Showing papers in "The EMBO Journal in 2021"

Autophagy in major human diseases

Abstract: Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

Molecular mechanisms and physiological functions of mitophagy.

Abstract: Degradation of mitochondria via a selective form of autophagy, named mitophagy, is a fundamental mechanism conserved from yeast to humans that regulates mitochondrial quality and quantity control. Mitophagy is promoted via specific mitochondrial outer membrane receptors, or ubiquitin molecules conjugated to proteins on the mitochondrial surface leading to the formation of autophagosomes surrounding mitochondria. Mitophagy-mediated elimination of mitochondria plays an important role in many processes including early embryonic development, cell differentiation, inflammation, and apoptosis. Recent advances in analyzing mitophagy in vivo also reveal high rates of steady-state mitochondrial turnover in diverse cell types, highlighting the intracellular housekeeping role of mitophagy. Defects in mitophagy are associated with various pathological conditions such as neurodegeneration, heart failure, cancer, and aging, further underscoring the biological relevance. Here, we review our current molecular understanding of mitophagy, and its physiological implications, and discuss how multiple mitophagy pathways coordinately modulate mitochondrial fitness and populations.

m 6 A RNA methylation: from mechanisms to therapeutic potential

Abstract: RNA carries a diverse array of chemical modifications that play important roles in the regulation of gene expression. N6 -methyladenosine (m6 A), installed onto mRNA by the METTL3/METTL14 methyltransferase complex, is the most prevalent mRNA modification. m6 A methylation regulates gene expression by influencing numerous aspects of mRNA metabolism, including pre-mRNA processing, nuclear export, decay, and translation. The importance of m6 A methylation as a mode of post-transcriptional gene expression regulation is evident in the crucial roles m6 A-mediated gene regulation plays in numerous physiological and pathophysiological processes. Here, we review current knowledge on the mechanisms by which m6 A exerts its functions and discuss recent advances that underscore the multifaceted role of m6 A in the regulation of gene expression. We highlight advances in our understanding of the regulation of m6 A deposition on mRNA and its context-dependent effects on mRNA decay and translation, the role of m6 A methylation of non-coding chromosomal-associated RNA species in regulating transcription, and the activities of the RNA demethylase FTO on diverse substrates. We also discuss emerging evidence for the therapeutic potential of targeting m6 A regulators in disease.

Dynamic EMT: a multi-tool for tumor progression.

Abstract: The process of epithelial-mesenchymal transition (EMT) is fundamental for embryonic morphogenesis. Cells undergoing it lose epithelial characteristics and integrity, acquire mesenchymal features, and become motile. In cancer, this program is hijacked to confer essential changes in morphology and motility that fuel invasion. In addition, EMT is increasingly understood to orchestrate a large variety of complementary cancer features, such as tumor cell stemness, tumorigenicity, resistance to therapy and adaptation to changes in the microenvironment. In this review, we summarize recent findings related to these various classical and non-classical functions, and introduce EMT as a true tumorigenic multi-tool, involved in many aspects of cancer. We suggest that therapeutic targeting of the EMT process will-if acknowledging these complexities-be a possibility to concurrently interfere with tumor progression on many levels.

TMPRSS2 expression dictates the entry route used by SARS-CoV-2 to infect host cells.

Abstract: SARS-CoV-2 is a newly emerged coronavirus that caused the global COVID-19 outbreak in early 2020. COVID-19 is primarily associated with lung injury, but many other clinical symptoms such as loss of smell and taste demonstrated broad tissue tropism of the virus. Early SARS-CoV-2-host cell interactions and entry mechanisms remain poorly understood. Investigating SARS-CoV-2 infection in tissue culture, we found that the protease TMPRSS2 determines the entry pathway used by the virus. In the presence of TMPRSS2, the proteolytic process of SARS-CoV-2 was completed at the plasma membrane, and the virus rapidly entered the cells within 10 min in a pH-independent manner. When target cells lacked TMPRSS2 expression, the virus was endocytosed and sorted into endolysosomes, from which SARS-CoV-2 entered the cytosol via acid-activated cathepsin L protease 40-60 min post-infection. Overexpression of TMPRSS2 in non-TMPRSS2 expressing cells abolished the dependence of infection on the cathepsin L pathway and restored sensitivity to the TMPRSS2 inhibitors. Together, our results indicate that SARS-CoV-2 infects cells through distinct, mutually exclusive entry routes and highlight the importance of TMPRSS2 for SARS-CoV-2 sorting into either pathway.

