Search

Retinol-binding protein-4 (RBP4) is an emerging candidate drug target for type 2 diabetes and lipofuscin-mediated macular degeneration. The retinoic acid derivative fenretinide (N-(4-hydroxyphenyl) retinamide; HPR) exerts therapeutic effects in mouse models of obesity, diabetes, and Stargardt's disease by targeting RBP4. Fenretinide competes with retinoids for RBP4 binding, disrupts RBP4-transthyretin (TTR) complexes, and results in urinary secretion of RBP4 and systemic depletion of retinol. To enable the search for nonretinoid molecules with fenretinide-like activities we developed a HTS-compatible homogeneous TR-FRET assay monitoring the displacement of retinoic acid derivatives from RBP4 in high-density 384-well and 1536-well microtiter plate formats. The retinoid displacement assay proved to be highly sensitive and robust after miniaturization with IC(50)s for fenretinide and retinol ranging around 50 and 100 nM, respectively, and Z'-factors around 0.7. In addition, a surface plasmon resonance (SPR)-based secondary assay was developed to interrogate small molecule RBP4 binders for their ability to modulate the RBP4-TTR interaction. Finally, a 1.6 x 10(6) compound library was screened against the retinoid displacement assay. Several potent retinoid competitors were identified that also appeared to disrupt RBP4-TTR complexes. Some of these compounds could potentially serve as valuable tools to further probe RBP4 biology in the future.

One of the most disabling forms of retinal degeneration occurs in Usher syndrome, since it affects patients who already suffer from deafness. Mutations in the myosin VIIa gene (MYO7A) cause a major subtype of Usher syndrome, type 1B. Owing to the loss of function nature of Usher 1B and the relatively large size of MYO7A, we investigated a lentiviral-based gene replacement therapy in the retinas of MYO7A-null mice. Among the different promoters tested, a CMV-MYO7A chimeric promoter produced wild-type levels of MYO7A in cultured RPE cells and retinas in vivo. Efficacy of the lentiviral therapy was tested by using cell-based assays to analyze the correction of previously defined, MYO7A-null phenotypes in the mouse retina. In vitro, defects in phagosome digestion and melanosome motility were rescued in primary cultures of RPE cells. In vivo, the normal apical location of melanosomes in RPE cells was restored, and the abnormal accumulation of opsin in the photoreceptor connecting cilium was corrected. These results demonstrate that a lentiviral vector can accommodate a large cDNA, such as MYO7A, and mediate correction of important cellular functions in the retina, a major site affected in the Usher syndrome. Therefore, a lentiviral-mediated gene replacement strategy for Usher 1B therapy in the retina appears feasible.

The protein content of melanosomes in the retinal pigment epithelium (RPE) was analyzed by mass spectrometry. More than 100 proteins were found to be common to two out of three variations of sample preparation. Some proteins normally associated with other organelles were detected. Several lysosomal enzymes were detected, with the presence of cathepsin D confirmed by immunoelectron microscopy, thus supporting the previously suggested notion that melanosomes may contribute to the degradation of ingested photoreceptor outer segment disks.

Myosin VIIa functions in the outer retina, and loss of this function causes human blindness in Usher syndrome type 1B (USH1B). In mice with mutant Myo7a, melanosomes in the retinal pigmented epithelium (RPE) are distributed abnormally. In this investigation we detected many proteins in RPE cells that could potentially participate in melanosome transport, but of those tested, only myosin VIIa and Rab27a were found to be required for normal distribution. Two other expressed proteins, melanophilin and myosin Va, both of which are required for normal melanosome distribution in melanocytes, were not required in RPE, despite the association of myosin Va with the RPE melanosome fraction. Both myosin VIIa and myosin Va were immunodetected broadly in sections of the RPE, overlapping with a region of apical filamentous actin. Some 70-80% of the myosin VIIa in RPE cells was detected on melanosome membranes by both subcellular fractionation of RPE cells and quantitative immunoelectron microscopy, consistent with a role for myosin VIIa in melanosome motility. Time-lapse microscopy of melanosomes in primary cultures of mouse RPE cells demonstrated that the melanosomes move in a saltatory manner, interrupting slow movements with short bursts of rapid movement (>1 RR01183m/second). In RPE cells from Myo7a-null mice, both the slow and rapid movements still occurred, except that more melanosomes underwent rapid movements, and each movement extended approximately five times longer (and further). Hence, our studies demonstrate the presence of many potential effectors of melanosome motility and localization in the RPE, with a specific requirement for Rab27a and myosin VIIa, which function by transporting and constraining melanosomes within a region of filamentous actin. The presence of two distinct melanosome velocities in both control and Myo7a-null RPE cells suggests the involvement of at least two motors other than myosin VIIa in melanosome motility, most probably, a microtubule motor and myosin Va.

