bims-mitrat 2024-11-17 papers

bims-mitrat

Biomed News

on Mitochondrial Transplantation and Transfer

Issue of 2024‒11‒17
sixteen papers selected by
Gökhan Burçin Kubat, Gulhane Health Sciences Institute

Mitochondria: great balls of fire.
From dysfunction to healing: advances in mitochondrial therapy for Osteoarthritis.
Mitophagy-Enhanced Nanoparticle-Engineered Mitochondria Restore Homeostasis of Mitochondrial Pool for Alleviating Pulmonary Fibrosis.
Mitochondrial transplantation following cardiopulmonary resuscitation improves neurological function in rats by inducing M2-type MG/MΦ polarization.
Molecular and cellular mechanisms of mitochondria transfer in models of central nervous system disease.
Xenotransplantation of Mitochondria: A Novel Strategy to Alleviate Ischemia-Reperfusion Injury during Ex Vivo Lung Perfusion.
Tunneling nanotubes enable intercellular transfer in zebrafish embryos.
Metabolic Phenotyping of Synaptic Mitochondria Using MitoPlates™ and Synaptoneurosomes.
Mitochondrial disorders are associated with morphological neuromuscular junction defects.
POLG p.A962T Mutation Leads to Neuronal Mitochondrial Dysfunction That is Restored After Mitochondrial Transplantation.
The whole-body and skeletal muscle metabolic response to 14 days of highly controlled low energy availability in endurance-trained females.
A PRKN-independent Mechanism Regulating Cardiac Mitochondrial Quality Control.
The mitochondrial mRNA-stabilizing protein SLIRP regulates skeletal muscle mitochondrial structure and respiration by exercise-recoverable mechanisms.
Up-regulated succinylation modifications induce a senescence phenotype in microglia by altering mitochondrial energy metabolism.
The Relation Between Mitochondrial Membrane Potential and Reactive Oxygen Species Formation.
PINK1-deficiency facilitates mitochondrial iron accumulation and colon tumorigenesis.

FEBS J. 2024 Nov 14.

Mitochondria: great balls of fire.

Howard T Jacobs, Pierre Rustin, Paule Bénit, Dan Davidi, Mügen Terzioglu.

  Recent experimental studies indicate that mitochondria in mammalian cells are maintained at temperatures of at least 50 °C. While acknowledging the limitations of current experimental methods and their interpretation, we here consider the ramifications of this finding for cellular functions and for evolution. We consider whether mitochondria as heat-producing organelles had a role in the origin of eukaryotes and in the emergence of homeotherms. The homeostatic responses of mitochondrial temperature to externally applied heat imply the existence of a molecular heat-sensing system in mitochondria. While current findings indicate high temperatures for the innermost compartments of mitochondria, those of the mitochondrial surface and of the immediately surrounding cytosol remain to be determined. We ask whether some aspects of mitochondrial dynamics and motility could reflect changes in the supply and demand for mitochondrial heat, and whether mitochondrial heat production could be a factor in diseases and immunity.

Keywords:  cold‐shock; eukaryote origins; heat‐shock; homeothermy; immunity; mitochondria; mitochondrial disease; mitochondrial dynamics; temperature gradients; thermogenesis

DOI:  https://doi.org/10.1111/febs.17316
J Transl Med. 2024 Nov 11. 22(1): 1013

From dysfunction to healing: advances in mitochondrial therapy for Osteoarthritis.

Minghang Zhang, Junfeng Wu, Kehan Cai, Yang Liu, Botao Lu, Jiaojiao Zhang, Jianzhong Xu, Chenxi Gu, Tao Chen.

