bims-minimp 2021-06-06 papers

bims-minimp

Biomed News

on Mitochondria, innate immunity, proteostasis

Issue of 2021–06–06
twenty papers selected by
Hanna Salmonowicz, Newcastle University

Mitochondrial Autophagy and Cell Survival is Regulated by the Circadian Clock Gene in Cardiac Myocytes during Ischemic Stress.
Melatonin reshapes the mitochondrial network and promotes intercellular mitochondrial transfer via tunneling nanotubes (TNTs) after ischemic-like injury in hippocampal HT22 cells.
Prohibitin 1 is essential to preserve mitochondria and myelin integrity in Schwann cells.
Nucleotide-binding domain and leucine-rich-repeat-containing protein X1 deficiency induces nicotinamide adenine dinucleotide decline, mechanistic target of rapamycin activation, and cellular senescence and accelerates aging lung-like changes.
The Mitochondrial Response to DNA Damage.
PINK1 and parkin shape the organism-wide distribution of a deleterious mitochondrial genome.
Efficient extra-mitochondrial aerobic ATP synthesis in neuronal membrane systems.
Modern cities modelled as "super-cells" rather than multicellular organisms: Implications for industry, goods and services.
Mitophagy and Oxidative Stress: The Role of Aging.
Type I interferon decreases macrophage energy metabolism during mycobacterial infection.
The ESCRT-III complex contributes to macromitophagy in yeast.
Loss of Fis1 impairs proteostasis during skeletal muscle aging in Drosophila.
The Function of KDEL Receptors as UPR Genes in Disease.
The NDUFS4 Knockout Mouse: A Dual Threat Model of Childhood Mitochondrial Disease and Normative Aging.
Acute systemic inflammation exacerbates neuroinflammation in Alzheimer's disease: IL-1β drives amplified responses in primed astrocytes and neuronal network dysfunction.
A proximity-dependent biotinylation map of a human cell.
Mitochondria-dependent synthetic small-molecule vaccine adjuvants for influenza virus infection.
Inflammasome regulation in driving COVID-19 severity in humans and immune tolerance in bats.
Suborganellar Localization of Mitochondrial Proteins and Transcripts in Human Cells.
Crosstalk between reactive oxygen species and Dynamin-related protein 1 in periodontitis.

Autophagy. 2021 Jun 04.

Mitochondrial Autophagy and Cell Survival is Regulated by the Circadian Clock Gene in Cardiac Myocytes during Ischemic Stress.

Inna Rabinovich-Nikitin, Mina Rasouli, Cristine J Reitz, Illana Posen, Victoria Margulets, Rimpy Dhingra, Tarak N Khatua, James A Thliveris, Tami A Martino, Lorrie A Kirshenbaum.

  Cardiac function is highly reliant on mitochondrial oxidative metabolism and quality control. The circadian Clock gene is critically linked to vital physiological processes including mitochondrial fission, fusion and bioenergetics; however, little is known of how the Clock gene regulates these vital processes in the heart. Herein, we identified a putative circadian CLOCK-mitochondrial interactome that gates an adaptive survival response during myocardial ischemia. We show by transcriptome and gene ontology mapping in CLOCK Δ19/Δ19 mouse that Clock transcriptionally coordinates the efficient removal of damaged mitochondria during myocardial ischemia by directly controlling transcription of genes required for mitochondrial fission, fusion and macroautophagy/autophagy. Loss of Clock gene activity impaired mitochondrial turnover resulting in the accumulation of damaged reactive oxygen species (ROS)-producing mitochondria from impaired mitophagy. This coincided with ultrastructural defects to mitochondria and impaired cardiac function. Interestingly, wild type CLOCK but not mutations of CLOCK defective for E-Box binding or interaction with its cognate partner ARNTL/BMAL-1 suppressed mitochondrial damage and cell death during acute hypoxia. Interestingly, the autophagy defect and accumulation of damaged mitochondria in CLOCK-deficient cardiac myocytes were abrogated by restoring autophagy/mitophagy. Inhibition of autophagy by ATG7 knockdown abrogated the cytoprotective effects of CLOCK. Collectively, our results demonstrate that CLOCK regulates an adaptive stress response critical for cell survival by transcriptionally coordinating mitochondrial quality control mechanisms in cardiac myocytes. Interdictions that restore CLOCK activity may prove beneficial in reducing cardiac injury in individuals with disrupted circadian CLOCK.

Keywords:  autophagy; clock; metabolism; mitochondrion; myocardial infarction

DOI:  https://doi.org/10.1080/15548627.2021.1938913
J Pineal Res. 2021 Jun 04. e12747

Melatonin reshapes the mitochondrial network and promotes intercellular mitochondrial transfer via tunneling nanotubes (TNTs) after ischemic-like injury in hippocampal HT22 cells.

