Skip to main content
SpringerLink
Log in

Pifithrin-α provides neuroprotective effects at the level of mitochondria independently of p53 inhibition

  • Original Paper
  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

Impaired mitochondrial integrity and function are key features of intrinsic death pathways in neuronal cells. Therefore, key regulators of intrinsic death pathways acting upstream of mitochondria are potential targets for therapeutic approaches of neuroprotection. The tumor suppressor p53 is a well-established regulator of cellular responses towards different kinds of lethal stress, including oxidative stress. Recent reports suggested that p53 may affect mitochondrial integrity and function through both, transcriptional activation of mitochondria-targeted pro-death proteins and direct effects at the mitochondrial membrane. In the present study, we compared the effects of pharmacological inhibition of p53 by pifithrin-α with those of selective p53 gene silencing by RNA interference. Using MTT assay and real-time cell impedance measurements we confirmed the protective effect of both strategies against glutamate-induced oxidative stress in immortalized mouse hippocampal HT-22 neurons. Further, we observed full restoration of mitochondrial membrane potential and inhibition of glutamate-induced mitochondrial fragmentation by pifithrin-α which was, in contrast, not achieved by p53 gene silencing. Downregulation of p53 by siRNA decreased p53 transcriptional activity and reduced expression levels of p21 mRNA, while pifithrin-α did not affect these endpoints. These results suggest a neuroprotective effect of pifithrin-α which occurred at the level of mitochondria and independently of p53 inhibition.

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
Fig. 6

Similar content being viewed by others

Abbreviations

AIF:

Apoptosis inducing factor

AD:

Alzheimer’s disease

ANOVA:

Analysis of variance

Bid:

BH3 interacting-domain death agonist

CCCP:

Carbonyl cyanide 3-chlorophenylhydrazone

DRP1:

Dynamin-related protein 1

FACS:

Fluorescence-activated cell sorting

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

LPS:

Lipopolysaccharide

MDM2:

Mouse double minute 2 homolog

MMP:

Mitochondrial membrane potential

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

PCD:

Programmed cell death

PD:

Parkinson’s disease

PFTα:

Pifithrin-α

pMCAO:

Permanent middle cerebral artery occlusion

PUMA:

p53 upregulated modulator of apoptosis

ROS:

Reactive oxygen species

TIF-IA:

Pol I-specific transcription initiation factor IA

TMRE:

Tetramethylrhodamin ethyl ester

References

  1. Culmsee C, Landshamer S (2006) Molecular insights into mechanisms of the cell death program: role in the progression of neurodegenerative disorders. Curr Alzheimer Res 3(4):269–283

    Article  CAS  PubMed  Google Scholar 

  2. Mattson MP (1998) Modification of ion homeostasis by lipid peroxidation: roles in neuronal degeneration and adaptive plasticity. Trends Neurosci 21(2):53–57

    Article  CAS  PubMed  Google Scholar 

  3. Lin MT, Beal MF (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443(7113):787–795

    Article  CAS  PubMed  Google Scholar 

  4. Grohm J, Plesnila N, Culmsee C (2010) Bid mediates fission, membrane permeabilization and peri-nuclear accumulation of mitochondria as a prerequisite for oxidative neuronal cell death. Brain Behav Immun 24(5):831–838

    Article  CAS  PubMed  Google Scholar 

  5. Tobaben S, Grohm J, Seiler A, Conrad M, Plesnila N, Culmsee C (2011) Bid-mediated mitochondrial damage is a key mechanism in glutamate-induced oxidative stress and AIF-dependent cell death in immortalized HT-22 hippocampal neurons. Cell Death Differ 18(2):282–292

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Galluzzi L, Blomgren K, Kroemer G (2009) Mitochondrial membrane permeabilization in neuronal injury. Nat Rev Neurosci 10(7):481–494

    Article  CAS  PubMed  Google Scholar 

  7. Zhang J, Cao Q, Li S, Lu X, Zhao Y, Guan J, Chen J, Wu Q, Chen G (2013) 3-hydroxybutyrate methyl ester as a potential drug against Alzheimer’s disease via mitochondria protection mechanism. Biomaterials 34(30):7552–7562

    Article  CAS  PubMed  Google Scholar 

  8. Marques-Aleixo I, Oliveira PJ, Moreira PI, Magalhães J, Ascensão A (2012) Physical exercise as a possible strategy for brain protection: evidence from mitochondrial-mediated mechanisms. Prog Neurobiol 99(2):149–162

