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

神经生物学现有的知识缺口：我和我的HQP研究了硫氧还蛋白（Trx）系统在神经元氧化应激（OS）管理中的作用。高水平的活性氧（ROS）在神经系统使神经元蛋白过度倾向于氧化损伤。溶酶体自噬是旧/氧化蛋白和受损细胞器的循环机制之一，然而，严重的OS导致自噬中断和细胞凋亡的促进。Trx1是一种细胞质蛋白，通过直接的蛋白-蛋白相互作用在挽救氧化蛋白中起重要作用。Trx1在此过程中被氧化，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Trx1的抑制可能作为抑制溶酶体自噬和诱导细胞凋亡的双向开关。超过30种蛋白质参与自噬的调节，然而，我们对Trx1系统与神经元中自噬相关蛋白的潜在相互作用的理解仍然很大程度上是未知的。我的长期研究目标是研究Trx系统在自噬-细胞凋亡串扰中的作用。我们已经为这项研究建立了必要的实验模型和方案。国际合作使我们能够获得Trx1敲除（KO）动物，这对这些短期计划在前五年至关重要：1)研究Trx和TrxR抑制/过表达对OS反应中神经元自噬的影响。2)探讨Trx在神经元自噬调节的性别差异中的作用。3)确定Trx与神经元正常和缺氧状态下自噬机制之间的潜在相互作用。将使用来自野生型、TrxKO和神经元特异性KO动物的转基因SH-SY5Y神经元和性别特异性神经元原代培养物。神经元将暴露于营养应激或缺氧条件下，自噬和凋亡标志物的表达变化将被检测。trx介导的氧化蛋白的还原，是通过瞬时和快速蛋白介导的。在Trx1活性位点产生点突变可导致稳定的Trx靶标结合。免疫沉淀法和质量分光光度法将使我们能够识别这样的底物。影响：本研究将研究神经元保护性自噬未被探索的方面，目的是发现Trx1与自噬相关蛋白之间新的分子相互作用。结合生物化学、分子生物学、细胞学和相关动物模型，研究神经元对OS反应的性别差异的分子基础。本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，并将扩展我们在神经生物学领域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.