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Dopamine system dysfunction is commonly implicated in adolescent-onset neuropsychiatric disorders. Although psychosis symptoms can be alleviated by antipsychotics, cognitive symptoms remain unresponsive to such pharmacological treatments and novel research paradigms investigating the circuit substrates underlying cognitive deficits are critically needed. The frontal cortex and its dopaminergic input from the midbrain are implicated in cognitive functions and undergo maturational changes during adolescence. Here, we used mice carrying mutations in theArcorDISC1genes to model mesofrontal dopamine circuit deficiencies and test circuit-based neurostimulation strategies to restore cognitive functions. We found that in a memory-guided spatial navigation task, frontal cortical neurons were activated coordinately at the decision-making point in wild-type but notArcmutant mice. Chemogenetic stimulation of midbrain dopamine neurons or optogenetic stimulation of frontal cortical dopamine axons in a limited adolescent period consistently reversed genetic defects in mesofrontal innervation, task-coordinated neuronal activity, and memory-guided decision-making at adulthood. Furthermore, adolescent stimulation of dopamine neurons also reversed the mesofrontal circuit and cognitive deficits inDISC1mutant mice. Our findings reveal common mesofrontal circuit alterations underlying the cognitive deficits caused by two different genes and demonstrate the feasibility of adolescent neurostimulation to reverse these circuit and behavioral deficits. These results may suggest developmental windows and circuit targets for treating cognitive deficits in neurodevelopmental disorders.

The versatility of somatosensation arises from heterogenous human dorsal root ganglion (DRG) neurons. The critical information to decipher their functions, i.e., the soma transcriptome, is lacking due to technical difficulties. Here we developed a novel approach to isolate individual human DRG neuron somas for deep RNA sequencing (RNA-seq). On average, >9000 unique genes per neuron were detected, and 16 neuronal types were identified. Cross-species analyses revealed that touch-, cold-, and itch-sensing neuronal types were relatively conserved, while the pain-sensing neurons displayed marked divergence. Soma transcriptomes of human DRG neurons predicted novel functional features, which were confirmed using single-cellin vivoelectrophysiological recordings. These results support a close relationship the between physiological properties of human sensory afferents and molecular profiles uncovered by the single-soma RNA-seq dataset. In summary, by conducting single-soma RNA-seq of human DRG neurons, we generated an unprecedented neural atlas for human somatosensation.

Traumatic brain injury (TBI) remains a prominent public health concern despite several decades of attempts to develop therapies for the associated neurological and cognitive deficits. Effective models of this condition are imperative for better defining its pathophysiology and testing therapeutics. Human brain organoids are stem cell–derived neural tissues that recapitulate many of the steps of normal neurodevelopment, resulting in the reproduction of a substantial degree of brain architecture. Organoids are highly relevant to clinical conditions because of their human nature and three-dimensional tissue structure, yet they are easier to manipulate and interrogate experimentally than animals. Thus, they have the potential to serve as a novel platform for studying TBI. In this article, we discuss available in vitro models of TBI, active areas of inquiry on brain organoids, and how these two concepts could be merged.

The emerging technology of brain organoids deriving from human pluripotent stem cells provides unprecedented opportunities to study human brain development and associated disorders. Various brain organoid protocols have been developed that can recapitulate some key features of cell type diversity, cytoarchitectural organization, developmental processes, functions, and pathologies of the developing human brain. In this review, we focus on patterning of human stem cell-derived brain organoids. We start with an overview of general procedures to generate brain organoids. We then highlight some recently developed brain organoid protocols and chemical cues involved in modeling development of specific human brain regions, subregions, and multiple regions together. We also discuss limitations and potential future improvements of human brain organoid technology.

The isocortex and hippocampal formation (HPF) in the mammalian brain play critical roles in perception, cognition, emotion and learning. We profiled ~1.3 million cells covering the entire adult mouse isocortex and HPF and derived a transcriptomic cell type taxonomy revealing a comprehensive repertoire of glutamatergic and GABAergic neuron types. Contrary to the traditional view of HPF as having a simpler cellular organization, we discover a complete set of glutamatergic types in HPF homologous to all major subclasses found in the six-layered isocortex, suggesting that HPF and isocortex share a common circuit organization. We also identify large-scale continuous and graded variation of cell types along isocortical depth, across isocortical sheet and in multiple dimensions in hippocampus and subiculum. Overall, our study establishes a molecular architecture of mammalian isocortex and hippocampal formation and begins to shed light on its underlying relationship with the development, evolution, connectivity and function of these brain structures.

Immature dentate granule cells (imGCs) arising from adult hippocampal neurogenesis contribute to plasticity and unique brain functions in rodents(1,2) and they are dysregulated in multiple human neurological disorders(3–5). Little is known about molecular characteristics of adult human hippocampal imGCs and even their existence is under debate(1,6–8). Here we performed single-nucleus RNA-sequencing (snRNA-seq) aided by a validated machine learning-based analytic approach to identify imGCs and quantify their abundance in the human hippocampus during infant, child, adolescent, adult, and aging stages. We identified common molecular hallmarks of human imGCs across the lifespan and discovered age-dependent transcriptional dynamics unique to human imGCs that suggest changes in cellular functionality, niche interactions, and disease relevance, in contrast to those in mice9. We also found a decreased number of imGCs with altered gene expression in Alzheimer’s disease (AD). Finally, we demonstrated the capacity for neurogenesis in the adult human hippocampus with the presence of rare dentate granule cell fate-specific proliferating neural progenitors and with cultured surgical specimens. Together, our findings suggest the presence of a significant number of imGCs in the adult human hippocampus via low frequency de novo generation and protracted maturation, and our study reveals their molecular properties across the lifespan and in AD.

