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1.
Cell Rep ; 43(8): 114637, 2024 Aug 27.
Article in English | MEDLINE | ID: mdl-39154337

ABSTRACT

Reactive changes of glial cells during neuroinflammation impact brain disorders and disease progression. Elucidating the mechanisms that control reactive gliosis may help us to understand brain pathophysiology and improve outcomes. Here, we report that adult ablation of autism spectrum disorder (ASD)-associated CHD8 in astrocytes attenuates reactive gliosis via remodeling chromatin accessibility, changing gene expression. Conditional Chd8 deletion in astrocytes, but not microglia, suppresses reactive gliosis by impeding astrocyte proliferation and morphological elaboration. Astrocyte Chd8 ablation alleviates lipopolysaccharide-induced neuroinflammation and septic-associated hypothermia in mice. Astrocytic CHD8 plays an important role in neuroinflammation by altering the chromatin landscape, regulating metabolic and lipid-associated pathways, and astrocyte-microglia crosstalk. Moreover, we show that reactive gliosis can be directly mitigated in vivo using an adeno-associated virus (AAV)-mediated Chd8 gene editing strategy. These findings uncover a role of ASD-associated CHD8 in the adult brain, which may warrant future exploration of targeting chromatin remodelers in reactive gliosis and neuroinflammation in injury and neurological diseases.


Subject(s)
Astrocytes , Gliosis , Animals , Gliosis/pathology , Gliosis/metabolism , Astrocytes/metabolism , Astrocytes/pathology , Mice , Chromatin/metabolism , Autism Spectrum Disorder/metabolism , Autism Spectrum Disorder/genetics , Autism Spectrum Disorder/pathology , Neuroinflammatory Diseases/metabolism , Neuroinflammatory Diseases/pathology , Chromatin Assembly and Disassembly , Microglia/metabolism , Microglia/pathology , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Mice, Inbred C57BL , Lipopolysaccharides/pharmacology , Humans , Mice, Knockout , Male , Cell Proliferation
2.
Trends Cell Biol ; 34(7): 547-565, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38180380

ABSTRACT

Astrocytes are predominant glial cells that tile the central nervous system (CNS). A cardinal feature of astrocytes is their complex and visually enchanting morphology, referred to as bushy, spongy, and star-like. A central precept of this review is that such complex morphological shapes evolved to allow astrocytes to contact and signal with diverse cells at a range of distances in order to sample, regulate, and contribute to the extracellular milieu, and thus participate widely in cell-cell signaling during physiology and disease. The recent use of improved imaging methods and cell-specific molecular evaluations has revealed new information on the structural organization and molecular underpinnings of astrocyte morphology, the mechanisms of astrocyte morphogenesis, and the contributions to disease states of reduced morphology. These insights have reignited interest in astrocyte morphological complexity as a cornerstone of fundamental glial biology and as a critical substrate for multicellular spatial and physiological interactions in the CNS.


Subject(s)
Astrocytes , Astrocytes/metabolism , Astrocytes/cytology , Humans , Animals , Cell Shape , Cell Communication , Central Nervous System/metabolism
3.
J Comp Neurol ; 532(1): e25589, 2024 01.
Article in English | MEDLINE | ID: mdl-38289192

ABSTRACT

Retinoic acid-induced 1 (RAI1) encodes a transcriptional regulator critical for brain development and function. RAI1 haploinsufficiency in humans causes a syndromic autism spectrum disorder known as Smith-Magenis syndrome (SMS). The neuroanatomical distribution of RAI1 has not been quantitatively analyzed during the development of the prefrontal cortex, a brain region critical for cognitive function and social behaviors and commonly implicated in autism spectrum disorders, including SMS. Here, we performed comparative analyses to uncover the evolutionarily convergent and divergent expression profiles of RAI1 in major cell types during prefrontal cortex maturation in common marmoset monkeys (Callithrix jacchus) and mice (Mus musculus). We found that while RAI1 in both species is enriched in neurons, the percentage of excitatory neurons that express RAI1 is higher in newborn mice than in newborn marmosets. By contrast, RAI1 shows similar neural distribution in adult marmosets and adult mice. In marmosets, RAI1 is expressed in several primate-specific cell types, including intralaminar astrocytes and MEIS2-expressing prefrontal GABAergic neurons. At the molecular level, we discovered that RAI1 forms a protein complex with transcription factor 20 (TCF20), PHD finger protein 14 (PHF14), and high mobility group 20A (HMG20A) in the marmoset brain. In vitro assays in human cells revealed that TCF20 regulates RAI1 protein abundance. This work demonstrates that RAI1 expression and protein interactions are largely conserved but with some unique expression in primate-specific cells. The results also suggest that altered RAI1 abundance could contribute to disease features in disorders caused by TCF20 dosage imbalance.


