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Optimized Quantities of GDNF Overexpressed by Engineered Astrocytes Are Critical for Protection of Neuroblastoma Cells Against 6-OHDA Toxicity

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Abstract

Optimized levels of glial cell line-derived neurotrophic factor glial cell line-derived neurotrophic factor (GDNF) are critical for protection of dopaminergic neurons against parkinsonian cell death. Recombinant lentiviruses harboring GDNF coding sequence were constructed and used to infect astrocytoma cell line 1321N1. The infected astrocytes overexpressed GDNF mRNA and secreted an average of 2.2 ng/mL recombinant protein as tested in both 2 and 16 weeks post-infection. Serial dilutions of GDNF-enriched conditioned medium from infected astrocytes added to growing neuroblastoma cell line SK-N-MC resulted in commensurate resistance against 6-OHDA toxicity. SK-N-MC cell survival rate rose from 51% in control group to 84% in the cells grown with astro-CM containing 453 pg secreted GDNF, an increase that was highly significant (P < 0.0001). However, larger volumes of the GDNF-enriched conditioned medium failed to improve cell survival and addition of volumes that contained 1,600 pg or more GDNF further reduced survival rate to below 70%. Changes in cell survival paralleled to changes in the percent of apoptotic cell morphologies. These data demonstrate the feasibility of using astrocytes as minipumps to stably oversecrete neurotrophic factors and further indicate that GDNF can be applied to neuroprotection studies in PD pending the optimization of its concentrations.

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Abbreviations

Astro-CM:

Astrocytic conditioned medium

GDNF:

Glial cell line-derived neurotrophic factor

6-OHDA:

6-Hydroxy dopamine

TH:

Tyrosine hydroxylase

SNpc:

Substantia nigra pars compacta

SN-ST:

Nigrostriatal

References

  • Airaksinen MS, Saarma M (2002) The GDNF family: signalling, biological functions and therapeutic value. Nat Rev Neurosci 3:383–394

    Article  PubMed  CAS  Google Scholar 

  • Akerud P, Canals JM, Snyder EY, Arenas E (2001) Neuroprotection through delivery of glial cell line-derived neurotrophic factor by neural stem cells in a mouse model of Parkinson's disease. J Neurosci 21:8108–8118

    PubMed  CAS  Google Scholar 

  • Arai K, Lo EH (2010) Astrocytes protect oligodendrocyte precursor cells via MEK/ERK and PI3K/Akt signaling. J Neurosci Res 88:758–763

    PubMed  CAS  Google Scholar 

  • Björklund A, Rosenblad C, Winkler C, Kirik D (1997) Studies on neuroprotective and regenerative effects of GDNF in a partial lesion model of Parkinson's disease. Neurobiol Dis 4:186–200

    Article  PubMed  Google Scholar 

  • Björklund A, Kirik D, Rosenblad C, Georgievska B, Lundberg C, Mandel RJ (2000) Towards a neuroprotective gene therapy for Parkinson's disease: use of adenovirus, AAV and lentivirus vectors for gene transfer of GDNF to the nigrostriatal system in the rat Parkinson model. Brain Res 886:82–98

    Article  PubMed  Google Scholar 

  • Bohn MC (2004) Motoneurons crave glial cell line-derived neurotrophic factor. Exp Neurol 190:263–275

    Article  PubMed  CAS  Google Scholar 

  • Böhr M (2004) Neuroprotection: models, mechanisms and therapies. Wiley, Weinheim

    Book  Google Scholar 

  • Brown DR (1999) Neurons depend on astrocytes in a coculture system for protection from glutamate toxicity. Mol Cell Neurosci 13:379–389

    Article  PubMed  CAS  Google Scholar 

  • Brundin P (2002) GDNF treatment in Parkinson’s disease: time for controlled clinical trials? Brain 125:2149–2151

    Article  PubMed  Google Scholar 

  • Capowski EE, Schneider BL, Ebert AD (2007) Lentiviral vector-mediated genetic modification of human neural progenitor cells for ex vivo gene therapy. J Neurosci Methods 163:338–349

    Article  PubMed  CAS  Google Scholar 

  • Choi-Lundberg DL, Bohn MC (1995) Ontogeny and distribution of glial cell line-derived neurotrophic factor (GDNF) mRNA in rat. Brain Res Dev Brain Res 85:80–88

