Study On The Role Of POU Domain Proteins In The Late Stage Of Neural Development Toxicity Induced By Pb

Lead (Pb) is widely used in industry and daily life, and it does not decompose but accumulate, so the pollution of Pb in environment has become more severe. Because of its multi-tissue affinity, Pb has an adverse impact on liver, brain, blood and immune system et al. Pb can affect many kinds of function in the nervous system, the neural development toxicity is always the focus of many researchers, especially it can influence the developmental central nervous system (CNS) of infants and children and lead to the changes of their neural behavior and intelligence quotient (IQ). However, the mechanism of the neural development toxicity of Pb has not been elucidated so far. Studies about some specific gene expression in the cells of mammal and nematode indicated that many POU domain proteins have close relationship with the development of nervous system, but it has not been reported that Pb may interfere the expression model of POU domain proteins in the late stage of neural development and the internal association between this changes and the neural development toxicity.This study attempts to reveal the possible molecular mechanism of Pb induced late neural development toxicity. We want to investigate the temporal and spatial expression model of POU proteins in the neural development of rat after low levels Pb exposure during perinatal stage (E15 to P21), and to probe the intrinsic relationship between the abnormal varieties of the expression model and the neural development toxicity. The main contents of this study consist of four parts as below: Part one Changes of the spatial learning and memory of rats afterlow levels Pb exposureObjective: The developing nervous system is preferentially vulnerable to lead (Pb) exposure with alterations in neuronal and glial cells of the brain, even if in low levels Pb exposure. Chronic exposure to Pb causes deficits of learning and memory and abnormal neural behavior in children. Epidemic study and animal experiment shows that in the perinatal period individual is susceptible to Pb exposure, so in this study we want to observe the deficits of learning and memory of offspring rats that exposed to Pb from maternal placenta and milk.Methods: The pregnant rats are housed separately and randomly divided into 4 groups, which are provided with distilled water, 0.5g/L, 1.0g/L, 2.0g/L lead acetate solution via drinking water respectively, the lead-exposure period of dams is limited from the gestational day 15 to postnatal day 21 when the offspring begin to weaned. We use littermates of those offspring rats to examine the effect on spatial learning and memory in Morris water maze, and we find the Pb exposure lead to adverse impact on the central nervous system.Results: The general time and pathway significantly increased in the lead exposed group (P<0.05 or P<0.01) compared with control group, and show dose-reaction relationship (values of r are 0.538 and 0.569 respectively, P<0.01). Based on the map of pathway, the lead exposed group rats had difficulties in finding the platform and they had bad orientation. On the other hand the control group showed better spatial learning ability. Conclusion: The Morris water-maze experiment shows that low levels Pb exposure impair the neural behavior and the ability of learning and memory of exposed rats, so the CNS of rats has been affected in some extent. Part two Changes of the expression model of POU ranscription factors in CNS after low levels Pb exposureObjective: The POU transcription factors have been showed that they play a critical role in the development of the mammalian nervous system. Studies involving the functional characterization of the Oct-2 and Brn-3a illustrate the critical role of them in the regulation of gene expression in neuronal cells and hence in the correct functioning of the developing nervous system. But it has not been reported whether the Pb exposure could interfere normal expression model of the two POU transcription factors in vivo, so we want to observe the gene transcription and protein expression level of them in rats after Pb exposure in order to reveal the possible molecular mechanism of Pb induced late neural development toxicity.Methods: Pups are weighed and sacrificed by decapitation at different days of age, and three brain regions are dissected from the skull to make the frozen sections. We detect the protein expression level of Oct-2 and Brn-3a by immunohistochemistry and the gene transcription level by in situ hybridization and polymerase chain reaction.Results: Immunohistochemistry