Subject(s)
Autism Spectrum Disorder , Smith-Magenis Syndrome , Trans-Activators , Animals , Mice , Autism Spectrum Disorder/genetics , Callithrix , GABAergic Neurons , High Mobility Group Proteins , Transcription Factors/genetics , Trans-Activators/genetics
4.
J Neurochem ; 167(1): 52-75, 2023 10.
Article in English | MEDLINE | ID: mdl-37525469

ABSTRACT

Astrocytes have essential roles in central nervous system (CNS) health and disease. During development, immature astrocytes show complex interactions with neurons, endothelial cells, and other glial cell types. Our work and that of others have shown that these interactions are important for astrocytic maturation. However, whether and how these cells work together to control this process remains poorly understood. Here, we test the hypothesis that cooperative interactions of astrocytes with neurons and endothelial cells promote astrocytic maturation. Astrocytes were cultured alone, with neurons, endothelial cells, or a combination of both. This was followed by astrocyte sorting, RNA sequencing, and bioinformatic analysis to detect transcriptional changes. Across culture configurations, 7302 genes were differentially expressed by 4 or more fold and organized into 8 groups that demonstrate cooperative and antagonist effects of neurons and endothelia on astrocytes. We also discovered that neurons and endothelial cells caused splicing of 200 and 781 mRNAs, respectively. Changes in gene expression were validated using quantitative PCR, western blot (WB), and immunofluorescence analysis. We found that the transcriptomic data from the three-culture configurations correlated with protein expression of three representative targets (FAM107A, GAT3, and GLT1) in vivo. Alternative splicing results also correlated with cortical tissue isoform representation of a target (Fibronectin 1) at different developmental stages. By comparing our results to published transcriptomes of immature and mature astrocytes, we found that neurons or endothelia shift the astrocytic transcriptome toward a mature state and that the presence of both cell types has a greater effect on maturation than either cell alone. These results increase our understanding of cellular interactions/pathways that contribute to astrocytic maturation. They also provide insight into how alterations to neurons and/or endothelial cells may alter astrocytes with implications for astrocytic changes in CNS disorders and diseases.


Subject(s)
Astrocytes , Transcriptome , Astrocytes/metabolism , Endothelial Cells/metabolism , Neurons/metabolism , Neurogenesis/physiology
5.
Curr Biol ; 33(5): 957-972.e5, 2023 03 13.
Article in English | MEDLINE | ID: mdl-36805126

ABSTRACT

Astrocytes are increasingly understood to be important regulators of central nervous system (CNS) function in health and disease; yet, we have little quantitative understanding of their complex architecture. While broad categories of astrocytic structures are known, the discrete building blocks that compose them, along with their geometry and organizing principles, are poorly understood. Quantitative investigation of astrocytic complexity is impeded by the absence of high-resolution datasets and robust computational approaches to analyze these intricate cells. To address this, we produced four ultra-high-resolution datasets of mouse cerebral cortex using serial electron microscopy and developed astrocyte-tailored computer vision methods for accurate structural analysis. We unearthed specific anatomical building blocks, structural motifs, connectivity hubs, and hierarchical organizations of astrocytes. Furthermore, we found that astrocytes interact with discrete clusters of synapses and that astrocytic mitochondria are distributed to lie closer to larger clusters of synapses. Our findings provide a geometrically principled, quantitative understanding of astrocytic nanoarchitecture and point to an unexpected level of complexity in how astrocytes interact with CNS microanatomy.


