Rosalba Maci

The parkinsonism producing neurotoxin MPP + affects microtubule dynamics by acting as a destabilising factor

Dysfunction of the microtubule system is emerging as a contributing factor in a number of neurodegenerative diseases. Looking for the potential role played by the microtubule cytoskeleton in neuron degeneration underlying Parkinsons disease (PD), we investigate the influence of the parkinsonism producing neurotoxin 1‐methyl‐4‐phenylpyridinium (MPP+) on microtubule dynamics. We find that it acts as a strong catastrophe promoter causing a decrease of the average length of microtubules assembled from purified tubulin. We also find that it reduces the number of microtubules nucleated from purified centrosomes. Finally, binding assays demonstrate that the neurotoxin binds specifically to tubulin in the microtubule lattice in a close to stoichiometric manner. This paper provides the first evidence that dynamic instability of microtubules is specifically affected by MPP+ and suggests that it could play a role in neuronal cell death underlying PD.

APOPTOSIS IN HUMAN LUNG EPITHELIAL CELLS: TRIGGERING BY PARAQUAT AND MODULATION BY ANTIOXIDANTS

Recent results have shown that apoptosis is an important feature of the normal and injured lung epithelium, but little conclusive evidence is available about the exact intracellular mechanisms involved. In this work, we studied apoptotic cell death in the established human lung epithelial cell line, A549, by evaluating the ability of the pulmonary toxin, paraquat (1,1′‐dimethyl‐4,4′‐bipyridylium dichloride), to act as a trigger, and assessing the ability of ascorbic acid and N‐acetyl‐cysteine (NAC) to modulate the process. The analysis of nuclear and cellular morphology along with TUNEL staining showed that paraquat is an inducer of apoptosis. A549 cells incubated with sublethal doses of paraquat for up to 24h showed no apoptotic features but, their following incubation in paraquat‐free medium resulted in a time‐dependent appearance of apoptosis. The antioxidants, ascorbic acid and NAC, proved effective in reducing paraquat‐induced apoptosis, and therefore were seen as protective agents. Finally, we propose an experimental model for investigating some of the key steps in the apoptotic programme in alveolar cells.

Protein tyrosine nitration is triggered by nerve growth factor during neuronal differentiation of PC12 cells

Nitric oxide (NO) is a signaling molecule implicated in a spectrum of cellular processes including neuronal differentiation. The signaling pathway triggered by NO in physiological processes involves the activation of soluble guanylate cyclase and S-nitrosylation of proteins, and, as recently proposed, nitration of tyrosine residues in proteins. However, little is known about the mechanisms involved and the target proteins for endogenous NO during the progression of neuronal differentiation. To address this question, we investigated the presence, localization, and subcellular distribution of nitrated proteins during neurotrophin-induced differentiation of PC12 cells. We find that some proteins show basal levels of tyrosine nitration in PC12 cells grown in the absence of nerve growth factor (NGF) and that nitration levels increase significantly after 2 days of incubation with this neurotrophin. Nitrated proteins accumulate over a period of several days in the presence of NGF. We demonstrate that this nitration is coupled to activation of nitric oxide synthase. The subcellular distribution of nitrated proteins changes during PC12 cell differentiation, displaying a shift from the cytosolic to the cytoskeletal fraction and we identified alpha-tubulin as the major target of nitration in PC12 cells by N-terminal sequence and MALDI-TOF analyses. We conclude that tyrosine nitration of proteins could be a novel molecular mechanism involved in the signaling pathway by which NO modulates NGF-induced differentiation in PC12 cells.

Influence of MPP+ on the state of tubulin polymerisation in NGF-differentiated PC12 cells

Cytoskeletal proteins have been reported as constituents of cytoplasmic inclusions typical of degenerated neurones in Parkinsons disease and, in addition, the involvement of cytoskeleton in the mechanism of action of the parkinsonism‐producing neurotoxin MPP+ is emerging. Here we investigate the influence of MPP+ on the dynamic behaviour of microtubules. Neurone‐like cells derived from a rat pheochromocytoma cell line (PC12) and differentiated with nerve growth factor are used as a model system. We found that sublethal doses of the neurotoxin markedly affect the state of tubulin polymerisation: polymerised tubulins significantly decreased, whereas an increase of unpolymerised α‐tubulin was observed. Since the concentration of unassembled tubulin directly regulates tubulin synthesis by a feedback mechanism, we studied α‐ and β–tubulin synthesis by metabolic labelling of PC12 cells with [35S] methionine and following immunoprecipitations. The results showed the significant decrease of labelling in both the microtubule subunits in cells exposed to the neurotoxin. We suggest that the MPP+‐induced imbalance of tubulin polymerisation and synthesis represents a novel early step in the mechanism of action of the neurotoxin. J. Neurosci. Res. 56:28–35, 1999.

Microtubule assembly is directly affected by MPP(+)in vitro.

