bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2022‒07‒03
forty-four papers selected by
Catalina Vasilescu
University of Helsinki

  1. Methods Mol Biol. 2022 ;2497 243-254
      I describe here a protocol for the analysis of mitochondrial protein synthesis as a useful tool to characterize the mitochondrial defects associated with mutations in mitochondrial tRNA genes. The yeast Saccharomyces cerevisiae mutants, bearing human equivalent pathogenic mutations, were used as a simple model for analysis. The mitochondrial proteins were labeled by L[35S]-methionine incorporation in growing cells, extracted from purified mitochondria, and fractionated by SDS-polyacrylamide gel electrophoresis followed by autoradiography. By this method, it is possible to distinguish different protein synthesis profiles in the analyzed mitochondrial tRNA mutants.
    Keywords:  Human equivalent mutations; In vivo L[35S]-methionine labeling; Mitochondria; Mitochondrial protein synthesis; Mitochondrial tRNA mutants; Saccharomyces cerevisiae
  2. Methods Mol Biol. 2022 ;2497 339-348
      Blue Native polyacrylamide gel electrophoresis (BN-PAGE) is a well-established technique for the isolation and separation of mitochondrial membrane protein complexes in a native conformation with high resolution. In combination with histochemical staining methods, BN-PAGE has been successfully used as clinical diagnostic tool for the detection of oxidative phosphorylation (OXPHOS) defects from small tissue biopsies from patients with primary mitochondrial disease. However, its application to patient-derived primary fibroblasts is difficult due to limited proliferation and high background staining. Here, we describe a rapid and convenient method to analyze the organization and activity of OXPHOS complexes from cultured skin fibroblasts.
    Keywords:  In-gel activity; Mitochondria; Oxidative phosphorylation; Primary fibroblasts; Supercomplex
  3. Methods Mol Biol. 2022 ;2497 97-106
      Mitochondrial calcium (Ca2+) plays a key role in regulating normal cardiac function. A physiological increase in mitochondrial matrix calcium [Ca2+]m drives mitochondrial ATP production to meet the high-energy demands during excitation-contraction coupling. However, a pathological increase in [Ca2+]m leads to increased oxidative stress, impaired bioenergetics, and the opening of mitochondrial permeability transition pore (mPTP), a hallmark of the failing heart. Therefore, a better understanding of the [Ca2+]m handling and its role in heart function and dysfunction is of great importance. Here, we describe a detailed protocol for measuring mitochondrial Ca2+ handling in the isolated functionally intact mitochondria from cardiac tissue of the guinea pig.
    Keywords:  Calcium retention capacity; Mitochondrial bioenergetics; Mitochondrial calcium handling; Mitochondrial membrane potential; Mitochondrial oxygen consumption
  4. Methods Mol Biol. 2022 ;2515 17-28
      Mitochondria are dynamic organelles that rely on a balance of opposing fission and fusion events to sustain mitochondrial function and efficiently meet the energy demands of a cell. As high-energy demanding cells, neurons rely heavily on optimally functional mitochondria with balanced mitochondrial dynamics, to ensure a sufficient energy supply required to maintain cell survival, establish membrane excitability and partake in processes of neurotransmission and plasticity. As such, many neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease) and stress conditions (e.g., stroke) leading to neuronal dysfunction or death are often associated with impaired mitochondrial function and dynamics, characterized by excessive mitochondrial fragmentation. For this reason, the assessment of mitochondrial morphology in neurons and within the brain can provide valuable information. The dynamic nature of mitochondria is not only observed in shape changes, but also changes in mitochondrial network connectivity and in cristae architecture. In this chapter, we will describe how mitochondrial morphology can be examined in vitro using hippocampal neuronal cultures and in vivo using mouse brain sections by immunocytochemistry, immunohistochemistry, and electron microscopy techniques.
    Keywords:  Confocal and electron microscopy; Cristae; Hippocampus; Mitochondrial dynamics; Mitochondrial dysfunction; Mitochondrial fission; Mitochondrial fusion; Mitochondrial morphology; Neurodegenerative diseases; Neuronal cultures
  5. Radiol Case Rep. 2022 Sep;17(9): 2915-2918
      Children with mitochondrial disease may present with diabetes mellitus (DM) without autoimmune antibodies as an initial manifestation, however, it is difficult to make a precise diagnosis in early stages. We present a 2-year-old male patient with mitochondrial disease who showed insulin-dependent DM without autoimmune antibodies as an initial symptom. He later presented with progressive motor deterioration, hearing disability, ptosis, external ophthalmoplegia, and retinitis pigmentosa at 6 years and 6 months. T2- and diffusion-weighted imaging revealed high signal lesions in the subcortical white matter, anterior thalamus, globus pallidus, and brainstem. MR spectroscopy showed elevated lactate and low N-acetylaspartate in the affected white matter. Genetic analysis revealed a single large-scale mitochondrial DNA deletion at 7117-13994, leading to a diagnosis of mitochondrial DNA deletion syndrome associated with insulin-dependent DM. Although the frequency of DM in pediatric mitochondrial disease is low, mitochondrial disease, especially due to mitochondrial DNA deletion, should be considered as a differential diagnosis in those with insulin-dependent DM without autoimmune antibodies, and MRI and MR spectroscopy are recommended for an early diagnosis.
    Keywords:  Diabetes mellitus; MR spectroscopy; MRI; Mitochondrial DNA deletion syndrome; Mitochondrial disease
  6. Methods Mol Biol. 2022 ;2497 291-299
      The ubiquinone (Q) pool represents a node in the mitochondrial electron transport chain (ETC) onto which the electrons of all respiratory dehydrogenases converge. The redox state of the Q pool correlates closely with the electron flux through the ETC and is thus a parameter of great metabolic value for both the mitochondrial and cellular metabolism. Here, we describe the simultaneous measurement of respiratory rates of isolated mouse heart mitochondria and the redox state of their Q pool using a custom-made combination of a Clark-type oxygen electrode and a Q electrode.