Brain organoids are self-organized three-dimensional aggregates generated from pluripotent stem cells. They exhibit complex cell diversities and organized architectures that resemble human brain development ranging from neural tube formation, neuroepithelium differentiation, neurogenesis and gliogenesis, to neural circuit formation. Rapid advancements in brain organoid culture technologies have allowed researchers to generate more accurate models of human brain development and neurological diseases. These models also allow for direct investigation of pathological processes associated with infectious diseases affecting the nervous system. In this review, we first briefly summarize recent advancements in brain organoid methodologies and neurodevelopmental processes that can be effectively modeled by brain organoids. We then focus on applications of brain organoids to investigate the pathogenesis of neurotropic viral infection. Finally, we discuss limitations of the current brain organoid methodologies as well as applications of other organ specific organoids in the infectious disease research.

As antiretroviral therapy (ART) becomes increasingly affordable and accessible to women of childbearing age across the globe, the number of children who are exposed to Human Immunodeficiency Viruses (HIV) but remain uninfected is on the rise, almost all of whom were also exposed to ART perinatally. Although ART has successfully aided in the decline of mother-to-child-transmission of HIV, the long-term effects of in utero exposure to ART on fetal and postnatal neurodevelopment remain unclear. Evaluating the safety and efficacy of therapeutic drugs for pregnant women is a challenge due to the historic limitations on their inclusion in clinical trials and the dynamic physiological states during pregnancy that can alter the pharmacokinetics of drug metabolism and fetal drug exposure. Thus, much of our data on the potential consequences of ART drugs on the developing nervous system comes from preclinical animal models and clinical observational studies. In this review, we will discuss the current state of knowledge and existing approaches to investigate whether ART affects fetal brain development, and describe novel human stem cell-based strategies that may provide additional information to better predict the impact of specific drugs on the human central nervous system. Graphical Abstract Approaches to evaluate the impact of drugs on the developing brain. Dysregulation of the developing nervous system can lead to long-lasting changes. Integration of data from animal models, clinical observations, and cell culture studies is needed to predict the safety of therapeutic antiretroviral drugs during pregnancy. New approaches include human induced pluripotent stem cell (iPSC)-based 2D and 3D models of neuronal networks and brain regions, as well as single cell profiling in response to drug exposure.

Psychiatric disorders are a collection of heterogeneous mental disorders arising from a contribution of genetic and environmental insults, many of which molecularly converge on transcriptional dysregulation, resulting in altered synaptic functions. The underlying mechanisms linking the genetic lesion and functional phenotypes remain largely unknown. Patient iPSC-derived neurons with a rare frame-shift DISC1 (Disrupted-in-schizophrenia 1) mutation have previously been shown to exhibit aberrant gene expression and deficits in synaptic functions. How DISC1 regulates gene expression is largely unknown. Here we show that activating transcription factor 4 (ATF4), a DISC1 binding partner, is more abundant in the nucleus of DISC1 mutant human neurons and exhibits enhanced binding to a collection of dysregulated genes. Functionally, overexpressing ATF4 in control neurons recapitulates deficits seen in DISC1 mutant neurons, whereas transcriptional and synaptic deficits are rescued in DISC1 mutant neurons with CRISPR-mediated heterozygous ATF4 knockout. By solving the high-resolution atomic structure of the DISC1-ATF4 complex, we show that mechanistically, the mutation of DISC1 disrupts normal DISC1-ATF4 interaction, and results in excessive ATF4 binding to DNA targets and deregulated gene expression. Together, our study identifies the molecular and structural basis of an DISC1-ATF4 interaction underlying transcriptional and synaptic dysregulation in an iPSC model of mental disorders.

Mutations in gene regulatory elements have been associated with a wide range of complex neuropsychiatric disorders. However, due to their cell-type specificity and difficulties in characterizing their regulatory targets, the ability to identify causal genetic variants has remained limited. To address these constraints, we perform an integrative analysis of chromatin interactions, open chromatin regions and transcriptomes using promoter capture Hi-C, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing, respectively, in four functionally distinct neural cell types: induced pluripotent stem cell (iPSC)-induced excitatory neurons and lower motor neurons, iPSC-derived hippocampal dentate gyrus-like neurons and primary astrocytes. We identify hundreds of thousands of long-range cis-interactions between promoters and distal promoter-interacting regions, enabling us to link regulatory elements to their target genes and reveal putative processes that are dysregulated in disease. Finally, we validate several promoter-interacting regions by using clustered regularly interspaced short palindromic repeats (CRISPR) techniques in human excitatory neurons, demonstrating that CDK5RAP3, STRAP and DRD2 are transcriptionally regulated by physically linked enhancers.

In 2015, public awareness of Zika virus (ZIKV) rose in response to alarming statistics of infants with microcephaly being born to women who were infected with the virus during pregnancy, triggering global concern over these potentially devastating consequences. Although we have discovered a great deal about the genome and pathogenesis of this reemergent flavivirus since this recent outbreak, we still have much more to learn, including the nature of the virus-host interactions and mechanisms that determine its tropism and pathogenicity in the nervous system, which are in turn shaped by the continual evolution of the virus. Inevitably, we will find out more about the potential long-term effects of ZIKV exposure on the nervous system from ongoing longitudinal studies. Integrating clinical and epidemiological data with a wider range of animal and human cell culture models will be critical to understanding the pathogenetic mechanisms and developing more specific antiviral compounds and vaccines.

The molecular diversity of glia in the human hippocampus and their temporal dynamics over the lifespan remain largely unknown. Here, we performed single-nucleus RNA sequencing to generate a transcriptome atlas of the human hippocampus across the postnatal lifespan. Detailed analyses of astrocytes, oligodendrocyte lineages, and microglia identified subpopulations with distinct molecular signatures and revealed their association with specific physiological functions, age-dependent changes in abundance, and disease relevance. We further characterized spatiotemporal heterogeneity of GFAP-enriched astrocyte subpopulations in the hippocampal formation using immunohistology. Leveraging glial subpopulation classifications as a reference map, we revealed the diversity of glia differentiated from human pluripotent stem cells and identified dysregulated genes and pathological processes in specific glial subpopulations in Alzheimer's disease (AD). Together, our study significantly extends our understanding of human glial diversity, population dynamics across the postnatal lifespan, and dysregulation in AD and provides a reference atlas for stem-cell-based glial differentiation.

