Pin1 knockdown delays cell death independent of Bim_EL. Sympathetic neurons from Bim (+/+) and Bim (−/−) mice (littermates) were microinjected with U6/Ctr or U6/Pin1 vectors and subjected to NGF deprivation as described in Fig. 3e. Cell viability was assessed 24, 48 and 72 hr after removing NGF. Results represent mean ± SEM from 6 independent experiments. In both Bim (+/+) and Bim (−/−) neurons, survival of U6/Pin1-injected neurons at 24 and 48 hr after NGF deprivation was significantly greater than the corresponding U6/Ctr-injected cells (p < 0.01, ANOVA with post-hoc Bonferroni test).

Knockdown of Pin1 expression delays loss of cytochrome c from mitochondria in NGF-deprived neurons. Sympathetic neurons were microinjected and treated as described in Fig. 3d except that immunofluorescence was performed with an antibody against cytochrome c. (a) Representative images obtained with confocal microscopy showing GFP-positive nuclei (green) and punctate cytochrome c immunofluorescence (red) present in only one of two U6/Ctr-injected cells and both U6/Pin1-injected cells 24 hr after NGF withdrawal. (b) The number of injected cells lacking the intense punctate immunofluorescence characteristic of mitochondrial-localized cytochrome c was expressed as a percentage of the total number of injected (GFP-positive) cells. Results are mean ± SEM from 3 independent experiments with 100–300 cells scored per condition in each experiment. U6/Luc (+NGF) vs U6/Luc (−NGF), p < 0.001; U6/Luc (−NGF) vs U6/Pin1 (−NGF), p < 0.001; one-way ANOVA followed by post-hoc Bonferroni test.

Modulating Pin1 levels influences c-Jun phosphorylation in NGF-dependent neurons. (a, b) Sympathetic neurons were microinjected with Pin1 plasmid or empty pcDNA₃ vector (100 μg/ml) together with pBOS-H2BGFP and maintained in NGF-containing media in the presence of BAF. After 48 hr, cells were subjected to immunofluorescence with an antibody against Ser⁶³-phosphorylated c-Jun. Nuclei were counterstained with Hoechst 33,258. (a) Representative images of vector-injected and Pin1-injected cells. (b) The percentage of injected cells with nuclei exhibiting phosphorylated c-Jun (P-c-Jun) immunofluorescence was determined (mean ± SEM of 3 independent experiments; *p < 0.05, Student’s t-test). (c) Sympathetic neurons were microinjected with U6/Luc or U6/Pin1 plasmids (75 μg/ml) together with pBOS-H2BGFP. The next day, cells were deprived of NGF in the presence of BAF or re-fed with fresh NGF-containing media and incubated for an additional 20 hr, after which immunofluorescence for phosphorylated c-Jun was performed. (d) The percentage of cells injected as described above with nuclei exhibiting phosphorylated c-Jun immunofluorescence was determined 20 hr after NGF withdrawal (mean ± SEM from 3 experiments; *p < 0.05, Student’s t-test). (e) Neurons from Bim (−/−) mice were injected with the U6/Luc or U6/Pin1 plasmid together with pBOS-H2BGFP and the next day deprived of NGF. Twenty hours after NGF withdrawal, cells were subjected to immunofluorescence with antibody against phosphorylated c-Jun and analyzed as described above (mean ± SEM, n = 3; *p < 0.05, Student’s t-test).

Pin1-induced cell death requires c-Jun activity. (a) Sympathetic neurons were injected with U6/Pin1 or U6/Ctr (75 μg/ml) together with a plasmid expressing dominant-negative c-Jun (c-Jun^bZIP) or empty vector (each at 100 μg/ml). The next day cells were deprived of NGF and survival was assessed 48 hr later. Results represent mean ± SEM of 3 experiments. Survival of neurons injected with U6/Pin1, c-Jun^bZIP, or both was significantly greater than U6/Ctr-injected neurons (p < 0.01, one-way ANOVA with post-hoc Bonferroni test). (b) NGF-maintained sympathetic neurons were injected with empty vector, Pin1 plasmid (75 μg/ml), or c-Jun^bZIP plasmid (100 μg/ml), or co-injected with Pin1 plus c-Jun^bZIP, along with pBOS-H2BGFP. The total number of GFP-positive cells was determined 12–16 hr after injection and the number of healthy GFP-expressing cells was assessed 24 hr later (36–40 hr post-injection). Results represent mean ± SEM from 4–6 independent injection experiments (*p < 0.01 compared to each of the other conditions, one-way ANOVA with post-hoc Bonferroni test).

