bims-nenemi 2022-10-23 papers

bims-nenemi

Biomed News

on Neuroinflammation, neurodegeneration and mitochondria

Issue of 2022‒10‒23
thirteen papers selected by
Marco Tigano
Thomas Jefferson University

Prohibitin 1 regulates mtDNA release and downstream inflammatory responses.
MTCH2 is a mitochondrial outer membrane protein insertase.
Mammalian oocytes store mRNAs in a mitochondria-associated membraneless compartment.
Human ZBP1 induces cell death-independent inflammatory signaling via RIPK3 and RIPK1.
The metabolite-controlled ubiquitin conjugase Ubc8 promotes mitochondrial protein import.
CRISPR/Cas9-mediated excision of ALS/FTD-causing hexanucleotide repeat expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro.
Mitochondrial DNA maintenance in Drosophila melanogaster.
Risk-associated single nucleotide polymorphisms of mitochondrial D-loop mediate imbalance of cytokines and redox in rheumatoid arthritis.
Silencing of the mitochondrial ribosomal protein L-24 gene activates the oxidative stress response in Caenorhabditis elegans.
Parkin coordinates mitochondrial lipid remodeling to execute mitophagy.
Double-Stranded RNA Induces Mortality in an MDA5-Mediated Type I Interferonopathy Model.
Pro-inflammatory RNA:DNA Hybrids Are p53 Independently Boosted by Hyperbaric Oxygen: a Subcellular Distribution Analysis by Automated Quantitative Imaging.
Protein synthesis inhibition and loss of homeostatic functions in astrocytes from an Alzheimer's disease mouse model: a role for ER-mitochondria interaction.

EMBO J. 2022 Oct 17. e111173

Prohibitin 1 regulates mtDNA release and downstream inflammatory responses.

Hao Liu, Hualin Fan, Pengcheng He, Haixia Zhuang, Xiao Liu, Meiting Chen, Wenwei Zhong, Yi Zhang, Cien Zhen, Yanling Li, Huilin Jiang, Tian Meng, Yiming Xu, Guojun Zhao, Du Feng.

  Exposure of mitochondrial DNA (mtDNA) to the cytosol activates innate immune responses. But the mechanisms by which mtDNA crosses the inner mitochondrial membrane are unknown. Here, we found that the inner mitochondrial membrane protein prohibitin 1 (PHB1) plays a critical role in mtDNA release by regulating permeability across the mitochondrial inner membrane. Loss of PHB1 results in alterations in mitochondrial integrity and function. PHB1-deficient macrophages, serum from myeloid-specific PHB1 KO (Phb1MyeKO) mice, and peripheral blood mononuclear cells from neonatal sepsis patients show increased interleukin-1β (IL-1β) levels. PHB1 KO mice are also intolerant of lipopolysaccharide shock. Phb1-depleted macrophages show increased cytoplasmic release of mtDNA and inflammatory responses. This process is suppressed by cyclosporine A and VBIT-4, which inhibit the mitochondrial permeability transition pore (mPTP) and VDAC oligomerization. Inflammatory stresses downregulate PHB1 expression levels in macrophages. Under normal physiological conditions, the inner mitochondrial membrane proteins, AFG3L2 and SPG7, are tethered to PHB1 to inhibit mPTP opening. Downregulation of PHB1 results in enhanced interaction between AFG3L2 and SPG7, mPTP opening, mtDNA release, and downstream inflammatory responses.

Keywords:  AFG3L2; MIMP; PHB; SPG7; mtDNA

DOI:  https://doi.org/10.15252/embj.2022111173
Science. 2022 Oct 21. 378(6617): 317-322

MTCH2 is a mitochondrial outer membrane protein insertase.

Alina Guna, Taylor A Stevens, Alison J Inglis, Joseph M Replogle, Theodore K Esantsi, Gayathri Muthukumar, Kelly C L Shaffer, Maxine L Wang, Angela N Pogson, Jeff J Jones, Brett Lomenick, Tsui-Fen Chou, Jonathan S Weissman, Rebecca M Voorhees.

