A virtual memory CD8+ T cell-originated subset causes alopecia areata through innate-like cytotoxicity.

Virtual memory T (TVM) cells are a T cell subtype with a memory phenotype but no prior exposure to foreign antigen. Although TVM cells have antiviral and antibacterial functions, whether these cells can be pathogenic effectors of inflammatory disease is unclear. Here we identified a TVM cell-originated CD44super-high(s-hi)CD49dlo CD8+ T cell subset with features of tissue residency. These cells are transcriptionally, phenotypically and functionally distinct from conventional CD8+ TVM cells and can cause alopecia areata. Mechanistically, CD44s-hiCD49dlo CD8+ T cells could be induced from conventional TVM cells by interleukin (IL)-12, IL-15 and IL-18 stimulation. Pathogenic activity of CD44s-hiCD49dlo CD8+ T cells was mediated by NKG2D-dependent innate-like cytotoxicity, which was further augmented by IL-15 stimulation and triggered disease onset. Collectively, these data suggest an immunological mechanism through which TVM cells can cause chronic inflammatory disease by innate-like cytotoxicity.

Coupling Neurogenesis to Circuit Formation.

A central question in neuroscience is how developmental programs instruct the formation of complex neural circuits with temporal, spatial, and numerical precision. Pinto-Teixeira et al. (2018) reveal simple developmental rules that govern sequential neurogenesis to concurrently establish highly organized retinotopic maps in the Drosophila visual system.

Stress induces behavioral abnormalities by increasing expression of phagocytic receptor MERTK in astrocytes to promote synapse phagocytosis.

Childhood neglect and/or abuse can induce mental health conditions with unknown mechanisms. Here, we identified stress hormones as strong inducers of astrocyte-mediated synapse phagocytosis. Using in vitro, in vivo, and human brain organoid experiments, we showed that stress hormones increased the expression of the Mertk phagocytic receptor in astrocytes through glucocorticoid receptor (GR). In post-natal mice, exposure to early social deprivation (ESD) specifically activated the GR-MERTK pathway in astrocytes, but not in microglia. The excitatory post-synaptic density in cortical regions was reduced in ESD mice, and there was an increase in the astrocytic engulfment of these synapses. The loss of excitatory synapses, abnormal neuronal network activities, and behavioral abnormalities in ESD mice were largely prevented by ablating GR or MERTK in astrocytes. Our work reveals the critical roles of astrocytic GR-MERTK activation in evoking stress-induced abnormal behaviors in mice, suggesting GR-MERTK signaling as a therapeutic target for stress-induced mental health conditions.

Temporal Control of Mammalian Cortical Neurogenesis by m6A Methylation.

N6-methyladenosine (m6A), installed by the Mettl3/Mettl14 methyltransferase complex, is the most prevalent internal mRNA modification. Whether m6A regulates mammalian brain development is unknown. Here, we show that m6A depletion by Mettl14 knockout in embryonic mouse brains prolongs the cell cycle of radial glia cells and extends cortical neurogenesis into postnatal stages. m6A depletion by Mettl3 knockdown also leads to a prolonged cell cycle and maintenance of radial glia cells. m6A sequencing of embryonic mouse cortex reveals enrichment of mRNAs related to transcription factors, neurogenesis, the cell cycle, and neuronal differentiation, and m6A tagging promotes their decay. Further analysis uncovers previously unappreciated transcriptional prepatterning in cortical neural stem cells. m6A signaling also regulates human cortical neurogenesis in forebrain organoids. Comparison of m6A-mRNA landscapes between mouse and human cortical neurogenesis reveals enrichment of human-specific m6A tagging of transcripts related to brain-disorder risk genes. Our study identifies an epitranscriptomic mechanism in heightened transcriptional coordination during mammalian cortical neurogenesis.

Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure.

