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Microglia are the resident macrophages of the central nervous system (CNS). Their phagocytic activity is central during brain development and homeostasis-and in a plethora of brain pathologies. However, little is known about the composition, dynamics, and function of human microglial phagosomes under homeostatic and pathological conditions. Here, we developed a method for rapid isolation of pure and intact phagosomes from human pluripotent stem cell-derived microglia under various in vitro conditions, and from human brain biopsies, for unbiased multiomic analysis. Phagosome profiling revealed that microglial phagosomes were equipped to sense minute changes in their environment and were highly dynamic. We detected proteins involved in synapse homeostasis, or implicated in brain pathologies, and identified the phagosome as the site where quinolinic acid was stored and metabolized for de novo nicotinamide adenine dinucleotide (NAD+) generation in the cytoplasm. Our findings highlight the central role of phagosomes in microglial functioning in the healthy and diseased brain.
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Cells must adapt to environmental changes to maintain homeostasis. One of the most striking environmental adaptations is entry into hibernation during which core body temperature can decrease from 37°C to as low at 4°C. How mammalian cells, which evolved to optimally function within a narrow range of temperatures, adapt to this profound decrease in temperature remains poorly understood. In this study, we conducted the first genome-scale CRISPR-Cas9 screen in cells derived from Syrian hamster, a facultative hibernator, as well as human cells to investigate the genetic basis of cold tolerance in a hibernator and a non-hibernator in an unbiased manner. Both screens independently revealed glutathione peroxidase 4 (GPX4), a selenium-containing enzyme, and associated proteins as critical for cold tolerance. We utilized genetic and pharmacological approaches to demonstrate that GPX4 is active in the cold and its catalytic activity is required for cold tolerance. Furthermore, we show that the role of GPX4 as a suppressor of cold-induced cell death extends across hibernating species, including 13-lined ground squirrels and greater horseshoe bats, highlighting the evolutionary conservation of this mechanism of cold tolerance. This study identifies GPX4 as a central modulator of mammalian cold tolerance and advances our understanding of the evolved mechanisms by which cells mitigate cold-associated damage-one of the most common challenges faced by cells and organisms in nature.
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BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disease, following Alzheimer's. It is characterized by the aggregation of α-synuclein into Lewy bodies and Lewy neurites in the brain. Microglia-driven neuroinflammation may contribute to neuronal death in PD, however the exact role of microglia remains unclear and has been understudied. The A53T mutation in the gene coding for α-synuclein has been linked to early-onset PD, and exposure to A53T-mutant human α-synuclein increases the potential for inflammation of murine microglia. To date, its effect has not been studied in human microglia. METHODS: Here, we used 2-dimensional cultures of human iPSC-derived microglia and transplantation of these cells into the mouse brain to assess the cell-autonomous effects of the A53T mutation on human microglia. RESULTS: We found that A53T-mutant human microglia had an intrinsically increased propensity towards pro-inflammatory activation upon inflammatory stimulus. Additionally, transplanted A53T mutant microglia showed a strong decrease in catalase expression in non-inflammatory conditions, and increased oxidative stress. CONCLUSIONS: Our results indicate that A53T mutant human microglia display cell-autonomous phenotypes that may worsen neuronal damage in early-onset PD.
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The heterogeneity of protein-rich inclusions and its significance in neurodegeneration is poorly understood. Standard patient-derived iPSC models develop inclusions neither reproducibly nor in a reasonable time frame. Here, we developed screenable iPSC "inclusionopathy" models utilizing piggyBac or targeted transgenes to rapidly induce CNS cells that express aggregation-prone proteins at brain-like levels. Inclusions and their effects on cell survival were trackable at single-inclusion resolution. Exemplar cortical neuron α-synuclein inclusionopathy models were engineered through transgenic expression of α-synuclein mutant forms or exogenous seeding with fibrils. We identified multiple inclusion classes, including neuroprotective p62-positive inclusions versus dynamic and neurotoxic lipid-rich inclusions, both identified in patient brains. Fusion events between these inclusion subtypes altered neuronal survival. Proteome-scale α-synuclein genetic- and physical-interaction screens pinpointed candidate RNA-processing and actin-cytoskeleton-modulator proteins like RhoA whose sequestration into inclusions could enhance toxicity. These tractable CNS models should prove useful in functional genomic analysis and drug development for proteinopathies.
