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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 20, 2004 as doi:10.1096/fj.03-1490fje. |
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Department of Pharmacology, Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA;
* Active Pass Pharmaceuticals Inc., Vancouver, BC Canada; and
Center for Neurodegenerative Disease Research, Institute on Aging and Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
1Correspondence: Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 173 Ashley Ave., PO Box 250505, Charleston, SC 29425, USA. E-mail: tewk{at}musc.edu
SPECIFIC AIMS
The ATP binding cassette, subfamily A transporter 2 (ABCA2) gene is localized in the endolysosomal compartment of the cell and expressed at high levels in brain tissue. ABCA2 expression has been associated with myelination, rat nervous system development, and response to cholesterol loading. Manipulation of the expression of ABCA2 by transfection or antisense treatments caused a corresponding change in resistance to estramustine. The aim of the present study is to identify gene products that may be altered in the cellular response to forced expression of high levels of ABCA2 and to study the roles of ABCA2 in response to oxidative stress and pathogenesis of Alzheimer’s disease (AD).
PRINCIPAL FINDINGS
1. Transport function and oxidative stress response gene clusters were identified
Amplified differential gene expression (ADGE) microarray was used to profile the gene expression of the ABCA2-transfected cell line. Developed in this laboratory, ADGE microarray magnifies the ratios of differential gene expression and improves detection sensitivity and fidelity while reducing the requirement for biological material and maintaining high throughput. The transfected cell line A16 was derived from the HEK293 cell line by transfection of a 7304 bp Hind III/EcoR1 ABCA2 fragment subcloned into the pcDNA 3.1 vector. The control cell line, C1, was parental HEK293 transfected with the pcDNA3.1 vector. Both cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 50 µg/mL streptomycin, 50 units/mL penicillin, 2 mM glutamine, 10% (v/v) fetal bovine serum, and 0.9 mg/mL G418. A16 and C1 cells were harvested at 
80% confluence and used for RNA isolation with RNeasy Maxi Kit. Total RNA was used for ADGE microarray. Details of ADGE microarray are described on Web sites http://www.fccc.edu/research/ADGEmicroarray/ and http://www.biomedcentral.com/1471-2164/4/28/abstract. Spots from the microarray images were quantified with ImaGene4.1. The Cy3 and Cy5 data were integrated into a dataset and transformed with GeneSight3.0 using the following sequence: local background correction, removal of flagged spots, logarithm of base 2, ratio calculation, and linear regression normalization. The transformed data were exported into Microsoft Excel. M values (log2(Cy5/Cy3)=log2Cy5-log2Cy3) were calculated for each gene. Differentially expressed genes were identified and their functions, where known, were annotated.
Seven genes (4 up-regulated, 3 down-regulated), along with the actin gene, were analyzed to verify the results of ADGE microarray with RT-PCR. Primers for RT-PCR were designed with Software OLIGO 4.0 based on sequences of the genes corresponding to the identified spots on the chip. Total RNA samples of C1 and A16 used for ADGE microarray were reverse-transcribed with Superscript II reverse transcriptase. Serial dilutions were made for templates to ensure amplification in the linear range. cDNA templates of the two samples were normalized to expression of ß-actin. For each gene, specific PCR cycle conditions were selected to optimize the levels of differential expression and quantified using the Kodak 1-D image analysis software program. Gene expression was also confirmed by real-time PCR. cDNA and primers were made and samples were run on the Cepheid Smart Cycler using the Qiagen Sybr Green detection system. Results of RT-PCR were consistent with those detected with microarray for the seven selected genes.
