Skip to main content

Advertisement

SpringerLink
Log in

Genetically Controlled Upregulation of Adenosine A1 Receptor Expression Enhances the Survival of Primary Cortical Neurons

  • Published:
Molecular Neurobiology Aims and scope Submit manuscript

Abstract

Adenosine has a key endogenous neuroprotective role in the brain, predominantly mediated by the adenosine A1 receptor (A1R). This has been mainly explored using pharmacological tools and/or receptor knockout mice strains. It has long been suggested that the neuroprotective effects of A1R are increased following receptor upregulation, thus attenuating neuronal damage in pathological conditions. We have previously shown that the neuroprotective and neuromodulatory actions of the cytokines IL-6 and oncostatin M are mediated by induction of neuronal A1R expression. In order to investigate the direct effects of A1R upregulation in neurons, we have generated a tetracycline-regulated expression system with a bidirectional promoter, directing the simultaneous expression of the mouse A1R and GFP/mCherry reporter genes. In a first step, we tested the efficacy of the system in transiently transfected human embryonic kidney 293 cells. In addition, we confirmed the functional integrity of the expressed A1R by whole-cell patch clamp recordings. We demonstrated that A1R-transfected primary neurons show enhanced survival against N-methyl-d-aspartate-induced excitotoxicity. Pretreatment with an A1R-selective agonist additionally strongly decreased neuronal cell death, while an A1R antagonist completely abolished the neuroprotective effects of A1R upregulation. The presented data provide for the first time direct evidence that the upregulation of A1R enhances neuronal survival.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Faden AI, Demediuk P, Panter SS, Vink R (1989) The role of excitatory amino acids and NMDA receptors in traumatic brain injury. Science 244(4906):798–800

    Article  PubMed  CAS  Google Scholar 

  2. Dirnagl U, Iadecola C, Moskowitz MA (1999) Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci 22(9):391–397

    Article  PubMed  CAS  Google Scholar 

  3. Vincent P, Mulle C (2009) Kainate receptors in epilepsy and excitotoxicity. Neuroscience 158(1):309–323

    Article  PubMed  CAS  Google Scholar 

  4. Dong XX, Wang Y, Qin ZH (2009) Molecular mechanisms of excitotoxicity and their relevance to pathogenesis of neurodegenerative diseases. Acta Pharmacol Sin 30(4):379–387

    Article  PubMed  CAS  Google Scholar 

  5. Dunwiddie TV, Masino SA (2001) The role and regulation of adenosine in the central nervous system. Annu Rev Neurosci 24:31–55

    Article  PubMed  CAS  Google Scholar 

  6. Barrie AP, Nicholls DG (1993) Adenosine A1 receptor inhibition of glutamate exocytosis and protein kinase C-mediated decoupling. J Neurochem 60(3):1081–1086

    Article  PubMed  CAS  Google Scholar 

  7. Trussell LO, Jackson MB (1985) Adenosine-activated potassium conductance in cultured striatal neurons. Proc Natl Acad Sci U S A 82(14):4857–4861

    Article  PubMed  CAS  Google Scholar 

  8. de Mendonca A, Sebastiao AM, Ribeiro JA (1995) Inhibition of NMDA receptor-mediated currents in isolated rat hippocampal neurones by adenosine A1 receptor activation. Neuroreport 6(8):1097–1100

    Article  PubMed  Google Scholar 

  9. Gomes CV, Kaster MP, Tome AR, Agostinho PM, Cunha RA (2011) Adenosine receptors and brain diseases: neuroprotection and neurodegeneration. Biochim Biophys Acta 1808(5):1380–1399

    Article  PubMed  CAS  Google Scholar 

  10. Dalpiaz A, Manfredini S (2002) Adenosine A(1) receptor: analysis of the potential therapeutic effects obtained by its activation in the central nervous system. Curr Med Chem 9(21):1923–1937

    PubMed  CAS  Google Scholar 

  11. Stone TW (2002) Purines and neuroprotection. Adv Exp Med Biol 513:249–280

    Article  PubMed  CAS  Google Scholar 

  12. Fredholm BB (2007) Adenosine, an endogenous distress signal, modulates tissue damage and repair. Cell Death Differ 14(7):1315–1323

    Article  PubMed  CAS  Google Scholar 

  13. Schubert P, Ogata T, Marchini C, Ferroni S, Rudolphi K (1997) Protective mechanisms of adenosine in neurons and glial cells. Ann N Y Acad Sci 825:1–10

