Kinesin-dependent motility generation as target mechanism of cadmium intoxication

Toxicology Letters 224 (2014) 356–361

Contents lists available at ScienceDirect

Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet

Kinesin-dependent motility generation as target mechanism of cadmium intoxication Konrad J. Böhm ∗ Leibniz Institute for Age Research – Fritz Lipmann Institute (FLI), Beutenbergstraße 11, D-07745 Jena, Germany

h i g h l i g h t s • • • •

Cadmium ions attack the kinesin/microtubule dependent transport system. Cadmium ions exert an inhibitory effect on microtubule formation. Cadmium ions cause a strong inhibition of the ATPase activity of kinesin. Cadmium ions slow down the movement of kinesin along microtubules.

a r t i c l e

i n f o

Article history: Received 23 September 2013 Received in revised form 1 November 2013 Accepted 4 November 2013 Available online 12 November 2013 Keywords: Cadmium Kinesin ATPase Motility KIF5A Tubulin

a b s t r a c t The anterograde vesicle transport within neurons critically depends on microtubules and on the activity of kinesin. The present study demonstrates that cadmium ions inhibit the in vitro assembly of microtubules from tubulin, whereby at high cadmium levels (∼500 ␮M) unstructured protein aggregates were formed. Cadmium ions also significantly lower both the ATPase and motility activity of neuron-specific kinesin KIF5A in concentration-dependent manner. For the inhibition of KIF5A ATPase activity, an IC50 value of 10.4 ± 1.5 ␮M was determined. Inhibition could be widely compensated by addition of EGTA, but not by addition of thiols. The inhibitory effect of cadmium on KIF5A was considerably weakened by increasing ATP concentration. As nucleoside triphosphate binding is known to be accompanied by conformational changes within the kinesin motor domain, it might be suggested that these changes protect the motor domain against cadmium. The effects of cadmium ions on the kinesin–microtubule motility generating system are considered to contribute to the development of neuronal disorders caused by cadmium intoxication. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Cadmium is a heavy metal, which accumulates in industrial and agricultural products. Actually, an evident tendency of elevation of environmental levels of cadmium in soil, water, and living organisms can be observed (Alexander et al., 2009). Beside cigarettes, food is a main source of cadmium uptake for human population. Cadmium uptake has been proved to be a serious health hazard, causing heavy damages of kidney, lung, liver, bone, and other organs (Godt et al., 2006). Recently, there is an increasing number of reports on toxic effects of cadmium on the neuronal system. So, cadmium exposure was discussed to cause neuropsychological disorders (Hart et al., 1989; Sarchielli et al., 2012) as e.g., amyotrophic lateral sclerosis (Bar-Sela et al., 2001) or the myalgic encephalomyelitis/chronic

∗ Tel.: +49 3641 656161; fax: +49 3641 656288. E-mail address: kboehm@fli-leibniz.de 0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.11.004

fatigue syndrome (Pacini et al., 2012). Moreover, cadmium has become known to damage striatum (O’Callaghan and Miller, 1986). Also Parkinson disease might be related to cadmium exposure (Okuda et al., 1997). The molecular mechanisms involved in neurotoxicity of cadmium are poorly understood. Oxidative stress seems to be one trigger for cadmium toxicity in tissues, including brain (Kumar et al., 1996). Cadmium can also affect proteasomal function and prion protein aggregation, which promote neurotoxicity (Kanthasamy et al., 2012). Basic physiological functions of eukaryotic cells essentially depend on microtubules and on the activity of kinesin and dynein, which are ATPase-active microtubule-binding motor proteins playing key roles in the anterograde (Hirokawa and Noda, 2008; Vale et al., 1985) and retrograde (Schnapp and Reese, 1989) vesicle transport. Particularly, the communication between the cell body of neurons and their extended processes, based on the so-called axonal transport, uniquely requires the activity of motor proteins. There is growing evidence that any disturbances of the

