bims-nenemi 2023-10-22 papers

bims-nenemi

Biomed News

on Neuroinflammation, neurodegeneration and mitochondria

Issue of 2023–10–22
eleven papers selected by
Marco Tigano, Thomas Jefferson University

mtFociCounter for automated single-cell mitochondrial nucleoid quantification and reproducible foci analysis.
The SARS-CoV-2 protein ORF3c is a mitochondrial modulator of innate immunity.
Long 3'UTRs predispose neurons to inflammation by promoting immunostimulatory double-stranded RNA formation.
Mitochondrial DNA is a key driver in cigarette smoke extract-induced IL-6 expression.
Two mitochondrial HMG-box proteins, Cim1 and Abf2, antagonistically regulate mtDNA copy number in Saccharomyces cerevisiae.
ATF5-regulated Mitochondrial Unfolded Protein Response Attenuates Neuronal Damage in Epileptic Rat by Reducing Endoplasmic Reticulum Stress Through Mitochondrial ROS.
ATP functions as a primary alarmin in allergen-induced type 2 immunity.
Long-term super-resolution inner mitochondrial membrane imaging with a lipid probe.
An early glycolysis burst in microglia regulates mitochondrial dysfunction in oligodendrocytes under neuroinflammation.
UvrD-like helicase Hmi1 Has an ATP independent role in yeast mitochondrial DNA maintenance.
A1 is induced by pathogen ligands to limit myeloid cell death and NLRP3 inflammasome activation.

Nucleic Acids Res. 2023 Oct 18. pii: gkad864. [Epub ahead of print]

mtFociCounter for automated single-cell mitochondrial nucleoid quantification and reproducible foci analysis.

Timo Rey, Luis Carlos Tábara, Julien Prudent, Michal Minczuk.

Mitochondrial DNA (mtDNA) encodes the core subunits for OXPHOS, essential in near-all eukaryotes. Packed into distinct foci (nucleoids) inside mitochondria, the number of mtDNA copies differs between cell-types and is affected in several human diseases. Currently, common protocols estimate per-cell mtDNA-molecule numbers by sequencing or qPCR from bulk samples. However, this does not allow insight into cell-to-cell heterogeneity and can mask phenotypical sub-populations. Here, we present mtFociCounter, a single-cell image analysis tool for reproducible quantification of nucleoids and other foci. mtFociCounter is a light-weight, open-source freeware and overcomes current limitations to reproducible single-cell analysis of mitochondrial foci. We demonstrate its use by analysing 2165 single fibroblasts, and observe a large cell-to-cell heterogeneity in nucleoid numbers. In addition, mtFociCounter quantifies mitochondrial content and our results show good correlation (R = 0.90) between nucleoid number and mitochondrial area, and we find nucleoid density is less variable than nucleoid numbers in wild-type cells. Finally, we demonstrate mtFociCounter readily detects differences in foci-numbers upon sample treatment, and applies to Mitochondrial RNA Granules and superresolution microscopy. mtFociCounter provides a versatile solution to reproducibly quantify cellular foci in single cells and our results highlight the importance of accounting for cell-to-cell variance and mitochondrial context in mitochondrial foci analysis.

DOI: https://doi.org/10.1093/nar/gkad864
iScience. 2023 Nov 17. 26(11): 108080

The SARS-CoV-2 protein ORF3c is a mitochondrial modulator of innate immunity.

Hazel Stewart, Yongxu Lu, Sarah O'Keefe, Anusha Valpadashi, Luis Daniel Cruz-Zaragoza, Hendrik A Michel, Samantha K Nguyen, George W Carnell, Nina Lukhovitskaya, Rachel Milligan, Yasmin Adewusi, Irwin Jungreis, Valeria Lulla, David A Matthews, Stephen High, Peter Rehling, Edward Emmott, Jonathan L Heeney, Andrew D Davidson, James R Edgar, Geoffrey L Smith, Andrew E Firth.

  The SARS-CoV-2 genome encodes a multitude of accessory proteins. Using comparative genomic approaches, an additional accessory protein, ORF3c, has been predicted to be encoded within the ORF3a sgmRNA. Expression of ORF3c during infection has been confirmed independently by ribosome profiling. Despite ORF3c also being present in the 2002-2003 SARS-CoV, its function has remained unexplored. Here we show that ORF3c localizes to mitochondria, where it inhibits innate immunity by restricting IFN-β production, but not NF-κB activation or JAK-STAT signaling downstream of type I IFN stimulation. We find that ORF3c is inhibitory after stimulation with cytoplasmic RNA helicases RIG-I or MDA5 or adaptor protein MAVS, but not after TRIF, TBK1 or phospho-IRF3 stimulation. ORF3c co-immunoprecipitates with the antiviral proteins MAVS and PGAM5 and induces MAVS cleavage by caspase-3. Together, these data provide insight into an uncharacterized mechanism of innate immune evasion by this important human pathogen.

