Toward an understanding of glucose metabolism in radial glial biology and brain development.

Decades of research have sought to determine the intrinsic and extrinsic mechanisms underpinning the regulation of neural progenitor maintenance and differentiation. A series of precise temporal transitions within progenitor cell populations generates all the appropriate neural cell types while maintaining a pool of self-renewing progenitors throughout embryogenesis. Recent technological advances have enabled us to gain new insights at the single-cell level, revealing an interplay between metabolic state and developmental progression that impacts the timing of proliferation and neurogenesis. This can have long-term consequences for the developing brain's neuronal specification, maturation state, and organization. Furthermore, these studies have highlighted the need to reassess the instructive role of glucose metabolism in determining progenitor cell division, differentiation, and fate. This review focuses on glucose metabolism (glycolysis) in cortical progenitor cells and the emerging focus on glycolysis during neurogenic transitions. Furthermore, we discuss how the field can learn from other biological systems to improve our understanding of the spatial and temporal changes in glycolysis in progenitors and evaluate functional neurological outcomes.

Profiling human brain vascular cells using single-cell transcriptomics and organoids.

Angiogenesis and neurogenesis are functionally interconnected during brain development. However, the study of the vasculature has trailed other brain cell types because they are delicate and of low abundance. Here we describe a protocol extension to purify prenatal human brain endothelial and mural cells with FACS and utilize them in downstream applications, including transcriptomics, culture and organoid transplantation. This approach is simple, efficient and generates high yields from small amounts of tissue. When the experiment is completed within a 24 h postmortem interval, these healthy cells produce high-quality data in single-cell transcriptomics experiments. These vascular cells can be cultured, passaged and expanded for many in vitro assays, including Matrigel vascular tube formation, microfluidic chambers and metabolic measurements. Under these culture conditions, primary vascular cells maintain expression of cell-type markers for at least 3 weeks. Finally, we describe how to use primary vascular cells for transplantation into cortical organoids, which captures key features of neurovascular interactions in prenatal human brain development. In terms of timing, tissue processing and staining requires ~3 h, followed by an additional 3 h of FACS. The transplant procedure of primary, FACS-purified vascular cells into cortical organoids requires an additional 2 h. The time required for different transcriptomic and epigenomic protocols can vary based on the specific application, and we offer strategies to mitigate batch effects and optimize data quality. In sum, this vasculo-centric approach offers an integrated platform to interrogate neurovascular interactions and human brain vascular development.

A single-cell multi-omic atlas spanning the adult rhesus macaque brain.

Cataloging the diverse cellular architecture of the primate brain is crucial for understanding cognition, behavior, and disease in humans. Here, we generated a brain-wide single-cell multimodal molecular atlas of the rhesus macaque brain. Together, we profiled 2.58 M transcriptomes and 1.59 M epigenomes from single nuclei sampled from 30 regions across the adult brain. Cell composition differed extensively across the brain, revealing cellular signatures of region-specific functions. We also identified 1.19 M candidate regulatory elements, many previously unidentified, allowing us to explore the landscape of cis-regulatory grammar and neurological disease risk in a cell type-specific manner. Altogether, this multi-omic atlas provides an open resource for investigating the evolution of the human brain and identifying novel targets for disease interventions.

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.

Astroglial toxicity promotes synaptic degeneration in the thalamocortical circuit in frontotemporal dementia with GRN mutations.

Mutations in the human progranulin (GRN) gene are a leading cause of frontotemporal lobar degeneration (FTLD). While previous studies implicate aberrant microglial activation as a disease-driving factor in neurodegeneration in the thalamocortical circuit in Grn-/- mice, the exact mechanism for neurodegeneration in FTLD-GRN remains unclear. By performing comparative single-cell transcriptomics in the thalamus and frontal cortex of Grn-/- mice and patients with FTLD-GRN, we have uncovered a highly conserved astroglial pathology characterized by upregulation of gap junction protein GJA1, water channel AQP4, and lipid-binding protein APOE, and downregulation of glutamate transporter SLC1A2 that promoted profound synaptic degeneration across the two species. This astroglial toxicity could be recapitulated in mouse astrocyte-neuron cocultures and by transplanting induced pluripotent stem cell-derived astrocytes to cortical organoids, where progranulin-deficient astrocytes promoted synaptic degeneration, neuronal stress, and TDP-43 proteinopathy. Together, these results reveal a previously unappreciated astroglial pathology as a potential key mechanism in neurodegeneration in FTLD-GRN.

