ABSTRACT
A recent case report described an individual who was a homozygous carrier of the APOE3 Christchurch (APOE3ch) mutation and resistant to autosomal dominant Alzheimer's Disease (AD) caused by a PSEN1-E280A mutation. Whether APOE3ch contributed to the protective effect remains unclear. We generated a humanized APOE3ch knock-in mouse and crossed it to an amyloid-ß (Aß) plaque-depositing model. We injected AD-tau brain extract to investigate tau seeding and spreading in the presence or absence of amyloid. Similar to the case report, APOE3ch expression resulted in peripheral dyslipidemia and a marked reduction in plaque-associated tau pathology. Additionally, we observed decreased amyloid response and enhanced microglial response around plaques. We also demonstrate increased myeloid cell phagocytosis and degradation of tau aggregates linked to weaker APOE3ch binding to heparin sulfate proteoglycans. APOE3ch influences the microglial response to Aß plaques, which suppresses Aß-induced tau seeding and spreading. The results reveal new possibilities to target Aß-induced tauopathy.
Subject(s)
Alzheimer Disease , Amyloid beta-Peptides , Apolipoprotein E3 , tau Proteins , Animals , Humans , Mice , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Amyloidogenic Proteins/metabolism , Apolipoprotein E3/genetics , Apolipoprotein E3/metabolism , Brain/metabolism , Disease Models, Animal , Mice, Transgenic , Microglia/metabolism , Plaque, Amyloid/metabolism , tau Proteins/genetics , tau Proteins/metabolism , Case Reports as TopicABSTRACT
Neurons build synaptic contacts using different protein combinations that define the specificity, function, and plasticity potential of synapses; however, the diversity of synaptic proteomes remains largely unexplored. We prepared synaptosomes from 7 different transgenic mouse lines with fluorescently labeled presynaptic terminals. Combining microdissection of 5 different brain regions with fluorescent-activated synaptosome sorting (FASS), we isolated and analyzed the proteomes of 18 different synapse types. We discovered â¼1,800 unique synapse-type-enriched proteins and allocated thousands of proteins to different types of synapses (https://syndive.org/). We identify shared synaptic protein modules and highlight the proteomic hotspots for synapse specialization. We reveal unique and common features of the striatal dopaminergic proteome and discover the proteome signatures that relate to the functional properties of different interneuron classes. This study provides a molecular systems-biology analysis of synapses and a framework to integrate proteomic information for synapse subtypes of interest with cellular or circuit-level experiments.
Subject(s)
Brain , Proteome , Synapses , Animals , Mice , Brain/metabolism , Mice, Transgenic , Proteome/metabolism , Proteomics , Synapses/metabolism , Synaptosomes/metabolismABSTRACT
Recent studies have begun to reveal critical roles for the brain's professional phagocytes, microglia, and their receptors in the control of neurotoxic amyloid beta (Aß) and myelin debris accumulation in neurodegenerative disease. However, the critical intracellular molecules that orchestrate neuroprotective functions of microglia remain poorly understood. In our studies, we find that targeted deletion of SYK in microglia leads to exacerbated Aß deposition, aggravated neuropathology, and cognitive defects in the 5xFAD mouse model of Alzheimer's disease (AD). Disruption of SYK signaling in this AD model was further shown to impede the development of disease-associated microglia (DAM), alter AKT/GSK3ß-signaling, and restrict Aß phagocytosis by microglia. Conversely, receptor-mediated activation of SYK limits Aß load. We also found that SYK critically regulates microglial phagocytosis and DAM acquisition in demyelinating disease. Collectively, these results broaden our understanding of the key innate immune signaling molecules that instruct beneficial microglial functions in response to neurotoxic material.
Subject(s)
Alzheimer Disease , Neurodegenerative Diseases , Animals , Mice , Alzheimer Disease/pathology , Amyloid beta-Peptides , Disease Models, Animal , Mice, Transgenic , Microglia/pathology , PhagocytosisABSTRACT
Although women experience significantly higher tau burden and increased risk for Alzheimer's disease (AD) than men, the underlying mechanism for this vulnerability has not been explained. Here, we demonstrate through in vitro and in vivo models, as well as human AD brain tissue, that X-linked ubiquitin specific peptidase 11 (USP11) augments pathological tau aggregation via tau deubiquitination initiated at lysine-281. Removal of ubiquitin provides access for enzymatic tau acetylation at lysines 281 and 274. USP11 escapes complete X-inactivation, and female mice and people both exhibit higher USP11 levels than males. Genetic elimination of usp11 in a tauopathy mouse model preferentially protects females from acetylated tau accumulation, tau pathology, and cognitive impairment. USP11 levels also strongly associate positively with tau pathology in females but not males. Thus, inhibiting USP11-mediated tau deubiquitination may provide an effective therapeutic opportunity to protect women from increased vulnerability to AD and other tauopathies.
