Apaf1 inhibition promotes cell recovery from apoptosis

Abstract

The protein apoptotic protease activating factor 1 (Apaf1) is the central component of the apoptosome, a multiprotein complex that activates procaspase-9 after cytochrome c release from the mitochondria in the intrinsic pathway of apoptosis. We have developed a vital method that allows fluorescence-activated cell sorting of cells at different stages of the apoptotic pathway and demonstrated that upon pharmacological inhibition of Apaf1, cells recover from doxorubicin- or hypoxia-induced early apoptosis to normal healthy cell. Inhibiting Apaf1 not only prevents procaspase-9 activation but delays massive mitochondrial damage allowing cell recovery.

Key words： Apaf1; Apaf1 inhibitors; apoptosis; apoptosome; autophagy; cell recovery

References

1 Albeck JG, Burke JM, Aldridge BB, Zhang M, Lauffenburger DA, Sorger PK (2008) Quantitative analysis of pathways controlling extrinsic apoptosis in single cells. Mol Cell 30: 11−25

2 Andreu-Fernandez V, Genoves A, Messeguer A, Orzaez M, Sancho M, Perez-Paya E (2013) BH3-mimetics- and cisplatin-induced cell death proceeds through different pathways depending on the availability of death-related cellular components. PLoS One 8: e56881

3 Boya P, Gonzalez-Polo RA, Casares N, Perfettini JL, Dessen P, Larochette N, Metivier D, Meley D, Souquere S, Yoshimori T (2005) Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol 25: 1025−1040

4 Brenner D, Mak TW (2009) Mitochondrial cell death effectors. Curr Opin Cell Biol 21: 871−877

5 Cheng Y, Deshmukh M, D’Costa A, Demaro JA, Gidday JM, Shah A, Sun Y, Jacquin MF, Johnson EM, Holtzman DM (1998) Caspase inhibitor affords neuroprotection with delayed administration in a rat model of neonatal hypoxic-ischemic brain injury. J Clin Invest 101: 1992−1999

6 Colell A, Ricci JE, Tait S, Milasta S, Maurer U, Bouchier-Hayes L, Fitzgerald P, Guio-Carrion A, Waterhouse NJ, Li CW (2007) GAPDH and autophagy preserve survival after apoptotic cytochrome c release in the absence of caspase activation. Cell 129: 983−997

7 D’Amelio M, Cavallucci V, Middei S, Marchetti C, Pacioni S, Ferri A, Diamantini A, De Zio D, Carrara P, Battistini L (2011) Caspase-3 triggers early synaptic dysfunction in a mouse model of Alzheimer’s disease. Nat Neurosci 14: 69−76

8 Debatin KMFS (2006) Apoptosis and cancer therapy. WILEY-VCH, Weinheim

9 Deshmukh M, Kuida K, Johnson EM Jr (2000) Caspase inhibition extends the commitment to neuronal death beyond cytochrome c release to the point of mitochondrial depolarization. J Cell Biol 150: 131−143

10 Dignam JD, Lebovitz RM, Roeder RG (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11: 1475−1489

11 Fearnhead HO (2001) Cell-free systems to study apoptosis. Methods Cell Biol 66: 167−185

12 Ferraro E, Pulicati A, Cencioni MT, Cozzolino M, Navoni F, di Martino S, Nardacci R, Carri MT, Cecconi F (2008) Apoptosome-deficient cells lose cytochrome c through proteasomal degradation but survive by autophagy-dependent glycolysis. Mol Biol Cell 19: 3576−3588

13 Gao Y, Liang W, Hu X, Zhang W, Stetler RA, Vosler P, Cao G, Chen J (2009) Neuroprotection against hypoxic-ischemic brain injury by inhibiting the apoptotic protease activating factor-1 pathway. Stroke 41: 166−172

14 Green DR, Kroemer G (2005) Pharmacological manipulation of cell death: clinical applications in sight? J Clin Invest 115: 2610−2617

15 Hoeppner DJ, Hengartner MO, Schnabel R (2001) Engulfment genes cooperate with ced-3 to promote cell death in Caenorhabditis elegans<?Pub Caret?>. Nature 412: 202−206

16 Hoglen NC, Chen LS, Fisher CD, Hirakawa BP, Groessl T, Contreras PC (2004) Characterization of IDN-6556 (3-[2-(2-tert-butyl-phenylaminooxalyl)-amino]-propionylamino]-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid): a liver-targeted caspase inhibitor. J Pharmacol Exp Ther 309: 634−640

17 Hotchkiss RS, Karl IE (2003) The pathophysiology and treatment of sepsis. N Engl J Med 348: 138−150

18 Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. Embo J 19: 5720−5728

19 Kabeya Y, Mizushima N, Yamamoto A, Oshitani-Okamoto S, Ohsumi Y, Yoshimori T (2004) LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. J Cell Sci 117: 2805−2812

20 Kunstle G, Leist M, Uhlig S, Revesz L, Feifel R, MacKenzie A, Wendel A (1997) ICE-protease inhibitors block murine liver injury and apoptosis caused by CD95 or by TNF-alpha. Immunol Lett 55: 5−10

21 Linton SD (2005) Caspase inhibitors: a pharmaceutical industry perspective. Curr Top Med Chem 5: 1697−1717

22 MacFarlane M, Merrison W, Bratton SB, Cohen GM (2002) Proteasome-mediated degradation of Smac during apoptosis: XIAP promotes Smac ubiquitination in vitro. J Biol Chem 277: 36611−36616

23 Madeo F, Tavernarakis N, Kroemer G (2010) Can autophagy promote longevity? Nat Cell Biol 12: 842−846

24 Malet G, Martin AG, Orzaez M, Vicent MJ, Masip I, Sanclimens G, Ferrer-Montiel A, Mingarro I, Messeguer A, Fearnhead HO (2006) Small molecule inhibitors of Apaf-1-related caspase- 3/-9 activation that control mitochondrial-dependent apoptosis. Cell Death Differ 13: 1523−1532

25 Martinou I, Desagher S, Eskes R, Antonsson B, Andre E, Fakan S, Martinou JC (1999) The release of cytochrome c from mitochondria during apoptosis of NGF-deprived sympathetic neurons is a reversible event. J Cell Biol 144: 883−889