OXPHOS Activity Research Articles

Evidence of acrylamide-induced behavioral deficit, mitochondrial dysfunction and cell death in Drosophila melanogaster

Acrylamide (ACR), a ubiquitous compound with diverse route of exposure, has been demonstrated to have detrimental effects on human and animal health. The mechanisms underlying its toxicity is multifaceted and not fully elucidated. This study aims to provide further insight into novel pathways underlying ACR toxicity by leveraging on Drosophila melanogaster as a model organism. The concentrations of acrylamide (25, 50 and 100 mg/kg) and period of exposure (7-days) used in this study was established through a concentration response curve. ACR exposure demonstrably reduced organismal viability, evidenced by decline in survival rate, offspring emergence and deficits in activity, sleep and locomotory behaviors. Using a high-resolution respirometry assay, the role of mitochondria respiratory system in ACR-mediated toxicity in the flies was investigated. Acrylamide caused dysregulation in mitochondrial bioenergetics and respiratory capacity leading to an impaired OXPHOS activity and electron transport, ultimately contributing to the pathological process of ACR-toxicity. Furthermore, ACR exacerbated apoptosis and induced oxidative stress in D. melanogaster. The up-regulation of mRNA transcription of Reaper, Debcl and Dark genes and down-regulation of DIAP1, an ubiquitylation catalyzing enzyme, suggests that ACR promotes apoptosis through disruption of caspase and pro-apoptotic protein ubiquitination and a mitochondria-dependent pathway in Drosophila melanogaster. Conclusively, this study provides valuable insights into the cellular mechanism underlying ACR-mediated toxicity. Additionally, our study reinforces the utility of D. melanogaster as a translational tool for elucidating the complex mechanisms of ACR toxicity.

Azathioprine promotes intestinal epithelial cell differentiation into Paneth cells and alleviates ileal Crohn’s disease severity

Paneth cells (PCs), a subset of intestinal epithelial cells (IECs) found at the base of small intestinal crypts, play an essential role in maintaining intestinal homeostasis. Altered PCs function is associated with diverse intestinal pathologies, including ileal Crohn’s disease (CD). CD patients with ileal involvement have been previously demonstrated to display impairment in PCs and decreased levels of anti-microbial peptides. Although the immunosuppressive drug Azathioprine (AZA) is widely used in CD therapy, the impact of AZA on IEC differentiation remains largely elusive. In the present study, we hypothesized that the orally administered drug AZA also exerts its effect through modulation of the intestinal epithelium and specifically via modulation of PC function. AZA-treated CD patients exhibited an ileal upregulation of AMPs on both mRNA and protein levels compared to non-AZA treated patients. Upon in vitro AZA stimulation, intestinal epithelial cell line MODE-K exhibited heightened expression levels of PC marker in concert with diminished cell proliferation but boosted mitochondrial OXPHOS activity. Moreover, differentiation of IECs, including PCs differentiation, was boosted in AZA-treated murine small intestinal organoids and was associated with decreased D-glucose consumption and decreased growth rates. Of note, AZA treatment strongly decreased Lgr5 mRNA expression as well as Ki67 positive cells. Further, AZA restored dysregulated PCs associated with mitochondrial dysfunction. AZA-dependent inhibition of IEC proliferation is accompanied by boosted mitochondria function and IEC differentiation into PC.

Abstract PO1-29-01: Single agent and combinatorial efficacy with imipridone ONC206 via inhibition of mitochondrial function in preclinical models of chemorefractory triple negative breast cancer

Abstract Breast cancer remains a major global health concern, with triple-negative breast cancer (TNBC) being one of the most aggressive subtypes associated with limited targeted therapies. Chemotherapy resistance in TNBC patients poses a significant clinical challenge and is associated with poor progression-free and overall survival. Thus, exploring novel approaches to improve chemotherapeutic efficacy is of paramount importance. We previously demonstrated a role for mitochondrial fusion in supporting oxidative phosphorylation (oxphos) activity and cell survival in residual TNBC (PMID: 30996079 and 36813854) cells refractory to chemotherapy treatments. Based on these observations, we sought to inhibit mitochondrial function to improve chemotherapeutic efficacy. We used ONC206, a novel agonist of mitochondrial serine protease, ClpP (Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit) to induce mitochondrial proteotoxic stress. ONC201, a parent molecule of ONC206, has shown efficacy in TNBC as a single agent or when combined with a MEK inhibitor in vitro (PMID: 34680527). We demonstrate treatment of TNBC cell lines with ONC206 results in reduced mitochondrial fusion and OXPHOS. Furthermore, ONC206 co-treatment enhanced chemo-sensitivity in vitro. Based on these promising findings, we tested this in orthotopic patient-derived xenograft (PDX) mouse preclinical trials. Three PDX models of TNBC were tested, BCM-2665, BCM-5471, and BCM-0002. ONC206 as a single agent (twice weekly for 4 weeks, oral gavage, 100mg/kg) significantly decreased tumor growth in all three PDX models. Remarkably, treatment ONC206 achieved nearly complete tumor regression as a single agent or when combined with carboplatin in BCM-2665. Regressed tumors did re-grow, but this was significantly delayed when combined with carboplatin. Treatment with ONC206 in BCM-5471 resulted in a modest but significant reduction in tumor growth rate as a single agent. However, ONC206 did not improve carboplatin (weekly, i.p., 50mg/kg) nor docetaxel (weekly, i.p., 20mg/kg) efficacy in this model. ONC206 treatment of BCM-0002 resulted in prolonged tumor stasis and a slight enhancement of docetaxel and carboplatin efficacy. Interestingly, the efficacy of ONC206 in these PDX models correlates with their relative mRNA expression of ClpP. The mice demonstrated tolerance to ONC206 as a single agent or when combined with chemotherapy during the course of treatment. Our study underscores the potentially crucial role of mitochondria in driving chemotherapy resistance in TNBC and provides novel insights into mitochondria-targeted therapeutic approaches. We report a novel targeting option for TNBC and anticipate that these findings will stimulate further research into innovative combination therapies for TNBC patients, and may lead us to a viable inhibitor. Citation Format: Lily Baek, Lacey Dobrolecki, Stefanie Faucher, Christina Sallas, Nia Griffith, Varun Prabhu, Bora Lim, Michael Lewis, Gloria Echeverria. Single agent and combinatorial efficacy with imipridone ONC206 via inhibition of mitochondrial function in preclinical models of chemorefractory triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-29-01.

