bims-nenemi Biomed News
on Neuroinflammation, neurodegeneration and mitochondria
Issue of 2022‒03‒13
23 papers selected by
Marco Tigano
Thomas Jefferson University

  1. Cells. 2022 Feb 23. pii: 771. [Epub ahead of print]11(5):
      Mitochondria are subcellular organelles that are a hub for key biological processes, such as bioenergetic, biosynthetic, and signaling functions. Mitochondria are implicated in all oncogenic processes, from malignant transformation to metastasis and resistance to chemotherapeutics. The harsh tumor environment constantly exposes cancer cells to cytotoxic stressors, such as nutrient starvation, low oxygen, and oxidative stress. Excessive or prolonged exposure to these stressors can cause irreversible mitochondrial damage, leading to cell death. To survive hostile microenvironments that perturb mitochondrial function, cancer cells activate a stress response to maintain mitochondrial protein and genome integrity. This adaptive mechanism, which is closely linked to mitochondrial function, enables rapid adjustment and survival in harsh environmental conditions encountered during tumor dissemination, thereby promoting cancer progression. In this review, we describe how the mitochondria stress response contributes to the acquisition of typical malignant traits and highlight the potential of targeting the mitochondrial stress response as an anti-cancer therapeutic strategy.
    Keywords:  mitochondrial dynamics; mitochondrial protein quality control; mitochondrial stress response; mitophagy; mtDNA
  2. Cell Rep. 2022 Mar 08. pii: S2211-1247(22)00208-X. [Epub ahead of print]38(10): 110475
      Mitochondrial cardiomyopathies are fatal diseases, with no effective treatment. Alterations of heart mitochondrial function activate the mitochondrial integrated stress response (ISRmt), a transcriptional program affecting cell metabolism, mitochondrial biogenesis, and proteostasis. In humans, mutations in CHCHD10, a mitochondrial protein with unknown function, were recently associated with dominant multi-system mitochondrial diseases, whose pathogenic mechanisms remain to be elucidated. Here, in CHCHD10 knockin mutant mice, we identify an extensive cardiac metabolic rewiring triggered by proteotoxic ISRmt. The stress response arises early on, before the onset of bioenergetic impairments, triggering a switch from oxidative to glycolytic metabolism, enhancement of transsulfuration and one carbon (1C) metabolism, and widespread metabolic imbalance. In parallel, increased NADPH oxidases elicit antioxidant responses, leading to heme depletion. As the disease progresses, the adaptive metabolic stress response fails, resulting in fatal cardiomyopathy. Our findings suggest that early interventions to counteract metabolic imbalance could ameliorate mitochondrial cardiomyopathy associated with proteotoxic ISRmt.
    Keywords:  1C metabolism; CHCHD10; coiled-coil-helix-coiled-coil-helix domain containing 10; heart, cardiomyopathy; heme; integrated stress response; metabolic rewiring; mitochondria
  3. RNA. 2022 Mar 07. pii: rna.079097.122. [Epub ahead of print]
      Mitochondria possess their own genome that encodes components of oxidative phosphorylation (OXPHOS) complexes, and mitochondrial ribosomes within the organelle translate the mRNAs expressed from the mitochondrial genome. Given the differential OXPHOS activity observed in diverse cell types, cell growth conditions, and other circumstances, cellular heterogeneity in mitochondrial translation can be expected. Although individual protein products translated in mitochondria have been monitored, the lack of techniques that address the variation in overall mitochondrial protein synthesis in cell populations poses analytic challenges. Here, we adapted mitochondrial-specific fluorescent noncanonical amino acid tagging (FUNCAT) for use with fluorescence-activated cell sorting (FACS) and developed mito-FUNCAT-FACS. The click chemistry-compatible methionine analog L-homopropargylglycine (HPG) enabled the metabolic labeling of newly synthesized proteins. In the presence of cytosolic translation inhibitors, HPG was selectively incorporated into mitochondrial nascent proteins and conjugated to fluorophores via the click reaction (mito-FUNCAT). The application of in situ mito-FUNCAT to flow cytometry allowed us to separate changes in net mitochondrial translation activity from those of the organelle mass and detect variations in mitochondrial translation in cancer cells. Our approach provides a useful methodology for examining mitochondrial protein synthesis in individual cells.
