No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Rubella virus tropism and single-cell responses in human primary tissue and microglia-containing organoids
Abstract
Rubella virus is an important human pathogen that can cause neurological deficits in a developing fetus when contracted during pregnancy. Despite successful vaccination programs in the Americas and many developed countries, rubella remains endemic in many regions worldwide and outbreaks occur wherever population immunity is insufficient. Intense interest since rubella virus was first isolated in 1962 has advanced our understanding of clinical outcomes after infection disrupts key processes of fetal neurodevelopment. Yet it is still largely unknown which cell types in the developing brain are targeted. We show that in human brain slices, rubella virus predominantly infects microglia. This infection occurs in a heterogeneous population but not in a highly microglia-enriched monoculture in the absence of other cell types. By using an organoid-microglia model, we further demonstrate that rubella virus infection leads to a profound interferon response in non-microglial cells, including neurons and neural progenitor cells, and this response is attenuated by the presence of microglia.
ResearchGate has not been able to resolve any citations for this publication.
The cerebral cortex forms early in development according to a series of heritable neurodevelopmental instructions. Despite deep evolutionary conservation of the cerebral cortex and its foundational six-layered architecture, significant variations in cortical size and folding can be found across mammals, including a disproportionate expansion of the prefrontal cortex in humans. Yet our mechanistic understanding of neurodevelopmental processes is derived overwhelmingly from rodent models, which fail to capture many human-enriched features of cortical development. With the advent of pluripotent stem cells and technologies for differentiating three-dimensional cultures of neural tissue in vitro, cerebral organoids have emerged as an experimental platform that recapitulates several hallmarks of human brain development. In this review, we discuss the merits and limitations of cerebral organoids as experimental models of the developing human brain. We highlight innovations in technology development that seek to increase its fidelity to brain development in vivo and discuss recent efforts to use cerebral organoids to study regeneration and brain evolution as well as to develop neurological and neuropsychiatric disease models.
Microglia, the brain’s resident macrophages, shape neural development and are key neuroimmune hubs in the pathological signatures of neurodevelopmental disorders. Despite the importance of microglia, their development has not been carefully examined in the human brain, and most of our knowledge derives from rodents. We aimed to address this gap in knowledge by establishing an extensive collection of 97 post-mortem tissues in order to enable quantitative, sex-matched, detailed analysis of microglia across the human lifespan. We identify the dynamics of these cells in the human telencephalon, describing waves in microglial density across gestation, infancy, and childhood, controlled by a balance of proliferation and apoptosis, which track key neurodevelopmental milestones. These profound changes in microglia are also observed in bulk RNA-seq and single-cell RNA-seq datasets. This study provides a detailed insight into the spatiotemporal dynamics of microglia across the human lifespan and serves as a foundation for elucidating how microglia contribute to shaping neurodevelopment in humans.
<br/
Microglia play a role in the emergence and preservation of a healthy brain microenvironment. Dysfunction of microglia has been associated with neurodevelopmental and neurodegenerative disorders. Investigating the function of human microglia in health and disease has been challenging due to the limited models of the human brain available. Here, we develop a method to generate functional microglia in human cortical organoids (hCOs) from human embryonic stem cells (hESCs). We apply this system to study the role of microglia during inflammation induced by amyloid-β (Aβ). The overexpression of the myeloid-specific transcription factor PU.1 generates microglia-like cells in hCOs, producing mhCOs (microglia containing hCOs), that we engraft in the mouse brain. Single-cell transcriptomics reveals that mhCOs acquire a microglia cell cluster with an intact complement and chemokine system. Functionally, microglia in mhCOs protect parenchyma from cellular and molecular damage caused by Aβ. Furthermore, in mhCOs, we observed reduced expression of Aβ-induced expression of genes associated with apoptosis, ferroptosis, and Alzheimer’s disease (AD) stage III. Finally, we assess the function of AD-associated genes highly expressed in microglia
in response to Aβ using pooled CRISPRi coupled with single-cell RNA sequencing in mhCOs. In summary, we provide a protocol to generate mhCOs that can be used in fundamental and translational studies as a model to investigate the role of microglia in neurodevelopmental and neurodegenerative disorders.
Induced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enable highly resolved descriptions of cell states within these systems; however, approaches are needed to directly measure lineage relationships. Here we establish iTracer, a lineage recorder that combines reporter barcodes with inducible CRISPR–Cas9 scarring and is compatible with single-cell and spatial transcriptomics. We apply iTracer to explore clonality and lineage dynamics during cerebral organoid development and identify a time window of fate restriction as well as variation in neurogenic dynamics between progenitor neuron families. We also establish long-term four-dimensional light-sheet microscopy for spatial lineage recording in cerebral organoids and confirm regional clonality in the developing neuroepithelium. We incorporate gene perturbation (iTracer-perturb) and assess the effect of mosaic TSC2 mutations on cerebral organoid development. Our data shed light on how lineages and fates are established during cerebral organoid formation. More broadly, our techniques can be adapted in any iPSC-derived culture system to dissect lineage alterations during normal or perturbed development.
