Investigating the role of the GCH1/AP-1 interaction and tetrahydrobiopterin in the regulation of oxidative stress within neurons

Under conditions of stress, Reactive Oxygen Species (ROS) are generated. ROS are high energy damaging forms of oxygen molecules. Dysfunction of the organism's ability to adapt to changing levels of ROS, and accumulated ROS-related damage through the life-history can lead to the ageing and senescence, which has been suggested to contribute to the development of Parkinson’s Disease and ALS. In neurons, ROS activates numerous cellular responses, including the activation of the Jun-N-terminal kinase (JNK) pathway, together with its target Activator Protein 1 (AP-1). How this response is regulated within neurons has yet to be clearly defined. In our model synapse, the Drosophila 3rd instar neuromuscular junction (NMJ), AP-1 positively regulates growth and strength while regulating ROS levels. A previous investigation into proteins that differentially bind to AP-1 during oxidative stress in Drosophila neurons identified Punch (Pu) (GTP cyclohydrolase-1 ). Punch is the first step in the synthesis for tetrahydrobiopterin (BH4), a crucial oxidation sensitive cofactor with an important role in neurotransmitter synthesis and regulation of ROS. We hypothesise that in conditions of normoxia Punch is inhibited by high levels of BH4 which promotes binding to and inhibition of AP-1. Punch was found bound to AP-1 in normoxic conditions, but not bound under conditions of oxidative stress, releasing AP-1 to function. Reducing the levels of Punch allows the release of AP-1 which drives synaptic overgrowth within the Drosophila NMJ under conditions of oxidative stress which can be reduced with anti-oxidant treatment or by raising BH4 levels using Dihydrofolate Reductase (DHFR) overexpression, an enzyme that converts BH2, the oxidised form of BH4, back to BH4. Our work dissects a novel mechanism tuning the response of AP-1 to increasing levels of ROS.