bims-nenemi 2022-01-09 papers

bims-nenemi

Biomed News

on Neuroinflammation, neurodegeneration and mitochondria

Issue of 2022‒01‒09
ten papers selected by
Marco Tigano
Thomas Jefferson University

Methods to Study the Mitochondrial Unfolded Protein Response (UPRmt) in Caenorhabditis elegans.
Temporal Analysis of Protein Ubiquitylation and Phosphorylation During Parkin-dependent Mitophagy.
AMPK-PERK axis represses oxidative metabolism and enhances apoptotic priming of mitochondria in acute myeloid leukemia.
Novel C19orf12 loss-of-function variant leading to neurodegeneration with brain iron accumulation.
Targeting Mitochondrial Metabolism and RNA Polymerase POLRMT to Overcome Multidrug Resistance in Cancer.
Restoration of mitophagy ameliorates cardiomyopathy in Barth syndrome.
TREX1 Deficiency Induces ER Stress-Mediated Neuronal Cell Death by Disrupting Ca2+ Homeostasis.
Assays to Study IRE1 Activation and Signaling.
The Transcriptional Response to Oxidative Stress is Independent of Stress-Granule Formation.
Detection of PERK Signaling in the Central Nervous System.

Methods Mol Biol. 2022 ;2378 249-259

Methods to Study the Mitochondrial Unfolded Protein Response (UPRmt) in Caenorhabditis elegans.

Simon Haeussler, Barbara Conradt.

  The nematode Caenorhabditis elegans is a powerful model to study cellular stress responses. Due to its transparency and ease of genetic manipulation, C. elegans is especially suitable for fluorescence microscopy. As a result, studies of C. elegans using different fluorescent reporters have led to the discovery of key players of cellular stress response pathways, including the mitochondrial unfolded protein response (UPRmt). UPRmt is a protective retrograde signaling pathway that ensures mitochondrial homeostasis. The nuclear genes hsp-6 and hsp-60 encode mitochondrial chaperones and are highly expressed upon UPRmt induction. The transcriptional reporters of these genes, hsp-6::gfp and hsp-60::gfp, have been instrumental for monitoring this pathway in live animals. Additional tools for studying UPRmt include fusion proteins of ATFS-1 and DVE-1, ATFS-1::GFP and DVE-1::GFP, key players of the UPRmt pathway. In this protocol, we discuss advantages and limitations of currently available methods and reporters, and we provide detailed instructions on how to image and quantify reporter expression.

Keywords:  UPRmt reporters; atfs-1; dve-1; gfp; hsp-6; hsp-60

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_16
Mol Cell Proteomics. 2021 Dec 30. pii: S1535-9476(21)00163-8. [Epub ahead of print] 100191

Temporal Analysis of Protein Ubiquitylation and Phosphorylation During Parkin-dependent Mitophagy.

Katharina I Zittlau, Anna Lechado Terradas, Nicolas Nalpas, Sven Geisler, Philipp J Kahle, Boris Macek.

  Mitophagy, the selective degradation of mitochondria by autophagy, affects defective mitochondria following damage or stress. At the onset of mitophagy, parkin ubiquitylates proteins on the mitochondrial outer membrane (MOM). While the role of parkin at the onset of mitophagy is well understood, less is known about its activity during later stages of the process. Here we used HeLa cells expressing catalytically active or inactive parkin to perform temporal analysis of the proteome, ubiquitylome and phosphoproteome during 18 hours after induction of mitophagy by mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP). Abundance profiles of proteins downregulated in parkin-dependent manner revealed a stepwise, "outside-in" directed degradation of mitochondrial subcompartments. While ubiquitylation of MOM proteins was enriched among early parkin-dependent targets, numerous mitochondrial inner membrane, matrix and cytosolic proteins were also found ubiquitylated at later stages of mitophagy. Phosphoproteome analysis revealed a possible cross-talk between phosphorylation and ubiquitylation during mitophagy on key parkin targets, such as VDAC1/2.

Keywords:  Mitochondria; Mitophagy; Parkin; Quantitative proteomics; Ubiquitin

DOI:  https://doi.org/10.1016/j.mcpro.2021.100191
Cell Rep. 2022 Jan 04. pii: S2211-1247(21)01701-0. [Epub ahead of print]38(1): 110197

AMPK-PERK axis represses oxidative metabolism and enhances apoptotic priming of mitochondria in acute myeloid leukemia.

