bims-nenemi Biomed News
on Neuroinflammation, neurodegeneration and mitochondria
Issue of 2022‒07‒03
eight papers selected by
Marco Tigano
Thomas Jefferson University

  1. Elife. 2022 Jun 27. pii: e77780. [Epub ahead of print]11
      Cells encountering stressful situations activate the integrated stress response (ISR) pathway to limit protein synthesis and redirect translation to better cope. The ISR has also been implicated in cancers, but redundancies in the stress-sensing kinases that trigger the ISR have posed hurdles to dissecting physiological relevance. To overcome this challenge, we targeted the regulatory node of these kinases, namely the S51 phosphorylation site of eukaryotic translation initiation factor eIF2α and genetically replaced eIF2α with eIF2α-S51A in mouse squamous cell carcinoma (SCC) stem cells of skin. While inconsequential under normal growth conditions, the vulnerability of this ISR-null state was unveiled when SCC stem cells experienced proteotoxic stress. Seeking mechanistic insights into the protective roles of the ISR, we combined ribosome profiling and functional approaches to identify and probe the functional importance of translational differences between ISR-competent and ISR-null SCC stem cells when exposed to proteotoxic stress. In doing so, we learned that the ISR redirects translation to centrosomal proteins that orchestrate the microtubule dynamics needed to efficiently concentrate unfolded proteins at the microtubule organizing center so that they can be cleared by the perinuclear degradation machinery. Thus, rather than merely maintaining survival during proteotoxic stress, the ISR also functions in promoting cellular recovery once the stress has subsided. Remarkably, this molecular program is unique to transformed skin stem cells hence exposing a vulnerability in cancer that could be exploited therapeutically.
    Keywords:  cancer biology; cell biology; mouse
  2. Mol Cell. 2022 Jun 16. pii: S1097-2765(22)00540-8. [Epub ahead of print]
      Protein import into mitochondria is a highly regulated process, yet how cells clear mitochondria undergoing dysfunctional protein import remains poorly characterized. Here we showed that mitochondrial protein import stress (MPIS) triggers localized LC3 lipidation. This arm of the mitophagy pathway occurs through the Nod-like receptor (NLR) protein NLRX1 while, surprisingly, without the engagement of the canonical mitophagy protein PINK1. Mitochondrial depolarization, which itself induces MPIS, also required NLRX1 for LC3 lipidation. While normally targeted to the mitochondrial matrix, cytosol-retained NLRX1 recruited RRBP1, a ribosome-binding transmembrane protein of the endoplasmic reticulum, which relocated to the mitochondrial vicinity during MPIS, and the NLRX1/RRBP1 complex in turn controlled the recruitment and lipidation of LC3. Furthermore, NLRX1 controlled skeletal muscle mitophagy in vivo and regulated endurance capacity during exercise. Thus, localization and lipidation of LC3 at the site of mitophagosome formation is a regulated step of mitophagy controlled by NLRX1/RRBP1 in response to MPIS.
    Keywords:  NLRX1; Nod-like receptors; mitochondria; mitochondrial protein import; mitophagy
  3. Nature. 2022 Jun 29.
      Aggressive and metastatic cancers show enhanced metabolic plasticity1, but the precise underlying mechanisms of this remain unclear. Here we show how two NOP2/Sun RNA methyltransferase 3 (NSUN3)-dependent RNA modifications-5-methylcytosine (m5C) and its derivative 5-formylcytosine (f5C) (refs.2-4)-drive the translation of mitochondrial mRNA to power metastasis. Translation of mitochondrially encoded subunits of the oxidative phosphorylation complex depends on the formation of m5C at position 34 in mitochondrial tRNAMet. m5C-deficient human oral cancer cells exhibit increased levels of glycolysis and changes in their mitochondrial function that do not affect cell viability or primary tumour growth in vivo; however, metabolic plasticity is severely impaired as mitochondrial m5C-deficient tumours do not metastasize efficiently. We discovered that CD36-dependent non-dividing, metastasis-initiating tumour cells require mitochondrial m5C to activate invasion and dissemination. Moreover, a mitochondria-driven gene signature in patients with head and neck cancer is predictive for metastasis and disease progression. Finally, we confirm that this metabolic switch that allows the metastasis of tumour cells can be pharmacologically targeted through the inhibition of mitochondrial mRNA translation in vivo. Together, our results reveal that site-specific mitochondrial RNA modifications could be therapeutic targets to combat metastasis.
