Arsenic induces autophagy-dependent apoptosis via Akt inactivation and AMPK activation signaling pathways leading to neuronal cell death

Highlights

• As³⁺ induced a concomitant activation of apoptosis and autophagy in neuronal cell death.

• Akt inactivation-regulated autophagy was involved in As³⁺-induced neuronal apoptosis.

• AMPK activation played an important role in the As³⁺-triggered neuronal cell autophagy contributing to apoptosis.

Abstract

Inorganic arsenic (As³⁺), a well-known worldwide industrial and environmental pollutant, has been linked to neurodegenerative disorders (NDs). Autophagy plays an important role in controlling neuronal cell survival/death. However, limited information is available regarding the toxicological mechanism at the interplay between autophagy and As³⁺-induced neurotoxicity. The present study found that As³⁺ exposure induced a concomitant activation of apoptosis and autophagy in Neuro-2a cells, which was accompanied with the increase of phosphatidylserine exposure on outer membrane leaflets and apoptotic cell population, and the activation of caspase-3, -7, and PARP as well as the elevation of protein expressions of LC3-II, Atg-5, and Beclin-1, and the accumulation of autophagosome. Pretreatment of cells with autophagy inhibitor 3-MA, but not that of Z-VAD-FMK (a pan-caspase inhibitor), effectively prevented the As³⁺-induced autophagic and apoptotic responses, indicating that As³⁺-triggered autophagy was contributing to neuronal cell apoptosis. Furthermore, As³⁺ exposure evoked the dephosphorylation of Akt. Pretreatment with SC79, an Akt activator, could significantly attenuated As³⁺-induced Akt inactivation as well as autophagic and apoptotic events. Expectedly, inhibition of Akt signaling with LY294002 obviously enhanced As³⁺-triggered autophagy and apoptosis. Exposure to As³⁺ also dramatically increased the phosphorylation level of AMPKα. Pretreatment of AMPK inhibitor (Compound C) could markedly abrogate the As³⁺-induced phosphorylated AMPKα expression, and autophagy and apoptosis activation. Taken together, these results indicated that As³⁺ exerted its cytotoxicity in neuronal cells via the Akt inactivation/AMPK activation downstream-regulated autophagy-dependent apoptosis pathways, which ultimately lead to cell death. Our findings suggest that the regulation of Akt/AMPK signals may be a promising intervention to against As³⁺-induced neurotoxicity and NDs.

Introduction

Arsenic, the 33^rd element of the Periodic Table of chemical elements, is a toxic metalloid and ubiquitous in the environment, which exists in air, water and soil (Mandal and Suzuki, 2002). Chronic exposure to As is still a global health problem in human (Ratnaike, 2003). Arsenic possesses both organic and inorganic forms that the inorganic form of arsenic such as trivalent arsenic (As³⁺), which is predominant in surface and groundwater reservoirs, is more toxic than the organic form (Gupta and Chatterjee, 2017). As³⁺ contamination supplied by natural deposits or industrial pollution is being reported from various countries, including India, Bangladesh, Myanmar, Mexico, the United States, and others (Camacho et al., 2011; Mochizuki et al., 2019; Natasha et al., 2020). Unfortunately, As³⁺ exposure via contaminated food chain, particularly at a nonlethal level in drinking water consumed over a period of time, is the major source of exposure to arsenic in human, resulting in the manifestations of toxicity in almost all of systems of the body (Mitra et al., 2020). Epidemiological studies have indicated a higher correlation between As³⁺ exposure and the development of neurodegenerative disorders (NDs), which could detect the higher levels of As³⁺ in the plasma and cerebrospinal fluid in mental health burden, Alzheimer's disease (AD), and Parkinson's disease (PD), leading to the neurobehavioral impairments and neuronal cell degeneration and death (Gong and O’Bryant, 2010; Miguel et al., 2015; Mitra et al., 2020; Mochizuki et al., 2019). In the experimental in vivo models, As³⁺ caused the serious neurological and neurobehavioral disorders as well as the neuronal cell apoptosis (Lu et al., 2014; Prakash et al., 2016; Wang et al., 2015a,b,c; Yen et al., 2011). Studies of Pellacani and Costa (2018) and Zhang et al. (2016) have also reported that exposure to toxic metals (such as MeHg, Pb, Cu, Cd, and Mn) can induce autophagy and neurotoxicity, which are correlated with the development of NDs. A few studies have indicated that As³⁺ exposure can induce autophagy in developing mouse brain, accompanied with the disappearances of axons, irregular shrinkage of cells, and karyolysis, pyknosis, and loss of neurons (Manthari et al., 2018a; and 2018b). However, limited information is available regarding the toxicological mechanism at the interplay between autophagy and As³⁺-induced neurotoxicity/neuronal cell apoptosis.