Maria Gemma Nasoni, Silvia Carloni, Barbara Canonico, Sabrina Burattini, Erica Cesarini, Stefano Papa, Marica Pagliarini, Patrizia Ambrogini, Walter Balduini, Francesca Luchetti.

  Mitochondrial dysfunction is considered one of the hallmarks of ischemia/reperfusion injury. Mitochondria are plastic organelles that undergo continuous biogenesis, fusion and fission. They can be transferred between cells through tunneling nanotubes (TNTs), dynamic structures that allow the exchange of proteins, soluble molecules and organelles. Maintaining mitochondrial dynamics is crucial to cell function and survival. The present study aimed to assess the effects of melatonin on mitochondrial dynamics, TNT formation and mitochondria transfer in HT22 cells exposed to oxygen/glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin treatment during the reoxygenation phase reduced mitochondrial reactive oxygen species (ROS) production, improved cell viability and increased the expression of PGC1α and SIRT3. Melatonin also preserved the expression of the membrane translocase proteins TOM20 and TIM23, and of the matrix protein HSP60, which are involved in mitochondrial biogenesis. Moreover, it promoted mitochondrial fusion and enhanced the expression of MFN2 and OPA1. Remarkably, melatonin also fostered mitochondrial transfer between injured HT22 cells through TNT connections. These results provide new insights into the effect of melatonin on mitochondrial network reshaping and cell survival. Fostering TNTs formation represents a novel mechanism mediating the protective effect of melatonin in ischemia/reperfusion injury.

Keywords:  HT22; Melatonin; mitochondrial network; oxygen-glucose deprivation; tunneling nanotubes

DOI:  https://doi.org/10.1111/jpi.12747
Nat Commun. 2021 06 02. 12(1): 3285

Prohibitin 1 is essential to preserve mitochondria and myelin integrity in Schwann cells.

Gustavo Della-Flora Nunes, Emma R Wilson, Leandro N Marziali, Edward Hurley, Nicholas Silvestri, Bin He, Bert W O'Malley, Bogdan Beirowski, Yannick Poitelon, Lawrence Wrabetz, M Laura Feltri.

In peripheral nerves, Schwann cells form myelin and provide trophic support to axons. We previously showed that the mitochondrial protein prohibitin 2 can localize to the axon-Schwann-cell interface and is required for developmental myelination. Whether the homologous protein prohibitin 1 has a similar role, and whether prohibitins also play important roles in Schwann cell mitochondria is unknown. Here, we show that deletion of prohibitin 1 in Schwann cells minimally perturbs development, but later triggers a severe demyelinating peripheral neuropathy. Moreover, mitochondria are heavily affected by ablation of prohibitin 1 and demyelination occurs preferentially in cells with apparent mitochondrial loss. Furthermore, in response to mitochondrial damage, Schwann cells trigger the integrated stress response, but, contrary to what was previously suggested, this response is not detrimental in this context. These results identify a role for prohibitin 1 in myelin integrity and advance our understanding about the Schwann cell response to mitochondrial damage.

DOI: https://doi.org/10.1038/s41467-021-23552-8
Aging Cell. 2021 Jun 04. e13410

Nucleotide-binding domain and leucine-rich-repeat-containing protein X1 deficiency induces nicotinamide adenine dinucleotide decline, mechanistic target of rapamycin activation, and cellular senescence and accelerates aging lung-like changes.

Hyeon Jun Shin, Sang-Hun Kim, Hong-Jai Park, Min-Sun Shin, Insoo Kang, Min-Jong Kang.

  Mitochondrial dysfunction has long been implicated to have a causative role in organismal aging. A mitochondrial molecule, nucleotide-binding domain and leucine-rich-repeat-containing protein X1 (NLRX1), represents the only NLR family member that targets this cellular location, implying that NLRX1 probably establishes a fundamental link between mitochondrial functions and cellular physiology. However, the significance of NLRX1 function in cellular senescence, a key conceptual constituent in aging biology, is yet to be defined. Here, we demonstrate that molecular hallmarks involved in aging biology including NAD+ decline, and activation of mTOR, p53, and p16INK4A are significantly enhanced in NLRX1 deficiency in vitro. Mechanistic studies of replicative cellular senescence in the presence or absence of NLRX1 in vitro reveal that NLRX1-deficient fibroblasts fail to maintain optimal NAD+ /NADH ratio, which instigates the decline of SIRT1 and the activation of mTOR, p16INK4A , and p53, leading to the increase in senescence-associated beta-galactosidase (SA-β-gal)-positive cells. Importantly, the enhanced cellular senescence response in NLRX1 deficiency is significantly attenuated by pharmacological inhibition of mTOR signaling in vitro. Finally, our in vivo murine studies reveal that NLRX1 decreases with age in murine lungs and NLRX1 deficiency in vivo accelerates pulmonary functional and structural changes that recapitulate the findings observed in human aging lungs. In conclusion, the current study provides evidence for NLRX1 as a crucial regulator of cellular senescence and in vivo lung aging.

