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Development of an In Vitro Model to Evaluate the Regenerative Capacity of Adult Brain-Derived Tyrosine Hydroxylase-Expressing Dopaminergic Neurons

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Abstract

The loss of nigral dopaminergic (DA) neurons is the disease-defining pathological change responsible for progressive motor dysfunction in Parkinson’s disease. In this study, we sought to establish a culture method for adult rat tyrosine hydroxylase (TH)-immunoreactive DA neurons. In this context, we investigated the role of fibroblast growth factor 2 (FGF2), brain-derived neurotrophic factor (BDNF), transforming growth factor-β3 (TGF-β3), glial-derived neurotrophic factor (GDNF) and dibutyryl-cyclic AMP (dbcAMP) in these cultures. Culturing in the presence of FGF2, BDNF and GDNF enhanced the survival of DA neurons by 15-fold and promoted neurite growth. In contrast, dbcAMP promoted neurite growth in all neurons but did not enhance DA cell survival. This study demonstrates that long-term cultures of DA neurons can be established from the mature rat brain and that survival and regeneration of DA neurons can be manipulated by epigenetic factors such as growth factors and intracellular cAMP pathways.

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References

  1. Woolf CJ (2001) Turbocharging neurons for growth: accelerating regeneration in the adult CNS. Nat Neurosci 4:7–9

    Article  PubMed  CAS  Google Scholar 

  2. Sapieha PS, Duplan L, Uetani N et al (2005) Receptor protein tyrosine phosphatase sigma inhibits axon regrowth in the adult injured CNS. Mol Cell Neurosci 28:625–635

    Article  PubMed  CAS  Google Scholar 

  3. Smith PD, Sun F, Park KK et al (2009) SOCS3 deletion promotes optic nerve regeneration in vivo. Neuron 64:617–623

    Article  PubMed  CAS  Google Scholar 

  4. Chesselet MF (2003) Dopamine and Parkinson’s disease: is the killer in the house? Mol. Psychiatry. 8:369–370

    Article  PubMed  CAS  Google Scholar 

  5. Uversky VN, Eliezer D (2009) Biophysics of Parkinson’s disease: structure and aggregation of alpha-synuclein. Curr Protein Pept Sci 10:483–499

    Article  PubMed  CAS  Google Scholar 

  6. Bishop NA, Lu T, Yankner BA (2010) Neural mechanisms of ageing and cognitive decline. Nature 464:529–535

    Article  PubMed  CAS  Google Scholar 

  7. Liu J, Head E, Gharib AM et al (2002) Memory loss in old rats is associated with brain mitochondrial decay and RNA/DNA oxidation: partial reversal by feeding acetyl-L-carnitine and/or R-alpha -lipoic acid. Proc Natl Acad Sci USA 99:2356–2361

    Article  PubMed  CAS  Google Scholar 

  8. Aliev G, Palacios HH, Walrafen B et al (2009) Brain mitochondria as a primary target in the development of treatment strategies for Alzheimer disease. Int J Biochem Cell Biol 41:1989–2004

    Article  PubMed  CAS  Google Scholar 

  9. Terman A, Gustafsson B, Brunk UT (2006) Mitochondrial damage and intralysosomal degradation in cellular aging. Mol Aspects Med 27:471–482

    Article  PubMed  CAS  Google Scholar 

  10. Terman A, Kurz T, Navratil M et al (2010) Mitochondrial turnover and aging of long-lived postmitotic cells: the mitochondrial-lysosomal axis theory of aging. Antioxid Redox Signal 12:503–535

    Article  PubMed  CAS  Google Scholar 

  11. Bronzetti E, Felici L, Ferrante F et al (1988) Age-related changes of the metabolic profile of rat cerebellar cortex: enzyme histochemical study. Mech Ageing Dev 44:277–286

