bims-nenemi Biomed News
on Neuroinflammation, neurodegeneration and mitochondria
Issue of 2022‒04‒17
eleven papers selected by
Marco Tigano
Thomas Jefferson University

  1. Methods Mol Biol. 2022 ;2431 291-310
      Mitochondria are highly dynamic organelles which form intricate networks with complex dynamics. Mitochondrial transport and distribution are essential to ensure proper cell function, especially in cells with an extremely polarised morphology such as neurons. A layer of complexity is added when considering mitochondria have their own genome, packaged into nucleoids. Major mitochondrial morphological transitions, for example mitochondrial division, often occur in conjunction with mitochondrial DNA (mtDNA) replication and changes in the dynamic behaviour of the nucleoids. However, the relationship between mtDNA dynamics and mitochondrial motility in the processes of neurons has been largely overlooked. In this chapter, we describe a method for live imaging of mitochondria and nucleoids in differentiated SH-SY5Y cells by instant structured illumination microscopy (iSIM). We also include a detailed protocol for the differentiation of SH-SY5Y cells into cells with a pronounced neuronal-like morphology and show examples of coordinated mitochondrial and nucleoid motility in the long processes of these cells.
    Keywords:  Axonal transport; Instant structured illumination microscopy (iSIM); Mitochondria; Mitochondrial DNA; Mitochondrial fission; Neuronal differentiation; Nucleoids; SH-SY5Y cells; Superresolution
  2. Mol Cell Oncol. 2021 ;8(6): 2007028
      How oxidative stress promotes aging-related human diseases like cancer and neurodegeneration remains unclear. Here, we discuss the origins and implications of an oxidative-stress response recently reported to destabilize the mitochondrial (mt) genome via unscheduled RNA/DNA hybrid (R-loop) accumulation, by impairing the recruitment of RNAseH1 to the regulatory regions of mtDNA.
    Keywords:  BRCA2; Oxidative stress; PRPF8; R-loops; RNA-DNA hybrid; SETX; cancer; genomic instability; mitochondria; mtDNA replication; neurodegeneration
  3. Elife. 2022 Apr 13. pii: e76171. [Epub ahead of print]11
      In eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Previously we showed that translational control is primarily exerted through a conformational switch in eIF2's nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2 (Schoof et al. 2021). Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B's β subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed A/I-State model of allosteric ISR regulation.
    Keywords:  biochemistry; chemical biology; human; molecular biophysics; structural biology
  4. Biochim Biophys Acta Gen Subj. 2022 Apr 10. pii: S0304-4165(22)00065-4. [Epub ahead of print] 130147
      Severe ethanol stress (>9% v/v) induces pronounced translation repression in yeast cells. However, some proteins, which are exceptionally synthesized even under translation repression, play important roles in ethanol tolerance. These proteins are expected to provide important clues for elucidating the survival strategies of yeast cells under severe ethanol stress. In this study, we identified Hsp78 as a protein effectively synthesized under severe ethanol stress. As Hsp78 is involved in mitochondrial protein quality control, we investigated the effect of severe ethanol stress on mitochondrial proteins and found that Ilv2, Kgd1, and Aco1 aggregated with Hsp78 under severe ethanol stress, forming mitochondrial deposition sites for denatured proteins, called DUMPs (Deposits of Unfolded Mitochondrial Proteins). Aggregation of mitochondrial proteins and formation of DUMPs were accelerated in hsp78∆ cells compared with those in wild-type cells. During the recovery process after ethanol removal, aggregated Ilv2 and DUMP levels rapidly decreased in wild-type cells but were maintained for a long time (>180 min) in hsp78Δ cells. Furthermore, the frequency of respiration-deficient mutants caused by severe ethanol stress was higher in hsp78∆ cells than in wild-type cells. These results indicate that severe ethanol stress damaged mitochondrial proteins and that Hsp78 was preferentially synthesized to cope with the damage, thereby suppressing the rapid increase in aggregated protein levels under stress and achieving proper clearance of aggregated proteins during the recovery process. This study provides novel insights into the adverse effects of ethanol on mitochondria and yeast response to severe ethanol stress.
