Neuroseience Letters, 129 (1991) 149-152 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100419M

NSL 07936

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N-Methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP) induces cytoskeletal alterations on 'Swiss 3T3' mouse fibroblasts

Graziella Cappelletti, Marina Camatini, Elena Brambilla and Rosalba Maci

Dipartimento di Biologia, Universit~ degli Studi di Milano, Milan (Italy)

(Received 13 March 1991; Revised version received 6 May 1991; Accepted 6 May 1991)

Key words: MPTP; Actin; Tubulin; Swiss 3T3 cell; Neurotoxin; Cytoskeleton

The effect of the neurotoxin MPTP (l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine) on Swiss 3T3 cells was investigated. Cell morphology alter- ations were observed when 3T3 cell cultures were exposed for 6 h to 1.5 mM MPTP. Using indirect immunofluorescence technique, cytoskeletal elements' organization of microfilaments and microtubules, has been analysed. MPTP modified both actin and tubulin networks, stress fibers appeared less sharp and microtubules were disorganized. The effect of MPTP was completely reversible and cell viability was unaffected. These results suggest that changes in cytoskeletal organization may be the first visible effect related to biochemical alterations induced by MPTP.

The concept that environmental agents can be etiologic factors in Parkinson disease has been strengthened by the ability of lrmethyl-4-phenyl-l,2,3,6-tetrahydropyri- dine (MPTP) to destroy central dopaminergic neurons in man [6] and monkey [1], and to damage dopamine termi- nals in the mouse [3]. The mechanism through which MPTP damages neurons is still under investigation.

Apparently MPTP is not directly toxic to the neurons, but, once inside the glial cells, the molecule is converted into the toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP ÷) by monoamine oxidase [7, 9]. Intraneuronal events evoked by MPP + are not clear. Ca 2 + homeostasis may be disturbed; in fact it has been shown in isolated mitochondria that the oxidative stress due to products derived from dopamine and MPTP may induce Ca 2 ÷ re- lease [2]. Moreover, the inhibition of mitochondrial res- piration and of NADH oxidase by MPP ÷ have been reported as terminal events leading to nigrostriatal cell death [11, 12]. Recently, it has been suggested that the neuronal injury may be associated with alterations in the expression of glial fibrillary acid proteins, the glial speci- fic intermediate filament protein [13]. The present paper also indicates that MPTP interferes with the cytoskeletal organization of cultured cells.

Swiss 3T3, an established cell line of mouse embryonic origin [14], was maintained at 37°C in minimal essential medium supplemented with 10% calf serum and antibio-

Correspondence: G. Cappelletti, Dipartimento di Biologia, Universitfi degli Studi di Milano, via Celoria 26, 20133 Milano, Italy.

tics in an atmosphere of 95% air/5% CO 2. Cells were plated on glass coverslips at a density of 5 x 10 a cells/cm 2 and treated with different MPTP concentrations (from 0.3 to 1.5 mM) 3 days after. The viability test on treated cells was performed by examination of Trypan Blue dye (0.4%) exclusion. Cells were counted with a haemocyt- ometer counter. The actin stress fibers and the microtu- bules' distribution were analyzed by indirect immuno- fluorescence technique. Actin was detected by staining

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Fig. 1. The effect of 24 h exposure to various concentrations of MPTP on Swiss 3T3 cell line. Cell viability test was performed by examination of Trypan Blue dye (0.4%) exclusion. The data are the means of 3

experiments.

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Fig. 2. Indirect immunofluorescence of actin and tubulin on Swiss 3T3 cells. Actin stress fibers (a-c) and microtubules (d-f) were detected by incubat- ing the cells with monoclonal anti-actin and anti-~-tubulin antibodies, respectively, as described in text. a,d: control cells; b,e: cells after 6 h exposure

to 1.5 mM MPTP; c,f: cells incubated with the control medium for 24 h after treatment with MPTP. Magnification x 1814.

