bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2022–03–20
23 papers selected by
Catalina Vasilescu, University of Helsinki



  1. Front Cell Dev Biol. 2022 ;10 786268
      Mitochondria are complex organelles containing 13 proteins encoded by mitochondrial DNA and over 1,000 proteins encoded on nuclear DNA. Many mitochondrial proteins are associated with the inner or outer mitochondrial membranes, either peripherally or as integral membrane proteins, while others reside in either of the two soluble mitochondrial compartments, the mitochondrial matrix and the intermembrane space. The biogenesis of the five complexes of the oxidative phosphorylation system are exemplars of this complexity. These large multi-subunit complexes are comprised of more than 80 proteins with both membrane integral and peripheral associations and require soluble, membrane integral and peripherally associated assembly factor proteins for their biogenesis. Mutations causing human mitochondrial disease can lead to defective complex assembly due to the loss or altered function of the affected protein and subsequent destabilization of its interactors. Here we couple sodium carbonate extraction with quantitative mass spectrometry (SCE-MS) to track changes in the membrane association of the mitochondrial proteome across multiple human knockout cell lines. In addition to identifying the membrane association status of over 840 human mitochondrial proteins, we show how SCE-MS can be used to understand the impacts of defective complex assembly on protein solubility, giving insights into how specific subunits and sub-complexes become destabilized.
    Keywords:  OXPHOS (oxidative phosphorylation); carbonate extraction; membrane protein; mitochondria; proteomic analyses; respiratory chain assembly
    DOI:  https://doi.org/10.3389/fcell.2022.786268
  2. IUBMB Life. 2022 Mar 18.
      Multiple mitochondrial matrix enzymes playing key roles in metabolism require cofactors for their action. Due to the high impermeability of the mitochondrial inner membrane, these cofactors need to be synthesized within the mitochondria or be imported, themselves or one of their precursors, into the organelles. Transporters belonging to the protein family of mitochondrial carriers have been identified to transport the coenzymes: thiamine pyrophosphate, coenzyme A, FAD and NAD+ , which are all structurally similar to nucleotides and derived from different B-vitamins. These mitochondrial cofactors bind more or less tightly to their enzymes and, after having been involved in a specific reaction step, are regenerated, spontaneously or by other enzymes, to return to their active form, ready for the next catalysis round. Disease-causing mutations in the mitochondrial cofactor carrier genes compromise not only the transport reaction but also the activity of all mitochondrial enzymes using that particular cofactor and the metabolic pathways in which the cofactor-dependent enzymes are involved. The mitochondrial transport, metabolism and diseases of the cofactors thiamine pyrophosphate, coenzyme A, FAD and NAD+ are the focus of this review.
    Keywords:  coenzyme; coenzyme A; flavin adenine dinucleotide; mitochondria; mitochondrial carrier family SLC25; nicotinamide adenine dinucleotide; thiamine pyrophosphate
    DOI:  https://doi.org/10.1002/iub.2612
  3. Mol Cell. 2022 Mar 17. pii: S1097-2765(22)00168-X. [Epub ahead of print]82(6): 1086-1088
      Li et al. (2022) discover that Toxoplasma infection triggers remodeling of the mitochondrial outer membrane through generation of a mitochondrial subdomain termed "structure positive for outer mitochondrial membrane" (SPOT).
    DOI:  https://doi.org/10.1016/j.molcel.2022.02.030
  4. Cell Cycle. 2022 Mar 17. 1-16
      We showed previously that POLG mutations cause major changes in mitochondrial function, including loss of mitochondrial respiratory chain (MRC) complex I, mitochondrial DNA (mtDNA) depletion and an abnormal NAD+/NADH ratio in both neural stem cells (NSCs) and astrocytes differentiated from induced pluripotent stem cells (iPSCs). In the current study, we looked at mitochondrial remodeling as stem cells transit pluripotency and during differentiation from NSCs to both dopaminergic (DA) neurons and astrocytes comparing the process in POLG-mutated and control stem cells. We saw that mitochondrial membrane potential (MMP), mitochondrial volume, ATP production and reactive oxygen species (ROS) changed in similar ways in POLG and control NSCs, but mtDNA replication, MRC complex I and NAD+ metabolism failed to remodel normally. In DA neurons differentiated from NSCs, we saw that POLG mutations caused failure to increase MMP and ATP production and blunted the increase in mtDNA and complex I. Interestingly, mitochondrial remodeling during astrocyte differentiation from NSCs was similar in both POLG-mutated and control NSCs. Further, we showed downregulation of the SIRT3/AMPK pathways in POLG-mutated cells, suggesting that POLG mutations lead to abnormal mitochondrial remodeling in early neural development due to the downregulation of these pathways. [Figure: see text].
