Developmental lead (Pb)-induced deficits in hippocampal protein translation at the synapses are ameliorated by ascorbate supplementation

Figures & data

Figure 1 Ascorbate supplementation reduces the elevation in blood Pb levels induced by chronic Pb treatment through drinking.

Notes: Significant increase in blood Pb levels of pups of the Pb group (665.5±384.6; mean ± SD) was observed when compared to those of the Ctrl group (13.07±1.332; mean ± SD). Ascorbate supplementation reduced the blood Pb levels of Pb exposed pups to 131.8±39.53 (mean ± SD). Data are represented as mean ± SEM (n=6 rats per group). ^*,#Statistical significance when compared to Ctrl and Pb-Asc groups, respectively (P<0.0001; F=36.27; ANOVA with Newman–Keuls correction).
Abbreviations: Asc, ascorbic acid; SEM, standard error of the mean.

Figure 2 Hippocampal SNs isolated by the sequential filtration protocol are highly enriched in PSD-95 protein.

Notes: (A) Protein levels of PSD-95, a synaptic protein marker, were analyzed by immunoblotting to estimate the purity of the SN. (B) Immunoreactivity of PSD-95 in SN was increased by 4.731±1.150 (mean ± SD) fold when compared with the initial Hgt. Sup = supernatant obtained after the centrifugation step. F1 = filtrate 1 obtained after filtration using 100 µm filters. Data are represented as mean ± SEM (n=3 independent samples from three rats). ^*,&,#Statistical significance in the Sup, Hgt, and F1 groups (P<0.0001; F=36.27; ANOVA with Newman–Keuls correction).
Abbreviations: Hgt, homogenate; PSD-95, post-synaptic density 95; SEM, standard error of the mean; SN, synaptoneurosome.

Figure 3 Ascorbate rescues the Pb-induced deficits in localized protein translation at hippocampal synapses.

Notes: (A) De novo protein translation rate under basal and KCl-stimulated conditions in the synaptoneurosomes was evaluated using the immunoblotting-based puromycin incorporation assay. (B) While depolarization in the presence of KCl significantly stimulated protein translation in synaptoneurosomes of the pups of the Ctrl (1.615±0.3992 for stimulated compared to 1.000±0.0 for unstimulated; mean ± SD) and Pb-Asc groups (1.443±0.3342 for stimulated compared to 1.026±0.3871 for unstimulated; mean ± SD), it was ineffective in increasing the rate of translation in Pb-exposed rats (0.6641±0.2668 for stimulated compared to 0.7546±0.1227 for unstimulated; mean ± SD). In addition, Pb treatment resulted in a small but insignificant reduction in the basal translation rates in the synaptoneurosomal samples when compared to both Ctrl and Pb-Asc pups. Data are represented as mean ± SEM (n=6 rats per group). *Statistical significance between the stimulated translation and the respective basal controls and ^#,&Statistical significance in the decrease of stimulated translation of Pb-exposed pups when compared to stimulated translation of Ctrl and Pb-Asc pups, respectively (P<0.0001; F=10.01; ANOVA with Newman–Keuls correction).
Abbreviations: Asc, ascorbic acid; SEM, standard error of the mean; US, unstimulated; St, stimulated.

Figure 4 The decrease in the ratio of stimulated to basal protein translation in synaptoneurosomes is significantly correlated with increases in blood Pb levels.

Notes: (A) Pb induced a marked reduction in the ratio of the rates of depolarization-induced KCl-stimulated protein synthesis to those of the corresponding basal (unstimulated) samples (0.8836±0.3639; mean ± SD) when compared to control values (1.615±0.3992; mean ± SD). Pb-induced reduction in the ratio of the translation rates was rescued by ascorbate supplementation (1.570±0.5975; mean ± SD). (B) Significant correlation was observed between depletion in the ratio of KCl-stimulated to basal de novo protein translation at the synapse and the increase in the blood Pb levels of the pups. Data (n=6 rats per group) were analyzed using Pearson’s correlation analysis. Data are represented as mean ± SEM (n=6 rats per group). ^*,#Statistical significance when compared to the Ctrl and Pb-Asc groups, respectively (P=0.0267; F=4.656; ANOVA with Newman–Keuls correction).
Abbreviations: Asc, ascorbic acid; SEM, standard error of the mean.

Figure S1 Ascorbate supplementation alone does not alter localized protein translation at hippocampal synapses.

Notes: (A) Basal and KCl-stimulated synaptosomal protein translation was evaluated in hippocampal samples of Ctrl and Asc pups. (B) Depolarization in the presence of KCl induced an increase in the protein translation in synaptoneurosomes of the pups of the Ctrl (1.555±0.6100 for stimulated compared to 1.000±0.0 for unstimulated; mean ± SD). A similar elevation in protein translation was observed in pups supplemented with ascorbate alone (1.720±0.4584 for stimulated compared to 1.126±0.2650 for unstimulated; mean ± SD). Data are represented as mean ± SEM (n=6 rats per group). *Statistical significance between the stimulated translation and the respective basal controls (P=0.0108; F=4.778; ANOVA with Newman–Keuls correction).

Abbreviations: Asc, ascorbic acid; NS, nonsignificant; SEM, standard error of the mean; US, unstimulated; St, stimulated.

Figure S2 Developmental Pb exposure does not influence global protein translation in the hippocampi of rat pups.

Notes: (A) De novo global protein translation rates were assessed in hippocampal homogenate samples from the pups of Ctrl, Pb, and Pb-Asc groups. (B) No alteration in global translation was observed upon developmental exposure of pups to Pb with (0.9294±0.5748 compared to the Ctrl; mean ± SD) or without ascorbate supplementation (0.9816±0.4963 compared to the Ctrl; mean ± SD). Data are represented as mean ± SEM (n=6 rats per group) (P=0.9616; F=0.03927; ANOVA with Newman–Keuls correction).

Abbreviations: Asc, ascorbic acid; SEM, standard error of the mean.