After screening 40,000 genes on three sets of chips, 152 genes were detected with a >3-fold changes (absolute value of M >1.5) in replicate samples of control and ABCA2-transfected cells. Among them, 100 genes were up-regulated and 52 down-regulated. Among 68 annotated genes, 22 were associated with transport functions and 6 with oxidative stress response and/or pathogenesis of AD. The 22 genes from the transport-related group have plausible roles in transport, membrane composition, substrate binding, and metabolism. For example, syntaxin 11 may act as a cofactor for ABCA2 and interact with the OCSYN protein, which is among the 22 genes. CACNA2D2 is a voltage-dependent calcium channel. DOC2, with double C2-like domains, interacts with Ca2+ and phospholipids and may have a role in Ca2+-dependent intracellular vesicle trafficking in cells. Stomatin is a major lipid raft component of platelet 
granules. Lipid rafts have cholesterol and sphingolipid-rich domains also involved in intracellular trafficking. Copine I is a Ca2+-dependent phospholipid binding protein. Expression of fatty acid binding protein 7 was increased while fatty acid binding protein 5 was decreased. Autotaxin is involved in the synthesis of the bioactive phospholipid lysophosphatidic acid. SULT1A3 and SULT1A2 catalyze sulfate conjugation in the metabolism of drugs and hormones, and angiopoietin-like 3 has a role in regulating lipid metabolism.
The six genes linked with oxidative stress response and pathogenesis of AD include seladin-1, vimentin, Slc23a1, APP, low-density lipoprotein receptor-related protein, and calcineurin. Seladin-1 is an oxido-reductase that has been shown to confer resistance to oxidative stress; its expression is down-regulated in affected areas of the brain in AD. Vimentin is an oxidation sensitive protein and is known to be up-regulated in cells exposed to Aß. Slc23a1 is a sodium-dependent vitamin C transporter. APP and low-density lipoprotein, receptor-related protein have been linked with the pathogenesis of AD. Calcineurin is a calcium- and calmodulin-dependent protein phosphatase and has been linked with tau hyperphosphorylation in AD.
2. ABCA2 conferred resistance to oxidative stress
Although the observed changes in transport-related gene expression after ABCA2 transfection were not unexpected, the apparent connection between ABCA2 and oxidative stress/AD was interesting enough to merit further study. Responses of the A16 and C1 cells to an oxidative stress inducer, AAPH (2,2'-azobis-(2-amidinopropane), were analyzed using the SRB colorimetric cytotoxicity assay. The results showed that ABCA2-transfected cells were more resistant than mock transfected to oxidative stress induced by AAPH (Fig. 1
).
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3. ABCA2 was colocalized with APP and Aß
Localization of ABCA2, APP, and Aß in human BE(2)-M17 neuroblastoma cells was analyzed by indirect immunofluorescence and confocal microscopy. BE(2)-M17 neuroblastoma cells were maintained in supplemented 1:1 modified Eagle’s medium /Hams’ F12 at 37°C, 5% CO2. Cells were prepared for immunofluorescence by standard methods. Primary antibodies used were a polyclonal rabbit anti-ABCA2 and monoclonal antibodies against APP and Aß at dilutions of 1:100. Secondary antibodies were species specific for the primary antibodies and were conjugated with rhodamine red X (ABCA2) or Cy2. Immunofluorescently stained cells were imaged using a BioRad MRC-600 laser scanning confocal microscope (LSCM). Using the two photomultiplier settings on the LSCM, dual acquisition of Cy2 and rhodamine red X was accomplished. Optical sections were acquired with 60x and 100x objectives using a 0.5 µm step size. Immunofluorescence results showed that ABCA2 was colocalized with Aß and APP in distinct punctate vesicles endosome/lysosome in origin (Fig. 2
).
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4. Overexpression of ABCA2 increased intracellular protein levels of APP and media levels of Aß
Levels of APP and Aß were measured in ABCA2-transfected cells using Western blot techniques. 293 EBNA cells (Invitrogen) stably transfected with wild-type amyloid precursor protein-695 (WT6 cells) were cultured in supplemented DMEM. Cells were transfected with either ABCA2 in pCEP4, the pCEP-ßGal empty vector control, or an ABC transporter of approximately the size of ABCA2 cloned by Active Pass Pharmaceuticals and subcloned in the pCEP4 vector (serving as an ABC control).