    Article  PubMed  CAS  Google Scholar 

  14. Biber K, Pinto-Duarte A, Wittendorp MC, Dolga AM, Fernandes CC, Von Frijtag Drabbe Kunzel J, Keijser JN, de Vries R, Ijzerman AP, Ribeiro JA, Eisel U, Sebastiao AM, Boddeke HW (2008) Interleukin-6 upregulates neuronal adenosine A1 receptors: implications for neuromodulation and neuroprotection. Neuropsychopharmacology 33(9):2237–2250

    Article  PubMed  CAS  Google Scholar 

  15. Moidunny S, Dias RB, Wesseling E, Sekino Y, Boddeke HW, Sebastiao AM, Biber K (2010) Interleukin-6-type cytokines in neuroprotection and neuromodulation: oncostatin M, but not leukemia inhibitory factor, requires neuronal adenosine A1 receptor function. J Neurochem 114(6):1667–1677

    Article  PubMed  CAS  Google Scholar 

  16. Wittendorp MC, von Frijtag Drabbe Kunzel J, Ijzerman AP, Boddeke HW, Biber K (2004) The mouse brain adenosine A1 receptor: functional expression and pharmacology. Eur J Pharmacol 487(1–3):73–79

    Article  PubMed  CAS  Google Scholar 

  17. Jiang M, Deng L, Chen G (2004) High Ca(2+)-phosphate transfection efficiency enables single neuron gene analysis. Gene Ther 11(17):1303–1311

    Article  PubMed  CAS  Google Scholar 

  18. Nott A, Meislin SH, Moore MJ (2003) A quantitative analysis of intron effects on mammalian gene expression. RNA 9(5):607–617

    Article  PubMed  CAS  Google Scholar 

  19. Thomas P, Smart TG (2005) HEK293 cell line: a vehicle for the expression of recombinant proteins. J Pharmacol Toxicol Methods 51(3):187–200

    Article  PubMed  CAS  Google Scholar 

  20. Mynlieff M, Beam KG (1994) Adenosine acting at an A1 receptor decreases N-type calcium current in mouse motoneurons. J Neurosci 14(6):3628–3634

    PubMed  CAS  Google Scholar 

  21. Zhu Y, Ikeda SR (1993) Adenosine modulates voltage-gated Ca2+ channels in adult rat sympathetic neurons. J Neurophysiol 70(2):610–620

    PubMed  CAS  Google Scholar 

  22. Berjukow S, Doring F, Froschmayr M, Grabner M, Glossmann H, Hering S (1996) Endogenous calcium channels in human embryonic kidney (HEK293) cells. Br J Pharmacol 118(3):748–754

    Article  PubMed  CAS  Google Scholar 

  23. Vasquez C, Navarro-Polanco RA, Huerta M, Trujillo X, Andrade F, Trujillo-Hernandez B, Hernandez L (2003) Effects of cannabinoids on endogenous K + and Ca2+ currents in HEK293 cells. Can J Physiol Pharmacol 81(5):436–442

    Article  PubMed  CAS  Google Scholar 

  24. Cunha RA (2005) Neuroprotection by adenosine in the brain: from A(1) receptor activation to A (2A) receptor blockade. Purinergic Signal 1(2):111–134

    Article  PubMed  CAS  Google Scholar 

  25. Pagonopoulou O, Angelatou F, Kostopoulos G (1993) Effect of pentylentetrazol-induced seizures on A1 adenosine receptor regional density in the mouse brain: a quantitative autoradiographic study. Neuroscience 56(3):711–716

    Article  PubMed  CAS  Google Scholar 

  26. Vanore G, Giraldez L, de Lores R, Arnaiz G, Girardi E (2001) Seizure activity produces differential changes in adenosine A1 receptors within rat hippocampus. Neurochem Res 26(3):225–230

    Article  PubMed  CAS  Google Scholar 

  27. Angelatou F, Pagonopoulou O, Maraziotis T, Olivier A, Villemeure JG, Avoli M, Kostopoulos G (1993) Upregulation of A1 adenosine receptors in human temporal lobe epilepsy: a quantitative autoradiographic study. Neurosci Lett 163(1):11–14

