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Nov 28, 1994 - ABSTRACT. Retinitis pigmentosa (RP) is a group of hereditary human diseases that cause retinal degeneration and lead to eventual blindness.
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 3070-3074, March 1995 Genetics

Defective intracellular transport is the molecular basis of rhodopsin-dependent dominant retinal degeneration NANSI J. COLLEY*, J. AARON CASSILLt, ELIZABETH K. BAKER, AND CHARLES S. ZUKERt Departments of Biology and Neuroscience and Howard Hughes Medical Institute, University of California at San Diego, La Jolla, CA 92093-0649

Communicated by Michael G. Rosenfeld, University of California, San Diego, CA, November 28, 1994

example, it is possible to design comprehensive screens to carry out a genetic dissection of retinal degeneration and photoreceptor dysfunction (17, 23). (ii) The structure and development of the eye have been characterized in great detail. In addition, the eye is dispensable and thus genes encoding proteins specifically involved in photoreceptor function can be readily subjected to a genetic and molecular analysis without affecting viability (24, 25). (iii) Genes can be readily introduced into the germ line of Drosophila. Therefore, genes and the proteins they encode can be experimentally manipulated in vitro and their functions can be studied in vivo after reintroduction into the organism (26). Drosophila photoreceptor cells contain specialized portions of the plasma membrane, called rhabdomeres, that are composed of numerous tightly packed microvilli, containing rhodopsin photopigments and other components of the phototransduction cascade (23, 24). These are functionally equivalent to the phototransducing membranes present in the vertebrate photoreceptor outer segments. In Drosophila, as in vertebrates, rhodopsin is synthesized and core-glycosylated in the endoplasmic reticulum (ER), transported through the various compartments of the Golgi, and delivered to the rhabdomeres where it functions in phototransduction (27-29). Rhodopsin 1 (Rhl), the major rhodopsin in the Drosophila eye, displays 22% amino acid identity with human rhodopsin (30, 31). This laboratory has shown (28, 32-34) that Rhi rhodopsin requires the cyclophilin homolog NinaA as a chaperone for its exit from the ER and proper transport through the secretory pathway. In the absence of NinaA, immature Rhl accumulates in the ER and is eventually degraded, leading to progressive retinal degeneration. These findings demonstrated that Rhl transport through the secretory pathway is essential for the continued stability of the retina and suggests that other defects in rhodopsin biogenesis may produce similar phenotypes. We have now used a combination of genetic, cell biological, and molecular approaches to directly demonstrate that rhodopsin-based ADRP-like disorders in vivo result from interference in the maturation of the wild-type rhodopsin by the mutant proteins.

ABSTRACT Retinitis pigmentosa (RP) is a group of hereditary human diseases that cause retinal degeneration and lead to eventual blindness. More than 25% of all RP cases in humans appear to be caused by dominant mutations in the gene encoding the visual pigment rhodopsin. The mechanism by which the mutant rhodopsin proteins cause dominant retinal degeneration is still unclear. Interestingly, the great majority of these mutants appear to produce misfolded rhodopsin. We now report the isolation and characterization of 13 rhodopsin mutations that act dominantly to cause retinal degeneration in Drosophila; four of these correspond to identical substitutions in human autosomal dominant RP patients. We demonstrate that retinal degeneration results from interference in the maturation of wild-type rhodopsin by the mutant proteins.

Retinitis pigmentosa (RP) is a heterogeneous group of inherited disorders that cause progressive retinal degeneration. Patients affected by RP initially display abnormal electroretinograms followed by gradual loss of peripheral vision and eventual loss of central vision (1, 2). RP may be inherited as an autosomal dominant (ADRP), autosomal recessive, or X chromosome-linked trait and afflicts 1 in 4000 individuals in the U.S. population alone (3). Recent work into the biology of ADRP has demonstrated that mutations in rhodopsin represent a major cause of the disease (1, 2, 4-9); >60 distinct mutations in rhodopsin have now been reported in ADRP patients. However, the mechanism by which the mutant rhodopsin proteins cause dominant retinal dysfunction and photoreceptor degeneration is not yet known (9, 10). Two general mechanisms have been envisioned. One involves constitutive activation of the rhodopsin molecule leading to unregulated activity of the visual cascade (11-14). The other invokes defects in rhodopsin biogenesis (6, 15-18). Two types of experimental approaches have been used to define the basis for ADRP: expression of mutant forms of rhodopsin in tissue culture cells (11, 16, 18) and production of transgenic mice expressing mutant forms of rhodopsin (10, 19-22). Both types of studies have demonstrated that the great majority of rhodopsin mutations produce misfolded proteins that are improperly transported through the secretory pathway. However, it is still not clear why a single mutant allele can induce a phenotype even in the presence of the normal gene. To study the genetic, cell biological, and physiological basis of ADRP-like disorders in vivo, we developed a genetic screen to identify and isolate rhodopsin-based dominant retinal degeneration mutants in Drosophila. Drosophila is powerful as an experimental organism in that it allows a multidisciplinary approach to biological questions that not only will yield much information but also may well provide a type of information not currently obtainable in other experimental organisms. (i) The system is amenable to analysis using classical and sophisticated genetic manipulations. For