After the 48 h transient transfection with ABC Cntl and ABCA2, double-transfected cells were washed once with warm PBS (37°C), then exposed to DMEM supplemented with sodium pyruvate (1 mM) for 4 h. Cells were washed once with PBS, harvested in 100 µL of ice-cold lysis buffer containing 20 mM MOPS (pH 7.2), 5 mM EDTA, 0.01% Nonidet P-40, 75 mM ß-glycerol phosphate, and a cocktail of protease inhibitors (Boehringer-Mannheim), and sonicated on ice for 8 s using a probe sonicator. Cellular APP levels were quantitated by 10% Tris-glycine SDS-PAGE Western blot analysis using an anti-APP N-terminal antibody (22C11, Boehringer-Mannheim). Immunoreactive bands were visualized using ECL detection (Amersham) and quantified with standard densitometry.
To examine Aß levels, the harvested cells were subjected to trichloroacetic acid precipitation. The pellet was resuspended in Laemmli buffer and the normalized levels of protein were subjected to electrophoresis. Total Aß was measured using monoclonal antibody 6E10 (Senetek Research). Bands were visualized using ECL detection and analyzed by standard densitometric techniques.
Cellular protein levels of APP increased with the introduction of ABCA2, but not with the ß-gal or ABC control, in WT6 cells. Differences between ABCA2 and both the control vector and ABC control were significant with Tukey’s post hoc test at P< 0.05. However, the difference between ABC Cntl and the control vector was not statistically significant. Introduction of ABCA2 in WT6 cells raised the media levels of Aß significantly at P < 0.05 compared with the control vector and ABC Cntl. However, introduction of ABC Cntl did not change the Aß levels significantly. Therefore, the changes in protein expression level of APP and secretion of Aß were associated specifically with ABCA2.
5. ABCA2 levels were highest in temporal and frontal regions of the Alzheimer brain
To determine relative levels of ABCA2 in different brain regions, autopsy samples of human brain tissue from normal, Alzheimer’s, and schizophrenia samples were analyzed by SDS-PAGE immunoblot. Membrane protein preparations were made from the brain samples and the preps were separated by SDS-PAGE. SDS gels were transferred to nitrocellulose membranes, probed with a polyclonal anti-ABCA2 antibody and appropriate secondary antibody, and bands were detected using the ECL system. Membranes were also probed with an anti-actin primary and appropriate secondary antibody for quantitation/standardization. Images of the blots were scanned and densitometry measured using the Kodak 1-D image analysis software program. Quantitation showed that ABCA2 levels were highest in temporal and frontal regions of the AD brain, with lower but significant levels in parietal, occipital, and cerebellar regions. Similar ABCA2 protein expression patterns were observed in normal and schizophrenia samples.
CONCLUSIONS AND SIGNIFICANCE
Two identifiable cluster patterns emerged in the expression profile of the ABCA2-transfected HEK 293 cell line. The altered expression of transporter-related gene products support the hypothesis that an imbalance caused by the enhanced levels of ABCA2 may cause compensatory adaptations in the expression of these related gene products. Equally intriguing was the cluster of genes more directly related to oxidative stress and AD. A number of independent lines of enquiry point to a possible cause/effect relationship between ABCA2 expression and the etiology of AD (Fig. 3
): 1) we observe by gene expression profiling of ABCA2 transfectants that some accompanying changes involve genes linked with the disease, either through transport functions or in pathways dealing with reactive oxygen species; 2) colocalization of ABCA2 with Aß and APP in the endolysosomal compartment occurs; 3) overexpression of ABCA2 causes increased protein levels of APP and Aß; 4) Toxicity produced through oxidation of lipids or proteins is abrogated by enhanced ABCA2 expression; 5) ABCA2 expression is high in frontal and temporal regions of the brain, areas associated with Alzheimer's pathology.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1490fje; doi: 10.1096/fj.03-1490fje
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