Advertisement

Molecular Biology Reports

, Volume 41, Issue 10, pp 6365–6376 | Cite as

The alternative splicing of the apolipoprotein E gene is unperturbed in the brains of Alzheimer’s disease patients

  • James D. Mills
  • Pamela J. Sheahan
  • Donna Lai
  • Jillian J. Kril
  • Michael Janitz
  • Greg T. Sutherland
Article

Abstract

The prevalence of Alzheimer’s disease (AD) is increasing rapidly worldwide due to an ageing population and a lack of disease modifying therapeutics. In monogenic forms of AD mutations lead to the accumulation of neurotoxic peptides called beta-amyloid. Beta-amyloid accumulation is also postulated to precipitate sporadic AD although the pathogenesis of this common form remains largely unknown. The two leading risk factors for sporadic AD are ageing and the possession of the APOE epsilon 4 allele. Changes in APOE expression that are independent of the epsilon genotype have also been described in the AD brain including a recent RNA-Seq analysis that showed upregulation of a rare alternative splice isoform (APOE-005). To replicate these RNA-Seq findings we used quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) to compare APOE-005 and total APOE expression in the superior temporal gyrus of 14 AD cases and 16 neurologically normal controls. In AD, this area shows prominent beta-amyloid deposition but few neurofibrillary tangles and only moderate neuronal loss. As poorer RNA quality among the AD cases was a likely confounder in this study, the analysis was repeated in a RIN-matched sub-cohort of 17 individuals. Contrary to the original RNA-Seq study, we found no difference in total APOE, APOE-005 or the common isoform, APOE-001, between AD cases and controls. Our findings are consistent with ApoE acting largely at the protein level to increase the risk for sporadic AD.

Keywords

Alzheimer’s disease Autopsy brain tissue RNA integrity Apolipoprotein E Alternative splicing RT-qPCR 

Abbreviations

Amyloid-beta

ACTB

Beta-actin

AD

Alzheimer’s disease

ANOVA

Analysis of variance

APOE

Apolipoprotein E

APP

Amyloid precursor protein

AT8

A phospho-tau antibody

BACE1

Beta-site APP-cleaving enzyme 1

BLAST

Basic Local Alignment Search Tool

cDNA

Copy DNA

Cq

Quantification cycle

CLU

Clusterin

DAB

3,3′-Diaminobenzidine

DNA

Deoxyribonucleic acid

FFPE

Formalin-fixed paraffin-embedded

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

GFAP

Glial fibrillary acidic protein

GOI

Gene of interest

HMBS

Hydroxymethylbilane synthase

HPRT1

Hypoxanthine phosphoribosyltransferase 1

mRNA

Messenger RNA

HRP

Horseradish peroxidase

MAPT

Microtubule associated protein tau

NCBI

National Center for Biotechnology Information

NFT

Neurofibrillary tangle

NSWBB

New South Wales Brain Banks

PMI

Post mortem interval

PSEN1

Presenilin-1

RG

Reference gene

RNA

Ribonucleic acid

RNA-Seq

Next generation sequencing of the transcriptome

RT-qPCR

Quantitative reverse transcriptase polymerase chain reaction

SDHA

Succinate dehydrogenase complex, subunit A

STG

Superior temporal gyrus

SNP

Single nucleotide polymorphism

TSS

Transcriptional start site

UBC

Ubiquitin C

Notes

Acknowledgments

The authors would like to thank the donors and their families for their kind gift that has allowed this research to be undertaken. A special thanks to A/Prof David Sullivan and Terrance Foo from Department of Clinical Biochemistry, Royal Prince Alfred Hospital, Sydney, for the APOE genotyping. We also thank the New South Wales Brain Bank Network for providing tissue samples and assistance with clinical and pathological data. The Network is made up of the Sydney Brain Bank (SBB) and NSW Tissue Resource Centre (NSW TRC). The SBB is supported by Neuroscience Research Australia, the University of New South Wales, the National Health and Medical Research Council (NHMRC) and the Australian Brain Bank Network. NSW TRC is supported by the University of Sydney, NHMRC (#605210), the Schizophrenia Research Institute and the National Institutes of Alcoholism and Alcohol Abuse (NIAAA; R24 AA012725).

