Program

Mitochondrial redox metabolism is central to the life and death of the cell. For example, mitochondrial production of free radicals and subsequent oxidative damage has long been known to contribute to damage in conditions such as ischaemia-reperfusion (IR) injury in stroke and heart attack. More recently mitochondrial redox changes have also been implicated in redox signalling. Over the past years we have developed a series of mitochondria-targeted compounds designed to ameliorate or determine how these changes occur. I will outline some of this work, which suggested that ROS production in IR injury during stroke was mainly coming from complex I. This led us to investigate the mechanism of the ROS production and using a metabolomic approach we found that the ROS production in IR injury came from the accumulation of succinate during ischaemia that then drove mitochondrial ROS production by reverse electron transport at complex I during reperfusion. This surprising mechanism led up to develop further new therapeutic approaches to impact on the damage that mitochondrial ROS do in pathology and also to explore how mitochondrial ROS can act as redox signals. I will discuss how these unexpected mechanisms may lead to redox and metabolic signals from mitochondria in a range of conditions under both healthy and pathological conditions.

References

A unifying mechanism for mitochondrial superoxide production during ischemia reperfusion injury

Edward T. Chouchani, Victoria R. Pell, Andrew M. James, Lorraine M. Work, Kourosh Saeb-Parsy, Christian Frezza, Thomas Krieg and Michael P. Murphy

Cell Metabolism (2016) 23 254-263

Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS

Edward T. Chouchani, Victoria R. Pell, Edoardo Gaude, Dunja Aksentijević, Stephanie Y. Sundier, Ellen L. Robb, Angela Logan, Sergiy M. Nadtochiy, Emily N. J. Ord, Anthony C. Smith, Filmon Eyassu, Rachel Shirley, Chou-Hui Hu, Anna J. Dare, Andrew M. James, Sebastian Rogatti, Richard C. Hartley, Simon Eaton, Ana, S. H. Costa, Paul S. Brookes, Sean M. Davidson, Michael R. Duchen, Kourosh Saeb-Parsy, Michael J. Shattock, Alan J. Robinson, Lorraine M. Work, Christian Frezza, Thomas Krieg and Michael P. Murphy

Nature (2014) 515 431-435

Mitochondrial DNA integrity defines muscle satellite cells determination into skeletal muscle fibres 

Ayesha Sen (1), Rudolf J. Wiesner (1,2,3), David Pla-Martín (1,2) 

1- University Clinic Cologne, University of Cologne, Centre for Physiology and Pathophysiology

2- University of Cologne, CMMC

3-University of Cologne, CECAD 

Mutations in the mitochondrial DNA (mtDNA) have been linked to skeletal muscle atrophy and sarcopenia. Muscle satellite cells (MuSCs), responsible for regenerating new muscle fibres, are generally quiescent but become activated during muscle biogenesis, damage or exercise. To explore how mtDNA mutations affect the regeneration capacity of MuSCs we employ a mouse model expressing a dominant negative mutation of TWINKLE (p.K320E), the mtDNA helicase, under the control of tamoxifen, specifically in the MuSCs. This mutation is known to accelerate accumulation of mtDNA alterations. In quiescent stem cells, K320E-Twinkle is well tolerated, and we only observe mtDNA depletion in very old mice. Activation of muscle regeneration in adult mice by cardiotoxin injection in the Tibialis Anterior, induces the accumulation of fibres with mitochondrial dysfunction, mtDNA alterations and a prominent fibre type shift. In contrast, if K320E-Twinkle is activated in a more physiological manner during muscle biogenesis, we do not observe mitochondrial dysfunction but, again, a prominent shift in fibre type. In these mice, cardiotoxin injection one year after K320E-Twinkle activation, did not result in accumulation of fibres with mitochondrial dysfunction. In addition, organelle proteomics of C2C12 expressing K320E-Twinkle revealed lower accumulation of mitochondrial proteins during fibre differentiation. We propose that the fibre type shift observed during ageing might be the consequence of the accumulation of mtDNA damage; a process that has been observed in all tissues of organisms with a long lifespan, including humans. 

Overexpression of UCP4 in astrocytic mitochondria prevents multilevel dysfunctions in a mouse model of Alzheimer's disease

Nadia Rosenberg (1), Maria Reva (2), Francesca Binda (1), Leonardo Restivo (1), Pauline Depierre (1), Julien Puyal (1), Marc Briquet (1), Yann Bernardinelli (3), Anne-Bérengère Rocher (1), Henry Markram (2), Jean-Yves Chatton (1) 

1- Department of Fundamental Neurosciences,University of Lausanne, Lausanne, Switzerland

2- Blue Brain Project (BBP), Ecole polytechnique fédérale de Lausanne (EPFL), Geneva, Switzerland

3- Neonomia, Geneva, Switzerland 

Alzheimer's disease (AD) is becoming increasingly prevalent worldwide. It represents one of the greatest medical challenges as no pharmacologic treatments are available to prevent disease progression. Astrocytes play crucial functions within neuronal circuits by providing metabolic and functional support, regulating interstitial solute composition, and modulating synaptic transmission. In addition to these physiological functions, growing evidence points to an essential role of astrocytes in neurodegenerative diseases like AD. Early-stage AD is associated with hypometabolism and oxidative stress. Contrary to neurons that are vulnerable to oxidative stress, astrocytes are particularly resistant to mitochondrial dysfunction and are therefore more resilient cells. In our study, we leveraged astrocytic mitochondrial uncoupling and examined neuronal function in the 3xTg AD mouse model. We overexpressed the mitochondrial uncoupling protein 4 (UCP4), which has been shown to improve neuronal survival in vitro. We found that this treatment efficiently prevented alterations of hippocampal metabolite levels observed in AD mice, along with hippocampal atrophy and reduction of basal dendrite arborization of subicular neurons. This approach also averted aberrant neuronal excitability observed in AD subicular neurons and preserved episodic-like memory in AD mice assessed in a spatial recognition task. These findings show that targeting astrocytes and their mitochondria is an effective strategy to prevent the decline of neurons facing AD-related stress at the early stages of the disease. 

