Autism genes converge on asynchronous development of shared neuron classes.

Genetic risk for autism spectrum disorder (ASD) is associated with hundreds of genes spanning a wide range of biological functions1-6. The alterations in the human brain resulting from mutations in these genes remain unclear. Furthermore, their phenotypic manifestation varies across individuals7,8. Here we used organoid models of the human cerebral cortex to identify cell-type-specific developmental abnormalities that result from haploinsufficiency in three ASD risk genes-SUV420H1 (also known as KMT5B), ARID1B and CHD8-in multiple cell lines from different donors, using single-cell RNA-sequencing (scRNA-seq) analysis of more than 745,000 cells and proteomic analysis of individual organoids, to identify phenotypic convergence. Each of the three mutations confers asynchronous development of two main cortical neuronal lineages-γ-aminobutyric-acid-releasing (GABAergic) neurons and deep-layer excitatory projection neurons-but acts through largely distinct molecular pathways. Although these phenotypes are consistent across cell lines, their expressivity is influenced by the individual genomic context, in a manner that is dependent on both the risk gene and the developmental defect. Calcium imaging in intact organoids shows that these early-stage developmental changes are followed by abnormal circuit activity. This research uncovers cell-type-specific neurodevelopmental abnormalities that are shared across ASD risk genes and are finely modulated by human genomic context, finding convergence in the neurobiological basis of how different risk genes contribute to ASD pathology.

Individual brain organoids reproducibly form cell diversity of the human cerebral cortex.

Experimental models of the human brain are needed for basic understanding of its development and disease1. Human brain organoids hold unprecedented promise for this purpose; however, they are plagued by high organoid-to-organoid variability2,3. This has raised doubts as to whether developmental processes of the human brain can occur outside the context of embryogenesis with a degree of reproducibility that is comparable to the endogenous tissue. Here we show that an organoid model of the dorsal forebrain can reliably generate a rich diversity of cell types appropriate for the human cerebral cortex. We performed single-cell RNA-sequencing analysis of 166,242 cells isolated from 21 individual organoids, finding that 95% of the organoids generate a virtually indistinguishable compendium of cell types, following similar developmental trajectories and with a degree of organoid-to-organoid variability comparable to that of individual endogenous brains. Furthermore, organoids derived from different stem cell lines show consistent reproducibility in the cell types produced. The data demonstrate that reproducible development of the complex cellular diversity of the central nervous system does not require the context of the embryo, and that establishment of terminal cell identity is a highly constrained process that can emerge from diverse stem cell origins and growth environments.

Taming human brain organoids one cell at a time.

Human brain organoids are self-organizing three-dimensional structures that emerge from human pluripotent stem cells and mimic aspects of the cellular composition and functionality of the developing human brain. Despite their impressive self-organizing capacity, organoids lack the stereotypic structural anatomy of their in vivo counterpart, making conventional analysis techniques underpowered to assess cellular composition and gene network regulation in organoids. Advances in single cell transcriptomics have recently allowed characterization and improvement of organoid protocols, as they continue to evolve, by enabling identification of cell types and states along with their developmental origins. In this review, we summarize recent approaches, progresses and challenges in resolving brain organoid's complexity through single-cell transcriptomics. We then discuss emerging technologies that may complement single-cell RNA sequencing by providing additional readouts of cellular states to generate an organ-level view of developmental processes. Altogether, these integrative technologies will allow monitoring of global gene regulation in thousands of individual cells and will offer an unprecedented opportunity to investigate features of human brain development and disease across multiple cellular modalities and with cell-type resolution.

Cell diversity and network dynamics in photosensitive human brain organoids.

