Redefining transcriptional regulation of the APOE gene and its association with Alzheimer’s disease

Autoři: Eun-Gyung Lee aff001;  Jessica Tulloch aff001;  Sunny Chen aff001;  Lesley Leong aff001;  Aleen D. Saxton aff001;  Brian Kraemer aff001;  Martin Darvas aff003;  C. Dirk Keene aff003;  Andrew Shutes-David aff001;  Kaitlin Todd aff001;  Steve Millard aff001;  Chang-En Yu aff001
Působiště autorů: Geriatric Research, Education, and Clinical Center, VA Puget Sound Health Care System, Seattle, WA, United States of America aff001;  Department of Medicine, University of Washington, Seattle, WA, United States of America aff002;  Department of Pathology, University of Washington, Seattle, WA, United States of America aff003
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article


The apolipoprotein E gene (APOE) is the strongest genetic risk factor for late-onset Alzheimer’s disease (AD), yet the expression of APOE is not clearly understood. For example, it is unclear whether AD patients have elevated or decreased APOE expression or why the correlation levels of APOE RNA and the ApoE protein differ across studies. Likewise, APOE has a single CpG island (CGI) that overlaps with its 3’-exon, and this CGI’s effect is unknown. We previously reported that the APOE CGI is highly methylated in human postmortem brain (PMB) and that this methylation is altered in AD frontal lobe. In this study, we comprehensively characterized APOE RNA transcripts and correlated levels of RNA expression with DNA methylation levels across the APOE CGI. We discovered the presence of APOE circular RNA (circRNA) and found that circRNA and full-length mRNA each constitute approximately one third of the total APOE RNA, with truncated mRNAs likely constituting some of the missing fraction. All APOE RNA species demonstrated significantly higher expression in AD frontal lobe than in control frontal lobe. Furthermore, we observed a negative correlation between the levels of total APOE RNA and DNA methylation at the APOE CGI in the frontal lobe. When stratified by disease status, this correlation was strengthened in controls but not in AD. Our findings suggest a possible modified mechanism of gene action for APOE in AD that involves not only the protein isoforms but also an epigenetically regulated transcriptional program driven by DNA methylation in the APOE CGI.

Klíčová slova:

Alzheimer's disease – Cerebellum – DNA methylation – Gene expression – Messenger RNA – Polymerase chain reaction – Apolipoprotein genes – Frontal lobe


1. Marais AD. Apolipoprotein E in lipoprotein metabolism, health and cardiovascular disease. Pathology. 2019;51(2):165–76. doi: 10.1016/j.pathol.2018.11.002 30598326.

2. Bennet AM, Di Angelantonio E, Ye Z, Wensley F, Dahlin A, Ahlbom A, et al. Association of apolipoprotein E genotypes with lipid levels and coronary risk. JAMA. 2007;298(11):1300–11. doi: 10.1001/jama.298.11.1300 17878422.

3. Zannis VI, Just PW, Breslow JL. Human apolipoprotein E isoprotein subclasses are genetically determined. Am J HumGenet. 1981;33(1):11–24. 7468588

4. Rall SC Jr., Weisgraber KH, Innerarity TL, Mahley RW. Structural basis for receptor binding heterogeneity of apolipoprotein E from type III hyperlipoproteinemic subjects. Proc Natl Acad Sci U S A. 1982;79(15):4696–700. doi: 10.1073/pnas.79.15.4696 6289314; PubMed Central PMCID: PMC346743.

5. Stengard JH, Zerba KE, Pekkanen J, Ehnholm C, Nissinen A, Sing CF. Apolipoprotein E polymorphism predicts death from coronary heart disease in a longitudinal study of elderly Finnish men. Circulation. 1995;91(2):265–9. doi: 10.1161/01.cir.91.2.265 7805227

6. Kathiresan S, Melander O, Anevski D, Guiducci C, Burtt NP, Roos C, et al. Polymorphisms Associated with Cholesterol and Risk of Cardiovascular Events. New England Journal of Medicine. 2008;358(12):1240–9. doi: 10.1056/NEJMoa0706728 18354102.

7. Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE, Gaskell PC, Small GW, et al. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families. SCIENCE. 1993;261(5123):921–3. doi: 10.1126/science.8346443 8346443

8. Farrer LA, Cupples LA, Haines JL, Hyman B, Kukull WA, Mayeux R, et al. Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease. A meta-analysis. APOE and Alzheimer Disease Meta Analysis Consortium. JAMA: The Journal of the American Medical Association. 1997;278(16):1349–56. 9343467

9. Yu CE, Payami H, Olson JM, Boehnke M, Wijsman EM, Orr HT, et al. The apolipoprotein E/CI/CII gene cluster and late-onset Alzheimer disease. Am J Hum Genet. 1994;54(4):631–42. 8128960

10. Neu SC, Pa J, Kukull W, Beekly D, Kuzma A, Gangadharan P, et al. Apolipoprotein E Genotype and Sex Risk Factors for Alzheimer Disease: A Meta-analysis. JAMA neurology. 2017;74(10):1178–89. doi: 10.1001/jamaneurol.2017.2188 28846757; PubMed Central PMCID: PMC5759346.

11. Meyer MR, Tschanz JT, Norton MC, Welsh-Bohmer KA, Steffens DC, Wyse BW, et al. APOE genotype predicts when—not whether—one is predisposed to develop Alzheimer disease. NatGenet. 1998;19(4):321–2.

12. Alzheimer's association. 2019 AD Facts and Figures. 2019. Available from:

13. Shinohara M, Kanekiyo T, Yang L, Linthicum D, Shinohara M, Fu Y, et al. APOE2 eases cognitive decline during Aging: Clinical and preclinical evaluations. Ann Neurol. 2016. doi: 10.1002/ana.24628 26933942; PubMed Central PMCID: PMC5010530.


15. Liu CC, Kanekiyo T, Xu H, Bu G. Apolipoprotein E and Alzheimer disease: risk, mechanisms and therapy. Nat Rev Neurol. 2013;9(2):106–18. doi: 10.1038/nrneurol.2012.263 23296339; PubMed Central PMCID: PMC3726719.

16. Bu G. Apolipoprotein E and its receptors in Alzheimer's disease: pathways, pathogenesis and therapy. Nat Rev Neurosci. 2009;10(5):333–44. doi: 10.1038/nrn2620 19339974; PubMed Central PMCID: PMC2908393.

17. Mahley RW, Weisgraber KH, Huang Y. Apolipoprotein E: structure determines function, from atherosclerosis to Alzheimer's disease to AIDS. J Lipid Res. 2009;50 Suppl:S183–8. doi: 10.1194/jlr.R800069-JLR200 19106071; PubMed Central PMCID: PMC2674716.

18. Guo L, LaDu MJ, Van Eldik LJ. A dual role for apolipoprotein e in neuroinflammation: anti- and pro-inflammatory activity. J Mol Neurosci. 2004;23(3):205–12. doi: 10.1385/JMN:23:3:205 15181248.

19. Harris FM, Brecht WJ, Xu Q, Mahley RW, Huang Y. Increased tau phosphorylation in apolipoprotein E4 transgenic mice is associated with activation of extracellular signal-regulated kinase: modulation by zinc. J Biol Chem. 2004;279(43):44795–801. doi: 10.1074/jbc.M408127200 15322121.

20. Wahrle SE, Jiang H, Parsadanian M, Legleiter J, Han X, Fryer JD, et al. ABCA1 is required for normal central nervous system ApoE levels and for lipidation of astrocyte-secreted apoE. J Biol Chem. 2004;279(39):40987–93. doi: 10.1074/jbc.M407963200 15269217.

21. Huang Y, Mahley RW. Apolipoprotein E: structure and function in lipid metabolism, neurobiology, and Alzheimer's diseases. Neurobiol Dis. 2014;72 Pt A:3–12. doi: 10.1016/j.nbd.2014.08.025 25173806; PubMed Central PMCID: PMC4253862.

22. Fernandez CG, Hamby ME, McReynolds ML, Ray WJ. The Role of APOE4 in Disrupting the Homeostatic Functions of Astrocytes and Microglia in Aging and Alzheimer's Disease. Front Aging Neurosci. 2019;11:14. doi: 10.3389/fnagi.2019.00014 30804776; PubMed Central PMCID: PMC6378415.