Autophagy is also known as type II programmed cell death, which involves a sequential set of programs including double membrane formation, elongation, vesicle maturation and finally delivery of the targeted materials to the lysosome (Ghavami et al., 2014; Gump and Thorburn, 2011). Autophagy plays a crucial role in maintaining cellular homeostasis and physiological processes that protects cells from environmental or toxic stress-induced insults, such as aggregated and misfolded proteins and damaged organelles, which induces cellular stress, failure, and death (Doherty and Baehrecke, 2018; Schneider and Cuervo, 2014). Autophagy occurs in almost all cell types and is useful to maintain cellular homeostasis, allowing cellular differentiation, growth control, cell defense, tissue remodeling, and adaptation for adverse environments (Doherty and Baehrecke, 2018; Pellacani and Costa, 2018). Both pro-survival and pro-death roles of autophagy have been proposed in the pathophysiology in many human diseases, including cancer, metabolic dysfunction, and NDs (Choi et al., 2013; Das et al., 2012). Growing studies have implicated that disrupted/defected autophagy is one of the factors contributing to mammalian cell death (particularly in neuronal cells), pointing to autophagic dysfunction as a potential pathogenesis in NDs (Martinez-Vicente, 2015; Nixon and Yang, 2012; Schneider and Cuervo, 2014). For examples, the abundant autophagosome vesicles have been found in AD brains and autophagosome-like structures have been observed in the substantia nigra (SN) neurons of PD patients (Anglade et al., 1997; Boland et al., 2008). Recently, the alteration in autophagy process upon exposure to environmental pollutants/chemicals-induced neurotoxicity has been indicated to associate with neurodegeneration (Manthari et al., 2018b; Song et al., 2019).

Akt (protein kinase B), a serine/threonine protein kinase, plays an essential role in the regulation of multiple cellular functions including cell differentiation, proliferation, survival, and apoptosis (Franke et al., 2003). The regulation/dysregulation mechanism of Akt signaling is an important factor involved in several life-threatening diseases, including cancer, diabetes, and NDs (Xu et al., 2020). Studies have shown that Akt activity and Akt levels are decreased in the brains of AD and PD patients, linking Akt signal and NDs development (Anglade et al., 1997; Liu et al., 2011; Malagelada et al., 2008). On the other hand, adenosine monophosphate-activated protein kinase (AMPK), a highly conserved serine/threonine protein kinase, is considered to be an importantly intracellular sensor and regulator of metabolic homeostasis (Spasic et al., 2009). In neurons, AMPK plays a critical role in the energy and functional maintenance, survival, and apoptosis (Chen et al., 2010; Spasic et al., 2009). Over-expression of AMPK signaling has been detected in several brain diseases, including NDs, suggesting a correlation between AMPK activation and NDs (Domise and Vingtdeux, 2016). In ischemic brain injury, the AMPK activation-related neuronal cell death has been detected, which could be obviously attenuated by the inhibition of AMPK activity (Li et al., 2010; Nakatsu et al., 2008; Xu et al., 2014). The increasing studies have shown that the diminished Akt phosphorylation and/or the activation of AMPK signal contribute to neuronal cell apoptosis by exposure to chemicals (Chung et al., 2019; Eom et al., 2016; Xu et al., 2014). Furthermore, it has also been reported that Akt inhibits autophagy and AMPK stimulates apoptotic and autophagic signals under the pathological processes of cell damage (Maiese, 2016).

Even though both Akt and AMPK signals have been explored in chemicals-induced neuronal autophagic/apoptotic cell death (He et al., 2017; Zhao et al., 2019), the roles of both signals in As³⁺-induced neurotoxicity linked to autophagy/apoptosis pathway are still unclear. In this study, we aimed to investigate how As³⁺ influences both Akt and AMPK-regulated autophagy and apoptosis pathways contributing to neuronal cell death.