Laser Cell Ablation in Intact Drosophila Larvae Reveals Synaptic Competition.

The protocol describes single-neuron ablation with a 2-photon laser system in the central nervous system (CNS) of intact Drosophila melanogaster larvae. Using this non-invasive method, the developing nervous system can be manipulated in a cell-specific manner. Disrupting the development of individual neurons in a network can be used to study how the nervous system can compensate for the loss of synaptic input. Individual neurons were specifically ablated in the giant fiber system of Drosophila, with a focus on two neurons: the presynaptic giant fiber (GF) and the postsynaptic tergotrochanteral motor neuron (TTMn). The GF synapses with the ipsilateral TTMn, which is crucial to the escape response. Ablating one of the GFs in the 3rd instar brain, just after the GF starts axonal growth, permanently removes the cell during the development of the CNS. The remaining GF reacts to the absent neighbor and forms an ectopic synaptic terminal to the contralateral TTMn. This atypical, bilaterally symmetric terminal innervates both TTMns, as demonstrated by dye coupling, and drives both motor neurons, as demonstrated by electrophysiological assays. In summary, the ablation of a single interneuron demonstrates synaptic competition between a bilateral pair of neurons that can compensate for the loss of one neuron and restore normal responses to the escape circuit.

Indirect neurogenesis in space and time.

During central nervous system (CNS) development, neural progenitor cells (NPCs) generate neurons and glia in two different ways. In direct neurogenesis, daughter cells differentiate directly into neurons or glia, whereas in indirect neurogenesis, neurons or glia are generated after one or more daughter cell divisions. Intriguingly, indirect neurogenesis is not stochastically deployed and plays instructive roles during CNS development: increased generation of cells from specific lineages; increased generation of early or late-born cell types within a lineage; and increased cell diversification. Increased indirect neurogenesis might contribute to the anterior CNS expansion evident throughout the Bilateria and help to modify brain-region size without requiring increased NPC numbers or extended neurogenesis. Increased indirect neurogenesis could be an evolutionary driver of the gyrencephalic (that is, folded) cortex that emerged during mammalian evolution and might even have increased during hominid evolution. Thus, selection of indirect versus direct neurogenesis provides a powerful developmental and evolutionary instrument that drives not only the evolution of CNS complexity but also brain expansion and modulation of brain-region size, and thereby the evolution of increasingly advanced cognitive abilities. This Review describes indirect neurogenesis in several model species and humans, and highlights some of the molecular genetic mechanisms that control this important process.

Single-cell Profiling of Reprogrammed Human Neural Stem Cells Unveils High Similarity to Neural Progenitors in the Developing Central Nervous System.

BACKGROUND: Similar to induced pluripotent cells (iPSCs), induced neural stem cells (iNSCs) can be directly converted from human somatic cells such as dermal fibroblasts and peripheral blood monocytes. While previous studies have demonstrated the resemblance of iNSCs to neural stem cells derived from primary sources and embryonic stem cells, respectively, a comprehensive analysis of the correlation between iNSCs and their physiological counterparts remained to be investigated. METHODS: Nowadays, single-cell sequencing technologies provide unique opportunities for in-depth cellular benchmarking of complex cell populations. Our study involves the comprehensive profiling of converted human iNSCs at a single-cell transcriptomic level, alongside conventional methods, like flow cytometry and immunofluorescence stainings. RESULTS: Our results show that the iNSC conversion yields a homogeneous cell population expressing bona fide neural stem cell markers. Extracting transcriptomic signatures from published single cell transcriptomic atlas data and comparison to the iNSC transcriptome reveals resemblance to embryonic neuroepithelial cells of early neurodevelopmental stages observed in vivo at 5 weeks of development. CONCLUSION: Our data underscore the physiological relevance of directly converted iNSCs, making them a valuable in vitro system for modeling human central nervous system development and establishing translational applications in cell therapy and compound screening.

Spatial atlas of the mouse central nervous system at molecular resolution.

