Article Figures & Data
Figures
- Fig. 1.
BDNF-mediated protection from NO-induced growth-cone collapse requires PKA activity.A, Treatment with NO donors (10 min with 500 μm NOC-7) causes growth-cone collapse (left, arrowheads), and the collapse is prevented by previous treatment with BDNF (right, arrowheads; 1 hr with 40 nm BDNF followed by 10 min with 500 μmNOC-7). Scale bar, 50 μm. B, Pretreatment with the protein kinase A inhibitor KT5720 (200 nm) or Rp-cAMP (100 μm) for 30 min before the addition of BDNF blocks the initiation of BDNF-mediated protection against growth-cone collapse induced by 100 μm SIN-1 (30 min; p < 0.001; Welch's t test). Neither PKA inhibitor had an effect on NO-induced growth-cone collapse (p> 0.6; Welch's t test). C, Treatment with cAMP-analog activators of PKA is not sufficient to initiate protection from NO-induced growth-cone collapse (30 min with 100 μm SIN-1). cAMP analogs (100 μm Sp-cAMP or 2 mm db-cAMP), which activate PKA, were added to cultures for 1–2 hr and subsequently treated with NO. Protection was not observed at either 1 or 2 hr of treatment, so data were pooled. Control cultures (CNT) were treated with the vehicle for the PKA activators (DMSO). Neither Sp-cAMP nor db-cAMP blocked NO-induced growth-cone collapse (p > 0.6 in both cases; Welch's t test).
- Fig. 2.
BDNF transiently activates PKA. A, PKA activity was determined using the MESACUP kinase assay. For each run, PKA activity was determined in retinal ganglion cells treated with BDNF for various times (2–30 min) and normalized to PKA activity in untreated cells run in parallel. BDNF caused a statistically significant increase in PKA activity (+94%) only at the earliest time point studied, 2 min (p < 0.05; ANOVA with Bonferronipost hoc tests). B, Example of a dissociated retinal ganglion cell stained for PKARIIβ. PKA is distributed evenly throughout the cell. At the growth cone (inset), PKA appears to be localized primarily to the central domain (arrow). Scale bar, 10 μm.
- Fig. 3.
PKA activity is required only during the first 15–30 min of signaling for the initiation of BDNF-mediated protection from NO-induced growth-cone collapse. Cultures were treated with PKA inhibitors, 200 nm KT5720 (A) or 100 μm Rp-cAMP (B), for various periods of time relative to the addition of BDNF. Time = 0 minmeans that BDNF and the PKA inhibitor were added together;15 min and 30 min mean that the inhibitor was added at 15 or 30 min, respectively, after addition of BDNF. For these experiments, individual growth cones were followed by live videomicroscopy (n >15 in each group). Note that only the simultaneous addition of BDNF and the PKA inhibitors blocked the protective effects of BDNF against growth-cone collapse in response to NO (100 μm SIN-1). C, Cultures were treated with PKA inhibitors during either the first or last 30 min of BDNF treatment before exposure to NO (500 μm NOC-7 for 10 min). The time course of treatments before exposure to NO is shown below each bar. B, BDNF; KT, KT5720; Rp, Rp-cAMP; --, DMSO. Note that exposure to PKA inhibitors during the first 30 min of BDNF signaling, but not the last 30 min (30–60 min), blocked the protective effect of BDNF (p < 0.001 for both KT and Rp; Welch's t test).
- Fig. 4.
