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Protection from MPTP-induced neurotoxicity in differentiating mouse N2a neuroblastoma cells
L A De Girolamo1, A J Hargreaves, E E Billett
1Department of Life Sciences, Faculty of Science and Mathematics, Nottingham Trent University, Clifton, Nottingham, UK.
Abstract:
We have shown previously that subcytotoxic concentrations of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) inhibit axon outgrowth and are associated with increased neurofilament heavy chain (NF-H) phosphorylation in differentiating mouse N2a neuroblastoma cells while higher doses (> 100 microM) cause cell death. In this work we assessed the ability of potential neuroprotective agents to alleviate both MPTP-induced cell death (cytotoxicity) and MPTP-induced NF-H phosphorylation/reduction in axon outgrowth (neurotoxicity) in N2a cells induced to differentiate by dbcAMP. The neurotoxic effects of MPTP occurred in the absence of significant alterations in energy status or mitochondrial membrane potential. The hormone oestradiol (100 microM) reduced the cytotoxic effect of MPTP, but blocked di-butyryl cyclic AMP (dbcAMP)-induced differentiation, i.e. axon outgrowth. Both the cytotoxic and neurotoxic effects of MPTP were reduced by the monoamine oxidase (MAO) inhibitors deprenyl and, to a lesser extent, clorgyline. Alleviation of both neurotoxicity and cytotoxicity was also achieved by conditioned medium derived from rat C6 glioma cells. In contrast, whilst the p38 MAP kinase inhibitor, SB202190, protected cells against MPTP-induced neurotoxicity, it could not maintain cell viability at high MPTP exposures. In each case neuroprotection involved maintenance of the differentiating phenotype linked with attenuation of NF-H hyper-phosphorylation; the latter may represent a mechanism by which neuronal cells can moderate MPTP-induced neurotoxicity. The use of a simplified neuronal cell model, which expresses subtle biochemical changes following neurotoxic insult, could therefore provide a valuable tool for the identification of potential neuroprotective agents.
Insights
Neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) causes cell death and inhibits axon growth. Monoamine oxidase inhibitors and C6 glioma cell conditioned medium protected against MPTP neurotoxicity and cytotoxicity.
Area of Science:
- Neuroscience
- Neurobiology
- Toxicology
Background:
- 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at subcytotoxic doses inhibits axon outgrowth and increases neurofilament heavy chain (NF-H) phosphorylation in differentiating N2a cells.
- Higher MPTP concentrations (> 100 microM) induce cell death (cytotoxicity).
- MPTP neurotoxicity occurs without significant changes in cellular energy status or mitochondrial membrane potential.
Purpose of the Study:
- To assess neuroprotective agents against MPTP-induced cytotoxicity and neurotoxicity in differentiating N2a cells.
- To investigate the mechanisms underlying MPTP neuroprotection, focusing on NF-H phosphorylation and cell differentiation.
- To evaluate the utility of a simplified neuronal cell model for identifying neuroprotective compounds.
Main Methods:
- Differentiating mouse N2a neuroblastoma cells were treated with MPTP and various potential neuroprotective agents.
- Neuroprotection was assessed by measuring cell viability and axon outgrowth.
- NF-H phosphorylation levels were analyzed to understand the molecular mechanisms of neuroprotection.
Main Results:
- Oestradiol reduced MPTP cytotoxicity but blocked differentiation.
- Monoamine oxidase (MAO) inhibitors (deprenyl, clorgyline) and C6 glioma cell conditioned medium reduced both MPTP cytotoxicity and neurotoxicity.
- The p38 MAP kinase inhibitor SB202190 protected against neurotoxicity but not cytotoxicity.
- Neuroprotection was associated with maintained differentiation and reduced NF-H hyper-phosphorylation.
Conclusions:
- MAO inhibitors and C6 glioma conditioned medium show potential as neuroprotective agents against MPTP toxicity.
- Attenuation of NF-H hyper-phosphorylation may be a key mechanism in moderating MPTP-induced neurotoxicity.
- A simplified N2a cell model is valuable for screening neuroprotective agents against neurotoxins like MPTP.