Decrease In Oxygen Consumption Rate Research Articles

8071 Prevention Of Acute Kidney Injury By Reducing Mitochondrial Dysfunction In Diabetic Mice

Abstract Disclosure: M. Kim: None. C. Oh: None. A. Khang: None. J. Jeon: None. I. Lee: None. Background: Acute Kidney Injury (AKI) significantly contributes to the pathogenesis of diabetic kidney disease, often exacerbating chronic kidney disease (CKD) and accelerating the need for dialysis. Mitochondrial dysfunction, induced by an increase in reactive Oxygen Species (ROS) and inflammation, plays a crucial role in AKI. This study investigates the prevention of AKI by reducing mitochondrial dysfunction through pyruvate dehydrogenase kinase (PDK) inhibition in an ischemia-reperfusion (IR)-induced AKI model using streptozotocin (STZ)-induced diabetic mice. Methods: We utilized an IR-induced AKI model in STZ-induced diabetic C57BL6/J mice, subjecting them to IR by clamping both renal pedicles. Mitochondrial function tests, cellular apoptosis, and inflammatory markers were evaluated in NRK-52E cells and mouse primary tubular cells after hypoxia and reoxygenation using a hypoxia workstation. Results: In diabetic mice experiencing AKI following IR injury, there was a significant increase in pyruvate dehydrogenase E1α (PDHE1α) phosphorylation. This correlated with a notable increase in mitochondrial damage - TEM images of mice with IR-induced kidney injury revealed distension, loss of cristae, and fragmentation of the mitochondria. We also observed a decrease in oxygen consumption rate (OCR) accompanied by increased ROS, as well as increased production of inflammatory markers. Remarkably, the pan-PDK inhibitor, sodium dichloroacetate (DCA), mitigated apoptosis, attributable to the reduction of PDHE1α phosphorylation levels as well as normalized mitochondrial morphology and function. DCA treatment lessened oxidative stress and decreased the production of inflammatory markers following IR-induced AKI or hypoxia-reoxygenation injury. Conclusion: Restoration of mitochondrial function by DCA effectively alleviated renal injury by decreasing ROS production and inflammation, highlighting its critical role in IR-mediated damage. These results suggest another potential target for renal protection during AKI; therefore, the prevention of acute kidney injury through the inhibition of PDK could be a promising therapeutic approach for treating Diabetic Kidney Disease (DKD). Presentation: 6/1/2024

Intercalation modified nano zirconium phosphate inhibitor for inhibiting coal spontaneous combustion

The synthesis and characterization of a modified zirconium phosphate (MZrP) nano-inhibitor for spontaneous coal combustion inhibition were explored through isooctylamine intercalation. Using oxidation experiment, FTIR and SEM, the variation rules of the characteristic parameters of the different coal samples were investigated. It was observed that MZrP exhibited enhanced dispersion within the coal matrix post-intercalation modification. As the MZrP concentration increased, there was a notable decrease in oxygen consumption rate, gas production concentration, and active group content in the coal, alongside a significant increase in apparent activation energy and inhibition efficacy. This enhanced inhibition is attributed to two primary mechanisms. Firstly, the hydrophilic nature of MZrP allows for its uniform distribution on the coal surface and within internal pores, creating a dense carbonized layer. This layer effectively retains moisture and isolates oxygen, enhancing physical inhibition. Secondly, MZrP inhibitor is thermally decomposed into phosphoric acid and its phosphoric acid derivatives, which can effectively capture the H- and -OH in the coal, and strengthens the inactivation of its reactive free radicals. The optimal inhibition was observed in coal samples treated with 6 wt% MZrP, exhibiting an average inhibition rate of 62.2 %, coupled with the lowest rates of gas production and oxygen consumption.

Influence of O-GlcNAcylation on KGN cell function

O-GlcNAcylation is a unique form of post-translational glycosylation that affects a variety of cytoplasmic and nuclear proteins of cells. Aberrant O-GlcNAcylation is characteristic of many cancers, and impacts cell proliferation, tumorigenicity and metabolism. O-GlcNAcylation occurs in granulosa cells of ovarian follicles, its expression differs between small (3-5 mm) and large (>8.5 mm) antral follicles, and its manipulation in vitro alters granulosa cell proliferation and metabolism. Here, the aim was to assess whether O-GlcNAcylation similarly occurs in cells from a type of granulosa cell tumor, specifically KGN cells, knowing these cells share functional features of granulosa cells of mature, preovulatory follicles (e.g., FSH-responsiveness and estradiol production). The immortal KGN cell line was utilized to conduct cell culture experiments for the detection and manipulation of O-GlcNAcylation. The cells were grown to confluency in serum containing medium and then sub-cultured in serum-free conditions for immunodetection of O-GlcNAcylation (n = 8 expts.), for cell proliferation (n = 3 expts) and for metabolism assays (n = 12 expts.). The KGN cells were also treated without or with small molecule inhibitors to directly enhance or impair O-GlcNAcylation. Immunoblotting confirmed O-GlcNAc expression in KGN cells, as well as the efficacy of Thiamet-G and OSMI-1 to augment (P < 0.05) and inhibit O-GlcNAcylation (P < 0.05), respectively. Only the inhibition of O-GlcNAcylation compromised KGN cell proliferation (P < 0.05), resulting in a 25 % reduction in proliferation compared to control conditions over a 72 h culture period. Seahorse XFe96 analysis measured effects of O-GlcNAcylation on cellular respiration in the KGN cells. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) provided indices of glycolysis and oxidative phosphorylation, respectively. During a glycolysis stress test, high glucose increased ECAR and decreased OCR (P < 0.05); oligomycin did not further affect ECAR (P > 0.05), but impaired OCR (P < 0.05); and 2-deoxy-d-glucose decreased ECAR (P < 0.05) without affecting OCR (P > 0.05). Comparatively, a mitochondrial stress test revealed oligomycin increased ECAR (P < 0.05) with a compensatory decrease in OCR (P < 0.05); FCCP increased both ECAR and OCR (P < 0.05); and rotenone + antimycin A decreased both ECAR and OCR (P < 0.05). Manipulation of O-GlcNAcylation in the KGN cells had no effect on ECAR (P > 0.05), but inhibited OCR (P < 0.05). Collectively, the results indicate O-GlcNAcylation occurs in KGN cells, its inhibition impairs cell proliferation, and while KGN cells rely upon both glycolysis and oxidative phosphorylation for cellular respiration, manipulation of O-GlcNAcylation acutely perturbs only oxidative phosphorylation, an effect observed previously with granulosa cells of large antral follicles.