K.J. Böhm / Toxicology Letters 224 (2014) 356–361

axonal transport, including mutations in kinesin genes, blocking kinesin–microtubule interaction, microtubule disruption, or inhibition of ATPase, might contribute to the development of neurodegenerative diseases (Chevalier-Larsen and Holzbaur, 2006; De Vos et al., 2008; LaPointe et al., 2013; Reid et al., 2002). The effect of cadmium on the microtubule system has been discussed rather controversially. In optic nerves exposed to 200 ␮M cadmium ions, the microtubules were found to be disassembled (Fern et al., 1996). Moreover, cadmium salts were reported to inhibit microtubule formation in vitro (Liliom et al., 2000; Wallin et al., 1977). On the contrary, Brunner and co-workers did not find an inhibition of microtubule formation in vitro up to 1000 ␮M (Brunner et al., 1991). So far, no data have been available concerning the influence of cadmium on kinesin. Therefore, besides re-evaluating the effect of cadmium ions on microtubule formation in vitro, the present study is focussed on the ATPase activity of neuron-specific kinesin and on kinesin-mediated motility generation in cell-free environment. The results obtained might contribute to better understanding molecular mechanisms of cadmium intoxication in common and of cadmium neurotoxicity in particular. 2. Materials and methods 2.1. Isolation of microtubule protein and microtubule formation Microtubule protein was purified from porcine brain by two cycles of temperature-dependent disassembly/reassembly, according to principles described before (Shelanski et al., 1973; Vater et al., 1986), using a buffer containing 20 mM 1,4-piperazine diethane sulfonic acid (PIPES) (pH 6.8), 80 mM NaCl, 0.5 mM MgCl2 , 1 mM ethylene bis(oxyethylenenitrilo)tetraacetic acid (EGTA), and 1 mM dithiothreitol (DTT). The prepared microtubule protein contained about 85% tubulin and about 15% microtubule-associated proteins (MAPs), co-purifying with tubulin in stoichiometric ratio. For microtubule formation protein stocks, stored at −80 ◦ C, were diluted from 24.0 to 1.2 mg/ml (corresponding to about 10 ␮M tubulin) with the buffer lacking EGTA, transferred to glass cuvettes, and supplemented with GTP (0.5 mM final concentration) and cadmium chloride (final concentrations as indicated in Section 3). The resulting final concentration of EGTA was 50 ␮M. After shifting temperature to 37 ◦ C, the kinetics of microtubule formation was recorded by measurement of turbidity at 360 nm (Gaskin et al., 1974) in a Cary 100 spectrophotometer (Agilent Technologies Deutschland GmbH), equipped with a temperature-controlled multichannel cuvette holder. Corresponding control measurements without protein showed that cadmium chloride up to 500 ␮M did not change the turbidity signal compared to the cadmiumfree sample. 2.2. Expression and purification of motor protein constructs Human neuron-specific kinesin KIF5A (Niclas et al., 1994) was expressed in Escherchia coli as truncated construct containing amino acids 1–560 and purified as described formerly (Kalchishkova and Bohm, 2008), yielding a protein lacking artificial tags. The kinesin was adjusted in motility buffer (50 mM imidazole, 0.5 mM MgCl2 , 0.5 mM EGTA, 0.5 mM dithiothreitol, pH 6.8) and stored at −80 ◦ C. 2.3. ATPase activity measurement The KIF5A was diluted 100-fold with ATPase assay buffer containing 50 mM PIPES and 5 mM MgCl2 (pH 6.8). To measure the ATPase activity, the diluted KIF5A was mixed with paclitaxel-stabilized microtubules, reassembled from pure tubulin obtained by phosphocellulose column chromatography (Weingarten et al., 1975), and cadmium chloride (ultra dry, 99.999% Alfa Johnson Matthey GmbH, Germany). ATP hydrolysis was initiated by adding ATP (sodium salt, Roche Diagnostics GmbH, Germany). If not indicated otherwise, the resulting reaction mixture contained 1.5 ␮M tubulin, 168 nM KIF5A, 1.25 mM ATP, 0.5 ␮M dithiothreitol, and 30.4 ␮M EGTA. After 30-min incubation at 30 ◦ C the reaction was stopped by addition of HCl (0.1 N final concentration). The ATPase activity was determined by measuring the released free inorganic phosphate, using a Malachite green staining technique (Martin et al., 1985). For this, Biomol GreenTM Reagent (Enzo Life Sciences GmbH, Germany) was added according to instructions of the supplier, the samples (triple aliquots) were incubated 25 min in the dark, and the intensity of colour was read in a Cary 100 spectrophotometer at 650 nm. Calibration was done with a series of 20–80 ␮M disodium hydrogen phosphate using a commercial 800-␮M standard solution (Enzo Life Sciences). It has been proved that cadmium chloride up to 1 mM (final concentration) does not affect phosphate determination under the assay conditions used (result not shown).

