Deciphering the Global Proteomic Profile Involved in Methylmercury-Induced Cerebellar Neurodegeneration and Motor Dysfunction in Adult Rats

Abstract

Mercury is a ubiquitous pollutant in the environment with potential neurotoxic effects. Several populations are susceptible to mercurial exposure, especially methylmercury (MeHg) at low doses for long periods through food consumption. Given this, the present work aimed to assess the effects of long-term MeHg exposure on the cerebellum of rats from a translational perspective using a representative dose, assessing molecular, biochemical, morphological, and behavioral parameters. The model was produced by administering 40 µg/kg of MeHg for 60 days to adult male Wistar rats by oral gavage. As a result of this exposure, the animals presented motor deficits in open field and rotarod tests which were associated with an increase in total mercury content in cerebellar parenchyma, a reduction in antioxidant competence against peroxyl radicals, and increased nitrite and lipid peroxidation levels. The proteomic approach showed 317 modulated proteins. Such findings were associated with reductions in mature neuron and Purkinje cell densities and glial fibrillary acidic protein immunostained areas and increased microglial density. In addition, decreases in myelin basic protein and synaptophysin immunostaining were also observed. The results thus provided new evidence of the mechanisms underlying complex MeHg-induced neurodegeneration, especially the proteins underlying the biochemical and morphological features associated with motor dysfunction.

Keywords: microglia; neurodegeneration; neurotoxicology; organic mercury; oxidative stress; proteomic.

Figure 1

Methodological summary of the study. Wistar rats were divided into control (n = 27) and MeHg groups (dose of 40 µg/kg/day) (n = 27). After 60 days of MeHg exposure, the motor functions were assessed by open field and rotarod tests. Then, after cerebellum collection, the samples were processed for total mercury determination, oxidative biochemistry (antioxidant capacity against peroxyl radicals (ACAP), nitrites, and lipid peroxidation (LPO)), and proteomic and morphological analyses (Purkinje cell count, immunohistochemistry for mature neurons (NeuNs), glial fibrillary acidic protein (GFAP), microglial markers (Iba1), myelin basic protein (MBP), and synaptophysin (SYP)).

Figure 2

Effects of prolonged exposure to MeHg (40 µg/kg/day), for 60 days, on the oxidative biochemistry of the cerebellums of Wistar rats. (A) Antioxidant Capacity Against Peroxyl Radicals (ACAP). (B) Nitrite levels. (C) Lipid peroxidation (LPO) levels measured by malondialdehyde (MDA) levels. Results are expressed as means ± standard errors of the mean (SEMs) and converted to percentages of the control group. n = 10 per group. * p < 0.05. Student’s t-test.

Figure 3

Functional distribution of proteins identified with differential expression in the cerebellums of adult rats between the MeHg group and the control group (A). Protein categories are based on the Gene Ontology (GO) of biological processes. (B) Protein–protein interaction network. Red and dark green colors indicate proteins found in the control and exposed groups, respectively; light green and pink colors indicate upregulated and downregulated proteins, respectively; gray nodes represent those proteins that were not identified in this study but which entered into interactions in the network.

Figure 4

Circos plot of protein-protein interactions (PPIs) resulting from the over-representation analysis of the cerebellar proteomes of rats exposed to MeHg. The results are organized into the categories of energy homeostasis (orange), oxidative stress (light green), nervous system regulation (purple), and synaptic signaling (warm pink). Blue-scale proteins were downregulated and red proteins were upregulated in the MeHg group compared to the control group.

Figure 5

Effects of exposure to MeHg on the morphological integrity of cerebellums of Wistar rats. PC: Purkinje cells; NeuN: mature neurons; GFAP: glial fibrillary acidic protein; Iba1: microglia markers; MBP: myelin basic protein; Syp: synaptophysin. Results are expressed as means ± standard errors of the means (SEMs). The Student’s t-test was used to analyze the PC, NeuN, and Iba1 results, and the Mann–Whitney test was used to analyze the area fraction data. * p < 0.05.

Figure 6

Effects of long-term exposure to MeHg (40 µg/kg/day) for 60 days on spontaneous and forced motor locomotion in Wistar rats. (A) Tracking plot of horizontal locomotor activity, where blue dots are for the initial position and red dots, final position (B) mean total distance traveled, and (C) number of rearings in the open field test. (D) Number of falls in the rotarod test. Results are expressed as means ± standard errors of the means (SEMs). n = 10 per group. The open-field parameters were analyzed using the Student’s t-test and the number of falls in the rotarod test by two-way ANOVA and Sidak’s post hoc test. * p < 0.05.