23. Huang Y, Weisgraber KH, Mucke L, Mahley RW. Apolipoprotein E: diversity of cellular origins, structural and biophysical properties, and effects in Alzheimer's disease. J Mol Neurosci. 2004;23(3):189–204. doi: 10.1385/JMN:23:3:189 15181247.

24. Foley P. Lipids in Alzheimer's disease: A century-old story. Biochim Biophys Acta. 2010;1801(8):750–3. doi: 10.1016/j.bbalip.2010.05.004 20471492.

25. Arendt T, Schindler C, Bruckner MK, Eschrich K, Bigl V, Zedlick D, et al. Plastic neuronal remodeling is impaired in patients with Alzheimer's disease carrying apolipoprotein epsilon 4 allele. J Neurosci. 1997;17(2):516–29. doi: 10.1523/JNEUROSCI.17-02-00516.1997 8987775

26. Beffert U, Aumont N, Dea D, Lussier-Cacan S, Davignon J, Poirier J. Apolipoprotein E isoform-specific reduction of extracellular amyloid in neuronal cultures. Brain Res MolBrain Res. 1999;68(1–2):181–5.

27. Bales KR, Liu F, Wu S, Lin S, Koger D, DeLong C, et al. Human APOE isoform-dependent effects on brain beta-amyloid levels in PDAPP transgenic mice. J Neurosci. 2009;29(21):6771–9. doi: 10.1523/JNEUROSCI.0887-09.2009 19474305.

28. Sullivan PM, Han B, Liu F, Mace BE, Ervin JF, Wu S, et al. Reduced levels of human apoE4 protein in an animal model of cognitive impairment. Neurobiol Aging. 2011;32(5):791–801. doi: 10.1016/j.neurobiolaging.2009.05.011 19577821.

29. Gupta VB, Wilson AC, Burnham S, Hone E, Pedrini S, Laws SM, et al. Follow-up plasma apolipoprotein E levels in the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing (AIBL) cohort. Alzheimers Res Ther. 2015;7(1):16. doi: 10.1186/s13195-015-0105-6 25859282; PubMed Central PMCID: PMC4391582.

30. Wolters FJ, Koudstaal PJ, Hofman A, Duijn CM, Ikram MA. Serum apolipoprotein E is associated with long-term risk of Alzheimer's disease: The Rotterdam Study. Neurosci Lett. 2016;617:139–42. doi: 10.1016/j.neulet.2016.02.018 26876448.

31. Sihlbom C, Davidsson P, Sjogren M, Wahlund LO, Nilsson CL. Structural and quantitative comparison of cerebrospinal fluid glycoproteins in Alzheimer's disease patients and healthy individuals. Neurochem Res. 2008;33(7):1332–40. doi: 10.1007/s11064-008-9588-x 18288611.

32. Akram A, Schmeidler J, Katsel P, Hof PR, Haroutunian V. Association of ApoE and LRP mRNA levels with dementia and AD neuropathology. Neurobiol Aging. 2012;33(3):628 e1- e14. doi: 10.1016/j.neurobiolaging.2011.04.010 21676498; PubMed Central PMCID: PMC3234309.

33. Baker-Nigh AT, Mawuenyega KG, Bollinger JG, Ovod V, Kasten T, Franklin EE, et al. Human Central Nervous System (CNS) ApoE Isoforms Are Increased by Age, Differentially Altered by Amyloidosis, and Relative Amounts Reversed in the CNS Compared with Plasma. J Biol Chem. 2016;291(53):27204–18. doi: 10.1074/jbc.M116.721779 27793990; PubMed Central PMCID: PMC5207148.

34. Bertrand P, Poirier J, Oda T, Finch CE, Pasinetti GM. Association of apolipoprotein E genotype with brain levels of apolipoprotein E and apolipoprotein J (clusterin) in Alzheimer disease. Brain Res MolBrain Res. 1995;33(1):174–8.

35. Talwar P, Sinha J, Grover S, Agarwal R, Kushwaha S, Srivastava MV, et al. Meta-analysis of apolipoprotein E levels in the cerebrospinal fluid of patients with Alzheimer's disease. J Neurol Sci. 2016;360:179–87. doi: 10.1016/j.jns.2015.12.004 26723997.

