| Production of the Aβ peptide by the regulated intra-membrane proteolysis of the β-amyloid precursor protein (APP) by the, α, β and γ secretase enzymes is the central biological pathway in Alzheimer's disease (AD). A number of studies have shown that APP over-expression, resulting from either a genomic locus duplication or alteration in the APP regulatory sequences, can lead to development of early-onset Alzheimer's disease (AD). Therefore, understanding the regulation of APP expression could provide valuable insight in to the genetic basis of the disease and illuminate novel therapeutic avenues for AD. Here we test the hypothesis that APP protein levels can be regulated by miRNAs, evolutionarily conserved small non-coding RNA molecules that play an important role in regulating gene expression. Utilizing human cell lines, we demonstrate that brain expressed miRNAs predicted to target APP 3'UTR bind to their putative target sequences in the APP 3'UTR and negatively regulate reporter gene expression. Over-expression of these miRNAs, but not control miRNAs, results in translational repression of APP mRNA and significantly reduces APP protein levels. For all miRNA over-expressed, except one, APP mRNA levels are not altered suggesting that these miRNAs result in translational inhibition while one miRNA does result in decreased APP mRNA levels. We also demonstrate novel expression of microRNA in the brain. We show that mir-324-5p and miR-17-5p are expressed in neurons and microglial of the hippocampus of the human brain and miR-324-5p is also shows microglial expression in the brain white matter. Disruption of miRNA mediated expression regulation might lead to increased APP levels and increased disease risk. To test this hypothesis, we determined genetic variations within the APP 3'UTR of probands from early onset familial Alzheimer's disease families linked to the APP-region on chromosome 21. We identified a rare sequence variant predicted to be located in a putative miRNA target site. Our functional data suggests that the minor allele of this variant disrupts miRNA binding to its target, resulting in potential dysregulation of APP mRNA and protein expression in vitro. Our results demonstrate that human APP levels can be regulated by miRNAs in vitro, and that rare genetic variants within the 3'UTR of APP may alter miRNA mediated regulation of APP mRNA, thereby increasing APP levels and possibly increasing AD risk. |
