RESUMO
Developing neurons undergo a progression of morphological and gene expression changes as they transition from neuronal progenitors to mature neurons. Here we used RNA-seq and H3K4me3 and H3K27me3 ChIP-seq to analyze how chromatin modifications control gene expression in a specific type of CNS neuron: the mouse cerebellar granule cell (GC). We found that in proliferating GC progenitors (GCPs), H3K4me3/H3K27me3 bivalency is common at neuronal genes and undergoes dynamic changes that correlate with gene expression during migration and circuit formation. Expressing a fluorescent sensor for bivalent domains revealed subnuclear bivalent foci in proliferating GCPs. Inhibiting H3K27 methyltransferases EZH1 and EZH2 in vitro and in organotypic cerebellar slices dramatically altered the expression of bivalent genes, induced the down-regulation of migration-related genes and up-regulation of synaptic genes, inhibited glial-guided migration, and accelerated terminal differentiation. Thus, histone bivalency is required to regulate the timing of the progression from progenitor cells to mature neurons.
Assuntos
Epigênese Genética , Histonas , Animais , Camundongos , Histonas/metabolismo , Ativação Transcricional , Diferenciação Celular/genéticaRESUMO
Proteasome-mediated degradation of intracellular proteins is essential for cell function and survival. The proteasome-binding protein PI31 (Proteasomal Inhibitor of 31kD) promotes 26S assembly and functions as an adapter for proteasome transport in axons. As localized protein synthesis and degradation is especially critical in neurons, we generated a conditional loss of PI31 in spinal motor neurons (MNs) and cerebellar Purkinje cells (PCs). A cKO of PI31 in these neurons caused axon degeneration, neuronal loss, and progressive spinal and cerebellar neurological dysfunction. For both MNs and PCs, markers of proteotoxic stress preceded axonal degeneration and motor dysfunction, indicating a critical role for PI31 in neuronal homeostasis. The time course of the loss of MN and PC function in developing mouse central nervous system suggests a key role for PI31 in human neurodegenerative diseases.
Assuntos
Proteínas de Transporte/metabolismo , Neurônios Motores/fisiologia , Doenças Neurodegenerativas/genética , Proteostase/fisiologia , Células de Purkinje/fisiologia , Sinapses/fisiologia , Animais , Axônios/patologia , Axônios/fisiologia , Técnicas de Observação do Comportamento , Proteínas de Transporte/genética , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Knockout , Neurônios Motores/patologia , Mutação , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Células de Purkinje/patologia , Sinapses/patologiaRESUMO
Spontaneous and sensory-evoked activity sculpts developing circuits. Yet, how these activity patterns intersect with cellular programs regulating the differentiation of neuronal subtypes is not well understood. Through electrophysiological and in vivo longitudinal analyses, we show that C-X-C motif chemokine ligand 14 (Cxcl14), a gene previously characterized for its association with tumor invasion, is expressed by single-bouquet cells (SBCs) in layer I (LI) of the somatosensory cortex during development. Sensory deprivation at neonatal stages markedly decreases Cxcl14 expression. Additionally, we report that loss of function of this gene leads to increased intrinsic excitability of SBCs-but not LI neurogliaform cells-and augments neuronal complexity. Furthermore, Cxcl14 loss impairs sensory map formation and compromises the in vivo recruitment of superficial interneurons by sensory inputs. These results indicate that Cxcl14 is required for LI differentiation and demonstrate the emergent role of chemokines as key players in cortical network development.
RESUMO
Developing neurons undergo a progression of morphological and gene expression changes as they transition from neuronal progenitors to mature, multipolar neurons. Here we use RNA-seq and H3K4me3 and H3K27me3 ChIP-seq to analyze how chromatin modifications control gene expression in a specific type of CNS neuron, the mouse cerebellar granule cell (GC). We find that in proliferating GC progenitors (GCPs), H3K4me3/H3K27me3 bivalency is common at neuronal genes and undergoes dynamic changes that correlate with gene expression during migration and circuit formation. Expressing a fluorescent sensor for bivalent H3K4me3 and H3K27me3 domains revealed subnuclear bivalent foci in proliferating GCPs. Inhibiting H3K27 methyltransferases EZH1 and EZH2 in vitro and in organotypic cerebellar slices dramatically altered the expression of bivalent genes and induced the downregulation of migration-related genes and upregulation of synaptic genes, inhibited glial-guided migration, and accelerated terminal differentiation. Thus, histone bivalency is required to regulate the timing of the progression from progenitor cells to mature neurons.
