Dual function of Zika virus NS2B-NS3 protease.

Zika virus (ZIKV) serine protease, indispensable for viral polyprotein processing and replication, is composed of the membrane-anchored NS2B polypeptide and the N-terminal domain of the NS3 polypeptide (NS3pro). The C-terminal domain of the NS3 polypeptide (NS3hel) is necessary for helicase activity and contains an ATP-binding site. We discovered that ZIKV NS2B-NS3pro binds single-stranded RNA with a Kd of ~0.3 µM, suggesting a novel function. We tested various structural modifications of NS2B-NS3pro and observed that constructs stabilized in the recently discovered "super-open" conformation do not bind RNA. Likewise, stabilizing NS2B-NS3pro in the "closed" (proteolytically active) conformation using substrate inhibitors abolished RNA binding. We posit that RNA binding occurs when ZIKV NS2B-NS3pro adopts the "open" conformation, which we modeled using highly homologous dengue NS2B-NS3pro crystallized in the open conformation. We identified two positively charged fork-like structures present only in the open conformation of NS3pro. These forks are conserved across Flaviviridae family and could be aligned with the positively charged grove on NS3hel, providing a contiguous binding surface for the negative RNA strand exiting helicase. We propose a "reverse inchworm" model for a tightly intertwined NS2B-NS3 helicase-protease machinery, which suggests that NS2B-NS3pro cycles between open and super-open conformations to bind and release RNA enabling long-range NS3hel processivity. The transition to the closed conformation, likely induced by the substrate, enables the classical protease activity of NS2B-NS3pro.

Tracking induced pluripotent stem cell differentiation with a fluorescent genetically encoded epigenetic probe.

Epigenetic modifications (methylation, acetylation, etc.) of core histones play a key role in regulation of gene expression. Thus, the epigenome changes strongly during various biological processes such as cell differentiation and dedifferentiation. Classical methods of analysis of epigenetic modifications such as mass-spectrometry and chromatin immuno-precipitation, work with fixed cells only. Here we present a genetically encoded fluorescent probe, MPP8-Green, for detecting H3K9me3, a histone modification associated with inactive chromatin. This probe, based on the chromodomain of MPP8, allows for visualization of H3K9me3 epigenetic landscapes in single living cells. We used this probe to track changes in H3K9me3 landscapes during the differentiation of induced pluripotent stem cells (iPSCs) into induced neurons. Our findings revealed two major waves of global H3K9me3 reorganization during 4-day differentiation, namely on the first and third days, whereas nearly no changes occurred on the second and fourth days. The proposed method LiveMIEL (Live-cell Microscopic Imaging of Epigenetic Landscapes), which combines genetically encoded epigenetic probes and machine learning approaches, enables classification of multiparametric epigenetic signatures of single cells during stem cell differentiation and potentially in other biological models.

ETS1 loss in mice impairs cardiac outflow tract septation via a cell migration defect autonomous to the neural crest.

Ets1 deletion in some mouse strains causes septal defects and has been implicated in human congenital heart defects in Jacobsen syndrome, in which one copy of the Ets1 gene is missing. Here, we demonstrate that loss of Ets1 in mice results in a decrease in neural crest (NC) cells migrating into the proximal outflow tract cushions during early heart development, with subsequent malalignment of the cushions relative to the muscular ventricular septum, resembling double outlet right ventricle (DORV) defects in humans. Consistent with this, we find that cultured cardiac NC cells from Ets1 mutant mice or derived from iPS cells from Jacobsen patients exhibit decreased migration speed and impaired cell-to-cell interactions. Together, our studies demonstrate a critical role for ETS1 for cell migration in cardiac NC cells that are required for proper formation of the proximal outflow tracts. These data provide further insights into the molecular and cellular basis for development of the outflow tracts, and how perturbation of NC cells can lead to DORV.

The Compound SBI-0090799 Inhibits Zika Virus Infection by Blocking De Novo Formation of the Membranous Replication Compartment.