Enhanced glycolysis causes extracellular acidification and activates acid-sensing ion channel 1a in hypoxic pulmonary hypertension.

In pulmonary hypertension (PHTN), a metabolic shift to aerobic glycolysis promotes a hyperproliferative, apoptosis-resistant phenotype in pulmonary arterial smooth muscle cells (PASMCs). Enhanced glycolysis induces extracellular acidosis, which can activate proton-sensing membrane receptors and ion channels. We previously reported that activation of the proton-gated cation channel acid-sensing ion channel 1a (ASIC1a) contributes to the development of hypoxic PHTN. Therefore, we hypothesize that enhanced glycolysis and subsequent acidification of the PASMC extracellular microenvironment activate ASIC1a in hypoxic PHTN. We observed decreased oxygen consumption rate and increased extracellular acidification rate in PASMCs from chronic hypoxia (CH)-induced PHTN rats, indicating a shift to aerobic glycolysis. In addition, we found that intracellular alkalization and extracellular acidification occur in PASMCs following CH and in vitro hypoxia, which were prevented by the inhibition of glycolysis with 2-deoxy-d-glucose (2-DG). Inhibiting H+ transport/secretion through carbonic anhydrases, Na+/H+ exchanger 1, or vacuolar-type H+-ATPase did not prevent this pH shift following hypoxia. Although the putative monocarboxylate transporter 1 (MCT1) and -4 (MCT4) inhibitor syrosingopine prevented the pH shift, the specific MCT1 inhibitor AZD3965 and/or the MCT4 inhibitor VB124 were without effect, suggesting that syrosingopine targets the glycolytic pathway independent of H+ export. Furthermore, 2-DG and syrosingopine prevented enhanced ASIC1a-mediated store-operated Ca2+ entry in PASMCs from CH rats. These data suggest that multiple H+ transport mechanisms contribute to extracellular acidosis and that inhibiting glycolysis-rather than specific H+ transporters-more effectively prevents extracellular acidification and ASIC1a activation. Together, these data reveal a novel pathological relationship between glycolysis and ASIC1a activation in hypoxic PHTN.NEW & NOTEWORTHY In pulmonary hypertension, a metabolic shift to aerobic glycolysis drives a hyperproliferative, apoptosis-resistant phenotype in pulmonary arterial smooth muscle cells. We demonstrate that this enhanced glycolysis induces extracellular acidosis and activates the proton-gated ion channel, acid-sensing ion channel 1a (ASIC1a). Although multiple H+ transport/secretion mechanisms are upregulated in PHTN and likely contribute to extracellular acidosis, inhibiting glycolysis with 2-deoxy-d-glucose or syrosingopine effectively prevents extracellular acidification and ASIC1a activation, revealing a promising therapeutic avenue.

TDP-43 dysfunction leads to bioenergetic failure and lipid metabolic rewiring in human cells

The dysfunction of TAR DNA-binding protein 43 (TDP-43) is implicated in various neurodegenerative diseases, though the specific contributions of its toxic gain-of-function versus loss-of-function effects remain unclear. This study investigates the impact of TARDBP loss on cellular metabolism and viability using human-induced pluripotent stem cell-derived motor neurons and HeLa cells. TARDBP silencing led to reduced metabolic activity and cell growth, accompanied by neurite degeneration and decreased oxygen consumption rates in both cell types. Notably, TARDBP depletion induced a metabolic shift, impairing ATP production, increasing metabolic inflexibility, and elevating free radical production, indicating a critical role for TDP-43 in maintaining cellular bioenergetics. Furthermore, TARDBP loss triggered non-apoptotic cell death, increased ACSL4 expression, and reprogrammed lipid metabolism towards lipid droplet accumulation, while paradoxically enhancing resilience to ferroptosis inducers. Overall, our findings highlight those essential cellular traits such as ATP production, metabolic activity, oxygen consumption, and cell survival are highly dependent on TARDBP function.

Carryover effects of embryonic hypoxia exposure on adult fitness of the Pacific abalone

The widespread and severe drop in dissolved oxygen concentration in the open ocean and coastal waters has attracted much attention, but assessments of the impacts of environmental hypoxia on aquatic organisms have focused primarily on responses to current exposure. Past stress exposure might also affect the performance of aquatic organisms through carryover effects, and whether these effects scale from positive to negative based on exposure degree is unknown. We investigated the carryover effects of varying embryonic hypoxia levels (mediate hypoxia: 3.0−3.1 mg O2/L; severe hypoxia: 2.0−2.1 mg O2/L) on the fitness traits of adult Pacific abalone (Haliotis discus hannai), including growth, hypoxia tolerance, oxygen consumption, ammonia excretion rate, and biochemical responses to acute hypoxia. Moderate embryonic hypoxia exposure significantly improved the hypoxia tolerance of adult Pacific abalone without sacrificing growth and survival. Adult abalone exposed to embryonic hypoxia exhibited physiological plasticity, including decreased oxygen consumption rates under environmental stress, increased basal methylation levels, and a more active response to acute hypoxia, which might support their higher hypoxia tolerance. Thus, moderate oxygen declines in early life have persistent effects on the fitness of abalone even two years later, further affecting population dynamics. The results suggested that incorporating the carryover effects of embryonic hypoxia exposure into genetic breeding programs would be an important step toward rapidly improving the hypoxia tolerance of aquatic animals. The study also inspires the protection of endangered wild animals and other vulnerable species under global climate change.

