Conference Agenda

Background: Mitochondrial permeability transition (mPT) is a crucial cellular process associated with physiological and pathological conditions, leading to mitochondrial swelling, and compromised integrity. Light-scattering assay is considered a “golden standard” method for mPT detection but until now it was limited to the large population of isolated mitochondria in suspension. Approach: Here, we propose a non-invasive approach using dark-field light scattering microscopy to monitor real-time mPT dynamics in intact cells at a single mitochondrion level. Ferutinin, a potent calcium ionophore, was employed to activate mPT in Wt and mutant HAP1 cells, while concurrently tracking ΔΨ_M with TMRM and mitochondrial light scattering using dark-field imaging. Results: We confirmed that mPT activation causes a decrease in light scattering that can be monitored in high temporal and spatial resolution. We have detected a heterogeneity of mPT pore activity in the mitochondrial population within the single cell. Further, mitochondrial calcium overload under non-mPT conditions leads to the increase in light-scattering, which presumably is caused by the formation of Ca²⁺-phosphate precipitates. Conclusion: Overall, we propose that this novel approach is a powerful tool that can be used for performing classical mPT assay in living cells and has the potential to be used for monitoring the dynamics of calcium precipitates.

[1] G. Morciano et al., "The mitochondrial permeability transition pore: an evolving concept critical for cell life and death," Biological Reviews, vol. 96, no. 6, pp. 2489-2521, 2021/12// 2021, doi: 10.1111/brv.12764.

[2] A. Y. Baev, P. A. Elustondo, A. Negoda, and E. V. Pavlov, "Osmotic regulation of the mitochondrial permeability transition pore investigated by light scattering, fluorescence and electron microscopy techniques," Analytical Biochemistry, vol. 552, pp. 38-44, 2018/07/01/ 2018, doi: https://doi.org/10.1016/j.ab.2017.07.006.

Hydrogen sulfide (H₂S) is a multifaceted signaling molecule in mammalian physiology. In bioenergetics, H₂S is a two-faced molecule, serving as a stimulant or an inhibitor of the mitochondrial electron transfer chain (mETC), depending on its concentrations. H₂S is catabolized in the mitochondria by a sulfide oxidation pathway. Persulfide dioxygenase (PDO) is a non-heme iron mononuclear enzyme of the metallo-β-lactamase structural family, which converts glutathione persulfide (GSSH, derived from sulfide:quinone oxidoreductase) into sulfite and glutathione, using O₂ as co-substrate. GSSH-sustained O₂ consumption by PDO presents an auto-inhibitory profile attributed to glutathionylation of cysteine residue(s). Altered PDO expression, localization and function have been associated with different diseases, although the molecular mechanisms underlying its dysfunction remain to be clarified.

Herein we report the modulation of human PDO structure and function by redox active endogenous modulators with relevance for bioenergetics.

Employing high resolution respirometry and working on the recombinant enzyme, we observed potent and reversible inhibition of human PDO by nitric oxide (NO). At physiological O₂ levels and high electron flow (higher GSSH), the apparent IC₅₀ for PDO inhibition by NO is <100 nM. The >5-fold higher apparent IC₅₀ observed at supra-physiological O₂ levels indicates that NO competes with O₂ for the ferrous active site.

Since PDO contains an unusually high number of non-disulfide cysteine residues, we investigated the effect of PDO cysteinylation. By mass spectrometry, we identified seven cysteinylated cysteines. PDO cysteinylation slightly impaired PDO activity but abrogated the auto-inhibitory profile, thereby affording higher kinetic stability. To further investigate this regulatory mechanism, we produced serine-substituted PDO variants of each cysteine and compared their structural and functional properties with the WT enzyme. Cys34 and Cys139 were identified as the candidate residues for the auto-inhibitory mechanism.

Altogether the data highlight the control of H₂S metabolism through a fine regulation of one of its catabolic enzymes, particularly through a crosstalk with other redox mediators.

The liver is a hub of systemic energy metabolism, yet it is surprisingly rarely a major affected organ in mitochondrial diseases that compromise oxidative phosphorylation (OXPHOS). Complex III (CIII)-deficient Bcs1l^p.S78G knock-in mice, carrying a GRACILE syndrome patient mutation in the gene encoding the CIII assembly factor BCS1L, show juvenile-onset liver disease and kidney tubulopathy with growth restriction, genomic instability, segmental progeria and premature death. To interrogate the contribution of the liver to the systemic manifestations in this model of severe CIII deficiency, we performed serotype 9 rAAV-based gene replacement therapy in the Bcs1l^p.S78G mice using hepatocyte-specific or broadly active promoters to drive the expression of wild-type BCS1L. A single rAAV-Bcs1l i.p. injection at presymptomatic age (21 days) transduced the liver efficiently, restored hepatic CIII assembly and activity and prevented liver disease, hepatocyte senescence and senescence associated secretory phenotype. Restoration of OXPHOS in hepatocytes was sufficient to improve growth and prevent lethal hypoglycemia, extending survival by 100%. The mutant mice attempted a fasting-type metabolic switch from glucose to fatty acid uptake (Cd36 up) and oxidation (Pdk4 up) upon glycogen depletion, but gene expression suggested paradoxical downregulation β-oxidation (Ppara, Cpt1a, Hadhb down). rAAV-Bcs1l corrected these changes to wild-type level. We found that the Bcs1l^p.S78G mice became hypothermic (mean core temperature ~35^oC at P28) with torpor-like immobility during the daytime. The hypothermia was not corrected by housing at thermoneutrality (30-32^oC), suggesting that it was a regulated response to the OXPHOS deficiency. Indeed, we found that markers of brown adipose tissue (BAT) thermogenesis like adrenergic stimulation (β3ar), uncoupling (Ucp1) and mitochondrial mass (Ndufs3, Sdhb) were downregulated rather than induced, whereas a marker of skeletal muscle thermogenesis (Sln) was slightly increased. Thyroid hormone (T4, T3) levels in plasma and liver tissue were not changed, but hepatic thyroid hormone uptake (Slc16a2), metabolism (Dio1, Dio3) and signaling (Rxra, Thrsp) were downregulated, suggesting attempt to dampen thyroid hormone-related energy expenditure in peripheral tissues. Accordingly, the mutant mice were intolerant to increased thyroid hormone, becoming even more hypothermic after T3 injection. Surprisingly, restoring OXPHOS in hepatocytes via rAAV-Bcs1l was able to maintain near-normal body temperature without improvement in BAT or muscle thermogenesis. We thus conclude that basal liver thermogenesis is sufficient to maintain body temperature and reverse torpor-like state in CIII deficient mice. These results serve as a preclinical study showing broad systemic therapeutic effects of liver-targeted gene therapy in a multiorgan mitochondrial disease.

How mitochondria are regulated and adjusted remains poorly understood. The Src kinase, a protein involved in cell metabolism and survival, is localized in several cellular compartments including mitochondria where it regulates its activity and morphology [1]. We hypothesised that Src modulates mitochondria through phosphorylation of «architectural» proteins. We recently showed that Src interacts with the stomatin-like protein 2 (Slp2) [2]. Slp2 stabilizes the mitochondrial inner membrane, favoring the assembly of the supercomplexes of the electron transfert chain (ETC) [3]. The aim of this work is to evaluate how Src does modulate mitochondria through phosphorylation of Slp2. First, we characterized the impact of Slp2 on mitochondrial physiology in Slp2^+/+ and Slp2^-/- mouse embryonic fibroblasts (MEFs). Oxygen consumption rates and activity of ETC supercomplexes are reduced in Slp2^-/- MEFs. Deletion of Slp2 increased levels of mitochondrial reactive oxygen species, whereas mitochondrial size is decreased in Slp2^-/- MEFs. Then, we observed that Src phosphorylates Slp2-Y316. We are now generating MEFs expressing phosphomutants of Slp2-Y316 to examine how Src-dependent phosphorylation of Slp2 does impact on mitochondria. This project will determine if Slp2 phosphorylation is sufficient and/or necessary for the regulation of mitochondrial activity and morphology by Src.

[1] O. Lurette, H. Guedouari, J.L. Morris, R. Martín-Jiménez, J.-P. Robichaud, G. Hamel-Côté, M. Khan, N. Dauphinee, N. Pichaud, J. Prudent, E. Hebert-Chatelain, Mitochondrial matrix-localized Src kinase regulates mitochondrial morphology, Cell Mol Life Sci 79 (2022) 327. https://doi.org/10.1007/s00018-022-04325-y.

[2] H. Guedouari, Y. Ould Amer, N. Pichaud, E. Hebert-Chatelain, Characterization of the interactome of c-Src within the mitochondrial matrix by proximity-dependent biotin identification, Mitochondrion 57 (2021) 257–269.

[3] P. Mitsopoulos, Y.-H. Chang, T. Wai, T. König, S.D. Dunn, T. Langer, J. Madrenas, Stomatin-Like Protein 2 Is Required for In Vivo Mitochondrial Respiratory Chain Supercomplex Formation and Optimal Cell Function, Mol Cell Biol 35 (2015) 1838–1847.

NADH detection by autofluorescence is a technique used to assess mitochondrial NAD redox regulation. Under ultraviolet (UV) light exposure, the reduced form NAD(P)H fluoresces, while the oxidized form NAD(P)⁺ does not. This technique is incorporated into the Oroboros NextGen-O2k allowing for simultaneous measurement of respiration and NAD redox state.

Substrate, uncoupler, inhibitor titration (SUIT) protocols were adjusted to interrogate sequentially several respiratory/NAD redox states in mitochondria isolated from mouse liver. Pyruvate, glutamate, and malate were used as substrates for mitochondrial dehydrogenases, reducing NAD⁺ to NADH. Titrations of ADP or uncoupler were employed to analyze oxidative phosphorylation (OXPHOS) and electron transfer (ET) capacities, P and E, respectively. The fully reduced NADH state was obtained by titration of the Complex III inhibitor myxothiazol under anoxia. After reoxygenation, O₂ flux in the presence of myxothiazol was used to correct mitochondrial respiration for residual oxygen consumption. UV light intensities of 0, 1, 7.5, or 15 mW were pulsed with 2 s on and 2 s off.

Increasing UV light intensities stimulated LEAK respiration by dyscoupling. This caused a compensatory increase of OXPHOS capacity up to 7.5 mW at minimally declining ET capacity. Under these conditions, the net P-L respiration was maintained constant and the P-L control efficiency (P-L)/P was conserved at 0.91. At 15 mW, however, E and P were inhibited by 36 % and 37 % respectively, and the P-L control efficiency dropped to 0.81. Since the E-P control efficiency even increased from 0.27 to 0.34 at 15 mW, this indicates that not only electron transfer but independently the phosphorylation system was affected.

Pulsing the UV light at the lowest intensity of 1 mW mitigated the UV light effects on respiration. These results emphasize the importance of monitoring respiration simultaneously with NAD redox analysis.

Mitochondrial metabolism lays on the efficient exchange of substances (ATP/ADP, NAD+/NADH, ions) through the Voltage-Dependent Anion-selective Channels (VDAC), the most abundant pore-forming protein family of the outer membrane [1]. Among the three human isoforms, VDAC1 is the most expressed especially in cancers, where it protects cells from apoptosis and promotes the "Warburg effect", aiding the tumor cells to proliferate. Thus, VDAC1 stands out as a potential oncogene and a pharmacological target for cancer therapy [2]. Targeting cancer cell metabolism is, indeed, a powerful strategy to slow down glycolysis and shut the metabolic coupling with neighboring cells, reducing cancer cell survival.

To modulate VDAC1 function(s) in an anti-cancer approach, our efforts were focused on the development of small molecules aimed at modulating VDAC1 function to disrupt cell metabolism and induce apoptosis. The AI-based screening revealed a class of molecules built around a three-ring architecture that potentially binds VDAC1 in the NADH-anchoring pocket [3]. Rational mutagenesis and binding assays confirmed that selected molecules prevent the NADH binding hampering the mitochondrial respiration driven by complex I – the principal user of NADH – triggering metabolic unbalance. In parallel, we performed vitality experiments on organoids derived from intrahepatic cholangiocarcinoma (iCCA) patients. We found that these small molecules are extremely effective in killing cancer while they showed a lower impact on healthy cells than Gemcitabine [4], the standard therapeutic intervention for iCCA. Overall, our work uncovers a multifaceted approach to cancer treatment, involving meticulous targeting of metabolic gatekeepers like VDAC1. The new molecules proposed, albeit in the nascent stages, represent promising candidates for further optimization and development, potentially revolutionizing treatment modalities in cancer therapy through precise metabolic interventions.

[1] A. Messina,S. Reina,F. Guarino,V. De Pinto, VDAC isoforms in mammals. BBA,1818(6) (2012):1466-1476.[2] A. Magrì,S. Reina,V. De Pinto, VDAC1 as Pharmacological Target in Cancer and Neurodegeneration: Focus on Its Role in Apoptosis. Front-Chem,(2018)6:108. [3] R. Böhm,GF.Amodeo, S.Murlidaran,S.Chavali,G.Wagner,M.Winterhalter,G.Brannigan,S.Hiller. The Structural Basis for Low Conductance in the Membrane Protein VDAC upon β-NADH Binding and Voltage Gating. Structure.28(2)(2020):206-214.e4.[4]S.De-Siervi,C Turato,Liver Organoids as an In Vitro Model to Study Primary Liver Cancer.Int.J.Mol.Sci.25;24(5)(2023):4529.

Background and aims: Electroconvulsive therapy (ECT) is an effective clinical intervention to ameliorate depressive symptoms, especially for individuals diagnosed with severe, treatment-resistant major depressive disorder (MDD). However, the underlying biomolecular mechanisms of ECT remain poorly explored. Translational research suggests that impairments in mitochondrial bioenergetics contribute to the psychopathology of MDD and relate to the severity of depression. By applying a translational approach, the study MITO-ECT aims to investigate the effects of ECT treatment on mitochondrial bioenergetics in immune cells from patients with MDD. Here, we report preliminary longitudinal data on the first 11 study participants across ECT intervention.

Methods: Individuals diagnosed with MDD (n=11; 36% female; mean age=49.09, SD=11.44) received 10-12 ECT sessions over a period of 4-5 weeks. Bioenergetic and clinical assessments covered four time points: (1) before; (2) after the fifth ECT; (3) after the last ECT; and (4) three months follow-up. The severity of depressive symptoms and their change over time were evaluated using the Beck Depression Inventory (BDI-II; self-report) and the Montgomery-Åsberg Depression Rating Scale (MADRS; interview). Venous blood samples were collected to isolate peripheral blood mononuclear (PBMC), and cryopreserved samples were stored until analysis. Mitochondrial respiration was measured in freshly thawed PBMC using a standard substrate-uncoupler-inhibitor-titration (SUIT) protocol with the O2K oxygraph (Oroboros Instruments, Austria). Mixed linear models were applied to study changes in clinical symptoms and mitochondrial respiration and to correlate oxygen consumption to the clinical response over the course of treatment.

Results: Preliminary findings indicated significant decreases in the subjectively (BDI-II sum score, effect of time: F_{3, 22.62} = 13.01, p<.0001) and objectively (MADRS, effect of time: F_{3, 22.81} = 34.1, p<.001) assessed severity of depressive symptoms in response to ECT treatment. In addition, we observed significant increases in routine respiration (F_{3, 23.53}=7.47, p=.001), maximal respiratory capacity (F_{3, 24.7}=10.64, p<.001) and spare respiratory capacity (F_{3, 24.96}=11.29, p<.001). Results further suggested significant associations between depression severity (BDI-II sum) and maximal respiratory capacity (F_{1, 21.66}=5.77, p=.048) and spare respiratory capacity F_{1, 8.08}=8.22, p=.02) measured over all time points.

Conclusions: Electroconvulsive therapy may have a stimulating effect on mitochondrial bioenergetics, at least in immune cells. Furthermore, mitochondrial bioenergetics and its clinical application as a potential prognostic biomarker of ECT treatment effects and stability of clinical improvement may advance predictive, preventive and personalized medicine in translational psychiatry.

Cardiolipin (CL) is a mitochondria-specific glycerophospholipid, comprising about 10-15% of the total mitochondrial phospholipid content. It is primarily located within the inner mitochondrial membrane (IMM) where it plays central roles in several aspects of mitochondrial biology, including the stabilisation of OXPHOS protein complexes and supercomplexes. Molecular defects in the genes responsible for CL biosynthesis and remodelling have been linked to several human diseases, including severe myopathy, cardiomyopathy, neutropenia, and multisystemic disorders.

