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. Author manuscript; available in PMC: 2014 Sep 1.
Published in final edited form as: Mol Cell Neurosci. 2013 Jul 13;0:322–332. doi: 10.1016/j.mcn.2013.07.004

C-terminal Binding Proteins are Essential Pro-survival Factors that Undergo Caspase-dependent Downregulation during Neuronal Apoptosis

Trisha R Stankiewicz a,b,#, Emily K Schroeder a,#, Natalie A Kelsey b, Ron J Bouchard a, Daniel A Linseman a,b,c,#
PMCID: PMC3791214  NIHMSID: NIHMS512486  PMID: 23859824

Abstract

C-terminal binding proteins (CtBPs) are transcriptional co-repressors that are subject to proteasome-dependent downregulation during apoptosis. Alternative mechanisms that regulate CtBP expression are currently under investigation and the role of CtBPs in neuronal survival is largely unexplored. Here, we show that CtBPs are downregulated in cerebellar granule neurons (CGNs) induced to undergo apoptosis by a variety of stressors. Moreover, antisense-mediated downregulation of CtBP1 is sufficient to cause CGN apoptosis. Similarly, the CtBP inhibitor, 4-methylthio-2-oxobutyric acid, induces expression of the CtBP target Noxa and causes actinomycin-sensitive CGN apoptosis. Unexpectedly, we found that the mechanism of CtBP downregulation in CGNs undergoing apoptosis varies in a stimulus-specific manner involving either the proteasome or caspases. In the case of CGNs deprived of depolarizing potassium (5K apoptotic condition), caspases appear to play a dominant role in CtBP downregulation. However, incubation in 5K does not enhance the kinetics of CtBP1 degradation and recombinant CtBP1 is not cleaved in vitro by caspase-3. In addition, 5K has no significant effect on CtBP transcript expression. Finally, mouse embryonic stem cells display caspase-dependent downregulation of CtBP1 following exposure to staurosporine, an effect that is not observed in DGCR8 knockout cells which are deficient in miRNA processing. These data identify caspase-dependent downregulation of CtBPs as an alternative mechanism to the proteasome for regulation of these transcriptional co-repressors in neurons undergoing apoptosis. Moreover, caspases appear to regulate CtBP expression indirectly, at a post-transcriptional level, and via a mechanism that is dependent upon miRNA processing. We conclude that CtBPs are essential pro-survival proteins in neurons and their downregulation contributes significantly to neuronal apoptosis via the de-repression of pro-apoptotic genes.

Keywords: C-terminal binding proteins (CtBPs), caspase-3, miRNA, neuronal apoptosis

Introduction

C-terminal binding proteins (CtBPs) were originally identified as binding partners for the adenovirus E1A transforming proteins (Schaeper et al., 1995). CtBP1 and CtBP2 are highly homologous transcriptional co-repressors that function as partners for repressor proteins like ZEB and basic krüppel-like factor (BKLF) (Turner and Crossley, 1998; Postigo and Dean, 1999). CtBPs recruit a number of key chromatin modifying enzymes (e.g., histone deacetylases) to gene promoters primarily via PXDLS-dependent interactions with the CtBP hydrophobic cleft (Quinlan et al., 2006; Kuppuswamy et al., 2008). In a similar manner, CtBPs repress p300-dependent transcriptional activation by directly binding to a PXDLS motif in the bromodomain of this co-activator (Kim et al., 2005). CtBPs are directed to the nuclear compartment either via a nuclear localization signal (which is unique to CtBP2) or by binding to PXDLS motif-containing partners like BKLF (Verger et al., 2006). CtBPs are functional dehydrogenases which bind to NADH with greater than 100-fold higher affinity than NAD+ (Kumar et al., 2002; Zhang et al., 2002; Fjeld et al., 2003). Binding of NAD(H) appears to stabilize the protein and promotes dimerization of CtBPs which is required for transcriptional repression (Fjeld et al., 2003; Mani-Telang et al., 2007; Kuppuswamy et al., 2008). Thus, CtBPs act as redox-sensitive transcriptional co-repressors of a specific subset of target genes.

CtBPs are key transcriptional co-repressors of epithelial and pro-apoptotic gene expression programs (Grooteclaes et al., 2003, Bergman and Blaydes, 2006). By repressing epithelial cell adhesion (via repression of E-cadherin) and concomitantly suppressing apoptosis and anoikis, CtBPs promote cancer cell migration, invasion, and survival (Grooteclaes and Frisch, 2000, Straza et al., 2010). In the context of apoptosis, CtBPs act as co-repressors at several pro-apoptotic Bcl-2 family member gene promoters, such as Bax and the Bcl-2 homology-3 domain (BH3)-only proteins, Noxa, Bik, Bim, and Bmf (Grooteclaes et al., 2003; Bergman and Blaydes, 2006; Kovi et al., 2010). Murine embryonic fibroblasts (MEFs) isolated and immortalized from Ctbp1/Ctbp2 double knockout embryos show constitutive upregulation of Bax and Noxa, and demonstrate enhanced sensitivity to diverse apoptotic stimuli (Grooteclaes et al., 2003). Both the increased expression of Bax and Noxa, as well as the enhanced susceptibility to apoptosis, were reversed by Ctbp1 or Ctbp2 rescue expression.

To date, relatively few studies have examined the roles of CtBPs in CNS development or neuronal survival. Based largely on the results of genetic deletion experiments, it appears that Ctbp1 and Ctbp2 display both duplicative and independent roles in mouse development including maturation of the CNS (Hildebrand and Soriano, 2002). Ctbp2 homozygous null mice display delayed development of the forebrain and midbrain, and typically die by E10.5. In contrast, Ctbp1 homozygous null mice are viable and fertile. In a genetic interaction experiment, increasing the dosage of Ctbp1 decreased the severity of the Ctbp2 null phenotype. For instance, Ctbp1+/− Ctbp2−/− embryos did not complete neural tube closure and arrested at the turning stage while Ctbp1+/+ Ctbp2−/− embryos completed both processes. In the context of cell survival, CtBPs are targeted for proteasomal degradation in response to pro-apoptotic stimuli that induce p53-independent apoptosis in non-neuronal cells (Zhang et al., 2003; Zhang et al., 2005; Wang et al., 2006; Paliwal et al., 2006). In contrast, the role of CtBPs in neuronal apoptosis has not previously been explored. Here, we identify a novel caspase-dependent pathway for CtBP downregulation during neuronal apoptosis and further show that loss of CtBP function is sufficient to induce neuronal cell death.