Rim protein (RmP) is an ABC transporter of unknown function in rod outer segment discs. The human gene for RmP (ABCR) is affected in several recessive retinal degenerations. Here, we characterize the ocular phenotype in abcr knockout mice. Mice lacking RmP show delayed dark adaptation, increased all-trans-retinaldehyde (all-trans-RAL) following light exposure, elevated phosphatidylethanolamine (PE) in outer segments, accumulation of the protonated Schiff base complex of all-trans-RAL and PE (N-retinylidene-PE), and striking deposition of a major lipofuscin fluorophore (A2-E) in retinal pigment epithelium (RPE). These data suggest that RmP functions as an outwardly directed flippase for N-retinylidene-PE. Delayed dark adaptation is likely due to accumulation in discs of the noncovalent complex between opsin and all-trans-RAL. Finally, ABCR-mediated retinal degeneration may result from "poisoning" of the RPE due to A2-E accumulation, with secondary photoreceptor degeneration due to loss of the RPE support role.

Rim protein (RmP) is an integral membrane glycoprotein localized to the rims of photoreceptor outer-segment discs. It belongs to the ABC transporter superfamily, but its function in the retina has not been determined. The gene for human RmP (ABCR) is affected in several recessively inherited human retinal degenerations, including Stargardt's macular dystrophy, retinitis pigmentosa, and cone-rod dystrophy. The complete structure of ABCR has not been determined. Here, we report the cloning of the human ABCR gene and present its complete intron-exon structure. The gene contains 50 exons that range in size from 33 to 406 bp. Almost all of the splice junctions follow the AG/GT rule. We have identified the site of transcription initiation by 5' RACE. The first several hundred bases upstream of the transcription unit are relatively conserved between mouse and human and contain several predicted cis-regulatory elements including a TATA-like box at -27 bp, and two Ret-4-like elements that reportedly confer photoreceptor-specific gene expression. We also present a complete set of tested oligonucleotide primers for the amplification and analysis of exons 1-50 by the polymerase chain reaction. These data should help with the identification of new disease-causing mutations in ABCR.

Rim protein (RmP) is a high-Mr membrane glycoprotein that has been localized to the rims of photoreceptor outer segment discs, but its molecular identity is unknown. Here, we describe the purification of RmP and present the sequence of its mRNA. RmP is a new member of the ATP-binding cassette (ABC) transporter superfamily. We show that RmP is expressed specifically in photoreceptors and predominantly in outer segments. Further, RmP is identical to the protein recently shown to be affected in recessive Stargardt's disease. RmP is the first ABC transporter observed in photoreceptors and may play a role in the photoresponse.

We have isolated and characterized three homologs of mammalian rds/peripherin from Xenopus retinae. One (xrds38) is likely the Xenopus ortholog, while the other two (xrds36 and -35) are more distant relatives. By immunocytochemical analysis of retinal sections, xrds38 is distributed in both rod and cone photoreceptors, while xrds36 and xrds35 are present in rods only. At the EM level, xrds38 is present specifically in the rims and incisures of rod and cone outer segment discs. All are N-glycosylated and form covalent dimers. Immunoprecipitation analysis showed that in rods, these three proteins interact to form heterotetrameric or higher-order complexes. The pattern of sequence conservation among the xrds proteins, mammalian rds/peripherin, and mammalian rom-1 suggest that the central portion of the intradiscal D2 loop contains the interacting structural elements.