    Article  CAS  PubMed  Google Scholar 

  9. Checler F, da Costa CA (2014) p53 in neurodegenerative diseases and brain cancers. Pharmacol Ther 142(1):99–113

    Article  CAS  PubMed  Google Scholar 

  10. Tomasevic G, Shamloo M, Israeli D, Wieloch T (1999) Activation of p53 and its target genes p21WAF1/Cip1 and PAG608/Wig-1 in ischemic preconditioning. Mol Brain Res 70(2):304–313

    Article  CAS  PubMed  Google Scholar 

  11. Culmsee C, Mattson MP (2005) p53 in neuronal apoptosis. Biochem. Biophys. Res. Commun. 331(3):761–777

    Article  CAS  PubMed  Google Scholar 

  12. Vousden KH, Prives C (2009) Blinded by the light: the growing complexity of p53. Cell 137(3):413–431

    Article  CAS  PubMed  Google Scholar 

  13. Muller Patricia A J, Vousden KH (2013) p53 mutations in cancer. Nat Cell Biol 15(1):2–8

    Article  CAS  PubMed  Google Scholar 

  14. Xiang H, Hochman DW, Saya H, Fujiwara T, Schwartzkroin PA, Morrison RS (1996) Evidence for p53-mediated modulation of neuronal viability. J Neurosci 16(21):6753–6765

    CAS  PubMed  Google Scholar 

  15. Culmsee C, Siewe J, Junker V, Retiounskaia M, Schwarz S, Camandola S, El-Metainy S, Behnke H, Mattson MP, Krieglstein J (2003) Reciprocal inhibition of p53 and nuclear factor-kappaB transcriptional activities determines cell survival or death in neurons. J Neurosci 23(24):8586–8595

    CAS  PubMed  Google Scholar 

  16. Plesnila N, von Baumgarten L, Retiounskaia M, Engel D, Ardeshiri A, Zimmermann R, Hoffmann F, Landshamer S, Wagner E, Culmsee C (2007) Delayed neuronal death after brain trauma involves p53-dependent inhibition of NF-kappaB transcriptional activity. Cell Death Differ 14(8):1529–1541

    Article  CAS  PubMed  Google Scholar 

  17. Green DR, Kroemer G (2009) Cytoplasmic functions of the tumour suppressor p53. Nature 458(7242):1127–1130

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Vaseva AV, Marchenko ND, Ji K, Tsirka SE, Holzmann S, Moll UM (2012) p53 opens the mitochondrial permeability transition pore to trigger necrosis. Cell 149(7):1536–1548

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Nijboer CH, Heijnen CJ, van der Kooij MA, Zijlstra J, van Velthoven CTJ, Culmsee C, van Bel F, Hagberg H, Kavelaars A (2011) Targeting the p53 pathway to protect the neonatal ischemic brain. Ann Neurol 70(2):255–264

    Article  CAS  PubMed  Google Scholar 

  20. Culmsee C, Zhu X, Yu QS, Chan SL, Camandola S, Guo Z, Greig NH, Mattson MP (2001) A synthetic inhibitor of p53 protects neurons against death induced by ischemic and excitotoxic insults, and amyloid beta-peptide. J Neurochem 77(1):220–228

    Article  CAS  PubMed  Google Scholar 

  21. Zhu G, Wang X, Wu S, Li Q (2012) Involvement of activation of PI3K/Akt pathway in the protective effects of puerarin against MPP+-induced human neuroblastoma SH-SY5Y cell death. Neurochem Int 60(4):400–408

    Article  CAS  PubMed  Google Scholar 

  22. Komarov PG, Komarova EA, Kondratov RV, Christov-Tselkov K, Coon JS, Chernov MV, Gudkov AV (1999) A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy. Science 285(5434):1733–1737

    Article  CAS  PubMed  Google Scholar 

  23. Duan W, Zhu X, Ladenheim B, Yu Q, Guo Z, Oyler J, Cutler RG, Cadet JL, Greig NH, Mattson MP (2002) p53 inhibitors preserve dopamine neurons and motor function in experimental parkinsonism. Ann Neurol 52(5):597–606

    Article  CAS  PubMed  Google Scholar 

  24. Chou J, Greig NH, Reiner D, Hoffer BJ, Wang Y (2011) Enhanced survival of dopaminergic neuronal transplants in hemiparkinsonian rats by the p53 inactivator PFT-α. Cell Transpl 20(9):1351–1359

