Expression of Netrin-1 and Its Receptors DCC and UNC-5H2 after Axotomy and during Regeneration of Adult Rat Retinal Ganglion Cells Benjamin Ellezam,* Inmaculada Selles-Navarro,* ,1 Colleen Manitt,† Timothy E. Kennedy,† and Lisa McKerracher* *De ´partement de pathologie et biologie cellulaire, Universite ´ de Montre ´al, Montre ´al, Que ´bec H3C 3J7; and †Centre for Neuronal Survival, Montreal Neurological Institute, McGill University, Montre ´al, Que ´bec H3A 2B4, Canada Received March 22, 2000; accepted October 5, 2000 Netrins are a family of chemotropic factors that guide axon outgrowth during development; however, their function in the adult CNS remains to be estab- lished. We examined the expression of the netrin re- ceptors DCC and UNC5H2 in adult rat retinal ganglion cells (RGCs) after grafting a peripheral nerve (PN) to the transected optic nerve and following optic nerve transection alone. In situ hybridization revealed that both Dcc and Unc5h2 mRNAs are expressed by normal adult RGCs. In addition, netrin-1 was found to be con- stitutively expressed by RGCs. Quantitative analysis using in situ hybridization demonstrated that both Dcc and Unc5h2 were down-regulated by RGCs follow- ing axotomy. In the presence of an attached PN graft, Dcc and Unc5h2 were similarly down-regulated in sur- viving RGCs regardless of their success in regenerat- ing an axon. Northern blot analysis demonstrated ex- pression of netrin-1 in both optic and sciatic nerve, and Western blot analysis revealed the presence of netrin protein in both nerves. Immunohistochemical analysis indicated that netrin protein was closely as- sociated with glial cells in the optic nerve. These re- sults suggest that netrin-1, DCC, and UNC5H2 may contribute to regulating the regenerative capacity of adult RGCs. © 2001 Academic Press Key Words: netrin; peripheral nerve graft; DCC; Unc5h2; optic nerve; axon guidance; growth inhibitory protein. INTRODUCTION During development, an important mechanism for axon pathfinding is chemotropism, and in the past several years, a growing number of chemotropic factors have been identified. Some of these have been shown to be bifunctional molecules, with both chemoattractive and chemorepulsive activity (8, 36, 52). Netrins are chemotropic factors with chemoattractant and che- morepellant guidance activities that have been de- scribed in vitro and in vivo (reviewed in 36). A che- moattractive axon guidance function for netrin was first demonstrated for the circumferential projection of developing spinal commissural axons toward the floor plate (22, 44, 45). Subsequently, netrins were shown to have chemorepulsive effect on the axons of developing rat trochlear motor neurons that extend circumferen- tially in a dorsal direction away from the floor plate (4). Consistent with netrins acting as bifunctional guid- ance cues, netrin receptors that mediate attractive or repulsive responses have been identified. DCC (deleted in colorectal cancer) is a receptor or component of a receptor complex that signals a chemoattractive re- sponse to netrin (21). Mammalian homologs of the Cae- norhabditis elegans gene unc-5 encode receptors or components of a receptor complex that signals a che- morepulsive effect of netrin (5, 15, 27). However, ge- netic studies in C. elegans have shown that unc-40 (the ortholog of DCC) is required for ectopic unc-5 function, suggesting that UNC-5 and DCC functionally collabo- rate to signal a chemorepulsive response to netrin. (5, 13, 31). Moreover, in growth cone turning assays per- formed with Xenopus spinal neurons, expression of an unc-5 family member changed netrin-dependent che- moattraction into chemorepulsion. In this case, both the attractant and the repellant response to netrin required DCC function, suggesting that DCC and UNC-5 family members likely cooperate to mediate the chemorepellant response to netrin (15). In addition, DCC and the DCC homolog neogenin (21) as well as the mammalian UNC-5 homolog family members, UNC5H1, UNC5H2 (27), and UNC5H3 (27, 41), all bind netrin, supporting the conclusion that they func- tion as netrin receptors. During development, retinal ganglion cell (RGC) ax- ons grow to the optic disk to form the optic nerve. The growth of embryonic RGC axons into the optic nerve is mediated, at least in part, by a DCC-dependent re- sponse of the RGC axons to netrin-1 expressed at the 1 Present address: Departamento Oftalmologia, Universidad de Murcia, 30100 Murcia, Spain. Experimental Neurology 168, 105–115 (2001) doi:10.1006/exnr.2000.7589, available online at http://www.idealibrary.com on 105 0014-4886/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.