    Keywords:  DA neurons; Mitochondrial remodeling; NSCs; POLG; astrocytes; iPSCs
    DOI:  https://doi.org/10.1080/15384101.2022.2044136
  5. Front Neurol. 2022 ;13 857279
      The nuclear gene TK2 encodes the mitochondrial thymidine kinase, an enzyme involved in the phosphorylation of deoxycytidine and deoxythymidine nucleosides. Biallelic TK2 mutations are associated with a spectrum of clinical presentations mainly affecting skeletal muscle and featuring muscle mitochondrial DNA (mtDNA) instability. Current classification includes infantile- ( ≤ 1 year), childhood- (1-12 years), and late-onset (≥12 years) forms. In addition to age at onset, these forms differ for progression, life expectancy, and signs of mtDNA instability (mtDNA depletion vs. accumulation of multiple mtDNA deletions). Childhood-onset TK2 deficiency typically causes a rapidly progressive proximal myopathy, which leads to wheelchair-bound status within 10 years of disease onset, and severe respiratory impairment. Muscle biopsy usually reveals a combination of mitochondrial myopathy and dystrophic features with reduced mtDNA content. Here we report the case of an Italian patient presenting childhood-onset, slowly progressive mitochondrial myopathy, ptosis, hypoacusis, dysphonia, and dysphagia, harboring the TK2 variants c.278A>G and c.543del, the latter unreported so far. Compared to other childhood-onset TK2-patients, our case displays atypical features, including slowly progressive muscle weakness and absence of respiratory failure, which are usually observed in late-onset forms. This report extends the genetic background of TK2-related myopathy, highlighting the clinical overlap among different forms.
    Keywords:  TK2; deoxynucleosides; mitochondrial DNA; mtDNA maintenance defects; myopathy; thymidine kinase 2
    DOI:  https://doi.org/10.3389/fneur.2022.857279
  6. Redox Biol. 2022 Mar 02. pii: S2213-2317(22)00049-0. [Epub ahead of print]51 102277
      Glutaredoxin 2 (Grx2) is a glutathione-dependent oxidoreductase that facilitates glutathionylation/de-glutathionylation of target proteins. The main variants of Grx2 are the mitochondrial Grx2a and the cytosolic Grx2c. The aim of this study was to investigate the specific role of mitochondrial Grx2 in vivo using a mitochondrial Grx2 depleted (mGD) mouse model. mGD mice displayed an altered mitochondrial morphology and functioning. Furthermore, the lack of Grx2 in the mitochondrial compartment is responsible for increased blood lipid levels under a normal diet, a metabolic dysfunction-associated fatty liver disease (MAFLD) phenotype and a decreased glycogen storage capacity. In addition, depleting Grx2a leads to an alteration in abundance and in glutathionylation pattern of different mitochondrial enzymes, highlighting the selective role of Grx2 in the regulation of metabolic pathways. Overall, our findings identify the involvement of mitochondrial Grx2a in the regulation of cell metabolism and highlight a previously unknown association between Grx2 and MAFLD.