  Osteoarthritis (OA) is a chronic degenerative joint condition characterised by cartilage deterioration and changes in bone morphology, resulting in pain and impaired joint mobility. Investigation into the pathophysiological mechanisms underlying OA has highlighted the significance of mitochondrial dysfunction in its progression. Mitochondria, which are cellular organelles, play a crucial role in regulating energy metabolism, generating reactive oxygen species, and facilitating essential biological processes including apoptosis. In recent years, the utilisation of exogenous drugs and MT to improve mitochondrial function in chondrocytes has shown great promise in OA treatment. Numerous studies have investigated the potential of stem cells and extracellular vesicles in mitochondrial transfer. This review aims to explore the underlying mechanisms of mitochondrial dysfunction in OA and assess the progress in utilising mitochondrial transfer as a therapeutic approach for this disease.

Keywords:  Extracellular vesicles; Mitochondrial dysfunction; Mitochondrial transfer; Osteoarthritis; Stem cell

DOI:  https://doi.org/10.1186/s12967-024-05799-z
ACS Nano. 2024 Nov 15.

Mitophagy-Enhanced Nanoparticle-Engineered Mitochondria Restore Homeostasis of Mitochondrial Pool for Alleviating Pulmonary Fibrosis.

Yi Wang, Li-Fan Hu, Na-Hui Liu, Jing-Song Yang, Lei Xing, Jee-Heon Jeong, Ling Li, Hu-Lin Jiang.

  Pulmonary fibrosis (PF) is an interstitial lung disease tightly associated with the disruption of mitochondrial pool homeostasis, a delicate balance influenced by functional and dysfunctional mitochondria within lung cells. Mitochondrial transfer is an emerging technology to increase functional mitochondria via exogenous mitochondrial delivery; however, the therapeutic effect on mitochondrial transfer is hampered during the PF process by the persistence of dysfunctional mitochondria, which is attributed to impaired mitophagy. Herein, we reported engineering mitochondria mediated by mitophagy-enhanced nanoparticle (Mito-MEN), which promoted synchronal regulation of functional and dysfunctional mitochondria for treating PF. Mitophagy-enhanced nanoparticles (MENs) were fabricated through the encapsulation of Parkin mRNA, and the electrostatic interaction favored MENs to anchor isolated healthy mitochondria for the construction of Mito-MEN. Mito-MEN increased the load of functional exogenous mitochondria by enhancing mitochondrial delivery efficiency and promoted mitophagy of dysfunctional endogenous mitochondria. In a bleomycin (BLM)-induced PF mouse model, Mito-MEN repaired mitochondrial function and efficiently relieved PF-related phenotypes. This study provides a powerful tool for synchronal adjustment of mitochondrial pool homeostasis and offers a translational approach for pan-mitochondrial disease therapies.

Keywords:  alveolar epithelial cells; mitophagy; nanoengineered mitochondria; nanoparticle; pulmonary fibrosis

DOI:  https://doi.org/10.1021/acsnano.4c10328
J Transl Med. 2024 Nov 11. 22(1): 1014

Mitochondrial transplantation following cardiopulmonary resuscitation improves neurological function in rats by inducing M2-type MG/MΦ polarization.

Jie Zhu, Zhen Wang, Mengda Xu, Xuyuan Ma, Maozheng Shen, Jingyu Yan, Xiang Zhou.

  AIM: Explore the effects of mitochondrial transplantation (MT) after cardiopulmonary resuscitation (CPR) on the polarization of microglia/macrophages (MG/MΦ) and neurological function.METHODS: Seventy-five Sprague-Dawley rats were randomly divided into five groups: sham, normal saline (NS), vehicle, mitochondria (Mito), and non-functional mitochondria (N-Mito) group. Rats in sham group underwent surgical procedures without cardiac arrest, while the other four groups underwent cardiac arrest and CPR, and then received NS, respiration buffer, mitochondrial suspension or non-functional mitochondria, immediately after the restoration of spontaneous circulation (ROSC). The number of mitochondria in the hippocampus, the morphology and structure of mitochondria in MG/MΦ, the phenotype of MG/MΦ, and hippocampal tissue injury, neuroinflammation, and neuronal apoptosis were detected on days 1 and 3 after ROSC. Neurodeficit score (NDS) was performed on days 1, 3, 7, 15 and 30 after ROSC.
RESULTS: Compared with other groups, the number of mitochondria in the hippocampus was increased, and the morphology and structure of mitochondria in MG/MΦ were significantly improved in the Mito group. Our results show higher expression of M2-type markers in MG/MΦ and decreased hippocampal tissue damage in the Mito group. Levels of NSE and S100β in serum, and TNF-α, IL-6 in the hippocampus were decreased, while the levels of TGF-β and IL-10 were increased in the Mito group. Apoptosis rate of neurons in the Mito group was decreased and the NDS of the Mito group was higher than the other groups.
CONCLUSIONS: Exogenous MT can improve neurological function after CPR by promoting the polarization of MG/MΦ to M2-type cells, and this could be a potential method for brain protection after CPR.