Approaches to investigating metabolism in human neurodevelopment using organoids: insights from intestinal and cancer studies.

Interrogating the impact of metabolism during development is important for understanding cellular and tissue formation, organ and systemic homeostasis, and dysregulation in disease states. To evaluate the vital functions metabolism coordinates during human brain development and disease, pluripotent stem cell-derived models, such as organoids, provide tractable access to neurodevelopmental processes. Despite many strengths of neural organoid models, the extent of their replication of endogenous metabolic programs is currently unclear and requires direct investigation. Studies in intestinal and cancer organoids that functionally evaluate dynamic bioenergetic changes provide a framework that can be adapted for the study of neural metabolism. Validation of in vitro models remains a significant challenge; investigation using in vivo models and primary tissue samples is required to improve our in vitro model systems and, concomitantly, improve our understanding of human development.

Ensembles of endothelial and mural cells promote angiogenesis in prenatal human brain.

Interactions between angiogenesis and neurogenesis regulate embryonic brain development. However, a comprehensive understanding of the stages of vascular cell maturation is lacking, especially in the prenatal human brain. Using fluorescence-activated cell sorting, single-cell transcriptomics, and histological and ultrastructural analyses, we show that an ensemble of endothelial and mural cell subtypes tile the brain vasculature during the second trimester. These vascular cells follow distinct developmental trajectories and utilize diverse signaling mechanisms, including collagen, laminin, and midkine, to facilitate cell-cell communication and maturation. Interestingly, our results reveal that tip cells, a subtype of endothelial cells, are highly enriched near the ventricular zone, the site of active neurogenesis. Consistent with these observations, prenatal vascular cells transplanted into cortical organoids exhibit restricted lineage potential that favors tip cells, promotes neurogenesis, and reduces cellular stress. Together, our results uncover important mechanisms into vascular maturation during this critical period of human brain development.

How mechanisms of stem cell polarity shape the human cerebral cortex.

Apical-basal progenitor cell polarity establishes key features of the radial and laminar architecture of the developing human cortex. The unique diversity of cortical stem cell populations and an expansion of progenitor population size in the human cortex have been mirrored by an increase in the complexity of cellular processes that regulate stem cell morphology and behaviour, including their polarity. The study of human cells in primary tissue samples and human stem cell-derived model systems (such as cortical organoids) has provided insight into these processes, revealing that protein complexes regulate progenitor polarity by controlling cell membrane adherence within appropriate cortical niches and are themselves regulated by cytoskeletal proteins, signalling molecules and receptors, and cellular organelles. Studies exploring how cortical stem cell polarity is established and maintained are key for understanding the features of human brain development and have implications for neurological dysfunction.

Tropism of SARS-CoV-2 for human cortical astrocytes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. Neurological symptoms, which range in severity, accompany as many as one-third of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized stem-cell-derived cortical organoids as well as primary human cortical tissue, both from developmental and adult stages. We find significant and predominant infection in cortical astrocytes in both primary tissue and organoid cultures, with minimal infection of other cortical populations. Infected and bystander astrocytes have a corresponding increase in inflammatory gene expression, reactivity characteristics, increased cytokine and growth factor signaling, and cellular stress. Although human cortical cells, particularly astrocytes, have no observable ACE2 expression, we find high levels of coronavirus coreceptors in infected astrocytes, including CD147 and DPP4. Decreasing coreceptor abundance and activity reduces overall infection rate, and increasing expression is sufficient to promote infection. Thus, we find tropism of SARS-CoV-2 for human astrocytes resulting in inflammatory gliosis-type injury that is dependent on coronavirus coreceptors.

Challenges of Organoid Research.

Organoids are 3D cell culture systems derived from human pluripotent stem cells that contain tissue resident cell types and reflect features of early tissue organization. Neural organoids are a particularly innovative scientific advance given the lack of accessibility of developing human brain tissue and intractability of neurological diseases. Neural organoids have become an invaluable approach to model features of human brain development that are not well reflected in animal models. Organoids also hold promise for the study of atypical cellular, molecular, and genetic features that underscore neurological diseases. Additionally, organoids may provide a platform for testing therapeutics in human cells and are a potential source for cell replacement approaches to brain injury or disease. Despite the promising features of organoids, their broad utility is tempered by a variety of limitations yet to be overcome, including lack of high-fidelity cell types, limited maturation, atypical physiology, and lack of arealization, features that may limit their reliability for certain applications.

Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. It proves fatal for one percent of those infected. Neurological symptoms, which range in severity, accompany a significant proportion of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized primary human cortical tissue and stem cell-derived cortical organoids. We find significant and predominant infection in cortical astrocytes in both primary and organoid cultures, with minimal infection of other cortical populations. Infected astrocytes had a corresponding increase in reactivity characteristics, growth factor signaling, and cellular stress. Although human cortical cells, including astrocytes, have minimal ACE2 expression, we find high levels of alternative coronavirus receptors in infected astrocytes, including DPP4 and CD147. Inhibition of DPP4 reduced infection and decreased expression of the cell stress marker, ARCN1. We find tropism of SARS-CoV-2 for human astrocytes mediated by DPP4, resulting in reactive gliosis-type injury.

Are Organoids Ready for Prime Time?

Innovations in organoid-based models of human tissues have made them an exciting experimental platform for studying development and disease. However, these models require systematic benchmarking against primary tissue to establish their value. We discuss key parameters that impact the utility of organoid models, primarily focusing on cerebral organoids as examples.

mTOR signaling regulates the morphology and migration of outer radial glia in developing human cortex.

Outer radial glial (oRG) cells are a population of neural stem cells prevalent in the developing human cortex that contribute to its cellular diversity and evolutionary expansion. The mammalian Target of Rapamycin (mTOR) signaling pathway is active in human oRG cells. Mutations in mTOR pathway genes are linked to a variety of neurodevelopmental disorders and malformations of cortical development. We find that dysregulation of mTOR signaling specifically affects oRG cells, but not other progenitor types, by changing the actin cytoskeleton through the activity of the Rho-GTPase, CDC42. These effects change oRG cellular morphology, migration, and mitotic behavior, but do not affect proliferation or cell fate. Thus, mTOR signaling can regulate the architecture of the developing human cortex by maintaining the cytoskeletal organization of oRG cells and the radial glia scaffold. Our study provides insight into how mTOR dysregulation may contribute to neurodevelopmental disease.

Cell stress in cortical organoids impairs molecular subtype specification.

Cortical organoids are self-organizing three-dimensional cultures that model features of the developing human cerebral cortex1,2. However, the fidelity of organoid models remains unclear3-5. Here we analyse the transcriptomes of individual primary human cortical cells from different developmental periods and cortical areas. We find that cortical development is characterized by progenitor maturation trajectories, the emergence of diverse cell subtypes and areal specification of newborn neurons. By contrast, organoids contain broad cell classes, but do not recapitulate distinct cellular subtype identities and appropriate progenitor maturation. Although the molecular signatures of cortical areas emerge in organoid neurons, they are not spatially segregated. Organoids also ectopically activate cellular stress pathways, which impairs cell-type specification. However, organoid stress and subtype defects are alleviated by transplantation into the mouse cortex. Together, these datasets and analytical tools provide a framework for evaluating and improving the accuracy of cortical organoids as models of human brain development.

Human brain development through the lens of cerebral organoid models.

The brain is one of the most complex organs in the body, which emerges from a relatively simple set of basic 'building blocks' during early development according to complex cellular and molecular events orchestrated through a set of inherited instructions. Innovations in stem cell technologies have enabled modelling of neural cells using two- and three-dimensional cultures. In particular, cerebral ('brain') organoids have taken the center stage of brain development models that have the potential for providing meaningful insight into human neurodevelopmental and neurological disorders. We review the current understanding of cellular events during human brain organogenesis, and the events occurring during cerebral organoid differentiation. We highlight the strengths and weaknesses of this experimental model system. In particular, we explain evidence that organoids can mimic many aspects of early neural development, including neural induction, patterning, and broad neurogenesis and gliogenesis programs, offering the opportunity to study genetic regulation of these processes in a human context. Several shortcomings of the current culture methods limit the utility of cerebral organoids to spontaneously give rise to many important cell types, and to model higher order features of tissue organization. We suggest that future studies aim to improve these features in order to make them better models for the study of laminar organization, circuit formation and how disruptions of these processes relate to disease.

New perspectives on the mechanisms establishing the dorsal-ventral axis of the spinal cord.

