Elsevier

NeuroToxicology

Volume 48, May 2015, Pages 109-119
NeuroToxicology

ER stress and ER stress-mediated apoptosis are involved in manganese-induced neurotoxicity in the rat striatum in vivo

https://doi.org/10.1016/j.neuro.2015.02.007Get rights and content

Highlights

  • Mn could cause rat striatal neurocytes injury in a dose-dependent manner.

  • Mn could induce ER stress in rat striatal neurocytes.

  • ER stress-mediated apoptosis in rat striatal neurocytes is involved in Mn-induced neurotoxicity.

Abstract

Manganese (Mn) is an essential trace element found in many enzymes, however, excessive Mn-exposure can result in manganism which is similar to Parkinson's movement disorder. The mechanisms of manganism are not well-known. The present in vivo study was carried out to determine whether endoplasmic reticulum stress (ER stress) and ER stress-mediated apoptosis are involved in manganese-induced neurotoxicity. Sixty-four SD rats were randomly divided into four groups and were administered intraperitoneally with normal saline (NS, as control) or MnCl2 (7.5, 15 and 30 mg/kg body weight, respectively) for 4 weeks. We found that MnCl2 dose-dependently accumulate in striatal. HE staining and TUNEL assay results indicated that MnCl2 induced striatal neurocytes apoptosis in both male and female rats. The alterations of ultrastructures showed that MnCl2 resulted in chromatin condensation, mitochondria and ER tumefaction in rat striatal neurocytes. Furthermore, MnCl2 increased the expressions of p-IRE-1, ATF-6α, PERK, GRP78, Sigma-1R, CHOP, Bim, Bax, caspase-12 and caspase-3, and decreased the expression of Bcl-2 in rat striatal neurocytes. In conclusion, MnCl2 could induce ER stress and ER stress-mediated apoptosis in rat striatal neurocytes, which might be one of the important mechanisms of Mn-induced neurotoxicity.

Introduction

Manganese (Mn), an essential trace element found in many enzymes such as Mn-superoxide dismutase and pyruvate carboxylase, can promote the growth and development of central nervous system and is a substitute for magnesium or calcium in many enzyme-catalyzed kinase reactions (Stephenson et al., 2013). However, exposure to excessive levels of Mn can lead to Mn accumulation in basal ganglia, striatum and substantia nigra, and thereby cause manganism which manifests the Parkinson's-like movement disorder (Crossgrove and Zheng, 2004, Guilarte, 2010). Chronic and excessive Mn exposure can happen in vast occupations including mining, dyes, dry cell battery manufacturing and pesticides. In some countries, Mn-containing fuel additive methylcyclopentadienyl manganese tricarbonyl is introduced as an anti-knock agent, which could increase human exposure to Mn (Gerber et al., 2002, Li et al., 2010).

Several studies have reported some different aspects of Mn-induced neurotoxicity, and proposed the possible underlying mechanisms. Mn can promote reactive oxygen species (ROS) generation in vitro and in vivo, which significantly affects mitochondrial function and impairs energy metabolism (Jiao et al., 2008, Zhang et al., 2003, Zhang et al., 2004, Zhang et al., 2005). Mn can also cause the alteration of neurotransmitters levels (Blecharz-Klin et al., 2012, Moberly et al., 2012), and thereby induce excitotoxic cell death (Quintanar et al., 2012). In addition, Mn can induce protease activation (Quintanar et al., 2012) and apoptotic cell death (Alaimo et al., 2013, Cheng et al., 2005, Roth et al., 2012). However, the mechanisms of Mn-induced neurotoxicity are still not well understood.