Adrien Grenier, Laury Poulain, Johanna Mondesir, Arnaud Jacquel, Claudie Bosc, Lucille Stuani, Sarah Mouche, Clement Larrue, Ambrine Sahal, Rudy Birsen, Victoria Ghesquier, Justine Decroocq, Fetta Mazed, Mireille Lambert, Mamy Andrianteranagna, Benoit Viollet, Patrick Auberger, Andrew A Lane, Pierre Sujobert, Didier Bouscary, Jean-Emmanuel Sarry, Jerome Tamburini.

  AMP-activated protein kinase (AMPK) regulates the balance between cellular anabolism and catabolism dependent on energy resources to maintain proliferation and survival. Small-compound AMPK activators show anti-cancer activity in preclinical models. Using the direct AMPK activator GSK621, we show that the unfolded protein response (UPR) is activated by AMPK in acute myeloid leukemia (AML) cells. Mechanistically, the UPR effector protein kinase RNA-like ER kinase (PERK) represses oxidative phosphorylation, tricarboxylic acid (TCA) cycle, and pyrimidine biosynthesis and primes the mitochondrial membrane to apoptotic signals in an AMPK-dependent manner. Accordingly, in vitro and in vivo studies reveal synergy between the direct AMPK activator GSK621 and the Bcl-2 inhibitor venetoclax. Thus, selective AMPK-activating compounds kill AML cells by rewiring mitochondrial metabolism that primes mitochondria to apoptosis by BH3 mimetics, holding therapeutic promise in AML.

Keywords:  AML; AMPK; GSK621; PERK; mitochondrial apoptosis; unfolded protein response; venetoclax

DOI:  https://doi.org/10.1016/j.celrep.2021.110197
Neurocase. 2022 Jan 04. 1-3

Novel C19orf12 loss-of-function variant leading to neurodegeneration with brain iron accumulation.

Antonia Lefter, Iulia Mitrea, Dan Mitrea, Vasilica Plaiasu, Aida Bertoli-Avella, Christian Beetz, Liviu Cozma, Delia Tulbă, Cristina Elena Mitu, Bogdan Ovidiu Popescu.

  Neurodegeneration with brain iron accumulation (NBIA) is a group of inherited disorders characterised by cerebral iron overload mainly in the basal ganglia. Mitochondrial membrane protein-associated neurodegeneration (MPAN) is a form of NBIA caused by pathogenic C19orf12 gene variants. We report on a Romanian patient with MPAN confirmed through exome sequencing, revealing a homozygous nonsense variant in the C19orf12 gene, NM_001031726.3: c.215T>G (p.Leu72*), that co-segregates with disease in tested relatives: the patient`s parents, younger brother and paternal uncle are heterozygous carriers. This is a novel disease-causing variant in the C19orf12 gene and the first reported MPAN case in a Romanian patient.

Keywords:  C19orf12; mitochondrial membrane protein-associated neurodegeneration; neurodegeneration with brain iron accumulation

DOI:  https://doi.org/10.1080/13554794.2021.2022703
Front Chem. 2021 ;9 775226

Targeting Mitochondrial Metabolism and RNA Polymerase POLRMT to Overcome Multidrug Resistance in Cancer.

Hui-Jing Yu, Guan-Li Xiao, Yu-Ying Zhao, Xin-Xin Wang, Rongfeng Lan.

  Clinically, the prognosis of tumor therapy is fundamentally affected by multidrug resistance (MDR), which is primarily a result of enhanced drug efflux mediated by channels in the membrane that reduce drug accumulation in tumor cells. How to restore the sensitivity of tumor cells to chemotherapy is an ongoing and pressing clinical issue. There is a prevailing view that tumor cells turn to glycolysis for energy supply due to hypoxia. However, studies have shown that mitochondria also play crucial roles, such as providing intermediates for biosynthesis through the tricarboxylic acid (TCA) cycle and a plenty of ATP to fuel cells through the complete breakdown of organic matter by oxidative phosphorylation (OXPHOS). High OXPHOS have been found in some tumors, particularly in cancer stem cells (CSCs), which possess increased mitochondria mass and may be depends on OXPHOS for energy supply. Therefore, they are sensitive to inhibitors of mitochondrial metabolism. In view of this, we should consider mitochondrial metabolism when developing drugs to overcome MDR, where mitochondrial RNA polymerase (POLRMT) would be the focus, as it is responsible for mitochondrial gene expression. Inhibition of POLRMT could disrupt mitochondrial metabolism at its source, causing an energy crisis and ultimately eradicating tumor cells. In addition, it may restore the energy supply of MDR cells to glycolysis and re-sensitize them to conventional chemotherapy. Furthermore, we discuss the rationale and strategies for designing new therapeutic molecules for MDR cancers by targeting POLRMT.

