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1.
Nat Commun ; 11(1): 4038, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788587

RESUMO

Asparaginyl-tRNA synthetase1 (NARS1) is a member of the ubiquitously expressed cytoplasmic Class IIa family of tRNA synthetases required for protein translation. Here, we identify biallelic missense and frameshift mutations in NARS1 in seven patients from three unrelated families with microcephaly and neurodevelopmental delay. Patient cells show reduced NARS1 protein, impaired NARS1 activity and impaired global protein synthesis. Cortical brain organoid modeling shows reduced proliferation of radial glial cells (RGCs), leading to smaller organoids characteristic of microcephaly. Single-cell analysis reveals altered constituents of both astrocytic and RGC lineages, suggesting a requirement for NARS1 in RGC proliferation. Our findings demonstrate that NARS1 is required to meet protein synthetic needs and to support RGC proliferation in human brain development.


Assuntos
Aspartato-tRNA Ligase/deficiência , Aspartato-tRNA Ligase/genética , Córtex Cerebral/patologia , Microcefalia/genética , Células-Tronco Neurais/patologia , Organoides/patologia , Aminoacil-RNA de Transferência/genética , Adolescente , Adulto , Sequência de Bases , Diferenciação Celular , Proliferação de Células , Tamanho Celular , Sobrevivência Celular , Criança , Família , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Antígeno Ki-67/metabolismo , Masculino , Mutação/genética , Células-Tronco Neurais/metabolismo , Neuroglia/metabolismo , Linhagem , Adulto Jovem
2.
Virology ; 548: 17-24, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838939

RESUMO

The demyelinating disease progressive multifocal leukoencephalopathy (PML) is caused by the human polyomavirus, JCPyV, under conditions of prolonged immunosuppression. Initial infection is asymptomatic, and the virus establishes lifelong persistence in the host. Following the loss of immune surveillance, the virus can traffic to the central nervous system and infect oligodendrocytes to cause demyelination and PML. The mechanisms involved in glial cell infection are not completely understood. In a screen for N-glycosylated proteins that influence JCPyV pathology, we identified Adipocyte Plasma Membrane Associated Protein (APMAP) as a host cell modulator of JCPyV infection. The removal of APMAP by small interfering siRNA as well as by CRISPR-Cas9 gene editing resulted in a significant decrease in JCPyV infection. Exogenous expression of APMAP in APMAP knockout cell lines rescued susceptibility to infection. These data suggest that virus infection of glial cells is dependent on APMAP.


Assuntos
Vírus JC/fisiologia , Neuroglia/metabolismo , Infecções por Polyomavirus/metabolismo , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Vírus JC/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana , Neuroglia/virologia , Oligodendroglia/metabolismo , Oligodendroglia/virologia , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/virologia
3.
Mol Cell ; 79(3): 376-389.e8, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32640193

RESUMO

Activation of dual-specificity tyrosine-phosphorylation-regulated kinases 1A and 1B (DYRK1A and DYRK1B) requires prolyl hydroxylation by PHD1 prolyl hydroxylase. Prolyl hydroxylation of DYRK1 initiates a cascade of events leading to the release of molecular constraints on von Hippel-Lindau (VHL) ubiquitin ligase tumor suppressor function. However, the proline residue of DYRK1 targeted by hydroxylation and the role of prolyl hydroxylation in tyrosine autophosphorylation of DYRK1 are unknown. We found that a highly conserved proline in the CMGC insert of the DYRK1 kinase domain is hydroxylated by PHD1, and this event precedes tyrosine autophosphorylation. Mutation of the hydroxylation acceptor proline precludes tyrosine autophosphorylation and folding of DYRK1, resulting in a kinase unable to preserve VHL function and lacking glioma suppression activity. The consensus proline sequence is shared by most CMGC kinases, and prolyl hydroxylation is essential for catalytic activation. Thus, formation of prolyl-hydroxylated intermediates is a novel mechanism of kinase maturation and likely a general mechanism of regulation of CMGC kinases in eukaryotes.


Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Isoenzimas/genética , Prolina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Glioma/patologia , Células HEK293 , Xenoenxertos , Humanos , Hidroxilação , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Camundongos , Camundongos Nus , Proteína Quinase 14 Ativada por Mitógeno/química , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Mutação , Neuroglia/metabolismo , Neuroglia/patologia , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(29): 17269-17277, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32611818

RESUMO

Traumatic brain injury (TBI) is the strongest environmental risk factor for the accelerated development of neurodegenerative diseases. There are currently no therapeutics to address this due to lack of insight into mechanisms of injury progression, which are challenging to study in mammalian models. Here, we have developed and extensively characterized a head-specific approach to TBI in Drosophila, a powerful genetic system that shares many conserved genes and pathways with humans. The Drosophila TBI (dTBI) device inflicts mild, moderate, or severe brain trauma by precise compression of the head using a piezoelectric actuator. Head-injured animals display features characteristic of mammalian TBI, including severity-dependent ataxia, life span reduction, and brain degeneration. Severe dTBI is associated with cognitive decline and transient glial dysfunction, and stimulates antioxidant, proteasome, and chaperone activity. Moreover, genetic or environmental augmentation of the stress response protects from severe dTBI-induced brain degeneration and life span deficits. Together, these findings present a tunable, head-specific approach for TBI in Drosophila that recapitulates mammalian injury phenotypes and underscores the ability of the stress response to mitigate TBI-induced brain degeneration.


Assuntos
Lesões Encefálicas Traumáticas/metabolismo , Encéfalo/metabolismo , Drosophila/fisiologia , Neuroglia/metabolismo , Animais , Comportamento Animal , Lesões Encefálicas Traumáticas/diagnóstico por imagem , Lesões Encefálicas Traumáticas/patologia , Modelos Animais de Doenças , Cabeça , Humanos , Masculino , Doenças Neurodegenerativas/metabolismo , Neuroglia/patologia , Estresse Fisiológico
5.
Life Sci ; 258: 118099, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32682917

RESUMO

Although emerging evidence has highlighted the heterogeneities of astrocytes under physiological versus pathological conditions, little is known regarding these processes in different brain regions during stress. Thus, the present study established a mouse model of chronic social defeat stress (CSDS) and isolated astrocytes from the medial prefrontal cortex (mPFC) and hippocampus. The results revealed dramatic A1-specific (neurotoxic phenotype) astrocytic responses, depressive-like behaviors, and significant inhibition of neuronal activities in both the mPFC and hippocampus according to electrophysiological data. Subsequently, astrocytes in the mPFC and hippocampus of CSDS mice were suppressed and this reversed the astrocytic responses and rescued depressive-like behaviors. Furthermore, when astrocytes were activated in the mPFC and hippocampus in healthy mice, there was a non-specific phenotypic activation of astrocytes in the absence of depressive-like behaviors. Next, microglia were depleted and the mice subsequently performed in the CSDS model; this reduced astrocyte responses and restored depressive-like behaviors. On the other hand, when microglia were depleted but astrocytes were activated in CSDS mice, this abolished the restoration of microglia depletion-induced depressive-like behaviors. Taken together, these results indicate that neuronal inhibition by astrocytes in the mPFC and hippocampus contributed to depressive-like behaviors mediated by activated microglia. This study provides evidence regarding the interaction of microglia and astrocytes during stress and how that relationship can trigger depressive-like behaviors.


Assuntos
Astrócitos/patologia , Comportamento Animal , Depressão/psicologia , Neurônios/patologia , Estresse Psicológico/patologia , Animais , Doença Crônica , Hipocampo/patologia , Locomoção , Masculino , Camundongos , Inibição Neural , Neuroglia/metabolismo , Córtex Pré-Frontal/patologia
6.
PLoS Genet ; 16(6): e1008312, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32598344

RESUMO

Drosophila circadian behavior relies on the network of heterogeneous groups of clock neurons. Short- and long-range signaling within the pacemaker circuit coordinates molecular and neural rhythms of clock neurons to generate coherent behavioral output. The neurochemistry of circadian behavior is complex and remains incompletely understood. Here we demonstrate that the gaseous messenger nitric oxide (NO) is a signaling molecule linking circadian pacemaker to rhythmic locomotor activity. We show that mutants lacking nitric oxide synthase (NOS) have behavioral arrhythmia in constant darkness, although molecular clocks in the main pacemaker neurons are unaffected. Behavioral phenotypes of mutants are due in part to the malformation of neurites of the main pacemaker neurons, s-LNvs. Using cell-type selective and stage-specific gain- and loss-of-function of NOS, we also demonstrate that NO secreted from diverse cellular clusters affect behavioral rhythms. Furthermore, we identify the perineurial glia, one of the two glial subtypes that form the blood-brain barrier, as the major source of NO that regulates circadian locomotor output. These results reveal for the first time the critical role of NO signaling in the Drosophila circadian system and highlight the importance of neuro-glial interaction in the neural circuit output.