357

2.4. Microtubule gliding assay Stock solutions of paclitaxel-stabilized microtubules, kinesin, ATP, and cadmium chloride and were transferred into ATPase assay buffer (kept at room temperature), resulting in final concentrations of 40 ␮g/ml tubulin, 0.5 mM ATP, 70 ␮M EGTA (arising from microtubule and kinesin preparations) and final concentrations of cadmium chloride and KIF5A as indicated in Results. To study the effect of EGTA, additionally 500 ␮M of the chelating agent were added. After 10-min preincubation at room temperature, 10-␮l drops of this mixture were transferred onto microscopic 26 mm × 76 mm glass slides (Gerhard Menzel GmbH, Germany) pretreated with 5 mg/ml casein, covered with a 18 mm × 18 mm coverslip (Gerhard Menzel GmbH), and sealed with a mixture of vaseline, lanolin, and paraffin (Bohm et al., 2000a). The gliding microtubules were visualized by video-enhanced differential interference contrast microscopy (AVEC DIC microscopy) using an Axiophot microscope (Zeiss, Germany) and the image processes system Argus 20 (Hamamatsu Deutschland GmbH). Using the Argus 20 software, the gliding velocities were measured within the first 5 min after application of the kinesin–microtubule mixture onto the glass slide by tracing the leading end of microtubules over a distance of at least 10 ␮m. The arithmetic mean and SD were calculated from the data of at least 15 individual microtubules.

3. Results and discussion The anterograde axonal transport in neurons essentially depends on the functional integrity of the interplay between the motor protein kinesin and microtubules. Disorders of the sensitive system of kinesin-mediated motility generation can result in disruption of the axonal transport, which seems to be a cause of the development of various neuronal diseases (Franker and Hoogenraad, 2013; Niwa et al., 2013), among them the hereditary spastic paraplegias (Ebbing et al., 2008; Ikenaka et al., 2012). Tubulin and kinesin are known to be affected by different heavy metal ions, including mercury and lead (Bonacker et al., 2004, 2005; Thier et al., 2003). The central aim of the present study was to check whether cadmium ions affect main functional structural components of the motility-generating system underlying the anterograde vesicle transport. Following this intention, at first microtubule assembly was recorded using the standard time-dependent turbidity measurement (Gaskin et al., 1974), in which the turbidity signal at 360 nm is a measure of the quantity of microtubules formed. We found that up to 50 ␮M cadmium chloride there were no remarkable effects. At 50 ␮M, the final turbidity level measured after 45 min was lowered by 15%; at 100 ␮M by 58.5% respectively (Fig. 1A). Surprisingly, at higher cadmium ion concentrations the turbidity levels determined at time point 45 min increased again. Microtubules from mammalian brain are commonly known to be highly cold sensitive. Lowering temperature to 2 ◦ C results in more or less complete disassembly of microtubules as seen for the control without cadmium chloride (Fig. 1A). However, at high cadmium chloride there was no turbidity decrease upon cooling (Fig. 1A). The reason for this behaviour might be the formation of cold-stable microtubules. However, the corresponding microscopic controls revealed that at 500 ␮M cadmium chloride no microtubules were formed (Fig. 2). Therefore, we conclude that the turbidity curves recorded in the presence of cadmium chloride are the result of superimposition of two counteracting processes: a cadmium-induced inhibition of microtubule assembly, expressed by lowered turbidity, and formation of unstructured protein aggregates (see Fig. 2C) at high cadmium, expressed by increased turbidity. Taking into account only the data measured for cadmium ion concentrations below 100 ␮M and excluding possible protein aggregate formation in this concentration range, an IC50 value of about 90 ␮M was crudely estimated. Summarizing assembly measurements, our results confirm the report of Wallin et al. (1977). Using a viscosimetric assay to quantify microtubule formation, these authors determined an about 80% inhibition at 1000 ␮M cadmium chloride and 100 ␮M EGTA. In contrast, Brunner et al. (1991) reported that cadmium ions up

358

K.J. Böhm / Toxicology Letters 224 (2014) 356–361

Fig. 3. Effect of cadmium chloride on the ATPase activity of KIF5A. The data points represent mean values and the standard deviations calculated from five independent experiments. Fitting was done using the sigmoidal DoseResp function of Microcal Origin 9.0 software (Additive GmbH, Germany), yielding the IC50 value as LOGx0 (10.4 ␮M) together with LOGx0 standard deviation (SD).

