Chapter 17

The Interphotoreceptor Retinoid Binding (IRBP) Is Essential for Normal Retinoid Processing in Cone Photoreceptors

Ryan O. Parker and Rosalie K. Crouch

Abstract 11-cis Retinal is the light-sensitive component in rod and cone photoreceptors, and its isomerization to all-trans retinal in the presence of light initiates the visual response. For photoreceptors to function normally, all-trans retinal must be converted back into 11-cis retinal through the visual cycle. While rods are primarily responsible for dim light vision, the ability of cones to function in constant light is essential to human vision and may be facilitated by cone-specific visual cycle pathways. The interphotoreceptor retinoid-binding protein (IRBP) is a proposed retinoid transporter in the visual cycle, but rods in Irbp–/– mice have a normal visual cycle. However, there is evidence that IRBP has cone-specific functions. Cone electroretinogram (ERG) responses are reduced, despite having cone densities and opsin levels similar to C57Bl/6 (WT) mice. Treatment with 9-cis retinal rescues the cone response in Irbp–/– mice and shows that retinoid deficiency underlies cone dysfunction. These data indicate that IRBP is essential to normal cone function and demonstrate that differences exist in the visual cycle of rods and cones.

17.1 Introduction

11-cis Retinal covalently binds opsin to form the light-sensitive visual pigments in rod and cone photoreceptors. In the dark, 11-cis retinal functions as an opsin inverse agonist, but when light strikes a visual pigment, 11-cis retinal is isomerized to all- trans retinal, an opsin agonist (Wald 1935, 1955). The photoisomerization of retinal triggers the photoresponse of rods and cones, but constant function requires that new 11-cis retinal continuously replace the all-trans retinal photoproduct. The retina and adjacent retinal pigment epithelium (RPE) accomplish this by efficiently converting all-trans retinal back to 11-cis retinal in a series of enzymatic steps known as the

R.O. Parker (B)

Department of Neurosciences, Medical University of South Carolina, Charleston, SC, USA e-mail: parkerry@musc.edu

Medicine and Biology 664, DOI 10.1007/978-1-4419-1399-9_17,C Springer Science+Business Media, LLC 2010

flow specific retinoids in the appropriate direction, it requires that poorly soluble and potentially toxic retinoids traverse the aqueous sub-retinal space between the photoreceptors and the RPE. The Interphotoreceptor Retinoid Binding Protein (IRBP) is the most abundant soluble protein in the sub-retinal space (Loew and GonzalezFernandez 2002) and is thought to facilitate this process (Bunt-Milam and Saari 1983; Fong et al. 1984).

In vitro studies have shown that IRBP promotes the release of all-trans retinol from photoreceptors (Ala-Laurila et al. 2006; Wu et al. 2007) and facilitates its delivery to the RPE (Okajima et al. 1994). Additionally, IRBP can also enhance 11-cis retinal release from the RPE (Edwards and Adler 2000), prevent its isomerization in the sub-retinal space (Crouch et al. 1992), and transfer 11-cis retinal to photoreceptors (Jones et al. 1989). Each of these steps would appear to be important for normal visual cycle function, and were IRBP essential for any of its proposed roles, an 11-cis retinal deficiency would inevitably occur in its absence. The Irbp–/– mouse was expected to confirm IRBP’s importance to the visual cycle in vivo (Liou et al. 1998). Although rod function is diminished in Irbp–/– mice, the visual cycle in rods is surprisingly normal (Palczewski et al. 1999; Ripps et al. 2000), and rod dysfunction is thought to be secondary to degeneration (Liou et al. 1998). Cone function in Irbp–/– mice is also diminished (Ripps et al. 2000), but the underlying cause remains unclear.

