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Pharmaceutical Research

, Volume 24, Issue 5, pp 859–867 | Cite as

The Origin of Deoxynucleosides in Brain: Implications for the Study of Neurogenesis and Stem Cell Therapy

  • Reynold Spector
  • Conrad E. Johanson
Research Paper

Abstract

Abstract

Detection of DNA synthesis in brain employing (3H)thymidine ((3H)dT) or bromo deoxyuridine (BrdU) is widely used as a measure of the “birth” of cells in brain development, adult neurogenesis and neuronal stem cell replacement strategies. However, recent studies have raised serious questions about whether this methodology adequately measures the “birth” of cells in brain either quantitatively or in an interpretable way in comparative studies, or in stem cell investigations. To place these questions in perspective, we review deoxynucleoside synthesis and pharmacokinetics focusing on the barriers interfacing the blood-brain (cerebral capillaries) and blood-cerebrospinal fluid (choroid plexus), and the mechanisms, molecular biology and location of the deoxynucleoside transport systems in the central nervous system. Brain interstitial fluid and CSF nucleoside homeostasis depend upon the activity of concentrative nucleoside transporters (CNT) on the ‘central side’ of the barrier cells and equilibrative nucleoside transporters (ENT) on their ‘plasma side.’ With this information about nucleoside transporters, blood/CSF concentrations and metabolic pathways, we discuss the assumptions and weaknesses of using (3H)dT or BrdU methodologies alone for studying DNA synthesis in brain in the context of neurogenesis and potential stem cell therapy. We conclude that the use of (3H)dT and/or BrdU methodologies can be useful if their limitations are recognized and they are used in conjunction with independent methods.

Key words

brain DNA repair brain ribonucleosides and deoxyribonucleosides cerebral microvessels cerebrospinal fluid homeostasis choroid plexus epithelium CNT2 CNT3 ENT1 ENT2 nucleoside pharmacokinetics thymidine kinase thymidylate synthetase 

Notes

Acknowledgements

The authors thank Michiko Spector for her aid in the preparation of the manuscript and Julie Johanson for assistance with graphics.