Opposing activities of IFITM proteins in SARS-CoV-2 infection.

Abstract: Interferon-induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. We show that SARS-CoV-2 Spike-pseudotyped virus and genuine SARS-CoV-2 infections are generally restricted by human and mouse IFITM1, IFITM2, and IFITM3, using gain- and loss-of-function approaches. Mechanistically, SARS-CoV-2 restriction occurred independently of IFITM3 S-palmitoylation, indicating a restrictive capacity distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the IFITM3 endocytosis-promoting YxxФ motif converted human IFITM3 into an enhancer of SARS-CoV-2 infection, and cell-to-cell fusion assays confirmed the ability of endocytic mutants to enhance Spike-mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which increases plasma membrane fusion versus endosome fusion of SARS-CoV-2, attenuated IFITM3 restriction and converted amphipathic helix mutants into infection enhancers. In sum, we uncover new pro- and anti-viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity-based mechanisms used for endosomal SARS-CoV-2 restriction.

An Organoid-derived Bronchioalveolar Model for SARS-CoV-2 Infection of Human Alveolar-type II-like Cells.

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), may result in acute respiratory distress syndrome (ARDS), multi-organ failure and death. The alveolar epithelium is a major target of the virus, but representative models to study virus host interactions in more detail are currently lacking. Here, we describe a human 2D air-liquid interface culture system which was characterized by confocal-, electron-microscopy and single cell mRNA expression analysis. In this model, alveolar cells, but also basal cells and rare neuroendocrine cells, are grown from 3D self-renewing fetal lung bud tip organoids. These cultures were readily infected by SARS-CoV-2 with mainly surfactant protein C-positive alveolar type II-like cells being targeted. Consequently, significant viral titers were detected and mRNA expression analysis revealed induction of type I/III interferon response program. Treatment of these cultures with a low dose of interferon lambda 1 reduced viral replication. Hence, these cultures represent an experimental model for SARS-CoV-2 infection and can be applied for drug screens.

SARS-CoV-2 sensing by RIG-I and MDA5 links epithelial infection to macrophage inflammation.

Abstract: SARS-CoV-2 infection causes broad-spectrum immunopathological disease, exacerbated by inflammatory co-morbidities. A better understanding of mechanisms underpinning virus-associated inflammation is required to develop effective therapeutics. Here, we discover that SARS-CoV-2 replicates rapidly in lung epithelial cells despite triggering a robust innate immune response through the activation of cytoplasmic RNA sensors RIG-I and MDA5. The inflammatory mediators produced during epithelial cell infection can stimulate primary human macrophages to enhance cytokine production and drive cellular activation. Critically, this can be limited by abrogating RNA sensing or by inhibiting downstream signalling pathways. SARS-CoV-2 further exacerbates the local inflammatory environment when macrophages or epithelial cells are primed with exogenous inflammatory stimuli. We propose that RNA sensing of SARS-CoV-2 in lung epithelium is a key driver of inflammation, the extent of which is influenced by the inflammatory state of the local environment, and that specific inhibition of innate immune pathways may beneficially mitigate inflammation-associated COVID-19.

Cell death pathways: intricate connections and disease implications

Abstract: Various forms of cell death have been identified over the last decades with each relying on a different subset of proteins for the activation and execution of their respective pathway(s). In addition to the three best characterized pathways-apoptosis, necroptosis, and pyroptosis-other forms of regulated cell death including autophagy-dependent cell death (ADCD), mitochondrial permeability transition pore (MPTP)-mediated necrosis, parthanatos, NETosis and ferroptosis, and their relevance for organismal homeostasis are becoming better understood. Importantly, it is increasingly clear that none of these pathways operate alone. Instead, a more complex picture is emerging with many pathways sharing components and signaling principles. Finally, a number of cell death regulators are implicated in human diseases and represent attractive therapeutic targets. Therefore, better understanding of physiological and mechanistic aspects of cell death signaling should yield improved reagents for addressing unmet medical needs.

