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

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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.