Expression of Oxidative Phosphorylation Complexes and Mitochondrial Mass in Pediatric and Adult Inflammatory Bowel Disease

Introduction. Inflammatory bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis (UC), is a multifactorial intestinal disorder but its precise etiology remains elusive. As the cells of the intestinal mucosa have high energy demands, mitochondria may play a role in IBD pathogenesis. The present study is aimed at evaluating the expression levels of mitochondrial oxidative phosphorylation (OXPHOS) complexes in IBD. Material and Methods. 286 intestinal biopsy samples from the terminal ileum, ascending colon, and rectum from 124 probands (34 CD, 33 UC, and 57 controls) were stained immunohistochemically for all five OXPHOS complexes and the voltage-dependent anion-selective channel 1 protein (VDAC1 or porin). Expression levels were compared in multivariate models including disease stage (CD and UC compared to controls) and age (pediatric/adult). Results. Analysis of the terminal ileum of CD patients revealed a significant reduction of complex II compared to controls, and a trend to lower levels was evident for VDAC1 and the other OXPHOS complexes except complex III. A similar pattern was found in the rectum of UC patients: VDAC1, complex I, complex II, and complex IV were all significantly reduced, and complex III and V showed a trend to lower levels. Reductions were more prominent in older patients compared to pediatric patients and more marked in UC than CD. Conclusion. A reduced mitochondrial mass is present in UC and CD compared to controls. This is potentially a result of alterations of mitochondrial biogenesis or mitophagy. Reductions were more pronounced in older patients compared to pediatric patients, and more prominent in UC than CD. Complex I and II are more severely compromised than the other OXPHOS complexes. This has potential therapeutic implications, since treatments boosting biogenesis or influencing mitophagy could be beneficial for IBD treatment. Additionally, substances specifically stimulating complex I activity should be tested in IBD treatment.

The mitochondrial calcium uniporter regulates mitochondrial mass and dendritic localization in distinct hippocampal circuits

CA2 is an understudied subregion of the hippocampus that is critical for social memory. Previous studies identified multiple components of the mitochondrial calcium uniporter (MCU) complex as selectively enriched in CA2, however the functional significance of this enrichment remains unclear. The MCU complex regulates calcium entry into mitochondria, which in turn regulates mitochondrial transport and localization to active synapses. We found that MCU is strikingly enriched in CA2 distal apical dendrites, precisely where CA2 neurons receive entorhinal cortical input carrying social information. Further, MCU-enriched mitochondria in CA2 distal dendrites are larger compared to mitochondria in CA2 proximal apical dendrites and neighboring CA1 apical dendrites. Genetic knockdown of MCU in CA2 resulted in smaller mitochondria in CA2 distal dendrites, indicating that MCU expression plays a role in regulating mitochondrial mass in CA2. MCU overexpression in neighboring CA1 led to larger mitochondria preferentially in proximal dendrites compared to distal dendrites and GFP controls. Our findings demonstrate that mitochondria are molecularly and structurally diverse across hippocampal cell types and circuits, and that MCU expression cell-autonomously regulates mitochondrial mass, but layer-specific dendritic localization depends on cell type. Our data support the idea that CA2 mitochondria are functionally distinct from CA1 mitochondria, which may confer unique synaptic and circuit properties underlying CA2 function in social memory.

UVRAG-Knockdown Regulates Mitochondrial Autophagy in Chronic Myeloid Leukaemia Cells by Targeting BNIP3L

Objective: To explore whether UVRAG regulates mitochondrial autophagy via BNIP3L in K562 cellsMaterial and methods: We designed various assays to verify the relation between UVRAG and BNIP3L, we estabilished a mitochondrial autophagy model of K562 cells by CCCP, a mitochondrial autophagy inducer, and regulated the expression of UVRAG by cells transfection. Then we detected the expression of the BINP3L and autophagy-related proteins LC3-II/LC3-Ⅰ and P62 by Western blot. The changes of ROS, mitochondrial mass, and mitochondrial membrane potential (MMP) were detected by flow cytometry technology.Results: We found that CCCP could induce K562 cells mitochondrial autophagy, along with the change of MMP, mitochondrial mass and accumulation of ROS, also our experiment proved that UVRAG-Knockdown could reverse this phenomenon. Investigating the pathway of mitochondrial autophagy revealed UVRAG knockdown was accompanied by a decrease in BNIP3L and LC3 expression, a increase in P62 during mitochondrial autophagy. Conclusion: In our study, the results suggested that UVRAG may regulate mitochondrial autophagy of K562 cells via targeting BINP3L, which may be a potential target for the treatment of CML.