    Keywords:  Glutaredoxin 2; Lipid metabolism; MAFLD; Mitochondria; Mouse model; ROS production
    DOI:  https://doi.org/10.1016/j.redox.2022.102277
  7. Curr Biol. 2022 Mar 08. pii: S0960-9822(22)00328-1. [Epub ahead of print]
      Mitochondrial damage (MtD) represents a dramatic change in cellular homeostasis, necessitating metabolic changes and stimulating mitophagy. One rapid response to MtD is a rapid peri-mitochondrial actin polymerization termed ADA (acute damage-induced actin). The activation mechanism for ADA is unknown. Here, we use mitochondrial depolarization or the complex I inhibitor metformin to induce ADA. We show that two parallel signaling pathways are required for ADA. In one pathway, increased cytosolic calcium in turn activates PKC-β, Rac, WAVE regulatory complex, and Arp2/3 complex. In the other pathway, a drop in cellular ATP in turn activates AMPK (through LKB1), Cdc42, and FMNL formins. We also identify putative guanine nucleotide exchange factors for Rac and Cdc42, Trio and Fgd1, respectively, whose phosphorylation states increase upon mitochondrial depolarization and whose suppression inhibits ADA. The depolarization-induced calcium increase is dependent on the mitochondrial sodium-calcium exchanger NCLX, suggesting initial mitochondrial calcium efflux. We also show that ADA inhibition results in enhanced mitochondrial shape changes upon mitochondrial depolarization, suggesting that ADA inhibits these shape changes. These depolarization-induced shape changes are not fragmentation but a circularization of the inner mitochondrial membrane, which is dependent on the inner mitochondrial membrane protease Oma1. ADA inhibition increases the proteolytic processing of an Oma1 substrate, the dynamin GTPase Opa1. These results show that ADA requires the combined action of the Arp2/3 complex and formin proteins to polymerize a network of actin filaments around mitochondria and that the ADA network inhibits the rapid mitochondrial shape changes that occur upon mitochondrial depolarization.
    Keywords:  AMPK; Arp2/3 complex; CCCP; FMNL formins; OMA1; OPA1; PKCβ; actin; calcium; mitochondrial depolarization
    DOI:  https://doi.org/10.1016/j.cub.2022.02.058
  8. Methods Mol Biol. 2022 ;2474 11-19
      Mitochondrial function, a key indicator of cell health, can be assessed through monitoring changes in mitochondrial membrane potential (MMP). Cationic fluorescent dyes are commonly used tools to assess MMP. We used a water-soluble mitochondrial membrane potential indicator (m-MPI) to detect changes in MMP in various types of cells, such as HepG2, HepaRG, and AC16 cells. A homogenous cell-based MMP assay has been optimized and performed in a 1536-well plate format, which can be used to screen several compound libraries for mitochondrial toxicity by evaluating the effects of chemical compounds on MMP.
    Keywords:  1536-well plate; Mesoxalonitrile 4-trifluoromethoxyphenylhydrazone (FCCP); Mitochondrial membrane potential (MMP); Mitochondrial membrane potential indicator (m-MPI); Mitochondrial toxicity
    DOI:  https://doi.org/10.1007/978-1-0716-2213-1_2
  9. Mol Metab. 2022 Mar 09. pii: S2212-8778(22)00038-2. [Epub ahead of print] 101469
       OBJECTIVE: The mitochondrial pyruvate carrier (MPC) has emerged as a promising drug target for metabolic disorders, including non-alcoholic steatohepatitis and diabetes, metabolically dependent cancers and neurodegenerative diseases. A range of structurally diverse small molecule inhibitors have been proposed but the nature of their interaction with MPC is not understood. Moreover, the composition of the functional human MPC is still debated. The goal of this study was to characterize the human MPC protein in vitro, to understand the chemical features that determine binding of structurally diverse inhibitors and to develop novel higher affinity ones.
    METHODS: We have recombinantly expressed and purified human MPC hetero-complexes and have studied the composition, transport and inhibitor binding properties by establishing in vitro transport assays, high throughput thermostability shift assays and pharmacophore modeling.
    RESULTS: We have determined that the functional unit of human MPC is a hetero-dimer. We have compared all different classes of MPC inhibitors to find that three closely arranged hydrogen bond acceptors followed by an aromatic ring are shared characteristics of all inhibitors and represent the minimal requirement for high potency. We also demonstrate that high affinity binding is not attributed to covalent bond formation with MPC cysteines, as previously proposed. Following the basic pharmacophore properties, we identify 14 new inhibitors of MPC, one outperforming compound UK5099 by tenfold. Two of them are the commonly prescribed drugs entacapone and nitrofurantoin, suggesting an off-target mechanism associated with their adverse effects.