Spatially charting molecular cell types at single-cell resolution across the 3D volume is critical for illustrating the molecular basis of brain anatomy and functions. Single-cell RNA sequencing has profiled molecular cell types in the mouse brain1,2, but cannot capture their spatial organization. Here we used an in situ sequencing method, STARmap PLUS3,4, to profile 1,022 genes in 3D at a voxel size of 194 × 194 × 345 nm3, mapping 1.09 million high-quality cells across the adult mouse brain and spinal cord. We developed computational pipelines to segment, cluster and annotate 230 molecular cell types by single-cell gene expression and 106 molecular tissue regions by spatial niche gene expression. Joint analysis of molecular cell types and molecular tissue regions enabled a systematic molecular spatial cell-type nomenclature and identification of tissue architectures that were undefined in established brain anatomy. To create a transcriptome-wide spatial atlas, we integrated STARmap PLUS measurements with a published single-cell RNA-sequencing atlas1, imputing single-cell expression profiles of 11,844 genes. Finally, we delineated viral tropisms of a brain-wide transgene delivery tool, AAV-PHP.eB5,6. Together, this annotated dataset provides a single-cell resource that integrates the molecular spatial atlas, brain anatomy and the accessibility to genetic manipulation of the mammalian central nervous system.

Specialized astrocytes mediate glutamatergic gliotransmission in the CNS.

Multimodal astrocyte-neuron communications govern brain circuitry assembly and function1. For example, through rapid glutamate release, astrocytes can control excitability, plasticity and synchronous activity2,3 of synaptic networks, while also contributing to their dysregulation in neuropsychiatric conditions4-7. For astrocytes to communicate through fast focal glutamate release, they should possess an apparatus for Ca2+-dependent exocytosis similar to neurons8-10. However, the existence of this mechanism has been questioned11-13 owing to inconsistent data14-17 and a lack of direct supporting evidence. Here we revisited the astrocyte glutamate exocytosis hypothesis by considering the emerging molecular heterogeneity of astrocytes18-21 and using molecular, bioinformatic and imaging approaches, together with cell-specific genetic tools that interfere with glutamate exocytosis in vivo. By analysing existing single-cell RNA-sequencing databases and our patch-seq data, we identified nine molecularly distinct clusters of hippocampal astrocytes, among which we found a notable subpopulation that selectively expressed synaptic-like glutamate-release machinery and localized to discrete hippocampal sites. Using GluSnFR-based glutamate imaging22 in situ and in vivo, we identified a corresponding astrocyte subgroup that responds reliably to astrocyte-selective stimulations with subsecond glutamate release events at spatially precise hotspots, which were suppressed by astrocyte-targeted deletion of vesicular glutamate transporter 1 (VGLUT1). Furthermore, deletion of this transporter or its isoform VGLUT2 revealed specific contributions of glutamatergic astrocytes in cortico-hippocampal and nigrostriatal circuits during normal behaviour and pathological processes. By uncovering this atypical subpopulation of specialized astrocytes in the adult brain, we provide insights into the complex roles of astrocytes in central nervous system (CNS) physiology and diseases, and identify a potential therapeutic target.

Lactate limits CNS autoimmunity by stabilizing HIF-1α in dendritic cells.

Dendritic cells (DCs) have a role in the development and activation of self-reactive pathogenic T cells1,2. Genetic variants that are associated with the function of DCs have been linked to autoimmune disorders3,4, and DCs are therefore attractive therapeutic targets for such diseases. However, developing DC-targeted therapies for autoimmunity requires identification of the mechanisms that regulate DC function. Here, using single-cell and bulk transcriptional and metabolic analyses in combination with cell-specific gene perturbation studies, we identify a regulatory loop of negative feedback that operates in DCs to limit immunopathology. Specifically, we find that lactate, produced by activated DCs and other immune cells, boosts the expression of NDUFA4L2 through a mechanism mediated by hypoxia-inducible factor 1α (HIF-1α). NDUFA4L2 limits the production of mitochondrial reactive oxygen species that activate XBP1-driven transcriptional modules in DCs that are involved in the control of pathogenic autoimmune T cells. We also engineer a probiotic that produces lactate and suppresses T cell autoimmunity through the activation of HIF-1α-NDUFA4L2 signalling in DCs. In summary, we identify an immunometabolic pathway that regulates DC function, and develop a synthetic probiotic for its therapeutic activation.

Microglia regulate central nervous system myelin growth and integrity.

