Kingsbury, Ann Elizabeth; (2002) In situ hybridization studies of human substantia nigra: Pathophysiology of Parkinson's disease. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes the development of a protocol for of semi-quantitative in situ hybridization, to examine and quantify brain gene expression in Parkinson's disease (PD), particularly in dopaminergic neurons of the substantia nigra. mRNA was stable post-mortem and appeared unaffected by post-mortem delay and freezer storage time. Agonal status, assessed by measurement of brain pH, was the major determinant of mRNA preservation post-mortem, therefore brain pH was used to match case-control groups for semi-quantitative studies. mRNA encoding the rate-limiting dopamine synthetic enzyme, tyrosine hydroxylase (TH) was not upregulated in surviving and nigral neurons in PD, and there was no evidence for intrinsically higher levels of dopamine synthesis in the ventral neuronal tier. These findings indicate that compensatory changes in PD do not involve regulation of TH gene expression and that the preferential vulnerability of ventral neurons is not due to toxic effects of endogenous dopamine. Similarly, there was no evidence of any effect of therapeutically-administered L-DOPA on mRNA expression. mRNA encoding the ND1 subunit of mitochondrial complex I was reduced in surviving nigral neurons PD. mRNA encoding the glycolytic enzyme, aldolase C was less strongly expressed in nigral than other brain stem neurons. The combination of impaired energy metabolism and intrinsically low glycolytic capacity may predispose these neurons to degeneration in PD. Alpha synuclein mRNA expression was also reduced in nigral neurons in PD, indicating that alpha synuclein accumulation in nigral Lewy bodies was not caused by over-expression of the gene; down-regulation was also seen in frontal cortex, a region similarly prone to Lewy body formation. DNA fragmentation, a marker of apoptotic cell death was seen commonly in nigral neurons in hypoxic control cases, possible evidence for the metabolic vulnerability of these cells. However, there was no evidence that apoptosis was the primary mechanism of cell death in PD.

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