Disturbance Of Calcium/Calmodulin-dependent Signalingin The Rat Brain Regions After Lead Exposure

Nowadays the level of lead pollution is still very severely in China, Lead is a kind of heavy metal, and the most common industrial or environmental toxicant. Lead could accumulate in the environment for a long time, even food caused by lead pollution, and can do long-term harm to human. We know lead does not have any physiological function, lead can play a role of neurotoxicity when through the blood-brain barrier (BBB) into the brain lead to insufficient supply of nutrients and oxygen. In addition, compared to adults, children are more sensitive to the lead neurotoxicity. We have confirmed that lead can display strong neurodevelopmental toxicity. The younger exposed to lead, the higher susceptibile to lead neurotoxicity. Moreover, lead can through the placental barrier and milk secretion directly, fetal and infant are more sensitive to the lead neurotoxicity. Otherwise there is non-safety critical level of lead. Lead can damage the nervous system below the blood lead level according to the existing clinical testing standards, and the development of intellectual damage generated by early lead exposure in childhood can sustain to the adult stage. However, treatment still supplemented and prevention is a main way for lead exposure in clinical, we still lack excellent and effective drug to solve the lead neurotoxicity.Research on molecular mechanisms of lead neurotoxicity is essential in order to therapy lead neurotoxicity, Now the important purpose of studying on the molecular mechanisms of lead neurotoxicity is to explore calcium (Ca2+) and protein kinase function in central nervous system (CNS) signal transduction pathway by lead exposure. Lead can affect the normal flow of intracellular Ca2+, interfere with Ca2+uptake and release on nerve cell membrane, and play a toxicity role through interfering with intracellular Ca2+homeostasis. Lead can also bind to calmodulin (CaM) by Ca2+or directly bind with CaM, and causes it’s configuration change, then bind and activate CaM-dependent proteins to interfere with functions of the nerve cell. However, the complex Ca2+/CaM-dependent signaling pathway mediated lead neurotoxity is not fully clear, further study on Ca2+/CaM-dependent expression of signaling molecules by lead in the nervous system will play a significant to understand the molecular mechanisms of lead neurotoxicity.Many studies had indicated that reactive oxygen species (ROS) could be induced by lead, and then cell’s redox state would be changed to generate free radicals in tissue and cell, causing an increase of free radical, oxidative damage, which would play an important role in the pathology of lead toxicity. Also we have learned that when cell is damaged or calcium homeostasis is unbalanced, autolysosome will be activated, which can protect cell from toxic damage, but excessive autophagy can lead to programmed cell-death. It is reported that autophagy would increase in epididymal epithelial cells after lead exposure, but whether lead exposure can impact the level of autophagy on nerve cells have not been reported, and whether autophagy-lysosomal pathway is involved in signal transduction process of lead neurotoxicity is still not clear.In the present study, we establish rat models of acute lead exposure, then detect the concentration of lead in the blood and brain regions, examine the expression of Ca2+/CaM-dependent signaling molecules along the dorsoventral axis of the hippocampus; we also establish rat models of chronic lead exposure and drug regulation to measure the concentration of lead in the blood and brain regions of weaning period, learning and memory in rat offspring. Then we examine the expression of Ca2+/CaM-dependent signaling molecules and autophagy-related proteins in brain regions, through exploring the internal relations among them, we want to provide important clues for study on the molecular mechanisms of lead neurotoxicity and innovative drug treatment targets. For chronic lead exposure model, we use melatonin, acts as calmodulin antagonist, free radical scavenging and antioxidant, maintain for60days of regulatory intervention in the offspring rats. Chapter1Expression profiling of Ca2+/calmodulin-dependent signaling molecules in the rat dorsal and ventral hippocampus after acute lead exposure.Objective:To determine whether the distinct hippocampus regions, which differ remarkably in their anatomical organization spatially and temporally, exhibit differential neurochemical profiles underlying lead neurotoxicity.Methods:Establish rat models of acute lead exposure. Sprague Dawley (SD) rats were randomly divided into three groups:two lead-exposed groups and control group. The lead-exposed groups (Low lead-exposed group, High lead-exposed group) were administrated intraperitoneally lead acetate in doses of25mg/kg and50mg/kg once daily for a period of5days. The control group (Control group) was treated with distilled water. Blood and brain tissues collection were analyzed for lead content by graphite furnace atomic absorption spectrophotometry. Changes in the expression of Ca2+/CaM-dependent enzymes were examined by western blot, including spectrin that a specific substrate for calpain, calcineurin, phosphorylation of Ca2+/CaM-dependent protein kinase Ⅱ (p-CaMK Ⅱ) and neuronal nitric oxide synthase (nNOS), in the rat dorsal and ventral hippocampus after acute lead exposure. At last, the expression of calcineurin in the ventral hippocampus was checked by immunohistochemistry.Results:Five days after acute lead exposure, blood lead concentration and brain lead level significantly increased (P<0.01), which lead in the hippocampus is relatively higher than other brain tissues. We observed constitutively active forms of calcineurin (45kDa and48kDa) in ventral hippocampus. This result is consistent with the observed calpain activation which is indicated by the breakdown of spectrin in this region. Our data demonstrate that nNOS expression is significantly higher in the ventral hippocampus when compared to the dorsal hippocampus (P<0.05), whereas p-CaMK Ⅱ (Thr286) is less pronounced in the ventral hippocampus and more pronounced in dorsal hippocampus after acute lead exposure (P<0.05).Conclusion:It appears that ventral hippocampus is more vulnerable to the neurotoxic effects of