    Article  PubMed  CAS  Google Scholar 

  12. Rogers SW, Gahring LC, Collins AC et al (1998) Age-related changes in neuronal nicotinic acetylcholine receptor subunit alpha4 expression are modified by long-term nicotine administration. J Neurosci 18:4825–4832

    PubMed  CAS  Google Scholar 

  13. Pascale A, Govoni S, Battaini F (1998) Age-related alteration of PKC, a key enzyme in memory processes: physiological and pathological examples. Mol Neurobiol 16:49–62

    Article  PubMed  CAS  Google Scholar 

  14. Zhou W, Schaack J, Zawada WM et al (2002) Overexpression of human alpha-synuclein causes dopamine neuron death in primary human mesencephalic culture. Brain Res 926:42–50

    Article  PubMed  CAS  Google Scholar 

  15. Ren D, Miller JD (2003) Primary cell culture of suprachiasmatic nucleus. Brain Res Bull 61:547–553

    Article  PubMed  Google Scholar 

  16. Martinoia S, Bonzano L, Chiappalone M et al (2005) In vitro cortical neuronal networks as a new high-sensitive system for biosensing applications. Biosens Bioelectron 20:2071–2078

    Article  PubMed  CAS  Google Scholar 

  17. Ganser C, Papazoglou A, Just L et al (2010) Neuroprotective effects of erythropoietin on 6-hydroxydopamine-treated ventral mesencephalic dopamine-rich cultures. Exp Cell Res 316:737–746

    Article  PubMed  CAS  Google Scholar 

  18. Prang P, Del Turco D, Kapfhammer JP (2001) Regeneration of entorhinal fibers in mouse slice cultures is age dependent and can be stimulated by NT-4, GDNF, and modulators of G-proteins and protein kinase C. Exp Neurol 169:135–147

    Article  PubMed  CAS  Google Scholar 

  19. Blackmore M, Letourneau PC (2006) Changes within maturing neurons limit axonal regeneration in the developing spinal cord. J Neurobiol 66:348–360

    Article  PubMed  CAS  Google Scholar 

  20. Wanner IB, Deik A, Torres M et al (2008) A new in vitro model of the glial scar inhibits axon growth. Glia 56:1691–1709

    Article  PubMed  Google Scholar 

  21. Schwab ME (2002) Repairing the injured spinal cord. Science 295:1029–1031

    Article  PubMed  CAS  Google Scholar 

  22. Silver J, Miller JH (2004) Regeneration beyond the glial scar. Nat Rev Neurosci 5:146–156

    Article  PubMed  CAS  Google Scholar 

  23. Spencer T, Domeniconi M, Cao Z et al (2003) New roles for old proteins in adult CNS axonal regeneration. Curr Opin Neurobiol 13:133–139

    Article  PubMed  CAS  Google Scholar 

  24. Lu P, Tuszynski MH (2008) Growth factors and combinatorial therapies for CNS regeneration. Exp Neurol 209:313–320

    Article  PubMed  CAS  Google Scholar 

  25. Brewer GJ (1997) Isolation and culture of adult rat hippocampal neurons. J Neurosci Methods 71:143–155

    Article  PubMed  CAS  Google Scholar 

  26. Evans J, Sumners C, Moore J et al (2002) Characterization of mitotic neurons derived from adult rat hypothalamus and brain stem. J Neurophysiol 87:1076–1085

    PubMed  Google Scholar 

  27. Majd S, Zarifkar A, Rastegar K et al (2008) Different fibrillar Abeta 1–42 concentrations induce adult hippocampal neurons to reenter various phases of the cell cycle. Brain Res 1218:224–229

    Article  PubMed  CAS  Google Scholar 

  28. Thoenen H (1995) Neurotrophins and neuronal plasticity. Science 270:593–598

    Article  PubMed  CAS  Google Scholar 

  29. Song XY, Li F, Zhang FH et al (2008) Peripherally-derived BDNF promotes regeneration of ascending sensory neurons after spinal cord injury. PLoS One 3:e1707

