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1.
PLoS Biol ; 18(9): e3000852, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32931487

RESUMO

Olfaction in most animals is mediated by neurons bearing cilia that are accessible to the environment. Olfactory sensory neurons (OSNs) in chordates usually have multiple cilia, each with a centriole at its base. OSNs differentiate from stem cells in the olfactory epithelium, and how the epithelium generates cells with many centrioles is not yet understood. We show that centrioles are amplified via centriole rosette formation in both embryonic development and turnover of the olfactory epithelium in adult mice, and rosette-bearing cells often have free centrioles in addition. Cells with amplified centrioles can go on to divide, with centrioles clustered at each pole. Additionally, we found that centrioles are amplified in immediate neuronal precursors (INPs) concomitant with elevation of mRNA for polo-like kinase 4 (Plk4) and SCL/Tal1-interrupting locus gene (Stil), key regulators of centriole duplication. These results support a model in which centriole amplification occurs during a transient state characterized by elevated Plk4 and Stil in early INP cells. These cells then go on to divide at least once to become OSNs, demonstrating that cell division with amplified centrioles, known to be tolerated in disease states, can occur as part of a normal developmental program.


Assuntos
Divisão Celular/fisiologia , Centríolos/fisiologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Envelhecimento/fisiologia , Animais , Ciclo Celular/fisiologia , Células Cultivadas , Embrião de Mamíferos , Desenvolvimento Embrionário/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Córtex Olfatório/citologia , Córtex Olfatório/embriologia , Mucosa Olfatória/citologia , Mucosa Olfatória/embriologia , Mucosa Olfatória/ultraestrutura , Neurônios Receptores Olfatórios/citologia , Neurônios Receptores Olfatórios/ultraestrutura
2.
PLoS Biol ; 18(8): e3000762, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760088

RESUMO

Centrosomes, the main microtubule organizing centers (MTOCs) of metazoan cells, contain an older "mother" and a younger "daughter" centriole. Stem cells either inherit the mother or daughter-centriole-containing centrosome, providing a possible mechanism for biased delivery of cell fate determinants. However, the mechanisms regulating centrosome asymmetry and biased centrosome segregation are unclear. Using 3D-structured illumination microscopy (3D-SIM) and live-cell imaging, we show in fly neural stem cells (neuroblasts) that the mitotic kinase Polo and its centriolar protein substrate Centrobin (Cnb) accumulate on the daughter centriole during mitosis, thereby generating molecularly distinct mother and daughter centrioles before interphase. Cnb's asymmetric localization, potentially involving a direct relocalization mechanism, is regulated by Polo-mediated phosphorylation, whereas Polo's daughter centriole enrichment requires both Wdr62 and Cnb. Based on optogenetic protein mislocalization experiments, we propose that the establishment of centriole asymmetry in mitosis primes biased interphase MTOC activity, necessary for correct spindle orientation.


Assuntos
Proteínas de Ciclo Celular/genética , Centríolos/metabolismo , Centrossomo/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Mitose , Proteínas Serina-Treonina Quinases/genética , Animais , Animais Geneticamente Modificados , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centríolos/ultraestrutura , Centrossomo/ultraestrutura , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interfase , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Optogenética/métodos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais
3.
Nat Commun ; 11(1): 4067, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792493

RESUMO

The brain is organized morphologically and functionally into a columnar structure. According to the radial unit hypothesis, neurons from the same lineage form a radial unit that contributes to column formation. However, the molecular mechanisms that link neuronal lineage and column formation remain elusive. Here, we show that neurons from the same lineage project to different columns under control of Down syndrome cell adhesion molecule (Dscam) in the fly brain. Dscam1 is temporally expressed in newly born neuroblasts and is inherited by their daughter neurons. The transient transcription of Dscam1 in neuroblasts enables the expression of the same Dscam1 splice isoform within cells of the same lineage, causing lineage-dependent repulsion. In the absence of Dscam1 function, neurons from the same lineage project to the same column. When the splice diversity of Dscam1 is reduced, column formation is significantly compromised. Thus, Dscam1 controls column formation through lineage-dependent repulsion.


Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas de Drosophila/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Axônios/metabolismo , Moléculas de Adesão Celular/genética , Células Cultivadas , Proteínas de Drosophila/genética , Drosophila melanogaster , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Neurogênese/fisiologia , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
Nat Commun ; 11(1): 4063, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792525

RESUMO

The neuroendocrine hypothalamus is the central regulator of vital physiological homeostasis and behavior. However, the cellular and molecular properties of hypothalamic neural progenitors remain unexplored. Here, hypothalamic radial glial (hRG) and hypothalamic mantle zone radial glial (hmRG) cells are found to be neural progenitors in the developing mammalian hypothalamus. The hmRG cells originate from hRG cells and produce neurons. During the early development of hypothalamus, neurogenesis occurs in radial columns and is initiated from hRG cells. The radial glial fibers are oriented toward the locations of hypothalamic subregions which act as a scaffold for neuronal migration. Furthermore, we use single-cell RNA sequencing to reveal progenitor subtypes in human developing hypothalamus and characterize specific progenitor genes, such as TTYH1, HMGA2, and FAM107A. We also demonstrate that HMGA2 is involved in E2F1 pathway, regulating the proliferation of progenitor cells by targeting on the downstream MYBL2. Different neuronal subtypes start to differentiate and express specific genes of hypothalamic nucleus at gestational week 10. Finally, we reveal the developmental conservation of nuclear structures and marker genes in mouse and human hypothalamus. Our identification of cellular and molecular properties of neural progenitors provides a basic understanding of neurogenesis and regional formation of the non-laminated hypothalamus.


Assuntos
Hipotálamo/citologia , Hipotálamo/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Animais , Análise por Conglomerados , Feminino , Genes Supressores de Tumor , Proteína HMGA2/genética , Proteína HMGA2/metabolismo , Humanos , Hibridização In Situ , Mamíferos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neurogênese/genética , Neurogênese/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Gravidez
5.
Science ; 369(6505): 858-862, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32792401

RESUMO

The conversion of neural stem cells into neurons is associated with the remodeling of organelles, but whether and how this is causally linked to fate change is poorly understood. We examined and manipulated mitochondrial dynamics during mouse and human cortical neurogenesis. We reveal that shortly after cortical stem cells have divided, daughter cells destined to self-renew undergo mitochondrial fusion, whereas those that retain high levels of mitochondria fission become neurons. Increased mitochondria fission promotes neuronal fate, whereas induction of mitochondria fusion after mitosis redirects daughter cells toward self-renewal. This occurs during a restricted time window that is doubled in human cells, in line with their increased self-renewal capacity. Our data reveal a postmitotic period of fate plasticity in which mitochondrial dynamics are linked with cell fate.


Assuntos
Córtex Cerebral/crescimento & desenvolvimento , Mitocôndrias/fisiologia , Dinâmica Mitocondrial , Mitose , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Neurônios/citologia , Animais , Córtex Cerebral/citologia , Feminino , Células HEK293 , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Masculino , Camundongos , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Sirtuínas/metabolismo
6.
Nat Commun ; 11(1): 4275, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848155

RESUMO

New neurons are generated in adult mammals. Adult hippocampal neurogenesis is considered to play an important role in cognition and mental health. The number and properties of newly born neurons are regulatable by a broad range of physiological and pathological conditions. To begin to understand the underlying cellular mechanisms and functional relevance of adult neurogenesis, many studies rely on quantification of adult-born neurons. However, lack of standardized methods to quantify new neurons is impeding research reproducibility across laboratories. Here, we review the importance of stereology, and propose why and how it should be applied to the study of adult neurogenesis.


Assuntos
Encéfalo/citologia , Encéfalo/fisiologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Adulto , Células-Tronco Adultas/citologia , Células-Tronco Adultas/fisiologia , Animais , Giro Denteado/citologia , Giro Denteado/fisiologia , Humanos , Modelos Neurológicos , Plasticidade Neuronal
7.
Nat Commun ; 11(1): 3839, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737294

