Effects Of Lead On Alzheimer’s Disease-like Pathlogy And The Expression Of Signal Transduction Proteins

Lead is a well known neurotoxicant.Central nervous system is most vulnerable to lead exposure(especially the case of developmental brain),which can lead to severe neurological dysfunctions and learning cognitive impairments.Learning and memory,as well as cognition are the basic functions of the CNS and brain.Hippocampus is the very place where memory and cognition occurs,and also the main target of lead toxicity.Cellular processes,like proliferation,differentiation,apoptosis,migration are regulated by the intracellular signaling pathways,for instance Insulin signaling and MAPKs signal pathway.The abnormal expressions of MAPKs may lead to pathological changes,as well as the outbreak of certain diseases.Alzheimer’s disease is a primary progressive neurodegenerative disease which is affected by both environmental and genetic factors.Among all,aging is thought to be the major cause of AD.Progressive cognitive impairments,as well as dysfunctions in spatial learning and memory are typical clinical features of AD,during which neuronal apoptosis,synaptic degradation and abnormal expressions of memory-related and signaling pathway concerning proteins have taken place.Although the pathogenesis of AD still remains a mystery,many believe that the aberrant expression and extracellular accumulation of Aβ may be a common factor and key link of AD.Recent studies have shown that younger people are also suffering from AD,suggesting that AD may not be a conventional type of senile diseases.The hypothesis of Fetal Basis for Adult Disease(Fe BAD)holds the idea that adult diseases may have a developmental origin and therefore exposure to risk factors during embryonic development period may lead to the outbreak of certain adult diseases at an advanced age.A group of scholars believe that AD might be one of these diseases.Animal experiments have shown that lead exposure during pregnancy and lactation can cause learning and memory impairment,as well as cognitive decline of the offspring.In addition,the results of population based studies have also confirmed that lead exposure during early phase of brain development may result in intellectual impairment,spatial learning and memory disorders,cognitive decline of children in later lives.The mechanism of which is unclear.In order to establish the relationship between AD,Aβ expression,neurogenesisand lead exposure,and to verify the intracellular signaling pathways might be involved during the process,the current study was undertaken using both lead-exposed mice model and Pb2+-treated PC12 cell line.The lead levels in the blood and hippocampus of offspring and the protein expression of hippocampal IDE and NGF were examined to determine the role of Pb2+in AD-like effect;The interactions between lead and intracellular signaling pathways were explored in vitro to seek the potential target for therapeutic intervention.Objectives1 To investigate the effect of developmental lead exposure in vivo,the lead-poisoning animal model was established by treating the maternal mice with lead-containing drinking water during gestation and lactation.The learning and memory abilities of mouse pups were evaluated by Morris water maze test.The protein expression of hippocampal IDE and NGF of young mice were quantified to show lead neurotoxicity on Aβ expression and neurogenesis.2 To clarify the mechanism of lead neurotoxicity and to seek the intracellular signaling pathways involving in the course,the expression of AD-related proteins(APP,Aβs,Aβ oligmers,IDE),neurogenesis-relating proteins(IGF1/IGF1R)and signal transduction proteins(IR,p-Akt,ERK1/2,JNK1/2/3,P38)in the PC12 cells were quantified and compared between control and lead exposure groups.Materials and methods1 Subjects1.1 Animals model: Forty(40)pregnant females at day E0(embryonic day)were randomly assigned to one of four groups and caged separately.Lead acetate was dissolved in distilled water at four different concentrations 0%,0.1%,0.2% and 0.5%for control,low,moderate and high concentrations respectively.Lead exposure of pregnant mice was initiated from the beginning of gestation and lasted until weaning(the 21 st postnatal day).The pups were fed by mother until weaning.1.2 Cell line: The PC12 cell from Rattus norvegicus was used as the experimental object.The differentiated PC12 cells were treated with lead acetate at the final concentration of 0,20,100 and 500μM respectively.2 Methods2.1 Experiment in vivo2.1.1 The lead levels in blood and hippocampus tissue of the offspring were analyzed by graphite furnace atomic absorption spectrophotometer Z5000(HITACHI).2.1.2 The learning and memory functions of the mice offspring were evaluated by Morris water maze test.2.1.3 The expression of AD-related protein(IDE)and neurogenesis-relating protein(NGF)in the hippocampus of mice offspring(among 4 groups;0%,0.1%,0.2% and0.5% Pb Ac groups)were analyzed by Western blot.Immunohistochemistry staining and immunofluorescence analysis were carried to detect the intracellular location,distribution and expression of IDE and NGF.2.2 Experiment in vitro2.2.1 The proliferation of the PC12 cells were detected by MTT assay after Pb2+treatment.2.2.2 The expression of AD-related proteins(APP,Aβs,Aβ oligmers,IDE),neurogenesis-relating