Cytoskeletal Disruption as a Biomarker of Developmental Neurotoxicity

C H A P T E R

58 Cytoskeletal Disruption as a Biomarker of Developmental Neurotoxicity Alan J. Hargreaves1, Magdalini Sachana2, John Flaskos3 1

School of Science and Technology, Nottingham Trent University, Nottingham, United Kingdom; 2Organization for Economic Cooperation and Development (OECD), Paris, France; 3Laboratory of Biochemistry and Toxicology, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece

INTRODUCTION The eukaryotic cytoskeleton comprises a network of three interconnected protein filamentous arrays known as microtubules (MTs), microfilaments (MFs), and intermediate filaments (IFs). Many MT and MF arrays are dynamic structures that can undergo changes in organization, activity, and function at key stages in neural cell development. These phenomena are regulated by a variety of posttranslational modifications and interactions with a range of accessory proteins (Carlier, 1998; Joshi, 1998; Biernat et al., 2002; Ishikawa and Kohama, 2007; Akhmanova and Steinmetz, 2008; Janke and Kneussel, 2010; Svitkina, 2018). IFs, however, are relatively stable but may be modulated by cross-linking interactions with associated proteins or by their phosphorylation status (Herrmann and Aebi, 2000; Omary et al., 2006). The cytoskeleton is involved in the control of key cellular processes in nervous system development and maintenance, such as cell division, cell migration, cell differentiation, intracellular transport, and structural support. Its disruption by the interaction of neurotoxins with core proteins or cytoskeletal regulatory systems can therefore be detrimental to a wide range of phenomena including neural development (Hargreaves, 1997; Flaskos, 2014). There is a growing body of evidence for the induction of developmental neurotoxicity via disruption of cell signaling and cytoskeleton-dependent physiological processes by several groups of chemicals including organophosphorus esters (OPs), heavy metals, polybrominated diphenyl ethers (PBDEs), and solvents (Sachana et al., 2017; Pierozan et al., 2017). This chapter focuses on studies showing cytoskeletal disruption

Biomarkers in Toxicology, Second Edition https://doi.org/10.1016/B978-0-12-814655-2.00058-X

associated with impairment of neural development following exposure to such compounds, supporting the idea that cytoskeletal disruption can be a useful biomarker of developmental neurotoxicity.

MICROTUBULES The main component of the MT network is the heterodimeric protein tubulin, which is composed of a and b subunits that form head-to-tail protofilaments, which in turn come together to make the tubular structure with an external diameter of 25 nm (Amos, 2004). MT assembly requires GTP binding to tubulin and MT dynamics are dependent on GTP hydrolysis, which occurs shortly after subunit addition to the growing MT end (“plus” end) (Carlier et al., 1984). Both a- and b-tubulins have several isoforms encoded by different genes (Luduen˜a, 1998; Amos, 2004; Tischfield and Engle, 2010). Recent findings concerning congenital human neurological syndromes further emphasize the unique roles of specific a- and b-tubulin isoforms during nervous system development (Tischfield and Engle, 2010). For example, isotype III of b-tubulin (bIII-tubulin) appears almost exclusively in neuronal cells, plays an important role in neuritogenesis (Katsetos et al., 2003), and is among the earliest neuronal cytoskeletal proteins to be expressed during CNS development (Lee et al., 1990; Easter et al., 1993). Tubulin can be chemically modified by a variety of posttranslational adjustments, which may affect stability and location of MTs or act as a guidance cue for MT-binding proteins (Janke and Kneussel, 2010; Baas et al., 2016).

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Many proteins can potentially interact with MTs, including the most studied microtubule associated proteins (MAPs). In neurons, these interactions influence MT dynamics, and it is believed that they are necessary for neuronal migration, differentiation, and axon guidance (Wade, 2009; Penazzi et al., 2016). Phosphorylation events have been found to regulate the association of MAPs with MTs, suggesting their potential involvement in cascade events relevant to neuronal development and degeneration (Biernat et al., 2002; Baas and Qiang, 2005). An important member of the MAP family is the protein tau, which has tandem repeats of a tubulin-binding domain and contributes to tubulin assembly. Tau is abundant in neurons and is mainly located in axons, where it is closely associated with MTs. Changes in tau-protein levels and its phosphorylation state have been detected in numerous neurodegenerative diseases (Johnson and Stoothoff, 2004). Phosphorylation is also very important from a developmental point of view and is encountered extensively in fetal rather than adult tau (Watanabe et al., 1993). Indeed, increased phosphorylation of tau and dynamic MTs seem to coexist during brain development (Brion et al., 1994). It has also been shown that MAP-1B is an essential protein for the development and function of nervous system both in vitro (Brugg et al., 1993; Di Tella et al., 1996) and in vivo (Meixner et al., 2000; VillarroelCampos and Gonzalez-Billault, 2014). On the other hand, MAP1A dynamics are very closely associated to spine plasticity and any alterations in MAP1A may indicate changes in synaptic density (Jaworski et al., 2009). As a counter measure to MT-stabilizing MAPs, other groups of MAPs can bind to and destabilize MTs. An example is stathmin, which has been shown to regulate MT stability in the formation of dendritic branches in neuronal cells (Ohkawa et al., 2007). This low molecular weight protein can bind to and sequester tubulin heterodimers and hydrolyze GTP at the growing ends of MTs, leading to reduced MT stability (Howell et al., 1999). The key role of MTs in intracellular transport (e.g., along developing axons) is regulated by another group of MAPs, which act as ATPase motor proteins. Such MAPs include kinesin and dynein, which direct anterograde and retrograde transport along axonal MTs respectively (Vale, 2003), a process which is essential for neurite growth and development. The interaction of such proteins with MTs or their ATPase activities could potentially be affected under conditions where ATP levels are depleted. Several studies have dealt with the effects of established developmental neurotoxicants on MT assembly, organization, protein levels, posttranslational modifications, cell distribution, and gene expression. Altered status or intracellular distribution of MT proteins could reflect a range of adverse effects on the regulation of

neural development. As discussed in the following text, most of the available experimental evidence from cell culture studies suggests that several wellestablished developmental neurotoxicants cause alterations in MTs of neuronal and glial cells under culture conditions, a common finding being a reduction in the levels of MAPs.