In the mitochondrial outer membrane, α-helical transmembrane proteins play critical roles in cytoplasmic-mitochondrial communication. Using genome-wide CRISPR screens, we identified mitochondrial carrier homolog 2 (MTCH2), and its paralog MTCH1, and showed that it is required for insertion of biophysically diverse tail-anchored (TA), signal-anchored, and multipass proteins, but not outer membrane β-barrel proteins. Purified MTCH2 was sufficient to mediate insertion into reconstituted proteoliposomes. Functional and mutational studies suggested that MTCH2 has evolved from a solute carrier transporter. MTCH2 uses membrane-embedded hydrophilic residues to function as a gatekeeper for the outer membrane, controlling mislocalization of TAs into the endoplasmic reticulum and modulating the sensitivity of leukemia cells to apoptosis. Our identification of MTCH2 as an insertase provides a mechanistic explanation for the diverse phenotypes and disease states associated with MTCH2 dysfunction.

DOI: https://doi.org/10.1126/science.add1856
Science. 2022 Oct 21. 378(6617): eabq4835

Mammalian oocytes store mRNAs in a mitochondria-associated membraneless compartment.

Shiya Cheng, Gerrit Altmeppen, Chun So, Luisa M Welp, Sarah Penir, Torben Ruhwedel, Katerina Menelaou, Katarina Harasimov, Alexandra Stützer, Martyn Blayney, Kay Elder, Wiebke Möbius, Henning Urlaub, Melina Schuh.

Full-grown oocytes are transcriptionally silent and must stably maintain the messenger RNAs (mRNAs) needed for oocyte meiotic maturation and early embryonic development. However, where and how mammalian oocytes store maternal mRNAs is unclear. Here, we report that mammalian oocytes accumulate mRNAs in a mitochondria-associated ribonucleoprotein domain (MARDO). MARDO assembly around mitochondria was promoted by the RNA-binding protein ZAR1 and directed by an increase in mitochondrial membrane potential during oocyte growth. MARDO foci coalesced into hydrogel-like matrices that clustered mitochondria. Maternal mRNAs stored in the MARDO were translationally repressed. Loss of ZAR1 disrupted the MARDO, dispersed mitochondria, and caused a premature loss of MARDO-localized mRNAs. Thus, a mitochondria-associated membraneless compartment controls mitochondrial distribution and regulates maternal mRNA storage, translation, and decay to ensure fertility in mammals.

DOI: https://doi.org/10.1126/science.abq4835
EMBO Rep. 2022 Oct 21. e55839

Human ZBP1 induces cell death-independent inflammatory signaling via RIPK3 and RIPK1.

Ruoshi Peng, Chris Kedong Wang, Xuan Wang-Kan, Manja Idorn, Majken Kjaer, Felix Y Zhou, Berthe Katrine Fiil, Frederik Timmermann, Susana L Orozco, Julia McCarthy, Carol S Leung, Xin Lu, Katrin Bagola, Jan Rehwinkel, Andrew Oberst, Jonathan Maelfait, Søren R Paludan, Mads Gyrd-Hansen.

  ZBP1 is an interferon-induced cytosolic nucleic acid sensor that facilitates antiviral responses via RIPK3. Although ZBP1-mediated programmed cell death is widely described, whether and how it promotes inflammatory signaling is unclear. Here, we report a ZBP1-induced inflammatory signaling pathway mediated by K63- and M1-linked ubiquitin chains, which depends on RIPK1 and RIPK3 as scaffolds independently of cell death. In human HT29 cells, ZBP1 associated with RIPK1 and RIPK3 as well as ubiquitin ligases cIAP1 and LUBAC. ZBP1-induced K63- and M1-linked ubiquitination of RIPK1 and ZBP1 to promote TAK1- and IKK-mediated inflammatory signaling and cytokine production. Inhibition of caspase activity suppressed ZBP1-induced cell death but enhanced cytokine production in a RIPK1- and RIPK3 kinase activity-dependent manner. Lastly, we provide evidence that ZBP1 signaling contributes to SARS-CoV-2-induced cytokine production. Taken together, we describe a ZBP1-RIPK3-RIPK1-mediated inflammatory signaling pathway relayed by the scaffolding role of RIPKs and regulated by caspases, which may induce inflammation when ZBP1 is activated below the threshold needed to trigger a cell death response.

