Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

University of Virginia 

Neurotropenic fever.

Fi&egrave;vre Neurotropenic.

Neurotropenic febbre.

Huntington's disease (HD) is a fatal neurodegenerative disease characterized by progressive cognitive, behavioral, and motor deficits and caused by expansion of a polyglutamine repeat in the Huntingtin protein (Htt). Despite its monogenic nature, HD pathogenesis includes obligatory non-cell-autonomous pathways involving both the cortex and the striatum, and therefore effective recapitulation of relevant HD disease pathways in cell lines and primary neuronal monocultures is intrinsically limited. To address this, the authors developed an automated high-content imaging screen in high-density primary cultures of cortical and striatal neurons together with supporting glial cells. Cortical and striatal neurons are transfected separately with different fluorescent protein markers such that image-based high-content analysis can be used to assay these neuronal populations separately but still supporting their intercellular interactions, including abundant synaptic interconnectivity. This assay was reduced to practice using transfection of a mutant N-terminal Htt domain and validated via a screen of ~400 selected small molecules. Both expected as well as novel candidate targets for HD emerged from this screen; of particular interest were target classes with close relative proximity to clinical testing. These findings suggest that composite primary cultures incorporating increased levels of biological complexity can be used for high-content imaging and "high-context" screening to represent molecular targets that otherwise may be operant only in the complex tissue environment found in vivo during disease pathogenesis.

Composite primary neuronal high-content screening assay for Huntington's disease incorporating non-cell-autonomous interactions.

La maladie de Huntington (MH) est une maladie neurod&eacute;g&eacute;n&eacute;rative mortelle caract&eacute;ris&eacute;e par des d&eacute;ficits cognitifs, comportements et moteurs progressifs et caus&eacute;e par l&#39;expansion d&#39;un polyglutamine r&eacute;p&eacute;ter dans la prot&eacute;ine huntingtine (Htt). Malgr&eacute; son caract&egrave;re monog&eacute;nique, pathogen&egrave;se comprendrait des voies non-cellules autonomes obligatoires associant le striatum et le cortex, et donc efficace r&eacute;capitulation des voies de maladie HD pertinents dans les lign&eacute;es cellulaires et les monocultures neuronales primaires est intrins&egrave;quement limit&eacute;e. Pour y rem&eacute;dier, les auteurs ont mis au point un &eacute;cran d&#39;imagerie automatis&eacute; de haute teneur dans des cultures primaires haute densit&eacute;s des corticales et les neurones striataux accompagn&eacute;e des justificatifs des cellules gliales. Neurones corticale et striatale transfected s&eacute;par&eacute;ment avec des marqueurs de la prot&eacute;ine fluorescente diff&eacute;rents tels qu&#39;imageur haute teneur analyse peut &ecirc;tre utilis&eacute;e pour doser ces populations neuronales s&eacute;par&eacute;ment mais toujours soutenir leurs interactions intercellulaires, y compris une interconnectivit&eacute; synaptique abondante. Ce dosage a &eacute;t&eacute; r&eacute;duit &agrave; la pratique &agrave; l&#39;aide de la transfection d&#39;un domaine N-terminal Htt mutant et valid&eacute; via un &eacute;cran de ~ 400 certaines petites mol&eacute;cules. Les deux attendus ainsi que des cibles de nouveaux candidats pour HD &eacute;merg&eacute; de cet &eacute;cran ; un int&eacute;r&ecirc;t particulier ont &eacute;t&eacute; classes cibles &agrave; proximit&eacute; relative aux essais cliniques. Ces r&eacute;sultats sugg&egrave;rent que les cultures primaires composites incorporant l&#39;accroissement de la complexit&eacute; biologique peuvent &ecirc;tre utilis&eacute;s pour l&#39;imagerie de haute teneur et &laquo; haute-contexte &raquo; d&eacute;pistage pour repr&eacute;senter des cibles mol&eacute;culaires qui autrement peuvent &ecirc;tre op&eacute;rante uniquement dans l&#39;environnement complexe tissu trouv&eacute; in vivo au cours de la pathogen&egrave;se de la maladie.

Dosage de criblage de haute teneur neuronale primaire composite pour la maladie de Huntington incorporant les interactions cellules-non-autonomes.

La malattia di Huntington (HD) &egrave; una malattia neurodegenerativa irreversibile caratterizzata da deficit cognitivi, comportamentali e motore progressivo e causato dall&#39;espansione delle poliglutamine ripetute nella proteina huntingtina (Htt). Nonostante la sua natura monogenica, patogenesi HD include obbligatorie-cella-autonomi percorsi che coinvolgono sia la corteccia e il corpo striato, e quindi efficace ricapitolazione di rilevanti percorsi di malattia HD in linee cellulari e monocolture neuronale primarie &egrave; intrinsecamente limitato. Per risolvere questo problema, gli autori hanno sviluppato un automatizzato dello schermo immagine ad alto contenuto in colture primarie ad alta densit&agrave; della corticale e neuroni striatali insieme con supporto di cellule gliali. Neuroni corticale e striatal transfected separatamente con marcatori differenti della proteina fluorescente tale che image-based high-content analysis pu&ograve; essere utilizzato per analisi di queste popolazioni neuronali separatamente ma ancora sostenere loro interazioni intercellulari, compreso l&#39;interconnettivit&agrave; sinaptica abbondante. Questo dosaggio &egrave; stato ridotto alla pratica utilizzando la trasfezione di un dominio N-terminale Htt mutante e convalidato tramite uno schermo di ~ 400 selezionati piccole molecole. Sia previsto cos&igrave; come obiettivi nuovi candidati per HD emerse da questa schermata; di particolare interesse sono stati classi di destinazione con la relativa prossimit&agrave; a test clinici. Questi risultati suggeriscono che compositi colture primarie incorporando maggiori livelli di complessit&agrave; biologica possono essere utilizzati per imaging di alto contenuto e &quot;high-context&quot; screening per rappresentare bersagli molecolari che altrimenti potrebbero essere operante solo nell&#39;ambiente complesso tessuto trovato in vivo durante la patogenesi della malattia.

