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LIF signaling regulates outer radial glial to interneuron fate during human cortical development.
Radial glial (RG) development is essential for cerebral cortex growth and organization. In humans, the outer radial glia (oRG) subtype is expanded and gives rise to diverse neurons and glia. However, the mechanisms regulating oRG differentiation are unclear. oRG cells express leukemia-inhibitory factor (LIF) receptors during neurogenesis, and consistent with a role in stem cell self-renewal, LIF perturbation impacts oRG proliferation in cortical tissue and organoids. Surprisingly, LIF treatment also increases the production of inhibitory interneurons (INs) in cortical cultures. Comparative transcriptomic analysis identifies that the enhanced IN population resembles INs produced in the caudal ganglionic eminence. To evaluate whether INs could arise from oRGs, we isolated primary oRG cells and cultured them with LIF. We observed the production of INs from oRG cells and an increase in IN abundance following LIF treatment. Our observations suggest that LIF signaling regulates the capacity of oRG cells to generate INs.
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Transcriptional networks identify synaptotagmin-like 3 as a regulator of cortical neuronal migration during early neurodevelopment.
Human brain development is a complex process involving neural proliferation, differentiation, and migration that are directed by many essential cellular factors and drivers. Here, using the NetBID2 algorithm and developing human brain RNA sequencing dataset, we identify synaptotagmin-like 3 (SYTL3) as one of the top drivers of early human brain development. Interestingly, SYTL3 exhibits high activity but low expression in both early developmental human cortex and human embryonic stem cell (hESC)-derived neurons. Knockout of SYTL3 (SYTL3-KO) in human neurons or knockdown of Sytl3 in embryonic mouse cortex markedly promotes neuronal migration. SYTL3-KO causes an abnormal distribution of deep-layer neurons in brain organoids and reduces presynaptic neurotransmitter release in hESC-derived neurons. We further demonstrate that SYTL3-KO-accelerated neuronal migration is modulated by high expression of matrix metalloproteinases. Together, based on bioinformatics and biological experiments, we identify SYTL3 as a regulator of cortical neuronal migration in human and mouse developing brains.
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Modulating hepatic hypoxanthine metabolism relieve metabolic stress-related neurovascular resilience disturbance via the liver-brain axis.
BACKGROUND: Unhealthy lifestyles promote brain aging, but their mechanisms remain unclear. The liver-brain axis acts as a key mediator of brain dysfunction and warrants investigation in lifestyle-induced brain aging. PURPOSE: To elucidate how the liver-brain axis mediates lifestyle-triggered neurovascular resilience disturbance and associated brain aging phenotypes, and identify effective anti-aging intervention targets. STUDY DESIGN: A combination of in vivo animal models, multi-omics analyses, computational simulation, brain organoid experiments, and molecular biology techniques to explore the mechanism of liver-brain axis mediates lifestyle-triggered neurovascular resilience disturbance and associated brain aging phenotypes and verify the efficacy of Bazi Bushen capsule (BZBS) intervention. METHODS: A mouse model established using a combined high-fat/high-sugar diet and circadian disruption (HFHS/CD). Cognitive function and anxiety-like behaviors were evaluated. Multi-omics analyses were performed, and liver-brain axis/hypoxanthine signals were verified via computational simulation and brain organoids. Active ingredient targets were identified by molecular docking, drug affinity responsive target stability, and validated by biolayer interferometry. RESULTS: HFHS/CD induced cognitive decline and anxiety accompanied by apparent brain aging-related phenotypes, which were alleviated by nicotinamide mononucleotide (NMN) and BZBS. Dysregulated hepatic hypoxanthine metabolism under metabolic stress contributed to neurovascular homeostasis disturbance, characterized by compromised BBB integrity and excessive microglial activation. In vitro experiments further indicated that elevated hypoxanthine levels were involved in endothelial senescence, potentially through P2X7-dependent suppression of NRF2-governed glutathione metabolism. BZBS acted through multiple targets: imperatorin/isopimpinellin regulated hepatic purine nucleoside phosphorylase (PNP)/hypoxanthine phosphoribosyltransferase 1 (HPRT1) to reduce hypoxanthine, while osthole/schizandrin A maintained endothelial integrity. CONCLUSION: Aberrant hypoxanthine metabolism induced by unhealthy lifestyles may disrupt neurovascular homeostasis through the liver-brain axis and contribute to the occurrence of brain aging phenotypes. BZBS improves age-related brain dysfunction by targeting hypoxanthine metabolism and protecting endothelial function, representing a promising intervention.
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Methadone alters transcriptional programs associated with synapse formation in human cortical organoids.