    Article  PubMed  CAS  Google Scholar 

  • Cunningham LA, Su C (2002) Astrocyte delivery of glial cell line-derived neurotrophic factor in a mouse model of Parkinson's disease. Exp Neurol 174:230–242

    Article  PubMed  CAS  Google Scholar 

  • Deierborg T, Soulet D, Roybon L, Hall V, Brundin P (2008) Emerging restorative treatments for Parkinson’s disease. Progress Neurobiol 85:407–432

    Article  CAS  Google Scholar 

  • Dhandapani KM, Hadman M, De Sevilla L, Wade MF, Mahesh VB, Brann DW (2003) Astrocyte protection of neurons: role of transforming growth factor-beta signaling via a c-Jun-AP-1 protective pathway. J Biol Chem 278:43329–43339

    Article  PubMed  CAS  Google Scholar 

  • Ding J, Yu JZ, Li QY, Wang X, Lu CZ, Xiao BG (2009) Rho kinase inhibitor Fasudil induces neuroprotection and neurogenesis partially through astrocyte-derived G-CSF. Brain Behav Immun 23:1083–1088

    Article  PubMed  CAS  Google Scholar 

  • Dringen R, Hirrlinger J (2003) Glutathione pathways in the brain. Biol Chem 348:505–516

    Article  Google Scholar 

  • Duan D, Yang H, Zhang J, Zhang J, Xu Q (2005) Long-term restoration of nigrostriatal system function by implanting GDNF genetically modified fibroblasts in a rat model of Parkinson's disease. Exp Brain Res 161:316–324

    Article  PubMed  CAS  Google Scholar 

  • Ebert AD, Barber AE, Heins BM, Svendsen CN (2010) Ex vivo delivery of GDNF maintains motor function and prevents neuronal loss in a transgenic mouse model of Huntington's disease. Exp Neurol 224:155–162

    Article  PubMed  CAS  Google Scholar 

  • Ericson C, Georgievska B, Lundberg C (2005) Ex vivo gene delivery of GDNF using primary astrocytes transduced with a lentiviral vector provides neuroprotection in a rat model of Parkinson's disease. Eur J Neurosci 22:2755–2764

    Article  PubMed  Google Scholar 

  • Eslamboli A, Georgievska B, Ridley RM et al (2005) Continuous low-level glial cell line-derived neurotrophic factor delivery using recombinant adeno-associated viral vectors provides neuroprotection and induces behavioral recovery in a primate model of Parkinson's disease. J Neurosci 25:769–777

    Article  PubMed  CAS  Google Scholar 

  • Galan-Rodriguez B, del-Marco A, Flores JA, Ramiro-Fuentes S, Gonzalez-Aparicio R, Tunez I, Tasset I, Fernandez-Espejo E (2008) Grafts of extra-adrenal chromaffin cells as aggregates show better survival rate and regenerative effects on parkinsonian rats than dispersed cell grafts. Neurobiol Dis 29:529–542

    Article  PubMed  CAS  Google Scholar 

  • Gardaneh M, Gholami M, Maghsoudi N (2011) Synergy between glutathione peroxidase-1 and astrocytic growth factors suppresses free radical generation and protects dopaminergic neurons against 6-Hydroxydopamine. Rejuven Res 14(2):195–204

    Article  CAS  Google Scholar 

  • Georgievska B, Kirik D, Bjorklund A (2002) Aberrant sprouting and downregulation of tyrosine hydroxylase in lesioned nigrostriatal dopamine neurons induced by longlasting overexpression of glial cell line derived neurotrophic factor in the striatum by lentiviral gene transfer. Exp Neurol 177:461–474

    Article  PubMed  CAS  Google Scholar 

  • Gill SS, Patel NK, Hotton GR et al (2003) Direct brain infusion of glial cell line-derived neurotrophic factor in Parkinson disease. Nat Med 9:589–595

    Article  PubMed  CAS  Google Scholar 

  • Hirsch EC, Faucheux B, Damier P, Mouatt-Prigent A, Agid Y (1997) Neuronal vulnerability in Parkinson's disease. J Neural Transm Suppl 50:79–88

    PubMed  CAS  Google Scholar 

  • Hong M, Mukhida K, Mendez I (2008) GDNF therapy for Parkinson's disease. Expert Rev Neurother 8:1125–1139

    Article  PubMed  CAS  Google Scholar 

  • Horita Y, Honmou O, Harada K, Houkin K, Hamada H, Kocsis JD (2006) Intravenous administration of glial cell line-derived neurotrophic factor gene-modified human mesenchymal stem cells protects against injury in a cerebral ischemia model in the adult rat. J Neurosci Res 84:1495–1504

