Skip to main content

PKCγ and PKCε are Differentially Activated and Modulate Neurotoxic Signaling Pathways During Oxygen Glucose Deprivation in Rat Cortical Slices

Neurochemical Research volume 44, pages 2577–2589 (2019)Cite this article

Abstract

Cerebral ischemia is known to trigger a series of intracellular events such as changes in metabolism, membrane function and intracellular transduction, which eventually leads to cell death. Many of these processes are mediated by intracellular signaling cascades that involve protein kinase activation. Among all the kinases activated, the serine/threonine kinase family, protein kinase C (PKC), particularly, has been implicated in mediating cellular response to cerebral ischemic and reperfusion injury. In this study, using oxygen–glucose deprivation (OGD) in acute cortical slices as an in vitro model of cerebral ischemia, I show that PKC family of isozymes, specifically PKCγ and PKCε are differentially activated during OGD. Detecting the expression and activation levels of these isozymes in response to different durations of OGD insult revealed an early activation of PKCε and delayed activation of PKCγ, signifying their roles in response to different durations and stages of ischemic stress. Specific inhibition of PKCγ and PKCε significantly attenuated OGD induced cytotoxicity, rise in intracellular calcium, membrane depolarization and reactive oxygen species formation, thereby enhancing neuronal viability. This study clearly suggests that PKC family of isozymes; specifically PKCγ and PKCε are involved in OGD induced intracellular responses which lead to neuronal death. Thus isozyme specific modulation of PKC activity may serve as a promising therapeutic route for the treatment of acute cerebral ischemic injury.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. Matsumoto S, Shamloo M, Matsumoto E et al (2004) Protein kinase C-gamma and calcium/calmodulin-dependent protein kinase II-alpha are persistently translocated to cell membranes of the rat brain during and after middle cerebral artery occlusion. J Cereb Blood Flow Metab 24:54–61. https://doi.org/10.1097/01.WCB.0000095920.70924.F5

    CAS  Article  PubMed  Google Scholar 

  2. Baines CP (2010) Role of the mitochondrion in programmed necrosis. Front Physiol. https://doi.org/10.3389/fphys.2010.00156

    Article  PubMed  PubMed Central  Google Scholar 

  3. Newmeyer DD, Ferguson-Miller S (2003) Mitochondria Cell 112:481–490. https://doi.org/10.1016/S0092-8674(03)00116-8

    CAS  Article  PubMed  Google Scholar 

  4. Lan JY, Skeberdis VA, Jover T et al (2001) Protein kinase C modulates NMDA receptor trafficking and gating. Nat Neurosci. https://doi.org/10.1038/86028

    Article  PubMed  Google Scholar 

  5. Majewski H, Iannazzo L (1998) Protein kinase C: a physiological mediator of enhanced transmitter output. Prog Neurobiol 55:463–475. https://doi.org/10.1016/S0301-0082(98)00017-3

    CAS  Article  PubMed  Google Scholar 

  6. Martelli AM, Evangelisti C, Nyakern M, Manzoli FA (2006) Nuclear protein kinase C. Biochim Biophys Acta 1761:542–551. https://doi.org/10.1016/j.bbalip.2006.02.009

    CAS  Article  PubMed  Google Scholar 

  7. Johnson JE, Giorgione J, Newton AC (2000) The C1 and C2 domains of protein kinase C are independent membrane targeting modules, with specificity for phosphatidylserine conferred by the C1 domain. Biochemistry. https://doi.org/10.1021/bi000902c

    Article  PubMed  Google Scholar 

  8. Mochly-Rosen D (1995) Localization of protein kinases by anchoring proteins: a theme in signal transduction. Science. https://doi.org/10.1126/science.7716516

    Article  PubMed  Google Scholar 

  9. Padanilam BJ (2001) Induction and subcellular localization of protein kinase C isozymes following renal ischemia. Kidney Int. https://doi.org/10.1046/j.1523-1755.2001.0590051789.x

    Article  PubMed  Google Scholar 

  10. Piccoletti R, Bendinelli P, Arienti D, Bernelli-Zazzera A (1992) State and activity of protein kinase C in postischemic reperfused liver. Exp Mol Pathol. https://doi.org/10.1016/0014-4800(92)90038-D