Alterations in mitochondrial metabolism are major hallmarks of both ageing cells and cancer. Age is the biggest risk factor for the development of a significant number of cancer types and this therefore raises the question of whether there is a link between age-related mitochondrial dysfunction and the advantageous changes in mitochondrial metabolism prevalent in cancer cells. A common underlying feature of both ageing and cancer cells is the presence of somatic mutations of the mitochondrial genome (mtDNA). MtDNA mutations are particularly enriched in colorectal cancers [1] and we have previously shown that individual normal human colonic crypt stem cells also accumulate somatic mtDNA point mutations with age [2]. This shows that somatic mtDNA mutations and altered metabolic pathways are present in colonic crypts prior to malignant transformation, suggesting that mtDNA mutations may either increase the risk of malignant transformation, promote tumour progression, or are selectively propagated during tumour development. To investigate this we generated a mouse model in which we induced tumours specifically in intestinal stem cells with and without mtDNA mutation-induced mitochondrial dysfunction. We found that the mice with mitochondrial dysfunction had similar numbers of tumours to controls but they were growing significantly faster resulting in a shortened lifespan [3]. Multi-omics analysis revealed the underlying mechanism to be an upregulation of the de novo serine synthesis pathway and mitochondrial one-carbon metabolism in response to mitochondrial dysfunction. These anabolic pathways are important regulators of cellular biomass production and, excitingly, may represent metabolic vulnerabilities for therapeutic exploitation in human colorectal cancer.

References

1 - Gorelick, A. N. et al. Nat Metab 3, 558-570, (2021).

2 - Greaves, L. C. et al. Exp Gerontol 45, 573-579, (2010).

3 - Smith, A. L. M. et al. Nature Cancer 1, 976-989, (2020).

Mitochondrial DNA (mtDNA) encodes essential cellular machinery in almost all eukaryotes. But the mitochondrion is an awkward place to store genetic information safely; it's a physically and chemically damaging environment involving frequent and error-prone replication. Most genes for mitochondrial machinery have been transferred to the nucleus since endosymbiosis. So why do mitochondria retain any genes at all? Why do different species retain dramatically different mitochondrial gene counts? And how do they mitigate against the inevitable damage in these essential genes?

I'll talk about our work combining bioinformatics, modelling, heteroplasmy profiling across taxa, and microscopy to test long-standing and new hypotheses about why mtDNA retains genes, and how species in different kingdoms and with different lifestyles can use mitochondrial structure and dynamics to segregate mtDNA damage. In organisms that cannot sequester a protected germline and so use an animal-like mtDNA bottleneck, I'll describe theoretical and experimental results showing that mtDNA recombination can serve this purpose -- at the cost of requiring innovative cellular behaviour to deal with the consequences.

References

Giannakis, K., Arrowsmith, S.J., Richards, L., Gasparini, S., Chustecki, J.M., Røyrvik, E.C. and Johnston, I.G., 2022. Evolutionary inference across eukaryotes identifies universal features shaping organelle gene retention. Cell Systems, 13(11), pp.874-884.

García Pascual, B., Nordbotten, J.M. and Johnston, I.G., 2023. Cellular and environmental dynamics influence species-specific extents of organelle gene retention. Proceedings of the Royal Society B, 290(1994), p.20222140.

Edwards, D.M., Røyrvik, E.C., Chustecki, J.M., Giannakis, K., Glastad, R.C., Radzvilavicius, A.L. and Johnston, I.G., 2021. Avoiding organelle mutational meltdown across eukaryotes with or without a germline bottleneck. PLoS biology, 19(4), p.e3001153.

Broz, A.K., Keene, A., Fernandes Gyorfy, M., Hodous, M., Johnston, I.G. and Sloan, D.B., 2022. Sorting of mitochondrial and plastid heteroplasmy in Arabidopsis is extremely rapid and depends on MSH1 activity. Proceedings of the National Academy of Sciences, 119(34), p.e2206973119.

Acknowledgements

This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 805046 [EvoConBiO]). Support from the Peder Sather fund is gratefully acknowleged.

Mitochondrial ROS as a regulator of synaptic function

Irina Stavrovskaya (1), Bethany Kristi Morin (2), Pablo Peixoto (1,2)

1- Baruch College CUNY, New York, NY, USA,

2- Graduate Center, CUNY, New York, NY, USA 

Exacerbated emission of reactive oxygen species (ROS) from presynaptic mitochondria is a well-studied hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis. Outside of the context of disease, the potential role of mitochondrial ROS in presynaptic function and plasticity remains largely understudied. Here, we investigated this potential role by combining electrophysiological recordings, confocal imaging, immunohistochemical staining and optogenetics using a well-established protocol for induction and measurement of synaptic potentiation in drosophila neuromuscular preparations. We have previously found that optogenetic induction of ROS emission from presynaptic mitochondria expressing mitokiller red was accompanied by an increase in spontaneous mini junction potentials. The increase in electrical activity did not appear to be associated

with major synaptic structure alterations such as the formation of presynaptic filopodia or the growth of ghost boutons. However, in existing boutons, we observed an increase in active zone markers such as nc82/Brp. We have not detected Wnt1/Wg release from synaptic boutons suggesting the involvement of other signaling pathways that underlie observed changes in electrophysiological activity mediated by ROS emission. Future studies will further inquire the role of mitochondrial ROS in synaptic potentiation, as well as the potential signaling targets of mitochondrial ROS in the presynaptic structure. 