    Keywords:  Mitochondria; Redox state; Respiratory rates; Ubiquinone pool
  7. Methods Mol Biol. 2022 ;2497 221-242
      Numerous diseases in humans have been associated with mutations of the mitochondrial genome (mtDNA). This genome encodes 13 protein subunits of complexes involved in oxidative phosphorylation (OXPHOS), a process that provides aerobic eukaryotes with the energy-rich adenosine triphosphate molecule (ATP). Mutations of the mtDNA may therefore have dramatic consequences especially in tissues and organs with high energy demand. Evaluating the pathogenicity of these mutations may be difficult because they often affect only a fraction of the numerous copies of the mitochondrial genome (up to several thousands in a single cell), which is referred to as heteroplasmy. Furthermore, due to its exposure to reactive oxygen species (ROS) produced in mitochondria, the mtDNA is prone to mutations, and some may be simply neutral polymorphisms with no detrimental consequences on human health. Another difficulty is the absence of methods for genetically transforming human mitochondria. Face to these complexities, the yeast Saccharomyces cerevisiae provides a convenient model for investigating the consequences of human mtDNA mutations in a defined genetic background. Owing to its good fermentation capacity, it can survive the loss of OXPHOS, its mitochondrial genome can be manipulated, and genetic heterogeneity in its mitochondria is unstable. Taking advantage of these unique attributes, we herein describe a method we have developed for creating yeast models of mitochondrial ATP6 gene mutations detected in patients, to determine how they impact OXPHOS. Additionally, we describe how these models can be used to discover molecules with therapeutic potential.
    Keywords:  ATP synthase; Drug screening; MT-ATP6 gene; Mitochondrial DNA mutations; Mitochondrial diseases; Mitochondrial transformation; Yeast
  8. Mol Genet Metab Rep. 2022 Sep;32 100887
      Mitochondrial DNA (mtDNA) depletion syndromes are disorders characterized by infantile-onset, severe progression, and the drastic loss of mtDNA content in affected tissues. In a patient who showed severe hypotonia, proximal tubulopathy and sensorineural hearing loss after birth, we observed severe mtDNA depletion and impaired respiratory chain activity in muscle due to heterozygous variants c.686G > T and c.551-2A > G in RRM2B, encoding the p53R2 subunit of the ribonucleotide reductase.
    Keywords:  Mitochondrial DNA depletion; Mitochondrial encephalomyopathy; Next generation sequencing; RRM2B; Ribonucleotide reductase
  9. Methods Mol Biol. 2022 ;2497 107-115
      The mitochondrial respiratory chain which carries out the oxidative phosphorylation (OXPHOS) consists of five multi-subunit protein complexes. Emerging evidences suggest that the supercomplexes which further consist of multiple respiratory complexes play important role in regulating OXPHOS function. Dysfunction of the respiratory chain and its regulation has been implicated in various human diseases including neurodegenerative diseases and muscular disorders. Many mouse models have been established which exhibit mitochondrial defects in brain and muscles. Protocols presented here aim to help to analyze the structures of mitochondrial respiratory chain which include the preparation of the tissue samples, isolation of mitochondrial membrane proteins, and analysis of their respiratory complexes by Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) in particular.
    Keywords:  Assembly; Blue Native Gel; Brain; Muscle; Respiratory complex
  10. Mol Cell. 2022 Jun 16. pii: S1097-2765(22)00540-8. [Epub ahead of print]
      Protein import into mitochondria is a highly regulated process, yet how cells clear mitochondria undergoing dysfunctional protein import remains poorly characterized. Here we showed that mitochondrial protein import stress (MPIS) triggers localized LC3 lipidation. This arm of the mitophagy pathway occurs through the Nod-like receptor (NLR) protein NLRX1 while, surprisingly, without the engagement of the canonical mitophagy protein PINK1. Mitochondrial depolarization, which itself induces MPIS, also required NLRX1 for LC3 lipidation. While normally targeted to the mitochondrial matrix, cytosol-retained NLRX1 recruited RRBP1, a ribosome-binding transmembrane protein of the endoplasmic reticulum, which relocated to the mitochondrial vicinity during MPIS, and the NLRX1/RRBP1 complex in turn controlled the recruitment and lipidation of LC3. Furthermore, NLRX1 controlled skeletal muscle mitophagy in vivo and regulated endurance capacity during exercise. Thus, localization and lipidation of LC3 at the site of mitophagosome formation is a regulated step of mitophagy controlled by NLRX1/RRBP1 in response to MPIS.
    Keywords:  NLRX1; Nod-like receptors; mitochondria; mitochondrial protein import; mitophagy
  11. J Biol Chem. 2022 Jun 24. pii: S0021-9258(22)00638-X. [Epub ahead of print] 102196
      In human cells, ATP is generated using oxidative phosphorylation machinery, which is inoperable without proteins encoded by mitochondrial DNA (mtDNA). The DNA polymerase gamma (Polγ) repairs and replicates the multicopy mtDNA genome in concert with additional factors. The Polγ catalytic subunit is encoded by the POLG gene, and mutations in this gene cause mtDNA genome instability and disease. Barriers to studying the molecular effects of disease mutations include scarcity of patient samples and a lack of available mutant models; therefore, we developed a human SJCRH30 myoblast cell line model with the most common autosomal dominant POLG mutation, c.2864A>G/p.Y955C, as individuals with this mutation can present with progressive skeletal muscle weakness. Using on-target sequencing, we detected a 50% conversion frequency of the mutation, confirming heterozygous Y955C substitution. We found mutated cells grew slowly in a glucose-containing medium and had reduced mitochondrial bioenergetics compared to the parental cell line. Furthermore, growing Y955C cells in a galactose-containing medium to obligate mitochondrial function enhanced these bioenergetic deficits. Also, we show complex I NDUFB8 and ND3 protein levels were decreased in the mutant cell line, and the maintenance of mtDNA was severely impaired (i.e., lower copy number, fewer nucleoids, and an accumulation of Y955C-specific replication intermediates). Finally, we show the mutant cells have increased sensitivity to the mitochondrial toxicant 2'-3'-dideoxycytidine. We expect this POLG Y955C cell line to be a robust system to identify new mitochondrial toxicants and therapeutics to treat mitochondrial dysfunction.
    Keywords:  2′-3′-dideoxycytidine (ddC, zalcitabine); Mitochondrial DNA (mtDNA) maintenance; POLG c.2864A>G/p.Y955C; SJCRH30; autosomal dominant progressive external ophthalmoplegia (adPEO); cell line model of mitochondrial disease; mitochondrial toxicity
  12. Neurobiol Dis. 2022 Jun 25. pii: S0969-9961(22)00195-4. [Epub ahead of print] 105803
      INTRODUCTION: Mitochondrial defects have been implicated in Parkinson's disease (PD) since complex I poisons were found to cause accelerated parkinsonism in young people in the early 1980s. More evidence of mitochondrial involvement arose when many of the genes whose mutations caused inherited PD were discovered to be subcellularly localized to mitochondria or have mitochondrial functions. However, the details of how mitochondrial dysfunction might impact or cause PD remain unclear. The aim of our study was to better understand mitochondrial dysfunction in PD by evaluating mitochondrial respiratory complex mutations in a Drosophila melanogaster (fruit fly) model of PD.METHODS: We have conducted a targeted heterozygous enhancer/suppressor screen using Drosophila mutations within mitochondrial electron transport chain (ETC) genes against a null PD mutation in parkin. The interactions were assessed by climbing assays at 2-5 days as an indicator of motor function. A strong enhancer mutation in COX5A was examined further for L-dopa rescue, oxygen consumption, mitochondrial content, and reactive oxygen species. A later timepoint of 16-20 days was also investigated for both COX5A and a suppressor mutation in cyclope. Generalized Linear Models and similar statistical tests were used to verify significance of the findings.
    RESULTS: We have discovered that mutations in individual genes for subunits within the mitochondrial respiratory complexes have interactions with parkin, while others do not, irrespective of complex. One intriguing mutation in a complex IV subunit (cyclope) shows a suppressor rescue effect at early time points, improving the gross motor defects caused by the PD mutation, providing a strong candidate for drug discovery. Most mutations, however, show varying degrees of enhancement or slight suppression of the PD phenotypes. Thus, individual mitochondrial mutations within different oxidative phosphorylation complexes have different interactions with PD with regard to degree and direction. Upon further investigation of the strongest enhancer (COX5A), the mechanism by which these interactions occur initially does not appear to be based on defects in ATP production, but rather may be related to increased levels of reactive oxygen species.
    CONCLUSIONS: Our work highlights some key subunits potentially involved in mechanisms underlying PD pathogenesis, implicating ETC complexes other than complex I in PD.
    Keywords:  COX5A; Cyclope; Drosophila melanogaster; Fruit flies; Mitochondria; Oxidative phosphorylation; PINK1; Parkin; Parkinson's disease; electron transport chain
  13. Methods Mol Biol. 2022 ;2497 325-332
      Mitochondrial Ca2+ buffering is a hallmark of eukaryotic cellular physiology, contributing to the spatiotemporal shaping of the cytosolic Ca2+ signals and regulation of mitochondrial bioenergetics. Often, this process is altered in a pathological context; therefore, it can be scrutinized experimentally for therapeutic intervention. In this chapter, we describe fluorescence and bioluminescence measurement of mitochondrial Ca2+ in both isolated mitochondria and intact cells.
    Keywords:  Bioluminescence calcium sensing; Calcium-sensitive genetic probes; Fluorescence calcium imaging; Mitochondrial calcium
  14. Methods Mol Biol. 2022 ;2497 1-10
      Assessment of mitochondrial metabolism is multidimensional and time consuming, usually requiring specific training. Respiration, NADH generation, and mitochondrial membrane potential (ΔΨm) are dynamic readouts of the metabolism and bioenergetics of mitochondria. Methodologies available to determine functional parameters in isolated mitochondria and permeabilized cells are sometimes of limited use or inapplicable to studies in live cells. In particular, the sequential assessment of the activity of each complex in the electron transport chain has not been reported in intact cells. Here, we describe a novel approach to sequentially assess electron flow through all respiratory complexes in permeabilized and intact cells by respirometry. We also describe a highly sensitive and fast method to assess ΔΨm and NADH generation in live cells using plate reader assays. Thus, our combined method allows a relatively inexpensive and fast determination of three major readouts of mitochondrial function in a few hours, using equipment that is frequently available in many laboratories worldwide.
    Keywords:  Electron transport chain; Mitochondria; Mitochondrial membrane potential; Mitochondrial metabolism; NADH; Oxygen consumption; Respiratory complex; TMRM; Warburg Metabolism
  15. Methods Mol Biol. 2022 ;2497 255-267
      Proving with certainty that a GFP-tagged protein is imported inside mitochondria by visualizing its fluorescence emission with an epifluorescence microscope is currently impossible using regular GFP-tagging. This is particularly true for proteins dual localized in the cytosol and mitochondria, which have been estimated to represent up to one third of the established mitoproteomes. These proteins are usually composed of a surpassingly abundant pool of the cytosolic isoform compared to the mitochondrial isoform. As a consequence, when tagged with a regular GFP, the fluorescence emission of the cytosolic isoform will inevitably eclipse that of the mitochondrial one and prevent the detection of the mitochondrial echoform. To overcome this technical limit, we engineered a yeast strain expressing a new type of GFP called Bi-Genomic Mitochondrial-Split-GFP (BiG Mito-Split-GFP). In this strain, one moiety of the GFP is encoded by the mitochondrial DNA while the second moiety of the GFP can be tagged to any nuclear-encoded protein (suspected to be dual localized or bona fide mitochondrial). By doing so, only mitochondrial proteins or echoforms of dual localized proteins, regardless of their organismal origin, trigger GFP reconstitution that can be visualized by regular fluorescence microscopy. The strength of the BiG Mito-Split-GFP system is that proof of the mitochondrial localization of a given protein rests on a simple and effortless microscopy observation.
    Keywords:  BiG Mito-Split-GFP; Dual localized; Epifluorescence microscopy; Living cells; Localization; Mitochondria; Saccharomyces cerevisiae
  16. Sci Rep. 2022 Jun 30. 12(1): 11099
      Several mitochondrial DNA (mtDNA) mutations of Leber's hereditary optic neuropathy (LHON) have been reported in patients with multiple sclerosis (MS) from different ethnicities. To further study the involvement of LHON mtDNA mutations in MS in the Arab population, we analyzed sequencing data of the entire mitochondrial genome from 47 unrelated Saudi individuals, 23 patients with relapse-remitting MS (RRMS) and 24 healthy controls. Ten LHON mutations/variants were detected in the patients but were absent in the controls. Of them, the common primary pathogenic mutation m.14484T>C and the rare mutation m.10237T>C were found in one patient, whereas the rare mutation m.9101T>C was found in another patient. The remaining were secondary single nucleotide variants (SNVs) found either in synergy with the primary/rare mutations or individually in other patients. Patients carrying LHON variants also exhibited distinct mtDNA variants throughout the mitochondrial genome, eight were previously reported in patients with LHON. Moreover, five other LHON-related SNVs differed significantly in their prevalence among patients and controls (P < 0.05). This study, the first to investigate LHON mtDNA mutations/variants in a Saudi cohort may suggest a role of these mutations/variants in the pathogenesis or genetic predisposition to MS, a possibility which needs to be explored further in a large-scale.
  17. Mol Genet Genomic Med. 2022 Jun 28. e2010
      BACKGROUND: Succinate-CoA ligase/synthetase (SCS) deficiency is responsible for encephalomyopathy with mitochondrial DNA depletion and mild methylmalonic aciduria. Variants in SUCLG1, the nuclear gene encoding the alpha subunit of the SCS enzyme playing a pivotal role in maintaining mtDNA integrity and stability, are associated with mitochondrial DNA depletion syndrome 9 (MTDPS9).METHODS: In this study, we reported an infant with clinical features of MTDPS9 from China. Whole exome sequencing (WES) was used to identify the genetic cause. Bioinformatic analysis and mtDNA level detection were performed to assess pathogenicity.
    RESULTS: The proband manifested with hypotonia, lactic acidosis, mild methylmalonic aciduria, hearing loss and psychomotor retardation. WES identified new compound heterozygous SUCLG1 variants of c.601A>G (p.R201G) in exon 6 and c.871G>C (p.A291P) in exon 8. Computational analysis predicted that these missense variants might alter structure stability and mitochondrial translocation of SUCLG1. qRT-PCR showed 68% depletion of mtDNA content in proband as compared to controls.
    CONCLUSION: Novel compound heterozygous variants c.601A>G (p.R201G) and c.871G>C (p.A291P) in SUCLG1 may cause MTDPS9 in this family. Our finding should be helpful for molecular diagnosis, genetic counseling and clinical management of SCS deficiency disorders.
    Keywords:   SUCLG1 ; compound heterozygous variants; mitochondrial DNA depletion syndrome 9; mitochondrial encephlomyopathy; whole exome sequencing
  18. Methods Mol Biol. 2022 ;2497 141-172
      Mitochondrial energy production is crucial for normal daily activities and maintenance of life. Herein, the logic and execution of two main classes of measurements are outlined to delineate mitochondrial function: ATP production and oxygen consumption. Aerobic ATP production is quantified by phosphorus magnetic resonance spectroscopy (31PMRS) in vivo in both human subjects and animal models using the same protocols and maintaining the same primary assumptions. Mitochondrial oxygen consumption is quantified by oxygen polarography and applied in isolated mitochondria, cultured cells, and permeabilized fibers derived from human or animal tissue biopsies. Traditionally, mitochondrial functional measures focus on maximal oxidative capacity-a flux rate that is rarely, if ever, observed outside of experimental conditions. Perhaps more physiologically relevant, both measurement classes herein focus on one principal design paradigm; submaximal mitochondrial fluxes generated by graded levels of ADP to map the function for ADP sensitivity. We propose this function defines the bioenergetic role that mitochondria fill within the myoplasm to sense and match ATP demands. Any deficit in this vital role for ATP homeostasis leads to symptoms often seen in cardiovascular and cardiopulmonary diseases, diabetes, and metabolic syndrome.
    Keywords:  ADP sensitivity; Aerobic metabolism; Bioenergetics; Free energy homeostasis; Magnetic resonance; Oxygen consumption
  19. Methods Mol Biol. 2022 ;2497 129-140
      The loss of mitochondrial cristae integrity and mitochondrial swelling are hallmarks of multiple forms of necrotic cell death. One of the most well-studied and relevant inducers of mitochondrial swelling is matrix calcium (Ca2+). Respiring mitochondria will intake available Ca2+ into their matrix until a threshold is reached which triggers the opening of the mitochondrial permeability transition pore (MPTP). Upon opening of the pore, mitochondrial membrane potential dissipates and the mitochondria begin to swell, rendering them dysfunctional. The total amount of Ca2+ taken up by a mitochondrion prior to the engagement of the MPTP is referred to as mitochondrial Ca2+ retention capacity (CRC). The CRC/swelling assay is a useful tool for observing the dose-dependent event of mitochondrial dysfunction in real-time. In this technique, isolated mitochondria are treated with specific boluses of Ca2+ until they reach CRC and undergo swelling. A fluorometer is utilized to detect an increase in transmitted light passing through the sample as the mitochondria lose cristae density, and simultaneously measures calcium uptake by way of a Ca2+-specific membrane impermeable fluorescent dye. Here we provide a detailed protocol describing the mitochondrial CRC/swelling assay and we discuss how varying amounts of mitochondria and Ca2+ added to the system affect the dose-dependency of the assay. We also report how to validate the assay by using MPTP and calcium uptake inhibitors and troubleshooting common mistakes that occur with this approach.
    Keywords:  CRC; Calcium Green 5 N; Calcium retention capacity; Cell death; Fluorometry; Mitochondria; Mitochondrial dysfunction; Mitochondrial permeability transition pore (MPTP); Mitochondrial swelling
  20. Methods Mol Biol. 2022 ;2515 1-15
      Defects in mitochondrial oxidative phosphorylation have been observed in numerous neurodegenerative disorders and are linked to bioenergetic crises leading to neuronal death. The distinct metabolic profile of neurons is predominantly oxidative, which is characterized by the oxidation of glucose or its metabolites in the mitochondria to produce ATP. This process involves the tricarboxylic acid cycle, electron transfer in the respiratory chain, and oxygen consumption. Therefore, measurement of oxygen consumption rates (OCR) can be accurately applied to assess the rate of mitochondrial respiration. In this chapter, we describe our optimized protocol for the assessment of OCR specifically in primary mouse cerebellar granule neurons (CGN). The protocol includes isolation and manipulation of mouse CGNs followed by real-time assessment of mitochondrial OCR using a Seahorse XFe96 extracellular flux analyzer.
    Keywords:  Cerebellum; Mitochondrion; Oxidative phosphorylation; Primary neurons; Seahorse XFe96 extracellular flux analyzer
  21. Methods Mol Biol. 2022 ;2497 319-324
      The mitochondrial membrane potential (ΔΨm) generated by proton pumps (Complexes I, III, and IV) is an essential component in the process of energy generation during oxidative phosphorylation. Tetramethylrhodamine, methyl ester, perchlorate (TMRM) is one of the most commonly used fluorescent reporters of ΔΨm. TMRM is routinely employed in a steady state for the measurement of membrane potential. However, it can also be utilized with time-lapse fluorescence imaging to effectively monitor the changes in membrane potential in response to a given stimulus by analyzing the change in distribution of the dye with time.
