Neural Cells Research Articles

From diversity to disease: unravelling the role of enteric glial cells.

Enteric glial cells (EGCs) are an essential component of the enteric nervous system (ENS) and play key roles in gastrointestinal development, homeostasis, and disease. Derived from neural crest cells, EGCs undergo complex differentiation processes regulated by various signalling pathways. Being among the most dynamic cells of the digestive system, EGCs react to cues in their surrounding microenvironment and communicate with various cell types and systems within the gut. Morphological studies and recent single cell RNA sequencing studies have unveiled heterogeneity among EGC populations with implications for regional functions and roles in diseases. In gastrointestinal disorders, including inflammatory bowel disease (IBD), infections and cancer, EGCs modulate neuroplasticity, immune responses and tumorigenesis. Recent evidence suggests that EGCs respond plastically to the microenvironmental cues, adapting their phenotype and functions in disease states and taking on a crucial role. They exhibit molecular abnormalities and alter communication with other intestinal cell types, underscoring their therapeutic potential as targets. This review delves into the multifaceted roles of EGCs, particularly emphasizing their interactions with various cell types in the gut and their significant contributions to gastrointestinal disorders. Understanding the complex roles of EGCs in gastrointestinal physiology and pathology will be crucial for the development of novel therapeutic strategies for gastrointestinal disorders.

Proteome profiling of cerebrospinal fluid using machine learning shows a unique protein signature associated with APOE4 genotype.

Proteome changes associated with APOE4 variant carriage that are independent of Alzheimer's disease (AD) pathology and diagnosis are unknown. This study investigated APOE4 proteome changes in people with AD, mild cognitive impairment, and no impairment. Clinical, APOE genotype, and cerebrospinal fluid (CSF) proteome and AD biomarker data was sourced from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database. Proteome profiling was done using supervised machine learning. We found an APOE4-specific proteome signature that was independent of cognitive diagnosis and AD pathological biomarkers, and increased risk of progression to cognitive impairment. Proteins were enriched in brain regions including the caudate and cortex and cells including endothelial cells, oligodendrocytes, and astrocytes. Enriched peripheral immune cells included T cells, macrophages, and B cells. APOE4 carriers have a unique CSF proteome signature associated with a strong brain and peripheral immune and inflammatory phenotype that likely underlies APOE4 carriers' vulnerability to cognitive decline and AD.

Identification and characterization of intermediate states in mammalian neural crest cell epithelial to mesenchymal transition and delamination

Epithelial to mesenchymal transition (EMT) is a cellular process that converts epithelial cells to mesenchymal cells with migratory potential in developmental and pathological processes. Although originally considered a binary event, EMT in cancer progression involves intermediate states between a fully epithelial and a fully mesenchymal phenotype, which are characterized by distinct combinations of epithelial and mesenchymal markers. This phenomenon has been termed epithelial to mesenchymal plasticity (EMP), however, the intermediate states remain poorly described and it’s unclear whether they exist during developmental EMT. Neural crest cells (NCC) are an embryonic progenitor cell population that gives rise to numerous cell types and tissues in vertebrates, and their formation and delamination is a classic example of developmental EMT. However, whether intermediate states also exist during NCC EMT and delamination remains unknown. Through single-cell RNA sequencing of mouse embryos, we identified intermediate NCC states based on their transcriptional signature and then spatially defined their locations in situ in the dorsolateral neuroepithelium. Our results illustrate the importance of cell cycle regulation and functional role for the intermediate stage marker Dlc1 in facilitating mammalian cranial NCC delamination and may provide new insights into mechanisms regulating pathological EMP.

The ENCODE mouse postnatal developmental time course identifies regulatory programs of cell types and cell states.