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Diseases caused by mutations of the mitochondrial DNA show a surprisingly diverse array of variable physical manifestations, age of onset, and severity in patients – even for mutations within the same gene. This tissue and cell-specific variability in the disease presentation necessitates animal model research to understand these phenomena and lead to translational breakthroughs for mitochondrial disease patients. Despite advances in nuclear genome-engineering, animal mitochondrial-DNA has remained resistant to transgenic manipulation until quite recently. I will present out “phenotype first” method of screening for mouse models of heteroplasmic, pathological  mtDNA  mutations and contrast two models with a 5 bp difference between the mutational sites in the mt-tRNAAla gene.  

Mitochondrial DNA variation and stress responses

Aurora Gomez-Duran

Centro de Investigaciones Biológicas Margarita Salas. CSIC. 28040, Madrid, Spain
Centro Singular de Investigación en Medicina Molecular y Enfermedades Crónicas. CiMUS. Universidade de Santiago de Compostela. 15782. A Coruña. 

Mitochondrial DNA (mtDNA) variants influence the risk of rare and late-onset human diseases, but the reasons for this are poorly understood. Interestingly, the same variant exerts a great variability in disease penetrance in each individual, which suggests the existence of a complex system that does not necessarily imply the dysfunction of the energy synthesis. In here, through the combination of multi-omics approaches on several human models, we will describe how variations in oxidative phosphorylation system capacity (OXPHOS) driven by the mtDNA variants activate different types of stress responses and their possible role in late-onset disease. We will further show how these findings can be applied to pharmacogenomic discovery and search of new biomarkers.

MitoKO: A library of base editors for the precise ablation of all protein-coding genes in the mouse mitochondrial genome

Pedro Silva-Pinheiro (1), Christian D. Mutti (1), Lindsey Van Haute (1), Christopher A. Powell (1), PavelA. Nash (1), Keira Turner (1) & Michal Minczuk (1)
1- MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK

Mitochondria are cellular organelles that have their own genome; mitochondrial DNA (mtDNA) encodes 13 subunits of the oxidative phosphorylation system, all of which are essential for the cell’s energy supply. Mutations in mtDNA can result in mitochondrial diseases, which are a group of incurable conditions affecting approximately 1 in 8,000 humans (1). MtDNA mutations have also been associated with common multifactorial diseases, metabolic disease, heart failure, cancer, neurodegeneration and ageing. Our progress towards understanding mitochondrial biology in health and disease has been hindered by the inability to precisely manipulate or modify mtDNA in mammalian mitochondria in living cells. Consequently, only a few cellular and animal models are available for researching mitochondrial biology. This limitation highlights the need to develop new approaches to manipulate the mitochondrial genome that will enable mtDNA dysfunction to be modelled in vitro and in vivo (2). DNA base editing was previously adapted to operate within mitochondria owing to the development of DddA-derived cytosine base editors (DdCBEs) (3). DdCBEs are based on a modified toxin called DddAtox, which is derived from the bacterium Burkholderia cenocepacia and has been separated into non-toxic halves fused to two adjacent programmable DNA-binding transcription activator-like effector (TALE) proteins that specifically determine the editing site. The resulting protein is targeted to the mitochondrial matrix to catalyse site-specific C to T conversions in mtDNA (or G to A in the opposite strand). We sought to use mitochondrial base editing to generate ‘MitoKO’ — a library of highly specific DdCBEs that can knockout (KO) every protein-coding gene of mouse mtDNA by introducing premature stop codons; our overall intention was to expand the limited repertoire of meaningful in vitro and in vivo models. Our strategy to introduce these premature stops codons was to use DdCBEs to convert TGA codons (encoding Trp (tryptophan)) present early in the mitochondrial gene into TAA codons (encoding STOP) by deaminating the C on the opposite (non-coding) strand (5′ TCA > 5′ TTA). We assembled a library of eight DdCBE designs per open reading frame and screened them in mouse cells for high ontarget editing and low off-target effects. Our approach resulted in a library of 13 specific DdCBEs capable of introducing high levels of truncating mutations into the open reading frames of mouse mtDNA, therefore knocking out each mtDNA-encoded protein-coding gene. We then applied our MitoKO library, generating KO cells in vitro and creating a mouse model with a truncating mtDNA mutation in Atp6 (the gene encoding ATP synthase subunit a) that perturbed the assembly of the ATP synthase complex; this feature is characteristic of some mitochondrial diseases. The MitoKO library should allow the systematic and comprehensive investigation of mtDNA-related pathways by providing an ‘off-the-shelf’ solution for researchers interested in establishing inactivating mutations in mouse mtDNA. It should also help researchers to investigate the fundamental processes that occur in mitochondria and their impact on organismal homeostasis, and to generate clinically meaningful in vivo models of mtDNA dysfunction for drug discovery and pre-clinical investigations.

