bims-nenemi Biomed News
on Neuroinflammation, neurodegeneration and mitochondria
Issue of 2022‒11‒20
twenty-one papers selected by
Marco Tigano
Thomas Jefferson University

  1. J Cell Biol. 2023 Jan 02. pii: e202203019. [Epub ahead of print]222(1):
      Astrocytes, often considered as secondary responders to neurodegeneration, are emerging as primary drivers of brain disease. Here we show that mitochondrial DNA depletion in astrocytes affects their primary cilium, the signaling organelle of a cell. The progressive oxidative phosphorylation deficiency in astrocytes induces FOXJ1 and RFX transcription factors, known as master regulators of motile ciliogenesis. Consequently, a robust gene expression program involving motile cilia components and multiciliated cell differentiation factors are induced. While the affected astrocytes still retain a single cilium, these organelles elongate and become remarkably distorted. The data suggest that chronic activation of the mitochondrial integrated stress response (ISRmt) in astrocytes drives anabolic metabolism and promotes ciliary elongation. Collectively, our evidence indicates that an active signaling axis involving mitochondria and primary cilia exists and that ciliary signaling is part of ISRmt in astrocytes. We propose that metabolic ciliopathy is a novel pathomechanism for mitochondria-related neurodegenerative diseases.
  2. Proc Natl Acad Sci U S A. 2022 Nov 22. 119(47): e2210730119
      Mitochondria have their own DNA (mtDNA), which encodes essential respiratory subunits. Under live imaging, mitochondrial nucleoids, composed of several copies of mtDNA and DNA-binding proteins, such as mitochondrial transcription factor A (TFAM), actively move inside mitochondria and change the morphology, in concert with mitochondrial membrane fission. Here we found the mitochondrial inner membrane-anchored AAA-ATPase protein ATAD3A mediates the nucleoid dynamics. Its ATPase domain exposed to the matrix binds directly to TFAM and mediates nucleoid trafficking along mitochondria by ATP hydrolysis. Nucleoid trafficking also required ATAD3A oligomerization via an interaction between the coiled-coil domains in intermembrane space. In ATAD3A deficiency, impaired nucleoid trafficking repressed the clustered and enlarged nucleoids observed in mitochondrial fission-deficient cells resulted in dispersed distribution of small nucleoids observed throughout the mitochondrial network, and this enhanced respiratory complex formation. Thus, mitochondrial fission and nucleoid trafficking cooperatively determine the size, number, and distribution of nucleoids in mitochondrial network, which should modulate respiratory complex formation.
    Keywords:  ATAD3A; Drp1; mitochondrial fission; mtDNA nucleoid; respiratory complex
  3. EMBO J. 2022 Nov 18. e112920
      Mitochondria are key signaling hubs for innate immune responses. In this issue, Wu et al (2022) report that remodeling of the outer mitochondrial membrane by the linear ubiquiting chain assembly complex (LUBAC) facilitates transport of activated NF-κB to the nucleus in response to TNF signaling.
  4. Mol Cell. 2022 Nov 08. pii: S1097-2765(22)01054-1. [Epub ahead of print]
      Nutrient sensing and damage sensing are two fundamental processes in living organisms. While hyperglycemia is frequently linked to diabetes-related vulnerability to microbial infection, how body glucose levels affect innate immune responses to microbial invasion is not fully understood. Here, we surprisingly found that viral infection led to a rapid and dramatic decrease in blood glucose levels in rodents, leading to robust AMPK activation. AMPK, once activated, directly phosphorylates TBK1 at S511, which triggers IRF3 recruitment and the assembly of MAVS or STING signalosomes. Consistently, ablation or inhibition of AMPK, knockin of TBK1-S511A, or increased glucose levels compromised nucleic acid sensing, while boosting AMPK-TBK1 cascade by AICAR or TBK1-S511E knockin improves antiviral immunity substantially in various animal models. Thus, we identify TBK1 as an AMPK substrate, reveal the molecular mechanism coupling a dual sensing of glucose and nuclei acids, and report its physiological necessity in antiviral defense.
    Keywords:  AMPK; TBK1; antiviral immunity; cGAS-STING; coronavirus; glucose metabolism; hyperglycemia; innate immunity; viral infection; virus-host interactions
  5. EMBO J. 2022 Nov 18. e112006
      Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear.