Postnatal genomic regulation significantly influences tissue and organ maturation but is under-studied relative to existing genomic catalogs of adult tissues or prenatal development in mouse. The ENCODE4 consortium generated the first comprehensive single-nucleus resource of postnatal regulatory events across a diverse set of mouse tissues. The collection spans seven postnatal time points, mirroring human development from childhood to adulthood, and encompasses five core tissues. We identified 30 cell types, further subdivided into 69 subtypes and cell states across adrenal gland, left cerebral cortex, hippocampus, heart, and gastrocnemius muscle. Our annotations cover both known and novel cell differentiation dynamics ranging from early hippocampal neurogenesis to a new sex-specific adrenal gland population during puberty. We used an ensemble Latent Dirichlet Allocation strategy with a curated vocabulary of 2,701 regulatory genes to identify regulatory "topics," each of which is a gene vector, linked to cell type differentiation, subtype specialization, and transitions between cell states. We find recurrent regulatory topics in tissue-resident macrophages, neural cell types, endothelial cells across multiple tissues, and cycling cells of the adrenal gland and heart. Cell-type-specific topics are enriched in transcription factors and microRNA host genes, while chromatin regulators dominate mitosis topics. Corresponding chromatin accessibility data reveal dynamic and sex-specific regulatory elements, with enriched motifs matching transcription factors in regulatory topics. Together, these analyses identify both tissue-specific and common regulatory programs in postnatal development across multiple tissues through the lens of the factors regulating transcription.

Early transcriptional similarities between two distinct neural lineages during ascidian embryogenesis

In chordates, the central nervous system arises from precursors that have distinct developmental and transcriptional trajectories. Anterior nervous systems are ontogenically associated with ectodermal lineages while posterior nervous systems are associated with mesoderm. Taking advantage of the well-documented cell lineage of ascidian embryos, we asked to what extent the transcriptional states of the different neural lineages become similar during the course of progressive lineage restriction. We performed single-cell RNA sequencing (scRNA-seq) analyses on hand-dissected neural precursor cells of the two distinct lineages, together with those of their sister cell lineages, with a high temporal resolution covering five successive cell cycles from the 16-cell to neural plate stages. A transcription factor binding site enrichment analysis of neural specific genes at the neural plate stage revealed limited evidence for shared transcriptional control between the two neural lineages, consistent with their different ontogenies. Nevertheless, PCA analysis and hierarchical clustering showed that, by neural plate stages, the two neural lineages cluster together. Consistent with this, we identified a set of genes enriched in both neural lineages at the neural plate stage, including miR-124, Celf3.a, Zic.r-b, and Ets1/2. Altogether, the current study has revealed genome-wide transcriptional dynamics of neural progenitor cells of two distinct developmental origins. Our scRNA-seq dataset is unique and provides a valuable resource for future analyses, enabling a precise temporal resolution of cell types not previously described from dissociated embryos.

Pharmacological activation of TLR7 exerts inhibition on the replication of EV-D68 in respiratory cells.

The globally reemerging respiratory pathogen enterovirus D68 (EV-D68) is implicated in outbreaks of severe respiratory illness and associated with acute flaccid myelitis. However, there remains a lack of effective treatments for EV-D68 infection. In this work, we found that the host Toll-like receptor 7 (TLR7) proteins, which function as powerful innate immune sensors, were selectively elevated in expression in response to EV-D68 infection. Subsequently, we investigated the impact of Vesatolimod (GS-9620), a Toll-like receptor 7 agonist, on EV-D68 replication. Our findings revealed that EV-D68 infection resulted in increased mRNA levels of TLR7. Treatment with Vesatolimod significantly inhibited EV-D68 replication [half maximal effective concentration (EC50) = 0.1427 µM] without inducing significant cytotoxicity at virucidal concentrations. Although Vesatolimod exhibited limited impact on EV-D68 attachment, it suppressed RNA replication and viral protein synthesis after virus entry. Vesatolimod broadly inhibited the replication of circulating isolated strains of EV-D68. Furthermore, our findings demonstrated that treatment with Vesatolimod conferred resistance to both respiratory and neural cells against EV-D68 infection. Overall, these results present a promising strategy for drug development by pharmacologically activating TLR7 to initiate an antiviral state in EV-D68-infected cells selectively.IMPORTANCEConventional strategies for antiviral drug development primarily focus on directly targeting viral proteases or key components, as well as host proteins involved in viral replication. In this study, based on our intriguing discovery that enterovirus D68 (EV-D68) infection specifically upregulates the expression of immune sensor Toll-like receptor 7 (TLR7) protein, which is either absent or expressed at low levels in respiratory cells, we propose a potential antiviral approach utilizing TLR7 agonists to activate EV-D68-infected cells into an anti-viral defense state. Notably, our findings demonstrate that pharmacological activation of TLR7 effectively suppresses EV-D68 replication in respiratory tract cells through a TLR7/MyD88-dependent mechanism. This study not only presents a promising drug candidate and target against EV-D68 dissemination but also highlights the potential to exploit unique alterations in cellular innate immune responses induced by viral infections, selectively inducing a defensive state in infected cells while safeguarding uninfected normal cells from potential adverse effects associated with therapeutic interventions.