Keywords:  RIPK1; RIPK3; SARS-CoV-2; ZBP1; inflammatory signaling

DOI:  https://doi.org/10.15252/embr.202255839
Life Sci Alliance. 2023 Jan;pii: e202201526. [Epub ahead of print]6(1):

The metabolite-controlled ubiquitin conjugase Ubc8 promotes mitochondrial protein import.

Saskia Rödl, Fabian den Brave, Markus Räschle, Büsra Kizmaz, Svenja Lenhard, Carina Groh, Hanna Becker, Jannik Zimmermann, Bruce Morgan, Elke Richling, Thomas Becker, Johannes M Herrmann.

Mitochondria play a key role in cellular energy metabolism. Transitions between glycolytic and respiratory conditions induce considerable adaptations of the cellular proteome. These metabolism-dependent changes are particularly pronounced for the protein composition of mitochondria. Here, we show that the yeast cytosolic ubiquitin conjugase Ubc8 plays a crucial role in the remodeling process when cells transition from respiratory to fermentative conditions. Ubc8 is a conserved and well-studied component of the catabolite control system that is known to regulate the stability of gluconeogenic enzymes. Unexpectedly, we found that Ubc8 also promotes the assembly of the translocase of the outer membrane of mitochondria (TOM) and increases the levels of its cytosol-exposed receptor subunit Tom22. Ubc8 deficiency results in compromised protein import into mitochondria and reduced steady-state levels of mitochondrial proteins. Our observations show that Ubc8, which is controlled by the prevailing metabolic conditions, promotes the switch from glucose synthesis to glucose usage in the cytosol and induces the biogenesis of the mitochondrial TOM machinery to improve mitochondrial protein import during phases of metabolic transition.

DOI: https://doi.org/10.26508/lsa.202201526
Nat Commun. 2022 Oct 21. 13(1): 6286

CRISPR/Cas9-mediated excision of ALS/FTD-causing hexanucleotide repeat expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro.

Katharina E Meijboom, Abbas Abdallah, Nicholas P Fordham, Hiroko Nagase, Tomás Rodriguez, Carolyn Kraus, Tania F Gendron, Gopinath Krishnan, Rustam Esanov, Nadja S Andrade, Matthew J Rybin, Melina Ramic, Zachary D Stephens, Alireza Edraki, Meghan T Blackwood, Aydan Kahriman, Nils Henninger, Jean-Pierre A Kocher, Michael Benatar, Michael H Brodsky, Leonard Petrucelli, Fen-Biao Gao, Erik J Sontheimer, Robert H Brown, Zane Zeier, Christian Mueller.

A GGGGCC24+ hexanucleotide repeat expansion (HRE) in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), fatal neurodegenerative diseases with no cure or approved treatments that substantially slow disease progression or extend survival. Mechanistic underpinnings of neuronal death include C9ORF72 haploinsufficiency, sequestration of RNA-binding proteins in the nucleus, and production of dipeptide repeat proteins. Here, we used an adeno-associated viral vector system to deliver CRISPR/Cas9 gene-editing machineries to effectuate the removal of the HRE from the C9ORF72 genomic locus. We demonstrate successful excision of the HRE in primary cortical neurons and brains of three mouse models containing the expansion (500-600 repeats) as well as in patient-derived iPSC motor neurons and brain organoids (450 repeats). This resulted in a reduction of RNA foci, poly-dipeptides and haploinsufficiency, major hallmarks of C9-ALS/FTD, making this a promising therapeutic approach to these diseases.

DOI: https://doi.org/10.1038/s41467-022-33332-7
Biosci Rep. 2022 Oct 18. pii: BSR20211693. [Epub ahead of print]

Mitochondrial DNA maintenance in Drosophila melanogaster.