    Keywords:  HOIP; NEMO; OTULIN; PINK1; ubiquitin
  6. Elife. 2022 11 17. pii: e69916. [Epub ahead of print]11
      Having its genome makes the mitochondrion a unique and semiautonomous organelle within cells. Mammalian mitochondrial DNA (mtDNA) is a double-stranded closed circular molecule of about 16 kb coding for 37 genes. Mutations, including deletions in the mitochondrial genome, can culminate in different human diseases. Mapping the deletion junctions suggests that the breakpoints are generally seen at hotspots. '9-bp deletion' (8271-8281), seen in the intergenic region of cytochrome c oxidase II/tRNA<sup>Lys</sup>, is the most common mitochondrial deletion. While it is associated with several diseases like myopathy, dystonia, and hepatocellular carcinoma, it has also been used as an evolutionary marker. However, the mechanism responsible for its fragility is unclear. In the current study, we show that Endonuclease G, a mitochondrial nuclease responsible for nonspecific cleavage of nuclear DNA during apoptosis, can induce breaks at sequences associated with '9-bp deletion' when it is present on a plasmid or in the mitochondrial genome. Through a series of <i>in vitro</i> and intracellular studies, we show that Endonuclease G binds to G-quadruplex structures formed at the hotspot and induces DNA breaks. Therefore, we uncover a new role for Endonuclease G in generating mtDNA deletions, which depends on the formation of G4 DNA within the mitochondrial genome. In summary, we identify a novel property of Endonuclease G, besides its role in apoptosis and the recently described elimination of paternal mitochondria during fertilisation.
    Keywords:  E. coli; cell biology; genetics; genomics; human; rat
  7. Cell Death Dis. 2022 Nov 18. 13(11): 969
      Multiple myeloma (MM) causes approximately 20% of deaths from blood cancers. Notwithstanding significant therapeutic progress, such as with proteasome inhibitors (PIs), MM remains incurable due to the development of resistance. mTORC1 is a key metabolic regulator, which frequently becomes dysregulated in cancer. While mTORC1 inhibitors reduce MM viability and synergize with other therapies in vitro, clinically, mTORC1 inhibitors are not effective for MM. Here we show that the inactivation of mTORC1 is an intrinsic response of MM to PI treatment. Genetically enforced hyperactivation of mTORC1 in MM was sufficient to compromise tumorigenicity in mice. In vitro, mTORC1-hyperactivated MM cells gained sensitivity to PIs and hypoxia. This was accompanied by increased mitochondrial stress and activation of the eIF2α kinase HRI, which initiates the integrated stress response. Deletion of HRI elevated the toxicity of PIs in wt and mTORC1-activated MM. Finally, we identified the drug PMA as a robust inducer of mTORC1 activity, which synergized with PIs in inducing MM cell death. These results help explain the clinical inefficacy of mTORC1 inhibitors in MM. Our data implicate mTORC1 induction and/or HRI inhibition as pharmacological strategies to enhance MM therapy by PIs.
  8. Cell Death Differ. 2022 Nov 14.
      How BAK and BAX induce mitochondrial outer membrane (MOM) permeabilization (MOMP) during apoptosis is incompletely understood. Here we have used molecular dynamics simulations, surface plasmon resonance, and assays for membrane permeabilization in vitro and in vivo to assess the structure and function of selected BAK subdomains and their derivatives. Results of these studies demonstrate that BAK helical regions α5 and α6 bind the MOM lipid cardiolipin. While individual peptides corresponding to these helical regions lack the full biological activity of BAK, tandem peptides corresponding to α4-α5, α5-α6, or α6-α7/8 can localize exogenous proteins to mitochondria, permeabilize liposomes composed of MOM lipids, and cause MOMP in the absence of the remainder of the BAK protein. Importantly, the ability of these tandem helices to induce MOMP under cell-free conditions is diminished by mutations that disrupt the U-shaped helix-turn-helix structure of the tandem peptides or decrease their lipid binding. Likewise, BAK-induced apoptosis in intact cells is diminished by CLS1 gene interruption, which decreases mitochondrial cardiolipin content, or by BAK mutations that disrupt the U-shaped tandem peptide structure or diminish lipid binding. Collectively, these results suggest that BAK structural rearrangements during apoptosis might mobilize helices involved in specific protein-lipid interactions that are critical for MOMP.