Haloperidol, Olanzapine, and Risperidone Induce Morphological Changes in an In Vitro Model of Human Hippocampal Neurogenesis.

Induced pluripotent stem cell (iPSC) based neuronal differentiation is valuable for studying neuropsychiatric disorders and pharmacological mechanisms at the cellular level. We aimed to examine the effects of typical and atypical antipsychotics on human iPSC-derived neural progenitor cells (NPCs). Proliferation and neurite outgrowth were measured by live cell imaging, and gene expression levels related to neuronal identity were analyzed by RT-QPCR and immunocytochemistry during differentiation into hippocampal dentate gyrus granule cells following treatment of low- and high-dose antipsychotics (haloperidol, olanzapine, and risperidone). Antipsychotics did not modify the growth properties of NPCs after 3 days of treatment. However, the characteristics of neurite outgrowth changed significantly in response to haloperidol and olanzapine. After three weeks of differentiation, mRNA expression levels of the selected neuronal markers increased (except for MAP2), while antipsychotics caused only subtle changes. Additionally, we found no changes in MAP2 or GFAP protein expression levels as a result of antipsychotic treatment. Altogether, antipsychotic medications promoted neurogenesis in vitro by influencing neurite outgrowth rather than changing cell survival or gene expression. This study provides insights into the effects of antipsychotics on neuronal differentiation and highlights the importance of considering neurite outgrowth as a potential target of action.

An injectable hydrogel loaded with GMSCs-derived neural lineage cells promotes recovery after stroke.

Ischemic stroke is a devastating medical condition with poor prognosis due to the lack of effective treatment modalities. Transplantation of human neural stem cells or primary neural cells is a promising treatment approach, but this is hindered by limited suitable cell sources and low in vitro expansion capacity. This study aimed i) to use small molecules to reprogram gingival mesenchymal stem cells (GMSCs) commitment to the neural lineage cells in vitro, and ii) to use hyaluronic acid (HA) hydrogel scaffolds seeded with GMSCs-derived neural lineage cells to treat ischemic stroke invivo. Neural induction was carried out with a small molecule cocktail-based one-step culture protocol over a period of 24 hours. The induced cells were analyzed for expression of neural markers with immunocytochemistry and qRT-PCR. The SD rats (n=100) were subjected to the middle cerebral artery occlusion (MCAO) reperfusion ischemic stroke model. Then, after 8 days post-MCAO, the modelled rats were randomly assigned to six study groups (n=12 per group): (i) GMSCs, (ii) GMSCs-derived neural lineage cells, (iii) HA and GMSCs-derived neural lineage cells, (iv) HA, (v) PBS, and (vi) sham transplantation control, and received their respective transplantation. Evaluation of post-stroke recovery were performed by the behavioral tests and histological assessments. The morphologically altered nature of neural lineages has been observed of the GMSCs treated with small molecules compared to the untreated controls. As shown by the qRT-PCR and immunocytochemistry, small molecules further signifcantly enhanced the experession level of neural markers of GMSCs as compared with the untreated controls (all p<0.05). Intracerebral injection of self-assembling HA hydrogel carrying GMSCs-derived neural lineage cells promoted the recovery of neural function and reduced ischemic damage in rats with ischemic stroke, as demonstrated by histological examination and behavioral assessments (all p<0.05). In conclusion, the small molecule cocktail significantly enhanced the differentiation of GMSCs into neural lineage cells. The HA hydrogel was found to facilitate the proliferation and differentiation of GMSCs-derived neural lineage cells. Furthermore, HA hydrogel seeded with GMSCs-derived neural lineage cells could promote tissue repair and functional recovery in rats with ischemic stroke and may be a promising alternative treatment modality for stroke.