Fig. 1. Effect of cadmium chloride on microtubule formation in vitro. 1.2 mg/ml MTP, 0.5 mM GTP. (A) 0.05 mM EGTA; after 45 min the temperature was lowered to 2 ◦ C. (B) 1.0 mM EGTA.

to 1000 ␮M did not affect microtubule formation. However, it has to be mentioned that their experiments had been performed in the presence of 2 mM EGTA and that effects of cadmium, e.g., on the respiration of mitochondria, can be completely abolished by addition of EGTA (Sokolova, 2004). To prove whether EGTA is able to compensate cadmium chloride-induced inhibition of microtubule formation, we assembled microtubule protein in the presence of 1 mM EGTA. As expected, cadmium ions had no effect on microtubule formation under these conditions (Fig. 1B). The second set of experiments was performed to find out possible effects of cadmium ions on neuron-specific kinesin KIF5A.

Like motor proteins in general, KIF5A is equipped with an intrinsic ATPase to convert the chemical energy of ATP into mechanical energy required for the movement of cellular cargoes along microtubules. To test the influence of cadmium ions on the kinesin-based motility-generating system we measured the ATPase activity of KIF5A. It has been found that cadmium chloride decreases the ATPase activity in concentration-dependent manner (Fig. 3). Under the conditions used, an IC50 of 10.4 ± 1.5 ␮M was determined. As observed for microtubule assembly, the metal chelator EGTA compensated also the cadmium ion-induced inhibition of KIF5A ATPase activity (Fig. 4). As to be expected from the lowered ATPase activity, cadmium ions affected the motility activity of KIF5A. The velocity of gliding microtubules decreased with increasing cadmium ion concentration (Fig. 5). The inhibitory strength also depended on the EGTA concentration in the assay system (Fig. 5A). Many reports propose the interaction of cadmium ions with reduced sulfhydryl groups to account for inhibition of diverse biological functions (Stacey, 1986). Correspondingly, thiols have been reported to protect proteins against cadmium: for instance, glutathione and dithiothreitol were able to weaken the inhibitory effect on flavokinase (Bandyopadhyay et al., 1997) or mitochondrial isocitrate dehydrogenase (Kil et al., 2006), suggesting that cadmium ions bind to cysteine residues of the enzymes. The human KIF5A (NCBI Reference Sequence NP 004975.2) comprises seven cysteine residues in its motor domain, which is responsible for hydrolysing the ATP and for microtubule binding. But, unlike the examples mentioned above, the KIF5A ATPase was not protected by dithiothreitol,

Fig. 2. AVEC DIC microscopic control of assembly products formed from MTP in the presence of 0 ␮M (A), 175 ␮M (B), and 500 ␮M cadmium chloride, respectively. The microtubules in (A) and (B) appear as filamentous structures; (C) displays unstructured protein aggregates. The samples were taken from the corresponding assay mixtures (see Fig. 1A) at time point 45 min. 1.2 mg/ml MTP, 0.5 mM GTP, 0.05 mM EGTA.

K.J. Böhm / Toxicology Letters 224 (2014) 356–361

359

Fig. 4. Effect of cadmium chloride on the ATPase activity at elevated EGTA concentration. 168 nM KIF5A560 , IC50 11.8 ± 1.9 ␮M at 30 ␮M EGTA and 539 ± 58 ␮M at 1030 ␮M EGTA, respectively.

Fig. 7. KIF5A binding to microtubules in the presence of cadmium chloride. 117 ␮M EGTA, 2 mM AMP-PNP, 400 ␮M KIF5A560 , 0.5 mg/ml tubulin. 30-min incubation at 30 ◦ C. 30 min centrifugation at 40,000 rpm, Beckman Optima TLX, TLA55-rotor, 30 ◦ C. Sediments were resuspended in ice-cold buffer and prepared together with the corresponding supernatants for electrophoresis as described (Laemmli, 1970). Lanes: 1 – protein standard (BioLabs), 2 – sediment KIF5A plus tubulin, 3 – sediment KIF5A plus tubulin plus 500 ␮M CdCl2 , 4 – supernatant KIF5A without tubulin, 5 – supernatant KIF5A plus tubulin, 6–supernatant KIF5A plus tubulin plus 500 ␮M CdCl2 .

