bims-mireme 2021-07-04 papers

bims-mireme

Biomed News

on Mitochondria in regenerative medicine

Issue of 2021–07–04
eleven papers selected by
Brian Spurlock, The University of North Carolina at Chapel Hill

Differential localization patterns of pyruvate kinase isoforms in murine naïve, formative, and primed pluripotent states.
Ubiquitination and receptor-mediated mitophagy converge to eliminate oxidation-damaged mitochondria during hypoxia.
Mitochondrial Transplantation in Cardiomyocytes: foundation, methods, and outcomes.
Dependence of mitochondrial function on the filamentous actin cytoskeleton in cultured mesenchymal stem cells treated with cytochalasin B.
NADPH Oxidases: Redox Regulators of Stem Cell Fate and Function.
Metformin and Vitamin D Modulate Inflammation and Autophagy during Adipose-Derived Stem Cell Differentiation.
Modulation of mitochondrial nucleoid structure during aging and by mtDNA content in Drosophila.
Mechanisms of organelle elimination for lens development and differentiation.
Monitoring Mitophagy via the FRET Mechanism: Visualizing Mitochondria, Lysosomes, and Autolysosomes in Three Different Sets of Fluorescence Signals.
Inflammation, epigenetics, and metabolism converge to cell senescence and ageing: the regulation and intervention.
Human Trisomic iPSCs from Down Syndrome Fibroblasts Manifest Mitochondrial Alterations Early during Neuronal Differentiation.

Exp Cell Res. 2021 Jun 25. pii: S0014-4827(21)00246-9. [Epub ahead of print] 112714

Differential localization patterns of pyruvate kinase isoforms in murine naïve, formative, and primed pluripotent states.

Joshua G Dierolf, Andrew J Watson, Dean H Betts.

  Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) represent opposite ends of the pluripotency continuum, referred to as naïve and primed pluripotent states, respectively. These divergent pluripotent states differ in several ways including growth factor requirements, transcription factor expression, DNA methylation patterns, and metabolic profiles. Naïve cells employ both glycolysis and oxidative phosphorylation (OXPHOS), whereas primed cells preferentially utilize aerobic glycolysis, a trait shared with cancer cells referred to as the Warburg Effect. Until recently, metabolism has been regarded as a by-product of cell fate; however, evidence now supports metabolism as being a driver of stem cell state and fate decisions. Pyruvate kinase muscle isoforms (PKM1 and PKM2) are important for generating and maintaining pluripotent stem cells (PSCs) and mediating the Warburg effect. Both isoforms catalyze the last step of glycolysis generating adenosine triphosphate and pyruvate, however, the precise role(s) of PKM1/2 in naïve and primed pluripotency is not well understood. The primary objective of this study was to characterize the cellular expression and localization patterns of PKM1 and PKM2 in mESCs, chemically transitioned epiblast-like cells (mEpiLCs) representing formative pluripotency, and mEpiSCs using immunoblotting and confocal microscopy. The results indicate that PKM1 and PKM2 are not only localized to the cytoplasm but also accumulate in differential subnuclear regions of mESC, mEpiLCs and mEpiSCs as determined by a quantitative confocal microscopy employing orthogonal projections and airyscan processing. Importantly, we discovered that the subnuclear localization of PKM1/2 shifts during the transition from mESCs, mEpiLCs and mEpiSCs. Finally, we have comprehensively validated the appropriateness and power of the Pearson's correlation coefficient and Manders' overlap coefficient for assessing nuclear and cytoplasmic protein colocalization in PSCs by immunofluorescence confocal microscopy. We propose that nuclear PKM1/2 may assist with distinct pluripotency state maintenance and lineage priming by non-canonical mechanisms. These results advance our understanding of the overall mechanisms controlling naïve, formative, and primed pluripotency.

Keywords:  Colocalization; Confocal microscopy; Embryonic stem cells; Metabolism; Pluripotency; Pyruvate kinase muscle isoforms 1/2

DOI:  https://doi.org/10.1016/j.yexcr.2021.112714
Redox Biol. 2021 Jun 17. pii: S2213-2317(21)00206-8. [Epub ahead of print]45 102047

Ubiquitination and receptor-mediated mitophagy converge to eliminate oxidation-damaged mitochondria during hypoxia.

Prasad Sulkshane, Jonathan Ram, Anita Thakur, Noa Reis, Oded Kleifeld, Michael H Glickman.