Heat stress upregulates arachidonic acid to trigger autophagy in sertoli cells via dysfunctional mitochondrial respiratory chain function

As a key factor in determining testis size and sperm number, sertoli cells (SCs) play a crucial role in male infertility. Heat stress (HS) reduces SCs counts, negatively impacting nutrient transport and supply to germ cells, and leading to spermatogenesis failure in humans and animals. However, how HS affects the number of SCs remains unclear. We hypothesized that changes in SC metabolism contribute to the adverse effects of HS. In this study, we first observed an upregulation of arachidonic acid (AA), an unsaturated fatty acid after HS exposure by LC-MS/MS metabolome detection. By increasing ROS levels, expression of KEAP1 and NRF2 proteins as well as LC3 and LAMP2, 100 µM AA induced autophagy in SCs by activating oxidative stress (OS). We observed adverse effects of AA on mitochondria under HS with a decrease of mitochondrial number and an increase of mitochondrial membrane potential (MMP). We also found that AA alternated the oxygen transport and absorption function of mitochondria by increasing glycolysis flux and decreasing oxygen consumption rate as well as the expression of mitochondrial electron transport chain (ETC) proteins Complex I, II, V. However, pretreatment with 5 mM NAC (ROS inhibitor) and 2 µM Rotenone (mitochondrial ETC inhibitor) reversed the autophagy induced by AA. In summary, AA modulates autophagy in SCs during HS by disrupting mitochondrial ETC function, inferring that the release of AA is a switch-like response, and providing insight into the underlying mechanism of high temperatures causing male infertility.

Acyl-CoA synthetase 4 modulates mitochondrial function in breast cancer cells

Mitochondria are dynamic organelles that respond to cellular stress through changes in global mass, interconnection, and subcellular location. As mitochondria play an important role in tumor development and progression, alterations in energy metabolism allow tumor cells to survive and spread even in challenging conditions. Alterations in mitochondrial bioenergetics have been recently proposed as a hallmark of cancer, and positive regulation of lipid metabolism constitutes one of the most common metabolic changes observed in tumor cells. Acyl-CoA synthetase 4 (ACSL4) is an enzyme catalyzing the activation of long chain polyunsaturated fatty acids with a strong substrate preference for arachidonic acid (AA). High ACSL4 expression has been related to aggressive cancer phenotypes, including breast cancer, and its overexpression has been shown to positively regulate the mammalian Target of Rapamycin (mTOR) pathway, involved in the regulation of mitochondrial metabolism genes. However, little is known about the role of ACSL4 in the regulation of mitochondrial function and metabolism in cancer cells. In this context, our objective was to study whether mitochondrial function and metabolism, processes usually altered in tumors, are modulated by ACSL4 in breast cancer cells. Using ACSL4 overexpression in MCF-7 cells, we demonstrate that this enzyme can increase the mRNA and protein levels of essential mitochondrial regulatory proteins such as nuclear respiratory factor 1 (NRF-1), voltage-dependent anion channel 1 (VDAC1) and respiratory chain Complex III. Furthermore, respiratory parameters analysis revealed an increase in oxygen consumption rate (OCR) and in spare respiratory capacity (SRC), among others. ACSL4 knockdown in MDA-MB-231 cells led to the decrease in OCR and in SCR, supporting the role of ACSL4 in the regulation of mitochondrial bioenergetics. Moreover, ACSL4 overexpression induced an increase in glycolytic function, in keeping with an increase in mitochondrial respiratory activity. Finally, there was a decrease in mitochondrial mass detected in cells that overexpressed ACSL4, while the knockdown of ACSL4 expression in MDA-MB-231 cells showed the opposite effect. Altogether, these results unveil the role of ACSL4 in mitochondrial function and metabolism and expand the knowledge of ACSL4 participation in pathological processes such as breast cancer.

Sera from people with HIV and depression induce commensurate metabolic alterations in astrocytes: toward precision diagnoses and therapies.

People with HIV (PWH) have high rates of depression and neurocognitive impairment (NCI) despite viral suppression on antiretroviral therapy (ART). Mounting evidence suggests that immunometabolic disruptions may contribute to these conditions in some PWH. We hypothesized that metabolic dysfunction in astrocytes is associated with depressive symptoms and cognitive function in PWH. Human astrocytes were exposed to sera from PWH (n=40) with varying degrees of depressive symptomatology and cognitive function. MitoTrackerTM Deep Red FM (MT) was used to visualize mitochondrial activity and glial fibrillary acidic protein (GFAP) as an indicator of astrocyte reactivity using the high-throughput fluorescent microscopy and image analyses platform, CellInsight CX5 (CX5). The Seahorse platform was used to assess glycolytic and mitochondrial metabolism. More severe depression, as indexed by higher Beck's Depression Inventory (BDI-II) scores, was associated with lower MT signal measures. Better cognitive function, as assessed by neuropsychiatric testing t-scores, was associated with increased MT signal measures. GFAP intensity negatively correlated with several cognitive t-scores. Age positively correlated with (higher) MT signal measures and GFAP intensity. Worse depressive symptoms (higher BDI-II scores) were associated with decreased oxygen consumption rate and spare respiratory capacity, concomitant with increased extracellular acidification rate in astrocytes. These findings show that factors in the sera of PWH alter mitochondrial activity in cultured human astrocytes, suggesting that mechanisms that alter mitochondrial and astrocyte homeostasis can be detected peripherally. Thus, in vitro cultures may provide a model to identify neuropathogenic mechanisms of depression or neurocognitive impairment in PWH and test personalized therapeutics for neurologic and psychiatric disorders.