  4. J Biol Chem. 2022 Jun 22. pii: S0021-9258(22)00622-6. [Epub ahead of print] 102180
      The Integrated Stress Response (ISR) is a network of highly-orchestrated pathways activated when cells are exposed to various types of environmental stressors. While global repression of translation is a well-recognized hallmark of the ISR, less is known about the regulation of mRNA stability during stress. DEAD box proteins are a family of RNA unwinding/remodeling enzymes involved in every aspect of RNA metabolism. We previously showed that DEAD Box 1 (DDX1) protein accumulates at DNA double-strand breaks (DSBs) during genotoxic stress and promotes DSB repair via homologous recombination. Here, we examine the role of DDX1 in response to environmental stress. We show that DDX1 is recruited to stress granules (SGs) in cells exposed to a variety of environmental stressors including arsenite, hydrogen peroxide, and thapsigargin. We also show that DDX1 depletion delays resolution of arsenite-induced SGs. Using RNA immunoprecipitation sequencing (RIP-Seq), we identify RNA targets bound to endogenous DDX1, including RNAs transcribed from genes previously implicated in stress responses. We show the amount of target RNAs bound to DDX1 increases when cells are exposed to stress, and the overall levels of these RNAs is increased during stress in a DDX1-dependent manner. Even though DDX1's RNA-binding property is critical for maintenance of its target mRNA levels, we found RNA binding is not required for localization of DDX1 to SGs. Furthermore, DDX1 knockdown does not appear to affect RNA localization to SGs. Taken together, our results reveal a novel role for DDX1 in maintaining cytoplasmic mRNA levels in cells exposed to oxidative stress.
    Keywords:  DEAD Box 1; RNA binding protein; RNA helicase; RNA stability; RNA-protein interaction; oxidative stress; stress granules
  5. Glia. 2022 Jun 28.
      Oligodendrocytes (ODCs) are myelinating cells of the central nervous system (CNS) supporting neuronal survival. Oxidants and mitochondrial dysfunction have been suggested as the main causes of ODC damage during neuroinflammation as observed in multiple sclerosis (MS). Nonetheless, the dynamics of this process remain unclear, thus hindering the design of neuroprotective therapeutic strategies. To decipher the spatio-temporal pattern of oxidative damage and dysfunction of ODC mitochondria in vivo, we created a novel mouse model in which ODCs selectively express the ratiometric H2 O2 biosensor mito-roGFP2-Orp1 allowing for quantification of redox changes in their mitochondria. Using 2-photon imaging of the exposed spinal cord, we observed significant mitochondrial oxidation in ODCs upon induction of the MS model experimental autoimmune encephalomyelitis (EAE). This redox change became already apparent during the preclinical phase of EAE prior to CNS infiltration of inflammatory cells. Upon clinical EAE development, mitochondria oxidation remained detectable and was associated with a significant impairment in organelle density and morphology. These alterations correlated with the proximity of ODCs to inflammatory lesions containing activated microglia/macrophages. During the chronic progression of EAE, ODC mitochondria maintained an altered morphology, but their oxidant levels decreased to levels observed in healthy mice. Taken together, our study implicates oxidative stress in ODC mitochondria as a novel pre-clinical sign of MS-like inflammation and demonstrates that evolving redox and morphological changes in mitochondria accompany ODC dysfunction during neuroinflammation.
