TRENDS IN COMPARATIVE ENDOCRINOLOGY AND NEUROBIOLOGY

Growth Hormone-induced Neuroprotectionin the Neural Retina during Chick

EmbryogenesisSteve Harvey, Marie-Laure Baudet, and Esmond J. Sanders

Department of Physiology, University of Alberta, Edmonton, Alberta, Canada

Recent studies have established that retinal ganglion cells (RGCs) of the neural retinaare extrapituitary sites of growth hormone (GH) production and action in early chickembryos prior to the ontogeny of pituitary somatotrophs. The presence of GH in axons ofthe RGCs is, however, restricted to the period when the retinofugal neurons project to andsynapse with visual centers within the brain, suggesting roles for GH in axonal growth orguidance. Autocrine and/or paracrine actions of retinal GH are also neuroprotective forRGCs during developmental waves of apoptosis that characterize RGC differentiation.The anti-apoptotic actions of retinal GH use signaling mechanisms that are common toother established neurotrophins (e.g., brain-derived growth factor, insulin-like growthfactor-1, transforming growth factor β-1). Retinal GH is therefore a novel neurotrophinin the visual system during chick embryogenesis.

Key words: growth hormone; receptor; retinal ganglion cells; apoptosis

Introduction

It is now well established that the growthhormone (GH) gene is expressed in many ex-trapituitary tissues, including those of the cen-tral and peripheral nervous systems.1 Withinthe nervous system, GH may have roles ingrowth and development, cell survival, neuro-transmission, and angiogenesis.1,2 In this briefreview of our recent work, the functional sig-nificance of GH expression in the developingneural retina of embryonic chicks is consideredin relation to the neuroprotection of retinal gan-glion cells (RGCs). Exogenous GH has similarlybeen found to be neuroprotective within otherneural tissues of the brain.3,4

GH Expression in the ChickVisual System

GH mRNA, identical to that expressed in thepostnatal pituitary gland, is present in the chick

Address for correspondence: Steve Harvey, Department of Physiol-ogy, 7-41 Medical Sciences Building, University of Alberta, Edmonton,Alberta, T6G 2H7, Canada. Voice: 1 780 492-2809; fax: 1 780 492-3956.steve.harvey@ualberta.ca

neural retina within the first trimester days ofthe 21-day incubation period5,6 prior to its on-togenetic expression in pituitary somatotrophs(after embryonic day (ED) 157). Although thistranscript codes for a full-length translated pro-tein with a molecular mass of 22 kDa, it israpidly degraded into a 15-kDa moiety in reti-nal tissue extracts.8 Immunoreactivity for thismoiety is readily detectable in the neural retinaby ED 56,7 and is unlikely to reflect sequestra-tion from the bloodstream.

By ED 7, GH mRNA in the neural retinais primarily in the RGCs,6 whereas GH im-munoreactivity is most intense in the RGCaxons that comprise the overlying optic fiberlayer (OFL)6,9 and give rise to the optic nerve.GH immunoreactivity can be traced throughthe optic nerve head at the back of the eye,through the optic chiasm, and into the optictract that projects toward the optic tectum, thevisual center of the brain with which it synapsesby ED 10 and ED 12.9 GH immunoreactiv-ity is, however, present within the optic tec-tum by ED 49 prior to synaptogenesis of theseneurons, and tectal GH in the early embryo istherefore not a result of its anterograde trans-port in RCG axons. GH immunoreactivity in

Trends in Comparative Endocrinology and Neurobiology: Ann. N.Y. Acad. Sci. 1163: 414–416 (2009).doi: 10.1111/j.1749-6632.2008.03641.x C© 2009 New York Academy of Sciences.

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Harvey et al.: GH and Retinal Neurogenesis 415

the OFL is, however, lost between ED 14 andED 18,7,8 suggesting a role for GH in neuroge-nesis that is not required after synaptogenesisof the retinofugal fibers is complete. Indeed,the paucity of GH immunoreactivity, if any, inthe neural retina of juveniles and adults6 sug-gests roles for GH specifically during neuraldevelopment.