In vitro models of the developing brain such as three-dimensional brain organoids offer an unprecedented opportunity to study aspects of human brain development and disease. However, the cells generated within organoids and the extent to which they recapitulate the regional complexity, cellular diversity and circuit functionality of the brain remain undefined. Here we analyse gene expression in over 80,000 individual cells isolated from 31 human brain organoids. We find that organoids can generate a broad diversity of cells, which are related to endogenous classes, including cells from the cerebral cortex and the retina. Organoids could be developed over extended periods (more than 9 months), allowing for the establishment of relatively mature features, including the formation of dendritic spines and spontaneously active neuronal networks. Finally, neuronal activity within organoids could be controlled using light stimulation of photosensitive cells, which may offer a way to probe the functionality of human neuronal circuits using physiological sensory stimuli.

The MDM4/MDM2-p53-IGF1 axis controls axonal regeneration, sprouting and functional recovery after CNS injury.

Regeneration of injured central nervous system axons is highly restricted, causing neurological impairment. To date, although the lack of intrinsic regenerative potential is well described, a key regulatory molecular mechanism for the enhancement of both axonal regrowth and functional recovery after central nervous system injury remains elusive. While ubiquitin ligases coordinate neuronal morphogenesis and connectivity during development as well as after axonal injury, their role specifically in axonal regeneration is unknown. Following a bioinformatics network analysis combining ubiquitin ligases with previously defined axonal regenerative proteins, we found a triad composed of the ubiquitin ligases MDM4, MDM2 and the transcription factor p53 (encoded by TP53) as a putative central signalling complex restricting the regeneration program. Indeed, conditional deletion of MDM4 or pharmacological inhibition of MDM2/p53 interaction in the eye and spinal cord promote axonal regeneration and sprouting of the optic nerve after crush and of supraspinal tracts after spinal cord injury. The double conditional deletion of MDM4-p53 as well as MDM2 inhibition in p53-deficient mice blocks this regenerative phenotype, showing its dependence upon p53. Genome-wide gene expression analysis from ex vivo fluorescence-activated cell sorting in MDM4-deficient retinal ganglion cells identifies the downstream target IGF1R, whose activity and expression was found to be required for the regeneration elicited by MDM4 deletion. Importantly, we demonstrate that pharmacological enhancement of the MDM2/p53-IGF1R axis enhances axonal sprouting as well as functional recovery after spinal cord injury. Thus, our results show MDM4-MDM2/p53-IGF1R as an original regulatory mechanism for CNS regeneration and offer novel targets to enhance neurological recovery.media-1vid110.1093/brain/awv125_video_abstractawv125_video_abstract.

Modulation of GABAA receptor signaling increases neurogenesis and suppresses anxiety through NFATc4.

Correlative evidence suggests that GABAergic signaling plays an important role in the regulation of activity-dependent hippocampal neurogenesis and emotional behavior in adult mice. However, whether these are causally linked at the molecular level remains elusive. Nuclear factor of activated T cell (NFAT) proteins are activity-dependent transcription factors that respond to environmental stimuli in different cell types, including hippocampal newborn neurons. Here, we identify NFATc4 as a key activity-dependent transcriptional regulator of GABA signaling in hippocampal progenitor cells via an unbiased high-throughput genome-wide study. Next, we demonstrate that GABAA receptor (GABAAR) signaling modulates hippocampal neurogenesis through NFATc4 activity, which in turn regulates GABRA2 and GABRA4 subunit expression via binding to specific promoter responsive elements, as assessed by ChIP and luciferase assays. Furthermore, we show that selective pharmacological enhancement of GABAAR activity promotes hippocampal neurogenesis via the calcineurin/NFATc4 axis. Importantly, the NFATc4-dependent increase in hippocampal neurogenesis after GABAAR stimulation is required for the suppression of the anxiety response in mice. Together, these data provide a novel molecular insight into the regulation of the anxiety response in mice, suggesting that the GABAAR/NFATc4 axis is a druggable target for the therapy of emotional disorders.

Nuclear factor of activated T cells (NFATc4) is required for BDNF-dependent survival of adult-born neurons and spatial memory formation in the hippocampus.

