bims-nenemi 2023-05-28 papers

bims-nenemi

Biomed News

on Neuroinflammation, neurodegeneration and mitochondria

Issue of 2023–05–28
eight papers selected by
Marco Tigano, Thomas Jefferson University

The Fate of Oxidative Strand Breaks in Mitochondrial DNA.
Mitophagy restricts BAX/BAK-independent, Parkin-mediated apoptosis.
Phospholipase D3 degrades mitochondrial DNA to regulate nucleotide signaling and APP metabolism.
Genetically encoded photocatalytic protein labeling enables spatially-resolved profiling of intracellular proteome.
Mitochondrial Trafficking of MLKL, Bak/Bax, and Drp1 Is Mediated by RIP1 and ROS which Leads to Decreased Mitochondrial Membrane Integrity during the Hyperglycemic Shift to Necroptosis.
High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution.
Examining Sporadic Cancer Mutations Uncovers a Set of Genes Involved in Mitochondrial Maintenance.
Construction of Genetically Encoded Light-Up RNA Aptamers for Label-free and Ultrasensitive Detection of CircRNAs in Cancer Cells and Tissues.

Antioxidants (Basel). 2023 May 12. pii: 1087. [Epub ahead of print]12(5):

The Fate of Oxidative Strand Breaks in Mitochondrial DNA.

Genevieve Trombly, Afaf Milad Said, Alexei P Kudin, Viktoriya Peeva, Janine Altmüller, Kerstin Becker, Karl Köhrer, Gábor Zsurka, Wolfram S Kunz.

  Mitochondrial DNA (mtDNA) is particularly vulnerable to somatic mutagenesis. Potential mechanisms include DNA polymerase γ (POLG) errors and the effects of mutagens, such as reactive oxygen species. Here, we studied the effects of transient hydrogen peroxide (H2O2 pulse) on mtDNA integrity in cultured HEK 293 cells, applying Southern blotting, ultra-deep short-read and long-read sequencing. In wild-type cells, 30 min after the H2O2 pulse, linear mtDNA fragments appear, representing double-strand breaks (DSB) with ends characterized by short GC stretches. Intact supercoiled mtDNA species reappear within 2-6 h after treatment and are almost completely recovered after 24 h. BrdU incorporation is lower in H2O2-treated cells compared to non-treated cells, suggesting that fast recovery is not associated with mtDNA replication, but is driven by rapid repair of single-strand breaks (SSBs) and degradation of DSB-generated linear fragments. Genetic inactivation of mtDNA degradation in exonuclease deficient POLG p.D274A mutant cells results in the persistence of linear mtDNA fragments with no impact on the repair of SSBs. In conclusion, our data highlight the interplay between the rapid processes of SSB repair and DSB degradation and the much slower mtDNA re-synthesis after oxidative damage, which has important implications for mtDNA quality control and the potential generation of somatic mtDNA deletions.

Keywords:  mitochondrial DNA; mtDNA degradation; mtDNA double-strand breaks mtDNA single-strand breaks; oxidative damage

DOI:  https://doi.org/10.3390/antiox12051087
Sci Adv. 2023 May 24. 9(21): eadg8156

Mitophagy restricts BAX/BAK-independent, Parkin-mediated apoptosis.

Giovanni Quarato, Luigi Mari, Nicholas J Barrows, Mao Yang, Sebastian Ruehl, Mark J Chen, Cliff S Guy, Jonathan Low, Taosheng Chen, Douglas R Green.

Degradation of defective mitochondria is an essential process to maintain cellular homeostasis and it is strictly regulated by the ubiquitin-proteasome system (UPS) and lysosomal activities. Here, using genome-wide CRISPR and small interference RNA screens, we identified a critical contribution of the lysosomal system in controlling aberrant induction of apoptosis following mitochondrial damage. After treatment with mitochondrial toxins, activation of the PINK1-Parkin axis triggered a BAX- and BAK-independent process of cytochrome c release from mitochondria followed by APAF1 and caspase 9-dependent apoptosis. This phenomenon was mediated by UPS-dependent outer mitochondrial membrane (OMM) degradation and was reversed using proteasome inhibitors. We found that the subsequent recruitment of the autophagy machinery to the OMM protected cells from apoptosis, mediating the lysosomal degradation of dysfunctional mitochondria. Our results underscore a major role of the autophagy machinery in counteracting aberrant noncanonical apoptosis and identified autophagy receptors as key elements in the regulation of this process.

DOI: https://doi.org/10.1126/sciadv.adg8156
Nat Commun. 2023 May 24. 14(1): 2847

Phospholipase D3 degrades mitochondrial DNA to regulate nucleotide signaling and APP metabolism.