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Microglia play key roles in shaping synaptic connectivity during neural circuits development. Whether microglia display human-specific features of structural and functional maturation is currently unknown. We show that the ancestral gene SRGAP2A and its human-specific (HS) paralogs SRGAP2B/C are not only expressed in cortical neurons but are the only HS gene duplications expressed in human microglia. Here, using combination of xenotransplantation of human induced pluripotent stem cell (hiPSC)-derived microglia and mouse genetic models, we demonstrate that (1) HS SRGAP2B/C are necessary and sufficient to induce neotenic features of microglia structural and functional maturation in a cell-autonomous manner, and (2) induction of SRGAP2-dependent neotenic features of microglia maturation non-cell autonomously impacts synaptic development in cortical pyramidal neurons. Our results reveal that, during human brain evolution, human-specific genes SRGAP2B/C coordinated the emergence of neotenic features of synaptic development by acting as genetic modifiers of both neurons and microglia.
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Mutations in the methyl-DNA-binding protein MECP2 cause the neurodevelopmental disorder Rett syndrome (RTT). How MECP2 contributes to transcriptional regulation in normal and disease states is unresolved; it has been reported to be an activator and a repressor. We describe here the first integrated CUT&Tag, transcriptome, and proteome analyses using human neurons with wild-type (WT) and mutant MECP2 molecules. MECP2 occupies CpG-rich promoter-proximal regions in over four thousand genes in human neurons, including a plethora of autism risk genes, together with RNA polymerase II (RNA Pol II). MECP2 directly interacts with RNA Pol II, and genes occupied by both proteins showed reduced expression in neurons with MECP2 patient mutations. We conclude that MECP2 acts as a positive cofactor for RNA Pol II gene expression at many neuronal genes that harbor CpG islands in promoter-proximal regions and that RTT is due, in part, to the loss of gene activity of these genes in neurons.
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Proteína 2 de Unión a Metil-CpG , Neuronas , ARN Polimerasa II , Transcripción Genética , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Proteína 2 de Unión a Metil-CpG/genética , Humanos , Neuronas/metabolismo , Regiones Promotoras Genéticas , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Islas de CpG/genética , Mutación , Regulación de la Expresión Génica/genéticaRESUMEN
Bat-borne viruses have attracted considerable research, especially in relation to the Covid-19 pandemic. Although bats can carry multiple zoonotic viruses that are lethal to many mammalian species, they appear to be asymptomatic to viral infection despite the high viral loads contained in their bodies. There are several differences between bats and other mammals. One of the major differences between bats and other mammals is the bats' ability to fly, which is believed to have induced evolutionary changes. It may have also favoured them as suitable hosts for viruses. This is related to their tolerance to viral infection. Innate immunity is the first line of defence against viral infection, but bats have metamorphosed the type of responses induced by innate immunity factors such as interferons. The expression patterns of interferons differ, as do those of interferon-related genes such as interferon regulatory factors and interferon-stimulated genes that contribute to the antiviral response of infected cells. In addition, the signalling pathways related to viral infection and immune responses have been subject to evolutionary changes, including mutations compared to their homologues in other mammals and gene selection. This article discusses the differences in the interferon-mediated antiviral response in bats compared to that of other mammals and how these differences are correlated to viral tolerance in bats. The effect of bat interferons related genes on human antiviral response against bat-borne viruses is also discussed.