Assuntos
Relógios Circadianos , Proteínas de Drosophila/genética , Neuroglia/metabolismo , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Proteínas de Ligação a RNA/genética , Animais , Drosophila , Mutação com Ganho de Função , Locomoção , Mutação com Perda de Função
7.
PLoS One ; 15(6): e0232308, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32530962

RESUMO

Zebrafish have the ability to regenerate damaged cells and tissues by activating quiescent stem and progenitor cells or reprogramming differentiated cells into regeneration-competent precursors. Proliferation among the cells that will functionally restore injured tissues is a fundamental biological process underlying regeneration. Midkine-a is a cytokine growth factor, whose expression is strongly induced by injury in a variety of tissues across a range of vertebrate classes. Using a zebrafish Midkine-a loss of function mutant, we evaluated regeneration of caudal fin, extraocular muscle and retinal neurons to investigate the function of Midkine-a during epimorphic regeneration. In wildtype zebrafish, injury among these tissues induces robust proliferation and rapid regeneration. In Midkine-a mutants, the initial proliferation in each of these tissues is significantly diminished or absent. Regeneration of the caudal fin and extraocular muscle is delayed; regeneration of the retina is nearly completely absent. These data demonstrate that Midkine-a is universally required in the signaling pathways that convert tissue injury into the initial burst of cell proliferation. Further, these data highlight differences in the molecular mechanisms that regulate epimorphic regeneration in zebrafish.


Assuntos
Midkina/metabolismo , Regeneração/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Nadadeiras de Animais/fisiologia , Animais , Animais Geneticamente Modificados/metabolismo , Diferenciação Celular , Proliferação de Células , Midkina/genética , Mutagênese , Neuroglia/citologia , Neuroglia/metabolismo , Músculos Oculomotores/fisiologia , Neurônios Retinianos/fisiologia , Proteínas de Peixe-Zebra/genética
8.
Nat Commun ; 11(1): 2898, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32518258

RESUMO

The sequential generation of layer-specific cortical neurons requires radial glia cells (RGCs) to precisely balance self-renewal and lineage commitment. While specific cell-cycle phases have been associated with these decisions, the mechanisms linking the cell-cycle machinery to cell-fate commitment remain obscure. Using single-cell RNA-sequencing, we find that the strongest transcriptional signature defining multipotent RGCs is that of G2/M-phase, and particularly CYCLIN-B1/2, while lineage-committed progenitors are enriched in G1/S-phase genes, including CYCLIN-D1. These data also reveal cell-surface markers that allow us to isolate RGCs and lineage-committed progenitors, and functionally confirm the relationship between cell-cycle phase enrichment and cell fate competence. Finally, we use cortical electroporation to demonstrate that CYCLIN-B1/2 cooperate with CDK1 to maintain uncommitted RGCs by activating the NOTCH pathway, and that CYCLIN-D1 promotes differentiation. Thus, this work establishes that cell-cycle phase-specific regulators act in opposition to coordinate the self-renewal and lineage commitment of RGCs via core stem cell regulatory pathways.


Assuntos
Ciclina B1/fisiologia , Ciclina B2/fisiologia , Ciclina D1/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Animais , Proteína Quinase CDC2/fisiologia , Ciclo Celular , Diferenciação Celular , Linhagem da Célula , Separação Celular , Córtex Cerebral/embriologia , Feminino , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Células-Tronco/citologia
9.
Nature ; 582(7811): 246-252, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32499648