  The contribution of the Ubiquitin-Proteasome System (UPS) to mitophagy has been largely attributed to the E3 ubiquitin ligase Parkin. Here we show that in response to the oxidative stress associated with hypoxia or the hypoxia mimic CoCl2, the damaged and fragmented mitochondria are removed by Parkin-independent mitophagy. Mitochondria isolated from hypoxia or CoCl2-treated cells exhibited extensive ubiquitination, predominantly Lysine 48-linked and involves the degradation of key mitochondrial proteins such as the mitofusins MFN1/2, or the import channel component TOM20. Reflecting the critical role of mitochondrial protein degradation, proteasome inhibition blocked CoCl2-induced mitophagy. The five conserved ubiquitin-binding autophagy receptors (p62, NDP52, Optineurin, NBR1, TAX1BP1) were dispensable for the ensuing mitophagy, suggesting that the mitophagy step itself was independent of ubiquitination. Instead, the expression of two ubiquitin-independent mitophagy receptor proteins BNIP3 and NIX was induced by hypoxia or CoCl2-treatment followed by their recruitment to the oxidation-damaged mitochondria. By employing BNIP3/NIX double knockout and DRP1-null cell lines, we confirmed that mitochondrial clearance relies on DRP1-dependent mitochondrial fragmentation and BNIP3/NIX-mediated mitophagy. General antioxidants such as N-Acetyl Cysteine (NAC) or the mitochondria-specific Mitoquinone prevented HIF-1α stabilization, ameliorated hypoxia-related mitochondrial oxidative stress, and suppressed mitophagy. We conclude that the UPS and receptor-mediated autophagy converge to eliminate oxidation-damaged mitochondria.

Keywords:  HIF-1α; Hypoxia; Mitochondria; Mitophagy; Oxidative stress; Proteasome; Ubiquitin

DOI:  https://doi.org/10.1016/j.redox.2021.102047
Am J Physiol Cell Physiol. 2021 06 30.

Mitochondrial Transplantation in Cardiomyocytes: foundation, methods, and outcomes.

Paria Ali Pour, Sina Hosseinian, Arash Kheradvar.

  Mitochondrial Transplantation is emerging as a novel cellular biotherapy to alleviate mitochondrial damage and dysfunction. Mitochondria play a crucial role in establishing cellular homeostasis and providing cell with the energy necessary to accomplish its function. Owing to its endosymbiotic origin, mitochondria share many features with their bacterial ancestors. Unlike the nuclear DNA, which is packaged into nucleosomes and protected from adverse environmental effects; mitochondrial DNA are more prone to harsh environmental effects, in particular that of the reactive oxygen species (ROS). Mitochondrial damage and dysfunction are implicated in many diseases ranging from metabolic diseases to cardiovascular and neurodegenerative diseases, among others. While it was once thought that transplantation of mitochondria would not be possible due to its semi-autonomous nature and reliance on the nucleus, recent advances have shown that it is possible to transplant viable functional intact mitochondria from autologous, allogenic, and xenogeneic sources into different cell types. Moreover, current research suggests that the transplantation could positively modulate bioenergetics and improve disease outcome. Mitochondrial transplantation techniques and consequences of transplantation in cardiomyocytes are the theme of this review. First, we outline the different mitochondrial isolation and transfer techniques. Second, we detail the consequences of mitochondrial transplantation in the cardiovascular system, more specifically in the context of cardiomyopathies and ischemia. Lastly, we elaborate our vision on how mitochondrial transplantation may mitigate other cardiac conditions secondary to mitochondrial damage and dysfunction, such as atherosclerosis and ST-elevated myocardial infarction.

Keywords:  Bioenergetics; Ischemia Reperfusion Injury; Mitochondiral transplantation; Mitochondrial Cardiomyopathy; Mitochondrial Transfer

DOI:  https://doi.org/10.1152/ajpcell.00152.2021
J Biosci Bioeng. 2021 Jun 23. pii: S1389-1723(21)00145-6. [Epub ahead of print]

Dependence of mitochondrial function on the filamentous actin cytoskeleton in cultured mesenchymal stem cells treated with cytochalasin B.

Ágnes Kocsis, Markus Pasztorek, Eva Rossmanith, Zoran Djinovic, Torsten Mayr, Sarah Spitz, Helene Zirath, Peter Ertl, Michael B Fischer.