Rubella virus (RuV) has recently been found in association with granulomatous inflammation of the skin and several internal organs in patients with inborn errors of immunity (IEI). The cellular tropism and molecular mechanisms of RuV persistence and pathogenesis in select immunocompromised hosts are not clear. We provide clinical, immunological, virological, and histological data on a cohort of 28 patients with a broad spectrum of IEI and RuV-associated granulomas in skin and nine extracutaneous tissues to further delineate this relationship. Combined immunodeficiency was the most frequent diagnosis (67.8%) among patients. Patients with previously undocumented conditions, i.e., humoral immunodeficiencies, a secondary immunodeficiency, and a defect of innate immunity were identified as being susceptible to RuV-associated granulomas. Hematopoietic cell transplantation was the most successful treatment in this case series resulting in granuloma resolution; steroids, and TNF-α and IL-1R inhibitors were moderately effective. In addition to M2 macrophages, neutrophils were identified by immunohistochemical analysis as a novel cell type infected with RuV. Four patterns of RuV-associated granulomatous inflammation were classified based on the structural organization of granulomas and identity and location of cell types harboring RuV antigen. Identification of conditions that increase susceptibility to RuV-associated granulomas combined with structural characterization of the granulomas may lead to a better understanding of the pathogenesis of RuV-associated granulomas and discover new targets for therapeutic interventions.
Rubella virus is the most teratogenic virus known to science and is capable of causing large epidemics. The RA 27/3 rubella vaccine, usually combined with measles vaccine, has eliminated rubella and congenital rubella syndrome from much of the world, notably from the Western Hemisphere. Except in immunosuppressed individuals, it is remarkably safe. Together with rubella vaccine strains used in China and Japan, eradication of the rubella virus is possible, indeed more feasible than eradication of measles or mumps.
The human cortex comprises diverse cell types that emerge from an initially uniform neuroepithelium that gives rise to radial glia, the neural stem cells of the cortex. To characterize the earliest stages of human brain development, we performed single-cell RNA-sequencing across regions of the developing human brain, including the telencephalon, diencephalon, midbrain, hindbrain and cerebellum. We identify nine progenitor populations physically proximal to the telencephalon, suggesting more heterogeneity than previously described, including a highly prevalent mesenchymal-like population that disappears once neurogenesis begins. Comparison of human and mouse progenitor populations at corresponding stages identifies two progenitor clusters that are enriched in the early stages of human cortical development. We also find that organoid systems display low fidelity to neuroepithelial and early radial glia cell types, but improve as neurogenesis progresses. Overall, we provide a comprehensive molecular and spatial atlas of early stages of human brain and cortical development. Eze et al. use single-cell sequencing and immunohistochemical validation to create an atlas of early human brain development. In the telencephalon, they discover a diversity of progenitor subtypes, including two that are enriched in humans.
Maternal immune activation increases the risk of neurodevelopmental disorders. Elevated cytokines, such as interferon-γ (IFN-γ), in offspring’s brains play a central role. IFN-γ activates an antiviral cellular state, limiting viral entry and replication. Moreover, IFN-γ is implicated in brain development. We tested the hypothesis that IFN-γ signaling contributes to molecular and cellular phenotypes associated with neurodevelopmental disorders. Transient IFN-γ treatment of neural progenitors derived from human induced pluripotent stem cells increased neurite outgrowth. RNA sequencing analysis revealed that major histocompatibility complex class I (MHCI) genes were persistently up-regulated through neuronal differentiation—an effect that was mediated by IFN-γ-induced promyelocytic leukemia protein (PML) nuclear bodies. Critically, IFN-γ-induced neurite outgrowth required both PML and MHCI. We also found evidence that IFN-γ disproportionately altered the expression of genes associated with schizophrenia and autism, suggesting convergence between genetic and environmental risk factors. Together, these data implicate IFN-γ signaling in neurodevelopmental disorder etiology.
Single-cell RNA-seq (scRNA-seq) data exhibits significant cell-to-cell variation due to technical factors, including the number of molecules detected in each cell, which can confound biological heterogeneity with technical effects. To address this, we present a modeling framework for the normalization and variance stabilization of molecular count data from scRNA-seq experiments. We propose that the Pearson residuals from "regularized negative binomial regression," where cellular sequencing depth is utilized as a covariate in a generalized linear model, successfully remove the influence of technical characteristics from downstream analyses while preserving biological heterogeneity. Importantly, we show that an unconstrained negative binomial model may overfit scRNA-seq data, and overcome this by pooling information across genes with similar abundances to obtain stable parameter estimates. Our procedure omits the need for heuristic steps including pseudocount addition or log-transformation and improves common downstream analytical tasks such as variable gene selection, dimensional reduction, and differential expression. Our approach can be applied to any UMI-based scRNA-seq dataset and is freely available as part of the R package sctransform, with a direct interface to our single-cell toolkit Seurat.
Human cortical organoids (hCOs), derived from human embryonic stem cells (hESCs), provide a platform to study human brain development and diseases in complex three-dimensional tissue. However, current hCOs lack microvasculature, resulting in limited oxygen and nutrient delivery to the inner-most parts of hCOs. We engineered hESCs to ectopically express human ETS variant 2 (ETV2). ETV2-expressing cells in hCOs contributed to forming a complex vascular-like network in hCOs. Importantly, the presence of vasculature-like structures resulted in enhanced functional maturation of organoids. We found that vascularized hCOs (vhCOs) acquired several blood-brain barrier characteristics, including an increase in the expression of tight junctions, nutrient transporters and trans-endothelial electrical resistance. Finally, ETV2-induced endothelium supported the formation of perfused blood vessels in vivo. These vhCOs form vasculature-like structures that resemble the vasculature in early prenatal brain, and they present a robust model to study brain disease in vitro.