    CONCLUSION: This work defines the composition of human MPC and the essential MPC inhibitor characteristics. In combination with the functional assays we describe, this new understanding will accelerate the development of clinically relevant MPC modulators.
    DOI:  https://doi.org/10.1016/j.molmet.2022.101469
  10. J Neurol. 2022 Mar 14.
       BACKGROUND AND OBJECTIVE: TK2 deficiency (TK2d) is a rare mitochondrial disorder that manifests predominantly as a progressive myopathy with a broad spectrum of severity and age of onset. The rate of progression is variable, and the prognosis is poor due to early and severe respiratory involvement. Early and accurate diagnosis is particularly important since a specific treatment is under development. This study aims to evaluate the diagnostic value of lower limb muscle MRI in adult patients with TK2d.
    METHODS: We studied a cohort of 45 genetically confirmed patients with mitochondrial myopathy (16 with mutations in TK2, 9 with mutations in other nuclear genes involved in mitochondrial DNA [mtDNA] synthesis or maintenance, 10 with single mtDNA deletions, and 10 with point mtDNA mutations) to analyze the imaging pattern of fat replacement in lower limb muscles. We compared the identified pattern in patients with TK2d with the MRI pattern of other non-mitochondrial genetic myopathies that share similar clinical characteristics.
    RESULTS: We found a consistent lower limb muscle MRI pattern in patients with TK2d characterized by involvement of the gluteus maximus, gastrocnemius medialis, and sartorius muscles. The identified pattern in TK2 patients differs from the known radiological involvement of other resembling muscle dystrophies that share clinical features.
    CONCLUSIONS: By analyzing the largest cohort of muscle MRI from patients with mitochondrial myopathies studied to date, we identified a characteristic and specific radiological pattern of muscle involvement in patients with TK2d that could be useful to speed up its diagnosis.
    Keywords:  MRI; Mitochondrial myopathy; TK2
    DOI:  https://doi.org/10.1007/s00415-021-10957-0
  11. Nat Commun. 2022 Mar 17. 13(1): 1426
      Progressive degeneration of dopaminergic (DA) neurons in the substantia nigra is a hallmark of Parkinson's disease (PD). Dysregulation of developmental transcription factors is implicated in dopaminergic neurodegeneration, but the underlying molecular mechanisms remain largely unknown. Drosophila Fer2 is a prime example of a developmental transcription factor required for the birth and maintenance of midbrain DA neurons. Using an approach combining ChIP-seq, RNA-seq, and genetic epistasis experiments with PD-linked genes, here we demonstrate that Fer2 controls a transcriptional network to maintain mitochondrial structure and function, and thus confers dopaminergic neuroprotection against genetic and oxidative insults. We further show that conditional ablation of Nato3, a mouse homolog of Fer2, in differentiated DA neurons causes mitochondrial abnormalities and locomotor impairments in aged mice. Our results reveal the essential and conserved role of Fer2 homologs in the mitochondrial maintenance of midbrain DA neurons, opening new perspectives for modeling and treating PD.
    DOI:  https://doi.org/10.1038/s41467-022-29075-0
  12. J Biol Chem. 2022 Mar 09. pii: S0021-9258(22)00255-1. [Epub ahead of print] 101815
      Mitochondrial transcription factor A (TFAM) plays important roles in mitochondrial DNA (mtDNA) compaction, transcription initiation, and in the regulation of processes like transcription and replication processivity. It is possible that TFAM is locally regulated within the mitochondrial matrix via such mechanisms like phosphorylation by protein kinase A (PKA) and non-enzymatic acetylation by acetyl-CoA. Here we demonstrate that DNA-bound TFAM is less susceptible to these modifications. We confirmed using electrophoretic mobility shift assays that phosphorylated or acetylated TFAM compacted circular double-stranded DNA just as well as unmodified TFAM and provide an in-depth analysis of acetylated sites on TFAM. We show that both modifications of TFAM increase the processivity of mitochondrial RNA polymerase during transcription through TFAM-imposed barriers on DNA, but that TFAM bearing either modification retains its full activity in transcription initiation. We conclude that TFAM phosphorylation by PKA and non-enzymatic acetylation by acetyl-CoA are unlikely to occur at the mtDNA and that modified free TFAM retains its vital functionalities like compaction and transcription initiation while enhancing transcription processivity.
