bims-midysc 2024-12-15 papers

Issue of 2024–12–15
seven papers selected by
Papachristodoulou Lab

Anal Biochem. 2024 Dec 05. pii: S0003-2697(24)00289-6. [Epub ahead of print] 115745

Cryopreserved PBMCs can be used for the analysis of mitochondrial respiration and serve as a diagnostic tool for mitochondrial diseases.

Zuzana Korandová, Petr Pecina, Alena Pecinová, Eliška Koňaříková, Markéta Tesařová, Josef Houštěk, Hana Hansíková, Hana Ptáčková, Jiří Zeman, Tomáš Honzík, Tomáš Mráček.

Mitochondrial diseases are severe, inherited metabolic disorders that affect the paediatric population. They affect the functioning of mitochondrial oxidative phosphorylation (OXPHOS) apparatus either directly or indirectly. Since mutations in mtDNA are responsible for only 25% of paediatric cases and next-generation sequencing does not always provide a conclusive diagnosis, the biochemical approach still represents a valuable tool in diagnostics. Mitochondrial defects can be identified in tissue biopsies (muscle or skin). However, they also often manifest in peripheral blood cells. We developed a protocol for isolation and cryopreservation of peripheral blood mononuclear cells (PBMCs) from 5 ml of children's blood using Ficoll centrifugation which can be utilised for subsequent functional measurements on thawed samples. Furthermore, we evaluated the diagnostic utility of the optimised high-resolution oxygraphy protocol using digitonin-permeabilized cryopreserved PBMCs on 47 samples from patients with confirmed or suspected mitochondrial disease. Overall, the diagnosis was confirmed in 72% of cases, while the analysis of cryopreserved PBMCs provided a false negative outcome in 13% of cases. Our study demonstrates a sensitive, fast, and non-invasive approach for the diagnostics of various types of mitochondrial disorders, especially those of nuclear genetic origin manifesting in paediatric patients.

Keywords: OXPHOS; PBMC; cryopreservation; diagnostics; glycolysis; mitochondrial diseases; oxidative phosphorylation; peripheral blood mononuclear cells; respirometry

DOI: https://doi.org/10.1016/j.ab.2024.115745

Autophagy. 2024 Dec 12. 1-16

Inhibition of the PI3K-AKT-MTORC1 axis reduces the burden of the m.3243A>G mtDNA mutation by promoting mitophagy and improving mitochondrial function.

Chih-Yao Chung, Kritarth Singh, Preethi Sheshadri, Gabriel E Valdebenito, Anitta R Chacko, María Alicia Costa Besada, Xiao Fei Liang, Lida Kabir, Robert D S Pitceathly, Gyorgy Szabadkai, Michael R Duchen.

Mitochondrial DNA (mtDNA) encodes genes essential for oxidative phosphorylation. The m.3243A>G mutation causes severe disease, including myopathy, lactic acidosis and stroke-like episodes (MELAS) and is the most common pathogenic mtDNA mutation in humans. We have previously shown that the mutation is associated with constitutive activation of the PI3K-AKT-MTORC1 axis. Inhibition of this pathway in patient fibroblasts reduced the mutant load, rescued mitochondrial bioenergetic function and reduced glucose dependence. We have now investigated the mechanisms that select against the mutant mtDNA under these conditions. Basal macroautophagy/autophagy and lysosomal degradation of mitochondria were suppressed in the mutant cells. Pharmacological inhibition of any step of the PI3K-AKT-MTORC1 pathway activated mitophagy and progressively reduced m.3243A>G mutant load over weeks. Inhibition of autophagy with bafilomycin A1 or chloroquine prevented the reduction in mutant load, suggesting that mitophagy was necessary to remove the mutant mtDNA. Inhibition of the pathway was associated with metabolic remodeling - mitochondrial membrane potential and respiratory rate improved even before a measurable fall in mutant load and proved crucial for mitophagy. Thus, maladaptive activation of the PI3K-AKT-MTORC1 axis and impaired autophagy play a major role in shaping the presentation and progression of disease caused by the m.3243A>G mutation. Our findings highlight a potential therapeutic target for this otherwise intractable disease.Abbreviation: ΔΨm: mitochondrial membrane potential; 2DG: 2-deoxy-D-glucose; ANOVA: analysis of variance; ARMS-qPCR: amplification-refractory mutation system quantitative polymerase chain reaction; Baf A1: bafilomycin A1; BSA: bovine serum albumin; CQ: chloroquine; Cybrid: cytoplasmic hybrid; CYCS: cytochrome c, somatic; DCA: dichloroacetic acid; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethylsulfoxide; EGFP: enhanced green fluorescent protein; LC3B-I: carboxy terminus cleaved microtubule-associated protein 1 light chain 3 beta; LC3B-II: lipidated microtubule-associated protein 1 light chain 3 beta; LY: LY290042; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MELAS: mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes; MFC: mitochondrial fragmentation count; mt-Keima: mitochondrial-targeted mKeima; mtDNA: mitochondrial DNA/mitochondrial genome; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; OA: oligomycin+antimycin A; OxPhos: oxidative phosphorylation; DPBS: Dulbecco's phosphate-buffered saline; PPARGC1A/PGC-1α: PPARG coactivator 1 alpha; PPARGC1B/PGC-1β: PPARG coactivator 1 beta; PI3K: phosphoinositide 3-kinase; PINK1: PTEN induced kinase 1; qPCR: quantitative polymerase chain reaction; RNA-seq: RNA sequencing; RP: rapamycin; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; WT: wild-type.

