NEW YORK (GenomeWeb) – The Alzheimer's disease-associated protein ApoE4 acts as a transcription factor and binds the promoters of genes involved in a range of processes linked to disease pathogenesis, a new study has found.
Investigators led by the Buck Institute for Research on Aging's Rammohan Rao turned to a combination of cell-based studies and chromatin immunoprecipitation experiments, including ChIP-seq, to show that not only does ApoE4 enter the nucleus, it binds DNA there tightly, as they reported in the Journal of Neuroscience. ApoE4 in particular binds the promoter of genes involved in programmed cell death, insulin resistance, aging, and more, the researchers added.
"Even though the link between ApoE4 and Alzheimer's disease was established way back in the early 1990s, the exact mechanism by which ApoE4 causes the disease was not really clear," Rao told GenomeWeb.
"Apolipoprotein [acting] in the blood and then acting as a transcription factor is something nobody thought about," he added.
The apolipoprotein ε 4 allele, or ApoE4, is a key genetic risk factor for Alzheimer's disease development, though it also has roles in longevity, atherosclerotic cardiovascular disease, and inflammation, among other processes. It has previously been linked with reduced clearance of amyloid-β peptides in the brain.
In an earlier paper, Rao and his colleagues reported that ApoE4 — and not the other isoforms of the protein — leads to the reduced expression of the anti-aging gene Sirtuin 1 (SirT1), a direct regulator of the amyloid precursor protein (APP). Through SirT1, they reported that ApoE4 leads to Aβ production.
Now, Rao and his colleagues aimed to determine whether ApoE4 directly interacts with SirT1.
Through a series of experiment involving cells transfected with SirT1 constructs, the researchers found that neither ApoE4 nor ApoE3 directly associates with SirT1. They noted, though, that both affect SirT1 expression at the transcriptional level, though ApoE4 is more effective than ApoE3 at changing SirT1 expression levels.
A chromatin immunoprecipitation assay using an ApoE antibody, followed by PCR based on a SirT1 promoter-specific primer uncovered a band that corresponds to the SirT1 promoter, indicating that both ApoE isoforms bind the promoter.
"Ape4 actually enters the nucleus, binds to DNA, especially binds to the SirT1 promoter region and causes the reduction in SirT1 levels," Rao said. "Then the next question we asked is: does it bind to only SirT1 or it bind to several other genes?"
Using ChIP-seq, he and his colleagues found that ApoE4 interacts with some 1,700 promoter peaks, of which 76 have been previously linked to AD.
Functional annotation of these genes indicates many of them are involved in axon guidance, neuronal signaling and cell death, glucose and insulin regulation, inflammation, and more. These, the researchers noted, are key processes that contribute to Alzheimer's disease pathophysiology.
In particular, ApoE4 binds the promoters of MAP kinase-activating death domain (MADD), activity-dependent neuroprotective protein (ADNP), and copper metabolism gene MURR1domain 6 (COMMD6) to affect their expression
Using real-time qPCR, the researchers confirmed this finding by examining the expression of these genes in cell lines transfected with either ApoE4 or ApoE3, finding that the addition of ApoE4 — but not ApoE3 — led to a decrease in mRNA level of the genes. The researchers also noted a corresponding protein level decrease.
This, they added, suggests that ApoE4 acts as a repressor at the promoter of these three genes as well as of SirT1.
Rao said that this novel function of ApoE4 in a multitude of pathways indicates that complex diseases can't be addressed by focusing on one pathway alone, but instead need to be examined globally. He and his colleague are currently screening compounds to search for a combination of drugs that may block a number of ApoE4-linked pathways at the same time.