Materials and Methods

Reagents

Clostridium difficile Toxin B (ToxB) and Clostridium sordellii lethal toxin (LTox) were kindly provided by Dr. Klaus Aktories (Albert-Ludwigs-Universität Freiburg, Germany). The high-throughput immunoblotting screen was performed by BD Pharmingen (Palo Alto, CA, USA) and monoclonal antibodies used for subsequent western blotting of CtBP1 and CtBP2 were obtained from BD Biosciences (San Diego, CA, USA). Polyclonal antibody against actin was obtained from Cell Signaling (Berverly, MA, USA). The polyclonal antibody used to detect Noxa was from Abcam (Cambridge, MA, USA). Horseradish peroxidase-linked secondary antibodies and reagents for enhanced chemiluminescence detection were from Amersham Biosciences (Piscataway, NJ, USA). The polyclonal antibody used to detect active caspase-3 was from Promega (Madison, WI, USA). 4,6-Diamidino-2-phenylindole (DAPI), Hoescht dye 33258, monoclonal antibody against β-tubulin, 1-methyl-4-phenylpyridinium (MPP+), 6-hydroxydopamine (6-OHDA), 4-methylthio-2-oxobutyric (MTOB), staurosporine, actinomycin D, and recombinant PARP were from Sigma (St. Louis, MO, USA). Cy3- and FITC-conjugated secondary antibodies for immunofluorescence were from Jackson Immunoresearch Laboratories (West Grove, PA, USA). HA14-1 and BOC were obtained from Alexis (San Diego, CA, USA). MG-132, sodium nitroprusside (SNP), and recombinant caspase-3 were from Calbiochem (Darmstadt, Germany). The polyclonal antibody to PARP was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Morpholino-antisense oligonucleotides and the EndoPorter delivery reagent were obtained from Gene Tools (Philomath, PA, USA). Wild type and DGCR8 knockout mouse embryonic stem cells, as well as, recombinant CtBP1 were obtained from Novus Biologicals (Littleton, CO, USA).

Cerebellar Granule Neuron (CGN) Culture

Rat CGNs were isolated from 7-day-old Sprague-Dawley rat pups of both sexes (15-19 g) as previously described (Linseman et al., 2001). CGNs were plated on 35-mm diameter plastic dishes coated with poly-L-lysine at a density of 2.0×106 cells/ml in basal modified Eagle's medium containing 10% fetal bovine serum, 25 mM KCl, 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (Life Techonologies, Grand Island, NY, USA). Cytosine arabinoside (10 μM) was added to the culture medium 24 h after plating to limit the growth of non-neuronal cells. With use of this protocol, the cultures were approximately 95% pure for granule neurons. In general, experiments were performed after 6-7 days in culture.

BD Pharmingen PowerBlot™ Analysis

CGNs were incubated in either control medium or medium containing 40 ng/ml Clostridium difficile Toxin B (ToxB) for 24 h and subsequently lysed according to the manufacturer's protocol. Lysates from three independent experiments were pooled and subjected to high-throughput immunoblotting against a panel of 1009 purified monoclonal antibodies (BD PowerBlot™). Raw data was obtained from the manufacturer in the form of image files of the actual blots and densitometric measurements of the immunoreactive proteins. The blots shown for CtBP1, CtBP2, and G protein-coupled receptor kinase-interacting protein-z-short are representative of 2 × 2 comparisons of duplicate control and ToxB lysates.

Cell Lysis and Immunoblotting

Following treatment, whole cell lysates of CGNs were prepared essentially as previously described (Loucks et al., 2006). Briefly, protein concentrations were determined by a commercially available protein assay kit (BCA; Thermo Fisher Scientific, Waltham, MA, USA), and SDS-polyacrylamide gel electrophoresis was performed using equal amounts of protein followed by transfer to polyvinylidene difluoride membranes. Nonspecific binding sites were blocked in PBS-T (1X phosphate-buffered saline (PBS, pH 7.4) containing 0.1% Tween 20) containing 1% BSA and 0.01% sodium azide for 1 h at room temperature (22° C). Membranes were incubated for 1 h in primary antibody diluted in blocking solution. Membranes were subsequently washed 5 times over 30 min in PBS-T to remove excess primary antibody. Membranes were then incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies diluted in PBS-T. Following secondary incubation, membranes were washed 5 times over 30 min in PBS-T to remove excess secondary antibody. Immunoreactive proteins were detected by enhanced chemiluminescence. Blots shown are representative of a minimum of three independent experiments.

Immunofluorescence Microscopy

CGNs were plated at a density of 4.5×105 cells per ml on glass coverslips coated with polyethylene-imine. After treatment, cells were fixed in 4% paraformaldehyde, washed once in PBS, and permeabilized and blocked in 0.2% Triton X-100 and 5% BSA in PBS (pH 7.4). Cells were incubated for 16 h at 4° C in primary antibodies diluted in 2% BSA and 0.2% Triton X-100 in PBS. They were subsequently washed 5 times in PBS over 30 min and then incubated for 1 h at room temperature with Cy3- or FITC-conjugated secondary antibodies and DAPI diluted in 2% BSA and 0.2% Triton X-100 in PBS. The cells were washed five additional times over 30 min with PBS before mounting coverslips onto slides with anti-quench composed of 0.1% p-phenylenediamine in 75% glycerol in PBS. Fluorescent images were captured using a 63x oil immersion objective on a Zeiss Axioplan 2 epifluorescence microscope that was equipped with a Cooke Sensicam deep-cooled charge-coupled device (CCD) camera and a Slidebook software analysis program for digital deconvolution (Intelligent Imaging Innovations Inc., Denver, CO, USA).

Morpholino-antisense Oligonucleotide Treatment

CGNs were plated at a density of 4.5×105 cells per ml on glass coverslips coated with polyethylenimine. On day 6 in vitro, cells were treated with EndoPorter reagent (6 μM) alone or in combination with morpholino-antisense or inverse oligonucleotides (3 μM final concentration). After 72 h of treatment, cells were fixed with 4% paraformaldehyde and nuclei were stained with Hoechst dye. CGNs containing condensed and/or fragmented nuclei were scored as apoptotic.

N27 Dopaminergic Cell Culture

N27 cells were maintained in culture in RPMI medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin.