References:
1. Gorman, G. S. et al. Mitochondrial diseases. Nat. Rev. Dis. Primers 2, 16080 (2016). A review article presenting an overview of mitochondrial diseases.
2. Silva-Pinheiro, P. & Minczuk, M. The potential of mitochondrial genome engineering. Nat. Rev. Genet. 23, 199–214 (2022). A review article that presents the advances of mitochondrial DNA engineering.
3. Mok, B. Y. et al. A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature 583, 631–637 (2020). This paper reports the development of the mitochondrial DNA base editor, DdCBE."

Mitochondria are organelles known as the “powerhouses” of the cells, as they are responsible for producing 90% of the energy necessary to maintain and support cell survival. When mitochondria fail to produce enough energy to sustain cell life, degeneration of the cells, the tissue and ultimately the organ occurs.

As the brain is a high energy demanding organ, our overarching goal is to clarify the intimate crosstalk between the host cell – the neuron – and the powerhouse organelle – the mitochondria. Mitochondria at the synapse have a pivotal role in neurotransmitter release, but almost nothing is known about synaptic mitochondria composition or specific functions. Synaptic mitochondria compared to mitochondria in other cells, need to cope with increased calcium load, more oxidative stress, and high energy demands that sustain neurotransmitter release and synaptic plasticity. At present, we have deciphered the intrinsic properties of synaptic mitochondria. For this, we have assessed the protein fingerprint, the metabolic profile and fuel preference of synaptic mitochondria. Ultimately, we intend to unveil how disruption of these synapse-specific mechanisms are relevant for a healthy brain, and to scrutinize their relevance in neurodegenerative disorders.

GEMIN5 and CoQ10, a link between neurodevelopment and mitochondrial metabolism

María Victoria Cascajo Almenara (1,3), Ana María Sánchez Cuesta (1,3), Daniel M. Fernández Ayala (1,3), Elena García Díaz (1), Rafael Artuch (2,4), and Carlos Santos Ocaña (1,3) 

1-Universidad Pablo de Olavide, Sevilla, Spain

2-Hospital San Joan de Deu, Barcelona, España

3-CIBERER U729, 

4-CIBERER U703 

Introduction

In 2006 a case of CoQ10 deficiency with cerebellar ataxia that improved after treatment with CoQ10 was published (1). However, a definitive molecular diagnosis could not be obtained. Recently, exome sequencing has shown that the patient carries a biallelic mutation in GEMIN5, a component of the SMN (Survival Motor Neuron) complex involved in transcriptional regulation (2). Searching for similar patients has allowed us to find a second patient already included in an extensive study of patients with cerebellar atrophy and delayed motor development (3) and a third patient who died a few months after birth.

Results

Patient fibroblasts show CoQ10 deficiency, decreased mitochondrial mass, and mitochondrial dysfunction manifested by low oxygen consumption, decreased activity of complexes II, III, and II+III of the respiratory chain, mtDNA depletion, and decreased expression of COQ proteins. Incorporating the 13C-labeled 4-HB precursor made it possible to determine a decrease in CoQ10 synthesis by LC-MS. The transcriptomic analysis in fibroblasts shows repression of mitochondrial biogenesis, fatty acid oxidation, Krebs cycle, OXPHOS activity, and skeletal muscle development. Transcriptional repression specifically affects genes encoding proteins required for CoQ10 synthesis, PPTC7 phosphatase, and transcription factors such as PGC-1α.

Functional validation of GEMIN5 deficiency was addressed in a silencing model of its orthologous gene, Rigor Mortis (rig), in D. melanogaster. Silencing increases mortality, increases the number of non-motile flies, decreases climbing ability, and decreases CoQ levels (CoQ8, CoQ9, and CoQ10). Analysis of larval development shows that silencing reduces the number of adult flies hatched.

In order to address the use of CoQ10 as a therapeutic strategy, CoQ10 was administered to fibroblasts from GEMIN5 patients achieving a recovery of total CoQ10 levels and oxygen consumption. In the fly model, CoQ10 supplementation increases only CoQ10 levels but no CoQ8 and CoQ9. However, it increases survival and restores the number of adult flies hatched during larval development to control levels.

Conclusions

Patient-derived fibroblasts show impaired mitochondrial function with a CoQ10 deficiency that can be termed secondary by current clinical criteria (no COQ genes are affected) but show some characteristics of a primary CoQ10 deficiency. In the D. melanogaster model, GEMIN5 silencing recapitulates the defects observed in patients, and as in patient fibroblasts, CoQ10 supplementation significantly restores these defects. Therefore, CoQ10 may constitute a suitable therapeutic strategy for treating neuro-disorders caused by defects in the GEMIN5 gene.

References

1. Artuch R, et al (2006). J. Neurol. Sci. 246: 153–158. 10.1016/j.jns.2006.01.021

2. Martinez-Salas E, et al (2020). Int. Journal Mol, Sci. 21: 3868. 10.3390/ijms21113868

3. Kour S, et al (2021). Nat Commun 12: 2558, 10.1038/s41467-021-22627-w 

Alterations in the contacts between endoplasmic reticulum and mitochondria in Bipolar disorder

Ana Catarina Pereira (1,2,3), Ana Patrícia Marques (1,3,4), Rosa Resende (1,3,4), Laura Serrano-Cuñarro (1), Mariana Batista (5), António Macedo (2,3,6,7), Cláudia Pais (8), Joana B. Melo (2,3,8,9), Nuno Madeira (2,3,6,7), Maria Teresa Cruz (1,3,10), Cláudia Cavadas (1,3,10), Cláudia Pereira (1,2,3) 