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Quirópteros , Virosis , Virus , Animales , Humanos , Línea Celular , Pandemias , Interferones/genética , Virosis/tratamiento farmacológico , Virosis/genética , Antivirales/farmacología , Antivirales/uso terapéutico , Antivirales/metabolismo , GenómicaRESUMEN
Developing effective vaccines against viral infections have significant impacts on development, prosperity and well-being of human populations. Thus, successful vaccines such as smallpox and polio vaccines, have promoted global societal well-being. In contrast, ineffective vaccines may fuel arguments that retard scientific progress. We aim to stimulate a multilevel discussion on how to develop effective vaccines against recent and future pandemics by focusing on acquired immunodeficiency syndrome (AIDS), coronavirus disease (COVID) and other viral infections. We appeal to harnessing recent achievements in this field specifically towards a cure for current pandemics and prevention of the next pandemics. Among these, we propose to apply the HIV DNA in chromatin format - an end product of aborted HIV integration in episomal forms, i.e., the chromatin vaccines (cVacc), to elicit the epigenetic silencing and memory that prevent viral replication and infection.
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Infecciones por Coronavirus , Infecciones por VIH , Vacunas Virales , Humanos , Cromatina/genética , Pandemias/prevención & controlRESUMEN
A complete knockout of a single key pluripotency gene may drastically affect embryonic stem cell function and epigenetic reprogramming. In contrast, elimination of only one allele of a single pluripotency gene is mostly considered harmless to the cell. To understand whether complex haploinsufficiency exists in pluripotent cells, we simultaneously eliminated a single allele in different combinations of two pluripotency genes (i.e., Nanog+/-;Sall4+/-, Nanog+/-;Utf1+/-, Nanog+/-;Esrrb+/- and Sox2+/-;Sall4+/-). Although these double heterozygous mutant lines similarly contribute to chimeras, fibroblasts derived from these systems show a significant decrease in their ability to induce pluripotency. Tracing the stochastic expression of Sall4 and Nanog at early phases of reprogramming could not explain the seen delay or blockage. Further exploration identifies abnormal methylation around pluripotent and developmental genes in the double heterozygous mutant fibroblasts, which could be rescued by hypomethylating agent or high OSKM levels. This study emphasizes the importance of maintaining two intact alleles for pluripotency induction.
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Metilación de ADN , Células Madre Pluripotentes Inducidas , Metilación de ADN/genética , Reprogramación Celular/genética , Haploinsuficiencia , Fibroblastos/metabolismo , Células Madre Embrionarias/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismoRESUMEN
COVID-19 has impacted billions of people since 2019 and unfolded a major healthcare crisis. With an increasing number of deaths and the emergence of more transmissible variants, it is crucial to better understand the biology of the disease-causing virus, the SARS-CoV-2. Peripheral neuropathies appeared as a specific COVID-19 symptom occurring at later stages of the disease. In order to understand the impact of SARS-CoV-2 on the peripheral nervous system, we generated human sensory neurons from induced pluripotent stem cells that we infected with the SARS-CoV-2 strain WA1/2020 and the variants delta and omicron. Using single-cell RNA sequencing, we found that human sensory neurons can be infected by SARS-CoV-2 but are unable to produce infectious viruses. Our data indicate that sensory neurons can be infected by the original WA1/2020 strain of SARS-CoV-2 as well as the delta and omicron variants, yet infectability differs between the original strain and the variants.
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Parkinson's disease (PD) is characterized by the aggregation of α-synuclein into Lewy bodies and Lewy neurites in the brain. Microglia-driven neuroinflammation may contribute to neuronal death in PD, however the exact role of microglia remains unclear and has been understudied. The A53T mutation in the gene coding for α-synuclein has been linked to early-onset PD, and exposure to A53T-mutant human α-synuclein increases the potential for inflammation of murine microglia. To date, its effect has not been studied in human microglia. Here, we used 2-dimensional cultures of human iPSC-derived microglia and transplantation of these cells into the mouse brain to assess the effects of the A53T mutation on human microglia. We found that A53T-mutant human microglia had an intrinsically increased propensity towards pro-inflammatory activation upon inflammatory stimulus. Additionally, A53T mutant microglia showed a strong decrease in catalase expression in non-inflammatory conditions, and increased oxidative stress. Our results indicate that A53T mutant human microglia display cell-autonomous phenotypes that may worsen neuronal damage in early-onset PD.