The hippocampus is involved in the formation of memories that require associations among stimuli to construct representations of space and the items and events within that space. Neurons in the dentate gyrus (DG), an initial input region of the hippocampus, have robust spatial tuning, but it is unclear how nonspatial information may be integrated with spatial activity in this region. We recorded from the DG of 21 adult mice as they foraged for food in an environment that contained discrete objects. We found DG cells with multiple firing fields at a fixed distance and direction from objects (landmark vector cells) as well as cells that exhibited localized changes in spatial firing when objects in the environment were manipulated. By classifying recorded DG cells into putative dentate granule cells and mossy cells, we examined how the addition or displacement of objects affected the spatial firing of these DG cell types. Object-related activity was detected in a significant proportion of mossy cells. Although few granule cells with responses to object manipulations were recorded, likely due to the sparse nature of granule cell firing, there was generally no significant difference in the proportion of granule cells and mossy cells with object responses. When mice explored a second environment with the same objects, DG spatial maps completely reorganized and a different subset of cells responded to object manipulations. Together, these data reveal the capacity of DG cells to detect small changes in the environment, while preserving a stable spatial representation of the overall context.

Microglia are resident macrophages in the brain that emerge in early development and respond to local environment by altering their molecular and phenotypic states. Fundamental questions about microglia diversity and function during development remain unanswered as 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 decreases interferon signaling response genes. Microglia additionally accelerate the emergence of synchronized oscillatory network activity in brain organoids by modulating synaptic density.

Human brain development is an intricate process that involves precisely timed coordination of cell proliferation, fate specification, neuronal differentiation, migration, and integration of diverse cell types. Understanding of these fundamental processes, however, has been largely constrained by limited access to fetal brain tissue and the inability to prospectively study neurodevelopment in humans at the molecular, cellular and system levels. Although non-human model organisms have provided important insights into mechanisms underlying brain development, these systems do not fully recapitulate many human-specific features that often relate to disease. To address these challenges, human brain organoids, self-assembled three-dimensional neural aggregates, have been engineered from human pluripotent stem cells to model the architecture and cellular diversity of the developing human brain. Recent advancements in neural induction and regional patterning using small molecules and growth factors have yielded protocols for generating brain organoids that recapitulate the structure and neuronal composition of distinct brain regions. Here, we first provide an overview of early mammalian brain development with an emphasis on molecular cues that guide region specification. We then focus on recent efforts in generating human brain organoids that model the development of specific brain regions and highlight endeavors to enhance the cellular complexity to better mimic the in vivo developing human brain. We also provide examples of how organoid models have enhanced our understanding of human neurological diseases and conclude by discussing limitations of brain organoids with our perspectives on future advancements to maximize their potential.

Neural stem cells (NSCs) persist into adulthood in the subgranular zone (SGZ) of the dentate gyrus in the hippocampus and in the ventricular-subventricular zone (V-SVZ) of the lateral ventricles, where they generate new neurons and glia cells that contribute to neural plasticity. A better understanding of the developmental process that enables NSCs to persist beyond development will provide insight into factors that determine the size and properties of the adult NSC pool and thus the capacity for life-long neurogenesis in the adult mammalian brain. We review current knowledge regarding the developmental origins of adult NSCs and the developmental process by which embryonic NSCs transition into their adult form. We also discuss potential mechanisms that might regulate proper establishment of the adult NSC pool, and propose future directions of research that will be key to unraveling how NSCs transform to establish the adult NSC pool in the mammalian brain.

We previously identified a causal link between a rare patient mutation in DISC1 (disrupted-in-schizophrenia 1) and synaptic deficits in cortical neurons differentiated from isogenic patient-derived induced pluripotent stem cells (iPSCs). Here we find that transcripts related to phosphodiesterase 4 (PDE4) signaling are significantly elevated in human cortical neurons differentiated from iPSCs with the DISC1 mutation and that inhibition of PDE4 or activation of the cAMP signaling pathway functionally rescues synaptic deficits. We further generated a knock-in mouse line harboring the same patient mutation in the Disc1 gene. Heterozygous Disc1 mutant mice exhibit elevated levels of PDE4s and synaptic abnormalities in the brain, and social and cognitive behavioral deficits. Pharmacological inhibition of the PDE4 signaling pathway rescues these synaptic, social and cognitive behavioral abnormalities. Our study shows that patient-derived isogenic iPSC and humanized mouse disease models are integral and complementary for translational studies with a better understanding of underlying molecular mechanisms., Previous work has shown in iPSC derived neurons that synaptic impairments are associated with a 4bp DISC1 deletion. Here the authors demonstrate a role for the PDE4 signalling pathway in these synaptic impairments.

Neurological complications are common in patients with COVID-19. While SARS-CoV-2, the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function are not well understood. Here we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found that neurons and astrocytes were sparsely infected, but choroid plexus epithelial cells underwent robust infection. We optimized a protocol to generate choroid plexus organoids from hiPSCs and showed that productive SARS-CoV-2 infection of these organoids is associated with increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our findings provide evidence for selective SARS-CoV-2 neurotropism and support the use of hiPSC-derived brain organoids as a platform to investigate SARS-CoV-2 infection susceptibility of brain cells, mechanisms of virus-induced brain dysfunction, and treatment strategies., Graphical Abstract, HIGHLIGHTS • SARS-CoV-2 minimally infects human neurons and astrocytes in 2D and brain organoids • Model developed for hiPSC-derived choroid plexus organoids (CPOs) • SARS-CoV-2 productively infects CPOs and increases cell death • SARS-CoV-2 CPO infection leads to transcriptional upregulation of inflammatory genes, SARS-CoV-2 causes neurological symptoms in a significant portion of patients with COVID-19. Ming and colleagues tested SARS-CoV-2 neurotropism using monolayer neural cells and brain organoids generated from human pluripotent stem cells and show minimal neuron and astrocyte infection, but efficient choroid plexus infection, leading to cell death and functional deficits.

