ToxSci Advance Access originally published online on May 13, 2008
Toxicological Sciences 2008 104(2):352-361; doi:10.1093/toxsci/kfn092
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Rotenone-Induced Toxicity is Mediated by Rho-GTPases in Hippocampal Neurons
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* Unidad CEPROCOR, Agencia Cordoba Ciencia, Córdoba
Laboratory Neurobiologia, Instituto Investigacion Medica Mercedes y Martín Ferreyra (INIMEC-CONICET), Córdoba, Argentina
1 To whom correspondence should be addressed at Laboratory Neurobiologia, Instituto Investigacion Medica Mercedes y Martin Ferreyra (INIMEC-CONICET), Cordoba, Argentina. E-mail: acaceres{at}immf.uncor.edu.
Received February 21, 2008; accepted May 5, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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In this study, we have examined the effects of rotenone in primary cultures of hippocampal and dopaminergic neurons in order to obtain insights into the possible mechanisms underlying the neurotoxic effects of this pesticide. The results obtained indicate that a 48-h exposure to rotenone (0.1µM) produces a complete and selective suppression of axon formation. This effect was dose dependent, not accompanied by changes in microtubule organization, and reversible after washout of the agrochemical from the tissue culture medium. Interestingly, pull-down assays revealed that rotenone decreases Cdc42 and Rac activities, whereas increasing that of Rho. In accordance with this, treatment of neuronal cultures with cytochalasin D, an actin-depolymerizing drug, or with the Rho-kinase inhibitor Y27632, or overexpression of Tiam1, a guanosine nucleotide exchange factor for Rac, reverts the inhibitory effect of rotenone on axon formation. Taken together, our data suggest that at least some of the neurotoxic effects of rotenone are associated with an inhibition of actin dynamics through modifications of Rho-GTPase activity.
Key Words: rotenone; neurons; axons; polarity; small Rho-GTPases.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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It is now well established that environmental neurotoxins, among them pesticides and herbicides, play an important role in the etiology of several neurodegenerative diseases (Baldi et al., 2003
; Betarbet et al., 2000; Burns et al., 2001; Corrigan et al., 2000; Takahashi et al., 1989). It follows that farming, living in rural areas and exposure to agricultural chemicals are associated with an increased risk for neurodegenerative diseases. Among pesticides, rotenone is a selective, non-specific botanical insecticide with some acaricidal properties. Rotenone is routinely used in home gardens for insect control, for lice and tick control on pets, and for fish eradications as part of water body management. It has been demonstrated that rotenone induces central nervous system and systemic toxicity (Lapointe et al., 2004). In addition, chronic (Alam and Schmidt, 2002; Betarbet et al., 2000) and acute (Saravanan et al., 2005) rotenone infusion in rats reproduces some features of Parkinson's disease. The interaction of rotenone with the complex I in the mitochondrial respiratory chain (Chance et al., 1963) and with microtubules (MTs) (Brinkley et al., 1974; Marshall and Himes, 1978) is thought to be mechanistically linked with the toxic effects of the pesticide on dopaminergic (DA) and serotonergic neurons (Ren and Feng, 2007; Ren et al., 2005). Rotenone-induced MT-depolymerizing activity causes toxicity because it disrupts the MT-based transport of neurotransmitters vesicles (Eisenhofer et al., 2004; Floor et al., 1995). This result in vesicle accumulation in the soma, which leads to increased oxidative stress due to oxidation of neurotransmitters leaked from the vesicles (Ren et al., 2005). Rotenone also produces aggregation of 
-tubulin protein in mesencephalic neurons both in vitro and in situ, inducing the appearance of enlarged and multiple centrosomes that contain multiple aggregates of 
-synuclein protein; interestingly, rotenone-treated neurons with disorganized centrosomes exhibited neurite retraction and MT destabilization, and astrocytes showed disturbances of mitotic spindles (Diaz-Corrales et al., 2005). In addition, rotenone-induced inhibition of mitochondrial complex I produces reactive oxygen species in all types of cells (Barrientos and Moraes, 1999). It has been shown that rotenone activities would generate far more reactive oxygen species in catecholaminergic neurons than in other cell types, rendering these neurons particularly vulnerable to this environmental toxin (Ren and Feng, 2007; Ren et al., 2005). Although, the mechanism underlying the toxicity upon catecholaminergic neurons has been studied with some detail, very little efforts have been made to uncover additional targets of the pesticide and/or its effects on developing neurons. In the present study, we have evaluated the effects of rotenone in primary cultures of hippocampal and DA neurons in order to obtain novel insights into the mechanisms underlying the neurotoxic effects of this pesticide. Our results show that neurotoxic effects of rotenone also involve an inhibition of actin dynamics through modifications of Rho-GTPase activity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Preparation of neuronal cultures.