Longitudinal Magnetic Resonance Imaging Tracking of Transplanted Neural Progenitor Cells in the Spinal Cord Utilizing the Bright-Ferritin Mechanism.

Human neural progenitor cells (hNPCs) hold promise for treating spinal cord injury. Studies to date have focused on improving their regenerative potential and therapeutic effect. Equally important is ensuring successful delivery and engraftment of hNPCs at the injury site. Unfortunately, no current imaging solution for cell tracking is compatible with long-term monitoring in vivo. The objective of this study was to apply a novel bright-ferritin magnetic resonance imaging (MRI) mechanism to track hNPC transplants longitudinally and on demand in the rat spinal cord. We genetically modified hNPCs to stably overexpress human ferritin. Ferritin-overexpressing (FT) hNPCs labeled with 0.2 mM manganese provided significant T1-induced bright contrast on in vitro MRI, with no adverse effect on cell viability, morphology, proliferation, and differentiation. In vivo, 2 M cells were injected into the cervical spinal cord of Rowett nude rats. MRI employed T1-weighted acquisitions and T1 mapping on a 3 T scanner. Conventional short-term cell tracking was performed using exogenous Mn labeling prior to cell transplantation, which displayed transient bright contrast on MRI 1 day after cell transplantation and disappeared after 1 week. In contrast, long-term cell tracking using bright-ferritin allowed on-demand signal recall upon Mn supplementation and precise visualization of the surviving hNPC graft. In fact, this new cell tracking technology identified 7 weeks post-transplantation as the timepoint by which substantial hNPC integration occurred. Spatial distribution of hNPCs on MRI matched that on histology. In summary, bright-ferritin provides the first demonstration of long-term, on-demand, high-resolution, and specific tracking of hNPCs in the rat spinal cord.

Research progress of genetic research on Char syndrome

Char syndrome is a rare autosomal dominant genetic disorder characterized by patent ductus arteriosus, facial dysmorphism, and dysplasia of fingers/toes. It may also be associated with multiple papillae, dental dysplasia, and sleep disorders. TFAP2B has proven to be a pathogenic gene for neural crest derivation and development, and several variants of this gene have been identified. Bone morphogenetic protein signaling plays an important role in embryonic development by participating in limb growth and patterning, and regulation of neural crest cell development. TFAP2B is an upstream regulatory gene for bone morphogenetic proteins 2 and 4. Variants of the TFAP2B gene may lead to abnormal proliferation of neural crest cells by affecting the expression of bone morphogenetic proteins, resulting in multiple organ dysplasia syndrome. In addition, TFAP2B variants may only lead to patent ductus arteriosus instead of typical Char syndrome.

A STATISTICAL FRAMEWORK FOR THE MODELING OF THE CONTINUOUS PROGRESSION OF ALZHEIMER'S DISEASE.

Throughout an organism's life, a multitude of biological systems transition through complex biophysical processes. These processes serve as indicators of the underlying biological states. Inferring these latent unobserved states is a key problem in modern biology and neuroscience. Unfortunately, in many experimental setups we can at best obtain snapshots of the system at different times for different individuals, and one major challenge is the one of reconciling those measurements. This formalism is particularly relevant in the study of Alzheimer's Disease (AD) progression, in which we observe in brain donors the aggregation of pathological proteins but the underlying disease state is unknown. The progression of AD can be modeled by assigning a latent score - termed pseudotime - to each pathological state, creating a pseudotemporal ordering of donors based on their pathological burden. This paper proposes a hierarchical Bayesian framework to model AD progression using detailed quantification of multiple AD pathological proteins from the Seattle AD Brain Cell Atlas consortium (SEA-AD). Inspired by biophysical models, we model pathological burden as an exponential process. Theoretical properties of the model are studied, by using linearization to reveal convergence and identifiability properties. We provide Markov chain Monte Carlo estimation algorithms, and show the effectiveness of our approach with multiple simulation studies across data conditions. Applying the methodology to SEA-AD brain data, we infer pseudotime for each donor and order them by pathological burden. Finally, we analyze the information within each pathological feature and utilize it to refine the model by focusing on the most informative pathologies. This lays the groundwork for suggesting future experimental design approaches.