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Although respiratory symptoms are the most prevalent disease manifestation of infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), nearly 20% of hospitalized patients are at risk for thromboembolic events 1 . This prothrombotic state is considered a key factor in the increased risk of stroke, which has been observed clinically during both acute infection and long after symptoms have cleared 2 . Here we developed a model of SARS-CoV-2 infection using human-induced pluripotent stem cell-derived endothelial cells, pericytes, and smooth muscle cells to recapitulate the vascular pathology associated with SARS-CoV-2 exposure. Our results demonstrate that perivascular cells, particularly smooth muscle cells (SMCs), are a specifically susceptible vascular target for SARS-CoV-2 infection. Utilizing RNA sequencing, we characterized the transcriptomic changes accompanying SARS-CoV-2 infection of SMCs, and endothelial cells (ECs). We observed that infected human SMCs shift to a pro-inflammatory state and increase the expression of key mediators of the coagulation cascade. Further, we showed human ECs exposed to the secretome of infected SMCs produce hemostatic factors that can contribute to vascular dysfunction, despite not being susceptible to direct infection. The findings here recapitulate observations from patient sera in human COVID-19 patients and provide mechanistic insight into the unique vascular implications of SARS-CoV-2 infection at a cellular level.
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SARS-CoV-2 sequences can be reverse-transcribed and integrated into the genomes of virus-infected cells by a LINE1-mediated retrotransposition mechanism. Whole genome sequencing (WGS) methods detected retrotransposed SARS-CoV-2 subgenomic sequences in virus-infected cells overexpressing LINE1, while an enrichment method (TagMap) identified retrotranspositions in cells that did not overexpress LINE1. LINE1 overexpression increased retrotranspositions about 1,000-fold as compared to non-overexpressing cells. Nanopore WGS can directly recover retrotransposed viral and flanking host sequences but its sensitivity depends on the depth of sequencing (a typical 20-fold sequencing depth would only examine 10 diploid cell equivalents). In contrast, TagMap enriches for the host-virus junctions and can interrogate up to 20,000 cells and is able to detect rare viral retrotranspositions in LINE1 non-overexpressing cells. Although Nanopore WGS is 10 - 20-fold more sensitive per tested cell, TagMap can interrogate 1,000 - 2,000-fold more cells and therefore can identify infrequent retrotranspositions. When comparing SARS-CoV-2 infection and viral nucleocapsid mRNA transfection by TagMap, retrotransposed SARS-CoV-2 sequences were only detected in infected but not in transfected cells. Retrotransposition in virus-infected in contrast to transfected cells may be facilitated because virus infection in contrast to viral RNA transfection results in significantly higher viral RNA levels and stimulates LINE1-expression which causes cellular stress.
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Autophagy is a homeostatic process critical for cellular survival, and its malfunction is implicated in human diseases including neurodegeneration. Loss of autophagy contributes to cytotoxicity and tissue degeneration, but the mechanistic understanding of this phenomenon remains elusive. Here, we generated autophagy-deficient (ATG5-/-) human embryonic stem cells (hESCs), from which we established a human neuronal platform to investigate how loss of autophagy affects neuronal survival. ATG5-/- neurons exhibit basal cytotoxicity accompanied by metabolic defects. Depletion of nicotinamide adenine dinucleotide (NAD) due to hyperactivation of NAD-consuming enzymes is found to trigger cell death via mitochondrial depolarization in ATG5-/- neurons. Boosting intracellular NAD levels improves cell viability by restoring mitochondrial bioenergetics and proteostasis in ATG5-/- neurons. Our findings elucidate a mechanistic link between autophagy deficiency and neuronal cell death that can be targeted for therapeutic interventions in neurodegenerative and lysosomal storage diseases associated with autophagic defect.