Hippocampal cultures were prepared as described previously (Chuang et al., 2005
; Kunda et al., 2001; Rosso et al., 2004; Sosa et al., 2006). Hippocampi from 18 d fetal rats were dissected and treated with trypsin (0.25% for 15 min at 37°C) and dissociated by trituration with a Pasteur pipette. Cells were plated on glass coverslips coated with 1 mg/ml poly-L-lysine (Sigma Chemical Co. St Louis, MO) at a density of 
2000 cells/cm2 in minimum essential medium (MEM) containing 10% horse serum. After 2 h, the coverslips were transferred to dishes containing serum-free MEM with N2 supplements, 0.1% ovalbumin, and 0.1mM pyruvate. Primary cultures of rat ventral mesencephalon from embryonic day 14 were prepared as described by Grilli et al., (1991; see also De Erausquin et al., 1994). Following mechanical dissociation, cells were plated onto glass coverslips, coated with 1 mg/ml poly-lysine (Sigma Chemical Co.) plus 10 µg/ml laminin (Invitrogen, Carlsbad, CA). Cells were plated at densities ranging from 2000 to 5000 cells/cm2 and maintained in Dulbecco's Modified Eagle's Medium/HAM F12 defined medium in a 50/50 mix, supplemented with glucose (25mM), glutamine (2mM), and 10% horse serum.
Drug treatment.
Rotenone was added to the culture medium at concentrations ranging from 0.01 to 0.1µM. For some experiments cytochalasin D (1 µM; Sigma Chemical Co.) or Y27632 (37 nmol/ml; Calbiochem, La Jolla, CA) were also added to the culture medium, as described by Da Silva et al., (2003; see also Kunda et al., 2001). Rotenone and cytochalasin D were dissolved in dimethyl sulfoxide (DMSO) (0.01%), whereas Y27632 in distilled water. Control cultures were treated with DMSO (0.01%) alone.
Primary antibodies.
The following primary antibodies were used in this study: a monoclonal antibody (mAb) against tyrosinated -tubulin (clone TUB-1A2, mouse IgG; Sigma Chemical Co.) diluted 1:1000; a mAb against β-tubulin (Gonzalez-Billault et al., 2001); a mAb against Tau protein (clone Tau1, mouse IgG; Cáceres et al., 1992) diluted 1:100; a mAb against tyrosine hydroxylase (mAb 318, Chemicon International, Temecula, CA) diluted 1:800; an affinity-purified rabbit polyclonal antibody against detyrosinated -tubulin (Gonzalez-Billault et al., 2001); an affinity-purified rabbit polyclonal antibody raised against a peptide corresponding to an amino acid sequence mapping at the C terminus of Tiam1 of mouse origin (C16; Santa Cruz Biotechnology, Santa Cruz, CA; see also Kunda et al., 2001) diluted 1:1000.
Immunofluorescence.
Cells were fixed for 30 min at room temperature with 4% (wt/vol) paraformaldehyde in phosphate-buffered saline (PBS) containing 4% (wt/vol) sucrose. Cultures were washed with PBS, permeabilized with 0.2% (vol/vol) Triton X-100 in PBS for 5 min, and again washed in PBS. For some experiments, cells were permeabilized with Triton X-100 (0.2%) prior to fixation under MT-stabilizing conditions ("cytoskeletal preparations") as described previously (Gonzalez-Billault et al., 2001). Cell were incubated with primary antibodies (1–3 h at room temperature), washed with PBS, and then incubated with Alexa 488 (dilution 1:800) or Alexa 564 (dilution 1:800) secondary antibodies (1 h at 37°C), then washed with PBS and the coverslips mounted using FluorSave (Calbiochem). For some experiments rhodamine-phalloidin (1:1500; Molecular Probes, Eugene, OR) was used to stain filamentous actin (F-actin). Cells were visualized in either a conventional confocal (Zeiss Pascal, Carl Zeiss, Inc., Germany) or spectral confocal (Olympus, Olympus, Japan Fluoview 1000) microscopes and images processed using Adobe Photoshop, Adobe Systems Inc., San Jose, CA. For some experiments, the relative intensities of β-tubulin, tyrosinated -tubulin, and detyrosinated -tubulin were evaluated in detergent-extracted cytoskeletons using quantitative fluorescence techniques as described previously (Gonzalez-Billault et al., 2001). Cells were visualized with an inverted Zeiss microscope, and images (8 bites) were collected using a CCD camera (Orca 1000, Hamamatsu Corp., Middlesex, NJ) and Metamorph software (Molecular Devices). Fluorescence intensity measurements were performed within the cell body and neurites of identified neurons; with these data, we then calculated the average fluorescence intensity expressed in pixels (0 = black/255 = white) within the cell body, and inner, middle, and distal third of identified neurites. Background levels were those detected in unlabeled cells.
