APOE4 allele is a major risk factor for late-onset Alzheimer disease (AD). The mechanism of action of APOE in AD remains unclear. To study the effects of APOE alleles on gene expression in AD, we have analyzed the gene transcription patterns of human hippocampus from APOE3/3, APOE3/4, APOE4/4 AD patients and normal control using Serial Analysis of Gene Expression (SAGE). Using SAGE, we found gene expression patterns in hippocampus of APOE3/4 and APOE4/4 AD patients differ substantially from those of APOE3/3 AD patients. APOE3/4 and APOE4/4 allele expression may activate similar genes or gene pools with associated functions. APOE4 AD alleles activate multiple tumor suppressors, tumor inducers and negative regulator of cell growth or repressors that may lead to increased cell arrest, senescent and apoptosis. In contrast, there is decreased expression of large clusters of genes associated with synaptic plasticity, synaptic vesicle trafficking (metabolism) and axonal/neuronal outgrowth. In addition, reduction of neurotransmitter receptors and Ca++ homeostasis, disruption of multiple signal transduction pathways, and loss of cell protection and notably mitochondrial oxidative phosphorylation/energy metabolism are associated with APOE3/4 and APOE4/4 AD alleles. These findings help define the mechanisms that APOE4 contribute increased risk for AD and identify new candidate genes conferring susceptibility to AD.
Keywords: Serial Analysis of Gene Expression (SAGE); Apolipoprotein E (APOE3/3, APOE3/4, APOE4/4); Alzheimer disease; Hippocampus; apoptosis; signal pathways
Overall design: Human brain samples and pathological assessment
Human brain tissues were collected in the Kathleen Price Bryan Brain Bank, in the Duke University Alzheimer Disease Research Center, and the Center for Human Genetics’ Brain Bank, Duke University Medical Center, following the rapid autopsy protocol (Hulette et al., 1997). The hippocampus was dissected at the time of autopsy and matching 100-200 mg portions of CA 1-4 was removed and used for RNA isolation and expression studies. We chose AD patients of apolipoprotein E4/4 (APOE4/4), APOE3/4, and APOE3/3 genotypes and controls with the APOE 3/3 genotypes judged cognitively and pathologically normal. Clinical information on all patients was collected from clinic and hospital records. The pathological diagnosis of AD was established according to CERAD criteria (Mirra et al., 1991) and the degree of AD pathological changes was staged according to Braak (Braak and Braak, 1991). We analyzed selected AD patients with pathological changes at the Braak and Braak stage IV or V (B&B stage IV or V) and compared them with cognitively and pathologically normal controls (B&B stage I) (Table 1). We chose hippocampal samples with relatively short post-mortem delay.
Construction of human SAGE libraries
Total RNA was isolated from frozen hippocampal samples of AD patients and controls using TRIzol reagent (Invitrogen, Carlsbad, CA) as previously described ( Xu 2006). For SAGE library construction, we used standard protocols as described by Velculescu et al (1995) and Basrai and Hieter (2002) with minor modifications. Briefly, SAGE was performed with 10 ug total RNA isolated from human brain hippocampus samples as outlined above. The cDNA was prepared using the SuperscriptII cDNA synthesis kit (Invitrogen) with gel-purified 5’-biotinylated Oligo(dT)18 (Integrated DNA Technologies, Coralville, IA), according to the manufacturer’s protocol. NlaIII and BsmFI restriction enzymes (New England Biolab, Beverly, MA) were used for tag generation. BsmFI digestion was per¬formed at 37°C for 2.5 h (instead of 65 °C) using 40 units BsmFI in a 300 ul reaction volume with supplied buffer. After a 3-hour concatemerization step, concatemers were heated at 65 °C for 10 minutes, following by 2 minutes on ice to enhance cloning efficiency. Purified concatemers were subsequently cloned in the SphI site of pZero-1 (Invitrogen) and transformed in competent ElectroMax DH10B cells (Invitrogen) using a 0.1 cm cuvette and the Gene Pulser II (BioRad). Individual SAGE library clones were selected and PCR amplified using 96-well format Qiagen Real minipreps, and sequenced with ABI 3700 capillary sequencer using BigDye chemistry.
SAGE tag extract and statistical analysis
SAGE tags (10 bp) were extracted from the PHD files with eSAGE software using a threshold value of PHRED 20 for each base (Margulies and Innis, 2000). The SAGE tags were extracted from individual sequence files from each SAGE library (Table 2). The SAGE tag databases were compared with each other, such as AD vs. control and APOE4/4 AD vs. APOE3/3 AD SAGE tag databases, etc. using eSAGE software (Table 3). The compared SAGE databases were mapped to UniGene Homo Sapiens tag-to-gene mapping set (build 182, /UniGene).
The Chi-square test and Fisher exact test were employed to test the difference in tag counts between two samples as previously described (Man et al., 2000;Hauser et al., 2003).
Functional categorization
We assigned differentially expressed gene transcripts to functional categories by two methods. One method assigned transcripts to broad functional categories defined on the basis of gene function derived from literature searches. The other method used the software tool, EXPRESSION ANALYSIS SYSTEMATIC EXPLORER (EASE; http://david.niaid.nih.gov/david/ease.htm), to assign identified gene transcripts to “GO: molecular function” categories of the GeneOntology Consortium (www.geneontology.org). We used the GO system to quantify over- and under-represented (up- and down-regulation) transcripts relative to total genes within a functional category and compare between different SAGE libraries. Since the GO system does not provide a functional designation for all genes, our functional interpretations relied primarily on the extensive literature search in PubMed and sequence search in NCBI and other HGP databases.
Quantitative real-time PCR
We confirmed selected SAGE data in individual APOE allele-specific ADs and controls by quantitative real-time PCR (qRT-PCR). Hippocampal total RNA (10 ug) was converted to cDNA using the High Capacity cDNA Archive Kit (Applied Biosystems, ABI, Foster City, CA) according to the manufacturer’s protocol. Assays-on DemandTM Gene Expression primer sets for qRT-PCR were purchased from Applied Biosystems (Table 6). The primer sequences can be obtained from the authors upon request. Four housekeeping genes (GAPD, GUSB, HPRT1, APLPO) were used as endogenous controls (or references). Real-time PCR was performed using TaqMan Universal PCR Mater Mix according to TaqMan Gene Expression Assay protocol (ABI). Each reaction was performed in quadruplicates, and non-RT (without reverse transcriptase) and non-RNA template controls were included. The relative amounts of specifically amplified cDNAs in APOE allele-specific ADs compared with APOE3/3 controls were calculated using comparative CT method.
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