scholarly journals ubtor Mutation Causes Motor Hyperactivity by Activating mTOR Signaling in Zebrafish

2021 ◽  
Author(s):  
Tiantian Wang ◽  
Mingshan Zhou ◽  
Quan Zhang ◽  
Cuizhen Zhang ◽  
Gang Peng
Keyword(s):  
Neuronal Activity ◽  
Neurological Diseases ◽  
Epileptic Encephalopathy ◽  
Mtor Signaling ◽  
Negative Regulator ◽  
Zebrafish Embryos ◽  
Zebrafish Model ◽  
Spinal Interneurons ◽  
Motor Hyperactivity ◽  
Increased Sensitivity

AbstractMechanistic target of rapamycin (mTOR) signaling governs important physiological and pathological processes key to cellular life. Loss of mTOR negative regulators and subsequent over-activation of mTOR signaling are major causes underlying epileptic encephalopathy. Our previous studies showed that UBTOR/KIAA1024/MINAR1 acts as a negative regulator of mTOR signaling, but whether UBTOR plays a role in neurological diseases remains largely unknown. We therefore examined a zebrafish model and found that ubtor disruption caused increased spontaneous embryonic movement and neuronal activity in spinal interneurons, as well as the expected hyperactivation of mTOR signaling in early zebrafish embryos. In addition, mutant ubtor larvae showed increased sensitivity to the convulsant pentylenetetrazol, and both the motor activity and the neuronal activity were up-regulated. These phenotypic abnormalities in zebrafish embryos and larvae were rescued by treatment with the mTORC1 inhibitor rapamycin. Taken together, our findings show that ubtor regulates motor hyperactivity and epilepsy-like behaviors by elevating neuronal activity and activating mTOR signaling.

scholarly journals NaV1.1 and NaV1.6 selective compounds reduce the behavior phenotype in a novel zebrafish model for Dravet Syndrome

2019 ◽  
Author(s):  
Wout J. Weuring ◽  
Sakshi Singh ◽  
Linda Volkers ◽  
Martin Rook ◽  
Ruben H. van ‘t Slot ◽  
...  
Keyword(s):  
Treatment Strategies ◽  
Epileptic Seizures ◽  
Selective Inhibition ◽  
Epileptic Encephalopathy ◽  
Model Systems ◽  
Dravet Syndrome ◽  
Loss Of Function ◽  
Zebrafish Model ◽  
Neuronal Inhibition ◽  
Increased Sensitivity

AbstractDravet syndrome is caused by dominant loss-of-function mutations in SCN1A which cause reduced activity of Nav1.1 leading to lack of neuronal inhibition. On the other hand, gain-of-function mutations in SCN8A can lead to a severe epileptic encephalopathy subtype by over activating NaV1.6 channels. These observations suggest that Nav1.1 and Nav1.6 represent two opposing sides of the neuronal balance between inhibition and activation. Here, we hypothesize that Dravet syndrome may be treated by either enhancing Nav1.1 or reducing Nav1.6 activity. To test this hypothesis we generated and characterized a novel DS zebrafish model and tested new compounds that selectively activate or inhibit the human NaV1.1 or NaV1.6 channel respectively. We used CRISPR/Cas9 to generate two separate Scn1Lab knockout lines as an alternative to previous knock-down models. Using an optimized locomotor assay, spontaneous burst movements were detected that were unique to Scn1Lab knockouts and disappear when introducing human SCN1A mRNA. Besides the behavioral phenotype, Scn1Lab knockouts show sudden, electrical discharges in the brain that indicate epileptic seizures in zebrafish. Scn1Lab knockouts showed increased sensitivity to the convulsant pentylenetetrazole and a reduction in whole organism GABA levels. Drug screenings further validated a Dravet syndrome phenotype. We tested the NaV1.1 activator AA43279 and our newly synthesized NaV1.6 inhibitors MV1369 and MV1312 in the Scn1Lab knockouts. Both type of compounds significantly reduced the number of burst movements. Our results show that selective inhibition of NaV1.6 could be just as efficient as selective activation of NaV1.1 and these approaches could prove to be novel potential treatment strategies for Dravet syndrome and other (genetic) epilepsies. Compounds tested in zebrafish however, should always be further validated in other model systems, preferably human derived.

scholarly journals DDRE-27. IDH MUTATED GLIOMAS PROMOTE EPILEPTOGENESIS VIA D-2-HYDROXYGLUTARATE DEPENDENT MTOR HYPERACTIVATION

