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1.
Elife ; 102021 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-34612812

RESUMO

The Ca2+-dependence of the priming, fusion, and replenishment of synaptic vesicles are fundamental parameters controlling neurotransmitter release and synaptic plasticity. Despite intense efforts, these important steps in the synaptic vesicles' cycle remain poorly understood due to the technical challenge in disentangling vesicle priming, fusion, and replenishment. Here, we investigated the Ca2+-sensitivity of these steps at mossy fiber synapses in the rodent cerebellum, which are characterized by fast vesicle replenishment mediating high-frequency signaling. We found that the basal free Ca2+ concentration (<200 nM) critically controls action potential-evoked release, indicating a high-affinity Ca2+ sensor for vesicle priming. Ca2+ uncaging experiments revealed a surprisingly shallow and non-saturating relationship between release rate and intracellular Ca2+ concentration up to 50 µM. The rate of vesicle replenishment during sustained elevated intracellular Ca2+ concentration exhibited little Ca2+-dependence. Finally, quantitative mechanistic release schemes with five Ca2+ binding steps incorporating rapid vesicle replenishment via parallel or sequential vesicle pools could explain our data. We thus show that co-existing high- and low-affinity Ca2+ sensors mediate priming, fusion, and replenishment of synaptic vesicles at a high-fidelity synapse.


Assuntos
Cálcio/metabolismo , Neurotransmissores/metabolismo , Sinapses/metabolismo , Animais , Transporte Biológico , Cerebelo/citologia , Cerebelo/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Transmissão Sináptica , Vesículas Sinápticas/metabolismo
2.
Neuron ; 109(18): 2981-2994.e5, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34534455

RESUMO

The role of the cerebellum in predictive motor control and coordination has been thoroughly studied during movements of a single body part. In the real world, however, actions are often more complex. Here, we show that a small area in the rostral anterior interpositus nucleus (rAIN) of the mouse cerebellum is responsible for generating a predictive motor synergy that serves to protect the eye by precisely coordinating muscles of the eyelid, neck, and forelimb. Within the rAIN region, we discovered a new functional category of neurons with unique properties specialized for control of motor synergies. These neurons integrated inhibitory cutaneous inputs from multiple parts of the body, and their activity was correlated with the vigor of the defensive motor synergy on a trial-by-trial basis. We propose that some regions of the cerebellum are organized in poly-somatotopic "action maps" to reduce dimensionality and simplify motor control during ethologically relevant behaviors.


Assuntos
Piscadela/fisiologia , Núcleos Cerebelares/química , Núcleos Cerebelares/fisiologia , Extremidades/fisiologia , Movimento/fisiologia , Animais , Núcleos Cerebelares/citologia , Cerebelo/química , Cerebelo/citologia , Cerebelo/fisiologia , Previsões , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Optogenética/métodos , Gravação em Vídeo/métodos
3.
Science ; 373(6558)2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34446581

RESUMO

Organ development is orchestrated by cell- and time-specific gene regulatory networks. In this study, we investigated the regulatory basis of mouse cerebellum development from early neurogenesis to adulthood. By acquiring snATAC-seq (single-nucleus assay for transposase accessible chromatin using sequencing) profiles for ~90,000 cells spanning 11 stages, we mapped cerebellar cell types and identified candidate cis-regulatory elements (CREs). We detected extensive spatiotemporal heterogeneity among progenitor cells and a gradual divergence in the regulatory programs of cerebellar neurons during differentiation. Comparisons to vertebrate genomes and snATAC-seq profiles for ∼20,000 cerebellar cells from the marsupial opossum revealed a shared decrease in CRE conservation during development and differentiation as well as differences in constraint between cell types. Our work delineates the developmental and evolutionary dynamics of gene regulation in cerebellar cells and provides insights into mammalian organ development.


Assuntos
Evolução Biológica , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Neurônios/fisiologia , Elementos Reguladores de Transcrição , Animais , Cerebelo/embriologia , Cromatina/genética , Cromatina/metabolismo , DNA Intergênico , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Masculino , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Neurogênese , Gambás/genética
4.
J Histochem Cytochem ; 69(8): 495-509, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34261344

RESUMO

Cyclic AMP element modulator (CREM) is a transcription factor best known for its intricate involvement in spermatogenesis. The CREM gene encodes for multiple protein isoforms, which can enhance or repress transcription of target genes. Recent studies have identified fusion genes, with CREM as a partner gene in many neoplastic diseases. EWSR1-CREM fusion genes have been found in several mesenchymal tumors and in salivary gland carcinoma. These genes encode fusion proteins that include the C-terminal DNA-binding domain of CREM. We used a transcriptomic approach and immunohistochemistry to study the expression of CREM isoforms that include DNA-binding domains across human tissues. We found that CREM protein is widely expressed in almost all normal human tissues. A transcriptomic analysis of normal tissues and cancer showed that transcription of CREM can be altered in tumors, suggesting that also wild-type CREM may be involved in cancer biology. The wide expression of CREM protein in normal human tissues and cancer may limit the utility of immunohistochemistry for identification of tumors with CREM fusions.