    Article  CAS  Google Scholar 

  25. Engel T, Murphy BM, Hatazaki S, Jimenez-Mateos EM, Concannon CG, Woods I, Prehn JHM, Henshall DC (2010) Reduced hippocampal damage and epileptic seizures after status epilepticus in mice lacking proapoptotic Puma. FASEB J 24(3):853–861

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Hoagland MS, Hoagland EM, Swanson HI (2005) The p53 inhibitor pifithrin-alpha is a potent agonist of the aryl hydrocarbon receptor. J Pharmacol Exp Ther 314(2):603–610

    Article  CAS  PubMed  Google Scholar 

  27. Murphy PJM, Galigniana MD, Morishima Y, Harrell JM, Kwok RPS, Ljungman M, Pratt WB (2004) Pifithrin-alpha inhibits p53 signaling after interaction of the tumor suppressor protein with hsp90 and its nuclear translocation. J Biol Chem 279(29):30195–30201

    Article  CAS  PubMed  Google Scholar 

  28. Komarova EA, Neznanov N, Komarov PG, Chernov MV, Wang K, Gudkov AV (2003) p53 inhibitor pifithrin alpha can suppress heat shock and glucocorticoid signaling pathways. J Biol Chem 278(18):15465–15468

    Article  CAS  PubMed  Google Scholar 

  29. Schutte B, Nuydens R, Geerts H, Ramaekers F (1998) Annexin V binding assay as a tool to measure apoptosis in differentiated neuronal cells. J Neurosci Methods 86(1):63–69

    Article  CAS  PubMed  Google Scholar 

  30. Grohm J, Kim S, Mamrak U, Tobaben S, Cassidy-Stone A, Nunnari J, Plesnila N, Culmsee C (2012) Inhibition of Drp1 provides neuroprotection in vitro and in vivo. Cell Death Differ 19(9):1446–1458

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Tan S, Sagara Y, Liu Y, Maher P, Schubert D (1998) The regulation of reactive oxygen species production during programmed cell death. J Cell Biol 141(6):1423–1432

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  32. Li Y, Maher P, Schubert D (1997) A role for 12-lipoxygenase in nerve cell death caused by glutathione depletion. Neuron 19(2):453–463

    Article  CAS  PubMed  Google Scholar 

  33. Vousden KH (2000) p53: death star. Cell 103(5):691–694

    Article  CAS  PubMed  Google Scholar 

  34. Rocha S, Campbell KJ, Roche KC, Perkins ND (2003) The p53-inhibitor pifithrin-alpha inhibits firefly luciferase activity in vivo and in vitro. BMC Mol Biol 4:9

    Article  PubMed Central  PubMed  Google Scholar 

  35. Yonekura I, Takai K, Asai A, Kawahara N, Kirino T (2006) p53 potentiates hippocampal neuronal death caused by global ischemia. J Cereb Blood Flow Metab 26(10):1332–1340

    Article  CAS  PubMed  Google Scholar 

  36. Crumrine RC, Thomas AL, Morgan PF (1994) Attenuation of p53 expression protects against focal ischemic damage in transgenic mice. J Cereb Blood Flow Metab 14(6):887–891

    Article  CAS  PubMed  Google Scholar 

  37. Rieker C, Engblom D, Kreiner G, Domanskyi A, Schober A, Stotz S, Neumann M, Yuan X, Grummt I, Schütz G, Parlato R (2011) Nucleolar disruption in dopaminergic neurons leads to oxidative damage and parkinsonism through repression of mammalian target of rapamycin signaling. J Neurosci 31(2):453–460

    Article  CAS  PubMed  Google Scholar 

  38. Kreiner G, Bierhoff H, Armentano M, Rodriguez-Parkitna J, Sowodniok K, Naranjo JR, Bonfanti L, Liss B, Schütz G, Grummt I, Parlato R (2013) A neuroprotective phase precedes striatal degeneration upon nucleolar stress. Cell Death Differ 20(11):1455–1464

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  39. Mendjargal A, Odkhuu E, Koide N, Nagata H, Kurokawa T, Nonami T, Yokochi T (2013) Pifithrin-α, a pharmacological inhibitor of p53, downregulates lipopolysaccharide-induced nitric oxide production via impairment of the MyD88-independent pathway. Int Immunopharmacol 15(4):671–678

    Article  CAS  PubMed  Google Scholar 

  40. Gilman CP, Chan SL, Guo Z, Zhu X, Greig N, Mattson MP (2003) p53 is present in synapses where it mediates mitochondrial dysfunction and synaptic degeneration in response to DNA damage, and oxidative and excitotoxic insults. Neuromol Med 3(3):159–172