    Keywords:  FACS; FUNCAT; HPG; Mitochondria; Translation
  4. Int J Mol Sci. 2022 Mar 07. pii: 2888. [Epub ahead of print]23(5):
      Molecular hydrogen ameliorates pathological states in a variety of human diseases, animal models, and cell models, but the effects of hydrogen on cancer have been rarely reported. In addition, the molecular mechanisms underlying the effects of hydrogen remain mostly unelucidated. We found that hydrogen enhances proliferation of four out of seven human cancer cell lines (the responders). The proliferation-promoting effects were not correlated with basal levels of cellular reactive oxygen species. Expression profiling of the seven cells showed that the responders have higher gene expression of mitochondrial electron transport chain (ETC) molecules than the non-responders. In addition, the responders have higher mitochondrial mass, higher mitochondrial superoxide, higher mitochondrial membrane potential, and higher mitochondrial spare respiratory capacity than the non-responders. In the responders, hydrogen provoked mitochondrial unfolded protein response (mtUPR). Suppression of cell proliferation by rotenone, an inhibitor of mitochondrial ETC complex I, was rescued by hydrogen in the responders. Hydrogen triggers mtUPR and induces cell proliferation in cancer cells that have high basal and spare mitochondrial ETC activities.
    Keywords:  cancer cell lines; cellular proliferation; mitochondrial electron transfer chain; mitochondrial unfolded protein response; molecular hydrogen
  5. Epigenetics. 2022 Mar 07. 1-16
      While DNA methylation is established as a major regulator of gene expression in the nucleus, the existence of mitochondrial DNA (mtDNA) methylation remains controversial. Here, we characterized the mtDNA methylation landscape in the prefrontal cortex of neurological healthy individuals (n=26) and patients with Parkinson's disease (n=27), using a combination of whole-genome bisulphite sequencing (WGBS) and bisulphite-independent methods. Accurate mtDNA mapping from WGBS data required alignment to an mtDNA reference only, to avoid misalignment to nuclear mitochondrial pseudogenes. Once correctly aligned, WGBS data provided ultra-deep mtDNA coverage (16,723 ± 7,711) and revealed overall very low levels of cytosine methylation. The highest methylation levels (5.49 ± 0.97%) were found on CpG position m.545, located in the heavy-strand promoter 1 region. The m.545 methylation was validated using a combination of methylation-sensitive DNA digestion and quantitative PCR analysis. We detected no association between mtDNA methylation profile and Parkinson's disease. Interestingly, m.545 methylation correlated with the levels of mtDNA transcripts, suggesting a putative role in regulating mtDNA gene expression. In addition, we propose a robust framework for methylation analysis of mtDNA from WGBS data, which is less prone to false-positive findings due to misalignment of nuclear mitochondrial pseudogene sequences.
    Keywords:  NUMTs; epigenetics; mitochondria; mtDNA; parkinson’s disease
  6. Neurol Genet. 2022 Apr;8(2): e660
      Background and Objectives: We report the pathogenic sequence variant m.5789T>C in the anticodon stem of the mitochondrial tRNA for cysteine as a novel cause of neuropathy, ataxia, and retinitis pigmentosa (NARP), which is usually associated with pathogenic variants in the MT-ATP6 gene.Methods: To address the correlation of oxidative phosphorylation deficiency with mutation loads, we performed genotyping on single laser-dissected skeletal muscle fibers. Stability of the mitochondrial tRNACys was investigated by Northern blotting. Accompanying deletions of the mitochondrial genome were detected by long-range PCR and their breakpoints were determined by sequencing of single-molecule amplicons.
    Results: The sequence variant m.5789T>C, originating from the patient's mother, decreases the stability of the mitochondrial tRNA for cysteine by disrupting the anticodon stem, which subsequently leads to a combined oxidative phosphorylation deficiency. In parallel, we observed a prominent cluster of low-abundance somatic deletions with breakpoints in the immediate vicinity of the m.5789T>C variant. Strikingly, all deletion-carrying mitochondrial DNA (mtDNA) species, in which the corresponding nucleotide position was not removed, harbored the mutant allele, and none carried the wild-type allele.