Ana P C Rodrigues, Audrey C Novaes, Grzegorz L Ciesielski, Marcos T Oliveira.

  All 37 mitochondrial DNA (mtDNA)-encoded genes involved with oxidative phosphorylation and intramitochondrial protein synthesis, and several nuclear-encoded genes involved with mtDNA replication, transcription, repair and recombination are conserved between the fruit fly Drosophilamelanogaster and mammals. This, in addition to its easy genetic tractability, has made Drosophila a useful model for our understanding of animal mtDNA maintenance and human mtDNA diseases. However, there are key differences between the Drosophila and mammalian systems that feature the diversity of mtDNA maintenance processes inside animal cells. Here, we review what is known about mtDNA maintenance in Drosophila, highlighting areas for which more research is warranted and providing a perspective preliminary in silico and in vivo analyses of the tissue specificity of mtDNA maintenance processes in this model organism. Our results suggest new roles (or the lack thereof) for well-known maintenance proteins, such as the helicase Twinkle and the accessory subunit of DNA polymerase γ, and for other Drosophila gene products that may even aid in shedding light on mtDNA maintenance in other animals. We hope to provide the reader some interesting paths that can be taken to help our community show how Drosophila may impact future mtDNA maintenance research.

Keywords:  DNA synthesis and repair; Drosophila melanogaster; mitochondria; nucleic acids

DOI:  https://doi.org/10.1042/BSR20211693
Int J Rheum Dis. 2022 Oct 17.

Risk-associated single nucleotide polymorphisms of mitochondrial D-loop mediate imbalance of cytokines and redox in rheumatoid arthritis.

Jingnan Zhang, Jingjing Zhang, Ruixue Lai, Chenxing Peng, Zhanjun Guo, Cuiju Wang.

  BACKGROUND: We have identified rheumatoid arthritis (RA) risk-associated single nucleotide polymorphisms (SNPs) in the mitochondrial displacement loop (D-loop) including the major alleles of nucleotides 195T/C, 16260C/T, and 16519C/T as well as the minor alleles of nucleotides 146T/C and 150C/T previously.OBJECTIVE: We evaluated the potential relationships of these SNPs with status for oxidative stress and inflammation cytokines.
METHODS: The DNA was extracted from blood samples of RA patients, and the SNPs of DNA D-loop were verified by polymerase chain reaction amplification and sequence analysis. Serum levels of inflammatory cytokines including interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-6, IL-10, and tumor necrosis factor-α (TNF-α) were determined by cytometric bead array. Plasma reactive oxygen species (ROS) levels were measured by fluorescent probe technology.
RESULTS: The RA risk-related allele 16519C was significantly associated with high IFN-γ levels (100.576 ± 11.769 vs 64.268 ± 8.199, 95% confidence interval [CI] -66.317 to -6.299, P = 0.018). This allele also associated with ROS at borderline statistics level (619.295 ± 36.687 vs 526.979 ± 25.896, 95% CI -186.145 to -1.513, P = 0.054). The subsequent analysis also showed that the ROS levels were positively correlated with IFN-γ levels (R = 0.291, P = 0.002). Further analysis showed that RA patients with high C-reactive protein levels displayed a higher ROS level (P = 0.001).
CONCLUSION: Our results imply that the 16519C allele of the mtDNA D-loop might promote ROS and IFN-γ levels by altering the replication and transcription of mtDNA, thereby modifying RA development.
REMARK: The potential relationships of RA-associated SNPs in the mitochondrial D-loop with status for oxidative stress and inflammation were evaluated. The 16519C allele of the mtDNA D-loop might promote ROS and IFN-γ levels by altering the replication and transcription of mtDNA to modify RA development.

Keywords:  cytokines; displacement loop; mitochondrial DNA; oxidative stress; rheumatoid arthritis

DOI:  https://doi.org/10.1111/1756-185X.14465
Biochim Biophys Acta Gen Subj. 2022 Oct 17. pii: S0304-4165(22)00173-8. [Epub ahead of print] 130255

Silencing of the mitochondrial ribosomal protein L-24 gene activates the oxidative stress response in Caenorhabditis elegans.