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. i12-i12
Author(s):  
Armin Mortazavi ◽  
Islam Fayed ◽  
Muzna Bachani ◽  
Tyrone Dowdy ◽  
Joseph Steiner ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Neuronal Excitability ◽  
Neurological Diseases ◽  
Mtor Signaling ◽  
Neuronal Firing ◽  
Metabolic Reprogramming ◽  
Neuronal Cultures ◽  
Low Grade ◽  
Idh Mutations

Abstract INTRODUCTION Epileptic seizures in patients with low-grade, isocitrate dehydrogenase (IDH) mutated gliomas reach 90%, a major source of morbidity for these patients. Albeit there are multiple features that contribute to tumor related epileptogenesis, IDH mutations are determined to be an independent factor, although the pathogenesis remains poorly understood. We demonstrate IDH-mutated tumors promote epileptogenesis through D-2-hydroxyglutarate (D-2-HG) dependent mTOR hyperactivation and metabolic reprogramming. METHODS Human epileptic and nonepileptic cortex were identified via subdural electrodes in patients with IDH-mutated gliomas (n=5). An in vitro rat cortical neuronal model on microelectrode arrays were utilized to investigate the role of D-2-HG on neuronal excitability. mTOR and lysine demethylase (KDM) modulators were applied to elucidate the epileptogenic mechanism. Tetrodotoxin was utilized to evaluate the contribution of neuronal activity to mTOR signaling and metabolism. mTOR signaling was evaluated through western blot analysis and multiplex immunofluorescence. Metabolic function were analyzed via Seahorse assays and metabolomic analysis. RESULTS D-2-HG increased normalized bursting rate in the neuronal cultures (p<0.0001). Inhibition of mTOR with rapamycin corrected bursting levels to control levels. Furthermore, D-2-HG induced mTOR hyperactivation, independent of bursting activity, which correlated with upregulation of mTOR signaling in human epileptic tissue. KDM inhibition resulted in mTOR hyperactivation and neuronal hyperexcitability, which we demonstrated with D-2-HG, succinate, and PFI-90, a small molecule KDM inhibitor. Epileptic cortex and D-2-HG-treated neurons, have distinct metabolisms independent of neuronal activity compared to peritumoral nonepileptic cortex and control, respectively. CONCLUSION We demonstrate IDH-mutated gliomas promote epileptogenesis through a D-2-HG dependent mTOR hyperactivation via KDM inhibition, a putative mechanism and potential therapeutic targets. Furthermore, we argue mTOR hyperactivation results in metabolic reprogramming, independent of neuronal firing, which may contribute to epileptogenesis, a heretofore unrecognized aspect of pathologic mTOR signaling in neurological diseases.

scholarly journals Preliminary Results Regarding Sleep in a Zebrafish Model of Autism Spectrum Disorder

2021 ◽  
Vol 11 (5) ◽  
pp. 556
Author(s):  
Madalina Andreea Robea ◽  
Alin Ciobica ◽  
Alexandrina-Stefania Curpan ◽  
Gabriel Plavan ◽  
Stefan Strungaru ◽  
...  
Keyword(s):  
Autism Spectrum Disorder ◽  
Valproic Acid ◽  
Social Behavior ◽  
Antiepileptic Drug ◽  
Alternative Model ◽  
Neurological Diseases ◽  
Autism Spectrum ◽  
Spectrum Disorder ◽  
Zebrafish Model ◽  
Developmental Defects

Autism spectrum disorder (ASD) is one of the most salient developmental neurological diseases and remarkable similarities have been found between humans and model animals of ASD. A common method of inducing ASD in zebrafish is by administrating valproic acid (VPA), which is an antiepileptic drug that is strongly linked with developmental defects in children. In the present study we replicated and extended the findings of VPA on social behavior in zebrafish by adding several sleep observations. Juvenile zebrafish manifested hyperactivity and an increase in ASD-like social behaviors but, interestingly, only exhibited minimal alterations in sleep. Our study confirmed that VPA can generate specific ASD symptoms, indicating that the zebrafish is an alternative model in this field of research.

Abstract 151: A Novel Role for DDiT4L in Regulation of mTOR and Autophagy in the Heart

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Bridget Simonson ◽  
Hannabeth Franchino ◽  
Ashley Knight ◽  
Anthony Rosenzweig ◽  
Saumya Das
Keyword(s):  
Heart Failure ◽  
Protein Expression ◽  
Glucose Deprivation ◽  
Mtor Signaling ◽  
Confocal Imaging ◽  
Negative Regulator ◽  
Neonatal Rat ◽  
Pathological Hypertrophy ◽  
Gene And Protein Expression ◽  
Physiological Hypertrophy