RESUMO

Chromatin regulates spatiotemporal gene expression during neurodevelopment, but it also mediates DNA damage repair essential to proliferating neural progenitor cells (NPCs). Here, we uncover molecularly dissociable roles for nucleosome remodeler Ino80 in chromatin-mediated transcriptional regulation and genome maintenance in corticogenesis. We find that conditional Ino80 deletion from cortical NPCs impairs DNA double-strand break (DSB) repair, triggering p53-dependent apoptosis and microcephaly. Using an in vivo DSB repair pathway assay, we find that Ino80 is selectively required for homologous recombination (HR) DNA repair, which is mechanistically distinct from Ino80 function in YY1-associated transcription. Unexpectedly, sensitivity to loss of Ino80-mediated HR is dependent on NPC division mode: Ino80 deletion leads to unrepaired DNA breaks and apoptosis in symmetric NPC-NPC divisions, but not in asymmetric neurogenic divisions. This division mode dependence is phenocopied following conditional deletion of HR gene Brca2. Thus, distinct modes of NPC division have divergent requirements for Ino80-dependent HR DNA repair.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Proteína BRCA2/genética , Cromatina/química , Proteínas de Ligação a DNA/genética , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Reparo de DNA por Recombinação , ATPases Associadas a Diversas Atividades Celulares/deficiência , Animais , Apoptose/genética , Proteína BRCA2/deficiência , Divisão Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/deficiência , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Transgênicos , Neocórtex/citologia , Neocórtex/crescimento & desenvolvimento , Neocórtex/metabolismo , Células-Tronco Neurais/citologia , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo
8.
Int J Nanomedicine ; 15: 3903-3920, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606657

RESUMO

Background: Researchers are trying to study the mechanism of neural stem cells (NSCs) differentiation to oligodendrocyte-like cells (OLCs) as well as to enhance the selective differentiation of NSCs to oligodendrocytes. However, the limitation in nerve tissue accessibility to isolate the NSCs as well as their differentiation toward oligodendrocytes is still challenging. Purpose: In the present study, a hybrid polycaprolactone (PCL)-gelatin nanofiber scaffold mimicking the native extracellular matrix and axon morphology to direct the differentiation of bone marrow-derived NSCs to OLCs was introduced. Materials and Methods: In order to achieve a sustained release of T3, this factor was encapsulated within chitosan nanoparticles and chitosan-loaded T3 was incorporated within PCL nanofibers. Polyaniline graphene (PAG) nanocomposite was incorporated within gelatin nanofibers to endow the scaffold with conductive properties, which resemble the conductive behavior of axons. Biodegradation, water contact angle measurements, and scanning electron microscopy (SEM) observations as well as conductivity tests were used to evaluate the properties of the prepared scaffold. The concentration of PAG and T3-loaded chitosan NPs in nanofibers were optimized by examining the proliferation of cultured bone marrow-derived mesenchymal stem cells (BMSCs) on the scaffolds. The differentiation of BMSCs-derived NSCs cultured on the fabricated scaffolds into OLCs was analyzed by evaluating the expression of oligodendrocyte markers using immunofluorescence (ICC), RT-PCR and flowcytometric assays. Results: Incorporating 2% PAG proved to have superior cell support and proliferation while guaranteeing electrical conductivity of 10.8 × 10-5 S/cm. Moreover, the scaffold containing 2% of T3-loaded chitosan NPs was considered to be the most biocompatible samples. Result of ICC, RT-PCR and flow cytometry showed high expression of O4, Olig2, platelet-derived growth factor receptor-alpha (PDGFR-α), O1, myelin/oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP) high expressed but low expression of glial fibrillary acidic protein (GFAP). Conclusion: Considering surface topography, biocompatibility, electrical conductivity and gene expression, the hybrid PCL/gelatin scaffold with the controlled release of T3 may be considered as a promising candidate to be used as an in vitro model to study patient-derived oligodendrocytes by isolating patient's BMSCs in pathological conditions such as diseases or injuries. Moreover, the resulted oligodendrocytes can be used as a desirable source for transplanting in patients.


Assuntos
Materiais Biomiméticos/farmacologia , Células da Medula Óssea/citologia , Diferenciação Celular , Nanofibras/química , Células-Tronco Neurais/citologia , Oligodendroglia/citologia , Tecidos Suporte/química , Compostos de Anilina/química , Animais , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condutividade Elétrica , Gelatina/química , Grafite/química , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Nanofibras/ultraestrutura , Células-Tronco Neurais/metabolismo , Oligodendroglia/efeitos dos fármacos , Poliésteres/química , Ratos , Suínos , Tri-Iodotironina/farmacologia
9.
Nat Commun ; 11(1): 3369, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32632153

