Generation of mitochondrial reactive oxygen species is controlled by ATPase inhibitory factor 1 and regulates cognition.

The mitochondrial ATP synthase emerges as key hub of cellular functions controlling the production of ATP, cellular signaling, and fate. It is regulated by the ATPase inhibitory factor 1 (IF1), which is highly abundant in neurons. Herein, we ablated or overexpressed IF1 in mouse neurons to show that IF1 dose defines the fraction of active/inactive enzyme in vivo, thereby controlling mitochondrial function and the production of mitochondrial reactive oxygen species (mtROS). Transcriptomic, proteomic, and metabolomic analyses indicate that IF1 dose regulates mitochondrial metabolism, synaptic function, and cognition. Ablation of IF1 impairs memory, whereas synaptic transmission and learning are enhanced by IF1 overexpression. Mechanistically, quenching the IF1-mediated increase in mtROS production in mice overexpressing IF1 reduces the increased synaptic transmission and obliterates the learning advantage afforded by the higher IF1 content. Overall, IF1 plays a key role in neuronal function by regulating the fraction of ATP synthase responsible for mitohormetic mtROS signaling.

Ablation of Atp5if1 impairs metabolic reprogramming and proliferation of T lymphocytes and compromises mouse survival.

T cells experience metabolic reprogramming to an enhanced glycolysis upon activation. Herein, we have investigated whether ATPase Inhibitory Factor 1 (IF1), the physiological inhibitor of mitochondrial ATP synthase, participates in rewiring T cells to a particular metabolic phenotype. We show that the activation of naive CD4+ T lymphocytes both in vitro and in vivo is accompanied by a sharp upregulation of IF1, which is expressed only in Th1 effector cells. T lymphocytes of conditional CD4+-IF1-knockout mice display impaired glucose uptake and flux through glycolysis, reducing the biogenesis of mitochondria and cellular proliferation after activation. Consequently, mice devoid of IF1 in T lymphocytes cannot mount an effective Th1 response against bacterial infection compromising their survival. Overall, we show that the inhibition of a fraction of ATP synthase by IF1 regulates metabolic reprogramming and functionality of T cells, highlighting the essential role of IF1 in adaptive immune responses.

The splicing factor SF3B1 is involved in brown adipocyte thermogenic activation.

The ability of alternative splicing mechanisms to control gene expression is increasingly being recognized as relevant for adipose tissue function. The expression of SF3B1, a key component of the SF3B complex directly involved in spliceosome formation, was previously reported to be significantly induced in brown adipose tissue under cold-induced thermogenic activation. Here, we identify that noradrenergic cAMP-mediated thermogenic stimulation increases SF3B1 expression in brown and beige adipocytes. We further show that pladienolide-B, a drug that binds SF3B1 to inhibit pre-mRNA splicing by targeting the SF3B complex, down-regulates key components of the thermogenic machinery (e.g., UCP1 gene expression), differentially alters the expression of alternative splicing-regulated transcripts encoding molecular actors involved in the oxidative metabolism of brown adipocytes (e.g., peroxisome proliferator-activated receptor-gamma co-activator-alpha [PGC-1α] and cytochrome oxidase subunit 7a genes), and impairs the respiratory activity of brown adipocytes. Similar alterations were found in brown adipocytes with siRNA-mediated knockdown of SF3B1 protein levels. Our findings collectively indicate that SF3B1 is a key factor in the appropriate thermogenic activation of differentiated brown adipocytes. This work exemplifies the importance of splicing processes in adaptive thermogenesis and suggests that pharmacological tools, such as pladienolide-B, may be used to modulate brown adipocyte thermogenic activity.

IF1 promotes oligomeric assemblies of sluggish ATP synthase and outlines the heterogeneity of the mitochondrial membrane potential.

The coexistence of two pools of ATP synthase in mitochondria has been largely neglected despite in vitro indications for the existence of reversible active/inactive state transitions in the F1-domain of the enzyme. Herein, using cells and mitochondria from mouse tissues, we demonstrate the existence in vivo of two pools of ATP synthase: one active, the other IF1-bound inactive. IF1 is required for oligomerization and inactivation of ATP synthase and for proper cristae formation. Immunoelectron microscopy shows the co-distribution of IF1 and ATP synthase, placing the inactive "sluggish" ATP synthase preferentially at cristae tips. The intramitochondrial distribution of IF1 correlates with cristae microdomains of high membrane potential, partially explaining its heterogeneous distribution. These findings support that IF1 is the in vivo regulator of the active/inactive state transitions of the ATP synthase and suggest that local regulation of IF1-ATP synthase interactions is essential to activate the sluggish ATP synthase.