Cerebral organoids, three-dimensional cultures that model organogenesis, provide a new platform to investigate human brain development. High cost, variability, and tissue heterogeneity limit their broad applications. Here, we developed a miniaturized spinning bioreactor (SpinΩ) to generate forebrain-specific organoids from human iPSCs. These organoids recapitulate key features of human cortical development, including progenitor zone organization, neurogenesis, gene expression, and, notably, a distinct human-specific outer radial glia cell layer. We also developed protocols for midbrain and hypothalamic organoids. Finally, we employed the forebrain organoid platform to model Zika virus (ZIKV) exposure. Quantitative analyses revealed preferential, productive infection of neural progenitors with either African or Asian ZIKV strains. ZIKV infection leads to increased cell death and reduced proliferation, resulting in decreased neuronal cell-layer volume resembling microcephaly. Together, our brain-region-specific organoids and SpinΩ provide an accessible and versatile platform for modeling human brain development and disease and for compound testing, including potential ZIKV antiviral drugs.

Setting the clock of neural progenitor cells during mammalian corticogenesis.

Radial glial cells (RGCs) as primary neural stem cells in the developing mammalian cortex give rise to diverse types of neurons and glial cells according to sophisticated developmental programs with remarkable spatiotemporal precision. Recent studies suggest that regulation of the temporal competence of RGCs is a key mechanism for the highly conserved and predictable development of the cerebral cortex. Various types of epigenetic regulations, such as DNA methylation, histone modifications, and 3D chromatin architecture, play a key role in shaping the gene expression pattern of RGCs. In addition, epitranscriptomic modifications regulate temporal pre-patterning of RGCs by affecting the turnover rate and function of cell-type-specific transcripts. In this review, we summarize epigenetic and epitranscriptomic regulatory mechanisms that control the temporal competence of RGCs during mammalian corticogenesis. Furthermore, we discuss various developmental elements that also dynamically regulate the temporal competence of RGCs, including biochemical reaction speed, local environmental changes, and subcellular organelle remodeling. Finally, we discuss the underlying mechanisms that regulate the interspecies developmental tempo contributing to human-specific features of brain development.

Resolving m6A epitranscriptome with stoichiometry.

A recent study by Hu et al. describes N6-methyladenosine (m6A)-selective allyl chemical labeling and sequencing (m6A-SAC-seq), which allows for quantitative, stoichiometric, and positional analyses of m6A at single-nucleotide resolution across the whole transcriptome level. Information on the m6A stoichiometry will provide additional layers of gene regulatory pathways mediated by m6A modification during diverse molecular, cellular, and physiological events.

Schizophrenia-associated Mitotic Arrest Deficient-1 (MAD1) regulates the polarity of migrating neurons in the developing neocortex.

Although large-scale genome-wide association studies (GWAS) have identified an association between MAD1L1 (Mitotic Arrest Deficient-1 Like 1) and the pathology of schizophrenia, the molecular mechanisms underlying this association remain unclear. In the present study, we aimed to address these mechanisms by examining the role of MAD1 (the gene product of MAD1L1) in key neurodevelopmental processes in mice and human organoids. Our findings indicated that MAD1 is highly expressed during active cortical development and that MAD1 deficiency leads to impairments in neuronal migration and neurite outgrowth. We also observed that MAD1 is localized to the Golgi apparatus and regulates vesicular trafficking from the Golgi apparatus to the plasma membrane, which is required for the growth and polarity of migrating neurons. In this process, MAD1 physically interacts and collaborates with the kinesin-like protein KIFC3 (kinesin family member C3) to regulate the morphology of the Golgi apparatus and neuronal polarity, thereby ensuring proper neuronal migration and differentiation. Consequently, our findings indicate that MAD1 is an essential regulator of neuronal development and that alterations in MAD1 may underlie schizophrenia pathobiology.

LSM12-EPAC1 defines a neuroprotective pathway that sustains the nucleocytoplasmic RAN gradient.