Zika virus (ZIKV) is a mosquito-borne pathogen classified by the World Health Organization (WHO) as a public health emergency of international concern in 2016, and it is still identified as a priority disease. Although most infected individuals are asymptomatic or show mild symptoms, a risk of neurologic complications is associated with infection in adults. Additionally, infection during pregnancy is directly linked to microcephaly and other congenital malformations. Since there are no currently available vaccines or approved therapeutics for this virus, there is a critical unmet need in developing treatments to prevent future ZIKV outbreaks. Toward this end, we performed a large-scale cell-based high-content screen of 51,520 chemical compounds to identify potential antiviral drug candidates. The compound (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) was found to inhibit replication of multiple ZIKV strains and in different cell systems. SBI-0090799 did not affect viral entry or RNA translation but suppressed RNA replication by preventing the formation of the membranous replication compartment. Selection of drug-resistant viruses identified single-amino-acid substitutions in the N-terminal region of nonstructural protein NS4A, arguing this is the likely drug target. These resistance mutations rescued viral RNA replication and restored the formation of the membranous replication compartment. This mechanism of action is similar to clinically approved NS5A inhibitors for hepatitis C virus (HCV). Taken together, SBI-0090799 represents a promising lead candidate for the development of an antiviral treatment against ZIKV infection for the mitigation of severe complications and potential resurgent outbreaks of the virus. IMPORTANCE This study describes the elucidation of (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) as a selective and potent inhibitor of Zika virus (ZIKV) replication using a high-throughput screening approach. Mapping and resistance studies, supported by electron microscopy observations, indicate that the small molecule is functioning through inhibition of NS4A-mediated formation of ZIKV replication compartments in the endoplasmic reticulum (ER). Intriguingly, this defines a novel nonenzymatic target and chemical matter for the development of a new class of ZIKV antivirals. Moreover, chemical modulation affecting this nonstructural protein mirrors the identification and development of hepatitis C virus (HCV) NS5A inhibitor daclatasvir and its derivatives, similarly interfering with the formation of the viral replication compartment and also targeting a protein with no enzymatic activity, which have been part of a curative strategy for HCV.

SOX2 primes the epigenetic landscape in neural precursors enabling proper gene activation during hippocampal neurogenesis.

Newborn granule neurons generated from neural progenitor cells (NPCs) in the adult hippocampus play a key role in spatial learning and pattern separation. However, the molecular mechanisms that control activation of their neurogenic program remain poorly understood. Here, we report a novel function for the pluripotency factor sex-determining region Y (SRY)-related HMG box 2 (SOX2) in regulating the epigenetic landscape of poised genes activated at the onset of neuronal differentiation. We found that SOX2 binds to bivalently marked promoters of poised proneural genes [neurogenin 2 (Ngn2) and neurogenic differentiation 1 (NeuroD1)] and a subset of neurogenic genes [e.g., SRY-box 21 (Sox21), brain-derived neurotrophic factor (Bdnf), and growth arrest and DNA-damage-inducible, beta (Gadd45b)] where it functions to maintain the bivalent chromatin state by preventing excessive polycomb repressive complex 2 activity. Conditional ablation of SOX2 in adult hippocampal NPCs impaired the activation of proneural and neurogenic genes, resulting in increased neuroblast death and functionally aberrant newborn neurons. We propose that SOX2 sets a permissive epigenetic state in NPCs, thus enabling proper activation of the neuronal differentiation program under neurogenic cue.

SOX2-LIN28/let-7 pathway regulates proliferation and neurogenesis in neural precursors.

The transcription factor SRY (sex-determining region)-box 2 (SOX2) is an important functional marker of neural precursor cells (NPCs) and plays a critical role in self-renewal and neuronal differentiation; however, the molecular mechanisms underlying its functions are poorly understood. Using human embryonic stem cell-derived NPCs to model neurogenesis, we found that SOX2 is required to maintain optimal levels of LIN28, a well-characterized suppressor of let-7 microRNA biogenesis. Exogenous LIN28 expression rescued the NPC proliferation deficit, as well as the early but not the late stages of the neurogenic deficit associated with the loss of SOX2. We found that SOX2 binds to a proximal site in the LIN28 promoter region and regulates LIN28 promoter acetylation, likely through interactions with the histone acetyltransferase complex. Misexpression of let-7 microRNAs in NPCs reduced proliferation and inhibited neuronal differentiation, phenocopying the loss of SOX2. In particular, we identified let-7i as a novel and potent inhibitor of neuronal differentiation that targets MASH1 and NGN1, two well-characterized proneural genes. In conclusion, we discovered the SOX2-LIN28/let-7 pathway as a unique molecular mechanism governing NPC proliferation and neurogenic potential.