Droplet-based scRNA-seq assays are known to produce a significant amount of background RNA counts, the hallmark of which is non-zero transcript counts in presumably empty droplets. The presence of background RNA can lead to systematic biases and batch effects in various downstream analyses such as differential expression and marker gene discovery. In this paper, we explore the phenomenology and mechanisms of background RNA generation in droplet-based scRNA-seq assays and present a deep generative model of background-contaminated counts mirroring those mechanisms. The model is used for learning the background RNA profile, distinguishing cell-containing droplets from empty ones, and retrieving background-free gene expression profiles. We implement the model along with a fast and scalable inference algorithm as the remove-background module in CellBender, an open-source scRNA-seq data processing software package. Finally, we present simulations and investigations of several scRNA-seq datasets to show that processing raw data using CellBender significantly boosts the magnitude and specificity of differential expression across different cell types.
Sample multiplexing facilitates scRNA-seq by reducing costs and identifying artifacts such as cell doublets. However, universal and scalable sample barcoding strategies have not been described. We therefore developed MULTI-seq: multiplexing using lipid-tagged indices for single-cell and single-nucleus RNA sequencing. MULTI-seq reagents can barcode any cell type or nucleus from any species with an accessible plasma membrane. The method involves minimal sample processing, thereby preserving cell viability and endogenous gene expression patterns. When cells are classified into sample groups using MULTI-seq barcode abundances, data quality is improved through doublet identification and recovery of cells with low RNA content that would otherwise be discarded by standard quality-control workflows. We use MULTI-seq to track the dynamics of T-cell activation, perform a 96-plex perturbation experiment with primary human mammary epithelial cells and multiplex cryopreserved tumors and metastatic sites isolated from a patient-derived xenograft mouse model of triple-negative breast cancer.
Community detection is often used to understand the structure of large and complex networks. One of the most popular algorithms for uncovering community structure is the so-called Louvain algorithm. We show that this algorithm has a major defect that largely went unnoticed until now: the Louvain algorithm may yield arbitrarily badly connected communities. In the worst case, communities may even be disconnected, especially when running the algorithm iteratively. In our experimental analysis, we observe that up to 25% of the communities are badly connected and up to 16% are disconnected. To address this problem, we introduce the Leiden algorithm. We prove that the Leiden algorithm yields communities that are guaranteed to be connected. In addition, we prove that, when the Leiden algorithm is applied iteratively, it converges to a partition in which all subsets of all communities are locally optimally assigned. Furthermore, by relying on a fast local move approach, the Leiden algorithm runs faster than the Louvain algorithm. We demonstrate the performance of the Leiden algorithm for several benchmark and real-world networks. We find that the Leiden algorithm is faster than the Louvain algorithm and uncovers better partitions, in addition to providing explicit guarantees.
The mechanistic basis of gliogenesis, which occurs late in human development, is poorly understood. Here we identify nuclear factor IA (NFIA) as a molecular switch inducing human glial competency. Transient expression of NFIA is sufficient to trigger glial competency of human pluripotent stem cell-derived neural stem cells within 5 days and to convert these cells into astrocytes in the presence of glial-promoting factors, as compared to 3–6 months using current protocols. NFIA-induced astrocytes promote synaptogenesis, exhibit neuroprotective properties, display calcium transients in response to appropriate stimuli and engraft in the adult mouse brain. Differentiation involves rapid but reversible chromatin remodeling, glial fibrillary acidic protein (GFAP) promoter demethylation and a striking lengthening of the G1 cell cycle phase. Genetic or pharmacological manipulation of G1 length partially mimics NFIA function. We used the approach to generate astrocytes with region-specific or reactive features. Our study defines key mechanisms of the gliogenic switch and enables the rapid production of human astrocytes for disease modeling and regenerative medicine. A new method generates astrocytes from human pluripotent stem cells in 5 days.
CellProfiler has enabled the scientific research community to create flexible, modular image analysis pipelines since its release in 2005. Here, we describe CellProfiler 3.0, a new version of the software supporting both whole-volume and plane-wise analysis of three-dimensional (3D) image stacks, increasingly common in biomedical research. CellProfiler’s infrastructure is greatly improved, and we provide a protocol for cloud-based, large-scale image processing. New plugins enable running pretrained deep learning models on images. Designed by and for biologists, CellProfiler equips researchers with powerful computational tools via a well-documented user interface, empowering biologists in all fields to create quantitative, reproducible image analysis workflows.
Rubella virus (RuV) causes a systemic infection, and transplacental fetal infection causes congenital rubella syndrome. In this study, we showed that treatment of cells with sphingomyelinase inhibited RuV infection. Assays using inhibitors of serine palmitoyl transferase and ceramide transport protein demonstrated the contribution of sphingomyelin (SM) to RuV infection. Compelling evidence for direct binding of RuV to lipid membranes at neutral pH was obtained using liposome coflotation assays. The absence of either SM or cholesterol (Chol) abrogated the RuV-liposome interaction. SM and Chol (SM/Chol) were also critical for RuV binding to erythrocytes and lymphoid cells. Removal of Ca²⁺ from the assay buffer or mutation of RuV envelope E1 protein Ca²⁺-binding sites abrogated RuV binding to liposomes, erythrocytes, and lymphoid cells. However, RuV bound to various nonlymphoid adherent cell lines independently of extracellular Ca²⁺ or SM/Chol. Even in these adherent cell lines, both the E1 protein Ca²⁺-binding sites and cellular SM/Chol were essential for the early stage of RuV infection, possibly affecting envelope-membrane fusion in acidic compartments. Myelin oligodendrocyte glycoprotein (MOG) has recently been identified as a cellular receptor for RuV. However, RuV bound to MOG-negative cells in a Ca²⁺-independent manner. Collectively, our data demonstrate that RuV has two distinct binding mechanisms: one is Ca²⁺ dependent and the other is Ca²⁺ independent. Ca²⁺-dependent binding observed in lymphoid cells occurs by the direct interaction between E1 protein fusion loops and SM/Chol-enriched membranes. Clarification of the mechanism of Ca²⁺-independent RuV binding is an important next step in understanding the pathology of RuV infection.
