Document Type : Original Article
BIOLOGY DEPARTMENT, SCIENCES AND RESEARCH CAMPUS, AZAD UNIVERSITY,TEHRAN, IRAN
Introduction: DNA damage, as an important initiator of neuronal cell death, has been implicated in numerous neurodegenerative conditions. Previously we have delineated several pathways that control embryonic cortical neuronal cell death evoked by the DNA-damaging agent, camptothecin. The camptothecin, topoisomerase Ι inhibitor, has been shown to induce cortical neuronal cell death in a reproducible manner. It has been shown that expression of p53, Bax and JNK is involved in this model. Accordingly, it has become important to further identify the mechanisms by which DNA damage evokes neuronal cell death. In this regard, we have investigated the role of P38 and ATF-2 expression in this model. Our results indicate that the expression of P38 MAPK is up-regulated and is involved in the death signaling following DNA damage.
Material and Methods: In this experimental model, primary embryonic cortical neurons were prepared from E14-16 rat embryos. Then cells were formed to suspensions. A density of 1.2×105 cells/well plated on the wells , pre-coated with poly-D-lysine, and neurons were cultured in serum-free medium. Camptothecin (10-5 M)was added to neuronal culture after 24 hours. After 4,6,24 and 48 hours , expression of the P38 and ATF-2 was studied using primary antibody in the Immunocytochemistry technique. For evaluating nuclei, number of healthy and death nuclei were counted by cell lysis buffer. Neurons were counted and studied using Fluorescent and light microscope. Then percentage of the healthy , death and expression of the cells was analyzed by one way ANOVA followed by Tukey’s post test .
Results: After camptothecin treatment, percentage of the expression of P38 was 4%, 20%, 40% and 55%, and percentage of the survival was 95%, 85%, 64% and 50% for 4, 6, 24 and 48 hours, respectively. In the same manner, percentage of the expression of ATF-2 was also 3%, 20%, 30% , 45% and percentage of the survival was 97%, 85%, 64% and 50% , respectively. Percentage of the expression and survival of the P38 neurons for 24 hours were 40 and 64 percent and it was for ATF-2 , 30 and 64 percent respectively which these results compared to control were increased significantly ( p<0.05). Expression of the proteins at 4 hours was not changed significantly.
Conclusion: This study revealed that expression of the P38 and ATF-2 was increased simultaneously . Thus, in this model, Camptothecin induces neuronal death by stimulation P38-ATF-2 pathway.