    Discussion: In addition to providing evidence for the novel association of a tRNA sequence alteration with NARP syndrome, our observations support the hypothesis that single nucleotide changes can lead to increased occurrence of site-specific mtDNA deletions through the formation of an imperfect repeat. This finding might be relevant for understanding mechanisms of deletion generation in the human mitochondrial genome.
  7. Front Cell Dev Biol. 2022 ;10 781558
      Mitochondria are biosynthetic, bioenergetic, and signaling organelles with a critical role in cellular physiology. Dysfunctional mitochondria are associated with aging and underlie the cause of a wide range of diseases, from neurodegeneration to cancer. Through signaling, mitochondria regulate diverse biological outcomes. The maintenance of the mitochondrial membrane potential, for instance, is essential for proliferation, the release of mitochondrial reactive oxygen species, and oxygen sensing. The loss of mitochondrial membrane potential triggers pathways to clear damaged mitochondria and often results in cell death. In this study, we conducted a genome-wide positive selection CRISPR screen using a combination of mitochondrial inhibitors to uncover genes involved in sustaining a mitochondrial membrane potential, and therefore avoid cell death when the electron transport chain is impaired. Our screen identified genes involved in mitochondrial protein translation and ATP synthesis as essential for the induction of cell death when cells lose their mitochondrial membrane potential. This report intends to provide potential targets for the treatment of diseases associated with mitochondrial dysfunction.
    Keywords:  ATP synthase; CRISPR screen; cell death; mitochondria; mitochondrial membrane potential; mitochondrial protein translation
  8. Front Pharmacol. 2022 ;13 816551
      Mitophagy is an intracellular mechanism to maintain mitochondrial health by removing dysfunctional mitochondria. The E3 ligase Parkin ubiquitinates the membrane proteins on targeted mitochondria to initiate mitophagy, whereas USP30 antagonizes Parkin-dependent mitophagy by removing ubiquitin from Parkin substrates. The AKT/mTOR signaling is a master regulator of cell proliferation, differentiation, apoptosis, and autophagy. Although mounting evidence suggests that perturbations in the AKT/mTOR signaling pathway may contribute to mitophagy regulation, the specific mechanisms between Parkin/USP30 and AKT/mTOR signaling have not been elucidated. In this study, we employ a set of genetic reagents to investigate the role of Parkin and USP30 in regulating the AKT/mTOR signaling during mitophagy. We demonstrated that, in the setting of mitochondrial stress, the AKT/mTOR signaling is regulated, at least in part, by the activity of Parkin and USP30. Parkin inhibits AKT/mTOR signaling following an in vitro mitochondrial stress, thereby promoting apoptosis. However, USP30 overexpression antagonizes the activity of Parkin to sustain AKT/mTOR activity and inhibit apoptosis. These findings provide new insights into Parkin and USP30's role in apoptosis and suggest that inhibiting USP30 might provide a specific strategy to synergize with AKT/mTOR inhibitors in cancer treatment.
    Keywords:  USP30; akt; cancer; leukemia; mTOR; mitophagy; parkin
  9. PLoS Genet. 2022 Mar 11. 18(3): e1010103
      Monitoring mitochondrial function is crucial for organismal survival. This task is performed by mitochondrial surveillance or quality control pathways, which are activated by signals originating from mitochondria and relayed to the nucleus (retrograde response) to start transcription of protective genes. In Caenorhabditis elegans, several systems are known to play this role, including the UPRmt, MAPKmt, and the ESRE pathways. These pathways are highly conserved and their loss compromises survival following mitochondrial stress. In this study, we found a novel interaction between the box C/D snoRNA core proteins (snoRNPs) and mitochondrial surveillance and innate immune pathways. We showed that box C/D, but not box H/ACA, snoRNPs are required for the full function of UPRmt and ESRE upon stress. The loss of box C/D snoRNPs reduced mitochondrial mass, mitochondrial membrane potential, and oxygen consumption rate, indicating overall degradation of mitochondrial function. Concomitantly, the loss of C/D snoRNPs increased immune response and reduced host intestinal colonization by infectious bacteria, improving host resistance to pathogenesis. Our data may indicate a model wherein box C/D snoRNP machinery regulates a "switch" of the cell's activity between mitochondrial surveillance and innate immune activation. Understanding this mechanism is likely to be important for understanding multifactorial processes, including responses to infection and aging.