IMPORTANCE
Rubella has a significant impact on public health as infection during early pregnancy can result in babies being born with congenital rubella syndrome. Even though effective rubella vaccines are available, rubella outbreaks still occur in many countries. We studied the entry mechanism of rubella virus (RuV) and found that RuV binds directly to the host plasma membrane in the presence of Ca²⁺ at neutral pH. This Ca²⁺-dependent binding is specifically directed to membranes enriched in sphingomyelin and cholesterol and is critical for RuV infection. Importantly, RuV also binds to many cell lines in a Ca²⁺-independent manner. An unidentified RuV receptor(s) is involved in this Ca²⁺-independent binding. We believe that the data presented here may aid the development of the first anti-RuV drug.
Significance
Zika virus (ZIKV) is a mosquito-borne flavivirus that has rapidly spread through the Americas and has been associated with fetal abnormalities, including microcephaly. To understand how microcephaly develops, it is important to identify which cell types of the developing brain are susceptible to infection. We use primary human tissue to show that radial glia and astrocytes are more susceptible to infection than neurons, a pattern that correlates with expression of a putative viral entry receptor, AXL. We also perform a screen of Food and Drug Administration-approved compounds, with an emphasis on drugs known to be safe in pregnancy. We identify an antibiotic, azithromycin, that reduces viral proliferation in glial cells, and compare its activity with daptomycin and sofosbuvir, two additional drugs with anti-ZIKV activity.
Background
Ocular infections remain a major cause of blindness and morbidity worldwide. While prognosis is dependent on the timing and accuracy of diagnosis, the etiology remains elusive in ~50 % of presumed infectious uveitis cases. The objective of this study is to determine if unbiased metagenomic deep sequencing (MDS) can accurately detect pathogens in intraocular fluid samples of patients with uveitis. Methods
This is a proof-of-concept study, in which intraocular fluid samples were obtained from five subjects with known diagnoses, and one subject with bilateral chronic uveitis without a known etiology. Samples were subjected to MDS, and results were compared with those from conventional diagnostic tests. Pathogens were identified using a rapid computational pipeline to analyze the non-host sequences obtained from MDS. ResultsUnbiased MDS of intraocular fluid produced results concordant with known diagnoses in subjects with (n = 4) and without (n = 1) uveitis. Samples positive for Cryptococcus neoformans, Toxoplasma gondii, and herpes simplex virus 1 as tested by a Clinical Laboratory Improvement Amendments-certified laboratory were correctly identified with MDS. Rubella virus was identified in one case of chronic bilateral idiopathic uveitis. The subject’s strain was most closely related to a German rubella virus strain isolated in 1992, one year before he developed a fever and rash while living in Germany. The pattern and the number of viral identified mutations present in the patient’s strain were consistent with long-term viral replication in the eye. ConclusionsMDS can identify fungi, parasites, and DNA and RNA viruses in minute volumes of intraocular fluid samples. The identification of chronic intraocular rubella virus infection highlights the eye’s role as a long-term pathogen reservoir, which has implications for virus eradication and emerging global epidemics.
Background:
The burden of Congenital Rubella Syndrome (CRS) is typically underestimated in routine surveillance. Updated estimates are needed following the recent WHO position paper on rubella and recent GAVI initiatives, funding rubella vaccination in eligible countries. Previous estimates considered the year 1996 and only 78 (developing) countries.
Methods:
We reviewed the literature to identify rubella seroprevalence studies conducted before countries introduced rubella-containing vaccination (RCV). These data and the estimated vaccination coverage in the routine schedule and mass campaigns were incorporated in mathematical models to estimate the CRS incidence in 1996 and 2000-2010 for each country, region and globally.
Results:
The estimated CRS decreased in the three regions (Americas, Europe and Eastern Mediterranean) which had introduced widespread RCV by 2010, reaching <2 per 100,000 live births (the Americas and Europe) and 25 (95% CI 4-61) per 100,000 live births (the Eastern Mediterranean). The estimated incidence in 2010 ranged from 90 (95% CI: 46-195) in the Western Pacific, excluding China, to 116 (95% CI: 56-235) and 121 (95% CI: 31-238) per 100,000 live births in Africa and SE Asia respectively. Highest numbers of cases were predicted in Africa (39,000, 95% CI: 18,000-80,000) and SE Asia (49,000, 95% CI: 11,000-97,000). In 2010, 105,000 (95% CI: 54,000-158,000) CRS cases were estimated globally, compared to 119,000 (95% CI: 72,000-169,000) in 1996.
Conclusions:
Whilst falling dramatically in the Americas, Europe and the Eastern Mediterranean after vaccination, the estimated CRS incidence remains high elsewhere. Well-conducted seroprevalence studies can help to improve the reliability of these estimates and monitor the impact of rubella vaccination.
Single-cell transcriptomics reveals gene expression heterogeneity but suffers from stochastic dropout and characteristic bimodal expression distributions in which expression is either strongly non-zero or non-detectable. We propose a two-part, generalized linear model for such bimodal data that parameterizes both of these features. We argue that the cellular detection rate, the fraction of genes expressed in a cell, should be adjusted for as a source of nuisance variation. Our model provides gene set enrichment analysis tailored to single-cell data. It provides insights into how networks of co-expressed genes evolve across an experimental treatment. MAST is available at https://github.com/RGLab/MAST .