Subject(s)
Astrocytes , Synapses , Animals , Mice , Astrocytes/physiology , Synapses/physiology , Cerebral Cortex
6.
J Neurosci ; 43(9): 1509-1529, 2023 03 01.
Article in English | MEDLINE | ID: mdl-36669885

ABSTRACT

Astrocytes have complex structural, molecular, and physiological properties and form specialized microenvironments that support circuit-specific functions in the CNS. To better understand how astrocytes acquire their unique features, we transplanted immature mouse cortical astrocytes into the developing cortex of male and female mice and assessed their integration, maturation, and survival. Within days, transplanted astrocytes developed morphologies and acquired territories and tiling behavior typical of cortical astrocytes. At 35-47 d post-transplantation, astrocytes appeared morphologically mature and expressed levels of EAAT2/GLT1 similar to nontransplanted astrocytes. Transplanted astrocytes also supported excitatory/inhibitory (E/I) presynaptic terminals within their territories, and displayed normal Ca2+ events. Transplanted astrocytes showed initially reduced expression of aquaporin 4 (AQP4) at endfeet and elevated expression of EAAT1/GLAST, with both proteins showing normalized expression by 110 d and one year post-transplantation, respectively. To understand how specific brain regions support astrocytic integration and maturation, we transplanted cortical astrocytes into the developing cerebellum. Cortical astrocytes interlaced with Bergmann glia (BG) in the cerebellar molecular layer to establish discrete territories. However, transplanted astrocytes retained many cortical astrocytic features including higher levels of EAAT2/GLT1, lower levels of EAAT1/GLAST, and the absence of expression of the AMPAR subunit GluA1. Collectively, our findings demonstrate that immature cortical astrocytes integrate, mature, and survive (more than one year) following transplantation and retain cortical astrocytic properties. Astrocytic transplantation can be useful for investigating cell-autonomous (intrinsic) and non-cell-autonomous (environmental) mechanisms contributing to astrocytic development/diversity, and for determining the optimal timing for transplanting astrocytes for cellular delivery or replacement in regenerative medicine.SIGNIFICANCE STATEMENT The mechanisms that enable astrocytes to acquire diverse molecular and structural properties remain to be better understood. In this study, we systematically analyzed the properties of cortical astrocytes following their transplantation to the early postnatal brain. We found that immature cortical astrocytes transplanted into cerebral cortex during early postnatal mouse development integrate and establish normal astrocytic properties, and show long-term survival in vivo (more than one year). In contrast, transplanted cortical astrocytes display reduced or altered ability to integrate into the more mature cerebral cortex or developing cerebellum, respectively. This study demonstrates the developmental potential of transplanted cortical astrocytes and provides an approach to tease apart cell-autonomous (intrinsic) and non-cell-autonomous (environmental) mechanisms that determine the structural, molecular, and physiological phenotype of astrocytes.


Subject(s)
Astrocytes , Neuroglia , Mice , Male , Female , Animals , Astrocytes/metabolism , Cerebral Cortex
7.
Trends Neurosci ; 45(9): 692-703, 2022 09.
Article in English | MEDLINE | ID: mdl-35879116

ABSTRACT

Astrocytes play crucial roles in regulating brain circuit formation and physiology. Recent technological advances have revealed unprecedented levels of astrocyte diversity encompassing molecular, morphological, and functional differences. This diversification is initiated during embryonic specification events and (in rodents) continues into the early postnatal period where it overlaps with peak synapse development and circuit refinement. In fact, several lines of evidence suggest astrocyte diversity both influences and is a consequence of molecular crosstalk among developing astrocytes and other cell types, notably neurons and their synapses. Neurological disease states exhibit additional layers of astrocyte heterogeneity, which could help shed light on these cells' key pathological roles. This review highlights recent advances in clarifying astrocyte heterogeneity and molecular/cellular crosstalk and identifies key outstanding questions.


Subject(s)
Astrocytes , Synapses , Astrocytes/physiology , Brain/physiology , Neurons/physiology , Synapses/physiology
8.
J Neurosci Res ; 99(12): 3121-3147, 2021 12.
Article in English | MEDLINE | ID: mdl-34716617