RESUMO

Induced pluripotent stem cell (iPSC)-derived dopaminergic (DA) neurons are an expected source for cell-based therapies for Parkinson's disease (PD). The regulatory criteria for the clinical application of these therapies, however, have not been established. Here we show the results of our pre-clinical study, in which we evaluate the safety and efficacy of dopaminergic progenitors (DAPs) derived from a clinical-grade human iPSC line. We confirm the characteristics of DAPs by in vitro analyses. We also verify that the DAP population include no residual undifferentiated iPSCs or early neural stem cells and have no genetic aberration in cancer-related genes. Furthermore, in vivo studies using immunodeficient mice reveal no tumorigenicity or toxicity of the cells. When the DAPs are transplanted into the striatum of 6-OHDA-lesioned rats, the animals show behavioral improvement. Based on these results, we started a clinical trial to treat PD patients in 2018.


Assuntos
Neurônios Dopaminérgicos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/transplante , Doença de Parkinson/terapia , Transplante de Células-Tronco/métodos , Animais , Diferenciação Celular/genética , Linhagem Celular , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Macaca fascicularis , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Avaliação de Resultados em Cuidados de Saúde/métodos , Avaliação de Resultados em Cuidados de Saúde/estatística & dados numéricos , Ratos Nus , Transplante Heterólogo
10.
BMC Evol Biol ; 20(1): 84, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32664907

RESUMO

BACKGROUND: Diverse architectures of nervous systems (NSs) such as a plexus in cnidarians or a more centralized nervous system (CNS) in insects and vertebrates are present across Metazoa, but it is unclear what selection pressures drove evolution and diversification of NSs. One underlying aspect of this diversity lies in the cellular and molecular mechanisms driving neurogenesis, i.e. generation of neurons from neural precursor cells (NPCs). In cnidarians, vertebrates, and arthropods, homologs of SoxB and bHLH proneural genes control different steps of neurogenesis, suggesting that some neurogenic mechanisms may be conserved. However, data are lacking for spiralian taxa. RESULTS: To that end, we characterized NPCs and their daughters at different stages of neurogenesis in the spiralian annelid Capitella teleta. We assessed cellular division patterns in the neuroectoderm using static and pulse-chase labeling with thymidine analogs (EdU and BrdU), which enabled identification of NPCs that underwent multiple rounds of division. Actively-dividing brain NPCs were found to be apically-localized, whereas actively-dividing NPCs for the ventral nerve cord (VNC) were found apically, basally, and closer to the ventral midline. We used lineage tracing to characterize the changing boundary of the trunk neuroectoderm. Finally, to start to generate a genetic hierarchy, we performed double-fluorescent in-situ hybridization (FISH) and single-FISH plus EdU labeling for neurogenic gene homologs. In the brain and VNC, Ct-soxB1 and Ct-neurogenin were expressed in a large proportion of apically-localized, EdU+ NPCs. In contrast, Ct-ash1 was expressed in a small subset of apically-localized, EdU+ NPCs and subsurface, EdU- cells, but not in Ct-neuroD+ or Ct-elav1+ cells, which also were subsurface. CONCLUSIONS: Our data suggest a putative genetic hierarchy with Ct-soxB1 and Ct-neurogenin at the top, followed by Ct-ash1, then Ct-neuroD, and finally Ct-elav1. Comparison of our data with that from Platynereis dumerilii revealed expression of neurogenin homologs in proliferating NPCs in annelids, which appears different than the expression of vertebrate neurogenin homologs in cells that are exiting the cell cycle. Furthermore, differences between neurogenesis in the head versus trunk of C. teleta suggest that these two tissues may be independent developmental modules, possibly with differing evolutionary trajectories.


Assuntos
Neurogênese/genética , Filogenia , Poliquetos/citologia , Poliquetos/genética , Animais , Encéfalo/citologia , Ciclo Celular/genética , Divisão Celular , Proliferação de Células/genética , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Cinética , Modelos Biológicos , Placa Neural/citologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Fatores de Transcrição SOX/metabolismo
11.
Proc Natl Acad Sci U S A ; 117(30): 17842-17853, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32669437