Assuntos
Glândulas Suprarrenais/metabolismo , Modulador de Elemento de Resposta do AMP Cíclico/genética , Neoplasias/genética , Placenta/metabolismo , Testículo/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Glândulas Suprarrenais/citologia , Linhagem Celular Tumoral , Cerebelo/citologia , Cerebelo/metabolismo , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Endométrio/citologia , Endométrio/metabolismo , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Imuno-Histoquímica , Masculino , Melanócitos/metabolismo , Melanócitos/patologia , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Especificidade de Órgãos , Células PC-3 , Placenta/citologia , Gravidez , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , Testículo/citologia
5.
Molecules ; 26(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069330

RESUMO

Angiotensin (Ang) II is well-known to have potent pro-oxidant and pro-inflammatory effects in the brain. Extensive crosstalk between the primary Ang II receptor, Ang type 1 receptor (AT1R), and the cannabinoid type 1 receptor (CB1R) has been demonstrated by various groups in the last decade. Since activation of glial CB1R has been demonstrated to play a key role in the resolution of inflammatory states, we investigated the role of Ang II (100 nM) and/or ACEA (10 nM), a potent CB1R-specific agonist in the regulation of inflammatory markers in astrocytes from spontaneously hypertensive rats (SHR) and Wistar rats. Astrocytes were cultured from brainstems and cerebellums of SHR and Wistar rats and assayed for IL1ß and IL10 gene expression and secreted fraction, in treated and non-treated cells, by employing qPCR and ELISA, respectively. mRNA expression of both IL10 and IL1ß were significantly elevated in untreated brainstem and cerebellar astrocytes isolated from SHR when compared to Wistar astrocytes. No changes were observed in the secreted fraction. While ACEA-treatment resulted in a significant increase in IL10 gene expression in Wistar brainstem astrocytes (Log2FC ≥ 1, p < 0.05), its effect in SHR brainstem astrocytes was diminished. Ang II treatment resulted in a strong inhibitory effect on IL10 gene expression in astrocytes from both brain regions of SHR and Wistar rats (Log2FC ≤ -1, p < 0.05), and an increase in IL1ß gene expression in brainstem astrocytes from both strains (Log2FC ≥ 1, p < 0.05). Co-treatment of Ang II and ACEA resulted in neutralization of Ang II-mediated effect in Wistar brainstem and cerebellar astrocytes, but not SHR astrocytes. Neither Ang II nor ACEA resulted in any significant changes in IL10 or IL1ß secreted proteins. These data suggest that Ang II and ACEA have opposing roles in the regulation of inflammatory gene signature in astrocytes isolated from SHR and Wistar rats. This however does not translate into changes in their secreted fractions.


Assuntos
Angiotensina II/farmacologia , Ácidos Araquidônicos/farmacologia , Expressão Gênica/efeitos dos fármacos , Interleucina-10/genética , Interleucina-18/genética , Animais , Astrócitos/efeitos dos fármacos , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Masculino , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos SHR , Ratos Wistar
6.
Sci Rep ; 11(1): 12959, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34155230

RESUMO

Current protocols for producing cerebellar neurons from human pluripotent stem cells (hPSCs) often rely on animal co-culture and mostly exist as monolayers, limiting their capability to recapitulate the complex processes in the developing cerebellum. Here, we employed a robust method, without the need for mouse co-culture to generate three-dimensional cerebellar organoids from hPSCs that display hallmarks of in vivo cerebellar development. Single-cell profiling followed by comparison to human and mouse cerebellar atlases revealed the presence and maturity of transcriptionally distinct populations encompassing major cerebellar cell types. Encapsulation with Matrigel aimed to provide more physiologically-relevant conditions through recapitulation of basement-membrane signalling, influenced both growth dynamics and cellular composition of the organoids, altering developmentally relevant gene expression programmes. We identified enrichment of cerebellar disease genes in distinct cell populations in the hPSC-derived cerebellar organoids. These findings ascertain xeno-free human cerebellar organoids as a unique model to gain insight into cerebellar development and its associated disorders.