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NAD , Mononucleótido de Nicotinamida , Humanos , NAD/metabolismo , Mononucleótido de Nicotinamida/metabolismo , Neuronas/metabolismo , Mitocondrias/metabolismo , Autofagia , Niacinamida/metabolismoRESUMEN
SARS-CoV-2 sequences can be reverse-transcribed and integrated into the genomes of virus-infected cells by a LINE1-mediated retrotransposition mechanism. Whole-genome sequencing (WGS) methods detected retrotransposed SARS-CoV-2 subgenomic sequences in virus-infected cells overexpressing LINE1, while an enrichment method (TagMap) identified retrotranspositions in cells that did not overexpress LINE1. LINE1 overexpression increased retrotranspositions about 1000-fold as compared to non-overexpressing cells. Nanopore WGS can directly recover retrotransposed viral and flanking host sequences, but its sensitivity depends on the depth of sequencing (a typical 20-fold sequencing depth would only examine 10 diploid cell equivalents). In contrast, TagMap enriches the host-virus junctions and can interrogate up to 20,000 cells and is able to detect rare viral retrotranspositions in LINE1 non-overexpressing cells. Although Nanopore WGS is 10-20-fold more sensitive per tested cell, TagMap can interrogate 1000-2000-fold more cells and, therefore, can identify infrequent retrotranspositions. When comparing SARS-CoV-2 infection and viral nucleocapsid mRNA transfection by TagMap, retrotransposed SARS-CoV-2 sequences were only detected in infected but not in transfected cells. Retrotransposition in virus-infected cells, in contrast to transfected cells, may be facilitated because virus infection, in contrast to viral RNA transfection, results in significantly higher viral RNA levels and stimulates LINE1 expression by causing cellular stress.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Transcripción Reversa , ARN Mensajero/genética , ARN Viral/genética , ARN Viral/metabolismo , GenómicaRESUMEN
Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by loss-of-function heterozygous mutations of methyl CpG-binding protein 2 (MECP2) on the X chromosome in young females. Reactivation of the silent wild-type MECP2 allele from the inactive X chromosome (Xi) represents a promising therapeutic opportunity for female patients with RTT. Here, we applied a multiplex epigenome editing approach to reactivate MECP2 from Xi in RTT human embryonic stem cells (hESCs) and derived neurons. Demethylation of the MECP2 promoter by dCas9-Tet1 with target single-guide RNA reactivated MECP2 from Xi in RTT hESCs without detectable off-target effects at the transcriptional level. Neurons derived from methylation-edited RTT hESCs maintained MECP2 reactivation and reversed the smaller soma size and electrophysiological abnormalities, two hallmarks of RTT. In RTT neurons, insulation of the methylation-edited MECP2 locus by dCpf1-CTCF (a catalytically dead Cpf1 fused with CCCTC-binding factor) with target CRISPR RNA enhanced MECP2 reactivation and rescued RTT-related neuronal defects, providing a proof-of-concept study for epigenome editing to treat RTT and potentially other dominant X-linked diseases.
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Síndrome de Rett , Humanos , Femenino , Síndrome de Rett/genética , Síndrome de Rett/terapia , Epigenoma , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Neuronas/metabolismo , Mutación , Heterocigoto , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Oxigenasas de Función Mixta/uso terapéutico , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
COVID-19 has impacted billions of people in the world since 2019 and unfolded a major healthcare crisis. With an increasing number of deaths and the emergence of more transmissible variants, it is crucial to better understand the biology of the disease-causing virus, the SARS-CoV-2. Peripheral neuropathies appeared as a specific COVID-19 symptom occurring at later stages of the disease. In order to understand the impact of SARS-CoV-2 on the peripheral nervous system, we generated human sensory neurons from induced pluripotent stem cells that we infected with the SARS-CoV-2 strain WA1/2020 and the variants delta and omicron. Using single cell RNA sequencing, we found that human sensory neurons can be infected by SARS-CoV-2 but are unable to produce new viruses. Our data suggests that sensory neurons can be infected by the original WA1/2020 strain of SARS-CoV-2 as well as the delta and omicron variants.