    Keywords:  Acetylation; DNA compaction; Mitochondrial transcription; Phosphorylation
    DOI:  https://doi.org/10.1016/j.jbc.2022.101815
  13. Stem Cell Res. 2022 Mar 08. pii: S1873-5061(22)00091-5. [Epub ahead of print]61 102742
      We report the generation of four human iPSC lines (8993-A12, 8993-B12, 8993-C11, and 8993-D7) from fibroblasts of four patients affected by maternally inherited Leigh syndrome (MILS) carrying homoplasmic mutations m.8993T > G or m.8993T > C in the mitochondrial gene MT-ATP6. We used Sendai viruses to deliver reprogramming factors OCT4, SOX2, KLF4, and c-MYC. The established iPSC lines expressed pluripotency markers, exhibited a normal karyotype, were capable to form cells of the three germ layers in vitro, and retained the MT-ATP6 mutations at the same homoplasmic level of the parental fibroblasts.
    DOI:  https://doi.org/10.1016/j.scr.2022.102742
  14. EMBO Rep. 2022 Mar 17. e52606
      Mitochondrial dysfunction can either extend or decrease Caenorhabditis elegans lifespan, depending on whether transcriptionally regulated responses can elicit durable stress adaptation to otherwise detrimental lesions. Here, we test the hypothesis that enhanced metabolic flexibility is sufficient to circumvent bioenergetic abnormalities associated with the phenotypic threshold effect, thereby transforming short-lived mitochondrial mutants into long-lived ones. We find that CEST-2.2, a carboxylesterase mainly localizes in the intestine, may stimulate the survival of mitochondrial deficient animals. We report that genetic manipulation of cest-2.2 expression has a minor lifespan impact on wild-type nematodes, whereas its overexpression markedly extends the lifespan of complex I-deficient gas-1(fc21) mutants. We profile the transcriptome and lipidome of cest-2.2 overexpressing animals and show that CEST-2.2 stimulates lipid metabolism and fatty acid beta-oxidation, thereby enhancing mitochondrial respiratory capacity through complex II and LET-721/ETFDH, despite the inherited genetic lesion of complex I. Together, our findings unveil a metabolic pathway that, through the tissue-specific mobilization of lipid deposits, may influence the longevity of mitochondrial mutant C. elegans.
    Keywords:   Caenorhabditis elegans ; carboxylesterase CEST-2.2; epigenetics; lipid metabolism; mitochondria
    DOI:  https://doi.org/10.15252/embr.202152606
  15. iScience. 2022 Mar 18. 25(3): 103957
      Babies are born young, largely independent of the age of their mothers. Mother-daughter age asymmetry in yeast is achieved, in part, by inheritance of higher-functioning mitochondria by buds and retention of some high-functioning mitochondria in mother cells. The mitochondrial F box protein, Mfb1p, tethers mitochondria at both poles in a cell cycle-regulated manner: it localizes to and anchors mitochondria at the mother cell tip throughout the cell cycle and at the bud tip before cytokinesis. Here, we report that cell polarity and polarized localization of Mfb1p decline with age in Saccharomyces cerevisiae. Moreover, deletion of genes (BUD1, BUD2, and BUD5) that mediate symmetry breaking during establishment of cell polarity and asymmetric yeast cell division cause depolarized Mfb1p localization and defects in mitochondrial distribution and quality control. Our results support a role for the polarity machinery in lifespan through modulating Mfb1 function in asymmetric inheritance of mitochondria during yeast cell division.