Morphometric analysis of neuronal shape parameters.
Neuronal shape parameters were measured using maximal projection images and the morphometric menu of the confocal microscope. To measure neurite length or growth cone shape parameters, antibody-labeled cells were randomly selected and traced from the video screen using the morphometric menu of the Pascal (Chuang et al., 2005; Rosso et al., 2004; Sosa et al., 2006). Differences among groups were analyzed by the use of ANOVA and Student-Newman-Keuls test.
Cell viability assays.
For evaluating cell survival, cultures were incubated for 5 min at room temperature, with calcein AM (C-1430, Molecular Probes), a fluorogenic substrate that is cleaved only in viable cells to form a green fluorescent membrane-impermeant product, and with ethidium homodimer-1 (EthD-1, Molecular Probes), a high-affinity red fluorescent DNA stain that is only able to pass through the compromised membrane of dead cells. The number of live and dead cells was then counted in control and Rotenone-treated cells. Apoptosis was also quantified by the detection of condensed chromatin with the fluorochrome, Hoechst 33258, as described (James and Roberts, 1996). For such a purpose, cells were fixed with 4% paraformaldehyde (pH 7.4) for 20 min and then stained with Hoechst 33258 (5µM physiological saline) for 1 min and washed twice in PBS. Apoptosis was quantified by scoring the percentage of cells with apoptotic nuclear morphology at the single cell level. Condensed or fragmented nuclei were scored as apoptotic. At least 700 cells from randomly selected fields were counted per experimental condition.
Expression construct and transient transfection assays.
A Tiam1 cDNA (C1199) (Kunda et al., 2001) cloned as a BamHI/XhoI fragment into pcDNA3 containing a cytomegalovirus promoter and a hemagglutinin tag (Invitrogen), a generous gift of Dr John Collard (The Netherlands Cancer Institute, Amsterdam, The Netherlands), was used for transfection of primary neurons. Transient transfection of cultured neurons was performed using Lipofectamine 2000 (Invitrogen). Cells were analyzed 18-, 20-, or 30-h post-transfection as described (Chuang et al., 2005; Kunda et al., 2001; Rosso et al., 2004).
Rho-GTPase activity assays.
The activities of small GTPases were quantified by measuring the amounts of Cdc42, Rac, and Rho precipitated in a pull-down reaction from cell lysates, using glutathione S-transferase (GST)-agarose containing the Rac binding domain of Pak1 for GTP-bound Rac and GTP-bound Cdc42, or the Rho-binding domain (Rhotekin-RBD) of a Rho effector coupled to agarose beads for GTP-bound Rho following the manufacturer's instructions (Cytoskeleton, Denver, CO). Equal amounts of protein extracted from untreated or rotenone-treated (0.1µM) neurons were subjected to the activity assay. As negative and positive controls for the pull-down assay, we used lysates treated with 100µM GDP or GTPS, respectively. GST-Rhotekin-RBD beads or GST-PAK1-beads were added to each aliquot, and the reaction mixtures were incubated for 45 min at 4°C with gentle agitation. After the pull-down reaction, the supernatants were removed by brief centrifugation, and the precipitated proteins bound to the beads were subjected to immunoblot analysis with mAbs against Cdc42 or Rac or Rho. Western blots were developed using ECL as described (Chuang et al., 2005; Sosa et al., 2006). Quantification of western blot signals (optical density) was performed on scanned films by Scion Image for Windows (Scion Corporation, MD) analysis software.