Supplementary material

11033_2014_3516_MOESM1_ESM.xls (63 kb)
Supplementary Table S1. This table contains all demographic, clinical, pathological, RNA quality and RT-qRCR raw and normalized data (GFAP as the reference gene) for the total cohort (n = 29)
11033_2014_3516_MOESM2_ESM.xls (45 kb)
Supplementary Table S2. This table contains all demographic, clinical, pathological, RNA quality and RT-qRCR raw and normalized data (GFAP as the reference gene) in a RIN-matched sub-cohort of 17 AD cases and controls

References

  1. 1.
    Halliday GM, Double KL, Macdonald V, Kril JJ (2003) Identifying severely atrophic cortical subregions in Alzheimer’s disease. Neurobiol Aging 24(6):797–806CrossRefPubMedGoogle Scholar
  2. 2.
    Gomez-Isla T, Price JL, McKeel DW Jr, Morris JC, Growdon JH, Hyman BT (1996) Profound loss of layer II entorhinal cortex neurons occurs in very mild Alzheimer’s disease. J Neurosci 16(14):4491–4500PubMedGoogle Scholar
  3. 3.
    Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297(5580):353–356CrossRefPubMedGoogle Scholar
  4. 4.
    McKhann GM, Knopman DS, Chertkow H, Hyman BT, Jack CR Jr, Kawas CH, Klunk WE, Koroshetz WJ, Manly JJ, Mayeux R, Mohs RC, Morris JC, Rossor MN, Scheltens P, Carrillo MC, Thies B, Weintraub S, Phelps CH (2011) The diagnosis of dementia due to Alzheimer’s disease: recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement 7(3):263–269PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Lambert JC, Amouyel P (2011) Genetics of Alzheimer’s disease: new evidences for an old hypothesis? Curr Opin Genet Dev 21(3):295–301CrossRefPubMedGoogle Scholar
  6. 6.
    Sutherland GT, Siebert GA, Kril JJ, Mellick GD (2011) Knowing me, knowing you: can a knowledge of risk factors for Alzheimer’s disease prove useful in understanding the pathogenesis of Parkinson’s disease? J Alzheimers Dis 25(3):395–415PubMedGoogle Scholar
  7. 7.
    Ashford JW (2004) APOE genotype effects on Alzheimer’s disease onset and epidemiology. J Mol Neurosci 23(3):157–165CrossRefPubMedGoogle Scholar
  8. 8.
    Chapman J, Korczyn AD, Karussis DM, Michaelson DM (2001) The effects of APOE genotype on age at onset and progression of neurodegenerative diseases. Neurology 57(8):1482–1485CrossRefPubMedGoogle Scholar
  9. 9.
    Drzezga A, Grimmer T, Henriksen G, Muhlau M, Perneczky R, Miederer I, Praus C, Sorg C, Wohlschlager A, Riemenschneider M, Wester HJ, Foerstl H, Schwaiger M, Kurz A (2009) Effect of APOE genotype on amyloid plaque load and gray matter volume in Alzheimer disease. Neurology 72(17):1487–1494CrossRefPubMedGoogle Scholar
  10. 10.
    Ramanan VK, Risacher SL, Nho K, Kim S, Swaminathan S, Shen L, Foroud TM, Hakonarson H, Huentelman MJ, Aisen PS, Petersen RC, Green RC, Jack CR, Koeppe RA, Jagust WJ, Weiner MW, Saykin AJ (2014) APOE and BCHE as modulators of cerebral amyloid deposition: a florbetapir PET genome-wide association study. Mol Psychiatry 19(3):351–357PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Schmechel DE, Saunders AM, Strittmatter WJ, Crain BJ, Hulette CM, Joo SH, Pericak-Vance MA, Goldgaber D, Roses AD (1993) Increased amyloid beta-peptide deposition in cerebral cortex as a consequence of apolipoprotein E genotype in late-onset Alzheimer disease. Proc Natl Acad Sci USA 90(20):9649–9653PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Christensen DZ, Schneider-Axmann T, Lucassen PJ, Bayer TA, Wirths O (2010) Accumulation of intraneuronal Abeta correlates with ApoE4 genotype. Acta Neuropathol 119(5):555–566PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Sabbagh MN, Malek-Ahmadi M, Dugger BN, Lee K, Sue LI, Serrano G, Walker DG, Davis K, Jacobson SA, Beach TG (2013) The influence of Apolipoprotein E genotype on regional pathology in Alzheimer’s disease. BMC Neurol 13:44PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Liu CC, Kanekiyo T, Xu H, Bu