Characterisation of MPTP-induced neurotoxicity in a neuroblastoma cell model system

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De Girolamo, L.A., 2000. Characterisation of MPTP-induced neurotoxicity in a neuroblastoma cell model system. PhD, Nottingham Trent University.

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Abstract

Cell lines, as in vitro models, are useful tools in unravelling tlie detailed action and effects of many neurotoxic compounds. In this thesis, two specific CNS cell lines, mouse N2a neuroblastoma cells and rat C6 glioma cells, have been used to characterise the cytotoxic and neurotoxic effects of the Parkinsonian-inducing neurotoxin l-methyl-4-phenyl-l,2,3,6-tetraliydropyridine (MPTP). The terms cytotoxic and neurotoxic are used separately here to define (i) Cytotoxic; toxic insult which has caused apoptotic/necrotic cell death; or (ii) neurotoxic; sub-cytotoxic insult which may compromise molecular events, prior to any measurable cell death. The project will ultimately allow: (a) characterisation of the relative susceptibility of the two cell lines to the neurotoxin; (b) characterisation of N2a and C6 monoamine oxidase status (MAO); (c) provide a better understanding of tie molecular events which occur during MPTP-induced neuronal degeneration; (d) identify possible early markers of neuronal toxicity; (e) provide a model which can be used to study potential neuroprotective agents.

Cytotoxicity assessments revealed that N2a cells exhibited an increased sensitivity to MPTP compared to C6 cells. In addition, cytotoxicity was increased when N2a cells were induced to differentiate by serum withdrawal and the addition of di-butyryl cyclic AMP. This increase in MPTP-induced cytotoxicity was shown to occur alongside a concomitant increase in total MAO activity. RT-PCR studies with MAO-specific primers revealed a predominance of MAO-A mRNA which increased accordingly in the differentiated state.

The use of sub-cytotoxic concentrations of MPTP on N2a cells have directed studies towards an understanding of the subtle molecular changes that occur following insult which may have particular relevance to neurodegeneration in Parkinson's Disease. Treatment of differentiating and differentiated neuroblastoma cells with sub-cytotoxic levels of MPTP led to an inhibition of axon outgrowth and retraction of axons, respectively. Biochemical characterisation by Western blotting revealed that MPTP had no significant effects on the levels of actin, α-tubulin or total neurofilament-H (NF-H). However, the use of phosphorylation state specific antibodies RMd09 and Ta51 showed NF-H phosphorylation to increase following MPTP treatment. In addition, indirect immunoflourescence analysis revealed an accumulation of phosphorylated NF-H in the cell perikaryon, suggesting that altered NF-H distribution was associated with the observed effects of MPTP on cell morphology. Since these modifications occurred in the absence of a change in energy balance or mitochondrial membrane potential, and since NFs are components of Lewy bodies, aberrant NF-H hyper-phosphorylation could be considered to be an early marker of neurotoxicity.

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) was also investigated. Both the cytotoxic and neurotoxic effects of MPTP were reduced by the MAO inhibitors deprenyl and, to a lesser extent, clorgyline.Alleviation of both neurotoxicity and cytotoxicity was also achieved by glial conditioned medium, derived from C6 cells. In contrast, whilst the p38 mitogen-activated protein 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 hyperphosphorylation; 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/molecular changes following neurotoxic insult, could therefore provide a valuable tool for the identification of potential neuroprotective agents.

Item Type:

Thesis

Description:

This research programme was funded by Immutec, Nottingham, UK.

Creators:

De Girolamo, L.A.

Date:

2000

ISBN:

9781369323689

Identifiers: