bims-nenemi 2024-02-18 papers

bims-nenemi

Biomed News

on Neuroinflammation, neurodegeneration and mitochondria

Issue of 2024–02–18
ten papers selected by
Marco Tigano, Thomas Jefferson University

The HRI branch of the integrated stress response selectively triggers mitophagy.
Single-nucleoid architecture reveals heterogeneous packaging of mitochondrial DNA.
Mapping Stress-Responsive Signaling Pathways Induced by Mitochondrial Proteostasis Perturbations.
Mitochondrial haplotype and mito-nuclear matching drive somatic mutation and selection throughout ageing.
High-throughput single-cell, single-mitochondrial DNA assay using hydrogel droplet microfluidics.
Mitochondrial S-adenosylmethionine deficiency induces mitochondrial unfolded protein response and extends lifespan in Caenorhabditis elegans.
Oxidative stress induces release of mitochondrial DNA into the extracellular space in human placental villous trophoblast BeWo cells.
A system for inducible mitochondria-specific protein degradation in vivo.
Proline restores mitochondrial function and reverses aging hallmarks in senescent cells.
STING-dependent signaling in microglia or peripheral immune cells orchestrates the early inflammatory response and influences brain injury outcome.

Mol Cell. 2024 Feb 06. pii: S1097-2765(24)00052-2. [Epub ahead of print]

The HRI branch of the integrated stress response selectively triggers mitophagy.

Yogaditya Chakrabarty, Zheng Yang, Hsiuchen Chen, David C Chan.

  To maintain mitochondrial homeostasis, damaged or excessive mitochondria are culled in coordination with the physiological state of the cell. The integrated stress response (ISR) is a signaling network that recognizes diverse cellular stresses, including mitochondrial dysfunction. Because the four ISR branches converge to common outputs, it is unclear whether mitochondrial stress detected by this network can regulate mitophagy, the autophagic degradation of mitochondria. Using a whole-genome screen, we show that the heme-regulated inhibitor (HRI) branch of the ISR selectively induces mitophagy. Activation of the HRI branch results in mitochondrial localization of phosphorylated eukaryotic initiation factor 2, which we show is sufficient to induce mitophagy. The HRI mitophagy pathway operates in parallel with the mitophagy pathway controlled by the Parkinson's disease related genes PINK1 and PARKIN and is mechanistically distinct. Therefore, HRI repurposes machinery that is normally used for translational initiation to trigger mitophagy in response to mitochondrial damage.

Keywords:  autophagy; integrated stress response; iron metabolism; mitochondria; mitophagy; organelle quality control

DOI:  https://doi.org/10.1016/j.molcel.2024.01.016
Nat Struct Mol Biol. 2024 Feb 12.

Single-nucleoid architecture reveals heterogeneous packaging of mitochondrial DNA.

R Stefan Isaac, Thomas W Tullius, Katja G Hansen, Danilo Dubocanin, Mary Couvillion, Andrew B Stergachis, L Stirling Churchman.

Cellular metabolism relies on the regulation and maintenance of mitochondrial DNA (mtDNA). Hundreds to thousands of copies of mtDNA exist in each cell, yet because mitochondria lack histones or other machinery important for nuclear genome compaction, it remains unresolved how mtDNA is packaged into individual nucleoids. In this study, we used long-read single-molecule accessibility mapping to measure the compaction of individual full-length mtDNA molecules at near single-nucleotide resolution. We found that, unlike the nuclear genome, human mtDNA largely undergoes all-or-none global compaction, with most nucleoids existing in an inaccessible, inactive state. Highly accessible mitochondrial nucleoids are co-occupied by transcription and replication components and selectively form a triple-stranded displacement loop structure. In addition, we showed that the primary nucleoid-associated protein TFAM directly modulates the fraction of inaccessible nucleoids both in vivo and in vitro, acting consistently with a nucleation-and-spreading mechanism to coat and compact mitochondrial nucleoids. Together, these findings reveal the primary architecture of mtDNA packaging and regulation in human cells.

DOI: https://doi.org/10.1038/s41594-024-01225-6
bioRxiv. 2024 Feb 01. pii: 2024.01.30.577830. [Epub ahead of print]

Mapping Stress-Responsive Signaling Pathways Induced by Mitochondrial Proteostasis Perturbations.

Nicole Madrazo, Zinia Khattar, Evan T Powers, Jessica D Rosarda, R Luke Wiseman.

Imbalances in mitochondrial proteostasis are associated with pathologic mitochondrial dysfunction implicated in etiologically-diverse diseases. This has led to considerable interest in defining the biological mechanisms responsible for regulating mitochondria in response to mitochondrial stress. Numerous stress responsive signaling pathways have been suggested to regulate mitochondria in response to proteotoxic stress, including the integrated stress response (ISR), the heat shock response (HSR), and the oxidative stress response (OSR). Here, we define the specific stress signaling pathways activated in response to mitochondrial proteostasis stress by monitoring the expression of sets of genes regulated downstream of each of these signaling pathways in published Perturb-seq datasets from K562 cells CRISPRi-depleted of individual mitochondrial proteostasis factors. Interestingly, we find that the ISR is preferentially activated in response to mitochondrial proteostasis stress, with no other pathway showing significant activation. Further expanding this study, we show that broad depletion of mitochondria-localized proteins similarly shows preferential activation of the ISR relative to other stress-responsive signaling pathways. These results both establish our gene set profiling approach as a viable strategy to probe stress responsive signaling pathways induced by perturbations to specific organelles and identify the ISR as the predominant stress-responsive signaling pathway activated in response to mitochondrial proteostasis disruption.

DOI: https://doi.org/10.1101/2024.01.30.577830
Nat Ecol Evol. 2024 Feb 15.

Mitochondrial haplotype and mito-nuclear matching drive somatic mutation and selection throughout ageing.

Isabel M Serrano, Misa Hirose, Charles C Valentine, Sharon Roesner, Elizabeth Schmidt, Gabriel Pratt, Lindsey Williams, Jesse Salk, Saleh Ibrahim, Peter H Sudmant.

Mitochondrial genomes co-evolve with the nuclear genome over evolutionary timescales and are shaped by selection in the female germline. Here we investigate how mismatching between nuclear and mitochondrial ancestry impacts the somatic evolution of the mitochondrial genome in different tissues throughout ageing. We used ultrasensitive duplex sequencing to profile ~2.5 million mitochondrial genomes across five mitochondrial haplotypes and three tissues in young and aged mice, cataloguing ~1.2 million mitochondrial somatic and ultralow-frequency inherited mutations, of which 81,097 are unique. We identify haplotype-specific mutational patterns and several mutational hotspots, including at the light strand origin of replication, which consistently exhibits the highest mutation frequency. We show that rodents exhibit a distinct mitochondrial somatic mutational spectrum compared with primates with a surfeit of reactive oxygen species-associated G > T/C > A mutations, and that somatic mutations in protein-coding genes exhibit signatures of negative selection. Lastly, we identify an extensive enrichment in somatic reversion mutations that 're-align' mito-nuclear ancestry within an organism's lifespan. Together, our findings demonstrate that mitochondrial genomes are a dynamically evolving subcellular population shaped by somatic mutation and selection throughout organismal lifetimes.

DOI: https://doi.org/10.1038/s41559-024-02338-3
bioRxiv. 2024 Jan 31. pii: 2024.01.29.577854. [Epub ahead of print]

High-throughput single-cell, single-mitochondrial DNA assay using hydrogel droplet microfluidics.

Juhwan Park, Parnika S Kadam, Yasemin Atiyas, Bonirath Chhay, Andrew Tsourkas, James H Eberwine, David A Issadore.

There is growing interest in understanding the biological implications of single cell heterogeneity and intracellular heteroplasmy of mtDNA, but current methodologies for single-cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single-cell genomic, RNA, and protein analysis, their application to sub-cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose-based droplet microfluidic approach for single-cell, single-mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells. Our microfluidic chip encapsulates individual cells in agarose beads, designed to have a sufficiently dense hydrogel network to retain mtDNA after lysis and provide a robust scaffold for subsequent multi-step processing and analysis. To mitigate the impact of the high viscosity of agarose required for mtDNA retention on the throughput of microfluidics, we developed a parallelized device, successfully achieving ~95% mtDNA retention from single cells within our microbeads at >700,000 drops/minute. To demonstrate utility, we analyzed specific regions of the single mtDNA using a multiplexed rolling circle amplification (RCA) assay. We demonstrated compatibility with both microscopy, for digital counting of individual RCA products, and flow cytometry for higher throughput analysis.

DOI: https://doi.org/10.1101/2024.01.29.577854
Aging Cell. 2024 Feb 15. e14103

Mitochondrial S-adenosylmethionine deficiency induces mitochondrial unfolded protein response and extends lifespan in Caenorhabditis elegans.

Tse Yu Chen, Feng-Yung Wang, Pin-Jung Lee, Ao-Lin Hsu, Tsui-Ting Ching.

  S-adenosylmethionine (SAM), generated from methionine and ATP by S-adenosyl methionine synthetase (SAMS), is the universal methyl group donor required for numerous cellular methylation reactions. In Caenorhabditis elegans, silencing sams-1, the major isoform of SAMS, genetically or via dietary restriction induces a robust mitochondrial unfolded protein response (UPRmt ) and lifespan extension. In this study, we found that depleting SAMS-1 markedly decreases mitochondrial SAM levels. Moreover, RNAi knockdown of SLC-25A26, a carrier protein responsible for transporting SAM from the cytoplasm into the mitochondria, significantly lowers the mitochondrial SAM levels and activates UPRmt , suggesting that the UPRmt induced by sams-1 mutations might result from disrupted mitochondrial SAM homeostasis. Through a genetic screen, we then identified a putative mitochondrial tRNA methyltransferase TRMT-10C.2 as a major downstream effector of SAMS-1 to regulate UPRmt and longevity. As disruption of mitochondrial tRNA methylation likely leads to impaired mitochondrial tRNA maturation and consequently reduced mitochondrial translation, our findings suggest that depleting mitochondrial SAM level might trigger UPRmt via attenuating protein translation in the mitochondria. Together, this study has revealed a potential mechanism by which SAMS-1 regulates UPRmt and longevity.

Keywords:  S-adenosyl methionine; UPRmt; longevity; mitochondrial tRNA methyltransferase

DOI:  https://doi.org/10.1111/acel.14103
bioRxiv. 2024 Feb 04. pii: 2024.02.02.578433. [Epub ahead of print]

Oxidative stress induces release of mitochondrial DNA into the extracellular space in human placental villous trophoblast BeWo cells.

Jennifer J Gardner, Spencer C Cushen, Reneé de Nazaré Oliveira da Silva, Jessica L Bradshaw, Nataliia Hula, Isabelle K Gorham, Selina M Tucker, Zhengyang Zhou, Rebecca L Cunningham, Nicole R Phillips, Styliani Goulopoulou.

Circulating cell-free mitochondrial DNA (ccf-mtDNA) is an indicator of cell death, inflammation, and oxidative stress. ccf-mtDNA differs in pregnancies with placental dysfunction from healthy pregnancies and the direction of this difference depends on gestational age and method of mtDNA quantification. Reactive oxygen species (ROS) trigger release of mtDNA from non-placental cells; yet it is unknown whether trophoblast cells release mtDNA in response to oxidative stress, a common feature of pregnancies with placental pathology. We hypothesized that oxidative stress would induce cell death and release of mtDNA from trophoblast cells. BeWo cells were treated with antimycin A (10-320 μM) or rotenone (0.2-50 μM) to induce oxidative stress. A multiplex real-time quantitative PCR (qPCR) assay was used to quantify mtDNA and nuclear DNA in membrane bound, non-membrane bound, and vesicular-bound forms in cell culture supernatants and cell lysates. Treatment with antimycin A increased ROS (p<0.0001), induced cell necrosis (p=0.0004) but not apoptosis (p=0.6471) and was positively associated with release of membrane-bound and non-membrane bound mtDNA (p<0.0001). Antimycin A increased mtDNA content in exosome-like extracellular vesicles (vesicular-bound form; p=0.0019) and reduced autophagy marker expression (LC3A/B, p=0.0002; p62, p<0.001). Rotenone treatment did not influence mtDNA release or cell death (p>0.05). Oxidative stress induces release of mtDNA into the extracellular space and causes non-apoptotic cell death and a reduction in autophagy markers in BeWo cells, an established in vitro model of human trophoblast cells. Intersection between autophagy and necrosis may mediate the release of mtDNA from the placenta in pregnancies exposed to oxidative stress.
NEW & NOTEWORTHY: This is the first study to test whether trophoblast cells release mitochondrial DNA in response to oxidative stress and to identify mechanisms of release and biological forms of mtDNA from this cellular type. This research identifies potential cellular mechanisms that can be used in future investigations to establish the source and biomarker potential of circulating mitochondrial DNA in preclinical experimental models and humans.

DOI: https://doi.org/10.1101/2024.02.02.578433
Nat Commun. 2024 Feb 16. 15(1): 1454

A system for inducible mitochondria-specific protein degradation in vivo.

Swastika Sanyal, Anna Kouznetsova, Lena Ström, Camilla Björkegren.

Targeted protein degradation systems developed for eukaryotes employ cytoplasmic machineries to perform proteolysis. This has prevented mitochondria-specific analysis of proteins that localize to multiple locations, for example, the mitochondria and the nucleus. Here, we present an inducible mitochondria-specific protein degradation system in Saccharomyces cerevisiae based on the Mesoplasma florum Lon (mf-Lon) protease and its corresponding ssrA tag (called PDT). We show that mitochondrially targeted mf-Lon protease efficiently and selectively degrades a PDT-tagged reporter protein localized to the mitochondrial matrix. The degradation can be induced by depleting adenine from the medium, and tuned by altering the promoter strength of the MF-LON gene. We furthermore demonstrate that mf-Lon specifically degrades endogenous, PDT-tagged mitochondrial proteins. Finally, we show that mf-Lon-dependent PDT degradation can also be achieved in human mitochondria. In summary, this system provides an efficient tool to selectively analyze the mitochondrial function of dually localized proteins.

