Poly(ADP-ribose) (PAR) polymer is a death signal

Abstract

Excessive activation of the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP-1) plays a prominent role in various of models of cellular injury. Here, we identify poly(ADP-ribose) (PAR) polymer, a product of PARP-1 activity, as a previously uncharacterized cell death signal. PAR polymer is directly toxic to neurons, and degradation of PAR polymer by poly(ADP-ribose) glycohydrolase (PARG) or phosphodiesterase 1 prevents PAR polymer-induced cell death. PARP-1-dependent, NMDA excitotoxicity of cortical neurons is reduced by neutralizing antibodies to PAR and by overexpression of PARG. Neuronal cultures with reduced levels of PARG are more sensitive to NMDA excitotoxicity than WT cultures. Transgenic mice overexpressing PARG have significantly reduced infarct volumes after focal ischemia. Conversely, mice with reduced levels of PARG have significantly increased infarct volumes after focal ischemia compared with WT littermate controls. These results reveal PAR polymer as a signaling molecule that induces cell death and suggests that interference with PAR polymer signaling may offer innovative therapeutic approaches for the treatment of cellular injury.

Fig. 1.

PAR polymer induces neuronal cell death. (A) Representative fluorescent microscopic images of PI (red) and Hoechst (blue) staining after treatment of neurons with BioPorter-mediated delivery of PAR polymer, PAR polymer + PARG, PAR polymer + PD1, and poly(A). (B) Silver staining of purified PAR polymer, showing degradation of PAR polymer after incubation with PARG (PAR+PARG) and PD1 (PAR+PD1). These experiments (A and B) have been replicated in separate experiments at least three times with similar results. (C) Quantitative analysis of Hoechst- and PI-stained WT neurons after BioPorter-mediated delivery of PAR polymer, PAR polymer + PARG, PAR polymer + PD1, and poly(A). Purified PAR, cleaved PAR by either PARG or PD1 pretreatment, or synthesized poly(A) was diluted with PBS, mixed with BioPorter reagent, and added to neuronal cultures at a final concentration of 80 nM in serum-free MEM. The neurons were cultured for 3 h to allow the polymers to be imported into neurons and then subjected to fluorescent microscopy and quantitative computer-assisted cell counting after Hoechst and PI double staining 24 h later. Data are the mean ± SD, n = 3; ∗, P < 0.05. (D) PAR polymer can also induce cell death in PARP-1 KO neurons. PAR polymer (80 nM) and cleaved PAR (80 nM PAR polymer + PARG) was delivered into neurons by BioPorter-mediated delivery, and cell death was analyzed by Hoechst and PI staining. Data are the mean ± SD, n = 3; ∗, P < 0.05.

Fig. 2.

PAR polymer toxicity is length- and dose-dependent and caspase-independent. (A) Purified PAR polymer was fractionated into fractions of varying polymer length. Different PAR polymer fractions with average polymer size (16, 30, and 60 mer) were delivered into the cortical neurons by the BioPorter delivery reagent at a final concentration of 80 nM. The most abundant fraction containing PAR polymer of 40–100 (average polymer size 60 mer) ADP-ribose residues induces robust neuronal cell death. Neurons were treated with BioPorter alone and serve as a control. Data are the mean ± SEM, n = 6; ∗, P < 0.05. (B) PAR polymer-induced neuronal death is dose-dependent. Different concentrations of PAR polymer (20–160 nM) were administered to cortical neurons by the BioPorter reagent, and cell death was assessed by Hoechst and PI staining. Data are the mean ± SD, n = 4; ∗, P < 0.05. (C) Representative pictures of PI- (red) and Hoechst- (blue) stained neurons were treated with BioPorter alone (Control), purified PAR polymer (PAR), or purified PAR polymer in the presence of z-VAD (z-VAD + PAR), by using BioPorter reagent as a delivery system. PI-positive cells were considered dead cells. (D) Quantification of the PAR polymer-mediated cell death in the presence of z-VAD. z-VAD is unable to protect against PAR polymer-mediated cell death in cortical neurons. BioPorter alone serves as a control. Cell death was assessed by Hoechst and PI staining. Data are the mean ± SEM, n = 6; ∗, P < 0.05.

Fig. 3.

