Large-scale neurophysiology and single-cell profiling in human neuroscience.

Advances in large-scale single-unit human neurophysiology, single-cell RNA sequencing, spatial transcriptomics and long-term ex vivo tissue culture of surgically resected human brain tissue have provided an unprecedented opportunity to study human neuroscience. In this Perspective, we describe the development of these paradigms, including Neuropixels and recent brain-cell atlas efforts, and discuss how their convergence will further investigations into the cellular underpinnings of network-level activity in the human brain. Specifically, we introduce a workflow in which functionally mapped samples of human brain tissue resected during awake brain surgery can be cultured ex vivo for multi-modal cellular and functional profiling. We then explore how advances in human neuroscience will affect clinical practice, and conclude by discussing societal and ethical implications to consider. Potential findings from the field of human neuroscience will be vast, ranging from insights into human neurodiversity and evolution to providing cell-type-specific access to study and manipulate diseased circuits in pathology. This Perspective aims to provide a unifying framework for the field of human neuroscience as we welcome an exciting era for understanding the functional cytoarchitecture of the human brain.

Massively parallel characterization of regulatory elements in the developing human cortex.

Nucleotide changes in gene regulatory elements are important determinants of neuronal development and diseases. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 open chromatin regions, including thousands of sequences with cell type-specific accessibility and variants associated with brain gene regulation. In primary cells, we identified 46,802 active enhancer sequences and 164 variants that alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development.

Type-I-interferon-responsive microglia shape cortical development and behavior.

Microglia are brain-resident macrophages that shape neural circuit development and are implicated in neurodevelopmental diseases. Multiple microglial transcriptional states have been defined, but their functional significance is unclear. Here, we identify a type I interferon (IFN-I)-responsive microglial state in the developing somatosensory cortex (postnatal day 5) that is actively engulfing whole neurons. This population expands during cortical remodeling induced by partial whisker deprivation. Global or microglial-specific loss of the IFN-I receptor resulted in microglia with phagolysosomal dysfunction and an accumulation of neurons with nuclear DNA damage. IFN-I gain of function increased neuronal engulfment by microglia in both mouse and zebrafish and restricted the accumulation of DNA-damaged neurons. Finally, IFN-I deficiency resulted in excess cortical excitatory neurons and tactile hypersensitivity. These data define a role for neuron-engulfing microglia during a critical window of brain development and reveal homeostatic functions of a canonical antiviral signaling pathway in the brain.

Thalamocortical organoids enable in vitro modeling of 22q11.2 microdeletion associated with neuropsychiatric disorders.

Thalamic dysfunction has been implicated in multiple psychiatric disorders. We sought to study the mechanisms by which abnormalities emerge in the context of the 22q11.2 microdeletion, which confers significant genetic risk for psychiatric disorders. We investigated early stages of human thalamus development using human pluripotent stem cell-derived organoids and show that the 22q11.2 microdeletion underlies widespread transcriptional dysregulation associated with psychiatric disorders in thalamic neurons and glia, including elevated expression of FOXP2. Using an organoid co-culture model, we demonstrate that the 22q11.2 microdeletion mediates an overgrowth of thalamic axons in a FOXP2-dependent manner. Finally, we identify ROBO2 as a candidate molecular mediator of the effects of FOXP2 overexpression on thalamic axon overgrowth. Together, our study suggests that early steps in thalamic development are dysregulated in a model of genetic risk for schizophrenia and contribute to neural phenotypes in 22q11.2 deletion syndrome.

Use of Augmented Reality as a Tool to Support Cargo Handling Operations at the CARGO Air Terminal.

