Alzheimer-specific variants in the 3'UTR of Amyloid precursor protein affect microRNA function
© Delay et al; licensee BioMed Central Ltd. 2011
Received: 9 May 2011
Accepted: 7 October 2011
Published: 7 October 2011
APP expression misregulation can cause genetic Alzheimer's disease (AD). Recent evidences support the hypothesis that polymorphisms located in microRNA (miRNA) target sites could influence the risk of developing neurodegenerative disorders such as Parkinson's disease (PD) and frontotemporal dementia. Recently, a number of single nucleotide polymorphisms (SNPs) located in the 3'UTR of APP have been found in AD patients with family history of dementia. Because miRNAs have previously been implicated in APP expression regulation, we set out to determine whether these polymorphisms could affect miRNA function and therefore APP levels.
Bioinformatics analysis identified twelve putative miRNA bindings sites located in or near the APP 3'UTR variants T117C, A454G and A833C. Among those candidates, seven miRNAs, including miR-20a, miR-17, miR-147, miR-655, miR-323-3p, miR-644, and miR-153 could regulate APP expression in vitro and under physiological conditions in cells. Using luciferase-based assays, we could show that the T117C variant inhibited miR-147 binding, whereas the A454G variant increased miR-20a binding, consequently having opposite effects on APP expression.
Taken together, our results provide proof-of-principle that APP 3'UTR polymorphisms could affect AD risk through modulation of APP expression regulation, and set the stage for further association studies in genetic and sporadic AD.
KeywordsAmyloid precursor protein microRNA, single nucleotide polymorphism Alzheimer's disease miR-147, miR-20a
AD is the most common form of dementia worldwide. Pathologically, the disease is defined by the intracellular accumulation of aggregated and hyperphosphorylated protein tau and the extracellular deposition of Aβ peptides, derived by proteolytic processing of APP. In genetic AD, mutations in the genes coding for APP, PSEN1 and PSEN2 lead to APP processing dysregulation resulting in Aβ over-production, accumulation and deposition, which ultimately leads to neuronal death . Accumulating evidences also support the notion that increasing APP protein levels directly results in Aβ over-production , and that APP overexpression alone is sufficient to induce neurodegeneration an dementia [3–6].
miRNAs function as negative regulators of gene expression regulation, and play a critical role in neuronal function and survival . These small (~21nt) non-coding RNAs interact with the 3'UTR of their target messenger RNA (mRNA) transcripts by partial sequence complementarity resulting in mRNA destabilization and/or translational inhibition [8, 9]. This function is dependent on the miRNA seed region, comprising nucleotides 2-8 of the mature miRNA sequence. As changes in APP expression is intimately involved in AD development, several groups have now investigated the impact of miRNA modulation on APP expression. These studies identified a number of miRNAs capable of regulating APP expression in vitro and in vivo, including miR-20a, miR-17 (previously referred as miR-17-5p, http://www.mirbase.org), miR-106a, miR-106b, miR-101 and miR-16 [10–15]. Interestingly, miR-101, and miR-106b have been shown to be down-regulated in AD brain, therefore potentially contributing to increased APP expression and Aβ production [16, 17].
Polymorphisms located in or near miRNA target sites located in the 3'UTR of hAPP
Position in 3'UTR
In order to validate these observations in more physiological conditions, we transfected our candidate pre-miRs into mouse neuroblastoma Neuro2A cells, a model previously used to study neuronal APP expression regulation by miRNAs . Except for miR-147, each miRNA seed region was conserved between human and mouse APP 3'UTRs . For this reason, miR-147 was omitted from the mouse cell line experiments. In Neuro2A cells, all pre-miRs tested decreased endogenous APP expression when compared to the scrambled miRNA control (Figure 1C). We also transfected human HeLa cells with our candidate pre-miRs. All but one miRNA (miR-655) decreased endogenous APP expression when compared to the scrambled miRNA control (Figure 1D). Notably, miR-147 could efficiently down-regulate endogenous APP in these cells. Taken together, these data add to the growing list of miRNAs that could regulate endogenous APP expression in cultured cells, including neuronal-like cells. These include miR-147, miR-323-3p, miR-644 and miR-153, in addition to the previously identified miR-20a and miR-17.