Keywords:  OxPhos; POLRMT; RNA polymerase; cancer stem cell; multidrug resisitance

DOI:  https://doi.org/10.3389/fchem.2021.775226
Autophagy. 2022 Jan 05. 1-16

Restoration of mitophagy ameliorates cardiomyopathy in Barth syndrome.

Jun Zhang, Xueling Liu, Jia Nie, Yuguang Shi.

  Barth syndrome (BTHS) is an X-linked genetic disorder caused by mutations in the TAFAZZIN/Taz gene which encodes a transacylase required for cardiolipin remodeling. Cardiolipin is a mitochondrial signature phospholipid that plays a pivotal role in maintaining mitochondrial membrane structure, respiration, mtDNA biogenesis, and mitophagy. Mutations in the TAFAZZIN gene deplete mature cardiolipin, leading to mitochondrial dysfunction, dilated cardiomyopathy, and premature death in BTHS patients. Currently, there is no effective treatment for this debilitating condition. In this study, we showed that TAFAZZIN deficiency caused hyperactivation of MTORC1 signaling and defective mitophagy, leading to accumulation of autophagic vacuoles and dysfunctional mitochondria in the heart of Tafazzin knockdown mice, a rodent model of BTHS. Consequently, treatment of TAFAZZIN knockdown mice with rapamycin, a potent inhibitor of MTORC1, not only restored mitophagy, but also mitigated mitochondrial dysfunction and dilated cardiomyopathy. Taken together, these findings identify MTORC1 as a novel therapeutic target for BTHS, suggesting that pharmacological restoration of mitophagy may provide a novel treatment for BTHS.Abbreviations: BTHS: Barth syndrome; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CL: cardiolipin; EIF4EBP1/4E-BP1: eukaryotic translation initiation factor 4E binding protein 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; KD: knockdown; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; LV: left ventricle; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; OCR: oxygen consumption rate; PE: phosphatidylethanolamine; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PINK1: PTEN induced putative kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; qRT-PCR: quantitative real-time polymerase chain reaction; RPS6KB/S6K: ribosomal protein S6 kinase beta; SQSTM1/p62: sequestosome 1; TLCL: tetralinoleoyl cardiolipin; WT: wild-type.

Keywords:  BTHS; MTORC1; TAFAZZIN; cardiolipin; mitophagy; rapamycin

DOI:  https://doi.org/10.1080/15548627.2021.2020979
Mol Neurobiol. 2022 Jan 07.

TREX1 Deficiency Induces ER Stress-Mediated Neuronal Cell Death by Disrupting Ca2+ Homeostasis.

Debasish Halder, Su-Jin Jeon, Ji-Yong Yoon, Jeong-Ju Lee, Soo Young Jun, Min-Hyuk Choi, Bohyeon Jeong, Duk Hyun Sung, Da Yong Lee, Byoung Joon Kim, Nam-Soon Kim.

  TREX1 is an exonuclease that degrades extranuclear DNA species in mammalian cells. Herein, we show a novel mechanism by which TREX1 interacts with the BiP/GRP78 and TREX1 deficiency triggers ER stress through the accumulation of single-stranded DNA and activates unfolded protein response (UPR) signaling via the disruption of the TREX1-BiP/GRP78 interaction. In TREX1 knockdown cells, the activation of ER stress signaling disrupted ER Ca2+ homeostasis via the ERO1α-IP3R1-CaMKII pathway, leading to neuronal cell death. Moreover, TREX1 knockdown dysregulated the Golgi-microtubule network through Golgi fragmentation and decreased Ac-α-tubulin levels, contributing to neuronal injury. These alterations were also observed in neuronal cells harboring a TREX1 mutation (V91M) that has been identified in hereditary spastic paraplegia (HSP) patients in Korea. Notably, this mutation leads to defects in the TREX1-BiP/GRP78 interaction and mislocalization of TREX1 from the ER and possible disruption of the Golgi-microtubule network. In summary, the current study reveals TREX1 as a novel regulator of the BiP/GRP78 interaction and shows that TREX1 deficiency promotes ER stress-mediated neuronal cell death, which indicates that TREX1 may hold promise as a therapeutic target for neurodegenerative diseases such as HSP.

Keywords:  BiP/GRP78; Ca2+ homeostasis; ER stress; Hereditary spastic paraplegia; Neuronal cells; Three prime repair exonuclease 1

DOI:  https://doi.org/10.1007/s12035-021-02631-3
Methods Mol Biol. 2022 ;2378 141-168

Assays to Study IRE1 Activation and Signaling.