    Keywords:  Fluorescence microscopy; Mitochondria membrane potential; Primary skin fibroblasts; TMRM; Uncoupler
  22. Methods Mol Biol. 2022 ;2497 281-290
      Mitochondria are pivotal organelles in the cell that regulate a myriad of cellular functions, which eventually govern cellular physiology and homeostasis. Intriguingly, microbial infection is known to trigger morphological and functional alterations of mitochondria. In fact, a number of bacteria and viruses have been reported to hijack mitochondrial functions including cell death induction and regulation of immune signaling to evade detection, promote their intracellular growth and subsequent dissemination. Here we describe methodologies that can be applied to assess mitochondrial functions upon infection. More specifically, we outline experimental procedures used to evaluate different parameters including mitochondrial morphology, adenosine triphosphate (ATP) levels, reactive oxygen species (ROS) levels, and mitophagy. Together these parameters can help gauge the overall health of mitochondria upon infection.
    Keywords:  ATP levels; Bacterial and viral infection; Mitochondria; Mitochondrial fission and fusion; Mitophagy; ROS levels
  23. Methods Mol Biol. 2022 ;2523 281-301
      Mitochondria have co-evolved with eukaryotic cells for more than a billion years, becoming an important cog in their machinery. They are best known for being tasked with energy generation through the production of adenosine triphosphate, but they also have roles in several other cellular processes, for example, immune and inflammatory responses. Mitochondria have important functions in macrophages, key innate immune cells that detect pathogens and drive inflammation. Mitochondrial activity is influenced by the highly dynamic nature of the mitochondrial network, which alternates between interconnected tubular and fragmented forms. The dynamic balance between this interconnected fused network and fission-mediated mitochondrial fragmentation modulates inflammatory responses such as production of cytokines and mitochondrial reactive oxygen species. Here we describe methods to differentiate mouse bone marrow cells into macrophages and the use of light microscopy, electron microscopy, flow cytometry, and Western blotting to quantify regulated mitochondrial dynamics in these differentiated macrophages.
    Keywords:  Drp1; Fission; Fusion; Inflammation; Macrophages; Microscopy; Mitochondria; Mitochondrial dynamics
  24. Hum Mol Genet. 2022 Jun 29. pii: ddac146. [Epub ahead of print]
      Peroxiredoxin 3 (PRDX3) encodes a mitochondrial antioxidant protein which is essential for the control of reactive oxidative species (ROS) homeostasis. So far, PRDX3 mutations are involved in mild-to-moderate progressive juvenile onset cerebellar ataxia. We aimed to unravel the molecular bases underlying the disease in an infant suffering from cerebellar ataxia that started at 19 months old and presented severe cerebellar atrophy and peripheral neuropathy early in the course of disease. By whole exome sequencing, we identified a novel homozygous mutation, PRDX3 p.D163E, which impaired the mitochondrial ROS defense system. In mouse primary cortical neurons, the exogenous expression of PRDX3 p.D163E was reduced and triggered alterations in neurite morphology and in mitochondria. Mitochondrial computational parameters showed that p.D163E led to serious mitochondrial alterations. In transfected HeLa cells expressing the mutation, mitochondria accumulation was detected by correlative light electron microscopy (CLEM). Mitochondrial morphology showed severe changes, including extremely damaged outer and inner membranes with a notable cristae disorganization. Moreover, spherical structures compatible with lipid droplets were identified, which can be associated with a generalized response to stress and can be involved in the removal of unfolded proteins. In the patient's fibroblasts, PRDX3 expression was nearly absent. The biochemical analysis suggested that the mutation p.D163E would result in an unstable structure tending to form aggregates that trigger unfolded protein responses via mitochondria and endoplasmic reticulum. Altogether, our findings broaden the clinical spectrum of the recently described PRDX3-associated neurodegeneration and provide new insight into the pathological mechanisms underlying this new form of cerebellar ataxia.
  25. Glia. 2022 Jun 30.
      Leigh syndrome is a mitochondrial disease characterized by neurodegeneration, neuroinflammation, and early death. Mice lacking NDUFS4, a mitochondrial complex I subunit (Ndufs4 KO mice), have been established as a good animal model for studying human pathology associated with Leigh syndrome. As the disease progresses, there is an increase in neurodegeneration and neuroinflammation, thereby leading to deteriorating neurological symptoms, including motor deficits, breathing alterations, and eventually, death of the animal. However, despite the magnitude of neuroinflammation associated with brain lesions, the role of neuroinflammatory pathways and their main cellular components have not been addressed directly as relevant players in the disease pathology. Here, we investigate the role of microglial cells, the main immune cells of the CNS, in Leigh-like syndrome pathology, by pharmacologically depleting them using the colony-stimulating factor 1 receptor antagonist PLX3397. Microglial depletion extended lifespan and delayed motor symptoms in Ndufs4 KO mice, likely by preventing neuronal loss. Next, we investigated the role of the major cytokine interleukin-6 (IL-6) in the disease progression. IL-6 deficiency partially rescued breathing abnormalities and modulated gliosis but did not extend the lifespan or rescue motor decline in Ndufs4 KO mice. The present results show that microglial accumulation is pathogenic, in a process independent of IL-6, and hints toward a contributing role of neuroinflammation in the disease of Ndufs4 KO mice and potentially in patients with Leigh syndrome.
    Keywords:  IL-6; Leigh syndrome; Ndufs4 KO; microglia; neuroinflammation
  26. Science. 2022 Jul;377(6601): 47-56
      The mechanistic target of rapamycin complex 1 (mTORC1) kinase controls growth in response to nutrients, including the amino acid leucine. In cultured cells, mTORC1 senses leucine through the leucine-binding Sestrin proteins, but the physiological functions and distribution of Sestrin-mediated leucine sensing in mammals are unknown. We find that mice lacking Sestrin1 and Sestrin2 cannot inhibit mTORC1 upon dietary leucine deprivation and suffer a rapid loss of white adipose tissue (WAT) and muscle. The WAT loss is driven by aberrant mTORC1 activity and fibroblast growth factor 21 (FGF21) production in the liver. Sestrin expression in the liver lobule is zonated, accounting for zone-specific regulation of mTORC1 activity and FGF21 induction by leucine. These results establish the mammalian Sestrins as physiological leucine sensors and reveal a spatial organization to nutrient sensing by the mTORC1 pathway.
