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Alzheimer-specific variants in the 3'UTR of Amyloid precursor protein affect microRNA function

Charlotte Delay12, Frédéric Calon13, Paul Mathews4 and Sébastien S Hébert12*

Author Affiliations

1 Centre de recherche du CHUQ (CHUL), Axe Neurosciences, Québec (Qc), Canada

2 Université Laval, Faculté de médecine, Département de psychiatrie et de neurosciences, Québec (Qc), Canada

3 Université Laval, Faculté de pharmacie, Québec (Qc), Canada

4 NYU School of Medicine, Nathan Kline Institute, Orangeburg (NY), USA

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Molecular Neurodegeneration 2011, 6:70  doi:10.1186/1750-1326-6-70

The electronic version of this article is the complete one and can be found online at: http://www.molecularneurodegeneration.com/content/6/1/70


Received:9 May 2011
Accepted:7 October 2011
Published:7 October 2011

© 2011 Delay et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

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.

Results

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.

Conclusions

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.

Keywords:
Amyloid precursor protein; microRNA, single nucleotide polymorphism; Alzheimer's disease; miR-147, miR-20a

Findings

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 [1]. Accumulating evidences also support the notion that increasing APP protein levels directly results in Aβ over-production [2], 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 [7]. 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 webcite), 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].

Increasing evidence supports the hypothesis that genetic variants that either abolish existing miRNA binding sites or create illegitimate miRNA binding sites could contribute significantly to risk for neurodegenerative disorders. For instance, Wang et al. showed that a SNP located in the 3'UTR of the fibroblast growth factor 20 (FGF20) gene confers risk for developing PD, possibly by loss of miR-433 binding [18]. In addition, Rademakers et al. showed that increased binding of miR-659 to the 3'UTR of the progranulin (GRN) gene provides an important risk for TDP43-positive frontotemporal dementia [19]. More recently, Bettens et al. identified a number of AD-specific genetic mutations in the 3'UTRs of APP and BACE1 [20]. We extended these findings and established a detailed list of miRNAs with potential binding sites in or near polymorphisms located in the 3'UTR of human APP (hAPP) (Table 1). These include the APP variants T171C (unknown SNP ID), A454G (unknown SNP ID) and A833C (SNP ID rs3200120). These bioinformatics predictions were performed using logarithms available on-line, such as Microcosm [21], TargetScan [22] and microRNA.org[23]. In this study, we focused on polymorphisms predicted to abrogate (completely or partially) miRNA binding.

Table 1. Polymorphisms located in or near miRNA target sites located in the 3'UTR of hAPP

We initially set out to determine whether candidate miRNAs could regulate APP expression. To this end, a luciferase construct harboring the hAPP full-length (~1100 bp) 3'UTR (Figure 1A) [10] was co-transfected with precursor miRNAs (pre-miRs) for miR-20a, miR-655, miR-147, miR-323-3p, miR-644, miR-203, miR-383, miR-106b, miR-153, miR-17, miR-128, miR-199b-5p, miR-1245 in HEK293 cells (Figure 1B). We used a scrambled miRNA sequence as negative control (SCR). As previously observed, miR-20a and miR-17 could significantly down-regulate luciferase (APP) reporter expression. We could not detect, however, a consistent effect of miR-106b on luciferase expression. Other miRNAs including miR-655, miR-147, miR-323-3p, miR-644 and miR-153 could negatively regulate luciferase expression, which is consistent with the bioinformatics predictions (Table 1). Taken together, these data suggest that miRNAs miR-20a, miR-17, miR-655, miR-147, miR-323-3p, miR-644 and miR-153 could be APP expression regulators. The candidate miRNAs that did not affect the luciferase signal, or increased its expression, were excluded in further analyses.

thumbnailFigure 1. In vitro functional analysis of APP A. Schematic representation (not to scale) of the luciferase reporter construct used in this study. TK; thymidine kinase promoter, AAAA(n); PolyA site B. HEK293T cells were transfected with 50nM pre-miRs (as indicated) as well as a reporter construct containing the 3'UTR of hAPP. The cells were lysed 24h post-transfection and luciferase signal was measured. Signals were normalization for transfection efficiency and graph represents the luciferase signals compared to the scrambled control (SCR). Statistical significance was assessed by Student paired t-test. (* = p < 0.05, ** = p < 0.01, *** = p < 0.001) C. and D. Neuro2A cells (C) and HeLa cells (D) were transfected with 50nM pre-miRNAs (as indicated). The cells were lysed 48h post-transfection and western blotting was performed. Representative (n = 3, in triplicate) western blots are shown. The ratios of the APP/β-Actin signals are presented. Measurements were normalized to the scrambled control (SCR). Statistical significance was assessed by Student's paired t-test. (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).

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 [10]. Except for miR-147, each miRNA seed region was conserved between human and mouse APP 3'UTRs [22]. 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.

