PFOS-induced excitotoxicity is dependent on Ca²⁺ influx via NMDA receptors in rat cerebellar granule neurons

Highlights

• PFOS induces rapid excitotoxicity in rat cerebellar granule neurons (CGNs) in vitro

• PFOS-induced excitotoxicity is dependent on calcium influx via the NMDA receptor

• Extracellular, but not intracellular calcium chelators protects against cell death

• PFOS, but not PFOA increases cytosolic calcium after short term (1 h) exposure

• PFOS toxicity is lower in PC12 cells lacking functional NMDA receptor expression

Abstract

Perfluoroalkyl acids (PFAAs) are persistent compounds used in many industrial as well as consumer products. Despite restrictions, these compounds are found at measurable concentrations in samples of human and animal origin. In the present study we examined whether the effects on cell viability of two sulfonated and four carboxylated PFAAs in cultures of cerebellar granule neurons (CGNs), could be associated with deleterious activation of the N-methyl-d-aspartate receptor (NMDA-R).

PFAA-induced effects on viability in rat CGNs and unstimulated PC12 cells were examined using the MTT assay. Cells from the PC12 rat pheochromocytoma cell line lack the expression of functional NMDA-Rs and were used to verify lower toxicity of perfluorooctanesulfonic acid (PFOS) in cells not expressing NMDA-Rs. Protective effects of NMDA-R antagonists, and extracellular as well as intracellular Ca²⁺ chelators were investigated. Cytosolic Ca²⁺ ([Ca²⁺]_i) was measured using Fura-2.

In rat CGNs the effects of the NMDA-R antagonists MK-801, memantine and CPP indicated involvement of the NMDA-R in the decreased viability induced by PFOS and perfluorohexanesulfonic acid (PFHxS). No effects were associated with the four carboxylated PFAAs studied. Further, EGTA and CPP protected against PFOS-induced decreases in cell viability, whereas no protection was afforded by BAPTA-AM. [Ca²⁺]_i significantly increased after exposure to PFOS, and this increase was completely blocked by MK-801. In PC12 cells a higher concentration of PFOS was required to induce equivalent levels of toxicity as compared to in rat CGNs. PFOS-induced toxicity in PC12 cells was not affected by CPP.

In conclusion, PFOS at the tested concentrations induces excitotoxicity in rat CGNs, which likely involves influx of extracellular Ca²⁺ via the NMDA-R. This effect can be blocked by specific NMDA-R antagonists.

Introduction

Perfluoroalkyl acids (PFAAs) including perfluoroalkyl sulfonated (PFSA) and carboxylated (PFCA) acids are found in measurable concentrations in samples from humans and wildlife, due to widespread use in industrial as well as consumer products since the 1950’s (Buck et al., 2011). In the industry they are used amongst others as surfactants, anti-reflective coatings for photolithography processes, etchants, lubricants and in ion-exchange processes (OECD, 2009), whereas in consumer products they may be found in paint and inks, in food contact papers, impregnation sprays, ski waxes and outdoor clothing (IVF Swerea, 2009; Kotthoff et al., 2015). They are slowly degradable (Haug et al., 2010; Holzer et al., 2009; Nilsson et al., 2010; Olsen et al., 2007), cross the blood brain barrier, and accumulate in the brain to a variable degree depending on their carbon chain length and functional group(s) (Ahrens et al., 2009; Eggers Pedersen et al., 2015; Greaves et al., 2013; Maestri et al., 2006). Exposure to PFAAs has been associated with neurobehavioural effects in laboratory animals including mice, rats and zebrafish such as decreases in spontaneous motor ability, deranged spontaneous behavior such as rearing and locomotion, and lack of habituation, as well as increased swimming speed in fish (Chen et al., 2013; Johansson et al., 2008; Yang et al., 2009). In humans a recent study suggested that prenatal exposure to the PFAAs perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) may be associated with a small to moderate effect on the neurobehavioural development of children and specifically an increase in hyperactivity (Hoyer et al., 2015), however more human studies are required due to large variability shown in published literature (Roth and Wilks, 2014).

Glutamate is considered the major excitatory neurotransmitter in the mammalian brain and spinal cord (Paoletti et al., 2013). Excitotoxicity refers to the process whereby neuronal death is induced by excessive rapid or prolonged activation of glutamate receptors (Fonnum and Lock, 2004), resulting in sustained elevation of free cytoplasmic Ca²⁺, which in turn release more glutamate into the affected area. Excitotoxicity is assumed to be involved in several pathological conditions such as ischemic brain injury (after stroke or trauma) and neurodegenerative disorders (e.g. Alzheimer's disease and Huntington's disease) (Feng et al., 2004; Marini and Paul, 1992; Xia et al., 1995). In addition to the metabotropic glutamate receptors, there are three types of ionotropic glutamate receptors, the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), kainate and N-methyl-d-aspartate (NMDA) receptors (Fonnum and Lock, 2004; Li et al., 2004), which contain central ligand gated ion-channels (Fonnum and Lock, 2004), all three which have been implicated in glutamate neurotoxicity (Marini and Paul, 1992). Of these the NMDA receptor (NMDA-R) is assumed to play a major role in the mediation of neuronal death in primary cultures of nerve cells (Marini and Paul, 1992).

