Protein kinase A modulates retinal ganglion cell growth during development
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
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