RESUMO
In this issue of Neuron, Du et al. (2019) demonstrate that the bicistronic CACNA1A gene encodes a transcription factor α1ACT, mutations in which are associated with SCA6, that controls expression of genes important for cerebellar Purkinje cell development and excitability. Reduction of α1ACT in the adult is well tolerated, suggesting a potential new therapy for SCA6.
Assuntos
Canais de Cálcio , Ataxias Espinocerebelares , Adulto , Cerebelo , Regulação da Expressão Gênica , Humanos , Recém-Nascido , Células de PurkinjeRESUMO
Mutations in regulators and effectors of the Rho GTPases underlie various forms of mental retardation (MR). Among them, oligophrenin-1 (OPHN1), which encodes a Rho-GTPase activating protein, was one of the first Rho-linked MR genes identified. Upon characterization of OPHN1 in hippocampal brain slices, we obtained evidence for the requirement of OPHN1 in dendritic spine morphogenesis and neuronal function of CA1 pyramidal neurons. Organotypic hippocampal brain slice cultures are commonly used as a model system to investigate the morphology and synaptic function of neurons, mainly because they allow for the long-term examination of neurons in a preparation where the gross cellular architecture of the hippocampus is retained. In addition, maintenance of the trisynaptic circuitry in hippocampal slices enables the study of synaptic connections. Today, a multitude of gene transfer methods for postmitotic neurons in brain slices are available to easily manipulate and scrutinize the involvement of signaling molecules, such as Rho GTPases, in specific cellular processes in this system. This chapter covers techniques detailing the preparation and culturing of organotypic hippocampal brain slices, as well as the production and injection of lentivirus into brain slices.
Assuntos
Encéfalo/metabolismo , Proteínas do Citoesqueleto/fisiologia , Proteínas Ativadoras de GTPase/fisiologia , Proteínas Nucleares/fisiologia , Animais , Proteínas do Citoesqueleto/isolamento & purificação , Proteínas Ativadoras de GTPase/isolamento & purificação , Técnicas de Transferência de Genes , Vetores Genéticos , Hipocampo/fisiologia , Hipocampo/virologia , Humanos , Lentivirus , Proteínas Nucleares/isolamento & purificação , Técnicas de Cultura de Órgãos/métodos , RatosRESUMO
Of 11 genes involved in nonspecific X-linked mental retardation (MRX), three encode regulators or effectors of the Rho GTPases, suggesting an important role for Rho signaling in cognitive function. It remains unknown, however, how mutations in Rho-linked genes lead to MRX. Here we report that oligophrenin-1, a Rho-GTPase activating protein that is absent in a family affected with MRX, is required for dendritic spine morphogenesis. Using RNA interference and antisense RNA approaches, we show that knock-down of oligophrenin-1 levels in CA1 neurons in rat hippocampal slices significantly decreases spine length. This phenotype can be recapitulated using an activated form of RhoA and rescued by inhibiting Rho-kinase, indicating that reduced oligophrenin-1 levels affect spine length by increasing RhoA and Rho-kinase activities. We further demonstrate an interaction between oligophrenin-1 and the postsynaptic adaptor protein Homer. Our findings provide the first insight into how mutations in a Rho-linked MRX gene may compromise neuronal function.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Sinapses/metabolismo , Animais , Tamanho Celular/fisiologia , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Dendritos/metabolismo , Regulação para Baixo , Proteínas Ativadoras de GTPase/deficiência , Proteínas Ativadoras de GTPase/genética , Hipocampo/citologia , Deficiência Intelectual Ligada ao Cromossomo X/genética , Camundongos , Morfogênese , Neurônios/citologia , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Técnicas de Cultura de Órgãos , Interferência de RNA/fisiologia , RNA Antissenso/fisiologia , Ratos , TransfecçãoRESUMO
CNS cortical histogenesis depends on polarity signaling pathways that regulate cell adhesion and motility. Here we report that conditional deletion of the Rho GTPase Cdc42 in cerebellar granule cell precursors (GCPs) results in abnormalities in cerebellar foliation revealed by iDISCO clearing methodology, a loss of columnar organization of proliferating GCPs in the external germinal layer (EGL), disordered parallel fiber organization in the molecular layer (ML), and a failure to extend a leading process and form a neuron-glial junction during migration along Bergmann glia (BG). Notably, GCPs lacking Cdc42 had a multi-polar morphology and slowed migration rate. In addition, secondary defects occurred in BG development and organization, especially in the lateral cerebellar hemispheres. By phosphoproteomic analysis, affected Cdc42 targets included regulators of the cytoskeleton, cell adhesion and polarity. Thus, Cdc42 signaling pathways are critical regulators of GCP polarity and the formation of neuron-glial junctions during cerebellar development.
