We then investigated whether SHH activity is required for BDNF-dependent neuroprotection against 3-NP toxicity. Cyclopamine (CPM) is a steroid–alkaloid that inhibits the hedgehog signaling through interaction with the hedgehog
LEP 116-130 Smoothened [20]. To test if inhibition of SHH activity may abolish BDNF neuroprotection, rat cortical cultures were pretreated for 8 h with BDNF (100 ng/ml) in the presence or absence of CPM (5 μM) before subsequent exposure to 3-NP (2.5 mM for 24 h). Results derived from Hoechst staining shown in Fig. 2A indicated that BDNF-dependent neuroprotection (percent cell survival, 84.77 ± 6.29% versus 49.88 ± 7.70%; BDNF/3-NP versus 3-NP) was completely abolished by co-incubation with CPM during preconditioning phase (percent cell survival, 46.29 ± 7.60% versus 84.77 ± 6.29%; BDNF+CPM/3-NP versus BDNF/3-NP). Similarly, PI/Hoechst double staining shown in Fig. 2B revealed that BDNF-mediated
reduction of “Death Index” (0.09 ± 0.03 versus 0.64 ± 0.08; BDNF/3-NP versus 3-NP) was also reversed by 5 μM CPM (0.51 ± 0.05 versus 0.09 ± 0.03; BDNF+CPM/3-NP versus BDNF/3-NP). CPM at this dosage did not cause cell death (data not shown). Together these results establish the notion that SHH is required for BDNF-dependent neuroprotection.
