Developmental lead exposure alters synaptogenesis through inhibiting canonical Wnt pathway in vivo and in vitro

Abstract

Lead (Pb) exposure has been implicated in the impairment of synaptic plasticity in the developing hippocampus, but the mechanism remains unclear. Here, we investigated whether developmental lead exposure affects the dendritic spine formation through Wnt signaling pathway in vivo and in vitro. Sprague-Dawley rats were exposed to lead throughout the lactation period and Golgi-Cox staining method was used to examine the spine density of pyramidal neurons in the hippocampal CA1 area of rats. We found that lead exposure significantly decreased the spine density in both 14 and 21 days-old pups, accompanied by a significant age-dependent decline of the Wnt7a expression and stability of its downstream protein (β-catenin). Furthermore, in cultured hippocampal neurons, lead (0.1 and 1 µM lead acetate) significantly decreased the spine density in a dose-dependent manner. Exogenous Wnt7a application attenuated the decrease of spine density and increased the stability of the downstream molecules in Wnt signaling pathway. Together, our results suggest that lead has a negative impact on spine outgrowth in the developing hippocampus through altering the canonical Wnt pathway.

Figure 1. Lead accumulations in hippocampus in the control and chronic lead-exposed rats.

The lead levels in P14 and P21 rats with or without lead exposure were determined. Histograms were plotted by the mean of 8 rat hippocampus per group. (*p<0.05, **p<0.01).

Figure 2. The dendritic spine alteration of pyramidal neurons in the hippocampus of P14 and P21 rats after chronic lead exposure.

A) Golgi-Cox impregnated dendritic arborization and dendritic spines in four groups: control (Ctrl) P14, lead (Pb) P14, Ctrl P21, lead (Pb) P21. Scale bar = 10 µm; B) Representative sections (20 µm) of Golgi-Cox stained dendrites of pyramidal neurons in hippocampus in four groups; C) Histograms plot showing the alteration of dendritic spine density (spines/10 µm) after lead exposure in P14 and P21 rats. (***p<0.001).

Figure 3. Effect of developmental lead exposure on Wnt/β-catenin pathway in vivo.

A) Representative immunoblot and corresponding densitometric analysis showed expression of Wnt7a which was normalized to β-actin in the control and lead-treated groups in P14 and P21 rats. (#p>0.05 and *p<0.05). B) Representative immunoblot and corresponding densitometric analysis showed the ratio of expression of phosphorylated β-catenin (phospho-β-catenin) to total β-catenin in control and lead treated groups in P14 and P21 rats. (#p>0.05 and *p<0.05).

Figure 4. Effect of Wnt/β-catenin pathway on the dendritic spine density of hippocampal pyramidal neurons after lead treatment in vitro.

A) Representative EGFP-transfected hippocampal neurons in the control group (Ctrl), the lead-treated groups (0.1 and 1 µM from DIV7 to DIV12), and the lead-treated groups with 16 hours Wnt7a treatment in DIV12 (0.1 µM Pb, 0.1 µM Pb+Wnt7a, 1 µM Pb, 1 µM Pb+Wnt7a), Scale bar = 10 µm; B) Representative sections (20 µm) of dendritic spines in four groups; C) Histograms plot showing the alteration of dendritic spine density (spines/10 µm) after lead treatment with or without Wnt7a. (*p<0.05, **p<0.01, ***p<0.001 and #p>0.05). All experiments were performed by using three independent cultures.

Figure 5. Effect of lead on the β-catenin phosphorylation in vitro and effect of Wnt7a on the lead-induced change of the β-catenin phosphorylation.

Representative immunoblot and corresponding densitometric analysis showed the ratio of expression of phosphorylated β-catenin to total β-catenin in cultured hippocampal neurons of five groups: control (Ctrl), 0.1 µM lead (0.1 µM Pb), 0.1 µM lead with Wnt7a (100 ng/ml) (0.1 µM Pb+Wnt7a), 1 µM lead (1 µM Pb) and 1 µM lead with Wnt7a (100 ng/ml) (1 µM Pb+Wnt7a). (*p<0.05, **p<0.01, ***p<0.01 and #p>0.05). All experiments in vitro were performed by using three independent cultures.