ABSTRACT

Astrocytes are abundant cells of the central nervous system (CNS) and are involved in processes including synapse formation/function, ion homeostasis, neurotransmitter uptake, and neurovascular coupling. Recent evidence indicates that astrocytes show diverse molecular, structural, and physiological properties within the CNS. This heterogeneity is reflected in differences in astrocyte structure, gene expression, functional properties, and responsiveness to injury/pathological conditions. Deeper investigation of astrocytic heterogeneity is needed to understand how astrocytes are configured to enable diverse roles in the CNS. While much has been learned about astrocytic heterogeneity in rodents, much less is known about astrocytic heterogeneity in the primate brain where astrocytes have greater size and complexity. The common marmoset (Callithrix jacchus) is a promising non-human primate model because of similarities between marmosets and humans with respect to genetics, brain anatomy, and cognition/behavior. Here, we investigated the molecular and structural heterogeneity of marmoset astrocytes using an array of astrocytic markers, multi-label confocal microscopy, and quantitative analysis. We used male and female marmosets and found that marmoset astrocytes show differences in expression of astrocytic markers in cortex, hippocampus, and cerebellum. These differences were accompanied by intra-regional variation in expression of markers for glutamate/GABA transporters, and potassium and water channels. Differences in astrocyte structure were also found, along with complex interactions with blood vessels, microglia, and neurons. This study contributes to our knowledge of the cellular and molecular features of marmoset astrocytes and is useful for understanding the complex properties of astrocytes in the primate CNS.


Subject(s)
Astrocytes , Callithrix , Animals , Astrocytes/metabolism , Brain/metabolism , Central Nervous System , Female , Male , Neurons/metabolism
9.
Front Cell Neurosci ; 15: 702685, 2021.
Article in English | MEDLINE | ID: mdl-34483840

ABSTRACT

Down Syndrome (DS) is the most common genetic cause of intellectual disability in which delays and impairments in brain development and function lead to neurological and cognitive phenotypes. Traditionally, a neurocentric approach, focusing on neurons and their connectivity, has been applied to understanding the mechanisms involved in DS brain pathophysiology with an emphasis on how triplication of chromosome 21 leads to alterations in neuronal survival and homeostasis, synaptogenesis, brain circuit development, and neurodegeneration. However, recent studies have drawn attention to the role of non-neuronal cells, especially astrocytes, in DS. Astrocytes comprise a large proportion of cells in the central nervous system (CNS) and are critical for brain development, homeostasis, and function. As triplication of chromosome 21 occurs in all cells in DS (with the exception of mosaic DS), a deeper understanding of the impact of trisomy 21 on astrocytes in DS pathophysiology is warranted and will likely be necessary for determining how specific brain alterations and neurological phenotypes emerge and progress in DS. Here, we review the current understanding of the role of astrocytes in DS, and discuss how specific perturbations in this cell type can impact the brain across the lifespan from early brain development to adult stages. Finally, we highlight how targeting, modifying, and/or correcting specific molecular pathways and properties of astrocytes in DS may provide an effective therapeutic direction given the important role of astrocytes in regulating brain development and function.

10.
Nat Neurosci ; 24(3): 312-325, 2021 03.
Article in English | MEDLINE | ID: mdl-33589835

ABSTRACT

Reactive astrocytes are astrocytes undergoing morphological, molecular, and functional remodeling in response to injury, disease, or infection of the CNS. Although this remodeling was first described over a century ago, uncertainties and controversies remain regarding the contribution of reactive astrocytes to CNS diseases, repair, and aging. It is also unclear whether fixed categories of reactive astrocytes exist and, if so, how to identify them. We point out the shortcomings of binary divisions of reactive astrocytes into good-vs-bad, neurotoxic-vs-neuroprotective or A1-vs-A2. We advocate, instead, that research on reactive astrocytes include assessment of multiple molecular and functional parameters-preferably in vivo-plus multivariate statistics and determination of impact on pathological hallmarks in relevant models. These guidelines may spur the discovery of astrocyte-based biomarkers as well as astrocyte-targeting therapies that abrogate detrimental actions of reactive astrocytes, potentiate their neuro- and glioprotective actions, and restore or augment their homeostatic, modulatory, and defensive functions.


Subject(s)
Aging/pathology , Astrocytes/pathology , Brain/pathology , Spinal Cord/pathology , Animals , Brain Diseases/pathology , Brain Injuries/pathology , Humans , Spinal Cord Injuries/pathology
11.
Genes Brain Behav ; 20(1): e12686, 2021 01.
Article in English | MEDLINE | ID: mdl-32691490

ABSTRACT

Understanding the rules that govern neuronal dynamics throughout the brain to subserve behavior and cognition remains one of the biggest challenges in neuroscience research. Recent technical advances enable the recording of increasingly larger neuronal populations to produce increasingly more sophisticated datasets. Despite bold and important open-science and data-sharing policies, these datasets tend to include unique data acquisition methods, behaviors, and file structures. Discrepancies between experimental protocols present key challenges in comparing data between laboratories and across different brain regions and species. Here, we discuss our recent efforts to create a standardized and high-throughput research platform to address these issues. The McGill-Mouse-Miniscope (M3) platform is an initiative to combine miniscope calcium imaging with standardized touchscreen-based animal behavioral testing. The goal is to curate an open-source and standardized framework for acquiring, analyzing, and accessing high-quality data of the neuronal dynamics that underly cognition throughout the brain in mice, marmosets, and models of disease. We end with a discussion of future developments and a call for users to adopt this standardized approach.