Keywords:  Cardiac arrest; Macrophages; Microglia; Mitochondrial transplantation; Neurological function

DOI:  https://doi.org/10.1186/s12967-024-05815-2
J Cereb Blood Flow Metab. 2024 Nov 14. 271678X241300223

Molecular and cellular mechanisms of mitochondria transfer in models of central nervous system disease.

Takafumi Nakano, Keiichi Irie, Koichi Matsuo, Kenichi Mishima, Yoshihiko Nakamura.

  In the central nervous system (CNS), neuronal function and dysfunction are critically dependent on mitochondrial integrity and activity. In damaged or diseased brains, mitochondrial dysfunction reduces adenosine triphosphate (ATP) levels and impairs ATP-dependent neural firing and neurotransmitter dynamics. Restoring mitochondrial capacity to generate ATP may be fundamental in restoring neuronal function. Recent studies in animals and humans have demonstrated that endogenous mitochondria may be released into the extracellular environment and transported or exchanged between cells in the CNS. Under pathological conditions in the CNS, intercellular mitochondria transfer contributes to new classes of signaling and multifunctional cellular activities, thereby triggering deleterious effects or promoting beneficial responses. Therefore, to take full advantage of the beneficial effects of mitochondria, it may be useful to transplant healthy and viable mitochondria into damaged tissues. In this review, we describe recent findings on the mechanisms of mitochondria transfer and provide an overview of experimental methodologies, including tissue sourcing, mitochondrial isolation, storage, and modification, aimed at optimizing mitochondria transplantation therapy for CNS disorders. Additionally, we examine the clinical relevance and potential strategies for the therapeutic application of mitochondria transplantation.

Keywords:  Central nervous system; experimental disease models; mitochondria transplantation; therapeutic strategies; transfer mechanism

DOI:  https://doi.org/10.1177/0271678X241300223
J Heart Lung Transplant. 2024 Nov 11. pii: S1053-2498(24)01938-7. [Epub ahead of print]

Xenotransplantation of Mitochondria: A Novel Strategy to Alleviate Ischemia-Reperfusion Injury during Ex Vivo Lung Perfusion.

Nicholas B Bechet, Aybuke Celik, Margareta Mittendorfer, Qi Wang, Tibor Huzevka, Gunilla Kjellberg, Embla Boden, Gabriel Hirdman, Leif Pierre, Anna Niroomand, Franziska Olm, James D McCully, Sandra Lindstedt.

  Ischemia-reperfusion injury (IRI) plays a crucial role in the development of primary graft dysfunction (PGD) following lung transplantation. A promising novel approach to optimize donor organs before transplantation and reduce the incidence of PGD is mitochondrial transplantation. In this study, we explored the delivery of isolated mitochondria in 4 hour ex vivo lung perfusion (EVLP) before transplantation as a means to mitigate IRI. To provide a fresh and viable source of mitochondria, as well as to streamline the workflow without the need for donor muscle biopsies, we investigated the impact of autologous, allogeneic and xenogeneic mitochondrial transplantation. In the xenogeneic settings, isolated mitochondria from mouse liver were utilized while autologous and allogeneic sources came from pig skeletal muscle biopsies. Treatment with mitochondrial transplantation increased the P/F ratio and reduced pulmonary peak pressure of the lungs during EVLP, compared to lungs without any mitochondrial transplantation, indicating IRI mitigation. Extensive investigations using advanced light and scanning electron microscopy did not reveal evidence of acute rejection in any of the groups, indicating safe xenotransplantation of mitochondria. Future work is needed to further explore this novel therapy for combating IRI in lung transplantation, where xenotransplantation of mitochondria may serve as a fresh, viable source to reduce IRI.