Keywords:  Immunology; Microbiology; Molecular biology; Proteomics

DOI:  https://doi.org/10.1016/j.isci.2023.108080
Sci Immunol. 2023 Oct 27. 8(88): eadg2979

Long 3'UTRs predispose neurons to inflammation by promoting immunostimulatory double-stranded RNA formation.

Tyler J Dorrity, Heegwon Shin, Kenenni A Wiegand, Justin Aruda, Michael Closser, Emily Jung, Jake A Gertie, Amanda Leone, Rachel Polfer, Bruce Culbertson, Lisa Yu, Christine Wu, Takamasa Ito, Yuefeng Huang, Anna-Lena Steckelberg, Hynek Wichterle, Hachung Chung.

Loss of RNA homeostasis underlies numerous neurodegenerative and neuroinflammatory diseases. However, the molecular mechanisms that trigger neuroinflammation are poorly understood. Viral double-stranded RNA (dsRNA) triggers innate immune responses when sensed by host pattern recognition receptors (PRRs) present in all cell types. Here, we report that human neurons intrinsically carry exceptionally high levels of immunostimulatory dsRNAs and identify long 3'UTRs as giving rise to neuronal dsRNA structures. We found that the neuron-enriched ELAVL family of genes (ELAVL2, ELAVL3, and ELAVL4) can increase (i) 3'UTR length, (ii) dsRNA load, and (iii) activation of dsRNA-sensing PRRs such as MDA5, PKR, and TLR3. In wild-type neurons, neuronal dsRNAs signaled through PRRs to induce tonic production of the antiviral type I interferon. Depleting ELAVL2 in WT neurons led to global shortening of 3'UTR length, reduced immunostimulatory dsRNA levels, and rendered WT neurons susceptible to herpes simplex virus and Zika virus infection. Neurons deficient in ADAR1, a dsRNA-editing enzyme mutated in the neuroinflammatory disorder Aicardi-Goutières syndrome, exhibited intolerably high levels of dsRNA that triggered PRR-mediated toxic inflammation and neuronal death. Depleting ELAVL2 in ADAR1 knockout neurons led to prolonged neuron survival by reducing immunostimulatory dsRNA levels. In summary, neurons are specialized cells where PRRs constantly sense "self" dsRNAs to preemptively induce protective antiviral immunity, but maintaining RNA homeostasis is paramount to prevent pathological neuroinflammation.

DOI: https://doi.org/10.1126/sciimmunol.adg2979
Hypertens Res. 2023 Oct 17.

Mitochondrial DNA is a key driver in cigarette smoke extract-induced IL-6 expression.

Yusuke Kobayashi, Chiemi Sakai, Takafumi Ishida, Minako Nagata, Yukiko Nakano, Mari Ishida.

  Smoking is an independent risk factor for atherosclerosis, the primary pathogenesis of which is inflammation. We recently reported that cigarette smoke extract (CSE) causes cytosolic and extracellular accumulation of both nuclear (n) and mitochondrial (mt) DNA, which leads to inflammation in human umbilical vein endothelial cells (HUVECs). In this study, we examined whether inflammation induction depends more on cytosolic nDNA or mtDNA, and which chemical constituents of CSE are involved. Acrolein (ACR), methyl vinyl ketone (MVK), and 2-cyclopenten-1-one (CPO) were used in the experiments, as these are the major cytotoxic factors in CSE in various cell types. Stimulation with ACR, MVK, or CPO alone resulted in the accumulation of DNA double-strand breaks (DSBs), but not oxidative DNA damage, accumulation of cytosolic DNA, or increased expression of inflammatory cytokines. Simultaneous administration of all three constituents (ALL) resulted in oxidative DNA damage in both the nucleus and mitochondria, accumulation of DSBs, reduced mitochondrial membrane potential, induction of minority mitochondrial outer membrane permeabilization, accumulation of cytosolic free DNA, and increased expression of inflammatory cytokines such as IL-6 and IL-1α. Treatment with N-acetyl-L-cysteine, a reactive oxygen species scavenger, suppressed oxidative DNA damage and the increased expression of IL-6 and IL-1α induced by ALL or CSE. The ALL- or CSE-induced increase in IL-6 expression, but not that of IL-1α, was suppressed by mtDNA depletion. In conclusion, ACR, MVK, and CPO may strongly contribute to CSE-induced inflammation. More importantly, cytosolic free mtDNA is thought to play an important role in IL-6 expression, a central mediator of inflammation.