Effects of Organophosphorus Esters on Microtubules The effects of several OP pesticides have been studied in the distant past, mainly in relation to posttranslational modifications of MT proteins and their role in organophosphate-induced delayed neuropathy (Abou-Donia, 1993, 1995). More recently, chlorpyrifos (CPF) and diazinon (DZ), the two OPs for which there are supporting data for developmental neurotoxicity, have been investigated regarding their potential effect on MTs both in vitro and in vivo. MAP-2 levels decreased following challenge with chlorpyrifos oxon (CPO) in organotypic slice cultures of immature rat hippocampus (Prendergast et al., 2007), whereas the parent compound CPF caused similar effects in the prefrontal cortex of Wistar rats (Ruiz-Mun˜oz et al., 2011) and in the mouse embryonic stem cells (mESCs) that were differentiated into neuronal cells (Visan et al., 2012). However, a-tubulin levels were not altered by 1e10 mM CPO exposure, suggesting that the general structure of MTs was not modified (Prendergast et al., 2007). Similar findings were reported in the case of N2a cells exposed to CPF at the time of the induction of cell differentiation, as well as 16 h after the induction of differentiation (Sachana et al., 2005). Interestingly, levels of total a-tubulin were also found unaltered in the case of DZ or diazoxon (DZO)-treated differentiating N2a cells (Flaskos et al., 2007; Harris et al., 2009a; Sidiropoulou et al., 2009a), whereas MAP-1B levels were reduced after 10 mM DZ exposure (Flaskos et al., 2007). No change in the levels of b-tubulin isotypes I and III were detected in differentiating N2a cells following exposure to DZ (Harris et al., 2009b). In contrast, in the same cell model, DZO induced a significant reduction in the levels of the bIII-tubulin isotype but had no effect on total b-tubulin levels, suggesting a neuron-specific effect (Sachana et al., 2014) In animal models, oral administration of CPF on postnatal days 1e6 did not affect the expression of the genes coding for the neuronal-specific marker bIII-tubulin (Betancourt et al., 2006). The polymerization of bovine brain tubulin was inhibited by low concentrations of CPO (0.1e10 mM) (Prendergast et al., 2007). The ability of tubulin to polymerize is very important because, apart from contributing toward the maintenance of neuronal

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MICROTUBULES

morphology, MTs also support axonal transport of mitochondria, components of ion channels, receptors, and scaffolding proteins. Perturbation of assembly and transport mechanisms in neurons due to CPF and CPO reaction with tubulin and its organophosphorylation has been suggested from several experimental works both in vivo (Terry et al., 2007; Jiang et al., 2010) and in vitro (Gearhart et al., 2007; Grigoryan et al., 2009b; Grigoryan and Lockridge, 2009). In glial cells, 24 h exposure to CPF or CPO suppresses extension outgrowth in differentiating C6 cells (Sachana et al., 2008). CPO had a stronger morphological effect than CPF that has been associated with a significant decrease in the levels of tubulin and MAP1B (Sachana et al., 2008). Similarly, only the in vivo metabolite of DZ, DZO, triggered inhibition of the development of C6 cell extensions, an effect linked also to the reduction in the levels of tubulin (Sidiropoulou et al., 2009b). Immunofluorescence staining revealed normal MT networks in control and CPFtreated cells and, although there was no evidence for a major collapse of the MT network, there were increased levels of localized patchy staining compared to the control, particularly in CPO-treated cells (Sachana et al., 2008).

Effects of Heavy Metals on Microtubules MT proteins have also been investigated in relation to methylmercuryeinduced developmental abnormalities of the nervous system. Exposure of N2a neuroblastoma cells to methylmercury revealed significant disruption in MT organization after staining the cells with antibody that recognizes b-tubulin (Kromidas et al., 1990). The same research group further emphasized the predominant effect of methylmercury on MTs compared to IFs and MFs by using scanning electron microscopy (Trombetta and Kromidas, 1992). In the same cell line, cells demonstrated decreased reactivity against C-terminally tyrosinated a-tubulin following only 4 h exposure to a sublethal concentration of methylmercury chloride compared to controls, which was associated with inhibition of neurite outgrowth (Lawton et al., 2007). The importance of the cytoskeleton in methylmercury neurotoxicity was further emphasized in a study by Castoldi et al. (2000) using primary cultures. In this study, rat cerebellar granule cells exhibited MT depolymerization within 1.5 h of exposure to 1 mM methylmercury and long before disturbance of neurite processes (Castoldi et al., 2000). Methylmercury was found to suppress tubulin polymerization in vitro, to disrupt the MT network, and to reduce tubulin synthesis in mouse glioma cells (Miura et al., 1984; Miura and Imura, 1987). This corresponded

