bims-midmar Biomed News
on Mitochondrial DNA maintenance and replication
Issue of 2021‒08‒01
twenty papers selected by
Flavia Söllner
Ludwig-Maximilians University

  1. J Cell Sci. 2021 07 01. pii: jcs252197. [Epub ahead of print]134(13):
      The mitochondrial inner membrane is a protein-rich environment containing large multimeric complexes, including complexes of the mitochondrial electron transport chain, mitochondrial translocases and quality control machineries. Although the inner membrane is highly proteinaceous, with 40-60% of all mitochondrial proteins localised to this compartment, little is known about the spatial distribution and organisation of complexes in this environment. We set out to survey the arrangement of inner membrane complexes using stochastic optical reconstruction microscopy (STORM). We reveal that subunits of the TIM23 complex, TIM23 and TIM44 (also known as TIMM23 and TIMM44, respectively), and the complex IV subunit COXIV, form organised clusters and show properties distinct from the outer membrane protein TOM20 (also known as TOMM20). Density based cluster analysis indicated a bimodal distribution of TIM44 that is distinct from TIM23, suggesting distinct TIM23 subcomplexes. COXIV is arranged in larger clusters that are disrupted upon disruption of complex IV assembly. Thus, STORM super-resolution microscopy is a powerful tool for examining the nanoscale distribution of mitochondrial inner membrane complexes, providing a 'visual' approach for obtaining pivotal information on how mitochondrial complexes exist in a cellular context.
    Keywords:  COXIV; Mitochondria; Mitochondrial complexes; Nanoscopy; Protein import; STORM; TIM23
  2. Neurology. 2021 Jul 27. pii: 10.1212/WNL.0000000000012559. [Epub ahead of print]
  3. eNeuro. 2021 Jul 26. pii: ENEURO.0232-21.2021. [Epub ahead of print]
      Mitochondrial composition varies by organ and their constituent cell types. This mitochondrial diversity likely determines variations in mitochondrial function. However, the heterogeneity of mitochondria in the brain remains underexplored despite the large diversity of cell types in neuronal tissue. Here, we used molecular systems biology tools to address whether mitochondrial composition varies by brain region and neuronal cell type in mice. We reasoned that proteomics and transcriptomics of microdissected brain regions combined with analysis of single cell mRNA sequencing could reveal the extent of mitochondrial compositional diversity. We selected nuclear encoded gene products forming complexes of fixed stoichiometry, such as the respiratory chain complexes and the mitochondrial ribosome, as well as molecules likely to perform their function as monomers, such as the family of SLC25 transporters. We found that the proteome encompassing these nuclear-encoded mitochondrial genes and obtained from microdissected brain tissue segregated the hippocampus, striatum, and cortex from each other. Nuclear-encoded mitochondrial transcripts could only segregate cell types and brain regions when the analysis was performed at the single cell level. In fact, single cell mitochondrial transcriptomes were able to distinguish glutamatergic and distinct types of GABAergic neurons from one another. Within these cell categories, unique SLC25A transporters were able to identify distinct cell subpopulations. Our results demonstrate heterogeneous mitochondrial composition across brain regions and cell types. We postulate that mitochondrial heterogeneity influences regional and cell type specific mechanisms in health and disease.Significance StatementMitochondria are important organelles for maintaining brain health. The composition of proteins making up mitochondria is essential for their function. Disturbances to mitochondria are thought to contribute to neurodegeneration and neurodevelopmental disorders. These conditions typically affect specific brain regions or cell types. Despite the link between mitochondria and diseases with distinct anatomical and cellular patterns, how mitochondrial composition varies across brain regions and cell types remains poorly explored. Here, we analyze mitochondrial composition in different brain regions and cell types in adult mice, showing composition differs by region and cell lineage. Our work provides a resource of genes enriched in certain cell types or regions that improves our understanding of how mitochondrial composition influences brain function in health and disease.
