Adenoviral expression of N17Rac1 induces apoptosis of CGNs: dominant-negative mutants of RhoA or Cdc42 do not cause significant cell death. (a) On day 4 in vitro, CGN cultures were infected with adenovirus expressing either GFP alone or in combination with dominant-negative N17Rac1. At 72 h post-infection, CGNs were fixed in paraformaldehyde and nuclei were stained with Hoechst dye. Images shown represent composites of two fields each for GFP or GFP/N17Rac1. Infected cells are indicated by the arrows. CGNs infected with adenovirus expressing GFP alone showed large intact nuclei characteristic of healthy cells (upper panels). In contrast, CGNs infected with adenovirus expressing N17Rac1 displayed marked chromatin condensation and fragmentation indicative of apoptotic cell death (lower panels). Note that non-infected cells in the N17Rac1 condition remained healthy. Scale bar, 10 μm. (b) Quantitation of CGN apoptosis in GFP-expressing cells following 72 h of infection with adenoviruses expressing either GFP alone or in combination with dominant-negative mutants of Rac1, Cdc42, or RhoA at a final titer of 50 infectious particles per cell. Using these conditions, each of the adenoviruses infected approximately 15-20% of the CGNs in the cultures. The results shown represent the means ± SEM for four experiments, each conducted on triplicate coverslips in which approximately 100 GFP-positive CGNs were counted per condition per coverslip. *Significantly different from adenoviral GFP alone (p < 0.01).

ToxB and N17Rac1 each trigger cytochrome c release from mitochondria in CGNs. (a) CGNs were incubated for 24 h in complete medium (containing serum and 25 mm KCl) in the presence of either vehicle (Veh; 2 lg/mL BSA in PBS) or ToxB (40 ng/mL). Following incubation, cell lysates were fractionated into cytosolic (C) and mitochondrial (M) subcellular fractions as described in Experimental procedures. Equivalent amounts of cytosolic (20 μg) and mitochondrial (40 μg) fractions from Veh- or ToxB-treated cells were subjected to SDS-PAGE and immunoblotted for cytochrome c (cyt C). The membranes were then stripped and re-probed for the integral mitochondrial membrane protein, cyt C oxidase subunit IV (COXIV), to verify the purity of the subcellular fractions. Note that the amount of cyt C detectable in mitochondrial fractions was significantly less in ToxB-treated CGNs, although the amount of immunoreactive COXIV was similar to Veh. The cyt C released into the cytosolic fraction in the ToxB-treated cells did not appear at the same molecular weight as that detected in the mitochondrial fractions, but instead it displayed several higher molecular weight forms (indicated by <) which may reflect ubiquitination of the cytoplasmic cyt C. The blots shown are illustrative of data from two independent experiments. (b) CGNs were exposed to Veh or ToxB as described in (a). After 24 h, cells were fixed with paraformaldehyde, blocked and permeabilized in 5% BSA and 0.2% Triton X-100, and incubated with a polyclonal antibody to cyt C. Localization of cyt C was detected using a Cy3-conjugated secondary antibody and nuclei were stained with DAPI. Veh-treated CGNs showed healthy nuclei and cyt C localization that was mostly perinuclear consistent with mitochondria (upper panels). In contrast, a significant fraction of the CGNs exposed to ToxB displayed fragmented nuclei and a diffuse distribution of cyt C over the entire cell body (lower panels, see arrows). The images shown are indicative of those from three separate experiments. Scale bar, 10 μm. (c) CGNs were incubated for 72 h after infection with adenovirus expressing GFP and N17Rac1 (at a final titer of 50 infectious particles per cell). Following infection, cells were stained for cyt C, as described in (b). The merged image shows that the majority of GFP- and N17Rac1-positive cells (indicated by the arrows) had condensed or fragmented nuclear morphology and lacked detectable cyt C staining, whereas surrounding uninfected cells were largely healthy and displayed significant perinuclear cyt C. The essentially complete loss of cyt C staining in the N17Rac1-expressing cells likely reflects the diffuse distribution of the protein over such a prolonged time post-infection. The images shown are composites of three different fields of CGNs infected with adenoviral GFP/N17Rac1. Scale bar, 10 μm.