FFAR4 activation inhibits lung adenocarcinoma via blocking respiratory chain complex assembly associated mitochondrial metabolism

Despite notable advancements in the investigation and management of lung adenocarcinoma (LUAD), the mortality rate for individuals afflicted with LUAD remains elevated, and attaining an accurate prognosis is challenging. LUAD exhibits intricate genetic and environmental components, and it is plausible that free fatty acid receptors (FFARs) may bridge the genetic and dietary aspects. The objective of this study is to ascertain whether a correlation exists between FFAR4, which functions as the primary receptor for dietary fatty acids, and various characteristics of LUAD, while also delving into the potential underlying mechanism. The findings of this study indicate a decrease in FFAR4 expression in LUAD, with a positive correlation (P < 0.01) between FFAR4 levels and overall patient survival (OS). Receiver operating characteristic (ROC) curve analysis demonstrated a significant diagnostic value [area under the curve (AUC) of 0.933] associated with FFAR4 expression. Functional investigations revealed that the FFAR4-specific agonist (TUG891) effectively suppressed cell proliferation and induced cell cycle arrest. Furthermore, FFAR4 activation resulted in significant metabolic shifts, including a decrease in oxygen consumption rate (OCR) and an increase in extracellular acidification rate (ECAR) in A549 cells. In detail, the activation of FFAR4 has been observed to impact the assembly process of the mitochondrial respiratory chain complex and the malate–aspartate shuttle process, resulting in a decrease in the transition of NAD+ to NADH and the inhibition of LUAD. These discoveries reveal a previously unrecognized function of FFAR4 in the negative regulation of mitochondrial metabolism and the inhibition of LUAD, indicating its potential as a promising therapeutic target for the treatment and diagnosis of LUAD.

Important Constituents of Heavy Water-containing Solution for Cold Storage and Subsequent Reperfusion on an Isolated Perfused Rat Liver

The University of Wisconsin (UW) solution is the most effective preservation solution currently used; however, to safely use expanded-criteria donor grafts, a new cold storage solution that alleviates graft injury more effectively is required. We prepared a heavy water (D2O)-containing buffer, Dsol, and observed strong protective effects during extended cold storage of rat hearts and livers. In the current study, we modified Dsol (mDsol) and tested its efficacy. The aim of the present study was to determine whether mDsol could protect the rat liver more effectively than the UW solution and to clarify the roles of D2O and deferoxamine (DFX). Rat livers were subjected to cold storage for 48 hours in test solutions: UW, mDsol, mDsol without D2O or DFX (mDsol-D2O[-], mDsol-DFX[-]), and subsequently reperfused on an isolated perfused rat liver for 90 minutes at 37°C. In the UW group, the liver was dehydrated during cold storage and rapidly expanded during reperfusion. Accordingly, the cumulative weight change was the highest in the UW group, together with augmented portal veinous resistance and ALT leakage and decreased oxygen consumption rate and bile production. These changes were significantly suppressed in the mDsol-treated group. In the mDsol-D2O(-) and mDsol-DFX(-) groups offered partial protection. In conclusion, mDsol appeared to be superior to the UW solution for simple cold storage of the rat liver, presumably due to improved microcirculation in the early phase of reperfusion. Both heavy water and deferoxamine are essential for alleviating seamless organ swelling that occurs during cold storage and subsequent reperfusion.

FABP4 Suppresses the Progress of Multiple Myeloma through Enhancing Alarming Function of Bone Marrow Resident Memory T Lymphocytes

Multiple myeloma (MM) is a hematological malignancy which characterized by malignant plasma cells resident in the bone marrow (BM). The BM microenvironment plays critical roles in the invasion, proliferation and migration of myeloma cells. Despite the therapies of MM improved in recent years, this disease is still incurable and requires precise treatment. Resident memory (TRM) cells are a distinct tissue-localized T cell lineage that is crucial for protective immunity in peripheral tissues. Recent studies reveal TRM cells as a vital component of the host immune response to cancer. TRM-like tumor-infiltrating lymphocytes (TILs) can be found in a wide range of human cancers, where they portend improved prognosis. However, the precise role and mechanism of bone marrow TRM cells in MM still remain elusive. In present study we first found that the accumulation of T RM cells (CD8 +CD44 +CD69 +CD62L -) in BM newly diagnosed MM (NDMM) patients was significantly higher than that of relapse recurrent MM (RRMM) ( Figure A), which was further confirmed by the immunohistochemistry (IHC) staining analysis ( Figure B). By using Seahorse XF e analyzer we found a decrease of oxygen consumption rate (OCR) of T RM cells in RRMM comparing to that of NDMM ( Figure C). Since fatty acid metabolism is important in β oxidation, we further investigated the expression of fatty acid binding proteins (FABP4/FABP5) in T RM cells and showed that FABP4 was dominant in T RM cells of NDMM patients ( Figure D). FABP4 +/+ mice and their relative controls FABP4 -/- mice were adopted to further explore the role and mechanism of T RM cells in MM progression. The abundance of genes related fatty acids β oxidation was significantly decreased in FABP4 -/- T RM cells ( Figure E). The tumor cell viability was decreased in myeloma cells co-cultured with FABP4 -/- T RM cells in the presence of bortezomib ( Figure F). Moreover, the expression of cytotoxic cytokines including IFN γ, TNFα and CXCL10 was significantly higher in FABP4 -/- T RM cells ( Figure G). The alarming function of T RM cells was further enhanced via recruiting more effector T (CD8 +CXCR3 +) cells ( Figure H), therefore the apoptosis of tumor cells was elevated in BM of MM patients. In summary, T RM cells are necessary and sufficient for long-lived protection against tumors. T RM cells derived FABP4 exerts anti-tumor immunity through prolonging the survival of T RM cells and enhancing the recruitment of more effector T cells to suppress the tumor progression ( Figure I). This study suggests that T RM cells might serve as a novel therapeutic target in MM and treatment specific targeting FABP4 might shed light on the development of potential therapeutic strategies for treating MM especially relapse patients. Acknowledgement This project is supported by the Sun Yat-sen University Hundred Talents Program (PT19200101), Natural Science Foundation of Guangdong Province (2022A1515010290).

