bims-nenemi 2021-11-14 papers

bims-nenemi

Biomed News

on Neuroinflammation, neurodegeneration and mitochondria

Issue of 2021–11–14
23 papers selected by
Marco Tigano, Thomas Jefferson University

Apoptotic stress-induced FGF signalling promotes non-cell autonomous resistance to cell death.
TFB2M and POLRMT are essential for mammalian mitochondrial DNA replication.
Bcl-xL acts as an inhibitor of IP3R channels, thereby antagonizing Ca2+-driven apoptosis.
Disrupted mitochondrial homeostasis coupled with mitotic arrest generates antineoplastic oxidative stress.
GRP75 mediates Endoplasmic Reticulum-Mitochondria coupling during palmitate-induced pancreatic β-cell apoptosis.
Integrated stress response restricts macrophage necroptosis.
TNF leads to mtDNA release and cGAS/STING-dependent interferon responses that support inflammatory arthritis.
Mitochondrial Fission Factor Is a Novel Interacting Protein of the Critical B Cell Survival Regulator TRAF3 in B Lymphocytes.
Alteration of mitochondrial homeostasis is an early event in a C. elegans model of human tauopathy.
An Aicardi-Goutières Syndrome-Causative Point Mutation in Adar1 Gene Invokes Multiorgan Inflammation and Late-Onset Encephalopathy in Mice.
Aberrant Mitochondrial Dynamics and Exacerbated Response to Neuroinflammation in a Novel Mouse Model of CMT2A.
Selenoprotein DIO2 Is a Regulator of Mitochondrial Function, Morphology and UPRmt in Human Cardiomyocytes.
Transcriptional Control of Metastasis by Integrated Stress Response Signaling.
Selective oxidative stress induces dual damage to telomeres and mitochondria in human T cells.
Morpholinos meet mitochondria: Targeting organellar gene expression.
Regulation of Endoplasmic Reticulum-Mitochondria Tethering and Ca2+ Fluxes by TDP-43 via GSK3β.
The FASTK family proteins fine-tune mitochondrial RNA processing.
Dynamic regulation of mitochondrial pyruvate metabolism is necessary for orthotopic pancreatic tumor growth.
MitoScape: A big-data, machine-learning platform for obtaining mitochondrial DNA from next-generation sequencing data.
Contribution of endoplasmic reticulum stress, MAPK and PI3K/Akt pathways to the apoptotic death induced by a penicillin derivative in melanoma cells.
A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines.
Reduced nuclear NAD+ drives DNA damage and subsequent immune activation in the retina.
Targeting Tau Mitigates Mitochondrial Fragmentation and Oxidative Stress in Amyotrophic Lateral Sclerosis.

Nat Commun. 2021 Nov 12. 12(1): 6572

Apoptotic stress-induced FGF signalling promotes non-cell autonomous resistance to cell death.

Florian J Bock, Egor Sedov, Elle Koren, Anna L Koessinger, Catherine Cloix, Désirée Zerbst, Dimitris Athineos, Jayanthi Anand, Kirsteen J Campbell, Karen Blyth, Yaron Fuchs, Stephen W G Tait.

Damaged or superfluous cells are typically eliminated by apoptosis. Although apoptosis is a cell-autonomous process, apoptotic cells communicate with their environment in different ways. Here we describe a mechanism whereby cells under apoptotic stress can promote survival of neighbouring cells. We find that upon apoptotic stress, cells release the growth factor FGF2, leading to MEK-ERK-dependent transcriptional upregulation of pro-survival BCL-2 proteins in a non-cell autonomous manner. This transient upregulation of pro-survival BCL-2 proteins protects neighbouring cells from apoptosis. Accordingly, we find in certain cancer types a correlation between FGF-signalling, BCL-2 expression and worse prognosis. In vivo, upregulation of MCL-1 occurs in an FGF-dependent manner during skin repair, which regulates healing dynamics. Importantly, either co-treatment with FGF-receptor inhibitors or removal of apoptotic stress restores apoptotic sensitivity to cytotoxic therapy and delays wound healing. These data reveal a pathway by which cells under apoptotic stress can increase resistance to cell death in surrounding cells. Beyond mediating cytotoxic drug resistance, this process also provides a potential link between tissue damage and repair.

DOI: https://doi.org/10.1038/s41467-021-26613-0
Biochim Biophys Acta Mol Cell Res. 2021 Oct 30. pii: S0167-4889(21)00221-4. [Epub ahead of print]1869(1): 119167

TFB2M and POLRMT are essential for mammalian mitochondrial DNA replication.

Teppei Inatomi, Shigeru Matsuda, Takashi Ishiuchi, Yura Do, Masunari Nakayama, Shusaku Abe, Kazutoshi Kasho, Sjoerd Wanrooij, Kazuto Nakada, Kenji Ichiyanagi, Hiroyuki Sasaki, Takehiro Yasukawa, Dongchon Kang.

  Two classes of replication intermediates have been observed from mitochondrial DNA (mtDNA) in many mammalian tissue and cells with two-dimensional agarose gel electrophoresis. One is assigned to leading-strand synthesis in the absence of synchronous lagging-strand synthesis (strand-asynchronous replication), and the other has properties of coupled leading- and lagging-strand synthesis (strand-coupled replication). While strand-asynchronous replication is primed by long noncoding RNA synthesized from a defined transcription initiation site, little is known about the commencement of strand-coupled replication. To investigate it, we attempted to abolish strand-asynchronous replication in cultured human cybrid cells by knocking out the components of the transcription initiation complexes, mitochondrial transcription factor B2 (TFB2M/mtTFB2) and mitochondrial RNA polymerase (POLRMT/mtRNAP). Unexpectedly, removal of either protein resulted in complete mtDNA loss, demonstrating for the first time that TFB2M and POLRMT are indispensable for the maintenance of human mtDNA. Moreover, a lack of TFB2M could not be compensated for by mitochondrial transcription factor B1 (TFB1M/mtTFB1). These findings indicate that TFB2M and POLRMT are crucial for the priming of not only strand-asynchronous but also strand-coupled replication, providing deeper insights into the molecular basis of mtDNA replication initiation.