Protein tyrosine phosphatase mitochondrial 1 (PTPMT1) is a highly conserved mitochondrial tyrosine phosphatase that is crucial in the de novo CL biosynthesis. It localises to the matrix leaflet of the IMM, where it dephosphorylates phosphatidylglycerol phosphate to phosphatidylglycerol, a key intermediate in the CL biosynthetic pathway. Total body ablation of Ptpmt1 is embryonic lethal, suggesting the protein is essential for CL biosynthesis and the survival of cells during development. Although PTPMT1 has been linked to mitochondrial activity deficiencies in several in vitro and in vivo models, its pathogenic role in humans has not been described yet.

Here, we present six individuals from three unrelated families presenting with a neonatal/infantile-onset neurological and neurodevelopmental syndrome associated with CL abnormalities resulting from novel biallelic variants in the PTPMT1 gene. Using patient-derived fibroblasts and skeletal muscle tissue, combined with cellular rescue experiments, we characterise the molecular defects caused by mutant PTPMT1 and validate the pathogenicity of the reported variants. To further understand the effect of aberrant PTPMT1 and CL deficiency on the respiratory complexes, we performed BN-PAGE and mitochondrial respiratory chain enzyme activity measurements in a novel zebrafish model. Ablation of ptpmt1 had a profound effect on OXPHOS function, resulting in reduced Complex I and IV assembly and activity. These results were consistent with the findings from patient skeletal muscle tissue, indicating that PTPMT1 is essential for the optimal stability and enzymatic activity of these complexes. Together, our data suggest that loss of PTMPT1 function is associated with a new autosomal recessive primary mitochondrial disorder caused by impaired CL metabolism. These findings highlight the contribution of aberrant CL metabolism towards human disease and the importance of normal CL homeostasis in mitochondrial bioenergetics.

Dystonia is one of the most common motor disorders in childhood, characterized by a wide heterogeneity of disease-causing genes [1]. The multiplicity of genes involved causes frequent errors in diagnosis and often the correct therapeutic strategies are not carried out. A novel pathogenic variant was recently identified in the nuclear gene ATP5MC3 that encodes for the subunit c3 of ATP synthase (complex V) causing dystonia. This point mutation generates the N106K substitution in the mature subunit c3 [2]. In this study we showed that in patient-derived fibroblasts the reduced ATPase activity and mitochondrial respiration are associated with altered assembly of complex V, resulting into accumulation of subunit c in total cellular lysates and crude mitochondria. This is accompanied by a reduced propensity of mitochondria to form the permeability transition pore (PTP) that would be generated within the c-ring [3], despite an overproduction of ROS that would, in principle, favor PTP generation [3]. Importantly, by electron microscopy analysis of the patient's fibroblasts we observed the presence of damaged mitochondria and a significant alteration in cellular trafficking, characterized by the accumulation of aberrant autophagy-lysosome structures. Consistently, the levels of key autophagy markers (p62 and LC3 II) are found increased. Taken together, our results suggest that the substitution N106K in the subunit c3 impairs the ATP synthase assembly and propensity to form the PTP and results into alterations of subunit c degradation, leading to autophagosome-lysosome accumulation, which would thus open new perspectives to identify new pharmacological targets.

[1] M. Thomsen, L.M. Lange, M. Zech, K. Lohmann. Genetics and Pathogenesis of Dystonia. Annu Rev Pathol, 19 (2024) 99-131

[2] M. Zech, R. Kopajtich, K. Steinbrücker, et al., Variants in Mitochondrial ATP Synthase Cause Variable Neurologic Phenotypes, Ann Neurol, 91 (2022) 225

[3] P. Bernardi, M. Carraro, G. Lippe, The mitochondrial permeability transition: Recent progress and open questions, FEBS J, (2021) 16254

Mitochondrial cytochrome c oxidase or respiratory chain complex IV is composed of 14 different subunits. The complex IV contains two redox-active heme A moieties (a and a3) and two copper centers (CuA and CuB). The assembly factor heme A: farnesyltransferase (COX10) participates in the initial step of heme A biosynthesis and catalyses the farnesylation of the vinyl group at the C2 position of heme B and converts it to heme O.

Using whole exome sequencing, pathogenic variants in the COX10 gene were identified in two patients. Patient 1 with growth failure, hypotonia, lactic acidosis, failure to thrive, and hypertrophic cardiomyopathy is a compound heterozygote for the c.1238_1240delTCT (novel) and c.1259C>T variants. Patient 2 with microcephaly, mild hypertrophic cardiomyopathy, hypotonia and sideroblastic anemia is a compound heterozygote for the c.610A>G and c.674C>T variants.

The aim of this study was to prove the pathogenicity of the identified variants and analyze their impact on the OXPHOS complexes.

In cultured skin fibroblasts, determination of specific cytochrome c oxidase activity and respirometry confirmed a significant functional deficiency of complex IV. The level of native complex IV was significantly reduced in both patients to 20% and 10%, respectively, compared to controls. The amount of complex IV subunits was reduced below 20%. This deficit was also confirmed in the buccal swab sample of P1. Severe deficiency of complex IV was also detected in muscle and liver of P2. The mitochondrial ultrastructure in fibroblasts was impaired in both patients, with half of the mitochondria examined showing lower numbers of cristae and defective shapes. Combinations of the variants found in both patients lead to a significant deficiency of complex IV in all tested tissues.

Supported by AZV MZCR NU22-01-00499, RVO-VFN64165.

Fatty acid-stimulated insulin secretion (FASIS) at low, unstimulating glucose concentrations is controversial, and its mechanism is not fully understood. Using rat insulinoma INS-1E β-cells, we previously demonstrated that externally delivered palmitic acid stimulated FASIS, but this effect was absent following siRNA-ablation of the mitochondrial redox-sensitive phospholipase A2γ (iPLA2γ) [1]. By utilizing perifusion of isolated mouse pancreatic islets, we now show the ability of short-, medium-, and long-chain fatty acids (FAs) to stimulate insulin secretion under low glucose. The increase in insulin secretion was paralleled with increased H₂O₂ release, detected extracellularly. Both insulin secretion and H₂O₂ release were inhibited by mitochondrial antioxidant SkQ1 and an inhibitor of FA β-oxidation etomoxir in isolated islets and by silencing of carnitine palmitoyltransferase 1 and overexpression of catalase in INS-1E cells. In addition, the FASIS was inhibited by GW1100, an antagonist of free fatty acid receptor 1 (FFA1/GPR40). These data support our hypothesis that mitochondrial β-oxidation of FAs produces superoxide/H₂O₂ and initiates a redox signal required for insulin secretion. Our results are further consistent with a mechanism of FA β-oxidation-derived intramitochondrial redox signal activating mitochondrial iPLA2γ, which causes the release of endogenous FAs that consequently stimulate FFA1/GPR40.

Supported by the Czech Science Foundation grant 22-17173S.

[1] J. Ježek, A. Dlasková, J. Zelenka, M. Jabůrek, P. Ježek, H₂O₂-Activated Mitochondrial Phospholipase iPLA₂γ Prevents Lipotoxic Oxidative Stress in Synergy with UCP2, Amplifies Signaling via G-Protein-Coupled Receptor GPR40, and Regulates Insulin Secretion in Pancreatic β-Cells, Antioxid Redox Signal, 23 (2015) 958-972.

Introduction: Kynurenic acid (KYNA) is the end product of tryptophan metabolism and formed during the irreversible transamination of L-kynurenine (L-KYN) by kynurenine aminotransferases (KATs). The present study aimed to investigate the mitochondrial effects of KYNA synthesizing KAT enzymes in newly created kynurenine aminotransferase I, II and III gene knockout (KAT I KO, KAT II KO and KAT III KO) mouse strains.

Methods: 8-10-week-old, male C57BL/6N wild-type (wt), KAT I KO, KAT II KO and KAT III KO mice were used (n=6/group). KAT gene knockouts were carried out through indel mutations using the CRISPR/Cas9 method (Kyushu University, Japan). Complex I- and II-linked maximal capacities of oxidative phosphorylation (CI and CII OXPHOS), as well as complex IV activity (CIV) were measured using high-resolution respirometry (Oroboros O2k, Austria) from brain (cerebellum, hippocampus, striatum) and liver homogenates.

Results: Cerebellar CII OXPHOS and CIV activity were significantly reduced in all three KO strains compared to the wt group (KAT I-III KO CII OXPHOS: 264±41; 278±64 and 241±40 vs. 368±20; KAT I-III KO CIV: 538+179; 632±158 and 634+124 vs. 995±188 pmol∙s⁻¹∙ml⁻¹, P˂0.05). Lower basal respiration was measured in hippocampal samples, and CII-OXPHOS was significantly reduced as a result of KAT KO (KAT I-III KO CII OXPHOS: 191±34; 199±56 and 170±45 vs. 272±17). Striatal CII OXPHOS was significantly reduced in the KAT II KO and KAT III KO strains, while hepatic mitochondrial respiration (OXPHOS capacity) remained unchanged.

Summary and Conclusion: We assume that the lower cerebral mitochondrial respiration detected in KAT enzyme-deficient mice may affect the efficiency of ATP synthesis under physiological conditions. Decrease in energy production can be originated from the reduced level of endogenous KYNA or from the lower levels of reducing equivalents as a result of other KAT catalyzed reactions connected to the Szent-Györgyi–Krebs cycle.

This research was funded by SZTESZAOK-KKASZGYA2023/5S775, National Research, Development, and Innovation Office—NKFIH K138125, SZTE SZAOK-KKA No:2022/5S729, HUN-REN Hungarian Research Network, JSPS Joint Research Projects under Bilateral Programs Grant Number JPJSBP120203803.

The brain is the organ with the highest energy needs and mitochondria are crucial to maintain its physiology. Any defects of mitochondrial function can lead to brain-associated diseases such as neurodegenerative diseases and neuropsychiatric diseases.

Src is a tyrosine kinase involved in a wide range of cellular processes such as proliferation, differentiation, apoptosis and metabolism. Our group showed that Src regulates mitochondrial bioenergetics and dynamics. However, little is known about the role of Src kinase in higher brain functions such as memory and anxiety.

To address this, Src^-/- mice were generated using the Cre-lox system. To generate mice with no Src in the brain, adeno-associated viruses encoding Cre under the control of the ubiquitous promoter CAG were retro-orbitally injected in Src^lox/lox mice. To generate mice lacking Src only in neurons or astrocytes, Src^lox/lox mice were crossed with mice expressing Cre under the control of the synapsin 1 or the glial fibrillary acidic protein promoters, respectively. Mitochondrial activity and behavior were then analyzed.

Our results show that Src deletion throughout the brain increases anxiety-like behavior assessed by the open field and zero maze tests, and decreases memory performance assessed by the Y maze test. Also, oxygen consumption is reduced in the hippocampus but not in other brain regions. Interestingly, deletion of Src only in neurons reduces memory performance but has no impact on anxiety-like behavior. Inversely, deletion of Src only in astrocytes increases anxiety-like behavior but has no impact on memory.

Overall, our results indicate that Src modulates brain metabolism and behavior in a cell-type specific manner. This project will also identify new therapeutic targets for the treatment of anxiety.

α-ketoglutarate dehydrogenase complex (αKGDHc, or oxoglutarate dehydrogenase complex) is one of the most regulated enzymes in the tricarboxylic acid (TCA) cycle catalysing the production of NADH for oxidative phosphorylation and of succinyl-CoA for the substrate level phosphorylation; thus being a crucial point in the mitochondrial ATP production. Additionally, αKGDHc is a major producer of reactive oxygen species (ROS). The complex is built up by three types of subunits: α-KGDH (E1), dihydrolipoyl succinyltransferase (E2), and dihydrolipoyl dehydrogenase (E3).

In the present study, E2 and E3 heterozygous knock-out (DKO) male middle-aged (200-250 day old) mice have been used to examine the in vitro and in vivo effects of this mutation. Of note, homozygous KO mice die in utero emphasizing the important role of αKGDHc in oxidative metabolism. The mRNA levels of mitochondrial enzymes were determined from liver samples. Histological studies were carried out on skeletal muscle samples. Mitochondria isolated from brain and kidneys were used to measure mitochondrial ATP production, oxygen consumption, ROS generation and aconitase activity. In vivo experiments included behavioural (open field- and Morris water maze-) tests and endurance (trademill fatigue) test.

Our in vitro results show that in DKO animals both oxygen consumption and ATP production were decreased with α-ketoglutarate as substrate compared to the WT animals. DKO mice produced less ROS during succinate-induced reverse electron transfer (RET). In line with that, higher aconitase activity was measured in DKO brain mitochondria. In the behavioural tests, small differences were detectable between the two groups indicating a minor cognitive decline. Importantly, DKO animals showed a decreased performance in the trademill fatigue test, which could not be explained by fibrotic alterations in the skeletal muscle.

Taken together, these data show that heterozygous DKO mice have significant in vitro changes compared to the WT animals, which is not reflected in significant differences in the phenotype. This is the first study analysing the phenotype of the αKGDHc DKO mice.

Diabetes mellitus type II (T2DM) is a chronic disease with increasing prevalence in modern society due to an unprecedented rise in obesity in developed countries. The World Health Organization (WHO) estimated that more than 425 million people are currently affected by diabetes. Diabetes mellitus is a metabolic disorder which is characterized by high blood glucose levels. The underlying problem of T2DM is insulin resistance in peripheral tissue such as skeletal muscle, adipose tissue and liver. This peripheral insulin resistance leads to hyperglycaemia and hyperinsulinemia. Furthermore, insulin resistance is associated with impaired mitochondrial function. Insulin supports the integrity of the mitochondrial electron transport chain (ETC) by dampening FOXO11/HMOX12 and maintaining the mitochondrial NAD+/NADH ratio. Individuals with insulin resistance show a lowered ETC activity, reduced ATP production and a higher ROS production.

To date, the molecular mechanism that causes insulin resistance and the subsequent mitochondrial dysfunction has not been elucidated. Using C2C12 skeletal muscle cells in which we induced insulin resistance, we have seen that ROS production as well as membrane potential are increased. These results suggest that insulin resistance may have an effect on the functionality of the NADH dehydrogenase (Complex I of the ETC). We used state-of-the-art techniques such as stimulated emission depletion (STED) microscopy, seahorse extracellular flux analysis, as well as Blue Native Gel-Electrophoresis to investigate the impact of insulin resistance on mitochondrial ultrastructure, function and the role of Complex I.

We hope that our investigation into the intricate relationship between insulin resistance, mitochondrial dysfunction, and type II diabetes mellitus sheds new light on the underlying mechanisms driving this complex metabolic disorder.

Lon protease 1 (LONP1) represents one of the major mitochondrial quality control proteases, being responsible for proteolytic degradation of misfolded or damaged proteins. LONP1 is one of the most important factors during mitochondrial unfolded protein response (UPR^mt), a protective mechanism that mitigate mitochondrial stress. Besides of degradation of malfunctioning proteins, LONP1 plays also significant role in intracellular signaling and metabolic reprogramming. LONP1 was shown to have protective effects in the heart, but the role of LONP1 in the regulation of endothelial function is not understood. The aim of this study was to investigate the effect of LONP1 on endothelial mitochondrial function in IL-1β-induced endothelial inflammation and to test whether the activity of LONP1 might be beneficial in response of endothelium to inflammation.

The studies were conducted in the human aortic endothelial cells (HAEC) and in the isolated murine aorta. IL-1β (10 ng/ml) was used to induce endothelial inflammation. Down-regulation of LONP1 was performed using siRNA and activation using compound 84-B10. The mRNA levels was measured by qPCR and protein levels by Western-Blot. Mitochondrial potential and morphology was imaged with CQ1 spinning disc confocal microscope and analyzed with Columbus and CellProfiler software. Endothelium-dependent vasodilation was assessed in myograph.

In HAEC cells, IL-1β activated UPR^mt, increasing LONP1 and SOD2 expression. LONP1 silencing in IL-1β-treated HAEC cells significantly decreased expression of SOD2 and increased expression of the mitochondrial protein ND2, a component of complex I of the respiratory chain, without effect on the other complexes. Activation of LONP1 by 84-B10 in IL-1β-treated cells increased SOD2 expression and activity, while silencing of LONP1 changed mitochondrial morphology, leading to mitochondrial fragmentation. Activation of LONP1 increased mitochondrial potential in IL-1β-activated endothelial inflammation, opposite effect was observed when LONP1 was silenced. In isolated aortic rings taken from 6 month-old APOE/LDLR^-/- mice 84-B10 activator improved NO-dependent vasodilation.

This study demonstrated, that LONP1 supported mitochondrial integrity in the endothelium during inflammation. Our results suggest that modulation of LONP1 represents a novel target to mitigate inflammation-induced endothelial dysfunction.