The hypothalamus plays crucial roles in regulating endocrine, autonomic, and behavioral functions via its diverse nuclei and neuronal subtypes. The developmental mechanisms underlying ontogenetic establishment of different hypothalamic nuclei and generation of neuronal diversity remain largely unknown. Here, we show that combinatorial T-box 3 (TBX3), orthopedia homeobox (OTP), and distal-less homeobox (DLX) expression delineates all arcuate nucleus (Arc) neurons and defines four distinct subpopulations, whereas combinatorial NKX2.1/SF1 and OTP/DLX expression identifies ventromedial hypothalamus (VMH) and tuberal nucleus (TuN) neuronal subpopulations, respectively. Developmental analysis indicates that all four Arc subpopulations are mosaically and simultaneously generated from embryonic Arc progenitors, whereas glutamatergic VMH neurons and GABAergic TuN neurons are sequentially generated from common embryonic VMH progenitors. Moreover, clonal lineage-tracing analysis reveals that diverse lineages from multipotent radial glia progenitors orchestrate Arc and VMH-TuN establishment. Together, our study reveals cellular mechanisms underlying generation and organization of diverse neuronal subtypes and ontogenetic establishment of individual nuclei in the mammalian hypothalamus.

SUMMARY In addition to altered gene expression, pathological cytoskeletal dynamics in the axon are another key intrinsic barrier for axon regeneration in the central nervous system (CNS). Here, we show that knocking out myosin IIA and IIB (myosin IIA/B) in retinal ganglion cells alone, either before or after optic nerve crush, induces significant optic nerve regeneration. Combined Lin28a overexpression and myosin IIA/B knockout lead to an additive promoting effect and long-distance axon regeneration. Immunostaining, RNA sequencing, and western blot analyses reveal that myosin II deletion does not affect known axon regeneration signaling pathways or the expression of regeneration-associated genes. Instead, it abolishes the retraction bulb formation and significantly enhances the axon extension efficiency. The study provides clear evidence that directly targeting neuronal cytoskeleton is sufficient to induce significant CNS axon regeneration and that combining altered gene expression in the soma and modified cytoskeletal dynamics in the axon is a promising approach for long-distance CNS axon regeneration., Graphical Abstract, In Brief Although modulating the neuronal cytoskeleton has been deemed a promising approach to enhance mammalian axon regeneration, only a few studies have shown convincing results, especially in the central nervous system. Wang et al. demonstrate that the deletion of non-muscle myosin II sufficiently induces significant mammalian CNS axon regeneration in vivo.

Human brain organoids represent remarkable platforms for recapitulating features of human brain development and diseases. Existing organoid models do not resolve fine brain subregions, such as different nuclei in the hypothalamus. We report the generation of arcuate organoids (ARCOs) from human induced pluripotent stem cells (iPSCs) to model the development of the human hypothalamic arcuate nucleus. Single-cell RNA sequencing of ARCOs revealed significant molecular heterogeneity underlying different arcuate cell types, and machine learning-aided analysis based on the neonatal human hypothalamus single-nucleus transcriptome further showed a human arcuate nucleus molecular signature. We also explored ARCOs generated from Prader-Willi syndrome (PWS) patient iPSCs. These organoids exhibit aberrant differentiation and transcriptomic dysregulation similar to postnatal hypothalamus of PWS patients, indicative of cellular differentiation deficits and exacerbated inflammatory responses. Thus, patient iPSC-derived ARCOs represent a promising experimental model for investigating nucleus-specific features and disease-relevant mechanisms during early human arcuate development.

Zika virus (ZIKV)-infection is associated with neurological disorders of both the central and peripheral nervous systems (PNS), yet few studies have directly examined PNS-infection. Here we show that intraperitoneally or intraventricularly-injected ZIKV in the mouse could infect and impact peripheral neurons in vivo. Moreover, ZIKV productively infects stem cell-derived human neural crest cells and peripheral neurons in vitro, leading to increased cell death, transcriptional dysregulation and cell-type specific molecular pathology.

The suspected link between infection by Zika virus (ZIKV), a re-emerging flavivirus, and microcephaly is an urgent global health concern. The direct target cells of ZIKV in the developing human fetus are not clear. Here we show that a strain of the ZIKV MR766, serially passaged in monkey and mosquito cells, efficiently infects human cortical neural progenitor cells (hNPCs) derived from induced pluripotent stem cells. Infected hNPCs further release infectious ZIKV particles. Importantly, ZIKV infection increases cell death and dysregulates cell cycle progression, resulting in attenuated hNPC growth. Global gene expression analysis of infected hNPCs reveals transcriptional dysregulation, notably of cell cycle-related pathways. Our results identify human cortical neural precursor cells as a direct ZIKV target. In addition, we establish a tractable experimental model system to investigate the impact and mechanism of ZIKV on human brain development and provide a platform to screen therapeutic compounds.

Epitranscriptomic modification of mRNA affects its metabolism and has recently been shown to regulate brain development. Two studies in this issue of Neuron, Koranda et al. (2018) and Engel et al. (2018), uncover dynamic and critical roles of m(6)A/m RNA modifications in the adult mammalian brain in regulating physiological and stress-induced behaviors.

Recent demonstrations of human brain organoid transplantation in rodents have accentuated ethical concerns associated with these entities, especially as they relate to potential "humanization" of host animals. Consideration of established scientific principles can help define the realistic range of expected outcomes in such transplantation studies. This practical approach suggests that augmentation of discrete brain functions in transplant hosts is a more relevant ethical question in the near term than the possibility of "conscious" chimeric animals. We hope that this framework contributes to a balanced approach for proceeding with studies involving brain organoid transplantation and other forms of human-animal brain chimeras.