Background: An estimated 100,000 cases of congenital rubella syndrome (CRS) occur worldwide each year. The reported mortality rate for infants with CRS is up to 33%. The cellular mechanisms responsible for the multiple congenital defects in CRS are presently unknown. Here we identify cell types positive for rubella virus (RV) in CRS infants.
Methods: Cells and organs involved in RV replication were identified in paraffin-embedded autopsy tissues from three fatal case-patients by histopathologic examination and immunohistochemical (IHC) staining using a rabbit polyclonal RV antibody. Normal rabbit antisera and RV antisera preabsorbed with highly purified RV served as negative controls.
Results: RV antigen was found in interstitial fibroblasts in the heart, adventitial fibroblasts of large blood vessels, alveolar macrophages, progenitor cells of the outer granular layer of the brain, and in capillary endothelium and basal plate in the placenta. The antibody specificity was verified by IHC staining of multiple tissue sections from other infectious disease cases. RV infection of each cell type is consistent with abnormalities which have been identified in patients with CRS, in the heart, large blood vessels, and brain. Antigen distribution was consistent with inflammatory response to vascular injury and systemic spread of RV.
Conclusions: The identification of RV positive cell types in CRS is important to better understand the pathology and pathogenesis of CRS.
Importance:
Viral infections of the central nervous system can lead to debilitating disease and death. Moreover, it is becoming increasingly clear that non-renewable cells, including most central nervous system neurons, combat neurotropic viral infections in fundamentally different ways than other rapidly dividing and renewable cell populations. Here we identify type I interferon signaling as a key inducer of a known anti-viral protein (Bst2) in neurons. Unexpectedly, this gene is dispensable for clearance of neurotropic viral infection despite its well-defined contribution to limiting the spread of enveloped viruses in proliferating cells. A deeper appreciation of the importance of cell-type heterogeneity in antiviral immunity will aid in the identification of unique therapeutic targets for life-threatening viral infections.
Dissection of the genetic basis of Aicardi-Goutières syndrome has highlighted a fundamental link between nucleic acid metabolism, innate immune sensors and type I interferon induction. This had led to the concept of the human interferonopathies as a broader set of Mendelian disorders in which a constitutive upregulation of type I interferon activity directly relates to disease pathology. Here, we discuss the molecular and cellular basis of the interferonopathies, their categorization, future treatment strategies and the insights they provide into normal physiology.
The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. Here, we present a simple and reproducible 3D culture approach for generating a laminated cerebral cortex-like structure, named human cortical spheroids (hCSs), from pluripotent stem cells. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. These neurons are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events. These 3D cultures should allow a detailed interrogation of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro.
Development of congenital rubella syndrome associated with rubella virus infection during pregnancy is clinically important, but the pathogenicity of the virus remains unclear.
Pathological examination was conducted on 3 aborted fetuses with congenital rubella infection.
At autopsy, all 3 aborted fetuses showed congenital cataract confirmed by gross observation. Rubella virus infection occurred via systemic organs including circulating hematopoietic stem cells confirmed by immunohistochemical and molecular investigations, and major histopathogical changes were found in the liver. It is noteworthy that the virus infected the ciliary body of the eye, suggesting a possible cause of cataracts.
Our study based on the pathological examination demonstrated that the rubella virus infection occurred via systemic organs of human fetuses. This fact was confirmed by immunohistochemistry and direct detection of viral RNA in multiple organs. To the best of our knowledge, this study is the first report demonstrating that the rubella virus infection occurred via systemic organs of the human body. Importantly, virus infection of the ciliary body could play an important role in cataractogenesis.
The phylogenetically conserved nuclear factor I (NFI) family of transcription/replication proteins is essential both for adenoviral
DNA replication and for the transcription of many cellular genes. We showed previously that the four murine NFI genes (Nfia, Nfib, Nfic, and Nfix) are expressed in unique but overlapping patterns during mouse development and in adult tissues. Here we show that disruption
of the Nfia gene causes perinatal lethality, with >95% of homozygous Nfia−/− animals dying within 2 weeks after birth. Newborn Nfia−/− animals lack a corpus callosum and show ventricular dilation indicating early hydrocephalus. Rare surviving homozygous Nfia−/− mice lack a corpus callosum, show severe communicating hydrocephalus, a full-axial tremor indicative of neurological defects,
male-sterility, low female fertility, but near normal life spans. These findings indicate that while the Nfia gene appears nonessential for cell viability and DNA replication in embryonic stem cells and fibroblasts, loss of Nfia function causes severe developmental defects. This finding of an NFI gene required for a developmental process suggests that
the four NFI genes may have distinct roles in vertebrate development.
Rubella virus (RV) is a highly transmissible pathogenic agent that causes the disease rubella. Maternal RV infection during
early pregnancy causes the death of the fetus or congenital rubella syndrome in infants. However, the cellular receptor for
RV has not yet been identified. In this study, we found that the myelin oligodendrocyte glycoprotein (MOG) specifically bound
to the E1 envelope glycoprotein of RV, and an antibody against MOG could block RV infection. Most importantly, we also showed
that ectopic expression of MOG on the cell surface of 293T cells rendered this nonpermissive cell line permissive for RV entry
and replication. Thus, this study has identified a cellular receptor for RV and suggests that blocking the MOG attachment
site of RV may be a strategy for molecular intervention of RV infection.