17.2 The Cone Population in Irbp–/– Mice

Because the rod population in Irbp–/– mice is reported to degenerate (Liou et al. 1998), it is possible that cones are similarly affected, and ERGs and cone densities were used to look for cone degeneration. Cone ERGs from Irbp–/– mice were diminished as early as 1 month (Fig. 17.2a) but showed no evidence of decline through 9 months of age (p = 0.28) (Fig. 17.2a, b). Analysis of the cone densities of aging Irbp–/– mice produced similar results. Cone densities were calculated using retina flat-mounts stained with peanut agglutinin (PNA), a lectin that binds the glycoprotein sheath surrounding cones (Johnson et al. 1986). While a small drop in the cone densities was noted from 1 to 2 months (256 ± 4.3, n = 4; 222

± 5.3, n = 4; p = 0.03), the population remained stable between 1 and 9 months (p = 0.14) (Fig. 17.2b). Cone densities were also similar in the dorsal and ventral retina, suggesting that neither the MWS nor SWS cones were uniquely affected. Thus, both ERGs and cone densities in aging Irbp–/– mice suggest that a significant degenerative process does not underlie cone dysfunction.

While a degenerative process does not appear to be present, IRBP has proposed developmental functions (Gonzalez-Fernandez and Healy 1990), and its absence could impair normal cone development. Thus, reduced cone densities could account for the attenuated cone response in Irbp–/– mice. Again, PNA-stained retina flatmounts were used, and representative flat-mounts from Irbp–/– and WT mice at 1 and 8 months are shown in Fig. 17.3a. The similar densities found in Irbp–/– and

PNA staining allows the rapid calculation of cone densities, PNA binds the sheath surrounding cones and not the cones, themselves. To account for this, western blots

were used to compare cone opsin levels in Irbp–/– and WT mice. In agreement with the findings from flat-mounts, Irbp–/– and WT mice at 4 months of age had equiv-

alent levels of MWS and SWS opsin (Fig. 17.3c). Retina cross-sections confirmed the correct localization of cone opsins to the outer segments (not shown). Together, the normal cone densities and opsin levels suggest that cone development is not impaired in Irbp–/– mice.

Neither degeneration nor development account for cone dysfunction in Irbp–/– mice, but an altered cone response could result from visual cycle deficits in IRBP’s absence. We tested for 11-cis retinal deficiency in the cones of Irbp–/– mice by analyzing photopic ERGs before and after intraperitoneal (IP) injections of 9-cis retinal, a functional analogue of 11-cis retinal (Crouch and Katz 1980). Baseline responses from Irbp–/– mice were reduced relative to WT mice at all intensities but recovered dramatically after treatment with 9-cis retinal (0.375 mg, IP) (Fig. 17.4a). Intensity response plots from Irbp–/– mice (n = 8) show that cone responses increased significantly with 9-cis retinal treatment at intensities above –0.8 log cd s/m2 (p = 0.005) (Fig. 17.4b) and did not differ significantly from the responses of treated WT mice (p = 0.25) (Fig. 17.4c). 9-cis Retinal had no effect on rod function in Irbp–/– mice (a-wave, p = 0.70; b-wave, p = 0.55) and did not significantly alter the rod or cone responses in WT mice (not shown). Thus, the cones of Irbp–/– mice were uniquely

Fig. 17.4 Recovery of cone ERGs in Irbp–/– mice with exogenous 9-cis retinal; a. Representative ERG traces from 2 month old animals are shown. Control responses from the Irbp–/– mouse were reduced relative to WT at all intensities. After the intraperitoneal (IP) injection of 9-cis retinal (0.375 mg), responses from the same mouse recovered to WT levels. b. Intensity-response plots from ERG recordings of 2 month old Irbp–/– mice (n = 8) treated with 9-cis retinal (0.375 mg, IP) showed a significant recovery of cone responses at all intensities above –0.8 log cd s/m2 (p = 0.005, paired two-way ANOVA). c. Responses of WT (n = 4) and Irbp–/– mice after 9-cis retinal injections were not significantly different (p = 0.25, two-way ANOVA)