Fig. 5. Effect of cadmium chloride on microtubule gliding: (A) 70 ␮M EGTA: 840 nM KIF5A560 , IC50 47.8 ± 3.2 ␮M, (B) 570 ␮M EGTA, 330 nM KIF5A560 , IC50 479 ± 54 ␮M). Motility activity (measured as gliding velocity 1.2 ␮m/s ± 0.1 ␮m/s) without cadmium ions was set at 100%. The data points represent mean values and the standard deviations calculated from three independent experiments. Sigmoidal fitting as described in Fig. 3.

glutathione, or cysteine (Fig. 6), suggesting that the thiol groups in the motor domain do not essentially contribute to activity of KIF5A. That also means that another molecular reaction mechanism should underlay the inhibitory effect of cadmium on kinesin. Microtubule binding to kinesin is known to stimulate its ATPase activity (Hackney, 1994). Therefore, the cadmium ion-induced inhibition of KIF5A ATPase activity might be due to destruction of microtubules by cadmium. But, around the IC50 range determined for inhibition of ATPase activity cadmium ions had no remarkable effect on microtubules (see Fig. 1A). Even at the relatively high

Fig. 6. Effect of cadmium chloride on the ATPase activity in the presence of SH-group protecting or SH-group containing additives. 1 mM dithiothreitol, 1 mM glutathione, 1 mM cysteine. 168 nM KIF5A560 , EGTA 30.4 ␮M. The control value was taken from data in Fig. 3, the other ones are mean values from at least two independent experiments, respectively.

cadmium dose of 175 ␮M, microtubules persist (Fig. 2B). Within this context, it has to be emphasized that for both the ATPase and the gliding assay the microtubules were stabilized by paclitaxel (Taxol), which counteracts disassembly processes caused by numerous noxae. Another reason of decreased ATPase activity might be a disturbed kinesin–microtubule binding. The critical microtubuleinteracting residues of kinesin are primarily positively charged, which is consistent with a primarily electrostatic interaction of kinesin with the negatively charged microtubule surface (Tucker and Goldstein, 1997; Woehlke et al., 1997). So, it might be possible that cadmium ions prevent kinesin binding to the microtubule surface by neutralizing the negatively charges. To prove whether this reaction mechanism could explain the cadmium-induced ATPase inhibition, kinesin was incubated with microtubules in the presence of the non-hydrolysable ATP analogue adenosine 5 -(␤,␥-imido)triphosphate (AMP-PNP) instead of ATP. Under such conditions kinesin molecules were immobilized on microtubules (Schaap et al., 2011). By analysing sediments and supernatants after centrifugation of such stable microtubule–kinesin complexes formed with cadmium it could be demonstrated that cadmium up to 500 ␮M did not prevent kinesin binding (Fig. 7). The activity of kinesin critically depends on the availability of magnesium ions (Bohm et al., 2000b; Cohn et al., 1989). Magnesium ions favour ATP hydrolysis by binding to the nucleoside triphosphate and changing charge distribution around the phosphate groups in general, including the ␥-phosphate group to be released (Kendrick et al., 1992). It is known that not only magnesium ions are able to form complexes with ATP but also other metal ions, including cadmium (Pecoraro et al., 1984). Therefore, is could be possible that cadmium ions compete with magnesium ions for binding to ATP and that cadmium-ATP cannot be hydrolysed by kinesin. However, considering the ratios of cadmium (IC50 value ∼ 10 ␮M) to magnesium (5 mM) and of cadmium to ATP (1.25 mM) in the assay system this possibility can be widely excluded.

360

K.J. Böhm / Toxicology Letters 224 (2014) 356–361

Acknowledgements The author is very grateful to Helge Prinz, Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, for numerous fruitful discussions and to Mrs Marina Wollmann for her skilful excellent assistance in technical performing the experiments described in this study. References

Fig. 8. Dependence of the cadmium chloride effect on the ATPase activity on ATP. (A) ATPase activity at 1.25 mM ATP (IC50 10.3 ± 1.5 ␮M) and at 2.5 mM ATP (IC50 24.7 ± 0.9 ␮M), respectively. Sigmoidal fitting as described in Fig. 3. (B) Inhibition of ATPase activity by 15 ␮M CdCl2 in dependence on the ATP concentration. The percentages were calculated by setting the activities of corresponding samples without CdCl2 at 100%.