SARS-CoV-2 Alpha, Beta, and Delta variants display enhanced Spike-mediated syncytia formation.

Abstract: Severe COVID-19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS-CoV-2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighboring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha (B.1.1.7) and Beta (B.1.351) spread and fusion in cell cultures, compared with the ancestral D614G strain. Alpha and Beta replicated similarly to D614G strain in Vero, Caco-2, Calu-3, and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Variant spike proteins displayed higher ACE2 affinity compared with D614G. Alpha, Beta, and D614G fusion was similarly inhibited by interferon-induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes modified fusogenicity, binding to ACE2 or recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS-CoV-2 emerging variants display enhanced syncytia formation.

A single-cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast.

Abstract: To examine global changes in breast heterogeneity across different states, we determined the single-cell transcriptomes of > 340,000 cells encompassing normal breast, preneoplastic BRCA1+/- tissue, the major breast cancer subtypes, and pairs of tumors and involved lymph nodes. Elucidation of the normal breast microenvironment revealed striking changes in the stroma of post-menopausal women. Single-cell profiling of 34 treatment-naive primary tumors, including estrogen receptor (ER)+ , HER2+ , and triple-negative breast cancers, revealed comparable diversity among cancer cells and a discrete subset of cycling cells. The transcriptomes of preneoplastic BRCA1+/- tissue versus tumors highlighted global changes in the immune microenvironment. Within the tumor immune landscape, proliferative CD8+ T cells characterized triple-negative and HER2+ cancers but not ER+ tumors, while all subtypes comprised cycling tumor-associated macrophages, thus invoking potentially different immunotherapy targets. Copy number analysis of paired ER+ tumors and lymph nodes indicated seeding by genetically distinct clones or mass migration of primary tumor cells into axillary lymph nodes. This large-scale integration of patient samples provides a high-resolution map of cell diversity in normal and cancerous human breast.

The calcium transporter ANNEXIN1 mediates cold-induced calcium signaling and freezing tolerance in plants.

Abstract: The transient elevation of cytosolic free calcium concentration ([Ca2+ ]cyt ) induced by cold stress is a well-established phenomenon; however, the underlying mechanism remains elusive. Here, we report that the Ca2+ -permeable transporter ANNEXIN1 (AtANN1) mediates cold-triggered Ca2+ influx and freezing tolerance in Arabidopsis thaliana. The loss of function of AtANN1 substantially impaired freezing tolerance, reducing the cold-induced [Ca2+ ]cyt increase and upregulation of the cold-responsive CBF and COR genes. Further analysis showed that the OST1/SnRK2.6 kinase interacted with and phosphorylated AtANN1, which consequently enhanced its Ca2+ transport activity, thereby potentiating Ca2+ signaling. Consistent with these results and freezing sensitivity of ost1 mutants, the cold-induced [Ca2+ ]cyt elevation in the ost1-3 mutant was reduced. Genetic analysis indicated that AtANN1 acts downstream of OST1 in responses to cold stress. Our data thus uncover a cascade linking OST1-AtANN1 to cold-induced Ca2+ signal generation, which activates the cold response and consequently enhances freezing tolerance in Arabidopsis.

ATP and cancer immunosurveillance

Abstract: While intracellular adenosine triphosphate (ATP) occupies a key position in the bioenergetic metabolism of all the cellular compartments that form the tumor microenvironment (TME), extracellular ATP operates as a potent signal transducer. The net effects of purinergic signaling on the biology of the TME depend not only on the specific receptors and cell types involved, but also on the activation status of cis- and trans-regulatory circuitries. As an additional layer of complexity, extracellular ATP is rapidly catabolized by ectonucleotidases, culminating in the accumulation of metabolites that mediate distinct biological effects. Here, we discuss the molecular and cellular mechanisms through which ATP and its degradation products influence cancer immunosurveillance, with a focus on therapeutically targetable circuitries.