    Keywords:  GABA; Glutamate; Mitochondria; Mitochondrial Ribosome; Respiratory Chain; Solute transporter
  4. Methods Cell Biol. 2021 ;pii: S0091-679X(20)30193-X. [Epub ahead of print]165 153-161
      Selective elimination of damaged mitochondria via macroautophagy (mitophagy) is a conserved cellular process that plays an important role in organismal health. In recent years mitophagy has been studied in parallel to the more general, non-selective autophagy pathway induced in response to amino acid starvation with important similarities and differences noted between the two. The elaborate sequence of membrane rearrangements that give rise to autophagosomes in the non-selective pathway have their counterpart in mitophagy, but with the addition of other factors, such as a ubiquitin mark and mitophagy receptors, which mediate cargo recognition. In some types of mitophagy such as the one induced by ivermectin, the forming autophagosomal structure contains six different elements: the targeted mitochondrial fragment, a section of endoplasmic reticulum that provides a cradle, a ubiquitin layer, the mitophagy receptors and the early and late autophagosomal proteins/membranes. Super-resolution microscopy is ideally suited to investigate the spatial relationships between these elements that converge together but retain some distinctive localization, and we provide here a general protocol that can be used for mammalian cells.
    Keywords:  Autophagy; Endoplasmic reticulum; Ivermectin; Mitochondria; Mitophagy; Structured illumination microscopy
  5. Stem Cell Reports. 2021 Jul 15. pii: S2213-6711(21)00324-6. [Epub ahead of print]
      The generation of inducible pluripotent stem cells (iPSCs) is a revolutionary technique allowing production of pluripotent patient-specific cell lines used for disease modeling, drug screening, and cell therapy. Integrity of nuclear DNA (nDNA) is mandatory to allow iPSCs utilization, while quality control of mitochondrial DNA (mtDNA) is rarely included in the iPSCs validation process. In this study, we performed mtDNA deep sequencing during the transition from parental fibroblasts to reprogrammed iPSC and to differentiated neuronal precursor cells (NPCs) obtained from controls and patients affected by mitochondrial disorders. At each step, mtDNA variants, including those potentially pathogenic, fluctuate between emerging and disappearing, and some having functional implications. We strongly recommend including mtDNA analysis as an unavoidable assay to obtain fully certified usable iPSCs and NPCs.
    Keywords:  human iPSCs; iPSCs quality control; mtDNA deep sequencing; neuronal precursor cells
  6. Dev Cell. 2021 Jul 26. pii: S1534-5807(21)00546-3. [Epub ahead of print]56(14): 2014-2015
      Mechanisms by which cells remove damaged mitochondria extracellularly are unclear. Recent work by Jiao and colleagues in Cell shows that migrating cells expel dysfunctional mitochondria in membrane-bound structures called migrasomes to maintain mitochondrial homeostasis.
  7. Dis Model Mech. 2021 Jul 27. pii: dmm.048995. [Epub ahead of print]
      Mitochondrial dysfunction in different cell types is associated to several pathological processes and potentially contributes to chronic inflammatory and ageing-related diseases. Mitochondrial Transcription Factor A (TFAM) plays a critical role in maintaining mtDNA integrity and function. Taking advantage of the Tfamfl/fl UBC-Cre/ERT2+/+ mice, we sought to develop a cellular in vitro system to investigate the role of mitochondrial dysfunction in the stromal cell component. We describe an inducible model of mitochondrial dysfunction by stable depletion of TFAM in primary mouse skin fibroblast (SK-FB) after 4-hydroxytamoxifen (4-OHT) administration. Tfam gene deletion caused a sustained reduction of Tfam and mtDNA-encoded mRNA expression in Cre(+) cultured for low (LP) and high passages (HP). Ultimately, Tfam knockout translated into a loss of TFAM protein. TFAM depletion led to a substantial reduction of the mitochondrial respiratory chain (MRC) complexes that was exacerbated in HP SK-FB cultures. The assembly pattern showed that the respiratory complexes fail to reach the respirasome in 4-OHT Cre(+) SK-FB. Functionally, we determined the mitochondrial function and the glycolytic activity by mito-stress and glycolysis-stress test respectively. These analysis showed that mitochondrial dysfunction was developed after long-term 4-OHT treatment in HP Cre(+) SK-FB and was compensated by an increase in the glycolytic capacity. Finally, expression analysis revealed that 4-OHT-treated HP Cre(+) SK-FB showed a senescent and pro-inflammatory phenotype. In conclusion, we have generated and validated the first ex vivo model of fibroblast mitochondrial dysfunction that results in a pro-inflammatory phenotype applicable to explore this process in other cell types in a variety of pathological conditions.
    Keywords:  Cellular senescence; Fibroblasts; Inflammation; Mitochondrial dysfunction; TFAM
  8. Cell Death Dis. 2021 Jul 29. 12(8): 751
      POLRMT (RNA polymerase mitochondrial) is responsible for the transcription of mitochondrial genome encoding key components of oxidative phosphorylation. This process is important for cancer cell growth. The current study tested expression and potential functions of POLRMT in non-small cell lung cancer (NSCLC). TCGA cohorts and the results from the local lung cancer tissues showed that POLRMT is overexpressed in human lung cancer tissues. In both primary human NSCLC cells and A549 cells, POLRMT silencing (by targeted lentiviral shRNAs) or knockout (through CRSIPR/Cas9 gene editing method) potently inhibited cell viability, proliferation, migration, and invasion, and induced apoptosis activation. On the contrast, ectopic overexpression of POLRMT using a lentiviral construct accelerated cell proliferation and migration in NSCLC cells. The mtDNA contents, mRNA levels of mitochondrial transcripts, and subunits of respiratory chain complexes, as well as S6 phosphorylation, were decreased in POLRMT-silenced or -knockout NSCLC cells, but increased after ectopic POLRMT overexpression. In vivo, intratumoral injection of POLRMT shRNA adeno-associated virus (AAV) potently inhibited NSCLC xenograft growth in severe combined immune deficiency mice. The mtDNA contents, mRNA levels of mitochondria respiratory chain complex subunits, and S6 phosphorylation were decreased in POLRMT shRNA AAV-injected NSCLC xenograft tissues. These results show that POLRMT is a novel and important oncogene required for NSCLC cell growth in vitro and in vivo.
  9. Invest Ophthalmol Vis Sci. 2021 Jul 01. 62(9): 38
      Purpose: To investigate the molecular mechanism underlying the Leber's hereditary optic neuropathy (LHON)-linked MT-ND1 3460G>A mutation.Methods: Cybrid cell models were generated by fusing mitochondrial DNA-less ρ0 cells with enucleated cells from a patient carrying the m.3460G>A mutation and a control subject. The impact of m.3460G>A mutations on oxidative phosphorylation was evaluated using Blue Native gel electrophoresis, and measurements of oxygen consumption were made with an extracellular flux analyzer. Assessment of reactive oxygen species (ROS) production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Assays for apoptosis and mitophagy were undertaken via immunofluorescence analysis.
    Results: Nineteen Chinese Han pedigrees bearing the m.3460G>A mutation exhibited variable penetrance and expression of LHON. The m.3460G>A mutation altered the structure and function of MT-ND1, as evidenced by reduced MT-ND1 levels in mutant cybrids bearing the mutation. The instability of mutated MT-ND1 manifested as defects in the assembly and activity of complex I, respiratory deficiency, diminished mitochondrial adenosine triphosphate production, and decreased membrane potential, in addition to increased production of mitochondrial ROS in the mutant cybrids carrying the m.3460G>A mutation. The m.3460G>A mutation mediated apoptosis, as evidenced by the elevated release of cytochrome c into the cytosol and increasing levels of the apoptotic-associated proteins BAK, BAX, and PARP, as well as cleaved caspases 3, 7, and 9, in the mutant cybrids. The cybrids bearing the m.3460G>A mutation exhibited reduced levels of autophagy protein light chain 3, accumulation of autophagic substrate P62, and impaired PTEN-induced kinase 1/parkin-dependent mitophagy.
    Conclusions: Our findings highlight the critical role of m.3460G>A mutation in the pathogenesis of LHON, manifested by mitochondrial dysfunction and alterations in apoptosis and mitophagy.
  10. Clin Park Relat Disord. 2020 ;3 100056
      Huntington disease (HD) is a devastating monogenic autosomal dominant disorder. HD is caused by a CAG expansion in exon 1 of the gene coding for huntingtin, placed in the short arm of chromosome 4. Despite its well-defined genetic origin, the molecular and cellular mechanisms underlying the disease are unclear and complex. Here, we review some of the currently known functions of the wild-type huntingtin protein and discuss the deleterious effects that arise from the expansion of the CAG repeats, which are translated into an abnormally long polyglutamine tract. Also, we present a modern view on the molecular biology of HD as a representative of the group of polyglutamine diseases, with an emphasis on conformational changes of mutant huntingtin, disturbances in its cellular processing, and proteolytic stress in degenerating neurons. The main pathogenetic mechanisms of neurodegeneration in HD are discussed in detail, such as autophagy, impaired mitochondrial biogenesis, lysosomal dysfunction, organelle and protein transport, inflammation, oxidative stress, and transcription factor modulation. However, other unraveling mechanisms are still unknown. This practical and brief review summarizes some of the currently known functions of the wild-type huntingtin protein and the recent findings related to the mechanisms involved in HD pathogenesis.