ImAge quantitates aging and rejuvenation.

For efficient, cost-effective and personalized healthcare, biomarkers that capture aspects of functional, biological aging, thus predicting disease risk and lifespan more accurately and reliably than chronological age, are essential. We developed an imaging-based chromatin and epigenetic age (ImAge) that captures intrinsic age-related trajectories of the spatial organization of chromatin and epigenetic marks in single nuclei, in mice. We show that such trajectories readily emerge as principal changes in each individual dataset without regression on chronological age, and that ImAge can be computed using several epigenetic marks and DNA labeling. We find that interventions known to affect biological aging induce corresponding effects on ImAge, including increased ImAge upon chemotherapy treatment and decreased ImAge upon caloric restriction and partial reprogramming by transient OSKM expression in liver and skeletal muscle. Further, ImAge readouts from chronologically identical mice inversely correlated with their locomotor activity, suggesting that ImAge may capture elements of biological and functional age. In sum, we developed ImAge, an imaging-based biomarker of aging with single-cell resolution rooted in the analysis of spatial organization of epigenetic marks.

Allosteric Inhibitors of Zika Virus NS2B-NS3 Protease Targeting Protease in "Super-Open" Conformation.

The Zika virus (ZIKV), a member of the Flaviviridae family, is considered a major health threat causing multiple cases of microcephaly in newborns and Guillain-Barré syndrome in adults. In this study, we targeted a transient, deep, and hydrophobic pocket of the "super-open" conformation of ZIKV NS2B-NS3 protease to overcome the limitations of the active site pocket. After virtual docking screening of approximately seven million compounds against the novel allosteric site, we selected the top six candidates and assessed them in enzymatic assays. Six candidates inhibited ZIKV NS2B-NS3 protease proteolytic activity at low micromolar concentrations. These six compounds, targeting the selected protease pocket conserved in ZIKV, serve as unique drug candidates and open new opportunities for possible treatment against several flavivirus infections.

Imaging-based chromatin and epigenetic age, ImAge, quantitates aging and rejuvenation.

Biomarkers of biological age that predict the risk of disease and expected lifespan better than chronological age are key to efficient and cost-effective healthcare1-3. To advance a personalized approach to healthcare, such biomarkers must reliably and accurately capture individual biology, predict biological age, and provide scalable and cost-effective measurements. We developed a novel approach - image-based chromatin and epigenetic age (ImAge) that captures intrinsic progressions of biological age, which readily emerge as principal changes in the spatial organization of chromatin and epigenetic marks in single nuclei without regression on chronological age. ImAge captured the expected acceleration or deceleration of biological age in mice treated with chemotherapy or following a caloric restriction regimen, respectively. ImAge from chronologically identical mice inversely correlated with their locomotor activity (greater activity for younger ImAge), consistent with the widely accepted role of locomotion as an aging biomarker across species. Finally, we demonstrated that ImAge is reduced following transient expression of OSKM cassette in the liver and skeletal muscles and reveals heterogeneity of in vivo reprogramming. We propose that ImAge represents the first-in-class imaging-based biomarker of aging with single-cell resolution.

Primary cilia regulate proliferation of amplifying progenitors in adult hippocampus: implications for learning and memory.

