Axon outgrowth and neurofilament protein expression in mouse neuroblastoma cells exposed to the neurotoxin MPTP.

The compound I -methyl4phenyl-l,2,3,6tertrahydropyridme (MPTP) produces a CNS pathology in mammals similar to that obsewed in Parkinson's Disease [1,2]. MPTP itself, is found not to be toxic. The widely accepted mechanism of MPTP-induced neurotoxicity, in viw, appears to be through deamination by monoamine oxidase-B (MAO-B), localised in the external mitochondrial membrane, which results in the formation of I-methyl4phenylpyridinium (MPP? [3]. This toxic metabolite is selectively accumulated in dopminergic neurones by way of a high affinity dopamine re-uptake system [4]. It is hrther concentrated in mitochondria, where it generates free radicals, impairing mitochondrial respiration through the inhibition of Complex I of the electron transport chain [ 5 ] . This results in a depletion of ATP leading to cell death. In this study we have attempted to identify an early cellular marker of neurotoxicity. To achieve this we have examined the morphological and biochemical effect of sublethal doses of MPTP on immortalised mouse neuroblastoma N2a cells. Initially sublethal doses of MPTP were established through the use of the trypan blue exclusion assay. Cells were plated out at a density of 50,OOO celldml in growth medium (Dulbeccos Modified Eagles Medium supplemented with 1O?h foetal calf serum, 2 mM glutamine, 100 unitdml penicillin, 10 pe/ml streptomycin) prior to differentiation. Differentiation of N2a cells was induced by serum withdrawal and the addition of 0.3 mM CAMP. Cells were incubated for a W h e r 24 hours prior to fbrther experimental assessment. The projection of axons fiom N2a cell bodies is characteristic of healthy differentiated cells. Therefore the effect of sublethal doses of MPTP on these cells was morphologically assessed. This was achieved by fixing differentiated cells, exposed to the neurotoxin for 24 hours, with 90% (v/v) methanol in Tris buffered saline and staining with Coomassie Blue R250 Cells were then viewed microscopically and the number of axons counted in five random fields per well (Fig 1). An axon was defined as a cell projection greater than two cell bodies in length.