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to a decrease in tubulin mRNA levels but no effect on the transcription rates of b-tubulin genes were found, suggesting that exposure had disrupted the autoregulatory control of tubulin synthesis in a manner similar to that described for the antimitotic agent colchicine (Miura et al., 1998). It is well documented that lead exposure can cause learning and memory impairment, particularly in developing organisms. However, the molecular mechanisms are not fully understood, although several studies investigated the potential role of MTs in disruption of memory formation. In human primary cultures, exposure to biologically relevant concentrations of lead (5, 10, 20, and 40 mg/dL) was associated with hyperphosphorylation of tau protein, as determined by Western blotting and immunocytochemistry, due to upregulation of protein phosphatases (Rahman et al., 2011). These findings were also confirmed in both Wistar rat and mouse pups, emphasizing the importance of tau hyperphosphorylation in cognitive impairment (Li et al., 2010; Rahman et al., 2012). Recent data further support this as the pre- and neonatal exposure to lead causes a significant increase in the phosphorylation of tau and upregulates tau protein level in the rat brain cortex and cerebellum (Gąssowska et al., 2016). In a study by Scortegagna et al. (1998), the detectable levels of MAP-2b and MAP-2c were found to decrease 24 h after a 3 h exposure to 3 or 6 mM lead in serumfree medium maintained E14 mesencephalic rat primary cultures. However, in the same study, these protein levels remained similar to controls in serum-cultured cells, suggesting that a serum factor prevents cytoskeletal changes otherwise noted in this primary culture containing differentiating neurons and proliferating astrocytes (Scortegagna et al., 1998). On the other hand, lead (II) acetate reduced the number of MAP-2 stained cells and the mRNA levels of MAP-2 in a concentration-dependent manner, by applying default differentiation of mESCs (Beak et al., 2011; Visan et al., 2012). Inorganic lead had no effect on the in vitro assembly of MTs from porcine brain, whereas trimethyl lead blocked and completely inhibited MT assembly at 300 mM, as monitored by turbidity measurements and electron microscopy (Roderer and Doenges, 1983). In contrast, triethyl lead chloride inhibited MT assembly and depolymerized preformed MTs in porcine brain preparations (Zimmermann et al., 1988), whereas the same research group reported no change in MT network in mouse N2a neuroblastoma cells after exposure to the same organic lead compound (Zimmermann et al., 1987). Regarding arsenic, it was shown that tau gene expression can be increased in ST-8814 schwannoma and SK-N-SH neuroblastoma cell lines by exposure to inorganic trivalent and monomethyl pentavalent

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arsenic metabolites, respectively (Vahidnia et al., 2007b). In addition, the phosphorylated state of MAPtau has been found altered in both in vitro and in vivo studies (Vahidnia et al., 2007a). More specifically, Giasson et al. (2002) reported hyperphosphorylation of tauprotein in Chinese hamster ovary cells after treatment with inorganic trivalent arsenic. Similarly, subchronic exposure of rats to this arsenic metabolite increased the phosphorylation of tau in sciatic nerves (Vahidnia et al., 2008a). However, a more recent study shows that sodium arsenite causes neurite inhibition in N2a cells that was associated with alterations in cytoskeletal proteins. More specifically, sodium arsenite decreased the mRNA levels of tau and tubulin in a dosedependent manner but had no significant effect on the mRNA levels of MAP-2 (Aung et al., 2013). The inhibitory effect of arsenic trioxide on the migration of primary neurons established from the brains of neonatal rats has also been recently investigated; the study revealed that it was associated with a decrease in the protein levels of doublecortin, which is a MTassociated protein expressed during the neuronal migration (Zhou et al., 2015).

Effects of Organic Solvents and Polybrominated Diphenyl Ethers on Microtubules Ethanol is the only organic solvent studied in relation to its neurotoxic potential against MTs in in vitro models. Ethanol had no effect on the rate and extent of bovine tubulin polymerization in vitro, whereas the ethanol metabolite acetaldehyde inhibited MT formation (Jennett et al., 1980). Similarly, McKinnon et al. (1987) demonstrated that acetaldehyde had an inhibitory effect on the polymerization of MT protein derived from calf brains, further emphasizing the acetaldehydemediated alteration of cytoskeletal MTs. Continuous exposure of developing neural crest cells to ethanol has also been found to cause MT disruption (Hassler and Moran, 1986). In PC12 cells, chronic exposure to ethanol led to alterations in the balance between free tubulin in the cytoplasm and tubulin polymerized into MTs, enhancing the content of the latter, possibly through phosphorylation (Reiter-Funk and Dohrman, 2005). In contrast, chronic alcohol exposure was found to decrease the levels of polymerized tubulin in cultured hippocampal neurons and simultaneously to reduce the amount of MTs and the levels of MAP-2 in dendrites (Romero et al., 2010). The same research group also described impairment of MT dynamics and reassembly in primary cultures of rat astrocytes treated with ethanol (Tomas et al., 2003). However, in a short-term exposure of rat C6 glioma cells to acute levels of ethanol (50, 100, and