The microtubular system is emerging as a cell target in neurodegeneration evoked by the Parkinsonism‐inducing neurotoxin N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and its toxic metabolite N‐methyl‐4‐phenylpyridinium (MPP+). Looking for a direct effect of the neurotoxin on microtubules, we have undertaken an in vitro study by using microtubule protein purified from bovine brain. We show that MPP+, but not MPTP, modifies the initial rate and the critical concentration of assembly without affecting microtubule ultrastructure. These findings strengthen the hypothesis for the role of microtubules in the transduction of MPP+neurotoxic effect and, probably, in neuronal cell death.

The nitration of τ protein in neurone-like PC12 cells

Tyrosine nitration of proteins is emerging as a post‐translational modification playing a role in physiological conditions. Looking for the molecular events triggered by nitric oxide in nerve growth factor‐induced neuronal differentiation, we now find that nitration occurs on the microtubule‐associated protein τ. In differentiated PC12 cells, we have identified as τ a nitrated protein that co‐immunoprecipitates with α‐tubulin and indicated that the modified protein is associated with the cytoskeleton but it is confined to a restricted cell region. This paper supplies the first evidence that nitration of τ occurs in a physiological process and suggests that it could play a role in neuronal differentiation.

Paraquat induces irreversible actin cytoskeleton disruption in cultured human lung cells

The herbicide paraquat (PQ) induces the selective necrosis of type I and type II alveolar pneumocytes. We investigated the effect of PQ on human lung A549 cells to determine the possible role of cytoskeleton in lung cytotoxicity. At 80 μmol/L PQ, a concentration that did not affect cell viability, the organization of actin cytoskeleton network depended on incubation time with the herbicide. Microfilaments appeared less numerous in 30% of the cells treated for 1 h. After 24 h, all the treated cells displayed only short filaments in the periphery. The effect of PQ on actin cytoskeleton was irreversible. Moreover, no modification of microtubule network was observed in PQ-treated cells. Next, we studied the effect of PQ on Chang Liver, an epithelial cell line from human liver. These cells appeared less sensitive to the herbicide than A549, and no cytoskeletal alteration was observed. To verify whether actin filament modifications in A549 cells were related to intracellular alterations of ATP concentrations, nucleotide levels during incubation with PQ were determined. The intracellular levels of ATP were not different in control and treated cells. Our results indicate that PQ induces specifically an irreversible actin filament disorganization on A549 cells and that the observed effect is independent of intracellular concentration of ATP.

Protein tyrosine nitration is associated with cold- and drug-resistant microtubules in neuronal-like PC12 cells.

Among the myriad of cellular functions played by nitric oxide in the brain, there is increasing evidence that nitric oxide might be a primary player in the program of neurogenesis and neuronal differentiation. We have recently reported that tyrosine nitration of proteins is implicated in the signaling pathway triggered by nitric oxide during NGF-induced neuronal differentiation in PC12 cells. The cytoskeleton becomes the main cellular fraction containing nitrotyrosinated proteins, and the cytoskeletal proteins alpha-tubulin and tau are two of the targets. Here, we have studied the association of nitrated proteins with the cytoskeletal fraction in differentiating PC12 cells following exposure to microtubule depolymerising treatments and found that nitration of the cytoskeleton correlates with the increased microtubule stability underlying the progression of neuronal differentiation. These results suggest a novel functional role for nitrated cytoskeletal proteins in the stabilisation of neurites occurring in differentiated neuronal cells.

Involvement of tubulin in MPP+ neurotoxicity on NGF-differentiated PC12 cells.

In vivo, the neurotoxin MPTP is oxidated to MPP+, which is toxic to dopaminergic neurons. In this paper, we have used MPP+ as a tool to evoke neurotoxicity in the PC12 cell line and investigate the intracellular events that are involved. A cytotoxicity test, performed on undifferentiated and NGF‐differentiated PC12 cells, showed that MPP+ is much more toxic on differentiated cells and indicated the suitable range of concentrations for studying the starting events evoked by the neurotoxin. By indirect immunofluorescence we have shown that the localisation of α ‐ and β ‐tubulin in NGF‐differentiated cells was modified by a 24 h treatment with 15 μmol/l MPP+. A biochemical analysis was performed on cell extracts and the results showed that MPP+ treatment induced an increase in α ‐tubulin levels and a decrease in β ‐tubulin levels. These results suggest the involvement of the two microtubule proteins in MPP+ neurotoxicity on NGF‐differentiated PC12 cells.

Teratogenic effects of the fungicide maneb on chick embryos

The teratogenicity of a commercial formulation of the fungicide maneb (Maneb 80, containing 80% manganese ethylenebisdithiocarbamate and 20% inert ingredients) in chick embryos was evaluated. Unincubated eggs (157-207 per treatment group) were immersed in 0.5, 1.5, 4.5, or 13.5 g/liter maneb aqueous solutions for 30 sec. Two control groups were used: one group of 200 eggs was immersed in tap water and a second group of 205 eggs was immersed in a solution of the inert ingredients (sodium lignin sulfonate and n-butylnaphthalene sulfonate) at the concentration present in the 13.5 g/liter maneb solution. Eggs were then incubated for 19 days. A single treatment with maneb was teratogenic at all concentrations tested, producing mainly unilateral lower limb deformities. No adverse effects on development were noticed after exposure to the inert ingredients.