and IL-6 by Western blot and ELISA/EIA. Because cells import the essential ?-6 polyunsaturated fatty acid, arachidonic acid as a component of LDL, we will measure the delivery of arachidonic acid in these cells. Since arachidonic acid is substrate for the cyclooxygenase-2 enzyme for the production of the prostaglandin PGE2, we will measure its secretion into tissue culture supernatants by ELISA. We will identify the class of prostatoid receptors (EP1-EP4) engaged by PGE2 and the signaling pathways (protein kinase A or protein kinase C) activated by the use of selective inhibitors. Because of the close connection between cholesterol and sphingolipid metabolism, we will investigate the consequences of ABCA2 expression on sphingolipid signaling pathways. We will measure sphingomyelinase activation and production of the 2nd messenger ceramide. Since prostaglandin E2 and ceramide activate downstream signaling pathways to exert their inflammatory effects, we will investigate the pathways of MAPK/ERK, JNK, and p38 MAPK activation and the activation of transcription factors, including, the early growth response genes, c-fos, c-jun (AP1), Egr-1, and NF-kB. We will evaluate the effects ABCA2 expression on proliferation of glial cells by MTT assay. For Specific Aim 2, we will generate stable glial cell lines that express small inhibitory RNA (shRNA) under tetracycline-inducible control to modulate the down-regulation of ABCA2 expression. We perform similar studies to those described above and attempt to show a correlation with the level of expression of ABCA2 and these effects to establish its role as a physiological regulator of these processes. For Specific Aim 3, we will identify protein binding in vivo to the putative PDZ ligand-binding motif in the ABCA2 c-terminus. We will perform yeast 2-hybrid and immunoprecipitation experiments to identify ABCA2 protein-binding partners and their role in ABCA2 function. We will generate mutant forms of the ABCA2 protein that contain single amino acid substitutions or deletions of the c-terminal region of the protein containing the ABCA2-specific "XXC" putative PDZ ligand-binding motif and measure the effects of expression of the mutant forms of the protein in cells. We will generate peptide competitor fragments of the putative PDZ ligand-binding domain and introduce them into glial cells to evaluate the effects on ABCA2 function in modulating the inflammatory response.

Results: As a consequence of the experiments outlined above we expect to provide evidence that ABCA2 is implicated in the modulation of the inflammatory response in glial cells and that it may be considered as an experimental therapeutic target to reduce the consequences of chronic inflammation on neurodegeneration associated to Alzheimer's disease.