200 mM), there was no detectable change in the MT network (Loureiro et al., 2011). In whole cerebral hemisphere model, developing Layer 6 neurons exposed to ethanol exhibited diminished MAP-2 levels in dendritic processes (Powrozek and Olson, 2012). Similarly, MAP-2 immunostaining intensity was reduced in hippocampal slice cultures from neonatal Wistar rats subjected to ethanol treatment (200 mM) for up to 4 weeks (Noraberg and Zimmer, 1998). MAP-2 immunolabeling has also been used to determine the effect of ethanol (70 mM) on dendrites of rat embryonic hippocampal pyramidal neurons in culture, revealing decreases in both length and number of dendrites compared with controls (Lindsley et al., 2002; Lindsley and Clarke, 2004). Stimulation of MAP-2 phosphorylation has been detected in MT preparations from rat brain exposed to low and biologically relevant doses of ethanol (6, 12, and 24 mM), whereas higher doses (48, 96, 384, and 768 mM) decreased phosphorylation (Ahluwalia et al., 2000). In the same study, MAP-1 was found to show increased phosphorylation with only 12 and 24 mM of ethanol and tubulin only from the lower dose tested (6 mM) (Ahluwalia et al., 2000). More recently, the application of neural stem cell technology indicated disturbance of neuronal differentiation from noncytotoxic concentrations of alcohol (25e100 mM), as recorded by reduced immunostaining and levels of MAP-2 protein (Tateno et al., 2005). Immunolabeling of axons derived from rat hippocampal pyramidal neurons with a bIII-tubulin antibody was used to assess the effect of ethanol on the length of axons (VanDemark et al., 2009). Indeed, ethanol alone had no effect on axon length, whereas carbachol-treated cells in the presence of ethanol (50 and 75 mM) did cause shortening of axons compared to controls (VanDemark et al., 2009). In the P7 rodent model, which is extensively used for mechanistic elucidation of ethanol-induced neurodevelopmental toxicity, it was found that ethanol elevated the phosphorylation state of tau, as demonstrated by two different phospho-specific antibodies (Saito et al., 2010). Similarly, in a human neuroblastoma cell line after tau induction, ethanol caused a dose-dependent increase in tau levels and cell mortality (Gendron et al., 2008). A more extensive overview on the effects of ethanol on the neuronal cytoskeleton, covering both in vivo and in vitro studies, can be found in Evrard and Brusco (2011).

MICROFILAMENTS MFs are formed by the polymerization of monomeric G-actin into filaments with a diameter of 5 nm (Dominguez and Holmes, 2011). MF assembly is

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MICROFILAMENTS

ATP-dependent, and the dynamic properties of MFs are dependent on the hydrolysis of actin-bound ATP following incorporation at the filament plus end (Carlier, 1998). MF dynamics are also influenced by a wide variety of actin binding proteins (ABPs) that regulate its organization and function by acting as either nucleating factors (e.g., ARP 2/3, formin), crosslinking proteins (e.g., fascin), destabilizing factors (e.g., ADF/cofilin, gelsolin, fragmin), membrane cross-linkers (e.g., spectrin, GAP-43), or MFassociated motor proteins (e.g., myosin) (Ishikawa and Kohama, 2007; Dominguez, 2009; Lee and Dominguez, 2010; Dominguez and Holmes, 2011; Jansen et al., 2011; Svitkina, 2018). The interaction between actin and its ABPs is in turn regulated by cell signaling pathways (Endo et al., 2003; Ishikawa and Kohama, 2007). MFs play key roles in mitosis, neural cell differentiation, and the regulation of cell migration (Gungabissoon and Bamburg, 2003; Kunda and Baum, 2009). The inhibition of neurite outgrowth from explants of embryonic chick spinal cord cultured in the presence of the MT-stabilizing agent taxol and the MF-disrupting agent cytochalasin D clearly demonstrates the importance of both MT and MF integrity in the developmentally important process of neurite outgrowth (Ro¨sner and Vacun, 1997). Given the important roles played by MFs in cytokinesis, receptor trafficking, and neurite outgrowth, their disruption by toxin exposure could have major effects on neural development.

Effects of Organophosphorus Esters on Microfilaments In early work, Carlson and Ehrich (2001) tested the ability of several OPs including paraoxon, parathion, diisopropyl fluorophosphate (DFP), phenyl saligenin phosphate (PSP), triorthotolyl phosphate (TOTP), and triphenyl phosphite at 0.1e1 mM to disrupt the filamentous actin (F-actin) network in mitotic SH-SY5Y cells using a fluorescently labeled phalloidin probe. Significant decreases in the levels of F-actin were observed within 30 min exposure of cells treated with PSP and TOTP, whereas other OPs required longer exposure times and DFP had no observable effect. The data clearly support the idea that some OPs can disrupt the MF network, although the concentrations used were relatively high and cytotoxic within a few hours, as determined by protein assay (Carlson and Ehrich, 2001). However, proteomic studies on differentiating N2a neuroblastoma cells exposed to sublethal neurite inhibitory concentrations of the OP pesticide DZ showed that inhibition of neurite outgrowth by 10 mM DZ was associated with increased levels but decreased LIM kinaseemediated

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phosphorylation of the actin-destabilizing protein cofilin (Harris et al., 2009a). Although total actin levels were unaffected by DZ, the altered levels and phosphorylation status of cofilin, together with reduced staining intensity of neurites with antiactin antibody, suggest that DZ exposure leads to a reduction in the levels of F-actin in neurites due to disruption of MF dynamics caused by altered expression and phosphorylation of cofilin. Another important modulator of MF organization in the axonal growth cone of developing neurons is growth associated protein 43 (GAP43), which has been shown to exhibit reduced mRNA and/or protein levels following exposure to sublethal neurite outgrowth inhibitory concentrations of OPs (Sachana et al., 2003; Flaskos et al., 2011; Ta et al., 2014). A transient reduction in GAP43 protein levels was also observed to be associated with the retraction of neurites in predifferentiated N2a cells exposed to sublethal concentrations of CPF and CPO (Sindi et al., 2016). It has been suggested that these phenomena may, at least in part, be the result of upstream events such as OP-induced autophagy or disruption of Ca2þ homeostasis, which could impact on the activity of Ca2þ-dependent ABPs and other MF-regulatory proteins (Chen et al., 2013; Fernandes et al., 2017). Thus, OPs can disrupt MF organization and functions by interacting with ABPs and/or signaling pathways that modulate MF dynamics. However, in vitro studies by Grigoryan et al. (2009a) and Schopfer et al. (2010) demonstrated a covalent interaction of OPs with lysine and tyrosine residues on actin, raising the possibility that a direct interaction with actin might also be involved in the disruption of MFs by OPs. Further in vivo and in vitro studies to determine the role of OP-actin adduct formation would be worthwhile.