Parkinson's disease (PD) is characterized by the gradual decline of dopaminergic cells in the substantia nigra pars compacta (SN) and is associated with the accumulation and aggregation of alpha-synuclein (αSyn) within SN neurons. Mitochondrial dysfunction, particularly impairment of mitochondrial Complex-I, plays a pivotal role in both sporadic and familial PD pathogenesis. Our study aimed to investigate the correlation between αSyn accumulation and mitochondrial changes over time by examining key mitochondrial proteins in dopaminergic neurons of the SN in a mouse model with αSyn overexpression.

To induce αSyn overexpression, mice received AAVs encoding human αSyn injected into the SN, resulting in approximately 30% degeneration of dopaminergic neurons at 12 weeks post-injection. Immunofluorescent staining of mitochondrial proteins in surviving dopaminergic neurons expressing both αSyn and tyrosine hydroxylase (a dopaminergic marker) revealed a consistent level of the general mitochondrial marker Grp75, but a significant decrease in GRIM19:Grp75 and MTCO1:Grp75 ratios, indicating losses in Complex I and IV at 12 weeks, but not at 4 weeks post-injection. Additionally, a negative correlation was observed between neuronal αSyn expression levels and the GRIM19:Grp75 ratio. Proximity ligation assays (PLAs) demonstrated αSyn-mitochondria interactions at 12 weeks post-injection, but not at 4 weeks. Despite the lack of oxidative phosphorylation capacity, rate-limiting enzymes involved in glycolysis remained unchanged or decreased within these neurons.

These findings suggest that αSyn-induced abnormalities, such as specific mitochondrial alterations involving Complex-I and -IV loss, develop gradually over time, possibly due to a progressive burden of αSyn-mitochondria interactions. Furthermore, metabolic compensation through increased glycolysis does not appear to play a role in these αSyn-burdened cells. In conclusion, our results provide evidence supporting a relationship between αSyn accumulation and distinct mitochondrial alterations characterized by loss of Complex-I and -IV expression, highlighting the time-dependent nature of αSyn-induced abnormalities in PD pathogenesis.

Ketone Bodies influence mitochondrial functionality in various physiological conditions, and little is known about their ability to affect brown adipose tissue (BAT) mitochondrial functionality. To explore this aspect, we investigated whether the elevation of plasma levels of the ketone body β-hydroxybutyrate (β-OHB), achieved through the in vivo administration of its precursor 1,3-butanediol to rats, could impact interscapular BAT mitochondrial physiology. We examined its rapid effects within three hours as well as those observed after two weeks of treatment.

Following two weeks of treatment, BD affects interscapular iBAT morphology, since it induced a reduction in adipocytes average size, revealed by histological analysis. BD administration enhanced mitochondrial respiration rate both when using glycerol-3-phosphate and succinate as respiratory substrates. The stimulatory effect of BD was also evident when respiration was detected in the presence of GDP, thus suggesting an enhancement in the activity of the respiratory chain. This effect was already evident within three hours of BD administration and persisted after 2 weeks of treatment. A quantitative proteomics approach, utilizing liquid chromatography coupled with tandem mass spectrometry analysis, allowed us to evaluate the changes in mitochondrial protein expression induced by BD administration. BD influences the mitochondrial levels of specific subunits belonging to each of the five respiratory complexes as well as Uncoupling protein-1 (UCP1), with the effect observed within 3 hours of BD administration. The enhancement in β-OHB levels rapidly leads to post-translational modification of mitochondrial proteins, as revealed by western-blot employing an antibody against β-hydroxybutyryl-lysine residues. The proteomic approach indicated that post-translationally modified proteins include some subunits of the respiratory chain complexes and UCP1.

In conclusion, by exploiting the BD-induced increase in β-OHB, we suggest a role for β-OHB as a signaling molecule capable of rapidly affecting mitochondrial BAT physiology.

Background

Metabolic dysfunction associated steatotic liver disease (MASLD) is a chronic condition characterized by an excess accumulation of liver lipids, which can progress further to steatohepatitis and hepatocellular carcinoma. Although the prevalence of MASLD globally exceeds 40%, no specific drugs have yet been developed. Among potential candidates, agonists of PPARs regulating liver lipid metabolism, appear promising. Bilirubin (BR), the final product of heme catabolism, has been recognized as a signaling molecule, activating various cytoplasmic and nuclear receptors, including PPARα, which could partially explain the protective effects of mild hyperbilirubinemia against MASLD. The aim of this study was to examine the role of BR in MASLD and compare its effect to another PPARα agonist fenofibrate (FF).

Methods

The HepG2 cell line was challenged with oleic (OA) and palmitic acid (PA) in a 2:1 ratio at a final concentration of 500 µM and then treated with either BR or FF (5/50 µM). Lipid droplets were observed under a fluorescent microscope using Nile Red (Ex.: 515 /Em.: 585 nm) and quantified on a fluorescence spectrophotometer. Concentrations of OA and PA were measured by GC/MS. Cell viability was obtained using the MTT test. qPCR was used for the determination of PDK4 expression (downstream gene of PPARα, coding for pyruvate dehydrogenase kinase) and concentration of PPARα was measured by western blot.

Results

After fatty acids exposure followed by BR and FF treatment, the lipid content decreased by 17% and 10%, respectively, as well as the total concentrations of both OA (decreased by 21% and 34% for BR and FF, respectively) and PA (decreased by 3% and 13% for BR and FF, respectively); the cell viability was not affected. Simultaneously, PDK4 expression increased significantly by 163% (BR) and 167% (FF), respectively and PPARα concenration increased by 120% and 132%.

Conclusion

BR and FF reduce lipid content in the HepG2 cell exposed to fatty acids, presumably by increasing fatty acid oxidation via PPARα activation.

Historically, ATP depletion has been assumed in mitochondrial diseases but surprisingly rarely experimentally proven. In addition to ATP, the biosynthesis of all nucleotides and maintenance of their phosphorylation and redox statuses have essential links to mitochondria. Different forms of mitochondrial dysfunction induce a common translational and transcriptional program called mitochondrial integrated stress response (mt-ISR) [1]. This response involves upregulated pathways related to nucleotide biosynthesis. Moreover, several mouse models of OXPHOS deficiency show upregulation of the oncogene MYC, a notable function of which is to boost nucleotide biosynthesis [1,2]. To what degree mt-ISR and MYC can maintain nucleotide homeostasis upon compromised OXPHOS in vivo remains unclear. One challenge to addressing this question has been that the simultaneous quantification of different nucleotide species has largely been limited to specialized chromatographic techniques unavailable for most life science laboratories. To overcome this methodological limitation, I have developed convenient enzymatic methods to quantify all canonical ribonucleotides and their deoxy counterparts as well as NAD(H) and NADP(H) in microplates in a high-throughput manner, basically in any laboratory [3,4]. Here, I aim to link high-quality nucleotide data from mouse models of mitochondrial diseases to the degree of induction of mt-ISR and MYC, and to the presence or absence of a disease phenotype in a given tissue. Preliminary results suggest tissue-specific alterations in nucleotide pools and that MYC upregulation is not directly part of mt-ISR, and that the MYC upregulation can precede mt-ISR or upregulate nucleotide biosynthesis without involvement of mt-ISR, depending on the degree of OXPHOS deficiency or tissue/cell type.

[1] J. Purhonen, J. Klefström, J. Kallijärvi, MYC—an emerging player in mitochondrial diseases, Front. Cell Dev. Biol. 11. (2023) 1257651.

[2] J. Purhonen, R. Banerjee, V. Wanne, N. Sipari, M. Mörgelin, V. Fellman, J. Kallijärvi, Mitochondrial complex III deficiency drives c-MYC overexpression and illicit cell cycle entry leading to senescence and segmental progeria, Nat. Commun. 14 (2023) 2356.

[3] J. Purhonen, J. Kallijärvi, Quantification of all 12 canonical ribonucleotides by real-time fluorogenic in vitro transcription, Nucleic Acids Res. 52 (2023) e6.

[4] J. Purhonen, R. Banerjee, A.E. McDonald, V. Fellman, J. Kallijärvi, A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase, Nucleic Acids Res. 48 (2020) e87.

Peripheral blood mononuclear cells (PBMCs) have garnered attention as a diagnostic model due to their specificity and sensitivity as biomarker for overall health. Studies have linked PBMC mitochondrial respiration to conditions like depression and type 2 diabetes ,suggesting that PBMC’s energy profile could serve as biomarker. Like for other biomarkers, it is essential to consider the day-to-day variations in mitochondrial respiration of PBMCs.

We examined the variability over consecutive days of mitochondrial respiration in PBMCs isolated from three healthy female volunteers (aged 25 ± 6 years) over three consecutive days in Innsbruck (575 m) and Kühtai (2000 m). Blood was collected each morning, and PBMCs were isolated using density gradient centrifugation. After the initial wash with DPBS (25 mL, 120 g, RT), PBMC were washed two times with ice-cold DPBS + 2 % FBS (50 mL, 300 g, 4 °C).The final pellet were resuspend in 0.5 mL cold PBS and counted on a SYSMEX XN-350 haematology analyser. 3·10⁶ PBMCs were placed in the 2-mL chambers of the Oroboros O2k high-resolution respirometer (Oroboros Instruments, Innsbruck, Austria) containing mitochondrial respiration medium MiR05 with catalase at 37 °C.

The PBMCs showed consistent ROUTINE respiration within individuals across days. For participant 1 no significant day-to-day variations were exhibited in the electron transport capacity E per cell, either in Innsbruck (19.3 ± 1.6 amol·s^-1·x^-1) or Kühtai (21.6 ± 1.1 amol·s^-1·x^-1). Conversely, participant 2 showed a noticeable drop in E on day 3 in Innsbruck (17.08 ± 0.1 amol·s^-1·x^-1) compared to other days (22.9 ± 0.6 and 25.2 ± 1.9 amol·s^-1·x^-1). However, E remained consistent in Kühtai (18.3 ± 1.8 amol·s^-1·x^-1). Participant 3 also demonstrated fluctuations in ET capacity, with higher values on day 1 compared to subsequent days at both sites.

These findings indicate that while some individuals may show stable mitochondrial respiration, others exhibit significant daily variability. This underscores the need for larger studies to further explore this variability and validate PBMC mitochondrial respiration as a biomarker.

Background: The dimeric protein Nicotinamide nucleotide transhydrogenase (NNT) is located in the mitochondrial inner membrane. It converts NADH and NADP⁺ into NAD⁺ and NADPH, utilizing the proteon gradient generated by oxidative phosphorylation (OXPHOS). NNT is a major adaptive source of mitochondrial NADPH production, connecting redox homeostasis to OXPHOS. The role of NNT in physiological developmental processes has not been established. Decreased NNT and increased OXPHOS levels in skeletal muscle fibres in the first weeks of murine life suggest a critical role for NNT in early development. Our aim was to understand the adaptive changes in M. gastrocnemius caused by absence of NNT.

Methods: Congenic BL6JRcc.BL6J-Nnt^C57BL/6J/Wuhap (Nnt^mutJ) and B6JRcc(B6J)-Nnt⁺/Wuhap (Nnt^wt) mice were generated. Genome-wide transcriptome analysis was performed on M. gastrocnemius of 24-day-old male mice. Key results were validated using gene and protein expression, enzyme activity, and high-resolution respirometry.

Results: Longevity on a chow diet was not different between the mouse substrains. In M. gastrocnemius, energy metabolism was the most affected process. In Nnt^mut mice, mRNA levels of most OXPHOS genes were significantly decreased, especially Complex I (42.2% of subunits) and Complex V (50% of subunits) were affected. Key genes involved in fatty acid transportation (Cpt1b, Cpt2 and Slc25a20) were also decreased, accompanied by a decreased tendency of oxygen consumption rate on palmitoylcarnitine and octanoylcarnitine. Finally, CHRNA1, involved in neuromuscular junction (NMJ) signal transduction, was decreased in agreement with NMJ staining. Few changes were seen in redox homeostasis.

Conclusion: Our results suggests that NNT dysfunction lowers OXPHOS gene expression and β-oxidation, which may affect NMJ function in the M. gastrocnemius of 24-day-old mice.

Capacities of electron transfer (ET) and oxidative phosphorylation (OXPHOS) are fundamental for mitochondrial function. Since the plasma membrane is impermeable to compounds used in OXPHOS analysis, respirometry with living cells provides limited information compared to mitochondrial preparations (isolated mitochondria, tissue homogenates, and permeabilized tissue/cells).

To compare respiratory control in mitochondrial preparations, we applied two substrate-uncoupler-inhibitor titration reference protocols in parallel, interrogating 20 pathway and coupling-control states. We studied mouse mitochondria from brain as a highly glucose-dependent tissue, and heart which relies highly on fatty acid oxidation. HEK 293T cells were analyzed as a widely used experimental model, and human peripheral blood monocyte cells (PBMCs) and platelets for mitochondrial diagnostics from liquid biopsies.

Respiratory rates were reproducible between comparable respiratory states interrogated in both protocols. In general, octanoylcarnitine did not exert an additive effect on OXPHOS capacity in the NADH&Succinate (NS)-linked pathway, in agreement with the limited role of fatty acid oxidation in supporting V_O2max.

Both in mouse heart and human PBMC mitochondria, OXPHOS capacities were identical to ET capacities in every pathway state. Similarly, the phosphorylation capacity coped with the low ET capacity of the N-pathway in platelets. However, ET capacity was in excess of OXPHOS capacity when the phosphorylation system was driven to saturation using substrate combinations in platelets. N-linked respiration increased more than 2-fold from OXPHOS to ET in mouse brain mitochondria and, in contrast to platelets, there was a decrease in ET excess capacity using the NS-substrate combination. Different batches of HEK 293T cultures had variable ET capacities at constant OXPHOS capacity in all pathway states. In all models, incomplete additivity of the N- and S-pathway argued against tight channeling through supercomplexes CI-III₂-IV_n.

Our study highlights the importance of OXPHOS analysis. (1) Changes in respiratory capacity can be attributed to nutrient-specific pathways. (2) Metabolic adjustments can be distinguished from functional defects by formulating robust working hypotheses. (3) The most suitable models can be selected to address specific metabolic targets. Bioenergetic snapshots obtained by OXPHOS analysis can be interpreted on the basis of an extensive mitochondrial database.

Thioesters of coenzyme A (CoA) carrying different acyl chains (acyl-CoAs) are central molecules in metabolism and bioenergetics, mostly generated in mitochondria. While there is considerable evidence suggesting that hepatic ischemia impacts acyl-CoA levels, there remains significant disagreement regarding the extent and timing of these alterations. Acyl-CoA species largely differ in cellular concentrations and physico-chemical properties, rendering their analysis challenging. In this study, we utilized various techniques, including LC-MS/MS, to quantify cellular short- and medium-chain acyl-CoAs in normal rat liver compared to livers experiencing mild or advanced ischemia. Our findings reveal that ischemia induces distinct changes in the relative acyl-CoA abundance, depending on the severity of the insult. Mild ischemia (1-2 min), characterized by a moderate increase in ADP/ATP and AMP/ATP ratios, significantly decreases succinyl-CoA, but largely preserves acetyl-CoA and even increases some acyl-CoAs upstream of the TCA cycle. Only advanced ischemia (5-6 min), characterized by a drastic increase in ADP/ATP and AMP/ATP ratios, also decreases acetyl-CoA, albeit to a lesser degree compared to previous reports. Our study demonstrates the suitability of our workflow for accurately preserving and quantifying in vivo acyl-CoA levels, thus offering a non-biased and comprehensive approach to acyl-CoA-related metabolism.

[1] M. Tokarska-Schlattner, N. Zeaiter, V. Cunin, V., S. Attia, C. Meunier, L. Kay, A. Achouri, A. Le Gouellec, U. Schlattner, Multi-method quantification of acetyl-CoA and further acyl-CoA species in normal and ischemic rat liver, Int. J. Mol. Sci. 24 (2023) 14957.

Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial inner membrane protein that uses the proton gradient to convert NADH and NADP⁺ to NAD⁺ and NADPH, linking mitochondrial energy production and reactive oxygen species homeostasis. Experimental data indicate that NNT is not always active. Here, we determined the substrate conditions under which NNT is active, and how this is regulated. For this purpose, we generated two strains of congenic mice, one with a functional NNT gene (B6JRccHsd(B6J)-Nnt⁺/Wuhap; Nnt^wt) and another with a mutated NNT gene (missing exons 7-11; B6JRccHsd.B6J-Nnt^C57BL/6J/Wuhap; Nnt^mut). Whole body energy metabolism of these mice on a chow diet was identical, suggesting that NNT is not always active. For cellular analysis, primary fibroblasts were derived from both substrains. Relative to Nnt^wt cells, Nnt^mut fibroblasts displayed low/absent Nnt gene expression and NNT protein expression/ activity. Permeabilized Nnt^mut fibroblasts exhibited increased succinate-dependent and decreased pyruvate-dependent metabolism. Moreover, the transcript level of pyruvate dehydrogenase (PDH) was reduced, whereas mitochondrial pyruvate carrier and pyruvate carboxylase transcripts were increased in Nnt^mut fibroblasts. This was in line with observed changes in lactate release and oxygen consumption. It could be speculated that this PDH bypass reduces ROS production, while replenishing the TCA cycle (anaplerosis). Interestingly, when glutamine was present in the culture medium, the differences between Nnt^wt and Nnt^mut fibroblasts were less prominent. In summary, we successfully generated Nnt^wt and Nnt^mut congenic mouse and -derived fibroblast models. Our current data suggest a mechanism in which NNT activity depends on the available metabolic substrates.

As early as 1976, Ferguson-Miller et al found an effect of ATP binding to cytochrome c oxidase (CytOx) in the way oxygen binding and electron transfer by cytochrome c binding clearly differed from the reaction with ADP. The binding of ATP to the holoenzyme causes a conformational change, which influences the affinity of the binding site for cytochrome c . In 1999, the Kadenbach group was successful in demonstrating the ATP/ADP ratio in the mitochondria allosterically controls the activity of CytOx. This in turn has an affect on the H+/e- stoichiometry. On the basis of these findings, Kadenbach formulated his hypothesis of the existence of a second mechanism of mitochondrial respiratory control, which is ATP-dependent but independent of the mitochondrial membrane potential and is of crucial importance for the formation of oxygen radicals under stress conditions.

The focus of further research is on the interplay between ATP-dependent, allosteric inhibition of CytOx with sigmoidal enzyme kinetics in contrast to hyperbolic kinetics with ADP, oxygen consumption and mitochondrial membrane potential. The initial focus was on specific phosphorylations of the enzyme. So far, 14 phosphorylation sites on the enzyme have been identified. With the notable exception of Tyr 304, a direct assignment of these positions with different enzyme activity or kinetics is not clear.

In the course of further investigations, this effect was clearly reproduced. It should be noted that the inhibition of CytOx can only be demonstrated at a very high ATP/ADP ratio. A sigmodality of the kinetics is dependent on a cAMP/Ca++ mediated reversible dimerization. ATP -dependent inhibition of CytOx decreases mitochondrial ROS production. Thus, this mechanism is of medical importance for the understanding of degenerative diseases, such as myocardial infarction and heart failure. We are now aware of a large number of mechanisms that influence the enzyme kinetics of CytOx and highlight its central role in respiratory control.

Therefore, to answer the question posed at the beginning, the second mechanism of respiratory control, which in turn is based on the ATP-dependent inhibition of CytOx, is valid for modern bioenergetic research in basic science and medicine as well.

Friedreich’s ataxia (FA) is the most common monogenic mitochondrial disease. FA is caused by a depletion of the mitochondrial protein frataxin (FXN), an iron-sulfur (Fe-S) cluster biogenesis factor. To better understand the cellular consequences of FA, we performed quantitative proteome profiling of human cells depleted for FXN. Nearly every known Fe-S cluster-containing protein was depleted in the absence of FXN, indicating that as a rule, cluster binding confers stability to Fe-S proteins. Proteomic and genetic interaction mapping identified impaired mitochondrial translation downstream of FXN loss, and specifically highlighted the methyltransferase-like protein METTL17 as a candidate effector. Using comparative sequence analysis, mutagenesis, biochemistry and cryogenic electron microscopy we show that METTL17 binds to the mitoribosomal small subunit during late assembly and harbors a previously unrecognized [Fe₄S₄]²⁺ cluster required for its stability on the mitoribosome. Notably, METTL17 overexpression rescued the mitochondrial translation and bioenergetic defects, but not the cellular growth, of FXN depleted cells. Our data suggest that METTL17 serves as an Fe-S cluster checkpoint: promoting the translation and assembly of Fe-S cluster rich OXPHOS proteins only when Fe-S cluster levels are replete.

Mitochondria are considered to be involved in ischemic brain damage, therefore modulation of mitochondrial functions by pharmacological means may have beneficial effects protecting the brain against ischemic injury and following neurological deficit. There is accumulating evidence that some antiadiabetic drugs such as metformin and its derivatives may also exert neuroprotective effects. In our study we aimed to investigate whether imeglimin – a recently approved drug for treatment of diabetes, can protect against ischemic brain damage via modulation of mitochondrial functions.

For the experiments Wistar rats of 3 age groups were used: juvenile (2-3 months old), middle age (10 months) and advanced age (24 months). We observed that injection of imeglimin 24 h before simulated brain ischemia exerted protective effect by reducing infarct area in brains of juvenile and middle-age but not aged rats. We also found that imeglimin added acutely to isolated mitochondria inhibited activity of Complex I of the electron transfer system and supressed NADH-dependent phosphorylating respiration in juvenile and middle-age groups. Interestingly, we observed stimulating effect of imeglimin on Complex II activity in the same age groups as above. In the advanced age group (24 months), imeglimin had no significant effect on mitochondrial respiration and enzymatic activities of the complexes. In conclusion, our data suggest that protective effect of imeglimin against ischemic brain injury is age-dependent. The observed dual effect of imeglimin stimulating Complex II and inhibiting Complex I activity may be benficial during ischemia-reperfusion by potentially preventing accumulation of succinate and subsequent production of reactive oxygen species.

Arsenic-based compounds such as the European Medicines Agency-approved Trisenox and Darinaparsin are clinically used agents for cancer therapy yielding high rates of remission in cancers such as acute promyelocytic leukemia. Their site of action is incompletely understood. It is believed they target a fusion protein called PML-RARα thought to contribute to neoplasia, and/or disrupt oxidative phosphorylation (OXPHOS). HepG2 cells do not express PML-RARα and demonstrate significant oxygen consumption rates (OCR) and respiratory capacity. We have recently discovered that they remain viable even after complete inhibition of any respiratory complex. Here, we demonstrate that several arsenic-based compounds -at clinically relevant concentrations- induce significant HepG2 cell death regardless of concomitant OXPHOS inhibition. Considering that they inhibit enzymes critical for glutaminolysis, a hallmark pathway of cancer that is active even in the absence of OXPHOS, we compared their effects to KMV (3-keto-2-methylvaleric acid) and succinyl phosphonate, specific inhibitors of the ketoglutarate dehydrogenase complex (KGDHC), a critical node in the glutaminolysis pathway. We further report that HepG2 cells death recorded by high-content automated microscopy was not affected by either KMV or succinyl phosphonate, despite both conferring complete inhibition of KGDHC activity. By performing systematic searches for amino acids-to-metabolite conversions using public databases (Metabolic Atlas, BioCYC, and LCSB), we identified potential targets of arsenic-based compounds that could still allow the distal branch of the oxidative decarboxylation of glutamine to occur without being sensitive to either KMV or succinyl phosphonate. It is suggested that arsenic-based compounds kill cancer cells by inhibiting enzymes participating in glutaminolysis starting from glutamine or other amino acids.

Brain mitochondrial defects lead to cognitive impairment or neurodegenerative diseases. However, due to the lack of suitable tools, no direct link between acute mitochondrial activity and higher brain functions has been established so far. Heterotrimeric guanine nucleotide-binding (G) proteins are key players in brain metabolism, cognition and behavior. Our group and others previously demonstrated that G proteins are found within mitochondria. Thus, we hypothesized that stimulation of specific G protein signaling within the organelle could modulate brain mitochondrial activity and possibly rescue behavioral defects associated to brain metabolic disorders. We developed a Galpha_s-linked recombinant designer receptor exclusively activated by designer drugs targeted to mitochondria. This so-called mitoDREADD-Gs can acutely increase mitochondrial metabolism in different types of cells both in vitro and ex vivo. Strikingly, in vivo activation of mitoDREADD-Gs in specific brain circuits abolished cognitive impairments linked to mitochondrial alterations, including cannabinoid-induced motor and cognitive deficits, as well as memory impairment in different mouse models of dementia. MitoDREADD-Gs does not only benefit our understanding of how mitochondria are involved in biological functions, but also provides new potential therapeutic concepts for the treatment of brain diseases associated to impaired cell metabolism.

The mitochondrial ATP synthase or Complex V (CV) uses a rotary mechanism to synthesize ATP. Under conditions of impaired respiration, however, this mechanism can operate in reverse, pumping protons at the expense of ATP breakdown (hydrolysis). When CV attempts to reverse rotation, a peptide named ATPase inhibitory factor 1 (IF1) inserts itself into the complex to prevent ATP breakdown, leading to depolarization of the mitochondrion and its removal by mitophagy. Here we hypothesize that aging results in downregulation of IF1, leading to increased ATP hydrolysis by CV and to energy depletion. We propose that age-related decrease in IF1 expression and/or binding to CV impedes the removal of dysfunctional mitochondria, creating a vicious cycle that is driving the cumulative nature of the aging process.

Our results suggest that IF1 expression in the human brain declines with age. In the brain of aging mice and rats we detect increase in the ATP hydrolysis by CV. Remarkably, our data indicate that ATP hydrolysis increases with age specifically in the regions/tissues showing the largest IF1 decline. In agreement with the in vivo data, we also observe increased ATP hydrolysis in a cellular model of accelerated aging - primary skin fibroblasts of a progeroid syndrome patient. Moreover, increase in ATP hydrolysis coincides with reduced mitophagy in these cells. Importantly, blocking of ATP hydrolysis with a selective inhibitor, (+)-epicatechin, enhances mitophagy in these cells, possibly via a PINK1-Parkin independent pathway.

Taken together, these results suggest that increased consumption of ATP by mitochondria is a maladaptive response that drives energy depletion during aging, while preventing the removal of dysfunctional mitochondria by mitophagy. Since mitophagy is meant to eliminate ATP-hydrolyzing mitochondria, and ATP hydrolysis inhibits mitophagy, the two processes form a vicious cycle that may drive the progressive nature of aging. Therefore, these findings not only shed light on the role of ATP breakdown by mitochondria in aging, but also offer an avenue for therapeutic interventions to alleviate age-related mitochondrial dysfunction.

Type 2 Diabetes presents a great challenge to healthcare systems worldwide, with an increasing number of individuals affected by this disease. Its underlying cause is insulin resistance, but little is known about the onset and its implications for mitochondrial function. Insulin, which typically signals certain tissues to uptake glucose from the blood stream, elicits now only a damped response. Skeletal muscle is early affected in the pathogenesis and mitochondria play a central role within the metabolic disease. In this work, we investigated the effect of early insulin resistance on mitochondrial function in C2C12 myotubes and found increased metabolic activity. The induction of insulin resistance was achieved through exposure to palmitic acid and high insulin concentrations. Consequently, these insulin-resistant cells exhibited significantly decreased glycolysis. ROS production in the cells was elevated and mitochondria displayed a higher degree of fragmentation and a reduced mitochondrial footprint. In contrast to these findings, fluorescence microscopy revealed a higher membrane potential and increased ATP level in these cells. Single molecule tracking experiments demonstrated faster movement of the ATP-Synthase and coverage of greater distances in insulin-resistant cells. Additionally, high resolution microscopy revealed a decrease in cristae density in the resistant cells. Together, our results offer interesting insights into the complex mitochondrial alterations during early insulin resistance. Based on our data we hypothesise that mitochondrial overheating may be the driving force behind insulin resistance.

Colorectal cancer (CRC) is one of the leading causes of death. CRC chemotherapies promote cell death mechanisms, such as apoptosis. However, cells can follow alternative fates to death, like chemotherapy-induced senescence. Cellular senescence corresponds to a stable cell cycle arrest, accompanied by different key aging features like altered metabolic rates. In various cancers, low levels of the mitochondrial fission protein DRP1 promotes susceptibility to chemotherapeutics. It has been suggested that these effects are explained by a reduction in mitochondrial fission. However, there is no consensus regarding the mechanisms underlying this phenomenon, and whether it occurs in CRC. Interestingly, it has been reported that DRP1 interacts directly with the anti-apoptotic protein Bcl-xL, which is key for senescence induction in different models. It has been shown that Bcl-xL promotes mitochondrial dynamics. We hypothesize that DRP1-Bcl-xL interaction also could enhance Bcl-xL mitochondrial localization promoting its anti-apoptotic role and improves the establishment of cell senescence instead of death. To explore this, we used the CRC cell lines HCT-116 wild type, DRP1KO and MFFKO. As a senescence inducer, sublethal doses of Doxorubicin were used. Survival and cell senescence were evaluated by crystal violet staining; flow cytometry viability probes; staining for SA-β-galactosidase activity; accumulation of P53, P21 and P16, which were evaluated by Western blot. We also evaluated the pharmacological inhibition of DRP1 and Bcl-xL, as well as the proximity between both proteins by confocal microscopy. The DRP1 lack, but not its pharmacological inhibition or the absence of MFF (another mitochondrial fission protein), promote cell death during senescence induction. Additionally, a DRP1-Bcl-xL co-compartmentalization was evidenced during senescence induction. To unveiling the eventual importance of this interplay it will be evaluated the effects of disruption in DRP1-Bcl-xL interaction on cellular bioenergetics and survival. This will be done through the expression of previously designed recombinant DRP1, lacking the Bcl-xL interaction site.

Funding: ANID/Scholarship N°21212019(PMC); ANID/FONDECYT Regular N°1230195(VP) & N°1200255(CC); ANID/FONDAP N°15130011(VP) & N°15150012(CC).

Hydrogen sulfide (H₂S) is a substrate of mitochondrial sulfide:quinone reductase (SQR) reducing ubiquinone to ubiquinol, supporting electron transfer to complexes III and IV, ultimately generating a protonmotive force. Its catabolism also generates sulfite (SO₃^2-) reducing cytochrome c. Here we show that H₂S provided as sodium sulfide in a dose-dependent manner, rescued mouse liver mitochondrial bioenergetics when complex I was inhibited. Up to a concentration of 100 μM sulfide, H₂S generated was able to sustain the forward operation of both the F₁-F_O ATP-synthase and the adenine nucleotide translocase (ANT) when complex I was inhibited by rotenone. When complex III was blocked by myxothiazol, a moderately beneficial effect of sulfide was observed. In the presence of the complex IV inhibitor cyanide, or under anoxic conditions, H₂S could not prevent the reversal of the ANT. On the other hand, sulfite (SO₃^2-) was an efficient substrate maintaining mitochondrial membrane potential when either complex I or III were inhibited. We conclude that the catabolism of low concentrations of H₂S, similar to those found in pathophysiological conditions, can bypass complex I blockade and maintain mitochondrial ATP production.

Introduction: Mitochondrial function plays a crucial role in cellular metabolism, with alterations observed in various diseases, including several types of cancer. The study of mitochondrial respiration in human cells provides relevant insights into metabolic diseases and potential therapeutic targets. Our study aims to evaluate the effect of glucose concentration on culture media on cell respiration in hepatocellular carcinoma (HuH-7) cells.

Methods: Using high-resolution respirometry, we analyzed mitochondrial respiration in the living HuH-7 cells cultured under low glucose (LG, 5.5 mM) and high glucose (HG, 25 mM) conditions. A coupling control protocol assessed ROUTINE respiration, LEAK respiration, electron transfer (ET) capacity, and E-L coupling efficiency. The protocol was extended to estimate cell viability in terms of plasma membrane permeability.

Results: A decrease was observed in ROUTINE respiration in the HG group compared to the LG group, with no difference in ET-capacity and coupling efficiency. A notable Crabtree effect, indicative of metabolic reprogramming towards glycolysis, was observed exclusively in the LG group.

Discussion: These findings suggest that high glucose concentrations in culture media can induce metabolic reprogramming in hepatocellular carcinoma cells, affecting mitochondrial function. Further investigations into aerobic glycolysis under varying glucose conditions are warranted to expand upon these respirometric findings. Our study underscores the importance of considering culture conditions in mitochondrial research and functional diagnostics.