Subject(s)
Behavioral Research/instrumentation , Brain/physiology , User-Computer Interface , Animals , Behavioral Research/methods , Brain/cytology , Brain/metabolism , Calcium/metabolism , Cognition , High-Throughput Screening Assays/instrumentation , High-Throughput Screening Assays/methods , Mice , Microscopy, Fluorescence/instrumentation , Microscopy, Fluorescence/methods , Neurons/metabolism , Neurons/physiology
12.
Front Neuroanat ; 14: 31, 2020.
Article in English | MEDLINE | ID: mdl-32848635

ABSTRACT

Astrocytes are commonly identified by their expression of the intermediate filament protein glial fibrillary acidic protein (GFAP). GFAP-immunoreactive (GFAP-IR) astrocytes exhibit regional heterogeneity in density and morphology in the mouse brain as well as morphological diversity in the human cortex. However, regional variations in astrocyte distribution and morphology remain to be assessed comprehensively. This was the overarching objective of this postmortem study, which mainly exploited the immunolabeling of vimentin (VIM), an intermediate filament protein expressed by astrocytes and endothelial cells which presents the advantage of more extensively labeling cell structures. We compared the densities of vimentin-immunoreactive (VIM-IR) and GFAP-IR astrocytes in various brain regions (prefrontal and primary visual cortex, caudate nucleus, mediodorsal thalamus) from male individuals having died suddenly in the absence of neurological or psychiatric conditions. The morphometric properties of VIM-IR in these brain regions were also assessed. We found that VIM-IR astrocytes generally express the canonical astrocytic markers Aldh1L1 and GFAP but that VIM-IR astrocytes are less abundant than GFAP-IR astrocytes in all human brain regions, particularly in the thalamus, where VIM-IR cells were nearly absent. About 20% of all VIM-IR astrocytes presented a twin cell morphology, a phenomenon rarely observed for GFAP-IR astrocytes. Furthermore VIM-IR astrocytes in the striatum were often seen to extend numerous parallel processes which seemed to give rise to large VIM-IR fiber bundles projecting over long distances. Moreover, morphometric analyses revealed that VIM-IR astrocytes were more complex than their mouse counterparts in functionally homologous brain regions, as has been previously reported for GFAP-IR astrocytes. Lastly, the density of GFAP-IR astrocytes in gray and white matter were inversely correlated with vascular density, but for VIM-IR astrocytes this was only the case in gray matter, suggesting that gliovascular interactions may especially influence the regional heterogeneity of GFAP-IR astrocytes. Taken together, these findings reveal special features displayed uniquely by human VIM-IR astrocytes and illustrate that astrocytes display important region- and marker-specific differences in the healthy human brain.

13.
Nature ; 585(7823): 91-95, 2020 09.
Article in English | MEDLINE | ID: mdl-32788726

ABSTRACT

Signalling between cells of the neurovascular unit, or neurovascular coupling, is essential to match local blood flow with neuronal activity. Pericytes interact with endothelial cells and extend processes that wrap capillaries, covering up to 90% of their surface area1,2. Pericytes are candidates to regulate microcirculatory blood flow because they are strategically positioned along capillaries, contain contractile proteins and respond rapidly to neuronal stimulation3,4, but whether they synchronize microvascular dynamics and neurovascular coupling within a capillary network was unknown. Here we identify nanotube-like processes that connect two bona fide pericytes on separate capillary systems, forming a functional network in the mouse retina, which we named interpericyte tunnelling nanotubes (IP-TNTs). We provide evidence that these (i) have an open-ended proximal side and a closed-ended terminal (end-foot) that connects with distal pericyte processes via gap junctions, (ii) carry organelles including mitochondria, which can travel along these processes, and (iii) serve as a conduit for intercellular Ca2+ waves, thus mediating communication between pericytes. Using two-photon microscope live imaging, we demonstrate that retinal pericytes rely on IP-TNTs to control local neurovascular coupling and coordinate light-evoked responses between adjacent capillaries. IP-TNT damage following ablation or ischaemia disrupts intercellular Ca2+ waves, impairing blood flow regulation and neurovascular coupling. Notably, pharmacological blockade of Ca2+ influx preserves IP-TNTs, rescues light-evoked capillary responses and restores blood flow after reperfusion. Our study thus defines IP-TNTs and characterizes their critical role in regulating neurovascular coupling in the living retina under both physiological and pathological conditions.