The cadmium ion-induced inhibition of ATPase activity of kinesin was significantly weakened by increasing the ATP concentration in the assay (Fig. 8A and B). Elevation of the ATP concentration was found to result in a comparative IC50 increase. It is known that ATP binding to kinesin is accompanied by conformational changes in the motor domain of kinesin (Chang et al., 2013). Considering this, it might be suggested that binding of the nucleoside triphosphate causes structural alterations in the KIF5A protecting its motor domain against cadmium. 4. Conclusions The present study demonstrates that cadmium ions are able to inhibit microtubule assembly and to lower both the ATPase and motility activity of neuronal kinesin. As microtubules and kinesin are indispensable elements of the anterograde axonal transport in neurons it is suggested that the effects of cadmium ions on the kinesin–microtubule motility generating system might contribute to the development of neuronal disorders caused by cadmium intoxication. Funding The study was completely funded by basic financial sources of my institute (FLI Jena). Conflict of interest There is no conflict of interests.

Alexander, J., Benford, D., Cockburn, A., Cravedi, J.P., Dogliotti, E., Di Domenico, A., Férnandez-Cruz, M.L., Fürst, P., Fink-Gremmels, J., Galli, C.L., Grandjean, P., Gzyl, J., Heinemeyer, G., Johansson, N., Mutti, A., Schlatter, J., van Leeuwen, R., Van Peteghem, C.P.V., 2009. Cadmium in food. Scientific opinion of the panel on contaminants in the food chain. EFSA J. 980, 1–139. Bandyopadhyay, D., Chatterjee, A.K., Datta, A.G., 1997. Effect of cadmium on purified hepatic flavokinase: involvement of reactive SH group(s) in the inactivation of flavokinase by cadmium. Life Sci. 60, 1891–1903. Bar-Sela, S., Reingold, S., Richter, E.D., 2001. Amyotrophic lateral sclerosis in a battery-factory worker exposed to cadmium. Int. J. Occup. Environ. Health 7, 109–112. Bohm, K.J., Stracke, R., Baum, M., Zieren, M., Unger, E., 2000a. Effect of temperature on kinesin-driven microtubule gliding and kinesin ATPase activity. FEBS Lett. 466, 59–62. Bohm, K.J., Stracke, R., Unger, E., 2000b. Speeding up kinesin-driven microtubule gliding in vitro by variation of cofactor composition and physicochemical parameters. Cell Biol. Int. 24, 335–341. Bonacker, D., Stoiber, T., Bohm, K.J., Prots, I., Wang, M.S., Unger, E., Thier, R., Bolt, H.M., Degen, G.H., 2005. Genotoxicity of inorganic lead salts and disturbance of microtubule function. Environ. Mol. Mutagen. 45, 346–353. Bonacker, D., Stoiber, T., Wang, M.S., Bohm, K.J., Prots, I., Unger, E., Thier, R., Bolt, H.M., Degen, G.H., 2004. Genotoxicity of inorganic mercury salts based on disturbed microtubule function. Arch. Toxicol. 