Cytoskeletal disruption as a biomarker of developmental neurotoxicity

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50 Cytoskeletal disruption as a biomarker of developmental neurotoxicity Alan J Hargreaves, Magdalini Sachana, and John Flaskos

INTRODUCTION

processes by several groups of chemicals including organophosphorus esters (OPs), heavy metals, polybrominated diphenyl ethers (PBDEs), and solvents (Sachana et al., 2011). This chapter focuses on evidence from studies showing that cytoskeletal disruption following exposure to such compounds may be able to disrupt neural development, thereby supporting the notion that cytoskeletal disruption may represent a useful biomarker of developmental neurotoxicity.

The eukaryotic cytoskeleton is a network of three interconnected protein filamentous arrays termed microtubules (MTs), microfilaments (MFs), and intermediate filaments (IFs). MTs and MFs are typically very dynamic structures, undergoing important changes in organization activity and function at key stages in neural cell development. These phenomena are in turn regulated by a combination of posttranslational modifications and interactions with a range of MT- and MF-associated proteins (Carlier, 1998; Joshi, 1998; Biernat et al., 2002; Ishikawa and Kohama, 2007; Akhmanova and Steinmetz, 2008; Janke and Kneussel, 2010). By contrast, IFs are considered to be biochemically more stable than the other networks but may be modulated by crosslinking interactions with associated proteins or by posttranslational modifications such as phosphorylation at key points during the cell cycle (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 with cytoskeletal regulatory systems can therefore be detrimental to a wide range of phenomena including neural development (Hargreaves, 1997). There is a growing body of evidence for the induction of developmental neurotoxicity via disruption of cell signaling and cytoskeleton-dependent physiological R. Gupta (Ed): Biomarkers in Toxicology. ISBN: 978-0-12-404630-6

MICROTUBULES The main component of the MT network is the heterodimeric protein tubulin, which is composed of α and β 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 upon GTP hydrolysis, which occurs shortly after subunit addition to the growing MT end (“plus” end) (Carlier et al., 1984). Both α- and β-tubulins have a number of distinct 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 α- and β-tubulin isoforms during nervous system development (Tischfield and Engle, 2010). For example, isotype III of β-tubulin (βIII-tubulin) appears almost exclusively in

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© 2014 Elsevier Inc. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-404630-6.00050-6

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50. CYTOSKELETAL DISRUPTION AS A BIOMARKER OF DEVELOPMENTAL NEUROTOXICITY

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). A large number of 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. 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 tauprotein 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 co-exist during brain development (Brion et al., 1994). MAP-2 is the most abundant MAP in the brain and is expressed mainly in dendrites and neuronal cell bodies during dendritic branching and neurite outgrowth and contributes significantly to MT interactions with MFs. Thus, MAP-2 is a well-established dendritic marker (Dehmelt and Halpain, 2005; Penzes et al., 2009) frequently used in immunocytochemical staining. It has also been shown that MAP-1B is an essential protein for the development and function of the nervous system both in vitro (Brugg et al., 1993; DiTella et al., 1996) and in vivo (Meixner et al., 2000). On the other hand, MAP-1A dynamics are very closely associated to spine plasticity and any alterations in MAP-1A may indicate changes in synaptic density (Jaworski et al., 2009). As a countermeasure 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 has the ability to bind to and sequester tubulin heterodimers and to 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 a number of well-established 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 organophosphorus (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 with regard to 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). However, α-tubulin levels were not altered by 1
10 μM 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 α-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 μM DZ exposure (Flaskos et al., 2007). No change at the levels of β-tubulin isotypes I and III were detected

MICROTUBULES

in differentiating N2a cells following exposure to DZ (Harris et al., 2009b). In contrast, in differentiating rat C6 glioma cells, DZO induced a significant reduction in the levels of βIIItubulin isotype but had no effect on total β-tubulin levels (unpublished data). Immunofluorescence demonstrated that the staining of neurites in DZO-N2a treated cells was weaker than in the controls for both βIII-tubulin and MAP-1B (unpublished data). In animal models, oral administration of CPF on PNDs 1
6 did not affect the expression of the genes coding for the neuronalspecific marker βIII-tubulin (Betancourt et al., 2006). The polymerization of bovine brain tubulin was inhibited by low concentrations of CPO (0.1
10 μM) (Prendergast et al., 2007). The ability of tubulin to polymerize is very important because, apart from contributing towards the maintenance of neuronal 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, both CPF and CPO suppress within 24 hours 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 MAP-1B (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 CPF-treated 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 CPOtreated cells (Sachana et al., 2008).