Statistical analyses.
The unpaired two-tailed Student's t-test and one-way ANOVA were used in the statistical analysis when appropriate. Post hoc comparisons of individual groups were performed using the Tukey-Kramer test. A p-value less than 0.05 was considered significant. All results are expressed as mean ± SEM for the stated number of observations.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Rotenone Inhibits Axon Formation in Cultured Hippocampal Neurons
Hippocampal pyramidal neurons in culture undergo several distinct morphological changes during differentiation that lead to the extension of a single long axon and several short and branched dendrites. Shortly after plating there is no discernable polarity, as neurons elaborate lamellipodia (stage 1), and then a symmetrical array of short neurites (minor processes, stage 2). Later on, one of these neurites forms the axon by extending a large and highly dynamic growth cone with labile actin cytoskeleton and abundant dynamic MTs (stage 3) (Bradke and Dotti, 1999
; Chuang et al., 2005; Kunda et al., 2001); several days later, the remaining minor processes differentiate as dendrites (Dotti et al., 1988).
The effect of rotenone on neuronal polarization was tested by adding the agrochemical to primary hippocampal cell cultures 2 h after plating and analyzing neuronal morphology 48 h later. As expected, in nontreated cultures most neurons have reached stage 3 of neuritic development after 2 days in culture (Figs. 1A–C). By contrast, rotenone-treated neurons display a selective and significant inhibition of axon formation, with many of them arrested at stage 2 of neuritic development (Figs. 1D–G). A quantitative analysis of neurite outgrowth was made in the presence of two different concentrations of rotenone (Table 1). For this analysis, an axon was defined as any neurite longer than 50 µm and that stained positively with the Tau1 mAb (Chuang et al., 2005). Increasing concentrations of the agrochemical caused a significantly decrease in total neurite length per cell (Fig. 2A), which either reflect the lack of an axon or the presence of a short axon in the few neurons that reach stage 3 of neuritic development (Fig. 2B). The effect of rotenone on axon formation was completely reversible after washout of the agrochemical from the culture medium (Figs. 3A–C). The axons that extend from neurons after recovery from rotenone treatment were morphologically indistinguishable from control ones, displaying the typical proximo-distal Tau1 staining pattern (Figs. 3D and 3E). To test whether rotenone also inhibits axon elongation, 0.1µM rotenone was applied to cultured neurons 24 h after plating, a time at which the majority of cells had reached stage 3 of neuritic development. Relative to control cultures, which exhibited continuous axonal growth and approximate doubled axon length over the next 24 h, rotenone-treated cells showed cessation of axon growth and retraction (Supplementary Table 1). Thus, rotenone (0.1µM) appears to suppress both axon outgrowth and elongation, without affecting minor process formation. In addition, cell viability assays revealed that at this concentration, rotenone does not produce cell death in cultured hippocampal pyramidal neurons; thus, the number of viable cells (97 ± 0.8) in rotenone-treated cultures was similar to that of control nontreated cultures (98 ± 0.6; see also, Supplementary Fig. 1). We also evaluated apoptotic cell death by examining nuclear morphology with Hoechst staining; this analysis revealed no significant differences between nontreated and rotenone-treated neurons. Thus, the percentage of cells with condensed or fragmented nuclei was 2.5 ± 0.6% for control cultures and 3.4 ± 0.8% for the rotenone treated ones.