Assuntos
Diferenciação Celular , Cerebelo/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Organoides/citologia , Idoso , Animais , Biomarcadores , Técnicas de Cultura de Células , Linhagem Celular , Colágeno , Biologia Computacional/métodos , Combinação de Medicamentos , Feminino , Perfilação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Laminina , Proteoglicanas , Células de Purkinje/metabolismo
7.
Elife ; 102021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33973524

RESUMO

Distinct populations of Purkinje cells (PCs) with unique molecular and connectivity features are at the core of the modular organization of the cerebellum. Previously, we showed that firing activity of PCs differs between ZebrinII-positive and ZebrinII-negative cerebellar modules (Zhou et al., 2014; Wu et al., 2019). Here, we investigate the timing and extent of PC differentiation during development in mice. We found that several features of PCs, including activity levels, dendritic arborization, axonal shape and climbing fiber input, develop differentially between nodular and anterior PC populations. Although all PCs show a particularly rapid development in the second postnatal week, anterior PCs typically have a prolonged physiological and dendritic maturation. In line herewith, younger mice exhibit attenuated anterior-dependent eyeblink conditioning, but faster nodular-dependent compensatory eye movement adaptation. Our results indicate that specific cerebellar regions have unique developmental timelines which match with their related, specific forms of cerebellum-dependent behaviors.


Assuntos
Cerebelo/fisiologia , Células de Purkinje/fisiologia , Potenciais de Ação/fisiologia , Animais , Animais Recém-Nascidos , Axônios/fisiologia , Cerebelo/citologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL
8.
Nat Commun ; 12(1): 2265, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859199

RESUMO

Nerve-glia (NG2) glia or oligodendrocyte precursor cells (OPCs) are distributed throughout the gray and white matter and generate myelinating cells. OPCs in white matter proliferate more than those in gray matter in response to platelet-derived growth factor AA (PDGF AA), despite similar levels of its alpha receptor (PDGFRα) on their surface. Here we show that the type 1 integral membrane protein neuropilin-1 (Nrp1) is expressed not on OPCs but on amoeboid and activated microglia in white but not gray matter in an age- and activity-dependent manner. Microglia-specific deletion of Nrp1 compromised developmental OPC proliferation in white matter as well as OPC expansion and subsequent myelin repair after acute demyelination. Exogenous Nrp1 increased PDGF AA-induced OPC proliferation and PDGFRα phosphorylation on dissociated OPCs, most prominently in the presence of suboptimum concentrations of PDGF AA. These findings uncover a mechanism of regulating oligodendrocyte lineage cell density that involves trans-activation of PDGFRα on OPCs via Nrp1 expressed by adjacent microglia.


Assuntos
Doenças Desmielinizantes/patologia , Microglia/fisiologia , Neuropilina-1/metabolismo , Células Precursoras de Oligodendrócitos/fisiologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Remielinização , Animais , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Corpo Caloso/citologia , Corpo Caloso/efeitos dos fármacos , Corpo Caloso/crescimento & desenvolvimento , Corpo Caloso/patologia , Doenças Desmielinizantes/induzido quimicamente , Modelos Animais de Doenças , Feminino , Humanos , Lisofosfatidilcolinas/administração & dosagem , Lisofosfatidilcolinas/toxicidade , Masculino , Camundongos , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/ultraestrutura , Microscopia Eletrônica de Transmissão , Modelos Animais , Bainha de Mielina/metabolismo , Neuropilina-1/genética , Oligodendroglia/fisiologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Cultura Primária de Células
9.
Nat Commun ; 12(1): 2380, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33888718

RESUMO

Diverse signaling complexes are precisely assembled at the presynaptic active zone for dynamic modulation of synaptic transmission and synaptic plasticity. Presynaptic GABAB-receptors nucleate critical signaling complexes regulating neurotransmitter release at most synapses. However, the molecular mechanisms underlying assembly of GABAB-receptor signaling complexes remain unclear. Here we show that neurexins are required for the localization and function of presynaptic GABAB-receptor signaling complexes. At four model synapses, excitatory calyx of Held synapses in the brainstem, excitatory and inhibitory synapses on hippocampal CA1-region pyramidal neurons, and inhibitory basket cell synapses in the cerebellum, deletion of neurexins rendered neurotransmitter release significantly less sensitive to GABAB-receptor activation. Moreover, deletion of neurexins caused a loss of GABAB-receptors from the presynaptic active zone of the calyx synapse. These findings extend the role of neurexins at the presynaptic active zone to enabling GABAB-receptor signaling, supporting the notion that neurexins function as central organizers of active zone signaling complexes.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Receptores de GABA-B/metabolismo , Sinapses/metabolismo , Animais , Tronco Encefálico/citologia , Tronco Encefálico/metabolismo , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/fisiologia , Região CA3 Hipocampal/citologia , Região CA3 Hipocampal/metabolismo , Proteínas de Ligação ao Cálcio/genética , Cerebelo/citologia , Cerebelo/metabolismo , Camundongos , Camundongos Knockout , Modelos Animais , Proteínas do Tecido Nervoso/genética , Moléculas de Adesão de Célula Nervosa/genética , Plasticidade Neuronal/fisiologia , Técnicas de Patch-Clamp , Células Piramidais/metabolismo , Técnicas Estereotáxicas , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismo
10.
Int J Mol Sci ; 22(9)2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33919387