Zoë P Van Acker, Anika Perdok, Ruben Hellemans, Katherine North, Inge Vorsters, Cedric Cappel, Jonas Dehairs, Johannes V Swinnen, Ragna Sannerud, Marine Bretou, Markus Damme, Wim Annaert.

Phospholipase D3 (PLD3) polymorphisms are linked to late-onset Alzheimer's disease (LOAD). Being a lysosomal 5'-3' exonuclease, its neuronal substrates remained unknown as well as how a defective lysosomal nucleotide catabolism connects to AD-proteinopathy. We identified mitochondrial DNA (mtDNA) as a major physiological substrate and show its manifest build-up in lysosomes of PLD3-defective cells. mtDNA accretion creates a degradative (proteolytic) bottleneck that presents at the ultrastructural level as a marked abundance of multilamellar bodies, often containing mitochondrial remnants, which correlates with increased PINK1-dependent mitophagy. Lysosomal leakage of mtDNA to the cytosol activates cGAS-STING signaling that upregulates autophagy and induces amyloid precursor C-terminal fragment (APP-CTF) and cholesterol accumulation. STING inhibition largely normalizes APP-CTF levels, whereas an APP knockout in PLD3-deficient backgrounds lowers STING activation and normalizes cholesterol biosynthesis. Collectively, we demonstrate molecular cross-talks through feedforward loops between lysosomal nucleotide turnover, cGAS-STING and APP metabolism that, when dysregulated, result in neuronal endolysosomal demise as observed in LOAD.

DOI: https://doi.org/10.1038/s41467-023-38501-w
Nat Commun. 2023 May 23. 14(1): 2978

Genetically encoded photocatalytic protein labeling enables spatially-resolved profiling of intracellular proteome.

Fu Zheng, Xinyue Zhou, Peng Zou, Chenxin Yu.

Mapping the subcellular organization of proteins is crucial for understanding their biological functions. Herein, we report a reactive oxygen species induced protein labeling and identification (RinID) method for profiling subcellular proteome in the context of living cells. Our method capitalizes on a genetically encoded photocatalyst, miniSOG, to locally generate singlet oxygen that reacts with proximal proteins. Labeled proteins are conjugated in situ with an exogenously supplied nucleophilic probe, which serves as a functional handle for subsequent affinity enrichment and mass spectrometry-based protein identification. From a panel of nucleophilic compounds, we identify biotin-conjugated aniline and propargyl amine as highly reactive probes. As a demonstration of the spatial specificity and depth of coverage in mammalian cells, we apply RinID in the mitochondrial matrix, capturing 477 mitochondrial proteins with 94% specificity. We further demonstrate the broad applicability of RinID in various subcellular compartments, including the nucleus and the endoplasmic reticulum (ER). The temporal control of RinID enables pulse-chase labeling of ER proteome in HeLa cells, which reveals substantially higher clearance rate for secreted proteins than ER resident proteins.

DOI: https://doi.org/10.1038/s41467-023-38565-8
Int J Mol Sci. 2023 May 11. pii: 8609. [Epub ahead of print]24(10):

Mitochondrial Trafficking of MLKL, Bak/Bax, and Drp1 Is Mediated by RIP1 and ROS which Leads to Decreased Mitochondrial Membrane Integrity during the Hyperglycemic Shift to Necroptosis.

Matthew A Deragon, William D McCaig, Phillip V Truong, Kevin R Metz, Katherine A Carron, Keven J Hughes, Angeleigh R Knapp, Molly J Dougherty, Timothy J LaRocca.

  Apoptosis and necroptosis overlap in their initial signaling but diverge to produce non-inflammatory and pro-inflammatory outcomes, respectively. High glucose pushes signaling in favor of necroptosis producing a hyperglycemic shift from apoptosis to necroptosis. This shift depends on receptor-interacting protein 1 (RIP1) and mitochondrial reactive oxygen species (ROS). Here, we show that RIP1, mixed lineage kinase domain-like (MLKL) protein, Bcl-2 agonist/killer (Bak), Bcl-2 associated x (Bax) protein, and dynamin-related protein 1 (Drp1) traffic to the mitochondria in high glucose. RIP1 and MLKL appear in the mitochondria in their activated, phosphorylated states while Drp1 appears in its activated, dephosphorylated state in high glucose. Mitochondrial trafficking is prevented in rip1 KO cells and upon treatment with N-acetylcysteine. Induction of ROS replicated the mitochondrial trafficking seen in high glucose. MLKL forms high MW oligomers in the outer and inner mitochondrial membranes while Bak and Bax form high MW oligomers in the outer mitochondrial membrane in high glucose, suggesting pore formation. MLKL, Bax, and Drp1 promoted cytochrome c release from the mitochondria as well as a decrease in mitochondrial membrane potential in high glucose. These results indicate that mitochondrial trafficking of RIP1, MLKL, Bak, Bax, and Drp1 are key events in the hyperglycemic shift from apoptosis to necroptosis. This is also the first report to show oligomerization of MLKL in the inner and outer mitochondrial membranes and dependence of mitochondrial permeability on MLKL.