    Keywords:  Huntington disease; Mechanisms; Pathophysiology
  11. Neurochem Int. 2021 Jul 27. pii: S0197-0186(21)00193-5. [Epub ahead of print] 105147
      Huntington's disease (HD), as well as Parkinson's disease and Alzheimer's disease, belong to a group of neurodegenerative diseases characterized by common features, such as the progressive loss of neurons and the presence of pathogenic forms of misfolded protein aggregates. A quality control system such as autophagy is crucial for the clearance of protein aggregates and dysfunctional organelles and thus essential for the maintenance of neuronal homeostasis. The constant high energy demand of neuronal tissue links neurodegeneration to mitochondria. Inefficient removal of damaged mitochondria is thought to contribute to the pathogenesis of neurodegenerative diseases such as HD. In addition, direct involvement of the huntingtin protein in the autophagic machinery has been described. In this review, we focus on mitophagy, a selective form of autophagy responsible for mitochondrial turnover. We also discuss the relevance of pharmacological regulation of mitophagy in the future therapeutic approach to neurodegenerations, including HD.
    Keywords:  Huntington's disease; Mitochondria; Mitophagy; Mitophagy adaptors; Pharmacological induction of mitophagy
  12. ASN Neuro. 2021 Jan-Dec;13:13 17590914211028364
      Mitochondrial dysfunction is a well-established pathological event in Parkinson's disease (PD). Proteins misfolding and its impaired cellular clearance due to altered autophagy/mitophagy/pexophagy contribute to PD progression. It has been shown that mitochondria have contact sites with endoplasmic reticulum (ER), peroxisomes and lysosomes that are involved in regulating various physiological processes. In pathological conditions, the crosstalk at the contact sites initiates alterations in intracellular vesicular transport, calcium homeostasis and causes activation of proteases, protein misfolding and impairment of autophagy. Apart from the well-reported molecular changes like mitochondrial dysfunction, impaired autophagy/mitophagy and oxidative stress in PD, here we have summarized the recent scientific reports to provide the mechanistic insights on the altered communications between ER, peroxisomes, and lysosomes at mitochondrial contact sites. Furthermore, the manuscript elaborates on the contributions of mitochondrial contact sites and organelles dysfunction to the pathogenesis of PD and suggests potential therapeutic targets.
    Keywords:  Parkinson’s disease; endoplasmic reticulum; lysosome; mitochondria; mitochondrial contact sites; peroxisome
  13. Clin Sci (Lond). 2021 Jul 30. 135(14): 1767-1772
      Fetal exposure to an unfavorable intrauterine environment programs an individual to have a greater susceptibility later in life to non-communicable diseases, such as coronary heart disease, but the molecular processes are poorly understood. An article in Clinical Science recently reported novel details on the effects of maternal nutrient reduction (MNR) on fetal heart development using a primate model that is about 94% genetically similar to humans and is also mostly monotocous. MNR adversely impacted fetal left ventricular (LV) mitochondria in a sex-dependent fashion with a greater effect on male fetuses, although mitochondrial transcripts increased more so in females. Increased expression for several respiratory chain and adenosine triphosphate (ATP) synthase proteins were observed. However, fetal LV mitochondrial complex I and complex II/III activities were significantly decreased, likely contributing to a 73% decreased LV ATP content and increased LV lipid peroxidation. Moreover, MNR fetal LV mitochondria showed sparse and disarranged cristae. This study indicates that mitochondria are targets of the remodeling and imprinting processes in a sex-dependent manner. Mitochondrial ROS production and inadequate energy production add another layer of complexity. Altogether these observations raise the possibility that dysfunctional mitochondria in the fetus may contribute in turn to epigenetic memory of in utero stress in the adult. The role of mitoepigenetics and involvement of mitochondrial and genomic non-coding RNAs in mitochondrial functions and nuclei-mitochondria crosstalk with in utero stress awaits further investigation.