Protein kinases orchestrate signal transduction pathways involved in central nervous system functions ranging from neurodevelopment to synaptic transmission and plasticity. Abnormalities in kinase-mediated signaling are involved in the pathophysiology of neurological disorders, including neuropsychiatric disorders. Here, we expand on the hypothesis that kinase networks are dysregulated in schizophrenia. We investigated changes in serine/threonine kinase activity in cortical excitatory neurons differentiated from induced pluripotent stem cells (iPSCs) from a schizophrenia patient presenting with a 4 bp mutation in the disrupted in schizophrenia 1 (DISC1) gene and a corresponding control. Using kinome peptide arrays, we demonstrate large scale abnormalities in DISC1 cells, including a global depression of serine/threonine kinase activity, and changes in activity of kinases, including AMP-activated protein kinase (AMPK), extracellular signal-regulated kinases (ERK), and thousand-and-one amino acid (TAO) kinases. Using isogenic cell lines in which the DISC1 mutation is either introduced in the control cell line, or rescued in the schizophrenia cell line, we ascribe most of these changes to a direct effect of the presence of the DISC1 mutation. Investigating the gene expression signatures downstream of the DISC1 kinase network, and mapping them on perturbagen signatures obtained from the Library of Integrated Network-based Cellular Signatures (LINCS) database, allowed us to propose novel drug targets able to reverse the DISC1 kinase dysregulation gene expression signature. Altogether, our findings provide new insight into abnormalities of kinase networks in schizophrenia and suggest possible targets for disease intervention.

Graphical abstract, Zika virus has emerged as a potential threat to human health globally. A previous drug repurposing screen identified the approved anthelminthic drug niclosamide as a small molecule inhibitor of Zika virus infection. However, as antihelminthic drugs are generally designed to have low absorption when dosed orally, the very limited bioavailability of niclosamide will likely hinder its potential direct repurposing as an antiviral medication. Here, we conducted SAR studies focusing on the anilide and salicylic acid regions of niclosamide to improve physicochemical properties such as microsomal metabolic stability, permeability and solubility. We found that the 5-bromo substitution in the salicylic acid region retains potency while providing better drug-like properties. Other modifications in the anilide region with 2’-OMe and 2’-H substitutions were also advantageous. We found that the 4’-NO2 substituent can be replaced with a 4’-CN or 4’-CF3 substituents. Together, these modifications provide a basis for optimizing the structure of niclosamide to improve systemic exposure for application of niclosamide analogs as drug lead candidates for treating Zika and other viral infections. Indeed, key analogs were also able to rescue cells from the cytopathic effect of SARS-CoV-2 infection, indicating relevance for therapeutic strategies targeting the COVID-19 pandemic.

Neuronal activity-induced gene expression modulates the function and plasticity of the nervous system. It is unknown whether and to what extent neuronal activity may trigger changes in chromatin accessibility, a major mode of epigenetic regulation of gene expression. Here we compared chromatin accessibility landscapes of adult mouse dentate granule neurons in vivo before and after synchronous neuronal activation using an assay for transposase-accessible chromatin using sequencing (ATAC-seq). We found genome-wide changes 1 h after activation, with enrichment of gained-open sites at active enhancer regions and at binding sites for AP1-complex components, including c-Fos. Some changes remained stable for at least 24 h. Functional analysis further implicates a critical role of c-Fos in initiating, but not maintaining, neuronal activity-induced chromatin opening. Our results reveal dynamic changes of chromatin accessibility in adult mammalian brains and suggest an epigenetic mechanism by which transient neuronal activation leads to dynamic changes in gene expression via modifying chromatin accessibility.

Human in vitro models of brain neurophysiology are needed to investigate molecular and cellular mechanisms associated with neurological disorders and neurotoxicity. We have developed a reproducible iPSC-derived human 3D brain microphysiological system (BMPS), comprised of differentiated mature neurons and glial cells (astrocytes and oligodendrocytes) that reproduce neuronal-glial interactions and connectivity. BMPS mature over eight weeks and show the critical elements of neuronal function: synaptogenesis and neuron-to-neuron (e.g., spontaneous electric field potentials) and neuronal-glial interactions (e.g., myelination), which mimic the microenvironment of the central nervous system, rarely seen in vitro before. The BMPS shows 40% overall myelination after 8 weeks of differentiation. Myelin was observed by immunohistochemistry and confirmed by confocal microscopy 3D reconstruction and electron microscopy. These findings are of particular relevance since myelin is crucial for proper neuronal function and development. The ability to assess oligodendroglial function and mechanisms associated with myelination in this BMPS model provide an excellent tool for future studies of neurological disorders such as multiple sclerosis and other demyelinating diseases. The BMPS provides a suitable and reliable model to investigate neuron-neuroglia function as well as pathogenic mechanisms in neurotoxicology. published

The re-emergence of Zika virus (ZIKV) and its suspected link with various disorders in newborns and adults led the World Health Organization to declare a global health emergency. In response, the stem cell field quickly established platforms for modeling ZIKV exposure using human pluripotent stem cell-derived neural progenitors and brain organoids, fetal tissues, and animal models. These efforts provided significant insight into cellular targets, pathogenesis, and underlying biological mechanisms of ZIKV infection as well as platforms for drug testing. Here we review the remarkable progress in stem cell-based ZIKV research and discuss current challenges and future opportunities.

The blossoming field of epitranscriptomics has recently garnered attention across many fields by findings that chemical modifications on RNA have immense biological consequences. Methylation of nucleotides in RNA, including N6-methyladenosine (m(6)A), 2-O-dimethyladenosine (m(6)A(m)), N1-methyladenosine (m(1)A), and 5-methylcytosine (m(5)C) and isomerization of uracil to pseudouridine (Ψ), have the potential to alter RNA processing events and contribute to developmental processes and different diseases. Though the abundance and roles of some RNA modifications remain contentious, the epitranscriptome is thought to be especially relevant in stem cell biology and neurobiology. In particular, m(6)A occurs at the highest levels in the brain and plays major roles in embryonic stem cell differentiation, brain development, and neurodevelopmental disorders. However, studies in these areas have reported conflicting results on epitranscriptomic regulation of stem cell pluripotency and mechanisms in neural development. In this review we provide an overview of the current understanding of several RNA modifications and disentangle the various findings on epitranscriptomic regulation of stem cell biology and neural development.