  9. Neuroscience. 2022 Nov 09. pii: S0306-4522(22)00557-7. [Epub ahead of print]
      Developmental sevoflurane exposure leads to widespread neuronal cell death known as sevoflurane-induced neurotoxicity (SIN). Receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL)-driven necroptosis plays an important role in cell fate. Previous research has shown that inhibition of RIPK1 activity alone did not attenuate SIN. Since RIPK3/MLKL signaling could also be activated by Z-DNA/RNA binding protein 1 (ZBP1), the present study was designed to investigate whether ZBP1-mediated and RIPK3/MLKL-driven necroptosis is involved in SIN through in vitro and in vivo experiments. We found that sevoflurane priming triggers neuronal cell death and LDH release in a time-dependent manner. The expression levels of RIPK1, RIPK3, ZBP1 and membrane phosphorylated MLKL were also dramatically enhanced in SIN. Intriguingly, knockdown of RIPK3, but not RIPK1, abolished MLKL-mediated neuronal necroptosis in SIN. Additionally, inhibition of RIPK3-mediated necroptosis with GSK'872, rather than inhibition of apoptosis with zVAD, significantly ameliorated SIN. Further investigation showed that sevoflurane treatment causes mitochondrial DNA (mtDNA) release into the cytosol. Accordingly, ZBP1 senses cytosolic mtDNA and consequently activates RIPK3/MLKL signaling. This conclusion was reinforced by the evidence that knockdown of ZBP1 or depleting mtDNA with ethidium bromide remarkably improved SIN. Finally, the administration of the RIPK3 inhibitor GSK'872 relieved sevoflurane-induced spatial and emotional disorders without influence on locomotor activity. Altogether, these results illustrate that ZBP1 senses cytosolic mtDNA to induce RIPK3/MLKL-driven necroptosis in SIN. Elucidating the role of necroptosis in SIN will provide new insights into understanding the mechanism of anesthetic exposure in the developing brain.
    Keywords:  Developing brain; MLKL; Necroptosis; RIPK3; Sevoflurane; ZBP1
  10. J Biol Chem. 2022 Nov 12. pii: S0021-9258(22)01147-4. [Epub ahead of print] 102704
      The autophagic clearance of mitochondria has been defined as mitophagy, which is triggered by mitochondrial damage and serves as a major pathway for mitochondrial homeostasis and cellular quality control. PINK1 and Parkin-mediated mitophagy is the most extensively studied form of mitophagy, which has been linked to the pathogenesis of neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The current paradigm of this particular mitophagy pathway is that the ubiquitination of the outer mitochondrial membrane is the key step to enable the recognition of damaged mitochondria by the core autophagic component autophagosome. However, whether the inner mitochondrial membrane (IMM) is ubiquitinated by Parkin and its contribution to sufficient mitophagy remain unclear. Here, using molecular, cellular, and biochemical approaches, we report that prohibitin 2 (PHB2), an essential IMM receptor for mitophagy, is ubiquitinated by Parkin and thereby gains higher affinity to the autophagosome during mitophagy. Our findings suggest that Parkin directly binds to PHB2 through its RING1 domain and promotes K11- and K33-linked ubiquitination on K142/K200 sites of PHB2, thereby enhancing the interaction between PHB2 and MAP1LC3B/LC3B. Interestingly and importantly, our study allows us to propose a novel model in which IMM protein PHB2 serves as both a receptor and a ubiquitin-mediated base for autophagosome recruitment to ensure efficient mitophagy.
    Keywords:  MAP1LC3B/LC3B; PHB2; Parkin; mitophagy; ubiquitination
  11. Int J Radiat Biol. 2022 Nov 16. 1-10
      PURPOSE: In living organisms, sensitivity to radiation increases in the presence of oxygen (O2) compared with that under anoxic or hypoxic conditions. Here, we investigated whether O2 concentration affected the response of mitochondria to X-rays radiation, which is associated with tumor microenvironment formation via fibroblast activation in radiation-related tumors.MATERIALS AND METHODS: O2 concentrations were controlled at <5% (internal environmental oxygen condition) or anoxic levels during culture of normal human diploid lung fibroblasts TIG-3 and MRC-5. Protein expression associated with the response of mitochondria to radiation was assessed using immunostaining or western blotting.
    RESULTS: Induction of DNA damage (marker: γ-H2A histone family member X) and mitochondrial signaling (AMP-activated protein kinase), suppression of mitochondrial metabolic activity, and generation of reactive oxygen species occurred with radiation in cells cultured under 5% and 20% O2 conditions. However, reducing O2 concentration mitigated the effects of radiation on cell growth, mitochondrial damage (parkin), induction of antioxidant responses (nuclear factor E2-related factor 2), and fibroblast activation (α-smooth muscle actin). Radiation did not affect the markers used in this study in the absence of O2.