Mitochondrial tRNA Pseudouridylation Regulates Erythropoiesis Via the mTOR Signaling Pathway: Implications for Mlasa and Treatment Strategies

Pseudouridine, the most abundant RNA modification found in various RNA molecules, is crucial for cellular functions. Pseudouridylation is the process of converting uridine to pseudouridine, catalyzed by pseudouridine synthases (PUSs), and abnormal pseudouridylation has been linked to human diseases, including mitochondrial myopathy, lactic acidosis, and sideroblastic anemia syndrome (MLASA). MLASA is associated with genetic mutations in the PUS1 gene, leading to progressive exercise intolerance, sideroblastic anemia, hyperlactatemia, and muscle weakness. The underlying mechanism of anemia in MLASA remains unclear. In this study, we identified a novel homozygous frameshift deletion (c.523delC, p.P175fs*8) in the PUS1 gene in an MLASA patient with progressive anemia in our hospital. We investigated the role of pseudouridylation in erythropoiesis and its impact on MLASA using in vitro and in vivo approaches. We established an MLASA patient-derived inducible pluripotent stem cell (iPSC) line (referred to as MLASA-iPSCs) with the mutation, as well as a repaired line (MLASA-Res iPSC), and a corresponding mutant mouse model for the investigation. During erythroid differentiation, MLASA-iPSCs showed a significant decrease in the production of erythroblasts compared to the repaired line. Also, MLASA-iPSCs exhibited a lower ratio of mitochondrial membrane potential to mitochondrial mass, decreased oxygen consumption rates, reduced ATP production, and elevated levels of ROS. Attenuated activities of NADH dehydrogenase (Complex I) and cytochrome c reductase (Complex III) were also observed in MLASA-iPSCs, indicating compromised mitochondrial functions. Further analysis revealed a reduction in mitochondrial tRNA levels and abnormal mitochondrial translation in MLASA-iPSCs due to the specific loss of pseudouridylation. Screening of mitochondrial supplements, which could boost mitochondrial function or modulating protein synthesis, revealed that nicotinamide ribose (NR) and mitoquinone (MitoQ) improved mitochondrial function but failed to promote erythroid differentiation in MLASA-iPSCs. Interestingly, rapamycin, an mTOR inhibitor, improved erythroid differentiation in MLASA-iPSCs. Furthermore, MLASA-iPSCs showed activated mTOR signaling, increased protein synthesis and higher translation efficiency of mTOR1 targets, all of which could be rescued by rapamycin treatment, suggesting mTOR-associated translation stress. Consistently, the corresponding PUS1 mutant mouse model exhibited anemia with blocked erythropoiesis at four weeks of age. Serial competitive transplantation experiments revealed an intrinsic impairment in the reconstitution of red blood cells. Mutant erythroblasts showed impaired oxidative phosphorylation and aberrant activation of the mTOR signaling pathway. Rapamycin treatment partially alleviated the anemia phenotype in mice. Significantly, in the MLASA patient, sirolimus (rapamycin) administration under strict supervision and medical guidance effectively alleviated anemia symptoms and improved blood routine profile. In summary, our findings provide insights into the critical role of mitochondrial tRNA pseudouridylation in governing erythropoiesis via mTOR signaling pathway. We propose rapamycin as potential therapeutic strategies for anemia patients experiencing protein translation pressure, such as those with MLASA. #These authors contributed equally: Chu Y, Wang B, Shi D and Lian Y. Correspondence to: wpyuan@ihcams.ac.cn, shijun@ihcams.ac.cn Fig. 1 Schematic illustration of erythropoiesis regulated by PUS1-mediated pseudouridylation. Under physiological conditions (left), PUS1 regulates mt-tRNA abundance by catalyzing the pseudouridine synthesis of specific mt-tRNAs, thereby regulating mitochondrial translation, and ultimately affecting mitochondrial function and erythroid differentiation. In MLASA disease states (right), PUS1 deficiency causes the loss of pseudouridine modifications of specific tRNAs and thus affects the amount of PUS1 targeted tRNAs and its corresponding mitochondrial protein products, leading to mitochondrial dysfunction. The defects resulted in aberrant activation of the mTOR signaling pathway and accelerated global translation rate, ultimately leading to a blockage in erythroid differentiation, which could be relieved with rapamycin treatment.

Mitochondrial Pyruvate Carrier Inhibition Mitigates Murine Chronic Graft Versus Host Disease By Attenuating the Germinal Center Reaction