proteins(IGF1/IGF1R)and signal transduction proteins(IR,p-Akt,ERK1/2,JNK1/2/3,P38)in the PC12 cells were quantified by Western blot.The effect of Pb2+ on the m RNA levels of intracellular APP,INSR,IDE,IGF1 and IGF1 R were assessed by quantitative real-time PCR.3 Statistical analysis All data were analyzed for significance using SPSS 21.0.Statistical analysis between two groups was performed with independent sample t test.One-way ANOVA followed by Significant Difference test(LSD)were used for intergroup comparison of multiple groups.Values satisfy the normal distribution were represented as mean ± SD.The inspection level of alpha = 0.05.Results1 Results of in vivo study1.1 Effect of maternal lead exposure on blood and hippocampal lead concentrations and the learning and memory ability of mouse pups The results of GFAAS(graphite furnace atomic absorption spectrophotometry)indicated that both blood and hippocampus lead concentrations were significantly increased by maternal lead exposure during pregnancy and lactation(F= 159.239,P<0.05;F=100.15,P< 0.05)and the concentrations of lead were positively associated with drinking water lead concentration.The results of Morris water maze test showed that the escape latency and the times of errors in the moderate and high maternal lead exposure groups were significantly higher than those in control group(P<0.05).1.2 Effect of maternal lead exposure on the protein expression of hippocampal IDE and NGF of mouse pups1.2.1 The results of Western Blot showed that the expression of hippocampal IDE and NGF in the lead-exposed groups were significantly lower than those in control group(P<0.05).1.2.2 The results of Immunohistochemistry staining indicated that maternal exposure to 0.5%Pb Ac substantially reduced NGF expression in hippocampus of pups as compared to control pups1.2.3 The results of immunofluorescence analysis showed that the expression of IDE was significantly reduced in the 0.5%Pb Ac group relative to control group,as was the viability of neurons.2 Results of in vitro study2.1 Cytotoxicity of Pb2+The results of MTT showed that the viability of PC12 cell was significantly reduced by Pb Ac treatment.Obvious changes in morphology and reduction in the numbers of cells were also observed during the course.Based on the cell counts and the variation trend,the exposure concentrations of 0,20,100 and 500μM and exposure periods of 12 h,24h,and 72 h were selected for subsequent studies.2.2 Effect of Pb Ac on the expression of AD-related proteins(Aβ/Aβ oligomers)in cultured PC12 cells The results of Western Blot showed that the expression of intracellular Aβ in lead-exposed groups were lower than those in control groups(at all exposure periods;P<0.05).The expression of intracellular Aβ oligomers in lead-exposed groups of 24 h and 72 h were significantly higher than those in control groups(P<0.05).2.3 Effect of Pb Ac on the expression of neurogenesis-relating protein(IGF1/IGF1R)in cultured PC12 cells According to the results of Western Blot,there was an increase in IGF1 expression after treatment of 20μM Pb Ac for 12 h comparing to that of control group(P<0.05);The protein expression of IGF1 were higher in the rest of lead-exposed groups than its control(P<0.05).The results showed a decrease in IGF1 expression after Pb2+ exposure for 12 h,with significant drops observed at concentrations of100μM and 500μM(P<0.05);There was a decrease in IGF1 protein expression following Pb2+ exposure of 24 h,the expression of IGF1 in lead-exposed groups of20μM and 500μM were significantly lower than that in control(P<0.05);The results indicated a significant decrease in IGF1 protein expression after treatment of 100μM or 500μM Pb Ac for 72 h in comparison to the control group(P<0.05).2.4 Effect of Pb Ac on the expression of Insulin signaling-related proteins(IR,p-Akt,IDE)in cultured PC12 cells The results of Western Blot: After treatment of Pb Ac for 12 h,the expression of IR in the 20μM Pb Ac-exposed group was lower than that in control(P<0.05);The expression of IR in cultured PC12 cell was higher in the 100μM Pb Ac exposure group than control group(P<0.05).The expression of p-Akt was higher in the 100μM Pb Ac exposure group than control group(P<0.05);The expression of p-Akt in the 500μM Pb Ac exposure group was significantly lower than that in control(P<0.05).Comparing to the control group,the protein expression of IDE decreased significantly after Pb Ac treatment of 500μM(P<0.05).After exposure to Pb Ac for 24 h,the expression of IR in the 20μM and 100μM Pb Ac-exposed group were lower than that in control(P<0.05).The expression of p-Akt were higher in the 20μM and 100μM Pb Ac exposure groups than control group(P<0.05);The expression of p-Akt in the 500μM Pb Ac exposure group was significantly lower than that of control(P<0.05).The protein expression of IDE in the lead exposure groups were significantly lower than that in control group(P<0.05).After treatment of lead acetate for 72 h,the expression of IR in the 20μM and500μM Pb Ac-exposed group were lower than that in control(P<0.05).The expression of p-Akt in the 500μM Pb Ac exposure group was significantly lower than that in control(P<0.05).The protein expression of IDE in the 100μM and 500μM lead exposure groups were significantly lower than that in control group(P<0.05).2.5 Effect of Pb Ac on the expression of MAPK