Nerve growth factor-mediated inhibition of apoptosis post-caspase activation is due to removal of active caspase-3 in a lysosome-dependent manner.

K Mnich,E T Kavanagh,A M Gorman,K M Doyle,A Samali,L A Carleton

doi:10.1038/cddis.2014.173

Abstract

Nerve growth factor (NGF) is well characterised as an important pro-survival factor in neuronal cells that can inhibit apoptotic cell death upstream of mitochondrial outer membrane permeabilisation. Here we addressed the question of whether NGF can also protect against apoptosis downstream of caspase activation. NGF treatment promoted a rapid reduction in the level of the p17 subunit of active caspase-3 in PC12 cells that had been induced to undergo apoptosis by various cytotoxins. The mechanism involved TrkA-dependent activation of extracellular signal-regulated kinase (ERK1/2) but not phosphatidylinositol 3-kinase (PI3K)/Akt, and de novo protein synthesis. Involvement of inhibitor of apoptosis proteins (IAPs) and proteasomal degradation were ruled out. In contrast, inhibition of lysosome function using chloroquine and concanamycin A reversed NGF-induced removal of p17. Moreover, in NGF-treated cells, active caspases were found to be localised to lysosomes. The involvement of macroautophagy and chaperone-mediated autophagy were ruled out. Taken together, these findings suggest an anti-apoptotic mechanism by which NGF induces removal of active caspase-3 in a lysosome-dependent manner.

Highlights

mitochondrial outer membrane permeabilisation (MOMP) and cytochrome c release are commonly regarded as the commitment point in apoptosis, such that once it occurs the cell is irretrievably destined to die
To establish a suitable time for studying Nerve growth factor (NGF) treatment post-caspase activation, we investigated the kinetics of thapsigargin (TG)-induced apoptosis in PC12 cells
We describe the ability of NGF to protect PC12 cells downstream of MOMP and post-caspase activation via a mechanism that involves the removal of active caspase-3 and requires functional lysosomes

Summary

Introduction

MOMP and cytochrome c release are commonly regarded as the commitment point in apoptosis, such that once it occurs the cell is irretrievably destined to die. Regulation of apoptosis occurs mainly upstream of mitochondrial changes, through the altered expression and/or posttranslational modification of pro- and anti-apoptotic Bcl-2 family members.[6,7] there exist mechanisms for the regulation of caspases downstream of mitochondria. We have previously shown that NGF activation of phosphatidylinositol 3-kinase (PI3K)/Akt signalling can protect cells upstream of MOMP through regulation of pro-apoptotic Bcl-2 family members.[13]. We explored whether NGF could interfere with apoptosis downstream of MOMP, that is, post-caspase activation, using the NGF-responsive cell line PC12. Received 01.8.13; revised 18.2.14; accepted 19.2.14; Edited by A Verkhratsky activation via extracellular signal-regulated kinase (ERK)dependent removal of active caspases to lysosomes

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