Novel miRNA-inducing drugs enable differentiation of retinoic acid-resistant neuroblastoma cells.

Tumor cell heterogeneity in neuroblastoma, a pediatric cancer arising from neural crest-derived progenitor cells, poses a significant clinical challenge. In particular, unlike adrenergic (ADRN) neuroblastoma cells, mesenchymal (MES) cells are resistant to chemotherapy and retinoid therapy and thereby significantly contribute to relapses and treatment failures. Previous research suggested that overexpression or activation of miR-124, a neurogenic microRNA with tumor suppressor activity, can induce the differentiation of retinoic acid-resistant neuroblastoma cells. Leveraging our established screen for miRNA modulatory small molecules, we validated PP121, a dual inhibitor of tyrosine and phosphoinositide kinases, as a robust inducer of miR-124. A combination of PP121 and miR-132-inducing bufalin synergistically arrests proliferation, induces differentiation, and prolongs the survival of differentiated MES SK-N-AS cells for 8 weeks. RNA- seq and deconvolution analyses revealed a collapse of the ADRN core regulatory circuitry (CRC) and the emergence of novel CRCs associated with chromaffin cells and Schwann cell precursors. Using a similar protocol, we differentiated and maintained other MES neuroblastoma, as well as glioblastoma cells, over 16 weeks. In conclusion, our novel protocol suggests a promising treatment for therapy-resistant cancers of the nervous system. Moreover, these long-lived, differentiated cells provide valuable models for studying mechanisms underlying differentiation, maturation, and senescence.

Revealing brain cell-stratified causality through dissecting causal variants according to their cell-type-specific effects on gene expression

The human brain has been implicated in the pathogenesis of several complex diseases. Taking advantage of single-cell techniques, genome-wide association studies (GWAS) have taken it a step further and revealed brain cell-type-specific functions for disease loci. However, genetic causal associations inferred by Mendelian randomization (MR) studies usually include all instrumental variables from GWAS, which hampers the understanding of cell-specific causality. Here, we developed an analytical framework, Cell-Stratified MR (csMR), to investigate cell-stratified causality through colocalizing GWAS signals with single-cell eQTL from different brain cells. By applying to obesity-related traits, our results demonstrate the cell-type-specific effects of GWAS variants on gene expression, and indicate the benefits of csMR to identify cell-type-specific causal effect that is often hidden from bulk analyses. We also found csMR valuable to reveal distinct causal pathways between different obesity indicators. These findings suggest the value of our approach to prioritize target cells for extending genetic causation studies.

Inhalation of hydrogen gas mitigates sevoflurane‐induced neuronal apoptosis in the neonatal cortex and is associated with changes in protein phosphorylation

AbstractInhalation of hydrogen (H2) gas is therapeutically effective for cerebrovascular diseases, neurodegenerative disorders, and neonatal brain disorders including pathologies induced by anesthetic gases. To understand the mechanisms underlying the protective effects of H2 on the brain, we investigated the molecular signals affected by H2 in sevoflurane‐induced neuronal cell death. We confirmed that neural progenitor cells are susceptible to sevoflurane and undergo apoptosis in the retrosplenial cortex of neonatal mice. Co‐administration of 1–8% H2 gas for 3 h to sevoflurane‐exposed pups suppressed elevated caspase‐3‐mediated apoptotic cell death and concomitantly decreased c‐Jun phosphorylation and activation of the c‐Jun pathway, all of which are induced by oxidative stress. Anesthesia‐induced increases in lipid peroxidation and oxidative DNA damage were alleviated by H2 inhalation. Phosphoproteome analysis revealed enriched clusters of differentially phosphorylated proteins in the sevoflurane‐exposed neonatal brain that included proteins involved in neuronal development and synaptic signaling. H2 inhalation modified cellular transport pathways that depend on hyperphosphorylated proteins including microtubule‐associated protein family. These modifications may be involved in the protective mechanisms of H2 against sevoflurane‐induced neuronal cell death.image

Schwannian and Perineuriomatous Differentiation in a Series of Giant Congenital Melanocytic Nevi.

