Role Of Developmental Exposure To Lead In The Process Of Alzheimer's Disease-like Effect And Epigenetic Modification In Aged Monkeys

Alzheimer's disease is a progressive primary neurodegenerative disease which is highly correlated with the age. Although the etiology and pathogenesis of AD is unclear, Aβis considered to be a common factor and key link of AD. Therefore, it is the recent hot point in the AD research regarding the exploration about the selected risk factors and its regulation mechanism in the onset and progression of AD pathogenesis.The health effects of environmental low-level lead (Pb) exposure are significant public issue in recent years. The developmental central nervous system is particularly sensitive to Pb toxicity which could induce a wide variety of long lasting neurologic impairment of children, such as mental retardation and cognitive and attentive dysfunction. Furthermore, there has been more convincing epidemiological evidence demonstrating the link between Pb and cognitive function in the aging population. In addition, the sporadic nature of LOAD also argues for an environmental-linked origin by the hypothesis of Fetal Basis for Adult Disease. The possible developmental origin of AD was proposed. Given the adverse effects of Pb in developing brains, these findings suggested that Pb might be involved in the onset of AD.Epigenetics is an important adaptation mechanism about gene expression due to genotype-environment interaction. The sporadic nature of most AD strongly argues that it is a kind of complex heterogeneous disease associated with gene and environmental factors. Some evidences also presumed that epigenetic abnormalities might be a potential mechanism of late-onset AD pathogenesis. Therefore, this study hypothesized that environmental Pb might be play an important role in the process of AD by modifying epigenetic effects.In order to verify above hypotheses, this study was undertaken to determine the role of Pb in AD-like effect using different model in vivo and in vitro and to discuss the potential epigenetic effect that could give scientific basis for the exploration of AD pathogenesis. Objectives1 To investigate the possible effects of developmental Pb exposure in vivo, we first explored the expression of AD-related proteins altered by developmental Pb exposure and aging using cynomolgus monkeys cohort.2 To explore the potential mechanism of Pb on AD-related gene expression in vitro, differentiated SH-SY5Y cells were exposed to different Pb concentrations. The possible epigenetic modification effects were monitored.Material and methods1 Subjects1.1 Non-human primate's model:a cohort of cynomolgus monkeys were randomly assigned at birth to one of two exposure groups:one received 1.5 mg·kg-1·d-1 of Pb-acetate from birth until 400 days of age via infant formula and vehicle after weaning, whereas the other group served as a control group and only received formula or vehicle. The cynomolgus monkeys were terminated at 23 years of age. A different cohort of cynomolgus monkeys aged 3-6 years and 12 years were used to plot the profiles of AD-related protein and across the lifespan.1.2 Cell line:The SH-SY5Y cells were employed as in-vitro study model. The differentiated SH-SY5Y cells were exposed to a series of Pb concentrations.2 Methods2.1 The expression of AD-related proteins in the cerebral cortex of monkeys and differentiated SH-SY5Y cells were analyzed by Western blot. The scope of changes in gene expression due to developmental exposure to Pb was determined using Atlas cDNA microarray. The changes in a variety of epigenetic intermediates associated with DNA methylation and histone modifications were determined by Western blot and immunofluorescence analysis respectively.2.2 The effects of Pb on intracellular AβPP and NEP mRNA levels were evaluated by quantitative real-time PCR. SH-SY5Y cells were exposed to Pb for 48 h and the Aβ1-40 secreted into the medium was measured by ELISA. NEP activity after exposure to various Pb concentrations was investigated by fluorometric assay.3 Statistical analysisAll results were represented as mean±SEM. Statistical analyses were performed with one-way ANOVA followed by significant difference LSD post hoc analysis and t-test with a threshold value of P< 0.05. Pearson correlation analysis was also used. All statistical analyses were performed using SPSS 12.0 software.Results1 Effect of aging and developmental exposure of Pb on the expression of AD-related proteins, DNMT1, DNMT3a and MeCP2The results