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Cadmium-induced apoptosis in primary rat cerebral cortical neurons culture is mediated by a calcium signaling pathway.

ABSTRACT: Cadmium (Cd) is an extremely toxic metal, capable of severely damaging several organs, including the brain. Studies have shown that Cd disrupts intracellular free calcium ([Ca(2+)]i) homeostasis, leading to apoptosis in a variety of cells including primary murine neurons. Calcium is a ubiquitous intracellular ion which acts as a signaling mediator in numerous cellular processes including cell proliferation, differentiation, and survival/death. However, little is known about the role of calcium signaling in Cd-induced apoptosis in neuronal cells. Thus we investigated the role of calcium signaling in Cd-induced apoptosis in primary rat cerebral cortical neurons. Consistent with known toxic properties of Cd, exposure of cerebral cortical neurons to Cd caused morphological changes indicative of apoptosis and cell death. It also induced elevation of [Ca(2+)]i and inhibition of Na(+)/K(+)-ATPase and Ca(2+)/Mg(2+)-ATPase activities. This Cd-induced elevation of [Ca(2+)]i was suppressed by an IP3R inhibitor, 2-APB, suggesting that ER-regulated Ca(2+) is involved. In addition, we observed elevation of reactive oxygen species (ROS) levels, dysfunction of cytochrome oxidase subunits (COX-I/II/III), depletion of mitochondrial membrane potential (??m), and cleavage of caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP) during Cd exposure. Z-VAD-fmk, a pan caspase inhibitor, partially prevented Cd-induced apoptosis and cell death. Interestingly, apoptosis, cell death and these cellular events induced by Cd were blocked by BAPTA-AM, a specific intracellular Ca(2+) chelator. Furthermore, western blot analysis revealed an up-regulated expression of Bcl-2 and down-regulated expression of Bax. However, these were not blocked by BAPTA-AM. Thus Cd toxicity is in part due to its disruption of intracellular Ca(2+) homeostasis, by compromising ATPases activities and ER-regulated Ca(2+), and this elevation in Ca(2+) triggers the activation of the Ca(2+)-mitochondria apoptotic signaling pathway. This study clarifies the signaling events underlying Cd neurotoxicity, and suggests that regulation of Cd-disrupted [Ca(2+)]i homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases.

SUBMITTER: Yuan Y

PROVIDER: S-EPMC3669330 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Cadmium-induced apoptosis in primary rat cerebral cortical neurons culture is mediated by a calcium signaling pathway.

Yuan Yan Y Jiang Chen-yang CY Xu Hui H Sun Ya Y Hu Fei-fei FF Bian Jian-chun JC Liu Xue-zhong XZ Gu Jian-hong JH Liu Zong-ping ZP

PloS one 20130531 5

Cadmium (Cd) is an extremely toxic metal, capable of severely damaging several organs, including the brain. Studies have shown that Cd disrupts intracellular free calcium ([Ca(2+)]i) homeostasis, leading to apoptosis in a variety of cells including primary murine neurons. Calcium is a ubiquitous intracellular ion which acts as a signaling mediator in numerous cellular processes including cell proliferation, differentiation, and survival/death. However, little is known about the role of calcium s ...[more]

