bims-nenemi Biomed News
on Neuroinflammation, neurodegeneration and mitochondria
Issue of 2023–12–03
nine papers selected by
Marco Tigano, Thomas Jefferson University
  1. Mitochondrial E3 ligase MARCH5 is a safeguard against DNA-PKcs-mediated immune signaling in mitochondria-damaged cells.
  2. Efficient elimination of MELAS-associated m.3243G mutant mitochondrial DNA by an engineered mitoARCUS nuclease.
  3. The poxvirus F17 protein counteracts mitochondrially orchestrated antiviral responses.
  4. Cytoplasmic Escape of Mitochondrial DNA Mediated by Mfn2 Downregulation Promotes Microglial Activation via cGas-Sting Axis in Spinal Cord Injury.
  5. Single-cell transcriptomic and neuropathologic analysis reveals dysregulation of the integrated stress response in progressive supranuclear palsy.
  6. Mitochondria possess a large, non-selective ionic current that is enhanced during cardiac injury.
  7. Data-driven discovery of innate immunomodulators via machine learning-guided high throughput screening.
  8. Pyroptosis inhibiting nanobodies block Gasdermin D pore formation.
  9. Dihydroartemisinin ameliorates innate inflammatory response induced by Streptococcussuis-derived muramidase-released protein via inactivation of TLR4-dependent NF-κB signaling.
  1. Cell Death Dis. 2023 Dec 01. 14(12): 788
    Mitochondrial E3 ligase MARCH5 is a safeguard against DNA-PKcs-mediated immune signaling in mitochondria-damaged cells.
    June Heo, Yeon-Ji Park, Yonghyeon Kim, Ho-Soo Lee, Jeongah Kim, Soon-Hwan Kwon, Myeong-Gyun Kang, Hyun-Woo Rhee, Woong Sun, Jae-Ho Lee, Hyeseong Cho.
      Mitochondrial dysfunction is important in various chronic degenerative disorders, and aberrant immune responses elicited by cytoplasmic mitochondrial DNA (mtDNA) may be related. Here, we developed mtDNA-targeted MTERF1-FokI and TFAM-FokI endonuclease systems to induce mitochondrial DNA double-strand breaks (mtDSBs). In these cells, the mtDNA copy number was significantly reduced upon mtDSB induction. Interestingly, in cGAS knockout cells, synthesis of interferon β1 and interferon-stimulated gene was increased upon mtDSB induction. We found that mtDSBs activated DNA-PKcs and HSPA8 in a VDAC1-dependent manner. Importantly, the mitochondrial E3 ligase MARCH5 bound active DNA-PKcs in cells with mtDSBs and reduced the type І interferon response through the degradation of DNA-PKcs. Likewise, mitochondrial damage caused by LPS treatment in RAW264.7 macrophage cells increased phospho-HSPA8 levels and the synthesis of mIFNB1 mRNA in a DNA-PKcs-dependent manner. Accordingly, in March5 knockout macrophages, phospho-HSPA8 levels and the synthesis of mIFNB1 mRNA were prolonged after LPS stimulation. Together, cytoplasmic mtDNA elicits a cellular immune response through DNA-PKcs, and mitochondrial MARCH5 may be a safeguard to prevent persistent inflammatory reactions.
    DOI:  https://doi.org/10.1038/s41419-023-06315-9
  2. Nat Metab. 2023 Nov 30.
    Efficient elimination of MELAS-associated m.3243G mutant mitochondrial DNA by an engineered mitoARCUS nuclease.
    Wendy K Shoop, Janel Lape, Megan Trum, Alea Powell, Emma Sevigny, Adam Mischler, Sandra R Bacman, Flavia Fontanesi, Jeff Smith, Derek Jantz, Cassandra L Gorsuch, Carlos T Moraes.
      Nuclease-mediated editing of heteroplasmic mitochondrial DNA (mtDNA) seeks to preferentially cleave and eliminate mutant mtDNA, leaving wild-type genomes to repopulate the cell and shift mtDNA heteroplasmy. Various technologies are available, but many suffer from limitations based on size and/or specificity. The use of ARCUS nucleases, derived from naturally occurring I-CreI, avoids these pitfalls due to their small size, single-component protein structure and high specificity resulting from a robust protein-engineering process. Here we describe the development of a mitochondrial-targeted ARCUS (mitoARCUS) nuclease designed to target one of the most common pathogenic mtDNA mutations, m.3243A>G. mitoARCUS robustly eliminated mutant mtDNA without cutting wild-type mtDNA, allowing for shifts in heteroplasmy and concomitant improvements in mitochondrial protein steady-state levels and respiration. In vivo efficacy was demonstrated using a m.3243A>G xenograft mouse model with mitoARCUS delivered systemically by adeno-associated virus. Together, these data support the development of mitoARCUS as an in vivo gene-editing therapeutic for m.3243A>G-associated diseases.