revealed that with advancing age there was an increase in AβPP expression which was significant at 23 years of age (P<0.01). Cynomolgus monkeys developmentally exposed to Pb displayed a further significant increase in AβPP protein expression at 23 years of age compared with their age matched control (P<0.05). There was an increase of NEP protein levels in 12-year-old cynomolgus monkeys group compared with other groups (P<0.01), however the levels were dropped at 23-year-old cynomolgus monkeys group and the addition of the Pb insult further inhibited its expression in comparison to the control (P<0.01).The results indicated that the expression of DNMT1, DNMT3a and MeCP2 protein was expressed at greater levels at 23-year-old monkeys group as aging occurs and that the addition of the Pb insult further enhanced that expression trend (P<0.05).2 Latent effect of developmental Pb exposure on the neurobiology-specific genes The expression profile of 22 genes changed due to early life exposure. Early life exposure to Pb resulted in up-regulation of 16 genes and down-regulation of only 6 genes. All the altered genes but two were abundant in CpG dinucleotides.3 Effect of aging and developmental exposure of Pb on the expression of histone modification biomarkersThere was a decrease in HDAC activity which was significant at 23 years of age (P<0.05) with advancing age. However, the levels of HDAC activity were raised at 23PbE group in comparison to 23CT group (P<0.01). The expression of H3K9ac and H4K12ac protein was expressed at greater levels at 23-year-old monkeys group as aging occurs (P<0.01), but was sharply dropped at 23PbE group in comparison to 23CT group (P<0.01).The results indicated that the expression of G9a protein was expressed at greater levels at 23-year-old monkeys group as aging occurs and that the addition of the Pb insult further enhances that expression trend (P<0.05). In addition, the expression of H3K9me and H3K27me2 had similar trend (P<0.01).4 Effect of Pb on the expression of AβPP, PSEN1, NEP and Aβ1-40The results showed an increase in AβPP protein expression within 48 h of Pb exposure (P<0.01). There was a decline in NEP protein expression following Pb exposure in these cells with the most significant decrease at 48 h for the 20μM-100μM Pb (P<0.01). There was an increase in AβPP mRNA levels following Pb exposure for 48 h which was observed to be significant in the range of 10μM-100μM (P<0.01). However for NEP, the mRNA expression was down-regulated following 5μM or 10μM of Pb exposure for 48 h (P<0.01). In addition, the results indicated a significant decrease in NEP activity after the addition of 5 or 10μM Pb for 48 h in comparison to the control group (P<0.01).The results indicated increased Aβ1-40 levels was in response to Pb exposure in a concentration-dependent manner (P<0.01). The level of Aβ1-40 was positively correlated with the expression of AβPP mRNA (P<0.01) and negatively correlated with the expression of NEP protein and activity (P<0.05) after Pb exposure for 48 h by Pearson correlation analysis.5 Effect of Pb exposure on the expression of histone modification biomarkersThe results indicated a decrease in H3K9ac and H4K12ac protein expression within 48 h of Pb exposure, with a significant elevation observed at 50μM and 100μM Pb concentrations (P<0.01). The HDAC activity was significantly induced by Pb at 100μM in comparison to the control. In addition, both HDAC activity and Aβ1-40 level were significant down-regulated with 100μM Pb exposure for 24 h followed by combined with 10μM VPA treatment for another 24 h in comparison to those exposed to 100μM Pb for 48 h (P<0.01).The results revealed an increase protein expression in H3K9me2 with 100μM Pb exposure and H3K27me2 with 50μM or 100μM Pb exposure concentrations for 48 h in comparison to the control (P<0.01). In addition, the significant elevation of G9a expression was observed by Pb at 100μM Pb concentrations in comparison to the control (P<0.01) and consistent with the results of intracellular G9a expression by immunofluorescence staining.Conclusions1 Age is an important impact factor on AD-related protein expression and epigenetic modification regulation.2 The expression of AD-related protein expression in aged cynomolgus monkeys after developmental Pb exposure was shown a latent effect and epigenetic pathway may be involved in this process.3 Pb can induce the change of AD-related biomarker in SH-SY5Y cells and the level of Aβ1-40 level was partly regulated by histone acetylation modification.