DOI: https://doi.org/10.1038/s41467-024-45819-6
Cell Rep. 2024 Feb 12. pii: S2211-1247(24)00066-4. [Epub ahead of print]43(2): 113738

Proline restores mitochondrial function and reverses aging hallmarks in senescent cells.

Debanik Choudhury, Na Rong, Hamsa Vardini Senthil Kumar, Sydney Swedick, Ronel Z Samuel, Pihu Mehrotra, John Toftegaard, Nika Rajabian, Ramkumar Thiyagarajan, Ashis K Podder, Yulun Wu, Shahryar Shahini, Kenneth L Seldeen, Bruce Troen, Pedro Lei, Stelios T Andreadis.

  Mitochondrial dysfunction is a hallmark of cellular senescence, with the loss of mitochondrial function identified as a potential causal factor contributing to senescence-associated decline in cellular functions. Our recent findings revealed that ectopic expression of the pluripotency transcription factor NANOG rejuvenates dysfunctional mitochondria of senescent cells by rewiring metabolic pathways. In this study, we report that NANOG restores the expression of key enzymes, PYCR1 and PYCR2, in the proline biosynthesis pathway. Additionally, senescent mesenchymal stem cells manifest severe mitochondrial respiratory impairment, which is alleviated through proline supplementation. Proline induces mitophagy by activating AMP-activated protein kinase α and upregulating Parkin expression, enhancing mitochondrial clearance and ultimately restoring cell metabolism. Notably, proline treatment also mitigates several aging hallmarks, including DNA damage, senescence-associated β-galactosidase, inflammatory cytokine expressions, and impaired myogenic differentiation capacity. Overall, this study highlights the role of proline in mitophagy and its potential in reversing senescence-associated mitochondrial dysfunction and aging hallmarks.

Keywords:  AMPKα; CP: Cell biology; CP: Metabolism; Parkin; aging; amino acid; autophagy; mitochondria; mitophagy; proline; senescence

DOI:  https://doi.org/10.1016/j.celrep.2024.113738
J Neurosci. 2024 Feb 15. pii: e0191232024. [Epub ahead of print]

STING-dependent signaling in microglia or peripheral immune cells orchestrates the early inflammatory response and influences brain injury outcome.

Lauren E Fritsch, Colin Kelly, John Leonard, Caroline de Jager, Xiaoran Wei, Samantha Brindley, Elizabeth A Harris, Alexandra M Kaloss, Nicole DeFoor, Swagatika Paul, Hannah O'Malley, Jing Ju, Michelle L Olsen, Michelle H Theus, Alicia M Pickrell.

While originally identified as an antiviral pathway, recent work has implicated cyclic GMP-AMP-synthase-Stimulator of Interferon Genes (cGAS-STING) signaling as playing a critical role in the neuroinflammatory response to traumatic brain injury (TBI). STING activation results in a robust inflammatory response characterized by the production of inflammatory cytokines called interferons, as well as hundreds of interferon stimulated genes (ISGs). Global knockout (KO) mice inhibiting this pathway display neuroprotection with evidence that this pathway is active days after injury; yet, the early neuroinflammatory events stimulated by STING signaling remain understudied. Furthermore, the source of STING signaling during brain injury is unknown. Using a murine controlled cortical impact (CCI) model of TBI, we investigated the peripheral immune and microglial response to injury utilizing male chimeric and conditional STING KO animals, respectively. We demonstrate that peripheral and microglial STING signaling contribute to negative outcomes in cortical lesion volume, cell death, and functional outcomes post injury. A reduction in overall peripheral immune cell and neutrophil infiltration at the injury site is STING dependent in these models at 24 hours. Transcriptomic analysis at 2 hours, when STING is active, reveals that microglia drive an early, distinct transcriptional program to elicit proinflammatory genes including interleukin 1-beta (IL-1β), which is lost in conditional knockout mice. The upregulation of alternative innate immune pathways also occurs after injury in these animals, which supports a complex relationship between brain-resident and peripheral immune cells to coordinate the proinflammatory response and immune cell influx to damaged tissue after injury.Significance Statement The innate immune STING pathway triggers harmful neuroinflammation after traumatic brain injury with support from human and preclinical models indicating that this pathway is active hours to days after injury. Our findings in a preclinical cortical contusion model suggest that STING signaling specifically from peripheral immune cells or microglia drive this process to affect injury outcome. Activation of STING in microglia as early as 2 hours post-injury drives a distinct transcriptional program that influences neutrophil and peripheral immune cell influx, which dictates injury outcomes. These findings shed light on the acute temporal changes in a cell-type specific manner where innate immunity drives subsequent events that affect secondary injury processes after insult.

DOI: https://doi.org/10.1523/JNEUROSCI.0191-23.2024