G (2013) Apolipoprotein E and Alzheimer disease: risk, mechanisms and therapy. Nat Rev Neurol 9(2):106–118PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Strittmatter WJ, Weisgraber KH, Huang DY, Dong LM, Salvesen GS, Pericak-Vance M, Schmechel D, Saunders AM, Goldgaber D, Roses AD (1993) Binding of human apolipoprotein E to synthetic amyloid beta peptide: isoform-specific effects and implications for late-onset Alzheimer disease. Proc Natl Acad Sci USA 90(17):8098–8102PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Huang Y, Liu XQ, Wyss-Coray T, Brecht WJ, Sanan DA, Mahley RW (2001) Apolipoprotein E fragments present in Alzheimer’s disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons. Proc Natl Acad Sci USA 98(15):8838–8843PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Bekris LM, Galloway NM, Montine TJ, Schellenberg GD, Yu CE (2010) APOE mRNA and protein expression in postmortem brain are modulated by an extended haplotype structure. Am J Med Genet B 153B(2):409–417Google Scholar
  18. 18.
    Bullido MJ, Artiga MJ, Recuero M, Sastre I, Garcia MA, Aldudo J, Lendon C, Han SW, Morris JC, Frank A, Vazquez J, Goate A, Valdivieso F (1998) A polymorphism in the regulatory region of APOE associated with risk for Alzheimer’s dementia. Nat Genet 18(1):69–71CrossRefPubMedGoogle Scholar
  19. 19.
    Lambert JC, Pasquier F, Cottel D, Frigard B, Amouyel P, Chartier-Harlin MC (1998) A new polymorphism in the APOE promoter associated with risk of developing Alzheimer’s disease. Hum Mol Genet 7(3):533–540CrossRefPubMedGoogle Scholar
  20. 20.
    Wang JC, Kwon JM, Shah P, Morris JC, Goate A (2000) Effect of APOE genotype and promoter polymorphism on risk of Alzheimer’s disease. Neurology 55(11):1644–1649CrossRefPubMedGoogle Scholar
  21. 21.
    Marioni JC, Mason CE, Mane SM, Stephens M, Gilad Y (2008) RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome Res 18(9):1509–1517PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Sutherland GT, Janitz M, Kril JJ (2011) Understanding the pathogenesis of Alzheimer’s disease: will RNA-Seq realize the promise of transcriptomics? J Neurochem 116(6):937–946CrossRefPubMedGoogle Scholar
  23. 23.
    Twine NA, Janitz K, Wilkins MR, Janitz M (2011) Whole transcriptome sequencing reveals gene expression and splicing differences in brain regions affected by Alzheimer’s disease. PLoS ONE 6(1):e16266PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Flicek P, Amode MR, Barrell D, Beal K, Brent S, Carvalho-Silva D, Clapham P, Coates G, Fairley S, Fitzgerald S, Gil L, Gordon L, Hendrix M, Hourlier T, Johnson N, Kahari AK, Keefe D, Keenan S, Kinsella R, Komorowska M, Koscielny G, Kulesha E, Larsson P, Longden I, McLaren W, Muffato M, Overduin B, Pignatelli M, Pritchard B, Riat HS, Ritchie GR, Ruffier M, Schuster M, Sobral D, Tang YA, Taylor K, Trevanion S, Vandrovcova J, White S, Wilson M, Wilder SP, Aken BL, Birney E, Cunningham F, Dunham I, Durbin R, Fernandez-Suarez XM, Harrow J, Herrero J, Hubbard TJ, Parker A, Proctor G, Spudich G, Vogel J, Yates A, Zadissa A, Searle SM (2012) Ensembl 2012. Nucleic Acids Res 40(Database issue):D84–D90PubMedCentralCrossRefPubMedGoogle Scholar
  25. 25.
    NIA-Reagan Institute Working Group (1997) Consensus recommendations for the postmortem diagnosis of Alzheimer’s disease. The National Institute on Aging, and Reagan Institute Working Group on Diagnostic Criteria for the Neuropathological Assessment of Alzheimer’s Disease. Neurobiol Aging 18(4 Suppl):S1–S2Google Scholar
  26. 26.
    Uchihara T, Nakamura A, Yamazaki M, Mori O (2001) Evolution from pretangle neurons to neurofibrillary tangles monitored by thiazine red combined with Gallyas method and double immunofluorescence. Acta Neuropathol 101(6):535–539PubMedGoogle Scholar
  27. 27.