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Rotenone Inhibits Axonal Elongation by Altering Rho-GTPase Activity
MTs and actin filaments are key components of the cytoskeleton that are crucial for the development of neuronal polarity (Arimura and Kaibuchi, 2007
; Wiggin et al., 2005). Because previous studies have demonstrated that rotenone inhibits MT assembly (Brinkley et al., 1974; Marshall and Himes, 1978) and that some of its toxic effects in central neurons (Ren and Feng, 2007; Ren et al., 2005) and in nonneuronal cells (Srivasta and Panda, 2007) involve MT depolymerization, the possibility exists that inhibition of axon formation by rotenone is a direct consequence of alterations in MT assembly, organization, or dynamics. Therefore, to determine whether rotenone affects MT assembly and/or stability in developing hippocampal neurons, we examined if minor processes or axons formed in the presence of the agrochemical differ from control ones in their content of MT polymer. For this experiment, cultures were fixed after detergent extraction performed under MT-stabilizing conditions and processed for immunofluorescence with antibodies against tubulin. This method removes unassembled tubulin from the cell, so that the tubulin staining remaining in such cells is attributable to MTs (Brown et al., 1992; Gonzalez-Billault et al., 2001). As described previously (Gonzalez-Billault et al., 2001), β-tubulin fluorescence intensity in "cytoskeletal preparations" was used as a relative measure of polymer mass because it is present at a constant stoichiometry in MTs (Brown et al., 1992); besides, the specific antibody to β-tubulin (Gonzalez-Billault et al., 2001) that we used stains all MTs uniformly within the cell body, axons, and minor processes. Quantitative measurements revealed no significant differences in the intensity of MT staining within the cell body or neurites between control and rotenone-treated cultures (Table 2). We also examined the relative amounts of two post-translationally modified tubulins in neuritic MTs of control and Rotenone-treated neurons. Several studies have shown that the relative abundance of tyrosinated (Tyr) and detyrosinated (Glu) -tubulin in MTs correlates with its stability properties such that tyr-tubulin is especially enriched in the more dynamic and recently assembled MT polymer, whereas detyr-tubulin is enriched in the more stable one (Baas et al., 1993; Brown et al., 1992; Gonzalez-Billault et al., 2001; Li and Black, 1996). Thus, the distribution and relative levels of these two posttranslational modifications of -tubulin can be used as an indirect, but reliable, assay of MT organization and dynamics. Spectral confocal microscopy and quantitative fluorescent measurements revealed that the balance between dynamic (Tyr-MT) and stable (Glu-MT) polymer is not significantly altered in neurites from rotenone-treated neurons (Supplementary Fig. 2 and Table 2). Because recent studies (Diaz-Corrales et al., 2004, 2005) have shown that rotenone induces aggregation of -tubulin, a key component of the centrosome, with the consequent disassembly of the Golgi apparatus in neurons of the substantia nigra, we decided to examine the morphology of the Golgi in cultured hippocampal neurons treated with rotenone. The results obtained showed no changes in Golgi morphology after treatment of cultured hippocampal pyramidal neurons with 0.1µM rotenone for 2 days (Supplementary Fig. 3). Taken together, these observations led us to hypothesize that rotenone may affect other components of the neuronal cytoskeleton, such as actin filaments and/or regulators of actin organization and dynamics.
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Axon formation is causally related to dramatic changes in the organization and dynamics of the growth cone actin cytoskeleton. This involves an expansion of the peripheral lamellipodial veil, a shortening of actin ribs, and disassembly of actin filaments in the central growth cone region, a phenomenon associated with increased actin dynamics and regulated by Rho-GTPase activity (Bradke and Dotti, 1999
; Kunda et al., 2001). It has also been established that all minor processes can generate an axon. This is dramatically illustrated after global application of low doses (0.5–1µM) of the actin-depolymerizing drug, cytochalasin D, which leads to multiple axon formation, a phenomenon preceded by the penetration of MTs within neuritic tips devoid of actin filaments (Bradke and Dotti, 1999; Kunda et al., 2001). Thus, rotenone-treated neurons may fail to elaborate an axon because regulation of actin dynamics is impaired. Therefore, as a first test of this idea we evaluated whether cytochalasin D was capable of inducing axon formation in Rotenone-treated neurons. The results obtained showed that cultured hippocampal pyramidal neurons treated for a short period (3–6 h) with cytochalasin D (1 µg/ml), in the presence or absence of rotenone (0.1µM) extends multiple long axon-like neurites (Figs. 4A–F). Interestingly, a similar effect was observed after treatment with cytochalasin E (Ruthel and Hollenbeck, 2000), a variety of cytochalasin believed to be free of cellular effects other than its inhibition of actin polymer assembly (not shown). In addition, confocal microscopy revealed the presence of many dynamic MTs at neuritic tips of neurons treated with rotenone plus cytochalasin D or E. Taken together these results demonstrate that after actin depolymerization, rotenone-treated neurons are not only able to elaborate axon-like neurites, but also capable of assembling MTs, just as control ones. It follows, that perhaps one primary target of rotenone, at the doses used in this study and during initial axon outgrowth, are regulators of actin organization, rather than tubulin itself or proteins controlling MT assembly.
