Effects of siRNA-Mediated Knockdown of GSK3β on Retinal Ganglion Cell Survival and Neurite/Axon Growth.

Ahmed Ahmed,Berry Berry,Morgan-Warren Morgan-Warren,Logan Logan,Scott Scott

doi:10.3390/cells8090956

Ahmed Ahmed, Berry Berry + Show 3 more

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https://doi.org/10.3390/cells8090956

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Abstract

There are contradictory reports on the role of the serine/threonine kinase isoform glycogen synthase kinase-3β (GSK3β) after injury to the central nervous system (CNS). Some report that GSK3 activity promotes axonal growth or myelin disinhibition, whilst others report that GSK3 activity prevents axon regeneration. In this study, we sought to clarify if suppression of GSK3β alone and in combination with the cellular-stress-induced factor RTP801 (also known as REDD1: regulated in development and DNA damage response protein), using translationally relevant siRNAs, promotes retinal ganglion cell (RGC) survival and neurite outgrowth/axon regeneration. Adult mixed retinal cell cultures, prepared from rats at five days after optic nerve crush (ONC) to activate retinal glia, were treated with siRNA to GSK3β (siGSK3β) alone or in combination with siRTP801 and RGC survival and neurite outgrowth were quantified in the presence and absence of Rapamycin or inhibitory Nogo-A peptides. In in vivo experiments, either siGSK3β alone or in combination with siRTP801 were intravitreally injected every eight days after ONC and RGC survival and axon regeneration was assessed at 24 days. Optimal doses of siGSK3β alone promoted significant RGC survival, increasing the number of RGC with neurites without affecting neurite length, an effect that was sensitive to Rapamycin. In addition, knockdown of GSK3β overcame Nogo-A-mediated neurite growth inhibition. Knockdown of GSK3β after ONC in vivo enhanced RGC survival but not axon number or length, without potentiating glial activation. Knockdown of RTP801 increased both RGC survival and axon regeneration, whilst the combined knockdown of GSK3β and RTP801 significantly increased RGC survival, neurite outgrowth, and axon regeneration over and above that observed for siGSK3β or siRTP801 alone. These results suggest that GSK3β suppression promotes RGC survival and axon initiation whilst, when in combination with RTP801, it also enhanced disinhibited axon elongation.

Highlights

The role of the serine/threonine kinase isoform glycogen synthase kinase-3β (GSK3β) [1,2,3,4], which is expressed in the central nervous system (CNS) [5], is controversial, since GSK3 activity is reported by some to promote axonal growth or myelin disinhibition, whilst other studies claim the opposite is true [6,7,8,9,10]
We determined if GSK3β-mediated retinal ganglion cell (RGC) survival and neurite outgrowth were mediated through mTORC1 activation using Rapamycin, which effectively blocks mammalian target of Rapamycin (mTOR) activation
SiGSKβ-treated animals than after siRTP801 treatment alone at each of the distances along the ON [21], the differences only reached statistical significance at 800 μm. These results demonstrated that the combination of Small Interfering RNA (siRNA) to GSK3β (siGSK3β) + siRTP801 is superior in promoting long distance RGC axon regeneration after optic nerve crush (ONC) than single administration of either siRNA

Summary

Introduction

The role of the serine/threonine kinase isoform glycogen synthase kinase-3β (GSK3β) [1,2,3,4], which is expressed in the central nervous system (CNS) [5], is controversial, since GSK3 activity is reported by some to promote axonal growth or myelin disinhibition, whilst other studies claim the opposite is true [6,7,8,9,10]. Used well-defined phosphorylation resistant GSK3αS21A /βS9A [GSK3(α/β)S/A ] double knock-in mice to show that elevated GSK3 activity accelerated peripheral nerve regeneration [9] This observed effect was based on phosphorylated MAP1B-associated inhibition of microtubule detyrosination and subsequent increase in microtubule dynamics in axon growth cones [9,17]. Using the transgenic as well as the conditional retinal ganglion cell (RGC) specific GSK3α and GSK3β knock out (KO) mice, the elevation of GSK3 activity in GSK3S/A mice was compromised, and GSK3β KO potentiated inflammatory stimulation-mediated RGC axon regeneration [10] These effects were associated with varying degrees of inactive CRMP2 in optic nerve axons, whereas no CRMP2 phosphorylation was observed in peripheral nerve regeneration [10]. Elevated GSK3 activity enhanced neurite outgrowth in RGC only when constant CRMP2 activity was maintained, suggesting that CRMP2 inhibition masks the positive effects of MAP1B activity in CNS neurons [10]

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