Nucleocytoplasmic transport (NCT) defects have been implicated in neurodegenerative diseases such as C9ORF72-associated amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). Here, we identify a neuroprotective pathway of like-Sm protein 12 (LSM12) and exchange protein directly activated by cyclic AMP 1 (EPAC1) that sustains the nucleocytoplasmic RAN gradient and thereby suppresses NCT dysfunction by the C9ORF72-derived poly(glycine-arginine) protein. LSM12 depletion in human neuroblastoma cells aggravated poly(GR)-induced impairment of NCT and nuclear integrity while promoting the nuclear accumulation of poly(GR) granules. In fact, LSM12 posttranscriptionally up-regulated EPAC1 expression, whereas EPAC1 overexpression rescued the RAN gradient and NCT defects in LSM12-deleted cells. C9-ALS patient-derived neurons differentiated from induced pluripotent stem cells (C9-ALS iPSNs) displayed low expression of LSM12 and EPAC1. Lentiviral overexpression of LSM12 or EPAC1 indeed restored the RAN gradient, mitigated the pathogenic mislocalization of TDP-43, and suppressed caspase-3 activation for apoptosis in C9-ALS iPSNs. EPAC1 depletion biochemically dissociated RAN-importin ß1 from the cytoplasmic nuclear pore complex, thereby dissipating the nucleocytoplasmic RAN gradient essential for NCT. These findings define the LSM12-EPAC1 pathway as an important suppressor of the NCT-related pathologies in C9-ALS/FTD.

Persistent Cyfip1 Expression Is Required to Maintain the Adult Subventricular Zone Neurogenic Niche.

Neural stem cells (NSCs) persist throughout life in the subventricular zone (SVZ) neurogenic niche of the lateral ventricles as Type B1 cells in adult mice. Maintaining this population of NSCs depends on the balance between quiescence and self-renewing or self-depleting cell divisions. Interactions between B1 cells and the surrounding niche are important in regulating this balance, but the mechanisms governing these processes have not been fully elucidated. The cytoplasmic FMRP-interacting protein (Cyfip1) regulates apical-basal polarity in the embryonic brain. Loss of Cyfip1 during embryonic development in mice disrupts the embryonic niche and affects cortical neurogenesis. However, a direct role for Cyfip1 in the regulation of adult NSCs has not been established. Here, we demonstrate that Cyfip1 expression is preferentially localized to B1 cells in the adult mouse SVZ. Loss of Cyfip1 in the embryonic mouse brain results in altered adult SVZ architecture and expansion of the adult B1 cell population at the ventricular surface. Furthermore, acute deletion of Cyfip1 in adult NSCs results in a rapid change in adherens junction proteins as well as increased proliferation and number of B1 cells at the ventricular surface. Together, these data indicate that Cyfip1 plays a critical role in the formation and maintenance of the adult SVZ niche; furthermore, deletion of Cyfip1 unleashes the capacity of adult B1 cells for symmetric renewal to increase the adult NSC pool.SIGNIFICANCE STATEMENT Neural stem cells (NSCs) persist in the subventricular zone of the lateral ventricles in adult mammals, and the size of this population is determined by the balance between quiescence and self-depleting or renewing cell division. The mechanisms regulating these processes are not fully understood. This study establishes that the cytoplasmic FMRP interacting protein 1 (Cyfip1) regulates NSC fate decisions in the adult subventricular zone and adult NSCs that are quiescent or typically undergo self-depleting divisions retain the ability to self-renew. These results contribute to our understanding of how adult NSCs are regulated throughout life and has potential implications for human brain disorders.

Neur1 and Neur2 are required for hippocampus-dependent spatial memory and synaptic plasticity.

Neur1 and Neur2, mouse homologs of the Drosophila neur gene, consist of two neuralized homology repeat domains and a RING domain. Both Neur1 and Neur2 are expressed in the whole adult brain and encode E3 ubiquitin ligases, which play a crucial role in the Notch signaling pathways. A previous study reported that overexpression of Neur1 enhances hippocampus-dependent memory, whereas the role of Neur2 remains largely unknown. Here, we aimed to elucidate the respective roles of Neur1 and Neur2 in hippocampus-dependent memory using three lines of genetically modified mice: Neur1 knock-out, Neur2 knock-out, and Neur1 and Neur2 double knock-out (D-KO). Our results showed that spatial memory was impaired when both Neur1 and Neur2 were deleted, but not in the individual knock-out of either Neur1 or Neur2. In addition, basal synaptic properties estimated by input-output relationships and paired-pulse facilitation did not change, but a form of long-term potentiation that requires protein synthesis was specifically impaired in the D-KO mice. These results collectively suggest that Neur1 and Neur2 are crucially involved in hippocampus-dependent spatial memory and synaptic plasticity.

Synaptic dysregulation in a human iPS cell model of mental disorders.