6-Hydroxydopamine Treatment

For treatment with 6-OHDA, the compound was diluted in a vehicle solution consisting of ddH2O containing 0.15% ascorbic acid and 10 mM DETAPAC. This solution was purged with nitrogen gas for 30 min while on ice. After removal of oxygen, 6-OHDA was added to the solution and used to treat N27 cells at a concentration of 25 μM or 50 μM for 24 h.

Pulse-Chase Assay

CGNs were incubated for 4 h in cysteine-methionine-free medium containing 35S-methionine. Following this labeling “pulse”, cells were washed and medium was replaced with control (25K) or apoptotic (5K) medium for the “chase” period. At the times indicated, cells were lysed, CtBP1 was immunoprecipitated, and immune complexes were resolved by SDS-PAGE followed by transfer to polyvinylidine difluoride membranes. Membranes were exposed directly to film and bands representing 35S-CtBP1 were quantified by densitometry.

In Vitro Caspase-3 Cleavage Assay

In separate microcentrifuge tubes, 5 μg of recombinant PARP or 1.5 μg of recombinant CtBP1 were incubated alone or in combination with 100 units of recombinant caspase-3. All samples were qued to 100 μl with caspase buffer (100 mM NaCl, 5 mM DTT, 50 mM EDTA, 20 mM PIPES, 1% Chaps, and 10% sucrose in ddH20). Samples were incubated for 4 h at 37°C in a thermomixer. Following incubation, proteins were resolved by SDS-PAGE, proteins transferred onto membranes, and immunoblotted as previously described.

RT-PCR

CGNs were incubated in control (25K) or apoptotic (5K) medium for 24 h. RNA was isolated using a miRCURY RNA isolation kit purchased from Exiqon (Woburn, MA, USA). cDNA was synthesized from the isolated RNA samples using an Omniscript reverse transcriptase kit that was purchased from Qiagen (Valencia, CA, USA). DNA was analyzed by polymerase chain reaction (PCR) using an Accuprime Pfx Supermix kit from Life Technologies (Grand Island, NY, USA). Primers were purchased from Integrative DNA Technologies (Coralville, IA, USA) correspond-ing to CtBP1, CtBP2, and beta-actin. The forward primer to CtBP1 was 5’-TTGGGCAT CATTGGACTAGGTCGT-3’ and the reverse primer was 5’-TCAGGTGGTCCTTGTT GACACAGT-3’. The forward primer to CtBP2 was 5’-TGTGATGCACAGTCCACTCAG GAA-3’ and the reverse primer was 5’-CCATTGAACACGGCATTGTCACCA-3’. The forward primer to beta-actin was 5’-CCATTGAACACGGCATTGTCACCA-3’ and the reverse primer was 5’-ACTCCTGCTTGCTGA TCCACATCT-3’. PCR was performed using the following conditions: 95°C for 5 min followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, and 68°C for 1 min.

Mouse Embryonic Stem Cell Culture

Wild type and DGCR8 knockout mouse embryonic stem cells were purchase from Novus Biologicals (Littleton, CO, USA). Cell culture plates were coated with a solution containing 1% gelatin that was purchased from Millipore (Billerica, MA, USA). Cells were maintained in basal modified Eagle's medium containing 10% fetal bovine serum, 1X non essential amino acids, 1% beta-mercaptoethanol, and 1000 units/ml leukemia inhibitory factor (Millipore). In general, the culture medium of both wild type and DGCR8 knockout cells was replaced daily.

Data Analysis

Results represent the mean ± S.E. for the number (n) of independent experiments performed. Statistical differences between the means of unpaired sets of data were evaluated by one-way analysis of variance with a post hoc Tukey's test. A p value of < 0.05 was considered statistically significant. Images and immunoblots shown are representative of at least three independent experiments.

Results

CtBPs are downregulated in CGNs exposed to diverse pro-death stimuli

In our initial studies using a high throughput immunoblotting screen (BD PowerBlot™), we identified CtBP1 and CtBP2 as two proteins that were significantly downregulated in CGNs following a 24 h incubation with the Rho family GTPase inhibitor, Clostridium difficile toxin B (Figure 1A). Toxin B is a monoglucosyltransferase that directly glucosylates and inhibits the small GTPases Rho, Rac, and Cdc42 (Just et al., 1995). We have previously shown that toxin B induces intrinsic apoptosis in CGNs that is largely dependent on its capacity to inhibit pro-survival signaling by Rac GTPase (Linseman et al., 2001; Le et al., 2005; Loucks et al., 2006; Stankiewicz et al., 2012). CtBP1 appeared on polyacrylamide gels as a single band of approximately 48 kDa and CtBP2 appeared as a doublet of approximately 48/50 kDa. In the initial PowerBlot™ analysis, the expression of each of these proteins was significantly decreased by at least two-fold in CGNs incubated for 24 h with toxin B (Figure 1B). In a separate experiment using a distinct preparation of primary CGN cultures, the toxin B-induced decrease in CtBP1 and CtBP2 expression was confirmed (Figure 1C).

Figure 1. CtBP1 and CtBP2 are downregulated in CGNs exposed to Clostridium difficile Toxin B, an inhibitor of Rho family GTPases.

Figure 1

A. CGNs were incubated for 24 h in control medium containing 25 mM KCl and 10% FBS (Con) in the absence or presence of Toxin B (Tox B; 40 ng/ml). Cells were then lysed and protein extracts were subjected to PowerBlot™ analysis. Incubation with Tox B resulted in the downregulation of both CtBP1 (indicated by the black arrows) and CtBP2 (a doublet indicated by the asterisks). G protein-coupled receptor kinase-interacting protein-z-short (GITz-short; white arrows) is shown to demonstrate equal protein loading of the PowerBlot™ gels. B. Densitometric quantification of CtBP1 and CtBP2 protein expression in Con and Tox B-treated CGNs. Results shown are the mean ± range from duplicate PowerBlot™ gels and are expressed as the fold change in CtBP expression between Tox B-treated cells and Con. C. To confirm the results of the Powerblot™, CGNs obtained from a different primary culture than those utilized in the PowerBlot™ analysis were incubated for 24 h in either Con medium or medium containing Tox B. After incubation, CtBP1 and CtBP2 expression was analyzed by western blotting. Blots were then stripped and re-probed for actin as a loading control.

