Modulating the Nrf2/KEAP1 pathway in models of Parkinson’s disease

Parkinson’s disease (PD) is the second most common neurodegenerative disease. Many mechanisms are thought to be involved in the pathogenesis of PD; the main pathways are thought to be mitochondrial dysfunction, protein aggregation, alterations of the autophagy pathway and oxidative stress. There is some overlap between these pathways in particular mitochondrial dysfunction and oxidative stress; as the mitochondria are the major source of reactive oxygen species in the cell.
Nrf2 is a transcription factor which is negatively regulated by Kelch-like ECH-associated protein 1 (KEAP1) in the cytoplasm. KEAP1 is a substrate adaptor for a Cul3 based E3 ubiquitin ligase, continuously targeting Nrf2 for destruction. Oxidative modification of sulphydryl groups on Keap1 during oxidative stress, leads to a conformational change in the complex, blocking ubiquitination of, and thereby stabilising, Nrf2. Nrf2 can then translocate to the nucleus, where it activates the transcription of a wide range of targets via antioxidant response elements (ARE) in their promoters. The Keap1-Nrf2 complex thus constitutes a cytoplasmic sensor for oxidative stress and electrophilic agents. There is evidence for suboptimal activation of Nrf2-ARE signalling in PD.
Hypothesis: Modulation of the Nrf2 antioxidant pathway using small molecules will be a useful therapeutic target in PD.
Aims: Using two patient derived models; fibroblasts from control and PD patients and dopaminergic neurons generated from these patient and controls cells to assess 1) levels of oxidative stress using state of the art redox sensitive probes targeted specifically to the mitochondria and expression levels of the Nrf2 pathway mediators in these models; 2) the ability of known activators of Nrf2 to modulate oxidative stress in these models; 3) the ability of known activators of mitochondrial function on the Nrf2 pathway and oxidative state of the cell; 4) the overlap of the mitochondrial and Nrf2 pathway via p62 and mitophagy.
Methods: The project will use primary fibroblasts from PD patients and controls, in addition dopaminergic neurons derived from the patient fibroblasts via conversion into induced neuronal progenitor cells will be used as a neuronal model of PD. Live imaging using state of the art high content imagers, western blotting, immunocytochemistry and qPCR will be used to assess protein content, mRNA expression levels and redox status of the cells. As well complex biochemical assays to assess mitochondrial function under various conditions.
Expected Results: It is expected from this project that we will be closer to understanding the link between mitochondrial dysfunction and the Nrf2 antioxidant pathway. In addition how these pathways can be modulated by small molecules to the benefit of the cells and further down the line, patients. This project builds upon several years of translational work at SITraN in drug screening of both the Nrf2 and mitochondrial pathways.