Keywords:  Ex Vivo Lung Perfusion; Ischemia-Reperfusion Injury; Mitochondria; Xenotransplantation

DOI:  https://doi.org/10.1016/j.healun.2024.10.033
Dev Cell. 2024 Nov 08. pii: S1534-5807(24)00635-X. [Epub ahead of print]

Tunneling nanotubes enable intercellular transfer in zebrafish embryos.

Olga Korenkova, Shiyu Liu, Inès Prlesi, Anna Pepe, Shahad Albadri, Filippo Del Bene, Chiara Zurzolo.

  Tunneling nanotubes (TNTs) are thin intercellular connections that facilitate the transport of diverse cargoes, ranging from ions to organelles. While TNT studies have predominantly been conducted in cell cultures, the existence of open-ended TNTs within live organisms remains unverified. Despite the observation of intercellular connections during embryonic development across various species, their functional role in facilitating material transfer between connected cells has not been confirmed. In this study, we performed mosaic labeling of gastrula cells in zebrafish embryos to demonstrate the coexistence of TNT-like structures alongside other cellular protrusions. These embryonic TNT-like connections exhibited a morphology similar to that of TNTs described in cell culture, appeared to have similar formation mechanisms, and could be induced by Eps8 overexpression and CK666 treatment. Most notably, we demonstrated their capability to transfer both soluble cargoes and organelles, thus confirming their open-endedness. This study demonstrates the existence of functional, open-ended TNTs in a living embryo.

Keywords:  TNT-like structures; TNTs; cytokinetic bridges; cytonemes; intercellular communication; intercellular connections; organelle transfer; tunneling nanotubes; zebrafish embryo; zebrafish gastrula

DOI:  https://doi.org/10.1016/j.devcel.2024.10.016
Methods Mol Biol. 2025 ;2878 67-74

Metabolic Phenotyping of Synaptic Mitochondria Using MitoPlates™ and Synaptoneurosomes.

Aleksandra Stawikowska, Magdalena Dziembowska, Bozena Kuzniewska.

  Mitochondrial functional assays using MitoPlates™ S-1 allow us to characterize mitochondria in terms of substrate metabolism. MitoPlates™ are 96-well microplates pre-coated with a diverse set of substrates. The electron flow from NADH and FADH2 producing mitochondrial substrates is measured based on the reduction of redox dye, that acts as a terminal electron acceptor. Here, we describe the application of MitoPlates™ to characterize the metabolism of synaptic mitochondria enclosed in isolated pre- and postsynaptic terminals (synaptoneurosomes).

Keywords:  MitoPlates™; Mitochondrial substrate metabolism; Synaptic mitochondria; Synaptoneurosomes

DOI:  https://doi.org/10.1007/978-1-0716-4264-1_4
Neuromuscul Disord. 2024 Oct 25. pii: S0960-8966(24)01731-0. [Epub ahead of print]45 105235

Mitochondrial disorders are associated with morphological neuromuscular junction defects.

Lola E R Lessard, Emmanuelle Girard, Nathalie Streichenberger, Philippe Petiot, Cécile Acquaviva, Cécile Pagan, Peter Mulligan, Françoise Bouhour, Laurent Schaeffer, Arnaud Jacquier.