Subject(s)
Nanotubes , Neurovascular Coupling , Pericytes/metabolism , Animals , Brain Ischemia/metabolism , Brain Ischemia/pathology , Calcium/metabolism , Calcium Signaling , Capillaries/physiopathology , Capillaries/radiation effects , Cell Communication , Female , Gap Junctions/metabolism , Hemodynamics , Male , Mice , Mitochondria/metabolism , Neurovascular Coupling/physiology , Pericytes/cytology , Pericytes/pathology , Retina/cytology , Retina/pathology
14.
Front Cell Neurosci ; 14: 36, 2020.
Article in English | MEDLINE | ID: mdl-32161521

ABSTRACT

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mature brain but has the paradoxical property of depolarizing neurons during early development. Depolarization provided by GABAA transmission during this early phase regulates neural stem cell proliferation, neural migration, neurite outgrowth, synapse formation, and circuit refinement, making GABA a key factor in neural circuit development. Importantly, depending on the context, depolarizing GABAA transmission can either drive neural activity or inhibit it through shunting inhibition. The varying roles of depolarizing GABAA transmission during development, and its ability to both drive and inhibit neural activity, makes it a difficult developmental cue to study. This is particularly true in the later stages of development when the majority of synapses form and GABAA transmission switches from depolarizing to hyperpolarizing. Here, we addressed the importance of depolarizing but inhibitory (or shunting) GABAA transmission in glutamatergic synapse formation in hippocampal CA1 pyramidal neurons. We first showed that the developmental depolarizing-to-hyperpolarizing switch in GABAA transmission is recapitulated in organotypic hippocampal slice cultures. Based on the expression profile of K+-Cl- co-transporter 2 (KCC2) and changes in the GABA reversal potential, we pinpointed the timing of the switch from depolarizing to hyperpolarizing GABAA transmission in CA1 neurons. We found that blocking depolarizing but shunting GABAA transmission increased excitatory synapse number and strength, indicating that depolarizing GABAA transmission can restrain glutamatergic synapse formation. The increase in glutamatergic synapses was activity-dependent but independent of BDNF signaling. Importantly, the elevated number of synapses was stable for more than a week after GABAA inhibitors were washed out. Together these findings point to the ability of immature GABAergic transmission to restrain glutamatergic synapse formation and suggest an unexpected role for depolarizing GABAA transmission in shaping excitatory connectivity during neural circuit development.

15.
Hum Mol Genet ; 29(5): 785-802, 2020 03 27.
Article in English | MEDLINE | ID: mdl-31943018

ABSTRACT

Down syndrome (DS), caused by the triplication of human chromosome 21, leads to significant alterations in brain development and is a major genetic cause of intellectual disability. While much is known about changes to neurons in DS, the effects of trisomy 21 on non-neuronal cells such as astrocytes are poorly understood. Astrocytes are critical for brain development and function, and their alteration may contribute to DS pathophysiology. To better understand the impact of trisomy 21 on astrocytes, we performed RNA-sequencing on astrocytes from newly produced DS human induced pluripotent stem cells (hiPSCs). While chromosome 21 genes were upregulated in DS astrocytes, we found consistent up- and down-regulation of genes across the genome with a strong dysregulation of neurodevelopmental, cell adhesion and extracellular matrix molecules. ATAC (assay for transposase-accessible chromatin)-seq also revealed a global alteration in chromatin state in DS astrocytes, showing modified chromatin accessibility at promoters of cell adhesion and extracellular matrix genes. Along with these transcriptomic and epigenomic changes, DS astrocytes displayed perturbations in cell size and cell spreading as well as modifications to cell-cell and cell-substrate recognition/adhesion, and increases in cellular motility and dynamics. Thus, triplication of chromosome 21 is associated with genome-wide transcriptional, epigenomic and functional alterations in astrocytes that may contribute to altered brain development and function in DS.


