Targeting a custom-engineered flavonoid to the mitochondria protects against acute oxidative stress
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Date
29/06/2015Author
Drummond, Nicola Jane
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Abstract
Oxidative stress is caused when there are more reactive oxygen species (ROS), than
antioxidants to scavenge them, resulting in damage to cellular components. It has been
implicated as a major player at multiple points in the disease process of Parkinson’s disease
(PD) and many other conditions. For example, evidence suggests oxidative damage to the
α-synuclein protein may affect its aggregation propensity. In addition, α-synuclein may
increase ROS production. However, how this oxidative stress relates to neurodegeneration is
not known. Therefore, there is a need for models of α-synucleinopathies and tools to assess
the involvement of oxidative stress in the disease process.
In order to model α-synucleinopathies, overexpression of the α-synuclein protein
was used. A BacMam viral expression system containing human α-synuclein was generated
and used to assess toxicity. α-Synuclein overexpression in undifferentiated or differentiated
SH-SY5Y cells failed to show toxicity. However, the stability of α-synuclein protein
expression and the cell line used may have influenced in the lack of toxicity. The current
work provides important guidance for future experimental design.
Flavonoids are found in plants and have antioxidant capability. AO-1-530 is a
synthetic compound with a flavonoid head group and a long hydrocarbon tail. It is highly
cell permeable and localises to the mitochondria. In order to investigate its protective
properties, toxin-induced oxidative stress cell assays were established. AO-1-530, in the low
micromolar range, was protective against high doses of tert-butyl hydroperoxide (tBHP),
whereas natural antioxidants, such as myricetin and quercetin, showed limited protection or
required at least 10-fold higher concentrations to achieve similar protection.
The ability of AO-1-530 to directly scavenge radicals was assessed cell-free in
solution and in a cell-based assay. In solution the mechanism of action was investigated by
electron paramagnetic resonance (EPR) spectroscopy. AO-1-530 had similar scavenging
ability to myricetin, but was a slightly stronger scavenger than quercetin. The intracellular
scavenging ability was quantified by CellROX® Deep Red live imaging. Although the
compounds had similar cell-free scavenging abilities, AO-1-530 significantly out-performed
both myricetin and quercetin in the intracellular assay, suggesting the mitochondrial
localisation is critical to its highly protective properties. AO-1-530 is a powerful, novel tool
to study the involvement of oxidative stress in diverse disease models.