  Owing to their self-renewal and multi-lineage differentiation capability, mesenchymal stem cells (MSCs) hold enormous potential in regenerative medicine. A prerequisite for a successful MSC therapy is the rigorous investigation of their function after in vitro cultivation. Damages introduced to mitochondria during cultivation adversely affect MSCs function and can determine their fate. While it has been shown that microtubules and vimentin intermediate filaments are important for mitochondrial dynamics and active mitochondrial transport within the cytoplasm of MSCs, the role of filamentous actin in this process has not been fully understood yet. To gain a deeper understanding of the interdependence between mitochondrial function and the cytoskeleton, we applied cytochalasin B to disturb the filamentous actin-based cytoskeleton of MSCs. In this study we combined conventional functional assays with a state-of-the-art oxygen sensor-integrated microfluidic device to investigate mitochondrial function. We demonstrated that cytochalasin B treatment at a dose of 16 μM led to a decrease in cell viability with high mitochondrial membrane potential, increased oxygen consumption rate, disturbed fusion and fission balance, nuclear extrusion and perinuclear accumulation of mitochondria. Treatment of MSCs for 48 h ultimately led to nuclear fragmentation, and activation of the intrinsic pathway of apoptotic cell death. Importantly, we could show that mitochondrial function of MSCs can efficiently recover from the damage to the filamentous actin-based cytoskeleton over a period of 24 h. As a result of our study, a causative connection between the filamentous actin-based cytoskeleton and mitochondrial dynamics was demonstrated.

Keywords:  Actin cytoskeleton; Cytochalasin B; Mesenchymal stem cell; Microfluidics; Mitochondrial function; Oxygen sensor

DOI:  https://doi.org/10.1016/j.jbiosc.2021.05.010
Antioxidants (Basel). 2021 Jun 17. pii: 973. [Epub ahead of print]10(6):

NADPH Oxidases: Redox Regulators of Stem Cell Fate and Function.

Tullia Maraldi, Cristina Angeloni, Cecilia Prata, Silvana Hrelia.

  One of the major sources of reactive oxygen species (ROS) generated within stem cells is the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes (NOXs), which are critical determinants of the redox state beside antioxidant defense mechanisms. This balance is involved in another one that regulates stem cell fate: indeed, self-renewal, proliferation, and differentiation are decisive steps for stem cells during embryo development, adult tissue renovation, and cell therapy application. Ex vivo culture-expanded stem cells are being investigated for tissue repair and immune modulation, but events such as aging, senescence, and oxidative stress reduce their ex vivo proliferation, which is crucial for their clinical applications. Here, we review the role of NOX-derived ROS in stem cell biology and functions, focusing on positive and negative effects triggered by the activity of different NOX isoforms. We report recent findings on downstream molecular targets of NOX-ROS signaling that can modulate stem cell homeostasis and lineage commitment and discuss the implications in ex vivo expansion and in vivo engraftment, function, and longevity. This review highlights the role of NOX as a pivotal regulator of several stem cell populations, and we conclude that these aspects have important implications in the clinical utility of stem cells, but further studies on the effects of pharmacological modulation of NOX in human stem cells are imperative.

Keywords:  NADPH oxidases; reactive oxygen species; stem cells

DOI:  https://doi.org/10.3390/antiox10060973
Int J Mol Sci. 2021 Jun 22. pii: 6686. [Epub ahead of print]22(13):

Metformin and Vitamin D Modulate Inflammation and Autophagy during Adipose-Derived Stem Cell Differentiation.

Sara Cruciani, Giuseppe Garroni, Renzo Pala, Maria Laura Cossu, Giorgio Carlo Ginesu, Carlo Ventura, Margherita Maioli.