Effects of Heavy Metals on Microfilaments Many cytoskeleton-related developmental neurotoxicity studies with heavy metals have focused on the MT network. However, micromolar concentrations of mercurial compounds can block SH groups on purified actin, thereby inhibiting its ability to interact with myosin and induce myosin ATPase activity in vitro (Perry and Cotterill, 1964; Martinez-Neira et al., 2005). The ability of mercury (as well as cadmium, copper, and zinc) ions to interfere with actineABP interactions has also been demonstrated by native gel electrophoresis (Kekic and dos Remedios, 1999), suggesting that direct binding of heavy metal ions to actin, ABPs, or other MF-regulatory proteins could induce toxic effects directed at the MF network. Studies on the inhibition of glioma cell migration by arsenic suggested that this heavy metal compound may be able to disrupt F-actin by interference with

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MF-regulatory cell signaling pathways such as protein kinase C (Lin et al., 2008). Sodium arsenite was also observed to inhibit neurite outgrowth in differentiating N2a neuroblastoma cells; although this was not found to involve alterations in mRNA levels for b-actin (Aung et al., 2013), the possibility that changes to MF dynamics may involve alterations to the levels and posttranslational modification of ABPs or other regulatory pathways cannot be ruled out and warrants further investigation. Changes were observed in the levels of mRNA for NFL and NFM (both elevated) and for tubulin and tau (both reduced) in arsenic-treated cells; the possibility that these changes, if they are reflected at the protein level, may cause disruption of F-actin organization could also be further investigated. In a study of the effects of methylmercury chloride on postnatal rat brain development, it was found that the degradation of the ABP a-spectrin by m-calpain was significantly greater in cerebral cortex extracts from treated animals than in controls at postnatal day 16 (Zhang et al., 2003). Calpain activation could also target a range of other cytoskeletal proteins that act as substrates for calpain. In this respect it is interesting to note that exposure to other metal compounds has also been linked to the activation of calpain in brain (Zhang et al., 2012), neural cells (Vahidnia et al., 2008b; Rocha et al., 2011), or in other tissues (Lee et al., 2007). Furthermore, studies in primary cultures of fetal mouse brainederived cerebellar granule cells and human placental tissue showed that submicromolar levels of methylmercury chloride induced a significant decrease in the phosphorylation of the ABP cofilin and the translocation of actin and cofilin to mitochondria (Vendrell et al., 2010; Caballero et al., 2017). The fact that these changes were associated with elevated levels of protein carbonylation (detected by immunoassay) and could be blocked by cotreatment with the antioxidant probucol, suggest that they were triggered by elevated levels of protein oxidation (Caballero et al., 2017). Moreover, evidence for the inhibition of both neurite outgrowth and neuronal cell migration via disruption of MFs through altered regulatory signaling pathways comes from a combination of cell culture and in vivo developing rodent brain studies following exposure to methylmercury chloride (Fujimura and Usuki, 2012; Usuki and Fujimura, 2012; Guo et al., 2013; Herna´ndez et al., 2018). Thus, direct binding to SH groups, elevated protein oxidation, and disruption of MF-regulatory signaling pathways have a major impact on the regulation of MF dynamics, suggesting that exposure to heavy metal compounds can disrupt the regulation of MF dynamics in a variety of ways.

Effects of Organic Solvents and Polybrominated Diphenyl Ethers on Microfilaments Chronic ethanol exposure has been shown to be associated with disruption of the MF network in cultured PC12 cells and in primary cultures of hippocampal neurons. In PC12 cells, chronic exposure was found to reduce dopamine release via protein kinase Ce dependent pathways, an effect that was attenuated by cotreatment with the MF-disrupting agent cytochalasin, indicating the need for MFs in this exocytotic process (Funk and Dohrman, 2007). In ethanol-exposed hippocampal neurons, a reduction was observed in the levels of F-actin compared to untreated control cell cultures, as determined by FITC-labeled phalloidin staining, which corresponded to reduced protein levels of total Rac1, RhoA, and cdc42 (small GTPases known to be involved in the regulation of MF assembly and dynamics), as determined by G-LISA assays (Romero et al., 2010). However, although the levels of activated (GTPbound) forms of Rac1 and cdc42 were reduced, ethanol had no significant effect on the levels of active RhoA, suggesting that only the inactive form of this GTPase was downregulated by ethanol treatment. Western blotting analysis indicated that the levels of total cofilin (which destabilizes MFs) were unchanged, although the levels of inactive (phospho-) cofilin were not assessed (Romero et al., 2010). Chronic exposure to ethanol was also found to inhibit endocytotic uptake of serum albumin and transferrin by cultured fetal rat hippocampal neurons (Marı´n et al., 2010) This effect was associated not only with altered levels of proteins involved in vesicle formation and docking but also with proteins involved in the regulation of MF assembly and dynamics, including Arf6 (upregulated), cdc42, and RhoA (both downregulated). Furthermore, the ability of ethanol to inhibit neural cell differentiation in a human embryonic stem cell model of early brain development was also associated with disruption of the MF network (Tale´ns-Visconti et al., 2011). In a study of chronic ethanol exposure on cultured rat astrocytes, the impairment of glucose uptake was associated with disassembly of actin stress fibers; the fact that lysophosphatidic acid attenuated these effects by stabilizing the MF network suggested that a major disruption of MF dynamics was caused by ethanol exposure (Tomas et al., 2003). Although there was no detectable change in the MT network following short-term exposure of rat C6 glioma cells to acute levels of ethanol, the resultant formation of reactive oxygen species was associated with major disruption of the MF network (Loureiro et al., 2011). Taken together, these data illustrate not only the fact that MFs are disrupted by this