    DOI:  https://doi.org/10.1038/s42255-023-00932-6
  3. Nat Commun. 2023 Nov 30. 14(1): 7889
    The poxvirus F17 protein counteracts mitochondrially orchestrated antiviral responses.
    Nathan Meade, Helen K Toreev, Ram P Chakrabarty, Charles R Hesser, Chorong Park, Navdeep S Chandel, Derek Walsh.
      Poxviruses are unusual DNA viruses that replicate in the cytoplasm. To do so, they encode approximately 100 immunomodulatory proteins that counteract cytosolic nucleic acid sensors such as cGAMP synthase (cGAS) along with several other antiviral response pathways. Yet most of these immunomodulators are expressed very early in infection while many are variable host range determinants, and significant gaps remain in our understanding of poxvirus sensing and evasion strategies. Here, we show that after infection is established, subsequent progression of the viral lifecycle is sensed through specific changes to mitochondria that coordinate distinct aspects of the antiviral response. Unlike other viruses that cause extensive mitochondrial damage, poxviruses sustain key mitochondrial functions including membrane potential and respiration while reducing reactive oxygen species that drive inflammation. However, poxvirus replication induces mitochondrial hyperfusion that independently controls the release of mitochondrial DNA (mtDNA) to prime nucleic acid sensors and enables an increase in glycolysis that is necessary to support interferon stimulated gene (ISG) production. To counter this, the poxvirus F17 protein localizes to mitochondria and dysregulates mTOR to simultaneously destabilize cGAS and block increases in glycolysis. Our findings reveal how the poxvirus F17 protein disarms specific mitochondrially orchestrated responses to later stages of poxvirus replication.
    DOI:  https://doi.org/10.1038/s41467-023-43635-y
  4. Adv Sci (Weinh). 2023 Nov 27. e2305442
    Cytoplasmic Escape of Mitochondrial DNA Mediated by Mfn2 Downregulation Promotes Microglial Activation via cGas-Sting Axis in Spinal Cord Injury.
    Fei-Long Wei, Tian-Fu Wang, Chao-Li Wang, Zhen-Peng Zhang, Jing-Wei Zhao, Wei Heng, Zhen Tang, Ming-Rui Du, Xiao-Dong Yan, Xiao-Xiang Li, Zheng Guo, Ji-Xian Qian, Cheng-Pei Zhou.
      Neuroinflammation is associated with poor outcomes in patients with spinal cord injury (SCI). Recent studies have demonstrated that stimulator of interferon genes (Sting) plays a key role in inflammatory diseases. However, the role of Sting in SCI remains unclear. In the present study, it is found that increased Sting expression is mainly derived from activated microglia after SCI. Interestingly, knockout of Sting in microglia can improve the recovery of neurological function after SCI. Microglial Sting knockout restrains the polarization of microglia toward the M1 phenotype and alleviates neuronal death. Furthermore, it is found that the downregulation of mitofusin 2 (Mfn2) expression in microglial cells leads to an imbalance in mitochondrial fusion and division, inducing the release of mitochondrial DNA (mtDNA), which mediates the activation of the cGas-Sting signaling pathway and aggravates inflammatory response damage after SCI. A biomimetic microglial nanoparticle strategy to deliver MASM7 (named MSNs-MASM7@MI) is established. In vitro, MSNs-MASM7@MI showed no biological toxicity and effectively delivered MASM7. In vivo, MSNs-MASM7@MI improves nerve function after SCI. The study provides evidence that cGas-Sting signaling senses Mfn2-dependent mtDNA release and that its activation may play a key role in SCI. These findings provide new perspectives and potential therapeutic targets for SCI treatment.
    Keywords:  biomimetic nanoparticles; mitofusin 2; mtDNA; neuroinflammation; spinal cord injury; stimulator of interferon genes (Sting)
    DOI:  https://doi.org/10.1002/advs.202305442
  5. bioRxiv. 2023 Nov 17. pii: 2023.11.17.567587. [Epub ahead of print]
    Single-cell transcriptomic and neuropathologic analysis reveals dysregulation of the integrated stress response in progressive supranuclear palsy.
    Kristen Whitney, Won-Min Song, Abhijeet Sharma, Diana K Dangoor, Kurt Farrell, Margaret M Krassner, Hadley W Ressler, Thomas D Christie, Ruth H Walker, Melissa J Nirenberg, Bin Zhang, Steven J Frucht, Giulietta M Riboldi, John F Crary, Ana C Pereira.