Close relationship between melanocytes and neural cells is accepted to reflect their common derivation from the neural crest and tumors combining both elements. We present a series of 10 patients with giant congenital melanocytic nevi (CMN) in which a secondary proliferation (11 lesions) with schwannian and/or perineuriomatous differentiation developed in the course of the disease. The age of the patients (4 male and 6 female) at the time of surgery and histological assessment varied from 3 months to 57 years. Histopathologically, the following subgroups were delineated: (1) nodular/tumoriform "neurotization" in CMN, (2) diffuse neurofibroma-like proliferation within CMN, (3) plexiform neurofibroma-like proliferation within CMN, and (4) diffuse perineuriomatous (hybrid schwannomatous-perineuriomatous) differentiation in CMN. We review the pertinent literature, including the role of recently identified Schwann cell precursors which are believed to represent the nerve-associated state of neural crest-like cells that persists into later developmental stages.

Cholesterol Metabolism in Neurodegenerative Diseases.

Cholesterol plays a crucial role in brain, where it is highly concentrated and tightly regulated to support normal brain functions. It serves as a vital component of cell membranes, ensuring their integrity, and acts as a key regulator of various brain processes. Dysregulation of cholesterol metabolism in the brain has been linked to impaired brain function and the onset of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). A significant advancement has been the identification of astrocyte-derived ApoE as a key regulator of de novo cholesterol biosynthesis in neurons, providing insights into how extracellular signals influence neuronal cholesterol levels. Additionally, the development of antibody-based therapies, particularly for AD, presents promising opportunities for therapeutic interventions. Despite significant research, the association between cholesterol and neurodegenerative diseases remains inconclusive. It is crucial to distinguish between plasma cholesterol and brain cholesterol, as these pools are relatively independent. This differentiation should be considered when evaluating statin-based treatment approaches. Assessing not only the total cholesterol content in the brain but also its distribution among different types of brain cells is essential. Establishing a causal link between changes in brain/plasma cholesterol levels and the onset of brain dysfunction/neurodegenerative diseases remains a key objective. Additionally, conducting cell-specific analyses of cholesterol homeostasis in various types of brain cells under pathological conditions will enhance our understanding of cholesterol metabolism in neurodegenerative diseases. Manipulating cholesterol levels to restore homeostasis may represent a novel approach for alleviating neurological symptom.

Edukasi Pencegahan Anemia pada Remaja Putri di PPTQ Al-Rasyid Kartasura

Adolescents experience a very dynamic period of physical development. Anemia is one of the problems experienced by young women. If iron intake in adolescent girls is insufficient, adolescents can experience problems with their needs and immune response. One of the causes of anemia in teenagers is a lack of knowledge about anemia. This service activity was for young women at the Tahfidz Quran Al-Rasyid Kartasura Islamic Boarding School. This service aims to educate young women about anemia to prevent anemia. The method for implementing this service program is to provide counseling, starting with a pre-test, delivering material and interactive discussions, and ending with a post-test. The results achieved from this service activity were increased teenagers' knowledge about anemia before and after the counseling. This shows the importance of health education or promotion to be carried out continuously to teenagers. Knowledge has a vital role in shaping a person's actions. It is hoped that teenagers with good knowledge about anemia can prevent anemia in themselves because it will affect learning ability and concentration, inhibiting the growth of both body and brain cells. Therefore, teenagers can increase their understanding of anemia through various media and improve their lifestyle.

