The cis-regulatory effect of an Alzheimer's disease-associated poly-T locus on expression of TOMM40 and apolipoprotein E genes

Abstract

Background: We investigated the genomic region spanning the Translocase of the Outer Mitochondrial Membrane 40-kD (TOMM40) and Apolipoprotein E (APOE) genes, that has been associated with the risk and age of onset of late-onset Alzheimer's disease (LOAD) to determine whether a highly polymorphic, intronic poly-T within this region (rs10524523; hereafter, 523) affects expression of the APOE and TOMM40 genes. Alleles of this locus are classified as S, short; L, long; and VL, very long based on the number of T residues.

Methods: We evaluated differences in APOE messenger RNA (mRNA) and TOMM40 mRNA levels as a function of the 523 genotype in two brain regions from APOE ε3/ε3 white autopsy-confirmed LOAD cases and normal controls. We further investigated the effect of the 523 locus in its native genomic context using a luciferase expression system.

Results: The expression of both genes was significantly increased with disease. Mean expression of APOE and TOMM40 mRNA levels were higher in VL homozygotes compared with S homozygotes in the temporal and occipital cortexes from normal and LOAD cases. Results of a luciferase reporter system were consistent with the human brain mRNA analysis; the 523 VL poly-T resulted in significantly higher expression than the S poly-T. Although the effect of poly-T length on reporter expression was the same in HepG2 hepatoma and SH-SY5Y neuroblastoma cells, the magnitude of the effect was greater in the neuroblastoma than in the hepatoma cells, which implies tissue-specific modulation of the 523 poly-T.

Conclusions: These results suggest that the 523 locus may contribute to LOAD susceptibility by modulating the expression of TOMM40 and/or APOE transcription.

Keywords: Alzheimer’s disease; Apolipoprotein E; Messenger RNA expression; Poly-T polymorphism; TOMM40; Transcription regulation.

Figure 1. Alzheimer's disease age of onset curves by TOMM40-523 genotypes

The figure is adapted from Crenshaw et al.[11] Kaplan-Meyer survival curves, where the Y axis shows the percent survival without cognitive impairment, and the X axis represents age. Data was obtained from the Duke Bryan ADRC cohort N=438 subjects: 106 diagnosed with dementia, 332 cognitively normal. N for each genotype: L,L:23; VL,L:54; S,L:72; S,S:100; S,VL:138; VL,VL:51. TOMM40 genotypes and the corresponding APOE genotypes are indicated on the figure. The red line corresponds to APOE ε4/4; the two green lines correspond to APOE ε3/4, and the three blue lines correspond to APOE ε3/3. The data for individuals who carried an APOE ε2 allele are indicated as points on the appropriate TOMM40 genotype curve; open arrowheads and filled diamonds indicate the age at onset of symptoms in the APOE ε2/4 and APOE ε2/3 groups, respectively.

Figure 2. The effect of disease state on TOMM40 and APOE mRNAs expression levels in human brain tissues from APOE ε3/3 donors

The study cohort consisted of LOAD and control brain tissues from Caucasian donors with APOE ε3/3 genotype. Fold levels of human TOMM40 mRNA (A and B) and APOE mRNA (C and D) in the temporal (A and C) and occipital (B and D) cortexes were assayed by real-time RT-PCR using TaqMan technology and calculated relative the geometric mean of GAPDH- and PPIA mRNAs reference control using the 2^−ΔΔCt method (i.e. results presented are relative to a specific brain RNA sample). The values presented here are means levels± SE adjusted for age, gender, PMI, and source. TC, temporal cortex; OCC, occipital cortex; Normal, clinically and neuropathologically healthy; LOAD, late onset Alzheimer's disease.