Cancer evolution: Darwin and beyond

Abstract: Clinical and laboratory studies over recent decades have established branched evolution as a feature of cancer. However, while grounded in somatic selection, several lines of evidence suggest a Darwinian model alone is insufficient to fully explain cancer evolution. First, the role of macroevolutionary events in tumour initiation and progression contradicts Darwin's central thesis of gradualism. Whole-genome doubling, chromosomal chromoplexy and chromothripsis represent examples of single catastrophic events which can drive tumour evolution. Second, neutral evolution can play a role in some tumours, indicating that selection is not always driving evolution. Third, increasing appreciation of the role of the ageing soma has led to recent generalised theories of age-dependent carcinogenesis. Here, we review these concepts and others, which collectively argue for a model of cancer evolution which extends beyond Darwin. We also highlight clinical opportunities which can be grasped through targeting cancer vulnerabilities arising from non-Darwinian patterns of evolution.

Neutrophils induce paracrine telomere dysfunction and senescence in ROS-dependent manner.

Abstract: Cellular senescence is characterized by an irreversible cell cycle arrest as well as a pro-inflammatory phenotype, thought to contribute to aging and age-related diseases. Neutrophils have essential roles in inflammatory responses; however, in certain contexts their abundance is associated with a number of age-related diseases, including liver disease. The relationship between neutrophils and cellular senescence is not well understood. Here, we show that telomeres in non-immune cells are highly susceptible to oxidative damage caused by neighboring neutrophils. Neutrophils cause telomere dysfunction both in vitro and ex vivo in a ROS-dependent manner. In a mouse model of acute liver injury, depletion of neutrophils reduces telomere dysfunction and senescence. Finally, we show that senescent cells mediate the recruitment of neutrophils to the aged liver and propose that this may be a mechanism by which senescence spreads to surrounding cells. Our results suggest that interventions that counteract neutrophil-induced senescence may be beneficial during aging and age-related disease.

Cell softness regulates tumorigenicity and stemness of cancer cells.

Abstract: Identifying and sorting highly tumorigenic and metastatic tumor cells from a heterogeneous cell population is a daunting challenge. Here, we show that microfluidic devices can be used to sort marker-based heterogeneous cancer stem cells (CSC) into mechanically stiff and soft subpopulations. The isolated soft tumor cells ( 700 Pa) can form a tumor in immunocompetent mice with 100 cells per inoculation. Notably, only the soft, but not the stiff cells, isolated from CD133+ , ALDH+ , or side population CSCs, are able to form a tumor with only 100 cells in NOD-SCID or immunocompetent mice. The Wnt signaling protein BCL9L is upregulated in soft tumor cells and regulates their stemness and tumorigenicity. Clinically, BCL9L expression is correlated with a worse prognosis. Our findings suggest that the intrinsic softness is a unique marker of highly tumorigenic and metastatic tumor cells.

SARS-CoV-2 nucleocapsid suppresses host pyroptosis by blocking Gasdermin D cleavage.

Abstract: SARS-CoV-2 is an emerging coronavirus that causes dysfunctions in multiple human cells and tissues. Studies have looked at the entry of SARS-CoV-2 into host cells mediated by the viral spike protein and human receptor ACE2. However, less is known about the cellular immune responses triggered by SARS-CoV-2 viral proteins. Here, we show that the nucleocapsid of SARS-CoV-2 inhibits host pyroptosis by blocking Gasdermin D (GSDMD) cleavage. SARS-CoV-2-infected monocytes show enhanced cellular interleukin-1s (IL-1s) expression, but reduced IL-1s secretion. While SARS-CoV-2 infection promotes activation of the NLRP3 inflammasome and caspase-1, GSDMD cleavage and pyroptosis are inhibited in infected human monocytes. SARS-CoV-2 nucleocapsid protein associates with GSDMD in cells and inhibits GSDMD cleavage in vitro and in vivo. The nucleocapsid binds the GSDMD linker region and hinders GSDMD processing by caspase-1. These insights into how SARS-CoV-2 antagonizes cellular inflammatory responses may open new avenues for treating COVID-19 in the future.

Flavonol-mediated stabilization of PIN efflux complexes regulates polar auxin transport.