RESUMO
Alternative polyadenylation (APA) regulates mRNA translation, stability, and protein localization. However, it is unclear to what extent APA regulates these processes uniquely in specific cell types. Using a new technique, cTag-PAPERCLIP, we discovered significant differences in APA between the principal types of mouse cerebellar neurons, the Purkinje and granule cells, as well as between proliferating and differentiated granule cells. Transcripts that differed in APA in these comparisons were enriched in key neuronal functions and many differed in coding sequence in addition to 3'UTR length. We characterize Memo1, a transcript that shifted from expressing a short 3'UTR isoform to a longer one during granule cell differentiation. We show that Memo1 regulates granule cell precursor proliferation and that its long 3'UTR isoform is targeted by miR-124, contributing to its downregulation during development. Our findings provide insight into roles for APA in specific cell types and establish a platform for further functional studies.
Assuntos
Regiões 3' não Traduzidas , Neurônios/fisiologia , Poliadenilação , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Cerebelo/citologia , CamundongosRESUMO
We review studies on the polarity of developing cerebellar granule, showing that the centrosome localizes to the pole of the neuron that extrudes the nascent axon, and the Rho GTPase Cdc42 (cell division cycle 42) activates the mPar6alpha/Par3 (Par for partitioning defective) complex to coordinate actin dynamics in the growth cone. Subsequently, mPar6alpha signaling controls the migration of immature granule neurons down the Bergmann glial fibers into the internal granule cell layer in which they establish synaptic connections.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Movimento Celular/fisiologia , Proteínas de Membrana/fisiologia , Neuroglia/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Cerebelo/citologia , Cerebelo/fisiologia , Humanos , Neuroglia/citologia , Isoformas de Proteínas , Proteínas/fisiologia , Sinapses/fisiologiaRESUMO
The formation and elaboration of axonal and dendritic morphologies are fundamental aspects of neuronal polarization critical for information processing. In general, developing CNS neurons elaborate one axon and multiple dendrites in response to intracellular and extracellular cues, so as to transmit and receive information, respectively. The molecular mechanisms underlying axon-dendrite polarity are complex and involve the integration of numerous signaling pathways that impinge on the cytoskeleton. One group of proteins, the Rho GTPases, has emerged as key integrators of environmental cues to regulate the underlying axonal and dendritic cytoskeletons. Here, we discuss the role of regulators of the Rac1 GTPase in axon development and highlight the importance of both actin and microtubule remodeling in this process.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/citologia , Neurônios/enzimologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Humanos , Neurônios/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Although mechanisms underlying early steps in cerebellar development are known, evidence is lacking on genetic and epigenetic changes during the establishment of the synaptic circuitry. Using metagene analysis, we report pivotal changes in multiple reactomes of epigenetic pathway genes in cerebellar granule cells (GCs) during circuit formation. During this stage, Tet genes are upregulated and vitamin C activation of Tet enzymes increases the levels of 5-hydroxymethylcytosine (5hmC) at exon start sites of upregulated genes, notably axon guidance genes and ion channel genes. Knockdown of Tet1 and Tet3 by RNAi in ex vivo cerebellar slice cultures inhibits dendritic arborization of developing GCs, a critical step in circuit formation. These findings demonstrate a role for Tet genes and chromatin remodeling genes in the formation of cerebellar circuitry.