Keywords:  NAD+ (nicotinamide adenine dinucleotide); NLRX1 (nucleotide-binding domain and leucine-rich-repeat-containing protein X1); cellular senescence; lung aging; mTOR (mechanistic target of rapamycin)

DOI:  https://doi.org/10.1111/acel.13410
Front Cell Dev Biol. 2021 ;9 669379

The Mitochondrial Response to DNA Damage.

Ziye Rong, Peipei Tu, Peiqi Xu, Yan Sun, Fangfang Yu, Na Tu, Lixia Guo, Yanan Yang.

  Mitochondria are double membrane organelles in eukaryotic cells that provide energy by generating adenosine triphosphate (ATP) through oxidative phosphorylation. They are crucial to many aspects of cellular metabolism. Mitochondria contain their own DNA that encodes for essential proteins involved in the execution of normal mitochondrial functions. Compared with nuclear DNA, the mitochondrial DNA (mtDNA) is more prone to be affected by DNA damaging agents, and accumulated DNA damages may cause mitochondrial dysfunction and drive the pathogenesis of a variety of human diseases, including neurodegenerative disorders and cancer. Therefore, understanding better how mtDNA damages are repaired will facilitate developing therapeutic strategies. In this review, we focus on our current understanding of the mtDNA repair system. We also discuss other mitochondrial events promoted by excessive DNA damages and inefficient DNA repair, such as mitochondrial fusion, fission, and mitophagy, which serve as quality control events for clearing damaged mtDNA.

Keywords:  DNA repair; mitochondrial DNA; mitochondrial fission; mitochondrial fusion; mitophagy

DOI:  https://doi.org/10.3389/fcell.2021.669379
Cell Rep. 2021 Jun 01. pii: S2211-1247(21)00552-0. [Epub ahead of print]35(9): 109203

PINK1 and parkin shape the organism-wide distribution of a deleterious mitochondrial genome.

Arnaud Ahier, Chuan-Yang Dai, Ina Kirmes, Nadia Cummins, Grace Ching Ching Hung, Jürgen Götz, Steven Zuryn.

  In multiple species, certain tissue types are prone to acquiring greater loads of mitochondrial genome (mtDNA) mutations relative to others, but the mechanisms that drive these heteroplasmy differences are unknown. We find that the conserved PTEN-induced putative kinase (PINK1/PINK-1) and the E3 ubiquitin-protein ligase parkin (PDR-1), which are required for mitochondrial autophagy (mitophagy), underlie stereotyped differences in heteroplasmy of a deleterious mitochondrial genome mutation (ΔmtDNA) between major somatic tissues types in Caenorhabditis elegans. We demonstrate that tissues prone to accumulating ΔmtDNA have lower mitophagy responses than those with low mutation levels. Moreover, we show that ΔmtDNA heteroplasmy increases when proteotoxic species that are associated with neurodegenerative disease and mitophagy inhibition are overexpressed in the nervous system. These results suggest that PINK1 and parkin drive organism-wide patterns of heteroplasmy and provide evidence of a causal link between proteotoxicity, mitophagy, and mtDNA mutation levels in neurons.

Keywords:  Alzheimer's disease; PINK1; heteroplasmy; mitochondria; mitophagy; mtDNA; parkin; polyglutamate; proteotoxicity; tau

DOI:  https://doi.org/10.1016/j.celrep.2021.109203
J Neurosci Res. 2021 Jun 03.

Efficient extra-mitochondrial aerobic ATP synthesis in neuronal membrane systems.

Silvia Ravera, Martina Bartolucci, Daniela Calzia, Alessandro M Morelli, Isabella Panfoli.