Next, we examined the effects of a number of diverse pro-death stimuli on the expression of CtBPs in CGNs. Primary CGN cultures require serum-derived growth factors and depolarizing extracellular potassium for their survival. Removal of depolarizing potassium (i.e., 5K conditions) induces apoptosis of CGNs through an intrinsic pathway (D'Mello et al., 1993, Linseman et al., 2002). Upon removal of serum and depolarizing potassium (5K conditions), the expression of both CtBP1 and CtBP2 was significantly decreased (Figure 2A). Similar reductions in CtBP expression were observed when CGNs were incubated with the BH3 mimetic, HA14-1, the nitric oxide donor, sodium nitroprusside (SNP), or the complex I inhibitor, 1-methyl-4-phenylpyridinium (MPP+) (Figure 2B). The expression of CtBP1 was also analyzed by immunofluorescent staining under control, 5K, and MPP+ treatment conditions. In control CGNs, CtBP1 was localized almost exclusively to the nucleus (Figure 2C, upper panels). However, whether CGNs were exposed to either 5K or MPP+, two completely distinct stressors, CtBP1 immunoreactivity essentially disappeared in cells that demonstrated condensed and/or fragmented chromatin indicative of apoptotic morphology (Figure 2C, middle and lower panels). These results demonstrate the downregulation of CtBP1 and CtBP2 in neurons undergoing apoptosis and further show that CtBPs are downregulated in response to a number of mechanistically distinct pro-death stimuli.

Figure 2. Multiple pro-death stimuli induce downregulation of CtBPs in CGNs.

Figure 2

A. CGNs were incubated for 24 h in either control medium containing 25 mM KCl and 10% FBS (Con) or apoptotic medium containing only 5 mM KCl and lacking FBS (5K). Cells were then lysed, proteins resolved by SDS-PAGE, and immunoblotted for CtBP1 and CtBP2. Blots were subsequently stripped and re-probed for actin to show equal loading. B. CGNs were incubated for 24 h in either Con medium alone or containing the Bcl-2 inhibitor HA14-1 (HA14; 15 μM), the nitric oxide donor sodium nitroprusside (SNP; 100 μM), or the complex I inhibitor 1-methyl-4-phenylpyridinium (MPP+; 150 μM). After incubation, protein extracts were western blotted for CtBP1 and CtBP2. Blots were also stripped and re-probed for actin as a loading control. C. CGNs were incubated for 24 h in either Con medium, 5K medium, or medium containing MPP+, as described above in (A) and (B). Following incubation, cells were fixed with 4% paraformaldehyde and their nuclei stained with DAPI. The microtubule network was stained with a polyclonal antibody to β-tubulin and a Cy3-conjugated secondary antibody. CtBP1 was detected using a monoclonal antibody to CtBP1 and a secondary antibody conjugated to FITC. White arrows indicate cells with condensed and/or fragmented nuclei indicative of apoptotic cell death. Note that regardless of the stimulus, CGNs with apoptotic nuclear morphology displayed essentially no detectable CtBP1. Scale bar=10 microns.

Antisense oligonucleotides to CtBP1 induce CGN apoptosis

To determine if forced downregulation of CtBP1 is sufficient to trigger CGN apoptosis, we transfected cells with morpholino-antisense oligonucleotides to rat CtBP1 using an EndoPorter delivery reagent. As negative controls, CGNs were transfected with inverse morpholino oligonucleotides or were exposed to the EndoPorter reagent alone. Transfections and subsequent 24 h incubations were performed in control medium containing 25 mM KCl and 10% FBS. CGNs transfected with morpholino-antisense oligonucleotides to rat CtBP1 underwent significant apoptosis characterized by nuclear condensation and fragmentation (Figures 3A and 3B). In contrast, CGNs exposed to the EndoPorter reagent alone or transfected with inverse morpholino oligonucleotides displayed a similar level of apoptosis to untreated control CGNs. Although the morpholino-antisense oligonucleotides were fluorescently labeled with fluorescein, we were unable to accurately assess the extent of CtBP1 downregulation within the transfected cell population using immunofluorescent staining for CtBP1. This was primarily because the cells that fluoresced positive for the morpholino-antisense oligonucleotides were most often apoptotic and their nuclei were essentially devoid of CtBP1 immunoreactivity. However, given that CtBP1 nuclear staining disappears in apoptotic cells (see Figure 2C), we cannot discern whether the CtBP1 staining is lost due to the antisense treatment or due to the fact that the cell is undergoing apoptosis. Given this limitation, our data suggest that antisense-mediated down-regulation of CtBP1 is sufficient to induce significant CGN apoptosis.

Figure 3. Morpholino-antisense oligonucleotides to CtBP1 induce CGN apoptosis.

Figure 3

A. CGNs were incubated for 24 h in Con medium alone or containing the vehicle Endoporter (EP; 6 μl/ml), EP plus morpholino-antisense oligonucleotides to CtBP1 (Anti; 10 μM), or EP plus inverse control morpholino-oligonucleotides (Inv; 10 μM). Apoptosis was quantified from 3 separate experiments each performed in triplicate. Antisense treatment induced significant CGN apoptosis (*p<0.05) when compared to Inv, EP, or Con treatments. B. Representative images of CGNs following incubation with morpholino-antisense oligonucleotides or appropriate controls, as described in (A). Cells were fixed and nuclei stained with Hoechst. CGNs containing nuclei that were visibly condensed and/or fragmented were scored as apoptotic.

The CtBP inhibitor, 4-methylthio-2-oxobutyric acid (MTOB), induces actinomycin D-sensitive apoptosis of CGNs

As an alternative means of knocking out CtBP function in CGNs, we next incubated cells with the putative CtBP inhibitor, MTOB. This compound is a CtBP dehydrogenase substrate which acts as a CtBP inhibitor and is toxic to cancer cells at high (1-10 mM) concentrations (Straza et al., 2010). In agreement with this previous study, incubation of CGNs with MTOB for 24 h revealed that significant apoptosis was induced at a concentration of 5 mM (Figure 4A). Moreover, the CGN apoptosis induced by this concentration of MTOB was delayed and required 24 h of incubation (Figure 4B). In accordance with MTOB inducing apoptosis of CGNs, its toxic effects were completely suppressed by blocking the transcription of new genes with actinomycin D (Figures 4C and 4D). Moreover, consistent with MTOB inhibiting the co-repressor function of CtBP, incubation with this compound induced a late induction of the CtBP target, the BH3-only protein Noxa, at 24 h post-treatment (Figure 4E). Collectively, these results demonstrate that inhibition of CtBP co-repressor function is capable of inducing actinomycin D-sensitive CGN apoptosis.