the cells on coverslips. They were washed with PBS (phosphate buffered saline), fixed for 1 h in paraformal- dehyde (3.7% in PBS) pH 7.4, at room temperature, and permeabilized for 10 min in acetone at -20°C. The cul- tures were washed with PBS, preincubated for 10 min with bovine serum albumin 1% in PBS (PBS+ 1% BSA) and incubated with a monoclonal anti-actin antibody (1:100 in PBS+ 1% BSA) in a humid atmosphere at 37°C for 1 h. After 5 washes with PBS, the cells were incu- bated with goat anti-mouse immunoglobulins (1:50 in PBS + 1% BSA) conjugated with Texas Red at 37°C for 45 min. Tubulin was detected with the same technique performed for actin except that cells were permeabilized for 10 min with methanol at -20°C. The cells were incu- bated with a monoclonal anti-0t-tubulin antibody (1:100 in PBS+ 1% BSA) in a humid atmosphere at 37°C for 1 h, then with goat anti-mouse immunoglobulins (1:50 in PBS+ 1% BSA) at 37°C for 45 min. The preparations were finally washed and mounted with 90% gly- cerol+ 10% PBS.

The coverslips were viewed with a Zeiss Axioplan mic- roscope equipped with epifluorescent optics. Pictures were taken with an oil immersion objective (63 x ) on Ektachrome 400 Kodak.

Fig. 1 shows the MPTP effect of concentrations rang- ing from 0.3 to 1.5 mM on cell viability of 3T3 cells incu- bated for 24 h. Cell viability was never significantly af- fected under our experimental conditions. MPTP expo- sures caused the fibroblastic shape to disappear and the cells became roundish; this suggests that cytoskeletal modifications were involved. These effects were present following incubation time ranging from 3 to 24 h; fol- lowing shorter exposures only few cells presented mor- phological alterations.

Immunofluorescence experiments with anti-actin and anti-tubulin antibodies were performed to visualise the cytoskeletal network alterations induced by MPTP. Fig. 2a,b illustrates microfilament distribution in control and treated cells. Control cells showed the conventional organization of stress fibers, while treated cells did not. MPTP treatment affected also microtubule organization: control cells (Fig. 2d), stained with anti-ct-tubulin anti- bodies, displayed a clear microtubule network, whereas treated cells (Fig. 2e) presented a completely different microtubule pattern. Microfilaments and microtubules of these cells resulted affected giving a drastically modi- fied cell profile. These alterations in cytoskeletal organi- zation recovered when treated cells were washed and incubated 24 h in the absence of MPTP. Fig. 2c illus- trates the recovery of actin filament organization and Fig. 2f the recovery of microtubule distribution.

Our results demonstrate that MPTP affects the organ- ization of microfilaments and microtubules on 3T3 cul-

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tured cells. The actin filament bundles and microtubules' network were modified by the neurotoxin treatment, as shown by the immunofluorescence staining.

There is no direct correlation between this effect and the terminal events leading to MPTP neurotoxicity. In fact, cell viability data showed no significant cell death evoked by MPTP. Moreover, the cytoskeletal alterations were completely reversible. Therefore, we suggest that cytoskeletal modifications may be a starting event of the MPTP cytotoxicity.

These modifications may be explained by the effects of MPTP on Ca 2 + homeostasis [2]. In fact, calcium ions in- duce microtubules' depolymerization and control the in- teraction between actin and its associated proteins [4, 5, 17]. Alternatively, MPTP effect may be explained by al- terations on nucleotide metabolism. The kinetics of actin and tubulin polymerization need nucleotide hydrolysis [10, 16]. Moreover an intracellular depletion of ATP may be the result of MPP+-induced inhibition of oxida- tive phosphorylation. In fact, MPP + inhibits the mito- chondrial oxidation of NAD+-linked substrates by the coenzyme 01o [l l , 12].

The cytoskeleton of 3T3 cells is well characterized [8, 15] and so these cells represent a good model to study cytoskeletal alterations.

This work was supported by CNR Project 90.01981.CT 11.