    Keywords:  Biological sciences; Cell biology; Genetics; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2022.103957
  16. Annu Rev Biochem. 2022 Feb 14.
      Mitochondria are central to energy production, metabolism and signaling, and apoptosis. To make new mitochondria from preexisting mitochondria, the cell needs to import mitochondrial proteins from the cytosol into the mitochondria with the aid of translocators in the mitochondrial membranes. The translocase of the outer membrane (TOM) complex, an outer membrane translocator, functions as an entry gate for most mitochondrial proteins. Although high-resolution structures of the receptor subunits of the TOM complex were deposited in the early 2000s, those of entire TOM complexes became available only in 2019. The structural details of these TOM complexes, consisting of the dimer of the β-barrel import channel Tom40 and four α-helical membrane proteins, revealed the presence of several distinct paths and exits for the translocation of over 1,000 different mitochondrial precursor proteins. High-resolution structures of TOM complexes now open up a new era of studies on the structures, functions, and dynamics of the mitochondrial import system. Expected final online publication date for the Annual Review of Biochemistry, Volume 91 is June 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
    DOI:  https://doi.org/10.1146/annurev-biochem-032620-104527
  17. Annu Rev Biochem. 2022 Feb 14.
      In the decade since the discovery of the innate immune cyclic GMP-AMP synthase (cGAS)- 2'3'-cyclic GMP-AMP (cGAMP)- stimulator of interferon genes (STING) pathway, its proper activation and dysregulation have been rapidly implicated in many aspects of human disease. Understanding the biochemical, cellular, and regulatory mechanisms of this pathway is critical to developing therapeutic strategies that either harness it to boost defense or inhibit it to prevent unwanted inflammation. In this review, we first discuss how the second messenger cGAMP is synthesized by cGAS in response to double-stranded DNA and cGAMP's subsequent activation of cell-type-dependent STING signaling cascades with differential physiological consequences. We then review how cGAMP as an immunotransmitter mediates tightly controlled cell-cell communication by being exported from producing cells and imported into responding cells via cell-type-specific transporters. Finally, we review mechanisms by which the cGAS-cGAMP-STING pathway responds to different sources of mislocalized double-stranded DNA in pathogen defense, cancer, and autoimmune diseases. Expected final online publication date for the Annual Review of Biochemistry, Volume 91 is June 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
    DOI:  https://doi.org/10.1146/annurev-biochem-040320-101629
  18. JIMD Rep. 2022 Mar;63(2): 114-122
      Cardiomyopathy is the most common presenting feature of Barth syndrome, often presenting in infancy with severe heart failure and cardiac dysfunction. Historically, affected infants commonly died early after presentation, sometimes before a diagnosis of Barth syndrome was made. With increases in awareness of Barth syndrome and in the care of infants with severe heart failure, survival of children with Barth syndrome and severe heart failure has improved. We describe our experience caring for five unrelated boys with Barth syndrome who presented with severe cardiomyopathy and heart failure prior to age 2 who have had marked improvement with long-term response to medical heart failure therapy.
    Keywords:  Barth syndrome; cardiomyopathy; heart failure; noncompaction; pediatric
    DOI:  https://doi.org/10.1002/jmd2.12264
  19. Sci Adv. 2022 Mar 18. 8(11): eabj4716
      Dysregulation of mitochondrial Ca2+ homeostasis has been linked to neurodegenerative diseases. Mitochondrial Ca2+ uptake is mediated via the calcium uniporter complex that is primarily regulated by MICU1, a Ca2+-sensing gatekeeper. Recently, human patients with MICU1 loss-of-function mutations were diagnosed with neuromuscular and cognitive impairments. While studies in patient-derived cells revealed altered mitochondrial calcium signaling, the neuronal pathogenesis was difficult to study. To fill this void, we created a neuron-specific MICU1-KO mouse model. These animals show progressive, abnormal motor and cognitive phenotypes likely caused by the degeneration of motor neurons in the spinal cord and the cortex. We found increased susceptibility to mitochondrial Ca2+ overload-induced excitotoxic insults and cell death in MICU1-KO neurons and MICU1-deficient patient-derived cells, which can be blunted by inhibiting the mitochondrial permeability transition pore. Thus, our study identifies altered neuronal mitochondrial Ca2+ homeostasis as causative in the clinical symptoms of MICU1-deficient patients and highlights potential therapeutic targets.