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potent therapy for many hematopoietic diseases and malignancies. However, the long-term efficacy of allo-HSCT is limited by the development of chronic graft versus host disease (cGVHD). During cGVHD, interactions between donor-derived T follicular helper cells (TFH) and germinal center B cells (GCB) result in the production of alloreactive class-switched affinity-matured antibodies that attack host tissues, causing multi-organ damage and tissue fibrosis. Novel treatment approaches are warranted to treat cGVHD since it is often refractory to current broad immune suppressive drugs. In this study we sought to delineate the metabolic dependencies of germinal center (GC) immune cells to discover new strategies to treat active murine cGVHD. We utilized a multi-organ mouse model of cGVHD that includes bronchiolitis obliterans (BO), a severe cGVHD manifestation seen in the clinic. Toward that end, B10.BR (H2 k) recipients received myeloablative conditioning with cyclophosphamide and total body irradiation before receiving MHC mismatched C57BL/6 (H2 b) donor T cell depleted bone marrow with (cGVHD) or without (BM only control) a low dose of splenic T cells. First, we sought to assess the metabolic dependencies of TFH during cGVHD progression through Seahorse metabolic flux assays. TFH sorted early (D28) from cGVHD mice were highly glycolytic with an increased extracellular acidification rate (ECAR) and less reliant on oxidative phosphorylation (OXPHOS) as reflected by a decreased oxygen consumption rate (OCR). However, TFH sorted later (D49) from cGVHD mice demonstrated a dramatic shift in their metabolic profile to a decreased ECAR and an increased OCR indicating a transition from glycolysis towards OXPHOS as disease progresses. To understand which substrates are driving increased OXPHOS and to identify promising metabolic targets, we performed unbiased metabolomics of sort purified TFH from BM only and cGVHD mice on D49 post-transplantation (Fig A). Among the top fifty differential metabolites, cGVHD TFH had a decreased abundance of anaplerotic amino acids (serine, glutamine) and an increased abundance of multiple breakdown products of anaplerotic amino acids (tryptophan, leucine), indicative of increased replenishment of tricarboxylic acid (TCA) cycle intermediates. Notably these metabolomics data showed a decreased abundance of glutamine which is consistent with an increase in glutaminolysis, validating our prior findings that TFH cells that cause cGVHD/BO depend on glutaminolysis ( Johnson MO, Cell 2018). Additionally, there was an increased abundance of pyruvate, which can be transported into mitochondria to fuel the TCA cycle through the mitochondrial pyruvate carrier (MPC). Based on these findings we chose to target the MPC since it has a central role in tying pyruvate to the TCA cycle and OXPHOS. We assessed the effect of an MPC inhibitor (7ACC1) on the GC reaction utilizing an ex vivo co-culture assay of TFH and B cells. 7ACC1 added to this co-culture system resulted in a significant reduction in the production of class-switched immunoglobulin compared to vehicle treatment. To assess the in vivo efficacy, we administered 7ACC1 daily through intraperitoneal injections after establishment of cGVHD (Days 28-49) in the cGVHD/BO mouse model. 7ACC1 treatment resulted in a significant reduction of the GC reaction with significantly decreased TFH and GCB frequencies. Additionally, there was a significant improvement in cGVHD disease severity as evidenced by assessing pulmonary function tests in anesthetized, intubated and mechanically ventilated mice using plethysmography. In contrast to the high resistance and low compliance seen in cGVHD/BO mice, 7ACC1 treated recipients had significantly improved lung function, comparable to the BM only controls (Fig B). In summary, inhibiting MPC mediated transport of pyruvate represents a novel approach to stifling the metabolic demands of TFH as their energy dependency shifts from glycolysis towards OXPHOS during the course of cGVHD/BO disease progression.

Targeting Sumoylation Sensitizes Acute Myeloid Leukemia to Venetoclax Treatment

Acute myeloid leukemia (AML) is a hematopoietic cancer characterized by aberrant immature myeloid progenitor blasts in bone marrow and peripheral blood. Venetoclax (VEN), a BCL-2 inhibitor, is FDA-approved for AML treatment with hypomethylating agents (HMA). Despite the improvement of VEN and HMA, not all patients respond, and most patients develop resistance. This presents an urgent need for new therapies to overcome VEN resistance and enhance VEN efficacy. We propose SUMOylation inhibition as a novel therapy to address this need. SUMOylation regulates protein function by covalently attaching Small Ubiquitin-like MOdifier (SUMO) proteins to target proteins. SUMOylation plays an important role in tumor progression and immune response. Our study aims to evaluate the effects of SUMOylation inhibition on anti-AML activity of VEN and the mechanism. We firstly generated VEN-resistant cell lines Molm13-VR and MV411-VR by culturing Molm13 and MV4-11 cells in increasing doses of VEN. SUMO E1 (SAE2) and global SUMOylation levels were remarkably upregulated in VEN-resistant cell lines, suggesting SUMOylation plays a role in VEN resistance. We evaluated the antileukemic effects of TAK-981 (subasumstat), a novel and specific SUMO E1 inhibitor, alone and in combination with VEN, using AML cell lines and primary blasts isolated from refractory/relapsed (R/R) AML patients. Different AML cell lines and blast samples showed various sensitivities to VEN (IC 50 10nM ~ 5,000nM), but significant synergism was observed with TAK-981 and VEN in cell viability assay. TAK-981 synergized with VEN at inducing AML cell apoptosis determined by annexin-V staining with flow cytometry as well as increased levels of cleaved-PARP and cleaved caspase-3 by western blotting. The mechanism of action of VEN is dependent on BCL-2 mediated mitochondrial function and metabolism. We assessed mitochondrial membrane potential using TMRM probe. TAK-981 reduced mitochondrial membrane potential in both VEN-sensitive and resistant AML lines, further in combination with VEN. Because mitochondrial membrane potential is maintained by normal mitochondrial structure, we performed electron microscopy to determine mitochondrial structure. VEN had limited effect on mitochondria in resistant cells. However, TAK-981 induced abnormal ultrastructure mitochondria with fewer cristae, worsened by TAK-981+VEN combination. Since TAK-981 treatment caused mitochondrial structure defects, we determine the mitochondrial function using Seahorse XF assay. VEN decreased oxygen consumption rate (OCR), indicating inhibition of oxidative phosphorylation (OXPHOS), further suppressed by TAK-981. Metabolomic profiling by mass spectrometry indicated TAK-981+VEN treated AML cells displayed striking decreases in TCA cycle intermediates and reduced electron transport chain (ETC) activity. Pathway analysis of the metabolomics data revealed significant enrichment in amino acids metabolism. Notably, RNA-sequencing and GSEA analysis showed consistent findings that TAK-981+VEN showed suppressed signaling pathways in mitochondrial function, glycolysis and amino-acid metabolisms, indicating TAK-981 targeted mitochondrial metabolism to enhance VEN activity. We then determined whether TAK-981 enhanced antileukemic effect of VEN ex vivo and in vivo. TAK-981 synergized with VEN at reducing colony formation capacity in colony formation assay using leukemia stem cells (LSCs) isolated from relapse AML blasts. More importantly, TAK-981 showed remarkable therapeutic effect in patient-derived xenograft (PDX) mouse model engrafted with blasts isolated from R/R AML patients, and significantly synergized with VEN at extending mice survival in vivo. In conclusion, TAK-981 enhances the antileukemic activity of VEN by targeting mitochondria function and metabolism, offering a potent therapy for refractory/relapsed AML.