Abstract: The transport of auxin controls the rate, direction and localization of plant growth and development. The course of auxin transport is defined by the polar subcellular localization of the PIN proteins, a family of auxin efflux transporters. However, little is known about the composition and regulation of the PIN protein complex. Here, using blue-native PAGE and quantitative mass spectrometry, we identify native PIN core transport units as homo- and heteromers assembled from PIN1, PIN2, PIN3, PIN4 and PIN7 subunits only. Furthermore, we show that endogenous flavonols stabilize PIN dimers to regulate auxin efflux in the same way as does the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). This inhibitory mechanism is counteracted both by the natural auxin indole-3-acetic acid and by phosphomimetic amino acids introduced into the PIN1 cytoplasmic domain. Our results lend mechanistic insights into an endogenous control mechanism which regulates PIN function and opens the way for a deeper understanding of the protein environment and regulation of the polar auxin transport complex.

Neutralization of SARS-CoV-2 by highly potent, hyperthermostable, and mutation-tolerant nanobodies.

Abstract: Monoclonal anti-SARS-CoV-2 immunoglobulins represent a treatment option for COVID-19. However, their production in mammalian cells is not scalable to meet the global demand. Single-domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein, we isolated 45 infection-blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS-CoV-2 at 17-50 pM concentration (0.2-0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X-ray and cryo-EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune-escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such 'fold-promoting' nanobodies may allow for simplified production of vaccines and their adaptation to viral escape-mutations.

Clock component OsPRR73 positively regulates rice salt tolerance by modulating OsHKT2;1-mediated sodium homeostasis.

Abstract: The roles of clock components in salt stress tolerance remain incompletely characterized in rice. Here, we show that, among OsPRR (Oryza sativa Pseudo-Response Regulator) family members, OsPRR73 specifically confers salt tolerance in rice. Notably, the grain size and yield of osprr73 null mutants were significantly decreased in the presence of salt stress, with accumulated higher level of reactive oxygen species and sodium ions. RNA sequencing and biochemical assays identified OsHKT2;1, encoding a plasma membrane-localized Na+ transporter, as a transcriptional target of OsPRR73 in mediating salt tolerance. Correspondingly, null mutants of OsHKT2;1 displayed an increased tolerance to salt stress. Immunoprecipitation-mass spectrometry (IP-MS) assays further identified HDAC10 as nuclear interactor of OsPRR73 and co-repressor of OsHKT2;1. Consistently, H3K9ac histone marks at OsHKT2;1 promoter regions were significantly reduced in osprr73 mutant. Together, our findings reveal that salt-induced OsPRR73 expression confers salt tolerance by recruiting HDAC10 to transcriptionally repress OsHKT2;1, thus reducing cellular Na+ accumulation. This exemplifies a new molecular link between clock components and salt stress tolerance in rice.

Molecular basis of accessible plasma membrane cholesterol recognition by the GRAM domain of GRAMD1b.

Abstract: Cholesterol is essential for cell physiology. Transport of the "accessible" pool of cholesterol from the plasma membrane (PM) to the endoplasmic reticulum (ER) by ER-localized GRAMD1 proteins (GRAMD1a/1b/1c) contributes to cholesterol homeostasis. However, how cells detect accessible cholesterol within the PM remains unclear. We show that the GRAM domain of GRAMD1b, a coincidence detector for anionic lipids, including phosphatidylserine (PS), and cholesterol, possesses distinct but synergistic sites for sensing accessible cholesterol and anionic lipids. We find that a mutation within the GRAM domain of GRAMD1b that is associated with intellectual disability in humans specifically impairs cholesterol sensing. In addition, we identified another point mutation within this domain that enhances cholesterol sensitivity without altering its PS sensitivity. Cell-free reconstitution and cell-based assays revealed that the ability of the GRAM domain to sense accessible cholesterol regulates membrane tethering and determines the rate of cholesterol transport by GRAMD1b. Thus, cells detect the codistribution of accessible cholesterol and anionic lipids in the PM and fine-tune the non-vesicular transport of PM cholesterol to the ER via GRAMD1s.

Sealing holes in cellular membranes.

Abstract: The compartmentalization of eukaryotic cells, which is essential for their viability and functions, is ensured by single or double bilayer membranes that separate the cell from the exterior and form boundaries between the cell's organelles and the cytosol. Nascent nuclear envelopes and autophagosomes, which both are enveloped by double membranes, need to be sealed during the late stage of their biogenesis. On the other hand, the integrity of cellular membranes such as the plasma membrane, lysosomes and the nuclear envelope can be compromised by pathogens, chemicals, radiation, inflammatory responses and mechanical stress. There are cellular programmes that restore membrane integrity after injury. Here, we review cellular mechanisms that have evolved to maintain membrane integrity during organelle biogenesis and after injury, including membrane scission mediated by the endosomal sorting complex required for transport (ESCRT), vesicle patching and endocytosis.