Assuntos
Encéfalo/metabolismo , Diferenciação Celular/genética , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Proto-Oncogênicas/genética , Animais , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Metilação de DNA , Dioxigenases , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologiaRESUMO
Although casein kinase 1δ (CK1δ) is at the center of multiple signaling pathways, its role in the expansion of CNS progenitor cells is unknown. Using mouse cerebellar granule cell progenitors (GCPs) as a model for brain neurogenesis, we demonstrate that the loss of CK1δ or treatment of GCPs with a highly selective small molecule inhibits GCP expansion. In contrast, CK1δ overexpression increases GCP proliferation. Thus, CK1δ appears to regulate GCP neurogenesis. CK1δ is targeted for proteolysis via the anaphase-promoting complex/cyclosome (APC/C(Cdh1)) ubiquitin ligase, and conditional deletion of the APC/C(Cdh1) activator Cdh1 in cerebellar GCPs results in higher levels of CK1δ. APC/C(Cdh1) also downregulates CK1δ during cell-cycle exit. Therefore, we conclude that APC/C(Cdh1) controls CK1δ levels to balance proliferation and cell-cycle exit in the developing CNS. Similar studies in medulloblastoma cells showed that CK1δ holds promise as a therapeutic target.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/biossíntese , Caseína Quinase Idelta/biossíntese , Proteínas Cdh1/biossíntese , Sistema Nervoso Central/crescimento & desenvolvimento , Neurogênese/genética , Ciclossomo-Complexo Promotor de Anáfase/genética , Animais , Caseína Quinase Idelta/genética , Proteínas Cdh1/genética , Ciclo Celular/genética , Proliferação de Células/genética , Sistema Nervoso Central/metabolismo , Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Humanos , Camundongos , Neurônios/metabolismo , Interferência de RNA , Transdução de SinaisRESUMO
During normal cerebellar development, the remarkable expansion of granule cell progenitors (GCPs) generates a population of granule neurons that outnumbers the total neuronal population of the cerebral cortex, and provides a model for identifying signaling pathways that may be defective in medulloblastoma. While many studies focus on identifying pathways that promote growth of GCPs, a critical unanswered question concerns the identification of signaling pathways that block mitogenic stimulation and induce early steps in differentiation. Here we identify WNT3 as a novel suppressor of GCP proliferation during cerebellar development and an inhibitor of medulloblastoma growth in mice. WNT3, produced in early postnatal cerebellum, inhibits GCP proliferation by down-regulating pro-proliferative target genes of the mitogen Sonic Hedgehog (SHH) and the bHLH transcription factor Atoh1. WNT3 suppresses GCP growth through a non-canonical Wnt signaling pathway, activating prototypic mitogen-activated protein kinases (MAPKs), the Ras-dependent extracellular-signal-regulated kinases 1/2 (ERK1/2) and ERK5, instead of the classical ß-catenin pathway. Inhibition of MAPK activity using a MAPK kinase (MEK) inhibitor reversed the inhibitory effect of WNT3 on GCP proliferation. Importantly, WNT3 inhibits proliferation of medulloblastoma tumor growth in mouse models by a similar mechanism. Thus, the present study suggests a novel role for WNT3 as a regulator of neurogenesis and repressor of neural tumors.
Assuntos
Transformação Celular Neoplásica/metabolismo , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células-Tronco Neurais/metabolismo , Proteína Wnt3/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Cerebelares/genética , Modelos Animais de Doenças , Ativação Enzimática , Expressão Gênica , Sistema de Sinalização das MAP Quinases , Meduloblastoma/genética , Camundongos , Camundongos Knockout , Células-Tronco Neurais/patologia , Transdução de Sinais , Transdução Genética , Transgenes , Proteína Wnt3/genéticaRESUMO
The architectonics of the mammalian brain arise from a remarkable range of directed cell migrations, which orchestrate the emergence of cortical neuronal layers and pattern brain circuitry. At different stages of cortical histogenesis, specific modes of cell motility are essential to the stepwise formation of cortical architecture. These movements range from interkinetic nuclear movements in the ventricular zone, to migrations of early-born, postmitotic polymorphic cells into the preplate, to the radial migration of precursors of cortical output neurons across the thickening cortical wall, and the vast, tangential migrations of interneurons from the basal forebrain into the emerging cortical layers. In all cases, actomyosin motors act in concert with cell adhesion receptor systems to provide the force and traction needed for forward movement. As key regulators of actin and microtubule cytoskeletons, cell polarity, and adhesion, the Rho GTPases play critical roles in CNS neuronal migration. This review will focus on the different types of migration in the developing neocortex and cerebellar cortex, and the role of the Rho GTPases, their regulators and effectors in these CNS migrations, with particular emphasis on their involvement in radial migration.
Assuntos
Encéfalo/crescimento & desenvolvimento , Movimento Celular/fisiologia , Neurogênese/fisiologia , Neurônios/citologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Encéfalo/enzimologia , Humanos , Neurônios/metabolismoRESUMO
Lamination of cortical regions of the vertebrate brain depends on glial-guided neuronal migration. The conserved polarity protein Par6alpha localizes to the centrosome and coordinates forward movement of the centrosome and soma in migrating neurons. The cytoskeletal components that produce this unique form of cell polarity and their relationship to polarity signaling cascades are unknown. We show that F-actin and Myosin II motors are enriched in the neuronal leading process and that Myosin II activity is necessary for leading process actin dynamics. Inhibition of Myosin II decreased the speed of centrosome and somal movement, whereas Myosin II activation increased coordinated movement. Ectopic expression or silencing of Par6alpha inhibited Myosin II motors by decreasing Myosin light-chain phosphorylation. These findings suggest leading-process Myosin II may function to "pull" the centrosome and soma forward during glial-guided migration by a mechanism involving the conserved polarity protein Par6alpha.