METHODS Genetic Screen. To isolate retinal degeneration mutants, ethyl methanesulfonate-treated rdgBEEl70 (retinal degeneration B) white-eyed males were mated to rdgBEEI70 virgin females and their progeny were grown in total darkness. Three days after eclosion, the flies were screened for retinal degeneration by using the deep pseudopupil as an assay. The deep pseudopupil is a sensitive eye phenotype that can be readily scored in live flies under blue light illumination (35, 36). A Abbreviations: RP, retinitis pigmentosa; ADRP, autosomal dominant RP; ER, endoplasmic reticulum; Rhl, rhodopsin 1. *Present address: Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792. tPresent address: Division of Life Sciences, The University of Texas, San Antonio, TX 78249. fTo whom reprint requests should be addressed.

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reduction in rhodopsin levels in the R1-6 photoreceptor cells leads to structural alterations in the photoreceptors and changes in the interommatidial angle that result in an attenuation of the deep pseudopupil (33). rdgBEEI70 mutants undergo severe and rapid retinal degeneration in the light but are quite normal in the dark (37, 38). Our choice of an rdgB stock for the mutagenesis allowed us to use a sensitized genetic background in which to do the screen. In essence, we were interested in obtaining two classes of mutants. One class would be mutations that cause constitutive activation of the rhodopsin molecule, or other phototransduction components, leading to unregulated activation of the visual cascade. In such mutants, rdgB flies should now degenerate in the dark (i.e., the constitutive activation mimics the effect of light). The other class could represent mutants with defects in rhodopsin biogenesis. We screened 67,200 flies and obtained 13 mutations that mapped to the third chromosome at the cytogenetic location of Rhl. Cloning, Sequencing, and DNA Manipulations. The Rhl gene from each mutant was amplified in two separate PCRs (12), subcloned into pBluescript KS- vector, and sequenced in its entirety. The rdgBEEl70 allele was removed from the genetic background before any of the dominant ninaE alleles were characterized. The hs-Rhl transgene was constructed by ligating the heat shock promoter from the Drosophila Hsp7O gene (39) into the Rhl structural gene (30). The 1D4 epitope tag (40,41) was added to the C-terminal tail of Rhl by using conventional site-directed mutagenesis techniques (42); this additional sequence does not affect the function of Rhl (E.KB. and C.S.Z., data not shown). P element-mediated germ-line transformations were carried out as described (43). Western Blot Analysis. Protein extracts and blots and were prepared from heads of the appropriate animals exactly as described (28, 34). In all cases extract from five heads was loaded per lane, and the different genetic stocks were assayed a minimum of four times. Animals used in Fig. 3 were harvested 2 weeks after eclosion. Animals used in the experiments described in Fig. 4 were heat-shocked at day 1 after eclosion. Electroretinograms and Physiological Recordings. All recordings were done in white eye flies. Retinal degeneration was assayed by electroretinogram analysis (44) and ultrastructural examination of mutant retinas (28). Electron microscopic studies and immunolabeling experiments were carried out exactly as described (28).

RESULTS AND DISCUSSION The Drosophila compound eye is composed of 800 individual eye units or ommatidia, each containing eight photoreceptor neurons, a lens, and accessory cells. Phototransduction in Drosophila utilizes an inositol phospholipid-mediated signaling cascade in which activation of rhodopsin leads to the opening of light-activated cation channels and the generation of a receptor potential (for reviews, see refs. 17, 23, and 45). To study the genetic and cell biological basis of ADRP-like disorders in vivo, we developed a genetic screen to identify and isolate rhodopsin-based dominant retinal degeneration mutants inDrosophila (Table 1). We screened

67,200 mutagenized chromosomes and recovered 13 mutations that mapped to Rhl. By using the PCR, we isolated the Rhl gene from each mutant and determined its entire nucleotide sequence (Fig. 1 and Table 1). All 13 mutations entail single nucleotide changes that together define 10 sites in the protein (there were 3 sites with two hits). Remarkably, 4 such missense mutations (G119E, P184L, E194K, and G195S) correspond to mutations in exactly the same amino acid residues found in known human ADRP (G106R, P171L, E181K, and G182S, respectively) (18). Animals heterozygous for a wild-type rhodopsin gene and a deletion of Rhl display normal retinal morphology, demonstrating that one copy of the rhodopsin gene is sufficient to maintain normal photoreceptor cell structure (Fig. 2A). In