RESUMO

The EBI2 receptor regulates the immune system and is expressed in various immune cells including B and T lymphocytes. It is also expressed in astrocytes in the central nervous system (CNS) where it regulates pro-inflammatory cytokine release, cell migration and protects from chemically induced demyelination. Its signaling and expression are implicated in various diseases including multiple sclerosis, where its expression is increased in infiltrating immune cells in the white matter lesions. Here, for the first time, the EBI2 protein in the CNS cells in the human brain was examined. The function of the receptor in MO3.13 oligodendrocytes, as well as its role in remyelination in organotypic cerebellar slices, were investigated. Human brain sections were co-stained for EBI2 receptor and various markers of CNS-specific cells and the human oligodendrocyte cell line MO3.13 was used to investigate changes in EBI2 expression and cellular migration. Organotypic cerebellar slices prepared from wild-type and cholesterol 25-hydroxylase knock-out mice were used to study remyelination following lysophosphatidylcholine (LPC)-induced demyelination. The data showed that EBI2 receptor is present in OPCs but not in myelinating oligodendrocytes in the human brain and that EBI2 expression is temporarily upregulated in maturing MO3.13 oligodendrocytes. Moreover, we show that migration of MO3.13 cells is directly regulated by EBI2 and that its signaling is necessary for remyelination in cerebellar slices post-LPC-induced demyelination. The work reported here provides new information on the expression and role of EBI2 in oligodendrocytes and myelination and provides new tools for modulation of oligodendrocyte biology and therapeutic approaches for demyelinating diseases.


Assuntos
Encéfalo/citologia , Cerebelo/citologia , Oligodendroglia/citologia , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/citologia , Animais , Encéfalo/metabolismo , Cerebelo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligodendroglia/metabolismo , Receptores Acoplados a Proteínas G/genética , Remielinização , Células-Tronco/metabolismo
11.
Sci Rep ; 11(1): 6323, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33737669

RESUMO

UBE3A is an E3 ubiquitin ligase encoded by the neurally imprinted UBE3A gene. The abundance and subcellular distribution of UBE3A has been the topic of many previous studies as its dosage and localization has been linked to neurodevelopmental disorders including Autism, Dup15q syndrome, and Angelman syndrome. While commercially available antibodies have been widely employed to determine UBE3A localization, an extensive analysis and comparison of the performance of different UBE3A antibodies has not been conducted. Here we evaluated the specificities of seven commercial UBE3A antibodies in two of the major experimental models used in UBE3A research, mouse and human pluripotent stem cell-derived neural cells and tissues. We tested these antibodies in their two most common assays, immunofluorescence and western blot. In addition, we also assessed the ability of these antibodies to capture dynamic spatiotemporal changes of UBE3A by utilizing human cerebral organoid models. Our results reveal that among the seven antibodies tested, three antibodies demonstrated substantial nonspecific immunoreactivity. While four antibodies show specific localization patterns in both mouse brain sections and human cerebral organoids, these antibodies varied significantly in background signals and staining patterns in undifferentiated human pluripotent stem cells.


Assuntos
Anticorpos/genética , Neurônios/metabolismo , Ubiquitina-Proteína Ligases/genética , Síndrome de Angelman/genética , Síndrome de Angelman/patologia , Animais , Anticorpos/imunologia , Transtorno Autístico/genética , Transtorno Autístico/patologia , Cerebelo/citologia , Modelos Animais de Doenças , Impressão Genômica , Humanos , Camundongos , Células-Tronco Neurais/metabolismo , Neurônios/patologia , Organoides/citologia , Células-Tronco Pluripotentes , Ubiquitina-Proteína Ligases/imunologia
12.
Int J Mol Sci ; 22(4)2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33671638

