Epigenetic signature and enhancer activity of the human APOE gene.

The human apolipoprotein E (APOE) gene plays an important role in lipid metabolism. It has three common genetic variants, alleles ε2/ε3/ε4, which translate into three protein isoforms of apoE2, E3 and E4. These isoforms can differentially influence total serum cholesterol levels; therefore, APOE has been linked with cardiovascular disease. Additionally, its ε4 allele is strongly associated with the risk of Alzheimer's disease (AD), whereas the ε2 allele appears to have a modest protective effect for AD. Despite decades of research having illuminated multiple functional differences among the three apoE isoforms, the precise mechanisms through which different APOE alleles modify diseases risk remain incompletely understood. In this study, we examined the genomic structure of APOE in search for properties that may contribute novel biological consequences to the risk of disease. We identify one such element in the ε2/ε3/ε4 allele-carrying 3'-exon of APOE. We show that this exon is imbedded in a well-defined CpG island (CGI) that is highly methylated in the human postmortem brain. We demonstrate that this APOE CGI exhibits transcriptional enhancer/silencer activity. We provide evidence that this APOE CGI differentially modulates expression of genes at the APOE locus in a cell type-, DNA methylation- and ε2/ε3/ε4 allele-specific manner. These findings implicate a novel functional role for a 3'-exon CGI and support a modified mechanism of action for APOE in disease risk, involving not only the protein isoforms but also an epigenetically regulated transcriptional program at the APOE locus driven by the APOE CGI.

MicroRNA in Alzheimer's disease: an exploratory study in brain, cerebrospinal fluid and plasma.

MicroRNA (miRNA) may be potential biomarkers of Alzheimer's disease (AD). The objective of this investigation was to demonstrate that miRNAs in human brain or biofluids are differentially expressed according to disease status, tissue type, neuritic plaque score or Braak stage. Post-mortem brain (PMB) miRNA were profiled using arrays and validated using quantitative RT-PCR (qRT-PCR). Five qRT-PCR-validated miRNAs were measured in an independent sample of PMB, cerebrospinal fluid and plasma from the same subjects. Plasma miR-15a was found to be associated with plaque score in the independent sample. In conclusion, miRNA present in human biofluids may offer utility as biomarkers of AD.

Functional analysis of APOE locus genetic variation implicates regional enhancers in the regulation of both TOMM40 and APOE.

Genetic variation within the apolipoprotein E gene (APOE) locus is associated with late-onset Alzheimer's disease risk and quantitative traits as well as apoE expression in multiple tissues. The aim of this investigation was to explore the influence of APOE locus cis-regulatory element enhancer region genetic variation on regional gene promoter activity. Luciferase reporter constructs containing haplotypes of APOE locus gene promoters; APOE, APOC1 and TOMM40, and regional putative enhancers; TOMM40 intervening sequence (IVS)2-4, TOMM40 IVS6 poly-T, as well as previously described enhancers; multienhancer 1 (ME1), or brain control region (BCR), were evaluated for their effects on luciferase activity in three human cell lines. Results of this investigation demonstrate that in SHSY5Y cells, the APOE promoter is significantly influenced by the TOMM40 IVS2-4 and ME1, and the TOMM40 promoter is significantly influenced by the TOMM40 IVS6 poly-T, ME1 and BCR. In HepG2 cells, theTOMM40 promoter is significantly influenced by all four enhancers, whereas the APOE promoter is not influenced by any of the enhancers. The main novel finding of this investigation was that multiple APOE locus cis-elements influence both APOE and TOMM40 promoter activity according to haplotype and cell type, suggesting that a complex transcriptional regulatory structure modulates regional gene expression.

Tau phosphorylation pathway genes and cerebrospinal fluid tau levels in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the presence in the brain of amyloid plaques, consisting predominately of the amyloid ß peptide (Aß), and neurofibrillary tangles, consisting primarily of tau. Hyper-phosphorylated-tau (p-tau) contributes to neuronal damage, and both p-tau and total-tau (t-tau) levels are elevated in AD cerebrospinal fluid (CSF) compared to cognitively normal controls. Our hypothesis was that increased ratios of CSF phosphorylated-tau levels relative to total-tau levels correlate with regulatory region genetic variation of kinase or phosphatase genes biologically associated with the phosphorylation status of tau. Eighteen SNPs located within 5' and 3' regions of 5 kinase and 4 phosphatase genes, as well as two SNPs within regulatory regions of the MAPT gene were chosen for this analysis. The study sample consisted of 101 AD patients and 169 cognitively normal controls. Rs7768046 in the FYN kinase gene and rs913275 in the PPP2R4 phosphatase gene were both associated with CSF p-tau and t-tau levels in AD. These SNPs were also differentially associated with either CSF t-tau (rs7768046) or CSF p-tau (rs913275) relative to t-tau levels in AD compared to controls. These results suggest that rs7768046 and rs913275 both influence CSF tau levels in an AD-associated manner.

Association of cerebrospinal fluid Aß42 with A2M gene in cognitively normal subjects.

Low cerebrospinal fluid (CSF) Aß(42) levels correlate with increased brain Aß deposition in Alzheimer's disease (AD), which suggests a disruption in the degradation and clearance of Aß from the brain. In addition, APOE ε4 carriers have lower CSF Aß(42) levels than non-carriers. The hypothesis of this investigation was that CSF Aß(42) levels would correlate with regulatory region variation in genes that are biologically associated with degradation or clearance of Aß from the brain. CSF Aß(42) levels were tested for associations with Aß degradation and clearance genes and APOE ε4. Twenty-four SNPs located within the 5' and 3' regions of 12 genes were analyzed. The study sample consisted of 99 AD patients and 168 cognitively normal control subjects. CSF Aß(42) levels were associated with APOE ε4 status in controls but not in AD patients; A2M regulatory region SNPs were also associated with CSF Aß(42) levels in controls but not in AD patients, even after adjusting for APOE ε4. These results suggest that genetic variation within the A2M gene influences CSF Aß(42) levels.

ADAM10 expression and promoter haplotype in Alzheimer's disease.

Alzheimer's disease is confirmed at autopsy according to the accumulation of brain neuritic plaques and neurofibrillary tangles in the brain. Neuritic plaques contain amyloid-ß (Aß) and lower levels of Aß correspond to an increase in ADAM10 α-secretase activity. ADAM10 α-secretase activity produces a soluble amyloid precursor protein (APP) alpha (sAPPα) product and negates the pathological production of Aß. In this investigation, it was hypothesized that genetic variation with the ADAM10 promoter is associated with ADAM10 expression levels as well as cerebrospinal fluid sAPPα levels. Results from this investigation suggest that the ADAM10 rs514049-rs653765 C-A promoter haplotype is associated with: (1) higher CSF sAPPα levels in cognitively normal controls compared with Alzheimer's disease (AD) patients, (2) higher postmortem brain hippocampus, but not cerebellum, ADAM10 protein levels in subjects with low plaque scores compared with those with high plaque scores, and (3) higher promoter activity for promoter-only reporter constructs compared with promoter 3' untranslated region (3'UTR) constructs in the human neuroblastoma SHSY5Y cell line, but not in HepG2 or U118 cell lines. Taken together, these findings suggest that ADAM10 expression is modulated according to a promoter haplotype that is influenced in a brain region- and cell type-specific manner.