  We aimed to evaluate whether inherited mitochondrial dysfunction is associated with neuromuscular junction remodeling in patients with mitochondrial disorders. Muscle biopsies from 15 patients with mitochondrial disorders and 10 control patients were analyzed through immunostaining for various neuromuscular junction components. The patient group, with a mean age of 49.9 years, exhibited various mitochondrial disorders including chronic progressive external ophthalmoplegia, Kearns-Sayre syndrome, and mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes. Patients with mitochondrial disorders had a high percentage of remodeled (p= 0.0001), neoformed (p= 0.0049) and dilated (p= 0.016) endplates. There was a trend toward an increased proportion of neuromuscular junctions with terminal Schwann cell extension in these patients (p= 0.052). No significant difference was found in myofiber diameter between the groups. The observed neuromuscular junction defects varied widely across different mitochondrial disorder phenotypes and were present even without accompanying muscle weakness or neuropathy. This suggest that mitochondrial disorders are associated with a primary NMJ remodeling independent of muscle structural damage. Pathomechanisms underpinning this remodeling of the neuromuscular junction, as well as clinical factors predictive of this remodeling, remain to be fully characterized.

Keywords:  Confocal microscopy; Denervation; Fatigability; Ptosis; Reinnervation; Remodeling

DOI:  https://doi.org/10.1016/j.nmd.2024.105235
Physiol Res. 2024 Nov 15. 73(5): 801-808

POLG p.A962T Mutation Leads to Neuronal Mitochondrial Dysfunction That is Restored After Mitochondrial Transplantation.

W Hu, C Shi, H Guo, B Zhang.

Mutations in DNA polymerase gamma (POLG) are known as the predominant cause of inherited mitochondrial disorders. But how these POLG mutations disturb mitochondrial function remains to be determined. Furthermore, no effective therapy, to date, has been reported for POLG diseases. Using differentiated SH-SY5Y cells, a human neuronal model cell line, the current study investigated whether the novel POLG variant p.A962T impairs mitochondrial function. This involved quantifying mitochondrial DNA (mtDNA) content using PCR and assessing the expression levels of the subunits of complex IV (COXI-IV), a complex I subunit NDUFV1 and Cytochrome C (Cyto C) release using Western blotting. Activities of mitochondrial complex I, II, and IV were measured using colorimetric assays. Mitochondrial membrane potential (delta Psim) and ATP were evaluated using fluorescence assays and luminescent assays, respectively. In addition, we investigated whether mitochondrial transplantation (MT) using Pep-1-conjugated mitochondria could compensate for mitochondrial defects caused by the variant in cells carrying mutant POLG. The results of this study showed that POLG p.A962T mutation resulted in mitochondrial defects, including mitochondrial DNA (mtDNA) depletion, membrane potential (delta Psim) depolarization and adenosine triphosphate (ATP) reduction. Mechanistically, POLG mutation-caused mtDNA depletion led to the loss of mtDNA-encoded subunits of complex I and IV and thus compromised their activities. POLG p.A962T mutation is a pathogenic mutation leading to mitochondrial malfunction and mtDNA depletion in neurons. Cell-penetrating peptide Pep-1-mediated MT treatment compensated for mitochondrial defects induced by these POLG variants, suggesting the therapeutic application of this method in POLG diseases.
FASEB J. 2024 Nov 15. 38(21): e70157

The whole-body and skeletal muscle metabolic response to 14 days of highly controlled low energy availability in endurance-trained females.

Hannah G Caldwell, Jan S Jeppesen, Lone O Lossius, Jesper P Atti, Cody G Durrer, Mikkel Oxfeldt, Anna K Melin, Mette Hansen, Jens Bangsbo, Lasse Gliemann, Ylva Hellsten.