  Adipose-derived stem cells (ADSCs) came out from the regenerative medicine landscape for their ability to differentiate into several phenotypes, contributing to tissue regeneration both in vitro and in vivo. Dysregulation in stem cell recruitment and differentiation during adipogenesis is linked to a chronic low-grade inflammation and macrophage infiltration inside the adipose tissue, insulin resistance, cardiovascular disease and obesity. In the present paper we aimed to evaluate the role of metformin and vitamin D, alone or in combination, in modulating inflammation and autophagy in ADSCs during adipogenic commitment. ADSCs were cultured for 21 days in the presence of a specific adipogenic differentiation medium, together with metformin, or vitamin D, or both. We then analyzed the expression of FoxO1 and Heat Shock Proteins (HSP) and the secretion of proinflammatory cytokines IL-6 and TNF-α by ELISA. Autophagy was also assessed by specific Western blot analysis of ATG12, LC3B I, and LC3B II expression. Our results showed the ability of the conditioned media to modulate adipogenic differentiation, finely tuning the inflammatory response and autophagy. We observed a modulation in HSP mRNA levels, and a significant downregulation in cytokine secretion. Taken together, our findings suggest the possible application of these molecules in clinical practice to counteract uncontrolled lipogenesis and prevent obesity and obesity-related metabolic disorders.

Keywords:  adipogenesis; adipose stem cells; autophagy; cell differentiation; conditioned media; epigenetic; gene expression; inflammation

DOI:  https://doi.org/10.3390/ijms22136686
Biol Open. 2021 Jun 15. pii: bio058553. [Epub ahead of print]10(6):

Modulation of mitochondrial nucleoid structure during aging and by mtDNA content in Drosophila.

Li-Jie Wang, Tian Hsu, Hsiang-Ling Lin, Chi-Yu Fu.

  Mitochondrial DNA (mtDNA) encodes gene products that are essential for oxidative phosphorylation. They organize as higher order nucleoid structures (mtNucleoids) that were shown to be critical for the maintenance of mtDNA stability and integrity. While mtNucleoid structures are associated with cellular health, how they change in situ under physiological maturation and aging requires further investigation. In this study, we investigated the mtNucleoid assembly at an ultrastructural level in situ using the TFAM-Apex2 Drosophila model. We found that smaller and more compact TFAM-nucleoids are populated in the mitochondria of indirect flight muscle of aged flies. Furthermore, mtDNA transcription and replication were cross-regulated in the mtTFB2-knockdown flies as in the mtRNAPol-knockdown flies that resulted in reductions in mtDNA copy numbers and nucleoid-associated TFAM. Overall, our study reveals that the modulation of TFAM-nucleoid structure under physiological aging, which is critically regulated by mtDNA content.

Keywords:  Mitochondrial DNA; Mitochondrial RNA polymerase (mtRNAPol); Mitochondrial nucleoid; Mitochondrial transcription factor B2 (mtTFB2); Transcription factor A (TFAM)

DOI:  https://doi.org/10.1242/bio.058553
Exp Eye Res. 2021 Jun 29. pii: S0014-4835(21)00248-7. [Epub ahead of print] 108682

Mechanisms of organelle elimination for lens development and differentiation.

Lisa Brennan, Joshua Disatham, Marc Kantorow.

  A hallmark feature of lens development and differentiation is the complete elimination of organelles from the center of the eye lens. A long unanswered question in lens biology is what are the mechanisms that control the elimination of organelles during the terminal remodeling program to form mature lens fiber cells? Recent advances have expanded our understanding of these mechanisms including newly discovered signaling pathways, proteasomal regulators, autophagy proteins, transcription factors and the hypoxic environment of the lens itself. These recent discoveries suggest that distinct mechanisms coordinate the elimination of the nucleus, mitochondria, endoplasmic reticulum and Golgi apparatus during lens fiber cell differentiation. Since regulation of organelle number and distribution is also a feature of the terminal remodeling programs of more complex cell-types and tissues, these advances are likely to impact a wide-variety of fields.

Keywords:  Autophagy; Chromatin; Development; Differentiation; Endoplasmic reticulum; Golgi apparatus; Hypoxia; Lens; Mitochondria; Nucleus; Organelle regulation; Transcriptional regulation

DOI:  https://doi.org/10.1016/j.exer.2021.108682
Anal Chem. 2021 Jun 28.

Monitoring Mitophagy via the FRET Mechanism: Visualizing Mitochondria, Lysosomes, and Autolysosomes in Three Different Sets of Fluorescence Signals.

Qingqing Lu, Wenpeng Li, Kaiyuan Chen, Minggang Tian.

