RESUMO
Inhibitory GABAergic interneurons migrate over long distances from their extracortical origin into the developing cortex. In humans, this process is uniquely slow and prolonged, and it is unclear whether guidance cues unique to humans govern the various phases of this complex developmental process. Here, we use fused cerebral organoids to identify key roles of neurotransmitter signaling pathways in guiding the migratory behavior of human cortical interneurons. We use scRNAseq to reveal expression of GABA, glutamate, glycine, and serotonin receptors along distinct maturation trajectories across interneuron migration. We develop an image analysis software package, TrackPal, to simultaneously assess 48 parameters for entire migration tracks of individual cells. By chemical screening, we show that different modes of interneuron migration depend on distinct neurotransmitter signaling pathways, linking transcriptional maturation of interneurons with their migratory behavior. Altogether, our study provides a comprehensive quantitative analysis of human interneuron migration and its functional modulation by neurotransmitter signaling.
Assuntos
Movimento Celular , Córtex Cerebral/fisiologia , Interneurônios/fisiologia , Neurotransmissores/metabolismo , Organoides/fisiologia , Córtex Cerebral/citologia , Células HEK293 , Humanos , Interneurônios/citologia , Neurogênese , Organoides/citologia , RNA-Seq , Análise de Célula ÚnicaRESUMO
The increasing prevalence of diabetes has resulted in a global epidemic1. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and amputation of lower limbs. These are often caused by changes in blood vessels, such as the expansion of the basement membrane and a loss of vascular cells2-4. Diabetes also impairs the functions of endothelial cells5 and disturbs the communication between endothelial cells and pericytes6. How dysfunction of endothelial cells and/or pericytes leads to diabetic vasculopathy remains largely unknown. Here we report the development of self-organizing three-dimensional human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks that are enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycaemia and inflammatory cytokines in vitro induces thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimic the microvascular changes found in patients with diabetes. DLL4 and NOTCH3 were identified as key drivers of diabetic vasculopathy in human blood vessels. Therefore, organoids derived from human stem cells faithfully recapitulate the structure and function of human blood vessels and are amenable systems for modelling and identifying the regulators of diabetic vasculopathy, a disease that affects hundreds of millions of patients worldwide.
Assuntos
Membrana Basal/patologia , Vasos Sanguíneos/patologia , Angiopatias Diabéticas/patologia , Modelos Biológicos , Organoides/patologia , Organoides/transplante , Proteínas Adaptadoras de Transdução de Sinal , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Artérias/citologia , Artérias/efeitos dos fármacos , Arteríolas/citologia , Arteríolas/efeitos dos fármacos , Membrana Basal/citologia , Membrana Basal/efeitos dos fármacos , Vasos Sanguíneos/citologia , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/crescimento & desenvolvimento , Proteínas de Ligação ao Cálcio , Angiopatias Diabéticas/enzimologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Humanos , Hiperglicemia/complicações , Técnicas In Vitro , Mediadores da Inflamação/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Organoides/citologia , Organoides/efeitos dos fármacos , Pericitos/citologia , Pericitos/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Receptor Notch3/metabolismo , Transdução de Sinais , Vênulas/citologia , Vênulas/efeitos dos fármacosRESUMO
A complex array of genetic factors regulates neuronal dendrite morphology. Epigenetic regulation of gene expression represents a plausible mechanism to control pathways responsible for specific dendritic arbor shapes. By studying the Drosophila dendritic arborization (da) neurons, we discovered a role of the double-bromodomain and extraterminal (BET) family proteins in regulating dendrite arbor complexity. A loss-of-function mutation in the single Drosophila BET protein encoded by female sterile 1 homeotic [fs(1)h] causes loss of fine, terminal dendritic branches. Moreover, fs(1)h is necessary for the induction of branching caused by a previously identified transcription factor, Cut (Ct), which regulates subtype-specific dendrite morphology. Finally, disrupting fs(1)h function impairs the mechanosensory response of class III da sensory neurons without compromising the expression of the ion channel NompC, which mediates the mechanosensitive response. Thus, our results identify a novel role for BET family proteins in regulating dendrite morphology and a possible separation of developmental pathways specifying neural cell morphology and ion channel expression. Since the BET proteins are known to bind acetylated histone tails, these results also suggest a role of epigenetic histone modifications and the "histone code," in regulating dendrite morphology.
Assuntos
Dendritos/genética , Dendritos/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Células Receptoras Sensoriais/citologia , Animais , Drosophila melanogaster , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Humanos , Mutação , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Células Receptoras Sensoriais/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In the originally published paper, the "before" image for the afatinib condition in Fig. 6c was incorrect. Instead of an image displaying a GBM-3 neoplastic organoid before afatinib treatment, this panel showed an image from the GBM-2 control (DMSO) group before treatment. This error has now been corrected in the HTML and PDF versions of the article; the "before, afatinib" panel in Fig. 6c now shows a representative image from the indicated experiment. The color of all error bars in Fig. 6 has also been changed to black, for consistency. All statistical analysis and all conclusions presented in the article are unaffected by this error. Nevertheless, we apologize for the mistake.