Figure 3. The effect of 523 genotypes on TOMM40 mRNA expression levels in human brain tissues from APOE ε3/3 donors

The study cohort consisted of brain tissues from Caucasian donors with APOE ε3/3 genotype. Cases and control subjects were genotyped for 523. Fold levels of human TOMM40 mRNA in the temporal (A and C) and occipital (B and D) cortexes from normal (A and B) and LOAD (C and D) donors were assayed by real-time RT-PCR using TaqMan technology and calculated relative the geometric mean of GAPDH- and PPIA mRNAs reference control using the 2^−ΔΔCt method (i.e. results presented are relative to a specific brain RNA sample). The values presented here are means levels± SE adjusted for age, gender, PMI, source, and Braak&Braak stage. TC, temporal cortex; OCC, occipital cortex; Normal, clinically and neuropathologically healthy; LOAD, late onset Alzheimer's disease.

Figure 4. The effect of 523 genotypes on APOE mRNA expression levels in human brain tissues from APOE ε3/3 donors

The study cohort consisted of brain tissues from Caucasian donors with APOE ε3/3 genotype. Cases and control subjects were genotyped for 523. Fold levels of human APOE mRNA in the temporal (A and C) and occipital (B and D) cortexes from normal (A and B) and LOAD (C and D) donors were assayed by real-time RT-PCR using TaqMan technology and calculated relative the geometric mean of GAPDH- and PPIA- mRNAs reference control using the 2^−ΔΔCt method (i.e. results presented are relative to a specific brain RNA sample). The values presented here are means levels± SE adjusted for age, gender, PMI, source, and Braak&Braak stage. TC, temporal cortex; OCC, occipital cortex; Normal, clinically and neuropathologically healthy; LOAD, late onset Alzheimer's disease.

Figure 5. The Luciferase reporter system for the TOMM40 523 locus

(A) Schematic representation of the luciferase reporter construct map. The constructs include a ~7 kb human region upstream of the APOE gene translational start site and extending 5’ of the 523 locus. The relative position of the 523 locus is marked in vertical line. The green boxes represent TOMM40 exons (7-10), the blue boxes represent APOE exons (1-2), the solid black line represents introns and intergenic region. The 5’ and 3’ indicate the human genes’ orientation. The arrow above indicates the transcription start site and direction for APOE. The translational start site is marked by ATG. The open box indicates the position of the luciferase reporter gene. The fold expression of luciferase activity derived by the constructs harboring different 523 alleles in (B) HepG2, and(C) SH-SY5Y cells. Cells were cotransfected with each of the 2 constructs harboring the different 523 alleles, S (p523S) or VL (p523VL), or pGL-4.10 and the Renilla reference control, pGL4.74. For each construct four experiments were performed each in triplicate. The relative activity with each construct was calculated by dividing the luminescence intensity of the Firefly luciferase by that of the cotransfected Renilla luciferase in each independent aliquot of cells and then averaging the three relative luciferase activities seen in each experiment. The fold expression for each construct, p523S and p523VL, was then determined by dividing the average relative activity of each construct to that of the average obtained with pGL-4.10. The average of the ‘fold expression’ of the four independent experiments performed on separate days was calculated. The data represented here are the ‘Fold Expression’ mean± SE. Tukey-Kramer HSD test comparing the ‘fold expression’ of each of the p523S and p523VL constructs revealed p= 0.0058 and <0.0001, in HepG2 (B) and SH-SY5Y (C) cells, respectively. We investigated the TOMM40-APOE genomic region that has been associated with the risk and age of onset of late-onset Alzheimer's disease (LOAD) to determine the functional effect of a polymorphic, intronic polyT within this region (rs10524523, hereafter 523). Differences in APOE-mRNA and TOMM40-mRNA levels as a function of 523 genotype were evaluated in two brain regions from APOEε3/3 Caucasian autopsy-confirmed LOAD cases and normal controls. The expression of both genes was significantly increased with disease. Mean expression of APOE and TOMM40-mRNA levels were significantly higher in VL-homozygotes compared to S-homozygotes in temporal and occipital cortexes from Normal and LOAD subjects. Results of a luciferase reporter system, in both in HepG2 hepatoma and SH-SY5Y neuroblastoma cells, were consistent with the human brain mRNA analysis: the 523-VL polyT resulted in significantly higher expression than the S-polyT. These results suggest that the 523 locus may contribute to LOAD susceptibility by modulating the expression of TOMM40 and/or APOE transcription.