  The nervous system displays high energy consumption, apparently not fulfilled by mitochondria, which are underrepresented therein. The oxidative phosphorylation (OxPhos) activity, a mitochondrial process that aerobically provides ATP, has also been reported also in the myelin sheath and the rod outer segment (OS) disks. Thus, commonalities and differences between the extra-mitochondrial and mitochondrial aerobic metabolism were evaluated in bovine isolated myelin (IM), rod OS, and mitochondria-enriched fractions (MIT). The subcellular fraction quality and the absence of contamination fractions have been estimated by western blot analysis. Oxygen consumption and ATP synthesis were stimulated by conventional (pyruvate + malate or succinate) and unconventional (NADH) substrates, observing that oxygen consumption and ATP synthesis by IM and rod OS are more efficient than by MIT, in the presence of both kinds of respiratory substrates. Mitochondria did not utilize NADH as a respiring substrate. When ATP synthesis by either sample was assayed in the presence of 10-100 µM ATP in the assay medium, only in IM and OS it was not inhibited, suggesting that the ATP exportation by the mitochondria is limited by extravesicular ATP concentration. Interestingly, IM and OS but not mitochondria appear able to synthesize ATP at a later time with respect to exposure to respiratory substrates, supporting the hypothesis that the proton gradient produced by the electron transport chain is buffered by membrane phospholipids. The putative transfer mode of the OxPhos molecular machinery from mitochondria to the extra-mitochondrial structures is also discussed, opening new perspectives in the field of neurophysiology.

Keywords:  RRID:AB_10696805; RRID:AB_11183050; RRID:AB_1845182; RRID:AB_2818988; RRID:AB_2851910; RRID:SCR_002798; RRID:SCR_014210; aerobic metabolism; bioenergetics; myelin sheath; oxidative phosphorylation; phototransduction; rod outer segments disks

DOI:  https://doi.org/10.1002/jnr.24865
Bioessays. 2021 Jun 03. e2100041

Modern cities modelled as "super-cells" rather than multicellular organisms: Implications for industry, goods and services.

Jie Chang, Ying Ge, Zhaoping Wu, Yuanyuan Du, Kaixuan Pan, Guofu Yang, Yuan Ren, Mikko P Heino, Feng Mao, Kang Hao Cheong, Zelong Qu, Xing Fan, Yong Min, Changhui Peng, Laura A Meyerson.

  The structure and "metabolism" (movement and conversion of goods and energy) of urban areas has caused cities to be identified as "super-organisms", placed between ecosystems and the biosphere, in the hierarchy of living systems. Yet most such analogies are weak, and render the super-organism model ineffective for sustainable development of cities. Via a cluster analysis of 15 shared traits of the hierarchical living system, we found that industrialized cities are more similar to eukaryotic cells than to multicellular organisms; enclosed systems, such as factories and greenhouses, paralleling organelles in eukaryotic cells. We further developed a "super-cell" industrialized city model: a "eukarcity" with citynucleus (urban area) as a regulating centre, and organaras (enclosed systems, which provide the majority of goods and services) as the functional components, and cityplasm (natural ecosystems and farmlands) as the matrix. This model may improve the vitality and sustainability of cities through planning and management.

Keywords:  hierarchical living system; industrial systems; scaling; sustainability; urban-rural system

DOI:  https://doi.org/10.1002/bies.202100041
Antioxidants (Basel). 2021 May 17. pii: 794. [Epub ahead of print]10(5):

Mitophagy and Oxidative Stress: The Role of Aging.

Anna De Gaetano, Lara Gibellini, Giada Zanini, Milena Nasi, Andrea Cossarizza, Marcello Pinti.

  Mitochondrial dysfunction is a hallmark of aging. Dysfunctional mitochondria are recognized and degraded by a selective type of macroautophagy, named mitophagy. One of the main factors contributing to aging is oxidative stress, and one of the early responses to excessive reactive oxygen species (ROS) production is the induction of mitophagy to remove damaged mitochondria. However, mitochondrial damage caused at least in part by chronic oxidative stress can accumulate, and autophagic and mitophagic pathways can become overwhelmed. The imbalance of the delicate equilibrium among mitophagy, ROS production and mitochondrial damage can start, drive, or accelerate the aging process, either in physiological aging, or in pathological age-related conditions, such as Alzheimer's and Parkinson's diseases. It remains to be determined which is the prime mover of this imbalance, i.e., whether it is the mitochondrial damage caused by ROS that initiates the dysregulation of mitophagy, thus activating a vicious circle that leads to the reduced ability to remove damaged mitochondria, or an alteration in the regulation of mitophagy leading to the excessive production of ROS by damaged mitochondria.

Keywords:  Alzheimer; PINK1; Parkinson; Reactive Oxygen Species; aging; mitochondria; mitophagy

DOI:  https://doi.org/10.3390/antiox10050794
Cell Rep. 2021 Jun 01. pii: S2211-1247(21)00541-6. [Epub ahead of print]35(9): 109195

Type I interferon decreases macrophage energy metabolism during mycobacterial infection.

Gregory S Olson, Tara A Murray, Ana N Jahn, Dat Mai, Alan H Diercks, Elizabeth S Gold, Alan Aderem.