Edukasi Pencegahan Anemia pada Remaja Putri di PPTQ Al-Rasyid Kartasura

Adolescents experience a very dynamic period of physical development. Anemia is one of the problems experienced by young women. If iron intake in adolescent girls is insufficient, adolescents can experience problems with their needs and immune response. One of the causes of anemia in teenagers is a lack of knowledge about anemia. This service activity was for young women at the Tahfidz Quran Al-Rasyid Kartasura Islamic Boarding School. This service aims to educate young women about anemia to prevent anemia. The method for implementing this service program is to provide counseling, starting with a pre-test, delivering material and interactive discussions, and ending with a post-test. The results achieved from this service activity were increased teenagers' knowledge about anemia before and after the counseling. This shows the importance of health education or promotion to be carried out continuously to teenagers. Knowledge has a vital role in shaping a person's actions. It is hoped that teenagers with good knowledge about anemia can prevent anemia in themselves because it will affect learning ability and concentration, inhibiting the growth of both body and brain cells. Therefore, teenagers can increase their understanding of anemia through various media and improve their lifestyle.

Human Cytomegalovirus Dysregulates Cellular Dual-Specificity Tyrosine Phosphorylation-Regulated Kinases and Sonic Hedgehog Pathway Proteins in Neural Astrocyte and Placental Models.

Human cytomegalovirus (CMV) infection is the leading non-genetic cause of congenital malformation in developed countries, causing significant fetal injury, and in some cases fetal death. The pathogenetic mechanisms through which this host-specific virus infects then damages both the placenta and the fetal brain are currently ill-defined. We investigated the CMV modulation of key signaling pathway proteins for these organs including dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) and Sonic Hedgehog (SHH) pathway proteins using human first trimester placental trophoblast (TEV-1) cells, primary human astrocyte (NHA) brain cells, and CMV-infected human placental tissue. Immunofluorescence demonstrated the accumulation and re-localization of SHH proteins in CMV-infected TEV-1 cells with Gli2, Ulk3, and Shh re-localizing to the CMV cytoplasmic virion assembly complex (VAC). In CMV-infected NHA cells, DYRK1A re-localized to the VAC and DYRK1B re-localized to the CMV nuclear replication compartments, and the SHH proteins re-localized with a similar pattern as was observed in TEV-1 cells. Western blot analysis in CMV-infected TEV-1 cells showed the upregulated expression of Rb, Ulk3, and Shh, but not Gli2. In CMV-infected NHA cells, there was an upregulation of DYRK1A, DYRK1B, Gli2, Rb, Ulk3, and Shh. These in vitro monoculture findings are consistent with patterns of protein upregulation and re-localization observed in naturally infected placental tissue and CMV-infected ex vivo placental explant histocultures. This study reveals CMV-induced changes in proteins critical for fetal development, and identifies new potential targets for CMV therapeutic development.

Long read subcellular fractionation and sequencing reveals the translational fate of full-length mRNA isoforms during neuronal differentiation.

Alternative splicing (AS) alters the cis-regulatory landscape of mRNA isoforms leading to transcripts with distinct localization, stability and translational efficiency. To rigorously investigate mRNA isoform-specific ribosome association, we generated subcellular fractionation and sequencing (Frac-seq) libraries using both conventional short reads and long reads from human embryonic stem cells (ESC) and neural progenitor cells (NPC) derived from the same ESC. We performed de novo transcriptome assembly from high-confidence long reads from cytosolic, monosomal, light and heavy polyribosomal fractions and quantified their abundance using short reads from their respective subcellular fractions. Thousands of transcripts in each cell type exhibited association with particular subcellular fractions relative to the cytosol. Of the multi-isoform genes, 27% and 19% exhibited significant differential isoform sedimentation in ESC and NPC respectively. Alternative promoter usage and internal exon skipping accounted for the majority of differences between isoforms from the same gene. Random forest classifiers implicated coding sequence (CDS) and UTR lengths as important determinants of isoform-specific sedimentation profiles, and motif analyses reveal potential cell type-specific and subcellular fraction-associated RNA-binding protein signatures. Taken together our data demonstrate that alternative mRNA processing within the CDS and UTRs impacts the translational control of mRNA isoforms during stem cell differentiation, and highlights the utility of using a novel long-read sequencing-based method to study translational control.