RESUMO

The vascular endothelial growth factor (VEGF) is well known for its wide-ranging functions, not only in the vascular system, but also in the central (CNS) and peripheral nervous system (PNS). To study the role of VEGF in neuronal protection, growth and maturation processes have recently attracted much interest. These effects are mainly mediated by VEGF receptor 2 (VEGFR-2). Current studies have shown the age-dependent expression of VEGFR-2 in Purkinje cells (PC), promoting dendritogenesis in neonatal, but not in mature stages. We hypothesize that microRNAs (miRNA/miR) might be involved in the regulation of VEGFR-2 expression during the development of PC. In preliminary studies, we performed a miRNA profiling and identified miR204-5p as a potential regulator of VEGFR-2 expression. In the recent study, organotypic slice cultures of rat cerebella (postnatal day (p) 1 and 9) were cultivated and VEGFR-2 expression in PC was verified via immunohistochemistry. Additionally, PC at age p9 and p30 were isolated from cryosections by laser microdissection (LMD) to analyse VEGFR-2 expression by quantitative RT-PCR. To investigate the influence of miR204-5p on VEGFR-2 levels in PC, synthetic constructs including short hairpin (sh)-miR204-5p cassettes (miRNA-mimics), were microinjected into PC. The effects were analysed by confocal laser scanning microscopy (CLSM) and morphometric analysis. For the first time, we could show that miR204-5p has a negative effect on VEGF sensitivity in juvenile PC, resulting in a significant decrease of dendritic growth compared to untreated juvenile PC. In mature PC, the overexpression of miR204-5p leads to a shrinkage of dendrites despite VEGF treatment. The results of this study illustrate, for the first time, which miR204-5p expression has the potential to play a key role in cerebellar development by inhibiting VEGFR-2 expression in PC.


Assuntos
MicroRNAs/genética , Células de Purkinje/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Cerebelo/citologia , Cerebelo/fisiologia , Dendritos/fisiologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Microdissecção e Captura a Laser , Masculino , Técnicas de Cultura de Órgãos , Células de Purkinje/efeitos dos fármacos , Ratos Wistar , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
13.
Nat Commun ; 12(1): 1029, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33589635

RESUMO

A primary challenge in single-cell RNA sequencing (scRNA-seq) studies comes from the massive amount of data and the excess noise level. To address this challenge, we introduce an analysis framework, named single-cell Decomposition using Hierarchical Autoencoder (scDHA), that reliably extracts representative information of each cell. The scDHA pipeline consists of two core modules. The first module is a non-negative kernel autoencoder able to remove genes or components that have insignificant contributions to the part-based representation of the data. The second module is a stacked Bayesian autoencoder that projects the data onto a low-dimensional space (compressed). To diminish the tendency to overfit of neural networks, we repeatedly perturb the compressed space to learn a more generalized representation of the data. In an extensive analysis, we demonstrate that scDHA outperforms state-of-the-art techniques in many research sub-fields of scRNA-seq analysis, including cell segregation through unsupervised learning, visualization of transcriptome landscape, cell classification, and pseudo-time inference.


Assuntos
Redes Neurais de Computação , Análise de Sequência de RNA/estatística & dados numéricos , Análise de Célula Única/estatística & dados numéricos , Aprendizado de Máquina não Supervisionado/estatística & dados numéricos , Animais , Teorema de Bayes , Benchmarking , Separação Celular/métodos , Cerebelo/química , Cerebelo/citologia , Embrião de Mamíferos , Humanos , Fígado/química , Fígado/citologia , Pulmão/química , Pulmão/citologia , Camundongos , Células-Tronco Embrionárias Murinas/química , Células-Tronco Embrionárias Murinas/citologia , Pâncreas/química , Pâncreas/citologia , Retina/química , Retina/citologia , Análise de Célula Única/métodos , Córtex Visual/química , Córtex Visual/citologia , Zigoto/química , Zigoto/citologia
14.
J Neurosci ; 41(13): 2854-2869, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33593855

