Metformin attenuates cadmium-induced neuronal apoptosis in vitro via blocking ROS-dependent PP5/AMPK-JNK signaling pathway

Highlights

• Metformin attenuates Cd-evoked apoptotic cell death in neuronal cells.

• Metformin suppresses Cd-activated JNK pathway, preventing neuronal apoptosis.

• Metformin prevents Cd-elicited mitochondrial ROS inactivation of PP5 and AMPK, thereby suppressing activation of JNK pathway.

• Metformin may be a promising drug for prevention of Cd-induced oxidative stress and neurodegenerative diseases.

Abstract

Cadmium (Cd), a toxic environment contaminant, induces reactive oxygen species (ROS)-mediated neuronal apoptosis and consequential neurodegenerative disorders. Metformin, an anti-diabetic drug, has recently received a great attention owing to its protection against neurodegenerative diseases. However, little is known regarding the effect of metformin on Cd-induced neurotoxicity. Here we show that metformin effectively prevented Cd-evoked apoptotic cell death in neuronal cells, by suppressing Cd activation of c-Jun N-terminal kinases (JNK), which was attributed to blocking Cd inactivation of protein phosphatase 5 (PP5) and AMP-activated protein kinase (AMPK). Inhibition of JNK with SP600125, knockdown of c-Jun, or overexpression of PP5 potentiated metformin's inhibitory effect on Cd-induced phosphorylation of JNK/c-Jun and apoptosis. Activation of AMPK with AICAR or ectopic expression of constitutively active AMPKα strengthened the inhibitory effects of metformin on Cd-induced phosphorylation of JNK/c-Jun and apoptosis, whereas expression of dominant negative AMPKα weakened these effects of metformin. Metformin repressed Cd-induced ROS, thereby diminishing cell death. N-acetyl-l-cysteine enhanced the inhibitory effects of metformin on Cd-induced ROS and apoptosis. Moreover, using Mito-TEMPO, we further demonstrated that metformin attenuated Cd-induced cell death by suppressing induction of mitochondrial ROS. Taken together, these results indicate that metformin prevents mitochondrial ROS inactivation of PP5 and AMPK, thus attenuating Cd-induced JNK activation and apoptosis in neuronal cells. Our data highlight that metformin may be a promising drug for prevention of Cd-induced oxidative stress and neurodegenerative diseases.

Introduction

Heavy metal contamination, one of the greatest global problems, not only negatively affects environment but also endangers humans and animals. Cadmium (Cd), an occupationally and environmentally heavy metal pollutant, can accumulate in multiple organs and tissues due to its long half-life (15–20 years) in the human body, thus resulting in a high incidence of related diseases, including impaired renal function, decreased bone mineral density, increased calcium excretion, reproductive toxicity, and neurotoxicity (Akesson et al., 2006; Johri et al., 2010; Wang and Du, 2013; Xu et al., 2017). Upon absorption into the bloodstream, Cd can easily traverse the blood-brain barrier and accumulate in the brain, manifesting with symptoms including headache and vertigo, olfactory dysfunction, slowed vasomotor function, peripheral neuropathy, decreased equilibrium, decreased ability to concentrate, and learning disabilities (Fowler, 2009; Okuda et al., 1997; Satarug and Nazar, 2010). Merging data have demonstrated that Cd exposure can induce increased levels of reactive oxygen species (ROS) and trigger neuronal apoptosis (Chen et al., 2011; Di Carlo et al., 2012; Yang et al., 2007). Excessive ROS induced by Cd have been recognized as an important pathogenic factor in the development of neuronal cell death and consequential neurodegenerative diseases, including Parkinson's disease (PD), Alzheimer's disease (AD) and Huntington's disease (HD) (Ben Mimouna et al., 2019; Mendez-Armenta and Rios, 2007; Okuda et al., 1997; Peng et al., 2017; Rigon et al., 2008; Williams et al., 2010).

Mitogen-activated protein kinases (MAPKs), a group of evolutionarily highly conserved cascade of dual (tyrosine and serine/threonine) protein kinases, can be activated by different extracellular stimuli such as cytokines, neurotransmitters, hormones and cellular stresses, and play a crucial role in signal transduction and regulate numerous cellular events such as cell growth and survival (Kyriakis and Avruch, 2012). MAPKs in mammalian cells possess at least three distinct groups, including the extracellular signal-regulated kinases (Erk1/2, Erk3/4, Erk5, and Erk7/8), the c-Jun N-terminal kinases (JNK1/2/3), and the p38 MAPKs (p38α/β/γ/δ) (Kyriakis and Avruch, 2012). It is well known that the phosphorylation of MAPKs is balanced by specific MAPK kinases and phosphatases (Kim and Choi, 2010; Kyriakis and Avruch, 2001). The protein phosphatase 5 (PP5) negatively regulates the JNK pathway involved in oxidative stress-induced apoptosis (Chen et al., 2008a, 2009; Han et al., 2012; Morita et al., 2001). AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis and cell survival in response to inflammation and oxidative stress (Chiang et al., 2018). Studies have documented that AMPK inactivation or activation due to oxidative stress is involved in neurodegenerative diseases (Chiang et al., 2018; Jiang et al., 2013; Vingtdeux et al., 2011). Our group has observed that Cd activates JNK pathway contributing to cell death by inducing ROS inactivation of PP5 in neuronal cells (Chen et al., 2008a; Xu et al., 2017). We have also demonstrated that Cd triggers ROS-dependent inactivation of AMPK leading to apoptosis of neuronal cells (Chen et al., 2011; Zhang et al., 2017). Hence, we postulated that a compound that can regulate ROS-dependent PP5-JNK and/or AMPK pathways might be useful to prevent Cd neurotoxicity.

Several studies have shown resveratrol, celastrol or nobiletin protection against Cd neurotoxicity by blocking ROS, involving AMPK or JNK pathways (Chen et al., 2014a; Qu et al., 2018; Shati, 2019; Zhang et al., 2017). Anti-diabetic drug metformin (N, N-dimethyl biguanide), a well-known AMPK activator, results in clear benefits in relation to glucose metabolism and diabetes mellitus-related complications (Barzilai et al., 2016; Lu et al., 2016; Singh et al., 2016). It is emerging that metformin may be also an effective drug to protect neurons from injuries and improve behavior in neurodegenerative diseases, such as memory function in a mouse model of Alzheimer's disease, the risk of Parkinson's disease in diabetes and MPTP-induced Parkinson's disease in mice (Patil et al., 2014; DiTacchio et al., 2015; El-Mir et al., 2008; Meng et al., 2016). Metformin has been demonstrated to exert protective effects against several aging-related diseases in humans by decreasing insulin levels, activating AMPK, inhibiting the mammalian target of rapamycin (mTOR), inhibiting mitochondrial complex 1 in the electron transport chain, reducing endogenous ROS production (Barzilai et al., 2016; Duca et al., 2015; Kickstein et al., 2010; Rotermund et al., 2018; Zheng et al., 2012). This prompted us to investigate whether and how metformin protects against Cd-induced neuronal apoptosis. Here, for the first time, we show that metformin ameliorates Cd-evoked neuronal apoptosis via blocking ROS-dependent PP5/AMPK-JNK signaling pathway. The results improve our understanding of the molecular mechanism by which metformin has a potential for the prevention of oxidative stress and neurodegeneration promoted by Cd toxicity.