    Keywords:  DUMPs; Hsp78; Mitochondrial protein damage; Respiration-deficient mutant; Severe ethanol stress; Translation repression
  5. Elife. 2022 Apr 11. pii: e76557. [Epub ahead of print]11
      High frequencies of mutant mitochondrial DNA (mtDNA) in human cells lead to cellular defects that are associated with aging and disease. Yet much remains to be understood about the dynamics of the generation of mutant mtDNAs and their relative replicative fitness that informs their fate within cells and tissues. To address this, we utilize long-read single-molecule sequencing to track mutational trajectories of mtDNA in the model organism Saccharomyces cerevisiae. This model has numerous advantages over mammalian systems due to its much larger mtDNA and ease of artificially competing mutant and wild-type mtDNA copies in cells. We show a previously unseen pattern that constrains subsequent excision events in mtDNA fragmentation in yeast. We also provide evidence for the generation of rare and contentious non-periodic mtDNA structures that lead to persistent diversity within individual cells. Finally, we show that measurements of relative fitness of mtDNA fit a phenomenological model that highlights important biophysical parameters governing mtDNA fitness. Altogether, our study provides techniques and insights into the dynamics of large structural changes in genomes that we show are applicable to more complex organisms like humans.
    Keywords:  S. cerevisiae; computational biology; genetics; genomics; systems biology
  6. Methods Mol Biol. 2022 ;2431 385-407
      Precise distribution of mitochondria is essential for maintaining neuronal homeostasis. Although detailed mechanisms governing the transport of mitochondria have emerged, it is still poorly understood how the regulation of transport is coordinated in space and time within the physiological context of an organism. How alteration in mitochondrial functionality may trigger changes in organellar dynamics also remains unclear in this context. Therefore, the use of genetically encoded tools to perturb mitochondrial functionality in real time would be desirable. Here we describe methods to interfere with mitochondrial function with high spatiotemporal precision with the use of photosensitizers in vivo in the intact wing nerve of adult Drosophila. We also provide details on how to visualize the transport of mitochondria and to improve the quality of the imaging to attain super-resolution in this tissue.
    Keywords:  Axonal transport; Drosophila; Intravital imaging; KillerRed; Mitochondria; Neurons; Reactive oxygen species (ROS); Super-resolution radial fluctuations (SRRF); SuperNova
  7. Cells. 2022 Mar 31. pii: 1175. [Epub ahead of print]11(7):
      CARD19 is a mitochondrial protein of unknown function. While CARD19 was originally reported to regulate TCR-dependent NF-κB activation via interaction with BCL10, this function is not recapitulated ex vivo in primary murine CD8+ T cells. Here, we employ a combination of SIM, TEM, and confocal microscopy, along with proteinase K protection assays and proteomics approaches, to identify interacting partners of CARD19 in macrophages. Our data show that CARD19 is specifically localized to the outer mitochondrial membrane. Through deletion of functional domains, we demonstrate that both the distal C-terminus and transmembrane domain are required for mitochondrial targeting, whereas the CARD is not. Importantly, mass spectrometry analysis of 3×Myc-CARD19 immunoprecipitates reveals that CARD19 interacts with the components of the mitochondrial intermembrane bridge (MIB), consisting of mitochondrial contact site and cristae organizing system (MICOS) components MIC19, MIC25, and MIC60, and MICOS-interacting proteins SAMM50 and MTX2. These CARD19 interactions are in part dependent on a properly folded CARD. Consistent with previously reported phenotypes upon siRNA silencing of MICOS subunits, absence of CARD19 correlates with irregular cristae morphology. Based on these data, we propose that CARD19 is a previously unknown interacting partner of the MIB and the MIC19-MIC25-MIC60 MICOS subcomplex that regulates cristae morphology.
    Keywords:  BinCARD; CARD proteins; CARD19; MIB; MICOS; cristae
  8. Cell Death Dis. 2022 Apr 11. 13(4): 334
      Autophagy-mediated mitochondrial degradation plays pivotal roles in both the acquisition and maintenance of pluripotency, but the molecular mechanisms that link autophagy-mediated mitochondrial homeostasis to pluripotency regulation are unclear. Here, we identified that the mitophagy receptor BNIP3 regulates pluripotency. In mouse ESCs, depletion of BNIP3 caused accumulation of aberrant mitochondria accompanied by decreased mitochondrial membrane potential, increased production of reactive oxygen species (ROS), and reduced ATP generation, which led to compromised self-renewal and differentiation. Impairment of mitophagy by knockdown of BNIP3 inhibited mitochondrial clearance during pluripotency induction, resulting in decreased reprogramming efficiency. These defects were rescued by reacquisition of wild-type but not LIR-deficient BNIP3 expression. Taken together, our findings highlight a critical role of BNIP3-mediated mitophagy in the induction and maintenance of pluripotency.
