Elsevier

Neuroscience

Volume 86, Issue 2, 1 June 1998, Pages 511-519
Neuroscience

Co-localization of P450 enzymes in the rat substantia nigra with tyrosine hydroxylase

https://doi.org/10.1016/S0306-4522(97)00649-0Get rights and content

Abstract

Susceptibility to develop Parkinson's disease has been linked to abnormalities of P450 enzyme function. Multiple P450 enzymes are expressed in brain but the relationship of these to Parkinson's disease is unknown. We have investigated the expression of P450 enzymes in the rat substantia nigra and their co-localization in tyrosine hydroxylase-positive neurons and astrocytes. Immunohistochemistry was performed using anti-peptide antisera against the following P450 enzymes: CYP1A1, CYP1A2, CYP2B1/2, CYP2C12, CYP2C13/2C6, CYP2D1, CYP2D4, CYP2E1, CYP3A1, CYP3A2 and NADPH-P450 oxidoreductase. Immunoreactivity in nigral cells was found only for CYP2E1 and CYP2C13/2C6. CYP2E1 immunoreactivity was localized to many midbrain nuclei including the substantia nigra pars compacta but not the substantia nigra pars reticulata while immunoreactivity to CYP2C13/2C6 was found in the substantia nigra pars compacta, substantia nigra pars reticulata and many other midbrain nuclei. Sections of rat midbrain double labelled for either CYP2E1 or CYP2C13/2C6 and tyrosine hydroxylase or glial fibrillary acidic protein were examined for co-localization by confocal laser scanning microscopy. CYP2E1 and CYP2C13/2C6 immunoreactivity was found in tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta but not in glial cells. CYP2C13/2C6, but not CYP2E1, was also found in non-glial, non-tyrosine hydroxylase-expressing cells in the substantia nigra pars reticulata. Isoniazid induction increased CYP2E1 fluorescence signal intensity from nigral dopaminergic neurons. At least two P450 enzymes are found in nigral dopamine containing cells and one, namely CYP2E1, is selectively localized to this cell population. CYP2E1 is a potent generator of free radicals which may contribute to nigral pathology in Parkinson's disease.

The expression of CYP2E1 in dopaminergic neurons in substantia nigra raises the possibility of a causal association with Parkinson's disease.

Section snippets

Materials

All chemicals were obtained from Sigma (Sigma Chemical Co. Ltd, Dorset, U.K.) unless otherwise stated.

Animals

Adult male adult Wistar rats (n=24; 150–200 g, Bantin and Kingman, Hull, U.K.) were housed six to a cage and given free access to pelleted food (Special Diet Services, Witham, U.K.) and water. All animal experiments were carried out in strict accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines.

Preparation of antibodies

Polyclonal rabbit anti-peptide antibodies directed against

Peroxidase immunohistochemistry

Liver sections, used as positive controls, produced immunoreactive centrilobular staining using antisera targeted against CYP1A2, CYP2B1/2, CYP2E1, CYP2C12, CYP2C13/2C6, CYP3A1, CYP3A2 and NADPH-P450 oxidoreductase. Antisera against CYP2D1 produced an intense uniform panlobular staining. No immunoreactivity to antisera against CYP1A1 or CYP2D4 was observed in liver sections. However, antisera against CYP1A1 stained the globus pallidus intensely, while antisera against CYP2D4 weakly stained

Discussion

This is the first comprehensive investigation of the expression of multiple P450 enzymes in the substantia nigra and also the first to use exclusively anti-peptide antisera. The antisera used were highly specific, as a result of using short synthetic peptide sequences unique to each P450 enzyme.[24]

CYP2E1 was previously reported in neurons in the rat substantia nigra but diffuse staining was also observed in the SNpr and in many other brain regions including cells identified as glia.[29]Others

Conclusion

The expression of CYP2E1 in nigral dopaminergic neurons in the rat raises important questions about its physiological function at this site and if expressed in humans, it could be involved in mechanisms of cell death including Parkinson's disease. The presence of CYP2E1 has been reported in the human brain, although there are no reports of it being found in the substantia nigra. CYP2E1 expression may vary between individuals, as a result of polymorphisms in CYP2E1 promoters. Clearly such

Acknowledgements

This work was supported by the Medical Research Council, the Wellcome Trust, the Parkinson's Disease Society and the National Parkinson Foundation, Miami, U.S.A.

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