Oxidative Stress Induces Mitochondrial Compromise in CD4 T Cells From Chronically HCV-Infected Individuals

We have previously shown that chronic Hepatitis C virus (HCV) infection can induce DNA damage and immune dysfunctions with excessive oxidative stress in T cells. Furthermore, evidence suggests that HCV contributes to increased susceptibility to metabolic disorders. However, the underlying mechanisms by which HCV infection impairs cellular metabolism in CD4 T cells remain unclear. In this study, we evaluated mitochondrial mass and intracellular and mitochondrial reactive oxygen species (ROS) production by flow cytometry, mitochondrial DNA (mtDNA) content by real-time qPCR, cellular respiration by seahorse analyzer, and dysregulated mitochondrial-localized proteins by Liquid Chromatography-Mass Spectrometry (LC-MS) in CD4 T cells from chronic HCV-infected individuals and health subjects. Mitochondrial mass was decreased while intracellular and mitochondrial ROS were increased, expressions of master mitochondrial regulators peroxisome proliferator-activated receptor 1 alpha (PGC-1α) and mitochondrial transcription factor A (mtTFA) were down-regulated, and oxidative stress was increased while mitochondrial DNA copy numbers were reduced. Importantly, CRISPR/Cas9-mediated knockdown of mtTFA impaired cellular respiration and reduced mtDNA copy number. Furthermore, proteins responsible for mediating oxidative stress, apoptosis, and mtDNA maintenance were significantly altered in HCV-CD4 T cells. These results indicate that mitochondrial functions are compromised in HCV-CD4 T cells, likely via the deregulation of several mitochondrial regulatory proteins.

Acetoacetate protects macrophages from lactic acidosis-induced mitochondrial dysfunction by metabolic reprograming

Lactic acidosis, the extracellular accumulation of lactate and protons, is a consequence of increased glycolysis triggered by insufficient oxygen supply to tissues. Macrophages are able to differentiate from monocytes under such acidotic conditions, and remain active in order to resolve the underlying injury. Here we show that, in lactic acidosis, human monocytes differentiating into macrophages are characterized by depolarized mitochondria, transient reduction of mitochondrial mass due to mitophagy, and a significant decrease in nutrient absorption. These metabolic changes, resembling pseudostarvation, result from the low extracellular pH rather than from the lactosis component, and render these cells dependent on autophagy for survival. Meanwhile, acetoacetate, a natural metabolite produced by the liver, is utilized by monocytes/macrophages as an alternative fuel to mitigate lactic acidosis-induced pseudostarvation, as evidenced by retained mitochondrial integrity and function, retained nutrient uptake, and survival without the need of autophagy. Our results thus show that acetoacetate may increase tissue tolerance to sustained lactic acidosis.

Lack of IL6 Improves Recovery from Anemia of Inflammation Which Gets Hampered in Presence of Excess Iron