      Progressive supranuclear palsy (PSP) is a sporadic neurodegenerative tauopathy variably affecting brainstem and cortical structures and characterized by tau inclusions in neurons and glia. The precise mechanism whereby these protein aggregates lead to cell death remains unclear. To investigate the contribution of these different cellular abnormalities to PSP pathogenesis, we performed single-nucleus RNA sequencing and analyzed 45,559 high quality nuclei targeting the subthalamic nucleus and adjacent structures from human post-mortem PSP brains with varying degrees of pathology compared to controls. Cell-type specific differential expression and pathway analysis identified both common and discrete changes in numerous pathways previously implicated in PSP and other neurodegenerative disorders. This included EIF2 signaling, an adaptive pathway activated in response to diverse stressors, which was the top activated pathway in vulnerable cell types. Using immunohistochemistry, we found that activated eIF2α was positively correlated with tau pathology burden in vulnerable brain regions. Multiplex immunofluorescence localized activated eIF2α positivity to hyperphosphorylated tau (p-tau) positive neurons and ALDH1L1-positive astrocytes, supporting the increased transcriptomic EIF2 activation observed in these vulnerable cell types. In conclusion, these data provide insights into cell-type-specific pathological changes in PSP and support the hypothesis that failure of adaptive stress pathways play a mechanistic role in the pathogenesis and progression of PSP.
    DOI:  https://doi.org/10.1101/2023.11.17.567587
  6. bioRxiv. 2023 Nov 16. pii: 2023.11.15.567241. [Epub ahead of print]
    Mitochondria possess a large, non-selective ionic current that is enhanced during cardiac injury.
    Enrique Balderas, Sandra H J Lee, Thirupura S Shankar, Xue Yin, Anthony M Balynas, Christos P Kyriakopoulos, Craig H Selzman, Stavros G Drakos, Dipayan Chaudhuri.
      Mitochondrial ion channels are essential for energy production and cell survival. To avoid depleting the electrochemical gradient used for ATP synthesis, channels so far described in the mitochondrial inner membrane open only briefly, are highly ion-selective, have restricted tissue distributions, or have small currents. Here, we identify a mitochondrial inner membrane conductance that has strikingly different behavior from previously described channels. It is expressed ubiquitously, and transports cations non-selectively, producing a large, up to nanoampere-level, current. The channel does not lead to inner membrane uncoupling during normal physiology because it only becomes active at depolarized voltages. It is inhibited by external Ca 2+ , corresponding to the intermembrane space, as well as amiloride. This large, ubiquitous, non-selective, amiloride-sensitive (LUNA) current appears most active when expression of the mitochondrial calcium uniporter is minimal, such as in the heart. In this organ, we find that LUNA current magnitude increases two- to threefold in multiple mouse models of injury, an effect also seen in cardiac mitochondria from human patients with heart failure with reduced ejection fraction. Taken together, these features lead us to speculate that LUNA current may arise from an essential protein that acts as a transporter under physiological conditions, but becomes a channel under conditions of mitochondrial stress and depolarization.
    DOI:  https://doi.org/10.1101/2023.11.15.567241
  7. Chem Sci. 2023 Nov 15. 14(44): 12747-12766
    Data-driven discovery of innate immunomodulators via machine learning-guided high throughput screening.
    Yifeng Tang, Jeremiah Y Kim, Carman K M Ip, Azadeh Bahmani, Qing Chen, Matthew G Rosenberger, Aaron P Esser-Kahn, Andrew L Ferguson.
      The innate immune response is vital for the success of prophylactic vaccines and immunotherapies. Control of signaling in innate immune pathways can improve prophylactic vaccines by inhibiting unfavorable systemic inflammation and immunotherapies by enhancing immune stimulation. In this work, we developed a machine learning-enabled active learning pipeline to guide in vitro experimental screening and discovery of small molecule immunomodulators that improve immune responses by altering the signaling activity of innate immune responses stimulated by traditional pattern recognition receptor agonists. Molecules were tested by in vitro high throughput screening (HTS) where we measured modulation of the nuclear factor κ-light-chain-enhancer of activated B-cells (NF-κB) and the interferon regulatory factors (IRF) pathways. These data were used to train data-driven predictive models linking molecular structure to modulation of the NF-κB and IRF responses using deep representational learning, Gaussian process regression, and Bayesian optimization. By interleaving successive rounds of model training and in vitro HTS, we performed an active learning-guided traversal of a 139 998 molecule library. After sampling only ∼2% of the library, we discovered viable molecules with unprecedented immunomodulatory capacity, including those capable of suppressing NF-κB activity by up to 15-fold, elevating NF-κB activity by up to 5-fold, and elevating IRF activity by up to 6-fold. We extracted chemical design rules identifying particular chemical fragments as principal drivers of specific immunomodulation behaviors. We validated the immunomodulatory effect of a subset of our top candidates by measuring cytokine release profiles. Of these, one molecule induced a 3-fold enhancement in IFN-β production when delivered with a cyclic di-nucleotide stimulator of interferon genes (STING) agonist. In sum, our machine learning-enabled screening approach presents an efficient immunomodulator discovery pipeline that has furnished a library of novel small molecules with a strong capacity to enhance or suppress innate immune signaling pathways to shape and improve prophylactic vaccination and immunotherapies.