  This study investigated the effects of 14 days low energy availability (LEA) versus optimal energy availability (OEA) in endurance-trained females on substrate utilization, insulin sensitivity, and skeletal muscle mitochondrial oxidative capacity; and the impact of metabolic changes on exercise performance. Twelve endurance-trained females (V̇O2max 55.2 ± 5.1 mL × min-1 × kg-1) completed two 14-day randomized, blinded, cross-over, controlled dietary interventions: (1) OEA (51.9 ± 2.0 kcal × kg fat-free mass (FFM)-1 × day-1) and (2) LEA (22.3 ± 1.5 kcal × kg FFM-1 × day-1), followed by 3 days OEA. Participants maintained their exercise training volume during both interventions (approx. 8 h × week-1 at 79% heart rate max). Skeletal muscle mitochondrial respiratory capacity, glycogen, and maximal activity of CS, HAD, and PFK were unaltered with LEA. 20-min time trial endurance performance was impaired by 7.8% (Δ -16.8 W, 95% CI: -23.3 to -10.4, p < .001) which persisted following 3 days refueling post-LEA (p < .001). Fat utilization was increased post-LEA as evidenced by: (1) 99.4% (p < .001) increase in resting plasma free fatty acids (FFA); (2) 270% (p = .007) larger reduction in FFA in response to acute exercise; and (3) 28.2% (p = .015) increase in resting fat oxidation which persisted during submaximal exercise (p < .001). These responses were reversed with 3 days refueling. Daily glucose control (via CGM), HOMA-IR, HOMA-β, were unaffected by LEA. Skeletal muscle O2 utilization and carbohydrate availability were not limiting factors for aerobic exercise capacity and performance; therefore, whether LEA per se affects aspects of training quality/recovery requires investigation.

Keywords:  caloric restriction; fat oxidation; insulin sensitivity; metabolism; mitochondrial oxidative capacity

DOI:  https://doi.org/10.1096/fj.202401780R
Autophagy. 2024 Nov 09.

A PRKN-independent Mechanism Regulating Cardiac Mitochondrial Quality Control.

Wenjuan Wang, Jinbao Liu, Jie Li, Huabo Su.

  PRKN-dependent mitophagy plays a crucial role in maintaining mitochondrial health. Yet, PRKN-deficient mice do not exhibit mitochondrial and cardiac phenotypes at baseline, suggesting the existence of other mitochondrial ubiquitin (Ub) ligases. Here, we discuss our recent work identifying RNF7/RBX2 as a novel mitochondrial Ub ligase. Upon mitochondrial depolarization, RNF7 proteins are recruited to the mitochondria, where they directly ubiquitinate mitochondrial proteins and stabilize PINK1 expression, thereby promoting the clearance of damaged mitochondria and regulating mitochondrial turnover in the heart. The actions of RNF7 in mitochondria do not require PRKN. Ablation of Rnf7 in mouse hearts results in severe mitochondrial dysfunction and heart failure. Our findings demonstrate that RNF7 is indispensable for mitochondrial turnover and cardiac homeostasis. These results open new avenues for exploring new PRKN-independent pathways that regulate mitophagy, which could have significant implications for developing therapeutic interventions for cardiac diseases.

Keywords:  Heart failure; RBX2/SAG; mitophagy; parkin; ubiquitination

DOI:  https://doi.org/10.1080/15548627.2024.2423329
Nat Commun. 2024 Nov 13. 15(1): 9826

The mitochondrial mRNA-stabilizing protein SLIRP regulates skeletal muscle mitochondrial structure and respiration by exercise-recoverable mechanisms.

Tang Cam Phung Pham, Steffen Henning Raun, Essi Havula, Carlos Henriquez-Olguín, Diana Rubalcava-Gracia, Emma Frank, Andreas Mæchel Fritzen, Paulo R Jannig, Nicoline Resen Andersen, Rikke Kruse, Mona Sadek Ali, Andrea Irazoki, Jens Frey Halling, Stine Ringholm, Elise J Needham, Solvejg Hansen, Anders Krogh Lemminger, Peter Schjerling, Maria Houborg Petersen, Martin Eisemann de Almeida, Thomas Elbenhardt Jensen, Bente Kiens, Morten Hostrup, Steen Larsen, Niels Ørtenblad, Kurt Højlund, Michael Kjær, Jorge L Ruas, Aleksandra Trifunovic, Jørgen Frank Pind Wojtaszewski, Joachim Nielsen, Klaus Qvortrup, Henriette Pilegaard, Erik Arne Richter, Lykke Sylow.