lead than dorsal hippocampus. Taken together, the present data suggest that acute lead exposure leads to differential expression patterns of Ca2+/calmodulin-dependent enzymes along the dorsoventral axis of the hippocampus.Chapter2Effect of chronic lead exposure and drug regulation on expression of Ca2+/calmodulin-dependent signaling molecules in the rat brain regionsObjective:To explore the expression of Ca2+/CaM-dependent signaling molecules in the rat brain regions after chronic lead exposure and conduct the drug regulation research.Methods:Firstly, establish chronic lead exposure model of rat pups. SD rats were randomly divided into four groups after conception:three lead-exposed groups and control group. Control group (Control) was given distilled water, three lead-exposed groups (L-lead, M-lead and H-lead) were given lead acetate solution via drinking water in doses of0.5g/L,1.0g/L and2.0g/L respectively. Since the pregnant16d, pregnant rats drinked until newborn pups weaning up to21d, pups were fed by the pregnant rats, and raised separately after weaning. Pups blood and brain tissues collection were analyzed for lead content by graphite furnace atomic absorption spectrophotometry. The remained rat pups start to drink the same lead content water as their mothers’until60d maturity. Secondly, establish five groups of chronic lead exposure models of rat offspring and drug regulation based on rat pups models of chronic lead exposure. Four treatment groups (L-lead or L, L-lead+Melatonin or L+M, H-lead or H, H-lead+Melatonin or H+M) were continued to drink the same as the original content of lead acetate drinking water, L+M group and H+M group began to take melatonin solution in dose of5mg/kg by intragastric administration daily, when control group (Control or C) was continued to give distilled water for2months. The ability of learning and memory in rat offspring was tested by Morris water maze. Offspring blood and brain collection were analyzed for lead content by graphite furnace atomic absorption spectrophotometry. The expression of Ca2+/CaM-dependent proteins in brain regions examined by western blot, including the proteins associate with CaMK II pathway and protein kinase C (PKC) pathway.Results:In the pups models of chronic lead exposure, compared with the control group, the content of lead was significantly increased in blood and brain in21d pups lead-exposed groups respectively (P<0.05). In the chronic lead exposure models and melatonin treatment, results were measured by Morris water maze show that escape latency, frequency and time in correct quadrant of lead-exposed groups has no significant difference compared with the control group respectively, there is also no significant difference compared between melatonin treatment groups and lead-exposed groups respectively. Compared with the same day control group, the first day short-term memory of L-Lead and L-lead+Melatonin, and the second day short-term memory of L-lead+Melatonin significantly increased (P<0.01). Compared with control group, the content of lead is significantly higher in blood and hippocampus, cortex, striatum in treatment groups respectively (P<0.05). But there is no significant difference compared with melatonin treatment groups and lead-exposed groups respectively. With increasing dose of lead exposure, we observed that p-CaMK II (Thr286) is less pronounced, whereas p-Synapsin I (Ser603) and p-GluRl (Ser831) is more pronounced in hippocampus, simultaneously, the expression of p-ERK (Thr202/Tyr204) and NMDAR1in hippocampus is increased, and p-MARCKS (Ser152/156) is decreased after chronic lead exposure. The expression of p-NRl(Ser896) and MARCKS in hippocampus is also increased following high lead exposure. Melatonin have certain reversal regulate function to hippocampal p-GluRl (Ser831)、p-ERK(Thr202/Tyr204)、 NMDAR1、p-MARCKS (Ser152/156) and MARCKS of high lead-exposed group. In addition, the expression of proteins associate with CaMKⅡ pathway and PKC pathway in striatum of lead-exposed groups have no significant difference compared with control group respectively.Conclusion:The results of this chapter shows that short-term memory of rats is influenced after chronic lead exposure. Disturbance of calcium/caimodulin-dependent signaling in rats hippocampus may be involved in the process of molecular mechanism of lead neurotoxicity. In addition, melatonin have certain reversal regulate function to hippocampal p-GluRl (Ser831)、p-ERK (Thr202/Tyr204)、NMDAR1、p-MARCKS (Serl52/156) and MARCKS of high lead-exposed group in our study.Chapter3Effect of chronic lead exposure and drug regulation on expression of autophagy associated genes in the rat brain regionsObjective:To explore whether autophagy-lysosomal signal transduction pathway is involved in process of molecular mechanisms of lead neurotoxicity, and research on drug regulation.Methods:Brain of chronic lead exposure and melatonin treatment in chapter2was collected for the present study. The expression of autophagy-related proteins in hippocampus and cortex were examined by western blot analysis, including Beclin-1, microtubule-associated protein1light chain3(LC3), nucleoporin (p62), lysosomal-associated membrane protein2(LAMP2), cathepsin B.Results:Western blot showed that, in the hippocampus nerve cells, compared with control group, the expression of Beclin1in low lead-exposed group descends significantly (P<0.05), but high lead-exposed group raises significantly(P<0.05); LC3raises very significantly (P<0.001);38kDa form of cathepsin B descends very significantly (P<0.001), while30kDa and25kDa forms have decline trend but no significant differences. In the cortex nerve cells, compared with control group, the expression of p62in low lead-exposed group raises significantly (P<0.05), while the LC3、p62and LAMP2expression raise significantly (P<0.05) in high lead-exposed group. But there were no significant differences compared with melatonin treatment groups and lead-exposed groups respectively.Conclusion:The results of this chapter shows that the level of autophagy in brain nerve cells may be affected by chronic lead exposure, we speculate that autophagy-lysosomal signal transduction pathway may be involved in the process of molecular mechanism of lead neurotoxicity. In addition, the level of autophagy in brain nerve cells is not affected by melatonin treatment in our study.