78, 575–583. Brunner, M., Albertini, S., Wurgler, F.E., 1991. Effects of 10 known or suspected spindle poisons in the in vitro porcine brain tubulin assembly assay. Mutagenesis 6, 65–70. Chang, Q., Nitta, R., Inoue, S., Hirokawa, N., 2013. Structural basis for the ATP-induced isomerization of kinesin. J. Mol. Biol. 425, 1869–1880. Chevalier-Larsen, E., Holzbaur, E.L.F., 2006. Axonal transport and neurodegenerative disease. Biochim. Biophys. Acta Mol. Basis Dis. 1762, 1094–1108. Cohn, S.A., Ingold, A.L., Scholey, J.M., 1989. Quantitative analysis of sea urchin egg kinesin-driven microtubule motility. J. Biol. Chem. 264, 4290–4297. De Vos, K.J., Grierson, A.J., Ackerley, S., Miller, C.C., 2008. Role of axonal transport in neurodegenerative diseases. Annu. Rev. Neurosci. 31, 151–173. Ebbing, B., Mann, K., Starosta, A., Jaud, J., Schols, L., Schule, R., Woehlke, G., 2008. Effect of spastic paraplegia mutations in KIF5A kinesin on transport activity. Hum. Mol. Genet. 17, 1245–1252. Fern, R., Black, J.A., Ransom, B.R., Waxman, S.G., 1996. Cd(2+)-induced injury in CNS white matter. J. Neurophysiol. 76, 3264–3273. Franker, M.A., Hoogenraad, C.C., 2013. Microtubule-based transport – basic mechanisms, traffic rules and role in neurological pathogenesis. J. Cell Sci. 126, 2319–2329. Gaskin, F., Cantor, C.R., Shelanski, M.L., 1974. Turbidimetric studies of the in vitro assembly and disassembly of porcine neurotubules. J. Mol. Biol. 89, 737–755. Godt, J., Scheidig, F., Grosse-Siestrup, C., Esche, V., Brandenburg, P., Reich, A., Groneberg, D.A., 2006. The toxicity of cadmium and resulting hazards for human health. J. Occup. Med. Toxicol. 1, 22. Hackney, D.D., 1994. The rate-limiting step in microtubule-stimulated Atp hydrolysis by dimeric kinesin head domains occurs while bound to the microtubule. J. Biol. Chem. 269, 16508–16511. Hart, R.P., Rose, C.S., Hamer, R.M., 1989. Neuropsychological effects of occupational exposure to cadmium. J. Clin. Exp. Neuropsychol. 11, 933–943. Hirokawa, N., Noda, Y., 2008. Intracellular transport and kinesin superfamily proteins, KIFs: structure, function, and dynamics. Physiol. Rev. 88, 1089–1118. Ikenaka, K., Katsuno, M., Kawai, K., Ishigaki, S., Tanaka, F., Sobue, G., 2012. Disruption of axonal transport in motor neuron diseases. Int. J. Mol. Sci. 13, 1225–1238. Kalchishkova, N., Bohm, K.J., 2008. The role of kinesin neck linker and neck in velocity regulation. J. Mol. Biol. 382, 127–135. Kanthasamy, A.G., Choi, C., Jin, H., Harischandra, D.S., Anantharam, V., Kanthasamy, A., 2012. Effect of divalent metals on the neuronal proteasomal system, prion protein ubiquitination and aggregation. Toxicol. Lett. 214, 288–295. Kendrick, M.J., May, M.T., Plishka, M.J., Robinson, K.D., 1992. Metals in Biological Systems: Chapter: Magnesium in Biological Systems. Ellis Horwood Ltd., New York. Kil, I.S., Shin, S.W., Yeo, H.S., Lee, Y.S., Park, J.W., 2006. Mitochondrial NADP+dependent isocitrate dehydrogenase protects cadmium-induced apoptosis. Mol. Pharmacol. 70, 1053–1061. Kumar, R., Agarwal, A.K., Seth, P.K., 1996. Oxidative stress-mediated neurotoxicity of cadmium. Toxicol. Lett. 89, 65–69. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. LaPointe, N.E., Morfini, G., Brady, S.T., Feinstein, S.C., Wilson, L., Jordan, M.A., 2013. Effects of eribulin, vincristine, paclitaxel and ixabepilone on fast

