Accumulating evidence suggests that increased expression of the amyloid precursor protein gene (APP) increases Alzheimer's disease (AD) risk. The resulting increase in APP protein levels results in increased Aβ levels, leading to synaptic dysfunction, neurodegeneration and, eventually, cognitive decline.
APP levels can be regulated at the genomic, transcriptional or translational level. At the genomic level, Down's Syndrome (Trisomy 21) patients have three copies of the APP
gene and develop AD symptoms early in life [1
]. Similarly, duplication of the APP
locus, in the absence of a full trisomy 21, also leads to early-onset AD [2
]. Dysregulation of APP
transcription can also increase the risk of AD. Genetic variants in the APP
promoter increase APP
transcription by ~2–3 fold and have been reported to increase AD risk [3
]. Growth factors have been reported to control APP
mRNA half-life [4
]. These growth factors effects are dependent on a 29 bp sequence in the APP
3' UTR [4
]. APP translation is also regulated; for example, IL-1 can induce an increase in APP translation [6
]. IL-1 is a pro-inflammatory cytokine and genetic variants have been linked to increased AD risk [7
]. Taken together, these findings provide strong evidence that increased APP levels increase AD risk.
MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression post-transcriptionally. Complementary binding between miRNAs and sequences within the 3' UTR of target genes results in repression of target gene expression by translational inhibition or mRNA degradation [9
]. Approximately 700 miRNA genes are encoded in the human genome and recent evidence demonstrates that some miRNAs are differentially expressed in AD patients compared to age-matched controls [10
]. These differences in miRNA expression may play an important role in AD pathogenesis. In an attempt to address this possibility, we test the hypothesis that miRNAs can regulate APP levels.
Bioinformatic analysis predicts that the 3' UTR of human APP
contains 28 unique miRNA target sites [11
]. To experimentally confirm that APP levels can be regulated by miRNAs, we chose to initially study miRNA hsa-mir-106a (mir-106a; Figure ) since (i) the putative target site in the APP
3'UTR is 100% complementary to the seed region of the miRNA, (ii) it has a large free energy of seed region binding, and (iii) it is expressed in human brain [13
]. To determine if the putative mir-106a target site in the APP
3'UTR is capable of regulating gene expression, we cloned it into the 3' UTR of firefly luciferase. We co-transfected this reporter into naïve HEK-293 cells along with a mir-106a over-expression vector [14
] and measured luciferase activity (Figure ). We observed a significant ~50% decrease (p < 0.0001) in luciferase activity when the putative mir-106a target site was included in the reporter compared to either a reporter lacking the putative target site or reporter carrying a seed-region mutant of the putative mir-106a target site. To determine if this effect was simply due to over-expressing miRNAs, we repeated the experiment while over-expressing mir-373, a miRNA not predicted to target the APP
3'UTR. We observed no change in luciferase activity. Another miRNA, mir-520c, shares the same seed region target sequence as mir-106a but is not expressed in human brain (Figure ) [13
]. Therefore we tested mir-520c, we observed that mir-520c over-expression significantly decreased luciferase activity when the putative mir-106a target site was included in the reporter compared to either a reporter lacking the putative target site or a reporter carrying a seed-region mutant of the putative mir-106a target site (Figure ). We repeated these experiments in the human neuroblastoma cell line SH-SY5Y and observed similar results (data not shown). To confirm that the miRNAs were being over-expressed, we utilized RT-QPCR to quantify miRNA levels and observed significant increases in both mir106a and mir-520c (Table ) levels (p < 0.0001).
Figure 1 mir-106a target sequence regulates reporter gene expression. (A) Predicted mir-106a and mir-520c target sites in the 3'UTR of APP. (B) Over-expression of mir-106a or mir-520c, but not mir-373, significantly reduced luciferase expression (p = 0.0006) controlled (more ...)
Having demonstrated that over-expression of mir-106a or mir-520c was capable of repressing reporter gene expression via interaction with its putative target site, we investigated whether over-expression of these miRNAs could decrease endogenous APP levels in human cell lines. We transfected naïve HEK-293 with mir-106a and mir-125b over-expression vectors and then performed quantitative Western blot analysis to determine APP steady state levels. We utilized mir-125b as a negative control since it is not predicted to target APP but has increased expression in AD brain [10
]. We observed that mir-106a over-expression significantly decreased APP levels (Figure ). Both APP isoforms expressed in this cell line, APP770
, were significantly and similarly affected. Mir-106a over-expression reduced APP levels by ~50% (p < 0.01) compared to cells transfected with the empty vector (Figure ). Over-expression of mir-125b had no significant effect on APP levels. Over-expression of mir-520c had a similar effect on APP levels as mir-106a (Figure and ).
Figure 2 mir-106a and mir-520c can regulate APP levels post-transcriptionally. APP770 and APP751 levels are reduced in cells over-expressing (A) mir-106a compared to cells expressing either mir-125b or the empty vector and (B) mir-520c compared to cells expressing (more ...)
Most human miRNAs repress gene expression by inhibiting translation and do not affect target gene mRNA levels [15
]. This seems to be the case in our experimental setting. We utilized RT-QPCR to determine if miRNA over-expression resulted in decreased APP
mRNA levels. Over-expression of mir-106a or mir-520c had no effect on APP
mRNA levels (Figure ). Mir-106a and mir-520c, therefore, appear to inhibit translation of the APP
Our results are the first to experimentally demonstrate that human APP levels can be regulated by miRNAs. In 2004, it was predicted that APP levels could be regulated by miRNAs [12
]; recently it was shown that expression of the C. elegans
orthologue of APP
, is regulated by developmentally-timed miRNAs [17
In human neurons the APP695
isoform is the predominant expressed isoform. All APP isoforms (APP695
) share the same 3' UTR [18
] therefore we expect that the mir-106a mediated regulation of APP levels that we observe in the HEK-293 cell line should also occur in neurons given that mir-106a is expressed in the brain. We do not expect mir-520c mediated APP regulation to occur in neurons since this miRNA is not expressed in brain. It is important to test if miRNA regulation is a normal aspect of APP metabolism in neurons. If so, it will be important to determine whether AD pathogenesis is affected by alterations in miRNA function and/or expression. It is possible that aging- or environment-induced changes in miRNA expression, and/or sequence variation in miRNAs or their targets, contribute to increased APP levels and increased AD risk. Recently, it was demonstrated that expression of the β-secretase BACE can be regulated by miR-29a/b-1 and mir-107; furthermore, increased BACE levels correlated with decreased miR-29a/b-1 and mir-107 levels in AD patients [19
Regardless of the biological roles of miRNA in APP metabolism, therapeutics based on miRNA-induced decrease in APP levels would offer a treatment targeting the underlying pathophysiology of the disease. In the near future, substantial progress will be made in understanding the role of miRNAs in AD pathogenesis and in therapeutic approaches to treating AD.