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Table 1. Mutations and phenotypes of the Rhl alleles Mutation Retinal degeneration Amino acid residue Nucleotide Severe G69D G377 A Severe S9SF C455 - T W116@ Reduced G519 >A rhabdomeres G119E* G527 A Greatly reduced rhabdomeres Reduced P184L* C722 - Tt rhabdomeres E194K* Reduced G751 A rhabdomeres Greatly reduced G195S* G754 - A rhabdomeres Reduced W209@ G797 - At rhabdomeres G299E Reduced G1067 - A rhabdomeres W313@ G1109 A Greatly reduced rhabdomeres W313@ Greatly reduced G1110 - A rhabdomeres Nucleotide and amino acid changes in each of the 13 dominant ninaE alleles are shown. Residues are numbered as in ref. 46. The retina was examined by transmission electron microscopy in heterozygous animals at days 1, 14, and 42. Severe, greatly reduced, and reduced refer to the morphology and structure of the rhabdomeres at day 42 (see text). @, Nonsense mutations. *Corresponds to human ADRP. tTwo independent mutations.

contrast, when any of the dominant mutations are placed with a wild-type gene, the resulting heterozygous animals display retinal defects that range from severe degeneration in which photoreceptor rhabdomeres are completely missing (G69D and S9SF), to retinas with greatly reduced rhabdomeres (G119E, G195S, and W313@, where @ refers to a nonsense mutation), to mild but reproducible defects in photoreceptor structure (W116@, P184L, E194K, W209@, and G299E)

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FIG. 1. Proposed secondary structure for Drosophila Rhl opsin (46) showing the location and nature of the 10 dominant mutations characterized in this study (for details, see Table 1). @ represents nonsense mutations. All mutations are in the proposed transmembrane-spanning regions or in the extracellular domain. The boxes highlight the four missense mutations that correspond to amino acid

substitutions found in human ADRP (fly/human residues: G119E/ G106R, P184L/P171L, E194K/E181K, and G195S/G182S).

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FIG.2. Dominant rhodopsin mutants accumulate Rhl rhodopsin in the ER, overproduce ER cisterae, and undergo retinal degeneration. (A-C) Electron micrographs of retinas from heterozygous animnals. (A) Animal with ninaE'17'(null allele of Rhl) and a wild-type Rhl gene. Note normal photoreceptor morphology at 6 weeks after eclosion. (R, rhabdomere). (B) All of the rhodopsi.n mutants in trans to a wild-type Rhl gene display large accumulations of rough ER. Shown is G195S in trans to wild-type Rhl 2 days after eclosion. (C) ninaEG9D in trans to wild-type Rhl undergoes dramatic retinal degeneration by 6 weeks after eclosion. Note the massive membrane buildup (M). (D) ninaEGll9E in trans to wild-type Rhl display a reduction in rhabdomere size, characteristic of rhodopsin mutants. The rhabdomere of cell R7, usually much smaller than Rl-6, is now significantly larger. (E and F) Immunolocalization of Rhl. Sections of photoreceptors embedded in either Lowicryl 4KM (E) or sucrose (F) were immunolabeled with a monoclonal antibody to Rhl (47), followed by 5-nm-gold-conjugated goat anti-mouse immunoglobulin (Jackson ImmunoRe'search) and silver enhancement (Janssen Silver enhancement kit). (E) Photoreceptors from wild-type flies immunolabel for Rhl predominantly in the rhabdomeres (R). (F) Photoreceptors of G69D in trans to a wild-type rhodopsin gene display large amounts of Rhl immunoreactivity in the ER. Samples were prepared and treated exactly as described (28). (Bars = 0.5 mm.) 2 C and D; see also Table 1). This range in variation mimics the clinical course of human ADRP (1, 2). All 13 mutations isolated in this study lead to retinal degeneration by a mechanism that is independent of activation of the visual cascade. This was demonstrated by preventing activation of this pathway by introducing a mutation that eliminated the effector molecule of this signaling cascade, a phospholipase C encoded by the norpA gene (48); norpA mutations did not protect any of these mutants from retinal degeneration (data not shown). Interestingly, when we examined the nature of the rhodopsin protein synthesized in the different mutants, all displayed accumulation of the high molecular weight, endoglycosidase H-sensitive, immature form of Rhl indicative of a defect in rhodopsin biogenesis (28) (Fig. 3A, see also ref. 18).

To understand the basis of their dominant phenotype, we assayed rhodopsin levels in flies heterozygous for the different mutant alleles. and either a complete deletion of the rhodopsin gene or a wild-type copy of the Rhl gene (i.e., mut/null or mut/wild type). When a wild-type rhodopsin gene is heterozygous over a deletion of the gene, the animals express '50% of the levels of wild-type rhodopsin (Fig. 3, lanes 1 and 6, and data not shown). This is consistent with the hypothesis that rhodopsin null mutations are recessive mutations and that protein levels reflect gene dosage. But, when we examined heterozygous animals carrying a wild-type copy and a mutant allele, all heterozygous combinations led to reductions in wild-type Rhl levels to