Paloma Moraga, Raul Aravena, Hery Urra, Claudio Hetz.

  The endoplasmic reticulum (ER) stress sensor IRE1 is a a major player of the unfolded protein response (UPR), the main pathway driving adaptation processes to restore proteostasis.  In addition, overactivation of IRE1 signaling contributes to a variety of pathologies including diabetes, neurodegenerative diseases, and cancer. Under ER stress, IRE1 auto-transphosphorylates and oligomerizes, triggering the activation of its endoribonuclease domain located in the cytosolic region. Active IRE1 catalyzes the splicing of the mRNA encoding for the XBP1 transcription factor, in addition to degrade several RNAs through a process known as regulated IRE1-dependent decay of mRNA (RIDD). Besides its role as an UPR transducer, several posttranslational modifications and protein-protein interactions can regulate IRE1 activity and modulate its signaling in the absence of stress. Thus, investigating the function of IRE1 in physiology and disease requires the use of complementary approaches. Here, we provide detailed protocols to perform four different assays to study IRE1 activation and signaling: (i) Phos-tag gels to evaluate the phosphorylation status of IRE1, (ii) microscopy using TREX-IRE1-GFP cells to measure IRE1 oligomerization, (iii) conventional RT-PCR to assess XBP1 mRNA processing, and (iv) quantitative PCR to determine the levels of canonical UPR target genes and the degradation of several mRNAs that are target of RIDD. We propose to use these experimental strategies as "gold standards" to study IRE1 signaling.

Keywords:  ER stress; IRE1 activation; IRE1 oligomerization; IRE1 phosphorylation; Regulated IRE1-dependent decay (RIDD); Unfolded protein response; XBP1 mRNA splicing

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_10
Mol Biol Cell. 2022 Jan 05. mbcE21080418

The Transcriptional Response to Oxidative Stress is Independent of Stress-Granule Formation.

Amanjot Singh, Arvind Reddy Kandi, Deepa Jayaprakashappa, Guillaume Thuery, Devam J Purohit, Joern Huelsmeier, Rashi Singh, Sai Shruti Pothapragada, Mani Ramaswami, Baskar Bakthavachalu.

Cells respond to stress with translational arrest, robust transcriptional changes, and transcription-independent formation of mRNP assemblies termed stress granules (SGs). Despite considerable interest in the role of SGs in oxidative, unfolded-protein and viral stress responses, whether and how SGs contribute to stress-induced transcription has not been rigorously examined. To address this, we characterized transcriptional changes in Drosophila S2 cells induced by acute oxidative-stress and assessed how these were altered under conditions that disrupted SG assembly. Oxidative stress for 3-hours predominantly resulted in induction or upregulation of stress-responsive mRNAs whose levels peaked during recovery after stress cessation. The stress-transcriptome is enriched in mRNAs coding for chaperones, including HSP70s, small heat shock proteins, glutathione transferases, and several non-coding RNAs. Oxidative stress also induced cytoplasmic SGs that disassembled 3-hours after stress cessation. As expected, RNAi-mediated knockdown of the conserved G3BP1/Rasputin protein inhibited SG assembly. However, this disruption had no significant effect on the stress-induced transcriptional response or stress-induced translational arrest. Thus, SG assembly and stress-induced gene expression alterations appear to be driven by distinctive signaling processes. We suggest that while SG assembly represents a fast, transient mechanism, the transcriptional response enables a slower, longer-lasting mechanism for adaptation to and recovery from cell stress.

DOI: https://doi.org/10.1091/mbc.E21-08-0418
Methods Mol Biol. 2022 ;2378 233-245

Detection of PERK Signaling in the Central Nervous System.

Zhixin Lei, Sarrabeth Stone, Wensheng Lin.

  In response to endoplasmic reticulum (ER) stress, activation of pancreatic ER kinase (PERK) signaling adapts cells to stressful conditions by phosphorylating eukaryotic translation initiation factor 2α (eIF2α). Phosphorylation of eIF2α inhibits global protein translation but stimulates the expression of numerous stress-responsive genes by inducing the transcription factor ATF4. A large number of studies have shown that activation of PERK signaling has beneficial or detrimental effects in various diseases of the central nervous system (CNS), including neurodegenerative diseases, myelin disorders, CNS injuries, among others. This chapter is devoted to describing the practical methods for the detection of PERK signaling in CNS diseases.

Keywords:  ATF4; CHOP; GADD34; Immunofluorescence; Neuron; Oligodendrocyte; PERK; Phosphorylated eIF2α; Real-time PCR; Western blot

DOI:  https://doi.org/10.1007/978-1-0716-1732-8_15