Wingless-type family member 3A triggers neuronal polarization via cross-activation of the insulin-like growth factor-1 receptor pathway

María E Bernis,Alvaro Nieto Guil,Emilio L Malchiodi,Mariana Oksdath,Santiago Quiroga,Marisa M Fernández,Sebastián Dupraz,Silvana B Rosso

doi:10.3389/fncel.2013.00194

Abstract

Initial axonal elongation is essential for neuronal polarization and requires polarized activation of IGF-1 receptors (IGF-1r) and the phosphatidylinositol 3 kinase (PI3k) pathway. Wingless-type family growth factors (Wnts) have also been implied in the regulation of axonal development. It is not known, however, if Wnts have any participation in the regulation of initial axonal outgrowth and the establishment of neuronal polarity. We used cultured hippocampal neurons and growth cone particles (GCPs) isolated from fetal rat brain to show that stimulation with the wingless family factor 3A (Wnt3a) was sufficient to promote neuronal polarization in the absence of IGF-1 or high insulin. We also show that Wnt3a triggered a strong activation of IGF-1r, PI3k, and Akt in developmental Stage 2 neurons and that the presence of activatable IGF-1r and PI3k activation were necessary for Wnt3a polarizing effects. Surface plasmon resonance (SPR) experiments show that Wnt3a did not bind specifically to the IGF-1r. Using crosslinking and immuno-precipitation experiments, we show that stimulation with Wnt3a triggered the formation of a complex including IGF-1r-Wnt3a-Frizzled-7. We conclude that Wnt3a triggers polarization of neurons via cross-activation of the IGF-1r/PI3k pathway upon binding to Fz7.

Highlights

The establishment of neuronal polarity requires the action of two interrelated processes: axon specification and elongation
To study the possible involvement of the Wingless-type family growth factors (Wnts) growth factor family wingless family factor 3A (Wnt3a) on this process, we cultured pyramidal hippocampal neurons in “control” conditions, and in the www.frontiersin.org maintenance, we scored the differentiation stages in neurons cultured for 20 h in the presence of 1.35 nM Wnt3a
We found that over 60% of the neurons cultured in the presence of Wnt3a for 44 h were in stage 3 of differentiation, in contrast less that 30% of the cells were in this stage when secreted frizzled-related protein 1 (sFRP-1) was added to the cultures (Figure 1D)

Summary

Introduction

The establishment of neuronal polarity requires the action of two interrelated processes: axon specification and elongation. Phosphatidylinositol-3 kinase (PI3k) and its product, phosphatidylinositol 3,4,5-trisphosphate (PIP3), accumulate in the distal region and growth cone of the neurite with the IGF-1r. These events are critical for the outgrowth of the future axon, together with activation of PI3k by IGF-1r (Shi et al, 2003; Sosa et al, 2006). It has been shown that IGF-1 produces a robust and long lasting activation of the PI3k-Akt pathway through activation of the IGF-1r in hippocampal neurons (Zheng and Quirion, 2004). Activation of IGF-1r by their cognate ligand (IGF-1) and by insulin is able to transactivate other receptor systems, such as epidermal growth factor receptors (EGFRs) (Roudabush et al, 2000)

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