    Article  PubMed  Google Scholar 

  30. Gao H, Qiao X, Hefti F et al (1997) Elevated mRNA expression of brain-derived neurotrophic factor in retinal ganglion cell layer after optic nerve injury. Invest Ophthalmol Vis Sci 38:1840–1847

    PubMed  CAS  Google Scholar 

  31. Loeliger MM, Briscoe T, Rees SM (2008) BDNF increases survival of retinal dopaminergic neurons after prenatal compromise. Invest Ophthalmol Vis Sci 49:1282–1289

    Article  PubMed  Google Scholar 

  32. Spenger C, Hyman C, Studer L et al (1995) Effects of BDNF on dopaminergic, serotonergic, and GABAergic neurons in cultures of human fetal ventral mesencephalon. Exp Neurol 133:50–63

    Article  PubMed  CAS  Google Scholar 

  33. Lin LF, Doherty DH, Lile JD et al (1993) GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 260:1130–1132

    Article  PubMed  CAS  Google Scholar 

  34. Nicole O, Ali C, Docagne F et al (2001) Neuroprotection mediated by glial cell line-derived neurotrophic factor: involvement of a reduction of NMDA-induced calcium influx by the mitogen-activated protein kinase pathway. J Neurosci 21:3024–3033

    PubMed  CAS  Google Scholar 

  35. Rahhal B, Heermann S, Ferdinand A et al (2009) In vivo requirement of TGF-beta/GDNF cooperativity in mouse development: focus on the neurotrophic hypothesis. Int J Dev Neurosci 27:97–102

    Article  PubMed  CAS  Google Scholar 

  36. Schaar DG, Sieber BA, Dreyfus CF et al (1993) Regional and cell-specific expression of GDNF in rat brain. Exp Neurol 124:368–371

    Article  PubMed  CAS  Google Scholar 

  37. Roussa E, von Bohlen und Halbach O, Krieglstein K (2009) TGF-beta in dopamine neuron development, maintenance and neuroprotection. Adv Exp Med Biol 651:81–90

    Article  PubMed  CAS  Google Scholar 

  38. Paxinos G, Watson C (2005) The rat brain in stereotaxic coordinates. Elsevier academic press, San Diego

    Google Scholar 

  39. Majd S, Rastegar K, Zarifkar A et al (2007) Fibrillar beta-amyloid (Abeta) (1–42) elevates extracellular Abeta in cultured hippocampal neurons of adult rats. Brain Res 1185:321–327

    Article  PubMed  CAS  Google Scholar 

  40. Majd S, Zarifkar A, Rastegar K et al (2008) Culturing adult rat hippocampal neurons with long-interval changing media. Iran Biomed J 12:101–107

    PubMed  Google Scholar 

  41. Huang EJ, Reichardt LF (2001) Neurotrophins: roles in neuronal development and function. Annu Rev Neurosci 24:677–736

    Article  PubMed  CAS  Google Scholar 

  42. Hyman C, Hofer M, Barde YA et al (1991) BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra. Nature 350:230–232

    Article  PubMed  CAS  Google Scholar 

  43. Engele J, Franke B (1996) Effects of glial cell line-derived neurotrophic factor (GDNF) on dopaminergic neurons require concurrent activation of cAMP-dependent signaling pathways. Cell Tissue Res 286:235–240

    Article  PubMed  CAS  Google Scholar 

  44. Kawano H, Li HP, Sango K et al (2005) Inhibition of collagen synthesis overrides the age-related failure of regeneration of nigrostriatal dopaminergic axons. J Neurosci Res 80:191–202

    Article  PubMed  CAS  Google Scholar 

  45. Goldberg JL (2004) Intrinsic neuronal regulation of axon and dendrite growth. Curr Opin Neurobiol 14:551–557

    Article  PubMed  CAS  Google Scholar 

  46. Kloos AD, Muller KJ, Modney BK (2007) Atypical embryonic synapses fail to regenerate in adulthood. J Comp Neurol 505:404–411