Opioid use disorder (OUD) among pregnant women has become an epidemic in the United States. Pharmacological interventions for maternal OUD most commonly involve methadone, a synthetic opioid analgesic that attenuates withdrawal symptoms and behaviors linked with drug addiction. However, evidence of methadone's ability to readily accumulate in neural tissue, and cause long-term neurocognitive sequelae, has led to concerns regarding its effect on prenatal brain development. We utilized human cortical organoid (hCO) technology to probe how this drug impacts the earliest mechanisms of cortico-genesis. Bulk mRNA sequencing of 2-month-old hCOs chronically treated with a clinically relevant dose of 1 μM methadone for 50 days revealed a robust transcriptional response to methadone associated with functional components of the synapse, the underlying extracellular matrix (ECM), and cilia. Co-expression network and predictive protein-protein interaction analyses demonstrated that these changes occurred in concert, centered around a regulatory axis of growth factors, developmental signaling pathways, and matricellular proteins (MCPs). TGFβ1 was identified as an upstream regulator of this network and appeared as part of a highly interconnected cluster of MCPs, of which thrombospondin 1 (TSP1) was most prominently downregulated and exhibited dose-dependent reductions in protein levels. These results demonstrate that methadone exposure during early cortical development alters transcriptional programs associated with synaptogenesis, and that these changes arise by functionally modulating extra-synaptic molecular mechanisms in the ECM and cilia. Our findings provide novel insight into the molecular underpinnings of methadone's putative effect on cognitive and behavioral development and a basis for improving interventions for maternal opioid addiction.
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Isotonic and minimally invasive optical clearing media for live cell imaging ex vivo and in vivo.
Tissue clearing has been widely used for fluorescence imaging of fixed tissues, but its application to live tissues has been limited by toxicity. Here we develop minimally invasive optical clearing media for fluorescence imaging of live mammalian tissues. Light scattering is minimized by adding spherical polymers with low osmolarity to the extracellular medium. A clearing medium containing bovine serum albumin (SeeDB-Live) is compatible with live cells, enabling structural and functional imaging of live tissues, such as spheroids, organoids, acute brain slices and the mouse brains in vivo. SeeDB-Live minimally affects neuronal electrophysiological properties and sensory responses in vivo, and facilitates fluorescence imaging of deep cortical layers in live animals without detectable toxicity to neurons or behavior. We further demonstrate its utility to epifluorescence voltage imaging in acute brain slices and in vivo preparations. Thus, SeeDB-Live expands both the depth and modality range of fluorescence imaging in live mammalian tissues.
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Persistent type I interferon signaling within the brain of people with HIV on ART with cognitive impairment.
To better understand the molecular mechanism that drives neuroinflammation, we analyzed the protein profiles of 27 brains from HIV with HIV (PWH) on antiretroviral therapy (ART), including various stages of HIV-associated neurocognitive disorders (HAND), and compared them to 9 HAND-negative controls. We found that most of the proteins that were increased-about 66.7%-were involved in immune response pathways. Of these, 23.3% were specifically related to type I interferon (IFN-I) signaling, which remains active in the brain through both HIV-related and unrelated mechanisms. Using single-cell RNA sequencing (scRNA-seq) on brain tissues collected during rapid autopsies from participants in the Last Gift cohort, we found that IFN-I signaling was especially strong in astrocytes, microglia (MG), and endothelial cells. In a mini-brain organoid model of acute HIV infection, IFN-I signaling was also highly active in astrocytes but less so in MG. Interestingly, IFN-I activation can happen without HIV being present-expression of human endogenous retrovirus-W1 (HERV-W1) Env can directly trigger this response in astrocytes, and it continues in glial cells even with effective ART. Together, our findings point to persistent IFN-I activation in glial and endothelial cells in the brain, which may contribute to neuroinflammation and cognitive disorders in PWH on ART.
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Engineering induction of singular neural rosette emergence within hPSC-derived tissues.
Human pluripotent stem cell (hPSC)-derived neural organoids display unprecedented emergent properties. Yet in contrast to the singular neuroepithelial tube from which the entire central nervous system (CNS) develops in vivo, current organoid protocols yield tissues with multiple neuroepithelial units, a.k.a. neural rosettes, each acting as independent morphogenesis centers and thereby confounding coordinated, reproducible tissue development. Here, we discover that controlling initial tissue morphology can effectively (>80%) induce single neural rosette emergence within hPSC-derived forebrain and spinal tissues. Notably, the optimal tissue morphology for observing singular rosette emergence was distinct for forebrain versus spinal tissues due to previously unknown differences in ROCK-mediated cell contractility. Following release of geometric confinement, the tissues displayed radial outgrowth with maintenance of a singular neuroepithelium and peripheral neuronal differentiation. Thus, we have identified neural tissue morphology as a critical biophysical parameter for controlling in vitro neural tissue morphogenesis furthering advancement towards biomanufacture of CNS tissues with biomimetic anatomy and physiology.