    Article  PubMed  Google Scholar 

  11. Speechly-Dick ME, Mocanu MM, Yellon DM (1994) Protein kinase C: its role in ischemic preconditioning in the rat. Circ Res. https://doi.org/10.1161/01.RES.75.3.586

    Article  PubMed  Google Scholar 

  12. Selvatici R, Marino S, Piubello C et al (2003) Protein kinase C activity, translocation, and selective isoform subcellular redistribution in the rat cerebral cortex after in vitro ischemia. J Neurosci Res. https://doi.org/10.1002/jnr.10464

    Article  PubMed  Google Scholar 

  13. Wang J, Bright R, Mochly-Rosen D, Giffard RG (2004) Cell-specific role for ε- And βi-protein kinase C isozymes in protecting cortical neurons and astrocytes from ischemia-like injury. Neuropharmacology. https://doi.org/10.1016/j.neuropharm.2004.03.009

    Article  PubMed  Google Scholar 

  14. Domańska-Janik K, Zalewska T (1992) Effect of Brain Ischemia on Protein Kinase C. J Neurochem. https://doi.org/10.1111/j.1471-4159.1992.tb11360.x

    Article  PubMed  Google Scholar 

  15. Gajkowska B, Domanska-Janik K, Viron A (1994) Protein kinase C-like immunoreactivity in gerbil hippocampus after a transient cerebral ischemia. Folia Histochem Cytobiol 32:71–77

    CAS  PubMed  Google Scholar 

  16. Felipo V, Miñana MD, Grisolía S (1993) Inhibitors of protein kinase C prevent the toxicity of glutamate in primary neuronal cultures. Brain Res. https://doi.org/10.1016/0006-8993(93)90368-W

    Article  PubMed  Google Scholar 

  17. Maiese K, Swiriduk M, TenBroeke M (1996) Cellular mechanisms of protection by metabotropic glutamate receptors during anoxia and nitric oxide toxicity. J Neurochem 66:2419–2428

    CAS  Article  PubMed  Google Scholar 

  18. Hara H, Onodera H, Yoshidomi M et al (1990) Staurosporine, a novel protein kinase C inhibitor, prevents postischemic neuronal damage in the gerbil and rat. J Cereb Blood Flow Metab. https://doi.org/10.1038/jcbfm.1990.117

    Article  PubMed  Google Scholar 

  19. Crumrine RC, Dubyak G, LaManna JC (1990) Decreased protein kinase C activity during cerebral ischemia and after reperfusion in the adult rat. J Neurochem. https://doi.org/10.1111/j.1471-4159.1990.tb05788.x

    Article  PubMed  Google Scholar 

  20. Hara H, Ayata G, Huang PL, Moskowitz MA (1997) Alteration of protein kinase C activity after transient focal cerebral ischemia in mice using in vitro [3H]phorbol-12,13-dibutyrate binding autoradiography. Brain Res 774:69–76

    CAS  Article  PubMed  Google Scholar 

  21. Chakravarthy BR, Wang J, Tremblay R et al (1998) Comparison of the changes in protein kinase C induced by glutamate in primary cortical neurons and by in vivo cerebral ischaemia. Cell Signal 10:291–295

    CAS  Article  PubMed  Google Scholar 

  22. Durkin JP, Tremblay R, Chakravarthy B et al (1997) Evidence that the early loss of membrane protein kinase C is a necessary step in the excitatory amino acid-induced death of primary cortical neurons. J Neurochem 68:1400–1412

    CAS  Article  PubMed  Google Scholar 

  23. Chen L, Hahn H, Wu G et al (2001) Opposing cardioprotective actions and parallel hypertrophic effects of PKC and PKC. Proc Natl Acad Sci. https://doi.org/10.1073/pnas.191369098

    Article  PubMed  Google Scholar 

  24. Ellrén K, Lehmann A (1989) Calcium dependency of n-methyl-d-aspartate toxicity in slices from the immature rat hippocampus. Neuroscience. https://doi.org/10.1016/0306-4522(89)90085-7