RESUMO

Stem cells are capable of unlimited proliferation but can be induced to form brain cells. Factors that specifically regulate human development are poorly understood. We found that human stem cells expressed high levels of the envelope protein of an endogenized human-specific retrovirus (HERV-K, HML-2) from loci in chromosomes 12 and 19. The envelope protein was expressed on the cell membrane of the stem cells and was critical in maintaining the stemness via interactions with CD98HC, leading to triggering of human-specific signaling pathways involving mammalian target of rapamycin (mTOR) and lysophosphatidylcholine acyltransferase (LPCAT1)-mediated epigenetic changes. Down-regulation or epigenetic silencing of HML-2 env resulted in dissociation of the stem cell colonies and enhanced differentiation along neuronal pathways. Thus HML-2 regulation is critical for human embryonic and neurodevelopment, while it's dysregulation may play a role in tumorigenesis and neurodegeneration.


Assuntos
Diferenciação Celular , Retrovirus Endógenos/fisiologia , Neurônios/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Biomarcadores , Diferenciação Celular/genética , Autorrenovação Celular/genética , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Regulação Viral da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Ligação Proteica , Células-Tronco/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas do Envelope Viral/genética
12.
J Vis Exp ; (160)2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32658201

RESUMO

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.


Assuntos
Isquemia Encefálica/cirurgia , Injeções Intra-Arteriais/métodos , Células-Tronco Neurais/metabolismo , Transplante de Células-Tronco/métodos , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Células-Tronco Neurais/citologia , Ratos , Ratos Wistar
13.
Proc Natl Acad Sci U S A ; 117(25): 13886-13895, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32522880

RESUMO

Elucidating the lineage relationships among different cell types is key to understanding human brain development. Here we developed parallel RNA and DNA analysis after deep sequencing (PRDD-seq), which combines RNA analysis of neuronal cell types with analysis of nested spontaneous DNA somatic mutations as cell lineage markers, identified from joint analysis of single-cell and bulk DNA sequencing by single-cell MosaicHunter (scMH). PRDD-seq enables simultaneous reconstruction of neuronal cell type, cell lineage, and sequential neuronal formation ("birthdate") in postmortem human cerebral cortex. Analysis of two human brains showed remarkable quantitative details that relate mutation mosaic frequency to clonal patterns, confirming an early divergence of precursors for excitatory and inhibitory neurons, and an "inside-out" layer formation of excitatory neurons as seen in other species. In addition our analysis allows an estimate of excitatory neuron-restricted precursors (about 10) that generate the excitatory neurons within a cortical column. Inhibitory neurons showed complex, subtype-specific patterns of neurogenesis, including some patterns of development conserved relative to mouse, but also some aspects of primate cortical interneuron development not seen in mouse. PRDD-seq can be broadly applied to characterize cell identity and lineage from diverse archival samples with single-cell resolution and in potentially any developmental or disease condition.


Assuntos
Linhagem da Célula , Córtex Cerebral/citologia , Neurogênese , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Acúmulo de Mutações , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Análise de Sequência de DNA , Análise de Célula Única
14.
Nat Commun ; 11(1): 2993, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32532970

RESUMO

The accumulation of protein aggregates is involved in the onset of many neurodegenerative diseases. Aggrephagy is a selective type of autophagy that counteracts neurodegeneration by degrading such aggregates. In this study, we found that LC3C cooperates with lysosomal TECPR1 to promote the degradation of disease-related protein aggregates in neural stem cells. The N-terminal WD-repeat domain of TECPR1 selectively binds LC3C which decorates matured autophagosomes. The interaction of LC3C and TECPR1 promotes the recruitment of autophagosomes to lysosomes for degradation. Augmented expression of TECPR1 in neural stem cells reduces the number of protein aggregates by promoting their autophagic clearance, whereas knockdown of LC3C inhibits aggrephagy. The PH domain of TECPR1 selectively interacts with PtdIns(4)P to target TECPR1 to PtdIns(4)P containing lysosomes. Exchanging the PH against a tandem-FYVE domain targets TECPR1 ectopically to endosomes. This leads to an accumulation of LC3C autophagosomes at endosomes and prevents their delivery to lysosomes.