Abstract 16488: Sestrin2 Protects Against Long-Term Cardiac Injury and Remodeling by Modulating mTORC1 Activation in Myocardial Ischemia/Reperfusion

Introduction: Myocardial ischemia/reperfusion (I/R) injury can lead to long-term cardiac dysfunction and heart failure with reduced ejection fraction (HFrEF) in aged hearts. However, young hearts may exhibit some resistance to this process and retain better cardiac function. Our previous studies have identified a novel protein called Sestrin2 (Sesn2), which is downregulated in aged hearts. Hypothesis: Sesn2 may protect hearts from progressing to HFrEF following myocardial I/R. Methods: We induced myocardial I/R injury by ligation/release of the left anterior descending coronary arteries (LAD) in both C57BL/6J mice and mice with cardiac-specific Sesn2 knockout (Sesn2 cKO ). Cardiac function was assessed, and samples were collected at baseline, one day post I/R, seven days post I/R, and 28 days post I/R. Results: In wild-type (WT) hearts (12-18 weeks), cardiac function showed a slight decrease 28 days post I/R (IR28D), but the ejection fraction (EF) remained above 50%. However, Sesn2 cKO mice exhibited a significant reduction in cardiac function at IR28D, with an about 27.5% decrease in EF compared to WT hearts. We also observed a significant increase in volume at diastole in Sesn2 cKO mice at IR28D compared to their baseline and corresponding WT. Furthermore, when comparing mitochondrial respiration in the area at risk (AAR) of the left ventricles between the two groups, we found a significant decrease in oxygen consumption rate (OCR) and a significant increase in H 2 O 2 production in Sesn2 cKO hearts. Additionally, the mammalian target of rapamycin complex 1 (mTORC1) was significantly upregulated in Sesn2 cKO mice compared to WT hearts. Conclusions: Sesn2 exerts a cardiac protective effect by downregulating mTORC1 activation, thereby reducing long-term cardiac injury following I/R, attenuating cardiac remodeling, and improving mitochondrial respiration in the AAR of the heart.

Abstract 13872: CD70 Modulates Vascular Smooth Muscle Phenotype and Metabolism by Regulating Cellular Redox State

Introduction: The tumor necrosis factor superfamily (TNFSF) displays a wide array of vascular effects; however, the role of the TNFSF member CD70 is still being elucidated in the vasculature. We have recently described how CD70 modulates endothelial cell function and redox status by altering nitric oxide bioavailability and intracellular reactive oxygen species (ROS). However, the role of CD70 in vascular smooth muscle cells (VSMCs) is not known. We hypothesized that CD70 can alter VSMC phenotype and mitochondrial function through regulation of intracellular ROS. Methods: Human aortic VSMCs (AoVSMCs) were transfected with control or CD70-directed siRNA (n = 5-6). Cellular phenotype was assessed by scratch assay and collagen matrix gel contraction assay. Cell lysates were analyzed by Western blot and quantitative RT-PCR. Intracellular cytosolic hydrogen peroxide levels were measured using the fluorescent biosensor Hyper7 following stimulation by auranofin (50-70 cells per group averaged over 4 experiments). Metabolic profiling was performed with Seahorse assay, and mitochondrial membrane potential was assessed using TMRM dye (120 cells per group averaged over 3 experiments). Results: Following CD70 knockdown, AoVSMCs demonstrated augmented wound closure and decreased collagen matrix contraction. Expression of synthetic/proliferative markers, including vimentin, alpha-1 type I collagen, osteopontin, caldesmon-1, and c-Myc, was increased by up to 50%, and contractile markers including smoothelin, synemin, and synaptopodin-2 were decreased by up to 50%. Cytosolic hydrogen peroxide production was increased, with expression of NADPH oxidase (NOX) 4 and gp91phox (the catalytic subunit of NOX2) both increased by over 2-fold. CD70 knockdown led to a pronounced alteration in VSMC metabolic profile, with significantly decreased oxygen consumption rate (25%), extracellular acidification rate (45%), and maximal respiratory capacity (46%), as well as a decrease in mitochondrial membrane potential (37%). Conclusions: CD70 exerts a novel regulatory effect on VSMC phenotype; loss of CD70 promotes transition to a synthetic phenotype characterized by increased ROS with consequent metabolic reprogramming and mitochondrial dysfunction.

Chronic Mitochondrial Dysfunction Renders Acute Myeloid Leukemia Resistant to Multipronged Inhibition of Mitochondrial Metabolism