Endoplasmic reticulum (ER) is an organelle in which membrane proteins are folded and assembled by ER-resident molecular chaperones in a secretory pathway. A number of cellular stresses and cytotoxic conditions can lead to ER stress via interfering protein folding, which can cause the accumulation of unfolded proteins and activate a complex signaling network termed unfolded protein response (UPR) (Jang et al., 2011, Ozcan et al., 2008, Zhong et al., 2012, Timmins et al., 2009). UPR contributes to two kinds of responses by adaptive and apoptotic pathways. UPR typically induces cell death through apoptotic pathway (Apostolou et al., 2008, Jang et al., 2011, Kaufman et al., 2002). Normally, three trans-membrane proteins inositol-requiring enzyme-1 (IRE-1), pancreatic ER kinase (PKR)-like ER kinase (PERK) and activating transcription factor-6 alpha (ATF-6α), can bind to 78-KDa glucose-regulationed protein (GRP78), which is an ER chaperone protein and can increase the activity of protein-folding and prevent the aggregation of proteins. When moderate and transient ER stress happens, the dissociation of GRP78 from UPR sensors PERK, ATF-6α and IRE-1 triggers the UPR to alleviate ER stress and maintain ER function. However, if ER stress is inordinate and cannot be alleviated, it eventually leads to cell death (Lakshmanan et al., 2013, Xu et al., 2012, Zhu et al., 2012). The death response might be induced by apoptotic signals which are produced at the ER. Several special mechanisms of ER stress-mediated apoptotic pathways are involved: the proapoptotic factor CHOP is induced by PERK/eIF2-dependent transcription and the cleavage and activation of procaspase-12 (Kadowaki et al., 2004, Malhotra and Kaufman, 2007).

Recently, multiple papers reported that ER stress and ER stress-mediated apoptosis were implicated in neurodegenerative diseases such as Parkinson disease (DeWitt et al., 2013, Kim et al., 2011, Sanchez-Betancourt et al., 2012). Lately, some studies showed that Mn increased the expression levels of GRP78 and caspase-12 in SK-M-NC human neuroblastoma (Yoon et al., 2011a, Yoon et al., 2011b) and DA SN4741 neuronal cells (Chun et al., 2001, Yoon et al., 2011a, Yoon et al., 2011b). Our previous studies demonstrated that MnCl2 could induce ER stress and ER stress-mediated apoptosis in human neuroblastoma SH-SY5Y cells. These available evidences obtained from in vitro studies and it is necessary to be further confirmed in vivo. The present in vivo study was conducted to further verify that ER stress and ER stress-mediated apoptosis were involved in Mn-induced injury of striatal neurocytes, and elucidate the cellular and molecular mechanism about the onset and progression of the disorder.

Section snippets

Materials

MnCl2·4H2O was purchased from Beijing Chemical Works (Beijing, China). The PERK, ATF-6α, GRP78, CHOP, Bax, Bcl-2, Bim antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the p-IRE-1, Sigma-1R, caspase-12 antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). The anti-β-actin and enhanced chemiluminescence (ECL) kit were purchased from Tiangen Biological Chemistry (Beijing, China). Horseradish peroxidase-labeled goat anti-rabbit IgG and

Increases of MnCl2 concentrations in rat striatum after MnCl2 treatment

As shown in Table 1, MnCl2 concentrations in rat striatum in MnCl2-treated groups were significantly increased (vs. control) both in female and male rats (P < 0.01) and the accumulation of MnCl2 in the striatum was in a dose-dependent manner.

Mn-induced striatal neurocytes injury

As shown in Fig. 1, Fig. 2, in control groups the striatum tissue sections had the normal morphology and nerve cells were big, full and irregular; the structures of nerve cells were clear; Nissl body could be seen in the cytoplasm; nuclear membranes were

Discussion

In this study, we performed an in vivo study to confirm that ER stress and ER stress-mediated apoptosis were involved in MnCl2-induced neurotoxicity.

Studies have shown that mild ER stress are beneficial to cell survival, but continued and excessive ER stress may induce apoptosis (Schroder and Kaufman, 2005). So we think only one treatment group is not sufficient to verify the role of ER stress. Furthermore, according to mild adjustment of the preliminary experiments, we de-sign three treatment

Conflict of interest

The authors declare that there are no conflicts of interest.

Transparency document

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Acknowledgements

This work was supported by National Natural Science Foundation of China (NSFC), Nos. 30972502 and 81172693.

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