RESUMO
Brain tumors are among the most lethal and devastating cancers. Their study is limited by genetic heterogeneity and the incompleteness of available laboratory models. Three-dimensional organoid culture models offer innovative possibilities for the modeling of human disease. Here we establish a 3D in vitro model called a neoplastic cerebral organoid (neoCOR), in which we recapitulate brain tumorigenesis by introducing oncogenic mutations in cerebral organoids via transposon- and CRISPR-Cas9-mediated mutagenesis. By screening clinically relevant mutations identified in cancer genome projects, we defined mutation combinations that result in glioblastoma-like and central nervous system primitive neuroectodermal tumor (CNS-PNET)-like neoplasms. We demonstrate that neoCORs are suitable for use in investigations of aspects of tumor biology such as invasiveness, and for evaluation of drug effects in the context of specific DNA aberrations. NeoCORs will provide a valuable complement to the current basic and preclinical models used to study brain tumor biology.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Organoides/patologia , Animais , Modelos Animais de Doenças , Genes myc , Engenharia Genética , Glioblastoma/genética , Glioblastoma/patologia , Xenoenxertos , Células-Tronco Embrionárias Humanas , Humanos , Masculino , Camundongos , Camundongos Nus , Mutação , Tumores Neuroectodérmicos Primitivos/genética , Tumores Neuroectodérmicos Primitivos/patologia , Oncogenes , Transcriptoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neurodegenerative disorders, such as Huntington's diseases and spinocerebellar ataxias (SCAs), are driven by proteins with expanded polyglutamine (polyQ) tracts. Recently, coiled-coil structures in polyQ regions of such proteins were shown to facilitate aggregate formation and ultimately lead to cell death. However, the molecular mechanism linking these structural domains to neuronal toxicity of polyQ proteins remains elusive. Here, we demonstrate that coiled-coil structures in the Q repeat region of SCA type 3 (SCA3) polyQ proteins confer protein toxicity in Drosophila neurons. To functionally characterize coiled-coil structures in the Q repeat regions, we generated three structural variants of SCA3 polyQ proteins: (i) MJDtr-76Q, containing both α-helical coiled-coil and ß-sheet hairpin structures in the Q repeat region; (ii) MJDtr-70Q_cc0, possessing only α-helical coiled-coil structures due to the incorporation of ß-sheet-breaking residues (Q-to-N or Q-to-E mutations); and (iii) MJDtr-70Q_pQp, with no secondary structure due to the introduced proline residues (Q-to-P mutations). Through comparative analysis of these variants, we found that coiled-coil structures facilitated nuclear localization of SCA3 polyQ proteins and induced dendrite defects in Drosophila dendritic arborization neurons. Furthermore, genetic and functional screening identified the transcription factor Foxo as a target of polyQ proteins, and coiled-coil-mediated interactions of Foxo and polyQ proteins in the nucleus resulted in the observed dendrite and behavioral defects in Drosophila These results demonstrate that coiled-coil structures of polyQ proteins are crucial for their neuronal toxicity, which is conferred through coiled-coil to coiled-coil interactions with the nuclear targets of these proteins.
Assuntos
Ataxina-3/química , Proteínas de Drosophila/química , Drosophila melanogaster/genética , Fatores de Transcrição Forkhead/química , Neurônios/metabolismo , Peptídeos/química , Ataxias Espinocerebelares/genética , Sequência de Aminoácidos , Animais , Ataxina-3/genética , Ataxina-3/metabolismo , Comportamento Animal , Sítios de Ligação , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Mutação , Neurônios/ultraestrutura , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/patologiaRESUMO
Human brain development involves complex interactions between different regions, including long-distance neuronal migration or formation of major axonal tracts. Different brain regions can be cultured in vitro within 3D cerebral organoids, but the random arrangement of regional identities limits the reliable analysis of complex phenotypes. Here, we describe a coculture method combining brain regions of choice within one organoid tissue. By fusing organoids of dorsal and ventral forebrain identities, we generate a dorsal-ventral axis. Using fluorescent reporters, we demonstrate CXCR4-dependent GABAergic interneuron migration from ventral to dorsal forebrain and describe methodology for time-lapse imaging of human interneuron migration. Our results demonstrate that cerebral organoid fusion cultures can model complex interactions between different brain regions. Combined with reprogramming technology, fusions should offer researchers the possibility to analyze complex neurodevelopmental defects using cells from neurological disease patients and to test potential therapeutic compounds.
