FISH Analysis of Aging-Associated Aneuploidy in Neurons and Nonneuronal Brain Cells

  • Grasiella A. Andriani
  • Cristina MontagnaEmail author
Part of the Neuromethods book series (NM, volume 131)


Aging is a ubiquitous complex process characterized by tissue degeneration and loss of cellular fitness. Genome instability (GIN) has long been implicated as a main causal factor in aging. The most severe form of genomic instability is whole chromosome instability (W-CIN), a state where dysfunction in chromosome segregation leads to whole chromosomes gains and losses. Aneuploidy is commonly linked to pathological states. It is a hallmark of spontaneous abortions and birth defects and it is observed virtually in every human tumor. There is mounting evidence that W-CIN increases with age, with the underlying hypothesis that some of the age-related loss of fitness phenotypes may be the result of W-CIN. Methodologically, the detection of stochastic W-CIN during the aging process poses unique challenges: aneuploid cells are scattered among diploid cells and, contrary to the cancer genome where aneuploidy is present in the background of massive ploidy changes, the number of aneuploid chromosome per cells is usually low (few per cell). Aging-associated aneuploidy is also largely stochastic or with limited clonal expansion. Therefore analysis at the single-cell level and the examination of a large number of cells is necessary. Here we describe a modification of the standard fluorescent in situ hybridization (FISH) protocol adapted for the detection of low-frequency mosaic aneuploidy in interphase cells isolated from the adult brain or within frozen tissue sections. This approach represents a straightforward method for the single-cell analysis of W-CIN in mammalian cells. It is based on the combination of four probes mapping to two different chromosomes and analysis of interphase cells, highly reducing false positives and enabling studying W-CIN also in post-mitotic tissues.

Key words

FISH (Fluorescent in situ hybridization) Fluorophores Aneuploidy Interphase FISH Genomic instability (GIN) Whole chromosome instability (W-CIN) Aging Brain NeuN+ NeuN− 


  1. 1.
    Täckholm G (1922) Zytologische studien über die gattung rosa. Acta Horti Bergiani 7:97–381Google Scholar
  2. 2.
    Santaguida S, Amon A (2015) Short- and long-term effects of chromosome mis-segregation and aneuploidy. Nat Rev Mol Cell Biol 16(8):473–485. doi: 10.1038/nrm4025 CrossRefPubMedGoogle Scholar
  3. 3.
    Holland AJ, Cleveland DW (2012) Losing balance: the origin and impact of aneuploidy in cancer. EMBO Rep 13(6):501–514. doi: 10.1038/embor.2012.55 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Siegel JJ, Amon A (2012) New insights into the troubles of aneuploidy. Annu Rev Cell Dev Biol 28:189–214. doi: 10.1146/annurev-cellbio-101011-155807 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Bakker B, van den Bos H, Lansdorp PM, Foijer F (2015) How to count chromosomes in a cell: an overview of current and novel technologies. BioEssays 37(5):570–577. doi: 10.1002/bies.201400218 CrossRefPubMedGoogle Scholar
  6. 6.
    Faggioli F, Vijg J, Montagna C (2011) Chromosomal aneuploidy in the aging brain. Mech Ageing Dev 132(8–9):429–436. doi: 10.1016/j.mad.2011.04.008 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Andriani GA, Vijg J, Montagna C (2016) Mechanisms and consequences of aneuploidy and chromosome instability in the aging brain. Mech Ageing Dev. doi: 10.1016/j.mad.2016.03.007
  8. 8.
    Faggioli F, Wang T, Vijg J, Montagna C (2012) Chromosome-specific accumulation of aneuploidy in the aging mouse brain. Hum Mol Genet 21(24):5246–5253. doi: 10.1093/hmg/dds375 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Rehen SK, McConnell MJ, Kaushal D, Kingsbury MA, Yang AH, Chun J (2001) Chromosomal variation in neurons of the developing and adult mammalian nervous system. Proc Natl Acad Sci U S A 98(23):13361–13366. doi: 10.1073/pnas.231487398 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Rehen SK, Yung YC, McCreight MP, Kaushal D, Yang AH, Almeida BS, Kingsbury MA, Cabral KM, McConnell MJ, Anliker B, Fontanoz M, Chun J (2005) Constitutional aneuploidy in the normal human brain. J Neurosci 25(9):2176–2180. doi: 10.1523/JNEUROSCI.4560-04.2005 CrossRefPubMedGoogle Scholar
  11. 11.