Neocortical development has been extensively studied and therefore is the basis of our understanding of mammalian brain development. One fundamental principle of neocortical development is that neurogenesis and gliogenesis are temporally segregated processes. However, it is unclear how neurogenesis and gliogenesis are coordinated in non-neocortical regions of the cerebral cortex, such as the hippocampus, also known as the archicortex. Here, we show that the timing of neurogenesis and astrogenesis in the Cornu Ammonis (CA) 1 and CA3 regions of mouse hippocampus mirrors that of the neocortex, neurogenesis occurs embryonically, followed by astrogenesis during early postnatal development. In contrast, we find that neurogenesis in the dentate gyrus begins embryonically but is a protracted process which peaks neonatally and continues at low levels postnatally. As a result, astrogenesis, which occurs during early postnatal development, overlaps with the process of neurogenesis in the dentate gyrus. During all stages, neurogenesis overwhelms astrogenesis in the dentate gyrus. In addition, we find that the timing of peak astrogenesis varies by hippocampal subregion. Together, our results show differential timing and coordination of neurogenesis and astrogenesis in developing mouse hippocampal subregions and suggest that neurogenesis and gliogenesis occur simultaneously during dentate gyrus development, challenging the conventional principle that neurogenesis and gliogenesis are temporally separated processes.

Genome-wide mapping of chromatin interactions at high-resolution remains experimentally and computationally challenging. Here we used a low-input “easy Hi-C” protocol to map the 3D genome architecture in human neurogenesis and brain tissues, and also demonstrated that a rigorous Hi-C bias-correction pipeline (HiCorr) can significantly improve the sensitivity and robustness of Hi-C loop identification at sub-TAD level, especially the enhancer-promoter (E-P) interactions. We used HiCorr to compare the high-resolution maps of chromatin interactions from 10 tissue- or cell-types with a focus on neurogenesis and brain tissues. We found that dynamic chromatin loops are better hallmarks for cellular differentiation than compartment switching. HiCorr allowed direct observation of cell type- and differentiation-specific E-P aggregates spanning large neighborhoods, suggesting a mechanism that stabilizes enhancer contacts during development. Interestingly, we concluded that Hi-C loop outperforms eQTL in explaining neurological GWAS results, revealing a unique value of high-resolution 3D genome maps in elucidating the disease etiology.

Human brain organoids provide unique platforms for modeling development and diseases by recapitulating the architecture of the embryonic brain. However, current organoid methods are limited by interior hypoxia and cell death due to insufficient surface diffusion, preventing generation of architecture resembling late developmental stages. Here, we report the sliced neocortical organoid (SNO) system, which bypasses the diffusion limit to prevent cell death over long-term cultures. This method leads to sustained neuro-genesis and formation of an expanded cortical plate that establishes distinct upper and deep cortical layers for neurons and astrocytes, resembling the third trimester embryonic human neocortex. Using the SNO system, we further identify a critical role of WNT/β-catenin signaling in regulating human cortical neuron subtype fate specification, which is disrupted by a psychiatric-disorder-associated genetic mutation in patient induced pluripotent stem cell (iPSC)-derived SNOs. These results demonstrate the utility of SNOs for investigating previously inaccessible human-specific, late-stage cortical development and disease-relevant mechanisms.

Summary Although congenital infection by human cytomegalovirus (HCMV) is well recognized as a leading cause of neurodevelopmental defects, HCMV neuropathogenesis remains poorly understood. A major challenge for investigating HCMV-induced abnormal brain development is the strict CMV species specificity, which prevents the use of animal models to directly study brain defects caused by HCMV. We show that infection of human-induced pluripotent-stem-cell-derived brain organoids by a “clinical-like” HCMV strain results in reduced brain organoid growth, impaired formation of cortical layers, and abnormal calcium signaling and neural network activity. Moreover, we show that the impeded brain organoid development caused by HCMV can be prevented by neutralizing antibodies (NAbs) that recognize the HCMV pentamer complex. These results demonstrate in a three-dimensional cellular biosystem that HCMV can impair the development and function of the human brain and provide insights into the potential capacity of NAbs to mitigate brain defects resulted from HCMV infection., Graphical Abstract, Highlights Human iPSC-derived brain organoids to model HCMV-induced brain malformation “Clinical-like” HCMV strain impairs human brain organoid growth and structure HCMV-infected brain organoids exhibit abnormal calcium signaling and neural network HCMV-induced brain organoid abnormality can be prevented by neutralizing antibodies, Sun et al. used human-iPSC-derived brain organoids to model the effects of HCMV infection on human brain development. They found that a “clinical-like” HCMV strain impairs brain organoid growth, structure, and neural network activity. Moreover, the abnormal brain organoid development caused by HCMV can be prevented by neutralizing antibodies.

The molecular mechanisms that govern the maturation of oligodendrocyte lineage cells remain unclear. Emerging studies have shown that N(6)-methyladenosine (m(6)A), the most common internal RNA modification of mammalian mRNA, plays a critical role in various developmental processes. Here, we demonstrate that oligodendrocyte lineage progression is accompanied by dynamic changes in m(6)A modification on numerous transcripts. In vivo conditional inactivation of an essential m(6)A writer component, METTL14, results in decreased oligodendrocyte numbers and CNS hypomyelination, although oligodendrocyte precursor cell (OPC) numbers are normal. In vitro Mettl14 ablation disrupts post-mitotic oligodendrocyte maturation and has distinct effects on OPC and oligodendrocyte transcriptomes. Moreover, the loss of Mettl14 in oligodendrocyte lineage cells causes aberrant splicing of myriad RNA transcripts, including that which encodes the essential paranodal component neurofascin 155 (NF155). Together, our findings indicate that dynamic RNA methylation plays an important regulatory role in oligodendrocyte development and CNS myelination.