    CONCLUSION: O2 concentration affected the response of mitochondria to radiation and reactive oxygen species-mediated fibroblast activation. Higher O2 concentrations enhanced the effects of radiation on mitochondria in human fibroblasts. In vitro studies may overestimate in vivo radiation effects due to high O2 concentrations.
    Keywords:  Oxygen; fibroblast activation; mitochondria; radiation; reactive oxygen species (ROS)
  12. iScience. 2022 Nov 18. 25(11): 105410
      Deletion of genes encoding ribosomal proteins extends lifespan in yeast. This increases translation of the functionally conserved transcription factor Gcn4, and lifespan extension in these mutants is GCN4-dependent. Gcn4 is also translationally upregulated by uncharged tRNAs, as are its C aenorhabditis elegans and mammalian functional orthologs. Here, we show that cytosolic tRNA synthetase inhibitors upregulate Gcn4 translation and extend yeast lifespan in a Gcn4-dependent manner. This cytosolic tRNA synthetase inhibitor is also able to extend the lifespan of C. elegans in an atf-4-dependent manner. We show that mitochondrial tRNA synthetase inhibitors greatly extend the lifespan of C. elegans, and this depends on atf-4. This suggests that perturbations of both cytosolic and mitochondrial translation may act in part via the same downstream pathway. These findings establish GCN4 orthologs as conserved longevity factors and, as long-lived mice exhibit elevated ATF4, leave open the possibility that tRNA synthetase inhibitors could also extend lifespan in mammals.
    Keywords:  Biochemistry; Biological sciences; Molecular biology
  13. Sci Adv. 2022 Nov 16. 8(46): eabq5234
      A stop codon within the mRNA facilitates coordinated termination of protein synthesis, releasing the nascent polypeptide from the ribosome. This essential step in gene expression is impeded with transcripts lacking a stop codon, generating nonstop ribosome complexes. Here, we use deep sequencing to investigate sources of nonstop mRNAs generated from the human mitochondrial genome. We identify diverse types of nonstop mRNAs on mitochondrial ribosomes that are resistant to translation termination by canonical release factors. Failure to resolve these aberrations by the mitochondrial release factor in rescue (MTRFR) imparts a negative regulatory effect on protein synthesis that is associated with human disease. Our findings reveal a source of underlying noise in mitochondrial gene expression and the importance of responsive ribosome quality control mechanisms for cell fitness and human health.
  14. Front Oncol. 2022 ;12 981332
      Recent studies suggest that inhibition of the ATR kinase can potentiate radiation-induced antitumor immune responses, but the extent and mechanisms of such responses in human cancers remain scarcely understood. We aimed to assess whether the ATR inhibitors VE822 and AZD6738, by abrogating the G2 checkpoint, increase cGAS-mediated type I IFN response after irradiation in human lung cancer and osteosarcoma cell lines. Supporting that the checkpoint may prevent IFN induction, radiation-induced IFN signaling declined when the G2 checkpoint arrest was prolonged at high radiation doses. G2 checkpoint abrogation after co-treatment with radiation and ATR inhibitors was accompanied by increased radiation-induced IFN signaling in four out of five cell lines tested. Consistent with the hypothesis that the cytosolic DNA sensor cGAS may detect DNA from ruptured micronuclei after G2 checkpoint abrogation, cGAS co-localized with micronuclei, and depletion of cGAS or STING abolished the IFN responses. Contrastingly, one lung cancer cell line showed no increase in IFN signaling despite irradiation and G2 checkpoint abrogation. This cell line showed a higher level of the exonuclease TREX1 than the other cell lines, but TREX1 depletion did not enhance IFN signaling. Rather, addition of a pan-caspase inhibitor restored the IFN response in this cell line and also increased the responses in the other cell lines. These results show that treatment-induced caspase activation can suppress the IFN response after co-treatment with radiation and ATR inhibitors. Caspase activation thus warrants further consideration as a possible predictive marker for lack of IFN signaling.