  10. Elife. 2022 Mar 08. pii: e73150. [Epub ahead of print]11
      Mechanical stress is known to fuel several hallmarks of cancer, ranging from genome instability to uncontrolled proliferation or invasion. Cancer cells are constantly challenged by mechanical stresses not only in the primary tumour but also during metastasis. However, this latter has seldom been studied with regards to mechanobiology, in particular resistance to anoikis, a cell death programme triggered by loss of cell adhesion. Here, we show in vitro that migrating breast cancer cells develop resistance to anoikis following their passage through microporous membranes mimicking confined migration (CM), a mechanical constriction that cancer cells encounter during metastasis. This CM-induced resistance was mediated by Inhibitory of Apoptosis Proteins, and sensitivity to anoikis could be restored after their inhibition using second mitochondria-derived activator of caspase (SMAC) mimetics. Anoikis-resistant mechanically stressed cancer cells displayed enhanced cell motility and evasion from natural killer cell-mediated immune surveillance, as well as a marked advantage to form lung metastatic lesions in mice. Our findings reveal that CM increases the metastatic potential of breast cancer cells.
    Keywords:  IAP; anoikis; cancer biology; caspases; cell biology; confinement; human; mechanobiology; metastasis; mouse
  11. Front Cell Dev Biol. 2022 ;10 833127
      Inorganic polyphosphate (polyP) is an ancient, ubiquitous, and well-conserved polymer which is present in all the studied organisms. It is formed by individual subunits of orthophosphate which are linked by structurally similar bonds and isoenergetic to those found in ATP. While the metabolism and the physiological roles of polyP have already been described in some organisms, including bacteria and yeast, the exact role of this polymer in mammalian physiology still remains poorly understood. In these organisms, polyP shows a co-localization with mitochondria, and its role as a key regulator of the stress responses, including the maintenance of appropriate bioenergetics, has already been demonstrated by our group and others. Here, using Wild-type (Wt) and MitoPPX (cells enzymatically depleted of mitochondrial polyP) SH-SY5Y cells, we have conducted a comprehensive study of the status of cellular physiology, using proteomics and metabolomics approaches. Our results suggest a clear dysregulation of mitochondrial physiology, especially of bioenergetics, in MitoPPX cells when compared with Wt cells. Moreover, the effects induced by the enzymatic depletion of polyP are similar to those present in the mitochondrial dysfunction that is observed in neurodegenerative disorders and in neuronal aging. Based on our findings, the metabolism of mitochondrial polyP could be a valid and innovative pharmacological target in these conditions.
    Keywords:  OXPHOS; SH-SY5Y cells; bioenergetics; inorganic polyphosphate; metabolomics; mitochondria; mitochondrial metabolism; proteomics
  12. Autophagy. 2022 Mar 08. 1-18
      Mitochondrial oxidative phosphorylation (OXPHOS) generates ATP, but OXPHOS also supports biosynthesis during proliferation. In contrast, the role of OXPHOS during quiescence, beyond ATP production, is not well understood. Using mouse models of inducible OXPHOS deficiency in all cell types or specifically in the vascular endothelium that negligibly relies on OXPHOS-derived ATP, we show that selectively during quiescence OXPHOS provides oxidative stress resistance by supporting macroautophagy/autophagy. Mechanistically, OXPHOS constitutively generates low levels of endogenous ROS that induce autophagy via attenuation of ATG4B activity, which provides protection from ROS insult. Physiologically, the OXPHOS-autophagy system (i) protects healthy tissue from toxicity of ROS-based anticancer therapy, and (ii) provides ROS resistance in the endothelium, ameliorating systemic LPS-induced inflammation as well as inflammatory bowel disease. Hence, cells acquired mitochondria during evolution to profit from oxidative metabolism, but also built in an autophagy-based ROS-induced protective mechanism to guard against oxidative stress associated with OXPHOS function during quiescence.Abbreviations: AMPK: AMP-activated protein kinase; AOX: alternative oxidase; Baf A: bafilomycin A1; CI, respiratory complexes I; DCF-DA: 2',7'-dichlordihydrofluorescein diacetate; DHE: dihydroethidium; DSS: dextran sodium sulfate; ΔΨmi: mitochondrial inner membrane potential; EdU: 5-ethynyl-2'-deoxyuridine; ETC: electron transport chain; FA: formaldehyde; HUVEC; human umbilical cord endothelial cells; IBD: inflammatory bowel disease; LC3B: microtubule associated protein 1 light chain 3 beta; LPS: lipopolysaccharide; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; mtDNA: mitochondrial DNA; NAC: N-acetyl cysteine; OXPHOS: oxidative phosphorylation; PCs: proliferating cells; PE: phosphatidylethanolamine; PEITC: phenethyl isothiocyanate; QCs: quiescent cells; ROS: reactive oxygen species; PLA2: phospholipase A2, WB: western blot.