    Article  PubMed  Google Scholar 

  25. Mackay K, Mochly-Rosen D (2001) Arachidonic acid protects neonatal rat cardiac myocytes from ischaemic injury through ε protein kinase C. Cardiovasc Res. https://doi.org/10.1016/S0008-6363(00)00322-9

    Article  PubMed  Google Scholar 

  26. O’Donnell BR, Bickler PE (1994) Influence of pH on calcium influx during hypoxia in rat cortical brain slices. Stroke. https://doi.org/10.1161/01.STR.25.1.171

    Article  PubMed  Google Scholar 

  27. Sweitzer SM, Wong SME, Peters MC et al (2004) Protein kinase C epsilon and gamma: involvement in formalin-induced nociception in neonatal rats. J Pharmacol Exp Ther. https://doi.org/10.1124/jpet.103.060350.et

    Article  PubMed  Google Scholar 

  28. Johnson JA, Gray MO, Chen CH, Mochly-Rosen D (1996) A protein kinase C translocation inhibitor as an isozyme-selective antagonist of cardiac function. J Biol Chem. https://doi.org/10.1074/jbc.271.40.24962

    Article  PubMed  Google Scholar 

  29. Crepel V, Krnjevic K, Ben-Ari Y (1992) Developmental and regional differences in the vulnerability of rat hippocampal slices to lack of glucose. Neuroscience 47:579–587

    CAS  Article  PubMed  Google Scholar 

  30. HARA H, SUKAMOTO T, KOGURE K (1993) Mechanism and pathogenesis of ischemia-induced neuronal damage. Prog Neurobiol 40:645–670. https://doi.org/10.1016/0301-0082(93)90009-H

    CAS  Article  PubMed  Google Scholar 

  31. Hawker K, Lang AE (1990) Hypoxic-ischemic damage of the basal ganglia case reports and a review of the literature. Mov Disord. https://doi.org/10.1002/mds.870050306

    Article  PubMed  Google Scholar 

  32. Bickler PE, Hansen BM (1994) Causes of calcium accumulation in rat cortical brain slices during hypoxia and ischemia: role of ion channels and membrane damage. Brain Res. https://doi.org/10.1016/0006-8993(94)91347-1

    Article  PubMed  Google Scholar 

  33. Lushnikova IV, Voronin KY, Malyarevskyy PY, Skibo GG (2004) Morphological and functional changes in rat hippocampal slice cultures after short-term oxygen-glucose deprivation. J Cell Mol Med. https://doi.org/10.1111/j.1582-4934.2004.tb00279.x

    Article  PubMed  PubMed Central  Google Scholar 

  34. Hong SS, Gibney GT, Esquilin M et al (2004) Effect of protein kinases on lactate dehydrogenase activity in cortical neurons during hypoxia. Brain Res. https://doi.org/10.1016/j.brainres.2004.03.004

    Article  PubMed  Google Scholar 

  35. Larsen GA, Berg-Johnsen J, Moe MC, Vinje ML (2004) Calcium-dependent protein kinase C activation in acutely isolated neurons during oxygen and glucose deprivation. Neurochem Res 29:1931–1937

    Article  PubMed  Google Scholar 

  36. Naik MU, Benedikz E, Hernandez I et al (2000) Distribution of protein kinase Mzeta and the complete protein kinase C isoform family in rat brain. J Comp Neurol 426:243–258

    CAS  Article  PubMed  Google Scholar 

  37. Saito N, Shirai Y (2002) Protein kinase C gamma (PKC gamma): function of neuron specific isotype. J Biochem 132:683–687

    CAS  Article  PubMed  Google Scholar 

  38. Di-Capua N, Sperling O, Zoref-Shani E (2003) Protein kinase C-epsilon is involved in the adenosine-activated signal transduction pathway conferring protection against ischemia-reperfusion injury in primary rat neuronal cultures. J Neurochem 84:409–412

    CAS  Article  PubMed  Google Scholar 

  39. Cardell M, Wieloch T (1993) Time course of the translocation and inhibition of protein kinase C during complete cerebral ischemia in the rat. J Neurochem 61:1308–1314

    CAS  Article  PubMed  Google Scholar 

  40. McNamara RK, Wees EA, Lenox RH (1999) Differential subcellular redistribution of protein kinase C isozymes in the rat hippocampus induced by kainic acid. J Neurochem. https://doi.org/10.1046/j.1471-4159.1999.721735.x