Abstract 659: TR-107 and erastin produce synergistic inhibition of colorectal cancer cell viability in vitro

Abstract Introduction: Ferroptosis is an emerging potential target for the treatment of colorectal cancer (CRC) and has been shown to sensitize cancer cells to combinatory chemotherapy. Our studies show that the TR-107 compound, an agonist of ClpP - the proteolytic subunit of mitochondrial maintenance complex - can synergize with Erastin, an inducer of ferroptosis cell death. TR-107 potently disrupts mitochondrial proteostasis and attenuates oxidative phosphorylation, resulting in excessive oxidative stress. Here, we show that Erastin synergizes with TR-107 activity by downregulating the cofactor of GPX4, a major intracellular antioxidant defense resulting in CRC cell apoptosis. Methods: We first evaluated the efficacy of TR-107 against eight CRC cell lines belonging to four established consensus molecular subtypes. Cells were treated with control (DMSO) or TR-107 (10 nM, 50 nM, 1 µM) for 24, 48, and 72 hours. We determined IC50 values with Cell Titer-Glo, cell proliferation with the Beckman Coulter cell counter, cell cycle with flow cytometry, OXPHOS activity with Seahorse Real-Time ATP Rate assay, mitochondrial protein levels with western blot and proteomics, and differential gene expression using RNA sequencing and RT-PCR. Subsequently, we assessed the efficacy of TR-107 and Erastin co-treatment. Cells were treated with DMSO, TR-107 (10, 50 nM), Erastin (1, 10 µM), or combinations of the abovementioned concentrations. Cell viability was measured 72 hours post-treatment with Cell Titer-Glo. Synergy scores (HSA) were calculated with Synergyfinder on RStudio. Ferroptosis-related protein expression 24 hours after co-treatment was assessed with western blot and densitometry. Results: TR-107 IC50 values ranged from 2-70 nM, producing a dose- and time-dependent inhibitory effect in cell proliferation. Western blot showed that 50 nM TR-107 abolished the expression of critical mitochondrial proteins ClpX, TFAM, and TUFM. Seahorse data showed a significant reduction in OXPHOS activity. Multi-omics revealed downregulation of respiratory chain complex subunits, mtDNA transcription/translation factors, and upregulation of mitophagy and ferroptosis-related pathways in treated cells. Co-treatment with Erastin produced synergistic HSA scores in 63% (5/8) of assessed CRC cell lines. RKO and NCI-H508 were highly sensitive to Erastin treatment, suggesting that lower Erastin concentrations may synergize with TR-107 and thus reduce dose toxicity. Western blot analysis 24 hours post-treatment showed a loss of GPX4 protein levels in co-treated cells compared to 50 nM TR-107 treatment. This suggests a rapid downregulation of intracellular antioxidant defense that may induce ferroptosis sooner and more effectively than single-agent treatment. Conclusion: In vitro results suggest that co-treating with TR-107 and Erastin produces a significant, synergistic reduction in CRC cell viability. Citation Format: Michael Giarrizzo, Joseph F. LaComb, Hetvi R. Patel, Agnieszka B. Bialkowska, Rohan Reddy, Lee M. Graves, Edwin J. Iwanowicz. TR-107 and erastin produce synergistic inhibition of colorectal cancer cell viability in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 659.

Platinum iodido drugs show potential anti-tumor activity, affecting cancer cell metabolism and inducing ROS and senescence in gastrointestinal cancer cells

Cisplatin-based chemotherapy has associated clinical disadvantages, such as high toxicity and resistance. Thus, the development of new antitumor metallodrugs able to overcome different clinical barriers is a public healthcare priority. Here, we studied the mechanism of action of the isomers trans and cis-[PtI2(isopropylamine)2] (I5 and I6, respectively) against gastrointestinal cancer cells. We demonstrate that I5 and I6 modulate mitochondrial metabolism, decreasing OXPHOS activity and negatively affecting ATP-linked oxygen consumption rate. Consequently, I5 and I6 generated Reactive Oxygen Species (ROS), provoking oxidative damage and eventually the induction of senescence. Thus, herein we propose a loop with three interconnected processes modulated by these iodido agents: (i) mitochondrial dysfunction and metabolic disruptions; (ii) ROS generation and oxidative damage; and (iii) cellular senescence. Functionally, I5 reduces cancer cell clonogenicity and tumor growth in a pancreatic xenograft model without systemic toxicity, highlighting a potential anticancer complex that warrants additional pre-clinical studies.