36. Cruchaga C, Kauwe JS, Nowotny P, Bales K, Pickering EH, Mayo K, et al. Cerebrospinal fluid APOE levels: an endophenotype for genetic studies for Alzheimer's disease. Hum Mol Genet. 2012;21(20):4558–71. doi: 10.1093/hmg/dds296 22821396; PubMed Central PMCID: PMC3459471.

37. Martinez-Morillo E, Hansson O, Atagi Y, Bu G, Minthon L, Diamandis EP, et al. Total apolipoprotein E levels and specific isoform composition in cerebrospinal fluid and plasma from Alzheimer's disease patients and controls. Acta Neuropathol. 2014;127(5):633–43. doi: 10.1007/s00401-014-1266-2 24633805.

38. Schmidt C, Becker H, Zerr I. Cerebrospinal fluid apolipoprotein E concentration and severity of cognitive impairment in patients with newly diagnosed Alzheimer's disease. Am J Alzheimers Dis Other Demen. 2014;29(1):54–60. doi: 10.1177/1533317513505133 24277910.

39. Zarow C, Victoroff J. Increased apolipoprotein E mRNA in the hippocampus in Alzheimer disease and in rats after entorhinal cortex lesioning. Exp Neurol. 1998;149(1):79–86. doi: 10.1006/exnr.1997.6709 9454617.

40. Matsui T, Ingelsson M, Fukumoto H, Ramasamy K, Kowa H, Frosch MP, et al. Expression of APP pathway mRNAs and proteins in Alzheimer's disease. Brain Res. 2007;1161:116–23. doi: 10.1016/j.brainres.2007.05.050 17586478.

41. Gottschalk WK, Mihovilovic M, Roses AD, Chiba-Falek O. The Role of Upregulated APOE in Alzheimer's Disease Etiology. J Alzheimers Dis Parkinsonism. 2016;6(1). doi: 10.4172/2161-0460.1000209 27104063; PubMed Central PMCID: PMC4836841.

42. Linnertz C, Anderson L, Gottschalk W, Crenshaw D, Lutz MW, Allen J, et al. The cis-regulatory effect of an Alzheimer's disease-associated poly-T locus on expression of TOMM40 and apolipoprotein E genes. Alzheimers Dement. 2014;10(5):541–51. doi: 10.1016/j.jalz.2013.08.280 24439168; PubMed Central PMCID: PMC4098029.

43. Lambert JC, Perez-Tur J, Dupire MJ, Galasko D, Mann D, Amouyel P, et al. Distortion of allelic expression of apolipoprotein E in Alzheimer's disease. Hum MolGenet. 1997;6(12):2151–4.

44. Growdon WB, Cheung BS, Hyman BT, Rebeck GW. Lack of allelic imbalance in APOE epsilon3/4 brain mRNA expression in Alzheimer's disease. NeurosciLett. 1999;272(2):83–6.

45. Bray NJ, Jehu L, Moskvina V, Buxbaum JD, Dracheva S, Haroutunian V, et al. Allelic expression of APOE in human brain: effects of epsilon status and promoter haplotypes. Hum Mol Genet. 2004;13(22):2885–92. doi: 10.1093/hmg/ddh299 15385439.

46. Yu CE, Cudaback E, Foraker J, Thomson Z, Leong L, Lutz F, et al. Epigenetic signature and enhancer activity of the human APOE gene. Hum Mol Genet. 2013;22(24):5036–47. doi: 10.1093/hmg/ddt354 23892237; PubMed Central PMCID: PMC3836480.

47. Foraker J, Millard SP, Leong L, Thomson Z, Chen S, Keene CD, et al. The APOE Gene is Differentially Methylated in Alzheimer's Disease. J Alzheimers Dis. 2015;48(3):745–55. doi: 10.3233/JAD-143060 26402071.

48. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–8. Epub 2002/02/16. doi: 10.1006/meth.2001.1262 [pii]. 11846609.

49. Holm S. A simple sequentially rejective multiple test procedure. Scandinavian Journal of Statistics 1979;6:65–70.

50. Nakagawa S, Cuthill IC. Effect size, confidence interval and statistical significance: a practical guide for biologists. Biol Rev Camb Philos Soc. 2007;82(4):591–605. doi: 10.1111/j.1469-185X.2007.00027.x 17944619.