    Keywords:  ATG4B; biosynthesis; cell death; electron transport chain; endothelial cells; mitochondria; oxidative phosphorylation; oxidative stress; reactive oxygen species
  13. J Exp Med. 2022 Apr 04. pii: e20211121. [Epub ahead of print]219(4):
      Aberrant induction of type I IFN is a hallmark of the inherited encephalopathy Aicardi-Goutières syndrome (AGS), but the mechanisms triggering disease in the human central nervous system (CNS) remain elusive. Here, we generated human models of AGS using genetically modified and patient-derived pluripotent stem cells harboring TREX1 or RNASEH2B loss-of-function alleles. Genome-wide transcriptomic analysis reveals that spontaneous proinflammatory activation in AGS astrocytes initiates signaling cascades impacting multiple CNS cell subsets analyzed at the single-cell level. We identify accumulating DNA damage, with elevated R-loop and micronuclei formation, as a driver of STING- and NLRP3-related inflammatory responses leading to the secretion of neurotoxic mediators. Importantly, pharmacological inhibition of proapoptotic or inflammatory cascades in AGS astrocytes prevents neurotoxicity without apparent impact on their increased type I IFN responses. Together, our work identifies DNA damage as a major driver of neurotoxic inflammation in AGS astrocytes, suggests a role for AGS gene products in R-loop homeostasis, and identifies common denominators of disease that can be targeted to prevent astrocyte-mediated neurotoxicity in AGS.
  14. Front Aging. 2022 ;pii: 805126. [Epub ahead of print]2
      Mitochondria are the main source of energy used to maintain cellular homeostasis. This aspect of mitochondrial biology underlies their putative role in age-associated tissue dysfunction. Proper functioning of the electron transport chain (ETC), which is partially encoded by the extra-nuclear mitochondrial genome (mtDNA), is key to maintaining this energy production. The acquisition of de novo somatic mutations that interrupt the function of the ETC have long been associated with aging and common diseases of the elderly. Yet, despite over 30 years of study, the exact role(s) mtDNA mutations play in driving aging and its associated pathologies remains under considerable debate. Furthermore, even fundamental aspects of age-related mtDNA mutagenesis, such as when mutations arise during aging, where and how often they occur across tissues, and the specific mechanisms that give rise to them, remain poorly understood. In this review, we address the current understanding of the somatic mtDNA mutations, with an emphasis of when, where, and how these mutations arise during aging. Additionally, we highlight current limitations in our knowledge and critically evaluate the controversies stemming from these limitations. Lastly, we highlight new and emerging technologies that offer potential ways forward in increasing our understanding of somatic mtDNA mutagenesis in the aging process.