Spheroid architecture strongly enhances miR-221/222 expression and promotes oxidative phosphorylation in an ovarian cancer cell line through a mechanism that includes restriction of miR-9 expression.

Tumor cell spheroids are organized multicellular structures that form during the expansive growth of carcinoma cells. Spheroids formation is thought to contribute to metastasis by supporting growth and survival of mobile tumor cell populations. We investigated how spheroid architecture affects OXPHOS activity, microRNA expression, and intraperitoneal survival of an ovarian carcinoma cell line using high resolution respirometry, quantitative RT-PCR, and a rodent intraperitoneal growth model. Rates of oxidative phosphorylation/respiration per cell of cells growing as spheroids were nearly double those of a variant of the same cell type growing in suspension as loosely aggregated cells. Further, inhibition of spheroid formation by treatment with CDH2 (N-cadherin) siRNA reduced the rate of OXPHOS to that of the non-spheroid forming variant. Cells growing as spheroids showed greatly enhanced expression of miR-221/222, an oncomiR that targets multiple tumor suppressor genes and promotes invasion, and reduced expression of miR-9, which targets mitochondrial tRNA-modification enzymes and inhibits OXPHOS. Consistent with greater efficiency of ATP generation, tumor cells growing as spheroids injected into the nutrient-poor murine peritoneum survived longer than cells growing in suspension as loosely associated aggregates. The data indicate that growth in spheroid form enhances the OXPHOS activity of constituent tumor cells. In addition, spheroid architecture affects expression of microRNA genes involved in growth control and mitochondrial function. During the mobile phase of metastasis, when ovarian tumor cells disperse through nutrient-poor environments such as the peritoneum, enhanced OXPHOS activity afforded by spheroid architecture would enhance survival and metastatic potential.

Sulforaphane impedes mitochondrial reprogramming and histone acetylation in polarizing M1 (LPS) macrophages

M1 (LPS) macrophages are characterized by a high expression of pro-inflammatory mediators, and distinct metabolic features that comprise increased glycolysis, a broken TCA cycle, or impaired OXPHOS with augmented mitochondrial ROS production. This study investigated whether the phytochemical sulforaphane (Sfn) influences mitochondrial reprogramming during M1 polarization, as well as to what extent this can contribute to Sfn-mediated inhibition of M1 marker expression in murine macrophages. The use of extracellular flux-, metabolite-, and immunoblot analyses as well as fluorescent dyes indicative for mitochondrial morphology, membrane potential or superoxide production, demonstrated that M1 (LPS/Sfn) macrophages maintain an unbroken TCA cycle, higher OXPHOS rate, boosted fusion dynamics, lower membrane potential, and less superoxide production in their mitochondria when compared to control M1 (LPS) cells. Sustained OXPHOS and TCA activity but not the concomitantly observed high dependency on fatty acids as fuel appeared necessary for M1 (LPS/Sfn) macrophages to reduce expression of nos2, il1β, il6 and tnfα. M1 (LPS/Sfn) macrophages also displayed lower nucleo/cytosolic acetyl-CoA levels in association with lower global and site-specific histone acetylation at selected pro-inflammatory gene promoters than M1 (LPS), evident in colorimetric coupled enzyme assays, immunoblot and ChIP-qPCR analyses, respectively. Supplementation with acetate or citrate was able to rescue both histone acetylation and mRNA expression of the investigated M1 marker genes in Sfn-treated cells. Overall, Sfn preserves mitochondrial functionality and restricts indispensable nuclear acetyl-CoA for histone acetylation and M1 marker expression in LPS-stimulated macrophages.

Loss of Fatty Acid Degradation by Astrocytic Mitochondria as a Mechanism of Neuroinflammation and Neurodegeneration

AbstractBackgroundAstrocytes provide key neuronal support, and their phenotypic transformation is strongly implicated in neurodegenerative disorders including Alzheimer’s disease (AD). Metabolically, astrocytes possess modest mitochondrial oxidative phosphorylation (OxPhos) activity, yet the pathological role of astrocytic OxPhos in neurodegeneration remains to be defined.MethodWe generated the TfamAKO mice, in which the transcription factor A mitochondrial (Tfam) is deleted selectively in astrocytes. Behavioral, electrophysiological, immunostaining, transcriptomics, metabolomics, and magnetic resonance imaging analyses were employed to characterize AD‐relevant phenotypes of TfamAKO mice, which were further compared to an AD mouse model (5xFAD). Primary cell cultures and co‐cultures were used to determine the cell autonomous and non‐autonomous mechanisms by which disrupted astrocytic OxPhos induces astrocyte reactivity, neuroinflammation and neurodegeneration.ResultHere we show that the brain critically depends on astrocytic OxPhos to degrade fatty acids (FAs) and maintain lipid homeostasis. Aberrant astrocytic OxPhos induces lipid droplet (LD) accumulation followed by neurodegeneration that recapitulates key features of AD including reactive astrogliosis, synaptic loss, microgliosis, demyelination, and cognitive impairment. Mechanistically, when FA load overwhelms astrocytic OxPhos capacity, elevated acetyl‐CoA levels induce astrocyte reactivity by enhancing STAT3 acetylation and activation. Intercellularly, lipid‐laden reactive astrocytes stimulate neuronal FA oxidation and oxidative stress, activate microglia via IL‐3 signaling, and inhibit the biosynthesis of FAs and phospholipids required for myelin replenishment. Moreover, the metabolic and transcriptional signatures of the hippocampus of TfamAKO mice highly overlap with that of 5xFAD mice.ConclusionWe reveal a lipid‐centric, AD‐resembling mechanism by which astrocytic mitochondrial dysfunction progressively induces neuroinflammation and neurodegeneration.