The control of RNA alternative splicing is critical for generating biological diversity. Despite emerging genome-wide technologies
to study RNA complexity, reliable and comprehensive RNA-regulatory networks have not been defined. Here, we used Bayesian
networks to probabilistically model diverse data sets and predict the target networks of specific regulators. We applied this
strategy to identify ~700 alternative splicing events directly regulated by the neuron-specific factor Nova in the mouse brain,
integrating RNA-binding data, splicing microarray data, Nova-binding motifs, and evolutionary signatures. The resulting integrative
network revealed combinatorial regulation by Nova and the neuronal splicing factor Fox, interplay between phosphorylation
and splicing, and potential links to neurologic disease. Thus, we have developed a general approach to understanding mammalian
RNA regulation at the systems level.
Rubella virus was capable of replicating in both unstimulated and phytohemagglutinin-stimulated cultures of human mononuclear blood cells. Monocyte-derived macrophages were the main cell type responsible for viral replication. The susceptibility of macrophages increased during cultivation. Phytohemagglutinin-stimulated lymphocytes were able to support replication to a limited degree. No viral replication was detected in unstimulated lymphocytes. Both stimulation and viral replication in phytohemagglutinin-treated lymphocyte cultures were enhanced by the addition of murine macrophages. Human leukocyte interferon depressed the production of virus in these combined cultures. The finding that rubella virus is able to replicate in human lymphocytes as well as in macrophages may contribute to understanding the mechanisms of the suppressive effect of the virus on in vitro lymphocyte phytohemagglutinin responsiveness and in vivo immune functions.
Microglia are resident macrophages in the brain that emerge in early development and respond to the local environment by altering their molecular and phenotypic states. Fundamental questions about microglia diversity and function during development remain unanswered because we lack experimental strategies to interrogate their interactions with other cell types and responses to perturbations ex vivo. We compared human microglia states across culture models, including cultured primary and pluripotent stem cell-derived microglia. We developed a "report card" of gene expression signatures across these distinct models to facilitate characterization of their responses across experimental models, perturbations, and disease conditions. Xenotransplantation of human microglia into cerebral organoids allowed us to characterize key transcriptional programs of developing microglia in vitro and reveal that microglia induce transcriptional changes in neural stem cells and decrease interferon signaling response genes. Microglia additionally accelerate the emergence of synchronized oscillatory network activity in brain organoids by modulating synaptic density.
A visualization suite for major forms of bulk and single-cell RNAseq data in R. dittoSeq is color blindness-friendly by default, robustly documented to power ease-of-use, and allows highly customizable generation of both daily-use and publication-quality figures.
Availability and implementation:
dittoSeq is an R package available through Bioconductor via an open source MIT license.
Supplementary information:
Supplementary Code and figures are available at Bioinformatics online. Full vignettes are available through Bioconductor, https://bioconductor.org/packages/dittoSeq/, and github, github.com/dtm2451/dittoSeq/.
The nuclear factor I (NFI) family of transcription factors play an important role in normal development of multiple organs. Three NFI family members are highly expressed in the brain, and deletions or sequence variants in two of these, NFIA and NFIX, have been associated with intellectual disability (ID) and brain malformations. NFIB, however, has not previously been implicated in human disease. Here, we present a cohort of 18 individuals with mild ID and behavioral issues who are haploinsufficient for NFIB. Ten individuals harbored overlapping microdeletions of the chromosomal 9p23-p22.2 region, ranging in size from 225 kb to 4.3 Mb. Five additional subjects had point sequence variations creating a premature termination codon, and three subjects harbored single-nucleotide variations resulting in an inactive protein as determined using an in vitro reporter assay. All individuals presented with additional variable neurodevelopmental phenotypes, including muscular hypotonia, motor and speech delay, attention deficit disorder, autism spectrum disorder, and behavioral abnormalities. While structural brain anomalies, including dysgenesis of corpus callosum, were variable, individuals most frequently presented with macrocephaly. To determine whether macrocephaly could be a functional consequence of NFIB disruption, we analyzed a cortex-specific Nfib conditional knockout mouse model, which is postnatally viable. Utilizing magnetic resonance imaging and histology, we demonstrate that Nfib conditional knockout mice have enlargement of the cerebral cortex but preservation of overall brain structure and interhemispheric connectivity. Based on our findings, we propose that haploinsufficiency of NFIB causes ID with macrocephaly.
Building a brain
The human brain is built in an inside-out manner as a series of layers. Although progenitor cells spin off new neurons in a seemingly organized fashion, the devil is in the details. Nowakowski et al. analyzed the transcriptomes of single cells from the developing brain to elucidate the hidden complexity of brain construction. For each cell, its position within the brain matters, as well as what type of neuron is being made at what point during overall development. These individual expression patterns result in organized diversity in the brain's cortex.
Science , this issue p. 1318
Background:
The unprecedented reemergence of Zika virus (ZIKV) has startled the world with reports of increased microcephaly in Brazil. ZIKV can infect human neural progenitors and impair brain growth. However, direct evidence of ZIKV infection in human fetal brain tissues remains elusive.
Methods:
Investigations were performed with brain cell preparations obtained from 9 donors. Virus infectivity was assessed by detection of virus antigen by flow cytometry together with various hematopoietic cell surface markers. Virus replication was determined by viral RNA quantification. Cytokine levels in supernatant obtained from virus-infected fetal brain cells were measured simultaneously in microbead-based immunoassays.
Results:
We also show that ZIKV infection was particularly evident in hematopoietic cells with microglia, the brain-resident macrophage population being one of the main targets. Infection induces high levels of proinflammatory immune mediators such as interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), and monocyte chemotactic protein 1 (MCP-1).
Conclusions:
Our results highlight an important role for microglia and neuroinflammation during congenital ZIKV pathogenesis.
