Deletion of the mitochondria-shaping protein Opa1 during early thymocyte maturation impacts mature memory T cell metabolism.

Optic atrophy 1 (OPA1), a mitochondria-shaping protein controlling cristae biogenesis and respiration, is required for memory T cell function, but whether it affects intrathymic T cell development is unknown. Here we show that OPA1 is necessary for thymocyte maturation at the double negative (DN)3 stage when rearrangement of the T cell receptor ß (Tcrß) locus occurs. By profiling mitochondrial function at different stages of thymocyte maturation, we find that DN3 cells rely on oxidative phosphorylation. Consistently, Opa1 deletion during early T cell development impairs respiration of DN3 cells and reduces their number. Opa1-deficient DN3 cells indeed display stronger TCR signaling and are more prone to cell death. The surviving Opa1-/- thymocytes that reach the periphery as mature T cells display an effector memory phenotype even in the absence of antigenic stimulation but are unable to generate metabolically fit long-term memory T cells. Thus, mitochondrial defects early during T cell development affect mature T cell function.

Developmental and Tumor Angiogenesis Requires the Mitochondria-Shaping Protein Opa1.

While endothelial cell (EC) function is influenced by mitochondrial metabolism, the role of mitochondrial dynamics in angiogenesis, the formation of new blood vessels from existing vasculature, is unknown. Here we show that the inner mitochondrial membrane mitochondrial fusion protein optic atrophy 1 (OPA1) is required for angiogenesis. In response to angiogenic stimuli, OPA1 levels rapidly increase to limit nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) signaling, ultimately allowing angiogenic genes expression and angiogenesis. Endothelial Opa1 is indeed required in an NFκB-dependent pathway essential for developmental and tumor angiogenesis, impacting tumor growth and metastatization. A first-in-class small molecule-specific OPA1 inhibitor confirms that EC Opa1 can be pharmacologically targeted to curtail tumor growth. Our data identify Opa1 as a crucial component of physiological and tumor angiogenesis.

The pro-oxidant adaptor p66SHC promotes B cell mitophagy by disrupting mitochondrial integrity and recruiting LC3-II.

Macroautophagy/autophagy has emerged as a central process in lymphocyte homeostasis, activation and differentiation. Based on our finding that the p66 isoform of SHC1 (p66SHC) pro-apoptotic ROS-elevating SHC family adaptor inhibits MTOR signaling in these cells, here we investigated the role of p66SHC in B-cell autophagy. We show that p66SHC disrupts mitochondrial function through its CYCS (cytochrome c, somatic) binding domain, thereby impairing ATP production, which results in AMPK activation and enhanced autophagic flux. While p66SHC binding to CYCS is sufficient for triggering apoptosis, p66SHC-mediated autophagy additionally depends on its ability to interact with membrane-associated LC3-II through a specific binding motif within its N terminus. Importantly, p66SHC also has an impact on mitochondria homeostasis by inducing mitochondrial depolarization, protein ubiquitination at the outer mitochondrial membrane, and local recruitment of active AMPK. These events initiate mitophagy, whose full execution relies on the role of p66SHC as an LC3-II receptor which brings phagophore membranes to mitochondria. Importantly, p66SHC also promotes hypoxia-induced mitophagy in B cells. Moreover, p66SHC deficiency enhances B cell differentiation to plasma cells, which is controlled by intracellular ROS levels and the hypoxic germinal center environment. The results identify mitochondrial p66SHC as a novel regulator of autophagy and mitophagy in B cells and implicate p66SHC-mediated coordination of autophagy and apoptosis in B cell survival and differentiation. Abbreviations: ACTB: actin beta; AMPK: AMP-activated protein kinase; ATP: adenosine triphosphate; ATG: autophagy-related; CYCS: cytochrome c, somatic; CLQ: chloroquine; COX: cyclooxygenase; CTR: control; GFP: green fluorescent protein; HIFIA/Hif alpha: hypoxia inducible factor 1 subunit alpha; IMS: intermembrane space; LIR: LC3 interacting region; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR/mTOR: mechanistic target of rapamycin kinase; OA: oligomycin and antimycin A; OMM: outer mitochondrial membrane; PHB: prohibitin; PBS: phosphate-buffered saline; PINK1: PTEN induced putative kinase 1; RFP: red fluorescent protein; ROS: reactive oxygen species; SHC: src Homology 2 domain-containing transforming protein; TMRM: tetramethylrhodamine, methyl ester; TOMM: translocase of outer mitochondrial membrane; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.