RESUMO

BK calcium-activated potassium channels have complex kinetics because they are activated by both voltage and cytoplasmic calcium. The timing of BK activation and deactivation during action potentials determines their functional role in regulating firing patterns but is difficult to predict a priori. We used action potential clamp to characterize the kinetics of voltage-dependent calcium current and BK current during action potentials in Purkinje neurons from mice of both sexes, using acutely dissociated neurons that enabled rapid voltage clamp at 37°C. With both depolarizing voltage steps and action potential waveforms, BK current was entirely dependent on calcium entry through voltage-dependent calcium channels. With voltage steps, BK current greatly outweighed the triggering calcium current, with only a brief, small net inward calcium current before Ca-activated BK current dominated the total Ca-dependent current. During action potential waveforms, although BK current activated with only a short (∼100 µs) delay after calcium current, the two currents were largely separated, with calcium current flowing during the falling phase of the action potential and most BK current flowing over several milliseconds after repolarization. Step depolarizations activated both an iberiotoxin-sensitive BK component with rapid activation and deactivation kinetics and a slower-gating iberiotoxin-resistant component. During action potential firing, however, almost all BK current came from the faster-gating iberiotoxin-sensitive channels, even during bursts of action potentials. Inhibiting BK current had little effect on action potential width or a fast afterhyperpolarization but converted a medium afterhyperpolarization to an afterdepolarization and could convert tonic firing of single action potentials to burst firing.SIGNIFICANCE STATEMENT BK calcium-activated potassium channels are widely expressed in central neurons. Altered function of BK channels is associated with epilepsy and other neuronal disorders, including cerebellar ataxia. The functional role of BK in regulating neuronal firing patterns is highly dependent on the context of other channels and varies widely among different types of neurons. Most commonly, BK channels are activated during action potentials and help produce a fast afterhyperpolarization. We find that in Purkinje neurons BK current flows primarily after the fast afterhyperpolarization and helps to prevent a later afterdepolarization from producing rapid burst firing, enabling typical regular tonic firing.


Assuntos
Potenciais de Ação/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Células de Purkinje/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Células Cultivadas , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Cerebelo/fisiologia , Feminino , Canais de Potássio Ativados por Cálcio de Condutância Alta/antagonistas & inibidores , Masculino , Camundongos , Células de Purkinje/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia
15.
FASEB J ; 35(2): e21329, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33484186

RESUMO

L1 syndrome is a rare developmental disorder characterized by hydrocephalus of varying severity, intellectual deficits, spasticity of the legs, and adducted thumbs. Therapy is limited to symptomatic relief. Numerous gene mutations in the L1 cell adhesion molecule (L1CAM, hereafter abbreviated L1) were identified in L1 syndrome patients, and those affecting the extracellular domain of this transmembrane type 1 glycoprotein show the most severe phenotypes. Previously analyzed rodent models of the L1 syndrome focused on L1-deficient animals or mouse mutants with abrogated cell surface expression of L1, making it difficult to test L1 function-triggering mimetic compounds with potential therapeutic value. To overcome this impasse, we generated a novel L1 syndrome mouse with a mutation of aspartic acid at position 201 in the extracellular part of L1 (p.D201N, hereafter termed L1-201) that displays a cell surface-exposed L1 accessible to the L1 mimetics. Behavioral assessment revealed an increased neurological deficit score and increased locomotor activity in male L1-201 mice carrying the mutation on the X-chromosome. Histological analyses of L1-201 mice showed features of the L1 syndrome, including enlarged ventricles and reduced size of the corpus callosum. Expression levels of L1-201 protein as well as extent of cell surface biotinylation and immunofluorescence labelling of cultured cerebellar neurons were normal. Importantly, treatment of these cultures with the L1 mimetic compounds duloxetine, crotamiton, and trimebutine rescued impaired cell migration and survival as well as neuritogenesis. Altogether, the novel L1 syndrome mouse model provides a first experimental proof-of-principle for the potential therapeutic value of L1 mimetic compounds.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/tratamento farmacológico , Deficiência Intelectual/tratamento farmacológico , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Peptidomiméticos/uso terapêutico , Paraplegia Espástica Hereditária/tratamento farmacológico , Animais , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Cerebelo/patologia , Ventrículos Cerebrais/metabolismo , Ventrículos Cerebrais/patologia , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Cloridrato de Duloxetina/farmacologia , Cloridrato de Duloxetina/uso terapêutico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Locomoção , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Molécula L1 de Adesão de Célula Nervosa/genética , Neurogênese , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Peptidomiméticos/farmacologia , Fenótipo , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/patologia , Toluidinas/farmacologia , Toluidinas/uso terapêutico , Trimebutina/farmacologia , Trimebutina/uso terapêutico
16.
Neural Netw ; 136: 72-86, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33450654

RESUMO

Recent evidence suggests that Golgi cells in the cerebellar granular layer are densely connected to each other with massive gap junctions. Here, we propose that the massive gap junctions between the Golgi cells contribute to the representational complexity of the granular layer of the cerebellum by inducing chaotic dynamics. We construct a model of cerebellar granular layer with diffusion coupling through gap junctions between the Golgi cells, and evaluate the representational capability of the network with the reservoir computing framework. First, we show that the chaotic dynamics induced by diffusion coupling results in complex output patterns containing a wide range of frequency components. Second, the long non-recursive time series of the reservoir represents the passage of time from an external input. These properties of the reservoir enable mapping different spatial inputs into different temporal patterns.