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Therefore, it become of interest to evaluate whether rotenone alters the activity of the small Rho-GTPases, Cdc42, Rac, and Rho, which regulate actin dynamics and are crucially implicated in axon formation (Arimura and Kaibuchi, 2007
; Bradke and Dotti, 1999; Garvalov et al., 2007; Kunda et al., 2001; Nishimura et al., 2005; Sosa et al., 2006). To this end, we used pull-down assays to determine levels of active Cdc42, Rac, and Rho in control and rotenone-treated hippocampal cell cultures. These assays revealed that rotenone induced a 2.5-fold increase in Rho activity, while significantly decreasing both Cdc42-GTP (40% reduction) and Rac-GTP (80% reduction) levels (Fig. 5). Based on current evidence (Arimura and Kaibuchi, 2007), an increase in Rho-GTP paralleled by decreased Cdc42-Rac activities could easily account for the inhibitory effect of rotenone. Therefore, to further test this idea we decided to evaluate whether inhibition of a downstream effector of Rho, such as Rho-kinase (RhoK; Da Silva et al., 2003) was capable of rescuing the inhibitory effect of rotenone on axon formation. The results obtained showed that treatment with Y27632, a RhoK inhibitor, was capable of inducing axon formation in rotenone-treated hippocampal pyramidal neurons (Figs. 6A and 6B). Because recent studies suggest that activation of RhoK could lead to inhibition of Cdc42-induced Rac activation by interfering with Tiam1/2 functioning (Nakayama et al., 2008), it became of interest to explore if the inhibitory effect of rotenone could also be rescued by overexpression of a guanosine nucleotide exchange factor (GEF) for Rac, such as Tiam1 (Kunda et al., 2001; Nishimura et al., 2005); as expected, ectopic expression of Tiam1 induces axon formation in rotenone-treated neurons.
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In the final set of experiments, we evaluated the effect of rotenone on process formation in cultured DA neurons obtained from E14 rat embryos and grown on a poly-lysine plus laminin coated substrate. The morphological differentiation of DA neurons, identified by tyrosine hydroxylase staining, was investigated at 6-h intervals during the first day after plating. As shown in Figures 7A–C, these neurons develop a bipolar asymmetric morphology, with one of its neurites exhibiting a fast growing rate and acquiring more than 100 µm in length within the first day after plating. Treatment of DA neurons with rotenone (0.1µM) alters this pattern by significantly reducing the length of the longest neurite (Figs. 7E–G). It is interesting to note that at this concentration of rotenone, we did not detect more than 5% of cell death; this percentage is much lower than that reported in previous studies for a similar dose of rotenone (Ren et al., 2005
). It may reflect the fact that we used serum, and because rotenone is lipophilic, the concentration available to the neurons may be lower than 0.1µM. Regardless of this, and as in the case of cultured hippocampal pyramidal neurons, the inhibitory effect of rotenone on the neurite outgrowth response of DA neurons was rescued by the RhoK inhibitor, Y27632 (Figs. 7H–J).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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We show here that treatment of cultured hippocampal or DA neurons with the pesticide rotenone inhibits their morphological differentiation. Thus, in young developing neurons, rotenone selectively prevented axon outgrowth and elongation. This effect was not associated or paralleled with cell death, because at the doses of rotenone used in this study and under the present culture conditions a high percentage of the cells were alive as determined by staining with calcein AM and Hoechst. Besides, the effect of rotenone on process outgrowth was reversible after washout of the pesticide from the culture medium. In addition, rotenone did not prevent initial neurite outgrowth, but the transformation of a minor neurite into an axon in the case of hippocampal neurons, or the initiation of a phase of rapid neurite elongation in the case of DA neurons. Taken together, these observations suggest that the effects of rotenone during early neuronal morphogenesis are rather specific and may involve signaling pathways directly linked with the development of neuronal polarity. These observations are in line with previous studies showing that rotenone affects process extension in PC12 cells (Tomaselli et al., 2005
) or primary cultured neurons (Bocklinger et al., 2003; Ren and Feng, 2007; Ren et al., 2005) by altering the mitochondrial complex I and/or MT organization.