Test di screening ad alto contenuto primario composito neuronale per la malattia di Huntington che incorporano le interazioni non-cella autonome.

Motor nerves play the critical role of shunting information out of the CNS to targets in the periphery. Their formation requires the coordinated development of distinct cellular components, including motor axons and the Schwann cells and perineurial glia that ensheath them. During nervous system assembly, these glial cells must migrate long distances and terminally differentiate, ensuring the efficient propagation of action potentials. Although we know quite a bit about the mechanisms that control Schwann cell development during this process, nothing is known about the mechanisms that mediate the migration and differentiation of perineurial glia. Using in vivo imaging in zebrafish, we demonstrate that Notch signaling is required for both perineurial migration and differentiation during nerve formation, but not regeneration. Interestingly, loss of Notch signaling in perineurial cells also causes a failure of Schwann cell differentiation, demonstrating that Schwann cells require perineurial glia for aspects of their own development. These studies describe a novel mechanism that mediates multiple aspects of perineurial development and reveal the critical importance of perineurial glia for Schwann cell maturation and nerve formation.

Perineurial glia require Notch signaling during motor nerve development but not regeneration.

Nerfs moteurs jouent le r&ocirc;le essentiel des man&oelig;uvres d&#39;informations hors de la CNS pour cibles dans la p&eacute;riph&eacute;rie. Leur formation n&eacute;cessite le d&eacute;veloppement coordonn&eacute; des composants cellulaires distincts, y compris les axones moteurs et les cellules de Schwann et de la glie p&eacute;rineurale cette forte eux. Lors du montage du syst&egrave;me nerveux, ces cellules gliales doivent migrer longues distances et se diff&eacute;rencient en phase terminale, assurant la propagation efficace des potentiels d&#39;action. M&ecirc;me si nous savons un peu sur les m&eacute;canismes qui contr&ocirc;lent le d&eacute;veloppement des cellules de Schwann pendant ce processus, on en sait rien sur les m&eacute;canismes qui assurent la m&eacute;diation la migration et la diff&eacute;renciation des cellules gliales p&eacute;rineurale. &Agrave; l&#39;aide d&#39;imagerie in vivo chez le poisson z&egrave;bre, nous d&eacute;montrons que la signalisation Notch est requise pour p&eacute;rineurale migration et diff&eacute;renciation au cours de la formation de nerf, mais pas de r&eacute;g&eacute;n&eacute;ration. Fait int&eacute;ressant, perte de l&#39;encoche de signalisation dans les cellules p&eacute;rineurale provoque &eacute;galement un &eacute;chec de la diff&eacute;renciation des cellules de Schwann, d&eacute;montrant que les cellules de Schwann n&eacute;cessitent glia p&eacute;rineurale des aspects de leur propre d&eacute;veloppement. Ces &eacute;tudes d&eacute;crivent un nouveau m&eacute;canisme qui intervient dans plusieurs aspects du d&eacute;veloppement p&eacute;rineurale et r&eacute;v&egrave;lent l&#39;importance cruciale des cellules gliales p&eacute;rineurale pour la maturation des cellules de Schwann et de la formation du nerf.

Glia p&eacute;rineurale n&eacute;cessitent de signalisation Notch pendant le d&eacute;veloppement du nerf moteur mais pas de r&eacute;g&eacute;n&eacute;ration.

Nervi motori giocano il ruolo critico di smistamento informazioni fuori SNC di bersagli in periferia. La loro formazione richiede lo sviluppo coordinato della distinte componenti cellulari, tra cui motore assoni e cellule di Schwann e glia perineurial che Sheath li. Durante il montaggio del sistema nervoso, devono eseguire la migrazione di queste cellule gliale lunghe distanze e differenziarsi terminalmente, assicurando l&#39;efficace propagazione dei potenziali di azione. Anche se sappiamo un po sui meccanismi che controllano lo sviluppo delle cellule di Schwann durante questo processo, niente &egrave; conosciuto circa i meccanismi che mediano la migrazione e la differenziazione di glia perineurial. Utilizzo di imaging in vivo in zebrafish, dimostriamo che tacca di segnalazione &egrave; necessaria per perineurial migrazione e la differenziazione durante la formazione del nervo, ma non la rigenerazione. &Egrave; interessante notare, perdita della tacca di segnalazione nelle cellule perineurial provoca anche una mancanza di differenziazione delle cellule di Schwann, dimostrando che le cellule di Schwann richiedono glia perineurial per gli aspetti del proprio sviluppo. Questi studi descrivono un nuovo meccanismo che media i molteplici aspetti dello sviluppo perineurial e rivelano l&#39;importanza critica di glia perineurial per la maturazione delle cellule di Schwann e la formazione del nervo.

Gwendolyn M. Lewis

Department of Biology

University of Virginia

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

Gwendolyn M. Lewis

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biology

University of Virginia

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

Department of Biology