    Article  PubMed  CAS  Google Scholar 

  • Kaddis FG, Zawada WM, Schaack J, Freed CR (1996) Conditioned medium from aged monkey fibroblasts stably expressing GDNF and BDNF improves survival of embryonic dopamine neurons in vitro. Cell Tissue Res 286:241–247

    Article  PubMed  CAS  Google Scholar 

  • Klein SM, Behrstock S, McHugh J, Hoffmann K, Wallace K, Suzuki M, Aebischer P, Svendsen CN (2005) GDNF delivery using human neural progenitor cells in a rat model of ALS. Hum Gene Ther 16:509–521

    Article  PubMed  CAS  Google Scholar 

  • Kobayashi T, Ahlenius H, Thored P, Kobayashi R, Kokaia Z, Lindvall O (2006) Intracerebral infusion of glial cell line-derived neurotrophic factor promotes striatal neurogenesis after stroke in adult rats. Stroke 37:2361–2367

    Article  PubMed  CAS  Google Scholar 

  • Kordower JH, Emborg ME, Bloch J et al (2000) Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease. Science 290:767–773

    Article  PubMed  CAS  Google Scholar 

  • Lang AE, Obeso JA (2004) Challenges in Parkinson's disease: restoration of the nigrostriatal dopamine system is not enough. Lancet Neurol 3:309–316

    Article  PubMed  Google Scholar 

  • Lang AE, Gill S, Patel NK et al (2006) Randomized controlled trial of intraputamenal glial cell line-derived neurotrophic factor infusion in Parkinson disease. Ann Neurol 59:459–466

    Article  PubMed  CAS  Google Scholar 

  • Lin LF, Doherty DH, Lile JD, Bektesh S (1993) Collins F (2010) GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 260:1130–1132

    Article  PubMed  CAS  Google Scholar 

  • Love S, Plaha P, Patel NK, Hotton GR, Brooks DJ, Gill SS (2005) Glial cell line-derived neurotrophic factor induces neuronal sprouting in human brain. Nat Med 11:703–704

    Article  PubMed  CAS  Google Scholar 

  • Mandel RJ, Burger C, Snyder RO (2008) Viral vectors for in vivo gene transfer in Parkinson's disease: properties and clinical grade production. Exp Neurol 209:58–71

    Article  PubMed  CAS  Google Scholar 

  • Matsushita N, Fujita Y, Tanaka M, Nagatsu T, Kiuchi K (1997) Cloning and structural organization of the gene encoding the mouse glial cell line-derived neurotrophic factor. GDNF Gene 203:149–157

    CAS  Google Scholar 

  • McBride JL, During MJ, Wuu J, Chen EY, Leurgans SE, Kordower JH (2003) Structural and functional neuroprotection in a rat model of Huntington's disease by viral gene transfer of GDNF. Exp Neurol 181:213–223

    Article  PubMed  CAS  Google Scholar 

  • McBride JL, Ramaswamy S, Gasmi M, Bartus RT, Herzog CD, Brandon EP, Zhou L, Pitzer MR, Berry-Kravis EM, Kordower JH (2006) Viral delivery of glial cell line-derived neurotrophic factor improves behavior and protects striatal neurons in a mouse model of Huntington's disease. Proc Natl Acad Sci USA 103:9345–9350

    Article  PubMed  CAS  Google Scholar 

  • Mena MA, Davila V, Sulzer D (2002) Neurotrophic effects of l-DOPA in postnatal midbrain dopamine neuron/cortical astrocyte cocultures. J Neurochem 69:1398–1408

    Article  Google Scholar 

  • Mohajeri MH, Figlewicz DA, Bohn MC (1999) Intramuscular grafts of myoblasts genetically modified to secrete glial cell line-derived neurotrophic factor prevent motoneuron loss and disease progression in a mouse model of familial amyotrophic lateral sclerosis. Hum Gene Ther 10:1853–1866

    Article  PubMed  CAS  Google Scholar 

  • Morrison PF, Lonser RR, Oldfield EH (2007) Convective delivery of glial cell line-derived neurotrophic factor in the human putamen. J Neurosurg 107:74–83