  Metabolic reprogramming powers and polarizes macrophage functions, but the nature and regulation of this response during infection with pathogens remain controversial. In this study, we characterize the metabolic and transcriptional responses of murine macrophages to Mycobacterium tuberculosis (Mtb) in order to disentangle the underlying mechanisms. We find that type I interferon (IFN) signaling correlates with the decreased glycolysis and mitochondrial damage that is induced by live, but not killed, Mtb. Macrophages lacking the type I IFN receptor (IFNAR) maintain glycolytic flux and mitochondrial function during Mtb infection in vitro and in vivo. IFNβ itself restrains the glycolytic shift of inflammatory macrophages and initiates mitochondrial stress. We confirm that type I IFN acts upstream of mitochondrial damage using macrophages lacking the protein STING. We suggest that a type I IFN-mitochondrial feedback loop controls macrophage responses to mycobacteria and that this could contribute to pathogenesis across a range of diseases.

Keywords:  STING; immunometabolism; macrophage; mitochondria; mycobacteria; tuberculosis; type I interferon

DOI:  https://doi.org/10.1016/j.celrep.2021.109195
Traffic. 2021 Jun 05.

The ESCRT-III complex contributes to macromitophagy in yeast.

Zulin Wu, Haiqian Xu, Junze Liu, Fan Zhou, Yongheng Liang.

  Mitochondria play important roles in energy generation and homeostasis maintenance in eukaryotic cells. The damaged or superfluous mitochondria can be nonselectively or selectively removed through the autophagy/lysosome pathway, which was referred as mitophagy. According to the molecular machinery for degrading mitochondria, the selectively removed mitochondria can occur through macromitophagy or micromitophagy. In this study, we show that the endosomal sorting complex required for transport III (ESCRT-III) in budding yeast regulates macromitophagy induced by nitrogen starvation, but not by the post-logarithmic phase growth in lactate medium by monitoring a mitochondrial marker, Om45. Firstly, loss of ESCRT-III subunit Snf7 or Vps4-Vta1 complex subunit Vps4, two representative subunits of the ESCRT complex, suppresses the delivery and degradation of Om45-GFP to vacuoles. Secondly, we show that the mitochondrial marker Om45 and mitophagy receptor Atg32 accumulate on autophagosomes marked with Atg8 (mitophagosomes, MPs) in ESCRT mutants. Moreover, the protease-protection assay indicates that Snf7 and Vps4 are involved in MP closure. Finally, Snf7 interacts with Atg11, which was detected by two ways, GST pulldown and BiFC, and this BiFC interaction happens on mitochondrial reticulum. Therefore, we proposed that the ESCRT-III machinery mediates nitrogen starvation-induced macromitophagy by the interaction between Snf7 and Atg11 so that Snf7 is recruited to Atg32 marked MPs by the known Atg11-Atg32 interaction to seal them. These results reveal that the ESCRT-III complex plays a new role in yeast on macromitophagy.

Keywords:  Atg11; Atg32; ESCRT; Macromitophagy; Micromitophagy; Mitophagosome; Mitophagy, Snf7; Vps4

DOI:  https://doi.org/10.1111/tra.12805
Aging Cell. 2021 Jun 01. e13379

Loss of Fis1 impairs proteostasis during skeletal muscle aging in Drosophila.

Tai-Ting Lee, Po-Lin Chen, Matthew P Su, Jian-Chiuan Li, Yi-Wen Chang, Rei-Wen Liu, Hsueh-Fen Juan, Jinn-Moon Yang, Shih-Peng Chan, Yu-Chen Tsai, Sophia von Stockum, Elena Ziviani, Azusa Kamikouchi, Horng-Dar Wang, Chun-Hong Chen.

  Increased levels of dysfunctional mitochondria within skeletal muscle are correlated with numerous age-related physiopathological conditions. Improving our understanding of the links between mitochondrial function and muscle proteostasis, and the role played by individual genes and regulatory networks, is essential to develop treatments for these conditions. One potential player is the mitochondrial outer membrane protein Fis1, a crucial fission factor heavily involved in mitochondrial dynamics in yeast but with an unknown role in higher-order organisms. By using Drosophila melanogaster as a model, we explored the effect of Fis1 mutations generated by transposon Minos-mediated integration. Mutants exhibited a higher ratio of damaged mitochondria with age as well as elevated reactive oxygen species levels compared with controls. This caused an increase in oxidative stress, resulting in large accumulations of ubiquitinated proteins, accelerated muscle function decline, and mitochondrial myopathies in young mutant flies. Ectopic expression of Fis1 isoforms was sufficient to suppress this phenotype. Loss of Fis1 led to unbalanced mitochondrial proteostasis within fly muscle, decreasing both flight capabilities and lifespan. Fis1 thus clearly plays a role in fly mitochondrial dynamics. Further investigations into the detailed function of Fis1 are necessary for exploring how mitochondrial function correlates with muscle health during aging.