Keywords:  Mitochondrial DNA; Mitochondrial RNA polymerase; Mitochondrial transcription factor; Replication initiation; Strand-asynchronous replication; Strand-coupled replication

DOI:  https://doi.org/10.1016/j.bbamcr.2021.119167
Cell Death Differ. 2021 Nov 08.

Bcl-xL acts as an inhibitor of IP3R channels, thereby antagonizing Ca2+-driven apoptosis.

Nicolas Rosa, Hristina Ivanova, Larry E Wagner, Justin Kale, Rita La Rovere, Kirsten Welkenhuyzen, Nikolaos Louros, Spyridoula Karamanou, Victoria Shabardina, Irma Lemmens, Elien Vandermarliere, Kozo Hamada, Hideaki Ando, Frederic Rousseau, Joost Schymkowitz, Jan Tavernier, Katsuhiko Mikoshiba, Anastassios Economou, David W Andrews, Jan B Parys, David I Yule, Geert Bultynck.

Anti-apoptotic Bcl-2-family members not only act at mitochondria but also at the endoplasmic reticulum, where they impact Ca2+ dynamics by controlling IP3 receptor (IP3R) function. Current models propose distinct roles for Bcl-2 vs. Bcl-xL, with Bcl-2 inhibiting IP3Rs and preventing pro-apoptotic Ca2+ release and Bcl-xL sensitizing IP3Rs to low [IP3] and promoting pro-survival Ca2+ oscillations. We here demonstrate that Bcl-xL too inhibits IP3R-mediated Ca2+ release by interacting with the same IP3R regions as Bcl-2. Via in silico superposition, we previously found that the residue K87 of Bcl-xL spatially resembled K17 of Bcl-2, a residue critical for Bcl-2's IP3R-inhibitory properties. Mutagenesis of K87 in Bcl-xL impaired its binding to IP3R and abrogated Bcl-xL's inhibitory effect on IP3Rs. Single-channel recordings demonstrate that purified Bcl-xL, but not Bcl-xLK87D, suppressed IP3R single-channel openings stimulated by sub-maximal and threshold [IP3]. Moreover, we demonstrate that Bcl-xL-mediated inhibition of IP3Rs contributes to its anti-apoptotic properties against Ca2+-driven apoptosis. Staurosporine (STS) elicits long-lasting Ca2+ elevations in wild-type but not in IP3R-knockout HeLa cells, sensitizing the former to STS treatment. Overexpression of Bcl-xL in wild-type HeLa cells suppressed STS-induced Ca2+ signals and cell death, while Bcl-xLK87D was much less effective in doing so. In the absence of IP3Rs, Bcl-xL and Bcl-xLK87D were equally effective in suppressing STS-induced cell death. Finally, we demonstrate that endogenous Bcl-xL also suppress IP3R activity in MDA-MB-231 breast cancer cells, whereby Bcl-xL knockdown augmented IP3R-mediated Ca2+ release and increased the sensitivity towards STS, without altering the ER Ca2+ content. Hence, this study challenges the current paradigm of divergent functions for Bcl-2 and Bcl-xL in Ca2+-signaling modulation and reveals that, similarly to Bcl-2, Bcl-xL inhibits IP3R-mediated Ca2+ release and IP3R-driven cell death. Our work further underpins that IP3R inhibition is an integral part of Bcl-xL's anti-apoptotic function.

DOI: https://doi.org/10.1038/s41418-021-00894-w
Oncogene. 2021 Nov 12.

Disrupted mitochondrial homeostasis coupled with mitotic arrest generates antineoplastic oxidative stress.

Xiaohe Hao, Wenqing Bu, Guosheng Lv, Limei Xu, Dong Hou, Jing Wang, Xiaojie Liu, Tingting Yang, Xiyu Zhang, Qiao Liu, Yaoqin Gong, Changshun Shao.

Reactive oxygen species (ROS) serve as critical signals in various cellular processes. Excessive ROS cause cell death or senescence and mediates the therapeutic effect of many cancer drugs. Recent studies showed that ROS increasingly accumulate during G2/M arrest, the underlying mechanism, however, has not been fully elucidated. Here, we show that in cancer cells treated with anticancer agent TH287 or paclitaxel that causes M arrest, mitochondria accumulate robustly and produce excessive mitochondrial superoxide, which causes oxidative DNA damage and undermines cell survival and proliferation. While mitochondrial mass is greatly increased in cells arrested at M phase, the mitochondrial function is compromised, as reflected by reduced mitochondrial membrane potential, increased SUMOylation and acetylation of mitochondrial proteins, as well as an increased metabolic reliance on glycolysis. CHK1 functional disruption decelerates cell cycle, spares the M arrest and attenuates mitochondrial oxidative stress. Induction of mitophagy and blockade of mitochondrial biogenesis, measures that reduce mitochondrial accumulation, also decelerate cell cycle and abrogate M arrest-coupled mitochondrial oxidative stress. These results suggest that cell cycle progression and mitochondrial homeostasis are interdependent and coordinated, and that impairment of mitochondrial homeostasis and the associated redox signaling may mediate the antineoplastic effect of the M arrest-inducing chemotherapeutics. Our findings provide insights into the fate of cells arrested at M phase and have implications in cancer therapy.

DOI: https://doi.org/10.1038/s41388-021-02105-9
J Biol Chem. 2021 Oct 28. pii: S0021-9258(21)01174-1. [Epub ahead of print] 101368

GRP75 mediates Endoplasmic Reticulum-Mitochondria coupling during palmitate-induced pancreatic β-cell apoptosis.

Shweta Tiwary, Arun Nandwani, Rukshar Khan, Malabika Datta.