    Article  PubMed  Google Scholar 

  47. Cohen-Cory S, Kidane AH, Shirkey NJ et al (2009) Brain-derived neurotrophic factor and the development of structural neuronal connectivity. Dev Neurobiol 70:271–288

    Google Scholar 

  48. Borghesani PR, Peyrin JM, Klein R et al (2002) BDNF stimulates migration of cerebellar granule cells. Development 129:1435–1442

    PubMed  CAS  Google Scholar 

  49. Liu X, Grishanin RN, Tolwani RJ et al (2007) Brain-derived neurotrophic factor and TrkB modulate visual experience-dependent refinement of neuronal pathways in retina. J Neurosci 27:7256–7267

    Article  PubMed  CAS  Google Scholar 

  50. Zeng F, Watson RP, Nash MS (2010) Glial cell-derived neurotrophic factor enhances synaptic communication and 5-hydroxytryptamine 3a receptor expression in enteric neurons. Gastroenterology 138:1491–1501

    Article  PubMed  CAS  Google Scholar 

  51. Alberch J, Perez-Navarro E, Canals JM (2002) Neuroprotection by neurotrophins and GDNF family members in the excitotoxic model of Huntington’s disease. Brain Res Bull 57:817–822

    Article  PubMed  CAS  Google Scholar 

  52. Alexi T, Borlongan CV, Faull RL et al (2000) Neuroprotective strategies for basal ganglia degeneration: Parkinson’s and Huntington’s diseases. Prog Neurobiol 60:409–470

    Article  PubMed  CAS  Google Scholar 

  53. Sharma HS (2010) Selected combination of neurotrophins potentiate neuroprotection and functional recovery following spinal cord injury in the rat. Acta Neurochir Suppl 106:295–300

    Article  PubMed  Google Scholar 

  54. Deister C, Schmidt CE (2006) Optimizing neurotrophic factor combinations for neurite outgrowth. J Neural Eng 3:172–179

    Article  PubMed  CAS  Google Scholar 

  55. Logan A, Ahmed Z, Baird A et al (2006) Neurotrophic factor synergy is required for neuronal survival and disinhibited axon regeneration after CNS injury. Brain 129:490–502

    Article  PubMed  Google Scholar 

  56. Wright DE, Snider WD (1995) Neurotrophin receptor mRNA expression defines distinct populations of neurons in rat dorsal root ganglia. J Comp Neurol 351:329–338

    Article  PubMed  CAS  Google Scholar 

  57. Numan S, Seroogy KB (1999) Expression of trkB and trkC mRNAs by adult midbrain dopamine neurons: a double-label in situ hybridization study. J Comp Neurol 403:295–308

    Article  PubMed  CAS  Google Scholar 

  58. Walker DG, Beach TG, Xu R et al (1998) Expression of the proto-oncogene Ret, a component of the GDNF receptor complex, persists in human substantia nigra neurons in Parkinson’s disease. Brain Res 792:207–217

    Article  PubMed  CAS  Google Scholar 

  59. He SM, Zhao ZW, Zhao L et al (2008) Nigrostriatal projecting neurons express GDNF receptor subunit RET in adult rats. Acta Neurobiol Exp (Wars) 68:347–353

    Google Scholar 

  60. Glazner GW, Mu X, Springer JE (1998) Localization of glial cell line-derived neurotrophic factor receptor alpha and c-ret mRNA in rat central nervous system. J Comp Neurol 391:42–49

    Article  PubMed  CAS  Google Scholar 

  61. Trupp M, Belluardo N, Funakoshi H et al (1997) Complementary and overlapping expression of glial cell line-derived neurotrophic factor (GDNF), c-ret proto-oncogene, and GDNF receptor-alpha indicates multiple mechanisms of trophic actions in the adult rat CNS. J Neurosci 17:3554–3567