  27. Nat Commun. 2022 Jul 01. 13(1): 3794
      The DEAD box protein DDX1, previously associated with 3'-end RNA processing and DNA repair, forms large aggregates in the cytoplasm of early mouse embryos. Ddx1 knockout causes stalling of embryos at the 2-4 cell stages. Here, we identify a DDX1-containing membrane-bound calcium-containing organelle with a nucleic acid core. We show that aggregates of these organelles form ring-like structures in early-stage embryos which we have named Membrane Associated RNA-containing Vesicles. We present evidence that DDX1 is required for the formation of Membrane Associated RNA-containing Vesicles which in turn regulate the spatial distribution of calcium in embryos. We find that Ddx1 knockout in early embryos disrupts calcium distribution, and increases mitochondria membrane potential, mitochondrial activity, and reactive oxygen species. Sequencing analysis of embryos from Ddx1 heterozygote crosses reveals downregulation of a subset of RNAs involved in developmental and mitochondrial processes in the embryos with low Ddx1 RNA. We propose a role for Membrane Associated RNA-containing Vesicles in calcium-controlled mitochondrial functions that are essential for embryonic development.
  28. Methods Mol Biol. 2022 ;2497 301-311
      The ability to sense and respond to acute changes in oxygen is essential for the viability of cells and organisms. To study molecular mechanisms of acute oxygen sensing, we established a setup for the adjustment of acute hypoxic conditions in cultured cells, exemplified here for the use of primary pulmonary arterial smooth muscle cells (PASMCs). The mitochondrial electron transport chain (ETC) is the main consumer of oxygen but recently also emerged as essential oxygen sensor suggesting that the ETC itself adapts its electron flux to oxygen availability. To test this assumption and to experimentally manipulate electron flux through the ETC, we used alternative oxidase (AOX), which bypasses the cytochrome pathway of the ETC when blocked. The described combination of our experimental setup and AOX allowed us in previous publications unprecedented insights into the role of the ETC in cellular oxygen sensing and cellular response mechanisms in living cells. Against this background, we here describe and discuss this method in detail, which will allow transfer to other cell types and research questions.
    Keywords:  Acute hypoxia; Alternative oxidase (AOX); Lung; Mitochondria; Oxygen sensing; Smooth muscle cells
  29. Methods Mol Biol. 2022 ;2497 207-220
      Doxorubicin (DOX)-induced cardiomyopathy constitutes dose-dependent cardiac toxicity, culminating in fatal heart failure progression. Cardiac toxicity limits effective and subsequent use of DOX in chemotherapy regimens in pediatric, adult, and recurrent cancer patients. DOX-induced profound alterations in mitochondrial morphology, dynamics, and defects in mitochondrial energy metabolism in the heart comprise key stressors in DOX-induced cardiotoxicity. Hence, the discovery of novel molecular targets and therapeutics to mitigate DOX-induced mitochondrial dysfunctions are imperative. Herein, we provided two laboratory protocols to monitor DOX-induced alterations in mitochondrial morphology and respiration in isolated primary neonatal rat cardiomyocytes. Neonatal rat cardiomyocytes are extensively used to monitor signaling mechanisms regulating cardiomyopathy in vitro. Therefore, these protocols will help researchers study the effects of novel pharmacological and genetic manipulations against DOX-induced alterations in mitochondrial morphology and energy metabolism in cardiomyocytes.
    Keywords:  Doxorubicin-induced cardiomyopathy; Mitochondrial morphology; Mitochondrial respiration; Oxygen consumption rates
  30. Commun Biol. 2022 Jul 01. 5(1): 649
      Mitochondrial ultrastructure represents a pinnacle of form and function, with the inner mitochondrial membrane (IMM) forming isolated pockets of cristae membrane (CM), separated from the inner-boundary membrane (IBM) by cristae junctions (CJ). Applying structured illumination and electron microscopy, a novel and fundamental function of MICU1 in mediating Ca2+ control over spatial membrane potential gradients (SMPGs) between CM and IMS was identified. We unveiled alterations of SMPGs by transient CJ openings when Ca2+ binds to MICU1 resulting in spatial cristae depolarization. This Ca2+/MICU1-mediated plasticity of the CJ further provides the mechanistic bedrock of the biphasic mitochondrial Ca2+ uptake kinetics via the mitochondrial Ca2+ uniporter (MCU) during intracellular Ca2+ release: Initially, high Ca2+ opens CJ via Ca2+/MICU1 and allows instant Ca2+ uptake across the CM through constantly active MCU. Second, MCU disseminates into the IBM, thus establishing Ca2+ uptake across the IBM that circumvents the CM. Under the condition of MICU1 methylation by PRMT1 in aging or cancer, UCP2 that binds to methylated MICU1 destabilizes CJ, disrupts SMPGs, and facilitates fast Ca2+ uptake via the CM.
  31. Methods Mol Biol. 2022 ;2497 363-422
      Mitochondria are complex organelles that use catabolic metabolism to produce ATP which is the critical energy source for cell function. Oxidative phosphorylation by the electron transport chain, which receives reducing equivalents (NADH and FADH2) from the tricarboxylic acid cycle, also produces reactive oxygen species (ROS) as a by-product at complex I and III. ROS play a significant role in health and disease. In order to better understand this process, a computational model of mitochondrial energy metabolism and the production of ROS has been developed. The model demonstrates the process regulating ROS production and removal and how different energy substrates can affect ROS production.
    Keywords:  Electron transport; Mitochondria; Reactive oxygen species
  32. Nat Commun. 2022 Jun 27. 13(1): 3669
      Very long-chain acyl-CoA dehydrogenase (VLCAD) is an inner mitochondrial membrane enzyme that catalyzes the first and rate-limiting step of long-chain fatty acid oxidation. Point mutations in human VLCAD can produce an inborn error of metabolism called VLCAD deficiency that can lead to severe pathophysiologic consequences, including cardiomyopathy, hypoglycemia, and rhabdomyolysis. Discrete mutations in a structurally-uncharacterized C-terminal domain region of VLCAD cause enzymatic deficiency by an incompletely defined mechanism. Here, we conducted a structure-function study, incorporating X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, computational modeling, and biochemical analyses, to characterize a specific membrane interaction defect of full-length, human VLCAD bearing the clinically-observed mutations, A450P or L462P. By disrupting a predicted α-helical hairpin, these mutations either partially or completely impair direct interaction with the membrane itself. Thus, our data support a structural basis for VLCAD deficiency in patients with discrete mutations in an α-helical membrane-binding motif, resulting in pathologic enzyme mislocalization.