    DOI:  https://doi.org/10.1126/sciadv.abj4716
  20. Prog Neurobiol. 2022 Mar 11. pii: S0301-0082(22)00050-8. [Epub ahead of print]213 102264
      The complexity of astrocyte morphology and syncytial coupling through gap junctions are crucial for astrocyte function in the brain. However, the ultrastructural details of astrocyte arborization and interactions between neighboring astrocytes remain unknown. While a prevailing view is that synapses selectively contact peripheral astrocyte processes, the precise spatial-location selectivity of synapses abutting astrocytes is unresolved. Additionally, knowing the location and quantity of vesicles and mitochondria are prerequisites to answer two emerging questions - whether astrocytes have a signaling role within the brain and whether astrocytes are highly metabolically active. Here, we provided structural context for these questions by tracing and 3D reconstructing three neighboring astrocytes using serial block-face scanning electron microscopy. Our reconstructions reveal a spongiform astrocytic morphology resulting from the abundance of reflexive and leaflet processes. At the interfaces, varying sizes of astrocyte-astrocyte contacts were identified. Inside an astrocyte domain, synapses contact the entire astrocyte, and synapse-astrocyte contacts increase from soma to terminal leaflets. In contrast to densely packed vesicles at synaptic boutons, vesicle-like structures were scant within astrocytes. Lastly, astrocytes contain dense mitochondrial networks with a mitochondrial volume ratio similar to that of neurites. Together, these ultrastructural details should expand our understanding of functional astrocyte-astrocyte and astrocyte-neuron interactions.
    Keywords:  Aldh1l1-eGFP; Astrocyte network; Mitochondria; Serial blockface scanning electron microscopy (SBF-SEM); Synapses; Synaptic-like microvesicles; Three-dimensional reconstruction
    DOI:  https://doi.org/10.1016/j.pneurobio.2022.102264
  21. Science. 2022 Mar 18. 375(6586): 1254-1261
      Copper is an essential cofactor for all organisms, and yet it becomes toxic if concentrations exceed a threshold maintained by evolutionarily conserved homeostatic mechanisms. How excess copper induces cell death, however, is unknown. Here, we show in human cells that copper-dependent, regulated cell death is distinct from known death mechanisms and is dependent on mitochondrial respiration. We show that copper-dependent death occurs by means of direct binding of copper to lipoylated components of the tricarboxylic acid (TCA) cycle. This results in lipoylated protein aggregation and subsequent iron-sulfur cluster protein loss, which leads to proteotoxic stress and ultimately cell death. These findings may explain the need for ancient copper homeostatic mechanisms.
    DOI:  https://doi.org/10.1126/science.abf0529
  22. Methods Mol Biol. 2022 ;2483 195-204
      The ubiquitous second messengers' 3',5'-cyclic adenosine monophosphate (cAMP ) and 3',5'-cyclic guanosine monophosphate (cGMP) are crucial in regulating cardiomyocyte function, as well as pathological processes, by acting in distinct subcellular microdomains and thus controlling excitation-contraction coupling. Spatio-temporal intracellular dynamics of cyclic nucleotides can be measured in living cells using fluorescence resonance energy transfer (FRET ) by transducing isolated cells with genetically encoded biosensors. While FRET experiments have been regularly performed in cardiomyocytes from different animal models, human-based translational experiments are very challenging due to the difficulty to culture and transduce adult human cardiomyocytes. Here, we describe a technique for obtaining human atrial and ventricular myocytes which allows to keep them alive in culture long enough to transduce them and visualize cAMP and cGMP in physiological and pathological human settings.
    Keywords:  Atrial myocytes; Biosensors; Culture; Fluorescence resonance energy transfer; Human; Isolation; Ventricular myocytes; cAMP; cGMP
    DOI:  https://doi.org/10.1007/978-1-0716-2245-2_12