Glioblastomas (GBMs) functionally integrate into diverse neuronal circuits within the central nervous system, which can promote tumor progression and affect neurons via neuron-to-glioma synapses. It remains challenging to identify and manipulate tumor-innervating neurons, which may remain localized or widely distributed throughout the brain. Building on GBM organoids (GBOs) derived from patient-resected surgical tissue, we present here detailed procedures for assessing interactions between tumors and neurons. We first discuss retrograde trans-monosynaptic tracing approaches to study the neuron-tumor connectome by using a rabies viral system in ex vivo human tissue and in xenogenic animal models. As a complementary approach, we then describe the use of anterograde transsynaptic tracing using herpes simplex virus in vivo and ex vivo to assess brain region-specific connectivity in GBMs. In addition, to facilitate the adaptability of these tracing methodologies in diverse systems, we provide procedures for the viral transduction into GBOs, the generation of assembloids comprising GBOs and human induced pluripotent stem cell-derived cortical organoids and the establishment of air-liquid interface cultures from surgical human brain tissue. Together, these techniques permit the flexible characterization and manipulation of tumor-neural circuits and can be easily adapted to other cancers with nervous system involvement. After the generation of GBOs and/or cortical organoids, transsynaptic tracing requires 12-35 d to complete ex vivo or in vivo. The procedure is suitable for users with expertise in human cell and organoid culture, viral production and transduction, rodent surgery and microscopy.

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, oligodendro-cyte lineages, and microglia identified subpopulations with distinct molecular signatures and revealed their association with specific physiological functions, age-dependent changes in abundance, and disease rele-vance. 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 identi-fied 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 dy-namics across the postnatal lifespan, and dysregulation in AD and provides a reference atlas for stem -cell-based glial differentiation.

BACKGROUND: Gene dosage imbalance caused by copy number variations (CNVs) is a prominent contributor to brain disorders. In particular, 15q11.2 CNV duplications and deletions have been associated with autism spectrum disorder and schizophrenia, respectively. The mechanism underlying these diametric contributions remains unclear.METHODS: We established both loss-of-function and gain-of-function mouse models of Cyfip1, one of four genes within 15q11.2 CNVs. To assess the functional consequences of altered CYFIP1 levels, we performed systematic investigations on behavioral, electrophysiological, and biochemical phenotypes in both mouse models. In addition, we utilized RNA immunoprecipitation sequencing (RIP-seq) analysis to reveal molecular targets of CYFIP1 in vivo.RESULTS: Cyfip1 loss-of-function and gain-of function mouse models exhibited distinct and shared behavioral abnormalities related to autism spectrum disorder and schizophrenia. RIP-seq analysis identified messenger RNA targets of CYFIP1 in vivo, including postsynaptic NMDA receptor (NMDAR) complex components. In addition, these mouse models showed diametric changes in levels of postsynaptic NMDAR complex components at synapses because of dysregulated protein translation, resulting in bidirectional alteration of NMDAR-mediated signaling. Importantly, pharmacological balancing of NMDAR signaling in these mouse models with diametric Cyfip1 dosages rescues behavioral abnormalities.CONCLUSIONS: CYFIP1 regulates protein translation of NMDAR and associated complex components at synapses to maintain normal synaptic functions and behaviors. Our integrated analyses provide insight into how gene dosage imbalance caused by CNVs may contribute to divergent neuropsychiatric disorders.

Chromatin is organized into multiscale three-dimensional structures, including chromosome territories, A/B compartments, topologically associating domains, and chromatin loops. This hierarchically organized genomic architecture regulates gene transcription, which, in turn, is essential for various biological pro-cesses during brain development and adult plasticity. Here, we review different aspects of spatial genome organization and their functions in regulating gene expression in the nervous system, as well as their dysre-gulation in brain disorders. We also highlight new technologies to probe and manipulate chromatin architec-ture and discuss how investigating spatial genome organization can lead to a better understanding of the nervous system and associated disorders.

Hippocampal neurogenesis has typically been studied during embryonic development or in adulthood, promoting the perception of two distinct phenomena. We propose a perspective that hippocampal neurogenesis in the mammalian brain is one continuous, lifelong developmental process. We summarize the common features of hippocampal neurogenesis that are maintained across the lifespan, as well as dynamic age-dependent properties. We highlight that while the progression of hippocampal neurogenesis across the lifespan is conserved between mammalian species, the timing of this progression is species-dependent. Finally, we discuss some current challenges in the hippocampus neurogenesis field, and future research directions to address them, such as time course analysis across the lifespan, mechanisms regulating neurogenesis progression, and interspecies comparisons. We hope that this new perspective of hippocampal neurogenesis will prompt fresh insight into previous research and inspire new directions to advance the field to identify biologically significant ways to harness the endogenous capacity for neurogenesis in the hippocampus.

Physiological blood-tissue barriers play a critical role in separating the circulation from immune-privileged sites and denying access to blood-borne viruses. The mechanism of virus restriction by these barriers is poorly understood. We utilize induced pluripotent stem cell (iPSC)-derived human brain microvascular endothelial cells (iBMECs) to study virus-blood-brain barrier (BBB) interactions. These iPSC-derived cells faithfully recapitulate a striking difference in in vivo neuroinvasion by two alphavirus isolates and are selectively permissive to neurotropic flaviviruses. A model of cocultured iBMECs and astrocytes exhibits high transendothelial electrical resistance and blocks non-neurotropic flaviviruses from getting across the barrier. We find that iBMECs constitutively express an interferon-induced gene, IFITM1, which preferentially restricts the replication of non-neurotropic flaviviruses. Barrier cells from blood-testis and blood-retinal barriers also constitutively express IFITMs that contribute to the viral resistance. Our application of a renewable human iPSC-based model for studying virus-BBB interactions reveals that intrinsic immunity at the barriers contributes to virus exclusion.

Human neurogenesis occurs mainly in embryonic, fetal, and neonatal stages and generates tremendously diverse neural cell types that constitute the human nervous system. Studies on human neurogenesis have been limited due to a lack of access to human embryonic and fetal tissues. Brain organoids derived from human pluripotent stem cells not only recapitulate major developmental processes during neurogenesis, but also exhibit human-specific features, thus providing an unprecedented opportunity to study human neurodevelopment. First, three-dimensional brain organoids resemble early human neurogenesis with diverse stem cell pools, including the presence of primate-enriched outer radial glia cells. Second, brain organoids recapitulate human neurogenesis at the cellular level, generating diverse neuronal cell types and forming stratified cortical layers. Third, brain organoids also capture gliogenesis with the presence of human-specific astrocytes. Fourth, combined with genome-editing technologies, brain organoids are promising models for investigating functions of human-specific genes at different stages of human neurogenesis. Finally, human organoids derived from patient iPSCs can recapitulate specific disease phenotypes, providing unique models for studying developmental brain disorders of genetic and environmental causes, and for mechanistic studies and drug screening. The aim of this review is to illustrate why brain organoids are good models to study various steps of human neurogenesis, with a focus on corticogenesis. We also discuss limitations of current brain organoid models and future improvements.