  33. Methods Mol Biol. 2022 ;2497 269-280
      During lymphocyte maturation and differentiation, cells undergo a series of proliferative stages interrupted with stages of low activity. The rapid proliferation stages are marked by changes in metabolic outputs-adapting to energy demands by either hindering or utilizing metabolic pathways. As such, it is necessary to view these changes in real time; however, current strategies for metabolomics are time consuming and very rarely provide a holistic profile of the cellular metabolism while also characterizing mitochondrial metabolism. Here, we devised a fluorescence lifetime imaging microscopy (FLIM) strategy to image mitochondrial metabolic profiles in lymphocytes as they go through changes in metabolic activity. Our method provides not only a comprehensive view of cellular metabolism but also narrow in mitochondrial contributions while also efficiently excluding non-viable cells with and without the use of a viability dye. Our novel imaging strategy offers a reliable tool to study changes in mitochondrial metabolism.
    Keywords:  FLIM; Immunology; Immunometabolism; Lymphocytes; Microscopy; Mitochondria
  34. Nat Commun. 2022 Jun 28. 13(1): 3702
      The endoplasmic reticulum (ER)-mitochondria contact site (ERMCS) is crucial for exchanging biological molecules such as phospholipids and Ca2+ ions between these organelles. Mitoguardin-2 (MIGA2), a mitochondrial outer membrane protein, forms the ERMCS in higher eukaryotic cells. Here, we report the crystal structures of the MIGA2 Lipid Droplet (LD) targeting domain and the ER membrane protein VAPB bound to the phosphorylated FFAT motif of MIGA2. These structures reveal that the MIGA2 LD targeting domain has a large internal hydrophobic pocket that accommodates phospholipids and that two phosphorylations of the FFAT motif are required for tight interaction of MIGA2 with VAPB, which enhances the rate of lipid transport. Further biochemical studies show that MIGA2 transports phospholipids between membranes with a strong preference for binding and trafficking phosphatidylserine (PS). These results provide a structural and molecular basis for understanding how MIGA2 mediates the formation of ERMCS and facilitates lipid trafficking at the ERMCS.
  35. Methods Mol Biol. 2022 ;2497 349-362
      The measurement of mitochondrial function has become imperative to understand and characterize diseases characterized by bioenergetic alterations. The advancement of automation and application of high-throughput technologies has propelled our understanding of biological complexity and facilitated drug discovery. Seahorse extracellular flux (XFe) technology measures changes in dissolved oxygen and proton concentration in cell culture media, providing kinetic measurements of oxidative phosphorylation and glycolytic metabolism. ImageXpress® Nano is an automated fluorescent microscope with the ability to perform high-content, fast, and robust imaging in multi-well formats. In this chapter, we present a comprehensive protocol to multiplex the Seahorse XFe24 analyzer with the ImageXpress® Nano high content imaging microscope to provide a comprehensive yet rigorous profile of bioenergetics and its correlation to neuronal function and morphology.
    Keywords:  Bioenergetics; ECAR; High-throughput imaging; Neurodegeneration; Neuron; OCR; Primary neuronal culture
  36. Nat Metab. 2022 Jun;4(6): 739-758
      Mitochondria are the main consumers of oxygen within the cell. How mitochondria sense oxygen levels remains unknown. Here we show an oxygen-sensitive regulation of TFAM, an activator of mitochondrial transcription and replication, whose alteration is linked to tumours arising in the von Hippel-Lindau syndrome. TFAM is hydroxylated by EGLN3 and subsequently bound by the von Hippel-Lindau tumour-suppressor protein, which stabilizes TFAM by preventing mitochondrial proteolysis. Cells lacking wild-type VHL or in which EGLN3 is inactivated have reduced mitochondrial mass. Tumorigenic VHL variants leading to different clinical manifestations fail to bind hydroxylated TFAM. In contrast, cells harbouring the Chuvash polycythaemia VHLR200W mutation, involved in hypoxia-sensing disorders without tumour development, are capable of binding hydroxylated TFAM. Accordingly, VHL-related tumours, such as pheochromocytoma and renal cell carcinoma cells, display low mitochondrial content, suggesting that impaired mitochondrial biogenesis is linked to VHL tumorigenesis. Finally, inhibiting proteolysis by targeting LONP1 increases mitochondrial content in VHL-deficient cells and sensitizes therapy-resistant tumours to sorafenib treatment. Our results offer pharmacological avenues to sensitize therapy-resistant VHL tumours by focusing on the mitochondria.
  37. Brain. 2022 Jun 30. 145(6): 1939-1948
      Biallelic pathogenic variants in SZT2 result in a neurodevelopmental disorder with shared features, including early-onset epilepsy, developmental delay, macrocephaly, and corpus callosum abnormalities. SZT2 is as a critical scaffolding protein in the amino acid sensing arm of the mTORC1 signalling pathway. Due to its large size (3432 amino acids), lack of crystal structure, and absence of functional domains, it is difficult to determine the pathogenicity of SZT2 missense and in-frame deletions, but these variants are increasingly detected and reported by clinical genetic testing in individuals with epilepsy. To exemplify this latter point, here we describe a cohort of 12 individuals with biallelic SZT2 variants and phenotypic overlap with SZT2-related neurodevelopmental disorders. However, the majority of individuals carried one or more SZT2 variants of uncertain significance (VUS), highlighting the need for functional characterization to determine, which, if any, of these VUS were pathogenic. Thus, we developed a novel individualized platform to identify SZT2 loss-of-function variants in the context of mTORC1 signalling and reclassify VUS. Using this platform, we identified a recurrent in-frame deletion (SZT2 p.Val1984del) which was determined to be a loss-of-function variant and therefore likely pathogenic. Haplotype analysis revealed that this single in-frame deletion is a founder variant in those of Ashkenazi Jewish ancestry. Moreover, this approach allowed us to tentatively reclassify all of the VUS in our cohort of 12 individuals, identifying five individuals with biallelic pathogenic or likely pathogenic variants. Clinical features of these five individuals consisted of early-onset seizures (median 24 months), focal seizures, developmental delay and macrocephaly similar to previous reports. However, we also show a widening of the phenotypic spectrum, as none of the five individuals had corpus callosum abnormalities, in contrast to previous reports. Overall, we present a rapid assay to resolve VUS in SZT2, identify a founder variant in individuals of Ashkenazi Jewish ancestry, and demonstrate that corpus callosum abnormalities is not a hallmark feature of this condition. Our approach is widely applicable to other mTORopathies including the most common causes of the focal genetic epilepsies, DEPDC5, TSC1/2, MTOR and NPRL2/3.