Subject(s)
Astrocytes/pathology , Cell Adhesion , Down Syndrome/pathology , Gene Expression Regulation , Genome, Human , Induced Pluripotent Stem Cells/pathology , Neural Stem Cells/pathology , Astrocytes/metabolism , Cell Differentiation , Cell Movement , Down Syndrome/genetics , Down Syndrome/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Neural Stem Cells/metabolism , Transcriptome
16.
Nat Genet ; 51(12): 1702-1713, 2019 12.
Article in English | MEDLINE | ID: mdl-31768071

ABSTRACT

Childhood brain tumors have suspected prenatal origins. To identify vulnerable developmental states, we generated a single-cell transcriptome atlas of >65,000 cells from embryonal pons and forebrain, two major tumor locations. We derived signatures for 191 distinct cell populations and defined the regional cellular diversity and differentiation dynamics. Projection of bulk tumor transcriptomes onto this dataset shows that WNT medulloblastomas match the rhombic lip-derived mossy fiber neuronal lineage and embryonal tumors with multilayered rosettes fully recapitulate a neuronal lineage, while group 2a/b atypical teratoid/rhabdoid tumors may originate outside the neuroectoderm. Importantly, single-cell tumor profiles reveal highly defined cell hierarchies that mirror transcriptional programs of the corresponding normal lineages. Our findings identify impaired differentiation of specific neural progenitors as a common mechanism underlying these pediatric cancers and provide a rational framework for future modeling and therapeutic interventions.


Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/pathology , Brain/embryology , Gene Expression Regulation, Developmental , Animals , Brain/pathology , Cell Line, Tumor , Humans , Infant , Medulloblastoma/genetics , Medulloblastoma/pathology , Mice , Neoplasms, Germ Cell and Embryonal/genetics , Neoplasms, Germ Cell and Embryonal/pathology , Nerve Fibers/pathology , Nerve Fibers/physiology , Prosencephalon/cytology , Prosencephalon/embryology , Rhabdoid Tumor/genetics , Rhabdoid Tumor/pathology , Single-Cell Analysis
17.
Glia ; 67(8): 1496-1509, 2019 08.
Article in English | MEDLINE | ID: mdl-30983036

ABSTRACT

The phenotypic changes of microglia in brain diseases are particularly diverse and their role in disease progression, beneficial, or detrimental, is still elusive. High-throughput molecular approaches such as single-cell RNA-sequencing can now resolve the high heterogeneity in microglia population for a specific physiological condition, however, the relation between the different microglial signatures and their surrounding brain microenvironment is barely understood. Thus, better tools to characterize the phenotypic variations of microglia in situ are needed, particularly for human brain postmortem samples analysis. To address this challenge, we developed MIC-MAC, a Microglia and Immune Cells Morphologies Analyser and Classifier pipeline that semiautomatically segments, extracts, and classifies all microglia and immune cells labeled in large three-dimensional (3D) confocal image stacks of mouse and human brain samples. Our imaging-based approach enables automatic 3D-morphology characterization and classification of thousands of individual microglia in situ and revealed species- and disease-specific morphological phenotypes in mouse aging, human Alzheimer's disease, and dementia with Lewy Bodie's samples. MIC-MAC is a precision diagnostic tool that allows a rapid, unbiased, and large-scale analysis of microglia morphological states in mouse models and patient brain samples.


Subject(s)
Brain/cytology , Imaging, Three-Dimensional , Microglia/cytology , Microscopy, Confocal , Pattern Recognition, Automated/methods , Aged , Aged, 80 and over , Aging/pathology , Alzheimer Disease/pathology , Animals , Brain/pathology , Cluster Analysis , Female , Humans , Imaging, Three-Dimensional/methods , Lewy Body Disease/pathology , Machine Learning , Male , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Transgenic , Microglia/classification , Microglia/pathology , Microscopy, Confocal/methods
18.
Sci Rep ; 9(1): 5236, 2019 03 27.
Article in English | MEDLINE | ID: mdl-30918286