  The endoplasmic reticulum (ER) and mitochondria are structurally connected with each other at specific sites termed mitochondria-associated membranes (MAM). These physical links are composed of several tethering proteins and are important during varied cellular processes, such as calcium homeostasis, lipid metabolism and transport, membrane biogenesis and organelle remodeling. However, the attributes of specific tethering proteins in these cellular functions remain debatable. Here, we present data to show that one such tether protein, GRP75, is essential in increasing ER-mitochondria contact during palmitate-induced apoptosis in pancreatic insulinoma cells. We demonstrate that palmitate increased GRP75 levels in mouse and rat pancreatic insulinoma cells as well as in mouse primary islet cells. This was associated with increased mitochondrial Ca2+ transfer, impaired mitochondrial membrane potential, increased ROS production, and enhanced physical coupling between the ER and mitochondria. Interestingly, GRP75 inhibition prevented these palmitate-induced cellular aberrations. Additionally, GRP75 overexpression alone was sufficient to impair mitochondrial membrane potential, increase mitochondrial Ca2+ levels and ROS generation, augment ER-mitochondria contact, and induce apoptosis in these cells. In vivo injection of palmitate induced hyperglycemia and hypertriglyceridemia, as well as impaired glucose and insulin tolerance in mice. These animals also exhibited elevated GRP75 levels accompanied by enhanced apoptosis within the pancreatic islets. Our findings suggest that GRP75 is critical in mediating palmitate-induced ER-mitochondrial interaction leading to apoptosis in pancreatic islet cells.

Keywords:  ER-mitochondria contact; GRP75; calcium apoptosis; palmitate

DOI:  https://doi.org/10.1016/j.jbc.2021.101368
Life Sci Alliance. 2022 Jan;pii: e202101260. [Epub ahead of print]5(1):

Integrated stress response restricts macrophage necroptosis.

David E Place, Parimal Samir, Rk Subbarao Malireddi, Thirumala-Devi Kanneganti.

The integrated stress response (ISR) regulates cellular homeostasis and cell survival following exposure to stressors. Cell death processes such as apoptosis and pyroptosis are known to be modulated by stress responses, but the role of the ISR in necroptosis is poorly understood. Necroptosis is an inflammatory, lytic form of cell death driven by the RIPK3-MLKL signaling axis. Here, we show that macrophages that have induced the ISR are protected from subsequent necroptosis. Consistent with a reduction in necroptosis, phosphorylation of RIPK1, RIPK3, and MLKL is reduced in macrophages pre-treated with ISR-inducing agents that are challenged with necroptosis-inducing triggers. The stress granule component DDX3X, which is involved in ISR-mediated regulation of pyroptosis, is not required for protecting ISR-treated cells from necroptosis. Disruption of stress granule assembly or knockdown of Perk restored necroptosis in pre-stressed cells. Together, these findings identify a critical role for the ISR in limiting necroptosis in macrophages.

DOI: https://doi.org/10.26508/lsa.202101260
Cell Rep. 2021 Nov 09. pii: S2211-1247(21)01456-X. [Epub ahead of print]37(6): 109977

TNF leads to mtDNA release and cGAS/STING-dependent interferon responses that support inflammatory arthritis.

Joschka Willemsen, Marie-Therese Neuhoff, Thomas Hoyler, Emma Noir, Clemence Tessier, Sophie Sarret, Tara N Thorsen, Amanda Littlewood-Evans, Juan Zhang, Maroof Hasan, James S Rush, Danilo Guerini, Richard M Siegel.

  Tumor necrosis factor (TNF) is a key driver of several inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis, in which affected tissues show an interferon-stimulated gene signature. Here, we demonstrate that TNF triggers a type-I interferon response that is dependent on the cyclic guanosine monophosphate-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. We show that TNF inhibits PINK1-mediated mitophagy and leads to altered mitochondrial function and to an increase in cytosolic mtDNA levels. Using cGAS-chromatin immunoprecipitation (ChIP), we demonstrate that cytosolic mtDNA binds to cGAS after TNF treatment. Furthermore, TNF induces a cGAS-STING-dependent transcriptional response that mimics that of macrophages from rheumatoid arthritis patients. Finally, in an inflammatory arthritis mouse model, cGAS deficiency blocked interferon responses and reduced inflammatory cell infiltration and joint swelling. These findings elucidate a molecular mechanism linking TNF to type-I interferon signaling and suggest a potential benefit for therapeutic targeting of cGAS/STING in TNF-driven diseases.

Keywords:  ISG; STING; TNF; arthritis; autoimmune; cGAS; interferon; mitophagy; mtDNA

DOI:  https://doi.org/10.1016/j.celrep.2021.109977
Front Immunol. 2021 ;12 670338

Mitochondrial Fission Factor Is a Novel Interacting Protein of the Critical B Cell Survival Regulator TRAF3 in B Lymphocytes.

Yingying Liu, Samantha Gokhale, Jaeyong Jung, Sining Zhu, Chang Luo, Debanjan Saha, Jessie Yanxiang Guo, Huaye Zhang, Saw Kyin, Wei-Xing Zong, Eileen White, Ping Xie.

  Proteins controlling mitochondrial fission have been recognized as essential regulators of mitochondrial functions, mitochondrial quality control and cell apoptosis. In the present study, we identified the critical B cell survival regulator TRAF3 as a novel binding partner of the key mitochondrial fission factor, MFF, in B lymphocytes. Elicited by our unexpected finding that the majority of cytoplasmic TRAF3 proteins were localized at the mitochondria in resting splenic B cells after ex vivo culture for 2 days, we found that TRAF3 specifically interacted with MFF as demonstrated by co-immunoprecipitation and GST pull-down assays. We further found that in the absence of stimulation, increased protein levels of mitochondrial TRAF3 were associated with altered mitochondrial morphology, decreased mitochondrial respiration, increased mitochondrial ROS production and membrane permeabilization, which eventually culminated in mitochondria-dependent apoptosis in resting B cells. Loss of TRAF3 had the opposite effects on the morphology and function of mitochondria as well as mitochondria-dependent apoptosis in resting B cells. Interestingly, co-expression of TRAF3 and MFF resulted in decreased phosphorylation and ubiquitination of MFF as well as decreased ubiquitination of TRAF3. Moreover, lentivirus-mediated overexpression of MFF restored mitochondria-dependent apoptosis in TRAF3-deficient malignant B cells. Taken together, our findings provide novel insights into the apoptosis-inducing mechanisms of TRAF3 in B cells: as a result of survival factor deprivation or under other types of stress, TRAF3 is mobilized to the mitochondria through its interaction with MFF, where it triggers mitochondria-dependent apoptosis. This new role of TRAF3 in controlling mitochondrial homeostasis might have key implications in TRAF3-mediated regulation of B cell transformation in different cellular contexts. Our findings also suggest that mitochondrial fission is an actionable therapeutic target in human B cell malignancies, including those with TRAF3 deletion or relevant mutations.