The Gene Expression Landscape of BTK Inhibitor Treatment in Chronic Lymphocytic Leukemia Exposed By Shallow Depth RNA Sequencing

Efforts to understand chronic lymphocytic leukemia (CLL) subgroups have focused on multi-omics profiling of patient cohorts (in steady-state) and linking biomarkers to responses. Here, we aim to combine ex-vivo drug screening and gene expression profiling (GEP) in primary CLL samples to dissect disease biology based on the observed phenotype as a qualitative and quantitative function of steady-state features and drug perturbation effects. As most differential gene expression changes in CLL are retrieved from highly expressed genes, we hypothesized that a relevant set of genes could still be reliably captured by reduced sequencing coverage. We profiled five samples with traditional whole transcript TruSeq and 3'-end QuantSeq (Moll et al., Nat Methods, 2014) library preparation with deep and shallow depth sequencing, respectively. We obtained 43.48-72.68 million mappable reads with TruSeq and 2.24-3.78 million with QuantSeq. Although the read coverage was reduced, QuantSeq was able to recover 71.17 ± 0.87% of all genes that were detected by TruSeq with reproducible quantification ( R > 0.85, P < 0.001 for all samples). Shallow sequencing of CLL samples reliably profiled intermediate to highly abundant genes with reduced sensitivity only for lowly expressed genes. We then selected 105 representative CLL patient samples from a well-characterized cohort, treated them ex-vivo with 100 nM ibrutinib for 48h and performed GEP using shallow depth QuantSeq. Sample viability was measured by flow cytometry before and after perturbation. Library preparation and sequencing was successful for 91% of samples. With a median coverage of 2.19 million mappable reads per sample, the principal components (PC) of transcriptional variation in DMSO-treated samples included IGHV (12.0%), methylation subgroup and trisomy 12 status (7.1%). These data show that the screening method reliably captured the transcriptional features of CLL at steady-state and thus support the use of shallow depth RNA sequencing. We were interested in the landscape of transcriptional change after perturbation of the B-cell receptor (BCR) pathway. Samples from the same patients clustered together and spread along a gradient of sample viability in the first PC. After correlation of gene counts from DMSO-treated samples with sample viability and subsequent gene set enrichment analysis, we found activation of the canonical apoptosis pathway via NFκB-mediated TNF-α signaling and cell stress to be associated with this signature of cell death. BTKi-treated samples showed the same signature of spontaneous apoptosis and additionally strong suppression of MYC and OXPHOS activity. To identify the ibrutinib-regulated transcriptional signature, we performed differential gene expression analysis between ibrutinib and DMSO. BTKi treatment induced 904 differentially expressed genes (DEGs) ( FDR < 0.005). These genes were negatively enriched in the hallmarks allograft rejection, MYC targets, TNF-α signaling via NFκB and KEGG pathways associated with BCR signaling, and immune cell response. Top DEGs included e. g. FCRL5, PTPN6, FILIP1L, LRMP and LAPTM5 ( FDR < 0.001) which were also significantly downregulated after in-vivo ibrutinib exposure (Rendeiro et al. Nat Commun, 2020). Inhibition of the BCR induces apoptosis in CLL in an IGHV status-dependent manner. Samples with unmutated IGHV (UM-CLL) were more sensitive to BTKi compared to samples with mutated IGHV (M-CLL) (83.1 vs. 92.3% relative viability, P < 0.001). On gene expression level, we observed more DEGs within UM-CLL (387) compared to M-CLL (182) ( FDR < 0.005). Response to BTKi was largely similar although the effect size in M-CLL tended to be smaller. To identify BCR-dependent genes that were significantly modulated by IGHV, we tested for the interaction between BTKi treatment and IGHV status in the differential expression models. We found the effects on the expression of CA12, FIG RPL5 and P2RY11 to be different upon BTK inhibition between UM- and M-CLL ( FDR < 0.1). In conclusion, we establish shallow depth sequencing in CLL and show it can be used to reliably capture disease biology and drug effects. By treating patient samples with ibrutinib, we show that abrogating BTK strongly suppresses BCR-dependent processes which are largely independent of IGHV status. By adding more pathway perturbations, this approach could be used for further functional dissection of CLL biology.

Mitochondrial metabolism in Drosophila macrophage-like cells regulates body growth via modulation of cytokine and insulin signaling.