Effects of heavy metals on microtubules MT proteins have also been investigated in relation to methyl mercury-induced developmental abnormalities of the nervous system. Exposure of N2a neuroblastoma cells to methyl mercury revealed significant disruption in MT organization after staining the cells with antibody that recognizes β-tubulin (Kromidas et al., 1990). The same research group further emphasized the predominant effect of methyl mercury on MTs compared to IFs and MFs by using scanning electron microscopy (Trombetta and Kromidas, 1992). In the same cell line, cells

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demonstrated decreased reactivity against C-terminally tyrosinated α-tubulin following only 4 h exposure to a sublethal concentration of methyl mercury chloride compared to controls, which was associated with inhibition of neurite outgrowth (Lawton et al., 2007). The importance of cytoskeleton in methyl mercury 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 μM methyl mercury and long before disturbance of neurite processes (Castoldi et al., 2000). Methyl mercury 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 to a decrease in tubulin mRNA levels but no effect on the transcription rates of β-tubulin genes were found, suggesting that exposure had disrupted the autoregulatory control of tubulin synthesis in a manner similar to that described for the anti-mitotic 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 μg/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). 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 μM lead in serum free 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 mouse embryonic stem cells (Beak et al., 2011). 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 μM, as monitored by turbidity measurements and electron microscopy (Roderer and

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Doenges 1983). In contrast, triethyl lead chloride inhibited microtubule assembly and depolymerized preformed microtubules in porcine brain preparations (Zimmermann et al., 1988), while 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 SKN-SH neuroblastoma cell lines by exposure to inorganic trivalent and monomethyl pentavalent arsenic metabolites, respectively (Vahidnia et al., 2007b). In addition, the phosphorylated state of MAP-tau 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 tau-protein in Chinese hamster ovary (CHO) 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).

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 acetaldehyde-mediated 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 MAP2 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 (25 to 100 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 βIII-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 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 filamentous F-actin, filaments with an external diameter of 5 nm (Dominguez and Holmes, 2011). MF assembly is ATP-dependent and the dynamic

MICROFILAMENTS

properties of MFs are dependent on the hydrolysis of actin-bound ATP following incorporation into the polymerizing filament (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), cross-linking proteins (e.g. fascin), destabilizing factors (e.g. ADF/cofilin, gelsolin, fragmin), membrane cross-linkers (e.g. spectrin, GAP-43), or MF-associated motor proteins (e.g. myosin) (Ishikawa and Kohama, 2007; Dominguez, 2009; Lee and Dominguez 2010; Dominguez and Holmes, 2011; Jansen et al., 2011). The interaction between actin and its ABPs is in turn regulated by cell signalling pathways (Endo et al., 2003; Ishikawa and Kohama, 2007). MFs play key roles in mitosis, in neural cell differentiation, and in 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 microfilaments 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 Carlson and Ehrich (2001) tested the ability of a number of OPs including paraoxon, parathion, diisopropyl fluorophosphate (DFP), phenyl saligenin phosphate (PSP), triorthotolyl phosphate (TOTP), and triphenyl phosphite at 0.1
1 mM to disrupt the filamentous actin (F-actin) network in mitotic human 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 microfilament 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 diazinon (DZ) showed that inhibition of neurite outgrowth by 10 μM DZ was associated with increased levels but decreased phosphorylation of the actin destabilizing protein cofilin (Harris et al.,

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2009a). Though total actin levels were unaffected by DZ, the altered levels and phosphorylation status of cofilin, together with reduced staining intensity of neurites with anti-actin antibody, suggests that DZ exposure leads to a reduction in the levels of F-actin in neurites due to disruption of dynamics caused by altered expression and phosphorylation of cofilin. However, the possibility of a direct covalent interaction of OPs with lysine or tyrosine residues on actin, which was raised by the in vitro binding studies of Grigoryan et al. (2009a) and Schopfer et al. (2010), deserves further investigation in cellular or animal models.

Effects of heavy metals on microfilaments As indicated earlier, the majority of cytoskeleton related developmental neurotoxicity studies with heavy metals have tended to focus mainly on the MT network. However, micromolar concentrations of mercurial compounds are capable of blocking SH groups on purified actin thereby inhibiting its ability to interact with myosin and to 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 actin-ABP binding has also been demonstrated by native gel electrophoreses (Kekic and dos Remedios, 1999), suggesting that direct binding of heavy metal ions to actin or ABPs could be one means of exerting toxic effects directed at the MF network. Studies on the inhibition of glioma cell migration by arsenic further suggest that heavy metal compounds may also be able to disrupt F-actin by interference with MF-regulatory cell signaling pathways such as protein kinase C (Lin et al., 2008). In a study of the effects of methylmercury on post natal rat brain development, it was found that the degradation of α-spectrin by μ-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, a study in rodent cerebellar granule cells clearly showed that submicromolar levels of methylmercury induced a significant decrease in the phosphorylation of the actin binding protein cofilin, which could have a major impact on the regulation of MF dynamics (Vendrell et al., 2010), suggesting that methylmercury has the ability to disrupt the levels and

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biochemical properties of MF proteins. As actin disruption has been observed following cadmium exposure in other cell types, further study of cadmium effects on the MF network in models of neural development would be worthwhile.