Dysfunction of mitochondria, which cause oxidative stress and failure in satisfying the brain's high energy needs, has been implicated in the pathogenesis of neurological and neurodegenerative diseases. The brain is particularly sensitive to oxidative stress, because of its high concentration of lipids that are vulnerable to oxidation, and its relatively low levels of antioxidants. Notably, mitochondria located in synapses supply energy to support synaptic functions and their defects may result in synaptic failure, a common feature of neuropathologies. Indeed, brain processes depend on synaptic plasticity, the great ability of the nervous system to change synaptic strength and neuronal connections in response to physiological stimuli and environmental changes. Brain plasticity and cognitive functions are tightly influenced by nutritional interventions, which determine a long-term metabolic and inflammatory modulation. We investigated the impact of the intake of milk, reference food for infant nutrition, on mitochondrial functions and redox state in the central nervous system. Male Wistar rats were used to explore the effects of isoenergetic supplementation of milk from cow, donkey or human on mitochondrial function, oxidative stress and inflammation in the brain cortex and synaptosomal fraction. We found that the administration of different milk modulates mitochondrial function, efficiency, and ROS production both in brain cortex and its purified synaptosomal fraction, where mitochondria provide energy to support synaptic functions and plasticity. Moreover, milk intake modulated the expression of two presynaptic proteins and the levels of main markers of neuroinflammation. These results emphasize the importance of nutrition in brain and synapse physiology, and highlight the key role played in this context by mitochondria, nutrient-sensitive organelles able to orchestrate metabolic and inflammatory responses.

Ferroptosis is a unique form of cell death driven by iron and lipid peroxidation, with mitochondria playing an influential role in the process [1,2]. While mitochondria are traditionally known for producing pro-ferroptotic reactive oxygen species (ROS), their involvement in ferroptosis extends to various other aspects, including the maintenance of membrane potential, mitochondrial fission and fusion, and mitophagy [2,3]. Our study has uncovered surprising insights into the complex roles mitochondria can play in ferroptosis.

Focusing on the inherited metabolic disorder Barth Syndrome, in which mitochondrial membrane lipids, particularly cardiolipins, are altered [4], we discovered a counterintuitive result: cells with altered cardiolipin composition were protected from ferroptosis, despite showing consistently high levels of mitochondrial ROS and fission. This unexpected resistance challenged existing assumptions about the relationship between mitochondrial activity, ROS production, and ferroptotic susceptibility.

Our investigation into this phenomenon revealed that iron availability and the respective lipid environment strongly impacted on ferroptotic outcomes, however, they did not explain the protective mechanism in Barth Syndrome. Further exploration showed that neither the mitochondrial electron transport chain function nor central carbon metabolism were the primary cause of the observed resistance. Instead, our results indicated that changes in cardiolipin composition hindered the formation of specific oligomeric structures in mitochondrial membrane transporter systems, especially when exposed to oxidative conditions. Furthermore, our study revealed that our Barth Syndrome model exhibited a reduction in ER-mitochondria contact sites dependent on the same transporters, suggesting that this altered mitochondrial signalling influencing cross-compartmental interactions and cell fate decisions.

These findings offer a new perspective on how mitochondrial membrane lipid composition can manipulate the ferroptosis susceptibility of cells, suggesting that the modulation of cell death pathways involves cross-compartmental interactions among lipids, membrane-interacting proteins, and lipid-metabolic enzymes.

[1] S. J. Dixon et al., “Ferroptosis: An iron-dependent form of nonapoptotic cell death,” Cell, vol. 149, no. 5, pp. 1060–1072, 2012, doi: 10.1016/j.cell.2012.03.042.

[2] M. Gao et al., “Role of Mitochondria in Ferroptosis,” Mol Cell, vol. 73, no. 2, pp. 354-363.e3, 2019, doi: 10.1016/j.molcel.2018.10.042.

[3] Y. Q. Wang et al., “The protective role of mitochondrial ferritin on erastin-induced ferroptosis,” Front Aging Neurosci, vol. 8, no. DEC, pp. 1–9, 2016, doi: 10.3389/fnagi.2016.00308.

[4] P. G. Barth et al., “An X-linked mitochondrial disease affecting cardiac muscle, skeletal muscle and neutrophil leucocytes,” J Neurol Sci, vol. 62, no. 1–3, pp. 327–355, Dec. 1983, doi: 10.1016/0022-510X(83)90209-5.

Daniel A. Arabie¹, Olivia G. Moncrief¹, Samantha M. Shirmer¹, Steven C. Hand¹

¹Department of Biological Sciences, Louisiana State University, Baton Rouge, USA

darabie7@lsu.edu

Defense against oxidative stress during mitochondrial transitions into anoxia for an invertebrate extremophile

Invertebrate extremophiles experience severe metabolic transitions promoted by anoxia and diapause [1-2]. For embryos of brine shrimp, Artemia franciscana, these reversible states are survived for months to years. In contrast, recovery from metabolic disruption in mammals is accompanied by generation of reactive oxygen species (ROS) that cause tissue damage during ischemia-reperfusion [3]. Isolated mitochondria were subjected to anoxia for 30 min while controls received continuous normoxia as in [3]. Samples were pelleted and resuspended in oxygenated buffer containing fresh substrate, ADP and Amplex Red assay components [3] and tested across 30 min of normoxic recovery. Parallel samples included auranofin and dinitrochlorobenzene (DNCB) to inhibit thioredoxin reductase and glutathione peroxidase, respectively. Remarkably, H₂O₂ accumulation did not increase significantly in mitochondria exposed to anoxia-reoxygenation compared to normoxic controls; extending anoxic exposures to 120 min actually lowered H₂O₂ accumulation upon recovery. By comparison, an 8-fold increase in H₂O₂ was reported for rat heart mitochondria given the same treatment [3]. As anticipated, inclusion of auranofin and DNCB statistically increased the H₂O₂ accumulation 2-3 fold in both control and experimental mitochondria. Consistent with the lack of elevated H₂O₂ after anoxia-reoxygenation, aconitase inactivation also was not detected compared to controls. Statistical increases were not observed in protein carbonyls or lipid hydroperoxides (even after extending recovery to 60 min for the latter). Evidence suggests mitochondria from A. franciscana embryos are well protected against ROS accumulation and oxidative damage during anoxia-reoxygenation. Mechanistic explanations for such multifaceted protection could be relevant for ischemia-reperfusion intervention [NSF grant IOS-1457061/IOS-1456809].

[1] S. Hand, D. Denlinger, J. Podrabsky, R. Roy, Mechanisms of animal diapause: Recent developments from nematodes, crustaceans, insects and fish. Amer. J. Physiol. 310 (2016) R1193-R1211.

[2] Y. Patil, E. Gnaiger, A. Landry, Z. Leno, S. Hand, OXPHOS capacity is diminished and the phosphorylation system inhibited during diapause in an extremophile, embryos of Artemia franciscana. J. Exp. Biol. 227 (2024) jeb245828.

[3] E. Chouchani, C. Methner, S. Nadtochiy, A. Logan, V. Pell, S. Ding, A. James, H. Cochemé, J. Reinhold, K. Lilley, L. Partridge, I. Fearnley, A. Robinson, R. Hartley, R. Smith, T. Krieg,

P. Brookes, M. Murphy, Cardioprotection by S-nitrosation of a cysteine switch on mitochondrial Complex I. Nature Medicine 19 (2013) 753-759.

Mitochondrial cytochrome c oxidase (COX) subunit 4 isoform 2 (Cox4i2) is essential for oxygen sensing in pulmonary arterial smooth muscle cells (PASMCs). It promotes mitochondrial superoxide production during acute hypoxia, which triggers hypoxic pulmonary vasoconstriction, thereby preventing systemic hypoxemia. We hypothesized that Cox4i2-dependent superoxide production in acute hypoxia is caused by alterations in supercomplex formation and/or regulation of the redox state of the electron transport system (ETS).

Oxygen consumption was determined by high-resolution respirometry simultaneously with the coenzyme Q (CoQ) redox state in isolated wild-type (WT) and Cox4i2^-/- mouse PASMCs. Complexome profiling and Raman spectroscopy were used to investigate complex assembly and redox changes of the ETS, respectively. The relevance of Cox4i2-specific cysteine residues for hypoxia-induced superoxide release was tested by analyzing superoxide production and oxygen affinity in a mouse epithelial cell line (CMT167) expressing either WT or mutant (C41S, C55A, C109S) Cox4i2.

Oxygen consumption, assembly of COX, and supercomplex formation were similar in WT and Cox4i2^-/-PASMCs, with Cox4i2 peptides detected only in the monomeric COX. Hypoxia-induced reduction of NAD⁺, CoQ, and cytochrome c was absent in Cox4i2^-/- PASMCs. Similarly, the reduced CoQ fraction generated by mitochondrial complex I, or complex II, or converging substrates at the CoQ-junction was lower in permeabilized Cox4i2^-/- mPASMCs compared to WT. CMT cells expressing mutant Cox4i2 lacked hypoxia-induced superoxide production, whereas oxygen affinity was not significantly different between Cox4i2 WT, Cox4i2 mutant, or Cox4i1 expressing cells.

In conclusion, our findings suggest that Cox4i2 sensitizes the ETS to a reduced state, which may promote superoxide release in acute hypoxia.

Supported by the German Research Foundation (DFG, project 268555672), joint U.S. National Science Foundation (NSF) and DFG (project SO 1237/4-1) and the O2k-Network award.

It is commonly accepted that neurons have essential energetic demands supplied by mitochondrial processes due to oxygen consumption and ATP production. Moreover, the level of metabolites is crucial to form the pathological processes under hypoxic conditions in a nervous tissue [1]. One of the most significant reasons to initiate cell death of neurons is so called excitotoxicity mediated by glutamate concentration excess in the interstitial fluid. The raise of glutamate concentration causes increase of intracellular Ca2+ level and cell swelling. Increased level of reactive oxygen species (ROS) production also occurs. It was recently demonstrated that overproduction of H2O2 initiated by glutamate in a cell causes an excess of its level in neighbour cells due to the influence of a tissue spatial heterogeneity [2]. In the present study 3D modelling of local nervous tissue area was fulfilled using a methodological approach described elsewhere [3] [4]. It was shown that a formed spatial ROS gradient has a direct impact on mitochondrial metabolic state, and it causes an appearance of local areas of ATP production and respiratory chain activity.

[1] N.V. Lobysheva, Selin, A. A., Yaguzhinsky, L.S., Nartsissov, Y.R., Diversity of neurodegenerative processes in the model of brain cortex tissue ischemia, Neurochemistry International, 54 (2009) 322-329.

[2] V.A. Selivanov, O.A. Zagubnaya, Y.R. Nartsissov, M. Cascante, Unveiling a key role of oxaloacetate-glutamate interaction in regulation of respiration and ROS generation in nonsynaptic brain mitochondria using a kinetic model, PLoS ONE, 16 (2021) e0255164.

[3] Y.R. Nartsissov, A novel algorithm of the digital nervous tissue phantom creation based on 3D Voronoi diagram application, Journal of Physics: Conference Series, 2090 (2021) 012009.

[4] Y.R. Nartsissov, Application of a multicomponent model of convectional reaction-diffusion to description of glucose gradients in a neurovascular unit, Frontiers in Physiology, 13 (2022).

Catalase (Ctl) dismutates hydrogen peroxide (H₂O₂) to O₂ and H₂O (catalytic reaction). Ctl plays dual roles in cellular and mitochondrial respirometry, as physiological antioxidant and for increasing the experimental O₂ concentration by H₂O₂ titrations. Substrate-uncoupler-inhibitor titration (SUIT) protocols are widely applied in OXPHOS analysis with multiple titrations in a respirometric assay, using several chemicals dissolved in ethanol [1]. Increasing ethanol concentrations exert an influence not only on mitochondrial function but also on the conversion of H₂O₂ by Ctl.

We studied mitochondrial function of HEK 293T cells in mitochondrial respiration medium supplemented with Ctl (MiR06). Ethanol concentrations of 135 to 425 mM (0.8 to 2.5 % v/v) led to a 40 % and 60 % reduction in O₂ production per amount of H₂O₂, respectively. These are common ethanol concentrations reached in standard SUIT protocols.

Besides the catalytic reaction generating O₂, Ctl oxidizes ethanol to acetaldehyde and H₂O (peroxidatic reaction without production of O₂) [2,3]. At increasing ethanol concentrations and low concentrations of H₂O₂, the peroxidatic reaction reduces the amount of O₂ produced per amount of H₂O₂ decomposed. Awareness of this blunted reoxygenation is critical for designing reoxygenation regimes and assessment of mitochondrial function in a range of ethanol concentrations that do not exert negative side effects. Importantly, respiratory dysfunction associated with ethanol may arise primarily through its oxidation to acetaldehyde.

References:

[1] Doerrier et al, 2018; High-resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Mitochondrial Bioenergetics: Methods and Protocols, 31-70

[2] Oshino et al, 1973; The characteristics of the ‘peroxidatic’ reaction of catalase in ethanol oxidation. Biochemical Journal, 131(3), 555-563

[3] Kremer, 1970; Peroxidatic activity of catalase. Biochimica et Biophysica Acta (BBA)-Enzymology, 198(2), 199-209

Characterizing the molecular architecture of the archaeal ACDS superassembly

Many anaerobic bacteria and archaea use the Wood-Ljungdahl pathway (WLP) or variants thereof to conserve energy and to fix CO₂. One of the key enzymes in this pathway is the bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS), which catalyzes the reversible condensation of carbon monoxide (CO), a methyl-group and coenzyme A (CoA) to form acetyl-CoA [1]. Archaea conducting methanogenesis, a variant of the WLP, possess a ~2 MDa CODH/ACS superassembly [2,3] distinct from its homologs in bacteria. Until today, despite decades of research only low-resolution structural information of the superassembly has been obtained. We employed an integrative structural biology approach to characterize subcomplexes of the superassembly and their interactions. Our results reveal that functional subcomplexes of the assembly show distinct homo-oligomerization patterns. Furthermore, we identified terminal disordered regions as likely being responsible for these patterns. Using cross-linking mass spectrometry, we resolved two catalytically relevant conformations of the acetyl-CoA synthase subcomplex bound to either one of the other two subcomplexes. With our work, we hope to gain further insights into the functional differences between the bacterial and archaeal CODH/ACS complex architectures.

[1] A. Biester, A.N. Marcano-Delgado, C.L. Drennan, Structural Insights into Microbial One-Carbon Metabolic Enzymes Ni−Fe−S-Dependent Carbon Monoxide Dehydrogenases and Acetyl-CoA Synthases, Biochemistry, 24 (2022) 2797-2805.

[2] K.C. Terlesky, M.J. Nelson, J.G. Ferry, Isolation of an enzyme complex with carbon monoxide dehydrogenase activity containing corrinoid and nickel from acetate-grown Methanosarcina thermophila, Journal of Bacteriology, 3 (1986) 1053-1058.

[3] D.A. Grahame, T.C. Stadtman, Carbon monoxide dehydrogenase from Methanosarcina barkeri. Disaggregation, purification, and physicochemical properties of the enzyme, Journal of Biological Chemistry, 8 (1987) 3706-3712.

Cytochrome c-dependent nitric oxide reductase (cNOR) is a heme-copper oxidase catalyzing the reduction of nitric oxide into nitrous oxide during denitrification. The active site of cNOR contains one heme b and one non-heme iron, both needed for catalytic function. We study the insertion of the non-heme iron, a process that in some denitrifiers, for example Paracoccus denitrificans, is dependent on two chaperone proteins, NorQ and NorD, encoded in the cNOR gene cluster. The two chaperone proteins, NorQ, a MoxR AAA+ (ATPases Associated with diverse cellular Activities) protein and NorD, a VWA (von Willebrand factor type A) domain protein, come together to form a cNOR activation complex. Through ATP hydrolysis, they stimulate conformational changes in cNOR, which opens up the cNOR active site and enables iron insertion. However, some denitrifying bacteria, such as Thermus thermophilus, lack the norQ and norD genes in their cNOR gene clusters. How come some cNORs need chaperons for iron insertion, and some don’t? The aim of our work is to shed light on the activation mechanisms of cNORs, using site-directed mutagenesis, enzymatic assays, cryo-EM and AlphaFold modelling.