Figure 4. The CtBP inhibitor, MTOB, induces CGN apoptosis.

Figure 4

A. MTOB dose response: CGNs were incubated for 24 h in Con medium containing increasing concentrations of MTOB. Apoptosis was quantified as the percentage of cells with condensed and/or fragmented nuclei. B. MTOB time course: CGNs were incubated for increasing durations with 5 mM MTOB and apoptosis was quantified. C. Representative fluorescence images of CGNs incubated for 24 h in Con medium alone or containing 5 mM MTOB ± actinomycin D (AD). D. Quantification of CGN apoptosis for the experiment described in (C). E. CGNs were incubated for increasing durations with 5 mM MTOB. Cell lysates were prepared and western blotted for Noxa and actin. For (A), (B), and (D); **p<0.01 compared to Con, ††p<0.01 compared to MTOB. The results shown represent the means ± SEM of three independent experiments each performed in duplicate.

In addition to the recognized proteasome degradation pathway, CtBPs are also downregulated through a novel caspase-dependent mechanism in CGNs

Several previous reports have demonstrated that CtBPs are downregulated via proteasomal degradation during p53-independent apoptosis in non-neuronal cells (Zhang et al., 2003; Zhang et al., 2005; Paliwal et al., 2006; Wang et al., 2006). However, the mechanism by which CtBPs are downregulated in neurons undergoing apoptosis has not previously been explored. To address this question, we analyzed the effects of inhibitors of the proteasome or caspases on the downregulation of CtBPs induced by various pro-death stimuli in CGNs. Downregulation of CtBP1 and CtBP2 induced under 5K apoptotic conditions in CGNs was completely prevented by the pan-caspase inhibitor, BOC, but was only partially attenuated by the proteasome inhibitor MG132 (Figure 5A). In response to apoptosis induced by toxin B or the related Clostridium sordellii lethal toxin which also inhibits Rac GTPase (Just et al., 1996), the observed downregulation of CtBP1 and CtBP2 was significantly blocked by caspase inhibition with BOC or zVAD but was unaffected by proteasome inhibition with MG132 (Figures 5B and 5C). In contrast to 5K and the Clostridial toxins which are each known to activate caspases in CGNs, we have previously shown that CGN death induced by MPP+ occurs through a caspase-independent mechanism (Harbison et al., 2011). In agreement with this, the downregulation of CtBP1 and CtBP2 induced by MPP+ in CGNs was unaltered by BOC but was significantly inhibited by MG132 (Figure 5D). These data indicate that distinct pro-death stimuli act through a proteasome-dependent pathway and/or an alternative caspase-dependent pathway to downregulate CtBPs in neurons. To our knowledge, this is the first demonstration of CtBP expression being regulated via a caspase-dependent mechanism.

Figure 5. Caspases and the proteasome contribute to CtBP downregulation in a stimulus-specific manner and CtBPs are downregulated in an exclusively proteasome-dependent manner following exposure to the complex I inhibitor MPP+.

Figure 5

A. CGNs were incubated for 24 h in Con medium or 5K medium ± the pan-caspase inhibitor BOC (50 μM) or the proteasome inhibitor MG132 (10 μM). B. CGNs were incubated for 24 h in Con medium alone or containing ToxB (40 ng/ml) ± either BOC or MG132. C. CGNs were incubated for 24 h in Con medium alone or containing Clostridium sordellii lethal toxin (400 μg/ml) ± the pan-caspase inhibitor zVAD (50 μM) or MG132. Following incubation, cells were lysed, proteins resolved by SDS-PAGE and transferred onto PVDF membranes. Blots were probed for CtBP1 and CtBP2 followed by stripping and re-probing for actin to indicate equal loading. D. CGNs were incubated for 24 h in Con medium alone or containing MPP+ (150 μM) ± BOC or MG132. Following incubation, cells were lysed, proteins resolved by SDS-PAGE, and immunoblotted using CtBP1 and CtBP2 antibodies. Blots were stripped and re-probed for actin to show equal loading.

6-hydroxydopamine (6-OHDA) induces caspase-dependent downregulation of CtBPs in the dopaminergic N27 cell line

In order to establish that the novel caspase-dependent mechanism of CtBP downregulation observed in CGNs undergoing apoptosis was not unique to this cell system, we next investigated the mode of CtBP downregulation in an in vitro model relevant to Parkinson's disease. The N27 cell line is a large T-antigen-immortalized, mesencephalon-derived cell line with characteristics of dopaminergic neurons (Zhou et al., 2000). Exposure of N27 cells to the neurotoxin, 6-OHDA, induces caspase-dependent apoptosis (Latchoumycandane et al., 2011). Incubation of N27 cells with 6-OHDA resulted in the downregulation of CtBP1 and CtBP2 in a dose-dependent manner (Figure 6A). In addition, the downregulation of CtBPs induced by 6-OHDA was largely prevented by the pan-caspase inhibitor, BOC, but was actually accentuated by the proteasome inhibitor MG132 (Figure 6B). Thus, caspase-dependent downregulation of CtBPs occurs in diverse neuronal cell systems and in response to multiple pro-apoptotic stressors.

Figure 6. Incubation of N27 dopaminergic cells with 6-OHDA induces downregulation of CtBP1 and CtBP2 through a caspase-dependent mechanism.

Figure 6

A. N27 cells were incubated for 24 h in Con medium alone or containing either 25 μM or 50 μM concentrations of 6-OHDA. Because of cell detachment induced by 6-OHDA, medium containing floating cells was obtained in a microcentrifuge tube and pelleted. The supernatant was then discarded and cells remaining in the 6-well dish were lysed, scraped, and added to the previously pelleted cells which had detached. Combined contents in the microcentrifuge tube were then lysed, and proteins were resolved by SDS-PAGE. Once transferred onto PVDF membranes, samples were immunoblotted with antibodies to CtBP1 and CtBP2. Membranes were stripped and re-probed for actin to show equal loading. B. N27 cells were incubated for 24 h in Con medium alone or containing 6-OHDA (25 μM) ± BOC (50 μM) or MG132 (10 μM). Cells lysates were immunoblotted as described in (A).