Graziella Ficociello, Emily Schifano, Michela Di Nottia, Alessandra Torraco, Rosalba Carrozzo, Daniela Uccelletti, Arianna Montanari.

  The mitochondrial translation machinery allows the synthesis of the mitochondrial-encoded subunits of the electron transport chain. Defects in this process lead to mitochondrial physiology failure; in humans, they are associated with early-onset, extremely variable and often fatal disorder. The use of a simple model to study the mitoribosomal defects is mandatory to overcome the difficulty to analyze the impact of pathological mutations in humans. In this paper we study in nematode Caenorhabditis elegans the silencing effect of the mrpl-24 gene, coding for the mitochondrial ribosomal protein L-24 (MRPL-24). This is a structural protein of the large subunit 39S of the mitoribosome and its effective physiological function is not completely elucidated. We have evaluated the nematode's fitness fault and investigated the mitochondrial defects associated with MRPL-24 depletion. The oxidative stress response activation due to the mitochondrial alteration has been also investigated as a compensatory physiological mechanism. For the first time, we demonstrated that MRPL-24 reduction increases the expression of detoxifying enzymes such as SOD-3 and GST-4 through the involvement of transcription factor SKN-1.BACKGROUND: In humans, mutations in genes encoding mitochondrial ribosomal proteins (MRPs) often cause early-onset, severe, fatal and extremely variable clinical defects. Mitochondrial ribosomal protein L-24 (MRPL24) is a structural protein of the large subunit 39S of the mitoribosome. It is highly conserved in different species and its effective physiological function is not completely elucidated.
METHODS: We characterized the MRPL24 functionality using the animal model Caenorhabditis elegans. We performed the RNA mediated interference (RNAi) by exposing the nematodes' embryos to double-stranded RNA (dsRNA) specific for the MRPL-24 coding sequence. We investigated for the first time in C. elegans, the involvement of the MRPL-24 on the nematode's fitness and its mitochondrial physiology.
RESULTS: Mrpl-24 silencing in C. elegans negatively affected the larval development, progeny production and body bending. The analysis of mitochondrial functionality revealed loss of mitochondrial network and impairment of mitochondrial functionality, as the decrease of oxygen consumption rate and the ROS production, as well as reduction of mitochondrial protein synthesis. Finally, the MRPL-24 depletion activated the oxidative stress response, increasing the expression levels of two detoxifying enzymes, SOD-3 and GST-4.
CONCLUSIONS: In C. elegans the MRPL-24 depletion activated the oxidative stress response. This appears as a compensatory mechanism to the alteration of the mitochondrial functionality and requires the involvement of transcription factor SKN-1.
GENERAL SIGNIFICANCE: C. elegans resulted in a good model for the study of mitochondrial disorders and its use as a simple and pluricellular organism could open interesting perspectives to better investigate the pathologic mechanisms underlying these devastating diseases.

Keywords:  Caenorhabditis elegans; MRPL-24 mitoribosomal protein; Mitochondrial translation; Oxidative stress response; SKN-1 transcription factor

DOI:  https://doi.org/10.1016/j.bbagen.2022.130255
EMBO Rep. 2022 Oct 18. e55191

Parkin coordinates mitochondrial lipid remodeling to execute mitophagy.

Chao-Chieh Lin, Jin Yan, Meghan D Kapur, Kristi L Norris, Cheng-Wei Hsieh, De Huang, Nicolas Vitale, Kah-Leong Lim, Ziqiang Guan, Xiao-Fan Wang, Jen-Tsan Chi, Wei-Yuan Yang, Tso-Pang Yao.