Integration of new neurons into the adult hippocampus has been linked to specific types of learning. Primary cilia were found to be required for the formation of adult neural stem cells (NSCs) in the hippocampal dentate gyrus during development. However, the requirement of cilia in maintenance of adult NSCs is unknown. We developed a genetic mouse model in which fetal/perinatal brain development is unaffected, but adult hippocampal neurogenesis is constantly reduced by conditional ablation of primary cilia in adult GFAP(+) neural stem/progenitor cells. We found that this approach specifically reduces the number of hippocampal amplifying progenitors (also called type 2a cells) without affecting the number of radial NSCs (or type 1 cells). Constant reduction of adult hippocampal neurogenesis produced a delay rather than a permanent deficiency in spatial learning without affecting the retention of long-term memories. Decreased neurogenesis also altered spatial novelty recognition and hippocampus-independent cue conditioning. Here, we propose that adult hippocampal newborn neurons increase the efficiency of generating the new representations of spatial memories and that reduction of adult hippocampal neurogenesis may be biased toward cue-based strategies. This novel mouse model provides evidences that cognitive deficits associated with ciliary defects (ciliopathies) might be, in part, mediated by the deficiency of primary cilia in adult hippocampal stem/progenitor cells.

High-content analysis and Kinetic Image Cytometry identify toxicity and epigenetic effects of HIV antiretrovirals on human iPSC-neurons and primary neural precursor cells.

INTRODUCTION: Despite viral suppression due to combination antiretroviral therapy (cART), HIV-associated neurocognitive disorders (HAND) continue to affect half of people with HIV, suggesting that certain antiretrovirals (ARVs) may contribute to HAND. METHODS: We examined the effects of nucleoside/nucleotide reverse transcriptase inhibitors tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) and the integrase inhibitors dolutegravir (DTG) and elvitegravir (EVG) on viability, structure, and function of glutamatergic neurons (a subtype of CNS neuron involved in cognition) derived from human induced pluripotent stem cells (hiPSC-neurons), and primary human neural precursor cells (hNPCs), which are responsible for neurogenesis. RESULTS: Using automated digital microscopy and image analysis (high content analysis, HCA), we found that DTG, EVG, and TDF decreased hiPSC-neuron viability, neurites, and synapses after 7 days of treatment. Analysis of hiPSC-neuron calcium activity using Kinetic Image Cytometry (KIC) demonstrated that DTG and EVG also decreased the frequency and magnitude of intracellular calcium transients. Longer ARV exposures and simultaneous exposure to multiple ARVs increased the magnitude of these neurotoxic effects. Using the Microscopic Imaging of Epigenetic Landscapes (MIEL) assay, we found that TDF decreased hNPC viability and changed the distribution of histone modifications that regulate chromatin packing, suggesting that TDF may reduce neuroprogenitor pools important for CNS development and maintenance of cognition in adults. CONCLUSION: This study establishes human preclinical assays that can screen potential ARVs for CNS toxicity to develop safer cART regimens and HAND therapeutics.

A Zika virus mutation enhances transmission potential and confers escape from protective dengue virus immunity.

Zika virus (ZIKV) and dengue virus (DENV) are arthropod-borne pathogenic flaviviruses that co-circulate in many countries. To understand some of the pressures that influence ZIKV evolution, we mimic the natural transmission cycle by repeating serial passaging of ZIKV through cultured mosquito cells and either DENV-naive or DENV-immune mice. Compared with wild-type ZIKV, the strains passaged under both conditions exhibit increased pathogenesis in DENV-immune mice. Application of reverse genetics identifies an isoleucine-to-valine mutation (I39V) in the NS2B proteins of both passaged strains that confers enhanced fitness and escape from pre-existing DENV immunity. Introduction of I39V or I39T, a naturally occurring homologous mutation detected in recent ZIKV isolates, increases the replication of wild-type ZIKV in human neuronal precursor cells and laboratory-raised mosquitoes. Our data indicate that ZIKV strains with enhanced transmissibility and pathogenicity can emerge in DENV-naive or -immune settings, and that NS2B-I39 mutants may represent ZIKV variants of interest.

The Rise of Induced Pluripotent Stem Cell Approach to Hair Restoration.

SUMMARY: The advent of pluripotent stem cells following the discovery of Shinya Yamanaka (2012 Nobel prize in Medicine) brought about a regenerative medicine approach to virtually every human condition including hair loss. It is now possible to reprogram somatic cells (eg, blood or skin cells) from a person experiencing hair loss to generate autologous induced pluripotent stem cells (iPSCs), which could be amplified and cryopreserved. Subsequently, these iPSCs could be differentiated into various cell types such as dermal papilla cells, epithelial cells, melanocytes, and other cell types constituting functional hair follicle. Transplantation of human iPSC-derived folliculogenic cells into the nude mice has successfully generated xenografts with hair outgrowth. Because iPSCs provide a virtually unlimited source of folliculogenic cells for de novo formation of hair follicles, this approach has major advantages over current surgical hair restoration procedures, which merely redistribute existing hair follicles from one part of the sculp to another. Combined with robotics and automation of the transplantation process, this novel regenerative medicine approach is well poised to make hair restoration a routine procedure affordable for everybody who can benefit from it.

Nicotinamide rescues human embryonic stem cell-derived neuroectoderm from parthanatic cell death.

Abundant cell death is observed when human embryonic stem cells (hESCs) undergo neuralization, a critical first step for future cell-based therapies addressing neurodegeneration. Using hESC neuralization as an in vitro model of human development, we demonstrated that the developing neuroepithelium acquires increased susceptibility to spontaneous cell death. We found that poly(ADP-ribose) polymerase-1 (PARP1)/apoptosis-inducing factor (AIF)-mediated cell death (parthanatos) is a dominant mechanism responsible for cell loss during hESC neuralization. The demise of neural progenitor cells, at least in part, is due to decreased endogenous antioxidant defenses and enhanced reactive oxygen species leakage from mitochondria fuelled by nonphysiological culture conditions. Under such conditions, PARP1 overactivation triggered cell death through the mitochondrial-nuclear translocation of AIF. Blocking PARP1 activity with small hairpin RNA interference or nicotinamide dramatically enhanced hESC neuralization, providing optimal survival of the developing neuroepithelium. Because nicotinamide is a physiological metabolite, our results raise the possibility that neural stem/progenitor cell survival in vivo requires a metabolic niche. We argue that small natural metabolites provide a powerful physiological tool to optimize hESC differentiation compatible with the requirements of regenerative medicine.

Maternal embryonic leucine zipper kinase (MELK) regulates multipotent neural progenitor proliferation.

Maternal embryonic leucine zipper kinase (MELK) was previously identified in a screen for genes enriched in neural progenitors. Here, we demonstrate expression of MELK by progenitors in developing and adult brain and that MELK serves as a marker for self-renewing multipotent neural progenitors (MNPs) in cultures derived from the developing forebrain and in transgenic mice. Overexpression of MELK enhances (whereas knockdown diminishes) the ability to generate neurospheres from MNPs, indicating a function in self-renewal. MELK down-regulation disrupts the production of neurogenic MNP from glial fibrillary acidic protein (GFAP)-positive progenitors in vitro. MELK expression in MNP is cell cycle regulated and inhibition of MELK expression down-regulates the expression of B-myb, which is shown to also mediate MNP proliferation. These findings indicate that MELK is necessary for proliferation of embryonic and postnatal MNP and suggest that it regulates the transition from GFAP-expressing progenitors to rapid amplifying progenitors in the postnatal brain.

Comprehensive spatiotemporal transcriptomic analyses of the ganglionic eminences demonstrate the uniqueness of its caudal subdivision.

The elucidation of mechanisms underlying telencephalic neural development has been limited by the lack of knowledge regarding the molecular and cellular aspects of the ganglionic eminence (GE), an embryonic structure that supplies the brain with diverse sets of GABAergic neurons. Here, we report a comprehensive transcriptomic analysis of this structure including its medial (MGE), lateral (LGE) and caudal (CGE) subdivisions and its temporal dynamics in 12.5 to 16 day-old rat embryos. Surprisingly, comparison across subdivisions showed that CGE gene expression was the most unique providing unbiased genetic evidence for its differentiation from MGE and LGE. The molecular signature of the CGE comprised a large set of genes, including Rwdd3, Cyp26b1, Nr2f2, Egr3, Cpta1, Slit3, and Hod, of which several encode cell signaling and migration molecules such as WNT5A, DOCK9, VSNL1 and PRG1. Temporal analysis of the MGE revealed differential expression of unique sets of cell specification and migration genes, with early expression of Hes1, Lhx2, Ctgf and Mdk, and late enrichment of Olfm3, SerpinE2 and Wdr44. These GE profiles reveal new candidate regulators of spatiotemporally governed GABAergic neuronogenesis.