    Yurov YB, Iourov IY, Monakhov VV, Soloviev IV, Vostrikov VM, Vorsanova SG (2005) The variation of aneuploidy frequency in the developing and adult human brain revealed by an interphase FISH study. J Histochem Cytochem 53(3):385–390. doi: 10.1369/jhc.4A6430.2005 CrossRefPubMedGoogle Scholar
  12. 12.
    Duncan AW, Hanlon Newell AE, Smith L, Wilson EM, Olson SB, Thayer MJ, Strom SC, Grompe M (2012) Frequent aneuploidy among normal human hepatocytes. Gastroenterology 142(1):25–28. doi: 10.1053/j.gastro.2011.10.029 CrossRefPubMedGoogle Scholar
  13. 13.
    Faggioli F, Vezzoni P, Montagna C (2011) Single-cell analysis of ploidy and centrosomes underscores the peculiarity of normal hepatocytes. PLoS One 6(10):e26080. doi: 10.1371/journal.pone.0026080 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Jacobs PA, Court Brown WM, Doll R (1961) Distribution of human chromosome counts in relation to age. Nature 191:1178–1180CrossRefPubMedGoogle Scholar
  15. 15.
    Jacobs KB, Yeager M, Zhou W, Wacholder S, Wang Z, Rodriguez-Santiago B, Hutchinson A, Deng X, Liu C, Horner MJ, Cullen M, Epstein CG, Burdett L, Dean MC, Chatterjee N, Sampson J, Chung CC, Kovaks J, Gapstur SM, Stevens VL, Teras LT, Gaudet MM, Albanes D, Weinstein SJ, Virtamo J, Taylor PR, Freedman ND, Abnet CC, Goldstein AM, Hu N, Yu K, Yuan JM, Liao L, Ding T, Qiao YL, Gao YT, Koh WP, Xiang YB, Tang ZZ, Fan JH, Aldrich MC, Amos C, Blot WJ, Bock CH, Gillanders EM, Harris CC, Haiman CA, Henderson BE, Kolonel LN, Le Marchand L, McNeill LH, Rybicki BA, Schwartz AG, Signorello LB, Spitz MR, Wiencke JK, Wrensch M, Wu X, Zanetti KA, Ziegler RG, Figueroa JD, Garcia-Closas M, Malats N, Marenne G, Prokunina-Olsson L, Baris D, Schwenn M, Johnson A, Landi MT, Goldin L, Consonni D, Bertazzi PA, Rotunno M, Rajaraman P, Andersson U, Beane Freeman LE, Berg CD, Buring JE, Butler MA, Carreon T, Feychting M, Ahlbom A, Gaziano JM, Giles GG, Hallmans G, Hankinson SE, Hartge P, Henriksson R, Inskip PD, Johansen C, Landgren A, McKean-Cowdin R, Michaud DS, Melin BS, Peters U, Ruder AM, Sesso HD, Severi G, Shu XO, Visvanathan K, White E, Wolk A, Zeleniuch-Jacquotte A, Zheng W, Silverman DT, Kogevinas M, Gonzalez JR, Villa O, Li D, Duell EJ, Risch HA, Olson SH, Kooperberg C, Wolpin BM, Jiao L, Hassan M, Wheeler W, Arslan AA, Bueno-de-Mesquita HB, Fuchs CS, Gallinger S, Gross MD, Holly EA, Klein AP, LaCroix A, Mandelson MT, Petersen G, Boutron-Ruault MC, Bracci PM, Canzian F, Chang K, Cotterchio M, Giovannucci EL, Goggins M, Hoffman Bolton JA, Jenab M, Khaw KT, Krogh V, Kurtz RC, McWilliams RR, Mendelsohn JB, Rabe KG, Riboli E, Tjonneland A, Tobias GS, Trichopoulos D, Elena JW, Yu H, Amundadottir L, Stolzenberg-Solomon RZ, Kraft P, Schumacher F, Stram D, Savage SA, Mirabello L, Andrulis IL, Wunder JS, Patino Garcia A, Sierrasesumaga L, Barkauskas DA, Gorlick RG, Purdue M, Chow WH, Moore LE, Schwartz KL, Davis FG, Hsing AW, Berndt SI, Black A, Wentzensen N, Brinton LA, Lissowska