Role of Thioredoxin system in regulation of autophagy-apoptosis cross talk in neurons: Uncovering Novel Molecular Interactions.

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The existing gap of knowledge in neurobiology: My HQP and I investigate the role of Thioredoxin (Trx) system in management of oxidative stress (OS) in neurons. High levels of reactive oxygen species (ROS) in the nervous system renders the neuronal proteins excessively prone to oxidative damage. Lysosomal autophagy is one of the recycling mechanisms for old/oxidized proteins and damaged organelles, however, severe OS leads to interruption of autophagy and promotion of apoptosis. Trx1 is a cytoplasmic protein that plays an important role in rescuing the oxidized proteins, through direct protein-protein interaction. Trx1 is oxidized in this process but can be reduced by Trx Reductase1 (TrxR1). Research by my team has highlighted novel mechanisms indicating that inhibition of Trx1 may work as a two-way switch that inhibits lysosomal autophagy and induces apoptosis. More than thirty proteins are involved in regulation of autophagy, however, our understanding from potential interaction of Trx1 system with the autophagy related proteins in neurons remain largely unknown. The long-term objective in my research program is to examine the role of Trx system in autophagy-apoptosis cross talk. We have established the necessary experimental models and protocols for this research. International collaborations have enabled us to receive Trx1 knockout (KO) animals that will be essential for these short-term plans during the first five years: 1) To examine the effect of Trx, and TrxR inhibition/ overexpression on neuronal autophagy in response to OS. 2) To investigate the role of Trx in sex- dependent differences in regulation of neuronal autophagy. 3) To identify the potential interaction between Trx and autophagy machinery in normal and OS conditions in neurons. Genetically modified SH-SY5Y neurons and sex specific primary cultures of neurons from wildtype, TrxKO and neuron-specific KO animals will be used. Neurons will be exposed to nutritional stress or hypoxic conditions and changes in expression of autophagy and apoptosis markers will be examined. Trx-mediated reduction of oxidized proteins, is mediated through a transient and fast protein-protein. Generation of a point mutation in Trx1 active site can result in stable Trx- target binding. Immunoprecipitation and mass spectrophotometry will enable us to identify such substrates. Impact: This research will examine the unexplored aspects of protective autophagy in neurons with the goal of finding novel molecular interactions between Trx1 and the autophagy related proteins. Using a combination of biochemistry, molecular biology, cytology and related animal models, our research will investigate the molecular basis of gender differences in neuronal response to OS. This proposed research will provide a rich training environment for at least two graduate (MSc, PhD), and five undergraduate students and will expand our basic knowledge of redox balance in the field of neurobiology.