    Keywords:  SZT2; epilepsy; genetics; mTOR; variant
  38. J Clin Invest. 2022 Jul 01. pii: e158447. [Epub ahead of print]132(13):
      Mitochondrial dysfunction and cell senescence are hallmarks of aging and are closely interconnected. Mitochondrial dysfunction, operationally defined as a decreased respiratory capacity per mitochondrion together with a decreased mitochondrial membrane potential, typically accompanied by increased production of oxygen free radicals, is a cause and a consequence of cellular senescence and figures prominently in multiple feedback loops that induce and maintain the senescent phenotype. Here, we summarize pathways that cause mitochondrial dysfunction in senescence and aging and discuss the major consequences of mitochondrial dysfunction and how these consequences contribute to senescence and aging. We also highlight the potential of senescence-associated mitochondrial dysfunction as an antiaging and antisenescence intervention target, proposing the combination of multiple interventions converging onto mitochondrial dysfunction as novel, potent senolytics.
  39. Methods Mol Biol. 2022 ;2497 313-318
      This section aims to describe the measurement of NADH and FAD2+ levels in intact cells using fluorescence microscopy. Both NADH and FADH2 are major electron donors for the electron transport chain through shifting of their redox status. Furthermore, within their redox couples, only NADH and FAD2+ are fluorescent. Therefore, calibration of the NADH and FAD2+ fluorescence signal is a crucial factor in accurately assessing mitochondrial function and redox status.
    Keywords:  Autofluorescence; ETC; FAD2+; NADH; Redox status
  40. Nature. 2022 Jun 29.
      Aggressive and metastatic cancers show enhanced metabolic plasticity1, but the precise underlying mechanisms of this remain unclear. Here we show how two NOP2/Sun RNA methyltransferase 3 (NSUN3)-dependent RNA modifications-5-methylcytosine (m5C) and its derivative 5-formylcytosine (f5C) (refs.2-4)-drive the translation of mitochondrial mRNA to power metastasis. Translation of mitochondrially encoded subunits of the oxidative phosphorylation complex depends on the formation of m5C at position 34 in mitochondrial tRNAMet. m5C-deficient human oral cancer cells exhibit increased levels of glycolysis and changes in their mitochondrial function that do not affect cell viability or primary tumour growth in vivo; however, metabolic plasticity is severely impaired as mitochondrial m5C-deficient tumours do not metastasize efficiently. We discovered that CD36-dependent non-dividing, metastasis-initiating tumour cells require mitochondrial m5C to activate invasion and dissemination. Moreover, a mitochondria-driven gene signature in patients with head and neck cancer is predictive for metastasis and disease progression. Finally, we confirm that this metabolic switch that allows the metastasis of tumour cells can be pharmacologically targeted through the inhibition of mitochondrial mRNA translation in vivo. Together, our results reveal that site-specific mitochondrial RNA modifications could be therapeutic targets to combat metastasis.
  41. J Proteome Res. 2022 Jun 27.
      We have developed the underrepresented post-translational modification (PTM) database (urPTMdb), a PTM gene set database to accelerate the discovery of enriched protein modifications in experimental data. urPTMdb provides curated lists of proteins reported to be substrates of underrepresented modifications. Their enrichment in proteomics datasets can reveal unexpected PTM regulations. urPTMdb can be implemented in existing workflows, or used in TeaProt, an online Shiny tool that integrates upstream transcription factor enrichment analysis with downstream pathway analysis through an easy-to-use interactive interface. TeaProt annotates user-uploaded data with drug-gene interactions, subcellular localizations, phenotypic functions, gene-disease associations, and enzyme-gene interactions. TeaProt enables gene set enrichment analysis (GSEA) to discover enrichments in gene sets from various resources, including MSigDB, CHEA, and urPTMdb. We demonstrate the utility of urPTMdb and TeaProt through the analysis of a previously published Western diet-induced remodeling of the tongue proteome, which revealed altered cellular processes associated with energy metabolism, interferon alpha/gamma response, adipogenesis, HMGylation substrate enrichment, and transcription regulation through PPARG and CEBPA. Additionally, we analyzed the interactome of ADP-ribose glycohydrolase TARG1, a key enzyme that removes mono-ADP-ribosylation. This analysis identified an enrichment of ADP-ribosylation, ribosomal proteins, and proteins localized in the nucleoli and endoplasmic reticulum. TeaProt and urPTMdb are accessible at
    Keywords:  PTM; bioinformatics; database; proteomics; software
  42. Methods Mol Biol. 2022 ;2497 117-128
      The Seahorse Extracellular Flux Analyzer enables the high-throughput characterization of oxidative phosphorylation capacity based on the electron transport chain organization and regulation with relatively small amount of material. This development over the traditional polarographic Clark-type electrode approaches make it possible to analyze the respiratory features of mitochondria isolated from tissue samples of particular animal models. Here we provide a description of an optimized approach to carry out multi-well measurement of O2 consumption, with the Agilent Seahorse XFe96 analyzer on mouse brain and muscles to determine the tissue-specific oxidative phosphorylation properties. Protocols include the preparation of the tissue samples, isolation of mitochondria, and analysis of their function; in particular, the preparation and optimization of the reagents and samples.
    Keywords:  Brain; Electron transport chain; Muscle; O2 consumption; Seahorse
  43. Nat Commun. 2022 Jun 28. 13(1): 3720
      PINK1-Parkin mediated mitophagy, a selective form of autophagy, represents one of the most important mechanisms in mitochondrial quality control (MQC) via the clearance of damaged mitochondria. Although it is well known that the conjugation of mammalian ATG8s (mATG8s) to phosphatidylethanolamine (PE) is a key step in autophagy, its role in mitophagy remains controversial. In this study, we clarify the role of the mATG8-conjugation system in mitophagy by generating knockouts of the mATG8-conjugation machinery. Unexpectedly, we show that mitochondria could still be cleared in the absence of the mATG8-conjugation system, in a process independent of lysosomal degradation. Instead, mitochondria are cleared via extracellular release through a secretory autophagy pathway, in a process we define as Autophagic Secretion of Mitochondria (ASM). Functionally, increased ASM promotes the activation of the innate immune cGAS-STING pathway in recipient cells. Overall, this study reveals ASM as a mechanism in MQC when the cellular mATG8-conjugation machinery is dysfunctional and highlights the critical role of mATG8 lipidation in suppressing inflammatory responses.