Do extracellularly released mitochondrial transcription factor A and cytochrome C function as intercellular signaling molecules of the brain?

大脑包含神经元和被称为神经胶质的非神经元细胞。神经胶质细胞对周围环境中出现的特定刺激有不同的反应。在某些情况下，神经胶质细胞可以分泌对神经元有益的分子，而在其他时候，它们会释放有毒物质。因此，识别诱导胶质细胞不同类型生物活性的分子，了解胶质细胞活化的分子机制具有重要意义。对于正常生理条件下发生在大脑中的神经元和神经胶质之间以及神经胶质细胞之间的信号传导（细胞间信号传导）知之甚少；因此，我的nserc资助的研究项目的长期目标是通过表征以前未识别的大脑细胞间信号分子并研究它们的生理作用来解决这一知识差距。我之前的NSERC发现项目的工作直接表明，细胞内通常发现的两种蛋白质，线粒体转录因子A （Tfam）和细胞色素C (cytC)，在释放到细胞外空间后会影响胶质细胞的功能，这可能是由细胞刺激或损伤引起的。我未来五年的研究计划的核心目标是研究Tfam和cytC在调节胶质细胞活性中的作用。我们的工作假设是，这两种线粒体蛋白可以作为关键的信号分子，通过特定的分子机制诱导神经胶质细胞的激活。我们计划：1)表征Tfam对大脑两种主要胶质细胞类型（星形胶质细胞和小胶质细胞）的影响。我们已经制备了人重组Tfam蛋白及其截短形式，这将使我们能够确定Tfam分子中负责与胶质细胞相互作用的部分。我们将使用人类胶质样细胞系和培养的原代胶质细胞来测量不同类型的细胞对Tfam的反应（如细胞因子和活性氧的产生）。我们是加拿大为数不多的实验室之一，拥有准备成人和猪神经胶质细胞培养模型的专业知识。实验还将进行，将Tfam注射到动物大脑中，研究神经胶质细胞对这种刺激的反应。2)使用类似的策略，研究细胞外细胞tc对小胶质细胞和星形胶质细胞的影响。我希望上述研究能够证实和扩展我们对Tfam和cytC激活神经胶质的初步观察。我还预计，我们将确定关键的胶质受体(s)和细胞内信号通路参与Tfam和cytC，以及这些蛋白质对一系列生物活性分子（细胞因子）的分泌的影响，已知影响免疫和脑功能。3)利用蛋白质组学技术比较细胞外施加Tfam和cytC时胶质细胞分泌的蛋白质混合物的整体变化。我期望获得的信息将对评估这两个候选信号分子对神经胶质功能的总体影响至关重要。可以确定暴露于Tfam和cytC的胶质细胞释放的其他特定信号分子。拟议的研究计划将建立一个以前未探索的研究途径，涉及线粒体蛋白在动物大脑中作为细胞间信号分子的作用，这将促进我们对大脑功能的理解。通过这个研究项目产生的关于细胞间信号分子的大脑网络的基础知识，可以通过识别改变或改善大脑功能的分子靶标来实现实际应用。

The brain contains neurons and non-neuronal cells called glia. Glial cells respond differently to particular stimuli appearing in their surrounding environment. In some instances, glia can secrete molecules that are beneficial to neurons, while at other times they release toxic substances. Therefore, it is important to identify molecules that induce different types of biological activity of glia and to understand the molecular mechanisms of glial activation. Very little is known about the signaling between neurons and glia as well as between glial cells themselves (intercellular signaling) that takes place in the brain under normal physiological conditions; therefore, the LONG-TERM OBJECTIVE of my NSERC-funded research program is to address this knowledge gap by characterizing previously unidentified intercellular signaling molecules in the brain and studying their physiological roles. Work directly stemming from my previous NSERC Discovery Program shows that two of the proteins that are normally found inside the cells, mitochondrial transcription factor A (Tfam) and cytochrome C (cytC), can affect glial functions after their release into the extracellular space, which can be caused by cell stimulation or damage. The CORE GOAL of my research program for the next five years is to examine the role of Tfam and cytC in regulating activity of glial cells. It is our working hypothesis that these two mitochondrial proteins can function as critical signaling molecules capable of inducing activation of glial cells via specific molecular mechanisms. We are planning to: 1) Characterize the effects of Tfam on the two main glial cell types of the brain (astrocytes and microglia). We have already prepared human recombinant Tfam protein as well as its truncated forms, which will allow us to identify the part of the Tfam molecule responsible for its interaction with glia. We will measure different types of cellular responses (such as production of cytokines and reactive oxygen species) to Tfam by using human glia-like cell lines as well as cultured primary glia. We are among the very few laboratories in Canada who possess expertise to prepare adult human and porcine glial cell culture models. Experiments will also be performed where Tfam will be injected into animal brains to study the responses of glial cells to this stimulus. 2) Using a similar strategy, examine the effects of extracellular cytC on microglia and astrocytes. I expect that the above studies will confirm and extend our preliminary observations of glial activation by Tfam and cytC. I also anticipate that we will identify the key glial receptor(s) and intracellular signaling pathways engaged by Tfam and cytC as well as the effects of these proteins on the secretion of a range of bioactive molecules (cytokines) that are known to affect immune and brain functions. 3) Employ proteomics techniques to compare the global change in the mixture of proteins secreted by glial cells in response to extracellularly applied Tfam and cytC. I expect that the obtained information will be critical for assessing the overall effect of these two candidate signaling molecules on glial function. Additional specific signaling molecules that are released by glia exposed to Tfam and cytC could be identified. The proposed research program will establish a previously unexplored avenue of research related to the role of mitochondrial proteins as intercellular signaling molecules in the animal brain, which will advance our understanding of brain function. Fundamental knowledge about the brain network of intercellular signaling molecules generated by this research program could lead to practical applications through identification of molecular targets for altering or improving brain function.