New neurons generated in the adult dentate gyrus are constantly integrated into the hippocampal circuitry and activated during encoding and recall of new memories. Despite identification of extracellular signals that regulate survival and integration of adult-born neurons such as neurotrophins and neurotransmitters, the nature of the intracellular modulators required to transduce those signals remains elusive. Here, we provide evidence of the expression and transcriptional activity of nuclear factor of activated T cell c4 (NFATc4) in hippocampal progenitor cells. We show that NFATc4 calcineurin-dependent activity is required selectively for survival of adult-born neurons in response to BDNF signaling. Indeed, cyclosporin A injection and stereotaxic delivery of the BDNF scavenger TrkB-Fc in the mouse dentate gyrus reduce the survival of hippocampal adult-born neurons in wild-type but not in NFATc4(-/-) mice and do not affect the net rate of neural precursor proliferation and their fate commitment. Furthermore, associated with the reduced survival of adult-born neurons, the absence of NFATc4 leads to selective defects in LTP and in the encoding of hippocampal-dependent spatial memories. Thus, our data demonstrate that NFATc4 is essential in the regulation of adult hippocampal neurogenesis and identify NFATc4 as a central player of BDNF-driven prosurvival signaling in hippocampal adult-born neurons.

The tumor suppressor p53 fine-tunes reactive oxygen species levels and neurogenesis via PI3 kinase signaling.

Mounting evidence points to a role for endogenous reactive oxygen species (ROS) in cell signaling, including in the control of cell proliferation, differentiation, and fate. However, the function of ROS and their molecular regulation in embryonic mouse neural progenitor cells (eNPCs) has not yet been clarified. Here, we describe that physiological ROS are required for appropriate timing of neurogenesis in the developing telencephalon in vivo and in cultured NPCs, and that the tumor suppressor p53 plays a key role in the regulation of ROS-dependent neurogenesis. p53 loss of function leads to elevated ROS and early neurogenesis, while restoration of p53 and antioxidant treatment partially reverse the phenotype associated with premature neurogenesis. Furthermore, we describe that the expression of a number of neurogenic and oxidative stress genes relies on p53 and that both p53 and ROS-dependent induction of neurogenesis depend on PI3 kinase/phospho-Akt signaling. Our results suggest that p53 fine-tunes endogenous ROS levels to ensure the appropriate timing of neurogenesis in eNPCs. This may also have implications for the generation of tumors of neurodevelopmental origin.

Waking up the sleepers: shared transcriptional pathways in axonal regeneration and neurogenesis.

In the last several years, relevant progress has been made in our understanding of the transcriptional machinery regulating CNS repair after acute injury, such as following trauma or stroke. In order to survive and functionally reconnect to the synaptic network, injured neurons activate an intrinsic rescue program aimed to increase their plasticity. Perhaps, in the attempt to switch back to a plastic and growth-competent state, post-mitotic neurons wake up and re-express a set of transcription factors that are also critical for the regulation of their younger brothers, the neural stem cells. Here, we review and discuss the transcriptional pathways regulating both axonal regeneration and neurogenesis highlighting the connection between the two. Clarification of their common molecular substrate may help simultaneous targeting of both neurogenesis and axonal regeneration with the hope to enhance functional recovery following CNS injury.

Human cerebellar organoids with functional Purkinje cells.

Research on human cerebellar development and disease has been hampered by the need for a human cell-based system that recapitulates the human cerebellum's cellular diversity and functional features. Here, we report a human organoid model (human cerebellar organoids [hCerOs]) capable of developing the complex cellular diversity of the fetal cerebellum, including a human-specific rhombic lip progenitor population that have never been generated in vitro prior to this study. 2-month-old hCerOs form distinct cytoarchitectural features, including laminar organized layering, and create functional connections between inhibitory and excitatory neurons that display coordinated network activity. Long-term culture of hCerOs allows healthy survival and maturation of Purkinje cells that display molecular and electrophysiological hallmarks of their in vivo counterparts, addressing a long-standing challenge in the field. This study therefore provides a physiologically relevant, all-human model system to elucidate the cell-type-specific mechanisms governing cerebellar development and disease.

p53 Regulates the neuronal intrinsic and extrinsic responses affecting the recovery of motor function following spinal cord injury.