    Garcia-Sierra F, Hauw JJ, Duyckaerts C, Wischik CM, Luna-Munoz J, Mena R (2000) The extent of neurofibrillary pathology in perforant pathway neurons is the key determinant of dementia in the very old. Acta Neuropathol 100(1):29–35CrossRefPubMedGoogle Scholar
  28. 28.
    Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4):611–622CrossRefPubMedGoogle Scholar
  29. 29.
    Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7). doi: 10.1186/gb-2002-3-7-research0034
  30. 30.
    Pattyn F, Speleman F, De Paepe A, Vandesompele J (2003) RTPrimerDB: the real-time PCR primer and probe database. Nucleic Acids Res 31(1):122–123PubMedCentralCrossRefPubMedGoogle Scholar
  31. 31.
    Uchihara T, Nakamura A, Yamazaki M, Mori O, Ikeda K, Tsuchiya K (2001) Different conformation of neuronal tau deposits distinguished by double immunofluorescence with AT8 and thiazin red combined with Gallyas method. Acta Neuropathol 102(5):462–466PubMedGoogle Scholar
  32. 32.
    Barrachina M, Castano E, Ferrer I (2006) TaqMan PCR assay in the control of RNA normalization in human post-mortem brain tissue. Neurochem Int 49(3):276–284CrossRefPubMedGoogle Scholar
  33. 33.
    Li JZ, Vawter MP, Walsh DM, Tomita H, Evans SJ, Choudary PV, Lopez JF, Avelar A, Shokoohi V, Chung T, Mesarwi O, Jones EG, Watson SJ, Akil H, Bunney WE Jr, Myers RM (2004) Systematic changes in gene expression in postmortem human brains associated with tissue pH and terminal medical conditions. Hum Mol Genet 13(6):609–616CrossRefPubMedGoogle Scholar
  34. 34.
    Bai B, Hales CM, Chen PC, Gozal Y, Dammer EB, Fritz JJ, Wang X, Xia Q, Duong DM, Street C, Cantero G, Cheng D, Jones DR, Wu Z, Li Y, Diner I, Heilman CJ, Rees HD, Wu H, Lin L, Szulwach KE, Gearing M, Mufson EJ, Bennett DA, Montine TJ, Seyfried NT, Wingo TS, Sun YE, Jin P, Hanfelt J, Willcock DM, Levey A, Lah JJ (2013) Peng J (2013) U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer’s disease. Proc Natl Acad Sci USA 110(41):16562–16567PubMedCentralCrossRefPubMedGoogle Scholar
  35. 35.
    Atz M, Walsh D, Cartagena P, Li J, Evans S, Choudary P, Overman K, Stein R, Tomita H, Potkin S, Myers R, Watson SJ, Jones EG, Akil H, Bunney WE Jr, Vawter MP (2007) Methodological considerations for gene expression profiling of human brain. J Neurosci Methods 163(2):295–309CrossRefPubMedGoogle Scholar
  36. 36.
    Durrenberger PF, Fernando S, Kashefi SN, Ferrer I, Hauw JJ, Seilhean D, Smith C, Walker R, Al-Sarraj S, Troakes C, Palkovits M, Kasztner M, Huitinga I, Arzberger T, Dexter DT, Kretzschmar H, Reynolds R (2010) Effects of antemortem and postmortem variables on human brain mRNA quality: a BrainNet Europe study. J Neuropathol Exp Neurol 69(1):70–81CrossRefPubMedGoogle Scholar
  37. 37.
    Weis S, Llenos IC, Dulay JR, Elashoff M, Martinez-Murillo F, Miller CL (2007) Quality control for microarray analysis of human brain samples: the impact of postmortem factors, RNA characteristics, and histopathology. J Neurosci Methods 165(2):198–209CrossRefPubMedGoogle Scholar
  38. 38.
    Barton AJ, Pearson RC, Najlerahim A, Harrison PJ (1993) Pre- and postmortem influences on brain RNA. J Neurochem 61(1):1–11CrossRefPubMedGoogle Scholar
  39. 39.
    Yates CM, Butterworth J, Tennant MC, Gordon A (1990) Enzyme activities in relation to pH and lactate in postmortem brain in Alzheimer-type and other dementias. J Neurochem 55(5):1624–1630CrossRefPubMedGoogle Scholar
  40. 40.
    Monoranu CM, Apfelbacher M, Grunblatt E, Puppe B, Alafuzoff I, Ferrer I, Al-Saraj S, Keyvani K, Schmitt A, Falkai P, Schittenhelm J, Halliday G, Kril J, Harper C, McLean C, Riederer P, Roggendorf W (2009) pH measurement as quality control on human post mortem brain tissue: a study of the BrainNet Europe consortium. Neuropathol Appl Neurobiol 35(3):329–337PubMedCentralCrossRefPubMedGoogle Scholar
  41. 41.
    Sutherland GT, Sheedy D (2014) Kril JJ (2013) Using autopsy brain tissue to study alcohol-related brain damage in the genomic age Alcoholism. Alcohol Clin Exp Res 38(1):1–8PubMedCentralCrossRefPubMedGoogle Scholar