  9. Hum Cell. 2022 Apr 16.
      Colon cancer cells rely on mitochondrial respiration as major source of energy for supporting their proliferation and invasion, thus promoting colon cancer malignancy and progression. In this study, we comprehensively investigated the prognostic significance of mitochondria-related genes in colon cancer and identified the hub genes that control colon cancer cell mitochondrial respiration and proliferation. We first systematically evaluated the prognostic significance of differentially expressed mitochondria-related genes in colon cancer specimens. Furthermore, a protein-protein interaction network was constructed to explore the hub genes. Eventually, five hub genes were identified, namely, POLG, FASTK, MRPS5, AARS2, and VARS2. Functional analyses showed that all these five hub genes are essential for maintaining mitochondrial respiration and proliferation of colon cancer cells. Mechanistic studies revealed the roles of these five hub genes in modulating mitochondrial DNA expression, that in turn influence mitochondrial respiration. In summary, our study demonstrated that POLG, FASTK, MRPS5, AARS2, and VARS2 may potentially serve as prognostic biomarkers and therapeutic targets for colon cancer.
    Keywords:  Colon cancer; Mitochondria; Mitochondria-related genes; Mitochondrial respiration; Proliferation; mtDNA expression
  10. Immunology. 2022 Apr 14.
      Cyclic GMP-AMP synthase (cGAS) is essential for fighting against viruses and bacteria, but how cGAS is involved in host immune response remains largely elusive. Here, we uncover the crucial role of cGAS in host immunity based on a Pseudomonas aeruginosa pulmonary infection model. cGAS-/- mice showed more heavy bacterial burdens and serious lung injury accompanied with exorbitant proinflammatory cytokines than wild-type mice. cGAS deficiency caused an accumulation of mitochondrial DNA in cytoplasm, which in turn induced excessive secretion of proinflammatory factors by activating inflammasome and TLR9 signaling. Mechanistically, cGAS deficiency inhibited the recruitment of LC3 by reducing the binding capacity of TBK-1 to p62, leading to impaired mitophagy and augmented release of mitochondrial DNA. Importantly, cytoplasmic mitochondrial DNA also acted as a feedback signal that induced the activation of cGAS. Altogether, these findings identify protective and homeostasis functions of cGAS against Pseudomonas aeruginosa infection, adding significant insight into the pathogenesis of bacterial infectious diseases.
    Keywords:  bacterial burdens; cGAS; cytokine secretion; mitochondrial DNA; pulmonary infection
  11. Nature. 2022 Apr 13.
      Chimeric antigen receptor (CAR) therapy has had a transformative effect on the treatment of haematologic malignancies1-6, but it has shown limited efficacy against solid tumours. Solid tumours may have cell-intrinsic resistance mechanisms to CAR T cell cytotoxicity. Here, to systematically identify potential resistance pathways in an unbiased manner, we conducted a genome-wide CRISPR knockout screen in glioblastoma, a disease in which CAR T cells have had limited efficacy7,8. We found that the loss of genes in the interferon-γ receptor (IFNγR) signalling pathway (IFNGR1, JAK1 or JAK2) rendered glioblastoma and other solid tumours more resistant to killing by CAR T cells both in vitro and in vivo. However, loss of this pathway did not render leukaemia or lymphoma cell lines insensitive to CAR T cells. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell-adhesion pathways after exposure to CAR T cells. We found that loss of IFNγR1 in glioblastoma cells reduced overall CAR T cell binding duration and avidity. The critical role of IFNγR signalling in susceptibility of solid tumours to CAR T cells is surprising, given that CAR T cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumours, IFNγR signalling was required for sufficient adhesion of CAR T cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumours differ in their interactions with CAR T cells and suggests that enhancing binding interactions between T cells and tumour cells may yield improved responses in solid tumours.