    PubMed  CAS  Google Scholar 

  62. Tsuzuki T, Takahashi M, Asai N et al (1995) Spatial and temporal expression of the ret proto-oncogene product in embryonic, infant and adult rat tissues. Oncogene 10:191–198

    PubMed  CAS  Google Scholar 

  63. Nosrat CA, Tomac A, Hoffer BJ et al (1997) Cellular and developmental patterns of expression of Ret and glial cell line-derived neurotrophic factor receptor alpha mRNAs. Exp Brain Res 115:410–422

    Article  PubMed  CAS  Google Scholar 

  64. Soderstrom S, Bengtsson H, Ebendal T (1996) Expression of serine/threonine kinase receptors including the bone morphogenetic factor type II receptor in the developing and adult rat brain. Cell Tissue Res 286:269–279

    Article  PubMed  CAS  Google Scholar 

  65. Charytoniuk DA, Traiffort E, Pinard E et al (2000) Distribution of bone morphogenetic protein and bone morphogenetic protein receptor transcripts in the rodent nervous system and up-regulation of bone morphogenetic protein receptor type II in hippocampal dentate gyrus in a rat model of global cerebral ischemia. Neuroscience 100:33–43

    Article  PubMed  CAS  Google Scholar 

  66. Remenyi J, Hunter CJ, Cole C et al (2010) Regulation of the miR-212/132 locus by MSK1 and CREB in response to neurotrophins. Biochem J 428:281–291

    Article  PubMed  CAS  Google Scholar 

  67. Cui Q, Yip HK, Zhao RC et al (2003) Intraocular elevation of cyclic AMP potentiates ciliary neurotrophic factor-induced regeneration of adult rat retinal ganglion cell axons. Mol Cell Neurosci 22:49–61

    Article  PubMed  CAS  Google Scholar 

  68. Meyer-Franke A, Kaplan MR, Pfrieger FW et al (1995) Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture. Neuron 15:805–819

    Article  PubMed  CAS  Google Scholar 

  69. Shen S, Wiemelt AP, McMorris FA et al (1999) Retinal ganglion cells lose trophic responsiveness after axotomy. Neuron 23:285–295

    Article  PubMed  CAS  Google Scholar 

  70. Rydel RE, Greene LA (1988) cAMP analogs promote survival and neurite outgrowth in cultures of rat sympathetic and sensory neurons independently of nerve growth factor. Proc Natl Acad Sci USA 85:1257–1261

    Article  PubMed  CAS  Google Scholar 

  71. Li M, Wang X, Meintzer MK et al (2000) Cyclic AMP promotes neuronal survival by phosphorylation of glycogen synthase kinase 3beta. Mol Cell Biol 20:9356–9363

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We would like to thank Jim Massalas for technical assistance. JD is an NHMRC Research Fellow. The funding for the conduct of the research was obtained from the NHMRC.

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  1. Shohreh Majd

    Present address: Department of Human Physiology, School of Medicine, Flinders University, Bedford Park, Adelaide, SA, 5042, Australia

Authors and Affiliations

  1. Florey Neuroscience Institutes, Royal Parade, University of Melbourne, Parkville, Melbourne, VIC, 3010, Australia

    Shohreh Majd, Arthur Smardencas, Clare L. Parish & John Drago

  2. Centre for Neuroscience, University of Melbourne, Parkville, Melbourne, VIC, Australia

    Clare L. Parish & John Drago

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  1. Shohreh Majd
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  2. Arthur Smardencas
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Correspondence to John Drago.

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Majd, S., Smardencas, A., Parish, C.L. et al. Development of an In Vitro Model to Evaluate the Regenerative Capacity of Adult Brain-Derived Tyrosine Hydroxylase-Expressing Dopaminergic Neurons. Neurochem Res 36, 967–977 (2011). https://doi.org/10.1007/s11064-011-0435-0

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