SUMMARY N6-methyladenosine (m6A) modification of mRNA is emerging as a vital mechanism regulating RNA function. Here, we show that fragile X mental retardation protein (FMRP) reads m6A to promote nuclear export of methylated mRNA targets during neural differentiation. Fmr1 knockout (KO) mice show delayed neural progenitor cell cycle progression and extended maintenance of proliferating neural progenitors into postnatal stages, phenocopying methyltransferase Mettl14 conditional KO (cKO) mice that have no m6A modification. RNA-seq and m6A-seq reveal that both Mettl14cKO and Fmr1KO lead to the nuclear retention of m6A-modified FMRP target mRNAs regulating neural differentiation, indicating that both m6A and FMRP are required for the nuclear export of methylated target mRNAs. FMRP preferentially binds m6A-modified RNAs to facilitate their nuclear export through CRM1. The nuclear retention defect can be mitigated by wild-type but not nuclear export-deficient FMRP, establishing a critical role for FMRP in mediating m6A-dependent mRNA nuclear export during neural differentiation., Graphical Abstract In Brief Edens et al. reveal fragile X mental retardation protein (FMRP) as an m6A reader that promotes the nuclear export of methylated mRNAs during neural differentiation. Loss of either Fmr1 or the m6A methyltransferase Mettl14 results in the nuclear accumulation of neural differentiation-related mRNAs, causing delayed neural progenitor differentiation in mice.

The convergence of nanoparticles and stem cell therapy holds great promise for the study, diagnosis, and treatment of neurodegenerative disorders. Researchers aim to harness the power of nanoparticles to regulate cellular microenvironment, improve the efficiency of cell and drug delivery to the brain, and enhance the survival of stem cell transplants. Understanding the various properties of different nanoparticles is key to applying them to clinical therapies; the many distinct types of nanoparticles offer unique capacities for medical imaging, diagnosis, and treatment of neurodegeneration disorders. In this review we introduce the biology of Alzheimer's, Parkinson's Disease, and amyotrophic lateral sclerosis, and discuss the potentials and shortcomings of metal, silica, lipid-based, polymeric, and hydrogel nanoparticles for diagnosis and treatment of neurodegenerative disorders. We then provide an overview of current strategies in stem cell therapies and how they can be combined with nanotechnology to improve clinical outcomes.

Brain organoids are an exciting new technology with the potential to significantly change how diseases of the brain are understood and treated. These three-dimensional neural tissues are derived from the self-organization of pluripotent stem cells, and they recapitulate the developmental process of the human brain, including progenitor zones and rudimentary cortical layers. Brain organoids have been valuable in investigating different aspects of developmental neurobiology and comparative biology. Several characteristics of organoids also make them attractive as models of brain disorders. Data generated from human organoids are more generalizable to patients because of the match in species background. Personalized organoids also can be generated from patient-derived induced pluripotent stem cells. Furthermore, the three-dimensionality of brain organoids supports cellular, mechanical, and topographical cues that are lacking in planar systems. In this review, we discuss the translational potential of brain organoids, using the examples of Zika virus, autism-spectrum disorder, and glioblastoma multiforme to consider how they could contribute to disease modeling, personalized medicine, and testing of therapeutics. We then discuss areas of improvement in organoid technology that will enhance the translational potential of brain organoids, as well as the possibility of their use as substrates for repairing cerebral circuitry after injury. Developmental Dynamics 248:53-64, 2019. © 2018 Wiley Periodicals, Inc.

The re-emergence of Zika virus (ZIKV) and Ebola virus (EBOV) poses serious and continued threats to the global public health. Effective therapeutics for these maladies is an unmet need. Here, we show that emetine, an anti-protozoal agent, potently inhibits ZIKV and EBOV infection with a low nanomolar half maximal inhibitory concentration (IC50) in vitro and potent activity in vivo. Two mechanisms of action for emetine are identified: the inhibition of ZIKV NS5 polymerase activity and disruption of lysosomal function. Emetine also inhibits EBOV entry. Cephaeline, a desmethyl analog of emetine, which may be better tolerated in patients than emetine, exhibits a similar efficacy against both ZIKV and EBOV infections. Hence, emetine and cephaeline offer pharmaceutical therapies against both ZIKV and EBOV infection.

Understanding how complex brain wiring is produced during development is a daunting challenge. In Drosophila, information from 800 retinal ommatidia is processed in distinct brain neuropiles, each subdivided into 800 matching retinotopic columns. In the Lobula Plate there are four T4 and four T5 neuronal subtypes. T4 neurons respond to bright edge motion while T5 neurons to dark edge motion, and each is tuned to motion in one of the four cardinal directions, effectively establishing eight concurrent retinotopic maps to support wide-field motion. We discovered a mode of neurogenesis where two sequential Notch-dependent divisions of either a horizontal or a vertical progenitor produces matching sets of two T4 and two T5 neurons retinotopically coincident with pair-wise opposite direction selectivity. We show that retinotopy is an emergent characteristic of this neurogenic program and derives directly from neuronal birth order. Our work illustrates how simple developmental rules can implement complex neural organization.

Psychiatric disorders are heterogeneous disorders characterized by complex genetics, variable symptomatology, and anatomically distributed pathology, all of which present challenges for effective treatment. Current treatments are often blunt tools used to ameliorate the most severe symptoms, often at the risk of disrupting functional neural systems, thus there is a pressing need to develop rational therapeutics. Induced pluripotent stem cells (iPSCs) reprogrammed from patient somatic cells offers an unprecedented opportunity to recapitulate both normal and pathologic human tissue and organ development, and provides new approaches for understanding disease mechanisms and for drug discovery with higher predictability of their effects in humans. Here we review recent progress and challenges in using human iPSCs for modeling neuropsychiatric disorders and developing novel therapeutic strategies.

Adult somatic stem cells in various organs maintain homeostatic tissue regeneration and enhance plasticity. Since its initial discovery five decades ago, investigations of adult neurogenesis and neural stem cells have led to an established and expanding field that has significantly influenced many facets of neuroscience, developmental biology, and regenerative medicine. Here we review recent progress and focus on questions related to adult mammalian neural stem cells that also apply to other somatic stem cells. We further discuss emerging topics that are guiding the field toward better understanding adult neural stem cells and ultimately applying these principles to improve human health.