IF1 ablation prevents ATP synthase oligomerization, enhances mitochondrial ATP turnover and promotes an adenosine-mediated pro-inflammatory phenotype.

ATPase Inhibitory Factor 1 (IF1) regulates the activity of mitochondrial ATP synthase. The expression of IF1 in differentiated human and mouse cells is highly variable. In intestinal cells, the overexpression of IF1 protects against colon inflammation. Herein, we have developed a conditional IF1-knockout mouse model in intestinal epithelium to investigate the role of IF1 in mitochondrial function and tissue homeostasis. The results show that IF1-ablated mice have increased ATP synthase/hydrolase activities, leading to profound mitochondrial dysfunction and a pro-inflammatory phenotype that impairs the permeability of the intestinal barrier compromising mouse survival upon inflammation. Deletion of IF1 prevents the formation of oligomeric assemblies of ATP synthase and alters cristae structure and the electron transport chain. Moreover, lack of IF1 promotes an intramitochondrial Ca2+ overload in vivo, minimizing the threshold to Ca2+-induced permeability transition (mPT). Removal of IF1 in cell lines also prevents the formation of oligomeric assemblies of ATP synthase, minimizing the threshold to Ca2+-induced mPT. Metabolomic analyses of mice serum and colon tissue highlight that IF1 ablation promotes the activation of de novo purine and salvage pathways. Mechanistically, lack of IF1 in cell lines increases ATP synthase/hydrolase activities and installs futile ATP hydrolysis in mitochondria, resulting in the activation of purine metabolism and in the accumulation of adenosine, both in culture medium and in mice serum. Adenosine, through ADORA2B receptors, promotes an autoimmune phenotype in mice, stressing the role of the IF1/ATP synthase axis in tissue immune responses. Overall, the results highlight that IF1 is required for ATP synthase oligomerization and that it acts as a brake to prevent ATP hydrolysis under in vivo phosphorylating conditions in intestinal cells.

The ATPase Inhibitory Factor 1 is a Tissue-Specific Physiological Regulator of the Structure and Function of Mitochondrial ATP Synthase: A Closer Look Into Neuronal Function.

The ATP synthase is an essential multifunctional enzyme complex of mitochondria that produces most of cellular ATP, shapes the structure of the inner membrane into cristae and regulates the signals that control cell fate or demise. The ATPase Inhibitory Factor 1 (IF1) functions in vivo as a physiological regulator of the ATP synthase and thereby controls mitochondrial structure and function, and the retrograde signaling pathways that reprogram nuclear gene expression. However, IF1 is not ubiquitously expressed in mammals, showing tissue-restricted expression in humans and mice and large expression differences between the two species in some tissues. Herein, we summarized key regulatory functions of IF1 for tissue homeostasis, with special emphasis on the deleterious effects that its genetic ablation in neurons has in learning. The development and characterization of tissue-specific mouse models with regulated expression of IF1 will be crucial to disentangle the contribution of the ATP synthase/IF1 axis in pathophysiology.

Analysis of the metabolic proteome of lung adenocarcinomas by reverse-phase protein arrays (RPPA) emphasizes mitochondria as targets for therapy.

Lung cancer is the leading cause of cancer-related death worldwide despite the success of therapies targeting oncogenic drivers and immune-checkpoint inhibitors. Although metabolic enzymes offer additional targets for therapy, the precise metabolic proteome of lung adenocarcinomas is unknown, hampering its clinical translation. Herein, we used Reverse Phase Protein Arrays to quantify the changes in enzymes of glycolysis, oxidation of pyruvate, fatty acid metabolism, oxidative phosphorylation, antioxidant response and protein oxidative damage in 128 tumors and paired non-tumor adjacent tissue of lung adenocarcinomas to profile the proteome of metabolism. Steady-state levels of mitochondrial proteins of fatty acid oxidation, oxidative phosphorylation and of the antioxidant response are independent predictors of survival and/or of disease recurrence in lung adenocarcinoma patients. Next, we addressed the mechanisms by which the overexpression of ATPase Inhibitory Factor 1, the physiological inhibitor of oxidative phosphorylation, which is an independent predictor of disease recurrence, prevents metastatic disease. We highlight that IF1 overexpression promotes a more vulnerable and less invasive phenotype in lung adenocarcinoma cells. Finally, and as proof of concept, the therapeutic potential of targeting fatty acid assimilation or oxidation in combination with an inhibitor of oxidative phosphorylation was studied in mice bearing lung adenocarcinomas. The results revealed that this therapeutic approach significantly extended the lifespan and provided better welfare to mice than cisplatin treatments, supporting mitochondrial activities as targets of therapy in lung adenocarcinoma patients.