Classical lissencephaly is a genetic neurological disorder associated with mental retardation and intractable epilepsy, and Miller-Dieker syndrome (MDS) is the most severe form of the disease. In this study, to investigate the effects of MDS on human progenitor subtypes that control neuronal output and influence brain topology, we analyzed cerebral organoids derived from control and MDS-induced pluripotent stem cells (iPSCs) using time-lapse imaging, immunostaining, and single-cell RNA sequencing. We saw a cell migration defect that was rescued when we corrected the MDS causative chromosomal deletion and severe apoptosis of the founder neuroepithelial stem cells, accompanied by increased horizontal cell divisions. We also identified a mitotic defect in outer radial glia, a progenitor subtype that is largely absent from lissencephalic rodents but critical for human neocortical expansion. Our study, therefore, deepens our understanding of MDS cellular pathogenesis and highlights the broad utility of cerebral organoids for modeling human neurodevelopmental disorders.
Autism spectrum disorder (ASD) involves substantial genetic contributions. These contributions are profoundly heterogeneous but may converge on common pathways that are not yet well understood. Here, through post-mortem genome-wide transcriptome analysis of the largest cohort of samples analysed so far, to our knowledge, we interrogate the noncoding transcriptome, alternative splicing, and upstream molecular regulators to broaden our understanding of molecular convergence in ASD. Our analysis reveals ASD-associated dysregulation of primate-specific long noncoding RNAs (lncRNAs), downregulation of the alternative splicing of activity-dependent neuron-specific exons, and attenuation of normal differences in gene expression between the frontal and temporal lobes. Our data suggest that SOX5, a transcription factor involved in neuron fate specification, contributes to this reduction in regional differences. We further demonstrate that a genetically defined subtype of ASD, chromosome 15q11.2-13.1 duplication syndrome (dup15q), shares the core transcriptomic signature observed in idiopathic ASD. Co-expression network analysis reveals that individuals with ASD show age-related changes in the trajectory of microglial and synaptic function over the first two decades, and suggests that genetic risk for ASD may influence changes in regional cortical gene expression. Our findings illustrate how diverse genetic perturbations can lead to phenotypic convergence at multiple biological levels in a complex neuropsychiatric disorder.
Rubella is usually a mild illness with febrile rash being its main symptom. However, serious consequences of rubella can result from an infection during the early stages of pregnancy. After a rubella outbreak in Japan observed from June 2012 to 2013, 45 infants were reportedly born with Congenital Rubella Syndrome (CRS). We followed up the 15 CRS cases reported in Tokyo to determine the virus shedding periods by utilizing nested RT-PCR to detect rubella virus genes. Out of the 15 cases, 4 cases became unavailable for follow-ups and were excluded from analysis. During the estimated virus shedding periods, positive test results for the virus gene were 79.4% (27/34) in throat swab samples, 50% (2/4) in urine samples, and 25% (1/4) in blood samples. The shortest virus shedding period was less than 1 month and the longest was 13 months. The proportion of the CRS cases shedding viruses were 100% (11/11) at 0 month and 9.1% (1/11) at 12 months. Our findings also suggested that the earlier the mother's onset of rubella was during pregnancy, the virus shedding periods of infants with CRS lasted longer. We believe a cutoff point for virus gene detections can be set for infants at 1 year of age.
Neural crest (NC) cells contribute to the development of many complex tissues of all three germ layers during embryogenesis, and its abnormal development accounts for several congenital birth defects. Generating NC cells-including specific subpopulations such as cranial, cardiac, and trunk NC cells-from human pluripotent stem cells will provide a valuable model system to study human development and disease. Here, we describe a rapid and robust NC differentiation method called "LSB-short" that is based on dual SMAD pathway inhibition. This protocol yields high percentages of NC cell populations from multiple human induced pluripotent stem and human embryonic stem cell lines in 8 days. The resulting cells can be propagated easily, retain NC marker expression over multiple passages, and can spontaneously differentiate into several NC-derived cell lineages, including smooth muscle cells, peripheral neurons, and Schwann cells. NC cells generated by this method represent cranial, cardiac and trunk NC subpopulations based on global gene expression analyses, are similar to in vivo analogues, and express a common set of NC alternative isoforms. Functionally, they are also able to migrate appropriately in response to chemoattractants such as SDF-1, FGF8b, and Wnt3a. By yielding NC cells that likely represent all NC subpopulations in a shorter time frame than other published methods, our LSB-short method provides an ideal model system for further studies of human NC development and disease.
Background: Premorbid neurocognitive, neuromotor, and behavioral function tends to be disturbed in schizophrenia. We previously demonstrated that a birth cohort clinically and serologically documented with prenatal rubella evidenced a marked increase in risk of nonaffective psychosis. In our study, we examined whether rubella-exposed subjects destined to develop schizophrenia and other schizophrenia spectrum disorders (SSD), compared with exposed control subjects, had greater impairment in several premorbid functions.Methods: Subjects were interviewed using a direct, comprehensive research assessment and diagnosed by consensus. We compared the degree of IQ decline, as well as premorbid neuromotor and behavioral dysfunction, between rubella-exposed subjects who developed schizophrenia spectrum psychosis (SSP) and exposed control subjects from the cohort. We also compared the gestational timing of rubella infection between the cases and control subjects.Results: This rubella-exposed birth cohort evidenced a markedly increased risk of SSD (20.4% or 11/53). Rubella-exposed SSP cases, compared with rubella-exposed control subjects, demonstrated a decline in IQ from childhood to adolescence, and increased premorbid neuromotor and behavioral abnormalities. Moreover, it appears that early gestational rubella exposure may represent a period of increased vulnerability for SSD.Conclusions: These findings link a known prenatal exposure, a deviant neurodevelopmental trajectory in childhood and adolescence, and SSP in adulthood within the same individuals.