References

  1. 1.
    G. Kempermann. Adult Neurogenesis. Oxford, New York, 2006.Google Scholar
  2. 2.
    H. A. Cameron and R. D. G. McKay. Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. J. Comp. Neurol. 435:406–417 (2001).PubMedCrossRefGoogle Scholar
  3. 3.
    T. C. Burns, X. R. Ortiz-González, M. Gutiérrez-Pérez, et al. Thymidine analogs are transferred from prelabeled donor to host cells in the central nervous system after transplantation: a word of caution. Stem Cells 24:1121–1127 (2006).PubMedCrossRefGoogle Scholar
  4. 4.
    K. I. Park, M. A. Hack, J. Ourednik, et al. Acute injury directs the migration, proliferation, and differentiation of solid organ stem cells: evidence from the effect of hypoxia-ischemia in the CNS on clonal “reporter” neural stem cells. Exp. Neurol. 199:156–178 (2006).PubMedCrossRefGoogle Scholar
  5. 5.
    P. Rakic. A century of progress in corticoneurogenesis: from silver impregnation to genetic engineering. Cereb. Cortex 16:i3–i17 (2006).PubMedCrossRefGoogle Scholar
  6. 6.
    K. Sato, J. Kanno, T. Tominaga, Y. Matsubara, and S. Kure. De novo and salvage pathways of DNA synthesis in primary cultured neural stem cells. Brain Res. 1071:24–33 (2006).PubMedCrossRefGoogle Scholar
  7. 7.
    J. Eells and R. Spector. Determination of ribonucleosides, deoxyribonucleosides and purine and pyrimidine bases in adult rabbit cerebrospinal fluid and plasma. Neurochem. Res. 8:1307–1320 (1983).PubMedCrossRefGoogle Scholar
  8. 8.
    J. Eells and R. Spector. Purine and pyrimidine base and nucleoside concentrations in human cerebrospinal fluid and plasma. Neurochem. Res. 8:1451–1457 (1983).PubMedCrossRefGoogle Scholar
  9. 9.
    A. Kornberg and T. A. Baker. DNA Replication, 2nd ed. Freeman, New York, 1992.Google Scholar
  10. 10.
    J. T. Eells and R. Spector. Identification, development and regional distribution of ribonucleotide reductase in adult rat brain. J. Neurochem. 40:1008–1012 (1983).PubMedCrossRefGoogle Scholar
  11. 11.
    S. A. Suleiman and R. Spector. Identification, development and regional distribution of thymidylate synthetase in adult rabbit brain. J. Neurochem. 38:392–396 (1982).PubMedCrossRefGoogle Scholar
  12. 12.
    L. Wang, A. Saada, and S. Eriksson. Kinetic properties of mutant human thymidine kinase 2 suggest a mechanism for mitochondrial DNA depletion myopathy. J. Biol. Chem. 278:6963–6968 (2003).PubMedCrossRefGoogle Scholar
  13. 13.
    V. Dolce, G. Fiermonte, M. J. Runswick, F. Palmieri, and J. E. Walker. The human mitochondrial deoxynucleotide carrier and its role in the toxicity of nucleoside antivirals. Proc. Natl. Acad. Sci. 98:2284–2288 (2001).PubMedCrossRefGoogle Scholar
  14. 14.
    G. Pontarin, P. Ferraro, M. L. Valentino, et al. Mitochondrial DNA depletion and thymidine phosphate pool dynamics in a cellular model of mitochondrial neurogastrointestinal encephalomyopathy. J. Biol. Chem. 281:22720–22728 (2006).PubMedCrossRefGoogle Scholar
  15. 15.
    A. Spinazzola, R. Marti, I. Nishino, et al. Altered thymidine metabolism due to defects of thymidine phosphorylase. J. Biol. Chem. 277:4128–4133 (2002).PubMedCrossRefGoogle Scholar
  16. 16.
    D. J. Begley and M. W. Brightman. Structural and functional aspects of the blood-brain barrier. Prog. Drug Res. 61:39–78 (2003).PubMedGoogle Scholar
  17. 17.
    C. E. Johanson, J. A. Duncan, E. G. Stopa, and A. Baird. Enhanced prospects for drug delivery and brain targeting by the choroid plexus-CSF route. Pharm. Res. 22:1011–1037 (2005).PubMedCrossRefGoogle Scholar
  18. 18.
    C. E. Johanson and D. M. Woodbury. Uptake of [14C]urea by the in vivo choroid plexus-cerebrospinal fluid-brain system: identification of sites of molecular sieving. J. Physiol. 275:167–176 (1978).PubMedGoogle Scholar