Subject(s)
Alzheimer Disease , Tauopathies , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Animals , Brain/metabolism , Brain/pathology , Disease Models, Animal , Female , Humans , Male , Mice , Mice, Transgenic , Sex Characteristics , Tauopathies/genetics , Tauopathies/pathology , Thiolester Hydrolases/genetics , Ubiquitin-Specific Proteases , tau Proteins/geneticsABSTRACT
The persistence of CD4+ T cells carrying latent human immunodeficiency virus-1 (HIV-1) proviruses is the main barrier to a cure. New therapeutics to enhance HIV-1-specific immune responses and clear infected cells will probably be necessary to achieve reduction of the latent reservoir. In the present study, we report two single-chain diabodies (scDbs) that target the HIV-1 envelope protein (Env) and the human type III Fcγ receptor (CD16). We show that the scDbs promoted robust and HIV-1-specific natural killer (NK) cell activation and NK cell-mediated lysis of infected cells. Cocultures of CD4+ T cells from people with HIV-1 on antiretroviral therapy (ART) with autologous NK cells and the scDbs resulted in marked elimination of reservoir cells that was dependent on latency reversal. Treatment of human interleukin-15 transgenic NSG mice with one of the scDbs after ART initiation enhanced NK cell activity and reduced reservoir size. Thus, HIV-1-specific scDbs merit further evaluation as potential therapeutics for clearance of the latent reservoir.
Subject(s)
Antibodies, Bispecific , HIV-1 , Animals , Mice , Humans , Killer Cells, Natural , Cytotoxicity, Immunologic , Cell Death , Mice, TransgenicABSTRACT
Affinity-matured plasma cells (PCs) of varying lifespans are generated through a germinal center (GC) response. The developmental dynamics and genomic programs of antigen-specific PC precursors remain to be elucidated. Here, using a model antigen in mice, we demonstrate biphasic generation of PC precursors, with those generating long-lived bone marrow PCs preferentially produced in the late phase of GC response. Clonal tracing using single-cell RNA sequencing and B cell antigen receptor sequencing in spleen and bone marrow compartments, coupled with adoptive transfer experiments, reveals a new PC transition state that gives rise to functionally competent PC precursors. The latter undergo clonal expansion, dependent on inducible expression of TIGIT. We propose a model for the proliferation and programming of precursors of long-lived PCs, based on extended antigen encounters in the GC.
Subject(s)
Cell Differentiation , Germinal Center , Plasma Cells , Animals , Plasma Cells/immunology , Plasma Cells/metabolism , Mice , Germinal Center/immunology , Receptors, Antigen, B-Cell/metabolism , Receptors, Antigen, B-Cell/genetics , Mice, Inbred C57BL , Receptors, Immunologic/metabolism , Receptors, Immunologic/genetics , Mice, TransgenicABSTRACT
The immunological mechanisms underlying chronic colitis are poorly understood. T follicular helper (TFH) cells are critical in helping B cells during germinal center reactions. In a T cell transfer colitis model, a lymphoid structure composed of mature dendritic cells (DCs) and TFH cells was found within T cell zones of colonic lymphoid follicles. TFH cells were required for mature DC accumulation, the formation of DC-T cell clusters and colitis development. Moreover, DCs promoted TFH cell differentiation, contributing to colitis development. A lineage-tracing analysis showed that, following migration to the lamina propria, TFH cells transdifferentiated into long-lived pathogenic TH1 cells, promoting colitis development. Our findings have therefore demonstrated the reciprocal regulation of TFH cells and DCs in colonic lymphoid follicles, which is critical in chronic colitis pathogenesis.