    Article  PubMed  Google Scholar 

  41. Koponen S, Kurkinen K, Åkerman KEO et al (2003) Prevention of NMDA-induced death of cortical neurons by inhibition of protein kinase Cζ. J Neurochem. https://doi.org/10.1046/j.1471-4159.2003.01846.x

    Article  PubMed  Google Scholar 

  42. Kurkinen K, Keinanen R, Li W, Koistinaho J (2001) Preconditioning with spreading depression activates specifically protein kinase Cdelta. Neuroreport 12:269–273

    CAS  Article  PubMed  Google Scholar 

  43. Raval AP, Dave KR, Mochly-Rosen D et al (2003) Epsilon PKC is required for the induction of tolerance by ischemic and NMDA-mediated preconditioning in the organotypic hippocampal slice. J Neurosci 23:384–391

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. Harada K, Maekawa T, Abu Shama KM et al (1999) Translocation and down-regulation of protein kinase C-alpha, -beta, and -gamma isoforms during ischemia-reperfusion in rat brain. J Neurochem 72:2556–2564

    CAS  Article  PubMed  Google Scholar 

  45. Lee HW, Smith L, Pettit GR, Smith JB (1997) Bryostatin 1 and phorbol ester down-modulate protein kinase C-alpha and -epsilon via the ubiquitin/proteasome pathway in human fibroblasts. Mol Pharmacol 51:439–447

    CAS  PubMed  Google Scholar 

  46. Choi DW (1995) Calcium: still center-stage in hypoxic-ischemic neuronal death. Trends Neurosci 18:58–60

    CAS  Article  PubMed  Google Scholar 

  47. Arundine M, Tymianski M (2004) Molecular mechanisms of glutamate-dependent neurodegeneration in ischemia and traumatic brain injury. Cell Mol Life Sci 61:657–668. https://doi.org/10.1007/s00018-003-3319-x

    CAS  Article  PubMed  Google Scholar 

  48. Gniel HM, Martin RL (2010) Changes in membrane potential and the intracellular calcium concentration during CSD and OGD in layer V and layer II/III mouse cortical neurons. J Neurophysiol 104:3203–3212. https://doi.org/10.1152/jn.00922.2009

    CAS  Article  PubMed  Google Scholar 

  49. Pisani A, Bonsi P, Calabresi P (2004) Calcium signaling and neuronal vulnerability to ischemia in the striatum. Cell Calcium 36:277–284. https://doi.org/10.1016/j.ceca.2004.02.010

    CAS  Article  PubMed  Google Scholar 

  50. Li H-F, Mochly-Rosen D, Kendig JJ (2005) Protein kinase Cgamma mediates ethanol withdrawal hyper-responsiveness of NMDA receptor currents in spinal cord motor neurons. Br J Pharmacol 144:301–307. https://doi.org/10.1038/sj.bjp.0706033

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. Sanchez-Perez AM, Felipo V (2005) Serines 890 and 896 of the NMDA receptor subunit NR1 are differentially phosphorylated by protein kinase C isoforms. Neurochem Int 47:84–91. https://doi.org/10.1016/j.neuint.2005.04.011

    CAS  Article  PubMed  Google Scholar 

  52. Jonas EA, Knox RJ, Smith TC et al (1997) Regulation by insulin of a unique neuronal Ca2+ pool and of neuropeptide secretion. Nature 385:343–346. https://doi.org/10.1038/385343a0

    CAS  Article  PubMed  Google Scholar 

  53. Mattson MP, LaFerla FM, Chan SL et al (2000) Calcium signaling in the ER: its role in neuronal plasticity and neurodegenerative disorders. Trends Neurosci 23:222–229

    CAS  Article  PubMed  Google Scholar 

  54. Ruiz A, Matute C, Alberdi E (2009) Endoplasmic reticulum Ca(2+) release through ryanodine and IP(3) receptors contributes to neuronal excitotoxicity. Cell Calcium 46:273–281. https://doi.org/10.1016/j.ceca.2009.08.005