CtBP downregulation induced by 5K apoptotic conditions in CGNs does not occur through an enhanced rate of protein degradation

The downregulation of CtBP1 observed under 5K apoptotic conditions occurred in a relatively protracted manner over the course of many hours (Figure 7A). One principal mechanism by which proteins are downregulated is by an enhanced rate of degradation. Given that the downregulation of CtBPs under 5K apoptotic conditions was sensitive to caspase inhibition, we utilized pulse-chase methodology to determine if CtBPs were being actively degraded during CGN apoptosis. Quantification of a discrete pool of 35S-labeled CtBP1 (pulse) following incubation in either 25K or 5K serum-free medium (chase) resulted in nearly identical t1/2 values for CtBP1 of approximately 10 h (Figures 7B and 7C). These data suggest that CtBP1 does not undergo an enhanced rate of degradation during 5K-induced apoptosis in CGNs. Instead, the downregulation of CtBPs under these conditions is likely due to decreased synthesis of new protein.

Figure 7. Pulse-chase analysis of the kinetics of CtBP1 downregulation in CGNs incubated in apoptotic (5K) medium.

Figure 7

A. CGNs were incubated in 5K medium for various times over the course of 56 h. CtBP1 protein levels were assessed by western blotting and membranes were stripped and re-probed for actin to show equal loading. B. CGNs were initially incubated for 4 h in cysteine-methionine-free medium containing 35S-methionine. Following this “pulse” incubation, cells were washed and medium was replaced with serum-free Con medium or 5K medium. At various time points during this “chase” incubation, cells were lysed and CtBP1 was immunoprecipated using a monoclonal antibody. Immunoprecipitated samples were resolved on a 10% polyacrylamide gel, transferred onto a PVDF membrane, and exposed directly to film. C. Quantification of 35S-CtBP1 densitometry from (B) was graphed to compare the kinetics of loss of CtBP1 during the 24 h “chase” incubation period. Values are plotted as a percentage of the time 0 band density. CtBP1 displayed a half-life of approximately 10 h in CGNs incubated in either Con or 5K medium.

CtBP1 is not directly cleaved by caspase-3 in vitro

Upon examination of the CtBP1 amino acid sequence, a consensus DXXD caspase-3 cleavage motif was noted in human CtBP1 at residues 103DNID106. This consensus caspase-3 cleavage site is highly conserved and is present in CtBP1 and CtBP2 proteins of human, mouse, and rat species (Figure 8A). An identical DNID motif present in the adenomatous polyposis coli protein was previously shown to be directly cleaved by caspase-3 (Webb et al., 1999). To determine if this 103DNID106 site serves as a viable caspase-3 cleavage site in CtBP1 we performed an in vitro proteolysis experiment with recombinant caspase-3 and recombinant CtBP1. As a positive control for caspase-3 activity, we utilized poly(ADP-ribose) polymerase (PARP) which is cleaved by caspase-3 to generate an 85 kDa fragment (Tewari et al., 1995). As expected, recombinant caspase-3 cleaved PARP to produce the 85 kDa fragment but it failed to cleave CtBP1 to any appreciable extent (Figure 8B). Thus, despite the presence of a consensus caspase-3 cleavage motif, CtBP1 is not a direct substrate of caspase-3. This result is in agreement with the above data and further suggests that the downregulation of CtBPs observed in CGNs undergoing apoptosis is not due to direct caspase-mediated degradation.

Figure 8. Despite the presence of a highly conserved, consensus caspase-3 cleavage site, CtBP1 is not directly cleaved by recombinant caspase-3 in vitro.

Figure 8

A. CtBP1 and CtBP2 sequences across numerous species indicate a highly conserved caspase-3 cleavage site (DXXD; shaded residues). B. Recombinant CtBP1 (1.5 μg) or recombinant PARP (5 μg) were incubated in the absence or presence of recombinant caspase-3 (rCasp3; 100 U) for 4 h at 37°C. Recombinant proteins were then run on 7.5% polyacrylamide gels and western blotted with monoclonal antibodies to CtBP1 and PARP. The full length CtBP1 and PARP proteins are indicated by “fl”. The expected 85 kDa PARP cleavage fragment produced by caspase-3-dependent proteolysis is denoted as “clv”.

CtBP transcript levels are not significantly decreased during 5K-induced apoptosis in CGNs

If the downregulation of CtBPs under 5K apoptotic conditions is due to decreased synthesis of new protein, then one possible point of regulation is at the level of CtBP gene transcription. To determine if CtBP1 and CtBP2 mRNA levels are reduced when CGNs are incubated in 5K apoptotic medium, we performed RT-PCR analysis. Surprisingly, even after 24 h incubation in 5K medium, no significant decrease in either CtBP1 or CtBP2 transcript expression was observed (Figures 9A and 9B). Similar results were also obtained using quantitative real-time PCR analysis (data not shown). These results indicate that the downregulation of CtBPs observed during CGN apoptosis is not due to decreased gene transcription.

Figure 9. Incubation of CGNs in apoptotic (5K) medium has no significant effect on CtBP1 or CtBP2 transcript expression.

Figure 9

A. CGNs were incubated in either Con or 5K medium for 24 h. Total RNA was isolated and subjected to RT-PCR using primers specific for CtBP1, CtBP2, and actin mRNA transcripts. Representative bands are shown. B. Quantification of three independent experiments performed in duplicate as described in (A). CtBP1 and CtBP2 transcript expression levels were divided by the corresponding actin signals for normalization.

