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

神经生物学知识的现有差距：我的HQP和我研究硫氧还蛋白（Trx）系统在神经元氧化应激（OS）管理中的作用。神经系统中高水平的活性氧（ROS）使神经元蛋白过度倾向于氧化损伤。溶酶体自噬是旧的/氧化的蛋白质和受损细胞器的再循环机制之一，然而，严重的OS导致自噬中断并促进凋亡。Trx 1是一种细胞质蛋白，通过直接的蛋白质-蛋白质相互作用在拯救氧化蛋白质中发挥重要作用。Trx 1在此过程中被氧化，但可以被Trx还原酶1（TrxR 1）还原。我的团队的研究强调了新的机制，表明抑制Trx 1可能作为一个双向开关，抑制溶酶体自噬和诱导凋亡。自噬的调控蛋白有30多种，但Trx 1系统与神经元自噬相关蛋白的相互作用尚不清楚。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。我们已经建立了必要的实验模型和协议，这项研究。国际合作已经使我们能够接收Trx 1敲除（KO）动物，这对于前五年期间的这些短期计划是必不可少的：1）检查Trx和TrxR抑制/过表达对响应于OS的神经元自噬的影响。2）研究Trx在神经元自噬调节中的性别依赖性差异中的作用。 3)确定在正常和OS条件下神经元中Trx和自噬机制之间的潜在相互作用。 将使用遗传修饰的SH-SY 5 Y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物。将神经元暴露于营养应激或缺氧条件，并检查自噬和凋亡标志物表达的变化。Trx介导的氧化蛋白质的还原是通过瞬时和快速的蛋白质-蛋白质介导的。在Trx 1活性位点产生点突变可导致稳定的Trx-靶标结合。免疫沉淀和质谱法将使我们能够识别这样的底物。影响力：这项研究将研究神经元中保护性自噬的未探索方面，目的是寻找Trx 1和自噬相关蛋白之间的新型分子相互作用。使用生物化学，分子生物学，细胞学和相关的动物模型相结合，我们的研究将调查神经元对OS的反应的性别差异的分子基础。这项拟议的研究将提供一个丰富的培训环境，至少有两个研究生（硕士，博士），和五个本科生，并将扩大我们在神经生物学领域的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.