Functional Analysis of Reggie Proteins during Neuronal Differentiation and Axon Regeneration

Munderloh, Christina

Functional Analysis of Reggie Proteins during Neuronal Differentiation and Axon Regeneration

Files

Diss_Munderloh.pdf(6.3 MB)

Date

2009

Authors

Munderloh, Christina

Title in another language

Funktionsanalyse der Reggie-Proteine während der neuronalen Differenzierung und Axon-Regeneration

Publication type

Dissertation

Abstract

The reggies are scaffolding proteins of membrane microdomains and involved in various cellular processes including neuronal differentiation. Reggie-1 and -2 were originally discovered as proteins upregulated during axon regeneration in retinal ganglion cells (RGCs) after optic nerve injury, suggesting a function of these proteins in axon regeneration. Loss-of-function studies via small interfering RNAs (siRNAs) or morpholino antisense oligonucleotides (Mos) against reggie-1 and -2 were performed to clarify, whether the reggies are causally linked to neuronal differentiation in cell cultures and/or axonal regeneration after optic nerve section (ONS) in zebrafish. Silencing of reggie-1 with siRNAs caused a simultaneous loss of reggie-2 protein by proteasomal degradation in N2a cells. Depletion of both reggies in differentiating N2a cells led to significantly shorter filopodia, more cells with lamellipodia and fewer with neurites, a defect which was rescued by a reggie-1 construct without siRNA binding sites. Furthermore, reggie knockdown strongly perturbed the balanced activation of the Rho family GTPases RhoA, Rac1 and cdc42 and affected activation of MAP kinases p38 and ERK1/2, Ras and focal adhesion kinase (FAK). Downregulation of zebrafish reggie-1a, -2a and -2b expression in vivo by application of reggie-specific Mos directly after ONS significantly reduced the capability of zebrafish RGCs to regenerate axons. In an outgrowth assay, the number of re-growing RGC axons in vitro from reggie Mo-treated retinae was markedly reduced compared to controls. Moreover, the number of axon regenerating RGCs in vivo, identified by insertion of A488-coupled dextran 7d after Mo-application, decreased by 69% in reggie Mo-retinae as opposed to controls. At 10 and 14 d, labeled RGCs decreased by 53 and 33%, respectively, in correlation with the gradual loss of the Mos. Thus, as suggested by their prominent re-expression upon lesion, the reggies represent neuron-intrinsic factors for axon outgrowth and regeneration in vitro and in vivo by coordinating signal transduction pathways to control cytoskeletal remodeling.

Summary in another language

Die Reggie-Proteine bilden die Basiskomponente spezieller Mikrodomänen (Reggie-Mikrodomänen) der Membran und sind an verschiedenen zellulären Prozessen beteiligt, u. a. an der neuronalen Differenzierung und Axon-Regeneration. Reggie-1 und -2 wurden als Proteine entdeckt, deren Expression nach Läsion des optischen Nervs im Goldfisch in retinalen Ganglienzellen (RGZ) hochreguliert wird. Diese Beobachtung implizierte eine Funktion der Reggie-Proteine in der neuronalen Regeneration. Untersuchungen, bei denen ein Funktionsverlust durch Herunterregulation der Expression (= Knockdown) der Reggie-Proteine herbeigeführt wurde, sollen Aufschluss über die ursächliche Beteilung der Reggie-Proteine an der neuronalen Differenzierung an Zellkulturmodellen in vitro und der axonalen Regeneration im optischen Nerv des Zebrafisch in vivo liefern. Knockdown von Reggie-1 führte zu einem gleichzeitigen Verlust von Reggie-2 durch proteosomalen Abbau von Reggie-2 in neuronalen (N2a) Zellen. Der Verlust von Reggie-1 und -2 führte weiterhin zu kürzeren Filopodien, weniger Zellen mit Neuriten und mehr Zellen mit Lamellipodien. Diese morphologischen Defekte konnten durch die ektopische Expression eines Reggie-1 Konstrukts aufgehoben werden, welches resistent gegenüber dem Knockdown ist. Außerdem führte der Knockdown der Reggie-Proteine zu einer gestörten Aktivierung der kleinen Rho GTPasen RhoA, Rac1 und cdc42, der MAP Kinasen p38 und ERK1/2, Ras und der Proteintyrosinkinase FAK. In vivo Knockdown von Reggie-1a, -2a and -2b im Zebrafisch durch das Einbringen von Morpholino Oligonukleotiden unmittelbar nach Durchtrennung des optischen Nervs führte zu einer signifikanten Reduktion regenerierender RGZ Axone in Retina Auswachs-Assays sowohl in vitro als auch in vivo. Die Anzahl Axon regenerierender RGZs, die durch Einbringung von fluoreszierendem Dextran sieben Tage nach der Morpholino Applikation identifiziert werden konnten, sank um 69% nach Reggie Knockdown im Vergleich zu den Kontrollen. Die gleiche Quantifizierung bei zehn bzw. 14 Tagen nach Morpholino Anwendung ergab Reduktionen in der Anzahl von RGZs mit regenerierenden Axonen von 53 bzw. 33%, was durch den allmählichen Verlust an Morpholinos bedingt ist. Demnach stellen die Reggie-Proteine neuron-intrinsische Faktoren für das Wachstum und die Regeneration von Axonen dar, wie bei ihrer Entdeckung vermutet. Diese Funktion der Reggie-Proteine wird durch Beeinflussung/Koordination der Aktivität verschiedener Signaltransduktions-Moleküle und folglich der Restrukturierung des Zytoskeletts hervorgerufen.

Subject (DDC)

570 Biosciences, Biology

Keywords

Reggie-Proteine,Axon-Regeneration,Rho-GTPasen,Zebrafisch,optischer Nerv,reggie proteins,axon regeneration,Rho GTPases,zebrafish,optic nerve

Cite This

ISO 690

MUNDERLOH, Christina, 2009. Functional Analysis of Reggie Proteins during Neuronal Differentiation and Axon Regeneration [Dissertation]. Konstanz: University of Konstanz

BibTex

@phdthesis{Munderloh2009Funct-6897,
  year={2009},
  title={Functional Analysis of Reggie Proteins during Neuronal Differentiation and Axon Regeneration},
  author={Munderloh, Christina},
  address={Konstanz},
  school={Universität Konstanz}
}

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Examination date of dissertation

December 16, 2009