Introduction: DDiT4L is a known negative regulator of mTOR signaling in skeletal muscle; however its role in the heart is unknown. We have recently showed increased DDiT4L mRNA in a murine transgenic model of pathological but not physiological hypertrophy. Here we test the hypothesis that DDiT4L is a regulator of mTOR signaling in the heart and may play a role in pathological hypertrophy and heart failure. Methods: We investigated the regulation of DDiT4L in murine models of hypertrophy and in cultured neonatal rat ventricular cardiomyocytes (NRVMs). Loss and gain of function of DDiT4L in mTOR regulation and autophagy was investigated using confocal imaging, immunoblotting, and qRT-PCR in NRVMs. Results: DDiT4L gene and protein expression was increased four-fold in pressure overload hypertrophy (n = 4-6, p<0.001), but not in a swim model of physiological hypertrophy. DDiT4L gene expression also significantly increased in a genetic model of dilated cardiomyopathy model (n = 4, p<0.001). In NRVMs, DDiT4L was induced by cardiac stressors such as pathological stretch, hypoxia, and glucose deprivation (n = 3-5 in duplicate, p<0.05-0.01). Increased DDiT4L expression correlated with inhibition of mTOR signaling, and an increase in autophagy markers. siRNA ablation of DDiT4L revealed that inhibition of mTOR signaling by DDiT4L was necessary for glucose deprivation induced autophagy, as determined by imaging of GFP-LC3 autophagosomes (n = 3 in duplicate, p<0.01), and immunoblotting of autophagy markers. Conversely, adenoviral-driven overexpression of DDiT4L inhibited mTOR signaling and significantly increased basal autophagy (n = 3 in duplicate, p<0.05). In TAC mice, the increase in DDiT4L protein expression correlated to inhibition of mTOR signaling, increases in autophagy markers (p<0.01), and preceded the transition to LV dilation and HF. Conclusion: Our data suggests that DDiT4L expression is altered in diverse models of pathological hypertrophy and precedes the development of LV dilatation and overt heart failure. DDiT4L inhibition of mTOR and modulation of autophagy may play a role in the progression to heart failure. DDiT4L may represent a novel therapeutic target to prevent this transition.

scholarly journals Lignans from Bursera fagaroides Affect In Vivo Cell Behavior by Disturbing the Tubulin Cytoskeleton in Zebrafish Embryos

Molecules ◽  
2018 ◽  
Vol 24 (1) ◽  
pp. 8 ◽  
Author(s):  
Mayra Antúnez-Mojica ◽  
Andrés Rojas-Sepúlveda ◽  
Mario Mendieta-Serrano ◽  
Leticia Gonzalez-Maya ◽  
Silvia Marquina ◽  
...  
Keyword(s):  
Cell Migration ◽  
Biological Effects ◽  
In Vitro Models ◽  
In Vivo Model ◽  
Zebrafish Embryos ◽  
Microtubule Cytoskeleton ◽  
Zebrafish Model ◽  
Bioassay Guided Fractionation

By using a zebrafish embryo model to guide the chromatographic fractionation of antimitotic secondary metabolites, seven podophyllotoxin-type lignans were isolated from a hydroalcoholic extract obtained from the steam bark of Bursera fagaroides. The compounds were identified as podophyllotoxin (1), β-peltatin-A-methylether (2), 5′-desmethoxy-β-peltatin-A-methylether (3), desmethoxy-yatein (4), desoxypodophyllotoxin (5), burseranin (6), and acetyl podophyllotoxin (7). The biological effects on mitosis, cell migration, and microtubule cytoskeleton remodeling of lignans 1–7 were further evaluated in zebrafish embryos by whole-mount immunolocalization of the mitotic marker phospho-histone H3 and by a tubulin antibody. We found that lignans 1, 2, 4, and 7 induced mitotic arrest, delayed cell migration, and disrupted the microtubule cytoskeleton in zebrafish embryos. Furthermore, microtubule cytoskeleton destabilization was observed also in PC3 cells, except for 7. Therefore, these results demonstrate that the cytotoxic activity of 1, 2, and 4 is mediated by their microtubule-destabilizing activity. In general, the in vivo and in vitro models here used displayed equivalent mitotic effects, which allows us to conclude that the zebrafish model can be a fast and cheap in vivo model that can be used to identify antimitotic natural products through bioassay-guided fractionation.

