Poly(ADP-Ribose) Polymerase-1 Causes Mitochondrial Damage and Neuron Death Mediated by Bnip3

Abstract

Excessive pathophysiological activity of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP1) causes neuron death in brain hypoxia/ischemia by inducing mitochondrial permeability transition and nuclear translocation of apoptosis-inducing factor (AIF). Bcl-2/adenovirus E1B 19 kDa-interacting protein (Bnip3) is a prodeath BH3-only Bcl-2 protein family member that is induced in hypoxia, and has effects on mitochondrial permeability and neuronal survival similar to those caused by PARP1 activation. We hypothesized that Bnip3 is a critical mediator of PARP1-induced mitochondrial dysfunction and neuron death. Hypoxic death of mouse cortical neuron cultures was mitigated by deletion of either PARP1 or Bnip3, indicating that both factors are involved. Direct normoxic PARP1 activation by a DNA alkylating agent enhanced Bnip3 expression, and caused Bnip3-dependent mitochondrial membrane permeability, AIF translocation, and neuron death. Hypoxia produced PARP1-dependent depletion of nicotinamide adenine dinucleotide (NAD ϩ ) and inhibition of the NAD ϩ -dependent class III histone deactelyase (HDAC) sirtuin-1 (SIRT1). This, in turn, led to hyperacetylation and nuclear localization of the forkhead box (Fox) protein FoxO3a, followed by enhanced association of FoxO3a with the Bnip3 upstream promoter region, increased levels of Bnip3 transcript, and elevated mitochondrial Bnip3 immunoreactivity. Finally, FoxO3a silencing using a lentiviral short hairpin RNA approach significantly reduced hypoxic Bnip3 expression, mitochondrial damage, and neuron death. Together, these data illustrate a direct PARP1-mediated hypoxic signaling pathway involving NAD ϩ depletion, SIRT1 inhibition, FoxO3a-driven Bnip3 generation, and mitochondrial AIF release.Key words: apoptosis-inducing factor; Bnip3; ischemia; mitochondrial permeability; PARP; sirtuin IntroductionPoly(ADP-ribose) polymerase-1 (PARP1) is a genomic stability enzyme that responds to DNA damage by using nicotinamide adenine dinucleotide (NAD ϩ ) as a substrate to synthesize protein-conjugated chains of ADP-ribose polymers (PARs; Rouleau et al., 2010). PARP1 plays a paradoxically destructive role in pathological DNA damage, causing detrimental cytosolic NAD ϩ consumption, glycolytic inhibition, ATP depletion, and cell death (Cohen and Barankiewicz, 1987). Accordingly, blocking PARP1 activity enhances cell survival in brain trauma (LaPlaca et al., 2001) and ischemia (Eliasson et al., 1997). In brain hypoxia/ ischemia, PARP1 causes mitochondrial permeability transition (MPT), leading to caspase-independent neuron death initiated by nuclear translocation of mitochondrial apoptosis-inducing factor (AIF; Yu et al., 2002;Culmsee et al., 2005). The mechanism by which nuclear PARP1 causes mitochondrial damage is the subject of continued study. There is a line of evidence that PAR binds directly to AIF, causing nuclear translocation (Andrabi et al., 2006). There are also reports that NAD ϩ depletion is a requisite step for AIF release (Alano et al., 2010; Won et al., 2012).Bcl-2/adenovi...

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