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INTERMEDIATE FILAMENTS

solvent but also that MF-dependent processes of importance in neural development are impaired when this occurs.

INTERMEDIATE FILAMENTS The most abundant IF proteins found in differentiating and mature neurons are the three neurofilament (NF) triplet proteins (NF-L, NF-M, and NF-H), whereas in glial cells the most important IF protein is GFAP. Nestin is the main IF specifically expressed in immature neural cells. Indeed, most of the genes coding for IF proteins are expressed in a tissue- or cell typeespecific manner, except for the nuclear lamins. Thus, the NF proteins are present only in neurons and GFAP only in glia (and more specifically in astrocytes). As a result, NF proteins and GFAP have been widely used in neurotoxicology as markers for specific effects on neurons and glia, respectively. In the context of developmental toxicology, changes in NF proteins and GFAP have been commonly employed as a measure of the capacity of a toxicant to interfere with neuronal and glial differentiation. Although NFs are typically considered to be more stable than MFs and MTs, their dynamic capability is demonstrated by their reorganization that occurs during several neurodevelopmental stages including proliferation (mitosis), apoptosis, and axonogenesis (Omary et al., 2006). NFs can facilitate axonal elongation by stabilizing the axonal cytoskeleton and inhibiting the retraction of long axons (Lariviere and Julien, 2003). Apart from their use in developmental neurotoxicity studies as a neuronal differentiation marker, NF proteins may constitute a mechanistically relevant marker for assessing specific biochemical cytoskeletal effects in some cases. This is particularly true for the studies on OPs and arsenic discussed in the following paragraphs, where NFs have been proposed to be a direct target for these neurotoxicants. NF parameters assessed in neurodevelopmental toxicity studies include NF protein levels, distribution, assembly, transport, phosphorylation, and expression of NF genes. A review of the available data on established developmental neurotoxicants (see the following) indicates that in most cases there is a decrease in the levels of at least one of the three NF proteins, whereas NF gene expression data are inconsistent, with both increases and decreases in NF mRNA levels caused by the toxicants. The distinct features of the three NF proteins in terms of structure, properties, and function and their differential expression during neuronal development imply that assessment of toxicant-induced changes in one NF protein cannot substitute for measurements in the other two. Finally, because NF protein phosphorylation is known to be the major factor in the

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regulation of the dynamics and function of NFs (Omary et al., 2006; Sihag et al., 2007), assessment of changes in NF phosphorylation-related parameters (determination of relevant kinases and upstream cell signaling molecules) may provide valuable markers for developmental neurotoxicity. In developmental neurotoxicity studies both protein and mRNA levels of GFAP have been assessed. These studies have usually reported decreases in these parameters, indicating specific repression of glial cell differentiation, whereas any increases obtained have been attributed to reactive gliosis because of high dosing and primary damage to neurons.

Effects of Organophosphorus Esters on Intermediate Filaments NF parameters have been assessed to a larger extent in toxicological studies involving OP pesticides than other developmental neurotoxicants. This may be partly because of the prior existence of data implicating NF (and other cytoskeletal) abnormalities as being etiologically important in delayed OP neurotoxicity (Abou-Donia, 1993; Jiang et al., 2010). In this context, in some OP neurodevelopmental studies NF parameters have not been employed as a common marker for neuronal cell-specific differentiation but as a mechanistically relevant marker for specific biochemical effects on the neuronal cytoskeleton. Parameters assessed in OP neurodevelopmental studies include the levels and intracellular distribution and posttranslational modification (phosphorylation) of NF proteins and the expression of NF genes. Exposure of both mitotic and differentiating rat PC12 pheochromocytoma cells to a sub-lethal concentration of CPF (30 mM), led to the upregulation of nfl and nef3 genes (which encode NF-L and NF-M, respectively), whereas expression of the nefh gene (which encodes NF-H) was unaffected (Slotkin and Seidler, 2009). On the other hand, exposure of PC12 cells to 30 mM DZ under the same conditions caused upregulation of the nef3 but had no effect on the expression of nfl and nefh genes (Slotkin and Seidler, 2009). The expression of genes encoding NF-L and NF-H, used as a marker for neuronal differentiation, was studied in primary neuronal cultures of cerebellar granule cells prepared from 7-dayold rat pups treated with parathion (Bal-Price et al., 2010). Exposure to this OP, used at concentrations of 10e50 mM, for up to 12 days caused a concentrationdependent decrease in mRNA levels for both NF-L and NF-H. A series of studies have assessed changes in several NF parameters following exposure of differentiating mouse N2a neuroblastoma cells to CPF and DZ, the two OPs for which there is the most evidence for