    Keywords:  aging; mitochondria; mtDNA; mutagenesis; sequencing; somatic mutations
  15. Sci Rep. 2022 Mar 09. 12(1): 3874
      Mitochondrial dysfunction is a key element in the progression of Parkinson's disease (PD). The inefficient operation of the electron transport chain (ETC) impairs energy production and enhances the generation of oxidative stress contributing to the loss of dopaminergic cells in the brain. ATPase inhibitory factor 1 (IF1) is a regulator of mitochondrial energy metabolism. IF1 binds directly to the F1Fo ATP synthase and prevents ATP wasting during compromised energy metabolism. In this study, we found treatment with IF1 protects mitochondria against PD-like insult in vitro. SH-SY5Y cells treated with IF1 were resistant to loss of ATP and mitochondrial inner membrane potential during challenge with rotenone, an inhibitor of complex I in the ETC. We further demonstrated that treatment with IF1 reversed rotenone-induced superoxide production in mitochondria and peroxide accumulation in whole cells. Ultimately, IF1 decreased protein levels of pro-apoptotic Bax, cleaved caspase-3, and cleaved PARP, rescuing SH-SY5Y cells from rotenone-mediated apoptotic death. Administration of IF1 significantly improved the results of pole and hanging tests performed by PD mice expressing human α-synuclein. This indicates that IF1 mitigates PD-associated motor deficit. Together, these findings suggest that IF1 exhibits a neuroprotective effect preventing mitochondrial dysfunction in PD pathology.
  16. J Neurosci. 2022 Mar 09. pii: JN-RM-1963-21. [Epub ahead of print]
      NF-κB proteins are well known as transcription factors important in immune system activation. In this highly conserved role, they contribute to changes in behavior in response to infection and in response to a variety of other insults and experiences. In some mammalian neurons, NF-κBs can be found at the synapse and translocate to the nucleus to alter gene expression when activated by synaptic activity. Here we demonstrate that, in Drosophila melanogaster, NF-κB action is important both inside and outside the nucleus and that the Dif gene has segregated nuclear and non-nuclear NF-κB action into different protein isoforms. The DifA isoform is a canonical nuclear-acting NF-κB protein that enters the nucleus and is important for combating infection. The DifB variant, but not the DifA variant, is found in the central nervous system (mushroom bodies and antennal lobes). DifB does not enter the nucleus and co-localizes with a synaptic protein. In males and females, a DifB mutant alters alcohol behavioral sensitivity without an obvious effect on combating infection, whereas a DifA mutant does not affect alcohol sensitivity but compromises the immune response. These data are evidence that the non-nuclear DifB variant contributes to alcohol behavioral sensitivity by a nongenomic mechanism that diverges from the NF-κB transcriptional effects used in the peripheral immune system. Enrichment of DifB in brain regions rich in synapses and biochemical enrichment of DifB in the synaptoneurosome fraction indicates that the protein may act locally at the synapse.Significance Statement:NF-κBs are transcription factors used by innate immune signaling pathways to protect against infection. Alcohol abuse also activates these pathways, which contributes to the addictive process and the health consequences associated with alcohol abuse. In the mammalian nervous system, NF-κBs localize to synapses, but it is axiomatic that they effect change by acting in the nucleus. However, for the Drosophila Dif gene, immune and neural function segregate into different protein isoforms. Whereas the nuclear isoform (DifA) activates immune genes in response to infection, the CNS isoform acts nongenomically to modulate alcohol sensitivity. Immunohistochemical and biochemical assays localize DifB to synapse-rich regions. Direct synaptic action would provide a novel and rapid way for NF-κB signaling to modulate behavior.
  17. Front Immunol. 2022 ;13 821816
      In solid tumors, as the tumor grows and the disease progresses, hypoxic regions are often generated, but in contrast to most normal cells which cannot survive under these conditions, tumour cells adapt to hypoxia by HIF-driven mechanisms. Hypoxia can further promote cancer development by generating an immunosuppressive environment within the tumour mass, which allows tumour cells to escape the immune system recognition. This is achieved by recruiting immunosuppressive cells and by upregulating molecules which block immune cell activation. Hypoxia can also confer resistance to antitumor therapies by inducing the expression of membrane proteins that increase drug efflux or by inhibiting the apoptosis of treated cells. In addition, tumor cells require an active interferon (IFN) signalling pathway for the success of many anticancer therapies, such as radiotherapy or chemotherapy. Therefore, hypoxic effects on this pathway needs to be addressed for a successful treatment.