Following spinal trauma, the limited physiological axonal sprouting that contributes to partial recovery of function is dependent upon the intrinsic properties of neurons as well as the inhibitory glial environment. The transcription factor p53 is involved in DNA repair, cell cycle, cell survival, and axonal outgrowth, suggesting p53 as key modifier of axonal and glial responses influencing functional recovery following spinal injury. Indeed, in a spinal cord dorsal hemisection injury model, we observed a significant impairment in locomotor recovery in p53(-/-) versus wild-type mice. p53(-/-) spinal cords showed an increased number of activated microglia/macrophages and a larger scar at the lesion site. Loss- and gain-of-function experiments suggested p53 as a direct regulator of microglia/macrophages proliferation. At the axonal level, p53(-/-) mice showed a more pronounced dieback of the corticospinal tract (CST) and a decreased sprouting capacity of both CST and spinal serotoninergic fibers. In vivo expression of p53 in the sensorimotor cortex rescued and enhanced the sprouting potential of the CST in p53(-/-) mice, while, similarly, p53 expression in p53(-/-) cultured cortical neurons rescued a defect in neurite outgrowth, suggesting a direct role for p53 in regulating the intrinsic sprouting ability of CNS neurons. In conclusion, we show that p53 plays an important regulatory role at both extrinsic and intrinsic levels affecting the recovery of motor function following spinal cord injury. Therefore, we propose p53 as a novel potential multilevel therapeutic target for spinal cord injury.

Present and Future Modeling of Human Psychiatric Connectopathies With Brain Organoids.

Brain organoids derived from human pluripotent stem cells are emerging as a powerful tool to model cellular aspects of neuropsychiatric disorders, including alterations in cell proliferation, differentiation, migration, and lineage trajectory. To date, most contributions in the field have focused on modeling cellular impairment of the cerebral cortex, with few studies probing dysfunction in local network connectivity. However, it is increasingly more apparent that these psychiatric disorders are connectopathies involving multiple brain structures and the connections between them. Therefore, the lack of reproducible anatomical features in these 3-dimensional cultures represents a major bottleneck for effectively modeling brain connectivity at the micro(cellular) level and at the macroscale level between brain regions. In this perspective, we review the use of current organoid protocols to model neuropsychiatric disorders with a specific emphasis on the potential and limitations of the current strategies to model impairments in functional connectivity. Finally, we discuss the importance of adopting interdisciplinary strategies to establish next-generation, multiregional organoids that can model, with higher fidelity, the dysfunction in the development and functionality of long-range connections within the brain of patients affected by psychiatric disorders.

Non-synaptic function of the autism spectrum disorder-associated gene SYNGAP1 in cortical neurogenesis.

Genes involved in synaptic function are enriched among those with autism spectrum disorder (ASD)-associated rare genetic variants. Dysregulated cortical neurogenesis has been implicated as a convergent mechanism in ASD pathophysiology, yet it remains unknown how 'synaptic' ASD risk genes contribute to these phenotypes, which arise before synaptogenesis. Here, we show that the synaptic Ras GTPase-activating (RASGAP) protein 1 (SYNGAP1, a top ASD risk gene) is expressed within the apical domain of human radial glia cells (hRGCs). In a human cortical organoid model of SYNGAP1 haploinsufficiency, we find dysregulated cytoskeletal dynamics that impair the scaffolding and division plane of hRGCs, resulting in disrupted lamination and accelerated maturation of cortical projection neurons. Additionally, we confirmed an imbalance in the ratio of progenitors to neurons in a mouse model of Syngap1 haploinsufficiency. Thus, SYNGAP1-related brain disorders may arise through non-synaptic mechanisms, highlighting the need to study genes associated with neurodevelopmental disorders (NDDs) in diverse human cell types and developmental stages.