Macrophages play critical roles in regulating and maintaining tissue and whole-body metabolism in normal and disease states. While the cell-cell signaling pathways that underlie these functions are becoming clear, less is known about how alterations in macrophage metabolism influence their roles as regulators of systemic physiology. Here, we investigate this by examining Drosophila macrophage-like cells called hemocytes. We used knockdown of TFAM, a mitochondrial genome transcription factor, to reduce mitochondrial OxPhos activity specifically in larval hemocytes. We find that this reduction in hemocyte OxPhos leads to a decrease in larval growth and body size. These effects are associated with a suppression of systemic insulin, the main endocrine stimulator of body growth. We also find that TFAM knockdown leads to decreased hemocyte JNK signaling and decreased expression of the TNF alpha homolog, Eiger in hemocytes. Furthermore, we show that genetic knockdown of hemocyte JNK signaling or Eiger expression mimics the effects of TFAM knockdown and leads to a non-autonomous suppression of body size without altering hemocyte numbers. Our data suggest that modulation of hemocyte mitochondrial metabolism can determine their non-autonomous effects on organismal growth by altering cytokine and systemic insulin signaling. Given that nutrient availability can control mitochondrial metabolism, our findings may explain how macrophages function as nutrient-responsive regulators of tissue and whole-body physiology and homeostasis.

Loss of Metabolic Control Beyond the Promyelocyte Stage Resolves Myeloid Maturation Arrest in Reticular Dysgenesis

Reticular Dysgenesis (RD) is a particularly grave syndromic form of severe combined immunodeficiency. The hematopoietic phenotype manifests in defective myeloid and lymphoid development with profound neutropenia and lymphopenia. RD is caused by biallelic mutations in the mitochondrial enzyme adenylate kinase 2 (AK2), which catalyzes the phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP) in the mitochondrial intermembrane space. ADP is then phosphorylated to adenosine triphosphate (ATP) during the OXPHOS process. While there are abundant other sources of APD, AK2 constitutes the only mitochondrial enzyme that contributes to AMP “recycling” by phosphorylation. Accordingly, AK2-deficient myeloid cells have been shown to exhibit increased AMP levels 1. Beyond this observation, the cellular basis for the combined hematopoietic defect in RD remains elusive. Using a CRISPR/Cas9 AK2 biallelic knock-out model in human hematopoietic stem and progenitor cells (HSPCs), we have previously shown that AK2-/- HSPCs recapitulate RD myelopoiesis defects in vitro. AK2-/- HSPCs exhibit a severe proliferative defect that becomes apparent at the myelocyte (MC) and neutrophil (NP) stages, while the promyelocyte (PM) stage appears relatively spared, consistent with observations in RD patients. Our prior metabolomic analysis showed that AK2-/- MCs not only have increased levels of AMP but up of 20-fold increased levels of IMP derived by converting excess AMP to IMP conversion via AMP deaminases (AMPDs). In addition, we have shown that beginning at the MC stage, AK2 -/- cells exhibit an increase in NADH:NAD+ ratio, consistent with reductive stress and decreased aspartate levels. Our new data revealed that expressing the aspartate transporter GLAST and/or the water-forming NADH oxidase LbNOX in AK2-/- HSPCs partially rescues differentiation but not the proliferation defect. It raises the possibility that reductive stress and aspartate deficiency compromise protein synthesis necessary for maturation, but are separate from the proliferative defect related to high IMP and AMP levels. There were no metabolomic abnormalities noted at the PM stage. PMs have a higher proliferative index than MCs and NPs. This raises the question why the most metabolically active stage is phenotypically spared by AK2 deficiency. To better understand why the metabolic abnormalities associated with RD selectively affect differentiation beyond the promyelocyte stage, we investigated key regulators of cellular metabolism and proliferation, i.e., AMPK, ACC1 and S6, a target of mTOR signaling, at PM, MC and NP stages. We found activation of AMPK, ACC1 and S6 only at the PM stage, while AMPK, ACC1 and S6 protein expression at MC and NP stages was virtually undetectable. Interestingly, AK2-/- PMs exhibited notably lower ATP synthesis rates compared to controls, while ATP synthesis in AK2-/- MCs and NPs was significantly higher than in controls of corresponding stages, paralleling the accumulation of AMP and IMP noted earlier. These findings raise the possibility that tight metabolic control at the promyelocyte stage prevents AMP levels from rising by curtailing OXPHOS metabolism in AK2-/- PMs. In contrast, lack of metabolic control beyond the PM stage permits unopposed OXPHOS activity, thereby allowing AMP and IMP levels to rise to toxic levels that interfere with proliferation. In our ongoing work, we are testing whether pharmacologically redirecting metabolism from OXPHOS to glycolysis with UK5099, could rescue AK2-/- myelopoiesis defects. UK5099 is a potent inhibitor of the mitochondrial pyruvate carrier, which facilitates pyruvate transport across the mitochondrial inner membrane to fuel the TCA cycle. Additionally, we developed a hematopoietic-specific and inducible Cre-mediated Ak2 knock-out mouse model, which recapitulates the profound neutropenia and lymphopenia observed in RD patients. This mouse model will allow us to corroborate our in vitro observations and to investigate if the same mechanistic underpinnings also apply to the lymphoid lineage. Rissone A, Weinacht KG, la Marca G, et al. Reticular dysgenesis-associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress. J Exp Med. Jul 27 2015;212(8):1185-202. doi:10.1084/jem.20141286

Training-Induced Increase in V·O2max and Critical Power, and Acceleration of V·O2 on-Kinetics Result from Attenuated Pi Increase Caused by Elevated OXPHOS Activity.