Keywords:   Drosophila melanogaster ; Fis1; aging; mitochondria

DOI:  https://doi.org/10.1111/acel.13379
Int J Mol Sci. 2021 May 21. pii: 5436. [Epub ahead of print]22(11):

The Function of KDEL Receptors as UPR Genes in Disease.

Emily S Wires, Kathleen A Trychta, Lacey M Kennedy, Brandon K Harvey.

  The KDEL receptor retrieval pathway is essential for maintaining resident proteins in the endoplasmic reticulum (ER) lumen. ER resident proteins serve a variety of functions, including protein folding and maturation. Perturbations to the lumenal ER microenvironment, such as calcium depletion, can cause protein misfolding and activation of the unfolded protein response (UPR). Additionally, ER resident proteins are secreted from the cell by overwhelming the KDEL receptor retrieval pathway. Recent data show that KDEL receptors are also activated during the UPR through the IRE1/XBP1 signaling pathway as an adaptive response to cellular stress set forth to reduce the loss of ER resident proteins. This review will discuss the emerging connection between UPR activation and KDEL receptors as it pertains to ER proteostasis and disease states.

Keywords:  ER resident proteins; KDEL receptor; disease; endoplasmic reticulum; exodosis; unfolded protein response

DOI:  https://doi.org/10.3390/ijms22115436
Methods Mol Biol. 2021 ;2277 143-155

The NDUFS4 Knockout Mouse: A Dual Threat Model of Childhood Mitochondrial Disease and Normative Aging.

Anthony S Grillo, Alessandro Bitto, Matt Kaeberlein.

  Mice missing the Complex I subunit NADH:Ubiquinone Oxidoreductase Fe-S Protein 4 (NDUFS4) of the electron transport chain are a leading model of the severe mitochondrial disease Leigh syndrome. These mice have enabled a better understanding of mitochondrial dysfunction in human disease, as well as in the discovery of interventions that can potentially suppress mitochondrial disease manifestations. In addition, increasing evidence suggests significant overlap between interventions that increase survival in NDUFS4 knockout mice and that extend life span during normative aging. This chapter discusses the practical aspects of handling and studying these mice, which can be challenging due to their severe disease phenotype. Common procedures such as breeding, genotyping, weaning, or treating these transgenic mice are also discussed.

Keywords:  Aging; Complex I; Electron transport chain; Hypoxia; Leigh syndrome; Mitochondrial disease; Mitochondrial dysfunction; NAD; NDUFS4; Rapamycin; mTOR

DOI:  https://doi.org/10.1007/978-1-0716-1270-5_10
Alzheimers Dement. 2021 Jun 03.

Acute systemic inflammation exacerbates neuroinflammation in Alzheimer's disease: IL-1β drives amplified responses in primed astrocytes and neuronal network dysfunction.

Ana Belen Lopez-Rodriguez, Edel Hennessy, Carol L Murray, Arshed Nazmi, Hugh J Delaney, Dáire Healy, Steven G Fagan, Michael Rooney, Erika Stewart, Anouchka Lewis, Niamh de Barra, Philip Scarry, Louise Riggs-Miller, Delphine Boche, Mark O Cunningham, Colm Cunningham.

  Neuroinflammation contributes to Alzheimer's disease (AD) progression. Secondary inflammatory insults trigger delirium and can accelerate cognitive decline. Individual cellular contributors to this vulnerability require elucidation. Using APP/PS1 mice and AD brain, we studied secondary inflammatory insults to investigate hypersensitive responses in microglia, astrocytes, neurons, and human brain tissue. The NLRP3 inflammasome was assembled surrounding amyloid beta, and microglia were primed, facilitating exaggerated interleukin-1β (IL-1β) responses to subsequent LPS stimulation. Astrocytes were primed to produce exaggerated chemokine responses to intrahippocampal IL-1β. Systemic LPS triggered microglial IL-1β, astrocytic chemokines, IL-6, and acute cognitive dysfunction, whereas IL-1β disrupted hippocampal gamma rhythm, all selectively in APP/PS1 mice. Brains from AD patients with infection showed elevated IL-1β and IL-6 levels. Therefore, amyloid leaves the brain vulnerable to secondary inflammation at microglial, astrocytic, neuronal, and cognitive levels, and infection amplifies neuroinflammatory cytokine synthesis in humans. Exacerbation of neuroinflammation to produce deleterious outcomes like delirium and accelerated disease progression merits careful investigation in humans.

Keywords:  APP/PS1; CCL2; IL-1β; astrocyte; chemokine; cytokine; delirium; dementia; gamma; memory; microglia; network dysfunction; neuroinflammation; primed; priming; vulnerability

DOI:  https://doi.org/10.1002/alz.12341
Nature. 2021 Jun 02.