The protective effects of BDNF are reversible, and PKA activity is not required for the maintenance of BDNF protection.A, Cultures were raised overnight in BDNF, and subsequently BDNF was washed out for 30–90 min before NO was added (500 μm NOC-7 for 10 min). Cultures treated overnight with BDNF exhibited protection from NO-induced collapse identical to that of cultures treated with BDNF for a shorter period (i.e., the 1 hr standard treatment for the previous experiments; p> 0.001; Welch's t test). Thirty minutes after BDNF washout, growth cones were still protected against NO-induced growth-cone collapse. At 60 min after washout, growth cones were significantly more collapsed by NO than growth cones in the continued presence of BDNF (p < 0.05; Welch'st test). At 90 min after washout of BDNF, NO-induced growth-cone collapse was fully restored (p< 0.001; Welch's t test). B, Cultures were raised overnight in BDNF and subsequently treated with PKA inhibitors (200 nm KT5720 and 100 μm Rp-cAMP) for 90 min in the continued presence of BDNF. Inhibition of PKA in cultures continuously exposed to BDNF did not block the maintenance of BDNF protection (p > 0.2 for all comparisons; Welch's t test), demonstrating further that PKA activity is not required for long-term maintenance of BDNF-mediated protection from NO-induced growth-cone collapse.
- Fig. 5.
Continuous activation of PKA does not block initiation of BDNF-mediated protection from NO-induced growth-cone collapse. Membrane-permeable phosphodiesterase-resistant forms of cAMP were used to activate PKA. Sp-cAMP (100 μm) or db-cAMP (2 mm) was added to cultures for 15 min before BDNF. Activating PKA independent of BDNF did not affect the initiation of BDNF mediated growth-cone protection from NO (p > 0.5; Welch's ttest).
- Fig. 6.
Activation of PKA is necessary but not sufficient for the initiation of BDNF-mediated F-actin stability. We have shown previously that BDNF induced the formation of CD-resistant F-actin in growth cones (Ernst et al., 2000). A, F-actin cytoskeleton (phalloidin staining) of a growth cone treated with BDNF followed by CD (30 min with 0.1 μg/ml). Notice that F-actin bundles persist after CD treatment (arrowheads).B, Treatment with PKA inhibitors (200 nmKT5720 or 100 μm Rp-cAMP) during the 1 hr period of exposure to BDNF blocked the formation of CD-resistant F-actin in response to BDNF. Notice the lack of F-actin bundles and the punctate appearance of the staining. PKA inhibitors alone did not alter the response of growth cones to CD (data not shown). C, Blocking PKA activity during only the first 15 min of BDNF signaling is sufficient to inhibit the F-actin-stabilizing effects of BDNF (compare with A, and note similarity to B).D, E, Growth cones treated for 1 hr with 100 μm Sp-cAMP and 2 mm db-cAMP, respectively.F, Image of a CD-treated growth cone without previous treatment with cAMP analogs. G, H, Images of growth cones pretreated with Sp-cAMP and db-cAMP, respectively, and subsequently treated with CD. Note that CD caused a similar extent of F-actin depolymerization regardless of treatment with cAMP analogs (compare F, G, andH).
Tables
- Table 1.
Protein synthesis is required for initiation and maintenance of BDNF-mediated protection from NO-induced growth-cone collapse
Treatment Axons with growth cones (%) Initiation Maintenance BDNF 84 ± 2 68 ± 4 BSA + NO 39 ± 4 37 ± 3 BDNF + NO 80 ± 4 62 ± 3 Cyclo + BDNF 83 ± 2 64 ± 9 Cyclo + BDNF + NO 43 ± 2* 34 ± 5* Puro + BDNF 77 ± 3 68 ± 2 Puro + BDNF + NO 36 ± 21-160 43 ± 2* To test the role of protein synthesis in initiation of BDNF-mediated protection, retinal explant cultures were pretreated with 2 μg/ml cycloheximide (Cyclo) or 7.5 μg/ml puromycin (Puro) for 1 hr before treatment with BDNF for 1 hr (Initiation column). To test the role of protein synthesis in maintenance of BDNF-mediated protection, cultures raised in continuous BDNF for 24 hr were treated with cycloheximide or puromycin for 2 hr (Maintenance column). A 10 min treatment with 750 μm NOC-7 was used to deliver NO to cultures. Cultures were then fixed and scored for growth-cone collapse (n ≥ 5 cultures scored in each group). Pretreatment or post-treatment with either protein synthesis inhibitor blocked the protective effects of BDNF.
↵* p < 0.01;
↵F1-160 p< 0.001; BDNF + NOC-7 versus BDNF + drug + NOC-7.