Myelin is required for the function of neuronal axons in the central nervous system, but the mechanisms that support myelin health are unclear. Although macrophages in the central nervous system have been implicated in myelin health1, it is unknown which macrophage populations are involved and which aspects they influence. Here we show that resident microglia are crucial for the maintenance of myelin health in adulthood in both mice and humans. We demonstrate that microglia are dispensable for developmental myelin ensheathment. However, they are required for subsequent regulation of myelin growth and associated cognitive function, and for preservation of myelin integrity by preventing its degeneration. We show that loss of myelin health due to the absence of microglia is associated with the appearance of a myelinating oligodendrocyte state with altered lipid metabolism. Moreover, this mechanism is regulated through disruption of the TGFß1-TGFßR1 axis. Our findings highlight microglia as promising therapeutic targets for conditions in which myelin growth and integrity are dysregulated, such as in ageing and neurodegenerative disease2,3.

Molecular basis of astrocyte diversity and morphology across the CNS in health and disease.

Astrocytes, a type of glia, are abundant and morphologically complex cells. Here, we report astrocyte molecular profiles, diversity, and morphology across the mouse central nervous system (CNS). We identified shared and region-specific astrocytic genes and functions and explored the cellular origins of their regional diversity. We identified gene networks correlated with astrocyte morphology, several of which unexpectedly contained Alzheimer's disease (AD) risk genes. CRISPR/Cas9-mediated reduction of candidate genes reduced astrocyte morphological complexity and resulted in cognitive deficits. The same genes were down-regulated in human AD, in an AD mouse model that displayed reduced astrocyte morphology, and in other human brain disorders. We thus provide comprehensive molecular data on astrocyte diversity and mechanisms across the CNS and on the molecular basis of astrocyte morphology in health and disease.

Parenchymal border macrophages regulate the flow dynamics of the cerebrospinal fluid.

Macrophages are important players in the maintenance of tissue homeostasis1. Perivascular and leptomeningeal macrophages reside near the central nervous system (CNS) parenchyma2, and their role in CNS physiology has not been sufficiently well studied. Given their continuous interaction with the cerebrospinal fluid (CSF) and strategic positioning, we refer to these cells collectively as parenchymal border macrophages (PBMs). Here we demonstrate that PBMs regulate CSF flow dynamics. We identify a subpopulation of PBMs that express high levels of CD163 and LYVE1 (scavenger receptor proteins), closely associated with the brain arterial tree, and show that LYVE1+ PBMs regulate arterial motion that drives CSF flow. Pharmacological or genetic depletion of PBMs led to accumulation of extracellular matrix proteins, obstructing CSF access to perivascular spaces and impairing CNS perfusion and clearance. Ageing-associated alterations in PBMs and impairment of CSF dynamics were restored after intracisternal injection of macrophage colony-stimulating factor. Single-nucleus RNA sequencing data obtained from patients with Alzheimer's disease (AD) and from non-AD individuals point to changes in phagocytosis, endocytosis and interferon-γ signalling on PBMs, pathways that are corroborated in a mouse model of AD. Collectively, our results identify PBMs as new cellular regulators of CSF flow dynamics, which could be targeted pharmacologically to alleviate brain clearance deficits associated with ageing and AD.

The dysregulation of autophagy and ER stress induced by HHV-6A infection activates pro-inflammatory pathways and promotes the release of inflammatory cytokines and cathepsin S by CNS cells.

HHV-6A is a neurotropic herpesvirus able to infect several CNS cells including astrocytes and primary neurons. Here we found that HHV-6A infection of astrocytoma cells, by reducing autophagy, increased ROS and induced ER stress, promoting the release of inflammatory cytokines such as IL-6 and IL-1ß and activating pathways such as STAT3, NF-kB and mTOR. Moreover, HHV-6A infection increased the production of CXCL13, a B lymphocyte attracting chemokine, whose recruitment in the CNS could further enhance neuroinflammation. Interestingly, HHV-6A also increased the release of cathepsin S by infected astrocytoma cells as well as by primary neurons. As this enzyme is involved in the degradation of MBP, this effect could contribute to the onset/progression of MS, a neurodegenerative disease that, besides inflammation, is characterized by a progressive demyelination process. In conclusion, this study unveils new molecular mechanisms through which HHV-6A may promote important aspects involved in several neurodegenerative diseases.

Oligodendroglia heterogeneity in the human central nervous system.

It is the centenary of the discovery of oligodendrocytes and we are increasingly aware of their importance in the functioning of the brain in development, adult learning, normal ageing and in disease across the life course, even in those diseases classically thought of as neuronal. This has sparked more interest in oligodendroglia for potential therapeutics for many neurodegenerative/neurodevelopmental diseases due to their more tractable nature as a renewable cell in the central nervous system. However, oligodendroglia are not all the same. Even from the first description, differences in morphology were described between the cells. With advancing techniques to describe these differences in human tissue, the complexity of oligodendroglia is being discovered, indicating apparent functional differences which may be of critical importance in determining vulnerability and response to disease, and targeting of potential therapeutics. It is timely to review the progress we have made in discovering and understanding oligodendroglial heterogeneity in health and neuropathology.