J, Peplonska B, McGlynn KA, Cook MB, Graubard BI, Kratz CP, Greene MH, Erickson RL, Hunter DJ, Thomas G, Hoover RN, Real FX, Fraumeni JF Jr, Caporaso NE, Tucker M, Rothman N, Perez-Jurado LA, Chanock SJ (2012) Detectable clonal mosaicism and its relationship to aging and cancer. Nat Genet 44(6):651–658. doi: 10.1038/ng.2270 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Jones KT (2008) Meiosis in oocytes: predisposition to aneuploidy and its increased incidence with age. Hum Reprod Update 14(2):143–158. doi: 10.1093/humupd/dmm043 CrossRefPubMedGoogle Scholar
  17. 17.
    Thomas P, Fenech M (2008) Chromosome 17 and 21 aneuploidy in buccal cells is increased with ageing and in Alzheimer’s disease. Mutagenesis 23(1):57–65. doi: 10.1093/mutage/gem044 CrossRefPubMedGoogle Scholar
  18. 18.
    Baker DJ, Dawlaty MM, Wijshake T, Jeganathan KB, Malureanu L, van Ree JH, Crespo-Diaz R, Reyes S, Seaburg L, Shapiro V, Behfar A, Terzic A, van de Sluis B, van Deursen JM (2013) Increased expression of BubR1 protects against aneuploidy and cancer and extends healthy lifespan. Nat Cell Biol 15(1):96–102. doi: 10.1038/ncb2643 CrossRefPubMedGoogle Scholar
  19. 19.
    Sunshine AB, Ong GT, Nickerson DP, Carr D, Murakami CJ, Wasko BM, Shemorry A, Merz AJ, Kaeberlein M, Dunham MJ (2016) Aneuploidy shortens replicative lifespan in Saccharomyces cerevisiae. Aging Cell 15(2):317–324. doi: 10.1111/acel.12443 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Baker DJ, Jeganathan KB, Cameron JD, Thompson M, Juneja S, Kopecka A, Kumar R, Jenkins RB, de Groen PC, Roche P, van Deursen JM (2004) BubR1 insufficiency causes early onset of aging-associated phenotypes and infertility in mice. Nat Genet 36(7):744–749. doi: 10.1038/ng1382 CrossRefPubMedGoogle Scholar
  21. 21.
    Baker DJ, Jeganathan KB, Malureanu L, Perez-Terzic C, Terzic A, van Deursen JM (2006) Early aging-associated phenotypes in Bub3/Rae1 haploinsufficient mice. J Cell Biol 172(4):529–540. doi: 10.1083/jcb.200507081 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Matsuyama M, Tanaka H, Inoko A, Goto H, Yonemura S, Kobori K, Hayashi Y, Kondo E, Itohara S, Izawa I, Inagaki M (2013) Defect of mitotic vimentin phosphorylation causes microophthalmia and cataract via aneuploidy and senescence in lens epithelial cells. J Biol Chem 288(50):35626–35635. doi: 10.1074/jbc.M113.514737 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Tanaka H, Goto H, Inoko A, Makihara H, Enomoto A, Horimoto K, Matsuyama M, Kurita K, Izawa I, Inagaki M (2015) Cytokinetic failure-induced Tetraploidy develops into aneuploidy, triggering skin aging in Phosphovimentin-deficient mice. J Biol Chem 290(21):12984–12998. doi: 10.1074/jbc.M114.633891 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Glisky EL (2007) Changes in cognitive function in human aging. In: Riddle DR (ed) Brain aging: models, methods, and mechanisms. Frontiers in Neuroscience, Boca Raton, FLGoogle Scholar
  25. 25.