Assuntos
Autofagossomos/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neurais/metabolismo , Autofagossomos/ultraestrutura , Autofagia/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular , Endossomos/metabolismo , Células HeLa , Humanos , Lisossomos/ultraestrutura , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microscopia Confocal , Microscopia Imunoeletrônica , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Células-Tronco Neurais/citologia , Doenças Neurodegenerativas/metabolismo , Agregados Proteicos , Agregação Patológica de Proteínas , Ligação Proteica , Transporte Proteico , Proteólise , Interferência de RNA
15.
J Vis Exp ; (160)2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32568233

RESUMO

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias Murinas/citologia , Neurônios/citologia , Animais , Corpos Embrioides/citologia , Camundongos , Células-Tronco Neurais/citologia , Neurogênese
16.
J Vis Exp ; (159)2020 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-32510488

RESUMO

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1-3-day-old (P1-3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6-12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.


Assuntos
Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Células-Tronco Neurais/citologia , Neurogênese , Animais , Astrócitos/citologia , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Hipocampo/citologia , Ventrículos Laterais/citologia , Camundongos , Neurônios/citologia , Oligodendroglia/citologia
17.
Mol Cell ; 79(3): 521-534.e15, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32592681

RESUMO

Genome-wide mapping of chromatin interactions at high resolution remains experimentally and computationally challenging. Here we used a low-input "easy Hi-C" protocol to map the 3D genome architecture in human neurogenesis and brain tissues and also demonstrated that a rigorous Hi-C bias-correction pipeline (HiCorr) can significantly improve the sensitivity and robustness of Hi-C loop identification at sub-TAD level, especially the enhancer-promoter (E-P) interactions. We used HiCorr to compare the high-resolution maps of chromatin interactions from 10 tissue or cell types with a focus on neurogenesis and brain tissues. We found that dynamic chromatin loops are better hallmarks for cellular differentiation than compartment switching. HiCorr allowed direct observation of cell-type- and differentiation-specific E-P aggregates spanning large neighborhoods, suggesting a mechanism that stabilizes enhancer contacts during development. Interestingly, we concluded that Hi-C loop outperforms eQTL in explaining neurological GWAS results, revealing a unique value of high-resolution 3D genome maps in elucidating the disease etiology.


Assuntos
Cromatina/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Genoma Humano , Neurogênese/genética , Regiões Promotoras Genéticas , Adulto , Linhagem Celular , Cérebro/citologia , Cérebro/crescimento & desenvolvimento , Cérebro/metabolismo , Cromatina/ultraestrutura , Mapeamento Cromossômico , Feto , Histonas/genética , Histonas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas do Tecido Nervoso/classificação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/citologia , Neurônios/metabolismo , Lobo Temporal/citologia , Lobo Temporal/crescimento & desenvolvimento , Lobo Temporal/metabolismo , Fatores de Transcrição/classificação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
Nature ; 581(7806): 77-82, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32376949

RESUMO

Grafts of spinal-cord-derived neural progenitor cells (NPCs) enable the robust regeneration of corticospinal axons and restore forelimb function after spinal cord injury1; however, the molecular mechanisms that underlie this regeneration are unknown. Here we perform translational profiling specifically of corticospinal tract (CST) motor neurons in mice, to identify their 'regenerative transcriptome' after spinal cord injury and NPC grafting. Notably, both injury alone and injury combined with NPC grafts elicit virtually identical early transcriptomic responses in host CST neurons. However, in mice with injury alone this regenerative transcriptome is downregulated after two weeks, whereas in NPC-grafted mice this transcriptome is sustained. The regenerative transcriptome represents a reversion to an embryonic transcriptional state of the CST neuron. The huntingtin gene (Htt) is a central hub in the regeneration transcriptome; deletion of Htt significantly attenuates regeneration, which shows that Htt has a key role in neural plasticity after injury.


Assuntos
Proliferação de Células/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Regeneração Nervosa/genética , Células-Tronco Neurais/citologia , Neurônios/metabolismo , Neurônios/patologia , Transcrição Genética , Animais , Axônios/patologia , Axônios/fisiologia , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Proteína Huntingtina/genética , Camundongos , Células-Tronco Neurais/transplante , Plasticidade Neuronal , Neurônios/citologia , Neurônios/transplante , Biossíntese de Proteínas , Tratos Piramidais/citologia , Tratos Piramidais/metabolismo , Tratos Piramidais/patologia , RNA-Seq , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologia , Transcriptoma
19.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(3): 248-254, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-32389173