Interfering with PAR signaling reduces NMDA-induced neuronal cell death. (A) Immunodepletion of purified PAR polymer (Upper) and SN(PAR) (Lower) by anti-PAR antibody. Supernatants after immunoprecipitation have reduced level of PAR polymers. NRS, normal rabbit serum; Pre-Im, preimmune serum; α-Par, anti-Par antibody. (B) Pretreatment with neutralizing PAR antiserum reduces NMDA-induced neuronal cell death. PAR antiserum, normal rabbit serum, or preimmune serum was delivered into cortical neurons with the BioPorter reagent. At 3 h after delivery, the neurons were treated with NMDA (500 μM for 5 min). Cell death was assessed 18–24 h later by Hoechst and PI staining. Data are the mean ± SEM, n = 6–8; ∗, P < 0.05. (C) Cortical neurons were infected with a cytosolic form (exon-1 deleted) PARG adenoviral construct (Av PARG WT) or catalytically inactive (Av PARG Mut) virus, and, after 48 h, the cells were harvested in lysis buffer containing protease inhibitors. Because of low basal levels of PARG, nondetectable levels of PARG are seen in adeno-GFP- and PBS-treated cultures, whereas high levels of PARG expression are seen in cultures that are treated with Av PARG WT or Av PARG Mut virus. (D) Overexpression of cytosolic, WT PARG decreases PAR levels in cortical neurons after NMDA treatment. Neurons [11 days in vitro (DIV)] were infected with an exon 1-deleted, cytosolic WT (Av PARG WT) and mutant (Av PARG mut) PARG adenoviral constructs, or a GFP-adenoviral control construct (Av GFP). After 48 h, the transduced cultures were treated with NMDA (500 μM for 5 min), and neurons were harvested at indicated time points and subjected to Western blotting analysis with anti-PAR antibody. These experiments (A, C, and D) have been replicated in separate experiments at least three times with similar results. (E) Overexpression of PARG reduces NMDA-induced neuronal cell death. Neurons (12 DIV) were infected with a Av PARG WT or catalytically inactive Av PARG Mut virus, GFP-adenoviral control construct (Adeno-GFP), or no-adenovirus (PBS). After 48 h, the neurons were treated with NMDA (500 μM for 5 min), and 18–24 h after NMDA administration, cells were stained with Hoechst and PI to assess cell death. Data are the mean ± SEM, n = 6; ∗, P < 0.05.

Fig. 4.

Enhanced PAR polymer formation and toxicity in PARG^+/− mice, and PAR polymer mediates PARP-1-dependent cell death in vivo. (A) Western blots showing PAR polymer formation in mouse cortical neurons prepared from PARG^+/− mice and their WT littermates. The neurons were subjected to NMDA stimulation for 5 min on DIV 14. Samples were harvested 1 h after NMDA stimulation and probed for PAR polymer by using anti-PAR antibody. PARG^+/− neurons that express reduced levels of PARG accumulate more PAR polymer than their WT littermates. PARG^+/− neurons also have higher basal PAR polymer levels. The same blot was stripped and probed for β-tubulin, serving as a loading control. This experiment has been replicated in separate experiments at least three times with similar results. (B) Cortical neurons from PARG^+/− KO mice and WT littermate cultures were treated with 500 μM NMDA for 5 min on DIV 14, and intracellular PAR polymer levels were determined at the indicated times points after NMDA administration by using an ELISA as described in Materials and Methods. Data are the mean ± SD, n = 4; ∗, P < 0.05. (C) Cortical neurons from PARG^+/− KO mice and WT littermate cultures were subjected to NMDA-stimulation (500 μM for 5 min) on DIV 14. PARG^+/− neurons that have reduced expression of PARG are more sensitive to NMDA toxicity than their WT littermates. Control cultures were treated with control salt solution (CSS) alone. Data are the mean ± SD, n = 6; ∗, P < 0.05. (D) PARG Tg mice overexpress PARG. (a) Expression of the PARG gene is under the control of the PrP promoter. (b) Northern blot analysis for PARG in WT vs. PARG Tg mice. β-actin was used as a loading control. (c) Immunoblotting of mouse PARG in whole-brain and testis homogenates of transgenic mice expressing the PARG and WT control mice. β-tubulin was used as a loading control. (E) Infarct volume (±SEM) in cerebral cortex, striatum, and total hemisphere (expressed as a percent of contralateral structure after correction for swelling) in 10 WT and 11 PARG Tg mice subjected to 120 min of MCAO. ∗, P < 0.05 between groups. (F) Decreased levels of PARG protein in PARG^+/− mice. Immunoblot analysis of full-length (110-kDa) PARG protein levels in brain and testis extracts from PARG^+/+ and PARG^+/− mice. These results have been replicated at least two separate times with similar results. (G) Infarct volume (± SEM) in cerebral cortex, striatum, and total hemisphere (expressed as a percent of contralateral structure after correction for swelling) in 15 WT and 16 PARG^+/− mice subjected to 90 min of MCAO. ∗, P < 0.05 between groups.