Assuntos
Cerebelo/citologia , Cerebelo/fisiologia , Rede Nervosa/citologia , Rede Nervosa/fisiologia , Dinâmica não Linear , Animais , Córtex Cerebelar/citologia , Córtex Cerebelar/fisiologia , Células Cerebelares de Golgi/fisiologia , Junções Comunicantes/fisiologia , Humanos
17.
Neurosci Lett ; 746: 135648, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33444672

RESUMO

Vitronectin, an extracellular matrix protein, controls the differentiation of cerebellar granule cell precursors (CGCPs) via αvß5 integrin, particularly in the initial stage of differentiation to granule cells. In this study, we determined whether vitronectin regulates axon specification in this initial differentiation stage of CGCPs. First, we analyzed whether vitronectin deficiency, ß5 integrin knockdown (KD), and ß5 integrin overexpression affect axon specification of primary cultured CGCPs. Vitronectin deficiency and ß5 integrin KD inhibited axon formation, while vitronectin administrated- and ß5 integrin overexpressed-neurons formed multiple axons. Moreover, KD of ß5 integrin suppressed vitronectin-induced multiple axon formation. These findings indicate that vitronectin contributes to regulating axon specification via αvß5 integrin in CGCPs. Next, we determined the signaling pathway involved in regulating vitronectin-induced axon specification. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited vitronectin-induced multiple axon specification, and lithium chloride, an inhibitor of glyocogen synthase kinase 3 beta (GSK3ß), attenuated the inhibitory effect of vitronectin-KO and ß5 integrin KD on the specification of CGCPs. In addition, vitronectin induced the phosphorylation of protein kinase B (Akt) and GSK3ß in neuroblastoma Neuro2a cells. Taken together, our results indicate that vitronectin plays an important factor in axon formation process in CGCPs via a ß5 integrin/PI3K/GSK3ß pathway.


Assuntos
Axônios/metabolismo , Diferenciação Celular/fisiologia , Cerebelo/metabolismo , Células-Tronco Neurais/metabolismo , Receptores de Vitronectina/metabolismo , Vitronectina/metabolismo , Animais , Axônios/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/efeitos dos fármacos , Gravidez , Vitronectina/farmacologia
18.
Anat Sci Int ; 96(1): 87-96, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32789737

RESUMO

Cerebellar abnormalities are commonly associated with hydrocephalus. However, the effect of hydrocephalus on the otherwise normal cerebellum has been largely neglected. This study assesses the morphological changes in the Purkinje cells in relation to cerebellar dysfunction observed in juvenile hydrocephalic rats. Fifty-five three-week old albino Wistar rats were used, hydrocephalus was induced by intracisternal injection of kaolin (n = 35) and others served as controls (n = 20). Body weight measurements, hanging wire, negative geotaxis, and open field tests were carried out at the onset and then weekly for 4 weeks, rats were killed, and their cerebella processed for Hematoxylin and Eosin, Cresyl violet and Golgi staining. Qualitative and quantitative studies were carried out; quantitative data were analyzed using two-way ANOVA and independent T tests at p < 0.05. Hydrocephalic rats weighed less than controls (p = 0.0247) but their cerebellar weights were comparable. The hydrocephalic rats had a consistently shorter latency to fall in the hanging wire test (F(4,112) = 18.63; p < 0.0001), longer latency to turn in the negative geotaxis test (F(4,112) = 22.2; p < 0.0001), and decreased horizontal (F(4,112) = 4.172, p = 0.0035) and vertical movements (F(4,112) = 4.397; p = 0.0024) in the open field test than controls throughout the 4 weeks post-induction. Cellular compression in the granular layer, swelling of Purkinje cells with vacuolations, reduced dendritic arborization and increased number of pyknotic Purkinje cells were observed in hydrocephalic rats. Hydrocephalus caused functional and morphological changes in the cerebellar cortex. Purkinje cell loss, a major pathological feature of hydrocephalus, may be responsible for some of the motor deficits observed in this condition.