Computer simulations using a dynamic model of the skeletal muscle bioenergetic system, involving the Pi-double-threshold mechanism of muscle fatigue, demonstrate that the training-induced increase in V·O2max, increase in critical power (CP) and acceleration of primary phase II of the V·O2 on kinetics (decrease in t0.63) is caused by elevated OXPHOS activity acting through a decrease in and slowing of the Pi (inorganic phosphate) rise during the rest-to-work transition. This change leads to attenuation of the reaching by Pi of Pipeak, peak Pi at which exercise is terminated because of fatigue. The delayed (in time and in relation to V·O2 increase) Pi rise for a given power output (PO) in trained muscle causes Pi to reach Pipeak (in very heavy exercise) after a longer time and at a higher V·O2; thus, exercise duration is lengthened, and V·O2max is elevated compared to untrained muscle. The diminished Pi increase during exercise with a given PO can cause Pi to stabilize at a steady state less than Pipeak, and exercise can continue potentially ad infinitum (heavy exercise), instead of rising unceasingly and ultimately reaching Pipeak and causing exercise termination (very heavy exercise). This outcome means that CP rises, as the given PO is now less than, and not greater than CP. Finally, the diminished Pi increase (and other metabolite changes) results in, at a given PO (moderate exercise), the steady state of fluxes (including V·O2) and metabolites being reached faster; thus, t0.63 is shortened. This effect of elevated OXPHOS activity is possibly somewhat diminished by the training-induced decrease in Pipeak.

Mild to moderate post-COVID-19 alters markers of lymphocyte activation, exhaustion, and immunometabolic responses that can be partially associated by physical activity level- an observational sub-analysis fit- COVID study.

This study aimed to evaluate if physical activity is associated with systemic and cellular immunometabolic responses, in young adults after mild-to-moderate COVID-19 infection. Mild- to- moderate post-COVID-19 patients (70.50 ± 43.10 days of diagnosis; age: 29.4 (21.9- 34.9) years; BMI: 25.5 ± 4.3 kg m2 n = 20) and healthy age-matched controls (age: 29.3 (21.2 - 32.6) years; BMI: 25.4 ± 4.7 kg m2; n = 20) were evaluated. Physical activity levels (PAL), body composition, dietary habits, muscular and pulmonary function, mental health, sleep quality, metabolic parameters, immune phenotypic characterization, stimulated whole blood and PBMC culture (cytokine production), mRNA, and mitochondrial respiration in PBMCs were evaluated. The post-COVID-19 group exhibited lower levels of moderate to vigorous physical activity (MVPA) (p = 0.038); therefore, all study comparisons were performed with adjustment for MVPA. Post-COVID-19 impacted the pulmonary function (FEV1, FEV1%pred, FVC, and FVC %pred) compared with the control (p adjusted by MVPA (p adj) <0.05). Post-COVID-19 exhibited lower levels of serum IL-6 (p adj <0.01), whereas it showed higher serum IL-10, triglyceride, leptin, IgG, ACE activity, TNFRSF1A, and PGE2 (p adj <0.05) levels compared with controls. Post-COVID-19 presented a lower percentage of Treg cells (p adj = 0.03) and altered markers of lymphocyte activation and exhaustion (lower CD28 expression in CD8+ T cells (p adj = 0.014), whereas CD4+T cells showed higher PD1 expression (p adj = 0.037)) compared with the control group. Finally, post- COVID-19 presented an increased LPS-stimulated whole- blood IL-10 concentration (p adj <0.01). When exploring mitochondrial respiration and gene expression in PBMCs, we observed a higher LEAK state value (p adj <0.01), lower OXPHOS activity (complex I) (p adj = 0.04), and expression of the Rev-Erb-α clock mRNA after LPS stimulation in the post-COVID-19 patients than in the control (p adj <0.01). Mainly, PAL was associated with changes in IL-10, triglyceride, and leptin levels in the plasma of post-COVID-19 patients. PAL was also associated with modulation of the peripheral frequency of Treg cells and the expression of PD-1 in CD8+ T cells, although it abrogated the statistical effect in the analysis of TNF-α and IL-6 production by LPS- and PMA-stimulated PBMC of post-COVID-19 patients. Young adults after mild-to-moderate SARS-CoV-2 infection appeared to have lower physical activity levels, which can be associated with clinical and immunometabolic responses in a complex manner.

Time-resolved proteomic analyses of senescence highlight metabolic rewiring of mitochondria.

Mitochondrial dysfunction and cellular senescence are hallmarks of aging. However, the relationship between these two phenomena remains incompletely understood. In this study, we investigated the rewiring of mitochondria upon development of the senescent state in human IMR90 fibroblasts. Determining the bioenergetic activities and abundance of mitochondria, we demonstrate that senescent cells accumulate mitochondria with reduced OXPHOS activity, resulting in an overall increase of mitochondrial activities in senescent cells. Time-resolved proteomic analyses revealed extensive reprogramming of the mitochondrial proteome upon senescence development and allowed the identification of metabolic pathways that are rewired with different kinetics upon establishment of the senescent state. Among the early responding pathways, the degradation of branched-chain amino acid was increased, whereas the one carbon folate metabolism was decreased. Late-responding pathways include lipid metabolism and mitochondrial translation. These signatures were confirmed by metabolic flux analyses, highlighting metabolic rewiring as a central feature of mitochondria in cellular senescence. Together, our data provide a comprehensive view on the changes in mitochondrial proteome in senescent cells and reveal how the mitochondrial metabolism is rewired in senescent cells.