Keywords: PI3K-AKT-MTORC1; m.3243A>G; mitochondria; mitophagy; mtDNA mutations; nutrient signaling

DOI: https://doi.org/10.1080/15548627.2024.2437908

Mitochondrion. 2024 Dec 09. pii: S1567-7249(24)00158-2. [Epub ahead of print] 102000

Mitochondria-targeted nanotherapeutics: A new Frontier in neurodegenerative disease treatment.

Nishad Keethedeth, Rajesh Anantha Shenoi.

Mitochondria are the seat of cellular energy and play key roles in regulating several cellular processes such as oxidative phosphorylation, respiration, calcium homeostasis and apoptotic pathways. Mitochondrial dysfunction results in error in oxidative phosphorylation, redox imbalance, mitochondrial DNA mutations, and disturbances in mitochondrial dynamics, all of which can lead to several metabolic and degenerative diseases. A plethora of studies have provided evidence for the involvement of mitochondrial dysfunction in the pathogenesis of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis. Hence mitochondria have been used as possible therapeutic targets in the regulation of neurodegenerative diseases. However, the double membranous structure of mitochondria poses an additional barrier to most drugs even if they are able to cross the plasma membrane. Most of the drugs acting on mitochondria also required very high doses to exhibit the desired mitochondrial accumulation and therapeutic effect which in-turn result in toxic effects. Mitochondrial targeting has been improved by direct conjugation of drugs to mitochondriotropic molecules like dequalinium (DQA) and triphenyl phosphonium (TPP) cations. But being cationic in nature, these molecules also exhibit toxicity at higher doses. In order to further improve the mitochondrial localization with minimal toxicity, TPP was conjugated with various nanomaterials like liposomes. inorganic nanoparticles, polymeric nanoparticles, micelles and dendrimers. This review provides an overview of the role of mitochondrial dysfunction in neurodegenerative diseases and various nanotherapeutic strategies for efficient targeting of mitochondria-acting drugs in these diseases.

Keywords: Blood brain barrier; Mitochondria targeting; Nanotherapeutics; Neurodegenerative diseases; Triphenyl Phosphonium

DOI: https://doi.org/10.1016/j.mito.2024.102000

bioRxiv. 2024 Nov 26. pii: 2024.11.24.625104. [Epub ahead of print]

Mitochondrial respiratory capacity in kidney podocytes is high, age-dependent, and sexually dimorphic.

Matthew D Campbell, Monica Sanchez-Contreras, Britta D Sibley, Phoebe Keiser, Carla Ruiz Sanchez, Carolyn N Mann, David J Marcinek, Behzad Najafian, Mariya T Sweetwyne.

Whether and how podocytes depend on mitochondria across their long post-mitotic lifespan is yet unclear. With limited cell numbers and broad kidney distribution, isolation of podocyte mitochondria typically requires first isolating podocytes themselves. Disassociation of podocytes from their basement membrane, however, recapitulates an injured state that may stress mitochondria. To address this, we crossed floxed hemagglutinin (HA) -mitochondria tagged (MITO-Tag) mice with those expressing Cre in either podocytes (NPHS2) or distal tubule and collecting duct (CDH16), thus allowing for rapid, kidney cell-specific, isolation of mitochondria via immunoprecipitation. Mitochondrial respiration in fresh isolates from young (4-7 mo) and aged (22-26 mo) mice of both sexes demonstrated several previously unreported significant differences between podocyte and tubule mitochondria. First, although podocytes contain fewer mitochondria than do tubule cells, mitochondria isolated from podocytes averaged twice the respiratory capacity of tubule mitochondria when normalized to mitochondrial content by citrate synthase (CS) levels. Second, age-related decline in respiration was detected only in podocyte mitochondria and only in aged male mice. Finally, disassociating podocytes for cell culture initiates functional decline in mitochondria as those from cultured primary podocytes have half the respiratory capacity, but twice the hydrogen peroxide production of podocyte mitochondria isolated directly from fresh kidneys. Thus, podocytes maintain sexually dimorphic mitochondria with greater oxidative phosphorylation capacity than mitochondria-dependent tubules per organelle. Previous studies may not have detected these differences due to reliance on podocyte cell culture conditions, which results in artifactual suppression of mitochondrial function.

DOI: https://doi.org/10.1101/2024.11.24.625104

iScience. 2024 Dec 20. 27(12): 111359

Crosstalk between metabolic and epigenetic modifications during cell carcinogenesis.

Yue Gao, Siyu Zhang, Xianhong Zhang, Yitian Du, Ting Ni, Shuailin Hao.