Anemia of inflammation (AI) is the second most common anemia after iron deficiency anemia. The predominant regulators of AI are the cytokine interleukin 6 (IL6) and the hormone hepcidin (HAMP). IL6 is an inflammatory cytokine which also limits iron absorption by inducing HAMP, which promotes the degradation of the iron exporter ferroportin. We hypothesized that knocking down both HAMP and IL6 simultaneously will help us to understand if their mode of action in AI is uniquely limited to iron absorption and erythroid iron intake or if they also have independent roles. Henceforth, we generated IL6/HampKO (DKO) mice and, unexpectedly, observed that IL6KO mice showed the best recovery in bone marrow (BM) erythropoiesis (using flow cytometry analysis and looking at the absolute number of erythroid progenitors) after BA administration when compared to wild type (WT), HampKO and DKO mice. The best differences were observed at 14 days post BA administration. In contrast, the extramedullary erythropoiesis in the spleen was more pronounced in HampKO and DKO mice compared to WT and IL6KO animals, indicating that the mechanism impairing erythropoiesis in the BM did not affect erythroid progenitors in the spleen. These observations suggest that HAMP and IL6 proteins contribute independently to AI, with IL6 having some effect on the erythropoiesis in the BM independent from the IL6-HAMP axis leading to iron restriction. Furthermore, these observations raised the question why both HampKO and DKO mice showed reduced BM erythropoiesis compared to IL6KO animals. We investigated inflammatory cytokines and altered iron parameters as potential mediators of impaired erythropoiesis. We compared several inflammatory cytokines, including IL6, TNFa and INFg following BA administration: cytokine levels were elevated 6 hrs, reduced 48hrs after BA administration and moderately increased again two weeks later. Interestingly, among all the cytokines the levels of IL1b were significantly attenuated in IL6KO mice at day 14 compared to WT and HampKO animals. Moreover, transferrin saturation and NTBI levels were higher in HampKO and DKO animals compared to IL6KO mice. These observations strongly suggested that BM erythropoiesis is more sensitive to inflammatory insult in presence of an excess of iron, while extramedullary erythropoiesis is mildly affected and can eventually thrive under supra-physiological transferrin saturation levels. To test if increased iron affects BM erythropoiesis in presence of inflammation, we treated both WT and IL6KO mice with combination of iron dextran and BA. Both WT and IL6KO mice were treated with a combination of BA and iron at day 0 followed by alternate day of iron injections showed the poorest erythropoiesis in the BM and became rapidly sick, although the effect was significantly more pronounced in WT animals, as suggested by their survival curve. Since mycobacterium infections lead to NLPR3 inflammasome activation and Caspase1 upregulation (Marim et al. Semin Immunopathology 2017), we investigated how erythroid progenitors were affected. By flow cytometry analyses, we observed a significantly higher upregulation of the Caspase1 protein in WT and DKO mice compared to IL6KO animals. This was also reproduced by culturing WT or IL6KO BM progenitor erythroid cells in presence of mouse serum derived from WT or IL6KO mice treated with BA. Most importantly, IL6KO mice treated with BA and iron showed the highest levels of Caspase1 compared to only BA treated IL6KO mice, indicating that excess of iron abrogates the beneficial effect of IL6 deficiency on erythropoiesis under conditions of AI. Furthermore, using flow cytometry, we observed in WT mice treated with BA or BA and iron a significant increase in mitochondrial mass, which is an indicator of mitochondrial stress. The mitochondrial mass was reduced in IL6KO mice treated with BA, but again increased in IL6KO mice treated with BA and iron. We have also observed an increase of mitochondrial superoxide by confocal microscopy in WT mice compared to IL6KO mice treated with BA. Altogether, these data support a model where inflammation in presence of an excess of iron impairs BM erythropoiesis through mechanisms at least in part mediated by Caspase1 and mitochondrial dysfunction, while iron excess itself is sufficient to boost extramedullary erythropoiesis to compensate and sustain RBC production. Disclosures Vinchi: PharmaNutra: Research Funding; Vifor Pharma: Research Funding; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding. Rivella: Ionis Pharmaceuticals: Consultancy; Meira GTx: Consultancy.

SATB1-dependent mitochondrial ROS production controls TCR signaling in CD4 T cells

Special AT-rich sequence binding protein-1 (SATB1) is localized to the nucleus and remodels chromatin structure in T cells. SATB1-deficient CD4 T cells cannot respond to TCR stimulation; however, the cause of this unresponsiveness is to be clarified. Here, we demonstrate that SATB1 is indispensable to proper mitochondrial functioning and necessary for the activation of signal cascades via the TCR in CD4 T cells. Naïve SATB1-deficient CD4 T cells contain fewer mitochondria than WT T cells, as the former do not express mitochondrial transcription factor A (TFAM). Impaired mitochondrial function in SATB1-deficient T cells subverts mitochondrial ROS production and SHP-1 inactivation by constitutive oxidization. Ectopic TFAM expression increases mitochondrial mass and mitochondrial ROS production and rescues defects in the antigen-specific response in the SATB1-deficient T cells. Thus, SATB1 is vital for maintaining mitochondrial mass and function by regulating TFAM expression, which is necessary for TCR signaling.

Mitochondrial Biogenesis, Mitochondrial Dynamics, and Mitophagy in the Maturation of Cardiomyocytes

Pluripotent stem cells (PSCs) can undergo unlimited self-renewal and can differentiate into all the cell types present in our body, including cardiomyocytes. Therefore, PSCs can be an excellent source of cardiomyocytes for future regenerative medicine and medical research studies. However, cardiomyocytes obtained from PSC differentiation culture are regarded as immature structurally, electrophysiologically, metabolically, and functionally. Mitochondria are organelles responsible for various cellular functions such as energy metabolism, different catabolic and anabolic processes, calcium fluxes, and various signaling pathways. Cells can respond to cellular needs to increase the mitochondrial mass by mitochondrial biogenesis. On the other hand, cells can also degrade mitochondria through mitophagy. Mitochondria are also dynamic organelles that undergo continuous fusion and fission events. In this review, we aim to summarize previous findings on the changes of mitochondrial biogenesis, mitophagy, and mitochondrial dynamics during the maturation of cardiomyocytes. In addition, we intend to summarize whether changes in these processes would affect the maturation of cardiomyocytes. Lastly, we aim to discuss unanswered questions in the field and to provide insights for the possible strategies of enhancing the maturation of PSC-derived cardiomyocytes.