ToxB elicits translocation of a GFP-Bax fusion protein to mitochondria and conformational activation of endogenous Bax in CGNs. (a) On day 5 in vitro, CGNs were transfected with either GFP alone or a GFP-Bax fusion protein using a helium-powered gene gun, as described in Experimental procedures. At 48 h post-transfection, cells were exposed for 24 h to either vehicle (Veh; 2 μg/mL BSA in PBS) or ToxB (40 ng/mL). CGNs were then fixed in paraformaldehyde and nuclei were stained with DAPI. The expressed GFP-Bax fusion protein had a diffuse distribution in the predominantly healthy CGNs incubated with Veh (upper panels). In contrast, cells exposed to ToxB demonstrated marked nuclear condensation and fragmentation, and GFP-Bax staining that was punctate and consistent with localization to mitochondria (middle panels). In CGNs transfected with GFP alone, ToxB still induced substantial apoptosis; however, GFP maintained a distribution that encompassed the entire cell unlike the more localized distribution of GFP-Bax under these conditions (lower panels). The images shown are representative of results from two experiments. Transfected cells in each panel are indicated by the arrows. Scale bar, 10 μm. (b) The areas demarcated by the boxes in (a) have been enlarged 300% to enhance the visualization of GFP-Bax or GFP. (c) Untransfected CGNs were incubated with either Veh or ToxB for 24 h and cells were fixed and immunostained with a monoclonal antibody (clone 6A7) that specifically detects the active conformation of Bax. Active endogenous Bax was visualized in a punctate mitochondrial distribution using a Cy3-conjugated anti-mouse secondary antibody (indicated by the arrows). Scale bar, 10 μm.

The pyridinyl imidazole, SB203580, inhibits mitochondrial apoptotic signaling induced by ToxB in CGNs. (a) CGNs were incubated for 24 h with either vehicle (Veh; 2 μg/mL BSA in PBS) or ToxB (40 ng/mL) ± SB203580 (SB; 20 μm). Following incubation, cells were immunostained with a monoclonal antibody to the active conformation of Bax (clone 6A7) and nuclei were stained with DAPI. The arrows indicate cells that stained positively for active Bax. Scale bar, 10 μm. (b) CGNs were incubated exactly as described in (a) and either mitochondrial fractions were immunoblotted (IB) for cyt C or whole cell lysates were probed for active caspase-9 or -3. The blots shown are each indicative of data obtained in three separate experiments.

ToxB induces apoptosis and elicits a selective caspase-mediated degradation of Rac1 GTPase in CGNs. (a) CGNs were incubated for 24 h in complete medium (containing serum and 25 mm KCl) in the presence of either vehicle (Veh; 2 μg/mL BSA in PBS) or ToxB (40 ng/mL). Following incubation, cells were fixed in paraformaldehyde and nuclei were stained with Hoechst dye. CGNs exposed to ToxB showed marked nuclear condensation and fragmentation characteristic of apoptosis. Scale bar, 10 μm. (b) Representative immunoblots of lysates obtained from CGNs incubated as described in (a). The blots shown were obtained from the BD PowerBlot™ (see Experimental procedures for details) using specific monoclonal antibodies against Rac1 (upper blots), RhoA (middle blots), or Cdc42 (lower blots). The immunoreactive Rho family GTPases are indicated by the arrows. Other protein bands apparent on the membranes represent adjacent lanes of the PowerBlot™ that were probed for distinct proteins and are shown to give an indication of the overall equality of protein loading. (c) The fold change in protein content induced by 24 h of ToxB treatment for each of the Rho family members blotted for in (b) is shown. Data represent the means ± range of duplicate PowerBlot™ comparisons for each GTPase. The dotted lines indicate no change (-1 to +1 fold). (d) CGNs were exposed for 8 h to ToxB in the absence or presence of either a pan-caspase inhibitor (Boc-Asp(OMe)-FMK; 200 μm) or zVAD-FMK; 200 μm) or a proteasome inhibitor (MG132; 10 μm). Following incubation, cell lysates were immunoblotted for Rac1.

Rac1 GTP-loading is maintained in CGNs by trophic support and integrin-mediated cell attachment. CGNs were incubated under either attached conditions (a) in trophic factor-deprived medium containing 5 mm KCl and lacking serum (5K-Ser) for up to 4 h, detached conditions (b) for up to 2 h in medium containing 25 mm KCl and serum (25K + Ser), or incubated with RAD or RGD peptides (c) for 8 h. Following incubation, CGNs were immediately lysed on ice into a high Mg²⁺ buffer. Cell debris was removed by brief centrifugation and a small aliquot of the resulting lysate was separated for quantitation of total Rac in each sample. The remaining cell lysates were incubated for 1 h with an excess of the protein binding domain of the Rac effector, PAK (PAK-PBD), bound to agarose beads. The pellet was washed three times with lysis buffer and samples were electrophoresed through 15% polyacrylamide gels. Proteins were transferred to PVDF membranes which were immunoblotted with a monoclonal antibody against Rac1 and an HRP-conjugated secondary antibody. Immunoreactive Rac1 protein was detected using standard ECL techniques. GTP-loaded Rac was brought down in the PAK-PBD precipitation (middle blots). The PAK-PBD is detected non-specifically by the secondary antibody and is shown as a control for equal loading (upper blots). The total Rac in each lysate is also shown as a loading control as no significant loss is detected in Rac protein under these conditions (bottom blots). The blots shown are representative of results obtained in three separate experiments. Att, attached control.