Distinct classes of neurons arise at different positions along the dorsal-ventral axis of the spinal cord leading to spinal neurons being segregated along this axis according to their physiological properties and functions. Thus, the neurons associated with motor control are generally located in, or adjacent to, the ventral horn whereas the interneurons (INs) that mediate sensory activities are present within the dorsal horn. Here, we review classic and recent studies examining the developmental mechanisms that establish the dorsal-ventral axis in the embryonic spinal cord. Intriguingly, while the cellular organization of the dorsal and ventral halves of the spinal cord looks superficially similar during early development, the underlying molecular mechanisms that establish dorsal vs ventral patterning are markedly distinct. For example, the ventral spinal cord is patterned by the actions of a single growth factor, sonic hedgehog (Shh) acting as a morphogen, i.e., concentration-dependent signal. Recent studies have shed light on the mechanisms by which the spatial and temporal gradient of Shh is transduced by cells to elicit the generation of different classes of ventral INs, and motor neurons (MNs). In contrast, the dorsal spinal cord is patterned by the action of multiple factors, most notably by members of the bone morphogenetic protein (BMP) and Wnt families. While less is known about dorsal patterning, recent studies have suggested that the BMPs do not act as morphogens to specify dorsal IN identities as previously proposed, rather each BMP has signal-specific activities. Finally, we consider the promise that elucidation of these mechanisms holds for neural repair.

Establishing Cerebral Organoids as Models of Human-Specific Brain Evolution.

Direct comparisons of human and non-human primate brains can reveal molecular pathways underlying remarkable specializations of the human brain. However, chimpanzee tissue is inaccessible during neocortical neurogenesis when differences in brain size first appear. To identify human-specific features of cortical development, we leveraged recent innovations that permit generating pluripotent stem cell-derived cerebral organoids from chimpanzee. Despite metabolic differences, organoid models preserve gene regulatory networks related to primary cell types and developmental processes. We further identified 261 differentially expressed genes in human compared to both chimpanzee organoids and macaque cortex, enriched for recent gene duplications, and including multiple regulators of PI3K-AKT-mTOR signaling. We observed increased activation of this pathway in human radial glia, dependent on two receptors upregulated specifically in human: INSR and ITGB8. Our findings establish a platform for systematic analysis of molecular changes contributing to human brain development and evolution.

BMPs direct sensory interneuron identity in the developing spinal cord using signal-specific not morphogenic activities.

The Bone Morphogenetic Protein (BMP) family reiteratively signals to direct disparate cellular fates throughout embryogenesis. In the developing dorsal spinal cord, multiple BMPs are required to specify sensory interneurons (INs). Previous studies suggested that the BMPs act as concentration-dependent morphogens to direct IN identity, analogous to the manner in which sonic hedgehog patterns the ventral spinal cord. However, it remains unresolved how multiple BMPs would cooperate to establish a unified morphogen gradient. Our studies support an alternative model: BMPs have signal-specific activities directing particular IN fates. Using chicken and mouse models, we show that the identity, not concentration, of the BMP ligand directs distinct dorsal identities. Individual BMPs promote progenitor patterning or neuronal differentiation by their activation of different type I BMP receptors and distinct modulations of the cell cycle. Together, this study shows that a 'mix and match' code of BMP signaling results in distinct classes of sensory INs.

A comparison of progestins within three classes: Differential effects on learning and memory in the aging surgically menopausal rat.

INTRODUCTION: For decades, progestins have been included in hormone therapies (HT) prescribed to women to offset the risk of unopposed estrogen-induced endometrial hyperplasia. However, the potential effects on cognition of subcategories of clinically used progestins have been largely unexplored. METHODS: In two studies, the present investigation evaluated the cognitive effects of norethindrone acetate (NETA), levonorgestrel (LEVO), and medroxyprogesterone acetate (MPA) on the water radial-arm maze (WRAM) and Morris water maze (MM) in middle-aged ovariectomized rats. RESULTS: In Study 1, six-weeks of a high-dose NETA treatment impaired learning and delayed retention on the WRAM, and impaired reference memory on the MM. Low-dose NETA treatment impaired delayed retention on the WRAM. In Study 2, high-dose NETA treatment was reduced to four-weeks and compared to MPA and LEVO. As previously shown, MPA impaired working memory performance during the lattermost portion of testing, at the highest working memory load, impaired delayed retention on the WRAM, and impaired reference memory on the MM. NETA also impaired performance on these WRAM and MM measures. Interestingly, LEVO did not impair performance, but instead enhanced learning on the WRAM. CONCLUSIONS: The current study corroborates previous evidence that the most commonly prescribed FDA-approved progestin for HT, MPA, impairs learning and memory in the ovariectomized middle-aged rat. When progestins from two different additional subcategories were investigated, NETA impaired learning and memory similarly to MPA, but LEVO enhanced learning. Future research is warranted to determine LEVO's potential as an ideal progestin for optimal health in women, including for cognition.