1-CNC - Center for Neuroscience and Cell Biology, CIBB - Centre for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal;

2-Faculty of Medicine, University of Coimbra, Coimbra, Portugal;

3-CACC - Clinical Academic Center of Coimbra, Coimbra, Portugal;

4-IIIUC - Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal;

5-CHUC - Centro Hospitalar e Universitário de Coimbra, Department of Dermatology, Coimbra, Portugal;

6-CHUC - Centro Hospitalar e Universitário de Coimbra, Department of Psychiatry Coimbra, Coimbra, Portugal;

7-CIBIT - Institute for Biomedical Imaging and Translational Research, University of Coimbra, Coimbra, Portugal;

8-Cytogenetics and Genomics Laboratory, Faculty of Medicine, University of Coimbra, Portugal;

9-iCBR - Coimbra Institute for Clinical and Biomedical Research, CIMAGO - Center of Investigation in Environment, Genetics and Oncobiology, Coimbra, Portugal;

10-Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal.  

Mitochondria-Associated Membranes (MAMs), which are the contact sites between the endoplasmic reticulum (ER) and mitochondria, are dynamic platforms that regulate several key processes, particularly mitochondrial bioenergetics and dynamics, as well as stress responses, due to the transfer of Ca2+ and lipids between both organelles. MAMs are modulated according to the cellular needs, and the impairment of these stress response structures has been linked to pathological conditions closely associated with mitochondrial dysfunction and ER stress, among other deleterious events. Since these molecular alterations have been closely associated with the structural brain changes and cognitive deficits reported in Bipolar disorder (BD), this study aimed to investigate the role of MAMs in BD physiopathology.

Dermal fibroblasts from BD patients versus healthy matched controls were used as an in vitro model of this mental illness. The ER-mitochondria coupling was assessed by confocal microscopy and transmission electron microscopy. Concomitantly, MAMs functional parameters such as Ca2+ fluxes, reactive oxygen species (ROS) production, and lipid droplets formation, were determined using the Fluo-4/Rhod-2, Mitopy/CellROX and LipidTOX fluorescent probes, respectively. Mitochondrial bioenergetics were evaluated by the Seahorse assay. Protein levels of ER stress-induced Unfolded Protein Response (UPR) markers were assessed by Western Blot, and autophagy was investigated by measuring the immunoreactivity of the autophagy-associated protein LC3B.

In BD patients-derived fibroblasts, it was observed an increase in the number of close ER-mitochondria contacts sites, or MAMs, which was shown to be correlated with functional alterations, such as deregulated ER-to-mitochondria Ca2+ transfer, enhanced ROS production, ER-to-mitochondria lipids transfer and formation of Lipid droplets, ER stress induction, as well as perturbation of mitochondrial metabolism and autophagy.

This study shows that the impaired communication between the ER and mitochondria at MAMs is implicated in BD physiopathology and might impact several crucial cellular and molecular events leading to the clinical manifestations of the pathology.

Acknowledgements: European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Programme under project CENTRO-01-0145-FEDER-000012 (HealthyAging2020) and through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia, under projects POCI-01-0145-FEDER-028214 (MAM4BD) and POCI-01-0145-FEDER-029369 and UIDB/04539/2020 and UIDP/04539/2020. PhD fellowship from FCT (SFRH/BD/148653/2019) to Ana Catarina Pereira. European Social Fund (Post-Doctoral Researcher Contract SFRH/BPD/101028/2014 to Rosa Resende). 

Coenzyme A (CoA) is a key metabolic cofactor in all living cells. CoA and its thioester derivatives (acetyl-CoA, malonyl-CoA, HMG-CoA etc.) participate in diverse anabolic and catabolic pathways, allosteric interactions, biosynthesis of neurotransmitters and the regulation of gene expression. Dysregulation of CoA/CoA derivatives biosynthesis and homeostasis has been associated with various human pathologies, including cancer and neurodegeneration and metabolic disorders.

We have recently discovered a novel mode of redox regulation, involving covalent modification of cellular proteins by CoA (protein CoAlation) in cellular response to oxidative or metabolic stress. To discover and study this novel post-translational modification, we have developed several novel reagents and methodologies, including: (a) anti-CoA mAb, which specifically recognize CoA in ELISA, WB, IP and IHC (no anti-CoA antibodies are commercially available); (b) a robust mass spectrometry-based methodology for the identification of CoAlated proteins; and (c) efficient in vitro CoAlation and deCoAlation assays. Cell-based and animal models have been employed to demonstrate that protein CoAlation is a reversible and widespread post-translational modification induced by oxidizing agents and metabolic stress in prokaryotic and eukaryotic cells. To date, we have identified using the developed methodology more than 2100 proteins, which are CoAlated under various experimental conditions in cell and tissues. We showed that protein CoAlation modulates the activity and subcellular localization of modified proteins. It can also protect oxidized cysteine residues from overoxidation and induce significant conformational changes. We have employed biochemical, biophysical, crystallographic and cellular approaches to study the mode of CoA binding to a panel of selected metabolic enzymes and signalling proteins. Over the last 5 years we have firmly established ourselves as a world-leading laboratory on protein CoAlation and the antioxidant function of CoA in health and pathologies associated with oxidative stress, including cancer and neurodegeneration.

Recent progress on molecular dissection of the CoAlation/deCoAlation cycle and investigating the antioxidant function of CoA in pathologies associated with oxidative stress will be presented. 