Keywords:  B cell malignancies; B lymphocytes; MFF; TRAF3; apoptosis; mitochondria

DOI:  https://doi.org/10.3389/fimmu.2021.670338
Aging (Albany NY). 2021 Nov 09. 13(undefined):

Alteration of mitochondrial homeostasis is an early event in a C. elegans model of human tauopathy.

Konstantinos Palikaras, Kavya Achanta, Seoyun Choi, Mansour Akbari, Vilhelm A Bohr.

  Tauopathies are a group of progressive neurodegenerative disorders characterized by the presence of insoluble intracellular tau filaments in the brain. Evidence suggests that there is a tight connection between mitochondrial dysfunction and tauopathies, including Alzheimer's disease. However, whether mitochondrial dysfunction occurs prior to the detection of tau aggregates in tauopathies remains elusive. Here, we utilized transgenic nematodes expressing the full length of wild type tau in neuronal cells and monitored mitochondrial morphology alterations over time. Although tau-expressing nematodes did not accumulate detectable levels of tau aggregates during larval stages, they displayed increased mitochondrial damage and locomotion defects compared to the control worms. Chelating calcium restored mitochondrial activity and improved motility in the tau-expressing larvae suggesting a link between mitochondrial damage, calcium homeostasis and neuronal impairment in these animals. Our findings suggest that defective mitochondrial function is an early pathogenic event of tauopathies, taking place before tau aggregation and undermining neuronal homeostasis and organismal fitness. Understanding the molecular mechanisms causing mitochondrial dysfunction early in tauopathy will be of significant clinical and therapeutic value and merits further investigation.

Keywords:  Alzheimer’s disease; C. elegans; aging; energy metabolism; mitochondria; tau; tauopathy

DOI:  https://doi.org/10.18632/aging.203683
J Immunol. 2021 Nov 12. pii: ji2100526. [Epub ahead of print]

An Aicardi-Goutières Syndrome-Causative Point Mutation in Adar1 Gene Invokes Multiorgan Inflammation and Late-Onset Encephalopathy in Mice.

Maal Inoue, Taisuke Nakahama, Ryuichiro Yamasaki, Toshiharu Shibuya, Jung In Kim, Hiroyuki Todo, Yanfang Xing, Yuki Kato, Eiichi Morii, Yukio Kawahara.

Aicardi-Goutières syndrome (AGS) is a congenital inflammatory disorder accompanied by overactivated type I IFN signaling and encephalopathy with leukodystrophy and intracranial calcification. To date, none of the mouse models carrying an AGS-causative mutation has mimicked such brain pathology. Here, we established a mutant mouse model carrying a K948N point mutation, corresponding to an AGS-causative K999N mutation, located in a deaminase domain of the Adar1 gene that encodes an RNA editing enzyme. Adar1K948N/K948N mice displayed postnatal growth retardation. Hyperplasia of splenic white pulps with germinal centers and hepatic focal inflammation were observed from 2 mo of age. Inflammation developed in the lungs and heart with lymphocyte infiltration in an age-dependent manner. Furthermore, white matter abnormalities with astrocytosis and microgliosis were detected at 1 y of age. The increased expression of IFN-stimulated genes was detected in multiple organs, including the brain, from birth. In addition, single-nucleus RNA sequencing revealed that this elevated expression of IFN-stimulated genes was commonly observed in all neuronal subtypes, including neurons, oligodendrocytes, and astrocytes. We further showed that a K948N point mutation reduced the RNA editing activity of ADAR1 in vivo. The pathological abnormalities found in Adar1K948N/K948N mice were ameliorated by either the concurrent deletion of MDA5, a cytosolic sensor of unedited transcripts, or the sole expression of active ADAR1 p150, an isoform of ADAR1. Collectively, such data suggest that although the degree is mild, Adar1K948N/K948N mice mimic multiple AGS phenotypes, including encephalopathy, which is caused by reduced RNA editing activity of the ADAR1 p150 isoform.

DOI: https://doi.org/10.4049/jimmunol.2100526
Int J Mol Sci. 2021 Oct 26. pii: 11569. [Epub ahead of print]22(21):

Aberrant Mitochondrial Dynamics and Exacerbated Response to Neuroinflammation in a Novel Mouse Model of CMT2A.

Filippos Stavropoulos, Irene Sargiannidou, Louiza Potamiti, Alexia Kagiava, Mihalis I Panayiotidis, Ji Hyun Bae, Su Cheong Yeom, Jae Young Lee, Kleopas A Kleopa.

  Charcot-Marie-Tooth disease type 2A (CMT2A) is the most common hereditary axonal neuropathy caused by mutations in MFN2 encoding Mitofusin-2, a multifunctional protein located in the outer mitochondrial membrane. In order to study the effects of a novel MFN2K357T mutation associated with early onset, autosomal dominant severe CMT2A, we generated a knock-in mouse model. While Mfn2K357T/K357T mouse pups were postnatally lethal, Mfn2+/K357T heterozygous mice were asymptomatic and had no histopathological changes in their sciatic nerves up to 10 months of age. However, immunofluorescence analysis of Mfn2+/K357T mice revealed aberrant mitochondrial clustering in the sciatic nerves from 6 months of age, in optic nerves from 8 months, and in lumbar spinal cord white matter at 10 months, along with microglia activation. Ultrastructural analyses confirmed dysmorphic mitochondrial aggregates in sciatic and optic nerves. After exposure of 6-month-old mice to lipopolysaccharide, Mfn2+/K357T mice displayed a higher immune response, a more severe motor impairment, and increased CNS inflammation, microglia activation, and macrophage infiltrates. Overall, ubiquitous Mfn2K357T expression renders the CNS and peripheral nerves of Mfn2+/K357T mice more susceptible to mitochondrial clustering, and augments their response to inflammation, modeling some cellular mechanisms that may be relevant for the development of neuropathy in patients with CMT2A.

