Activation of PI3-K/Akt pathway for thermal preconditioning to protect cultured cerebellar granule neurons against low potassium-induced apoptosis



This study was designed to investigate whether the activation of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway is required for thermal preconditioning to protect rat cerebellar granule neurons (CGN) against apoptosis induced by low potassium, and to explore the possibility of a link between the upregulated heat shock protein (HSP)70 expression and Akt activation in the acquisition of neuroprotection induced by thermal preconditioning.


CGN cultured for 8 d in vitro were switched to 5K medium for 24 h after thermal preconditioning (TP; 43.5°C for 90 min, then 37°C for 1 h). To study the role of the PI3-K/Akt pathway, a PI3-K inhibitor, LY294002 (20 μmol/L) was added into the cultures 1 h before TP. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay and fluorescein diacetate staining were used to determine cell viability. Hoechst 33258 staining and agar gel electrophoresis were used to test the morphological and biological characters of CGN. Western blot analysis was employed to detect the levels of phospho-Akt, phospho-glycogen synthase kinase 3β (GSK3β) Akt, GSK3β, and HSP70.


TP protected CGN against apoptosis induced by low potassium. LY294002 inhibited the neuroprotective effect on CGN induced by TP. TP induced a robust activation of Akt and the inactivation of GSK3β via PI3-K. Furthermore, the activation of the PI3-K/Akt pathway by TP persisted for 24 h in the 5K cultures. LY294002 (20 μmol/L) failed to inhibit the upregulated HSP70 expression induced by TP.


The activation of the PI3-K/Akt pathway is required for TP to protect CGN against apoptosis induced by low potassium, but the neuroprotective effect by Akt activation is not mediated through the downstream induction of HSP70 expression.


  1. 1

    Sastry PS, Subba RK . Apoptosis and the nervous system. J Neurochem 2000; 74: 1–20.

    CAS  Article  Google Scholar 

  2. 2

    Nijhawan D, Honarpour N, Wang X . Apoptosis in neural development and disease. Annu Rev Neurosci 2000; 23: 73–7.

    CAS  Article  Google Scholar 

  3. 3

    Li Y, Chopp M, Jiang N, Zhang ZG, Zaloga C . Induction of DNA fragmentation after 10 to 120 min focal cerebral ischemia in rats. Stroke 1995; 26: 1252–8.

    CAS  Article  Google Scholar 

  4. 4

    Ohtsuka K, Suzuki T . Roles of molecular chaperones in the nervous system. Brain Res Bull 2000; 53: 141–6.

    CAS  Article  Google Scholar 

  5. 5

    Mailhos C, Howard MK, Latchman DS . Heat shock proteins hsp90 and hsp70 protect neuronal cells from thermal stress but not from programmed cell death. J Neurochem 1994; 63: 1787–95.

    CAS  Article  Google Scholar 

  6. 6

    Bijur GN, Jope RS . Opposing actions of phosphatidylinositol 3-Kinase and glycogen synthase kinase-3 in the regulation of HSF-1 activity. J Neurochem 2000; 75: 2401–8.

    CAS  Article  Google Scholar 

  7. 7

    Maroni P, Bendinelli P, Tiberio L, Rovetta F, Piccoletti R . In vivo heat-shock response in the brain: signalling pathway and transcription factor activation. Brain Res Mol Brain Res 2003; 119: 90–9.

    CAS  Article  Google Scholar 

  8. 8

    Brazil DP, Hemmings BA . Ten years of protein kinase B signalling: a hard Akt to follow. Trends Biochem Sci 2001; 26: 657–64.

    CAS  Article  Google Scholar 

  9. 9

    Kaytor MD, Orr HT . The GSK3 beta signaling cascade and neurodegenerative disease. Curr Opin Neurobiol 2002; 12: 275–8.

    CAS  Article  Google Scholar 

  10. 10

    Goswami R, Kilkus J, Dawson SA, Dawson G . Overexpression of Akt (protein kinase B) confers protection against apoptosis and prevents formation of ceramide in response to pro-apoptotic stimuli. J Neurosci Res 1999; 57: 884–93.

    CAS  Article  Google Scholar 

  11. 11

    D'Mello SR, Galli C, Ciotti T, Calissano P . Induction of apoptosis in cerebellar granule neurons by low potassium: inhibition of death by insulin-like growth factor I and cAMP. Proc Natl Acad Sci USA 1993; 90: 10989–93.

    CAS  Article  Google Scholar 

  12. 12

    Yan GM, Ni B, Weller M, Wood KA, Paul SM . Depolarization or glutamate receptor activation blocks apoptotic cell death of cultured cerebellar granule neurons. Brain Res 1994; 656: 43–51.