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developmental neurotoxicity. In these mechanistic studies of OP-induced developmental neurotoxicity, determination of NF (and other cytoskeletal) parameters were extended to include the influence of CPO and DZO, the two in vivo oxon metabolites of CPF and DZ, because there is now considerable evidence to suggest that these compounds can interfere with neuronal differentiation and development (Flaskos, 2012). These studies have demonstrated decreases in the levels of NF-H protein by both CPF and DZ. Thus, exposure of N2a cells to 3 mM CPF for 8 h (Sachana et al., 2001) and to 10 mM DZ for 24 h (Flaskos et al., 2007) leads to reduced NF-H protein levels. Indirect immunofluorescence studies also showed that, apart from an alteration in the expression of NF-H, there was a change in the intracellular distribution of this protein, with NF-H located mainly in cell body aggregates of DZ-treated N2a cells (Flaskos et al., 2007). NF parameters were also affected by exposure to CPO and DZO. For example, at a concentration of 10 mM, CPO exposure for 24 h reduced the total levels of NFH and disrupted NF-H intracellular distribution in differentiating N2a cells (Flaskos et al., 2011). On the other hand, CPO had no effect on the levels of phosphorylated NF-H under these experimental conditions, in which the OP was added at the point of induction of cell differentiation. However, in N2a cells induced to differentiate for 20 h prior to OP exposure, CPO induced a transient increase in ERK 1/2 activation and NF-H phosphorylation after 2 h, which preceded neurite retraction (Sindi et al., 2016), suggesting a mechanistic link between NF-H hyperphosphorylation and neurite destabilization. In contrast, the oxon metabolite DZO, applied at concentrations of 5 and 10 mM for 24 h, had no effect on total NF-H levels, but increased phosphorylated NF-H levels in differentiating N2a cells compared to the control (Sidiropoulou et al., 2009a). Under these conditions, DZO had no effect on the total levels of NF-L and NF-M (Sachana et al., 2014). Further work is required to determine the molecular basis of these effects in more detail. Determination of GFAP in neurodevelopmental studies of OPs has involved measurement of both its protein and mRNA levels. Following developmental exposure to OPs, GFAP protein and mRNA levels exhibited both decreases and increases, the latter being attributed to the occurrence of reactive gliosis because of high dosing. Thus, in aggregating brain cells of fetal rat telencephalon, GFAP levels were found to be increased following parathion treatment, indicative of gliosis (Zurich et al., 2000). Postnatal exposure of rats to CPF for 4 days initially decreased GFAP levels, indicating specific repression of normal glial (astrocytic) development. At a later stage, however, increases in the levels of GFAP occurred, typical of reactive gliosis

following neuronal cell damage (Garcia et al., 2002). Postnatal administration of CPF to rats for 6 days at doses high enough to cause significant cholinesterase inhibition also led to increased GFAP mRNA levels, reflecting increased astrocyte reactivity (Betancourt et al., 2006). On the other hand, administration of DZ to neonatal rats for 4 days resulted in a decrease in the expression of the gene coding for GFAP (Slotkin and Seidler, 2007). Similarly, the in vivo metabolite of DZ, DZO (at 1e10 mM) caused, after 24 h in N2a cells, a reduction in GFAP protein levels, indicating repression of specific glial cell/astrocytic differentiation (Sidiropoulou et al., 2009b).

Effects of Heavy Metals on Intermediate Filaments Both NF-L and NF-M levels were found to be altered in a number of mammalian cell lines after exposure to mercuric oxide. Thus, exposure of differentiating human SK-N-SH neuroblastoma cells for 6 days to mercuric oxide decreased particularly NF-L levels (Abdulla et al., 1995). Because this effect correlated well with effects on neurite outgrowth, it was suggested that determination of NF-L might afford a rapid measure of effects on neuronal differentiation. More recently, methylmercury exposure was also found to decrease the mRNA levels for NF-L and NF-H in primary cultures of rat cerebellar granule cells, whereas GFAP mRNA expression was unaffected (Hogberg et al., 2010). NF organization was disrupted following exposure of N2a cells to triethyl lead (Zimmermann et al., 1987). NF assembly was also disrupted. These effects led to suggestions that interaction of triethyl lead with NFs may be responsible for triethyl lead neurotoxicity in vivo. In addition, in rats exposed for 13 weeks to lead acetate, transport of NF proteins was retarded, indicating impairment of slow axonal transport (Yokoyama and Araki, 1992). Apart from NF organization, assembly, and transport, NF protein phosphorylation was also affected by lead; exposure of mice to lead acetate throughout gestation and postnatally led to increased phosphorylation of both NF-M and NF-H in auditory brainstem nuclei (Jones et al., 2008). Lead also affects GFAP, as suggested by decreased GFAP expression in four human and two rat glioma cell lines, indicating interference with glial cell differentiation (Stark et al., 1992). The mRNA levels for GFAP were also reduced after lead treatment of primary cultures of rat cerebellar granule cells for 12 days (Bal-Price et al., 2010). In neurotoxicity studies involving arsenic, NF parameters constitute a mechanistically relevant marker because NFs (and other cytoskeletal elements) have been suggested to represent a possible target in arsenic neuropathy. Although these studies have been not