Emerging roles for CNS fibroblasts in health, injury and disease.

Recent transcriptomic, histological and functional studies have begun to shine light on the fibroblasts present in the meninges, choroid plexus and perivascular spaces of the brain and spinal cord. Although the origins and functions of CNS fibroblasts are still being described, it is clear that they represent a distinct cell population, or populations, that have likely been confused with other cell types on the basis of the expression of overlapping cellular markers. Recent work has revealed that fibroblasts play crucial roles in fibrotic scar formation in the CNS after injury and inflammation, which have also been attributed to other perivascular cell types such as pericytes and vascular smooth muscle cells. In this Review, we describe the current knowledge of the location and identity of CNS perivascular cell types, with a particular focus on CNS fibroblasts, including their origin, subtypes, roles in health and disease, and future areas for study.

Neuronal exosome proteins: novel biomarkers for predicting neonatal response to therapeutic hypothermia.

OBJECTIVE: Central nervous system (CNS) derived exosomes can be purified from peripheral blood and have been used widely in adult neurological disease. Application to neonatal neurological disease deserves investigation in the setting of hypoxic-ischaemic encephalopathy (HIE). DESIGN: Observational cohort. SETTING: Level III neonatal intensive care unit. PARTICIPANTS: Term/near-term neonates undergoing therapeutic hypothermia (TH) for HIE. INTERVENTIONS: Blood samples were collected at 0-6, 12, 24, 48 and 96 hours of life. MAIN OUTCOMES AND MEASURES: CNS exosomes were purified from serum using previously described methods. Biomarker protein levels were quantified using standard ELISA methods and normalised to exosome marker CD-81. The slope of change for biomarker levels was calculated for each time interval. Our primary outcome was MRI basal ganglia/watershed score of ≥3. RESULTS: 26 subjects were included (umbilical artery pH range 6.6-7.29; 35% seizures). An increasing MRI injury score was significantly associated with decreasing levels of synaptopodin between 0-6 and 12 hours (p=0.03) and increasing levels of lipocalin-2 (NGAL) between 12 and 48 hours (p<0.0001). Neuronal pentraxin was not significant. The negative predictive values for increasing synaptopodin and decreasing NGAL was 70.0% and 90.9%, respectively. CONCLUSIONS AND RELEVANCE: Our results indicate that CNS exosome cargo has the potential to act as biomarkers of the severity of brain injury and response to TH as well as quantify pharmacological response to neuroactive therapeutic/adjuvant agents. Rigorous prospective trials are critical to evaluate potential clinical use of exosome biomarkers.

Glial Cells as Possible Targets of Neuroprotection through Neurotrophic and Antioxidative Molecules in the Central and Enteric Nervous Systems in Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. The loss of nigrostriatal dopaminergic neurons produces its characteristic motor symptoms, but PD patients also have non-motor symptoms such as constipation and orthostatic hypotension. The pathological hallmark of PD is the presence of α-synuclein-containing Lewy bodies and neurites in the brain. However, the PD pathology is observed in not only the central nervous system (CNS) but also in parts of the peripheral nervous system such as the enteric nervous system (ENS). Since constipation is a typical prodromal non-motor symptom in PD, often preceding motor symptoms by 10-20 years, it has been hypothesized that PD pathology propagates from the ENS to the CNS via the vagal nerve. Discovery of pharmacological and other methods to halt this progression of neurodegeneration in PD has the potential to improve millions of lives. Astrocytes protect neurons in the CNS by secretion of neurotrophic and antioxidative factors. Similarly, astrocyte-like enteric glial cells (EGCs) are known to secrete neuroprotective factors in the ENS. In this article, we summarize the neuroprotective function of astrocytes and EGCs and discuss therapeutic strategies for the prevention of neurodegeneration in PD targeting neurotrophic and antioxidative molecules in glial cells.

Complex Organ Construction from Human Pluripotent Stem Cells for Biological Research and Disease Modeling with New Emerging Techniques.