  19. 19.
    D. E. Smith, C. E. Johanson, and R. F. Keep. Peptide and peptide analog transport systems at the blood-CSF barrier. Adv. Drug Deliv. Rev. 56:1765–1791 (2004).PubMedCrossRefGoogle Scholar
  20. 20.
    R. Spector and C. E. Johanson. The mammalian choroid plexus. Sci. Am. 261:68–74 (1989).PubMedCrossRefGoogle Scholar
  21. 21.
    R. Spector and C. Johanson. Micronutrient and urate transport in choroid plexus and kidney: implications for drug therapy. Pharm. Res. 23:2515–2524 (2006).PubMedCrossRefGoogle Scholar
  22. 22.
    Z. B. Redzic. Homeostasis of nucleosides and nucleobases in the brain: the role of flux between the CSF and the brain ISF, transport across the choroid plexus and the blood-brain barrier, and cellular uptake. In W. Zheng and A. Chodobski (eds)., The Blood-Cerebrospinal Fluid Barrier, CRC, Boca Raton, 2005, pp. 175–208.Google Scholar
  23. 23.
    E. M. Cornford and W. H. Oldendorf. Independent blood-brain barrier transport systems for nucleic acid precursors. Biochim. Biophys. Acta 394:211–219 (1975).PubMedCrossRefGoogle Scholar
  24. 24.
    R. Spector. Thymidine transport in the central nervous system. J. Neurochem. 35:1092–1098 (1980).PubMedCrossRefGoogle Scholar
  25. 25.
    R. Spector and W. G. Berlinger. Localization and mechanism of thymidine transport in the central nervous system. J. Neurochem. 39:837–841 (1982).PubMedCrossRefGoogle Scholar
  26. 26.
    R. Spector. Development and localization of the thymidine phosphorylating systems in the brain. J. Neurochem. 36:2019–2024 (1981).PubMedCrossRefGoogle Scholar
  27. 27.
    R. Spector. Thymidine accumulation by choroid plexus in vitro. Arch. Biochem. Biophys. 205:85–93 (1980).PubMedCrossRefGoogle Scholar
  28. 28.
    R. Spector. Nucleoside transport in choroid plexus: mechanism and specificity. Arch. Biochem. Biophys. 216:693–703 (1982).PubMedCrossRefGoogle Scholar
  29. 29.
    R. Spector and S. Huntoon. Specificity and sodium-dependence of the active nucleoside transport system in choroid plexus. J. Neurochem. 42:1048–1052 (1984).PubMedCrossRefGoogle Scholar
  30. 30.
    R. Spector and S. Huntoon. Deoxycytidine transport metabolism in the central nervous system. Neurochemistry 40:1474–1480 (1983).Google Scholar
  31. 31.
    R. Spector and S. Huntoon. Characterization, development, and localization of the deoxycytidine phosphorylating systems in mammalian brain. J. Neurochem. 40:1481–1486 (1983).PubMedGoogle Scholar
  32. 32.
    R. Spector and S. Huntoon. Deoxycytidine transport and metabolism in the central nervous system. J. Neurochem. 41:1131–1136 (1983).PubMedCrossRefGoogle Scholar
  33. 33.
    S. A. Suleiman and R. Spector. Metabolism of deoxyuridine in rabbit brain. J. Neurochem. 39:824–830 (1982).PubMedCrossRefGoogle Scholar
  34. 34.
    J. H. Gray, R. P. Owen, and K. M. Giacomini. The concentrative nucleoside transporter family, SLC28. Eur. J. Physiol. 447:728–734 (2004).CrossRefGoogle Scholar
  35. 35.
    S. A. Baldwin, P. R. Beal, S. Y. M. Yao, et al. The equilibrative nucleoside transport family SLC29. Eur. J. Physiol. 447:735–743 (2004).CrossRefGoogle Scholar
  36. 36.
    M. W. L. Ritzel, A. M. L. Ng, S. Y. M. Yao, et al. Molecular identification and characterization of novel human and mouse concentrative Na+-nucleoside cotransporter proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib). J. Biol. Chem. 276:2914–2927 (2001).PubMedCrossRefGoogle Scholar
  37. 37.
    D. Wu, J. G. Clement, and W. M. Pardridge. Low blood-brain barrier permeability to azidothymidine (AZT), 3TC™, and thymidine in the rat. Brain Res. 791:313–316 (1998).PubMedCrossRefGoogle Scholar
  38. 38.