    Keywords:  IFN; cancer; hypoxia; therapy; type I IFN
  18. Front Physiol. 2022 ;13 800171
      MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) is an OXPHOS disease mostly caused by the m.3243A>G mutation in the mitochondrial tRNALeu(UUR) gene. Recently, we have shown that the mutation significantly changes the expression pattern of several mitochondrial tRNA-derived small RNAs (mt tsRNAs or mt tRFs) in a cybrid model of MELAS and in fibroblasts from MELAS patients versus control cells. Among them are those derived from mt tRNA LeuUUR containing or not the m.3243A>G mutation (mt 5'-tRF LeuUUR-m.3243A>G and mt 5'-tRF LeuUUR), whose expression levels are, respectively, increased and decreased in both MELAS cybrids and fibroblasts. Here, we asked whether mt 5'-tRF LeuUUR and mt 5'-tRF LeuUUR-m.3243A>G are biologically relevant and whether these mt tRFs are detected in diverse patient samples. Treatment with a mimic oligonucleotide of mt tRNA LeuUUR fragment (mt 5'-tRF LeuUUR) showed a therapeutic potential since it partially restored mitochondrial respiration in MELAS cybrids. Moreover, these mt tRFs could be detected in biofluids like urine and blood. We also investigated the participation of miRNA pathway components Dicer and Ago2 in the mt tRFs biogenesis process. We found that Dicer and Ago2 localize in the mitochondria of MELAS cybrids and that immunoprecipitation of these proteins in cytoplasm and mitochondria fractions revealed an increased mt tRF/mt tRNA ratio in MELAS condition compared to WT. These preliminary results suggest an involvement of Dicer and Ago2 in the mechanism of mt tRF biogenesis and action.
    Keywords:  mitochondrial dysfunction; retrograde signaling; sncRNAs; tRF and tiRNA; tRNA fragment
  19. Nature. 2022 Mar 09.
      The tricarboxylic acid (TCA) cycle is a central hub of cellular metabolism, oxidizing nutrients to generate reducing equivalents for energy production and critical metabolites for biosynthetic reactions. Despite the importance of the products of the TCA cycle for cell viability and proliferation, mammalian cells display diversity in TCA-cycle activity1,2. How this diversity is achieved, and whether it is critical for establishing cell fate, remains poorly understood. Here we identify a non-canonical TCA cycle that is required for changes in cell state. Genetic co-essentiality mapping revealed a cluster of genes that is sufficient to compose a biochemical alternative to the canonical TCA cycle, wherein mitochondrially derived citrate exported to the cytoplasm is metabolized by ATP citrate lyase, ultimately regenerating mitochondrial oxaloacetate to complete this non-canonical TCA cycle. Manipulating the expression of ATP citrate lyase or the canonical TCA-cycle enzyme aconitase 2 in mouse myoblasts and embryonic stem cells revealed that changes in the configuration of the TCA cycle accompany cell fate transitions. During exit from pluripotency, embryonic stem cells switch from canonical to non-canonical TCA-cycle metabolism. Accordingly, blocking the non-canonical TCA cycle prevents cells from exiting pluripotency. These results establish a context-dependent alternative to the traditional TCA cycle and reveal that appropriate TCA-cycle engagement is required for changes in cell state.
  20. Int J Mol Sci. 2022 Feb 27. pii: 2627. [Epub ahead of print]23(5):
      In the past decade, anti-tumour immune responses have been successfully exploited to improve the outcome of patients with different cancers. Significant progress has been made in taking advantage of different types of T cell functions for therapeutic purposes. Despite these achievements, only a subset of patients respond favorably to immunotherapy. Therefore, there is a need of novel approaches to improve the effector functions of immune cells and to recognize the major targets of anti-tumour immunity. A major hallmark of cancer is metabolic rewiring associated with switch of mitochondrial functions. These changes are a consequence of high energy demand and increased macromolecular synthesis in cancer cells. Such adaptations in tumour cells might generate novel targets of tumour therapy, including the generation of neoantigens. Here, we review the most recent advances in research on the immune response to mitochondrial proteins in different cellular conditions.