PMID: 23741317

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Project description:Cortical rat neuronal culture, lysis and proteolytic digestion Primary rat cortical neurons were generated from E18 Sprague-Dawley rat embryos with minor modifications (Swanger, Mattheyses et al. 2015, Henderson, Greathouse et al. 2019). Neurons were cultured at a density of 4x105 cells per well in 12-well culture plates (Fisher Scientific, catalog no. 353043). Neurons were cultured in Neurobasal medium (Fisher Scientific, catalog no. 21103-049) supplemented with B27 (Fisher Scientific, catalog no.17504-044). Culture maintenance included a half media change every 2-3 days. At DIV 14, neurons were either treated with 150 ng/mL recombinant NRN1 protein (Abcam, ab69755) or vehicle treated with diH2O for 6 hours. NRN1 concentration was chosen based on published data that identified a plateau in exogenous NRN1 induced effects on transient potassium currents at 150 ng/mL (Yao et al., 2012). After 6 hours neurons were washed 2x with 1 mL 1X phosphate-buffered saline (PBS). To harvest cells, 1 mL 1X PBS + protease inhibitor (Fisher Scientific, catalog no. 78426) was added and cells were centrifuged for 2300rpm for 5 minutes at 4°C. Cell pellets were lysed in 200uL 8M urea buffer and HALT protease and phosphatase inhibitor cocktail (1x final concentration). Lysates were sonicated with a probe sonicator 3 times for 10 s with 10 s intervals at 30% amplitude and cleared of cellular debris by centrifugation in a tabletop centrifuge at 18,000 rcf for 3 minutes at 4° C. Protein concentration was determined by BCA assay and one-dimensional SDS-PAGE gels were run followed by Coomassie blue staining as quality control for protein integrity and equal loading before proceeding to protein digestion. Protein homogenates (50 µg) were diluted with 50 mM NH4HCO3 to a final concentration of less than 2 M urea and then treated with 1 mM dithiothreitol (DTT) at 25°C for 30 minutes, followed by 5 mM iodoacetimide (IAA) at 25°C for 30 minutes in the dark. Protein was digested with 1:100 (w/w) lysyl endopeptidase (Wako) at 25°C for 2 hours and further digested overnight with 1:50 (w/w) trypsin (Pierce) at 25°C. Resulting peptides were desalted with a Sep-Pak C18 column (Waters) and dried under vacuum.

Project description:Enterovirus D68 (EV-D68) is an emerging pathogen associated with mild to severe respiratory disease. Since 2014, EV-D68 is also linked to acute flaccid myelitis (AFM), causing paralysis and muscle weakness in children. However, it remains unclear whether this is due to an increased pathogenicity of contemporary EV-D68 clades or increased awareness and detection of this virus. Here, we describe an infection model of primary rat cortical neurons to study the entry, replication, and functional consequences of different EV-D68 strains, including historical and contemporary strains. We demonstrate that sialic acids are important (co)receptors for infection of both neurons and respiratory epithelial cells. Using a collection of glycoengineered isogenic HEK293 cell lines, we show that sialic acids on either N-glycans or glycosphingolipids can be used for infection. Additionally, we show that both excitatory glutamatergic and inhibitory GABA-ergic neurons are susceptible and permissive to historical and contemporary EV-D68 strains. EV-D68 infection of neurons leads to the reorganization of the Golgi-endomembranes forming replication organelles, first in the soma and later in the processes. Finally, we demonstrate that the spontaneous neuronal activity of EV-D68-infected neuronal network cultured on microelectrode arrays (MEA) is decreased, independent of the virus strain. Collectively, our findings provide novel insights into neurotropism and -pathology of different EV-D68 strains, and argue that it is unlikely that increased neurotropism is a recently acquired phenotype of a specific genetic lineage. IMPORTANCE Acute flaccid myelitis (AFM) is a serious neurological illness characterized by muscle weakness and paralysis in children. Since 2014, outbreaks of AFM have emerged worldwide, and they appear to be caused by nonpolio enteroviruses, particularly enterovirus-D68 (EV-D68), an unusual enterovirus that is known to mainly cause respiratory disease. It is unknown whether these outbreaks reflect a change of EV-D68 pathogenicity or are due to increased detection and awareness of this virus in recent years. To gain more insight herein, it is crucial to define how historical and circulating EV-D68 strains infect and replicate in neurons and how they affect their physiology. This study compares the entry and replication in neurons and the functional consequences on the neural network upon infection with an old "historical" strain and contemporary "circulating" strains of EV-D68.

Dataset Information

Cadmium-induced apoptosis in primary rat cerebral cortical neurons culture is mediated by a calcium signaling pathway.

Publications

Cadmium-induced apoptosis in primary rat cerebral cortical neurons culture is mediated by a calcium signaling pathway.

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