A proximity-dependent biotinylation map of a human cell.

Christopher D Go, James D R Knight, Archita Rajasekharan, Bhavisha Rathod, Geoffrey G Hesketh, Kento T Abe, Ji-Young Youn, Payman Samavarchi-Tehrani, Hui Zhang, Lucie Y Zhu, Evelyn Popiel, Jean-Philippe Lambert, Étienne Coyaud, Sally W T Cheung, Dushyandi Rajendran, Cassandra J Wong, Hana Antonicka, Laurence Pelletier, Alexander F Palazzo, Eric A Shoubridge, Brian Raught, Anne-Claude Gingras.

Compartmentalization is a defining characteristic of eukaryotic cells, and partitions distinct biochemical processes into discrete subcellular locations. Microscopy1 and biochemical fractionation coupled with mass spectrometry2-4 have defined the proteomes of a variety of different organelles, but many intracellular compartments have remained refractory to such approaches. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach to define the composition of cellular compartments in living cells5-7. Here we present a BioID-based map of a human cell on the basis of 192 subcellular markers, and define the intracellular locations of 4,145 unique proteins in HEK293 cells. Our localization predictions exceed the specificity of previous approaches, and enabled the discovery of proteins at the interface between the mitochondrial outer membrane and the endoplasmic reticulum that are crucial for mitochondrial homeostasis. On the basis of this dataset, we created humancellmap.org as a community resource that provides online tools for localization analysis of user BioID data, and demonstrate how this resource can be used to understand BioID results better.

DOI: https://doi.org/10.1038/s41586-021-03592-2
Proc Natl Acad Sci U S A. 2021 Jun 08. pii: e2025718118. [Epub ahead of print]118(23):

Mitochondria-dependent synthetic small-molecule vaccine adjuvants for influenza virus infection.

Fumi Sato-Kaneko, Shiyin Yao, Fitzgerald S Lao, Jason Nan, Jonathan Shpigelman, Annette Cheng, Tetsuya Saito, Karen Messer, Minya Pu, Nikunj M Shukla, Howard B Cottam, Michael Chan, Anthony J Molina, Maripat Corr, Tomoko Hayashi, Dennis A Carson.

  Vaccine adjuvants enhance and prolong pathogen-specific protective immune responses. Recent reports indicate that host factors-such as aging, pregnancy, and genetic polymorphisms-influence efficacies of vaccines adjuvanted with Toll-like receptor (TLR) or known pattern-recognition receptor (PRR) agonists. Although PRR independent adjuvants (e.g., oil-in-water emulsion and saponin) are emerging, these adjuvants induce some local and systemic reactogenicity. Hence, new TLR and PRR-independent adjuvants that provide greater potency alone or in combination without compromising safety are highly desired. Previous cell-based high-throughput screenings yielded a small molecule 81 [N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide], which enhanced lipopolysaccharide-induced NF-κB and type I interferon signaling in reporter assays. Here compound 81 activated innate immunity in primary human peripheral blood mononuclear cells and murine bone marrow-derived dendritic cells (BMDCs). The innate immune activation by 81 was independent of TLRs and other PRRs and was significantly reduced in mitochondrial antiviral-signaling protein (MAVS)-deficient BMDCs. Compound 81 activities were mediated by mitochondrial dysfunction as mitophagy inducers and a mitochondria specific antioxidant significantly inhibited cytokine induction by 81. Both compound 81 and a derivative obtained via structure-activity relationship studies, 2F52 [N-benzyl-N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide] modestly increased mitochondrial reactive oxygen species and induced the aggregation of MAVS. Neither 81 nor 2F52 injected as adjuvants caused local or systemic toxicity in mice at effective concentrations for vaccination. Furthermore, vaccination with inactivated influenza virus adjuvanted with 2F52 demonstrated protective effects in a murine lethal virus challenge study. As an unconventional and safe adjuvant that does not require known PRRs, compound 2F52 could be a useful addition to vaccines.

Keywords:  NF-κB; influenza virus; mitochondrial reactive oxygen species; mitochondrial stress; vaccine adjuvant

DOI:  https://doi.org/10.1073/pnas.2025718118
J Leukoc Biol. 2021 May 31.

Inflammasome regulation in driving COVID-19 severity in humans and immune tolerance in bats.

Sahana Nagaraja, Disha Jain, Sannula Kesavardhana.