  42. 42.
    Tomita H, Vawter MP, Walsh DM, Evans SJ, Choudary PV, Li J, Overman KM, Atz ME, Myers RM, Jones EG, Watson SJ, Akil H, Bunney WE Jr (2004) Effect of agonal and postmortem factors on gene expression profile: quality control in microarray analyses of postmortem human brain. Biol Psychiatry 55(4):346–352PubMedCentralCrossRefPubMedGoogle Scholar
  43. 43.
    Broniscer A, Baker JN, Baker SJ, Chi SN, Geyer JR, Morris EB, Gajjar A (2010) Prospective collection of tissue samples at autopsy in children with diffuse intrinsic pontine glioma. Cancer 116(19):4632–4637PubMedCentralCrossRefPubMedGoogle Scholar
  44. 44.
    Trabzuni D, Ryten M, Walker R, Smith C, Imran S, Ramasamy A, Weale ME, Hardy J (2011) Quality control parameters on a large dataset of regionally dissected human control brains for whole genome expression studies. J Neurochem 119(2):275–282PubMedCentralCrossRefPubMedGoogle Scholar
  45. 45.
    Roth RB, Hevezi P, Lee J, Willhite D, Lechner SM, Foster AC, Zlotnik A (2006) Gene expression analyses reveal molecular relationships among 20 regions of the human CNS. Neurogenetics 7(2):67–80CrossRefPubMedGoogle Scholar
  46. 46.
    Vermeulen J, De Preter K, Lefever S, Nuytens J, De Vloed F, Derveaux S, Hellemans J, Speleman F, Vandesompele J (2011) Measurable impact of RNA quality on gene expression results from quantitative PCR. Nucleic Acids Res 39(9):e63PubMedCentralCrossRefPubMedGoogle Scholar
  47. 47.
    Mexal S, Berger R, Adams CE, Ross RG, Freedman R, Leonard S (2006) Brain pH has a significant impact on human postmortem hippocampal gene expression profiles. Brain Res 1106(1):1–11CrossRefPubMedGoogle Scholar
  48. 48.
    Vawter MP, Tomita H, Meng F, Bolstad B, Li J, Evans S, Choudary P, Atz M, Shao L, Neal C, Walsh DM, Burmeister M, Speed T, Myers R, Jones EG, Watson SJ, Akil H, Bunney WE (2006) Mitochondrial-related gene expression changes are sensitive to agonal-pH state: implications for brain disorders. Mol Psychiatry 11(7):615, 663-679Google Scholar
  49. 49.
    Sutherland GT, Sheedy D, Sheahan PJ, Kaplan W, Kril JJ (2014) Comorbidities, confounders, and the white matter transcriptome in chronic alcoholism. Alcohol Clin Exp Res 38(4):994–1001Google Scholar
  50. 50.
    Coulson DT, Brockbank S, Quinn JG, Murphy S, Ravid R, Irvine GB, Johnston JA (2008) Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue. BMC Mol Biol 9:46PubMedCentralCrossRefPubMedGoogle Scholar
  51. 51.
    Wang Q, Ishikawa T, Michiue T, Zhu BL, Guan DW, Maeda H (2012) Stability of endogenous reference genes in postmortem human brains for normalization of quantitative real-time PCR data: comprehensive evaluation using geNorm, NormFinder, and BestKeeper. Int J Legal Med 126(6):943–952CrossRefPubMedGoogle Scholar
  52. 52.
    Coulson DT, Beyer N, Quinn JG, Brockbank S, Hellemans J, Irvine GB, Ravid R, Johnston JA (2010) BACE1 mRNA expression in Alzheimer’s disease postmortem brain tissue. J Alzheimers Dis 22(4):1111–1122PubMedGoogle Scholar
  53. 53.
    Rhinn H, Fujita R, Qiang L, Cheng R, Lee JH, Abeliovich A (2013) Integrative genomics identifies APOE epsilon4 effectors in Alzheimer’s disease. Nature 500(7460):45–50CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • James D. Mills
    • 1
  • Pamela J. Sheahan
    • 2
  • Donna Lai
    • 4
  • Jillian J. Kril
    • 2
    • 3
  • Michael Janitz
    • 1
  • Greg T. Sutherland
    • 2
  1. 1.School of Biotechnology and Biomolecular SciencesUniversity of New South WalesSydneyAustralia
  2. 2.Discipline of Pathology, Sydney Medical SchoolUniversity of SydneyNSWAustralia
  3. 3.Discipline of Medicine, Sydney Medical SchoolUniversity of SydneySydneyAustralia
  4. 4.Bosch Research InstituteUniversity of SydneySydneyAustralia

Personalised recommendations