signaling proteins(ERK1/2,JNK1/2/3,P38)in cultured PC12 cells The results of Western Blot: After exposure to Pb Ac for 12 h,the expression of ERK1 in cultured PC12 cell were significantly higher in the lead-exposed groups than the control(P<0.05).The expression of ERK2 in the lead-exposed groups were significantly lower than that in control group(P<0.05).The expression of JNK1 in the500μM Pb Ac treatment group was significantly lower than that in control group(P<0.05).The expression of JNK2 in the 20μM and 100μM Pb Ac treatment groups were higher than that in control group(P<0.05).The expression of JNK3 in cultured PC12 cell were lower in 100μM and 500μM Pb Ac treatment groups than the control(P<0.05).The expression of P38 in the lead-exposed groups were significantly lower than that in control group(P<0.05).After treatment of lead acetate for 24 h,the protein expression of ERK1 in the20μM Pb Ac-exposed group was significantly higher than that in control(P<0.05);ERK1 protein expression of the 500μM Pb Ac exposure group was lower than that of control group(P<0.05).The expression of ERK2 in the lead-exposed groups were significantly lower than that in control group(P<0.05).The expression of JNK1 in the100μM and 500μM lead-exposed groups were significantly lower than that in control group(P<0.05).The protein expression of JNK2 in the 500μM lead-exposed groups was significantly lower than that in control group(P<0.05).The expression of JNK3 in cultured PC12 cell were lower in the 20μM and 500μM Pb Ac treatment groups than the control(P<0.05);P38 protein expression of the 100μM and 500μM Pb Ac exposure group were significantly lower than that of control(P<0.05).After exposure to Pb Ac for 72 h,the protein expression of ERK1 in the 500μM Pb Ac-exposed group was significantly lower than that in control(P<0.05).The expression of ERK2 in cultured PC12 cell were significantly lower in the lead-exposed groups than control group(P<0.05).The expression of JNK1 in the lead-exposed groups were significantly lower than that in control group(P<0.05).JNK2 protein expression of 500μM lead-exposed group was lower than that of control(P<0.05).JNK3 protein expression of 100μM and 500μM lead-exposed group were lower than that of control(P<0.05).P38 protein expression of the 100μM and 500μM Pb Ac exposure group were significantly lower than that of control(P<0.05)2.6 Effect of Pb Ac on the expression of APP,INSR,AKT,IDE and IGF1/IGF1 R m RNA in cultured PC12 cells The results of RT-PCR: After exposure to lead acetate for 12 h,the expression of APP m RNA in the 20 and 100μM Pb Ac-exposed groups were higher than that in conttrol,the differences were not statistically significant(P<0.05).The m RNA level of INSR in the 20μM Pb Ac-exposed group was lower than that in control group(P<0.05).The expression of AKT m RNA in cultured PC12 cell were lower in lead-exposed groups(both the 20μM Pb Ac-exposed group and the 100μM Pb Ac-exposed group)than control group(P<0.05).No significant change in IDE m RNA level was detected between control case and lead exposure groups(P>0.05).The expressions of IGF1 and IGF1 R m RNA in the 20μM and 100μM Pb Ac-exposed groups were lower than those in control groups(P<0.05).After treatment of lead acetate for 24 h,the expression of APP m RNA in the20μM and 100μM Pb Ac-exposed groups were higher than that in control(P<0.05).The m RNA level of INSR in the 100μM Pb Ac-exposed group was lower than that in control(P<0.05).The expression of AKT m RNA in cultured PC12 cell were lower in lead-exposed groups(both the 20μM Pb Ac-exposed group and the 100μM Pb Ac-exposed group)than control group(P<0.05).The m RNA expression level of IDE in the 20μM Pb Ac treatment group was lower than that in control(P<0.05).The expression of IGF1 m RNA in the 100μM Pb Ac-exposed group was lower than that in control group(P<0.05).There was no significant difference between the expression of IGF1 R m RNA in lead-exposed groups and the control(P>0.05).After exposure to Pb Ac for 72 h,the expression of APP m RNA in the 20μM and100μM Pb Ac-exposed groups were significantly lower than that in control(P<0.05).The expression of INSR m RNA in the 20μM Pb Ac-exposed group was lower than that in control(P<0.05).The expression of AKT m RNA in cultured PC12 cell were lower in lead-exposed groups(both the 20μM Pb Ac-exposed group and the 100μM Pb Ac-exposed group)than control group(P<0.05).There was no significant difference between the expression of IDE m RNA in lead-exposed groups and the control(P>0.05).The expressions of IGF1 and IGF1 R m RNA in the 20μM and100μM Pb Ac-exposed groups were lower than those in control groups(P<0.05).Conclusion1 Maternal lead exposure during gestation and lactation led to increased blood and hippocampal lead concentrations in mouse pups.The protein expression of hippocampal IDE and NGF of the pups were inhibited by maternal lead exposure.The learning and memory abilities of the young mice were impaired by maternal lead exposure in a dose-dependent manner,suggesting that the learning and memory capacities of offspring,AD-like pathological changes and maternal lead exposure are closely related.2 Changes in expressions of AD-related biomarkers and neurogenesis-relating proteins could be triggered by Pb2+ exposure,during which the abnormal expression of intracellular signaling pathways may take place.