The NMDA-Rs consist of several subunits forming heterotetrameric complexes (Feng et al., 2004; Tarnok et al., 2008), and contain a channel largely permeable to Ca²⁺ (Paoletti et al., 2013). Functional NMDA-Rs are formed by the combination of GluN1 and at least one GluN2 subunit, or GluN1 in combination with GluN2 and GluN3 subunits (Iacobucci and Popescu, 2018; Kew and Kemp, 2005). Similar to the cerebellum in vivo, the expression of subunits in cultures of cerebellar granule neurons (CGNs) in vitro changes with time, and maturation of the neuronal cultures (Cebers et al., 2001; Vallano et al., 1996). Due to these by others well documented and previously reported time-dependent changes in NMDA-R subunit expression, experiments in the present study were conducted in CGNs at various time-points, and neuronal cells exposed at days in vitro (DIV) 14 were assumed to express more mature receptors than cells exposed at DIV 0 or 8. This was however not verified by gene or protein analysis.

Granule cells are the most abundant neuronal cell type in the cerebellum (Gallo et al., 1982), they are easy to isolate, and to use in in vitro studies. They have been in use for several decades, and have been applied in many studies examining mechanisms of excitotoxicity. CGNs may be cultured to a high purity, express excitatory glutamate receptors, and produce and release L-glutamate (Kramer and Minichiello, 2010) and thus make, in addition to hippocampal and cortical cultures, a good model for the study of glutamatergic functions. Whereas the cerebellum earlier was assumed to be involved mainly in the planning and execution of movements, studies have shown that it is largely associated with cerebral networks involved in cognition (Buckner, 2013). Studies of children with malformations of the cerebellum find associations with neurophsychological deficits affecting executive functions, visuospatial and linguistic abilities, as well as with affective and social disorders, and autistic syndromes (Volpe, 2009). Exposure to certain POPs have been associated with an increased risk of autism spectrum disorders (Schmidt et al., 2014). Changes in the expression of ionotropic glutamate receptors have been observed in autism, and an upregulation of NMDA-R GluN1 subunit protein levels has been observed in post-mortem samples from the human cerebellum (Rojas, 2014). In animal models of autism overexpression of GluN2B has also been observed (Rojas, 2014). In the cerebellum the migration of granule cells from the external germinative layer to the internal granule cell layer is dependent on the NMDA-Rs, where the GluN2B subunits are especially important (Akazawa et al., 1994; Llansola et al., 2005; Tarnok et al., 2008; Watanabe et al., 1994).

Previous studies using inhibitors of the NMDA-R function have indicated an involvement of this receptor in death of cultured CGNs induced by halogenated hydrocarbons such as the polychlorinated biphenyls (PCBs) (Mariussen et al., 2002), as well as the brominated flame retardant, tetrabromobisphenol A (TBBPA) (Reistad et al., 2007). We have previously reported that viability in cultures of CGNs exposed to six different PFAAs (perfluorohexanesulfonic acid (PFHxS), PFOS, PFOA, perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA) and perfluoroundecanoic acid (PFUnDA)) is affected by carbon chain length and the functional groups attached to the perfluorinated molecular backbone (Berntsen et al., 2017). In the present study the initial aim was to investigate the possible involvement of the AMPA/kainate- and NMDA-Rs in PFAA-induced reductions in viability of CGNs using the same six compounds.

After the initial studies conducted with the six different PFAAs we chose to focus our remaining studies on PFOS. For comparative purposes we also included PFOA in some, but not all of the experiments. PFOS and PFOA both have 8 carbon atoms in the molecular backbone structure, but have different functional groups attached. Whereas PFOS has a sulfonate group, PFOA contains a carboxyl group (Fig. 1).

We have previously reported that CGNs exposed at DIV 8 to concentrations of PFOS and PFOA inducing equipotent effects after 24 h exposure, show very different time-curves for cytotoxicity induction. Whereas PFOS induced most of its toxicity within 1 h of exposure, PFOA did not induce significant effects on viability until after 12 h exposure (Berntsen et al., 2017), which may indicate different mechanisms of action. For the second part of the experiments the aim was to investigate how PFOS and PFOA affected the viability of CGNs at different stages of maturation. Further, time-dependent effects of intracellular and extracellular calcium chelation on PFOS- and PFOA-induced effects on viability were investigated. Finally, the effects of PFOS and PFOA exposure on cytosolic calcium concentration were assessed. Cells from the PC12 rat pheochromocytoma cell line reportedly lack the expression of functional NMDA-Rs (Edwards et al., 2007) and were exposed to concentrations within the same range as used in the CGN experiments, to verify lower toxicity of PFOS in cells not expressing NMDA-Rs.