ToxB and N17Rac1 each promote activation of caspases-9 and -3 in CGNs. (a) CGNs were incubated for 24 h in complete medium (containing serum and 25 mm KCl) in the presence of either vehicle (Veh; 2 μg/mL BSA in PBS) or ToxB (40 ng/mL). After incubation, cell lysates were resolved by SDS-PAGE on 15% polyacrylamide gels and proteins were transferred to PVDF membranes. The blots were then probed with an antibody that specifically detects the cleaved (active) form of caspase-9. (b) CGNs were treated exactly as described in (a) and lysates were western blotted with an antibody that recognizes both the pro- and cleaved (active) forms of caspase-3. The blots shown in (a) and (b) are representative of results obtained in three separate experiments. (c) CGNs were incubated for 72 h after infection with adenovirus expressing GFP and N17Rac1 (at a final titer of 50 infectious particles per cell). Following infection, cells were fixed in paraformaldehyde, blocked and permeabilized in 5% BSA and 0.2% Triton X-100, and immunostained with an antibody that specifically detects the active (cleaved) form of caspase-9 [Casp-9 (a)] and a Cy3-conjugated secondary antibody. Nuclei were stained with DAPI. The merged image shows that GFP- and N17Rac1-positive cells (indicated by the arrows) had condensed or fragmented nuclear morphology and displayed significant immunoreactivity for active caspase-9, whereas surrounding uninfected cells were largely healthy and devoid of active caspase-9. (d) CGNs were infected as described in (c) and were then immunostained for active (cleaved) caspase-3 [Casp-3 (a)]. As described above, most of the GFP- and N17Rac1-positive cells (indicated by the arrows) showed marked chromatin condensation and fragmentation and stained positively for active caspase-3. The images shown in (c) and (d) are composites of three to four different fields of CGNs infected with adenoviral GFP/N17Rac1. Ad-GFP control data not shown. Scale bars, 10 μm.

ToxB and N17Rac1 each stimulate c-Jun activation and induction of Bim in CGNs: inhibition of ToxB-induced Bim expression by the pyridinyl imidazole, SB203580. (a) CGNs were incubated for up to 8 h with ToxB (40 ng/mL) and cell lysates were immunoblotted (IB) with a polyclonal antibody to c-Jun. The decreased mobility of c-Jun observed with increasing duration of ToxB exposure is indicative of activating phosphorylation. (b) CGNs were incubated for 6 h with either BSA/PBS vehicle (Veh) or ToxB ± SB203580 (SB; 20 μm). Following treatment, CGN lysates were immunoblotted for c-Jun. (c) CGNs were incubated for 6 h as described in (b). Cell lysates were then immunoblotted with a polyclonal antibody that detects an approximately 15-kDa isoform of Bim, Bim short (Bim_s). (d) CGNs were infected with either adenoviral GFP alone or in combination with N17Rac1. At 96 h post-infection, cell lysates were obtained and immunoblotted for c-Jun, Bim_s, and actin (as a loading control). NS, non-specific protein bands that are detected by the antibodies and are shown as loading controls.

The JNK-specific inhibitor, SP600125, significantly blocks c-Jun phosphorylation, Bim induction, and apoptosis in ToxB-treated neurons. (a) CGNs were incubated for 8 h with ToxB (40 ng/mL) ± SP600125 (20 μm). Cell lysates were then immunoblotted with a polyclonal antibody for c-Jun. Activation of phosphorylation is seen by the decreased mobility of c-Jun in a time-dependent manner upon incubation with ToxB. (b) CGNs were incubated as above in (a) for 8 h. The cell lysates were then immunoblotted with a polyclonal antibody that detects Bim_s, approximately 15 kDa. (c) CGNs were incubated as described above for 24 h. The cells were then fixed with paraformal-dehyde and stained with Hoechst to visualize nuclei. The cells incubated with SP600125 showed a significant decrease in apoptosis. The results shown represent the means ± SEM of three experiments with approximately 400 cells counted per condition in each experiment. *Significantly different from Veh-treated cells (p< 0.01). †Significantly different from ToxB-treated cells (p< 0.01).