shows that the protein level of Brn-3a decreases in the cortex, the hippocampus and the cerebellum of Pb-exposed rat (P<0.05 or P<0.01), but the level of Oct-2 increases in the various brain region (P<0.05 or P<0.01). In situ hybridization shows in some brain regions that mRNA expression level of Brn-3a also decreases (P<0.05) accordingly. We have the same result by means of the RT-PCR about expression level of Brn-3a (P<0.05). In our study we could not detect mRNA of Oct-2.Conclusion: The results indicate that POU-domain proteins, as a transcription regular factor, may participate in the neurological toxicity damaging the learning and memory ability induced by lead. The study provided experimental basis for revealing the molecular neurotoxic mechanism of lead. Pb-exposure may interfere the normal model of expression of Oct-2 and Brn-3a, so it interfere the differentiation of neuronal cells. Part three Changes of expression level of some specific proteins in CNS after low levels Pb exposureObjective: It is clear that Brn-3a play a key role in the differentiation of some neural cells to a mature neuronal-like phenotype, with enhanced Brn-3a expression promoting the activation of a number of neuronal specific genes and the induction of neurite outgrowth. We are interested in whether some neuronal specific genes, such as those encoding neurofilaments(NF), have changes in transcription or translating level in rats after low levels Pb exposure.Methods: Pups are weighed and sacrificed by decapitation and three brain regions are dissected from the skull to make the frozen sections. We detect the protein expression level of neurofilaments (NF) and glial fiber acid protein (GFAP) by immunohisto chemistry and the gene transcription level of brain-derived neurotrophic factor (BDNF) by polymerase chain reaction.Results: Immunohistochemistry results show that neural cells specific proteins, such as the protein level of neurofilaments (NFs) decreases in the cortex, the hippocampus and the cerebellum of high-dose Pb-exposed rats (P<0.05 or P<0.01), glial fiber acid protein (GFAP) also change as it. By means of the RT-PCR, we find that in hippocampus mRNA expression level of Brain-derived neurotrophic factor (BDNF) also decreases (P<0.05).Conclusion: The ability of Brn-3a to activate target genes such as those encoding the neurofilaments and its ability to induce neurite outgrowth are therefore both dependent upon the POU domain of the protein. Glial fiber acid protein and Brain-derived neurotrophic factor may involve in the pathway of neuronal differentiation.Part Four Apoptosis of the CNS cells after low levels Pb exposureObjective: It has been suggested that programmed cell death (PCD) is significant in the developing nervous system. Some findings indicate that Brn-3a has a direct effect promoting neuronal survival, acting at least in part by inducing increased levels of the antiapoptotic Bcl-2 protein. We want to know whether apoptosis of the CNS cells in rats change after low levels Pb exposure.Methods: In order to investigate Pb-induced apoptosis in the brain of rats, HE staining for morphological, Annexin V-PI apoptosis detection by flow cytometry, TdT-mediated dUTP nick end labeling (TUNEL) and immunofluorescence detection of Bcl-2 protein expression are utilized.Results: The rates of apoptosis in neural cells from hippocampus in high level treatment group is siginificiantly higher than that of control (P<0.01) by means of Annexin V-PI staining. The expression of Bcl-2 is siginificiantly decreased in neural cells from cerebral cortex, hippocampus and cerebellum in medium and high level treatment groups compared with the control group ((P<0.05 or P<0.01), and show dose-reaction relationship (values of r are -0.897, -0.825 and -0.849 respectively, P<0.01). By means of TUNEL, We get identical result in high level treatment group.Conclusion: It demonstrates that Pb could induce apoptosis in CNS cells, moreover, it possibly implement this through the decreased level of Brn-3a to interfere with the activation of promoter of anti-apoptosis gene Bcl-2 in part.In summary, the results indicate that disturbance of expression model of POU domain proteins in CNS may be one of the mechanisms of neural development toxicity induced by Pb. The changes of expression model of POU domain proteins may produce changes in the regulation of downstream genes expression in neuronal cells, thereby they affect the normal differentiation of neuronal cells, the neurite outgrowth, the correct functioning of the dopaminergic neuron and the information transfer process, and the protection of neuronal cells from Pb which induce apoptosis.