RESUMO

Objective To explore the impact of various conditions during cryopreservation on the survival rate of oligodendrocyte precursor cells (OPCs) derived from human fetal neural stem cells. Methods We compared the cell viability of oligodendrocyte precursors harvested with or without digestion. Then we tested the impact of 3 factors during cryopreservation, freezing solutions (solution with 70 mL/L DMSO and 930 mL/L FBS; solution with 70 mL/L DMSO, 300 mL/L FBS and OPC culture medium; solution with 70 mL/L DMSO, 300 mL/L FBS, 0.2 mol/mL trehalose and OPC culture medium; solution with 70 mL/L DMSO, 300 mL/L FBS, 300 mL/L HES and OPC culture medium), freezing methods (the step-wised freezing or rapid freezing within liquid nitrogen) and storage durations for the better survival rate of OPCs. The optimized method with the best survival rate of OPCs was implemented and at day 7 after recovery, the viability, OPCs specific markers [platelet derived growth factor receptor alpha (PDGFRα), ST8 alpha-N-acetyl-neuraminide alpha-2, 8-sialyltransferase 1 (ST8SIA1/A2B5), chondroitin sulfate proteoglycan 4 (CSPG4/NG2), ki67 were tested and compared with immunofluorescent cytochemical staining. Results Harvesting with digestion contributed to higher OPCs survival rate. OPCs of rapid freezing had survival rates less than 30% and couldn't be re-cultured. The step-wised freezing group showed higher recovery rate. Harvesting with digestion, preservation solution with trehalose, using 2.0×106/mL of cell number, step-wised freezing, contributed to the highest OPCs survival rate reaching (75.73±6.66)%. Compared with the fresh cultured group, cell proliferation, ki67 antigen, PDGFRα, A2B5 and NG2 expression of OPCs were similar in the recovered cells. Storage duration didn't affect OPCs survival rate. Conclusion Harvesting with digestion, step-wised freezing, preservation solution with trehalose contribute to higher OPCs survival rate during cryopreservation and cell-thawing. Storage time doesn't affect phenotypes and viability of OPCs.


Assuntos
Criopreservação/métodos , Células-Tronco Neurais/citologia , Células Precursoras de Oligodendrócitos/citologia , Encéfalo/citologia , Diferenciação Celular , Sobrevivência Celular , Feto , Humanos
20.
Ecotoxicol Environ Saf ; 200: 110733, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32450442

RESUMO

Paraquat (1,1'-dimethyl-4,4'-bipyridium dichloride, PQ), a non-selective and efficient herbicide, causes neuroinflammation, neurodegeneration and memory dysfunction. However, adverse effects of PQ on the neuroimmune interactions have rarely been investigated. Female adult C57/BL6 mice were divided into 3 groups and treated with PQ (intraperitoneal injection, 1 mg/kg or 5 mg/kg) or the vehicle (an equivalent volume of 0.9% saline) every two days, at day 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, for a total of 14 doses. We evaluated blood-brain barrier (BBB) integrity and PQ concentrations during the course of PQ exposure and tested interleukin-1ß (IL-1ß) concentrations in dentate gyrus (DG) after 28 days PQ exposure. In addition, memory function, neural stem cells (NSCs) proliferation, neurogenesis and microglia polarization were analyzed after PQ exposure. Furthermore, mice were intraperitoneal injections of anti-IL-1ß during 5 mg/kg PQ exposure to test the rule of IL-1ß. Blood-brain barrier (BBB) permeability and PQ concentrations increased gradually during PQ exposure (n = 6). Moreover, memory function, NSCs proliferation and neurogenesis were impaired after 5 mg/kg PQ exposure (n = 6). Further analyses revealed that 'classically' activated (M1) microglia and IL-1ß concentrations in DG were increased after 5 mg/kg PQ treatment (n = 6). Moreover, we found that neutralization of IL-1ß partly restored PQ-induced NSCs impairments and memory dysfunction (n = 6). In conclusion, our results revealed that PQ induced NSCs impairments and memory dysfunction in adult mice, which was related to the release of IL-1ß by M1-polarized microglia in DG. These findings may help understand the neurotoxic effect of PQ.


Assuntos
Giro Denteado/efeitos dos fármacos , Herbicidas/toxicidade , Interleucina-1beta/metabolismo , Neurogênese/efeitos dos fármacos , Paraquat/toxicidade , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Giro Denteado/citologia , Feminino , Memória/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos
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