Keywords:  Cytosolic DNA; Endothelial cells; Inflammation; Mitochondrial DNA; Smoking

DOI:  https://doi.org/10.1038/s41440-023-01463-z
Nucleic Acids Res. 2023 Oct 18. pii: gkad849. [Epub ahead of print]

Two mitochondrial HMG-box proteins, Cim1 and Abf2, antagonistically regulate mtDNA copy number in Saccharomyces cerevisiae.

Simon Schrott, Christof Osman.

The mitochondrial genome, mtDNA, is present in multiple copies in cells and encodes essential subunits of oxidative phosphorylation complexes. mtDNA levels have to change in response to metabolic demands and copy number alterations are implicated in various diseases. The mitochondrial HMG-box proteins Abf2 in yeast and TFAM in mammals are critical for mtDNA maintenance and packaging and have been linked to mtDNA copy number control. Here, we discover the previously unrecognized mitochondrial HMG-box protein Cim1 (copy number influence on mtDNA) in Saccharomyces cerevisiae, which exhibits metabolic state dependent mtDNA association. Surprisingly, in contrast to Abf2's supportive role in mtDNA maintenance, Cim1 negatively regulates mtDNA copy number. Cells lacking Cim1 display increased mtDNA levels and enhanced mitochondrial function, while Cim1 overexpression results in mtDNA loss. Intriguingly, Cim1 deletion alleviates mtDNA maintenance defects associated with loss of Abf2, while defects caused by Cim1 overexpression are mitigated by simultaneous overexpression of Abf2. Moreover, we find that the conserved LON protease Pim1 is essential to maintain low Cim1 levels, thereby preventing its accumulation and concomitant repressive effects on mtDNA. We propose a model in which the protein ratio of antagonistically acting Cim1 and Abf2 determines mtDNA copy number.

DOI: https://doi.org/10.1093/nar/gkad849
Neurochem Res. 2023 Oct 17.

ATF5-regulated Mitochondrial Unfolded Protein Response Attenuates Neuronal Damage in Epileptic Rat by Reducing Endoplasmic Reticulum Stress Through Mitochondrial ROS.

Xiaolei Lian, Xiaoyi Wang, Yinyin Xie, Hanqing Sheng, Jiao He, Tingting Peng, Nanchang Xie, Cui Wang, Yajun Lian.

  Endoplasmic reticulum (ER) dysfunction caused by excessive ER stress is a crucial mechanism underlying seizures-induced neuronal injury. Studies have shown that mitochondrial reactive oxygen species (ROS) are closely related to ER stress, and our previous study showed that activating transcription factor 5 (ATF5)-regulated mitochondrial unfolded protein response (mtUPR) modulated mitochondrial ROS generation in a hippocampal neuronal culture model of seizures. However, the effects of ATF5-regulated mtUPR on ER stress and the underlying mechanisms remain uncertain in epilepsy. In this study, ATF5 upregulation by lentivirus infection attenuated seizures-induced neuronal damage and apoptosis in a rat model of pilocarpine-induced epilepsy, whereas ATF5 downregulation by lentivirus infection had the opposite effects. ATF5 upregulation potentiated mtUPR by increasing the expression of mitochondrial chaperone heat shock protein 60 (HSP60) and caseinolytic protease proteolytic subunit (ClpP) and reducing mitochondrial ROS generation in pilocarpine-induced seizures in rats. Additionally, upregulation of ATF5 reduced the expression of glucose-regulated protein 78 (GRP78), protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP), suggesting suppression of ER stress; Moreover, ATF5 upregulation attenuated apoptosis-related proteins such as B-cell lymphoma-2 (BCL2) downregulation, BCL2-associated X (BAX) and cleaved-caspase-3 upregulation. However, ATF5 downregulation exerted the opposite effects. Furthermore, pretreatment with the mitochondria-targeted antioxidant mito-TEMPO attenuated the harmful effects of ATF5 downregulation on ER stress and neuronal apoptosis by reducing mitochondrial ROS generation. Overall, our study suggested that ATF5-regulated mtUPR exerted neuroprotective effects against pilocarpine-induced seizures in rats and the underlying mechanisms might involve mitochondrial ROS-mediated ER stress.