1 Burns, R.S., Chiuech, C.C., Markey, S.P., Ebert, M.H., Jacobow- itz, D.M. and Kopin, I., A primate model of Parkinsonism: selec- tive destruction of dopaminergic neurons in the pars compacta of the substantia nigra by N-methyl-4-phenyl-l,2,3,6-tetrahydropyri- dine, Proc. Natl. Acad. Sci. U.S.A., 80 0983) 4546-4550.

2 Frei, B. and Richter, C., N-Methyl-4-phenylpyridine (MPP ÷) to- gether with 6-hydroxydopamine or dopamine stimulates Ca 2 ÷ re- lease from mitochondria, FEBS Lett., 198 (1986) 99-102.

3 Heikkila, R.E., Hess, A. and Duvoisin, R.C., Dopaminergic neuro- toxicity of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine in mice, Science, 224 (1984) 1451-1453.

4 Hwo, S. and Bryan, J., Immuno-identification of Ca 2 ÷ induced conformational changes in human gelsolin and brevin, J. Cell. Biol., 102 (1986) 227-236.

5 Kilhoffer, M.C. and Gerard, D., Fluorescence study of brevin, the Mr 92000 actin-capping and -fragmenting protein isolated from se- rum. Effect of Ca 2 + on protein conformation, Biochemistry, 24 (1985) 5653-5660.

6 Langston, J.W., Ballard, P., Tetrud, J.W. and Irwin, I., Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis, Science, 219 (1983) 979-980.

7 Langston, J.W., Irwin, I., Langston, E.B. and Forno, L.S., N- Methyl-4-phenylpiridinium ion (MPP÷): identification of a meta- bolite of MPTP, a toxin selective to the substantia nigra, Neurosci. Lett., 48 0984) 87-92.

8 Lazarides, E. and Weber, K., Actin antibody: the specific visuali- zation of actin filaments in non muscle cells, Proc. Natl. Acad. Sci. U.S.A., 71 (1974) 2268-2272.

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9 Markey, S.P., Johannessen, J.N., Chiuech, C.C., Burns, R.S. and Herkenham, M.A., Intraneuronal generation of a pyridinium metabolite may cause drug-induced parkinsonism, Nature, 311 (1984) 464-467.

10 Pantaloni, D. and Carlier, M.F., Involvement of guanosine tri- phosphate (GTP) hydrolysis in the mechanism of tubulin polymeri- zation: regulation of microtubule dynamics at steady state by a GTP Cap, Ann. N.Y. Acad. Sci., 466 (1986) 496-509.

11 Ramsay, R.R., Salach, J.I., Dadgar, J. and Singer, T.P., Inhibition of mitochondrial NADH dehydrogenase by pyridine derivatives and its possible relation to experimental and idiopathic parkinso- nism, Biochem. Biophys. Res. Commun., 135 (1986) 269 275.

12 Ramsay, R.R., Youngster, S.K., Nicklas, W.J., McKeown, K.A., Jin, Y., Heikkila, R.E. and Singer, T.P., Structural dependence of the inhibition of mitochondrial respiration and of NADH oxidase by 1-methyl-4-phenylpyridinium ion (MPP +) analogs and their energized accumulation by mitochondria, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 9108-9178.

13 Reinhard, J.F., Jr., Miller, D.B. and O'Callaghan, J.P., The neuro- toxicant MPTP (1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine) in- creases glial fibrillary acid protein and decreases dopamine levels of the mouse striatum: evidence for glial response to injury, Neur- osci. Lett., 95 (1988) 245-251.

14 Todaro, G.J. and Green, H., Quantitative studies of the growth of mouse embryo cells in culture and their development into estab- lished lines, J. Cell. Biol., 17 (1963) 299-313.

15 Weber, K., Pollack, R. and Bibring, T., Antibody against tubulin: the specific visualization of cytoplasmic microtubules in tissue cul- ture cells, Proc. Natl. Acad. Sci. U.S.A., 72 (1975) 459-463.

16 Wegner, A., Head to tail polymerization of actin, J. Mol. Biol., 108 (1976) 139-150.

17 Yin, H.L., Zaner, K.S. and Stossel T.P., Ca z÷ control of actin gela- tion, J. Biol. Chem., 255 (1980) 9494-9500.