REDOX CONTROL OF THE SYNAPTIC VESICLE CYCLE

Redox balance plays a crucial role in the development of several pathological conditions, including cancer, cardiovascular diseases, and neurodegenerative disorders. The Thioredoxin/Thioredoxin Reductase (Trx/TrxR) system is a key antioxidant defense involved in cellular homeostasis, and its dysfunction has been associated with synaptic failure and neurodegenerative diseases, such as Alzheimer’s, Parkinson’s and Huntington’s diseases. However, the molecular mechanisms linking redox control to synaptic vesicle (SV) cycling remain poorly understood. This thesis investigated the role of cytosolic Thioredoxin 1 (Trx1) at presynaptic terminals, using synaptosomes as an experimental model, with particular focus on the SV cycle. A mechanism-based kinetic trapping approach was performed in synaptosomes and combined with mass spectrometry to identify neuronal proteins interacting with Trx1 through mixed disulphide bonds. Known targets, such as peroxiredoxins, were confirmed, while previously uncharacterized candidates involved in SV trafficking and neurotransmission were identified. The effect of Trx/TrxR inhibition on SV trafficking were assessed through a synaptotagmin uptake assay using PX-12, a Trx inhibitor, and Ebselen, a dual inhibitor of the Trx/TrxR system. In addition, a glutamate assay was performed to evaluate neurotransmitter release under stimulation and in response to treatment with Auranofin, a TrxR inhibitor. Finally, Trx1 redox states were examined by redox WB, while TrxR enzymatic activity was evaluated using a DTNB-based assay. Together, these findings support the hypothesis that the Trx/TrxR system acts as a redox-dependent regulator of SV cycling and neurotransmitter release, providing new insight into how altered redox homeostasis may contribute to early synaptic dysfunction in neurodegeneration and age-related brain disorders.