Mitophagy is a vital biological process playing central roles in the regulation of metabolic activity and quality control of mitochondria. The presented dual-color fluorescent probes to directly monitor mitophagy were based on the optical response to pH change during mitophagy, but pH fluctuation may lead to interference. To overcome this, herein, two fluorescent probes (G-Mito, R-Lyso) were rationally designed to visualize mitophagy directly in a dual-color manner, relying on the Förster resonance energy transfer (FRET) process for the first time. Green emissive G-Mito targeted and anchored the mitochondria via reaction with protein thiols. Red-emissive R-Lyso exclusively targeted lysosomes. Live cells loaded with the two probes demonstrated strong fluorescence in only the green channel with excitation at 405 nm. After mitophagy, G-Mito in mitochondria was delivered into the lysosomes, and red fluorescence evidently increased due to the FRET process. With the probes, mitochondria, lysosomes, and autolysosomes could be discriminatively visualized in three different sets of signals. Mitophagy induced by starvation and in normal physiological status were successfully observed. The probes revealed that a certain amount of H2O2 could induce mitophagy. We expect that the two probes can serve as molecular tools for validation of mitophagy and promote the development of related areas.

DOI: https://doi.org/10.1021/acs.analchem.1c01237
Signal Transduct Target Ther. 2021 Jun 28. 6(1): 245

Inflammation, epigenetics, and metabolism converge to cell senescence and ageing: the regulation and intervention.

Xudong Zhu, Zhiyang Chen, Weiyan Shen, Gang Huang, John M Sedivy, Hu Wang, Zhenyu Ju.

Remarkable progress in ageing research has been achieved over the past decades. General perceptions and experimental evidence pinpoint that the decline of physical function often initiates by cell senescence and organ ageing. Epigenetic dynamics and immunometabolic reprogramming link to the alterations of cellular response to intrinsic and extrinsic stimuli, representing current hotspots as they not only (re-)shape the individual cell identity, but also involve in cell fate decision. This review focuses on the present findings and emerging concepts in epigenetic, inflammatory, and metabolic regulations and the consequences of the ageing process. Potential therapeutic interventions targeting cell senescence and regulatory mechanisms, using state-of-the-art techniques are also discussed.

DOI: https://doi.org/10.1038/s41392-021-00646-9
Biology (Basel). 2021 Jun 30. pii: 609. [Epub ahead of print]10(7):

Human Trisomic iPSCs from Down Syndrome Fibroblasts Manifest Mitochondrial Alterations Early during Neuronal Differentiation.

Nunzia Mollo, Matteo Esposito, Miriam Aurilia, Roberta Scognamiglio, Rossella Accarino, Ferdinando Bonfiglio, Rita Cicatiello, Maria Charalambous, Claudio Procaccini, Teresa Micillo, Rita Genesio, Gaetano Calì, Agnese Secondo, Simona Paladino, Giuseppe Matarese, Gabriella De Vita, Anna Conti, Lucio Nitsch, Antonella Izzo.

   BACKGROUND: The presence of mitochondrial alterations in Down syndrome suggests that it might affect neuronal differentiation. We established a model of trisomic iPSCs, differentiating into neural precursor cells (NPCs) to monitor the occurrence of differentiation defects and mitochondrial dysfunction.
METHODS: Isogenic trisomic and euploid iPSCs were differentiated into NPCs in monolayer cultures using the dual-SMAD inhibition protocol. Expression of pluripotency and neural differentiation genes was assessed by qRT-PCR and immunofluorescence. Meta-analysis of expression data was performed on iPSCs. Mitochondrial Ca2+, reactive oxygen species (ROS) and ATP production were investigated using fluorescent probes. Oxygen consumption rate (OCR) was determined by Seahorse Analyzer.
RESULTS: NPCs at day 7 of induction uniformly expressed the differentiation markers PAX6, SOX2 and NESTIN but not the stemness marker OCT4. At day 21, trisomic NPCs expressed higher levels of typical glial differentiation genes. Expression profiles indicated that mitochondrial genes were dysregulated in trisomic iPSCs. Trisomic NPCs showed altered mitochondrial Ca2+, reduced OCR and ATP synthesis, and elevated ROS production.
CONCLUSIONS: Human trisomic iPSCs can be rapidly and efficiently differentiated into NPC monolayers. The trisomic NPCs obtained exhibit greater glial-like differentiation potential than their euploid counterparts and manifest mitochondrial dysfunction as early as day 7 of neuronal differentiation.

Keywords:  Down syndrome; induced pluripotent stem cells; mitochondrial dysfunction; neural differentiation

DOI:  https://doi.org/10.3390/biology10070609