Protein kinase A modulates retinal ganglion cell growth during development

https://doi.org/10.1016/j.expneurol.2008.02.014Get rights and content

Abstract

During development, retinal ganglion cells (RGCs) extend their axons toward their thalamic and mesencephalic targets. Their navigation is largely directed by guidance cues present in their environment. Since cAMP is an important second messenger that mediates the neural response to guidance molecules and its intracellular levels seem to decrease significantly following birth, we tested whether modulation of the cAMP/protein kinase A (PKA) pathway would affect the normal development of RGC axons. At postnatal day 1, hamsters received a unilateral intraocular injection of either 0.9% saline solution, 12 mM of the membrane-permeable cAMP analogue (dibutyryl cAMP; db-cAMP), or 10 µM of the PKA inhibitor KT5720. Intraocular elevation of cAMP significantly accelerated RGC axonal growth while inhibition of PKA activity decreased it. Moreover, when highly purified RGC cultures were treated with forskolin (an activator of adenylate cyclase) or cAMP analogues (db-cAMP and Sp-cAMP), neurite length, growth cone (GC) surface area and GC filopodia number were significantly increased. This indicates that intraocular elevation of cAMP acts directly on RGCs. Since these effects were prevented by PKA inhibitors, it demonstrates that cAMP also exerts its action via the PKA pathway. Taken together, these results suggest that the cAMP/PKA cascade is essential for the normal development of retinothalamic projections.

Introduction

Following differentiation, RGCs extend their axons to form functional connections with their target cells in the thalamus and the superior colliculus (SC). These axons, tipped at their distal end by the GC, navigate through relatively long distances in a highly directed manner in order to establish functional synapses with thalamic and SC neurons. This is achieved with the help of extracellular guidance molecules which steer RGC axon growth by regulating GC morphology by means of attractive and/or repulsive mechanisms. Consequently, upon binding of a guidance molecule to its receptor, it relays its signal to intracellular second messengers which modulate the GC's response accordingly (Huber et al., 2003). The cyclic nucleotide cAMP is a key intracellular second messenger that plays an essential role in numerous neuronal functions such as cell survival, axon regeneration, and GC behaviour to guidance cues. For example, elevated intracellular cAMP levels enhance the survival rate of CNS neurons (Meyer-Franke et al., 1995, Hanson et al., 1998, Hu et al., 2007). Various mechanisms have been proposed to explain this phenomenon. In RGCs, an increase in survival rate is attributed in part by cAMP's ability to facilitate the cells' responsiveness to trophic factors (Meyer-Franke et al., 1998). cAMP's role in axon regeneration is well documented (Spencer and Filbin, 2004, Teng and Tang, 2006). Furthermore, treatment of injured neurons with a cocktail of trophic factors and cell permeable analogues of cAMP induces elevated numbers of regenerating axons, even promoting their re-growth through inhibitory environment (Cui et al., 2003, Lu et al., 2004, Rodger et al., 2005, Hu et al., 2007). In addition, in vitro development of dissociated and highly purified rat dorsal root ganglion cells (DRGs) and RGCs depend upon endogenous cAMP levels (Cai et al., 2001). cAMP also regulates neurite growth by influencing GC behaviour to guidance cues. This is supported by the fact that manipulating the cAMP signalling pathway can alter the response of the GC to a given guidance molecule (Song and Poo, 1999, Bouchard et al., 2004, Nicol et al., 2007).

Despite a large body of evidence describing the role played by cAMP and its downstream effector, PKA, in cell survival and axon regeneration, their role during development in vivo remains unknown. The aims of the present study are to determine how the cAMP/PKA pathway modulates neonatal development of RGCs, and to test the hypothesis that modification of retinal cAMP levels will alter retinal axon growth. Our results demonstrate that in vivo retinal cAMP levels decrease abruptly following birth. We also show for the first time that an increase in endogenous cAMP levels during development accelerates physiological projection growth by directly acting on RGCs and modulating the cAMP/PKA pathway.

Section snippets

Materials and methods

All animal experiments were approved by the Comité de déontologie de l'expérimentation sur les animaux from the University of Montreal and were handled in accordance with the recommendations provided by the Canadian Council on Animal Care. The distribution of the animals in the different experimental groups appears in Table 1.

Results

The objective of the present study was to investigate the implication of the cAMP/PKA pathway in axon guidance during RGC development. First, we examined the effects of modulating retinal cAMP levels in vivo on RGC projections development during the first neonatal week. Then, using highly purified RGC culture, we investigated the direct action of cAMP on RGCs by stimulating the cAMP-dependent-PKA.

Discussion

The present study demonstrates that retinal cAMP levels decrease during early postnatal development. Interestingly, increasing retinal concentration with a single intraocular injection of db-cAMP accelerates RGC growth by activating directly PKA in RGCs. Conversely, inhibition of PKA slows RGC axon branch development in the thalamus (LTN and DTN).

cAMP is an important second messenger for axon growth (Meyer-Franke et al., 1995, Meyer-Franke et al., 1998, Bouchard et al., 2004) and segregation of

Acknowledgments

This work is supported by a grant to J.F.B. from the Natural Sciences and Engineering Research Council of Canada (NSERC). A.A. is supported by a doctoral research award from E.A. Baker Foundation and The Institute of Neuroscience, Mental Health and Addiction (INMHA) of the Canadian Institutes of Health Research (CIHR). G.D. is supported by a fellowship from the Réseau Santé-Vision (FRSQ). M.P. holds the Harland Sanders Chair for Visual Science. J.F.B. is a scholar of Rx&D-CIHR Health Research

References (37)

  • QiuJ. et al.

    Spinal axon regeneration induced by elevation of cyclic AMP

    Neuron

    (2002)
  • RodgerJ. et al.

    cAMP regulates axon outgrowth and guidance during optic nerve regeneration in goldfish

    Mol. Cell. Neurosci.

    (2005)
  • SongH-j. et al.

    Signal transduction underlying growth cone guidance by diffusible factors

    Curr. Opin. Neurobiol.

    (1999)
  • StellwagenD. et al.

    Dynamics of retinal waves are controlled by cyclic

    AMP

    (1999)
  • AmatoM.A. et al.

    Hedgehog signaling in vertebrate eye development: a growing puzzle

    Cell. Mol. Life Sci. (CMLS)

    (2004)
  • BaiW. et al.

    Postnatal development of the retinal projection to the nucleus of the

    Arch. Histol. Cytol.

    (2001)
  • BhideP.G. et al.

    Stages of growth of hamster retinofugal axons: implications for developing axonal pathways with multiple targets

    J. Neurosci.

    (1991)
  • BhideP.G. et al.

    Intrinsic determinants of retinal axon collateralization and arborization

    J. Comp. Neurol.

    (1999)
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