    Keywords:  experimental autoimmune encephalomyelitis; mitochondria; multiple sclerosis; myelin; neurodegeneration; oligodendrocyte; reactive species
  6. Methods Mol Biol. 2022 ;2497 255-267
      Proving with certainty that a GFP-tagged protein is imported inside mitochondria by visualizing its fluorescence emission with an epifluorescence microscope is currently impossible using regular GFP-tagging. This is particularly true for proteins dual localized in the cytosol and mitochondria, which have been estimated to represent up to one third of the established mitoproteomes. These proteins are usually composed of a surpassingly abundant pool of the cytosolic isoform compared to the mitochondrial isoform. As a consequence, when tagged with a regular GFP, the fluorescence emission of the cytosolic isoform will inevitably eclipse that of the mitochondrial one and prevent the detection of the mitochondrial echoform. To overcome this technical limit, we engineered a yeast strain expressing a new type of GFP called Bi-Genomic Mitochondrial-Split-GFP (BiG Mito-Split-GFP). In this strain, one moiety of the GFP is encoded by the mitochondrial DNA while the second moiety of the GFP can be tagged to any nuclear-encoded protein (suspected to be dual localized or bona fide mitochondrial). By doing so, only mitochondrial proteins or echoforms of dual localized proteins, regardless of their organismal origin, trigger GFP reconstitution that can be visualized by regular fluorescence microscopy. The strength of the BiG Mito-Split-GFP system is that proof of the mitochondrial localization of a given protein rests on a simple and effortless microscopy observation.
    Keywords:  BiG Mito-Split-GFP; Dual localized; Epifluorescence microscopy; Living cells; Localization; Mitochondria; Saccharomyces cerevisiae
  7. Proc Natl Acad Sci U S A. 2022 Jul 05. 119(27): e2111262119
      All cells contain specialized signaling pathways that enable adaptation to specific molecular stressors. Yet, whether these pathways are centrally regulated in complex physiological stress states remains unclear. Using genome-scale fitness screening data, we quantified the stress phenotype of 739 cancer cell lines, each representing a unique combination of intrinsic tumor stresses. Integrating dependency and stress perturbation transcriptomic data, we illuminated a network of genes with vital functions spanning diverse stress contexts. Analyses for central regulators of this network nominated C16orf72/HAPSTR1, an evolutionarily ancient gene critical for the fitness of cells reliant on multiple stress response pathways. We found that HAPSTR1 plays a pleiotropic role in cellular stress signaling, functioning to titrate various specialized cell-autonomous and paracrine stress response programs. This function, while dispensable to unstressed cells and nematodes, is essential for resilience in the presence of stressors ranging from DNA damage to starvation and proteotoxicity. Mechanistically, diverse stresses induce HAPSTR1, which encodes a protein expressed as two equally abundant isoforms. Perfectly conserved residues in a domain shared between HAPSTR1 isoforms mediate oligomerization and binding to the ubiquitin ligase HUWE1. We show that HUWE1 is a required cofactor for HAPSTR1 to control stress signaling and that, in turn, HUWE1 feeds back to ubiquitinate and destabilize HAPSTR1. Altogether, we propose that HAPSTR1 is a central rheostat in a network of pathways responsible for cellular adaptability, the modulation of which may have broad utility in human disease.
    Keywords:  network; signaling; stress
  8. Nat Commun. 2022 Jun 28. 13(1): 3720
      PINK1-Parkin mediated mitophagy, a selective form of autophagy, represents one of the most important mechanisms in mitochondrial quality control (MQC) via the clearance of damaged mitochondria. Although it is well known that the conjugation of mammalian ATG8s (mATG8s) to phosphatidylethanolamine (PE) is a key step in autophagy, its role in mitophagy remains controversial. In this study, we clarify the role of the mATG8-conjugation system in mitophagy by generating knockouts of the mATG8-conjugation machinery. Unexpectedly, we show that mitochondria could still be cleared in the absence of the mATG8-conjugation system, in a process independent of lysosomal degradation. Instead, mitochondria are cleared via extracellular release through a secretory autophagy pathway, in a process we define as Autophagic Secretion of Mitochondria (ASM). Functionally, increased ASM promotes the activation of the innate immune cGAS-STING pathway in recipient cells. Overall, this study reveals ASM as a mechanism in MQC when the cellular mATG8-conjugation machinery is dysfunctional and highlights the critical role of mATG8 lipidation in suppressing inflammatory responses.