Expression of wild type but not catalytically inactive Pin1 promotes death of NGF-maintained sympathetic neurons. (a) Sympathetic neurons maintained in the presence of NGF for 5–6 days were microinjected with either Pin1 expression plasmid or empty pcDNA₃ vector (each at 100 μg/ml) together with pBOS-H2BGFP, which expresses a GFP-histone fusion protein for visualizing the nuclei of injected cells. Shown are phase-contrast and epifluorescence images of injected (GFP-positive) cells and neighboring uninjected cells 72 hr after microinjection. Breakdown of the nuclear membrane and initial chromatin condensation are evident in the Pin1-expressing neuron in the middle panels while the right panels show the remnant of a Pin1-expressing cell with condensed chromatin. (b) Quantitation of Pin1-induced cell death 72 and 96 hr post-injection. The percentage of healthy injected cells was scored as described in Experimental procedures. Results represent mean ± SEM of 3 independent experiments with a minimum of 150–200 cells injected/condition in each experiment. Death of Pin1-injected cells was significantly greater than controls at both 72 and 96 hr (Student’s t-test, p < 0.05). (c) Pin1 catalytic activity is required for cell death. Microinjections were performed as outlined above except that additional neurons were injected with plasmids expressing Pin1(H59A) and Pin1(C113A). Viability was assessed 72 hr after microinjection. Results represent mean ± SEM of 3 independent experiments. Death of Pin1-injected neurons was significantly greater than that of neurons injected with empty vector, Pin1(H59A), or Pin1(C113A) [*p < 0.05, one-way analysis of variance (ANOVA) with Bonferroni post-hoc test].

Pin1-induced death is reduced by a caspase inhibitor and in the absence of Bax. (a) Sympathetic neurons maintained in the presence of NGF for 5–6 days were microinjected with 100 μg/ml Pin1 expression plasmid or empty pcDNA₃ vector together with pBOS-H2BGFP. Immediately following the injections, the culture medium was replaced with medium containing the general caspase inhibitor BAF (50 μmol/L). Fresh BAF was added to the media again at 36–40 hr and cell survival was assessed at 60–70 hr after microinjection. Results represent mean ± SEM of 3–6 independent experiments. The death of Pin1-injected neurons was significantly greater than that of control-injected neurons or Pin1-injected, BAF-treated neurons (p < 0.001, one-way ANOVA with Bonferroni post-hoc test). Death of Pin1-injected, BAF-treated neurons was not significantly different from control (p > 0.05). (b and c) Sympathetic neurons isolated from Bax (+/+) and Bax (−/−) mice (littermates) were microinjected with either Pin1 expression plasmid or empty vector as described above. (b) Phase-contrast and epifluorescence images show injected (GFP-positive) and neighboring uninjected cells 60 hr post-injection. (c) The percentage of injected cells that remained healthy was determined 36 and 60 hr post-injection. Results represent the mean ± SEM from 3 injection experiments with a minimum of 150–200 cells injected per condition in each experiment. Note: For several data points the error bars do not extend outside the symbols. Survival of Pin1-injected Bax (+/+) neurons was significantly less than vector-injected Bax (+/+) and Bax (−/−) neurons (p < 0.001), and Pin1-injected Bax (−/−) neurons (p < 0.05, 60 hr time) (one-way ANOVA with Bonferroni post-hoc test).

Knockdown of Pin1 expression delays death of NGF-deprived neurons. (a) Expression of Pin1 in mouse sympathetic neurons. Sympathetic neurons were incubated with fresh NGF-containing media or deprived of NGF for 7.5 hr. Whole cell lysates were immunoblotted with antibodies against Pin1, c-Jun, phosphorylated c-Jun (Ser⁶³), Bim_EL, and actin. (b) shRNA-mediated knockdown of Pin1 in HEK293 cells. Cells were mock-transfected or transfected with U6/Pin1 or U6/Ctr plasmids. After 48 hr, whole cell lysates were prepared and immunoblotted with antibodies against Pin1 and actin. The graph depicts the relative amounts of Pin1 (normalized to actin) determined by densitometric analyses of blots from 3 experiments (mean ± SEM). (c) Knockdown of Pin1 in mouse sympathetic neurons. Neurons were microinjected with U6/Pin1 or U6/Ctr (each at 75 μg/ml), along with pBOS-H2BGFP. After 48 hr, immunofluorescence was performed using anti-Pin1 antibody. Representative phase-contrast and epifluorescence images show Pin1 immunofluorescence in uninjected and injected (arrows) cells. (d) Effect of Pin1 knockdown on survival of NGF-deprived sympathetic neurons. Neurons were microinjected with U6/Ctr, U6/Luc, or U6/Pin1 (75 μg/ml) together with pBOS-H2BGFP. Twenty to 24 hr later, the number of successfully injected (GFP-expressing) cells was counted after which half of the dishes were deprived of NGF while the others were switched into fresh NGF-containing media. After 24 hr, the number of healthy injected cells that remained was determined and expressed relative to the number of injected cells counted just prior to NGF withdrawal. The survival of NGF-deprived neurons was normalized to that of the corresponding NGF-maintained cells (mean ± SEM of 3 independent experiments). (e) Neurons microinjected with U6/Ctr or U6/Pin1 were deprived of NGF or maintained in fresh NGF-containing media for 24–48 hr. Cell survival at each time point was expressed relative to the number of injected cells counted immediately before NGF withdrawal (mean ± SEM of 3 independent experiments). Survival of NGF-deprived cells injected with U6/Pin1 was significantly greater than that of control-injected NGF-deprived cells at both 24 hr and 48 hr (p < 0.05, ANOVA with post-hoc Bonferroni test).