    Peterson SE, Yang AH, Bushman DM, Westra JW, Yung YC, Barral S, Mutoh T, Rehen SK, Chun J (2012) Aneuploid cells are differentially susceptible to caspase-mediated death during embryonic cerebral cortical development. J Neurosci 32(46):16213–16222. doi: 10.1523/JNEUROSCI.3706-12.2012 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Andriani GA, Almeida VP, Faggioli F, Mauro M, Tsai WL, Santambrogio L, Maslov A, Gadina M, Campisi J, Vijg J, Montagna C (2016) Whole chromosome instability induces senescence and promotes SASP. Sci Rep 6:35218. doi: 10.1038/Srep35218 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Franceschi C, Campisi J (2014) Chronic inflammation (inflammaging) and its potential contribution to age-associated diseases. J Gerontol A: Biol 69:S4–S9. doi: 10.1093/gerona/glu057 CrossRefGoogle Scholar
  28. 28.
    Bhatia-Dey N, Kanherkar RR, Stair SE, Makarev EO, Csoka AB (2016) Cellular senescence as the causal nexus of aging. Front Genet 7:13CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Thompson SL, Compton DA (2010) Proliferation of aneuploid human cells is limited by a p53-dependent mechanism. J Cell Biol 188(3):369–381. doi: 10.1083/jcb.200905057 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Flowers A (2000) Brain tumors in the older person. Cancer Control 7(6):523–538CrossRefPubMedGoogle Scholar
  31. 31.
    Kaushal D, Contos JJA, Treuner K, Yang AH, Kingsbury MA, Rehen SK, McConnell MJ, Okabe M, Barlow C, Chun J (2003) Alteration of gene expression by chromosome loss in the postnatal mouse brain. J Neurosci 23(13):5599–5606PubMedGoogle Scholar
  32. 32.
    Lopez-Sanchez N, Ovejero-Benito MC, Borreguero L, Frade JM (2011) Control of neuronal ploidy during vertebrate development. Results Probl Cell Differ 53:547–563. doi: 10.1007/978-3-642-19065-0_22 CrossRefPubMedGoogle Scholar
  33. 33.
    Darmanis S, Sloan SA, Zhang Y, Enge M, Caneda C, Shuer LM, Gephart MGH, Barres BA, Quake SR (2015) A survey of human brain transcriptome diversity at the single cell level. Proc Natl Acad Sci U S A 112(23):7285–7290. doi: 10.1073/pnas.1507125112 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Rose CR, Kirchhoff F (2015) Glial heterogeneity: the increasing complexity of the brain. e-Neuroforum 6(3):59–62. doi: 10.1007/s13295-015-0012-0 CrossRefGoogle Scholar
  35. 35.
    McCaffrey JB (2015) The brain’s heterogeneous functional landscape. Philos Sci 82(5):1010–1022. doi: 10.1086/683436 CrossRefGoogle Scholar
  36. 36.
    Andriani GA, Faggioli F, Baker D, Dolle ME, Sellers RS, Hebert JM, Van Steeg H, Hoeijmakers J, Vijg J, Montagna C (2016) Whole chromosome aneuploidy in the brain of Bub1bH/H and Ercc1−/Delta7 mice. Hum Mol Genet 25(4):755–765. doi: 10.1093/hmg/ddv612 CrossRefPubMedGoogle Scholar
  37. 37.
    McConnell MJ, Lindberg MR, Brennand KJ, Piper JC, Voet T, Cowing-Zitron C, Shumilina S, Lasken RS, Vermeesch JR, Hall IM, Gage FH (2013) Mosaic copy number variation in human neurons. Science 342(6158):632–637. doi: 10.1126/science.1243472 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Regan CM (1988) Neuronal and glial markers of the central nervous system. Experientia 44(8):695–697. doi: 10.1007/bf01941031 CrossRefPubMedGoogle Scholar
  39. 39.