Keywords:  Charcot-Marie-Tooth disease type 2A; knock-in mouse model; lipopolysaccharide; mitochondria; mitofusin-2; neuroinflammation; peripheral neuropathy

DOI:  https://doi.org/10.3390/ijms222111569
Int J Mol Sci. 2021 Nov 02. pii: 11906. [Epub ahead of print]22(21):

Selenoprotein DIO2 Is a Regulator of Mitochondrial Function, Morphology and UPRmt in Human Cardiomyocytes.

Nils Bomer, Mario G Pavez-Giani, Frederik E Deiman, Annet N Linders, Martijn F Hoes, Christiane L J Baierl, Silke U Oberdorf-Maass, Rudolf A de Boer, Herman H W Silljé, Eugene Berezikov, Warner S Simonides, B Daan Westenbrink, Peter van der Meer.

  Members of the fetal-gene-program may act as regulatory components to impede deleterious events occurring with cardiac remodeling, and constitute potential novel therapeutic heart failure (HF) targets. Mitochondrial energy derangements occur both during early fetal development and in patients with HF. Here we aim to elucidate the role of DIO2, a member of the fetal-gene-program, in pluripotent stem cell (PSC)-derived human cardiomyocytes and on mitochondrial dynamics and energetics, specifically. RNA sequencing and pathway enrichment analysis was performed on mouse cardiac tissue at different time points during development, adult age, and ischemia-induced HF. To determine the function of DIO2 in cardiomyocytes, a stable human hPSC-line with a DIO2 knockdown was made using a short harpin sequence. Firstly, we showed the selenoprotein, type II deiodinase (DIO2): the enzyme responsible for the tissue-specific conversion of inactive (T4) into active thyroid hormone (T3), to be a member of the fetal-gene-program. Secondly, silencing DIO2 resulted in an increased reactive oxygen species, impaired activation of the mitochondrial unfolded protein response, severely impaired mitochondrial respiration and reduced cellular viability. Microscopical 3D reconstruction of the mitochondrial network displayed substantial mitochondrial fragmentation. Summarizing, we identified DIO2 to be a member of the fetal-gene-program and as a key regulator of mitochondrial performance in human cardiomyocytes. Our results suggest a key position of human DIO2 as a regulator of mitochondrial function in human cardiomyocytes.

Keywords:  DIO2; fetal-gene-program; heart failure; human cardiomyocytes; mitochondrial function; mtUPR; selenoproteins

DOI:  https://doi.org/10.3390/ijms222111906
Front Oncol. 2021 ;11 770843

Transcriptional Control of Metastasis by Integrated Stress Response Signaling.

Si Lu, Li-Xian Yang, Zi-Jian Cao, Jiang-Sha Zhao, Jia You, Yu-Xiong Feng.

  As a central cellular program to sense and transduce stress signals, the integrated stress response (ISR) pathway has been implicated in cancer initiation and progression. Depending on the genetic mutation landscape, cellular context, and differentiation states, there are emerging pieces of evidence showing that blockage of the ISR can selectively and effectively shift the balance of cancer cells toward apoptosis, rendering the ISR a promising target in cancer therapy. Going beyond its pro-survival functions, the ISR can also influence metastasis, especially via proteostasis-independent mechanisms. In particular, ISR can modulate metastasis via transcriptional reprogramming, in the help of essential transcription factors. In this review, we summarized the current understandings of ISR in cancer metastasis from the perspective of transcriptional regulation.

Keywords:  EMT; cancer; integrated stress response (ISR); metastasis; transcription

DOI:  https://doi.org/10.3389/fonc.2021.770843
Aging Cell. 2021 Nov 09. e13513

Selective oxidative stress induces dual damage to telomeres and mitochondria in human T cells.

Ling Wang, Zeyuan Lu, Juan Zhao, Madison Schank, Dechao Cao, Xindi Dang, Lam Nhat Nguyen, Lam Ngoc Thao Nguyen, Sushant Khanal, Jinyu Zhang, Xiao Y Wu, Mohamed El Gazzar, Shunbin Ning, Jonathan P Moorman, Zhi Q Yao.

  Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere erosion and mitochondrial injury, leading to impaired cellular functions and cell death. Whether oxidative stress-mediated telomere erosion induces mitochondrial injury, or vice versa, in human T cells-the major effectors of host adaptive immunity against infection and malignancy-is poorly understood due to the pleiotropic effects of ROS. Here we employed a novel chemoptogenetic tool that selectively produces a single oxygen (1 O2 ) only at telomeres or mitochondria in Jurkat T cells. We found that targeted 1 O2 production at telomeres triggered not only telomeric DNA damage but also mitochondrial dysfunction, resulting in T cell apoptotic death. Conversely, targeted 1 O2 formation at mitochondria induced not only mitochondrial injury but also telomeric DNA damage, leading to cellular crisis and apoptosis. Targeted oxidative stress at either telomeres or mitochondria increased ROS production, whereas blocking ROS formation during oxidative stress reversed the telomeric injury, mitochondrial dysfunction, and cellular apoptosis. Notably, the X-ray repair cross-complementing protein 1 (XRCC1) in the base excision repair (BER) pathway and multiple mitochondrial proteins in other cellular pathways were dysregulated by the targeted oxidative stress. By confining singlet 1 O2 formation to a single organelle, this study suggests that oxidative stress induces dual injury in T cells via crosstalk between telomeres and mitochondria. Further identification of these oxidation pathways may offer a novel approach to preserve mitochondrial functions, protect telomere integrity, and maintain T cell survival, which can be exploited to combat various immune aging-associated diseases.