In the healthy gut, the little oxygen diffusing from the blood into the mammalian colon is quickly consumed by coloncytes for fatty acid oxidation, keeping the gut lumen strictly anaerobic. If the composition of the gut microbiome is altered by e.g. antibiotics or immune response, oxygen levels might rise as a consequence, allowing facultative anaerobes like pathogenic Salmonella and E. coli to expand, leading to gut dysbiosis or inflammatory bowel disease.[1] In addition, it has been found that facultative anaerobes use reactive oxygen species originating from the immune response as a source for oxygen or as electron acceptor, enabling advantageous oxidative phosphorylation even under low-oxygen conditions. This process involves cytoplasmic catalase, which converts hydrogen peroxide (H₂O₂) to oxygen, or cytochrome c peroxidase, which can directly reduce H₂O₂.[2]

Here, we developed a minimal bottom-up system containing all relevant enzymes. Purified E. coli quinol oxidases and F₁F_O ATP synthase were co-reconstituted and commercially available bovine liver catalase was encapsulated into small unilamellar liposomes. This bottom-up system enables the measurement of individual enzymatic functions and their interplay at different peroxide concentrations and oxygen levels. Using a Clark-type electrode, we show that catalase-produced oxygen inside the liposomes is readily consumed by terminal oxidases driven by ubiquinol and that the resulting proton motive force can be used to drive ATP synthesis. Unlike oxygen, the concentration of H₂O₂ is not limited by solubility, enabling increased ATP synthesis by the addition of more H₂O₂. Using a controlled experimental system with a minimal set of enzymes, our results suggest that the co-localization of oxygen production by catalase in the liposome lumen and oxygen consumption in the liposomal membrane is an efficient strategy of facultative anaerobes to exploit host-derived peroxide as terminal electron acceptor. The developed system further expands the toolbox for efficient ATP production for synthetic biology applications.

Continuous electron flux from cellular reduction equivalents such as NADH to final electron acceptors such as oxygen is essential to keep the out-of-equilibrium state of a living cell. During this process, respiratory oxidases utilize reduction equivalents to establish and maintain the proton motive force (pmf) that plays a critical role as universal energy intermediate that energizes many cellular processes such as nutrient uptake, waste excretion and most notably, ATP synthesis via the F₁F_O ATP synthase. Genomic depletion of the latter in E. coli leads to a reduced growth, accompanied by membrane hyperpolarization.

Here, we describe the essential role of cytochrome b₅₆₁ (CybB) that counteracts membrane hyperpolarization and ensures continuous electron flux in the absence of the main pmf consumer ATP synthase. In its role, the enzyme oxidizes membranous ubiquinol and reduces the membrane potential by electrogenic electron transfer from the cytoplasmic to periplasmic side of the membrane. Oxygen acts as electron acceptor and periplasmic superoxide is produced, which has been described earlier but was attributed to unspecific quinol autooxidation. In other words, cybB uses the same substrates as the terminal bd or bo oxidases (quinol and oxygen) but differs in its electrogenic directionality. Next to growth data and in vitro functional measurements, we share data on the altered expression levels of cybB in an Δatp knockout strain and discuss the functional roles of two orthologous proteins found in E. coli.

Mitochondrial reactive oxygen species (mtROS) are essential for T cell activation and thus can regulate the host response to viral infection. However, the exact mechanism of mtROS release during T cell activation and relevance for viral infection is unknown. We thus investigated the contribution of the regulatory subunit cytochrome c oxidase subunit 4 isoform 2 (Cox4i2) for mtROS release during T cell activation and viral defense. We determined T cell activation, mtROS levels and mitochondrial respiration in vitro in isolated spleen T cells of wild type (WT) and Cox4i2^-/- mice by flow cytometry or high-resolution respirometry, respectively. Viral load, inflammation, and T cell characteristics were analyzed in vivo after H1N1 influenza (PR8) infection. Cox4i2^-/- T cells showed decreased expression of the late T cell activation marker CD25 after in vitro activation by αCD3/αCD28 antibodies compared to WT T cells. Importantly, mtROS levels increased in WT T cells after in vitro activation but not in Cox4i2^-/- T cells, while mitochondrial respiration was increased in both intact T cells of WT and Cox4i2^-/- mice after activation. Measurement of oxygen consumption using a substrate-inhibitor protocol in permeabilized T cells revealed increased complex IV activity after in vitro activation of WT T cells, which was absent in Cox4i2^-/- T cells. In contrast to the in vitro results, in vivo, viral infection did not affect the T cell activation marker CD25 or viral clearance in Cox4i2^-/- mice. However, PR8-induced interferon γ release, as a marker for antigen-specific T cell numbers, was largely decreased in spleen T cells of Cox4i2^-/- mice, as well as bronchoalveolar lavage fluid levels of chemokine C-X-C motif chemokine ligand 1 (CXCL1) and infiltration of neutrophils. In conclusion, Cox4i2 regulates mtROS release and T cell activation but is not essential for viral defense during sublethal PR8 infection in vivo.

Funded by KFO309

Background

Normothermic machine perfusion (NMP) of the liver is a dynamic organ preservation method, which offers the ability to assess organ function ex situ but requires reliable biomarkers. Since bioenergetic competency is central for cellular maintenance and thus, organ functionality, we herein aimed to characterize mitochondrial function using high-resolution respirometry (HRR) during liver NMP.

Methods

Human livers (N=50) considered for transplantation were applied to clinical NMP for up to 24 hours. Serial perfusate and biopsy samples were collected at the end of cold storage and longitudinal during NMP. Conventional perfusate parameters and histopathology was performed. HRR was applied to assess LEAK respiration, OXPHOS capacity, efficiency of ATP production (P-L control efficiency) and integrity of the mitochondrial outer membrane (cytochrome c control efficiency). Livers declined for transplantation during NMP were considered for experimental long-term perfusion up to 7 days.

Results

A considerable variability of the individual liver grafts was observed at the end of cold storage, which remained stable after the start of NMP. For the 35 livers deemed suitable for transplantation the area under curve (AUC) during 6 hours of NMP was calculated. For those livers the AUC of LEAK respiration, cytochrome c control efficiency and P-L control efficiency correlated with the early clinical outcome (L-GrAFT score). Four of the declined livers could be preserved for 7 days by application of a novel perfusion protocol. The bioenergetic function in the liver biopsies was stable as indicated by adequate P-L control efficiency (0.780 ± 0.099) and outer mitochondrial membrane integrity (0.108 ± 0.047) and was in line with conventional assessment methods.

Conclusion

Evaluation of the bioenergetic function in livers undergoing NMP can be included in liver functionality assessment methods and predict the clinical outcome after transplantation. In addition, NMP can preserve bioenergetic competency of the liver during long-term preservation.

Background:

Drugs that specifically kill senescent cells (senolytics) can delay and in some cases even cure age-associated diseases and disabilities in pre-clinical settings, and first results from ongoing clinical trials appear encouraging. However, so far applications of current senolytics are limited due to narrow therapeutic windows and low sensitivity and specificity. We hypothesized that co-targeting the functionally compromised mitochondria in senescent cells could enhance the efficacy of senolytics that works through inducing mitochondrial apoptosis pathways, BH3 mimetics.

Results:

Senescence-associated mitochondrial dysfunction was assessed by multiple bioenergetics and cell biological methods. We establish specific loss of complex I-linked coupled respiration and the inability to maintain mitochondrial membrane potential upon respiratory stimulation as a specific vulnerability of senescent cells. Further decreasing mitochondrial membrane potential of senescent cells with a mitochondrial uncoupler synergistically enhances the in vitro senolytic efficacy of BH3 mimetic drugs, including Navitoclax, by up two orders of magnitude, whereas non-senescent cells remain unaffected. Moreover, in an in vivo mouse model, a short-term intervention combining the mitochondrial uncoupler BAM15 with Navitoclax at a dose two orders of magnitude lower than typically used rescues the radiation-induced premature ageing phenotype.

Conclusions:

In senescent cells, compromised mitochondrial functional capacity is a specific vulnerability distinct from non-senescent cells, which can be targeted by mild uncoupling in vitro and in vivo to greatly enhance the efficacy of BH3 mimetic senolytics. BH3 mimetics are amongst the most potent senolytics, but their translation into clinical use has been hindered by severe side effects including thrombocytopenia. These risks may be massively reduced by combination with a clinically safe amount of mitochondrial uncoupler such as BAM15.

There is an increasing number of studies focusing on the long-term toxic effects and risks associated with alcohol consumption ^[1]. "Dry January" is a campaign which is encouraging individuals to abstain from alcohol for the entirety of January ^[2]. Participants report numerous benefits such as improved sleep and increased energy levels. This success has inspired individuals worldwide to take up the "dry January" challenge. Conversely, the month of February presents a contrast to the moderation of January, particularly in carnival cities across Germany. During carnival festivities, characterized by parades, music, and entertainment, there is a notable increase in alcohol consumption alongside indulgent street food offerings.

When blood alcohol levels rise significantly during binge drinking, there is a notable decrease in NAD+ levels. This reduction leads to liver injury since the enzymes reliant on NAD+ as a cofactor are affected ^[3].The intracellular NAD+/NADH ratio governs the rate of ATP synthesis ^[4]. Additionally, alcohol consumption impacts mitochondria through various pathways beyond NAD+ regulation ^[5].

We examined the alterations in NAD+ levels (by Q-NADMED Blood NAD+ assay kit, NADMED) and mitochondrial respiration parameters (by Seahorse XF Cell Mito Stress Test Kit, Agilent) in four individuals who abstained from alcohol for four weeks in January and subsequently engaged in a binge-drinking episode in February (consuming 10 or more units of alcohol in less than three days). Additionally, we assessed baseline values for three individuals who are non-drinkers. We observed a reduction in NAD+ levels in all four individuals during the drinking phase. The decrease in NAD+ levels was positively correlated with the amount of alcohol consumed. Additionally, total NAD+ levels showed a positive correlation with the Bioenergetics Health Index, calculated from mitochondrial respiration parameters.

Despite the small sample size, we aim to continue examining mitochondrial parameters and NAD+ levels pre- and post-binge drinking to understand alcohol's short-term toxic effects.

[1] GBD 2020 Alcohol Collaborators. Lancet. 2022 Jul 16;400(10347):185-235.

[2] Ballard J. Br J Gen Pract. 2016 Jan;66(642):32.

[3] French SW. Exp Mol Pathol. 2016 Apr;100(2):303-6.

[4] Walker MA, Tian R. Curr Opin Physiol. 2018 Jun;3:101-109.

[5] Manzo-Avalos S, Saavedra-Molina A. Int J Environ Res Public Health. 2010 Dec;7(12):4281-304.

Inherited pathogenic mutations affecting mitochondrial bioenergetics can give rise to a wide spectrum of metabolic disorders with variable ages of onset and clinical manifestations. Even for disease-causing mutations within the same gene, patients may develop heterogeneous clinical symptoms that cannot be easily reconciled with a certain genetic makeup or simply linked to the loss of mitochondrial fitness and function. While genetic modifiers may account for some of these phenotypic variabilities, new evidence suggests that environmental factors can also contribute to disease outcomes and progression. Here we investigate the impact of diets on the pathogenic phenotypes associated with a disease-causing mutation within the gene encoding apoptosis-inducing factor (AIF or AIFM1). In invertebrates and higher organisms, hypomorphic AIFM1 mutations and/or AIF downregulation inhibit the assembly of Complex I and alter mitochondrial oxidative phosphorylation. We report that high-lipid diets can stimulate animal survival, whereas low-lipid diets compromise longevity pathways by promoting the expression of leucine-rich repeat kinase (LRRK1-2) and dynamin-related protein 1 (DRP1). Together, our findings mechanistically demonstrate that a hypomorphic AIFM1mutation can have different downstream effects on pro-longevity mitochondrial network remodeling and lipid metabolism, with disease presentations and severity that vary depending on the dietary interventions as well as on the nutritional state of the organism.

Myoclonus, Epilepsy and Ragged-Red Fibers (MERRF) is a rare form of mitochondrial encephalomyopathy associated with the m.8344A>G mutation in the tRNALys gene, which resides in the mitochondrial DNA (mtDNA). Defective tRNALys impairs mitochondrial protein synthesis when heteroplasmy crosses the threshold for full-blown clinical phenotype. Cellular models thereby display a plethora of metabolic dysfunctions ranging from declined cellular respiration, raising oxidative stress and abnormal calcium homeostasis according to the wt:mutant ratio of mtDNA copies. Because of the limited symptomatic treatment and the scarcity of relevant animal models, effective in vitro cellular models are needed that recapitulate the MERRF features to identify pertinent therapeutic compounds. However, this task is complicated by the multi-copy nature of the mtDNA, which possibly drives the wild type-mediated rescue of disease-related impairment. In this work, we first established 3D cortical organoids (CORs) from patient-derived human induced pluripotent stem cells (iPSCs) bearing the m.8344A>G mutation at 20%. As expected, the generation was successful but yielded organoids which lacked an overt mutant phenotype due to the low heteroplasmy load. MERRF CORs grew comparably to healthy organoids and displayed neither decreased oxygen consumption nor excessive extracellular acidification. The expression of common markers of progenitor and mature neurons was not affected either. The only positive score was fluorescence imaging, which revealed an irregular morphology of mutant CORs, deformed neural rosettes and a markedly decreased GFAP-positive astrocyte population. Because of the poor accuracy of this model, we set out to artificially raise the MERRF heteroplasmy in iPSCs through ethidium bromide-mediated mtDNA depletion and repopulation, which remarkably increased the 8344A>G load to 70%. Hopefully, this newly generated high heteroplasmy iPSC line will serve as a more suitable substrate for MERRF characterization, differentiation into cortical organoids, phenotyping and drug testing.

Mitochondrial diseases are clinically and genetically heterogeneous conditions characterized by defects in oxidative phosphorylation. The accumulation of damaged mitochondria can be a common pathophysiological mechanism and a target for treatment. Mitophagy, the selective degradation of mitochondria by autophagy, is a cellular mechanism used to target and degrade damaged mitochondria. In our screen of mitochondrial disease patient-derived fibroblasts, we show impaired mitophagy in most of the cells analyzed. Given that a hallmark of mitochondrial disease is the accumulation of damaged mitochondria and that mitophagy is typically impaired, we sought to determine if inducing mitophagy is a viable treatment option. Here, we report a novel mitophagy inducer CAP-1902, an agonist of the MAS G-Protein Coupled Receptor, in a CIII-deficient patient fibroblast cell line. Analysis of CAP-1902 efficacy was performed in fibroblasts carrying a BCS1L mutation, which impairs CIII assembly. These CIII-deficient cells show reduced CIII activity and impaired respiration, accumulation of depolarized and fragmented mitochondria, and defects in lysosomal distribution. Analysis using the mito-QC reporter showed an accumulation of mitolysosomes in CIII deficient cells when compared to control; however, the accumulation was cleared after treatment with CAP-1902. We demonstrate that CAP-1902 induces mitophagy via MasR and downstream through the AMPK/ULK1/FUNDC1 signaling pathway. RNA-seq analysis revealed that treatment with CAP-1902 effected mitochondrial biogenesis and mitophagy related targets. The induction of mitochondrial biogenesis via PGC1α activation coupled with increased mitophagic flux after CAP-1902 treatment suggest it promotes mitochondrial turnover. Clearance of damaged mitochondria through CAP-1902 treatment was accompanied by an improvement of bioenergetic function, decreased mitochondrial stress, and corrected lysosomal distribution. In summary, we have described the first potential GPCR-mediated treatment of a mitochondrial disease model through mitophagy induction.

Parkinson's disease is a progressive neurodegenerative disorder that affects the entire system and manifests in a wide range of symptoms, the severity of which escalates over time and significantly impacts the patient's independence. The worldwide prevalence ranges from 10 to 800 patients per 100,000 people, with average onset age of 60 years. The disease is characterized by progressive neurodegeneration affecting dopaminergic neurons in the substantia nigra pars compacta, leading to neuronal loss. Despite the multitude of symptoms and multifactorial etiopathogenesis, there is currently no suitable marker for disease confirmation, so doctors must wait for the appearance of specific motor symptoms, which typically occur 10 to 20 years after the onset of neurodegeneration. Prolonged diagnosis, lasting 6 to 12 months, is also problematic. The discovery of a specific marker confirming the disease is crucial for objective diagnosis, monitoring disease progression, and initiating effective treatment, especially when diagnosed early.

Recent studies have shown that mitochondrial dysfunction plays a key role in the pathomechanism of Parkinson's disease. Mitochondria, as energy-producing powerhouses through oxidative phosphorylation, are interest due to their role in various processes, particularly energy metabolism, calcium homeostasis, and the generation of reactive oxygen species. Increasing evidence suggests that changes in mitochondrial respiration, associated with subsequent energy failure, significantly contribute to neuronal degradation.

In our study, we investigate changes in mitochondrial respiration and mitochondrial membrane permeability in patients with Parkinson's disease compared to healthy controls. Mitochondria from peripheral leukocytes are suitable for studying intact mitochondria and represent readily available material. Studies also demonstrate that changes in mitochondrial respiration parameters, such as complex activity, membrane permeability, and reactive oxygen species production by the respiratory chain, are also evident in peripheral leukocytes in patients with Parkinson's disease, indicating systemic dysfunction in cellular bioenergetics. Examining the dynamics of changes in mitochondrial respiration in peripheral leukocytes appears to be an effective part of a cumulative marker, akin to a fingerprint for Parkinson's disease. In the study, we monitored changes in parameters in 30 patients with Parkinson's disease, classified according to the Hoehn-Yahr scale into stages 2.0 to 2.5, and in 30 healthy controls matched by age and gender. The study is part of the project MITOPAT APVV-19-0222, where initially over 200 parameters were evaluated. Using machine learning, we filtered out more than 135 parameters. Changes in cellular and mitochondrial respiration proved to be significant, with several of them making it into the narrower selection of 15 parameters.

