NAD+ depletion or PAR polymer formation: which plays the role of executioner in ischaemic cell death?

Abstract

Multiple cell death pathways are activated in cerebral ischaemia. Much of the initial injury, especially in the core of the infarct where cerebral blood flow is severely reduced, is necrotic and secondary to severe energy failure. However, there is considerable evidence that delayed cell death continues for several days, primarily in the penumbral region. As reperfusion therapies grow in number and effectiveness, restoration of blood flow early after injury may lead to a shift towards apoptosis. It is important to elucidate what are the key mediators of apoptotic cell death after stroke, as inhibition of apoptosis may have therapeutic implications. There are two well described pathways that lead to apoptotic cell death; the caspase pathway and the more recently described caspase-independent pathway triggered by poly-ADP-ribose polymers (PARP) activation. Caspase-induced cell death is initiated by release of mitochondrial cytochrome c, formation of the cytosolic apoptosome, and activation of endonucleases leading to a multitude of small randomly cleaved DNA fragments. In contrast caspase-independent cell death is secondary to activation of apoptosis inducing factor (AIF). Mitochondrial AIF translocates to the nucleus, where it induces peripheral chromatin condensation, as well as characteristic high-molecular-weight (50 kbp) DNA fragmentation. Although caspase-independent cell death has been recognized for some time and is known to contribute to ischaemic injury, the upstream triggering events leading to activation of this pathway remain unclear. The two major theories are that ischaemia leads to nicotinamide adenine dinucleotide (NAD+) depletion and subsequent energy failure, or alternatively that cell death is directly triggered by a pro-apoptotic factor produced by activation of the DNA repair enzyme PARP. PARP activation is robust in the ischaemic brain producing variable lengths of poly-ADP-ribose (PAR) polymers as byproducts of PARP activation. PAR polymers may be directly toxic by triggering mitochondrial AIF release independently of NAD+ depletion. Recently, sex differences have been discovered that illustrate the importance of understanding these molecular pathways, especially as new therapeutics targeting apoptotic cell death are developed. Cell death in females proceeds primarily via caspase activation whereas caspase-independent mechanisms triggered by the activation of PARP predominate in the male brain. This review summarizes the current literature in an attempt to clarify the roles of NAD+ and PAR polymers in caspase-independent cell death, and discuss sex specific cell death to provide an example of the possible importance of these downstream mediators.

Figure 1

Caspase-Independent Cell Death Cascade. Ischemia causes calcium influx through multiple channels including, NMDA, ASIC, and TRPM7, which leads to increased neuronal nitric oxide synthase (nNOS), and generation of peroxynitrite (ONOO⁻) from nitric oxide (NO) and superoxide (O₂⁻), damagings DNA. O₂⁻ is generated from NMDA receptor activation of NADPH oxidase. The DNA repair enzyme PARP-1 is activated utilizing cellular NAD+ forming PAR polymers, which cause nuclear translocation of AIF further damaging DNA. Cell death occurs from AIF-mediated DNA degradation and NAD+ depletion causing energy failure. What plays the role of executioner?

Figure 2

PAR Polymer Formation and Degradation. (A) PARP-1 encounters DNA strand breaks. PARP-1 breaks down NAD+ into Nicotinamide (NA) and forms PAR polymers while repairing DNA. PAR polymers are toxic, and attach to neighboring proteins and PARP-1 itself. PARG degrades PAR Polymers, and under normal conditions keeps toxic PAR polymers at low levels. (B) Western blot illustrating sex differences in PAR polymer formation. Male mice have increased PAR Polymer formation compared to shams and females 2, 6, 12, and 24 hours after 90 minute MCAO (Yuan et. al., 2009). Note AIF translocation is equivalent between the sexes after injury.

Figure 3

NAD+ Biosynthesis. De Novo NAD+ synthesis starts with the conversion of Nicotinic Acid to Nicotinic Acid Mononucleotide (NAMN) by nicotinic acid phosphoribosyltransferase (npt). NAMN is then converted to Nicotinic acid adenine dinucleotide (NAAD) by nicotinic acid/nicotinamide mononucleotide adenylyltransferase (Nmnat). NAAD is finally converted to NAD+ by NAD synthetase. The salvage pathway utilizes Nicotinamide, which is converted to Nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyltranferase (Nampt). Nmnat then converts NMN to NAD+. The salvage pathway is continually active as NAD+ is degraded by PARP-1 and other proteins (adapted from Yang and Sauve, 2006).

Figure 4

NAD+ vs. PAR Polymers in Caspase-Independent Cell Death. This cartoon depicts the dichotomy in the caspase-independent cell death cascade (exaggerated for clarity as significant overlap likely occurs in vivo). There is cross talk between PARP-1 and NAD+ depletion, but NAD+ depletion caused glycolytic inhibition, mitochondrial depolarization, AIF translocation, and cell death without PARP-1 activation. Although PARP-1 activation causes PAR polymer formation, AIF translocation and cell death does not necessarily follow. The reviewed literature suggests that NAD+ depletion is necessary and plays the role of executioner in the caspase-independent cell death pathway.