RESUMO

A wealth of specialized neuroendocrine command systems intercalated within the hypothalamus control the most fundamental physiological needs in vertebrates1,2. Nevertheless, we lack a developmental blueprint that integrates the molecular determinants of neuronal and glial diversity along temporal and spatial scales of hypothalamus development3. Here we combine single-cell RNA sequencing of 51,199 mouse cells of ectodermal origin, gene regulatory network (GRN) screens in conjunction with genome-wide association study-based disease phenotyping, and genetic lineage reconstruction to show that nine glial and thirty-three neuronal subtypes are generated by mid-gestation under the control of distinct GRNs. Combinatorial molecular codes that arise from neurotransmitters, neuropeptides and transcription factors are minimally required to decode the taxonomical hierarchy of hypothalamic neurons. The differentiation of γ-aminobutyric acid (GABA) and dopamine neurons, but not glutamate neurons, relies on quasi-stable intermediate states, with a pool of GABA progenitors giving rise to dopamine cells4. We found an unexpected abundance of chemotropic proliferation and guidance cues that are commonly implicated in dorsal (cortical) patterning5 in the hypothalamus. In particular, loss of SLIT-ROBO signalling impaired both the production and positioning of periventricular dopamine neurons. Overall, we identify molecular principles that shape the developmental architecture of the hypothalamus and show how neuronal heterogeneity is transformed into a multimodal neural unit to provide virtually infinite adaptive potential throughout life.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hipotálamo/citologia , Hipotálamo/embriologia , Morfogênese , Animais , Diferenciação Celular , Linhagem da Célula , Dopamina/metabolismo , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Ectoderma/citologia , Ectoderma/metabolismo , Feminino , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Ácido Glutâmico/metabolismo , Hipotálamo/metabolismo , Masculino , Camundongos , Morfogênese/genética , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Neuropeptídeos/metabolismo , Neurotransmissores/metabolismo , Receptores Imunológicos/metabolismo , Regulon/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ácido gama-Aminobutírico/metabolismo
10.
J Vis Exp ; (160)2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32568252

RESUMO

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.


Assuntos
Sistema Nervoso Central/metabolismo , Neuroglia/metabolismo , Animais , Técnicas de Cultura de Células , Camundongos , Neuroglia/citologia
11.
Science ; 369(6503): 546-550, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32554627

RESUMO

The neocortex has expanded during mammalian evolution. Overexpression studies in developing mouse and ferret neocortex have implicated the human-specific gene ARHGAP11B in neocortical expansion, but the relevance for primate evolution has been unclear. Here, we provide functional evidence that ARHGAP11B causes expansion of the primate neocortex. ARHGAP11B expressed in fetal neocortex of the common marmoset under control of the gene's own (human) promoter increased the numbers of basal radial glia progenitors in the marmoset outer subventricular zone, increased the numbers of upper-layer neurons, enlarged the neocortex, and induced its folding. Thus, the human-specific ARHGAP11B drives changes in development in the nonhuman primate marmoset that reflect the changes in evolution that characterize human neocortical development.


Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Neocórtex/embriologia , Animais , Animais Geneticamente Modificados , Callithrix , Feto , Proteínas Ativadoras de GTPase/genética , Humanos , Ventrículos Laterais/embriologia , Ventrículos Laterais/metabolismo , Neocórtex/anatomia & histologia , Neocórtex/metabolismo , Células-Tronco Neurais/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Tamanho do Órgão , Regiões Promotoras Genéticas
12.
J Vis Exp ; (159)2020 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-32510511

RESUMO

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague-Dawley rats. In this method, the neurons and glia were incubated in a 37 °C, 5% CO2 incubator for 5-7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by ß-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.


Assuntos
Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Neuroglia/citologia , Neurônios/citologia , Bexiga Urinária/citologia , Animais , Neuroglia/metabolismo , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley
13.
Nat Commun ; 11(1): 2123, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358558

RESUMO

Mammals differ in their regeneration potential after traumatic injury, which might be caused by species-specific regeneration programs. Here, we compared murine and human Schwann cell (SC) response to injury and developed an ex vivo injury model employing surgery-derived human sural nerves. Transcriptomic and lipid metabolism analysis of murine SCs following injury of sural nerves revealed down-regulation of lipogenic genes and regulator of lipid metabolism, including Pparg (peroxisome proliferator-activated receptor gamma) and S1P (sphingosine-1-phosphate). Human SCs failed to induce similar adaptations following ex vivo nerve injury. Pharmacological PPARg and S1P stimulation in mice resulted in up-regulation of lipid gene expression, suggesting a role in SCs switching towards a myelinating state. Altogether, our results suggest that murine SC switching towards a repair state is accompanied by transcriptome and lipidome adaptations, which are reduced in humans.


Assuntos
Metabolismo dos Lipídeos/fisiologia , Células de Schwann/citologia , Células de Schwann/metabolismo , Animais , Feminino , Humanos , Lisofosfolipídeos/metabolismo , Masculino , Camundongos , Bainha de Mielina/metabolismo , Regeneração Nervosa/genética , Regeneração Nervosa/fisiologia , Sistema Nervoso/citologia , Sistema Nervoso/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , PPAR gama/metabolismo , Sistema Nervoso Periférico/citologia , Sistema Nervoso Periférico/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo
14.
Nat Commun ; 11(1): 2138, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358570

RESUMO

Signaling molecules that regulate neurodevelopmental processes in the early postnatal subventricular zone (SVZ) are critical for proper brain development yet remain poorly characterized. Here, we report that Endothelin-1 (ET-1), a molecular component of the postnatal SVZ, promotes radial glial cell maintenance and proliferation in an autocrine manner via Notch signaling. Loss of ET-1 signaling increases neurogenesis and reduces oligodendrocyte progenitor cell proliferation (OPC) in the developing SVZ, thereby altering cellular output of the stem cell niche. We also show that ET-1 is required for increased neural stem cell and OPC proliferation in the adult mouse SVZ following demyelination. Lastly, high levels of ET-1 in the SVZ of patients with Cathepsin A-related arteriopathy with strokes and leukoencephalopathy correlate with an increased number of SVZ OPCs, suggesting ET-1's role as a regulator of glial progenitor proliferation may be conserved in humans. ET-1 signaling therefore presents a potential new therapeutic target for promoting SVZ-mediated cellular repair.


Assuntos
Endotelina-1/metabolismo , Sistema Nervoso/citologia , Sistema Nervoso/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Nicho de Células-Tronco/fisiologia , Animais , Proliferação de Células/fisiologia , Endotelina-1/genética , Humanos , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Nicho de Células-Tronco/genética
15.
Nat Commun ; 11(1): 1674, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32245946

RESUMO

Neurodevelopment requires precise regulation of gene expression, including post-transcriptional regulatory events such as alternative splicing and mRNA translation. However, translational regulation of specific isoforms during neurodevelopment and the mechanisms behind it remain unknown. Using RNA-seq analysis of mouse neocortical polysomes, here we report translationally repressed and derepressed mRNA isoforms during neocortical neurogenesis whose orthologs include risk genes for neurodevelopmental disorders. We demonstrate that the translation of distinct mRNA isoforms of the RNA binding protein (RBP), Elavl4, in radial glia progenitors and early neurons depends on its alternative 5' UTRs. Furthermore, 5' UTR-driven Elavl4 isoform-specific translation depends on upstream control by another RBP, Celf1. Celf1 regulation of Elavl4 translation dictates development of glutamatergic neurons. Our findings reveal a dynamic interplay between distinct RBPs and alternative 5' UTRs in neuronal development and underscore the risk of post-transcriptional dysregulation in co-occurring neurodevelopmental disorders.


Assuntos
Proteínas CELF1/metabolismo , Proteína Semelhante a ELAV 4/genética , Regulação da Expressão Gênica no Desenvolvimento , Neocórtex/crescimento & desenvolvimento , Neurogênese/genética , Regiões 5' não Traduzidas/genética , Processamento Alternativo , Animais , Linhagem Celular Tumoral , Feminino , Ácido Glutâmico/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Neocórtex/citologia , Células-Tronco Neurais/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Polirribossomos/metabolismo , Cultura Primária de Células , Biossíntese de Proteínas/genética , Isoformas de RNA/genética , RNA-Seq
16.
J Neurosci ; 40(17): 3374-3384, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32229518