    Keywords:  ATR; TREX1; cGAS; caspase; cell cycle checkpoints; micronuclei (MN); radiation therapy (radiotherapy); type I interferon (IFN) signaling
  15. Cell Metab. 2022 Nov 08. pii: S1550-4131(22)00489-2. [Epub ahead of print]
      Impairment of translation can lead to collisions of ribosomes, which constitute an activation platform for several ribosomal stress-surveillance pathways. Among these is the ribotoxic stress response (RSR), where ribosomal sensing by the MAP3K ZAKα leads to activation of p38 and JNK kinases. Despite these insights, the physiological ramifications of ribosomal impairment and downstream RSR signaling remain elusive. Here, we show that stalling of ribosomes is sufficient to activate ZAKα. In response to amino acid deprivation and full nutrient starvation, RSR impacts on the ensuing metabolic responses in cells, nematodes, and mice. The RSR-regulated responses in these model systems include regulation of AMPK and mTOR signaling, survival under starvation conditions, stress hormone production, and regulation of blood sugar control. In addition, ZAK-/- male mice present a lean phenotype. Our work highlights impaired ribosomes as metabolic signals and demonstrates a role for RSR signaling in metabolic regulation.
    Keywords:  AMPK; FGF21; ZAK-alpha; amino acid starvation; mTOR; metabolic regulation; mouse models; ribosome collision; ribotoxic stress response
  16. Cell Syst. 2022 Nov 16. pii: S2405-4712(22)00402-1. [Epub ahead of print]13(11): 911-923.e9
      Morphological and gene expression profiling can cost-effectively capture thousands of features in thousands of samples across perturbations by disease, mutation, or drug treatments, but it is unclear to what extent the two modalities capture overlapping versus complementary information. Here, using both the L1000 and Cell Painting assays to profile gene expression and cell morphology, respectively, we perturb human A549 lung cancer cells with 1,327 small molecules from the Drug Repurposing Hub across six doses, providing a data resource including dose-response data from both assays. The two assays capture both shared and complementary information for mapping cell state. Cell Painting profiles from compound perturbations are more reproducible and show more diversity but measure fewer distinct groups of features. Applying unsupervised and supervised methods to predict compound mechanisms of action (MOAs) and gene targets, we find that the two assays not only provide a partially shared but also a complementary view of drug mechanisms. Given the numerous applications of profiling in biology, our analyses provide guidance for planning experiments that profile cells for detecting distinct cell types, disease phenotypes, and response to chemical or genetic perturbations.
    Keywords:  Cell Painting; L1000; benchmark; drug discovery; high-dimensional profiling; images; systems biology
  17. Mater Today Bio. 2022 Dec 15. 17 100475
      Recent advances in microfluidic engineering allow the creation of microenvironments in which human cells can be cultured under (patho-)physiological conditions with greater reality than standard plastic tissue culture plates. Microfluidic devices, also called Organs-on-Chip (OoC), allow complex engineering of the cellular compartment, yielding designs in which microfluidic flow can be precisely controlled. However, it is important that cellular physiology is not only controlled but can also be monitored in these devices. Here, we integrated oxygen and pH sensors into microfluidics, allowing close monitoring of the extracellular flux from the cells, enabling constant assessment of features such as glycolysis and mitochondrial oxidative phosphorylation in situ. Using human-induced pluripotent stem cells (hiPSCs) as an exemplar of a highly metabolic and relatively challenging cell type to maintain, we showed that monitoring the extracellular environment allowed rapid optimization of the seeding protocol. Based on the measurements, we implemented earlier and more frequent media refreshment to counteract the rapid acidification and depletion of oxygen. The integrated sensors showed that hiPSCs in the devices exhibited mitochondrial and glycolytic capacity similar to that measured with the Seahorse extracellular flux system, the most widely used standard for these types of assays in conventional cell culture. Under both conditions, hiPSCs showed greater reliance on glycolysis than mitochondrial OXPHOS and the absolute values obtained were similar. These results thus pave the way for the assessment of cell metabolism in situ under conditions of fluidic flow with the same precision and relevance as current standard static cell cultures.
    Keywords:  Extracellular flux; Human stem cells; Metabolism; Organ-on-Chip; Oxygen; Sensors; pH
  18. Nat Commun. 2022 Nov 17. 13(1): 7031
      An enhanced NADH/NAD+ ratio, termed reductive stress, is associated with many diseases. However, whether a downstream sensing pathway exists to mediate pathogenic outcomes remains unclear. Here, we generate a soluble pyridine nucleotide transhydrogenase from Escherichia coli (EcSTH), which can elevate the NADH/NAD+ ratio and meantime reduce the NADPH/NADP+ ratio. Additionally, we fuse EcSTH with previously described LbNOX (a water-forming NADH oxidase from Lactobacillus brevis) to resume the NADH/NAD+ ratio. With these tools and by using genome-wide CRISPR/Cas9 library screens and metabolic profiling in mammalian cells, we find that accumulated NADH deregulates PRPS2 (Ribose-phosphate pyrophosphokinase 2)-mediated downstream purine biosynthesis to provoke massive energy consumption, and therefore, the induction of energy stress. Blocking purine biosynthesis prevents NADH accumulation-associated cell death in vitro and tissue injury in vivo. These results underscore the pathophysiological role of deregulated purine biosynthesis in NADH accumulation-associated disorders and demonstrate the utility of EcSTH in manipulating NADH/NAD+ and NADPH/NADP+.