    CAS  Article  Google Scholar 

  13. 13

    Chen LJ, Su XW, Qiu PX, Huang YJ, Yan GM . Thermal preconditioning protected cerebellar granule neurons of rat by modulating HSP70 expression. Acta Pharmacol Sin 2004; 25: 458–61.

    CAS  PubMed  Google Scholar 

  14. 14

    Castoldi AF, Barni S, Randine G, Costa LG, Manzo L . Ethanol selectively interferes with the trophic action of NMDA and car-bachol on cultured cerebellar granule neurons undergoing apoptosis. Brain Res Dev Brain Res 1998; 111: 279–89.

    CAS  Article  Google Scholar 

  15. 15

    Zhao LZ, Su XW, Huang YJ, Qiu PX, Yan GM . Activation of c-Jun and suppression of phospho-p44/42 were involved in diphenylhydantoin-induced apoptosis of cultured rat cerebellar granule neurons. Acta Pharmacol Sin 2003; 24: 539–48.

    CAS  PubMed  Google Scholar 

  16. 16

    McAlister L, Finkelstein DB . Heat shock proteins and thermal resistance in yeast. Biochem Biophys Res Commun 1980; 93: 819–24.

    CAS  Article  Google Scholar 

  17. 17

    Wang JL, Ke DS, Lin MT . Heat shock pretreatment may protect against heatstroke-induced circulatory shock and cerebral ischemia by reducing oxidative stress and energy depletion. Shock 2005; 23: 161–7.

    Article  Google Scholar 

  18. 18

    Ikeda T, Ikenoue T, Xia XY, Xia YX . Important role of 72-kd heat shock protein expression in the endothelial cell in acquisition of hypoxic-ischemic tolerance in the immature rat. Am J Obstet Gynecol 2000; 182: 380–6.

    CAS  Article  Google Scholar 

  19. 19

    Basaran M, Kafali E, Sayin O, Ugurlucan M, Us MH, Bayindir C, et al. Heat stress increases the effectiveness of early ischemic preconditioning in spinal cord protection. Eur J Cardiothorac Surg. 2005; 28: 467–72.

    Article  Google Scholar 

  20. 20

    Kwong JM, Lam TT, Caprioli J . Hyperthermic pre-conditioning protects retinal neurons from N-methyl-D-aspartate (NMDA)-induced apoptosis in rat. Brain Res 2003; 970: 119–30.

    CAS  Article  Google Scholar 

  21. 21

    Rordorf G, Koroshetz WJ, Bonventre JV . Heat shock protects cultured neurons from glutamate toxicity. Neuron 1991; 7: 1043–51.

    CAS  Article  Google Scholar 

  22. 22

    Fan GH, Qi C, Chen SD . Heat shock proteins reduce toxicity of 1-methyl-4-phenylpyridinium ion in SK-N-SH cells. J Neurosci Res 2005; 82: 551–62.

    CAS  Article  Google Scholar 

  23. 23

    Edwards MJ . Apoptosis, the heat shock response, hyperthermia, birth defects, disease and cancer. Where are the common links?*. Cell Stress Chaperones 1998; 3: 213–20.

    CAS  Article  Google Scholar 

  24. 24

    Jaattela M . Heat shock proteins as cellular lifeguards. Ann Med 1999; 31: 261–71.

    CAS  Article  Google Scholar 

  25. 25

    Pap M, Cooper GM . Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-kinase/Akt cell survival pathway. J Biol Chem 1998; 273: 19929–32.

    CAS  Article  Google Scholar 

  26. 26

    Magrane J, Rosen KM, Smith RC, Walsh K, Gouras GK, Querfurth HW . Intraneuronal beta-amyloid expression downregulates the Akt survival pathway and blunts the stress response. J Neurosci 2005; 25: 10960–9.

    CAS  Article  Google Scholar 

  27. 27

    Zhou J, Schmid T, Frank R, Brune B . PI3K/Akt is required for heat shock proteins to protect hypoxia-inducible factor 1α from pVHL-independent degradation. J Biol Chem 2004; 279: 13506–13.

    CAS  Article  Google Scholar 

Download references

Author information



Corresponding author

Correspondence to Guang-mei Yan.

Additional information

Project supported by the National Natural Science Foundation of China (No 30472010), the team project grants of the Natural Scientific Foundation of Guangdong Province (No 039191), and the major project grants from the Department of Science and Technology of Guangdong Province (No 2003A10905).

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Cao, L., Cao, Dx., Su, Xw. et al. Activation of PI3-K/Akt pathway for thermal preconditioning to protect cultured cerebellar granule neurons against low potassium-induced apoptosis. Acta Pharmacol Sin 28, 173–179 (2007).

Download citation


  • thermal preconditioning
  • heat
  • cerebellum
  • neurons
  • apoptosis
  • LY294002
  • phospho-Akt
  • phospho-GSK3β
  • HSP70

Further reading