Genetic mutations arising from various internal and external factors drive cells to become cancerous. Cancerous cells undergo numerous changes, including metabolic reprogramming and epigenetic modifications, to support their abnormal proliferation. This metabolic reprogramming leads to the altered expression of many metabolic enzymes and the accumulation of metabolites. Recent studies have shown that these enzymes and metabolites can serve as substrates or cofactors for chromatin-modifying enzymes, thereby participating in epigenetic modifications and promoting carcinogenesis. Additionally, epigenetic modifications play a role in the metabolic reprogramming and immune evasion of cancer cells, influencing cancer progression. This review focuses on the origins of cancer, particularly the metabolic reprogramming of cancer cells and changes in epigenetic modifications. We discuss how metabolites in cancer cells contribute to epigenetic remodeling, including lactylation, acetylation, succinylation, and crotonylation. Finally, we review the impact of epigenetic modifications on tumor immunity and the latest advancements in cancer therapies targeting these modifications.

Keywords: Epigenetics; Molecular genetics

DOI: https://doi.org/10.1016/j.isci.2024.111359

PLoS One. 2024 ;19(12): e0312352

Mitochondrial fission is required for thermogenesis in brown adipose tissue.

Yuta Ibayashi, Nao Hasuzawa, Seiji Nomura, Masaharu Kabashima, Ayako Nagayama, Shimpei Iwata, Miyuki Kitamura, Kenji Ashida, Yoshinori Moriyama, Ken Yamamoto, Masatoshi Nomura, Lixiang Wang.

Brown adipose tissue (BAT) thermogenesis is pivotal for maintaining body temperature and energy balance. Mitochondrial morphology is dynamically controlled by a balance between fusion and fission, which is crucial for cell differentiation, response to metabolic insults, and heat production. Dynamin-related protein 1 (Drp1) is a key regulator of mitochondrial fission. This study investigates the role of Drp1 in BAT development and thermogenesis by generating Drp1-deficient mice. These mice were created by crossing Drp1 floxed mice with fatty acid-binding protein 4-Cre (aP2-Cre) transgenic mice, resulting in aP2-Cre+/-Drp1flox/flox (aP2-Drp1f/f) mice. The aP2-Drp1f/f mice exhibited severe BAT and brain hypoplasia, with the majority dying within 48 hours postnatally, highlighting Drp1's crucial role in neonatal survival. Impaired thermogenic responses were observed in aP2-Drp1f/f mice, characterized by significantly decreased expression of thermogenic and lipogenic genes in BAT. Ultrastructural analysis revealed disrupted mitochondrial morphology and reduced lipid droplet content in BAT. The few surviving adult aP2-Drp1f/f mice also showed impaired BAT and brain development, along with BAT thermogenesis dysfunction during cold exposure. Our findings underscore the essential role of Drp1-mediated mitochondrial fission in BAT thermogenesis and neonatal survival, providing insights into potential therapeutic approaches for metabolic disorders.

DOI: https://doi.org/10.1371/journal.pone.0312352

Comp Biochem Physiol A Mol Integr Physiol. 2024 Dec 09. pii: S1095-6433(24)00217-4. [Epub ahead of print]300 111790

Modulation of mitochondrial dynamics genes and mtDNA during embryonic development and under UVB exposure.

Thaline de Quadros, Michael Lorenz Jaramillo, Cairé Barreto, Rafael Diego da Rosa, Madson Silveira de Melo, Evelise Maria Nazari.

Studies using the embryos of the freshwater prawn Macrobrachium olfersii have reported changes in embryonic cells after exposure to ultraviolet B (UVB) radiation, such as DNA damage and apoptosis activation. Considering the importance of mitochondria in embryonic cells, this study aimed to characterize the aspects of mitochondrial morphofunctionality in M. olfersii embryos and mitochondrial responses to UVB radiation exposure. The coding sequences of genes Tfam, Nrf1, Mfn1, and Drp1 were identified from the transcriptome of M. olfersii embryos. The phylogenetic relationship showed strong amino acid identity and a highly conserved nature of the sequences. Additionally, the number of mitochondrial DNA (mtDNA) copies were higher in the early embryonic days. The results showed that the expression of the analyzed genes was highly regulated during embryonic development, increasing their levels near hatching. Furthermore, when embryos were exposed to UVB radiation, mitochondrial biogenesis was activated, recognized by higher levels of transcripts of genes Tfam and Nrf1, accompanied by mitochondrial fission. Additionally, these mitochondrial events were supported by an increase of mtDNA copies. Our results showed that UVB radiation was able to change the mitochondrial morphofunctionality, and under the current knowledge, certainly compromise embryonic cellular integrity. Additionally, mitochondria is an important cellular target of this radiation and its responses can be used to assess environmental stress caused by UVB radiation in embryos of aquatic species.

Keywords: Bioinformatic tool; Embryo; Embryotoxicity; Fusion/fission mitochondrial; Macrobrachium prawn; Ultraviolet radiation

DOI: https://doi.org/10.1016/j.cbpa.2024.111790