(1) Background: A current trend observed in the logistics sector is the use of Industry 4.0 tools to improve and enhance the efficiency of cargo handling processes. One of the popular solutions is an augmented reality system that supports operators in everyday tasks. The article aims to present design assumptions for implementing an augmented reality system to support air cargo handling at the warehouse. (2) Methods: Research was carried out based on a five-stage analytical procedure, aiming to analyze the current state and identify the potential for implementing the AR system. The following methods were used to collect data: co-participant observations, process analysis, direct interviews, analysis of internal documentation, and applicable legal regulations. (3) Results: The conducted research allowed for identifying information flows accompanying cargo flows and developing a project to automate selected information flows. The obtained results made it possible to identify operations for which the AR system's implementation will increase their effectiveness and efficiency. (4) Conclusions: The obtained results identified the need to develop a hybrid algorithm for arranging cargo in the warehouse and to build a system supporting self-verification of markings on air cargo.

Improving cellular diversity in human cerebellar organoids.

The developing human cerebellum has a greater diversity of progenitor types than that of the mouse, necessitating a human-based model for studying cerebellar development and disease. Atamian et al.1 developed a 3D organoid model of cerebellar development, which recapitulates many cell types found in the developing human cerebellum, including Purkinje-neuron-like cells.

Optimal trade-off control in machine learning-based library design, with application to adeno-associated virus (AAV) for gene therapy.

Adeno-associated viruses (AAVs) hold tremendous promise as delivery vectors for gene therapies. AAVs have been successfully engineered-for instance, for more efficient and/or cell-specific delivery to numerous tissues-by creating large, diverse starting libraries and selecting for desired properties. However, these starting libraries often contain a high proportion of variants unable to assemble or package their genomes, a prerequisite for any gene delivery goal. Here, we present and showcase a machine learning (ML) method for designing AAV peptide insertion libraries that achieve fivefold higher packaging fitness than the standard NNK library with negligible reduction in diversity. To demonstrate our ML-designed library's utility for downstream engineering goals, we show that it yields approximately 10-fold more successful variants than the NNK library after selection for infection of human brain tissue, leading to a promising glial-specific variant. Moreover, our design approach can be applied to other types of libraries for AAV and beyond.

A foundational atlas of autism protein interactions reveals molecular convergence.

Translating high-confidence (hc) autism spectrum disorder (ASD) genes into viable treatment targets remains elusive. We constructed a foundational protein-protein interaction (PPI) network in HEK293T cells involving 100 hcASD risk genes, revealing over 1,800 PPIs (87% novel). Interactors, expressed in the human brain and enriched for ASD but not schizophrenia genetic risk, converged on protein complexes involved in neurogenesis, tubulin biology, transcriptional regulation, and chromatin modification. A PPI map of 54 patient-derived missense variants identified differential physical interactions, and we leveraged AlphaFold-Multimer predictions to prioritize direct PPIs and specific variants for interrogation in Xenopus tropicalis and human forebrain organoids. A mutation in the transcription factor FOXP1 led to reconfiguration of DNA binding sites and altered development of deep cortical layer neurons in forebrain organoids. This work offers new insights into molecular mechanisms underlying ASD and describes a powerful platform to develop and test therapeutic strategies for many genetically-defined conditions.

Astrocytic ß-catenin signaling via TCF7L2 regulates synapse development and social behavior.

The Wnt/ß-catenin pathway contains multiple high-confidence risk genes that are linked to neurodevelopmental disorders, including autism spectrum disorder. However, its ubiquitous roles across brain cell types and developmental stages have made it challenging to define its impact on neural circuit development and behavior. Here, we show that TCF7L2, which is a key transcriptional effector of the Wnt/ß-catenin pathway, plays a cell-autonomous role in postnatal astrocyte maturation and impacts adult social behavior. TCF7L2 was the dominant Wnt effector that was expressed in both mouse and human astrocytes, with a peak during astrocyte maturation. The conditional knockout of Tcf7l2 in postnatal astrocytes led to an enlargement of astrocytes with defective tiling and gap junction coupling. These mice also exhibited an increase in the number of cortical excitatory and inhibitory synapses and a marked increase in social interaction by adulthood. These data reveal an astrocytic role for developmental Wnt/ß-catenin signaling in restricting excitatory synapse numbers and regulating adult social behavior.

Spatiotemporal molecular dynamics of the developing human thalamus.