Enhanced Ih Depresses Rat Entopeduncular Nucleus Neuronal Activity From High-Frequency Stimulation or Raised Ke+

2008 ◽  
Vol 99 (5) ◽  
pp. 2203-2219 ◽  
Author(s):  
D. S. Shin ◽  
P. L. Carlen
Keyword(s):  
Neuronal Activity ◽  
High Frequency ◽  
Neurological Diseases ◽  
Input Resistance ◽  
High Frequency Stimulation ◽  
Channel Activity ◽  
Entopeduncular Nucleus ◽  
Inhibitory Effects ◽  
Frequency Stimulation ◽  
Spontaneous Action

High-frequency stimulation (HFS) is used to treat a variety of neurological diseases, yet its underlying therapeutic action is not fully elucidated. Previously, we reported that HFS-induced elevation in [K+]e or bath perfusion of raised Ke+ depressed rat entopeduncular nucleus (EP) neuronal activity via an enhancement of an ionic conductance leading to marked depolarization. Herein, we show that the hyperpolarization-activated ( Ih) channel mediates the HFS- or K+-induced depression of EP neuronal activity. The perfusion of an Ih channel inhibitor, 50 μM ZD7288 or 2 mM CsCl, increased input resistance by 23.5 ± 7% (ZD7288) or 35 ± 10% (CsCl), hyperpolarized cells by 3.4 ± 1.7 mV (ZD7288) or 2.3 ± 0.9 mV (CsCl), and decreased spontaneous action potential (AP) frequency by 51.5 ± 12.5% (ZD7288) or 80 ± 13.5% (CsCl). The Ih sag was absent with either treatment, suggesting a block of Ih channel activity. Inhibition of the Ih channel prior to HFS or 6 mM K+ perfusion not only prevented the previously observed decrease in AP frequency, but increased neuronal activity. Under voltage-clamp conditions, Ih currents were enhanced in the presence of 6 mM K+. Calcium is also involved in the depression of EP neuronal activity, since its removal during raised Ke+ application prevented this attenuation and blocked the Ih sag. We conclude that the enhancement of Ih channel activity initiates the HFS- and K+-induced depression of EP neuronal activity. This mechanism could underlie the inhibitory effects of HFS used in deep brain stimulation in output basal ganglia nuclei.

scholarly journals High-resolution cardiovascular function confirms functional orthology of myocardial contractility pathways in zebrafish

2010 ◽  
Vol 42 (2) ◽  
pp. 300-309 ◽  
Author(s):  
Jordan T. Shin ◽  
Eugene V. Pomerantsev ◽  
John D. Mably ◽  
Calum A. MacRae
Keyword(s):  
High Speed ◽  
Dynamic Range ◽  
Model Organism ◽  
Cardiovascular Physiology ◽  
Zebrafish Embryos ◽  
Cardiovascular Development ◽  
Zebrafish Model ◽  
Ventricular Performance ◽  
Pharmacological Agents

Phenotype-driven screens in larval zebrafish have transformed our understanding of the molecular basis of cardiovascular development. Screens to define the genetic determinants of physiological phenotypes have been slow to materialize as a result of the limited number of validated in vivo assays with relevant dynamic range. To enable rigorous assessment of cardiovascular physiology in living zebrafish embryos, we developed a suite of software tools for the analysis of high-speed video microscopic images and validated these, using established cardiomyopathy models in zebrafish as well as modulation of the nitric oxide (NO) pathway. Quantitative analysis in wild-type fish exposed to NO or in a zebrafish model of dilated cardiomyopathy demonstrated that these tools detect significant differences in ventricular chamber size, ventricular performance, and aortic flow velocity in zebrafish embryos across a large dynamic range. These methods also were able to establish the effects of the classic pharmacological agents isoproterenol, ouabain, and verapamil on cardiovascular physiology in zebrafish embryos. Sequence conservation between zebrafish and mammals of key amino acids in the pharmacological targets of these agents correlated with the functional orthology of the physiological response. These data provide evidence that the quantitative evaluation of subtle physiological differences in zebrafish can be accomplished at a resolution and with a dynamic range comparable to those achieved in mammals and provides a mechanism for genetic and small-molecule dissection of functional pathways in this model organism.

scholarly journals mTORC1 is necessary but mTORC2 and GSK3β are inhibitory for AKT3-induced axon regeneration in the central nervous system

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Linqing Miao ◽  
Liu Yang ◽  
Haoliang Huang ◽  
Feisi Liang ◽  
Chen Ling ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Axon Regeneration ◽  
Nodal Point ◽  
Mtor Signaling ◽  
Negative Regulator ◽  
Molecular Dissection ◽  
Parallel Pathways ◽  
Downstream Effectors ◽  
The Central Nervous System

