TOMM40 RNA Transcription in Alzheimer's Disease Brain and Its Implication in Mitochondrial Dysfunction

Abstract

Increasing evidence suggests that the Translocase of Outer Mitochondria Membrane 40 (TOMM40) gene may contribute to the risk of Alzheimer's disease (AD). Currently, there is no consensus as to whether TOMM40 expression is up- or down-regulated in AD brains, hindering a clear interpretation of TOMM40's role in this disease. The aim of this study was to determine if TOMM40 RNA levels differ between AD and control brains. We applied RT-qPCR to study TOMM40 transcription in human postmortem brain (PMB) and assessed associations of these RNA levels with genetic variants in APOE and TOMM40. We also compared TOMM40 RNA levels with mitochondrial functions in human cell lines. Initially, we found that the human genome carries multiple TOMM40 pseudogenes capable of producing highly homologous RNAs that can obscure precise TOMM40 RNA measurements. To circumvent this obstacle, we developed a novel RNA expression assay targeting the primary transcript of TOMM40. Using this assay, we showed that TOMM40 RNA was upregulated in AD PMB. Additionally, elevated TOMM40 RNA levels were associated with decreases in mitochondrial DNA copy number and mitochondrial membrane potential in oxidative stress-challenged cells. Overall, differential transcription of TOMM40 RNA in the brain is associated with AD and could be an indicator of mitochondrial dysfunction.

Keywords: Alzheimer’s disease; RNA transcription; TOMM40 gene; mitochondrial dysfunction; pseudogene.

Figure 1

Phylogenetic tree evolution and end-point RT-PCR amplification of TOMM40 pseudogenes. (A) Phylogenetic tree structure of TOMM40-related genes and pseudogenes. Numbers above the lines are bootstrap value (or node), and numbers below the lines are substitution rate. (B) Capillary gel electrophoresis images of RT-PCR amplified pseudogenes from total RNA of three human cell lines (HepG2, U-87, and SH-SY5Y). Amplicons for pseudogenes P1, P1b, P2, P3, and P4 are shown. Genomic DNA (lane 1) served as a positive control, TOMM40 cDNA (lane 2) served as a reference for cross-amplification, and RT- RNA (lane 6) served as a negative control.

Figure 2

Map of the TOMM40 gene, RNA transcripts, and RT-PCR assays. (A) Structure and position of the exons, CpG island (CGI), and genetic variants (SNP). (B) Structure of the six mRNA transcripts defined by the Ensembl database. (C) Targets of the RT-PCR assays used in this study.

Figure 3

Comparison between a conventional cDNA assay and primary transcript-targeted cDNA assays. Capillary gel electrophoresis images of the RT-PCR amplified TOMM40 pseudogene amplicons. (A) TOMM40 primer set spans Ex4 and Ex5 that mimics a commercial TaqMan gene expression assay (Thermo Fisher, Waltham, MA, USA, Hs01587378_mH). (B) TOMM40 primer sets targeting primary transcripts of exon 6 to intron 6 (EX6-IVS6) and intron 9 (IVS9). Genomic DNA (lane 1) served as a positive control; no-template control (NTC, lane 2) and RT- RNA (lane 11) served as a negative control.

Figure 4

TOMM40 RNA transcript levels in human PMB tissues by RT-qPCR quantification. Transcription levels of TOMM40 RNAs are plotted as values of ΔC_T (mean of C_T triplicates (target)—mean of ACTB C_T triplicates) and compared between control (Ctrl) (blue) and AD (red) frontal lobes. In this setting, smaller ΔC_T values indicate higher RNA levels. (A) Comparison of the two TOMM40 primary transcript-targeted assays (Ex6-IVS6 vs. IVS9) as the pilot study. (B) Comparison of a commercial TaqMan cDNA assay (Thermo Fisher, assay Hs01587378_mH) and the primary transcript-targeted TaqMan IVS9 assay with expanded samples. Numbers in parentheses denote sample size. The t-test p values are shown where significant differences between Ctrl and AD were detected. Boxplot shows quartiles and median.

Figure 5

Associations of PMB TOMM40 RNA levels with genetic variants. Transcription levels of PMB TOMM40 RNA were measured by the IVS9 assay and compared across six genetic variants. The t-test p values are shown where significant associations with TOMM40 intron 6 poly-T SNP rs10524523 (p < 0.02) and intron 8 SNP rs741780 (p < 0.01) were detected. For rs10524523, the S group includes S/S homozygotes and S/L heterozygotes, and the VL group includes VL/VL homozygotes and VL/L heterozygotes. Numbers in parentheses denote sample size. Boxplot shows quartiles and median.

Figure 6

Comparison of TOMM40 RNA levels with mitochondrial functions in human cell lines. TOMM40 RNA levels were measured by the IVS9 assay and mitochondrial function assays including MtDNA copy number and Mt membrane potential were performed. The fold change of H₂O₂-treated cells was compared to the untreated cells (set as baseline of 1.0). Graph shows the relationship between the three measurements in response to the oxidative stress. Standard deviation error bars are shown.