    Article  PubMed  Google Scholar 

  • Nakao N, Yokote H, Nakai K, Itakura T (2000) Promotion of survival and regeneration of nigral dopamine neurons in a rat model of Parkinson’s disease after implantation of embryonal carcinoma-derived neurons genetically engineered to produce glial cell line-derived neurotrophic factor. J Neurosurg 92:659–670

    Article  PubMed  CAS  Google Scholar 

  • Nutt JG, Burchiel KJ, Comella CL, Jankovic J, Lang AE, Laws ER Jr, Lozano AM, Penn RD, Simpson RK Jr, Stacy M, Wooten GF (2003) Randomized, double-blind trial of glial cell line-derived neurotrophic factor (GDNF) in PD. Neurology 60:69–73

    PubMed  CAS  Google Scholar 

  • Parsadanian A, Pan Y, Li W, Myckatyn TM, Brakefield D (2006) Astrocyte-derived transgene GDNF promotes complete and long-term survival of adult facial motoneurons following avulsion and differentially regulates the expression of transcription factors of AP-1 and ATF/CREB families. Exp Neurol 200:26–37

    Article  PubMed  CAS  Google Scholar 

  • Patel NK, Gill SS (2007) GDNF delivery for Parkinson's disease. Acta Neurochir Suppl 97:135–154

    Article  PubMed  CAS  Google Scholar 

  • Patel NK, Bunnage M, Plaha P, Svendsen CN, Heywood P, Gill SS (2005) Intraputamenal infusion of glial cell line-derived neurotrophic factor in PD: a two-year outcome study. Ann Neurol 57:298–302

    Article  PubMed  CAS  Google Scholar 

  • Pertusa M, Garcia-Matas S, Mammeri H, Adell A, Rodrigo T, Mallet J, Cristofol R, Sarkis C, Sanfeliu C (2007) Expression of GDNF transgene in astrocytes improves cognitive deficits in aged rats. Neurobiol Aging 29:1366–1379

    Article  PubMed  Google Scholar 

  • Pluta K, Luce ML, Bao L, Agha-Mohammadi S, Reiser J (2005) Tight control of transgene expression by lentivirus vectors containing second-generation tetracycline-responsive promoters. J Gene Med 7:803–817

    Article  PubMed  CAS  Google Scholar 

  • Ponce S, Orive G, Gascón AR, Hernández RM, Pedraz JL (2005) Microcapsules prepared with different biomaterials to immobilize GDNF secreting 3T3 fibroblasts. Int J Pharm 293:1–10

    Article  PubMed  CAS  Google Scholar 

  • Riederer P, Wuketich S (1976) Time course of nigrostriatal degeneration in Parkinson’s disease. J Neural Transm Park Dis Dement Sect 38:277–301

    Article  CAS  Google Scholar 

  • Rosenblad C, Georgievska B, Kirik D (2003) Long-term striatal overexpression of GDNF selectively downregulates tyrosine hydroxylase in the intact nigrostriatal dopamine system. Eur J Neurosci 17:260–270

    Article  PubMed  Google Scholar 

  • Saijo K, Winner B, Carson CT, Collier JG, Boyer L, Rosenfeld MG, Gage FH, Glass CK (2009) A Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic neurons from inflammation-induced death. Cell 137:47–59

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Sandhu JK, Gardaneh M, Iwasiow R, Lanthier P, Gangaraju S, Ribecco-Lutkiewicz M, Tremblay R, Kiuchi K, Sikorska M (2009) Astrocyte-secreted GDNF and glutathione antioxidant system protect neurons against 6OHDA cytotoxicity. Neurobiol Dis 33:405–414

    Article  PubMed  CAS  Google Scholar 

  • Sariola H, Saarma M (2003) Novel functions and signalling pathways for GDNF. J Cell Sci 116:3855–3862

    Article  PubMed  CAS  Google Scholar 

  • Schneider B, Zufferey R, Aebischer P (2008) Viral vectors, animal models and new therapies for Parkinson's disease. Parkinsonism Relat Disord 2:S169–S171

    Article  Google Scholar 

  • Sherer TB, Fiske BK, Svendsen CN, Lang AE, Langston JW (2006) Crossroads in GDNF therapy for Parkinson's disease. Mov Disord 21:136–141

    Article  PubMed  Google Scholar 

  • Slevin JT, Gerhardt GA, Smith CD, Gash DM, Kryscio R, Young B (2005) Improvement of bilateral motor functions in patients with Parkinson disease through the unilateral intraputaminal infusion of glial cell line-derived neurotrophic factor. J Neurosurg 102:216–222

