Bid-induced release of AIF from mitochondria causes immediate neuronal cell death

Abstract

Mitochondrial dysfunction and release of pro-apoptotic factors such as cytochrome c or apoptosis-inducing factor (AIF) from mitochondria are key features of neuronal cell death. The precise mechanisms of how these proteins are released from mitochondria and their particular role in neuronal cell death signaling are however largely unknown. Here, we demonstrate by fluorescence video microscopy that 8-10 h after induction of glutamate toxicity, AIF rapidly translocates from mitochondria to the nucleus and induces nuclear fragmentation and cell death within only a few minutes. This markedly fast translocation of AIF to the nucleus is preceded by increasing translocation of the pro-apoptotic bcl-2 family member Bid (BH3-interacting domain death agonist) to mitochondria, perinuclear accumulation of Bid-loaded mitochondria, and loss of mitochondrial membrane integrity. A small molecule Bid inhibitor preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated glutamate-induced neuronal cell death, as shown by experiments using Bid small interfering RNA (siRNA). Cell death induced by truncated Bid was inhibited by AIF siRNA, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that although caspase-3 was activated, specific caspase-3 inhibition did not protect neuronal cells against glutamate toxicity. In conclusion, Bid-mediated mitochondrial release of AIF followed by rapid nuclear translocation is a major mechanism of glutamate-induced neuronal death. Progressive degeneration and death of neurons are the major features of several acute and chronic neurodegenerative diseases such as ischemic stroke, Alzheimer's disease, or Parkinson's disease. 1 The main mechanisms of neuronal cell death are, for example, disturbed calcium homeostasis, oxidative stress, breakdown of the mitochondrial membrane potential, and release of mitochondrial factors that initiate downstream apoptotic cell death programs.2 In particular, mitochondrial membrane permeabilization is considered as a critical step for the release of pro-apoptotic proteins such as cytochrome c, Smac/Diablo (second mitochondria-derived activator of caspase/direct IAP binding protein with low pI), HtrA2/Omi, apoptosis-inducing factor (AIF), or endonuclease G, which trigger caspase-dependent or caspase-independent mechanisms of DNA degradation and cell death. 3,4 An increasing number of recent studies provide evidence that AIF is a major factor for an alternative post-mitochondrial cell death pathway, for example, following hypoxia, 5,6 ischemia, 7,8 or excitotoxic lesions. 9,10 AIF is a 63 kDa flavoprotein located at the inner mitochondrial membrane that is released early after oxygen-glucose deprivation in vitro or cerebral ischemia in vivo.7 Using harlequin (Hq) mutant mice expressing low AIF levels and small interfering RNA (siRNA) approaches, we recently demonstrated a causal role of AIF in neuronal cell death in models of i...

Inhibition of Drp1 provides neuroprotection in vitro and in vivo

“…Further, depolarization of the MOM and mitochondrial fragmentation are both important key events of apoptosis induced by oxidative stress, and both events were apparently Bid dependent ( Supplementary Figures 8a-d). 5,19 The pivotal role for Bid upstream of such mitochondrial dysfunction is substantiated by experiments demonstrating pronounced neuroprotective effects of the Bid inhibitor BI-6c9 in models of glutamate-induced excitotoxicity and OGD in primary cultured neurons, and similar studies in HT-22 neurons using Bid siRNA. 19,20,30,31 Further, reduced Bid expression attenuated neuronal death in a model of OGD in vitro and reduced brain damage in models of cerebral ischemia and brain trauma in vivo.…”

“…5,19 The pivotal role for Bid upstream of such mitochondrial dysfunction is substantiated by experiments demonstrating pronounced neuroprotective effects of the Bid inhibitor BI-6c9 in models of glutamate-induced excitotoxicity and OGD in primary cultured neurons, and similar studies in HT-22 neurons using Bid siRNA. 19,20,30,31 Further, reduced Bid expression attenuated neuronal death in a model of OGD in vitro and reduced brain damage in models of cerebral ischemia and brain trauma in vivo. 21 Recent studies showed that tBid and full-length Bid exert similar effects on mitochondria and AIF-dependent cell death in neurons.…”

“…21 Recent studies showed that tBid and full-length Bid exert similar effects on mitochondria and AIF-dependent cell death in neurons. 19,32 Mitochondrial translocation of full-length Bid was sufficient to induce Bax oligomerization and integration into the outer mitochondrial membrane in Hela cells 33 and to induce mitochondrial dysfunction in neurons. 32 Activated tBid induces rapid mitochondrial demise in caspase-dependent death receptor mediated pathways, while full-length Bidmediated caspase-independent cell death pathways, however with slower kinetics compared with tBid.…”

“…Our recent data demonstrated a role for the proapoptotic BH3-only protein Bid for enhanced mitochondrial fragmentation in the present model systems of HT-22 cell oxytosis and primary neuron cell death induced by glutamate or OGD. 5,19 We documented that Bid is involved in the loss of MMP and release of mitochondrial AIF, which mediates delayed neuronal death after cerebral ischemia. [19][20][21] Here, we examined the role of Drp1 in Bid-mediated neuronal cell death and observed that treatment of cells with mdiviA or mdiviB also prevented tBid-induced mitochondrial fission and preserved cell viability ( Figure 5).…”

“…5,19 We documented that Bid is involved in the loss of MMP and release of mitochondrial AIF, which mediates delayed neuronal death after cerebral ischemia. [19][20][21] Here, we examined the role of Drp1 in Bid-mediated neuronal cell death and observed that treatment of cells with mdiviA or mdiviB also prevented tBid-induced mitochondrial fission and preserved cell viability ( Figure 5). Vector-induced expression of tBid reduced cell viability to 30% of control levels, whereas cell viability was restored to control levels when the Drp1 inhibitors were applied to tBid expressing cells ( Figure 5c).…”