Abstract
Lead is a common environmental heavy metal that is probably carcinogenic to human. Lead also has toxic effect on the central nervous system and impairs learning and memory. In Drosophila model organism, long-term memory (LTM) formation is dependent on the spaced training effect which is related to the regulation of ERK activation. Previous reports of our laboratory indicate that lead acetate (Pb(II)) would induce sustained ERK activation to prevent its genotoxicity in mammalian cells. Little is known regarding how does the biological signal transduction pathway elicited by Pb interfere with brain nervous system, this thesis is thus designed to adopt the Drosophila model system for investigating the role of ERK activation regulation in learning and memory following Pb(II) exposure. Fruit flies were fed with 0-500 μM Pb(II) from larva to adult eclosion stages, and then subjected to the Pavlovian olfactory learning experiment. Results showed that Pb(II) at 100 and 250 μM could markedly reduce LTM, while it has little effect on anesthesia-resistance memory (ARM) and learning ability. Immunoblotting showed that during the untrained state, Pb(II) could elicit a dose-dependent increase (100 μM to 250 μM) of the phospho-ERK levels; however, Pb(II) blocks the spaced training-induced ERK activation. Additionally, treatment with antioxidant drug N-acetyl-L-cysteine (NAC) could prevent the Drosophila LTM impaired by Pb(II). Interestingly, NAC alone could enhance the LTM by shortening sessions of spaced training from ten to three times. On the other hand, Drosophila Rrp1 and human APE1/Ref-1 have high homology. Our recently finding indicates that the redox function of APE1 plays an essential role in maintaining ERK activation, promoting us to explore whether Rrp1 affects Drosophila learning and memory. Immunoblotting showed that Pb(II) could increase Rrp1 levels in Drosophila brain. Flies carrying Rrp1 RNAi in whole brain or P element-derived insertion mutants have significant impaired LTM and reduced learning abilities. Immunoblotting showed that the Rrp1-depleted flies exhibit no ERK activation in response to spaced training. Moreover, the Rrp1 RNAi knockdown line had higher level of S-nitrosylation of ERK as determined by using SNO-RAC in combined with immunoblotting. This suggests that Rrp1 may participate in redox regulation of ERK kinase. In summary, this research points out that Drosophila is a good model system to study neurotoxicity of Pb. Results obtained from this thesis should provide basis for the future study of the impact of S-nitrosylation on ERK signaling during LTM formation.