    Redwine JM, Evans CF (2002) Markers of central nervous system glia and neurons in vivo during normal and pathological conditions. Curr Top Microbiol Immunol 265:119–140PubMedGoogle Scholar
  40. 40.
    Yuan SH, Martin J, Elia J, Flippin J, Paramban RI, Hefferan MP, Vidal JG, Mu Y, Killian RL, Israel MA, Emre N, Marsala S, Marsala M, Gage FH, Goldstein LSB, Carson CT (2011) Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PLoS One 6(3):e17540. doi: 10.1371/journal.pone.0017540 CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Lopez-Sanchez N, Frade JM (2015) Flow cytometric analysis of DNA synthesis and apoptosis in central nervous system using fresh cell nuclei. Methods Mol Biol 1254:33–42. doi: 10.1007/978-1-4939-2152-2_3 CrossRefPubMedGoogle Scholar
  42. 42.
    Slaninova I, Lopez-Sanchez N, Sebrlova K, Vymazal O, Frade JM, Taborska E (2016) Introduction of macarpine as a novel cell-permeant DNA dye for live cell imaging and flow cytometry sorting. Biol Cell 108(1):1–18. doi: 10.1111/boc.201500047 CrossRefPubMedGoogle Scholar
  43. 43.
    Chiu K, Lau WM, Lau HT, So KF, Chang RCC (2007) Micro-dissection of rat brain for RNA or Protein extraction from specific brain region. J Vis Exp 7:269Google Scholar
  44. 44.
    Freshney RI (2010) Cell Separation. In: Culture of animal cells. Wiley, Hoboken, NJ, pp 227–237. doi: 10.1002/9780470649367.ch14 CrossRefGoogle Scholar
  45. 45.
    Matevossian A, Akbarian S (2008) Neuronal nuclei isolation from human postmortem brain tissue. J Vis Exp 20:914Google Scholar
  46. 46.
    Peters SR (2010) Embedding of tissue for frozen section. In: Peters SR (ed) A practical guide to frozen section technique. Springer, New York, NY, pp 37–74. doi: 10.1007/978-1-4419-1234-3_3 CrossRefGoogle Scholar
  47. 47.
    Peters SR (2010) Cutting the frozen section. In: Peters SR (ed) A practical guide to frozen section technique. Springer, New York, NY, pp 75–95. doi: 10.1007/978-1-4419-1234-3_4 CrossRefGoogle Scholar
  48. 48.
    Knouse KA, Wu J, Whittaker CA, Amon A (2014) Single cell sequencing reveals low levels of aneuploidy across mammalian tissues. Proc Natl Acad Sci U S A 111(37):13409–13414. doi: 10.1073/pnas.1415287111 CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    van den Bos H, Spierings DC, Taudt AS, Bakker B, Porubsky D, Falconer E, Novoa C, Halsema N, Kazemier HG, Hoekstra-Wakker K, Guryev V, den Dunnen WF, Foijer F, Tatche MC, Boddeke HW, Lansdorp PM (2016) Single-cell whole genome sequencing reveals no evidence for common aneuploidy in normal and Alzheimer’s disease neurons. Genome Biol 17(1):116. doi: 10.1186/s13059-016-0976-2 CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Cai X, Evrony GD, Lehmann HS, Elhosary PC, Mehta BK, Poduri A, Walsh CA (2014) Single-cell, genome-wide sequencing identifies clonal somatic copy-number variation in the human brain. Cell Rep 8(5):1280–1289. doi: 10.1016/j.celrep.2014.07.043 CrossRefPubMedPubMedCentralGoogle Scholar

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© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of GeneticsAlbert Einstein College of Medicine, Yeshiva UniversityBronxUSA
  2. 2.Department of PathologyAlbert Einstein College of Medicine, Yeshiva UniversityBronxUSA

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