Mitochondrial dynamics as a hub in the control of muscle inflammation

Andrea Irazoki (1,2,3), Isabel Gordaliza-Alaguero (1, 2, 3), Emma Frank (4), Nikolaos Nikiforos Giakoumakis (1), Jordi Seco (1), Manuel Palacín (1, 2, 5), Anna Gumà (2,3,6), Lykke Sylow (4,7), David Sebastián (1,2,3*) and Antonio Zorzano (1,2,3*) 

1-Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Baldiri Reixac, 10-12, 08028 Barcelona, Spain. 2-Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, 08028 Barcelona, Spain. 3-Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) Instituto de Salud Carlos III, Spain. 4-Department of Biomedical Sciences (BMI), University of Copenhagen, Denmark, 5-CIBER de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Spain. 6-Institute of Biomedicine of the University of Barcelona (IBUB), Barcelona, Spain. 7-Department of Nutrition, Exercise and Sports, University of Copenhagen, Copenhagen, Denmark. *Corresponding and supervising authors.

Some forms of mitochondrial dysfunction induce sterile inflammation through mitochondrial DNA (mtDNA) recognition by intracellular DNA sensors. However, the involvement of mitochondrial dynamics mitigating such processes and their impact on muscle fitness remain unaddressed. Here we report that opposite mitochondrial morphologies induce distinct inflammatory signatures, caused by differential activation of DNA sensors TLR9 or cGAS. In the context of mitochondrial fragmentation, we demonstrate that mitochondria-endosome contacts mediated by the endosomal protein RAB5C are required in TLR9 activation in cells. Skeletal muscle mitochondrial fragmentation promotes TLR9-dependent inflammation, muscle atrophy, reduced physical performance and enhanced IL6 response to exercise, which improved upon chronic anti-inflammatory treatment. Taken together, our data demonstrate that mitochondrial dynamics is key in preventing sterile inflammatory responses, which precede the development of muscle atrophy and impaired physical performance. Thus, we propose the targeting of mitochondrial dynamics as an approach to treating disorders characterized by chronic inflammation and mitochondrial dysfunction.

Non-canonical nitrogen metabolism in Fumarate Hidratase-deficient cells. 

Marc Segarra-Mondejar (1), Ming Yang (1), Agnieszka Sokol (1), Christian Frezza (1) 

1- CECAD Research Centre, University of Cologne, Cologne, Germany

Fumarate hydratase (FH) is the enzyme in the Tricarboxylic Acid (TCA) Cycle that catalyses the reversible conversion of succinate to fumarate. Germline mutations of FH has been shown to predispose to leiomyomatosis and renal cancer (HLRCC), a cancer syndrome characterized by the development of benign skin and uterine lesions and of an aggressive form of type II papillary renal cell cancer (pRCC2).

We have shown that FH-deficient cells depend on glutamine as the main source of carbons for the TCA cycle and for the synthesis a glutathione, via glutamate, to compensate for the increased oxidative stress that we reported. This reaction is usually catalysed by glutaminase (GLS), which release glutamine amide group in the form of NH4+, a toxic metabolite. However, how FH-deficient cells overcome ammonia toxicity is currently unknown.

Here we found that FH-deficient mouse cells depend on de novo nucleotide and asparagine synthesis pathways to maintain high rates of glutamine catabolism minimising ammonia production. Of note, despite exhibiting a reduction in total pyrimidine levels, FH-deficient cells exhibit a significant increase in early intermediate in pyrimidine synthesis, including carbamoyl-aspartate, and a higher incorporation of glutamine-derived nitrogen into GMP and IMP, and into asparagine. Since all these pathways use the amide-group of glutamine as a source of nitrogen, we conclude that FH-deficient cells rely on transamidases as an alternative mechanism of glutamine deamination. Our results shed some light into FH-deficient tumour metabolism and indicate transamidases inhibitors as a possible therapy against one of the most aggressive forms of kidney cancer.

AntiOxCIN4 protects against brain and skeletal muscle oxidative/nitrosative stress in the amyotrophic lateral sclerosis SOD1G93A mouse

Débora Mena (1,2,3), Fernando Cagide (4), Sofia Benfeito (4), Pedro Soares (4), Luís Grilo (1,2,3), Daniela F. Silva (1), Paulo Pinheiro (1), Elisabete Ferreiro (1,2), José Teixeira (1,2), Filomena Silva (1), Fernanda Borges (4), Paulo J. Oliveira (1,2,3), Ana I. Duarte (1,2,3*) 

1- CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal;

2- CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal;

3- IIIUC - Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal;

4- CIQUP-IMS/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Porto, Portugal.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease. It is characterized by progressive motor neuron loss, muscle weakness, atrophy and paralysis, ultimately leading to death. Although ALS pathophysiology remains poorly understood, evidence suggests a pivotal role for mitochondrial dysfunction and oxidative stress. The currently available treatments for ALS are only symptomatic and with limited efficacy. Hence, the improvement of mitochondrial function and/or antioxidant capacity may constitute potential therapeutic approaches to delay ALS progression. We hypothesized that the mitochondria-targeted antioxidant AntiOxCIN4 can counteract brain and skeletal muscle oxidative/nitrosative stress in SOD1G93A ALS mice.

Early adult SOD1G93A ALS mice were subcutaneously injected with AntiOxCIN4 (0.1 mg/Kg/day), for 2 months. We obtained brain cortical and skeletal muscle homogenates and used colorimetry/fluorimetry-based methods to assess the effect of AntiOxCIN4 in oxidative/nitrosative stress markers nitrites and hydroperoxides, as well for measuring the activities of the antioxidant enzymes superoxide dismutase (total SOD, SOD-2), and glutathione peroxidase (GPx).