Subject(s)
Cell Differentiation , Colitis , Dendritic Cells , T Follicular Helper Cells , Animals , Dendritic Cells/immunology , Colitis/immunology , Colitis/pathology , T Follicular Helper Cells/immunology , Mice , Cell Differentiation/immunology , Mice, Inbred C57BL , Disease Models, Animal , Th1 Cells/immunology , Colon/immunology , Colon/pathology , Mice, Knockout , Germinal Center/immunology , Mice, TransgenicABSTRACT
Current prophylactic human immunodeficiency virus 1 (HIV-1) vaccine research aims to elicit broadly neutralizing antibodies (bnAbs). Membrane-proximal external region (MPER)-targeting bnAbs, such as 10E8, provide exceptionally broad neutralization, but some are autoreactive. Here, we generated humanized B cell antigen receptor knock-in mouse models to test whether a series of germline-targeting immunogens could drive MPER-specific precursors toward bnAbs. We found that recruitment of 10E8 precursors to germinal centers (GCs) required a minimum affinity for germline-targeting immunogens, but the GC residency of MPER precursors was brief due to displacement by higher-affinity endogenous B cell competitors. Higher-affinity germline-targeting immunogens extended the GC residency of MPER precursors, but robust long-term GC residency and maturation were only observed for MPER-HuGL18, an MPER precursor clonotype able to close the affinity gap with endogenous B cell competitors in the GC. Thus, germline-targeting immunogens could induce MPER-targeting antibodies, and B cell residency in the GC may be regulated by a precursor-competitor affinity gap.
Subject(s)
Antibody Affinity , B-Lymphocytes , Germinal Center , HIV Antibodies , HIV-1 , Germinal Center/immunology , Animals , Mice , Humans , B-Lymphocytes/immunology , HIV-1/immunology , HIV Antibodies/immunology , Antibody Affinity/immunology , Antibodies, Neutralizing/immunology , HIV Infections/immunology , AIDS Vaccines/immunology , Receptors, Antigen, B-Cell/metabolism , Receptors, Antigen, B-Cell/immunology , Gene Knock-In Techniques , Mice, Transgenic , Broadly Neutralizing Antibodies/immunology , Mice, Inbred C57BLABSTRACT
Upregulation of diverse self-antigens that constitute components of the inflammatory response overlaps spatially and temporally with the emergence of pathogen-derived foreign antigens. Therefore, discrimination between these inflammation-associated self-antigens and pathogen-derived molecules represents a unique challenge for the adaptive immune system. Here, we demonstrate that CD8+ T cell tolerance to T cell-derived inflammation-associated self-antigens is efficiently induced in the thymus and supported by redundancy in cell types expressing these molecules. In addition to thymic epithelial cells, this included thymic eosinophils and innate-like T cells, a population that expressed molecules characteristic for all major activated T cell subsets. We show that direct T cell-to-T cell antigen presentation by minute numbers of innate-like T cells was sufficient to eliminate autoreactive CD8+ thymocytes. Tolerance to such effector molecules was of critical importance, as its breach caused by decreased thymic abundance of a single model inflammation-associated self-antigen resulted in autoimmune elimination of an entire class of effector T cells.
Subject(s)
Antigen Presentation , Autoantigens , CD8-Positive T-Lymphocytes , Inflammation , Thymocytes , Thymus Gland , Animals , Autoantigens/immunology , CD8-Positive T-Lymphocytes/immunology , Mice , Thymus Gland/immunology , Inflammation/immunology , Antigen Presentation/immunology , Thymocytes/immunology , Thymocytes/metabolism , Mice, Inbred C57BL , Immunity, Innate , Autoimmunity/immunology , Immune Tolerance/immunology , Mice, Transgenic , Mice, Knockout , Lymphocyte Activation/immunology , Eosinophils/immunologyABSTRACT
The durability of an antitumor immune response is mediated in part by the persistence of progenitor exhausted CD8+ T cells (Tpex). Tpex serve as a resource for replenishing effector T cells and preserve their quantity through self-renewal. However, it is unknown how T cell receptor (TCR) engagement affects the self-renewal capacity of Tpex in settings of continued antigen exposure. Here we use a Lewis lung carcinoma model that elicits either optimal or attenuated TCR signaling in CD8+ T cells to show that formation of Tpex in tumor-draining lymph nodes and their intratumoral persistence is dependent on optimal TCR engagement. Notably, attenuated TCR stimulation accelerates the terminal differentiation of optimally primed Tpex. This TCR-reinforced Tpex development and self-renewal is coupled to proximal positioning to dendritic cells and epigenetic imprinting involving increased chromatin accessibility at Egr2 and Tcf1 target loci. Collectively, this study highlights the critical function of TCR engagement in sustaining Tpex during tumor progression.