    S. A. Thomas and M. B. Segal. Saturation kinetics, specificity and NBMPR sensitivity of thymidine entry into the central nervous system. Brain Res. 760:59–67 (1997).PubMedCrossRefGoogle Scholar
  39. 39.
    J. Y. Li, R. J. Boado, and W. M. Pardridge. Cloned blood-brain barrier adenosine transporter is identical to the rat concentrative Na+ nucleoside cotransporter CNT2. J. Cereb. Blood Flow Metab. 21:929–936 (2001).PubMedCrossRefGoogle Scholar
  40. 40.
    A. J. Isakovic, M. B. Segal, B. A. Milojkovic, et al. The efflux of purine nucleobases and nucleosides from the rat brain. Neurosci. Lett. 318:65–68 (2002).PubMedCrossRefGoogle Scholar
  41. 41.
    Z. B. Redzic, J. Biringer, K. Barnes, et al. Polarized distribution of nucleoside transporters in rat brain endothelial and choroid plexus epithelial cells. J. Neurochem. 94:1420–1426 (2005).PubMedCrossRefGoogle Scholar
  42. 42.
    L. Alanko, T. Porkka-Heiskanen, and S. Soinila. Localization of equilibrative nucleoside transporters in the rat brain. J. Chem. Neuroanat. 31:162–168 (2006).PubMedCrossRefGoogle Scholar
  43. 43.
    M. E. Schaner, K. M. Gerstin, J. Wang, and K. M. Giacomini. Mechanisms of transport of nucleosides and nucleoside analogues in choroid plexus. Adv. Drug Deliv. Rev. 39:51–62 (1999).PubMedCrossRefGoogle Scholar
  44. 44.
    B. Tavazzi, G. Lazzarino, P. Leone, et al. Simultaneous high performance liquid chromatographic separation of purines, pyrimidines, N-acetylated amino acids, and dicarboxylic acids for the chemical diagnosis of inborn errors of metabolism. Clin. Biochem. 38:997–1008 (2005).PubMedCrossRefGoogle Scholar
  45. 45.
    J. Eells, R. Spector, and S. Huntoon. Nucleoside and oxypurine homeostasis in adult rabbit cerebrospinal fluid and plasma. J. Neurochem. 42:1620–1624 (1984).PubMedCrossRefGoogle Scholar
  46. 46.
    R. Stene and R. Spector. Effect of a 400-kilocalorie carbohydrate diet on human plasma uridine and hypoxanthine concentrations. Biochem. Med. Metabol. Biol. 38:44–46 (1987).CrossRefGoogle Scholar
  47. 47.
    G. Kempermann, H. G. Kuhn, and F. H. Gage. Genetic influence on neurogenesis in the dentate gyrus of adult mice. Proc. Natl. Acad. Sci. USA 94:10409–10414 (1997).PubMedCrossRefGoogle Scholar
  48. 48.
    B. Leuner, E. Gould, and T. J. Shors. Is there a link between adult neurogenesis and learning? Hippocampus 16:216–224 (2006).PubMedCrossRefGoogle Scholar
  49. 49.
    A. Harman, P. Meyer, and A. Ahmat. Neurogenesis in the hippocampus of an adult marsupial. Brain Behav. Evol. 62:1–12 (2003).PubMedCrossRefGoogle Scholar
  50. 50.
    N. Kee, S. Sivalingam, R. Boonstra, and J. M. Wojtowics. The utility of Ki-67 and BrdU as proliferative markers of adult neurogenesis. J. Neurosci. Methods 115:97–105 (2002).PubMedCrossRefGoogle Scholar
  51. 51.
    H. Zhu, Z. Wang, and H. Hansson. Visualization of proliferating cells in the adult mammalian brain with the aid of ribonucleotide reductase. Brain Res. 977:180–189 (2003).PubMedCrossRefGoogle Scholar
  52. 52.
    J. R. Selden, F. Dolbeare, J. H. Clair, et al. Statistical confirmation that immunofluorescent detection of DNA repair in human fibroblasts by measurement of bromodeoxyuridine incorporation is stoichiometric and sensitive. Cytometry 14:154–167 (1993).PubMedCrossRefGoogle Scholar
  53. 53.
    T. D. Palmer, A. R. Willhoite, and F. H. Gage. Vascular niche for adult hippocampal neurogenesis. J. Comp. Neurol. 425:479–494 (2000).PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  1. 1.Robert Wood Johnson Medical SchoolNew BrunswickUSA
  2. 2.Harvard-MIT Program in the Health SciencesCambridgeUSA
  3. 3.Brown Medical SchoolProvidenceUSA
  4. 4.Dept. of NeurosurgeryRhode Island HospitalProvidenceUSA

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