Improving drug discovery using image-based multiparametric analysis of the epigenetic landscape.

High-content phenotypic screening has become the approach of choice for drug discovery due to its ability to extract drug-specific multi-layered data. In the field of epigenetics, such screening methods have suffered from a lack of tools sensitive to selective epigenetic perturbations. Here we describe a novel approach, Microscopic Imaging of Epigenetic Landscapes (MIEL), which captures the nuclear staining patterns of epigenetic marks and employs machine learning to accurately distinguish between such patterns. We validated the MIEL platform across multiple cells lines and using dose-response curves, to insure the fidelity and robustness of this approach for high content high throughput drug discovery. Focusing on noncytotoxic glioblastoma treatments, we demonstrated that MIEL can identify and classify epigenetically active drugs. Furthermore, we show MIEL was able to accurately rank candidate drugs by their ability to produce desired epigenetic alterations consistent with increased sensitivity to chemotherapeutic agents or with induction of glioblastoma differentiation.

Zika Virus Protease Cleavage of Host Protein Septin-2 Mediates Mitotic Defects in Neural Progenitors.

Zika virus (ZIKV) targets neural progenitor cells in the brain, attenuates cell proliferation, and leads to cell death. Here, we describe a role for the ZIKV protease NS2B-NS3 heterodimer in mediating neurotoxicity through cleavage of a host protein required for neurogenesis. Similar to ZIKV infection, NS2B-NS3 expression led to cytokinesis defects and cell death in a protease activity-dependent fashion. Among binding partners, NS2B-NS3 cleaved Septin-2, a cytoskeletal factor involved in cytokinesis. Cleavage of Septin-2 occurred at residue 306 and forced expression of a non-cleavable Septin-2 restored cytokinesis, suggesting a direct mechanism of ZIKV-induced neural toxicity. VIDEO ABSTRACT.

Efficient propagation of single cells Accutase-dissociated human embryonic stem cells.

Human embryonic stem cells (hESCs) hold great promise for cell-based therapies and drug screening applications. However, growing and processing large quantities of undifferentiated hESCs is a challenging task. Conventionally, hESCs are passaged as clusters, which can limit their growth efficiency and use in downstream applications. This study demonstrates that hESCs can be passaged as single cells using Accutase, a formulated mixture of digestive enzymes. In contrast to trypsin treatment, Accutase treatment does not significantly affect the viability and proliferation rate of hESC dissociation into single cells. Accutase-dissociated single cells can be separated by FACS and proliferate as fully pluripotent hESCs. An Oct4-eGFP reporter construct engineered into hESCs was used to monitor the pluripotency of hESCs passaged with Accutase. Compared to collagenase-passaged hESCs, Accutase-treated cultures contained a larger proportion of undifferentiated (Oct4-positive) cells. Additionally, Accutase-passaged undifferentiated hESCs could be grown as monolayers without the need for monitoring and/or selection for quality hESC colonies.

Zika Virus: Origins, Pathological Action, and Treatment Strategies.

The Zika virus (ZIKV) global epidemic prompted the World Health Organization to declare it a 2016 Public Health Emergency of International Concern. The overwhelming experience over the past several years teaches us that ZIKV and the associated neurological complications represent a long-term world-wide challenge to public health. Although the number of ZIKV cases in the Western Hemisphere has dropped since 2016, the need for basic research and anti-ZIKV drug development remains strong. Re-emerging viruses like ZIKV are an ever-present threat in the 21st century where fast transcontinental travel lends itself to viral epidemics. Here, we first present the origin story for ZIKV and review the rapid progress researchers have made toward understanding of the ZIKV pathology and in the design, re-purposing, and testing-particularly in vivo-drug candidates for ZIKV prophylaxis and therapy ZIKV. Quite remarkably, a short, but intensive, drug-repurposing effort has already resulted in several readily available FDA-approved drugs that are capable of effectively combating the virus in infected adult mouse models and, most importantly, in both preventing maternal-fetal transmission and severe microcephaly in newborns in pregnant mouse models.