It is now well established that axon formation is preceded and accompanied by dramatic rearrangements of growth cone MTs and actin filaments (Bradke and Dotti, 1997, 1999; Kunda et al., 2001). A direct interaction of rotenone with tubulin and the subsequent inhibition of MT assembly have been suggested as a mechanism responsible for at least some of the neurotoxic effects of the pesticide (Ren and Feng, 2007; Ren et al., 2005; Srivasta and Panda, 2007). Rotenone-induced aggregation of -tubulin and Golgi disorganization has also been implicated as a cause of MT disassembly and neurite retraction (Diaz-Corrales et al., 2005). Because MT assembly and stabilization are directly implicated in axon formation (Gonzalez-Billault et al., 2001; Witte et al., 2008), rotenone-induced alteration in MT organization and dynamics could easily explain it effects on the development of neuronal polarity. However, in cultured hippocampal pyramidal neurons the selective inhibition of axon outgrowth induced by rotenone (0.1µM) was not accompanied by detectable changes in the distribution and/or content of both dynamic and stable MTs along neurites and within growth cones, and/or in the localization or morphology of the Golgi apparatus. Therefore, our data suggest that additional events may explain the selective inhibition of axon formation induced by rotenone.
In cultured hippocampal neurons, axon formation is preceded by the appearance in one of the multiple growing neurites of a large and highly dynamic growth cone containing a very labile actin network (Bradke and Dotti, 1997, 1999; Kunda et al., 2001; Paglini et al., 1998). It follows that regulation of actin organization and activity within selected growth cones may be one of the major factors underlying the establishment of neuronal polarity. Our results show that cytochalasin D, an actin-depolymerizing drug, reverts the inhibitory effect of rotenone on axon formation; as a matter of fact, in the presence of cytochalasin D, rotenone-treated neurons behave as control ones extending multiple axon-like neurites, filled with dynamic MTs at their tips. Therefore, rotenone may exert its inhibitory effect on axon formation by stabilizing growth cone actin filaments. Actin cytoskeleton dynamics and activity during neuronal polarization are critically regulated by the small GTPases of the Rho/Rac/Cdc42 superfamily (Arimura and Kaibuchi, 2007); then, the pesticide could exert its toxic effect altering the activity of the distinct GTPases. Our findings support this view, revealing that rotenone decreases Cdc42 and Rac activities, while increasing that of Rho. Previous studies have shown that suppression of either Cdc42 or Rac does not affect the initial extension of neurites by hippocampal pyramidal neurons (Bradke and Dotti, 1999; Kunda et al., 2001; Sosa et al., 2006); Therefore, it was not surprising that rotenone had no effect on the initial extension of neurites. By contrast, Cdc42 and Rac are crucially required for axon formation (Kunda et al., 2001; Nishimura et al., 2005; Sosa et al., 2006), and therefore a decrease in their activities, like the one induced by rotenone may prevent axon formation.
It has also been demonstrated that increased activity of Rho pathways produces axon retraction and arrest growth in neurons due to stabilization of the actin cytoskeleton (Bito et al., 2000; Kozma et al., 1997). These effects may involve activation of LIMK1 followed by phosphorylation and inhibition of cofilin, with the consequent reduction in actin dynamics and prevention of axon outgrowth (Rosso et al., 2004). However, and perhaps more important, Rho also antagonizes Cdc42-Rac activities in certain types of cells, including neurons (Takefuji et al., 2007), a phenomenon involving activation of RhoK, and the subsequent phosphorylation and inactivation of the polarity protein Par3 (Nakayama et al., 2008) and the Rac GEFs, Tiam1/2 (Takefuji et al., 2007). Consistent with this, inhibition of RhoK activity by Y27632, or enhanced Rac activation induced by ectopic expression of Tiam1, reverts the inhibitory effect of rotenone on axon formation. A similar mechanism appears to operate in DA neurons, despite their morphological differences with hippocampal pyramidal neurons, because Y2763 was also able to induce neurite elongation in rotenone-treated cultures.
In summary, we have identified Rho-GTPases as a new target of rotenone action, and demonstrate that at least some of the neurotoxic effects of rotenone in developing neurons are associated with an inhibition of actin dynamics through a modification of Rho-GTPase activity.
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SUPPLEMENTARY DATA |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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ANPCyT and CONICET, to A.C.
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ACKNOWLEDGMENTS |
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Monica Sanchez, Carlos Landa and Alfredo Caceres are members of the National Research Council of Argentina.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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