The thalamus plays a central coordinating role in the brain. Thalamic neurons are organized into spatially distinct nuclei, but the molecular architecture of thalamic development is poorly understood, especially in humans. To begin to delineate the molecular trajectories of cell fate specification and organization in the developing human thalamus, we used single-cell and multiplexed spatial transcriptomics. We show that molecularly defined thalamic neurons differentiate in the second trimester of human development and that these neurons organize into spatially and molecularly distinct nuclei. We identified major subtypes of glutamatergic neuron subtypes that are differentially enriched in anatomically distinct nuclei and six subtypes of γ-aminobutyric acid-mediated (GABAergic) neurons that are shared and distinct across thalamic nuclei.

Single-cell analysis of prenatal and postnatal human cortical development.

We analyzed >700,000 single-nucleus RNA sequencing profiles from 106 donors during prenatal and postnatal developmental stages and identified lineage-specific programs that underlie the development of specific subtypes of excitatory cortical neurons, interneurons, glial cell types, and brain vasculature. By leveraging single-nucleus chromatin accessibility data, we delineated enhancer gene regulatory networks and transcription factors that control commitment of specific cortical lineages. By intersecting our results with genetic risk factors for human brain diseases, we identified the cortical cell types and lineages most vulnerable to genetic insults of different brain disorders, especially autism. We find that lineage-specific gene expression programs up-regulated in female cells are especially enriched for the genetic risk factors of autism. Our study captures the molecular progression of cortical lineages across human development.

Spatiotemporal molecular dynamics of the developing human thalamus.

The thalamus plays a central coordinating role in the brain. Thalamic neurons are organized into spatially-distinct nuclei, but the molecular architecture of thalamic development is poorly understood, especially in humans. To begin to delineate the molecular trajectories of cell fate specification and organization in the developing human thalamus, we used single cell and multiplexed spatial transcriptomics. Here we show that molecularly-defined thalamic neurons differentiate in the second trimester of human development, and that these neurons organize into spatially and molecularly distinct nuclei. We identify major subtypes of glutamatergic neuron subtypes that are differentially enriched in anatomically distinct nuclei. In addition, we identify six subtypes of GABAergic neurons that are shared and distinct across thalamic nuclei. One-Sentence Summary: Single cell and spatial profiling of the developing thalamus in the first and second trimester yields molecular mechanisms of thalamic nuclei development.

Rubella virus tropism and single-cell responses in human primary tissue and microglia-containing organoids.

Rubella virus is an important human pathogen that can cause neurological deficits in a developing fetus when contracted during pregnancy. Despite successful vaccination programs in the Americas and many developed countries, rubella remains endemic in many regions worldwide and outbreaks occur wherever population immunity is insufficient. Intense interest since rubella virus was first isolated in 1962 has advanced our understanding of clinical outcomes after infection disrupts key processes of fetal neurodevelopment. Yet it is still largely unknown which cell types in the developing brain are targeted. We show that in human brain slices, rubella virus predominantly infects microglia. This infection occurs in a heterogeneous population but not in a highly microglia-enriched monoculture in the absence of other cell types. By using an organoid-microglia model, we further demonstrate that rubella virus infection leads to a profound interferon response in non-microglial cells, including neurons and neural progenitor cells, and this response is attenuated by the presence of microglia.

Three-dimensional genome rewiring in loci with human accelerated regions.

Human accelerated regions (HARs) are conserved genomic loci that evolved at an accelerated rate in the human lineage and may underlie human-specific traits. We generated HARs and chimpanzee accelerated regions with an automated pipeline and an alignment of 241 mammalian genomes. Combining deep learning with chromatin capture experiments in human and chimpanzee neural progenitor cells, we discovered a significant enrichment of HARs in topologically associating domains containing human-specific genomic variants that change three-dimensional (3D) genome organization. Differential gene expression between humans and chimpanzees at these loci suggests rewiring of regulatory interactions between HARs and neurodevelopmental genes. Thus, comparative genomics together with models of 3D genome folding revealed enhancer hijacking as an explanation for the rapid evolution of HARs.

Transcriptomic cell type structures in vivo neuronal activity across multiple timescales.