Subject(s)
CD8-Positive T-Lymphocytes , Carcinoma, Lewis Lung , Hepatocyte Nuclear Factor 1-alpha , Mice, Inbred C57BL , Receptors, Antigen, T-Cell , Animals , CD8-Positive T-Lymphocytes/immunology , Receptors, Antigen, T-Cell/metabolism , Receptors, Antigen, T-Cell/immunology , Mice , Carcinoma, Lewis Lung/immunology , Carcinoma, Lewis Lung/pathology , Carcinoma, Lewis Lung/metabolism , Hepatocyte Nuclear Factor 1-alpha/metabolism , Cell Differentiation/immunology , Dendritic Cells/immunology , Signal Transduction/immunology , Mice, Knockout , Lymphocyte Activation/immunology , Cell Self Renewal , Mice, Transgenic , Early Growth Response Protein 2ABSTRACT
Lung-resident macrophages, which include alveolar macrophages and interstitial macrophages (IMs), exhibit a high degree of diversity, generally attributed to different activation states, and often complicated by the influx of monocytes into the pool of tissue-resident macrophages. To gain a deeper insight into the functional diversity of IMs, here we perform comprehensive transcriptional profiling of resident IMs and reveal ten distinct chemokine-expressing IM subsets at steady state and during inflammation. Similar IM subsets that exhibited coordinated chemokine signatures and differentially expressed genes were observed across various tissues and species, indicating conserved specialized functional roles. Other macrophage types shared specific IM chemokine profiles, while also presenting their own unique chemokine signatures. Depletion of CD206hi IMs in Pf4creR26EYFP+DTR and Pf4creR26EYFPCx3cr1DTR mice led to diminished inflammatory cell recruitment, reduced tertiary lymphoid structure formation and fewer germinal center B cells in models of allergen- and infection-driven inflammation. These observations highlight the specialized roles of IMs, defined by their coordinated chemokine production, in regulating immune cell influx and organizing tertiary lymphoid tissue architecture.
Subject(s)
Chemokines , Macrophages , Animals , Mice , Chemokines/metabolism , Macrophages/immunology , Macrophages/metabolism , Lung/immunology , Mice, Inbred C57BL , Inflammation/immunology , Macrophages, Alveolar/immunology , Macrophages, Alveolar/metabolism , Organ Specificity/immunology , Gene Expression Profiling , Mice, Transgenic , Tertiary Lymphoid Structures/immunology , TranscriptomeABSTRACT
The isocortex and hippocampal formation (HPF) in the mammalian brain play critical roles in perception, cognition, emotion, and learning. We profiled â¼1.3 million cells covering the entire adult mouse isocortex and HPF and derived a transcriptomic cell-type taxonomy revealing a comprehensive repertoire of glutamatergic and GABAergic neuron types. Contrary to the traditional view of HPF as having a simpler cellular organization, we discover a complete set of glutamatergic types in HPF homologous to all major subclasses found in the six-layered isocortex, suggesting that HPF and the isocortex share a common circuit organization. We also identify large-scale continuous and graded variations of cell types along isocortical depth, across the isocortical sheet, and in multiple dimensions in hippocampus and subiculum. Overall, our study establishes a molecular architecture of the mammalian isocortex and hippocampal formation and begins to shed light on its underlying relationship with the development, evolution, connectivity, and function of these two brain structures.
