Molecular characterisation of SGCE-associated myoclonus-dystonia ...

Discussion 79
paraquat, only cells harbouring the p.V170G mutation showed a loss of potential, consistent
with the abnormal mitochondrial function in the sample. This might suggest that mechanisms
compensating for the dysfunction of complex I and IV can no longer be perpetuated under
influence of additional oxidative stress. Valente and colleagues reported similar results. When
they stressed SH-SY5Y cells, which were transfected with p.G309D mutant PINK1, ∆ψm
decreased significantly (2004a). In the future, it would be interesting to see whether paraquat
treatment reduces the observed complex II and III hyperactivity in the missense mutant
fibroblasts. Such an experiment would investigate the relevance of the up-regulation for the
maintenance of ∆ψm under basal conditions in the PINK1 p.V170G mutant.
In this thesis, the results gained from the ATP quantification approach appear to be in conflict
with the outcome from the enzyme activity assays. The cellular ATP concentration in the
missense mutant was comparable with the control level. Apparently, the functional deficits of
complex I and IV in the p.V170G mutant did not result in a detectable cellular ATP variation.
By contrast, a significant reduction in the level of ATP was determined in the nonsense
mutant cultures. Since the mitochondrial function was “normal” in these samples this result
can only be explained by increased cellular ATP consumption. Piccoli and colleagues report
reduced cellular ATP levels in a PD patient harbouring the p.W437X mutation when the
respective fibroblasts were cultured in a galactose medium. In a glucose cultivation medium
the ATP level was, on the contrary, higher than in control cells (2008).
As a further step it would be interesting to examine the consequences of overexpressed
p.V170G and p.Q456X PINK1 on the cellular ATP concentration in fibroblasts and to
compare these results with the findings in patient cells.
4.3.3 Elevated oxidative stress in PINK1 mutant cells
Signs of oxidative stress are a common finding in the substantia nigra of patients with PD.
Post-mortem studies of PD patient brains revealed increased damage of lipids and proteins by
ROS (Bowling and Beal, 1995).
SOD2 scavenges superoxide radicals in the mitochondrial matrix. In the missense fibroblasts,
where inhibition of the respiratory chain was observed, levels of SOD2 were elevated.
Assuming that respiratory chain dysfunction increases the production of ROS the enhanced
antioxidant release could be a protective mechanism to prevent the mitochondria from further
damage. Alternatively, the cellular SOD2 levels may be increased to compensate for the loss
of a PINK1-dependent antioxidant pathways. High superoxide production in PINK1
missense-mutant fibroblasts was also seen by Hoepken et al (2007).