  Autophagy has emerged as the prime machinery for implementing organelle quality control. In the context of mitophagy, the ubiquitin E3 ligase Parkin tags impaired mitochondria with ubiquitin to activate autophagic degradation. Although ubiquitination is essential for mitophagy, it is unclear how ubiquitinated mitochondria activate autophagosome assembly locally to ensure efficient destruction. Here, we report that Parkin activates lipid remodeling on mitochondria targeted for autophagic destruction. Mitochondrial Parkin induces the production of phosphatidic acid (PA) and its subsequent conversion to diacylglycerol (DAG) by recruiting phospholipase D2 and activating the PA phosphatase, Lipin-1. The production of DAG requires mitochondrial ubiquitination and ubiquitin-binding autophagy receptors, NDP52 and optineurin (OPTN). Autophagic receptors, via Golgi-derived vesicles, deliver an autophagic activator, EndoB1, to ubiquitinated mitochondria. Inhibition of Lipin-1, NDP52/OPTN, or EndoB1 results in a failure to produce mitochondrial DAG, autophagosomes, and mitochondrial clearance, while exogenous cell-permeable DAG can induce autophagosome production. Thus, mitochondrial DAG production acts downstream of Parkin to enable the local assembly of autophagosomes for the efficient disposal of ubiquitinated mitochondria.

Keywords:  Lipin-1; PLD2; Parkin; diacylglycerol; mitophagy

DOI:  https://doi.org/10.15252/embr.202255191
J Immunol. 2022 Oct 17. pii: ji2200367. [Epub ahead of print]

Double-Stranded RNA Induces Mortality in an MDA5-Mediated Type I Interferonopathy Model.

Francine Lianne Emralino, Saya Satoh, Nobuhiro Sakai, Masamichi Takami, Fumihiko Takeuchi, Nan Yan, Frank Rutsch, Takashi Fujita, Hiroki Kato.

Gain-of-function mutations in the viral dsRNA sensor melanoma differentiation-associated protein 5 (MDA5) lead to autoimmune IFNopathies, including Singleton-Merten syndrome (SMS) and Aicardi-Goutières syndrome. However, much remains unclear regarding the mechanism of disease progression and how external factors such as infection or immune stimulation with vaccination can affect the immune response. With this aim, we generated mice with human MDA5 bearing the SMS-associated mutation R822Q (hM-R822Q). hM-R822Q transgenic (Tg) mice developed SMS-like heart fibrosis, aortic valve enlargement, and aortic calcification with a systemic IFN-stimulated gene signature resulting in the activation of the adaptive immune response. Although administration of the viral dsRNA mimic polyinosinic-polycytidylic acid [poly(I:C)] did not have remarkable effects on the cardiac phenotype, dramatic inflammation was observed in the intestines where IFN production was most elevated. Poly(I:C)-injected hM-R822Q Tg mice also developed lethal hypercytokinemia marked by massive IL-6 levels in the serum. Interrupting the IFN signaling through mitochondrial antiviral signaling protein or IFN-α/β receptor alleviated hM-R822Q-induced inflammation. Furthermore, inhibition of JAK signaling with tofacitinib reduced cytokine production and ameliorated mucosal damage, enabling the survival of poly(I:C)-injected hM-R822Q Tg mice. These findings demonstrate that the MDA5 R822Q mutant introduces a critical risk factor for uncontrollable inflammation on viral infection or vaccination.

DOI: https://doi.org/10.4049/jimmunol.2200367
Mol Imaging Biol. 2022 Oct 19.

Pro-inflammatory RNA:DNA Hybrids Are p53 Independently Boosted by Hyperbaric Oxygen: a Subcellular Distribution Analysis by Automated Quantitative Imaging.

Ilaria De Santis, Michele Zanoni, Sara Pignatta, Pasquale Longobardi, Anna Tesei, Alessandro Bevilacqua.