We observed that AntiOxCIN4 upregulates brain GPx activity (P=0.07), decreases skeletal muscle hydroperoxides (P=0.009), and brain and skeletal muscle nitrites levels (P=0.002, P0.001, respectively) in ALS mice. This was accompanied by a slight increase in brain total SOD and SOD-2 activities, as well as by a reduction in total SOD and SOD-2 activities in skeletal muscle (P=0.07, P=0.08, respectively) of AntiOxCIN4-treated ALS mice.

Our results suggest that the peripheral administration of AntiOxCIN4 may counteract ALS brain and skeletal muscle oxidative/nitrosative stress. However, further studies are needed to determine whether this protection delays ALS progression.

This work was funded by European Regional Development Fund: Centro2020 Operational Programme (POCI-01-0145-FEDER-029391 (Mito4ALS)), COMPETE 2020 and FCT- Fundação para a Ciência e a Tecnologia (POCI-01-0145-FEDER-029391, PTDC/MED-FAR/29391/2017, EXPL/BTM-TEC/1407/2021, UIDB/04539/2020, UIDP/04539/2020, LA/P/0058/2020, UIDB/00081/2020); European Social Fund: 2021.04707.BD and Mito4ALS-PTDC/MED-FAR/29391/2017 of D.M.; FCT and DL57/2016 of F.C.; DL57/2016 SFRH/BPD/84473/2012 of A.I.D.; FCT/P2020/COMPETE PTDC/MED-QUI/29164/2017 and PT-OPENSCREEN-NORTE-01-0145-FEDER-085468 of S.B.; EU Horizon2020 R&I Program/Marie Sklodowska-Curie grant 895144 of P.S.; FCT Contract 2020.01560.CEECIND of J.T.; SFRH/BD/5539/2020 of L.G. 

Mitochondrial replication at the periphery of neurons requires eEF1A1 and local translation of nuclear-encoded proteins

Carlos Cardanho-Ramos (1), Rúben A. Simões (1), Andreia Faria-Pereira (1), Vanessa A. Morais (1) 

1- Instituto de Medicina Molecular - João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Portugal

Neurons rely on mitochondria for ATP production and Ca2+ homeostasis, particularly at the synapse. It is assumed that mitochondria are generated in the cell body and travel to the synapse to exert their functions. However, considering the rate of mitochondrial transport in neurons, the time it would take for a single mitochondrion to travel from the cell body to the synapse exceeds the half-life of most mitochondrial proteins. Deficits in mitochondrial replication have been linked to several neurodegenerative diseases, such as Parkinson’s and Huntington’s disease, however whether mitochondria can replicate locally in the periphery of neurons is still unknown.

We developed a technique to assess mitochondrial replication in mouse primary neuron cultures using EdU-labelling, a thymidine analogue that is incorporated into mtDNA upon replication. Most EdU staining was observed in the cell body, but staining was also present to a lower extent in the periphery. Using microfluidic devices, where axons can be isolated from the cell body, we were able to add EdU only to the axonal side, without interfering with the cell body. In these conditions, EdU staining was only present in axons, confirming that mitochondrial replication in neurons can occur away from the cell body. We hypothesized that mRNA and local translation must be at place in the periphery of neurons in order to provide all the necessary machinery for mitochondria to replicate. To test this, we assessed mitochondrial replication upon inhibition of both nuclear-encoded and mitochondrial-encoded protein translation. Mitochondrial replication in neurons decreased when nuclear-encoded protein translation was inhibited. However, no differences were observed upon inhibition of mitochondrial-encoded protein translation.

Taking advantage of a proteomic screen comparing synaptic with non-synaptic mitochondria, two candidate proteins related with protein translation were found upregulated in the synaptic fraction – eEFF1a1, involved in nuclear-encoded protein translation; and TUFM, involved in mitochondrial-encoded protein translation. We performed loss and gain of function assays with our candidate proteins and assessed their impact on mitochondrial replication. When eEF1A1 was downregulated, we observed a decrease in mitochondrial replication in the periphery of neurons. This effect was rescued by re-introducing eEF1A1. Regarding TUFM, no differences were observed.

Our results confirm that mitochondrial replication can occur in the periphery of neurons, and that this process requires nuclear-encoded protein translation, mediated by eEF1A1. Understanding how mitochondrial replication occurs in neurons, particularly at the level of the synapse, provides novel lines of research to tackle the pathological mechanisms underlying neurodegenerative diseases.  

Mitochondria damage-associated molecular patterns associated to extracellular microvesicles promote microglia-mediated proinflammatory response

Henrique Tavares (1), Cláudia M. Deus (1), Margarida Beatriz  (1), Paulo Oliveira(1), Carla Lopes (1,2) 

1- CNC – Center for Neuroscience and Cell Biology, CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal.

2- Multidisciplinary Institute of Ageing, MIA – Portugal, University of Coimbra; Portugal

Mitochondrial and autophagy dysfunction play important roles in the pathogenesis of neurodegenerative diseases. Mitochondrial quality control mechanisms (QCM) are essential to maintain the mitochondrial structure and function. However, these mechanisms can be disrupted by the impairment of the mitochondrial-lysosomal axis [1]. Mitochondria-derived vesicles (MDVs) can dispose mildly damaged mitochondrial components in a complementary degradative pathway [2]. MDVs are particularly enriched in mitochondria-associated damage-associated molecular patterns (mitDAMPs) which can be released into the extracellular space as extracellular vesicles (EVs) and induce microglia-mediated immune response.