    Article  PubMed  CAS  Google Scholar 

  • Slevin JT, Gash DM, Smith CD, Gerhardt GA, Kryscio R, Chebrolu H, Walton A, Wagner R, Young AB (2007) Unilateral intraputamenal glial cell line-derived neurotrophic factor in patients with Parkinson disease: response to 1 year of treatment and 1 year of withdrawal. J Neurosurg 106:614–620

    Article  PubMed  CAS  Google Scholar 

  • Wallén-Mackenzie Å, de Urquiza AM, Petersson S, Rodriguez FJ, Friling S, Wagner J, Ordentlich P, Lengqvist J, Heyman RA, Arenas A, Perlmann T (2003) Nurr1-RXR heterodimers mediate RXR ligand-induced signaling in neuronal cells. Genes Dev 17:3036–3047

    Article  PubMed  Google Scholar 

  • Wang L, Muramatsu S, Lu Y, Ikeguchi K, Fujimoto K, Okada T, Mizukami H, Hanazono Y, Kume A, Urano F, Ichinose H, Nagatsu T, Nakano I, Ozawa K (2002) Delayed delivery of AAV-GDNF prevents nigral neurodegeneration and promotes functional recovery in a rat model of Parkinson's disease. Gene Ther 9:381–389

    Article  PubMed  CAS  Google Scholar 

  • Yacoubian TA, Standaert DG (2009) Targets for neuroprotection in Parkinson's disease. Biochim Biophys Acta 1792:676–687

    PubMed  CAS  Google Scholar 

  • Yang D, Peng C, Li X, Fan X, Li L, Ming M, Chen S, Le W (2008) Pitx3-transfected astrocytes secrete brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor and protect dopamine neurons in mesencephalon cultures. J Neurosci Res 86:3393–3400

    Article  PubMed  CAS  Google Scholar 

  • Yasuhara T, Shingo T, Date I (2007) Glial cell line-derived neurotrophic factor (GDNF) therapy for Parkinson's disease. Acta Med Okayama 61:51–56

    PubMed  CAS  Google Scholar 

  • Yoshida M, Saito H, Katsuki H (1995) Neurotrophic effects of conditioned media of astrocytes isolated from different brain regions on hippocampal and cortical neurons. Experientia 51:133–136

    Article  PubMed  CAS  Google Scholar 

  • Zhao Z, Alam S, Oppenheim RW, Prevette DM, Evenson A, Parsadanian A (2004) Overexpression of glial cell line-derived neurotrophic factor in the CNS rescues motoneurons from programmed cell death and promotes their long-term survival following axotomy. Expt Neurol 190:356–372

    Article  CAS  Google Scholar 

  • Zhu ZH, Yang R, Fu X, Wang YQ, Wu GC (2006) Astrocyte-conditioned medium protecting hippocampal neurons in primary cultures against corticosterone-induced damages via PI3-K/Akt signal pathway. Brain Res 1114:1–10

    Article  PubMed  CAS  Google Scholar 

  • Ziv I, Barzilai A, Offen D, Nardi N, Melamed E (1997) Nigrostriatal neuronal death in Parkinson's disease—a passive or an active genetically-controlled process? J Neural Transm Suppl 49:69–76

    PubMed  CAS  Google Scholar 

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Acknowledgments

We are grateful to Dr J. Reiser of the Louisiana State University for his kind gifts of lentivirus vectors. This study was supported in part by a grant from NRC (project-133).

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Authors and Affiliations

  1. Molecular Genetics Group, National Institute of Genetic Engineering and Biotechnology (NIGEB), Pajoohesh Blvd, Tehran-Karaj HWY, Kilometer 15, PO Box 14965/161, Tehran, Iran

    Roya Safi, Mossa Gardaneh, Yasin Panahi, Mohammad Zaefizadeh & Ehsan Gharib

  2. Neuroscience Research Center (NRC), Shahid Beheshti University of Medical Sciences, Tehran, IR, Iran

    Mossa Gardaneh & Nader Maghsoudi

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  1. Roya Safi
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  2. Mossa Gardaneh
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Safi, R., Gardaneh, M., Panahi, Y. et al. Optimized Quantities of GDNF Overexpressed by Engineered Astrocytes Are Critical for Protection of Neuroblastoma Cells Against 6-OHDA Toxicity. J Mol Neurosci 46, 654–665 (2012). https://doi.org/10.1007/s12031-011-9654-8

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