  PURPOSE: RNA:DNA hybrids are co-transcriptional products with acknowledged cytoplasmic pro-inflammatory role as activators of the cGAS-STING pathway. We recently proved them also as radiation-induced senescence messages for the abscopal effect mediation, demonstrating the need for a functional p53 for their production and release in A549 and H1299 tumour cells. However, little is known about their role under different stress conditions, especially in cancer cells.METHODS: In this work, we open the investigation making use of automated quantitative imaging to characterize the hybrid subcellular distribution in HeLa cells grown under different oxygen pressures or exposed to different ionizing radiation doses. After cell imaging by confocal fluorescent microscopy, we apply automated imaging methods developed on purpose to quantify hybrid foci and nuclear cluster intensity, regional and local density and dimension.
RESULTS: We show that alteration of culture oxygenation increases hybrid cytoplasmic presence, especially when caused by an hyperoxic environment, with evident hybrid gathering at the cell membrane. Ionizing radiations always fail to increase hybrids, in accordance with the absence of functional p53 in HeLa cells. However, dose-dependent effects are still evident and suggest a threshold dose of 7.5 Gy for remarkable hybrid reduction.
CONCLUSION: Together with our previous results, these data demonstrate for the first time that different types of stress can increase hybrid production in cancer cells and by at least two different pathways, one p53-dependent triggerable by ionizing radiations and one p53-independent triggerable by oxidative stress. Together, our findings provide a starting point for understanding hybrid role in tumour stress response.

Keywords:  Automated quantitative imaging; Cancer; Inflammation; Oxidative stress; Oxygen therapy; Radiotherapy; Tumour microenvironment

DOI:  https://doi.org/10.1007/s11307-022-01778-2
Cell Death Dis. 2022 Oct 18. 13(10): 878

Protein synthesis inhibition and loss of homeostatic functions in astrocytes from an Alzheimer's disease mouse model: a role for ER-mitochondria interaction.

Laura Tapella, Giulia Dematteis, Marianna Moro, Beatrice Pistolato, Elisa Tonelli, Virginia Vita Vanella, Daniele Giustina, Aleida La Forgia, Elena Restelli, Elettra Barberis, Tito Cali, Marisa Brini, Salvatore Villani, Erika Del Grosso, Mariagrazia Grilli, Marcello Manfredi, Marco Corazzari, Ambra A Grolla, Armando A Genazzani, Dmitry Lim.

Deregulation of protein synthesis and ER stress/unfolded protein response (ER stress/UPR) have been reported in astrocytes. However, the relationships between protein synthesis deregulation and ER stress/UPR, as well as their role in the altered homeostatic support of Alzheimer's disease (AD) astrocytes remain poorly understood. Previously, we reported that in astrocytic cell lines from 3xTg-AD mice (3Tg-iAstro) protein synthesis was impaired and ER-mitochondria distance was reduced. Here we show that impaired protein synthesis in 3Tg-iAstro is associated with an increase of p-eIF2α and downregulation of GADD34. Although mRNA levels of ER stress/UPR markers were increased two-three-fold, we found neither activation of PERK nor downstream induction of ATF4 protein. Strikingly, the overexpression of a synthetic ER-mitochondrial linker (EML) resulted in a reduced protein synthesis and augmented p-eIF2α without any effect on ER stress/UPR marker genes. In vivo, in hippocampi of 3xTg-AD mice, reduced protein synthesis, increased p-eIF2α and downregulated GADD34 protein were found, while no increase of p-PERK or ATF4 proteins was observed, suggesting that in AD astrocytes, both in vitro and in vivo, phosphorylation of eIF2α and impairment of protein synthesis are PERK-independent. Next, we investigated the ability of 3xTg-AD astrocytes to support metabolism and function of other cells of the central nervous system. Astrocyte-conditioned medium (ACM) from 3Tg-iAstro cells significantly reduced protein synthesis rate in primary hippocampal neurons. When added as a part of pericyte/endothelial cell (EC)/astrocyte 3D co-culture, 3Tg-iAstro, but not WT-iAstro, severely impaired formation and ramification of tubules, the effect, replicated by EML overexpression in WT-iAstro cells. Finally, a chemical chaperone 4-phenylbutyric acid (4-PBA) rescued protein synthesis, p-eIF2α levels in 3Tg-iAstro cells and tubulogenesis in pericyte/EC/3Tg-iAstro co-culture. Collectively, our results suggest that a PERK-independent, p-eIF2α-associated impairment of protein synthesis compromises astrocytic homeostatic functions, and this may be caused by the altered ER-mitochondria interaction.

DOI: https://doi.org/10.1038/s41419-022-05324-4