A longitudinal study was conducted of 243 children with congenital rubella. In this sample a high rate of autism and a high rate of recovery were observed. Examination of the data suggested that the rubella virus was the primary etiologic agent. It is hypothesized that the course of autism was that of a chronic infection in which recovery, chronicity, improvement, worsening, and delayed appearance of the autistic syndrome all were found. Other rubella consequences such as blindness, deafness, and cardiac and neuromuscular defects remained present except as modified by operations and prostheses. Degree of mental retardation initially was related to the outcome of autism but shifts in mental retardation over time did not correlate significantly for the group with shift in the autistic symptoms.
Membrane receptors for rubella virus (RV) in Vero cells were studied by means of two different approaches: (i) by enzyme treatment of the whole cell membrane and (ii) by testing the ability of isolated plasma membrane molecules to compete with cells for virus binding. The replication of RV was studied with both indirect immunofluorescence assay and molecular hybridization techniques. Phospholipases A2 and C digestion of cells greatly reduced the infectivity by the virus, pointing towards the involvement of lipid structures as receptor sites for RV. Furthermore, susceptibility of Vero cells to virus infection was also reduced after beta-N-acetyl-D-glucosaminidase, alpha-glucosidase and beta-galactosidase treatment, suggesting that carbohydrate residues may participate in a complex cellular receptor structure for RV. When the major membrane lipids were examined separately for their ability to inhibit viral infectivity, several phospholipids (phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin) and glycolipids (gangliosides, lactosylceramide, cerebroside sulphate) showed a strong neutralizing activity, confirming the role of membrane lipid moiety in the cell surface receptor for RV.
The Time relations between maternal rubella infection in pregnancy and the presence and type of defects in the children were determined from the records of 422 children with confirmed congenital rubella, registered in the National Congenital Rubella Surveillance Programme. In the 106 children born after laboratory-proven maternal infection, no defects were recorded following infection after the 17th week of pregnancy, but in the remaining 316 children defects followed infection reported to be as late as 33 weeks. The striking difference underlines the importance of serological investigation of pregnant women who present with a rash or a history of contact with rubella. With proven infection later than the 16th week the risk of fetal damage seems to be very small. Of 148 children followed up to school age, 40 (27%) attended normal schools.
We examined the effect of interferon-gamma (IFN gamma) on expression of the major histocompatibility proteins on cultured human islet cells isolated from adult and fetal pancreas and from an insulinoma. While the pancreatic beta-cells from different sources varied in their responses to IFN gamma, in all instances the expression of HLA-A,B,C protein was increased. Pancreatic beta-cells did not express HLA-DR protein, before or after culture of the islets in IFN gamma, although HLA-DR protein expression was induced on some non-beta-cells. These findings are at variance with those reported with thyroid follicular cells, in which IFN gamma induced expression of HLA-DR. We, therefore, conclude that the interaction between the immune and the endocrine systems may be endocrine cell specific. The up-regulation of HLA-A,B,C protein on beta-cells by IFN gamma provides a mechanism for enhanced targetting to the beta-cells of autoreactive cytotoxic T-lymphocytes and, hence, for amplifying beta-cell destruction.
Vascular lesions were reviewed in 13 autopsied infants with congenital defects of maternal rubella. The characteristic finding was intimal fibromuscular proliferation. The internal elastic lamella and media were almost always unchanged, even in the presence of a greatly thickened intima. These arterial changes appeared to be unlike other vascular lesions described in infancy.
Severe and extensive lesions were found in four infants, and lesser changes in another five. The lesions in the pulmonary artery were probably related to clinically diagnosed peripheral pulmonary artery stenosis. Ostial stenosis of a renal artery was found in an infant with documented systemic hypertension.
HLA class I and II molecules play a central role in regulating host immune responses against microbial infections because they present foreign antigens to CD8+ and CD4+ T lymphocytes, respectively. Many cytokines, especially interferons (IFN), are known to upregulate human leucocyte antigen (HLA) class I and II gene expression, but the kinetics, expression levels and viral regulation of HLA genes in primary human cells have not been well documented. Stimulation of peripheral blood mononuclear cells (PBMC) with IFN-alpha and IFN-gamma resulted in a 1.5- to twofold increase in HLA class I and beta 2-microglobulin expression in lymphocytes and monocytes. Lymphocytes did not express any detectable HLA class II either basally or after IFN induction. In monocytes, instead, a high basal class II expression was found and it was further induced by IFN-alpha (up to twofold) and especially by IFN-gamma (up to fivefold). In granulocyte-macrophage colony-stimulating factor (GM-CSF) differentiated human macrophages, basal HLA class I and II protein expression levels were high but IFN-gamma stimulation was able to further enhance their expression. Accordingly, class I and II mRNA expression was elevated by IFN-gamma, whereas IFN-alpha practically had no effect on HLA class I mRNA levels. Influenza A virus infection of macrophages resulted in temporary increases in HLA class I, beta 2-microglobulin and class II antigen expression. Neutralization of virus-induced IFN production by antibodies against type I and II IFNs prevented the virus-induced upregulation of HLA antigens. At late times of infection, as analysed by steady-state mRNA expression, both HLA class I and II mRNA were strongly reduced. These results suggest that IFNs are important regulators of HLA genes and responsible for a temporary increase in HLA antigen expression during influenza A virus infection.