We next tested whether the "APP-positive" miRNAs were affected by the APP 3'UTR polymorphisms (Table 1). For these experiments, we focused on AD-specific SNPs, and generated hAPP 3'UTR luciferase constructs with T171C or A454G mutations. Our screens indicated that miR-147, but not miR-644 or miR-323-3p, was significantly affected by the T171C mutation when compared to the wild-type (WT) construct (Figure 2B, upper panel). In a similar set of experiments, we could show that miR-20a, but not miR-153 or miR-17, was affected by the A454G mutation (Figure 2B, lower panel). As expected, miR-147 is a less potent inhibitor of APP expression in the presence of T171C. On the other hand, and surprisingly, miR-20a further decreased the expression of APP in the presence of A454G. For miR-147, the effect may be explained by the fact that T171C is located immediately adjacent to the miR-147 seed region, therefore directly inhibiting miRNA binding (Figure 2C, upper panel). This effect can also be explained as the mutation increased the binding energy (ΔG = -18.9 kCal/mol vs. -16.8 kCal/mol) between miR-147 and the 3'UTR of hAPP, which makes the binding less favorable. Although we observe a net gain of function of miR-20a towards the SNP A454G form compared to the WT 3'UTR, A454G is not predicted to change the binding energy (ΔG = -22.8 kCal/mol) between miR-20a and the 3'UTR of hAPP, and the SNP is also not located within the seed region (Figure 2C, lower panel). Taken together, we identified two miRNAs, that is, miR-147 and miR-20a, affected by AD-specific 3'UTR SNPs.

thumbnailFigure 2. SNPs are located near predicted miRNA binding sites on the APP 3'UTR and affect the function of miR-147 and miR-20a A. Schematic representation (not to scale) of SNP localization and predicted miRNA binding sites in the 3'UTR of hAPP. Red mutations represent the AD-specific SNPs, while the black mutations are not yet tested for their disease specificity. B. HEK293T cells were transfected with 5nM pre-miRs (as indicated) as well as a reporter construct containing the WT, T171C or A454G mutated 3'UTR of hAPP. The cells were lysed 24h post-transfection and luciferase signal was measured. After normalization for transfection efficiency, the signals were compared to the WT. Representative results (n = 3, performed in triplicate) are shown. Statistical significance was assessed by 2-way ANOVA (* = p < 0.05, ** = p < 0.01, *** = p < 0.001). SCR; scrambled, A.U.; Arbitrary Unit. C. Schematic representation of base pair matching between miRNAs and the 3'UTR of hAPP. The seed region of the miRNAs is indicated. The red bases represents the SNPs T171C (upper panel) or A454G (lower panel).

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 [20] 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 [10], 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.

Methods

Cell culture

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.

cDNA constructs

The full-length hAPP 3'UTR luciferase construct was described previously [10]. 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).

Statistics

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, APP; single nucleotide polymorphism, SNP; 3'UTR; 3'untranslated region, nt; nucleotide;

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

CD and SH participated in the design of the study. CD performed the experiments and statistical analysis. PM and FC provided material. CD and SH wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

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.

References

  1. De Strooper B: Proteases and proteolysis in Alzheimer disease: a multifactorial view on the disease process.

    Physiological reviews 2010, 90:465-94. PubMed Abstract | Publisher Full Text OpenURL

  2. Mucke L, Masliah E, Yu GQ, Mallory M, Rockenstein EM, Tatsuno G, Hu K, Kholodenko D, Johnson-Wood K, McConlogue L: High-level neuronal expression of abeta 1-42 in wild-type human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation.

    The Journal of neuroscience: the official journal of the Society for Neuroscience 2000, 20:4050-8. OpenURL

  3. D'Hooge R, Nagels G, Westland CE, Mucke L, De Deyn PP: Spatial learning deficit in mice expressing human 751-amino acid beta-amyloid precursor protein.

    Neuroreport 1996, 7:2807-11. PubMed Abstract | Publisher Full Text OpenURL

  4. Theuns J, Brouwers N, Engelborghs S, Sleegers K, Bogaerts V, Corsmit E, De Pooter T, van Duijn CM, De Deyn PP, Van Broeckhoven C: Promoter mutations that increase amyloid precursor-protein expression are associated with Alzheimer disease.

    American journal of human genetics 2006, 78:936-46. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  5. Podlisny MB, Lee G, Selkoe DJ: Gene dosage of the amyloid beta precursor protein in Alzheimer's disease.

    Science (New York, N.Y.) 1987, 238:669-71. Publisher Full Text OpenURL

  6. Rovelet-Lecrux A, Hannequin D, Raux G, Le Meur N, Laquerrière A, Vital A, Dumanchin C, Feuillette S, Brice A, Vercelletto M, Dubas F, Frebourg T, Campion D: APP locus duplication causes autosomal dominant early-onset Alzheimer disease with cerebral amyloid angiopathy.