Keywords:  Activating transcription factor 5; Endoplasmic reticulum stress; Mitochondrial reactive oxygen species; Mitochondrial unfolded protein response; Seizures

DOI:  https://doi.org/10.1007/s11064-023-04042-3
Am J Physiol Cell Physiol. 2023 Oct 16.

ATP functions as a primary alarmin in allergen-induced type 2 immunity.

Scott M O'Grady, Hirohito Kita.

  Environmental allergens that interact with the airway epithelium can activate cellular stress pathways that lead to the release of danger signals known as alarmins. The mechanisms of alarmin release are distinct from Damage-Associated Molecular Patterns (DAMPs), which typically escape from cells following loss of plasma membrane integrity. Oxidative stress represents a form of allergen-induced cellular stress that stimulates oxidant-sensing mechanisms coupled to pathways, which facilitate alarmin mobilization and efflux across the plasma membrane. In this review we highlight examples of alarmin release and discuss their role in the initiation of type 2 immunity and allergic airway inflammation. In addition, we discuss the concept of alarmin amplification where "primary" alarmins, which are directly released in response to a specific cellular stress, stimulate additional signaling pathways that lead to secretion of "secondary" alarmins that include pro-inflammatory cytokines, such as IL-33 as well as genomic and mitochondrial DNA that coordinate or amplify type 2 immunity. Accordingly, allergen-evoked cellular stress can elicit a hierarchy of alarmin signaling responses from the airway epithelium that trigger local innate immune reactions, impact adaptive immunity and exacerbate diseases including asthma and other chronic inflammatory conditions that affect airway function.

Keywords:  DNA; IL-33; asthma; inflammation; oxidative stress

DOI:  https://doi.org/10.1152/ajpcell.00370.2023
Nat Chem Biol. 2023 Oct 19.

Long-term super-resolution inner mitochondrial membrane imaging with a lipid probe.

Shuai Zheng, Neville Dadina, Deepto Mozumdar, Lauren Lesiak, Kayli N Martinez, Evan W Miller, Alanna Schepartz.

The inner mitochondrial membrane (IMM) generates power to drive cell function, and its dynamics control mitochondrial health and cellular homeostasis. Here, we describe the cell-permeant, lipid-like small molecule MAO-N3 and use it to assemble high-density environmentally sensitive (HIDE) probes that selectively label and image the IMM in live cells and multiple cell states. MAO-N3 pairs with strain-promoted azide-alkyne click chemistry-reactive fluorophores to support HIDE imaging using confocal, structured illumination, single-molecule localization and stimulated emission depletion microscopy, all with significantly improved resistance to photobleaching. These probes generate images with excellent spatial and temporal resolution, require no genetic manipulations, are non-toxic in model cell lines and primary cardiomyocytes (even under conditions that amplify the effects of mitochondrial toxins) and can visualize mitochondrial dynamics for 12.5 h. This probe will enable comprehensive studies of IMM dynamics with high temporal and spatial resolution.

DOI: https://doi.org/10.1038/s41589-023-01450-y
iScience. 2023 Oct 20. 26(10): 107921

An early glycolysis burst in microglia regulates mitochondrial dysfunction in oligodendrocytes under neuroinflammation.

Hamid Suhail, Mohammad Nematullah, Faraz Rashid, Mir Sajad, Mena Fatma, Jaspreet Singh, Insha Zahoor, Wing Lee Cheung, Nivedita Tiwari, Kameshwar Ayasolla, Ashok Kumar, Nasrul Hoda, Ramandeep Rattan, Shailendra Giri.