IX. SPECIAL TOPICS

CONCLUDING REMARKS AND FUTURE DIRECTIONS

always carried out in a developmental context, the known significance of NFs in neurodevelopment implies that any neurotoxic effects on NFs obtained in adult animals or differentiated neurons may be of potential relevance in neurodevelopment. Both acute (Vahidnia et al., 2006) and subchronic (Vahidnia et al., 2008a) administration of arsenite to adult rats induced a reduction in NF-L levels in the sciatic nerve. In contrast, NF-M and NF-H expressions remained unchanged. In addition, NF-L was found to be hyperphosphorylated (Vahidnia et al., 2008a). These NF-L changes have been proposed to contribute to the disruption of the NF network, ultimately leading (in combination with other cytoskeletal changes) to the axonal degeneration seen in arsenic neuropathy (Vahidnia et al., 2007a, 2008a). However, in a study by the same group involving cell lines and assessment of NF gene expression, the results obtained were not compatible with the above data. In this case, exposure of SK-N-SH neuroblastoma and ST-8814 schwannoma cells to arsenite for up to 48 h had no effect on the expression of genes coding for NF-L and NF-M (Vahidnia et al., 2007b). However, the metabolites monomethyl- and dimethyl-arsenic induced alterations in the expression of NF genes in both cell lines and particularly in the expression of the gene coding for NF-H. Mechanistically important NF parameters assessed under the influence of arsenite also included axonal transport and phosphorylated NF distribution. For example, exposure of differentiated mouse NB2/dl neuroblastoma cells and dorsal root ganglion neurons cultured from embryonic day 12 chicks to arsenite decreased NF transport into axons and caused accumulation of phosphorylated NFs in the perikaryon leading to changes in NF dynamics that may contribute to arsenic neuropathy (De Furia and Shea, 2007).

Effects of Organic Solvents and Polybrominated Diphenyl Ethers on Intermediate Filaments Ethanol exposure inhibited the expression of NF proteins in N2a cells, indicating disruption of neuronal differentiation (Chen et al., 2009). In contrast, at a concentration of 100 mM, ethanol induced an increase in GFAP levels in differentiating neural stem cells, which was thought to imply increased glial differentiation as a compensatory mechanism to repair the impaired neuronal differentiation (Tateno et al., 2005). However, exposure to environmentally relevant low levels of toluene (down to 0.2 ppb), decreased GFAP levels during differentiation of mouse embryo cells into an astrocytic lineage in serum-free medium (Yamaguchi et al., 2002, 2003).

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In a study that adopted a proteomic approach, exposure of neonatal mice to 2,20 ,4,40 ,5-pentabromodiphenyl ether (PBDE 99) induced significant alterations in several cytoskeletal and other proteins in the cerebral cortex. However, one of the greatest changes noted was an increase in the levels of NF-L (Alm et al., 2008).

CONCLUDING REMARKS AND FUTURE DIRECTIONS It is well established that the cytoskeleton plays a key role in a range of cellular processes involved in neural development. From the mechanistic studies of developmental neurotoxicity to date, there is now a significant body of evidence pointing to the disruption of one or more of the cytoskeletal networks following exposure to a range of developmental neurotoxicants, with changes at the protein level currently being more consistent than those at the level of gene expression. Taken together, the findings from studies discussed in this chapter strongly suggest that cytoskeletal disruption is a common feature of adverse outcome pathways associated with chronic exposure to many developmental neurotoxicants. Thus, despite the diversity of molecular initiating events associated with exposure to different developmental neurotoxins, subsequent molecular changes invariably converge on pathways that regulate the cytoskeleton, causing cytoskeletal disruption. In some cases, the molecular initiating event may be direct binding to cytoskeletal proteins themselves. A schematic view of cytoskeletal disruption as a convergence point in developmental neurotoxicity is summarized in Fig. 58.1. Further work to characterize the ability of wellestablished developmental neurotoxins to disrupt cytoskeletal elements would be worthwhile, as this would help to identify the key events in each adverse outcome pathway in more detail. This could, for example, involve the study of upstream events such as kinase/phosphatase activities in cases where the phosphorylation status of cytoskeletal proteins is disrupted, or RT-PCR and/or proteolytic enzymes (calpain, proteasomes, etc.) cases where protein levels are significantly affected by exposure to toxin. The monitoring of these molecular events in high throughput screening platforms would help to establish a more comprehensive battery of rapid tests. In summary, the monitoring of cytoskeletal disruption is an integral part of mechanistic studies of developmental neurotoxicity and is becoming an increasingly important component in a battery of in vitro tests to rapidly screen large numbers of compounds for their ability to induce developmental neurotoxicity.

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Cytoskeletal disruption as a convergence point in developmental neurotoxicity pathways Developmental neurotoxicants Mitochondrial dysfunction Elevated ROS Multiple molecular initiating events

CYTOSKELETAL DISRUPTION

Disrupted Ca2+ homeostasis Disruption of cell signaling

Impairment of cytoskeleton-mediated processes:

Direct binding to cytoskeletal proteins

Intracellular transport Membrane trafficking Neurite outgrowth Synaptogenesis Mitosis Cell migration

FIGURE 58.1 Simplified schematic representation of the involvement of cytoskeletal disruption in developmental neurotoxicity. Developmental neurotoxicants may act via many different molecular initiating events, such as direct binding to membrane or nuclear receptors and disruption of gene expression patterns. They may also induce the generation of increased ROS and/or cause disruption of mitochondrial function, both of which could interfere with a range of cellular activities, including intracellular transport, protein folding, and degradation. Similarly, the disruption of Ca2þ homeostasis could interfere with a host of Ca2þ dependent activities, including cell signaling pathways, proteolytic degradation (e.g., by calpain), and cytoskeletal dynamics. Finally, direct binding of toxins to cytoskeletal proteins could induce conformational changes that affect subunit assembly and/or the interaction of subunits with accessory proteins that regulate cytoskeletal dynamics.

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