Keywords:  DNA damage and repair; T cell senescence; mitochondria; oxidative stress; telomeres

DOI:  https://doi.org/10.1111/acel.13513
Cell. 2021 Nov 11. pii: S0092-8674(21)01235-6. [Epub ahead of print]184(23): 5693-5695

Morpholinos meet mitochondria: Targeting organellar gene expression.

Heike Rampelt, Nikolaus Pfanner.

The mitochondrial genome encodes proteins central to mitochondrial function; however, transcript-specific mechanistic studies of mitochondrial gene products have been difficult because of challenges in their experimental manipulation. Cruz-Zaragoza et al. provide a solution to this challenge, introducing an elegant system for efficient translational silencing of transcripts in human mitochondria.

DOI: https://doi.org/10.1016/j.cell.2021.10.019
Int J Mol Sci. 2021 Nov 01. pii: 11853. [Epub ahead of print]22(21):

Regulation of Endoplasmic Reticulum-Mitochondria Tethering and Ca2+ Fluxes by TDP-43 via GSK3β.

Caterina Peggion, Maria Lina Massimino, Raphael Severino Bonadio, Federica Lia, Raffaele Lopreiato, Stefano Cagnin, Tito Calì, Alessandro Bertoli.

  Mitochondria-ER contacts (MERCs), tightly regulated by numerous tethering proteins that act as molecular and functional connections between the two organelles, are essential to maintain a variety of cellular functions. Such contacts are often compromised in the early stages of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). TDP-43, a nuclear protein mainly involved in RNA metabolism, has been repeatedly associated with ALS pathogenesis and other neurodegenerative diseases. Although TDP-43 neuropathological mechanisms are still unclear, the accumulation of the protein in cytoplasmic inclusions may underlie a protein loss-of-function effect. Accordingly, we investigated the impact of siRNA-mediated TDP-43 silencing on MERCs and the related cellular parameters in HeLa cells using GFP-based probes for MERCs quantification and aequorin-based probes for local Ca2+ measurements, combined with targeted protein and mRNA profiling. Our results demonstrated that TDP-43 down-regulation decreases MERCs density, thereby remarkably reducing mitochondria Ca2+ uptake after ER Ca2+ release. Thorough mRNA and protein analyses did not highlight altered expression of proteins involved in MERCs assembly or Ca2+-mediated ER-mitochondria cross-talk, nor alterations of mitochondrial density and morphology were observed by confocal microscopy. Further mechanistic inspections, however, suggested that the observed cellular alterations are correlated to increased expression/activity of GSK3β, previously associated with MERCs disruption.

Keywords:  ER–mitochondria contacts; GSK3β; SPLICS; TDP-43; amyotrophic lateral sclerosis; calcium homeostasis; neurodegenerative disorders

DOI:  https://doi.org/10.3390/ijms222111853
PLoS Genet. 2021 Nov 08. 17(11): e1009873

The FASTK family proteins fine-tune mitochondrial RNA processing.

Akira Ohkubo, Lindsey Van Haute, Danielle L Rudler, Maike Stentenbach, Florian A Steiner, Oliver Rackham, Michal Minczuk, Aleksandra Filipovska, Jean-Claude Martinou.

Transcription of the human mitochondrial genome and correct processing of the two long polycistronic transcripts are crucial for oxidative phosphorylation. According to the tRNA punctuation model, nucleolytic processing of these large precursor transcripts occurs mainly through the excision of the tRNAs that flank most rRNAs and mRNAs. However, some mRNAs are not punctuated by tRNAs, and it remains largely unknown how these non-canonical junctions are resolved. The FASTK family proteins are emerging as key players in non-canonical RNA processing. Here, we have generated human cell lines carrying single or combined knockouts of several FASTK family members to investigate their roles in non-canonical RNA processing. The most striking phenotypes were obtained with loss of FASTKD4 and FASTKD5 and with their combined double knockout. Comprehensive mitochondrial transcriptome analyses of these cell lines revealed a defect in processing at several canonical and non-canonical RNA junctions, accompanied by an increase in specific antisense transcripts. Loss of FASTKD5 led to the most severe phenotype with marked defects in mitochondrial translation of key components of the electron transport chain complexes and in oxidative phosphorylation. We reveal that the FASTK protein family members are crucial regulators of non-canonical junction and non-coding mitochondrial RNA processing.

DOI: https://doi.org/10.1371/journal.pgen.1009873
Cancer Metab. 2021 Nov 08. 9(1): 39

Dynamic regulation of mitochondrial pyruvate metabolism is necessary for orthotopic pancreatic tumor growth.

Nancy P Echeverri Ruiz, Vijay Mohan, Jinghai Wu, Sabina Scott, McKenzie Kreamer, Martin Benej, Tereza Golias, Ioanna Papandreou, Nicholas C Denko.