Injured mature CNS axons do not regenerate in mammals. Deletion of PTEN, the negative regulator of PI3K, induces CNS axon regeneration through the activation of PI3K-mTOR signaling. We have conducted an extensive molecular dissection of the cross-regulating mechanisms in axon regeneration that involve the downstream effectors of PI3K, AKT and the two mTOR complexes (mTORC1 and mTORC2). We found that the predominant AKT isoform in CNS, AKT3, induces much more robust axon regeneration than AKT1 and that activation of mTORC1 and inhibition of GSK3β are two critical parallel pathways for AKT-induced axon regeneration. Surprisingly, phosphorylation of T308 and S473 of AKT play opposite roles in GSK3β phosphorylation and inhibition, by which mTORC2 and pAKT-S473 negatively regulate axon regeneration. Thus, our study revealed a complex neuron-intrinsic balancing mechanism involving AKT as the nodal point of PI3K, mTORC1/2 and GSK3β that coordinates both positive and negative cues to regulate adult CNS axon regeneration.

scholarly journals EDS1 Contributes to Nonhost Resistance of Arabidopsis thaliana Against Erwinia amylovora

2012 ◽  
Vol 25 (3) ◽  
pp. 421-430 ◽  
Author(s):  
Manon Moreau ◽  
Alexandre Degrave ◽  
Régine Vedel ◽  
Frédérique Bitton ◽  
Oriane Patrit ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Protein Synthesis ◽  
Erwinia Amylovora ◽  
Gene Activation ◽  
Negative Regulator ◽  
Nonhost Resistance ◽  
Callose Deposition ◽  
In The Wild ◽  
Increased Sensitivity ◽  
Main Components

Erwinia amylovora causes fire blight in rosaceous plants. In nonhost Arabidopsis thaliana, E. amylovora triggers necrotic symptoms associated with transient bacterial multiplication, suggesting either that A. thaliana lacks a susceptibility factor or that it actively restricts E. amylovora growth. Inhibiting plant protein synthesis at the time of infection led to an increase in necrosis and bacterial multiplication and reduced callose deposition, indicating that A. thaliana requires active protein synthesis to restrict E. amylovora growth. Analysis of the callose synthase–deficient pmr4-1 mutant indicated that lack of callose deposition alone did not lead to increased sensitivity to E. amylovora. Transcriptome analysis revealed that approximately 20% of the genes induced following E. amylovora infection are related to defense and signaling. Analysis of mutants affected in NDR1 and EDS1, two main components of the defense-gene activation observed, revealed that E. amylovora multiplied ten times more in the eds1-2 mutant than in the wild type but not in the ndr1-1 mutant. Analysis of mutants affected in three WRKY transcription factors showing EDS1-dependent activation identified WRKY46 and WRKY54 as positive regulators and WRKY70 as a negative regulator of defense against E. amylovora. Altogether, we show that EDS1 is a positive regulator of nonhost resistance against E. amylovora in A. thaliana and hypothesize that it controls the production of several effective defenses against E. amylovora through the action of WRKY46 and WRKY54, while WRKY70 acts as a negative regulator.

scholarly journals The Genome-Wide Impact of Nipblb Loss-of-Function on Zebrafish Gene Expression

2020 ◽  
Vol 21 (24) ◽  
pp. 9719
Author(s):  
Marco Spreafico ◽  
Eleonora Mangano ◽  
Mara Mazzola ◽  
Clarissa Consolandi ◽  
Roberta Bordoni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Developmental Stages ◽  
System Development ◽  
Expression Patterns ◽  
Nervous System Development ◽  
Zebrafish Embryos ◽  
Loss Of Function ◽  
Zebrafish Model ◽  
Genome Wide ◽  
Transcriptional Effect

Transcriptional changes normally occur during development but also underlie differences between healthy and pathological conditions. Transcription factors or chromatin modifiers are involved in orchestrating gene activity, such as the cohesin genes and their regulator NIPBL. In our previous studies, using a zebrafish model for nipblb knockdown, we described the effect of nipblb loss-of-function in specific contexts, such as central nervous system development and hematopoiesis. However, the genome-wide transcriptional impact of nipblb loss-of-function in zebrafish embryos at diverse developmental stages remains under investigation. By RNA-seq analyses in zebrafish embryos at 24 h post-fertilization, we examined genome-wide effects of nipblb knockdown on transcriptional programs. Differential gene expression analysis revealed that nipblb loss-of-function has an impact on gene expression at 24 h post fertilization, mainly resulting in gene inactivation. A similar transcriptional effect has also been reported in other organisms, supporting the use of zebrafish as a model to understand the role of Nipbl in gene regulation during early vertebrate development. Moreover, we unraveled a connection between nipblb-dependent differential expression and gene expression patterns of hematological cell populations and AML subtypes, enforcing our previous evidence on the involvement of NIPBL-related transcriptional dysregulation in hematological malignancies.

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