Subject(s)
Hippocampus/cytology , Neocortex/cytology , Transcriptome/genetics , Animals , GABAergic Neurons/cytology , GABAergic Neurons/metabolism , Glutamic Acid/metabolism , Mice, Inbred C57BL , Mice, TransgenicABSTRACT
The mesencephalic locomotor region (MLR) is a key midbrain center with roles in locomotion. Despite extensive studies and clinical trials aimed at therapy-resistant Parkinson's disease (PD), debate on its function remains. Here, we reveal the existence of functionally diverse neuronal populations with distinct roles in control of body movements. We identify two spatially intermingled glutamatergic populations separable by axonal projections, mouse genetics, neuronal activity profiles, and motor functions. Most spinally projecting MLR neurons encoded the full-body behavior rearing. Loss- and gain-of-function optogenetic perturbation experiments establish a function for these neurons in controlling body extension. In contrast, Rbp4-transgene-positive MLR neurons project in an ascending direction to basal ganglia, preferentially encode the forelimb behaviors handling and grooming, and exhibit a role in modulating movement. Thus, the MLR contains glutamatergic neuronal subpopulations stratified by projection target exhibiting roles in action control not restricted to locomotion.
Subject(s)
Locomotion/physiology , Mesencephalon/anatomy & histology , Animals , Basal Ganglia/metabolism , Behavior, Animal , Female , Integrases/metabolism , Male , Mice, Inbred C57BL , Mice, Transgenic , Neurons/metabolism , Optogenetics , Retinol-Binding Proteins, Plasma/metabolism , Spinal Cord/metabolism , Transgenes , Vesicular Glutamate Transport Protein 2/metabolismABSTRACT
In motor neuroscience, state changes are hypothesized to time-lock neural assemblies coordinating complex movements, but evidence for this remains slender. We tested whether a discrete change from more autonomous to coherent spiking underlies skilled movement by imaging cerebellar Purkinje neuron complex spikes in mice making targeted forelimb-reaches. As mice learned the task, millimeter-scale spatiotemporally coherent spiking emerged ipsilateral to the reaching forelimb, and consistent neural synchronization became predictive of kinematic stereotypy. Before reach onset, spiking switched from more disordered to internally time-locked concerted spiking and silence. Optogenetic manipulations of cerebellar feedback to the inferior olive bi-directionally modulated neural synchronization and reaching direction. A simple model explained the reorganization of spiking during reaching as reflecting a discrete bifurcation in olivary network dynamics. These findings argue that to prepare learned movements, olivo-cerebellar circuits enter a self-regulated, synchronized state promoting motor coordination. State changes facilitating behavioral transitions may generalize across neural systems.
Subject(s)
Movement/physiology , Nerve Net/physiology , Action Potentials/physiology , Animals , Calcium/metabolism , Cerebellum/physiology , Cortical Synchronization , Forelimb/physiology , Interneurons/physiology , Learning , Mice, Inbred C57BL , Mice, Transgenic , Models, Neurological , Motor Activity/physiology , Olivary Nucleus/physiology , Optogenetics , Purkinje Cells/physiology , Stereotyped Behavior , Task Performance and AnalysisABSTRACT
How early events in effector T cell (TEFF) subsets tune memory T cell (TMEM) responses remains incompletely understood. Here, we systematically investigated metabolic factors in fate determination of TEFF and TMEM cells using in vivo pooled CRISPR screening, focusing on negative regulators of TMEM responses. We found that amino acid transporters Slc7a1 and Slc38a2 dampened the magnitude of TMEM differentiation, in part through modulating mTORC1 signaling. By integrating genetic and systems approaches, we identified cellular and metabolic heterogeneity among TEFF cells, with terminal effector differentiation associated with establishment of metabolic quiescence and exit from the cell cycle. Importantly, Pofut1 (protein-O-fucosyltransferase-1) linked GDP-fucose availability to downstream Notch-Rbpj signaling, and perturbation of this nutrient signaling axis blocked terminal effector differentiation but drove context-dependent TEFF proliferation and TMEM development. Our study establishes that nutrient uptake and signaling are key determinants of T cell fate and shape the quantity and quality of TMEM responses.