   BACKGROUND: Pyruvate dehydrogenase complex (PDC) plays a central role in carbohydrate metabolism, linking cytoplasmic glycolysis to the mitochondrial tricarboxylic acid (TCA) cycle. PDC is a conserved E1-E2-E3 dehydrogenase with a PDHA1 and PDHB heterotetramer functioning as the E1 subunit. PDHA1 contains three serine residues that can be reversibly phosphorylated by a dedicated family of four inhibitory pyruvate dehydrogenase kinases (PDHK1-4) and two reactivating phosphatases (PDP1, 2). Hypoxia induces the expression of PDHK1 and PDHK3 and hyperphosphorylates PDHA1. The role of PDC in metabolic reprogramming and tumor progression appears to be for the integration of oncogenic and environmental signals which supports tumor growth.
METHODS: To isolate the function of the serine-dependent regulation of PDC, we engineered MiaPaca2 cells to express PDHA1 protein with either intact serines at positions 232, 293, and 300 or all the combinations of non-phosphorylatable alanine substitution mutations. These lines were compared in vitro for biochemical response to hypoxia by western blot, metabolic activity by biochemical assay and Seahorse XF flux analysis, and growth in media with reduced exogenous metabolites. The lines were also tested for growth in vivo after orthotopic injection into the pancreata of immune-deficient mice.
RESULTS: In this family of cells with non-phosphorylatable PDHA1, we found reduced hypoxic phosphorylation of PDHA1, decreased PDH enzymatic activity in normoxia and hypoxia, decreased mitochondrial function by Seahorse flux assay, reduced in vitro growth of cells in media depleted of lipids, and reduced growth of tumors after orthotopic transplantation of cells into the pancreata of immune-deficient mice.
CONCLUSIONS: We found that any substitution of alanine for serine at regulatory sites generated a hypomorphic PDC. However, the reduced PDC activity was insensitive to further reduction in hypoxia. These cells had a very modest reduction of growth in vitro, but failed to grow as tumors indicating that dynamic PDC adaptation to microenvironmental conditions is necessary to support pancreatic cancer growth in vivo.

Keywords:  Glucose metabolism; Hypoxia; Mitochondria; Orthotopic pancreatic tumors; Pyruvate dehydrogenase

DOI:  https://doi.org/10.1186/s40170-021-00275-4
PLoS Comput Biol. 2021 Nov 11. 17(11): e1009594

MitoScape: A big-data, machine-learning platform for obtaining mitochondrial DNA from next-generation sequencing data.

Regeneron Genetics Center

The growing number of next-generation sequencing (NGS) data presents a unique opportunity to study the combined impact of mitochondrial and nuclear-encoded genetic variation in complex disease. Mitochondrial DNA variants and in particular, heteroplasmic variants, are critical for determining human disease severity. While there are approaches for obtaining mitochondrial DNA variants from NGS data, these software do not account for the unique characteristics of mitochondrial genetics and can be inaccurate even for homoplasmic variants. We introduce MitoScape, a novel, big-data, software for extracting mitochondrial DNA sequences from NGS. MitoScape adopts a novel departure from other algorithms by using machine learning to model the unique characteristics of mitochondrial genetics. We also employ a novel approach of using rho-zero (mitochondrial DNA-depleted) data to model nuclear-encoded mitochondrial sequences. We showed that MitoScape produces accurate heteroplasmy estimates using gold-standard mitochondrial DNA data. We provide a comprehensive comparison of the most common tools for obtaining mtDNA variants from NGS and showed that MitoScape had superior performance to compared tools in every statistically category we compared, including false positives and false negatives. By applying MitoScape to common disease examples, we illustrate how MitoScape facilitates important heteroplasmy-disease association discoveries by expanding upon a reported association between hypertrophic cardiomyopathy and mitochondrial haplogroup T in men (adjusted p-value = 0.003). The improved accuracy of mitochondrial DNA variants produced by MitoScape will be instrumental in diagnosing disease in the context of personalized medicine and clinical diagnostics.

DOI: https://doi.org/10.1371/journal.pcbi.1009594
Apoptosis. 2021 Nov 12.

Contribution of endoplasmic reticulum stress, MAPK and PI3K/Akt pathways to the apoptotic death induced by a penicillin derivative in melanoma cells.

Yanina Bellizzi, Juan Manuel Anselmi Relats, Patricia G Cornier, Carina M L Delpiccolo, Ernesto G Mata, Florencia Cayrol, Graciela A Cremaschi, Viviana C Blank, Leonor P Roguin.

  We have previously examined the in vitro and in vivo antitumor action of TAP7f, a synthetic triazolylpeptidyl penicillin, on murine melanoma cells. In this work, we explored the signal transduction pathways modulated by TAP7f in murine B16-F0 and human A375 melanoma cells, and the contribution of some intracellular signals to the apoptotic cell death. TAP7f decreased ERK1/2 phosphorylation and increased phospho-p38, phospho-JNK and phospho-Akt levels. ERK1/2 blockage suppressed cell growth, while inhibition of p38, JNK and PI3K-I pathways reduced the antitumor effect of TAP7f. Pharmacological inhibition of p38 and JNK, or blockage of PI3K-I/Akt cascade with a dominant negative PI3K-I mutant diminished Bax expression levels and PARP-1 cleavage, indicating the involvement of these pathways in apoptosis. PI3K-I/Akt inhibition also favored an autophagic response, as evidenced by the higher expression levels of Beclin-1 and LC3-II detected in transfected cells exposed to TAP7f. However, although PI3K-I/Akt blockage promoted an autophagic survival response, this mechanism appears not to be critical for TAP7f antitumor action. It was also shown that TAP7f induced ER stress by enhancing the expression of ER stress-related genes and proteins. Downregulation of CHOP protein with specific siRNA increased cell growth and decreased cleavage of PARP-1, supporting its role in apoptosis. Furthermore, it was found that activation of p38, JNK and Akt occurred downstream ER perturbation. In summary, our results showed that TAP7f triggers an apoptotic cell death in melanoma cells through induction of ER stress and activation of p38, JNK and PI3K-I/Akt pathways.

Keywords:  Apoptosis; Cell signalling; Endoplasmic reticulum stress; Melanoma cells; Triazolylpeptidyl penicillin

DOI:  https://doi.org/10.1007/s10495-021-01697-7
Sci Rep. 2021 Nov 12. 11(1): 22154

A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines.

Reto Eggenschwiler, Thomas Gschwendtberger, Christian Felski, Christopher Jahn, Florian Langer, Jared Sterneckert, Andreas Hermann, Jonathan Lühmann, Doris Steinemann, Alexandra Haase, Ulrich Martin, Susanne Petri, Tobias Cantz.