  Metabolism and energy processes governing oligodendrocyte function during neuroinflammatory disease are of great interest. However, how varied cellular environments affect oligodendrocyte activity during neuroinflammation is unknown. We demonstrate that activated microglial energy metabolism controls oligodendrocyte mitochondrial respiration and activity. Lipopolysaccharide/interferon gamma promote glycolysis and decrease mitochondrial respiration and myelin protein synthesis in rat brain glial cells. Enriched microglia showed an early burst in glycolysis. In microglia-conditioned medium, oligodendrocytes did not respire and expressed less myelin. SCENITH revealed metabolic derangement in microglia and O4-positive oligodendrocytes in endotoxemia and experimental autoimmune encephalitogenic models. The early burst of glycolysis in microglia was mediated by PDPK1 and protein kinase B/AKT signaling. We found that microglia-produced NO and itaconate, a tricarboxylic acid bifurcated metabolite, reduced mitochondrial respiration in oligodendrocytes. During inflammation, we discovered a signaling pathway in microglia that could be used as a therapeutic target to restore mitochondrial function in oligodendrocytes and induce remyelination.

Keywords:  Cellular neuroscience; Immune response; Neuroscience

DOI:  https://doi.org/10.1016/j.isci.2023.107921
DNA Repair (Amst). 2023 Oct 11. pii: S1568-7864(23)00136-2. [Epub ahead of print]132 103582

UvrD-like helicase Hmi1 Has an ATP independent role in yeast mitochondrial DNA maintenance.

Sirelin Sillamaa, Vlad-Julian Piljukov, Iris Vaask, Tiina Sedman, Priit Jõers, Juhan Sedman.

  Hmi1 is a UvrD-like DNA helicase required for the maintenance of the yeast Saccharomyces cerevisiae mitochondrial DNA (mtDNA). Deletion of the HMI1 ORF leads to the formation of respiration-deficient petite mutants, which either contain a short fragment of mtDNA arranged in tandem repeats or lack mtDNA completely. Here we characterize point mutants of the helicase designed to target the ATPase or ssDNA binding activity and show that these mutations do not separately lead to complete loss of the Hmi1 function. The mutant strains support ATP production via oxidative phosphorylation and enable us to directly analyze the impact of both activities on the stability of wild-type mtDNA in this petite-positive yeast. Our data reveal that Hmi1 mutants affecting ssDNA binding display a stronger defect in the maintenance of mtDNA compared to the mutants of ATP binding/hydrolysis. Hmi1 mutants impaired in ssDNA binding demonstrate sensitivity to UV irradiation and lower levels of Cox2 encoded by the mitochondrial genome. This suggests a complex and multifarious role for Hmi1 in mtDNA maintenance-linked transactions, some of which do not require the ATP-dependent helicase activity.

Keywords:  DNA helicase; Mitochondrial DNA repair; Saccharomyces cerevisiae; Yeast mitochondrial DNA

DOI:  https://doi.org/10.1016/j.dnarep.2023.103582
EMBO Rep. 2023 Oct 17. e56865

A1 is induced by pathogen ligands to limit myeloid cell death and NLRP3 inflammasome activation.

Mary Speir, Hazel Tye, Timothy A Gottschalk, Daniel S Simpson, Tirta M Djajawi, Pankaj Deo, Rebecca L Ambrose, Stephanie A Conos, Jack Emery, Gilu Abraham, Ashlyn Pascoe, Sebastian A Hughes, Ashley Weir, Edwin D Hawkins, Isabella Kong, Marco J Herold, Jaclyn S Pearson, Najoua Lalaoui, Thomas Naderer, James E Vince, Kate E Lawlor.

  Programmed cell death pathways play an important role in innate immune responses to infection. Activation of intrinsic apoptosis promotes infected cell clearance; however, comparatively little is known about how this mode of cell death is regulated during infections and whether it can induce inflammation. Here, we identify that the pro-survival BCL-2 family member, A1, controls activation of the essential intrinsic apoptotic effectors BAX/BAK in macrophages and monocytes following bacterial lipopolysaccharide (LPS) sensing. We show that, due to its tight transcriptional and post-translational regulation, A1 acts as a molecular rheostat to regulate BAX/BAK-dependent apoptosis and the subsequent NLRP3 inflammasome-dependent and inflammasome-independent maturation of the inflammatory cytokine IL-1β. Furthermore, induction of A1 expression in inflammatory monocytes limits cell death modalities and IL-1β activation triggered by Neisseria gonorrhoeae-derived outer membrane vesicles (NOMVs). Consequently, A1-deficient mice exhibit heightened IL-1β production in response to NOMV injection. These findings reveal that bacteria can induce A1 expression to delay myeloid cell death and inflammatory responses, which has implications for the development of host-directed antimicrobial therapeutics.

Keywords:  BCL-2A1; NLRP3; TLR ligation; mitochondrial apoptosis; myeloid cells

DOI:  https://doi.org/10.15252/embr.202356865