GH Action in the Chick VisualSystem

The possibility that GH is involved in earlychick neurogenesis is supported by the presenceof GH receptor (GHR) immunoreactivity inthe optic vesicle of ED-2 embryos10 and GHRmRNA and GHR immunoreactivity in theneural retinas of first trimester embryos.5,6,9 Ofinterest, the distribution of GHR immunoreac-tivity in the neural retina mirrors that of GH,with which it is colocalized.9 Actions of GHwithin the developing visual system are thuslikely to reflect autocrine and/or paracrine ac-tions within RGCs or their projecting neuronsor target sites. This possibility is supported, inpart, by the expression of a GH-response gene,GH-response gene-1, a specific marker of GHaction in chickens,11 in the neural retina andoptic tract of embryonic chicks.9 The fivefoldinduction of insulin-like growth factor (IGF)-1by exogenous GH in ED-7 chick neural retinaexplants similarly demonstrates GH action inthe visual system,6 as IGF-1 is commonly in-duced in GH signaling.

GH Action: Neuroprotection

Developmental waves of apoptosis occur inthe RGCs during development; one wave thatpeaks at ED 6–8 that involves an exit of cellsfrom the cell cycle and initiation of differentia-tion and a second wave that peaks around ED10–12 that reflects RGCs that fail to synapsein the optic tectum.12 Retinal GH may pro-mote the survival of retinal cells because exoge-

nous chicken GH, at 1.0 μmol/L, significantlyreduced the number of apoptotic (TUNEL-labeled) cells in ED 6 neural retina explantsafter 24 h (unpublished data) and 48 h ofculture.12 This neuroprotective action of GHwas accompanied by decreased expression ofcaspase-3 and decreased expression of apop-tosis inducing factor-1, which is caspase in-dependent.13 These actions were specific asother genes involved in apoptotic signaling(bcl-2, bcl-x, bid, and inhibitor of apoptosisprotein-1) were unaffected.13 The neuropro-tection of RGCs was also accompanied by areduction in the cleavage of caspase-914 andby increased Akt phosphorylation15 and in-creased activation of cytosolic tyrosine kinases(Trks) and extracellular-signal-related kinases(Erks) (all anti-apoptotic events), which resultin the activation of cAMP response elementbinding protein (CREB) and alterations inthe transcription of genes involved in the celldeath cascade.14 The neuroprotective actionsof GH are therefore common to other estab-lished neurotrophic factors, including brain-derived neurotrophic factor, IGF-1, and trans-forming growth factor β-1. This is of interestas these may be downstream mediators of GHaction.16,17 Indeed, the immunoneutralizationof endogenous IGF-1 in cultured RGCs dupli-cates the apoptotic action of GH immunoneu-tralization (unpublished observations).

A functional role of endogenous retinal GHin retinal cell survival is supported by its specificimmunoneutralization in retinal tissues andcells in vitro and in vivo. The immunoneutrali-zation of GH in culture immunopanned RGCsquadrupled the number of apoptotic cells15

by inhibiting Akt phosphorylation, increas-ing caspase-3 activation and PARP-1 cleav-age, and by blocking CREB activation.14,15

Microinjections of GH antibodies into the op-tic cup of ED-2.5 embryos similarly increasedthe number of apoptotic retinal cells at ED 5–8.15 This was particularly marked for antibod-ies that preferentially recognize the 15-kDa GHmoiety.15

416 Annals of the New York Academy of Sciences

Concluding Remarks

The colocalization of GH in RGCs in chickembryos during the neurogenesis of the visualsystem has functional relevance because it oc-curs during developmental waves of apoptosisand the loss of endogenous GH by immunoneu-tralization results in RGC death. RGC survivalduring the development of the visual systemmay be necessary for the anterograde produc-tion of neurotrophins required for the growthand synapsing of retinofugal neurons with vi-sual centers of the brain. The loss of GH fromthe OFL after the narrow window of retinofugaldevelopment strongly supports this possibilityand suggests additional autocrine or paracrineroles for GH as a neurotrophic factor involvedin axonal growth or guidance, especially as aloss of GH signaling in GHR-knockout micealters the expression of proteins involved in neu-rite development.18

Acknowledgments

This work was supported by the Natural Sci-ences and Engineering Research Council ofCanada.

Conflicts of Interest

The authors declare no conflicts of interest.

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