CtBP1 undergoes caspase-dependent downregulation in staurosporine-treated, wild type (WT) mouse embryonic stem cells (mESCs) but not DGCR8 knockout (KO) mESCs

The above findings indicate that CtBP function is essential for CGN survival and furthermore, during CGN apoptosis CtBPs undergo an indirect, caspase-dependent downregulation that occurs via a post-transcriptional mechanism. Micro RNAs (miRNAs) are small noncoding RNAs that act as negative post-transcriptional regulators by binding to the 3’ UTRs of target mRNAs (Lai, 2002). Mature miRNAs are produced by a series of tightly regulated processing events. First, the primary miRNA transcript (pri-miRNA) is cleaved by the Microprocessor, a protein complex consisting of the ribonuclease Drosha and its essential cofactor, DiGeorge Critical Region 8 (DGCR8) (Gregory and Schiekhattar, 2005). Microprocessor cleavage of pri-miRNAs generates intermediate precursor miRNAs (pre-miRNAs) which are in turn, processed by Dicer to produce the mature miRNAs (Triboulet and Gregory, 2010). As a first step to determine if the caspase inhibitor-sensitive downregulation of CtBPs induced during apoptosis might be mediated via a miRNA-dependent pathway, we compared the expression of CtBP1 in WT mESCs to DGCR8 KO mESCs which are deficient in miRNA biogenesis (Wang et al., 2007). Incubation of WT mESCs with the classical apoptosis inducer, staurosporine, caused a marked downregulation of CtBP1 that was prevented by the pan-caspase inhibitor QVD (Figure 10A, left blot). In contrast, incubation of DGCR8 KO mESCs with staurosporine failed to have any significant effect on the expression of CtBP1 (Figure 10A, right blot). It is important to note that staurosporine treatment caused significant cell death in both cell lines that was partially blunted by co-treatment with QVD (Figure 10B). These data indicate that the caspase-dependent downregulation of CtBP1 induced under apoptotic conditions requires intact miRNA processing and biogenesis machinery.

Figure 10. DGCR8 knockout mouse embryonic stem cells (mESCs) do not display caspase-dependent downregulation of CtBP1 in response to staurosporine treatment.

Figure 10

A. Wild-type (WT) and DGCR8 knockout (KO) mESCs were incubated for 24 h in Con medium alone or containing staurosporine (STS; 100 nM) ± the pan-caspase inhibitor QVD (20 μM). Following incubation, cell lysates were resolved by SDS-PAGE and western blotted for CtBP1. Membranes were subsequently stripped and reprobed for actin as a loading control. B. WT or KO mESCs were treated as described in (A). Representative bright field images are shown following a 24 h treatment period. Scale bar=10 microns.

Discussion

CtBPs act as transcriptional co-repressors of a number of pro-apoptotic genes including the Bcl-2 family members Bax, Noxa, Bik, Bim, and Bmf (Grooteclaes et al., 2003, Bergman and Blaydes, 2006, Kovi et al., 2010). Therefore, it is not surprising that CtBPs might be downregulated in cells undergoing apoptosis. However, to our knowledge the present study is the first to document this effect during neuronal apoptosis. Previous studies of CtBP function in the nervous system have been mostly limited to the role of these proteins in development. For instance, the Drosophila CtBP (dCtBP) significantly impacts development of the fly peripheral nervous system by negatively regulating formation of mechanosensory bristles, perhaps by influencing extra sensory organ precursor cell fate (Biryukova and Heitzler, 2008; Stern et al., 2009). CtBPs are expressed throughout the developing avian CNS, often in overlapping regions but sometimes in unique localizations such as CtBP1 expression in dorsal root ganglia and CtBP2 expression in emigrating neural crest cells (Van Hateren et al., 2006). The functional significance of CtBP expression in the developing chick CNS is demonstrated by the key role that these proteins play in regulating the transition of neural precursor cells in the ventricular zone of the dorsal spinal cord from a proliferative to a differentiated state (Xie et al., 2011). In a similar manner, CtBP1 and CtBP2 display both duplicative and independent roles in mouse CNS development including maturation of the forebrain and midbrain (Hildebrand and Soriano, 2002). Additional studies have demonstrated interactions of CtBPs with a number of neuronal proteins including neuronal nitric oxide synthase, actin-related protein alpha, and calsenilin, although the physiological significance of these interactions is not well defined (Riefler and Firestein, 2001; Oma et al., 2003; Zaidi et al., 2006). Finally, CtBPs have been loosely associated with some types of CNS injury. For example, CtBP expression has been shown to decline rapidly following spinal cord injury in the mouse (Cai et al., 2012). In another study, the glycolytic inhibitor, 2-deoxy-D-glucose, suppressed seizure activity in a rat kindling model of temporal lobe epilepsy via an NRSF/CtBP-dependent repression of the BDNF gene promoter (Garriga-Canut et al., 2006). These studies demonstrate that CtBPs are key factors in CNS and peripheral nervous system development; however, the role of CtBPs in determining neuronal survival and death has not been explicitly investigated.

In the present study, we have identified CtBPs as essential pro-survival proteins in CGNs. The expression of CtBP1 and CtBP2 was significantly downregulated in CGNs exposed to a number of pro-apoptotic stressors. Moreover, forced downregulation of CtBP1 using morpholino-antisense oligonucleotides was sufficient to induce CGN apoptosis. In a similar manner, incubation of CGNs with the CtBP inhibitor, MTOB, induced the upregulation of pro-apoptotic Noxa and triggered actinomycin D-sensitive CGN apoptosis. These results are consistent with CtBPs functioning in CGNs as pro-survival factors which are subject to downregulation during neuronal apoptosis. One may consider the downregulation of CtBPs as a major factor in determining whether neurons succumb to apoptosis since loss of CtBP function ultimately leads to the de-repression of pro-apoptotic genes.

In non-neuronal cells, CtBPs have previously been shown to be targeted for proteasomal degradation in response to pro-apoptotic stimuli that induce p53-independent apoptosis (Zhang et al., 2003; Zhang et al., 2005; Paliwal et al., 2006; Wang et al., 2006). In contrast, CGNs exposed to Rac GTPase-inhibitory Clostridial toxins displayed downregulation of CtBP1 and CtBP2 that was insensitive to proteasome inhibition but sensitive to caspase inhibition. This novel caspase-dependent downregulation of CtBPs also predominated under 5K apoptotic conditions in CGNs and in N27 dopaminergic cells exposed to 6-OHDA. In contrast, the downregulation of CtBPs induced in CGNs by the complex I inhibitor, MPP+, was completely insensitive to caspase inhibition but was significantly attenuated by proteasome inhibition. These results demonstrate that the downregulation of CtBPs during neuronal apoptosis occurs via a stimulus-specific mechanism with both proteasome-dependent and caspase-dependent modes of downregulation.