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In vivo mapping of cellular resolution neuropathology in brain ischemia with diffusion MRI.
Noninvasive mapping of cellular pathology can provide critical diagnostic and prognostic information. Recent advances in diffusion magnetic resonance imaging enabled in vivo examination of tissue microstructures well beyond the imaging resolution. Here, we proposed to use diffusion time-dependent diffusion kurtosis imaging (tDKI) to simultaneously assess cellular morphology and transmembrane permeability in hypoxic-ischemic (HI) brain injury. Through numerical simulations and organoid imaging, we demonstrated the feasibility of capturing effective size and permeability changes using tDKI. In vivo MRI of HI-injured mouse brains detected a shift of the tDKI peak to longer diffusion times, suggesting swelling of the cellular processes. Furthermore, we observed a faster decrease of the tDKI tail, reflecting increased transmembrane permeability associated with up-regulated water exchange or necrosis. Such information, unavailable from a single diffusion time, can predict salvageable tissues. Preliminary applications of tDKI in patients with ischemic stroke suggested increased transmembrane permeability in stroke regions, illustrating tDKI's potential for detecting pathological changes in the clinics.
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De novo genes with an lncRNA origin encode unique human brain developmental functionality.
Human de novo genes can originate from neutral long non-coding RNA (lncRNA) loci and are evolutionarily significant in general, yet how and why this all-or-nothing transition to functionality happens remains unclear. Here, in 74 human/hominoid-specific de novo genes, we identified distinctive U1 elements and RNA splice-related sequences accounting for RNA nuclear export, differentiating mRNAs from lncRNAs, and driving the origin of de novo genes from lncRNA loci. The polymorphic sites facilitating the lncRNA-mRNA conversion through regulating nuclear export are selectively constrained, maintaining a boundary that differentiates mRNAs from lncRNAs. The functional new genes actively passing through it thus showed a mode of pre-adaptive origin, in that they acquire functions along with the achievement of their coding potential. As a proof of concept, we verified the regulations of splicing and U1 recognition on the nuclear export efficiency of one of these genes, the ENSG00000205704, in human neural progenitor cells. Notably, knock-out or over-expression of this gene in human embryonic stem cells accelerates or delays the neuronal maturation of cortical organoids, respectively. The transgenic mice with ectopically expressed ENSG00000205704 showed enlarged brains with cortical expansion. We thus demonstrate the key roles of nuclear export in de novo gene origin. These newly originated genes should reflect the novel uniqueness of human brain development.
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Cerebellar Differentiation from Human Stem Cells Through Retinoid, Wnt, and Sonic Hedgehog Pathways.
Differentiating cerebellar organoids can be challenging due to complex cell organization and structure in the cerebellum. Different approaches were investigated to recapitulate differentiation process of the cerebellum from human-induced pluripotent stem cells (hiPSCs) without high efficiency. This study was carried out to test the hypothesis that the combination of different signaling factors including retinoic acid (RA), Wnt activator, and sonic hedgehog (SHH) activator promotes the cerebellar differentiation of hiPSCs. Wnt, RA, and SHH pathways were activated by CHIR99021 (CHIR), RA, and purmorphamine (PMR), respectively. Different combinations of the morphogens (RA/CHIR, RA/PMR, CHIR/PMR, and RA/CHIR/PMR) were utilized, and the spheroids (day 35) were characterized for the markers of three cerebellum layers (the molecular layer, the Purkinje cell layer, and the granule cell layer). Of all the combinations tested, RA/CHIR/PMR promoted both the Purkinje cell layer and the granule cell layer differentiation. The cells also exhibited electrophysiological characteristics using whole-cell patch clamp recording, especially demonstrating Purkinje cell electrophysiology. This study should advance the understanding of different signaling pathways during cerebellar development to engineer cerebellum organoids for drug screening and disease modeling. Impact statement This study investigated the synergistic effects of retinoic acid, Wnt activator, and sonic hedgehog activator on cerebellar patterning of human-induced pluripotent stem cell (hiPSC) spheroids and organoids. The results indicate that the combination promotes the differentiation of the Purkinje cell layer and the granule cell layer. The cells also exhibit electrophysiological characteristics using whole-cell patch clamp recording, especially demonstrating Purkinje cell electrophysiology. The findings are significant for understanding the biochemical signaling of three-dimensional microenvironment on neural patterning of hiPSCs for applications in organoid engineering, disease modeling, and drug screening.