    DOI:  https://doi.org/10.1039/d3sc03613h
  8. Nat Commun. 2023 Dec 01. 14(1): 7923
    Pyroptosis inhibiting nanobodies block Gasdermin D pore formation.
    Anja Kopp, Gregor Hagelueken, Isabell Jamitzky, Jonas Moecking, Lisa D J Schiffelers, Florian I Schmidt, Matthias Geyer.
      Human Gasdermin D (GSDMD) is a key mediator of pyroptosis, a pro-inflammatory form of cell death occurring downstream of inflammasome activation as part of the innate immune defence. Upon cleavage by inflammatory caspases in the cytosol, the N-terminal domain of GSDMD forms pores in the plasma membrane resulting in cytokine release and eventually cell death. Targeting GSDMD is an attractive way to dampen inflammation. In this study, six GSDMD targeting nanobodies are characterized in terms of their binding affinity, stability, and effect on GSDMD pore formation. Three of the nanobodies inhibit GSDMD pore formation in a liposome leakage assay, although caspase cleavage was not perturbed. We determine the crystal structure of human GSDMD in complex with two nanobodies at 1.9 Å resolution, providing detailed insights into the GSDMD-nanobody interactions and epitope binding. The pore formation is sterically blocked by one of the nanobodies that binds to the oligomerization interface of the N-terminal domain in the multi-subunit pore assembly. Our biochemical and structural findings provide tools for studying inflammasome biology and build a framework for the design of GSDMD targeting drugs.
    DOI:  https://doi.org/10.1038/s41467-023-43707-z
  9. J Pharm Anal. 2023 Oct;13(10): 1183-1194
    Dihydroartemisinin ameliorates innate inflammatory response induced by Streptococcussuis-derived muramidase-released protein via inactivation of TLR4-dependent NF-κB signaling.
    Yun Ji, Kaiji Sun, Ying Yang, Zhenlong Wu.
      Muramidase-released protein (MRP) is now being recognized as a critical indicator of the virulence and pathogenicity of Streptococcus suis (S. suis). However, the identification of viable therapeutics for S. suis infection was hindered by the absence of an explicit mechanism for MRP-actuated inflammation. Dihydroartemisinin (DhA) is an artemisinin derivative with potential anti-inflammatory activity. The modulatory effect of DhA on the inflammatory response mediated by the virulence factor MRP remains obscure. This research aimed to identify the signaling mechanism by which MRP triggers the innate immune response in mouse spleen and cultured macrophages. With the candidate mechanism in mind, we investigated DhA for its ability to dampen the pro-inflammatory response induced by MRP. The innate immune response in mice was drastically triggered by MRP, manifesting as splenic and systemic inflammation with splenomegaly, immune cell infiltration, and an elevation in pro-inflammatory cytokines. A crucial role for Toll-like receptor 4 (TLR4) in coordinating the MRP-mediated inflammatory response via nuclear factor-kappa B (NF-κB) activation was revealed by TLR4 blockade. In addition, NF-κB-dependent transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinases (MAPKs) activation was required for the inflammatory signal transduction engendered by MRP. Intriguingly, we observed an alleviation effect of DhA on the MRP-induced immune response, which referred to the suppression of TLR4-mediated actuation of NF-κB-STAT3/MAPK cascades. The inflammatory response elicited by MRP is relevant to TLR4-dependent NF-κB activation, followed by an increase in the activity of STAT3 or MAPKs. DhA mitigates the inflammation process induced by MRP via blocking the TLR4 cascade, highlighting the therapeutic potential of DhA in targeting S. suis infection diseases.
    Keywords:  Dihydroartemisinin; Inflammation; Muramidase-released protein; Streptococcus suis; Toll-like receptor 4
    DOI:  https://doi.org/10.1016/j.jpha.2023.05.013
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