Decline in mitochondrial function is linked to decreased muscle mass and strength in conditions like sarcopenia and type 2 diabetes. Despite therapeutic opportunities, there is limited and equivocal data regarding molecular cues controlling muscle mitochondrial plasticity. Here we uncovered that the mitochondrial mRNA-stabilizing protein SLIRP, in complex with LRPPRC, is a PGC-1α target that regulates mitochondrial structure, respiration, and mtDNA-encoded-mRNA pools in skeletal muscle. Exercise training effectively counteracts mitochondrial defects caused by genetically-induced LRPPRC/SLIRP loss, despite sustained low mtDNA-encoded-mRNA pools, by increasing mitoribosome translation capacity and mitochondrial quality control. In humans, exercise training robustly increases muscle SLIRP and LRPPRC protein across exercise modalities and sexes, yet less prominently in individuals with type 2 diabetes. SLIRP muscle loss reduces Drosophila lifespan. Our data points to a mechanism of post-transcriptional mitochondrial regulation in muscle via mitochondrial mRNA stabilization, offering insights into how exercise enhances mitoribosome capacity and mitochondrial quality control to alleviate defects.

DOI: https://doi.org/10.1038/s41467-024-54183-4
J Neuroinflammation. 2024 Nov 14. 21(1): 296

Up-regulated succinylation modifications induce a senescence phenotype in microglia by altering mitochondrial energy metabolism.

Xinnan Zhao, Xiaohan Yang, Cong Du, Huimin Hao, Shuang Liu, Gang Liu, Guangyin Zhang, Kai Fan, Jianmei Ma.

  The aging of the central nervous system(CNS) is a primary contributor to neurodegenerative diseases in older individuals and significantly impacts their quality of life. Neuroinflammation, characterized by activation of microglia(MG) and release of cytokines, is closely associated with the onset of these neurodegenerative diseases. The activated status of MG is modulated by specifically programmed metabolic changes under various conditions. Succinylation, a novel post-translational modification(PTM) mainly involved in regulating mitochondrial energy metabolism pathways, remains unknown in its role in MG activation and aging. In the present study, we found that succinylation levels were significantly increased both during aging and upon lipopolysaccharide-induced(LPS-induced) MG activation undergoing metabolic reprogramming. Up-regulated succinylation induced by sirtuin 5 knockdown(Sirt5 KD) in microglial cell line BV2 resulted in significant up-regulation of aging-related genes, accompanied by impaired mitochondrial adaptability and a shift towards glycolysis as a major metabolic pathway. Furthermore, after LPS treatment, Sirt5 KD BV2 cells exhibited increased generation of reactive oxygen species(ROS), accumulation of lipid droplets, and elevated levels of lipid peroxidation. By employing immunoprecipitation, introducing point mutation to critical succinylation sites, and conducting enzyme activity assays for succinate dehydrogenase(SDH) and trifunctional enzyme subunit alpha(ECHA), we demonstrated that succinylation plays a regulatory role in modulating the activities of these mitochondrial enzymes. Finally, down-regulation the succinylation levels achieved through administration of succinyl phosphonate(SP) led to amelioration of MG senescence in vitro and neuroinflammation in vivo. To our knowledge, our data provide preliminary evidence indicating that up-regulated succinylation modifications elicit a senescence phenotype in MG through alterations in energy metabolism. Moreover, these findings suggest that manipulation of succinylation levels may offer valuable insights into the treatment of aging-related neuroinflammation.

Keywords:  Aging; Metabolic reprogramming; Microglia; Neuroinflammation; Succinylation

DOI:  https://doi.org/10.1186/s12974-024-03284-4
Methods Mol Biol. 2025 ;2878 133-162

The Relation Between Mitochondrial Membrane Potential and Reactive Oxygen Species Formation.