Dysregulated neurodevelopment with altered structural and functional connectivity is believed to underlie many neuropsychiatric disorders, and 'a disease of synapses' is the major hypothesis for the biological basis of schizophrenia. Although this hypothesis has gained indirect support from human post-mortem brain analyses and genetic studies, little is known about the pathophysiology of synapses in patient neurons and how susceptibility genes for mental disorders could lead to synaptic deficits in humans. Genetics of most psychiatric disorders are extremely complex due to multiple susceptibility variants with low penetrance and variable phenotypes. Rare, multiply affected, large families in which a single genetic locus is probably responsible for conferring susceptibility have proven invaluable for the study of complex disorders. Here we generated induced pluripotent stem (iPS) cells from four members of a family in which a frameshift mutation of disrupted in schizophrenia 1 (DISC1) co-segregated with major psychiatric disorders and we further produced different isogenic iPS cell lines via gene editing. We showed that mutant DISC1 causes synaptic vesicle release deficits in iPS-cell-derived forebrain neurons. Mutant DISC1 depletes wild-type DISC1 protein and, furthermore, dysregulates expression of many genes related to synapses and psychiatric disorders in human forebrain neurons. Our study reveals that a psychiatric disorder relevant mutation causes synapse deficits and transcriptional dysregulation in human neurons and our findings provide new insight into the molecular and synaptic etiopathology of psychiatric disorders.

Multiplexed Biomarker Panels Discriminate Zika and Dengue Virus Infection in Humans.

Zika virus (ZIKV) and dengue virus (DENV) are closely related flaviviruses that cause widespread, acute febrile illnesses, notably microcephaly for fetuses of infected pregnant women. Detecting the viral cause of these illnesses is paramount to determine risks to patients, counsel pregnant women, and help fight outbreaks. A combined diagnostic algorithm for ZIKV and DENV requires Reverse transcription polymerase chain reaction (RT-PCR) and IgM antibody detection. RT-PCR differentiates between DENV and ZIKV infections during the acute phases of infection, but differentiation based on IgM antibodies is currently nearly impossible in endemic areas. We have developed a ZIKV/DENV protein array and tested it with serum samples collected from ZIKV- and DENV-infected patients and healthy subjects in Puerto Rico. Our analyses reveal a biomarker panel that are capable of discriminating ZIKV and DENV infections with high accuracy, including Capsid protein from African ZIKV strain MR766, and other 5 pair of proteins (NS1, NS2A, NS3, NS4B and NS5) from ZIKV and DENV respectively. Both sensitivity and specificity of the test for ZIKV from DENV are around 90%. We propose that the ZIKV/DENV protein array will be used in future studies to discriminate patients infected with ZIKV from DENV.

Molecular signatures associated with ZIKV exposure in human cortical neural progenitors.

Zika virus (ZIKV) infection causes microcephaly and has been linked to other brain abnormalities. How ZIKV impairs brain development and function is unclear. Here we systematically profiled transcriptomes of human neural progenitor cells exposed to Asian ZIKVC, African ZIKVM, and dengue virus (DENV). In contrast to the robust global transcriptome changes induced by DENV, ZIKV has a more selective and larger impact on expression of genes involved in DNA replication and repair. While overall expression profiles are similar, ZIKVC, but not ZIKVM, induces upregulation of viral response genes and TP53. P53 inhibitors can block the apoptosis induced by both ZIKVC and ZIKVM in hNPCs, with higher potency against ZIKVC-induced apoptosis. Our analyses reveal virus- and strain-specific molecular signatures associated with ZIKV infection. These datasets will help to investigate ZIKV-host interactions and identify neurovirulence determinants of ZIKV.

Guidelines for Manufacturing and Application of Organoids: Brain.

This study offers a comprehensive overview of brain organoids for researchers. It combines expert opinions with technical summaries on organoid definitions, characteristics, culture methods, and quality control. This approach aims to enhance the utilization of brain organoids in research. Brain organoids, as three-dimensional human cell models mimicking the nervous system, hold immense promise for studying the human brain. They offer advantages over traditional methods, replicating anatomical structures, physiological features, and complex neuronal networks. Additionally, brain organoids can model nervous system development and interactions between cell types and the microenvironment. By providing a foundation for utilizing the most human-relevant tissue models, this work empowers researchers to overcome limitations of two-dimensional cultures and conduct advanced disease modeling research.