Assuntos
Cerebelo/patologia , Cerebelo/fisiopatologia , Hidrocefalia/patologia , Hidrocefalia/fisiopatologia , Caulim/efeitos adversos , Desempenho Psicomotor , Células de Purkinje/fisiologia , Animais , Cerebelo/citologia , Modelos Animais de Doenças , Hidrocefalia/induzido quimicamente , Movimento , Ratos Wistar
19.
Cancer Lett ; 499: 188-200, 2021 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-33253789

RESUMO

The deregulation of epigenetic pathways has been implicated as a critical step in tumorigenesis including in childhood brain tumor medulloblastoma. The H3K27me3 demethylase UTX/KDM6A plays important roles in development and is frequently mutated in various types of cancer. However, how UTX regulates tumor development remains largely unclear. Here, we report the generation of a UTX-deleted mouse model of SHH medulloblastoma that demonstrates the tumor suppressor functions of UTX, which could be antagonized by the deletion of another H3K27me3 demethylase JMJD3/KDM6B. Intriguingly, UTX deletion in cancerous cerebellar granule neuron precursors (CGNPs) resulted in the impaired recruitment of host CD8+ T cells to the tumor microenvironment through a non-cell autonomous mechanism. In both mouse medulloblastoma models and in human medulloblastoma cells, we showed that UTX activates Th1-type chemokines, which are responsible for T cell migration. Surprisingly, our results showed that the depletion of cytotoxic CD8+ T cells did not affect mouse medulloblastoma growth. Nevertheless, the UTX/chemokine/T cell recruitment pathway we identified may be applied to many other cancers and may be important for improving cancer immunotherapy. In addition, UTX is required for the expression of NeuroD2 in precancerous progenitors, which encodes a potent proneural transcription factor. Overexpression of NEUROD2 in CGNPs decreased cell proliferation and increased neuron differentiation. We showed that UTX deletion led to impaired neural differentiation, which could coordinate with active SHH signaling to accelerate medulloblastoma development. Thus, UTX regulates both cell-intrinsic oncogenic processes and the tumor microenvironment in medulloblastoma. Our study provides insights into both medulloblastoma development and context dependent functions of UTX in tumorigenesis.


Assuntos
Neoplasias Cerebelares/genética , Histona Desmetilases/metabolismo , Meduloblastoma/genética , Proteínas Supressoras de Tumor/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Neoplasias Cerebelares/imunologia , Neoplasias Cerebelares/patologia , Cerebelo/citologia , Cerebelo/patologia , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Histona Desmetilases/genética , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Masculino , Meduloblastoma/imunologia , Meduloblastoma/patologia , Camundongos , Camundongos Transgênicos , Células-Tronco Neoplásicas/patologia , Neurônios/patologia , Neuropeptídeos/genética , Cultura Primária de Células , Receptor Smoothened/genética , Linfócitos T Citotóxicos/imunologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Proteínas Supressoras de Tumor/genética
20.
Neural Netw ; 134: 173-204, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33316723

RESUMO

We consider a cerebellar ring network for the optokinetic response (OKR), and investigate the effect of diverse recoding of granule (GR) cells on OKR by varying the connection probability pc from Golgi to GR cells. For an optimal value of pc∗(=0.06), individual GR cells exhibit diverse spiking patterns which are in-phase, anti-phase, or complex out-of-phase with respect to their population-averaged firing activity. Then, these diversely-recoded signals via parallel fibers (PFs) from GR cells are effectively depressed by the error-teaching signals via climbing fibers from the inferior olive which are also in-phase ones. Synaptic weights at in-phase PF-Purkinje cell (PC) synapses of active GR cells are strongly depressed via strong long-term depression (LTD), while those at anti-phase and complex out-of-phase PF-PC synapses are weakly depressed through weak LTD. This kind of "effective" depression (i.e., strong/weak LTD) at the PF-PC synapses causes a big modulation in firings of PCs, which then exert effective inhibitory coordination on the vestibular nucleus (VN) neuron (which evokes OKR). For the firing of the VN neuron, the learning gain degree Lg, corresponding to the modulation gain ratio, increases with increasing the learning cycle, and it saturates at about the 300th cycle. By varying pc from pc∗, we find that a plot of saturated learning gain degree Lg∗ versus pc forms a bell-shaped curve with a peak at pc∗ (where the diversity degree in spiking patterns of GR cells is also maximum). Consequently, the more diverse in recoding of GR cells, the more effective in motor learning for the OKR adaptation.


Assuntos
Cerebelo/citologia , Cerebelo/fisiologia , Rede Nervosa/fisiologia , Plasticidade Neuronal/fisiologia , Nistagmo Optocinético/fisiologia , Adaptação Fisiológica/fisiologia , Animais , Depressão Sináptica de Longo Prazo/fisiologia , Células de Purkinje/fisiologia , Sinapses/fisiologia
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