Immunolocalization of Ciliary Neurotrophic Factor Receptor α (CNTFRα) in Mammalian Photoreceptor Cells

PURPOSE: To characterize the site of expression of the α subunit of the receptor for ciliary neurotrophic factor (CNTFRα) in the retina of a variety of mammalian species, and determine whether CNTFRα is localized to photoreceptor cells. METHODS: The cellular distribution of CNTFRα (protein) was examined by immunocytochemistry in the adult retinas of several mammalian species that included mouse, rat, dog, cat, sheep, pig, horse, monkey, and human. Developing retinas from 3-day-old and 6-day-old rats were also included in this study. The molecular weight of CNTFRα in rat, dog, cat, pig, and human retinas was determined by immunoblotting. RESULTS: CNTFRα immunolabeling was present in the retina of all species. A common pattern was observed in all species, and represented labeling of the nerve fiber layer (NFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), and outer plexiform layer (OPL). CNTFRα did not immunolocalize to photoreceptor cells in both adult and developing rodent retinas, but was consistently observed in both rods and cones of non-rodent species. The molecular weight of CNTFRα in mammalian retinas was approximately 61-64 kDa. CONCLUSIONS: These findings highlight a significant difference in the expression of CNTFRα in the retina of rodent and nonrodent mammalian species. The expression of CNTFRα by rods and cones in non-rodent species may suggest a direct mechanism of action if CNTF administration results in photoreceptor rescue. Disciplines Comparative and Laboratory Animal Medicine | Medicine and Health Sciences | Ophthalmology | Veterinary Medicine Comments PMID: 15827545 http://www.molvis.org/molvis/v11/a27 This journal article is available at ScholarlyCommons: http://repository.upenn.edu/vet_papers/34 Molecular Vision 2005; 11:232-44 Received 23 January 2004 | Accepted 17 December 2004 | Published 1 April 2005 Ciliary neurotrophic factor (CNTF) rescues photoreceptors in several genetic [1-7] and in light induced models of retinal degeneration [1,8]. Its photoreceptor survival effect was demonstrated in vivo in a variety of animal species that include mouse [1-5], rat [4,8], cat [6], and dog [7], and in mouse retinal explants [9,10]. Although its mechanism of action on photoreceptor cells is not fully understood, CNTF is thought to initiate a survival response by binding to the plasma membrane of retinal cells that express its receptor, ciliary neurotrophic factor receptor (CNTFR). CNTFR is composed of an α subunit (CNTFRα) that specifically binds CNTF, and two β subunits (LIFR, gp-130) that are shared by other members of the IL-6 R family [11]. Binding of CNTF to CNTFRα causes heterodimerization of the α and β subunits and activation of various signaling pathways that promote cell survival [12]. It has been shown that in the rat retina, CNTFRα mRNA is expressed in horizontal cells and subpopulations of amacrine and ganglion cells, but not in photoreceptors [13]. In addition, intravitreal delivery of CNTF to the rodent eye activates signaling pathways predominantly in Müller cells and other inner nuclear layer (INL) cells, ganglion cells, and astrocytes, yet fails to activate signaling pathways in photoreceptors [14-16]. These studies suggest that in the rat and mouse retina, the CNTF photoreceptor rescue effect is mediated through an indirect mechanism of action. It has been proposed that microglia derived CNTF could prevent photoreceptor cells from undergoing degeneration by promoting the release of direct acting photoreceptor survival factors such as bFGF and GDNF by Müller cells [17]. We have recently shown that in the normal adult canine retina both the CNTFRα transcript and protein are expressed by photoreceptors, INL cells, and ganglion cells [18]. The immunolocalization of CNTFRα to rods and cones suggests that, at least in the dog, the photoreceptor rescue effect observed with CNTF in the rcd1 model of retinal degeneration [7] may be mediated through a direct mechanism of action. Determining whether photoreceptors are the direct targets of CNTF has become increasingly important since this survival factor is currently being tested in Phase 1 clinical trials in humans with retinitis pigmentosa. To address the differences between dogs and rodents and determine if CNTFRα is localized to photoreceptors in other species, we performed immunocytochemical studies on retinas from a variety of mammalian species. METHODS Animals and tissue fixation: Normal adult retinas from the following mammalian species were used for the study: mouse (Balb/c, 6 months), rat (AO derived, 6 months), dog (Beagle, adult), cat (DSH, adult), sheep (adult), horse (Pony of America, 7 years), pig (adult), monkey (cynomolgus and rhesus macaques, adult), and human (52 year old male). In addition, we also collected immature retinas from 3-day-old (PD3) Lewis rats, and 6-day-old (PD6) AO rats. With the exception ©2005 Molecular Vision Immunolocalization of ciliary neurotrophic factor receptor α (CNTFRα) in mammalian photoreceptor cells William A. Beltran,1,2 Hermann Rohrer,3 Gustavo D. Aguirre2 James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY; Section of Medical Genetics, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA; Max-Planck-Institut für Hirnforschung,