    Nature genetics 2006, 38:24-6. PubMed Abstract | Publisher Full Text OpenURL

  7. Hébert SS, De Strooper B: Alterations of the microRNA network cause neurodegenerative disease.

    Trends in neurosciences 2009, 32:199-206. PubMed Abstract | Publisher Full Text OpenURL

  8. Ambros V: The functions of animal microRNAs.

    Nature 2004, 431:350-5. PubMed Abstract | Publisher Full Text OpenURL

  9. Djuranovic S, Nahvi A, Green R: A Parsimonious Model for Gene Regulation by miRNAs.

    Science 2011, 331:550-553. PubMed Abstract | Publisher Full Text OpenURL

  10. Hébert SS, Horré K, Nicolaï L, Bergmans B, Papadopoulou AS, Delacourte A, De Strooper B: MicroRNA regulation of Alzheimer's Amyloid precursor protein expression.

    Neurobiology of disease 2009, 33:422-8. PubMed Abstract | Publisher Full Text OpenURL

  11. Fan X, Liu Y, Jiang J, Ma Z, Wu H, Liu T, Liu M, Li X, Tang H: miR-20a promotes proliferation and invasion by targeting APP in human ovarian cancer cells.

    Acta biochimica et biophysica Sinica 2010, 42:318-24. PubMed Abstract | Publisher Full Text OpenURL

  12. Patel N, Hoang D, Miller N, Ansaloni S, Huang Q, Rogers JT, Lee JC, Saunders AJ: MicroRNAs can regulate human APP levels.

    Molecular neurodegeneration 2008, 3:10. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  13. Vilardo E, Barbato C, Ciotti MT, Cogoni C, Ruberti F: MicroRNA-101 regulates amyloid precursor protein expression in hippocampal neurons.

    The Journal of biological chemistry 2010, 285:18344-18351. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  14. Long JM, Lahiri DK: MicroRNA-101 downregulates Alzheimer's amyloid-β precursor protein levels in human cell cultures and is differentially expressed.

    Biochemical and biophysical research communications 2010. OpenURL

  15. Liu W, Liu C, Zhu J, Shu P, Yin B, Gong Y, Qiang B, Yuan J, Peng X: MicroRNA-16 targets amyloid precursor protein to potentially modulate Alzheimer's-associated pathogenesis in SAMP8 mice.

    Neurobiology of aging 2010. OpenURL

  16. Hébert SS, Horré K, Nicolaï L, Papadopoulou AS, Mandemakers W, Silahtaroglu AN, Kauppinen S, Delacourte A, De Strooper B: Loss of microRNA cluster miR-29a/b-1 in sporadic Alzheimer's disease correlates with increased BACE1/beta-secretase expression.

    Proceedings of the National Academy of Sciences of the United States of America 2008, 105:6415-20. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  17. Nunez-Iglesias J, Liu CC, Morgan TE, Finch CE, Zhou XJ: Joint genome-wide profiling of miRNA and mRNA expression in Alzheimer's disease cortex reveals altered miRNA regulation.

    PloS one 2010, 5:e8898. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  18. Wang G, van Der Walt JM, Mayhew G, Li YJ, Züchner S, Scott WK, Martin ER, Vance JM: Variation in the miRNA-433 binding site of FGF20 confers risk for Parkinson disease by overexpression of alpha-synuclein.

    American journal of human genetics 2008, 82:283-9. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  19. Rademakers R, Eriksen JL, Baker M, Robinson T, Ahmed Z, Lincoln SJ, Finch N, Rutherford NJ, Crook RJ, Josephs KA, Boeve BF, Knopman DS, Petersen RC, Parisi JE, Caselli RJ, Wszolek ZK, Uitti RJ, Feldman H, Hutton ML, Mackenzie IR, Graff-Radford NR, Dickson DW: Common variation in the miR-659 binding-site of GRN is a major risk factor for TDP43-positive frontotemporal dementia.

    Human molecular genetics 2008, 17:3631-42. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  20. Bettens K, Brouwers N, Engelborghs S, Van Miegroet H, De Deyn PP, Theuns J, Sleegers K, Van Broeckhoven C: APP and BACE1 miRNA genetic variability has no major role in risk for Alzheimer disease.

    Human mutation 2009, 30:1207-13. PubMed Abstract | Publisher Full Text OpenURL

  21. Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ: miRBase: tools for microRNA genomics.

    Nucleic acids research 2008, 36:D154-8. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  22. Lewis BP, Burge CB, Bartel DP: Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.

    Cell 2005, 120:15-20. PubMed Abstract | Publisher Full Text OpenURL

  23. Enright AJ, John B, Gaul U, Tuschl T, Sander C, Marks DS: MicroRNA targets in Drosophila.

    Genome biology 2003, 5:R1. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  24. Bimonte H a, Granholm A-CE, Seo H, Isacson O: Spatial memory testing decreases hippocampal amyloid precursor protein in young, but not aged, female rats.

    Neuroscience letters 2002, 328:50-4. PubMed Abstract | Publisher Full Text OpenURL

  25. Nishimura I: Upregulation and antiapoptotic role of endogenous Alzheimer amyloid precursor protein in dorsal root ganglion neurons.

    Experimental Cell Research 2003, 286:241-251. PubMed Abstract | Publisher Full Text OpenURL