Dominant Optic Atrophy (DOA) is the most common childhood form of mitochondrial optic neuropathy, characterized by the degeneration of retinal ganglion cells leading to blindness. Like other mitochondrial diseases (MDs), DOA is genetically heterogeneous with about 60% of patients harboring mutations in OPA1. Moreover, over 400 OPA1 mutations have been identified, generating either haploinsufficiency or gain-of-function effects [1]. Owing to its complexity, developing effective therapies for DOA is significantly challenging. Therefore, gene/mutation-independent approaches may be valid alternatives. microRNAs represent promising therapeutic tools since they can simultaneously modulate multiple pathways involved in disease pathogenesis and progression. We focused on the study of miR-181a/b, involved in the modulation of mitochondrial function and whose downregulation ameliorates the phenotype of different MDs models [2]. We first evaluated the effect of miR-181a/b inactivation in OPA1-deficient cells. In this model, a miR-181a/b sponge, able to specifically bind miR-181a/b and inhibit their activity, promoted mitochondrial biogenesis by increasing PGC1α, NRF1, and TFAM levels, and ameliorated mitochondria fragmentation, by decreasing mitochondrial translocation of DRP1, a main regulator of mitochondrial fission. Furthermore, we found a reduction of ROS production and a decrease in cell death in sponge-expressing OPA1-deficient cells. Based on these promising results, we applied sponge-strategy also in a DOA in vivo model. Interestingly intravitreally injections of AAV2/2 vectors expressing miR181a/b sponge in Opa1^+/Q285STOP mice [3] induce an amelioration of visual function with respect to the untreated mice. Altogether our data indicate that miR-181a/b may be considered new therapeutic targets for treating the ADOA.

[1] Yu-Wai-Man, Patrick, and Patrick F Chinnery. “Dominant optic atrophy: novel OPA1 mutations and revised prevalence estimates.” Ophthalmology vol. 120,8 (2013): 1712-1712.e1.

[2] Indrieri, Alessia et al. “miR-181a/b downregulation exerts a protective action on mitochondrial disease models.” EMBO molecular medicine vol. 11,5 (2019): e8734.

[3] Davies, V. J. et al. Opa1 deficiency in a mouse model of autosomal dominant optic atrophy impairs mitochondrial morphology, optic nerve structure and visual function. Hum Mol Genet 16, 1307–1318 (2007).

Protein kinases play an important role in numerous signaling pathways regulating cell proliferation, differentiation, cell death, and metabolism. Deregulation of kinase functions have been connected to various human diseases, such as cancer [1, 2]. In recent years, kinase inhibitors have gained recognition by aiming to block single or multiple oncogenic kinase pathways, which is underlined by 49 FDA-approved kinase inhibitor drugs. Besides kinases also mitochondrial metabolism has emerged as a central drug target. Mitochondria are dynamic cell organelles, orchestrating cellular energy production and cellular signalling. Blockade of kinase activities has been shown to converge on mitochondria [3].

In our study, we have applied defined pharmacological perturbations to manipulate kinase pathways involved in both, mitochondrial functions and oncogenesis. We set out to systematically profile the impact of broad and specific kinase drugs on mitochondrial respiration in several cancer cell models using High-resolution respirometry. We observed that the impact of kinase inhibitors depends on the mutational background of the tested cancer cell lines as well as on cell culture medium formulations [4]. First, we detected off-target effects of sunitinib, an FDA-approved multikinase blocker, only in a more physiological cell culture medium as compared with classical formulations. Second, mitochondrial profiling of the glycolytic kinase inhibitor PFK158 revealed off-target mitochondrial dysfunction. Third, we were able to show that inhibition of kinase signaling is connected to mitochondrial reactive oxygen species (ROS), which can be influenced by protein kinase modulators. In summary, examining drug-induced mitochondrial dysfunctions offers a deeper understanding of the apparent off-target effects of kinase inhibitors. Consequently, utilizing cell-based diagnostics to analyze mitochondrial bioenergetic profiles emerges as a promising approach for identifying on-target effects or predicting potential off-target effects of drugs that disrupt cell metabolism.

[1] Deribe, Y. L., Pawson, T. & Dikic, I (2010). Nat. Struct. Mol. Biol.

[2] Zhang R, Loughran TP, Jr (2011). Leukemia & Lymphoma.

[3] Torres-Quesada O, Strich S, Stefan E (2022).Bioenerg. Commun.

[4] Torres-Quesada, O, Doerrier, C, Strich, S, Gnaiger, E, Stefan, E (2022). Cancers.

Leigh syndrome (LS), also known as subacute necrotizing encephalomyelopathy, is one of the most frequent mitochondrial disorders affecting around 1 in 40,000 newborns. This condition is caused by mutations in over 115 genes, with mitochondrial respiratory chain complexes being the most affected. Early onset and rapid deterioration of cognitive and motor functions, leading to death within months or years, characterize the disease. Due to LS biochemical and genetic heterogeneity, effective treatments have not yet been developed. Gene/mutation-independent strategies are thus needed.
We hypothesized that a synergic and fine-tuned modulation of pathways acting on mitochondrial homeostasis may ensure effective neuroprotection in mitochondrial disorders. In this respect, microRNA (miRNA) modulation is an attractive therapeutic strategy that has reached the preclinical and clinical stages. We demonstrated that miR-181a and miR-181b (miR-181a/b) regulate key genes involved in mitochondrial biogenesis and function and that their downregulation ameliorates the phenotype of different animal models of mitochondrial diseases [1]. Therefore, we decided to test if miR-181a/b downregulation may be exploited as a therapeutic strategy for LS using the Ndufs4 KO murine model. Our results show that genetic and Adeno Associated Viral (AAV) vector-mediated inactivation of miR-181a/b improves survival rate, ameliorates locomotor activity, and increases mitochondrial biogenesis. Based on the evidence above, we propose targeting miR-181a/b may represent a promising gene-independent therapeutic strategy for LS and other neurodegenerative disorders caused by mitochondrial dysfunction.

[1] A. Indrieri, S. Carrella, A. Romano, A. Spaziano, E. Marrocco, E. Fernandez‐Vizarra, S. Barbato, M. Pizzo, Y. Ezhova, F.M. Golia, L. Ciampi, R. Tammaro, J. Henao‐Mejia, A. Williams, R.A. Flavell, E. De Leonibus, M. Zeviani, E.M. Surace, S. Banfi, B. Franco, miR-181a/b downregulation exerts a protective action on mitochondrial disease models, EMBO Mol Med 11 (2019). https://doi.org/10.15252/EMMM.201708734.

Neurodegeneration with Brain Iron Accumulation (NBIA) is a rare disease (1–3 patients / 1000000 people) expressed by an excessive iron accumulation in the brain. NBIA is usually associated with slowly progressive pyramidal and extrapyramidal symptoms, axonal motor neuropathy, optic nerve atrophy, cognitive impairment and neuropsychiatric disorders. The most common from eleven NBIA subtypes include pantothenate kinase-associated neurodegeneration (PKAN), PLA2G6-associated neurodegeneration (PLAN), mitochondrial membrane protein-associated neurodegeneration (MPAN) and 35 beta-propeller protein-associated neurodegeneration (BPAN). Interestingly, MPAN is a dominant subtype among Polish population and is associated with the mutation in C19orf12 gene. The mechanism of how the loss of protein’s function results in the disease development remines unclear, hence, there are no pharmacological therapies to date. However, few pharmacological approaches are recently investigated with the hope to attenuate symptoms of the disease.

The goal of our study is to examine whether the intervention based on influencing of metabolic pathways could have a positive effect on mitochondrial bioenergetic and cell fate of fibroblasts of MPAN patients. Our experimental approach covers both, basal and OXPHOS promoting conditions, in order to better visualize mitochondrial metabolic defect in MPAN fibroblasts.

We want to clarify whether changes observed in a cellular proteome are accompanied by the alterations in functional parameters such as: metabolic (dehydrogenase) activity, glycolysis, activity of Krebs cycle enzymes, as well as mitochondrial respiratory chain activity (oxygen consumption). This could potentially explain a beneficial effect of the pharmacological approach used in our study.

The study is co-financed from the state budget from the Education and Science Ministry program entitled “Science for Society”. Project number NdS/537386/2021/2022, the amount of co-financing 1 900 000 PLN, total value of the project 1 900 000 PLN. Poland

The diatom Phaeodactylum tricornutum is an aquatic microalga that lives in brackish and marine water[1] and exhibits CO₂ concentration mechanism (CCM) to ensures an adequate supply of CO₂ for Calvin Benson Bassham cycle (CBB). Biophysical CCM relies on the transport of HCO₃^- across the different cell compartments and the catalysis of the reversible conversion of HCO₃ ^- to CO₂ by carbonic anhydrase. These carbonic anhydrases are divided into eight subclasses[2] . Among these subclasses is the Iota-Carbonic anhydrase (ιCA), a distinctive enzyme that is not a metalloenzyme[3] and tends to be overexpressed in low abundance of CO₂ or zinc. This latter phenotype is surprising since most carbonic anhydrases use zinc as a cofactor[4][5]. We are aiming to investigate the function, regulation, and interaction of ιCA with other enzymes of CCM by comparing the behavior of ιCA-deletion mutant strains to wild type in low CO₂ and zinc concentrations conditions. We also aim to characterize the mode of action by purifying the enzyme, characterizing its structural state and measuring its activity. This study will allow us to unravel the photosynthetic key elements in diatoms.

[1] Alessandra De Martino et al., “Genetic and Phenotypic Characterization of Phaeodactylum Tricornutum (Bacillariophyceae) Accessions1,” Journal of Phycology 43, no. 5 (2007): 992–1009, https://doi.org/10.1111/j.1529-8817.2007.00384.x.

[2] Erik L. Jensen et al., “A New Widespread Subclass of Carbonic Anhydrase in Marine Phytoplankton,” The ISME Journal 13, no. 8 (August 2019): 2094–2106, https://doi.org/10.1038/s41396-019-0426-8.

[3] Romain Clement et al., “Responses of the Marine Diatom Thalassiosira Pseudonana to Changes in CO2 Concentration: A Proteomic Approach,” Scientific Reports 7 (February 9, 2017): 42333, https://doi.org/10.1038/srep42333.

[4] Yoshihisa Hirakawa et al., “Characterization of a Novel Type of Carbonic Anhydrase That Acts without Metal Cofactors,” BMC Biology 19, no. 1 (May 18, 2021): 105, https://doi.org/10.1186/s12915-021-01039- 8

[5] Maria Giulia Lionetto et al., “The Complex Relationship between Metals and Carbonic Anhydrase: New Insights and Perspectives,” International Journal of Molecular Sciences 17, no. 1 (January 2016): 127, https://doi.org/10.3390/ijms17010127.

Background: Excessive intake of saturated fats disrupts hypothalamic neuronal function, crucial for maintaining body energy balance. Mitochondrial activity within hypothalamic neurons is pivotal in regulating food intake and energy expenditure, dynamically adapting to cellular energy demands and nutrient availability. While neuronal activity is commonly studied in energy homeostasis, glial cells, particularly microglia, also sense brain environmental changes, providing structural and metabolic support and acting as the brain's immune system. In parallel, Nicotinamide Adenosine Dinucleotide (NAD+) plays a crucial role in mitochondrial function, yet its involvement in microglial-neuronal mitochondrial dynamics remains unclear. Aim: In this study, we examined the morpho functional aspects of mitochondria in the arcuate nucleus of the hypothalamus of obese mice treated with Nicotinamide Riboside (NR), a NAD+ booster. Methods: The cellular model was used to explore specifically microglial and neuron responses to palmitate treatment. We also examined the arcuate nucleus of the hypothalamus in HFD-fed and NR-treated mice, assessing physiological and molecular parameters through different techniques including RNAseq analysis, Western blot, mitochondrial respirometry and electron microscopy. Results: Our findings reveal that palmitate affected NAD synthesis and stimulates mitochondrial fragmentation in both, microglia (BV2 cells) and neurons (mHypho A2-29). Similarly, high fat diet consumption suppressed NAD synthesis pathway proteins, induced mitochondrial fragmentation and reduced mitochondrial respiration in the arcuate nucleus of mice. Conversely, the gene ontology biological processes analysis from RNAseq of arcuate nucleus revealed that NR treatment stimulated several mitochondrial-related pathways including NAD metabolism and oxidative phosphorylation. Finally, oral NR treatment effectively restored NAD markers, stimulated mitochondrial fusion and increased mitochondrial respiration capacity in the arcuate nucleus of mice. Conclusion: These preliminary data suggest that while saturated fatty acid affect NAD synthesis and mitochondrial metabolism in microglial cells neurons, NAD+ precursor may recover mitochondrial morpho functional aspects of these cells in the arcuate nucleus of obese mice.

Bacterial cell geometry is highly regulated and constrains the surface area available for acquiring nutrients as well as the volume available for synthesizing proteins. The surface area to volume (SA:V) ratio of all documented bacteria decreases with growth rate due to increases in cell size. However, the membrane protein content of Escherichia coli increases with growth rate creating a positive correlation between membrane enzyme capacity and growth rate. Despite its central role in cell biology, the intersection of membrane protein capacity, cell geometry, and central metabolism has not been defined with a predictive and quantitative theory. Here, we present a biophysical basis for maximum growth rate, overflow metabolism, electron transport chain efficiency, and maintenance energy flux. The theory successfully predicts the phenotypes of two E. coli K-12 strains, MG1655 and NCM3722, which are genetically similar but have different SA:V ratios, different maximum growth rates, and different overflow phenotypes. These analyses do not consider the cytosolic proteome, demonstrating the predictive power of the surface area and membrane protein crowding alone. Analyses suggest E. coli does not maximize growth rate nor biomass yield. Instead, E. coli phenotypes operate at intermediate growth rates and yields while maximizing the areal density of ATP synthase complexes, maximizing the rate of substrate energy dissipation. Cell geometry and membrane protein crowding plays a central role in phenotype from prokaryotes to eukaryotic organelles.

Mitochondria, essential for cellular viability, are the primary source of free radicals, crucial molecules with high reactivity and dynamic nature, making their detection challenging. This work presents a robust and straightforward quantum sensing method for selective free radical detection throughout biological processes. We employ negative charge state of Nitrogen-Vacancy (NV) colour centres in diamond exploiting their spin-dependent photoluminescence. We employ a low optical power method to harness the NV ground state spin triplet's sensitivity to paramagnetic species, like free radicals, that preserves the NV charge state. Using simple modifications to an inverted fluorescent microscope we perform Optically Detected Magnetic Resonance (ODMR) and Microwave Modulation (MM) of the NV photoluminescence and evaluate changes ODMR and MM contrast, as surrogate measures of the NV optical polarizability and hence paramagnetic species. We introduce a methodology using the spin probe 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) for selective free radical identification. TEMPOL, a cell-permeable probe, scavenges free radicals, as a Superoxide dismutase 2 (SOD2) mimic, quenching the contrast in both ODMR and MM. Restoration of contrast is observed upon free radical species that preferentially react with TEMPOL (hydroxyl radical, superoxide radicals). Validation experiments with irradiation of H₂O₂ corroborated the findings.
The aforementioned methodology was employed in biological studies encompassing neuroblastoma cells, and wildtype versus PINK1 (PINK189/Y) whole Drosophila Melanogaster. PINK1 mutations exhibit a set of relevant phenotypes of Parkinson’s Disease such as impaired locomotor activity, dopaminergic neuron degradation and mitochondrial abnormalities. High-resolution respirometry alongside specific substrate-uncoupler-inhibitor titrations with TEMPOL was performed to monitor mitochondrial function. This approach effectively detected free radicals across the electron transport system (ETS). Furthermore, the method distinguished enhanced free radical expression in PINK1 flies across the ETS compared to wildtype flies. This simple protocol offers significant advantages for cell biologists and medical discovery, lowering the barrier to entry for quantum sensing applications. This approach allows for studying the entire oxidative phosphorylation process and spin-active intermediates within biological systems. The proposed technology has the potential to help elucidate the biophysical parameters underlying mitochondrial function and dysfunction.

