Role of PARP1 and PAR Polymer in Excitotoxicity

Poly (ADP-ribose) polymerases (PARPs), are known to play key roles in DNA repair (Jeggo 1998; Poirier et al. 1982). PARP-1 is the founding member of PARP family, which includes 18 different isoforms based on protein sequence homology to the PARP-1 catalytic domain (Ame et al. 2004; D'Amours et al. 1999; Hong et al. 2004; Smith 2001; Virag and Szabo 2002). PARP-1 accounts for more than 90% of PARP activity in living cells. In response to DNA damage, PARP-1 uses NAD+ as a substrate and attaches polymers of PAR on different acceptor proteins (hetero-modification) or on PARP-1 itself (auto-modification)

  1. 5.1 PARP-1 and PAR mediated cell death in excitotoxicity. Over-stimulation of NMDA receptors by glutamate results in the influx of Ca2+, which binds calmodulin and activates nNOS, to convert L-arginine to NO and L-citrulline. Even though NO is an essential molecule in neuronal signal transduction, excess NO can be neurotoxic. Neuronal toxicity by excess NO is mediated by peroxynitrite, a reaction product from NO and superoxide anion (O2-). Peroxynitrite causes severe damage to DNA, which results in over activation of PARP (PARPf), depletion of NAD+, and generation of PAR polymer, leading to neuronal death
  2. 5.1 PARP-1 and PAR mediated cell death in excitotoxicity. Over-stimulation of NMDA receptors by glutamate results in the influx of Ca2+, which binds calmodulin and activates nNOS, to convert L-arginine to NO and L-citrulline. Even though NO is an essential molecule in neuronal signal transduction, excess NO can be neurotoxic. Neuronal toxicity by excess NO is mediated by peroxynitrite, a reaction product from NO and superoxide anion (O2-). Peroxynitrite causes severe damage to DNA, which results in over activation of PARP (PARPf), depletion of NAD+, and generation of PAR polymer, leading to neuronal death

(D'Amours et al. 1999; Virag and Szabo 2002). PARP-1 is considered a "genome guardian," because it takes part in DNA repair under physiological conditions (Jeggo 1998; Poirier et al. 1982). Under mild genomic stress, PARP-1 is activated to induce DNA repair, whereas severe cell stress induces massive PARP-1 activation that ultimately leads to cell death (Virag and Szabo 2002). Both gene deletion and pharmacological inhibition studies have shown that PARP-1 activation plays a key role in cytotoxicity following ischemia/reperfusion, neurodegeneration, spinal cord injury, ischemic injury in heart, liver, and lungs, and in retinal degeneration, arthritis and diabetes (Virag and Szabo 2002). In the nervous system, massive PARP-1 activation is triggered by excitotoxic stimuli. It was originally presumed that cell death in PARP-1 toxicity was induced by the intracellular energy depletion from PARP-1's use of NAD+ (Virag and Szabo 2002). NAD+ is an important cellular molecule for many physiological processes. Energy-generating processes, like glycolysis, the Krebs cycle and the pentose phosphate pathway, utilize NAD+ as a cofactor (Belenky et al. 2007). While PARP-1 activation leads to decreased cellular NAD+ and energy levels (Ha and Snyder 1999), it is difficult to obtain evidence that proves that PARP-1 activation depletes enough cellular energy to kill the cell (Fossati et al. 2007; Moubarak et al. 2007). Numerous studies show that cellular ATP and NAD+ levels drop significantly following PARP-1 activation (Eliasson et al. 1997; Yu et al. 2002). The drop in cellular energy levels following PARP-1 activation may primarily be due to alterations in mitochondrial function and defective oxidative phosphorylation as opposed to PARP-1 mediated catabolism of NAD+ (Virag and Szabo 2002). Along these lines, it was shown by many studies that mito-chondrial depolarization, loss of mitochondrial function and increased mito-chondrial membrane permeability are required factors for PARP-1-dependent cell death (Alano et al. 2004). Conclusions that NAD+ utilization by PARP-1 is a death inducer were drawn from studies that used direct exogenous delivery of NAD+ or energy substrates as cytoprotective agents. It is important to note that the off-target effects of these substrates may contribute to the observed effects. For example, consumption of NAD+ by PARP-1 generates nicotinamide (NAM) as a by-product. NAM is a potent PARP-1 inhibitor, so the protective mechanism mediated by exogenous NAD+ should be interpreted with caution. Recent studies indicate that energy depletion following PARP-1 activation is not a critical factor for cell death. Following PARP-1 activation, we recently demonstrated that cells die as a result of toxic accumulation of PAR. PAR, generated by PARP-1 in the nucleus, travels to the cytosol to induce cell death. Neutralization of cyto-solic PAR by PAR-specific antibodies protects against NMDA-induced cell death in mouse primary neurons (Andrabi et al. 2006). Conversely, exogenous delivery of purified PAR kills cells (Andrabi et al. 2006). The toxic potential of PAR increases with dose and polymer complexity. Highly complex and long chain polymers are more toxic than shorter and less complex polymers (Andrabi et al. 2006). Among the PARP family members, there are at least six different PARPs that are confirmed to synthesize PAR. The heterogeneity in the complexity and structure of PAR may vary depending upon the PARP involved. This may contribute to the possible different contributions of individual PARP isoforms to cell survival or cell death. PARP-1-dependent cell death, known as parthanatos, is distinct from classic necrosis or apoptosis in its biochemical and morphological features, although many of the morphologic features are similar to those previously described in excitotoxic/necrotic neurons. The biochemical features of parthanatos are distinct from classically defined pathways of cell death, and include rapid PARP-1 activation, early PAR accumulation, mitochondrial depolarization, early nuclear AIF translocation, loss of cellular NAD and ATP, and late caspase activation. Caspase activation, which is a hallmark of apoptotic cell death, does not play a primary role in parthanatos, as broad-spectrum cas-pase inhibitors are unable to protect cells. Morphological features of parthanatos include shrunken and condensed nuclei, disintegrating membranes and cells becoming propidium iodide-positive within a few hours after the onset of parthana-tos (Figs. 5.2 and 5.3).

  1. 5.2 Parthanatos. PARP-1 utilizes NAD+ as a substrate for synthesis of PAR polymers. In the process of PAR formation, nicotinamide (NAM), a product of NAD+ hydrolysis, is first converted into nicotinamide mononucleotide (NMN) and then into NAD+ by nicotinamide phosphoribosyl transferase (NamPRT) and nicotinamide mononucleotide adenylyl transferases (Na/NMNAT-1, -2, and -3), respectively. Mild DNA damage or breaks activate the PARP proteins, where they play a role in the DNA repair process. Under conditions of severe DNA damage, parthanatos is initiated through excessive PAR polymer formation
  2. 5.2 Parthanatos. PARP-1 utilizes NAD+ as a substrate for synthesis of PAR polymers. In the process of PAR formation, nicotinamide (NAM), a product of NAD+ hydrolysis, is first converted into nicotinamide mononucleotide (NMN) and then into NAD+ by nicotinamide phosphoribosyl transferase (NamPRT) and nicotinamide mononucleotide adenylyl transferases (Na/NMNAT-1, -2, and -3), respectively. Mild DNA damage or breaks activate the PARP proteins, where they play a role in the DNA repair process. Under conditions of severe DNA damage, parthanatos is initiated through excessive PAR polymer formation

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