Fungal-induced glycolysis in macrophages promotes colon cancer by enhancing innate lymphoid cell secretion of IL-22.

Abstract: Incorporation of microbiome data has recently become important for prevention, diagnosis, and treatment of colorectal cancer, and several species of bacteria were shown to be associated with carcinogenesis. However, the role of commensal fungi in colon cancer remains poorly understood. Here, we report that mice lacking the c-type lectin Dectin-3 (Dectin-3-/- ) show increased tumorigenesis and Candida albicans burden upon chemical induction. Elevated C. albicans load triggered glycolysis in macrophages and interleukin-7 (IL-7) secretion. IL-7 induced IL-22 production in RORγt+ (group 3) innate lymphoid cells (ILC3s) via aryl hydrocarbon receptor and STAT3. Consistently, IL-22 frequency in tumor tissues of colon cancer patients positively correlated with fungal burden, indicating the relevance of this regulatory axis in human disease. These results establish a C. albicans-driven crosstalk between macrophages and innate lymphoid cells in the intestine and expand our understanding on how commensal mycobiota regulate host immunity and promote tumorigenesis.

Mapping the origin and fate of myeloid cells in distinct compartments of the eye by single-cell profiling.

Abstract: Similar to the brain, the eye is considered an immune-privileged organ where tissue-resident macrophages provide the major immune cell constituents However, little is known about spatially restricted macrophage subsets within different eye compartments with regard to their origin, function, and fate during health and disease Here, we combined single-cell analysis, fate mapping, parabiosis, and computational modeling to comprehensively examine myeloid subsets in distinct parts of the eye during homeostasis This approach allowed us to identify myeloid subsets displaying diverse transcriptional states During choroidal neovascularization, a typical hallmark of neovascular age-related macular degeneration (AMD), we recognized disease-specific macrophage subpopulations with distinct molecular signatures Our results highlight the heterogeneity of myeloid subsets and their dynamics in the eye that provide new insights into the innate immune system in this organ which may offer new therapeutic targets for ophthalmological diseases

BRCA2 promotes DNA-RNA hybrid resolution by DDX5 helicase at DNA breaks to facilitate their repair‡.

Abstract: The BRCA2 tumor suppressor is a DNA double-strand break (DSB) repair factor essential for maintaining genome integrity. BRCA2-deficient cells spontaneously accumulate DNA-RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood. Here we identified the DEAD-box RNA helicase DDX5 as a BRCA2-interacting protein. DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs, and this association is enhanced by BRCA2. Notably, BRCA2 stimulates the DNA-RNA hybrid-unwinding activity of DDX5 helicase. An impaired BRCA2-DDX5 interaction, as observed in cells expressing the breast cancer variant BRCA2-T207A, reduces the association of DDX5 with DNA-RNA hybrids, decreases the number of RPA foci, and alters the kinetics of appearance of RAD51 foci upon irradiation. Our findings are consistent with DNA-RNA hybrids constituting an impediment for the repair of DSBs by homologous recombination and reveal BRCA2 and DDX5 as active players in their removal.

Modulation of plant root growth by nitrogen source-defined regulation of polar auxin transport.

Abstract: Availability of the essential macronutrient nitrogen in soil plays a critical role in plant growth, development, and impacts agricultural productivity. Plants have evolved different strategies for sensing and responding to heterogeneous nitrogen distribution. Modulation of root system architecture, including primary root growth and branching, is among the most essential plant adaptions to ensure adequate nitrogen acquisition. However, the immediate molecular pathways coordinating the adjustment of root growth in response to distinct nitrogen sources, such as nitrate or ammonium, are poorly understood. Here, we show that growth as manifested by cell division and elongation is synchronized by coordinated auxin flux between two adjacent outer tissue layers of the root. This coordination is achieved by nitrate-dependent dephosphorylation of the PIN2 auxin efflux carrier at a previously uncharacterized phosphorylation site, leading to subsequent PIN2 lateralization and thereby regulating auxin flow between adjacent tissues. A dynamic computer model based on our experimental data successfully recapitulates experimental observations. Our study provides mechanistic insights broadening our understanding of root growth mechanisms in dynamic environments.