Human pluripotent stem cells (hPSCs) are grouped into two cell types; embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). hESCs have provided multiple powerful platforms to study human biology, including human development and diseases; however, there were difficulties in the establishment of hESCs from human embryo and concerns over its ethical issues. The discovery of hiPSCs has expanded to various applications in no time because hiPSCs had already overcome these problems. Many hPSC-based studies have been performed using two-dimensional monocellular culture methods at the cellular level. However, in many physiological and pathophysiological conditions, intra- and inter-organ interactions play an essential role, which has hampered the establishment of an appropriate study model. Therefore, the application of recently developed technologies, such as three-dimensional organoids, bioengineering, and organ-on-a-chip technology, has great potential for constructing multicellular tissues, generating the functional organs from hPSCs, and recapitulating complex tissue functions for better biological research and disease modeling. Moreover, emerging techniques, such as single-cell transcriptomics, spatial transcriptomics, and artificial intelligence (AI) allowed for a denser and more precise analysis of such heterogeneous and complex tissues. Here, we review the applications of hPSCs to construct complex organs and discuss further prospects of disease modeling and drug discovery based on these PSC-derived organs.

Human iPSC-Derived Glia as a Tool for Neuropsychiatric Research and Drug Development.

Neuropsychiatric disorders such as schizophrenia or autism spectrum disorder represent a leading and growing burden on worldwide mental health. Fundamental lack in understanding the underlying pathobiology compromises efficient drug development despite the immense medical need. So far, antipsychotic drugs reduce symptom severity and enhance quality of life, but there is no cure available. On the molecular level, schizophrenia and autism spectrum disorders correlate with compromised neuronal phenotypes. There is increasing evidence that aberrant neuroinflammatory responses of glial cells account for synaptic pathologies through deregulated communication and reciprocal modulation. Consequently, microglia and astrocytes emerge as central targets for anti-inflammatory treatment to preserve organization and homeostasis of the central nervous system. Studying the impact of neuroinflammation in the context of neuropsychiatric disorders is, however, limited by the lack of relevant human cellular test systems that are able to represent the dynamic cellular processes and molecular changes observed in human tissue. Today, patient-derived induced pluripotent stem cells offer the opportunity to study neuroinflammatory mechanisms in vitro that comprise the genetic background of affected patients. In this review, we summarize the major findings of iPSC-based microglia and astrocyte research in the context of neuropsychiatric diseases and highlight the benefit of 2D and 3D co-culture models for the generation of efficient in vitro models for target screening and drug development.

Microglia: Immune and non-immune functions.

As resident macrophages of the central nervous system (CNS), microglia are associated with diverse functions essential to the developing and adult brain during homeostasis and disease. They are aided in their tasks by intricate bidirectional communication with other brain cells under steady-state conditions as well as with infiltrating peripheral immune cells during perturbations. Harmonious cell-cell communication involving microglia are considered crucial to maintain the healthy state of the tissue environment and to overcome pathology such as neuroinflammation. Analyses of such intercellular pathways have contributed to our understanding of the heterogeneous but context-associated microglial responses to environmental cues across neuropathology, including inflammatory conditions such as infections and autoimmunity, as well as immunosuppressive states as seen in brain tumors. Here, we summarize the latest evidence demonstrating how these interactions drive microglia immune and non-immune functions, which coordinate the transition from homeostatic to disease-related cellular states.

Mechanisms governing activity-dependent synaptic pruning in the developing mammalian CNS.

Almost 60 years have passed since the initial discovery by Hubel and Wiesel that changes in neuronal activity can elicit developmental rewiring of the central nervous system (CNS). Over this period, we have gained a more comprehensive picture of how both spontaneous neural activity and sensory experience-induced changes in neuronal activity guide CNS circuit development. Here we review activity-dependent synaptic pruning in the mammalian CNS, which we define as the removal of a subset of synapses, while others are maintained, in response to changes in neural activity in the developing nervous system. We discuss the mounting evidence that immune and cell-death molecules are important mechanistic links by which changes in neural activity guide the pruning of specific synapses, emphasizing the role of glial cells in this process. Finally, we discuss how these developmental pruning programmes may go awry in neurodevelopmental disorders of the human CNS, focusing on autism spectrum disorder and schizophrenia. Together, our aim is to give an overview of how the field of activity-dependent pruning research has evolved, led to exciting new questions and guided the identification of new, therapeutically relevant mechanisms that result in aberrant circuit development in neurodevelopmental disorders.

Cholinesterase Research.

Cholinesterases are fundamental players in the peripheral and central nervous systems [...].