    Keywords:  epigenetics; intrauterine growth restriction; mitochondrial dysfunction; oxidative stress; sex
  14. Plant J. 2021 Jul 26.
      Plant defense against melon necrotic spot virus (MNSV) is triggered by the viral auxiliary replicase p29 that is targeted to mitochondrial membranes causing morphological alterations, oxidative burst, and necrosis. Here we show that MNSV coat protein (CP) was also targeted to mitochondria and mitochondrial-derived replication complexes (VRC), in close association with p29, in addition to chloroplasts. CP import resulted in the cleavage of the R/arm domain previously implicated in genome binding during encapsidation and RNA silencing suppression (RSS). We also show that CP organelle import inhibition enhanced RSS activity, CP accumulation, and VRC biogenesis but resulted in inhibition of systemic spreading, indicating that MNSV whole-plant infection requires CP organelle import. We hypothesize that to alleviate the p29 impact on host physiology, MNSV could moderate its replication and p29 accumulation by regulating CP RSS activity through organelle targeting and, consequently, eluding early-triggered antiviral response. Cellular and molecular events also suggested that S/P domains, which correspond to processed CP in chloroplast stroma or mitochondrion matrix, could mitigate host response inhibiting p29-induced necrosis. S/P deletion mainly resulted in a precarious balance between defense and counter-defense responses, generating either cytopathic alterations and MNSV cell-to-cell movement restriction or some degree of local movement. In addition, local necrosis and defense responses were dampened when RSS activity but not S/P organelle targeting was affected. Based on a robust biochemical and cellular analysis, we established that the mitochondrial and chloroplast dual targeting of MNSV CP profoundly impacts the viral infection cycle.
    Keywords:  MNSV; chloroplasts; coat protein; dual targeting; hypersensitive response; mitochondria; silencing
  15. Am J Phys Anthropol. 2021 Jul 26.
      OBJECTIVE: We aim to identify maternal genetic affinities between the Middle to Final Neolithic (3850-2300 BC) populations from present-day Poland and possible genetic influences from the Pontic steppe.MATERIALS AND METHODS: We conducted ancient DNA studies from populations associated with Złota, Globular Amphora, Funnel Beaker, and Corded Ware cultures (CWC). We sequenced genomic libraries on Illumina platform to generate 86 complete ancient mitochondrial genomes. Some of the samples were enriched for mitochondrial DNA using hybridization capture.
    RESULTS: The maternal genetic composition found in Złota-associated individuals resembled that found in people associated with the Globular Amphora culture which indicates that both groups likely originated from the same maternal genetic background. Further, these two groups were closely related to the Funnel Beaker culture-associated population. None of these groups shared a close affinity to CWC-associated people. Haplogroup U4 was present only in the CWC group and absent in Złota group, Globular Amphora, and Funnel Beaker cultures.
    DISCUSSION: The prevalence of mitochondrial haplogroups of Neolithic farmer origin identified in Early, Middle and Late Neolithic populations suggests a genetic continuity of these maternal lineages in the studied area. Although overlapping in time - and to some extent - in cultural expressions, none of the studied groups (Złota, Globular Amphora, Funnel Beaker), shared a close genetic affinity to CWC-associated people, indicating a larger extent of cultural influence from the Pontic steppe than genetic exchange. The higher frequency of haplogroup U5b found in populations associated with Funnel Beaker, Globular Amphora, and Złota cultures suggest a gradual maternal genetic influx from Mesolithic hunter-gatherers. Moreover, presence of haplogroup U4 in Corded Ware groups is most likely associated with the migrations from the Pontic steppe at the end of the Neolithic and supports the observed genetic distances.
    Keywords:  Central Europe; Neolithic; ancient DNA; human population; mitochondrial haplogroups
  16. Sci Rep. 2021 Jul 30. 11(1): 15564
      In mammalian ovaries, mitochondria are integral sites of energy production and steroidogenesis. While shifts in cellular activities and steroidogenesis are well characterized during the differentiation of large luteal cells in folliculogenesis and luteal formation, mitochondrial dynamics during this process have not been previously evaluated. In this study, we collected ovaries containing primordial follicles, mature follicles, corpus hemorrhagicum, or corpus luteum from goats at specific times in the estrous cycle. Enzyme histochemistry, ultrastructural observations, and 3D structural analysis of serial sections of mitochondria revealed that branched mitochondrial networks were predominant in follicles, while spherical and tubular mitochondria were typical in large luteal cells. Furthermore, the average mitochondrial diameter and volume increased from folliculogenesis to luteal formation. In primordial follicles, the signals of cytochrome c oxidase and ATP synthase were undetectable in most cells, and the large luteal cells from the corpus hemorrhagicum also showed low enzyme signals and content when compared with granulosa cells in mature follicles or large luteal cells from the corpus luteum. Our findings suggest that the mitochondrial enlargement could be an event during folliculogenesis and luteal formation, while the modulation of mitochondrial morphology and respiratory enzyme expressions may be related to tissue remodeling during luteal formation.
