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

神经生物学方面现有的知识差距：我和我的HQP研究硫氧还蛋白(TRX)系统在管理神经元氧化应激(OS)中的作用。神经系统中高水平的活性氧物种(ROS)使神经元蛋白质极易受到氧化损伤。溶酶体自噬是陈旧/氧化蛋白和受损细胞器的再循环机制之一，但严重的OS会导致自噬中断，促进细胞凋亡。Trx1是一种细胞质蛋白，通过蛋白质与蛋白质的直接相互作用，在抢救氧化蛋白中发挥重要作用。Trx1在这个过程中被氧化，但可以被Trx Reductase1(TrxR1)还原。*我的团队的研究强调了新的机制，表明抑制Trx1可能是一个双向开关，抑制溶酶体自噬并诱导细胞凋亡。超过30个蛋白质参与自噬的调节，然而，我们对Trx1系统与神经元中与自噬相关的蛋白质之间的潜在相互作用的了解仍然很少。*我的研究计划的长期目标是研究Trx系统在自噬-凋亡串扰中的作用。为本研究建立了必要的实验模型和实验方案。国际合作使我们能够获得在最初五年中对这些短期计划至关重要的Trx1基因敲除(KO)动物：*1)检测Trx的影响，以及TrxR抑制/过度表达对神经元自噬的响应。*2)研究TRX在调节神经元自噬中的性别差异中的作用。*3)确定正常和OS条件下神经元中Trx和自噬机制之间的潜在相互作用。*将使用转基因SH-SY5Y神经元和来自野生型、TrxKO和神经元特异性KO动物的神经元的性别特异性原代培养。神经元将暴露在营养应激或低氧条件下，自噬和凋亡标记物的表达变化将被检测。Trx介导的氧化蛋白质的还原，是通过一种瞬时的、快速的蛋白质-蛋白质来调节的。在Trx1活性部位产生点突变可以导致稳定的Trx-靶结合。免疫沉淀和质量分光光度将使我们能够识别这些底物。*影响：这项研究将检查神经元保护性自噬的未知方面，目的是找到Trx1和自噬相关蛋白之间的新的分子相互作用。我们的研究将结合生物化学、分子生物学、细胞学和相关动物模型，探讨神经元对OS反应的性别差异的分子基础。这项拟议的研究将为至少两名研究生(硕士、博士)和五名本科生提供丰富的培训环境，并将扩大我们在神经生物学领域关于氧化还原平衡的基础知识。**

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. **