RESUMO

Stress alters brain function by modifying the structure and function of neurons and astrocytes. The fine processes of astrocytes are critical for the clearance of neurotransmitters during synaptic transmission. Thus, experience-dependent remodeling of glial processes is anticipated to alter the output of neural circuits. However, the molecular mechanisms that underlie glial structural plasticity are not known. Here we show that a single exposure of male and female mice to an acute stress produced a long-lasting retraction of the lateral processes of cerebellar Bergmann glial cells. These cells express the GluA1 subunit of AMPA-type glutamate receptors, and GluA1 knockdown is known to shorten the length of glial processes. We found that stress reduced the level of GluA1 protein and AMPA receptor-mediated currents in Bergmann glial cells, and these effects were absent in mice devoid of CPEB3, a protein that binds to GluA1 mRNA and regulates GluA1 protein synthesis. Administration of a ß-adrenergic receptor blocker attenuated the reduction in GluA1, and deletion of adenylate cyclase 5 prevented GluA1 suppression. Therefore, stress suppresses GluA1 protein synthesis via an adrenergic/adenylyl cyclase/CPEB3 pathway, and reduces the length of astrocyte lateral processes. Our results identify a novel mechanism for GluA1 subunit plasticity in non-neuronal cells and suggest a previously unappreciated role for AMPA receptors in stress-induced astrocytic remodeling.SIGNIFICANCE STATEMENT Astrocytes play important roles in synaptic transmission by extending fine processes around synapses. In this study, we showed that a single exposure to an acute stress triggered a retraction of lateral/fine processes in mouse cerebellar astrocytes. These astrocytes express GluA1, a glutamate receptor subunit known to lengthen astrocyte processes. We showed that astrocytic structural changes are associated with a reduction of GluA1 protein levels. This requires activation of ß-adrenergic receptors and is triggered by noradrenaline released during stress. We identified adenylyl cyclase 5, an enzyme that elevates cAMP levels, as a downstream effector and found that lowering GluA1 levels depends on CPEB3 proteins that bind to GluA1 mRNA. Therefore, stress regulates GluA1 protein synthesis via an adrenergic/adenylyl cyclase/CPEB3 pathway in astrocytes and remodels their fine processes.


Assuntos
Adenilil Ciclases/metabolismo , Neuroglia/metabolismo , Plasticidade Neuronal/fisiologia , Angústia Psicológica , Proteínas de Ligação a RNA/metabolismo , Receptores de AMPA/metabolismo , Transdução de Sinais/fisiologia , Animais , Feminino , Masculino , Camundongos , Camundongos Knockout , Neuroglia/citologia , Neurônios/citologia , Neurônios/metabolismo , Proteínas de Ligação a RNA/genética , Transmissão Sináptica/fisiologia
17.
J Neurosci ; 40(17): 3360-3373, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32265259

RESUMO

The Drosophila nervous system is ensheathed by a layer of outer glial cells, the perineurial glia, and a specialized extracellular matrix, the neural lamella. The function of perineurial glial cells and how they interact with the extracellular matrix are just beginning to be elucidated. Integrin-based focal adhesion complexes link the glial membrane to the extracellular matrix, but little is known about integrin's regulators in the glia. The transmembrane Ig domain protein Basigin/CD147/EMMPRIN is highly expressed in the perineurial glia surrounding the Drosophila larval nervous system. Here we show that Basigin associates with integrin at the focal adhesions to uphold the structure of the glia-extracellular matrix sheath. Knockdown of Basigin in perineurial glia using RNAi results in significant shortening of the ventral nerve cord, compression of the glia and extracellular matrix in the peripheral nerves, and reduction in larval locomotion. We determined that Basigin is expressed in close proximity to integrin at the glial membrane, and that expression of the extracellular integrin-binding domain of Basigin is sufficient to rescue peripheral glial compression. We also found that a reduction in expression of integrin at the membrane rescues the ventral nerve cord shortening, peripheral glial compression, and locomotor phenotypes, and that reduction in the integrin-binding protein Talin can partially rescue glial compression. These results identify Basigin as a potential negative regulator of integrin in the glia, supporting proper glial and extracellular matrix ensheathment of the nervous system.SIGNIFICANCE STATEMENT The glial cells and extracellular matrix play important roles in supporting and protecting the nervous system, but the interactions between these components have not been well characterized. Our study identified expression of a conserved Ig superfamily protein, Basigin, at the glial membrane of Drosophila where it associates with the integrin-based focal adhesion complexes to ensure proper ensheathment of the CNS and PNS. Loss of Basigin in the glia results in an overall compression of the nervous system due to integrin dysregulation, which causes locomotor defects in the animals. This underlies the importance of glia-matrix communication for structural and functional support of the nervous system.