  19. EMBO Rep. 2022 Nov 16. e51800
      Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA (dsDNA) sensor that functions in the innate immune system. Upon binding dsDNA, cGAS and dsDNA form phase-separated condensates in which cGAS catalyzes the synthesis of 2'3'-cyclic GMP-AMP that subsequently triggers a STING-dependent, type I interferon (IFN-I) response. Here, we show that cytoplasmic RNAs regulate cGAS activity. We discover that RNAs do not activate cGAS but rather promote phase separation of cGAS in vitro. In cells, cGAS colocalizes with RNA and forms complexes with RNA. In the presence of cytoplasmic dsDNA, RNAs colocalize with phase-separated condensates of cGAS and dsDNA. Further in vitro assays showed that RNAs promote the formation of cGAS-containing phase separations and enhance cGAS activity when the dsDNA concentration is low. Cotransfection of RNA with a small amount of dsDNA into THP1 cells significantly enhances the production of the downstream signaling molecule interferon beta (IFNB). This enhancement can be blocked by a cGAS-specific inhibitor. Thus, cytoplasmic RNAs could regulate cGAS activity by modulating the formation of cGAS-containing condensates.
    Keywords:  RNA; cGAS; innate immunity; phase separation; regulation
  20. iScience. 2022 Nov 18. 25(11): 105447
      An increase in permeability of the mitochondrial inner membrane, mitochondrial permeability transition (PT), is the central event responsible for cell death and tissue damage in conditions such as stroke and heart attack. PT is caused by the cyclosporin A (CSA)-dependent calcium-induced pore, the permeability transition pore (PTP). The molecular details of PTP are incompletely understood. We utilized holographic and fluorescent microscopy to assess the contribution of ATP synthase and adenine nucleotide translocator (ANT) toward PTP. In cells lacking either ATP synthase or ANT, we observed CSA-sensitive membrane depolarization, but not high-conductance PTP. In wild-type cells, calcium-induced CSA-sensitive depolarization preceded opening of PTP, which occurred only after nearly complete mitochondrial membrane depolarization. We propose that both ATP synthase and ANT are required for high-conductance PTP but not depolarization, which presumably occurs through activation of the low-conductance PT, which has a molecular nature that is different from both complexes.
    Keywords:  Cell biology; Functional aspects of cell biology; Molecular biology
  21. Front Cell Dev Biol. 2022 ;10 1044672
      Mitochondrial dysfunction is strongly implicated in neurodegenerative diseases including age-related macular degeneration (AMD), which causes irreversible blindness in over 50 million older adults worldwide. A key site of insult in AMD is the retinal pigment epithelium (RPE), a monolayer of postmitotic polarized cells that performs essential functions for photoreceptor health and vision. Recent studies from our group and others have identified several features of mitochondrial dysfunction in AMD including mitochondrial fragmentation and bioenergetic defects. While these studies provide valuable insight at fixed points in time, high-resolution, high-speed live imaging is essential for following mitochondrial injury in real time and identifying disease mechanisms. Here, we demonstrate the advantages of live imaging to investigate RPE mitochondrial dynamics in cell-based and mouse models. We show that mitochondria in the RPE form extensive networks that are destroyed by fixation and discuss important live imaging considerations that can interfere with accurate evaluation of mitochondrial integrity such as RPE differentiation status and acquisition parameters. Our data demonstrate that RPE mitochondria show localized heterogeneities in membrane potential and ATP production that could reflect focal changes in metabolism and oxidative stress. Contacts between the mitochondria and organelles such as the ER and lysosomes mediate calcium flux and mitochondrial fission. Live imaging of mouse RPE flatmounts revealed a striking loss of mitochondrial integrity in albino mouse RPE compared to pigmented mice that could have significant functional consequences for cellular metabolism. Our studies lay a framework to guide experimental design and selection of model systems for evaluating mitochondrial health and function in the RPE.
    Keywords:  RPE; live imaging; mitochondria; pigmented and albino mice; retina