Magdalena Lebiedzinska-Arciszewska, Jan Suski, Massimo Bonora, Barbara Pakula, Paolo Pinton, Jerzy Duszynski, Patrycja Jakubek-Olszewska, Mariusz R Wieckowski.

  Mitochondria are considered one of the main sites of reactive oxygen species (ROS) production in the eukaryotic cells. For this reason, mitochondrial dysfunction associated with increased ROS production underlies various pathological conditions as well as promotes aging. Chronically increased rates of ROS production contribute to oxidative damage to macromolecules, i.e., DNA, proteins, and lipids. Accumulation of unrepaired oxidative damage may result in progressive cell dysfunction, which can finally trigger cell death. The main by-product of mitochondrial oxidative phosphorylation is superoxide, which is generated by the leak of electrons from the mitochondrial respiratory chain complexes leading to one-electron reduction of oxygen. Mitochondrial superoxide dismutase (MnSOD, SOD2) as well as cytosolic superoxide dismutase (Cu/ZnSOD, SOD1), whose smaller pool is localized in the mitochondrial intermembrane space, converts superoxide to H2O2, which can be then degraded by the catalase to harmless H2O.In this chapter, we focus on the relationship between one of the bioenergetic parameters, which is mitochondrial membrane potential, and the rate of ROS formation. We present a set of various methods enabling the characterization of these parameters applicable to isolated mitochondria or intact cells. We also present examples of experimental data demonstrating that the magnitude and direction (increase or decrease) of a change in mitochondrial ROS production depend on the mitochondrial metabolic state.

Keywords:  Confocal microscopy; Hydrogen peroxide; Mitochondria; Oxygen consumption; Resazurin; Superoxide

DOI:  https://doi.org/10.1007/978-1-0716-4264-1_8
Autophagy. 2024 Nov 08.

PINK1-deficiency facilitates mitochondrial iron accumulation and colon tumorigenesis.

Mariella Arcos, Lavanya Goodla, Hyeoncheol Kim, Sharina P Desai, Rui Liu, Kunlun Yin, Zhaoli Liu, David R Martin, Xiang Xue.

  Mitophagy, the process by which cells eliminate damaged mitochondria, is mediated by PINK1 (PTEN induced kinase 1). Our recent research indicates that PINK1 functions as a tumor suppressor in colorectal cancer by regulating cellular metabolism. Interestingly, PINK1 ablation activated the NLRP3 (NLR family pyrin domain containing 3) inflammasome, releasing IL1B (interleukin 1 beta). However, inhibiting the NLRP3-IL1B signaling pathway with an IL1R (interleukin 1 receptor) antagonist or NLRP3 inhibitor did not hinder colon tumor growth after PINK1 loss. To identify druggable targets in PINK1-deficient tumors, ribonucleic acid sequencing analysis was performed on colon tumors from pink1 knockout and wild-type mice. Gene Set Enrichment Analysis highlighted the enrichment of iron ion transmembrane transporter activity. Subsequent qualitative polymerase chain reaction and western blot analysis revealed an increase in mitochondrial iron transporters, including mitochondrial calcium uniporter, in PINK1-deficient colon tumor cells and tissues. Live-cell iron staining demonstrated elevated cellular and mitochondrial iron levels in PINK1-deficient cells. Clinically used drugs deferiprone and minocycline reduced mitochondrial iron and superoxide levels, resulting in decreased colon tumor cell growth in vitro and in vivo. Manipulating the mitochondrial iron uptake protein MCU (mitochondrial calcium uniporter) also affected cell and xenograft tumor growth. This study suggests that therapies aimed at reducing mitochondrial iron levels may effectively inhibit colon tumor growth, particularly in patients with low PINK1 expression.

Keywords:  Colorectal cancer; deferiprone; iron chelation; minocycline; mitochondrial iron; mitophagy

DOI:  https://doi.org/10.1080/15548627.2024.2425594