Assuntos
Proteínas de Drosophila/metabolismo , Integrinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Neuroglia/metabolismo , Nervos Periféricos/metabolismo , Animais , Adesão Celular/fisiologia , Drosophila melanogaster , Matriz Extracelular/metabolismo , Larva/metabolismo , Locomoção/fisiologia , Neuroglia/citologia , Nervos Periféricos/citologia , Interferência de RNA
18.
J Vis Exp ; (158)2020 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-32338650

RESUMO

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, and a ventricular zone niche, which generates the inhibitory GABAergic Purkinje cells, which are neurons that constitute the deep cerebellar nuclei and Bergman glia. Recently, a third stem cell niche has been described that arises as a secondary germinal zone from the ventricular zone niche. The cells of this niche are defined by the cell surface marker prominin-1 and are localized to the developing white matter of the postnatal cerebellum. This niche accounts for the late born molecular layer GABAergic interneurons along with postnatally generated cerebellar astrocytes. In addition to their developmental role, this niche is gaining translational importance in regards to its involvement in neurodegeneration and tumorigenesis. The biology of these cells has been difficult to decipher because of a lack of efficient techniques for their purification. Demonstrated here are efficient methods to purify, culture, and differentiate these postnatal cerebellar stem cells.


Assuntos
Antígeno AC133/metabolismo , Separação Celular/métodos , Cerebelo/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Astrócitos/metabolismo , Interneurônios/citologia , Camundongos , Neuroglia/metabolismo , Células de Purkinje/citologia
19.
Neuron ; 106(5): 743-758.e5, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32272058

RESUMO

The habenula complex is appreciated as a critical regulator of motivated and pathological behavioral states via its output to midbrain nuclei. Despite this, transcriptional definition of cell populations that comprise both the medial habenular (MHb) and lateral habenular (LHb) subregions in mammals remain undefined. To resolve this, we performed single-cell transcriptional profiling and highly multiplexed in situ hybridization experiments of the mouse habenula complex in naive mice and those exposed to an acute aversive stimulus. Transcriptionally distinct neuronal cell types identified within the MHb and LHb, were spatially defined, differentially engaged by aversive stimuli, and had distinct electrophysiological properties. Cell types identified in mice also displayed a high degree of transcriptional similarity to those previously described in zebrafish, highlighting the well-conserved nature of habenular cell types across the phylum. These data identify key molecular targets within habenular cell types and provide a critical resource for future studies.


Assuntos
Habenula/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Perfilação da Expressão Gênica , Ontologia Genética , Habenula/citologia , Camundongos , Microglia/citologia , Microglia/metabolismo , Neuroglia/citologia , Neurônios/citologia , Oligodendroglia/citologia , Oligodendroglia/metabolismo , RNA-Seq , Análise de Célula Única , Peixe-Zebra
20.
Nat Rev Neurol ; 16(4): 199-212, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203399

RESUMO

Most neurodegenerative diseases are characterized by the intracellular or extracellular aggregation of misfolded proteins such as amyloid-ß and tau in Alzheimer disease, α-synuclein in Parkinson disease, and TAR DNA-binding protein 43 in amyotrophic lateral sclerosis. Accumulating evidence from both human studies and disease models indicates that intercellular transmission and the subsequent templated amplification of these misfolded proteins are involved in the onset and progression of various neurodegenerative diseases. The misfolded proteins that are transferred between cells are referred to as 'pathological seeds'. Recent studies have made exciting progress in identifying the characteristics of different pathological seeds, particularly those isolated from diseased brains. Advances have also been made in our understanding of the molecular mechanisms that regulate the transmission process, and the influence of the host cell on the conformation and properties of pathological seeds. The aim of this Review is to summarize our current knowledge of the cell-to-cell transmission of pathological proteins and to identify key questions for future investigation.


Assuntos
Encéfalo/metabolismo , Doenças Neurodegenerativas/metabolismo , Agregação Patológica de Proteínas/metabolismo , Transporte Proteico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Transporte Axonal , Encéfalo/patologia , Comunicação Celular , Proteínas de Ligação a DNA/metabolismo , Endocitose , Exossomos/metabolismo , Predisposição Genética para Doença , Humanos , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Fusão de Membrana , Nanotubos , Doenças Neurodegenerativas/patologia , Neuroglia/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Agregação Patológica de Proteínas/patologia , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo
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