K.J. Böhm / Toxicology Letters 224 (2014) 356–361 axonal transport and kinesin-1 driven microtubule gliding: implications for chemotherapy-induced peripheral neuropathy. Neurotoxicology 37, 231–239. Liliom, K., Wagner, G., Pacz, A., Cascante, M., Kovacs, J., Ovadi, J., 2000. Organizationdependent effects of toxic bivalent ions microtubule assembly and glycolysis. Eur. J. Biochem. 267, 4731–4739. Martin, B., Pallen, C.J., Wang, J.H., Graves, D.J., 1985. Use of fluorinated tyrosine phosphates to probe the substrate specificity of the low molecular weight phosphatase activity of calcineurin. J. Biol. Chem. 260, 14932–14937. Niclas, J., Navone, F., Hom-Booher, N., Vale, R.D., 1994. Cloning and localization of a conventional kinesin motor expressed exclusively in neurons. Neuron 12, 1059–1072. Niwa, S., Takahashi, H., Hirokawa, N., 2013. Beta-tubulin mutations that cause severe neuropathies disrupt axonal transport. EMBO J. 32, 1352–1364. O’Callaghan, J.P., Miller, D.B., 1986. Diethyldithiocarbamate increases distribution of cadmium to brain but prevents cadmium-induced neurotoxicity. Brain Res. 370, 354–358. Okuda, B., Iwamoto, Y., Tachibana, H., Sugita, M., 1997. Parkinsonism after acute cadmium poisoning. Clin. Neurol. Neurosurg. 99, 263–265. Pacini, S., Fiore, M.G., Magherini, S., Morucci, G., Branca, J.J., Gulisano, M., Ruggiero, M., 2012. Could cadmium be responsible for some of the neurological signs and symptoms of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Med. Hypotheses 79, 403–407. Pecoraro, V.L., Hermes, J.D., Cleland, W.W., 1984. Stability constants of Mg2+ and Cd2+ complexes of adenine nucleotides and thionucleotides and rate constants for formation and dissociation of MgATP and MgADP. Biochemistry 23, 5262–5271. Reid, E., Kloos, M., shley-Koch, A., Hughes, L., Bevan, S., Svenson, I.K., Graham, F.L., Gaskell, P.C., Dearlove, A., Pericak-Vance, M.A., Rubinsztein, D.C., Marchuk, D.A., 2002. A kinesin heavy chain (KIF5A) mutation in hereditary spastic paraplegia (SPG10). Am. J. Hum. Genet. 71, 1189–1194.

361

Sarchielli, E., Pacini, S., Morucci, G., Punzi, T., Marini, M., Vannelli, G.B., Gulisano, M., 2012. Cadmium induces alterations in the human spinal cord morphogenesis. Biometals 25, 63–74. Schaap, I.A., Carrasco, C., de Pablo, P.J., Schmidt, C.F., 2011. Kinesin walks the line: single motors observed by atomic force microscopy. Biophys. J. 100, 2450–2456. Schnapp, B.J., Reese, T.S., 1989. Dynein is the motor for retrograde axonal transport of organelles. Proc. Natl. Acad. Sci. U.S.A. 86, 1548–1552. Shelanski, M.L., Gaskin, F., Cantor, C.R., 1973. Microtubule assembly in the absence of added nucleotides. Proc. Natl. Acad. Sci. U.S.A. 70, 765–768. Sokolova, I.M., 2004. Cadmium effects on mitochondrial function are enhanced by elevated temperatures in a marine poikilotherm, Crassostrea virginica Gmelin (Bivalvia: Ostreidae). J. Exp. Biol. 207, 2639–2648. Stacey, N.H., 1986. Protective effects of dithiothreitol on cadmium-induced injury in isolated rat hepatocytes. Toxicol. Appl. Pharmacol. 82, 224–232. Thier, R., Bonacker, D., Stoiber, T., Bohm, K.J., Wang, M., Unger, E., Bolt, H.M., Degen, G., 2003. Interaction of metal salts with cytoskeletal motor protein systems. Toxicol. Lett., 75–81, 140-141. Tucker, C., Goldstein, L.S.B., 1997. Probing the kinesin–microtubule interaction. J. Biol. Chem. 272, 9481–9488. Vale, R.D., Schnapp, B.J., Mitchison, T., Steuer, E., Reese, T.S., Sheetz, M.P., 1985. Different axoplasmic proteins generate movement in opposite directions along microtubules in vitro. Cell 43, 623–632. Vater, W., Bohm, K.J., Unger, E., 1986. A simple method to obtain brain microtubule protein poor in microtubule-associated proteins. Acta Histochem. 33, 123–129. Wallin, M., Larsson, H., Edstrom, A., 1977. Tubulin sulfhydryl groups and polymerization in vitro. Effects of di- and trivalent cations. Exp. Cell Res. 107, 219–225. Weingarten, M.D., Lockwood, A.H., Hwo, S.Y., Kirschner, M.W., 1975. A protein factor essential for microtubule assembly. Proc. Natl. Acad. Sci. U.S.A. 72, 1858–1862. Woehlke, G., Ruby, A.K., Hart, C.L., Ly, B., HomBooher, N., Vale, R.D., 1997. Microtubule interaction site of the kinesin motor. Cell 90, 207–216.