Acute Myeloid Leukemia (AML) is a genetically diverse and aggressive myeloid neoplasm that awaits novel therapeutics to improve patient survival. Targeting AML metabolic aberrations as a genotype-agnostic strategy has shown promise, evidenced by the clinical success of venetoclax and the rapid pre-clinical development of other mitochondrial (mt) metabolism targeted therapies. However, the mechanism of how AML develops resistance to drugs targeting key mt metabolic pathways is not fully understood. We previously identified sirtuin 5 (SIRT5), a deacylase that regulates several pathways of mt metabolism in AML including oxidative phosphorylation and glutamine metabolism. In AML, it functions as a master metabolic regulator that is necessary for leukemic stem cell survival. Genetic or pharmacological inhibition of SIRT5 leads to rapid AML cell apoptosis in vitro and disease regression in multiple AML mouse models. However, a subset of AML cell lines and primary samples can survive SIRT5 inhibition, indicating an unusual intrinsic resistance to mt dysfunction. To provide a mechanistic explanation, we first compared the molecular features between SIRT5-dependent and independent cases. Among the 25 primary AML samples we tested, NPM1c mutation, but not FLT3-ITD, TP53, WT1, NRAS, or IDH1 mutations, is significantly enriched in SIRT5-independent samples. Accordingly, among the panel of 22 AML cell lines we tested, the only NPM1 mutant cell line (OCI-AML3) sits at the most SIRT5-independent extreme of the spectrum. NPM1 mutant AML cells have increased cytoplasmic mitochondrial DNA (mtDNA) and fragmented mt morphology, indicating chronic mt damage (Wu H et al. Cancer Discovery. 2021). Because SIRT5 exerts its metabolic maintenance functions within the mt, we hypothesize that AML cells that can adapt to chronic, sublethal mt dysfunction will be capable of proliferation without SIRT5. To test this, we took three orthogonal approaches: (1) introducing NPM1c mutation, (2) using low concentrations of ethidium bromide (EtBr) to selectively block mtDNA replication, or (3) genetically inducing chronic mt dysfunction by silencing the essential mt transcription factor TFAM in 3 AML cell lines that are heavily dependent on SIRT5 - OCI-AML2, CMK, and SKM-1. SIRT5-dependent cells expressing ectopic NPM1c exhibit mt dysfunction as evidenced by decreased oxygen consumption rate (OCR) measured using the Seahorse metabolic flux assay. Also, the lethal effect of SIRT5 depletion is rescued in these cells. Pharmacologically blocking NPM1c nuclear export with selinexor in the typically SIRT5-independent OCI-AML3 cells sensitizes them to the lethal effect of SIRT5 depletion. After culturing in 100 ng/mL EtBr for 3, 5, or 11 days, cells had significantly decreased mt abundance as measured by mtDNA content relative to nuclear DNA and had significantly decreased OCR and extracellular acidification rate (ECAR). SIRT5-dependent cells cultured in EtBr survived shRNA-induced SIRT5 depletion, in contrast to the rapid lethality of cells not cultured in EtBr. Additionally, cells initially cultured in EtBr and allowed to recover in the absence of EtBr assume their original phenotype of SIRT5 dependence. However, inhibiting specific subunits of the mt using potent complex I and III inhibitors (rotenone and antimycin A) shows no effect on SIRT5 dependence. Together these data suggest that mt damage, rather than loss of function in one pathway, is needed to convert SIRT5-dependent cells to SIRT5 independence. TFAM, a nuclearly coded transcription factor for mtDNA, is necessary for the replication and transcription of mt genes. We used cas13d-mediated knockdown of TFAM to deplete mtDNA and induce mt dysfunction. Experiments that investigate how this method of inducing mt dysfunction affects OCR, ECAR, and SIRT5-dependent cell line proliferation are currently underway and will be presented at the ASH conference. In summary, we present a mechanism of how NPM1 mutation-associated or experimentally induced chronic sublethal mt dysfunction renders AML resistant to therapies targeting vital mt metabolic pathways, such as SIRT5 inhibitors, demonstrating AML's metabolic flexibility. Experiments are scheduled to validate these findings in primary samples and additional metabolomic and transcriptional studies will be done to deduce the mechanism for SIRT5 dependence in AML.

Sex differences in the neuronal transcriptome and synaptic mitochondrial function in the cerebral cortex of a multiple sclerosis model.

Multiple sclerosis (MS) affects the cerebral cortex, inducing cortical atrophy and neuronal and synaptic pathology. Despite the fact that women are more susceptible to getting MS, men with MS have worse disability progression. Here, sex differences in neurodegenerative mechanisms are determined in the cerebral cortex using the MS model, chronic experimental autoimmune encephalomyelitis (EAE). Neurons from cerebral cortex tissues of chronic EAE, as well as age-matched healthy control, male and female mice underwent RNA sequencing and gene expression analyses using RiboTag technology. The morphology of mitochondria in neurons of cerebral cortex was assessed using Thy1-CFP-MitoS mice. Oxygen consumption rates were determined using mitochondrial respirometry assays from intact as well as permeabilized synaptosomes. RNA sequencing of neurons in cerebral cortex during chronic EAE in C57BL/6 mice showed robust differential gene expression in male EAE compared to male healthy controls. In contrast, there were few differences in female EAE compared to female healthy controls. The most enriched differential gene expression pathways in male mice during EAE were mitochondrial dysfunction and oxidative phosphorylation. Mitochondrial morphology in neurons showed significant abnormalities in the cerebral cortex of EAE males, but not EAE females. Regarding function, synaptosomes isolated from cerebral cortex of male, but not female, EAE mice demonstrated significantly decreased oxygen consumption rates during respirometry assays. Cortical neuronal transcriptomics, mitochondrial morphology, and functional respirometry assays in synaptosomes revealed worse neurodegeneration in male EAE mice. This is consistent with worse neurodegeneration in MS men and reveals a model and a target to develop treatments to prevent cortical neurodegeneration and mitigate disability progression in MS men.

Impact of Exposure to Pyraclostrobin and to a Pyraclostrobin/Boscalid Mixture on the Mitochondrial Function of Human Hepatocytes.

Fungicides are widely used in agriculture for crop protection. Succinate dehydrogenase inhibitors (SDHIs) and strobilurins inhibit mitochondria electron transport chain (ETC) in fungi, by blocking complex II and complex III, respectively. Questions regarding their selectivity of action for fungi have been raised in the literature, and we previously showed that boscalid and bixafen (SDHIs) alter the mitochondrial function of human hepatocytes. Here, we analyzed the impact of the exposure of human hepatocytes to pyraclostrobin, a fungicide belonging to the class of strobilurins. Using electron paramagnetic resonance (EPR), we observed a decrease in oxygen consumption rate (OCR) and an increase in mitochondrial superoxide levels after 24 h exposure to 0.5 µM concentration. As a consequence, the content in ATP amount in the cells was reduced, the ratio reduced/oxidized glutathione was decreased, and a decrease in cell viability was observed using three different assays (PrestoBlue, crystal violet, and annexin V assays). In addition, as SDHIs and strobilurins are commonly associated in commercial preparations, we evaluated a potential "cocktail" toxic effect. We selected low concentrations of boscalid (0.5 µM) and pyraclostrobin (0.25 µM) that did not induce a mitochondrial dysfunction in liver cells when used separately. In sharp contrast, when both compounds were used in combination at the same concentration, we observed a decrease in OCR, an increase in mitochondrial superoxide production, a decrease in the ratio reduced/oxidized glutathione, and a decrease in cell viability in three different assays.