CRISPR prime-editors are emergent tools for genome editing and offer a versatile alternative approach to HDR-based genome engineering or DNA base-editors. However, sufficient prime-editor expression levels and availability of optimized transfection protocols may affect editing efficiencies, especially in hard-to-transfect cells like hiPSC. Here, we show that piggyBac prime-editing (PB-PE) allows for sustained expression of prime-editors. We demonstrate proof-of-concept for PB-PE in a newly designed lentiviral traffic light reporter, which allows for estimation of gene correction and defective editing resulting in indels, based on expression of two different fluorophores. PB-PE can prime-edit more than 50% of hiPSC cells after antibiotic selection. We also show that improper design of pegRNA cannot simply be overcome by extended expression, but PB-PE allows for estimation of effectiveness of selected pegRNAs after few days of cultivation time. Finally, we implemented PB-PE for efficient editing of an amyotrophic lateral sclerosis-associated mutation in the SOD1-gene of patient-derived hiPSC. Progress of genome editing can be monitored by Sanger-sequencing, whereas PB-PE vectors can be removed after editing and excised cells can be enriched by fialuridine selection. Together, we present an efficient prime-editing toolbox, which can be robustly used in a variety of cell lines even when non-optimized transfection-protocols are applied.

DOI: https://doi.org/10.1038/s41598-021-01689-2
Hum Mol Genet. 2021 Nov 08. pii: ddab324. [Epub ahead of print]

Reduced nuclear NAD+ drives DNA damage and subsequent immune activation in the retina.

Emily E Brown, Michael J Scandura, Sudeep Mehrotra, Yekai Wang, Jianhai Du, Eric A Pierce.

Mutations in NMNAT1, a key enzyme involved in the synthesis of NAD+ in the nucleus, lead to an early onset severe inherited retinal degeneration (IRD). We aimed to understand the role of nuclear NAD+ in the retina and to identify the molecular mechanisms underlying NMNAT1-associated disease, using a mouse model that harbors the p.V9M mutation in Nmnat1 (Nmnat1V9M/V9M). We identified temporal transcriptional reprogramming in the retinas of Nmnat1V9M/V9M mice prior to retinal degeneration, which begins at 4 weeks of age, with no significant alterations in gene expression at 2 weeks of age and over 2600 differentially expressed genes by 3 weeks of age. Expression of the primary consumer of NAD+ in the nucleus, PARP1, an enzyme involved in DNA damage repair and transcriptional regulation, as well as 7 other PARP family enzymes, was elevated in the retinas of Nmnat1V9M/V9M. This was associated with elevated levels of DNA damage, PARP-mediated NAD+ consumption, and migration of Iba1+/CD45+ microglia/macrophages to the subretinal space in the retinas of Nmnat1V9M/V9M mice. These findings suggest that photoreceptor cells are especially sensitive to perturbation of genome homeostasis, and that PARP-mediated cell death may play a role in other genetic forms of IRDs, and potentially other forms of neurodegeneration.

DOI: https://doi.org/10.1093/hmg/ddab324
Mol Neurobiol. 2021 Nov 10.

Targeting Tau Mitigates Mitochondrial Fragmentation and Oxidative Stress in Amyotrophic Lateral Sclerosis.

Tiziana Petrozziello, Evan A Bordt, Alexandra N Mills, Spencer E Kim, Ellen Sapp, Benjamin A Devlin, Abigail A Obeng-Marnu, Sali M K Farhan, Ana C Amaral, Simon Dujardin, Patrick M Dooley, Christopher Henstridge, Derek H Oakley, Andreas Neueder, Bradley T Hyman, Tara L Spires-Jones, Staci D Bilbo, Khashayar Vakili, Merit E Cudkowicz, James D Berry, Marian DiFiglia, M Catarina Silva, Stephen J Haggarty, Ghazaleh Sadri-Vakili.

  Understanding the mechanisms underlying amyotrophic lateral sclerosis (ALS) is crucial for the development of new therapies. Previous studies have demonstrated that mitochondrial dysfunction is a key pathogenetic event in ALS. Interestingly, studies in Alzheimer's disease (AD) post-mortem brain and animal models link alterations in mitochondrial function to interactions between hyperphosphorylated tau and dynamin-related protein 1 (DRP1), the GTPase involved in mitochondrial fission. Recent evidence suggest that tau may be involved in ALS pathogenesis, therefore, we sought to determine whether hyperphosphorylated tau may lead to mitochondrial fragmentation and dysfunction in ALS and whether reducing tau may provide a novel therapeutic approach. Our findings demonstrated that pTau-S396 is mis-localized to synapses in post-mortem motor cortex (mCTX) across ALS subtypes. Additionally, the treatment with ALS synaptoneurosomes (SNs), enriched in pTau-S396, increased oxidative stress, induced mitochondrial fragmentation, and altered mitochondrial connectivity without affecting cell survival in vitro. Furthermore, pTau-S396 interacted with DRP1, and similar to pTau-S396, DRP1 accumulated in SNs across ALS subtypes, suggesting increases in mitochondrial fragmentation in ALS. As previously reported, electron microscopy revealed a significant decrease in mitochondria density and length in ALS mCTX. Lastly, reducing tau levels with QC-01-175, a selective tau degrader, prevented ALS SNs-induced mitochondrial fragmentation and oxidative stress in vitro. Collectively, our findings suggest that increases in pTau-S396 may lead to mitochondrial fragmentation and oxidative stress in ALS and decreasing tau may provide a novel strategy to mitigate mitochondrial dysfunction in ALS. pTau-S396 mis-localizes to synapses in ALS. ALS synaptoneurosomes (SNs), enriched in pTau-S396, increase oxidative stress and induce mitochondrial fragmentation in vitro. pTau-S396 interacts with the pro-fission GTPase DRP1 in ALS. Reducing tau with a selective degrader, QC-01-175, mitigates ALS SNs-induced mitochondrial fragmentation and increases in oxidative stress in vitro.

Keywords:  Amyotrophic lateral sclerosis; Hyperphosphorylated tau; Mitochondrial dynamics; Mitochondrial dysfunction; Tau degrader

DOI:  https://doi.org/10.1007/s12035-021-02557-w