Baf-mediated transcriptional regulation of teashirt is essential for the development of neural progenitor cell lineages.

Accumulating evidence hints heterochromatin anchoring to the inner nuclear membrane as an upstream regulatory process of gene expression. Given that the formation of neural progenitor cell lineages and the subsequent maintenance of postmitotic neuronal cell identity critically rely on transcriptional regulation, it seems possible that the development of neuronal cells is influenced by cell type-specific and/or context-dependent programmed regulation of heterochromatin anchoring. Here, we explored this possibility by genetically disrupting the evolutionarily conserved barrier-to-autointegration factor (Baf) in the Drosophila nervous system. Through single-cell RNA sequencing, we demonstrated that Baf knockdown induces prominent transcriptomic changes, particularly in type I neuroblasts. Among the differentially expressed genes, our genetic analyses identified teashirt (tsh), a transcription factor that interacts with beta-catenin, to be closely associated with Baf knockdown-induced phenotypes that were suppressed by the overexpression of tsh or beta-catenin. We also found that Baf and tsh colocalized in a region adjacent to heterochromatin in type I NBs. Notably, the subnuclear localization pattern remained unchanged when one of these two proteins was knocked down, indicating that both proteins contribute to the anchoring of heterochromatin to the inner nuclear membrane. Overall, this study reveals that the Baf-mediated transcriptional regulation of teashirt is a novel molecular mechanism that regulates the development of neural progenitor cell lineages.

Adolescent neurostimulation of dopamine circuit reverses genetic deficits in frontal cortex function.

Dopamine system dysfunction is implicated in adolescent-onset neuropsychiatric disorders. Although psychosis symptoms can be alleviated by antipsychotics, cognitive symptoms remain unresponsive and novel 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 Arc or Disc1 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 not Arc-/- 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 same cognitive deficits in Disc1+/- 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.

Adolescent neurostimulation of dopamine circuit reverses genetic deficits in frontal cortex function.

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 the Arc or DISC1 genes 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 not Arc mutant 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 same cognitive deficits in DISC1 mutant 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.

Trametinib activates endogenous neurogenesis and recovers neuropathology in a model of Alzheimer's disease.

Enhancing adult neurogenesis in the brain has been suggested as a potential therapeutic strategy for AD. We developed a screening platform, ATRIVIEW®, for molecules that activate neuronal differentiation of adult mouse NSCs. The most potent hit from an FDA-approved drug library was SNR1611 (trametinib), a selective MEK1/2 inhibitor. We found that trametinib increases the levels of P15INK4b and Neurog2, suggesting a mechanism by which MEK1/2 inhibition induces neuronal differentiation. Oral administration of trametinib increased adult neurogenesis in the dentate gyrus and subventricular zone of the 5XFAD AD mouse model. Surprisingly, we also found that trametinib enhanced adult neurogenesis in the cortex. Consequently, trametinib rescued AD pathologies such as neuronal loss and cognitive impairment in 5XFAD mice. Finally, trametinib induced neurogenic differentiation of NSCs derived from AD patient iPSCs, which suggests its potential therapeutic application. Altogether, we suggest that restoration of endogenous adult neurogenesis by trametinib may be a promising therapeutic approach to AD.

Exploring the brain epitranscriptome: perspectives from the NSAS summit.

Increasing evidence reinforces the essential function of RNA modifications in development and diseases, especially in the nervous system. RNA modifications impact various processes in the brain, including neurodevelopment, neurogenesis, neuroplasticity, learning and memory, neural regeneration, neurodegeneration, and brain tumorigenesis, leading to the emergence of a new field termed neuroepitranscriptomics. Deficiency in machineries modulating RNA modifications has been implicated in a range of brain disorders from microcephaly, intellectual disability, seizures, and psychiatric disorders to brain cancers such as glioblastoma. The inaugural NSAS Challenge Workshop on Brain Epitranscriptomics hosted in Crans-Montana, Switzerland in 2023 assembled a group of experts from the field, to discuss the current state of the field and provide novel translational perspectives. A summary of the discussions at the workshop is presented here to simulate broader engagement from the general neuroscience field.