Organoids as tools for fundamental discovery and translation-a Keystone Symposia report.

Complex three-dimensional in vitro organ-like models, or organoids, offer a unique biological tool with distinct advantages over two-dimensional cell culture systems, which can be too simplistic, and animal models, which can be too complex and may fail to recapitulate human physiology and pathology. Significant progress has been made in driving stem cells to differentiate into different organoid types, though several challenges remain. For example, many organoid models suffer from high heterogeneity, and it can be difficult to fully incorporate the complexity of in vivo tissue and organ development to faithfully reproduce human biology. Successfully addressing such limitations would increase the viability of organoids as models for drug development and preclinical testing. On April 3-6, 2022, experts in organoid development and biology convened at the Keystone Symposium "Organoids as Tools for Fundamental Discovery and Translation" to discuss recent advances and insights from this relatively new model system into human development and disease.

Upgrading the Physiological Relevance of Human Brain Organoids.

The recent advent of human pluripotent stem cell (PSC)-derived 3D brain organoids has opened a window into aspects of human brain development that were not accessible before, allowing tractable monitoring and assessment of early developmental processes. However, their broad and effective use for modeling later stages of human brain development and disease is hampered by the lack of a stereotypic anatomical organization, which limits maturation processes dependent upon formation of unique cellular interactions and short- and long-range network connectivity. Emerging methods and technologies aimed at tighter regulatory control through bioengineering approaches, along with newer unbiased organoid analysis readouts, should resolve several of the current limitations. Here, we review recent advances in brain organoid generation and characterization with a focus on highlighting future directions utilizing interdisciplinary strategies that will be important for improving the physiological relevance of this model system.

Impaired adult neurogenesis associated with short-term memory defects in NF-kappaB p50-deficient mice.

Neurogenesis proceeds throughout adulthood in the brain of most mammalian species, but the molecular mechanisms underlying the regulation of stem/progenitor cell proliferation, survival, maturation, and differentiation have not been completely unraveled. We have studied hippocampal neurogenesis in NF-kappaB p50-deficient mice. Here we demonstrate that in absence of p50, the net rate of neural precursor proliferation does not change, but some of the steps leading to the final neuron differentiation status are hampered, resulting in approximately 50% reduction in the number of newly born neurons in the adult mutant hippocampus. Additionally, in p50(-/-) mice, we observed a selective defect in short-term spatial memory performance without impairment of hippocampal-dependent spatial long-term memory and learning. Our results highlight the role of NF-kappaB p50 in hippocampal neurogenesis and in short-term spatial memory.

Studying the Brain in a Dish: 3D Cell Culture Models of Human Brain Development and Disease.

The study of the cellular and molecular processes of the developing human brain has been hindered by access to suitable models of living human brain tissue. Recently developed 3D cell culture models offer the promise of studying fundamental brain processes in the context of human genetic background and species-specific developmental mechanisms. Here, we review the current state of 3D human brain organoid models and consider their potential to enable investigation of complex aspects of human brain development and the underpinning of human neurological disease.

Present and future of modeling human brain development in 3D organoids.

Three-dimensional (3D) brain organoids derived from human pluripotent stem cells hold great potential to investigate complex human genetic states and to model aspects of human brain development and pathology. However, the field of brain organoids is still in its infancy, and their use has been limited by their variability and their inability to differentiate into 3D structures with reproducible anatomical organization. Here, starting from a review of basic principles of in vitro 'brain organogenesis', we discuss which aspects of human brain development and disease can be faithfully modeled with current brain organoid protocols, and discuss improvements that would allow them to become reliable tools to investigate complex features of human brain development and disease.