Ythdf is a N6‐methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila

Abstract: N6-methyladenosine (m6 A) regulates a variety of physiological processes through modulation of RNA metabolism. This modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m6 A alters fly behavior, albeit the underlying molecular mechanism and the role of m6 A during nervous system development have remained elusive. Here we find that impairment of the m6 A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. We identify Ythdf as the main m6 A reader in the nervous system, being required to limit axonal growth. Mechanistically, we show that the m6 A reader Ythdf directly interacts with Fmr1, the fly homolog of Fragile X mental retardation RNA binding protein (FMRP), to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m6 A pathway controls development of the nervous system and modulates Fmr1 target transcript selection.

Viral nucleoprotein antibodies activate TRIM21 and induce T cell immunity.

Abstract: Nucleoprotein (N) is an immunodominant antigen in many enveloped virus infections. While the diagnostic value of anti-N antibodies is clear, their role in immunity is not. This is because while they are non-neutralising, they somehow clear infection by coronavirus, influenza and LCMV in vivo. Here, we show that anti-N immune protection is mediated by the cytosolic Fc receptor and E3 ubiquitin ligase TRIM21. Exploiting LCMV as a model system, we demonstrate that TRIM21 uses anti-N antibodies to target N for cytosolic degradation and generate cytotoxic T cells (CTLs) against N peptide. These CTLs rapidly eliminate N-peptide-displaying cells and drive efficient viral clearance. These results reveal a new mechanism of immune synergy between antibodies and T cells and highlights N as an important vaccine target.

Best practices for the visualization, mapping, and manipulation of R-loops

Abstract: R-loops represent an abundant class of large non-B DNA structures in genomes. Even though they form transiently and at modest frequencies, interfering with R-loop formation or dissolution has significant impacts on genome stability. Addressing the mechanism(s) of R-loop-mediated genome destabilization requires a precise characterization of their distribution in genomes. A number of independent methods have been developed to visualize and map R-loops, but their results are at times discordant, leading to confusion. Here, we review the main existing methodologies for R-loop mapping and assess their limitations as well as the robustness of existing datasets. We offer a set of best practices to improve the reproducibility of maps, hoping that such guidelines could be useful for authors and referees alike. Finally, we propose a possible resolution for the apparent contradictions in R-loop mapping outcomes between antibody-based and RNase H1-based mapping approaches.

Gut inflammation triggers C/EBPβ/δ-secretase-dependent gut-to-brain propagation of Aβ and Tau fibrils in Alzheimer's disease.

Abstract: Inflammation plays an important role in the pathogenesis of Alzheimer's disease (AD). Some evidence suggests that misfolded protein aggregates found in AD brains may have originated from the gut, but the mechanism underlying this phenomenon is not fully understood. C/EBPβ/δ-secretase signaling in the colon was investigated in a 3xTg AD mouse model in an age-dependent manner. We applied chronic administration of 1% dextran sodium sulfate (DSS) to trigger gut leakage or colonic injection of Aβ or Tau fibrils or AD patient brain lysates in 3xTg mice and combined it with excision/cutting of the gut-brain connecting vagus nerve (vagotomy), in order to explore the role of the gut-brain axis in the development of AD-like pathologies and to monitor C/EBPβ/δ-secretase signaling under those conditions. We found that C/EBPβ/δ-secretase signaling is temporally activated in the gut of AD patients and 3xTg mice, initiating formation of Aβ and Tau fibrils that spread to the brain. DSS treatment promotes gut leakage and facilitates AD-like pathologies in both the gut and the brain of 3xTg mice in a C/EBPβ/δ-secretase-dependent manner. Vagotomy selectively blunts this signaling, attenuates Aβ and Tau pathologies, and restores learning and memory. Aβ or Tau fibrils or AD patient brain lysates injected into the colon propagate from the gut into the brain via the vagus nerve, triggering AD pathology and cognitive dysfunction. The results indicate that inflammation activates C/EBPβ/δ-secretase and initiates AD-associated pathologies in the gut, which are subsequently transmitted to the brain via the vagus nerve.