  Coronaviruses (CoVs) are RNA viruses that cause human respiratory infections. Zoonotic transmission of the SARS-CoV-2 virus caused the recent COVID-19 pandemic, which led to over 2 million deaths worldwide. Elevated inflammatory responses and cytotoxicity in the lungs are associated with COVID-19 severity in SARS-CoV-2-infected individuals. Bats, which host pathogenic CoVs, operate dampened inflammatory responses and show tolerance to these viruses with mild clinical symptoms. Delineating the mechanisms governing these host-specific inflammatory responses is essential to understand host-virus interactions determining the outcome of pathogenic CoV infections. Here, we describe the essential role of inflammasome activation in determining COVID-19 severity in humans and innate immune tolerance in bats that host several pathogenic CoVs. We further discuss mechanisms leading to inflammasome activation in human SARS-CoV-2 infection and how bats are molecularly adapted to suppress these inflammasome responses. We also report an analysis of functionally important residues of inflammasome components that provide new clues of bat strategies to suppress inflammasome signaling and innate immune responses. As spillover of bat viruses may cause the emergence of new human disease outbreaks, the inflammasome regulation in bats and humans likely provides specific strategies to combat the pathogenic CoV infections.

Keywords:  Bat immunity; COVID-19; Coronaviruses; Immune tolerance; Inflammasome; NLRP3; SARS-CoV-2

DOI:  https://doi.org/10.1002/JLB.4COVHR0221-093RR
Methods Mol Biol. 2021 ;2277 157-173

Suborganellar Localization of Mitochondrial Proteins and Transcripts in Human Cells.

Smirnova Anna, Richert Ludovic, Smirnov Alexandre, Mély Yves, Tarassov Ivan.

  Mitochondria have complex ultrastructure which includes continuous subcompartments, such as matrix, intermembrane space, and two membranes, as well as focal structures, such as nucleoids, RNA granules, and mitoribosomes. Comprehensive studies of the spatial distribution of proteins and RNAs inside the mitochondria are necessary to understand organellar gene expression processes and macromolecule targeting pathways. Here we give examples of distribution analysis of mitochondrial proteins and transcripts by conventional microscopy and the super-resolution technique 3D STORM. We provide detailed protocols and discuss limitations of immunolabeling of mitochondrial proteins and newly synthesized mitochondrial RNAs by bromouridine incorporation and single-molecule RNA FISH in hepatocarcinoma cells.

Keywords:  3D STORM; Colocalization analysis; Confocal microscopy; Immunolabeling; RNA in situ hybridization; Submitochondrial ultrastructure

DOI:  https://doi.org/10.1007/978-1-0716-1270-5_11
Free Radic Biol Med. 2021 May 27. pii: S0891-5849(21)00323-3. [Epub ahead of print]

Crosstalk between reactive oxygen species and Dynamin-related protein 1 in periodontitis.

Lixi Shi, Yinghui Ji, Shufan Zhao, Houxuan Li, Yun Jiang, Jiajie Mao, Yang Chen, Xiaorong Zhang, Yixin Mao, Xiaoyu Sun, Panpan Wang, Jianfeng Ma, Shengbin Huang.

  Excessive generation of reactive oxygen species (ROS) have great impacts on the development of periodontitis. Dynamin-related protein 1 (Drp1) mediated mitochondrial fission is the main reason and the result of excessive ROS generation. However, whether Drp1 and crosstalk between ROS and Drp1 contribute to the process of periodontitis remains elusive. We herein investigated the role and functional significance of crosstalk between ROS and Drp1 in periodontitis. Firstly, human periodontal ligament cells (hPDLCs) were treated with hydrogen peroxide (H2O2) and ROS inhibitor N-acetyl-cysteine (NAC) or Drp1 inhibitor mitochondrial division inhibitor 1 (Mdivi-1). Cell viability, apoptosis, osteogenic differentiation, expression of Drp1, and mitochondrial function were investigated. Secondly, mice with periodontitis were treated with NAC or Mdivi-1. Finally, gingival tissues were collected from periodontitis patients and healthy individuals to evaluate ROS and Drp1 levels. H2O2 induced cellular injury and inflammation, excessive ROS production, mitochondrial abnormalities, and increased expression of p-Drp1 and Drp1 in hPDLCs, which could be reversed by NAC and Mdivi-1. Moreover, both NAC and Drp1 ameliorated tissue damage and inflammation, and decreased expression of p-Drp1 and Drp1 in mice with periodontitis. More importantly, patients with periodontitis presented significantly higher levels of ROS-induced oxidative damage and p-Drp1 than that in healthy individuals and correlated with clinical parameters. In summary, ROS-Drp1 crosstalk greatly promotes the development of periodontitis. Pharmacological blockade of this crosstalk might be a novel therapeutic strategy for periodontitis.

Keywords:  Drp1; ROS; inflammation; mitochondrial dysfunction; periodontitis; tissue damage

DOI:  https://doi.org/10.1016/j.freeradbiomed.2021.05.031