    Article  PubMed  CAS  Google Scholar 

  28. Fredholm BB (1982) Adenosine actions and adenosine receptors after 1 week treatment with caffeine. Acta Physiol Scand 115(2):283–286

    Article  PubMed  CAS  Google Scholar 

  29. Lupica CR, Berman RF, Jarvis MF (1991) Chronic theophylline treatment increases adenosine A1, but not A2, receptor binding in the rat brain: an autoradiographic study. Synapse 9(2):95–102

    Article  PubMed  CAS  Google Scholar 

  30. Lupica CR, Jarvis MF, Berman RF (1991) Chronic theophylline treatment in vivo increases high affinity adenosine A1 receptor binding and sensitivity to exogenous adenosine in the in vitro hippocampal slice. Brain Res 542(1):55–62

    Article  PubMed  CAS  Google Scholar 

  31. Tsutsui S, Schnermann J, Noorbakhsh F, Henry S, Yong VW, Winston BW, Warren K, Power C (2004) A1 adenosine receptor upregulation and activation attenuates neuroinflammation and demyelination in a model of multiple sclerosis. J Neurosci 24(6):1521–1529

    Article  PubMed  CAS  Google Scholar 

  32. Scholz KP, Miller RJ (1991) Analysis of adenosine actions on Ca2+ currents and synaptic transmission in cultured rat hippocampal pyramidal neurones. J Physiol 435:373–393

    PubMed  CAS  Google Scholar 

  33. McCool BA, Farroni JS (2001) A1 adenosine receptors inhibit multiple voltage-gated Ca2+ channel subtypes in acutely isolated rat basolateral amygdala neurons. Br J Pharmacol 132(4):879–888

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The study was funded by grants from the German Research Council (DFG) (CA 115/5-4) to D.v.C. and K.B. and from the EU FP7 program “MoodInflame” to D.v.C.

Author information

Authors and Affiliations

  1. Department of Psychiatry and Psychotherapy, University of Freiburg, Hauptstrasse 5, 79104, Freiburg, Germany

    Tsvetan Serchov, Hasan-Cem Atas, Claus Normann, Dietrich van Calker & Knut Biber

  2. Department of Neuroscience, University Medical Center Groningen, Ant. Deusinglaan 1, 9713 AV, Groningen, The Netherlands

    Knut Biber

Authors
  1. Tsvetan Serchov
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hasan-Cem Atas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Claus Normann
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dietrich van Calker
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Knut Biber
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Knut Biber.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Suppl. Fig. 1

Representative fluorescence photomicrographs demonstrating the effect of transfection and NMDA challenge (10 μM, 50 μM and 100 μM) on the number of PI-stained dead nuclei (red) of GFP-expressing cells (green) (indicated by arrowheads) vs. the number of unaffected GFP-positive cells. Cell nuclei stained with DAPI (blue). (JPEG 1886 kb)

High resolution image (EPS 3313 kb)

Suppl. Fig. 2

IV-curve of endogenous Ca2+ channels in HEK293 cells. Current-voltage relations were averaged from 11 cells and fitted. The Ba2+ currents reached maximal amplitude around a membrane potential of 0 mV. Both IV-curve and channel kinetics correspond to earlier findings describing characteristics of endogenous Ca2+ channels in HEK293 cells [22, 23]. (JPEG 1825 kb)

High resolution image (EPS 481 kb)

Suppl. Fig. 3

A1R mediated inhibition of voltage-gated Ca2+ channels in transfected primary neurons expressing upregulated A1R. (a) Representative whole-cell voltage-gated Ba2+ currents of the pTet-Off and pTRE-tight-BI-AcGFP (mock transfected) (black triangles; dotted trendline) or pTRE-tight-BI-AcGFP-A1R-BGH (A1R transfected) (red squares; solid trendline) co-transfected primary neurons; (b) Current inhibition by 100 nM CPA in mock (n=2) and A1R (n=2) transfected primary neurons (averaged peak current amplitude in percent ±SEM of baseline currents). (JPEG 1854 kb)

High resolution image (EPS 678 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Serchov, T., Atas, HC., Normann, C. et al. Genetically Controlled Upregulation of Adenosine A1 Receptor Expression Enhances the Survival of Primary Cortical Neurons. Mol Neurobiol 46, 535–544 (2012). https://doi.org/10.1007/s12035-012-8321-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12035-012-8321-6

Keywords

Search

Navigation