Keywords:  MLKL; RIP1; apoptosis; glucose; hyperglycemia; necroptosis; pores; reactive oxygen species

DOI:  https://doi.org/10.3390/ijms24108609
J Vis Exp. 2023 05 05.

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution.

Lukasz S Borowski, Karolina Kasztelan, Jolanta Czerwinska-Kostrzewska, Roman J Szczesny.

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

DOI: https://doi.org/10.3791/65236
Genes (Basel). 2023 Apr 29. pii: 1009. [Epub ahead of print]14(5):

Examining Sporadic Cancer Mutations Uncovers a Set of Genes Involved in Mitochondrial Maintenance.

Armando Moreno, Allison Taffet, Elissa Tjahjono, Quinton L Anderson, Natalia V Kirienko.

  Mitochondria are key organelles for cellular health and metabolism and the activation of programmed cell death processes. Although pathways for regulating and re-establishing mitochondrial homeostasis have been identified over the past twenty years, the consequences of disrupting genes that regulate other cellular processes, such as division and proliferation, on affecting mitochondrial function remain unclear. In this study, we leveraged insights about increased sensitivity to mitochondrial damage in certain cancers, or genes that are frequently mutated in multiple cancer types, to compile a list of candidates for study. RNAi was used to disrupt orthologous genes in the model organism Caenorhabditis elegans, and a series of assays were used to evaluate these genes' importance for mitochondrial health. Iterative screening of ~1000 genes yielded a set of 139 genes predicted to play roles in mitochondrial maintenance or function. Bioinformatic analyses indicated that these genes are statistically interrelated. Functional validation of a sample of genes from this set indicated that disruption of each gene caused at least one phenotype consistent with mitochondrial dysfunction, including increased fragmentation of the mitochondrial network, abnormal steady-state levels of NADH or ROS, or altered oxygen consumption. Interestingly, RNAi-mediated knockdown of these genes often also exacerbated α-synuclein aggregation in a C. elegans model of Parkinson's disease. Additionally, human orthologs of the gene set showed enrichment for roles in human disorders. This gene set provides a foundation for identifying new mechanisms that support mitochondrial and cellular homeostasis.

Keywords:  Caenorhabditis elegans; bioinformatics; cancer; development; mitochondria; mitophagy; neurodegeneration; pps-1

DOI:  https://doi.org/10.3390/genes14051009
Anal Chem. 2023 May 23.

Construction of Genetically Encoded Light-Up RNA Aptamers for Label-free and Ultrasensitive Detection of CircRNAs in Cancer Cells and Tissues.

Ning-Ning Zhao, Feng-Zheng Li, Xinyi Zhang, Meng Liu, Hua Cao, Chun-Yang Zhang.

Circular RNAs (circRNAs) as endogenous non-coding RNAs are characterized by covalently closed circular structures, and they widely exist in mammalian cells. The aberrant expression of circRNAs may result in various diseases. Herein, we demonstrate the construction of genetically encoded light-up RNA aptamers for ultrasensitive and label-free detection of circRNA mitochondrial tRNA translation optimization 1 (circMTO1) in cancer cells and tissues. The light-up RNA aptamers are generated by proximity ligation-activated recombinase polymerase amplification (RPA)-assisted transcription amplification. When circMTO1 is present, it initiates the proximity ligation reaction, activating RPA to produce numerous long double-stranded DNAs containing T7 promoters. Subsequently, the RPA products are identified by T7 RNA polymerase, initiating the transcription amplification reaction to generate abundant Spinach RNA aptamers. Spinach RNA aptamers can bind with DFHBI (3,5-difluoro-4-hydroxybenzylidene imidazolidinone) dye to produce a distinct fluorescence signal with near-zero background. This biosensor exhibits excellent selectivity and high sensitivity with a limit of detection of 2.54 aM. It can accurately monitor cellular circMTO1 at the single-cell level and discriminate the expression of circMTO1 between breast cancer patient tissues and healthy tissues. Notably, this biosensor can be employed to measure other nucleic acids by altering the corresponding target recognition sequences, providing a valuable platform for cancer diagnosis and biomedical study.

DOI: https://doi.org/10.1021/acs.analchem.3c01624