    Article  CAS  Google Scholar 

  41. Duan W, Li Q, Xia M, Tashiro S, Onodera S, Ikejima T (2011) Silibinin activated p53 and induced autophagic death in human fibrosarcoma HT1080 cells via reactive oxygen species-p38 and c-Jun N-terminal kinase pathways. Biol Pharm Bull 34(1):47–53

    Article  CAS  PubMed  Google Scholar 

  42. Strom E, Sathe S, Komarov PG, Chernova OB, Pavlovska I, Shyshynova I, Bosykh DA, Burdelya LG, Macklis RM, Skaliter R, Komarova EA, Gudkov AV (2006) Small-molecule inhibitor of p53 binding to mitochondria protects mice from gamma radiation. Nat Chem Biol 2(9):474–479

    Article  CAS  PubMed  Google Scholar 

  43. Beckerman R, Prives C (2010) Transcriptional regulation by p53. Cold Spring Harb Perspect Biol 2(8):a000935

    Article  PubMed Central  PubMed  Google Scholar 

  44. Toledo F, Wahl GM (2006) Regulating the p53 pathway: in vitro hypotheses, in vivo veritas. Nat Rev Cancer 6(12):909–923

    Article  CAS  PubMed  Google Scholar 

  45. Yu J, Wang Z, Kinzler KW, Vogelstein B, Zhang L (2003) PUMA mediates the apoptotic response to p53 in colorectal cancer cells. Proc Natl Acad Sci USA 100(4):1931–1936

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  46. Yu J, Zhang L (2005) The transcriptional targets of p53 in apoptosis control. Biochem Biophys Res Commun 331(3):851–858

    Article  CAS  PubMed  Google Scholar 

  47. Leker RR, Aharonowiz M, Greig NH, Ovadia H (2004) The role of p53-induced apoptosis in cerebral ischemia: effects of the p53 inhibitor pifithrin alpha. Exp Neurol 187(2):478–486

    Article  CAS  PubMed  Google Scholar 

  48. Nakano K, Vousden KH (2001) PUMA, a novel proapoptotic gene, is induced by p53. Mol Cell 7(3):683–694

    Article  CAS  PubMed  Google Scholar 

  49. Niizuma K, Endo H, Nito C, Myer DJ, Chan PH (2009) Potential role of PUMA in delayed death of hippocampal CA1 neurons after transient global cerebral ischemia. Stroke 40(2):618–625

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  50. Jansson M, Durant ST, Cho E, Sheahan S, Edelmann M, Kessler B, La T, Nicholas B (2008) Arginine methylation regulates the p53 response. Nat Cell Biol 10(12):1431–1439

    Article  CAS  PubMed  Google Scholar 

  51. Diemert S, Dolga AM, Tobaben S, Grohm J, Pfeifer S, Oexler E, Culmsee C (2012) Impedance measurement for real time detection of neuronal cell death. J Neurosci Methods 203(1):69–77

    Article  CAS  PubMed  Google Scholar 

  52. Hermeking H, Lengauer C, Polyak K, He TC, Zhang L, Thiagalingam S, Kinzler KW, Vogelstein B (1997) 14-3-3 sigma is a p53-regulated inhibitor of G2/M progression. Mol Cell 1(1):3–11

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank the excellent technical support by Mrs. Katharina Elsässer and Eileen Daube. Moreover, we thank Mrs. Emma Esser for careful editing of the manuscript and Roche Diagnostics GmbH for providing support with the xCELLigence system.

Conflict of interest

The authors declare that they have no conflict of interests.

Author information

Authors and Affiliations

  1. Institut für Pharmakologie und Klinische Pharmazie, Fachbereich Pharmazie, Philipps-Universität Marburg, Karl-von-Frisch-Straße 1, 35032, Marburg, Germany

    Sandra Neitemeier, Goutham K. Ganjam, Sebastian Diemert & Carsten Culmsee

Authors
  1. Sandra Neitemeier
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Goutham K. Ganjam
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sebastian Diemert
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Carsten Culmsee
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Carsten Culmsee.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (TIFF 783 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Neitemeier, S., Ganjam, G.K., Diemert, S. et al. Pifithrin-α provides neuroprotective effects at the level of mitochondria independently of p53 inhibition. Apoptosis 19, 1665–1677 (2014). https://doi.org/10.1007/s10495-014-1048-2

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-014-1048-2

Keywords

Search

Navigation