Dysfunction of cortical synapse-specific mitochondria in developing rats exposed to lead and its amelioration by ascorbate supplementation

Abstract

Background: Lead (Pb) is a widespread environmental neurotoxin and its exposure even in minute quantities can lead to compromised neuronal functions. A developing brain is particularly vulnerable to Pb mediated toxicity and early-life exposure leads to permanent alterations in brain development and neuronal signaling and plasticity, culminating into cognitive and behavioral dysfunctions and elevated risk of neuropsychiatric disorders later in life. Nevertheless, the underlying biochemical mechanisms have not been completely discerned.

Methods: Because of their ability to fulfill high energy needs and to act as calcium buffers in events of high intensity neuronal activity as well as their adaptive regulatory capability to match the requirements of the dynamicity of synaptic signaling, synapse-specific or synaptic mitochondria (SM) are critical for synaptic development, function and plasticity. Our aim for the present study hence was to characterize the effects of early-life Pb exposure on the functions of SM of prepubertal rats. For this purpose, employing a chronic model of Pb neurotoxicity, we exposed rat pups perinatally and postnatally to Pb and used a plethora of colorimetric and fluorometric assays for assessing redox and bioenergetic properties of SM. In addition, taking advantage of its ability as an antioxidant and as a metal chelator, we employed ascorbic acid (vitamin C) supplementation as an ameliorative therapeutic strategy against Pb-induced neurotoxicity and dysfunction of SM.

Results: Our results suggest that early-life exposure to Pb leads to elevated oxidative stress in cortical SM with consequent compromises in its energy metabolism activity. Ascorbate supplementation resulted in significant recovery of Pb-induced oxidative stress and functional compromise of SM.

Conclusion: Alterations in redox status and bioenergetic properties of SM could potentially contribute to the synaptic dysfunction observed in events of Pb neurotoxicity. Additionally, our study provides evidence for suitability of ascorbate as a significant ameliorative agent in tacking Pb neurotoxicity.

Keywords: heavy metal neurotoxicity; mitochondrial bioenergetics; mitochondrial membrane potential; neuropsychiatric; oxidative damage; synaptic.

Figure 1

Isolation of purified synapse-specific mitochondrial fraction from rat brain cortices. Notes: Enrichment in activity of mitochondrial marker SDH was observed for SM fraction isolated by discontinuous Ficoll medium-based gradient ultracentrifugation method. SDH activity levels in SM fraction were increased by 7.2±0.4 (mean ± SEM) times compared to homogenate. Negative control comprising SM fraction preincubated with malonate was used to confirm validity of the assay. Data are represented as mean ± SEM (n=4 samples from 4 rats). Abbreviations: SDH, succinate dehydrogenase; SM, synapse-specific mitochondria; malo, malonate; PNS, post-nuclear supernatant; PMS, post-mitochondrial supernatant; CMP, crude mitochondrial pellet.

Figure 2

SM of rats developmentally exposed to Pb have increased levels of ROS and RNS and the increase is ameliorated by ascorbate. Notes: (A) DCFH-DA-based fluorometric analysis of ROS species show elevated ROS levels in SM of Pb-exposed rats. The increase is attenuated by ascorbic supplementation. (B) Increase in levels of RNS was observed in SM from brain cortices of rats chronically exposed to Pb. This Pb-induced increase in RNS levels was not observed in rats supplemented with ascorbic acid. Data are represented as mean ± SEM (n=5 pups from 3 litters) and * and # represent statistical significance (p<0.05) when compared to Ctrl and Pb+Asc groups, respectively (ANOVA with Newman-Keuls correction). Abbreviations: SM, synapse-specific mitochondria; Pb, lead; ROS, reactive oxygen species; RNS, reactive nitrogen species; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; Ctrl, control; Asc, ascorbic acid; SEM, standard error of the mean; NOx, total nitrites and nitrates.

Figure 3

SM from cerebral cortices of rat pups exposed to Pb show increased oxidative damage to lipids and proteins and have reduced antioxidant capacity and their redox status is rescued by ascorbic acid supplementation. Notes: Increased oxidative damage to lipids and proteins was observed in SM fractions of Pb-exposed rats as evidenced from elevated TBARS (A), increased protein carbonylation (B) and increased protein thiol oxidation (C). Moreover, ascorbic acid supplementation could rescue the oxidative damage in SM fraction induced by Pb exposure. (D) Antioxidant capacity of SM fraction, as measured by ABTS radical decolorization assay, was found to be significantly lowered upon chronic Pb exposure but was recovered to control levels upon ascorbate supplementation. Data are represented as mean ± SEM (n=5 pups from 3 litters) and * and # represent statistical significance (p<0.05) when compared to Ctrl and Pb+Asc groups, respectively (ANOVA with Newman-Keuls correction). Abbreviations: SM, synapse-specific mitochondria; Pb, lead; TBARS, thiobarbituric acid reactive substances; ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); Ctrl, control; Asc, ascorbic acid; SEM, standard error of the mean.

Figure 4

SM from cerebral cortices of rat pups exposed to Pb show alterations in basal mitochondrial membrane potential as well as energization-induced generation of mitochondrial membrane potential and ascorbic acid abolishes these alterations. Notes: MMP at both basal states (A) and in presence of substrates (B) was reduced in SM fraction isolated from Pb-exposed rats. Pb-induced alterations in MMP and substrates-induced MMP generation were rescued upon ascorbate supplementation. Data are represented as mean ± SEM (n=5 pups from 3 litters) and * and # represent statistical significance (p<0.05) when compared to Ctrl and Pb+Asc groups, respectively (ANOVA with Newman-Keuls correction). Abbreviations: SM, synapse-specific mitochondria; Pb, lead; MMP, mitochondrial membrane potential; Ctrl, control; Asc, ascorbic acid.

Figure 5

Pb induced alternations in the bioenergetic properties of cortical SM are mitigated by ascorbate supplementation. Notes: While activities of complex I (A) and IV (D) of the ETC were unaltered in SM of Pb-exposed rats when compared to aged matched controls, activities of complex II (B) and III (C) were found to be significantly reduced in SM fraction isolated from Pb-exposed rats. However, ascorbate supplementation rescued the loss in activity of complex II alone without affecting loss in activity of complex III. (E) Significant decrease in the rate of ATP synthesis was observed for SM of Pb-exposed rats when compared to aged matched controls and the reduction was ameliorated by ascorbic acid supplementation. Data are represented as mean ± SEM (n=5 pups from 3 litters) and * and # represent statistical significance (p<0.05) when compared to Ctrl and Pb+Asc groups, respectively (ANOVA with Newman-Keuls correction). Abbreviations: SM, synapse-specific mitochondria; Pb, lead; ETC, electron transport chain; Ctrl, control; Asc, ascorbic acid.