Assuntos
Actinas/metabolismo , Movimento Celular/fisiologia , Centrossomo/fisiologia , Miosina Tipo II/metabolismo , Neuroglia/fisiologia , Neurônios/citologia , Actinas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Recém-Nascidos , Proteínas de Ciclo Celular/metabolismo , Movimento Celular/genética , Polaridade Celular/fisiologia , Células Cultivadas , Cerebelo/citologia , Recuperação de Fluorescência Após Fotodegradação/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Miosina Tipo II/genética , Neurônios/fisiologia , Transporte Proteico/genética , RNA Interferente Pequeno/farmacologia , Transfecção/métodos , Tubulina (Proteína)/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
The diversity of neuronal morphologies and the complexity of synaptic connections in the mammalian brain provide striking examples of cell polarity. Over the past decade, the identification of the PAR (for partitioning-defective) proteins, their function in polarity in the Caenorhabditis elegans zygote, and the conservation of polarity proteins related to the PAR polarity complex in Drosophila and vertebrates, kindled intense interest in polarity pathways. Although the existence of a conserved polarity protein complex does not prove that these proteins function the same way in different systems, the emergence of an evolutionarily conserved mechanism that regulates cell polarity provides an exciting opportunity to define the role of polarity proteins in the generation of the diverse array of cell types and patterns of connections in the developing mammalian brain. This review addresses emerging genetic, molecular genetic, biochemical, and cell biological approaches and mechanisms that control neuronal polarity, focusing on recent studies using the neonatal cerebellum and hippocampus as model systems.
Assuntos
Polaridade Celular/fisiologia , Sistema Nervoso Central/embriologia , Neurônios/fisiologia , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Humanos , Modelos Biológicos , Proteínas Serina-Treonina QuinasesRESUMO
Our brain serves as a center for cognitive function and neurons within the brain relay and store information about our surroundings and experiences. Modulation of this complex neuronal circuitry allows us to process that information and respond appropriately. Proper development of neurons is therefore vital to the mental health of an individual, and perturbations in their signaling or morphology are likely to result in cognitive impairment. The development of a neuron requires a series of steps that begins with migration from its birth place and initiation of process outgrowth, and ultimately leads to differentiation and the formation of connections that allow it to communicate with appropriate targets. Over the past several years, it has become clear that the Rho family of GTPases and related molecules play an important role in various aspects of neuronal development, including neurite outgrowth and differentiation, axon pathfinding, and dendritic spine formation and maintenance. Given the importance of these molecules in these processes, it is therefore not surprising that mutations in genes encoding a number of regulators and effectors of the Rho GTPases have been associated with human neurological diseases. This review will focus on the role of the Rho GTPases and their associated signaling molecules throughout neuronal development and discuss how perturbations in Rho GTPase signaling may lead to cognitive disorders.
Assuntos
Neurônios/citologia , Proteínas rho de Ligação ao GTP/fisiologia , Animais , Extensões da Superfície Celular , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/genética , Humanos , Doença dos Neurônios Motores/etiologia , Doença dos Neurônios Motores/genética , Neurônios/ultraestrutura , Proteínas rho de Ligação ao GTP/genéticaRESUMO
A consistent feature of neurons in patients with mental retardation is abnormal dendritic structure and/or alterations in dendritic spine morphology. Deficits in the regulation of the dendritic cytoskeleton affect both the structure and function of dendrites and synapses and are believed to underlie mental retardation in some instances. In support of this, there is good evidence that alterations in signaling pathways involving the Rho family of small GTPases, key regulators of the actin and microtubule cytoskeletons, contribute to both syndromic and nonsyndromic mental retardation disorders. Because the Rho GTPases have been shown to play increasingly well-defined roles in determining dendrite and dendritic spine development and morphology, Rho signaling has been suggested to be important for normal cognition. The purpose of this review is to summarize recent data on the Rho GTPases pertaining to dendrite and dendritic spine morphogenesis, as well as to highlight their involvement in mental retardation resulting from a variety of genetic mutations within regulators and effectors of these molecules.