Final Report Summary - DYNA-MITO IN THYMUS (Role of mitochondria-shaping proteins in T cell development and migration in vivo.) SUMMARY A correct repertoire of functional and mature T cells in the organism depends on different selective processes that take place in the Thymus. In particular, the so called Activation-Induced Cell Death (AICD), defined as a second stimulation of the T cell receptor (TCR), is present at almost every step of the T cell development and maturation by removing unfunctional cells through apoptosis. Evidence is accumulating on the role of mitochondrial morphology and of autophagy, a key cellular pro-survival mechanism of response to stress in T cell physiology, but whether these processes participate in AICD was still unclear.In this work, we show that AICD indeed depends on early mitochondrial fragmentation and inhibition of autophagy. Mitochondrial fragmentation is one of the earliest hallmarks of AICD and its genetic inhibition reduces cytochrome c release and death. Interestingly, during AICD, fragmentation cross-talks with autophagy, which is molecularly inhibited, leading to accumulation of fragmented dysfunctional mitochondria. Of note, pharmacological induction of autophagy counteracts the pro-apoptotic features, and protect the cells from death. Accordingly, AICD is increased in T cells from mouse models where key components of the autophagic machinery have been ablated. In conclusion, efficient AICD and thus efficient lymphocyte maturation depend on the accumulation of fragmented and dysfunctional mitochondria that are excluded from autophagy after restimulation of TCR, and that lead "undesired" and dangerous T cells to death.INTRODUCTION AND AIM OF THE WORK Mitochondria are highly dynamic organelles that continuously move, divide and fuse in a highly regulated fashion. The balance between the opposing processes of mitochondrial fusion and fission is controlled by a growing family of "mitochondria-shaping" proteins. Evidence is accumulating on the role of these proteins in several functions, from apoptosis to Ca2+ signaling, to regulation of migration of leukocytes. The knowledge of the regulation of mitochondrial dynamics, as well as its impact on tissue development and homeostasis is currently limited. The process Activation-Induced Cell Death (AICD) is necessary and crucial to control the development of lymphocytes and to maintain their homeostasis and peripheral tolerance. It is induced by re-stimulation of the T cell receptor (TCR) in pre-activated T cells and culminates in a physiological programmed cell death. This process is present at almost every step of the T cell development and maturation, thus the identification of the role of mitochondrial dynamics in this process would be crucial on understanding the regulation of T cell development. Based on the evidence that mitochondrial fragmentation could play a crucial role in the modulation of AICD (mitochondria-derived ROS are key mediators of AICD and production of ROS from mitochondria has been functionally linked to DRP1-dependent fragmentation of the organelle), we hypothesized that changes in mitochondrial morphology could play a key role in the amplification of AICD and thus could be crucial events in determining T cell development.RESULTS In order to analyze whether or not mitochondrial shape changes during AICD we used Jurkat cells (a good model for AICD in vitro studies: they are able to undergo apoptosis by direct cross-linking of CD3) and primary T cells isolated from mouse thymi or spleens. All the results here elucidated refer for simplicity to Jurkat cells but they were obtained in both sample types. A time-course of AICD induction in cells transfected with the fluorescent protein targeted to mitochondria mtYFP, followed by the analysis of the organelle morphology by confocal microscopy, showed indeed a TCR-activation-specific mitochondrial fragmentation early in the process of AICD, before the onset of any detectable death (24h after cross-linking of CD3, while the decrease in viability is between 28h and 48h). This Ca2+-Drp1-dependent fragmentation is accompanied by another morphological alteration in the ultrastructure of mitochondria during AICD: a significant disorganisation of the mitochondrial cristae called "cristae-remodeling". Together, these modifications lead to the release of the majority of the Cytochrome c usually stored in the cristae and to the activation of caspases and amplification of cell death. We observed that the two morphological alterations are crucial in the modulation of AICD, since their genetic inhibition (through overexpression of the pro-fusion mitochondria-shaping proteins Opa1 and the dominant negative Drp1-K38A) rescues the normal morphology and ultrastructure of the mitochondrial network but, more interestingly, it reduces the release of cytochrome c and cell death.Thus, we observed that during AICD there is an accumulation of fragmented dysfunctional mitochondria that leads the cell to its fatal destiny. This is possible since autophagy, a key pro-survival mechanism of response to stress that usually protect the cell by removal of damaged mitochondria, important and active in T cell physiology, is efficiently inhibited early during AICD. Indeed, we could observe a reduction in the activity of some autophagic key players around 4-6 hours from AICD induction in Jurkat cells. On the contrary, pharmacological induction of autophagy with Rapamycin counteracts the pro-apoptotic AICD features, and protects the cells from death through clearance of damaged mitochondria and block of cytochrome c release. Accordingly, AICD is increased in T cells from mouse models where key components of the autophagic machinery, such as Ambra1 and Beclin 1, have been ablated. During thymic development, early T cells progenitors arriving from the blood stream undergo a series of migration, proliferation and differentiation events in the thymus before returning to the circulation as mature T cells, defined by the acquisition of maturation markers such as CD4, CD8 and the T cell receptor complex (TCR). In this way, it is possible to distinguish thymocytes subpopulations corresponding to different maturation stages [immature CD4-CD8-double-negative (DN), immature CD4+ CD8+ double-positive (DP), and CD4+ CD8-and CD4-CD8+ single-positive (SP) ]. In thymocytes isolated from mice lacking the pro-fission protein Drp1 (transgenic mice with Drp1 depleted only in T cells), we observed a significant reduction of DP cells compared to control wild-type mice. This discrepancy is rescued in later stages of development (SP cells), where we cannot detect any difference between the two phenotypes. Since this step of maturation is characterised by a "positive selection" process, where AICD is crucial (where not correctly developed cells are removed through apoptosis), we think that this difference is due to a non-functional AICD because of the Drp1 deficiency. Thus, the unfunctional developing cells would not be correctly removed. This point needs anyway a deeper analysis.In conclusion, we discovered how an efficient AICD, and thus efficient lymphocyte selection and maturation, depend on the accumulation of fragmented and dysfunctional mitochondria that are excluded from autophagy after restimulation of TCR. This, in turn, allows the elimination of all the "undesired" T cells that, at the end of their maturation, would result unfunctional or self-reactive, and highly dangerous for the organism once in periphery. We finally unraveled a new crucial role for mitochondrial dynamics and autophagy in a cross-talk of AICD modulation.These findings have a high impact in the socio-economic and health system community, since mitochondria and mitochondrial dynamics could be veiled as an important therapeutic target to modulate T cell development and function in common autoimmune diseases, graft-versus-host diseases and infections. We identified a potential role of mitochondrial dynamics on modulating deletion of auto-reactive and unfunctional T cells. By this approach, we unraveled a field of research completely unexplored yet, that will permit to modulate the peripheral tolerance state of an organism. This could, in the long term, allow for the identification of novel therapeutic tools to interfere with common autoimmune diseases, as Multiple Sclerosis, by targeting mitochondrial dynamics for therapy development against these diseases