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BACKGROUND: Fragile X syndrome (FXS) is characterized by physical abnormalities, anxiety, intellectual disability, hyperactivity, autistic behaviors, and seizures. Abnormal neuronal development in FXS is poorly understood. Data on patients with FXS remain scarce, and FXS animal models have failed to yield successful therapies. In vitro models do not fully recapitulate the morphology and function of human neurons. METHODS: To mimic human neuron development in vivo, we coinjected neural precursor cells derived from FXS patient-derived induced pluripotent stem cells and neural precursor cells derived from corrected isogenic control induced pluripotent stem cells into the brain of neonatal immune-deprived mice. RESULTS: The transplanted cells populated the brain and a proportion differentiated into neurons and glial cells. Immunofluorescence and single and bulk RNA sequencing analyses showed accelerated maturation of FXS neurons after an initial delay. Additionally, we found increased percentages of Arc- and Egr-1-positive FXS neurons and wider dendritic protrusions of mature FXS striatal medium spiny neurons. CONCLUSIONS: This transplantation approach provides new insights into the alterations of neuronal development in FXS by facilitating physiological development of cells in a 3-dimensional context.
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Síndrome del Cromosoma X Frágil , Células-Madre Neurales , Humanos , Ratones , Animales , Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Fenotipo , Encéfalo/metabolismo , Ratones NoqueadosRESUMEN
Multielectrode arrays would benefit from intimate engagement with neural cells, but typical arrays do not present a physical environment that mimics that of neural tissues. It is hypothesized that a porous, conductive hydrogel scaffold with appropriate mechanical and conductive properties could support neural cells in 3D, while tunable electrical and mechanical properties could modulate the growth and differentiation of the cellular networks. By incorporating carbon nanomaterials into an alginate hydrogel matrix, and then freeze-drying the formulations, scaffolds which mimic neural tissue properties are formed. Neural progenitor cells (NPCs) incorporated in the scaffolds form neurite networks which span the material in 3D and differentiate into astrocytes and myelinating oligodendrocytes. Viscoelastic and more conductive scaffolds produce more dense neurite networks, with an increased percentage of astrocytes and higher myelination. Application of exogenous electrical stimulation to the scaffolds increases the percentage of astrocytes and the supporting cells localize differently with the surrounding neurons. The tunable biomaterial scaffolds can support neural cocultures for over 12 weeks, and enable a physiologically mimicking in vitro platform to study the formation of neuronal networks. As these materials have sufficient electrical properties to be used as electrodes in implantable arrays, they may allow for the creation of biohybrid neural interfaces and living electrodes.
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Tejido Nervioso , Células-Madre Neurales , Hidrogeles/farmacología , Diferenciación Celular , Astrocitos , Andamios del Tejido , Ingeniería de TejidosRESUMEN
Insulin receptor (IR) signaling is central to normal metabolic control and is dysregulated in metabolic diseases such as type 2 diabetes. We report here that IR is incorporated into dynamic clusters at the plasma membrane, in the cytoplasm and in the nucleus of human hepatocytes and adipocytes. Insulin stimulation promotes further incorporation of IR into these dynamic clusters in insulin-sensitive cells but not in insulin-resistant cells, where both IR accumulation and dynamic behavior are reduced. Treatment of insulin-resistant cells with metformin, a first-line drug used to treat type 2 diabetes, can rescue IR accumulation and the dynamic behavior of these clusters. This rescue is associated with metformin's role in reducing reactive oxygen species that interfere with normal dynamics. These results indicate that changes in the physico-mechanical features of IR clusters contribute to insulin resistance and have implications for improved therapeutic approaches.