Parkinson's disease is a neurodegenerative disorder characterized by the loss of nigrostriatal dopaminergic neurons.

Experimentally, Parkinsonis's disease can be induced in animals by administration of the heroin analogue MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine). MPTP is metabolized into MPP+ (1-methyl-4-phenylpyridinium) which is taken up by dopamine transporter of dopaminergic neurons where it inhibits the mitochondrial NADH dehydrogenase complex and intoxicates specifically the dopaminergic neurons.

In this example, the neuroprotective effect of Brain Derived Neurotrophic Factor (BDNF) and Glial cell line-Derived Neurotrophic Factor (GDNF) against MPP+ intoxication was investigated.

Biological model:
Rat dopaminergic neurons isolated from midbrains.

Culture conditions:
Cells were cultured with defined medium (without FBS) in 96-wells microplates.

Treatment:
The conditions tested in this study were:

• medium alone
• medium with MPP+
• medium with BDNF and GDNF
• medium with BDNF and GDNF with MPP+
  

Six wells per experimental condition were carried out. After 48 hours of MPP+ intoxication, cultures were washed and placed in fresh culture medium without MPP+.

Immunohistochemistry:
The nuclei of the cells were labeled by a fluorescent marker (Hoechst dye solution). Dopaminergic neurons were labeled by an anti-tyrosine hydroxylase antibody. This antibody labels the neurites and the cell bodies of dopaminergic neurons.

Analysis:
Analysis of the number of neurons was carried out using In Cell Analyzer 1000. The results were expressed as number of cell bodies per picture. Statistical analysis was performed with the Student unpaired t-test.

3.1. Effect of BDNF and GDNF on the survival of dopaminergic neurons after MPP+ intoxication.

The results are shown in table 1 and figure 1.

After MPP+ intoxication, the number of dopaminergic neurons decreased compared to the control medium without intoxication (-50%). MPP+ had a dose dependent effect (data not show) with a IC50 value of 10 µM. In this condition, BDNF and GNDF had a protective effect on dopaminergic neurons.

Table 1: effects of BDNF & GDNF on Dopaminergic neurons survival after MPP+ intoxication

Photograph 1: protective effect of BDNF & GDNF on MPP+ induced damages (Green: dopaminergic neurons labeled with Tyrosine Hydroxylase antibody. Red: astrocytes labeled with GFAP antibody. Blue: nuclei stained with Hoechst dye).

3.2. Conclusion

The BDNF and GDNF had a neuroprotective effect against MPP+ intoxication. These compounds can be used as a positive control.

This model of MPP+ intoxication is adapted to test several compounds.

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