Cellular allostatic load is linked to increased energy expenditure and accelerated biological aging

Stress triggers anticipatory physiological responses that promote survival, a phenomenon termed allostasis. However, the chronic activation of energy-dependent allostatic responses results in allostatic load, a dysregulated state that predicts functional decline, accelerates aging, and increases mortality in humans. The energetic cost and cellular basis for the damaging effects of allostatic load have not been defined. Here, by longitudinally profiling three unrelated primary human fibroblast lines across their lifespan, we find that chronic glucocorticoid exposure increases cellular energy expenditure by ∼60%, along with a metabolic shift from glycolysis to mitochondrial oxidative phosphorylation (OxPhos). This state of stress-induced hypermetabolism is linked to mtDNA instability, non-linearly affects age-related cytokines secretion, and accelerates cellular aging based on DNA methylation clocks, telomere shortening rate, and reduced lifespan. Pharmacologically normalizing OxPhos activity while further increasing energy expenditure exacerbates the accelerated aging phenotype, pointing to total energy expenditure as a potential driver of aging dynamics. Together, our findings define bioenergetic and multi-omic recalibrations of stress adaptation, underscoring increased energy expenditure and accelerated cellular aging as interrelated features of cellular allostatic load.

Colorectal polyps increase the glycolytic activity.

In colorectal cancer (CRC) energy metabolism research, the precancerous stage of polyp has remained rather unexplored. By now, it has been shown that CRC has not fully obtained the glycolytic phenotype proposed by O. Warburg and rather depends on mitochondrial respiration. However, the pattern of metabolic adaptations during tumorigenesis is still unknown. Understanding the interplay between genetic and metabolic changes that initiate tumor development could provide biomarkers for diagnosing cancer early and targets for new cancer therapeutics. We used human CRC and polyp tissue material and performed high-resolution respirometry and qRT-PCR to detect changes on molecular and functional level with the goal of generally describing metabolic reprogramming during CRC development. Colon polyps were found to have a more glycolytic bioenergetic phenotype than tumors and normal tissues. This was supported by a greater GLUT1, HK, LDHA, and MCT expression. Despite the increased glycolytic activity, cells in polyps were still able to maintain a highly functional OXPHOS system. The mechanisms of OXPHOS regulation and the preferred substrates are currently unclear and would require further investigation. During polyp formation, intracellular energy transfer pathways become rearranged mainly by increasing the expression of mitochondrial adenylate kinase (AK) and creatine kinase (CK) isoforms. Decreased glycolysis and maintenance of OXPHOS activity, together with the downregulation of the CK system and the most common AK isoforms (AK1 and AK2), seem to play a relevant role in CRC development.

Abstract 1374: Small cell lung cancer predominantly expressing ASCL1 shows a distinct oxidative phenotype

Abstract Small cell lung cancer (SCLC) accounts for approximately 15% of lung cancers and has a poor prognosis due to a high proliferation rate and early metastasis. The current standard treatment, a combination of a platinum-based chemotherapy and etoposide, does not consider the underlying molecular profiles and, therefore, displays limited success. Tailoring treatment to the specific attributes of the 4 SCLC subtypes characterized by the predominant expression of either ASCL1, NeuroD1, POU2F3 or YAP1 (SCLC-A/N/P/Y) might improve patient outcomes. Here, we investigated the proteomic landscape of SCLC subsets with the aim to identify novel subtype-specific characteristics of diagnostic and therapeutic relevance. MS-based proteomics on 26 human SCLC cell lines was performed to decipher pathway-level differences between the subtypes. Mitochondria and lipid droplets were stained with Mitotracker Red CMXROS and Bodipy 493/503, respectively, and quantified by flow cytometry and confocal imaging. Differential responses to inhibitors of complex 1 (metformin, IACS-010759), the CPT1 inhibitor perhexiline, the glutaminolysis inhibitor BPTES and the aerobic glycolysis inhibitor UK5099 were tested using MTT-based cell viability assays. Pathway analyses based on proteomic data identified several metabolic pathways including “oxidative phosphorylation” (OXPHOS) to be significantly upregulated in SCLC-A compared to the other subtypes. Furthermore, multiple proteins essential for the electron transport chain (COX4I1, COX5B, NDUFA5) were highly expressed. Analysis of mitochondria showed a significantly higher number of mitochondria in cells (n=3) from the SCLC-A subtype, which also exhibited a more elongated shape. Lipid droplets were more abundant in the other, low OXPHOS subtypes (n=3). Perhexiline treatment confirmed higher OXPHOS activity solely in the SCLC-A subtype. In contrast, low OXPHOS cells were more sensitive to glutaminolysis inhibition, while no difference was seen when glycolysis was inhibited. Suggesting a differential response to clinically used OXPHOS inhibitors, SCLC cell lines with high and low oxidative activity were treated with metformin and IACS-010759 and response to treatment correlated positively with oxidative state. In this study, we demonstrate distinct differences in metabolic processes in the SCLC-A subtype compared to the other subtypes. Specifically, our data show upregulated oxidative phosphorylation as a potential therapeutic target. Understanding the variances in the metabolic phenotype between the molecular subtypes is integral to achieving precision medicine and advancing SCLC patient treatment. Citation Format: Karin Schelch, Anna Schwendenwein, Kristiina Boettiger, Dominik Kirchhofer, Christian Lang, Zsolt Megyesfalvi, Beata Szeitz, Konrad Hoetzenecker, Lukas Unger, Melinda Rezeli, Balazs Dome. Small cell lung cancer predominantly expressing ASCL1 shows a distinct oxidative phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1374.

Loss of fatty acid degradation by astrocytic mitochondria triggers neuroinflammation and neurodegeneration.

Astrocytes provide key neuronal support, and their phenotypic transformation is implicated in neurodegenerative diseases. Metabolically, astrocytes possess low mitochondrial oxidative phosphorylation (OxPhos) activity, but its pathophysiological role in neurodegeneration remains unclear. Here, we show that the brain critically depends on astrocytic OxPhos to degrade fatty acids (FAs) and maintain lipid homeostasis. Aberrant astrocytic OxPhos induces lipid droplet (LD) accumulation followed by neurodegeneration that recapitulates key features of Alzheimer's disease (AD), including synaptic loss, neuroinflammation, demyelination and cognitive impairment. Mechanistically, when FA load overwhelms astrocytic OxPhos capacity, elevated acetyl-CoA levels induce astrocyte reactivity by enhancing STAT3 acetylation and activation. Intercellularly, lipid-laden reactive astrocytes stimulate neuronal FA oxidation and oxidative stress, activate microglia through IL-3 signalling, and inhibit the biosynthesis of FAs and phospholipids required for myelin replenishment. Along with LD accumulation and impaired FA degradation manifested in an AD mouse model, we reveal a lipid-centric, AD-resembling mechanism by which astrocytic mitochondrial dysfunction progressively induces neuroinflammation and neurodegeneration.

Perturbation of Autophagy by a Beclin 1-Targeting Stapled Peptide Induces Mitochondria Stress and Inhibits Proliferation of Pancreatic Cancer Cells.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, with a dismal five-year survival rate of less than 10%. PDAC possesses prominent genetic alterations in the oncogene KRAS and tumor suppressors p53, SMAD4 and CDKN2A. However, efforts to develop targeted drugs against these molecules have not been successful, and novel therapeutic modalities for PDAC treatment are urgently needed. Autophagy is an evolutionarily conserved self-degradative process that turns over intracellular components in a lysosome-dependent manner. The role of autophagy in PDAC is complicated and context-dependent. Elevated basal autophagy activity has been detected in multiple human PDAC cell lines and primary tumors resected from patients. However, clinical trials using chloroquine (CQ) to inhibit autophagy failed to show therapeutic efficacy. Here we show that a Beclin 1-targeting stapled peptide (Tat-SP4) developed in our lab further enhanced autophagy in multiple PDAC cell lines possessing high basal autophagy activity. Tat-SP4 also triggered faster endolysosomal degradation of EGFR and induced significant mitochondria stress as evidenced by partial loss of Δψ, increased level of ROS and reduced OXPHOS activity. Tat-SP4 exerted a potent anti-proliferative effect in PDAC cell lines in vitro and prohibited xenograft tumor growth in vivo. Intriguingly, excessive autophagy has been reported to trigger a unique form of cell death termed autosis. Tat-SP4 does induce autosis-like features in PDAC cells, including mitochondria stress and non-apoptotic cell death. Overall, our study suggests that autophagy perturbation by a Beclin 1-targeting peptide and the resulting autosis may offer a new strategy for PDAC drug discovery.

A Mutation in Mouse MT-ATP6 Gene Induces Respiration Defects and Opposed Effects on the Cell Tumorigenic Phenotype.

As the last step of the OXPHOS system, mitochondrial ATP synthase (or complex V) is responsible for ATP production by using the generated proton gradient, but also has an impact on other important functions linked to this system. Mutations either in complex V structural subunits, especially in mtDNA-encoded ATP6 gene, or in its assembly factors, are the molecular cause of a wide variety of human diseases, most of them classified as neurodegenerative disorders. The role of ATP synthase alterations in cancer development or metastasis has also been postulated. In this work, we reported the generation and characterization of the first mt-Atp6 pathological mutation in mouse cells, an m.8414A&gt;G transition that promotes an amino acid change from Asn to Ser at a highly conserved residue of the protein (p.N163S), located near the path followed by protons from the intermembrane space to the mitochondrial matrix. The phenotypic consequences of the p.N163S change reproduce the effects of MT-ATP6 mutations in human diseases, such as dependence on glycolysis, defective OXPHOS activity, ATP synthesis impairment, increased ROS generation or mitochondrial membrane potential alteration. These observations demonstrate that this mutant cell line could be of great interest for the generation of mouse models with the aim of studying human diseases caused by alterations in ATP synthase. On the other hand, mutant cells showed lower migration capacity, higher expression of MHC-I and slightly lower levels of HIF-1α, indicating a possible reduction of their tumorigenic potential. These results could suggest a protective role of ATP synthase inhibition against tumor transformation that could open the door to new therapeutic strategies in those cancer types relying on OXPHOS metabolism.