In conclusion, we provide evidence for the first time that polymorphisms located in the 3'UTR of hAPP may affect its expression, at least in the experimental conditions tested here. Indeed, we show that two AD-specific 3'UTR variants previously identified by Bettens and colleagues  affect the modulating activity of miR-147 and miR-20a on the expression of APP. SNP T171C decreases the ability of miR-147 to down-regulate APP, theoretically leading to increased APP and Aβ production. On the other hand, SNP A454G increases the effect of miR-20a, suggesting that APP expression is reduced in these patients. Although these data seem to contradict with the main hypothesis that increased APP levels lead to AD, some reports indicate that decreasing the APP levels might have deleterious consequences in the brain [24, 25]. Another possibility is that miR-20a levels (or function) vary depending on brain region or disease state, therefore only locally affecting APP. In line with this hypothesis, our preliminary data suggest that certain "APP-positive" miRNAs are differently expressed between human regions (Delay et al., not shown). Finally, we cannot exclude at this stage of investigation that the second, less functional miR-20a binding site located at position 709-715 in the hAPP 3'UTR , could become more prone to miRNA regulation in the presence of SNP A454G. While follow-up studies are required to evaluate the incidence of these variants in other populations, our results suggest that 3'UTR mutations may contribute to risk for AD development. These studies also set the stage for validation studies regarding APP expression regulation by specific miRNAs in vivo in the brain, and further evaluation of 3'UTR variants in AD-related genes in general.
Human HEK293 and HeLa cells, as well as mouse Neuro2A cells, were cultured in DMEM medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum. One day before transfection, HEK293 cells were plated at 100,000 cells per well in 24-well plates, Neuro2A cells at 192,000 cells per well in 6-well plates, while HeLa cells were plated at a 20% confluence in 6-well plates. Transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturers instructions.
The full-length hAPP 3'UTR luciferase construct was described previously . Mutagenesis was performed by TOPgene technologies (Montreal, Quebec, Canada) and validated by sequencing.
Luciferase assay and protein analysis
Cells were transfected with 5 or 50 nM (see text) pre-miRs (Applied Biosystems, USA), 2.5 ng/cm2 pRL control vector, and 50ng/cm2 pGL3_HSV TK_3'UTR hAPP WT or T171C or A454G plasmids. Twenty-four hours post-transfection, cells were lysed, and luciferase activity was measured according to the manufacturer's instructions (Promega, USA). For western blots, cells were lysed in RIPA buffer [50mM Tris Hcl, 1% NP40, 0.9% NaCl, 0.25% Na-deoxycholate, 1mM EDTA, 1x proteinase inhibitors (Roche, Basel, Switzerland), 1mM PMSF, 1mM Na3VO4 and 1mM NaF], mixed with LDS sample buffer (Invitrogen, Carlsbad, CA, USA) containing 5% beta-mercapto-ethanol and boiled at 95°C for 8 min. Crude protein lysates (10 μg) were immunoblotted with the APP C1.61 (for human APP), the APP C-ter (Sigma Aldrich, St-Louis, MO, USA) (for mouse APP) or β-Actin (Sigma Aldrich, St-Louis, MO, USA) antibodies, and detected using the ECL detection kit (Millipore, Billerica, MA, USA). Quantifications were performed using the Multi Gauge software (FUJIFILM, Minato-ku, Tokyo, Japan).
Statistical significance of western blots and luminescence quantifications were determined using 1-way ANOVA, 2-way ANOVA or Student's paired t-test as indicated in the text. Calculations were made using the GraphPad Prism 5 software.
List of Abbreviations
- Amyloid precursor protein:
- single nucleotide polymorphism:
We would like to thank Claudia Goupil and Joanie Baillargeon for technical expertise. We would also like to thank Johanne Girard and Pascal Smith for helpful discussions. This work was supported by the Alzheimer Society of Canada, including a fellowship for CD.
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