Activation of the Keap1/Nrf2 pathway as a potential therapy for Parkinson's disease and the role of neuron-astrocyte crosstalk
Elske H.P. Franssen1, Robyn L. McAdam1, Oliver J. Freeman1, Sophie Clayton1, Damian C. Crowther1, Mercedes Vazquez-Chantada2, Andrew Billinton1, Roland W. Bürli1 1Neuroscience, IMED Biotech Unit, AstraZeneca, Granta Park, Cambridge, UK; 2Discovery Sciences MBP, IMED Biotech Unit, AstraZeneca, Cambridge Science Park, Cambridge, UK
Oxidative stress is thought to play a significant role in the progression of Parkinson's disease. Neurons rely heavily on the presence of astrocytes to combat oxidative insults, as astrocytes robustly activate the Keap1-Nrf2 pathway leading to expression of several antioxidant factors. Under normal conditions, the nuclear erythroid 2-related factor 2 (Nrf2) is silenced by the Kelch-like ECH-associated protein 1 (Keap1) whereby it is sequestered, ubiquitinated by the CuI3-containing E3 ubiquitin ligase complex and targeted for degradation. In state of oxidative stress, cysteine residues of Keap1 are oxidized, ultimately leading to release and translocation of Nrf2 to the nucleus where it promotes transcription of cyto-protective genes. Our ultimate aim is to boost cellular defence mechanisms and attenuate oxidative damage in Parkinson's disease by modulating the Nrf2-regulatory network. Since the Keap1-Nrf2 pathway is mainly active in astrocytes, we hypothesise that an increase in neuronal viability will be through a non-cell-autonomous effect of astrocytes on neurons. We have developed inhibitors of Keap1 that induce Nrf2 nuclear translocation and expression of downstream genes, such as HO1, Nqo1 and Gclc in astrocytes. We have studied the effect of the compounds on the viability of H2O2-stressed astrocytes as well as on markers of oxidative damage and we show that Keap1 inhibitors can rescue astrocytes from H2O2-induced oxidative damage. Neuron-astrocyte co-cultures have been established in combination with ViewRNA in-situ-hybridisation assays to visualise gene expression in each cell type. Data from co-cultures treated with Keap1 inhibitors indicate that the Keap1-Nrf2 pathway is activated differently in neurons versus astrocytes. This approach is being used to study the molecular mechanism of Keap1 inhibitors at the level of neuron-astrocyte crosstalk. These data will support the design and interpretation of in vivo animal studies aimed at reducing oxidative damage in neurodegenerative disease.
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