According to the fluidity model, complexes of the mammalian mitochondrial electron transport chain (ETC) partition between free complexes and quaternary assemblies known as supercomplexes (SCs). However, the physiological requirement for SCs in oxidative metabolism is a matter of ongoing intense debate, and the mechanisms regulating their formation remain enigmatic. Here, we show that genetic perturbations affecting the biogenesis or maturation of mammalian ETC Complex III (CIII) stimulates the formation of a specialized extra-large SC (SC-XL) with a structure of I₂+III_2, resolved to a nominal resolution of 3.7 Å by cryogenic electron microscopy. SC-XL formation increases mitochondrial cristae density and sustains normal ETC output despite a 70% reduction in electron flow through CIII, effectively rescuing mild to moderate CIII deficiency. Increasing the SC-XL:free III₂ ratio significantly reduced CIII ROS production and propensity for CI ROS triggered by reverse electron transport, whereas inhibiting SC-XL formation via the Uqcrc1^{DEL:E258-D260} mutation increased CIII ROS production and led to respiratory decompensation in CIII mutants. Furthermore, higher SC-XL:free III₂ ratio reprogrammed mitochondria towards fatty acid oxidation and protected against ischemic heart failure in mice. Our study reveals an unanticipated plasticity in the mammalian ETC to buttress against intrinsic perturbations via structural adaptations, and suggests that ETC reprogramming via controlled regulation of SC-XL formation is a potential therapeutic strategy for remediating diseases characterized by a decline in ETC bioenergetics and oxidative damage.

The mitochondrial oxidative phosphorylation (OXPHOS) system consists of four enzymatic multiprotein complexes that collaborate to facilitate the transfer of electrons from reducing equivalents to molecular oxygen. Given the central role of OXPHOS in cellular energy metabolism, deficiencies in the enzymes catalyzing these processes contribute to human diseases. Understanding the assembly, regulation, and functioning of these complexes is crucial for unraveling the mechanisms underlying cellular energy metabolism and its implications for various health conditions.

In this study, the human respirasome CICIII2CIV was isolated from human cells, and high-resolution structures of the individual complexes were determined by cryo-EM. Intriguingly, we identified the presence of the HIGD2A protein, which binds to complex IV within the respirasome. Detailed structural analyses uncovered the mutually exclusive appearance of HIGD2A and NDUFA4 in complex IV. Our data propose a role for HIGD2A in the final stages of complex IV assembly, including the regulation of NDUFA4 incorporation, providing insights into the temporal dynamics of the assembly process.

Background:

In humans, three enzymes; cystathionine-β-synthase(CBS), cystathionine gamma-lyase(CSE), and 3-mercaptopyruvate sulfurtransferase(3-MST) mainly synthesize the gasotransmitter hydrogen sulphide(H₂S). The expression of these enzymes was found to be significantly upregulated in breast cancer(BC). A key target of H₂S is the mitochondria, where H₂S has been demonstrated to affect mitochondrial respiration, morphology, function and biogenesis.H₂S was shown to upregulate or activate key players in the mitochondrial biogenesis pathway such as increasing PGC1α expression and activating NRF2. These effects were reported in hepatocytes and cardiac tissue. However, the role of H₂S on mitochondrial biogenesis in BC was not thoroughly examined. Thus, the aim of this study was to elucidate the role of mitochondrial biogenesis in BC patients and the impact of knocking down of H₂S synthesizing enzymes on mitochondria in BC cell lines.

Methodology:

Breast tissues were collected from 26 female BC patients. TNBC MDA-MB-231 cells were transfected with CBS, and CSE siRNAs. RNA extraction was followed by reverse transcription into cDNA using reverse transcriptase.PGC1-α, Nrf1, NRF-2, TFAM and ND-1 expression was quantified using q-RT-PCR. Mitochondrial membrane potential was measured using rhodamine123 and ATP using luminescence kit.

Results:

CBS, CSE, 3-MST and ND-1 were upregulated in BC tissues compared to the surrounding non-cancerous tissue. Knocking down of CBS in MDA-MB-231 cells resulted in an increased expression of PGC1α, NRF1/2, TFAM and ND-1. Similar results were observed with CSE knocking down except TFAM levels were not altered. Silencing of CBS and CSE reduced the mitochondrial membrane potential and decreased ATP levels in TNBC cells.

Conclusion:

This study highlights the supportive role of H₂S in mitochondrial biogenesis and function in BC.

Background: Acute alveolar hypoxia triggers hypoxic pulmonary vasoconstriction (HPV) which is essential to optimize arterial oxygenation. In contrast, chronic hypoxia induces pulmonary vascular remodelling and consequently pulmonary hypertension (PH). HPV, but not PH, is triggered by increased superoxide release from mitochondrial complex III. As ubiquinol-cytochrome c reductase hinge protein (Uqcrh) deficiency reduces complex III activity, we hypothesized that Uqcrh knockout (Uqcrh^-/-) may impair acute oxygen sensing in the pulmonary vasculature.

Methods: HPV was quantified in isolated perfused and ventilated lungs from Uqcrh^-/- and wild type (WT) mice. Acute hypoxia-induced cellular membrane depolarization was determined in pulmonary arterial smooth muscle cells (PASMC) from Uqcrh^-/- and WT mice using patch clamp. To assess the role of Uqcrh in chronic hypoxic signalling, protein levels of the hypoxia-inducible factor 1α (HIF-1α) and cellular proliferation were determined in Uqcrh^-/- and WT PASMC after exposure to normoxia or 1% oxygen (for 24h or 72h).

Results: HPV was inhibited in lungs from Uqcrh^-/- mice, while pulmonary vasoconstriction induced by the thromboxane analogue U46619 was preserved. Acute hypoxia-induced cellular membrane depolarization was decreased in Uqcrh^-/- PASMC compared to WT PASMC. In contrast, chronic hypoxic exposure increased HIF-1α protein expression and cellular proliferation to similar levels in Uqcrh^-/- and WT PASMC. Furthermore, Uqcrh expression was not altered in WT PASMC after chronic hypoxic exposure.

Conclusion: These results support previous findings that acute and chronic hypoxic signalling is triggered by different mechanisms in PASMC. Electron flow through complex III is involved in acute, but not chronic oxygen sensing of the pulmonary vasculature.

The powerhouse of the cells, mitochondria, are highly dynamic double-membrane organelles. The protein-rich nature of mitochondrial inner membrane and its important functions in respiration and mitochondrial biogenesis require a high degree of organization and flexibility. The inner membrane is home to several well-established protein complexes that are subject to compartmentalization, including the respiratory chain (super)complexes, the prohibitin complex, and TIM translocases. These protein complexes play fundamental roles in mitochondrial biogenesis, homeostasis, regulation, and physiology. Here we present a recently identified inner membrane mega-assembly, mitochondrial multifunctional assembly (MIMAS) [1]. Striking features of the MIMAS complex include its large size of around 3 MDa, as well as its dependence on the phospholipid phosphatidylethanolamine. By combining multiple methods, like biochemical approaches, analysis of the high-resolution mitochondrial complexome [2], and mass spectrometry, we were able to identify MIMAS components involved in diverse functions such as respiratory chain assembly, lipid biosynthesis and metabolic processes. Our results suggest that this protein-lipid assembly functions as a biogenesis platform that may organize the inner membrane by integrating different aspects of mitochondrial physiology.

[1] P. Horten, K. Song, J. Garlich, R. Hardt, L. Colina-Tenorio, S.E. Horvath, U. Schulte, B. Fakler, M. van der Laan, T. Becker, R.A. Stuart, N. Pfanner, H. Rampelt, Identification of MIMAS, a multifunctional mega-assembly integrating metabolic and respiratory biogenesis factors of mitochondria, Cell Reports 43 (2024) 113772.

[2] U. Schulte, F. den Brave, A. Haupt, A. Gupta, J. Song, C.S. Müller, J. Engelke, S. Mishra, C. Mårtensson, L. Ellenrieder, C. Priesnitz, S.P. Straub, K.N. Doan, B. Kulawiak, W. Bildl, H. Rampelt, N. Wiedemann, N. Pfanner, B. Fakler, T. Becker, Mitochondrial complexome reveals quality-control pathways of protein import, Nature 614 (2023) 153–159.

EGFR pathway involved in cancer cell migration, proliferation and survival drives a lots attentions of cancer biologist in searching the therapeutic targets. Tyrosine kinase inhibitor (TKI)-resistant

small lung carcinoma cells and recurrent cancer stem cell sub population with EGFR mutations have been quite frustrated approaches by anti-EGFR based therapy. In the synthetic EGFR mutant axils

enlightening Mitochondria-desRed small lung carcinoma CL1-0 cell line revealing an interesting findings well correlating the active EGFR or spontaneous active EGFR ^T790M/L858R mutant with high energy

demanding status. In our EGFR axes-mitochondria synthetic cell based model, we first observe a phenomenon that EGF treatment enhances the amount of mitochondria per cell which is correlated to

up-regulation of MMP-7 expression. The MMP-7 mediated proteolytic processing substrates could potentially activate mitochondria proliferation. EGFR can be cleaved by MMP-7 and release

proteolytic EGFR c-terminal fragments further translocated to mitochondria and initiate mitochondria proliferation. Over expression of C-terminal fragments of EGFR ^T790M/L858R mutant in

CL1-0 can translocate into mitochondria, because through MMP-7 cleaving EGFR reveal the cryptic mitochondria targeting sequences which can drive C-terminal EGFR ^T790M/L858R trafficking

into mitochondria and lead the bonifying proliferation of mitochondria. Over expression of C-terminal fragments of EGFR ^T790M/L858R mutant in CL1-0 can increase the capacity of soft-agar growth

and also increasing the sphere growth in the floating colonies. All of these evidences indicate that both MMP-7 mediated proteolytic processing EGFR ^T790M/L858R releasing C-terminal fragments

or over expression of C-terminal EGFR ^T790M/L858R translocating into mitochondria confirm the anchorage-independent growth in soft agar and sphere suspension growth capacity

and implicates the cancer stem cell transformation. The platelet purified mitochondria transplantation into the C-terminal EGFR ^T790M/L858R mutant CL1-0 can induce cancer/cancer stem cell apoptosis.

Mitochondria targeting compound 007 can also sensitize EGFR ^T790M/L858R mutant CL1-0 to gefitinib chemotherapy.

Ubiquinone is an essential metabolite that greatly aids mitochondrial respiratory function. It is responsible for carrying electrons from respiratory complexes I and II to respiratory complex III, which leads to ATP production by oxidative phosphorylation. Ubiquinone is also known to act as an anti-oxidant by preventing damages from various reactive oxygen species. With its hydrophilic benzoquinone head and hydrophobic polyprenyl tail, ubiquinone mainly exists within the inner mitochondrial membrane.

The protein subunits of respiratory complexes are encoded by both mitochondrial DNA (mtDNA) and nuclear DNA. Thus the proper function and maintenance of mtDNA is indispensable for governing both mitochondrial and cellular activity. In live imaging of cultured mammalian cells, these mtDNA molecules are observed as dynamic complexes known as mitochondrial nucleoids. We previously showed that the dynamic nature of nucleoids, regulated in cooperation with mitochondrial membrane fusion and fission, is essential for maintaining mitochondrial respiratory complex formation. However, molecular details and the pathophysiological roles of the mitochondrial nucleoid dynamics remained ambiguous.

In this work, we revealed that ubiquinone has a unique function in regulating the functional expression of mtDNA in mitochondrial inner membrane. Loss of ubiquinone synthesis not only causes mitochondrial respiratory failure, as expected, but also results in the incomplete formation of respiratory complexes and deformation of mitochondrial nucleoid structures, which is unrelated to its traditional electron carrying and anti-oxidising roles. As a result, we found a non-canonical role of ubiquinone and propose the exploitation of this phenomenon towards therapeutic advantage in clinical conditions arising from mitochondrial defects due to insufficient mtDNA activity.

In the ancient microbial Wood-Ljungdahl pathway, CO₂ is fixed in a multi-step process ending with acetyl-CoA synthesis at the bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase complex (CODH/ACS). Here, we present catalytic snapshots of the CODH/ACS from the gas-converting acetogen Clostridium autoethanogenum, characterizing the molecular choreography of the overall reaction including electron transfer to the CODH for CO₂ reduction, methyl transfer from the corrinoid iron-sulfur protein (CoFeSP) partner to the ACS active site and the acetyl-CoA production. Unlike CODH, the multidomain ACS undergoes large conformational changes to form an internal connection to the CODH active site, accommodate the CoFeSP for methyl transfer and protect the reaction intermediates. Altogether, the structures allow us to draw a detailed reaction mechanism of this enzyme crucial for CO₂ fixation in anaerobic organisms.

Long-COVID is a clinical condition characterized by long-term consequences of severe respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which poses a persistent public health concern worldwide. It impacts multiple body functions, including immunological, respiratory, cardiovascular, gastrointestinal, neuropsychological, musculoskeletal, and other important systems. Affected individuals frequently describe symptoms such as “mental fog” and persistent fatigue, among other various symptoms. Despite intensified research, the biological mechanisms causing this multitude of symptoms are not fully understood, sparking a quest for innovative research directions, particularly in the combined area of bioenergetics and psychoneuroimmunology. This interdisciplinary field focusses on how (immune) cells generate energy, exploring possible bioenergetic disruptions in individuals with Long-COVID, especially focusing on the role of mitochondrial functioning in peripheral blood mononuclear cells (PBMC) isolated from whole blood.

This research adopts a comprehensive approach, starting with clinical evaluations to measure both mental and physical impairments in those impacted. Using a thorough methodology, it merges clinical assessments of psychological issues and the influence of body weight, with the gathering of blood samples for isolating PBMC. These cells are being analyzed applying O2K high-resolution respirometry to evaluate mitochondrial function, providing insights into cellular energy dynamics and potential malfunctions in Long-COVID.

Our findings indicates significant disruptions in the mitochondrial activity in intact PBMC collected from patients with Long-COVID compared to non-affected controls, indicating a major loss of cellular energy processes, at least in immune cells. Being under current investigation, these issues might also correlate with the clinical symptom severity of Long-COVID, such as severe fatigue and cognitive impairments. The final findings will be shared during the presentation.

We will highlight the essential importance of mitochondrial health in understanding and managing Long-COVID more broadly. Evidence for mitochondrial perturbations in PBMC points towards bioenergetic health as a key area for both tracking and addressing the wide range of Long-COVID symptoms. Expanding our grasp of the disease's biological basis not only deepens our understanding of Long-COVID, but also unveils new paths for developing specific interventions aimed at reducing the long-lasting impact of the ailment and asks for a holistic approach.

Since the discovery of mitochondria in 1857, research focus and funding have varied, particularly due to challenges in reaching these organelles. Notably, funding the protein unfolding in mitochondria, holds potential promise for cancer cures.

CoR (Cofactor Raphanin) has successfully penetrated mitochondria, enhancing ATP production and activating FAD in the Electron Transport Chain.

Applied Methods: Utilize permeating molecular (CoR) vectors to target mitochondrial enzymes, with a specific focus on its impact on ATP and FAD production at the intracellular level.

Cell Lines Utilized: SK-MEL-31: 15,000 cells/wellA204: 15,000 cells/wellPANC-1: 12,000 cells/well. Assay Protocol: Perform a cell viability assay using the CTG Assay (Promega Cat#G7571). Assess the effect of CoR to permeate mitochondria, focus on its influence on ATP production. Finding: CoR treatment results in significant increased ATP levels within the mitochondrial electron transport chain compared to the control, as indicated by cellular bioenergetics analysis.

Cell Line Used: A-204 – Muscle. Assay Protocol: Utilizing the ATP Assay and FAD Assay. Evaluate the effect of CoR to target mitochondria, focus on its impact. Finding: the levels of ATP and FAD in response to CoR treatment are significant enhanced in the mitochondrial electron transport chain compared to the control. as observed based on cellular bioenergetics analysis.

Cortechnology can penetrate cell membranes and lipid layers to activate FAD, which plays a crucial role in facilitating 90% of redox reactions and encodes 89% of the human genome, the key features include:

An innovative approach to aging-related diseases: Mitigating the harmful byproducts of mitochondrial dysfunction, including free radicals, apoptosis, protein misfolding, and calcium overload.

Advanced tools for cell and gene therapies: This enables the direct delivery of any agent into targeted cells, including cancer cells or genes, via the mitochondrial pathway.

Reverse aging: Regenerate ATP through cellular rejuvenation and repair across all cell types, rather than focusing solely on a single cell.

Innovative drug delivery strategy: Approach that bypasses the need for traditional nanomaterials by utilizing the endocytosis process for cellular entry.