51. R Core Team. The R project for statistical computing. 2017. Available from:

52. Schloerke B CJ, Cook D, Briatte F, Marbach M, Thoen E, Elberg A, Larmarange J,. GGally: Extension to 'ggplot2'. R package version 1.4.0. 2018. Available from:

53. Wickham H. tidyverse: Easily Install and Load the 'Tidyverse'. R package version 1.2.1. 2017. Available from:

54. Millard SP. EnvStats: An R Package for Environmental Statistics.: Springer, New York.; 2013.

55. Zerbino DR, Achuthan P, Akanni W, Amode MR, Barrell D, Bhai J, et al. Ensembl 2018. Nucleic Acids Res. 2018;46(D1):D754–D61. doi: 10.1093/nar/gkx1098 29155950; PubMed Central PMCID: PMC5753206.

56. Hendrich B, Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins. Mol Cell Biol. 1998;18(11):6538–47. Epub 1998/10/17. doi: 10.1128/mcb.18.11.6538 9774669; PubMed Central PMCID: PMC109239.

57. Ballestar E, Wolffe AP. Methyl-CpG-binding proteins. Targeting specific gene repression. Eur J Biochem. 2001;268(1):1–6. doi: 10.1046/j.1432-1327.2001.01869.x 11121095.

58. Christman JK. 5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene. 2002;21(35):5483–95. doi: 10.1038/sj.onc.1205699 12154409.

59. Chahrour M, Jung SY, Shaw C, Zhou X, Wong ST, Qin J, et al. MeCP2, a key contributor to neurological disease, activates and represses transcription. Science. 2008;320(5880):1224–9. doi: 10.1126/science.1153252 18511691; PubMed Central PMCID: PMC2443785.

60. Kruusvee V, Lyst MJ, Taylor C, Tarnauskaite Z, Bird AP, Cook AG. Structure of the MeCP2-TBLR1 complex reveals a molecular basis for Rett syndrome and related disorders. Proc Natl Acad Sci U S A. 2017;114(16):E3243–E50. doi: 10.1073/pnas.1700731114 28348241; PubMed Central PMCID: PMC5402415.

61. Seitz A, Gourevitch D, Zhang XM, Clark L, Chen P, Kragol M, et al. Sense and antisense transcripts of the apolipoprotein E gene in normal and ApoE knockout mice, their expression after spinal cord injury and corresponding human transcripts. Hum Mol Genet. 2005;14(18):2661–70. doi: 10.1093/hmg/ddi296 16091415.

62. Salzman J, Chen RE, Olsen MN, Wang PL, Brown PO. Cell-type specific features of circular RNA expression. PLoS Genet. 2013;9(9):e1003777. doi: 10.1371/journal.pgen.1003777 24039610; PubMed Central PMCID: PMC3764148.

63. Rybak-Wolf A, Stottmeister C, Glazar P, Jens M, Pino N, Giusti S, et al. Circular RNAs in the Mammalian Brain Are Highly Abundant, Conserved, and Dynamically Expressed. Mol Cell. 2015;58(5):870–85. doi: 10.1016/j.molcel.2015.03.027 25921068.

64. Jeck WR, Sorrentino JA, Wang K, Slevin MK, Burd CE, Liu J, et al. Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA. 2013;19(2):141–57. doi: 10.1261/rna.035667.112 23249747; PubMed Central PMCID: PMC3543092.

65. Pasman Z, Been MD, Garcia-Blanco MA. Exon circularization in mammalian nuclear extracts. RNA. 1996;2(6):603–10. 8718689; PubMed Central PMCID: PMC1369399.

66. Guo JU, Agarwal V, Guo H, Bartel DP. Expanded identification and characterization of mammalian circular RNAs. Genome Biol. 2014;15(7):409. doi: 10.1186/s13059-014-0409-z 25070500; PubMed Central PMCID: PMC4165365.

67. Zhang XO, Wang HB, Zhang Y, Lu X, Chen LL, Yang L. Complementary sequence-mediated exon circularization. Cell. 2014;159(1):134–47. doi: 10.1016/j.cell.2014.09.001 25242744.

68. Mathews DH. RNAstructure Web Servers 2017. Available from:

Článek vyšel v časopise


2020 Číslo 1
Nejčtenější tento týden