ABSTRACT

Epileptogenesis is the gradual process by which the healthy brain develops epilepsy. However, the neuronal circuit changes that underlie epileptogenesis are not well understood. Unfortunately, current chemically or electrically induced epilepsy models suffer from lack of cell specificity, so it is seldom known which cells were activated during epileptogenesis. We therefore sought to develop an optogenetic variant of the classical kindling model of epilepsy in which activatable cells are both genetically defined and fluorescently tagged. We briefly optogenetically activated pyramidal cells (PCs) in awake behaving mice every two days and conducted a series of experiments to validate the effectiveness of the model. Although initially inert, brief optogenetic stimuli eventually elicited seizures that increased in number and severity with additional stimulation sessions. Seizures were associated with long-lasting plasticity, but not with tissue damage or astrocyte reactivity. Once optokindled, mice retained an elevated seizure susceptibility for several weeks in the absence of additional stimulation, indicating a form of long-term sensitization. We conclude that optokindling shares many features with classical kindling, with the added benefit that the role of specific neuronal populations in epileptogenesis can be studied. Links between long-term plasticity and epilepsy can thus be elucidated.


Subject(s)
Epilepsy/genetics , Epilepsy/physiopathology , Kindling, Neurologic/genetics , Neocortex/physiopathology , Optogenetics , Animals , Electroencephalography , Male , Mice , Mice, Inbred C57BL
19.
Methods Mol Biol ; 1938: 85-95, 2019.
Article in English | MEDLINE | ID: mdl-30617974

ABSTRACT

Astrocytes are among the most numerous cells in the brain and fulfill diverse functions in homeostasis and regulation of neuronal activity. Astrocytes also dramatically change their properties in response to brain injury or disease, a process called reactive gliosis. Precisely how astrocytes contribute to healthy brain function and play differential roles in brain pathology and regeneration remain important areas of investigation. To better understand the properties of astrocytes, more sophisticated approaches for probing their rich and complex anatomical and molecular features are needed to fully determine their contribution to brain physiology. Here we present an efficient and straightforward immunolabeling protocol to obtain high-resolution fluorescence-based images from fixed nonhuman primate (common marmoset Callithrix jacchus) and human brain samples. Importantly, the protocol is useful for obtaining images from samples that have been stored in fixative solutions (such as formalin) for years. This approach is especially useful for three-dimensional, multichannel confocal microscopy and can be optimized for super-resolution techniques such as stimulated emission depletion (STED) microscopy. We also present a strategy for using specific combinations of markers to define the phenotypic variations and cellular/subcellular properties of astrocytes to better predict the function of these cells on their surrounding brain microenvironment.


Subject(s)
Astrocytes/cytology , Brain/cytology , Imaging, Three-Dimensional/methods , Animals , Astrocytes/metabolism , Brain/metabolism , Callithrix , Hippocampus/cytology , Humans , Immunohistochemistry , Microscopy, Confocal/methods
20.
eNeuro ; 5(4)2018.
Article in English | MEDLINE | ID: mdl-30225353

ABSTRACT

Leucine-rich glioma-inactivated protein 1 (LGI1) is a secreted neuronal protein and a Nogo receptor 1 (NgR1) ligand. Mutations in LGI1 in humans causes autosomal dominant lateral temporal lobe epilepsy and homozygous deletion of LGI1 in mice results in severe epileptic seizures that cause early postnatal death. NgR1 plays an important role in the development of CNS synapses and circuitry by limiting plasticity in the adult cortex via the activation of RhoA. These relationships and functions prompted us to examine the effect of LGI1 on synapse formation in vitro and in vivo. We report that application of LGI1 increases synaptic density in neuronal culture and that LGI1 null hippocampus has fewer dendritic mushroom spines than in wild-type (WT) littermates. Further, our electrophysiological investigations demonstrate that LGI1 null hippocampal neurons possess fewer and weaker synapses. RhoA activity is significantly increased in cortical cultures derived from LGI1 null mice and using a reconstituted system; we show directly that LGI1 antagonizes NgR1-tumor necrosis factor receptor orphan Y (TROY) signaling. Our data suggests that LGI1 enhances synapse formation in cortical and hippocampal neurons by reducing NgR1 signaling.


Subject(s)
Excitatory Postsynaptic Potentials/physiology , Hippocampus/physiology , Neocortex/physiology , Neuronal Plasticity/physiology , Neurons/physiology , Nogo Receptor 1/metabolism , Proteins/physiology , Receptors, Tumor Necrosis Factor/metabolism , Signal Transduction/physiology , Synapses/physiology , rho GTP-Binding Proteins/metabolism , Animals , Embryo, Mammalian , Epilepsy , Female , Intracellular Signaling Peptides and Proteins , Male , Mice , Mice, Knockout , Patch-Clamp Techniques , rhoA GTP-Binding Protein
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