Cell type is hypothesized to be a key determinant of a neuron's role within a circuit. Here, we examine whether a neuron's transcriptomic type influences the timing of its activity. We develop a deep-learning architecture that learns features of interevent intervals across timescales (ms to >30 min). We show that transcriptomic cell-class information is embedded in the timing of single neuron activity in the intact brain of behaving animals (calcium imaging and extracellular electrophysiology) as well as in a bio-realistic model of the visual cortex. Further, a subset of excitatory cell types are distinguishable but can be classified with higher accuracy when considering cortical layer and projection class. Finally, we show that computational fingerprints of cell types may be universalizable across structured stimuli and naturalistic movies. Our results indicate that transcriptomic class and type may be imprinted in the timing of single neuron activity across diverse stimuli.

Massively parallel characterization of psychiatric disorder-associated and cell-type-specific regulatory elements in the developing human cortex.

Nucleotide changes in gene regulatory elements are important determinants of neuronal development and disease. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 sequences, including differentially accessible cell-type specific regions in the developing cortex and single-nucleotide variants associated with psychiatric disorders. In primary cells, we identified 46,802 active enhancer sequences and 164 disorder-associated variants that significantly alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning, we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development.

Type I interferon responsive microglia shape cortical development and behavior.

Microglia are brain resident phagocytes that can engulf synaptic components and extracellular matrix as well as whole neurons. However, whether there are unique molecular mechanisms that regulate these distinct phagocytic states is unknown. Here we define a molecularly distinct microglial subset whose function is to engulf neurons in the developing brain. We transcriptomically identified a cluster of Type I interferon (IFN-I) responsive microglia that expanded 20-fold in the postnatal day 5 somatosensory cortex after partial whisker deprivation, a stressor that accelerates neural circuit remodeling. In situ, IFN-I responsive microglia were highly phagocytic and actively engulfed whole neurons. Conditional deletion of IFN-I signaling (Ifnar1fl/fl) in microglia but not neurons resulted in dysmorphic microglia with stalled phagocytosis and an accumulation of neurons with double strand DNA breaks, a marker of cell stress. Conversely, exogenous IFN-I was sufficient to drive neuronal engulfment by microglia and restrict the accumulation of damaged neurons. IFN-I deficient mice had excess excitatory neurons in the developing somatosensory cortex as well as tactile hypersensitivity to whisker stimulation. These data define a molecular mechanism through which microglia engulf neurons during a critical window of brain development. More broadly, they reveal key homeostatic roles of a canonical antiviral signaling pathway in brain development.

New York Stem Cell Foundation Robertson Investigators.

As the stem cell community mourns the loss of New York Stem Cell Foundation founder Susan Solomon, we also look to celebrate her legacy. In this Voices, members of the 2022 class of NYSCF Roberston Investigators share how NYSCF community support will impact them and the bold ideas they will pursue as a result.

Integrated gene analyses of de novo variants from 46,612 trios with autism and developmental disorders.

Most genetic studies consider autism spectrum disorder (ASD) and developmental disorder (DD) separately despite overwhelming comorbidity and shared genetic etiology. Here, we analyzed de novo variants (DNVs) from 15,560 ASD (6,557 from SPARK) and 31,052 DD trios independently and also combined as broader neurodevelopmental disorders (NDDs) using three models. We identify 615 NDD candidate genes (false discovery rate [FDR] < 0.05) supported by ≥1 models, including 138 reaching Bonferroni exome-wide significance (P < 3.64e-7) in all models. The genes group into five functional networks associating with different brain developmental lineages based on single-cell nuclei transcriptomic data. We find no evidence for ASD-specific genes in contrast to 18 genes significantly enriched for DD. There are 53 genes that show mutational bias, including enrichments for missense (n = 41) or truncating (n = 12) DNVs. We also find 10 genes with evidence of male- or female-bias enrichment, including 4 X chromosome genes with significant female burden (DDX3X, MECP2, WDR45, and HDAC8). This large-scale integrative analysis identifies candidates and functional subsets of NDD genes.