Effects of ageing on the vulnerability of the substantia nigra pars compacta dopaminergic neurons: implications in Parkinson’s disease

Abstract

The degeneration of dopaminergic neurons (DAn) in the midbrain substantia nigra pars compacta (SNpc) is a definitive feature of Parkinson's disease (PD). Although age has been established as one of the main risk factors, the role that ageing has in the development of the disease is not yet fully understood. The aim of this Thesis, therefore, was to enhance our understanding of how ageing may increase the vulnerability of SNpc DAn.
To achieve this aim, isobaric tags for relative and absolute quantitation (iTRAQ) labelling combined with liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to quantitatively compare the SNpc proteome of rats during ageing. Western blot and immunofluorescence analyses were subsequently conducted to verify some of the differentially expressed proteins from the analysis (e.g., GFAP), while immunohistochemistry analyses were performed to quantitatively and morphologically characterize the DAn of the rat SNpc themselves during ageing. From a total of 1,953 proteins that were identified and quantified in the proteomic study, the expression levels of 66 proteins were altered throughout ageing. Bioinformatic analysis revealed that proteins related to glial cells (e.g., GFAP) and the extracellular matrix (ECM) were differentially expressed in the old rat SNpc. Importantly, an unusual form of the GFAP protein (i.e., GFAPd) was showed for the first time to be differentially expressed during ageing. In addition to this, the level of tyrosine hydroxylase (TH) expression in the SNpc throughout ageing was maintained. This was somewhat surprising as it appears that, independent of the proteomic changes, there was a general decrease in the density of rat SNpc DAn together with an increase of their soma size with ageing, which might indicate that the remaining DAn are able to maintain the level of tyrosine hydroxylase (TH) expression in the SNpc throughout ageing.
These results were followed by an in vitro investigation of the role astrocytes play in the vulnerability of DAn. To do this, primary cultures of embryonic DAn were challenged with the toxin 6-hydroxidopamine (6-OHDA) after reducing the number of astrocytes in the cultures in a unique way (i.e., by using the anti-mitotic drug paclitaxel). Though the anti-mitotic drug was successful in reducing astrocytes in the cultures, it was difficult to test the effect this had on combating the effects of 6-OHDA on DAn because the toxin also affected the viability of the remaining astrocytes in culture.
Lastly, multiple multi-study proteomic comparisons of published studies on the ageing nervous system and PD demonstrated that metabolism, oxidation-reduction mechanisms, mitochondrial function and immune system were biological processes and pathways enriched in both ageing and PD. Because some of these biological processes were the same as those found differentially expressed in the proteomics study of ageing conducted here, this support the idea that they may be key toward understanding how ageing is involved in the development of PD.
In conclusion, this Thesis showed that ageing alters the metabolic support associated with mitochondrial and oxidation-reduction functions (as it happens in PD) and suggests that this might have considerable repercussions on highly reactive oxygen species (ROS) sensitive neurons such as SNpc DAn. Adding to this, the alteration of proteins related to glial cells (e.g., astrocytes) might affect their protective function in the SNpc during a time when they are become even more essential to the survival of DAn.