  17. Front Cell Dev Biol. 2021 ;9 698658
      Mitochondrial protein biogenesis relies almost exclusively on the expression of nuclear-encoded polypeptides. The current model postulates that most of these proteins have to be delivered to their final mitochondrial destination after their synthesis in the cytoplasm. However, the knowledge of this process remains limited due to the absence of proper experimental real-time approaches to study mitochondria in their native cellular environment. We developed a gentle microinjection procedure for fluorescent reporter proteins allowing a direct non-invasive study of protein transport in living cells. As a proof of principle, we visualized potential-dependent protein import into mitochondria inside intact cells in real-time. We validated that our approach does not distort mitochondrial morphology and preserves the endogenous expression system as well as mitochondrial protein translocation machinery. We observed that a release of nascent polypeptides chains from actively translating cellular ribosomes by puromycin strongly increased the import rate of the microinjected pre-protein. This suggests that a substantial amount of mitochondrial translocase complexes was involved in co-translational protein import of endogenously expressed pre-proteins. Our protein microinjection method opens new possibilities to study the role of mitochondrial protein import in cell models of various pathological conditions as well as aging processes.
    Keywords:  GFP; SNAP-tag; fluorescence microscopy; microinjection; mitochondria; mitochondrial protein import
  18. Dev Cell. 2021 Jul 26. pii: S1534-5807(21)00529-3. [Epub ahead of print]56(14): 2010-2012
      Cancers are dependent on mitochondria, the powerhouse of the cell, and autophagy, the mechanism to preserve mitochondrial quality and function. In this issue of Developmental Cell, Towers et al. identify mitochondria-derived vesicles (MDVs) as a new adaptive mechanism enabling cancer cells to compensate for autophagy loss and to maintain mitochondrial function.
  19. Front Neurosci. 2021 ;15 680572
      Pathogenic variants in SPG11 are the most frequent cause of autosomal recessive complicated hereditary spastic paraplegia (HSP). In addition to spastic paraplegia caused by corticospinal degeneration, most patients are significantly affected by progressive weakness and muscle wasting due to alpha motor neuron (MN) degeneration. Mitochondria play a crucial role in neuronal health, and mitochondrial deficits were reported in other types of HSPs. To investigate whether mitochondrial pathology is present in SPG11, we differentiated MNs from induced pluripotent stem cells derived from SPG11 patients and controls. MN derived from human embryonic stem cells and an isogenic SPG11 knockout line were also included in the study. Morphological analysis of mitochondria in the MN soma versus neurites revealed specific alterations of mitochondrial morphology within SPG11 neurites, but not within the soma. In addition, impaired mitochondrial membrane potential was indicative of mitochondrial dysfunction. Moreover, we reveal neuritic aggregates further supporting neurite pathology in SPG11. Correspondingly, using a microfluidic-based MN culture system, we demonstrate that axonal mitochondrial transport was significantly impaired in SPG11. Overall, our data demonstrate that alterations in morphology, function, and transport of mitochondria are an important feature of axonal dysfunction in SPG11 MNs.
    Keywords:  SPG11; alpha motor neuron; axonal transport; hereditary spastic paraplegia; induced pluripotent stem cells; mitochondria
  20. Methods Mol Biol. 2021 ;2369 301-317
      Extracellular vesicles (EVs) or exovesicles are a heterogeneous group of small cell-derived membranous structures that carry complex cargoes including lipids, proteins, RNA, and DNA. Emerging evidence suggest that EVs secreted by kinetoplastid parasites play a cardinal role in the pathogenesis of diseases they cause, becoming valuable structures for understanding parasite-host interactions. Moreover, the characterization of EVs molecular cargo may provide a new approach to develop alternative tools for diagnosis and therapy of infectious diseases. EVs have a potential use as biomarkers since it contains a repertoire of DNA species that could be detected at different stages of infection by PCR-based assays. Here, we provide a detailed protocol for the isolation of Trypanosoma cruzi-derived EVs and purification of its DNA cargo for subsequent characterization. The methods described here are transferrable to other medically important parasites that are well adapted to grow in vitro and, therefore, suitable volume of EVs-containing supernatants can be obtained.
    Keywords:  DNA; Exosome; Extracellular vesicles; Isolation; Leishmania; Parasites; Trypanosoma; Trypanosomatid