Assuntos
Córtex Cerebral/fisiologia , Interneurônios/fisiologia , Organoides/fisiologia , Animais , Encéfalo/embriologia , Comunicação Celular , Técnicas de Cultura de Células , Movimento Celular , Córtex Cerebral/citologia , HumanosRESUMO
The dendritic arbors of the larval Drosophila peripheral class IV dendritic arborization neurons degenerate during metamorphosis in an ecdysone-dependent manner. This process-also known as dendrite pruning-depends on the ubiquitin-proteasome system (UPS), but the specific processes regulated by the UPS during pruning have been largely elusive. Here, we show that mutation or inhibition of Valosin-Containing Protein (VCP), a ubiquitin-dependent ATPase whose human homolog is linked to neurodegenerative disease, leads to specific defects in mRNA metabolism and that this role of VCP is linked to dendrite pruning. Specifically, we find that VCP inhibition causes an altered splicing pattern of the large pruning gene molecule interacting with CasL and mislocalization of the Drosophila homolog of the human RNA-binding protein TAR-DNA-binding protein of 43 kilo-Dalton (TDP-43). Our data suggest that VCP inactivation might lead to specific gain-of-function of TDP-43 and other RNA-binding proteins. A similar combination of defects is also seen in a mutant in the ubiquitin-conjugating enzyme ubcD1 and a mutant in the 19S regulatory particle of the proteasome, but not in a 20S proteasome mutant. Thus, our results highlight a proteolysis-independent function of the UPS during class IV dendritic arborization neuron dendrite pruning and link the UPS to the control of mRNA metabolism.
Assuntos
Adenosina Trifosfatases/fisiologia , Dendritos/metabolismo , Proteínas de Drosophila/fisiologia , Regulação da Expressão Gênica , RNA Mensageiro/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Ecdisona/metabolismo , Mutação , Neurônios/metabolismo , Fenótipo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Proteína com ValosinaRESUMO
Whereas the neurodegeneration associated with various polyglutamine (polyQ) diseases has prompted extensive studies of polyQ-induced cell death, the neuronal loss that typically appears during late stages of the diseases may not account for the preceding movement and mental disorders. The cellular basis for polyQ-induced neuronal dysfunction preceding neuronal cell death remains largely unknown. Here we report defective dendrite morphogenesis within a specific subset of neurons due to polyQ toxicity that can be dissociated from caspase-dependent cell death. Expressing pathogenic spinocerebellar ataxia type 1 (SCA1) or type 3 (SCA3) proteins in Drosophila larval dendritic arborization neurons caused neuronal type-specific dendrite phenotypes primarily affecting terminal branches. We further show that expression of pathogenic polyQ proteins in adult flies after the formation of neuronal dendrites also greatly reduced dendritic complexity. These defects are associated with disruption of dendritic F-actin structures that can be partially mitigated by increasing Rac-PAK signaling. Together, these findings suggest that specific actin cytoskeletal alterations that alter dendrite morphology and function may contribute to the pathogenesis of at least a subset of polyQ disorders, including SCA3 and SCA1.
Assuntos
Dendritos/metabolismo , Drosophila/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Peptídeos/metabolismo , Actinas/metabolismo , Animais , Ataxinas , Citoesqueleto/metabolismo , Drosophila/genética , Drosophila/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Larva/metabolismo , Larva/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Peptídeos/genética , Imagem com Lapso de TempoRESUMO
Loss-of-function (LOF) screens provide a powerful approach to identify regulators in biological processes. Pioneered in laboratory animals, LOF screens of human genes are currently restricted to two-dimensional cell cultures, which hinders the testing of gene functions requiring tissue context. Here, we present CRISPR-lineage tracing at cellular resolution in heterogeneous tissue (CRISPR-LICHT), which enables parallel LOF studies in human cerebral organoid tissue. We used CRISPR-LICHT to test 173 microcephaly candidate genes, revealing 25 to be involved in known and uncharacterized microcephaly-associated pathways. We characterized IER3IP1, which regulates the endoplasmic reticulum (ER) function and extracellular matrix protein secretion crucial for tissue integrity, the dysregulation of which results in microcephaly. Our human tissue screening technology identifies microcephaly genes and mechanisms involved in brain-size control.
Assuntos
Encéfalo/crescimento & desenvolvimento , Proteínas de Transporte/fisiologia , Retículo Endoplasmático/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Testes Genéticos/métodos , Proteínas de Membrana/fisiologia , Microcefalia/genética , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Proteínas de Transporte/genética , Linhagem Celular , Linhagem da Célula , Técnicas de Inativação de Genes , Humanos , Proteínas de Membrana/genética , Tamanho do Órgão , Organoides/crescimento & desenvolvimento , Organoides/metabolismoRESUMO
Disruptions in lipid homeostasis have been observed in many neurodevelopmental disorders that are associated with dendrite morphogenesis defects. However, the molecular mechanisms of how lipid homeostasis affects dendrite morphogenesis are unclear. We find that easily shocked (eas), which encodes a kinase with a critical role in phospholipid phosphatidylethanolamine (PE) synthesis, and two other enzymes in this synthesis pathway are required cell autonomously in sensory neurons for dendrite growth and stability. Furthermore, we show that the level of Sterol Regulatory Element-Binding Protein (SREBP) activity is important for dendrite development. SREBP activity increases in eas mutants, and decreasing the level of SREBP and its transcriptional targets in eas mutants largely suppresses the dendrite growth defects. Furthermore, reducing Ca2+ influx in neurons of eas mutants ameliorates the dendrite morphogenesis defects. Our study uncovers a role for EAS kinase and reveals the in vivo function of phospholipid homeostasis in dendrite morphogenesis.