    Keywords:  T cell response; cancer neoantigens; mitochondria; mtDNA mutations; post translational modifications
  21. Clin Cancer Res. 2022 Mar 04. pii: clincanres.0833.2021. [Epub ahead of print]
      PURPOSE: To investigate the anti-tumor activity of a mitochondrial-localized HSP90 inhibitor, Gamitrinib, in multiple glioma models, and to elucidate the anti-tumor mechanisms of Gamitrinib in gliomas.EXPERIMENTAL DESIGN: A broad panel of primary and temozolomide (TMZ)-resistant human glioma cell lines were screened by cell viability assays, flow cytometry and crystal violet assays to investigate the therapeutic efficacy of Gamitrinib. Seahorse assays were used to measure the mitochondrial respiration of glioma cells. Integrated analyses of RNA sequencing (RNAseq) and reverse phase protein array (RPPA) data were performed to reveal the potential anti-tumor mechanisms of Gamitrinib. Neurospheres, patient-derived organoids (PDOs), cell line-derived xenografts (CDX) and patient-derived xenografts (PDX) models were generated to further evaluate the therapeutic efficacy of Gamitrinib.
    RESULTS: Gamitrinib inhibited cell proliferation and induced cell apoptosis and death in 17 primary glioma cells, 6 TMZ-resistant glioma cells, 4 neurospheres and 3 PDOs. Importantly, Gamitrinib significantly delayed the tumor growth and improved survival of mice in both CDX and PDX models in which tumors were either subcutaneously or intracranially implanted. Integrated computational analyses of RNAseq and RPPA data revealed that Gamitrinib exhibited its anti-tumor activity via (1) suppressing mitochondrial biogenesis, OXPHOS and cell cycle progression and (2) activating the energy-sensing AMP-activated kinase, DNA damage and stress response.
    CONCLUSIONS: These preclinical findings established the therapeutic role of Gamitrinib in gliomas and revealed the inhibition of mitochondrial biogenesis and tumor bioenergetics as the primary anti-tumor mechanisms in gliomas.
  22. Cell Rep. 2022 Mar 08. pii: S2211-1247(22)00226-1. [Epub ahead of print]38(10): 110493
      Unlike most cell types, many cancer cells survive at low extracellular pH (pHe), a chemical signature of tumors. Genes that facilitate survival under acid stress are therefore potential targets for cancer therapies. We performed a genome-wide CRISPR-Cas9 cell viability screen at physiological and acidic conditions to systematically identify gene knockouts associated with pH-related fitness defects in colorectal cancer cells. Knockouts of genes involved in oxidative phosphorylation (NDUFS1) and iron-sulfur cluster biogenesis (IBA57, NFU1) grew well at physiological pHe, but underwent profound cell death under acidic conditions. We identified several small-molecule inhibitors of mitochondrial metabolism that can kill cancer cells at low pHe only. Xenografts established from NDUFS1-/- cells grew considerably slower than their wild-type controls, but growth could be stimulated with systemic bicarbonate therapy that lessens the tumoral acid stress. These findings raise the possibility of therapeutically targeting mitochondrial metabolism in combination with acid stress as a cancer treatment option.
    Keywords:  CRISPR-Cas9 screen; acidosis; oxidative phosphorylation; tumor acidity
  23. Cell Rep. 2022 03 08. pii: S2211-1247(22)00161-9. [Epub ahead of print]38(10): 110434
      Type I interferons (IFN-I) are essential to establish antiviral innate immunity. Unanchored (or free) polyubiquitin (poly-Ub) has been shown to regulate IFN-I responses. However, few unanchored poly-Ub interactors are known. To identify factors regulated by unanchored poly-Ub in a physiological setting, we developed an approach to isolate unanchored poly-Ub from lung tissue. We identified the RNA helicase DHX16 as a potential pattern recognition receptor (PRR). Silencing of DHX16 in cells and in vivo diminished IFN-I responses against influenza virus. These effects extended to members of other virus families, including Zika and SARS-CoV-2. DHX16-dependent IFN-I production requires RIG-I and unanchored K48-poly-Ub synthesized by the E3-Ub ligase TRIM6. DHX16 recognizes a signal in influenza RNA segments that undergo splicing and requires its RNA helicase motif for direct, high-affinity interactions with specific viral RNAs. Our study establishes DHX16 as a PRR that partners with RIG-I for optimal activation of antiviral immunity requiring unanchored poly-Ub.
    Keywords:  DHX16; RIG-I; SARS-CoV-2; TRIM6; influenza A virus; innate immunity; splicing; tripartite motif (TRIM) protein; type I interferon; unanchored ubiquitin