Previous studies on the regulation of CtBP expression have focused largely on the proteasome-dependent degradation of these transcriptional co-repressors. During p53-independent apoptosis of non-neuronal cells, CtBPs are phosphorylated on Ser422 or Ser 428 (CtBP1 or CtBP2, respectively) by either homeodomain interacting protein kinase-2 or c-Jun NH2-terminal kinase, targeting these proteins for subsequent ubiquitinylation and proteasomal degradation (Zhang et al., 2003; Zhang et al., 2005; Wang et al., 2006). Under some conditions, targeting of CtBP to the proteasome may require interaction with additional proteins such as the tumor suppressor ARF (Paliwal et al., 2006). Recently, additional pathways have been suggested to regulate CtBP expression via the proteasome. For instance, AKT1 has recently been shown to cooperate with the SUMO E3 ligase Pc2 to induce phosphorylation and ubiquitinylation of CtBP resulting in its enhanced degradation (Merrill et al., 2010). Another pathway for CtBP degradation involves its interaction with the X-linked inhibitor of apoptosis protein which directly ubiquitinylates CtBP and targets it for proteasomal degradation (Lee et al., 2012). On the other hand, B-cell lymphoma-3 (Bcl-3) is a proto-oncogene that has recently been shown to interact with and stabilize CtBP by preventing its ubiquitinylation and subsequent proteasomal degradation (Choi et al., 2010). Interestingly, CtBP1 protein levels were downregulated in HEK293T cells incubated with the apoptosis inducer etoposide, but this effect was prevented by overexpression of Bcl-3. Thus, proteasome-dependent degradation appears to be a dominant pathway for turnover of CtBPs in non-neuronal cells undergoing apoptosis.

In marked contrast to these previous studies, we have identified a novel caspase-dependent pathway for CtBP downregulation in neurons undergoing apoptosis. Despite the presence of a conserved caspase-3 consensus cleavage site in the CtBP1 and CtBP2 proteins, CtBPs do not appear to be direct caspase-3 substrates in vitro. Although it cannot be ruled out that other caspase family members may directly cleave CtBPs in neurons undergoing apoptosis, the rate of degradation of CtBP1 in CGNs is not significantly altered in 5K (apoptotic) medium suggesting that the downregulation of CtBPs observed under these conditions is not due to enhanced proteolysis. Intriguingly, CtBP1 and CtBP2 mRNA transcripts are not significantly decreased in CGNs subjected to 5K apoptotic conditions indicating that the downregulation of CtBPs likely occurs via a post-transcriptional mechanism. Consistent with this idea, the caspase-dependent downregulation of CtBP1 observed in mESCs exposed to staurosporine does not occur in DGCR8 KO cells that are deficient in miRNA biogenesis. Collectively, these data suggest that the caspase-dependent downregulation of CtBPs observed in neurons undergoing apoptosis may occur via a miRNA-dependent mechanism.

Several recent studies add support to the hypothesis that CtBPs are subject to significant post-transcriptional regulation by miRNAs. For instance, the expression of miR-137 was found to inversely correlate with CtBP1 expression in melanoma cell lines. Moreover, miR-137 suppressed CtBP1 3’ UTR luciferase-reporter activity and overexpression of miR-137 decreased CtBP1 levels and caused a corresponding increase in expression of the CtBP1 target Bax (Deng et al., 2011). Interestingly, the miR-137 gene is found on chromosome 1p22 which is a known susceptibility region for melanoma and furthermore, miR-137 was found to be under expressed in a subset of patient-derived melanomas (Walker et al., 2004; Bemis et al., 2008; Chan et al., 2011). In a similar manner, the miR-141-200c cluster was recently shown to downregulate the expression of CtBP2 and its transcriptional repressor partner, ZEB, in PANC-1 human pancreatic carcinoma cells (Sass et al., 2011). ZEB is a key inducer of the epithelial-to-mesenchymal transition which is thought to promote malignant tumor progression, particularly in pancreatic, colorectal, and breast cancer (Burk et al., 2008). These studies indicate that specific miRNAs may act as tumor suppressors in part by targeting CtBPs for translational repression. It is noteworthy that miR-137 has recently been shown to act as a key regulator of embryonic neural stem cell fate (Sun et al., 2011). However, whether miR-137 regulates CtBP expression in the CNS is presently unknown. In the future, it will be important to determine if downregulation of CtBPs is associated with particular neurodegenerative diseases, particularly those for which caspases are implicated in the underlying pathogenesis.

Finally, elucidating the mechanism by which caspases indirectly influence CtBP expression during neuronal apoptosis will require additional study. One possibility is that caspases degrade a protein that under healthy conditions acts as a suppressor of specific miRNAs. Once this suppressor is degraded, miRNAs are induced and target CtBPs for translational repression. This in turn, leads to the de-repression of a subset of CtBP target pro-apoptotic genes that contribute to the execution of neuronal apoptosis. The existence of “RNA silencing suppressor” (RSS) proteins is evidenced by the HIV-1 Tat and Rex proteins which suppress specific siRNAs or miRNAs by competing for their binding with Dicer or other proteins of the RNA-induced silencing complex (Houzet and Jeang, 2011; Rawlings et al., 2011). Thus, it is reasonable to hypothesize that neurons may possess intrinsic RSS proteins that are degraded by caspases during neuronal apoptosis. Identification of these putative neuronal RSS proteins may be necessary to resolve the mechanism underlying caspase-dependent CtBP downregulation during neuronal apoptosis.

Acknowledgements

This study was supported by a Merit Review grant from the Department of Veterans Affairs (to D.A.L.) and a R01 grant NS062766 from the National Institutes of Health (to D.A.L.).

The abbreviations used are

6-OHDA

6-hydroxydopamine

AD

actinomycin D

Anti

morpholino-antisense oligonucleotides

Bcl-3

B-cell lymphoma-3

BKLF

basic krüppel-like factor

CCD

charge-coupled device

CGNs

cerebellar granule neurons

Con

control

CtBP

C-terminal binding protein

DAPI

4,6-diamidino-2-phenylidole

dCtBP

drosophila CtBP

DGCR8

DiGeorge critical region 8

EP

endoporter

GITz-short

G protein-coupled receptor kinase-interacting protein-z-short

Inv

inverse

KO

knockout

LTox

lethal toxin

MEFs

murine embryonic fibroblasts

mESCs

mouse embryonic stem cells

miRNA

micro RNA

MPP+

methyl-4-phenylpyridinium

MTOB

4-methylthio-2-oxobutyric

PARP

poly-(ADP-ribose) polymerase

PBS

phosphate buffer saline

PCR

polymerase chain reaction

rCasp3

recombinant caspase-3

RSS

RNA silencing suppressor

SNP

sodium nitroprusside

STS

staurosporine

Tox B

Toxin B

WT

wild type

Footnotes

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