    CAS  Article  PubMed  Google Scholar 

  55. Karunasinghe RN, Lipski J (2013) Oxygen and glucose deprivation (OGD)-induced spreading depression in the Substantia Nigra. Brain Res 1527:209–221. https://doi.org/10.1016/j.brainres.2013.06.016

    CAS  Article  PubMed  Google Scholar 

  56. Lipski J, Park TIH, Li D et al (2006) Involvement of TRP-like channels in the acute ischemic response of hippocampal CA1 neurons in brain slices. Brain Res 1077:187–199. https://doi.org/10.1016/j.brainres.2006.01.016

    CAS  Article  PubMed  Google Scholar 

  57. Henrich M, Buckler KJ (2008) Effects of anoxia and aglycemia on cytosolic calcium regulation in rat sensory neurons. J Neurophysiol 100:456–473. https://doi.org/10.1152/jn.01380.2007

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  58. Schuchmann S, Luckermann M, Kulik A et al (2000) Ca(2+)- and metabolism-related changes of mitochondrial potential in voltage-clamped CA1 pyramidal neurons in situ. J Neurophysiol 83:1710–1721. https://doi.org/10.1152/jn.2000.83.3.1710

    CAS  Article  PubMed  Google Scholar 

  59. Sun X, Budas GR, Xu L et al (2013) Selective activation of protein kinase C in mitochondria is neuroprotective in vitro and reduces focal ischemic brain injury in mice. J Neurosci Res 91:799–807. https://doi.org/10.1002/jnr.23186

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  60. Dave KR, DeFazio RA, Raval AP et al (2008) Ischemic preconditioning targets the respiration of synaptic mitochondria via protein kinase C epsilon. J Neurosci 28:4172–4182. https://doi.org/10.1523/JNEUROSCI.5471-07.2008

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  61. Pivovarova NB, Stanika RI, Watts CA et al (2008) Reduced calcium-dependent mitochondrial damage underlies the reduced vulnerability of excitotoxicity-tolerant hippocampal neurons. J Neurochem 104:1686–1699. https://doi.org/10.1111/j.1471-4159.2007.05080.x

    CAS  Article  PubMed  Google Scholar 

  62. Starkov AA, Chinopoulos C, Fiskum G (2004) Mitochondrial calcium and oxidative stress as mediators of ischemic brain injury. Cell Calcium 36:257–264. https://doi.org/10.1016/j.ceca.2004.02.012

    CAS  Article  PubMed  Google Scholar 

  63. Nicholls DG (2009) Mitochondrial calcium function and dysfunction in the central nervous system. Biochim Biophys Acta 1787:1416–1424. https://doi.org/10.1016/j.bbabio.2009.03.010

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  64. Moncada S, Erusalimsky JD (2002) Does nitric oxide modulate mitochondrial energy generation and apoptosis? Nat Rev Mol Cell Biol 3:214–220. https://doi.org/10.1038/nrm762

    CAS  Article  PubMed  Google Scholar 

  65. Barnett ME, Madgwick DK, Takemoto DJ (2007) Protein kinase C as a stress sensor. Cell Signal 19:1820–1829. https://doi.org/10.1016/j.cellsig.2007.05.014

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Support and critical comments from Nanda B. Joshi, Former Professor and, Head of the Department is highly acknowledged. This work was supported by a Research Fellowship awarded to Dayana Surendran by Council of Scientific and Industrial Research (CSIR), Government of India.

Author information

Affiliations

  1. Department of Biophysics, National Institute of Mental Health and Neurosciences, Bengaluru, 560 029, India

    Dayana Surendran

Authors
  1. Dayana Surendran
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dayana Surendran.

Ethics declarations

Conflict of interest

The author declares no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 203 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Surendran, D. PKCγ and PKCε are Differentially Activated and Modulate Neurotoxic Signaling Pathways During Oxygen Glucose Deprivation in Rat Cortical Slices. Neurochem Res 44, 2577–2589 (2019). https://doi.org/10.1007/s11064-019-02876-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11064-019-02876-4

Keywords

  • Cerebral ischemia
  • Oxygen glucose deprivation (OGD)
  • Protein kinase C (PKC)
  • Intracellular calcium ([Ca2+]i)
  • Membrane potential
  • Reactive oxygen species (ROS)