SummarySomatic stem cells contribute to tissue ontogenesis, homeostasis, and regeneration through sequential processes. Systematic molecular analysis of stem cell behavior is challenging because classic approaches cannot resolve cellular heterogeneity or capture developmental dynamics. Here we provide a comprehensive resource of single-cell transcriptomes of adult hippocampal quiescent neural stem cells (qNSCs) and their immediate progeny. We further developed Waterfall, a bioinformatic pipeline, to statistically quantify singe-cell gene expression along a de novo reconstructed continuous developmental trajectory. Our study reveals molecular signatures of adult qNSCs, characterized by active niche signaling integration and low protein translation capacity. Our analyses further delineate molecular cascades underlying qNSC activation and neurogenesis initiation, exemplified by decreased extrinsic signaling capacity, primed translational machinery, and regulatory switches in transcription factors, metabolism, and energy sources. Our study reveals the molecular continuum underlying adult neurogenesis and illustrates how Waterfall can be used for single-cell omics analyses of various continuous biological processes.

A group of papers investigates functional regulatory elements in genomes from human tissue samples and cell lines. What can neuroscientists learn from the gigantic data set and how will it affect the direction of neuroepigenetics?

Contrary to the long-held belief that DNA methylation of terminally differentiated cells is permanent and essentially immutable, post-mitotic neurons exhibit extensive DNA demethylation. The cellular function of active DNA demethylation in neurons, however, remains largely unknown. Tet family proteins oxidize 5-methylcytosine to initiate active DNA demethylation through the base-excision repair pathway. Here, we show that synaptic activity bi-directionally regulates neuronal Tet3 expression. Functionally, knockdown of Tet or inhibition of base-excision repair in hippocampal neurons elevates excitatory glutamatergic synaptic transmission, whereas overexpressing Tet3 or Tet1 catalytic domain decreases it. Furthermore, dysregulation of Tet3 signalling prevents homeostatic synaptic plasticity. Mechanistically, Tet3 dictates neuronal surface GluR1 levels. RNA-seq analyses further revealed a pivotal role of Tet3 in regulating gene expression in response to global synaptic activity changes. Thus, Tet3 serves as a synaptic activity sensor to epigenetically regulate fundamental properties and meta-plasticity of neurons via active DNA demethylation.

Human brain organoids, 3D self-assembled neural tissues derived from pluripotent stem cells, are important tools for studying human brain development and related disorders. Suspension cultures maintained by spinning bioreactors allow for the growth of large organoids despite the lack of vasculature, but commercially available spinning bioreactors are bulky in size and have low throughput. Here, we describe the procedures for building the miniaturized multiwell spinning bioreactor SpinΩ from 3D-printed parts and commercially available hardware. We also describe how to use SpinΩ to generate forebrain, midbrain and hypothalamus organoids from human induced pluripotent stem cells (hiPSCs). These organoids recapitulate key dynamic features of the developing human brain at the molecular, cellular and structural levels. The reduction in culture volume, increase in throughput and reproducibility achieved using our bioreactor and region-specific differentiation protocols enable quantitative modeling of brain disorders and compound testing. This protocol takes 14-84 d to complete (depending on the type of brain region-specific organoids and desired developmental stages), and organoids can be further maintained over 200 d. Competence with hiPSC culture is required for optimal results.

Summary Mutations of DISC1 (disrupted-in-schizophrenia 1) have been associated with major psychiatric disorders. Despite the hundreds of DISC1-binding proteins reported, almost nothing is known about how DISC1 interacts with other proteins structurally to impact human brain development. Here we solved the high-resolution structure of DISC1 C-terminal tail in complex with its binding domain of Ndel1. Mechanistically, DISC1 regulates Ndel1's kinetochore attachment, but not its centrosome localization, during mitosis. Functionally, disrupting DISC1/Ndel1 complex formation prolongs mitotic length and interferes with cell-cycle progression in human cells, and it causes cell-cycle deficits of radial glial cells in the embryonic mouse cortex and human forebrain organoids. We also observed similar deficits in organoids derived from schizophrenia patient induced pluripotent stem cells (iPSCs) with a DISC1 mutation that disrupts its interaction with Ndel1. Our study uncovers a new mechanism of action for DISC1 based on its structure, and it has implications for how genetic insults may contribute to psychiatric disorders.

Summary N6-methyladenosine (m6A), the most prevalent internal RNA modification on mammalian messenger RNAs (mRNAs), regulates fates and functions of modified transcripts through m6A-specific binding proteins1–5. m6A is abundant in the nervous system and modulates various neural functions6–11. While m6A marks groups of mRNAs for coordinated degradation in various physiological processes12–15, the relevance of m6A in mRNA translation remains largely unknown in vivo. Here we show that, through its binding protein Ythdf1, m6A promotes protein synthesis of target transcripts in response to neuronal stimuli in the adult mouse hippocampus, thereby facilitating learning and memory. Mice with genetic deletion of Ythdf1 (Ythdf1-KO) exhibit learning and memory defects as well as impaired hippocampal synaptic transmission and long-term potentiation. Ythdf1 re-expression in the hippocampus of adult Ythdf1-KO mice rescues behavioral and synaptic defects, while hippocampus-specific acute knockdown of Ythdf1 or Mettl3, the catalytic component of m6A methyltransferase complex, recapitulates the hippocampal deficiency. Transcriptome-wide mapping of Ythdf1 binding sites and m6A sites on hippocampal mRNAs uncovered key neuronal genes. Nascent protein labeling and tether reporter assays in hippocampal neurons revealed that Ythdf1 enhances protein synthesis in a neuronal-stimulus-dependent manner. Collectively, our results uncover a pathway of mRNA m6A methylation in learning and memory, which is mediated through Ythdf1 in response to stimuli.

Modification of messenger RNAs through a process called m6A methylation facilitates dynamic temporal regulation of RNA levels in neural precursor cells, enabling fine-tuning of developing neuronal circuits in the brain.