Subject(s)
Amino Acids/metabolism , CD8-Positive T-Lymphocytes/cytology , Immunologic Memory , Signal Transduction , Amino Acid Transport Systems/metabolism , Animals , CD8-Positive T-Lymphocytes/immunology , CRISPR-Cas Systems , Cell Cycle , Cell Differentiation , Disease Models, Animal , Female , Gene Knock-In Techniques , Lymphocytic Choriomeningitis/immunology , Male , Mice , Mice, Transgenic , Precursor Cells, T-Lymphoid/cytologyABSTRACT
Gasdermin B (GSDMB) belongs to a large family of pore-forming cytolysins that execute inflammatory cell death programs. While genetic studies have linked GSDMB polymorphisms to human disease, its function in the immunological response to pathogens remains poorly understood. Here, we report a dynamic host-pathogen conflict between GSDMB and the IpaH7.8 effector protein secreted by enteroinvasive Shigella flexneri. We show that IpaH7.8 ubiquitinates and targets GSDMB for 26S proteasome destruction. This virulence strategy protects Shigella from the bacteriocidic activity of natural killer cells by suppressing granzyme-A-mediated activation of GSDMB. In contrast to the canonical function of most gasdermin family members, GSDMB does not inhibit Shigella by lysing host cells. Rather, it exhibits direct microbiocidal activity through recognition of phospholipids found on Gram-negative bacterial membranes. These findings place GSDMB as a central executioner of intracellular bacterial killing and reveal a mechanism employed by pathogens to counteract this host defense system.
Subject(s)
Biomarkers, Tumor/metabolism , Host-Pathogen Interactions , Killer Cells, Natural/immunology , Neoplasm Proteins/metabolism , Pore Forming Cytotoxic Proteins/metabolism , Shigella flexneri/physiology , Ubiquitination , Animals , Bacterial Proteins/metabolism , Cardiolipins/metabolism , Cell Line , Cell Membrane/metabolism , Female , Granzymes/metabolism , Humans , Lipid A/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Microbial Viability , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Proteolysis , Substrate SpecificityABSTRACT
Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50 < 0.1 µg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Binding Sites, Antibody , CHO Cells , Chlorocebus aethiops , Cricetulus , Epitopes , Female , HEK293 Cells , Humans , Male , Mice , Mice, Transgenic , Models, Molecular , Protein Binding , Protein Structure, Tertiary , SARS-CoV-2/immunology , Vero CellsABSTRACT
The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro, and in vivo analyses, we report that topoisomerase 1 (TOP1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of topotecan (TPT), an FDA-approved TOP1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as 4 days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of TOP1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing TOP1 inhibitors for severe coronavirus disease 2019 (COVID-19) in humans.
Subject(s)
COVID-19 Drug Treatment , DNA Topoisomerases, Type I/metabolism , SARS-CoV-2/metabolism , Topoisomerase I Inhibitors/pharmacology , Topotecan/pharmacology , Animals , COVID-19/enzymology , COVID-19/pathology , Chlorocebus aethiops , Humans , Inflammation/drug therapy , Inflammation/enzymology , Inflammation/pathology , Inflammation/virology , Mesocricetus , Mice , Mice, Transgenic , THP-1 Cells , Vero CellsABSTRACT
In vivo cell fate conversions have emerged as potential regeneration-based therapeutics for injury and disease. Recent studies reported that ectopic expression or knockdown of certain factors can convert resident astrocytes into functional neurons with high efficiency, region specificity, and precise connectivity. However, using stringent lineage tracing in the mouse brain, we show that the presumed astrocyte-converted neurons are actually endogenous neurons. AAV-mediated co-expression of NEUROD1 and a reporter specifically and efficiently induces reporter-labeled neurons. However, these neurons cannot be traced retrospectively to quiescent or reactive astrocytes using lineage-mapping strategies. Instead, through a retrograde labeling approach, our results reveal that endogenous neurons are the source for these viral-reporter-labeled neurons. Similarly, despite efficient knockdown of PTBP1 in vivo, genetically traced resident astrocytes were not converted into neurons. Together, our results highlight the requirement of lineage-tracing strategies, which should be broadly applied to studies of cell fate conversions in vivo.
Subject(s)
Astrocytes/cytology , Cell Differentiation , Cell Lineage , Neurons/cytology , Animals , Astrocytes/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Brain/pathology , Brain Injuries/pathology , Cell Line, Tumor , Cellular Reprogramming , Dependovirus/metabolism , Down-Regulation , Gene Expression Regulation , Genes, Reporter , Glial Fibrillary Acidic Protein/genetics , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Homeodomain Proteins/metabolism , Humans , Integrases/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Neurons/metabolism , Polypyrimidine Tract-Binding Protein/metabolism , Promoter Regions, Genetic/genetics , Transcription Factors/metabolismABSTRACT
The biosafety level 3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research. Here, we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing ORF3 and envelope gene deletions, as well as mutated transcriptional regulator sequences, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. Thus, the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development.