Visual arrestin (48 kDa) plays a role in the deactivation of rhodopsin by binding to the light-activated, phosphorylated form of the receptor. In bovine rod outer segments that were prepared in the presence of protease inhibitors, two faster migrating forms of arrestin, with apparent molecular masses of 46 and 44 kDa, were observed by Western blot analysis. The 46-kDa form was more evident in rod outer segments of eyes kept in the light than those placed in darkness and was found to be identical to that generated by in vitro proteolysis of arrestin by pure retinal calpain II. In vitro analysis showed that arrestin was proteolyzed only when bound to rhodopsin; soluble arrestin was not significantly cleaved by calpain. Proteolysis involves sequential cleavage at two, possibly three sites, resulting in the removal of 27 amino acids from the COOH terminus. The remaining 46-kDa protein was resistant to further proteolysis by calpain. Unlike intact arrestin, the 46-kDa truncated arrestin was not readily released from the receptor after the receptor had lost its chromophore, nor was it released upon the addition of 11-cis-retinal to regenerate the receptor. Truncated arrestin was found to inhibit receptor dephosphorylation to the same extent as intact arrestin. In conclusion, these results provide evidence that a 46-kDa form of arrestin in rod outer segments is a product of selective proteolysis by calpain. Furthermore, they suggest that this proteolysis may provide a mechanism for prolonging the phosphorylated state of the visual receptor.

Calpains are calcium-activated proteinases which have been implicated in tissue differentiation and degeneration. The aims of the present study were: (1) to determine the relationship between postnatal age and calpain activity in the rat retina; (2) to test if calpain activity was aberrant in the RCS retina at different postnatal ages. Calpain activity was measured by a standard in vitro assay in fractions of retinas of rats, ranging in postnatal age of 2 to 42 days. Most retinal calpain activity was in the cytosolic fraction. Specific calpain activity declined with age. In the Long Evans rat, it was 8-fold higher on postnatal day 2 than on postnatal day 42. Comparison between RCS rats and their congenic controls showed that calpain activity was lower in the retinas of neonatal RCS rats. Specific calpain activity in RCS rat retinas was 46% lower on postnatal day 2 and 22% lower on postnatal day 3. It is concluded that during postnatal development of the retina, marked changes occurred in calpain activity. In addition, calpain activity is abnormal in the retina of the neonatal RCS rat--well before the onset of any morphological deterioration and preceding any other previously detected abnormality. Aberrant calpain activity appears to be a manifestation of very early events in processes that lead to retinal degeneration in the RCS rat.

Calpain II was purified to apparent homogeneity from bovine neural retinas. It was found to be biochemically similar to brain calpain II, purified by the same procedure, with respect to: subunit mobility in SDS-polyacrylamide gel electrophoresis; Ca2+ sensitivity; inhibition by calpeptin and other cysteine protease inhibitors; and optimal pH. Semithin cryosections were immuno-labeled with antibodies specific for the catalytic subunit of calpain II. Calpain II was detected in most layers of the retina, with the most pronounced label present in the plexiform layers (synaptic regions) and the photoreceptor outer segments. In dark-adapted retinas, the label was distributed throughout the outer segments. In light-adapted retinas, outer segment labeling was concentrated in the connecting cilium, and the inner segments were labeled. A partially pure preparation of calpain II from isolated rod outer segments was found to have the same biochemical characteristics as calpain II prepared in the same way from the whole retina. The enzyme was distributed fairly evenly between the cytosolic and cytoskeletal fractions of isolated rod outer segments. Immunoblots of the rod outer segment cytoskeleton were used to determine the susceptibility of known components of the actin-based cytoskeleton to proteolysis by calpain II in vitro. Actin was not proteolyzed at all, alpha-actinin was only slowly degraded, but myosin II heavy chain was rapidly proteolyzed. Actin filaments have been shown previously to be associated with myosin II and alpha-actinin in a small domain within the connecting cilium, where they play an essential role in the morphogenesis of new disk membranes. The localization of calpain II in the connecting cilium after light exposure, combined with the in vitro proteolysis of myosin II, suggests that calpain II could be involved in light-dependent regulation of disk membrane morphogenesis by proteolysis of myosin II.