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 C-dependent pathways, an effect that was attenuated by co-treatment with the MF disrupting agent cytochalasin, indicating the need for dynamic 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 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, while the levels of activated (GTP-bound) 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 solvent but also that MF-dependent processes of importance in neural development are impaired when this occurs.

INTERMEDIATE FILAMENTS The most abundant intermediate filament (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 tissueor cell type-specific manner, with the exception of 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 which 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 in some cases constitute a mechanistically relevant marker for assessing specific biochemical cytoskeletal effects. 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

INTERMEDIATE FILAMENTS

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, since NF protein phosphorylation is known to be the major factor in the 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 as a result 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 due to 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 cellspecific 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. Thus, up to 72 h exposure of proliferating or differentiating rat PC12 pheochromocytoma cells to CPF, used at a noncytotoxic concentration of 30 μM, induced upregulation of nfl (which encodes NF-L) and nef3 (coding for NF-M) genes, whereas the nefh gene (coding for NF-H) was unaffected (Slotkin and Seidler, 2009). On the other hand, exposure of PC12 cells to 30 μM 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). More recently, the

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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-day old rat pups treated with parathion (Bal-Price et al., 2010). Exposure to this OP, used at concentrations of 10
50 μM, for up to 12 days caused a concentration-dependent decrease in mRNA levels for both NF-L and NF-H. Of particular interest are a series of studies that have assessed changes in a number of 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 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, since there is now considerable evidence to suggest that these compounds can interfere potently 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 μM CPF for 8 h (Sachana et al., 2001) and to 10 μM DZ for 24 h (Flaskos et al., 2007) leads to reduced NF-H 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 μM, CPO exposure 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. In contrast, the oxon metabolite DZO, at concentrations of 5 and 10 μM, had no effect on total NF-H levels, but increased phosphorylated NF-H levels compared to the control (Sidiropoulou et al., 2009a). Under these conditions, DZO had no effects on the total levels of NF-L and NF-M (unpublished data). 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 as a result 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

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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 concentrations of 1
10 μM, 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). Since 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, methyl mercury exposure was also found to decrease the mRNA levels for NF-L and NF-H in primary cultures of rat cerebellar granule cells, whereas mRNA GFAP 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, since NFs (and other cytoskeletal elements) have been

suggested to represent a possible target in arsenic neuropathy. Although these studies have been not 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 expression 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 a number of 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 dose of 100 mM, ethanol induced in differentiating neural stem cells an increase in GFAP levels, which was thought to imply increased glial differentiation as a compensatory mechanism in order 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

CONCLUDING REMARKS AND FUTURE DIRECTIONS

of mouse embryo cells into an astrocytic lineage in serum-free medium (Yamaguchi et al., 2002, 2003). 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 a number of 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 The key role played by the cytoskeleton in a range of phenomena of importance in neural development is well established. 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 considered

841

to be 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 may be a common feature of adverse outcome pathways associated with chronic exposure to many developmental neurotoxicants. This implies that, despite the diverse nature of molecular initiating events associated with different developmental neurotoxins, subsequent molecular events may converge on pathways that regulate the cytoskeleton, leading to cytoskeletal disruption. Indeed, 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 Figure 50.1. Further work is warranted to characterize the ability of well-established developmental neurotoxins to disrupt cytoskeletal elements, 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

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

Disrupted Ca2+ homeostasis

CYTOSKELETAL DISRUPTION

Disruption of cell signaling Direct binding to cytoskeletal proteins

Impairment of cytoskeleton-mediated processes: Intracellular transport Membrane trafficking Neurite outgrowth Synaptogenesis Mitosis Cell migration

FIGURE 50.1 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 Ca21 homeostasis could interfere with a host of Ca21 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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50. CYTOSKELETAL DISRUPTION AS A BIOMARKER OF DEVELOPMENTAL NEUROTOXICITY

proteolytic enzymes (calpain, proteasomes, etc.) cases where protein levels are significantly affected by exposure to toxin. The possibility that these molecular events could be monitored in high throughput screening platforms should also be investigated, as this could help to establish a more comprehensive battery of tests. In summary, the monitoring of cytoskeletal disruption is an integral part of mechanistic studies of developmental neurotoxicity and, with further method development, could become an 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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