We here evaluated if an impairment of the mitochondrial-lysosomal axis, in normal human dermal fibroblasts or in fibroblasts from sporadic Parkinson Disease (PD), regulate the extracellular release of MDV and induce a proinflammatory microglial profile. Moreover, we evaluated if treating fibroblasts with AntiOxCIN4, a mitochondria-targeted antioxidant, can prevent this phenotype.

Microglia was stimulated with cell-free mitochondria, mtDNA, EVs and EV-isolated DNA from AntiOxCIN4-treated and non-treated fibroblasts.

Fibroblasts treated with FCCP/Chloroquine (CQ) showed increased mitochondrial fission and reactive oxygen species levels along with decreased mitochondrial biogenesis factors and mitochondrial DNA copies. Also, the inhibition of the mitochondrial-lysosomal axis resulted in an increase of EV secretion, with higher mtDNA copies and increased DNA oxidation (8-OHdG levels). An alteration in microglia transcriptional profile for several inflammatory factors (IL-1β, TNF-α, Arg1, and HMGB1) and NF-kβ activation was measured upon treatment with different stimuli. FCCP/CQ-treated fibroblasts displayed greater oxidative DNA damage and mediated the most significant mRNA transcriptional fold changes in microglia. AntiOxCIN4 reverted the mitochondrial abnormalities found in FCCP/CQ-treated cells and decreased the number of mtDNA copies associated to EVs. We observed an increase in the EVs-associated mtDNA copies in PD fibroblasts, although no alteration was observed in cells. Surprisingly, EVs isolated from PD fibroblasts treated with AntiOxCIN4 appear to induce a lower microglia proinflammatory response.

Our data highlighted how a dysfunctional mitochondrial-lysosomal axis may influence EVs role as a trigger for microglia activation, associating mitochondria dysfunction, EV secretion and mitochondria-driven immune responses.

European Regional Development Fund (ERDF), through the COMPETE2020–Operational Programme for Competitiveness and Internationalization and Portuguese national funds via FCT- Fundação para a Ciência e a Tecnologia, under project UIDB/04539/2020, UIDP/04539/2020 and LA/P/0058/2020.

1. Beatriz, M., et al., Journal of Extracellular Biology, 2022. 1(10): p. e65.

2. Soubannier, V., et al., Curr Biol, 2012. 22(2): p. 135-41.

Defective autophagy masquerading as mitochondrial disease

 Kritarth Singh and Michael R Duchen 

UCL Consortium for Mitochondrial Research and Department of Cell and Developmental Biology, University College London, London UK

Vici Syndrome, a congenital disease of childhood is caused by mutations of a protein known as EPG5 [1], a protein which is important in mediating autophagosome/lysosome fusion – a key step in autophagy and mitophagy [2].  This multisystem disorder includes developmental defects involving the heart, CNS and immune system, but also involves progressive neurodegeneration and epilepsy. Most affected children do not survive into teenage years and there is no treatment.  We have asked whether defective autophagy/mitophagy leads to significant mitochondrial dysfunction, to what extent this might contribute to the presentation and progression of the disease and whether the mechanisms involved might present novel therapeutic targets.

We have found that in fibroblasts from children with Vici syndrome, mitochondrial function is profoundly disordered, with impaired respiratory chain function and reduced membrane potential. We have also found that mitochondrial calcium handling is abnormal, with unexpected changes in the expression of components of the calcium uptake machinery that lead to mitochondrial calcium overload and increased vulnerability to opening of the permeability transition pore (mPTP). Remarkably, some children have been misdiagnosed with mitochondrial disease before genomic identification of an EPG5 mutation [3]. Indeed, Vici syndrome appears to a paradigm for an increasingly recognised group of neurodevelopmental and neurodegenerative disorders involving impaired autophagy [4]. It seems plausible that affected children may benefit from pharmacological interventions targeting mitochondrial function, especially the mPTP or autophagy, and preliminary data suggest that pharmacological stimulation of autophagy in the cell model leads to improved bioenergetic function.

References

1 - Byrne S, ……., Fanto M, Gautel M, Jungbluth H et al  EPG5-related Vici syndrome: a paradigm of neurodevelopmental disorders with defective autophagy., Brain. 2016 Mar;139(Pt 3):765-81.

2 - Wang Z, Miao G, Xue X, Guo X, Yuan C, Wang Z, Zhang G, Chen Y, Feng D, Hu J, Zhang H. The Vici Syndrome Protein EPG5 Is a Rab7 Effector that Determines the Fusion Specificity of Autophagosomes with Late Endosomes/Lysosomes. Mol Cell. 2016 Sep 1;63(5):781-95.

3 - Deneubourg C, Ramm M, Smith LJ, Baron O, Singh K, Byrne SC, Duchen MR, Gautel M, Eskelinen EL, Fanto M, Jungbluth H The spectrum of neurodevelopmental, neuromuscular and neurodegenerative disorders due to defective autophagy. Autophagy. 2022 Mar;18(3):496-517. 

4 - Balasubramaniam S, Riley LG, Vasudevan A, Cowley MJ, Gayevskiy V, Sue CM, Edwards C, Edkins E, Junckerstorff R, Kiraly-Borri C, Rowe P, Christodoulou J  EPG5-Related Vici Syndrome: A Primary Defect of Autophagic Regulation with an Emerging Phenotype Overlapping with Mitochondrial Disorders. JIMD Rep. 2018;42:19-29. doi: 10.1007/8904_2017_71.