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.

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 μm/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 a high-M r 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.

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.

For more than 2 centuries active immunotherapy has been at the forefront of efforts to prevent infectious disease [Waldmann TA (2003) Nat Med 9:269 -277]. However, the decreased ability of the immune system to mount a robust immune response to selfantigens has made it more difficult to generate therapeutic vaccines against cancer or chronic degenerative diseases. Recently, we showed that the site-specific incorporation of an immunogenic unnatural amino acid into an autologous protein offers a simple and effective approach to overcome self-tolerance. Here, we characterize the nature and durability of the polyclonal IgG antibody response and begin to establish the generality of p-nitrophenylalanine (pNO 2Phe)-induced loss of self-tolerance. Mutation of several surface residues of murine tumor necrosis factor-␣ (mTNF-␣) independently to pNO2Phe leads to a T cell-dependent polyclonal and sustainable anti-mTNF-␣ IgG autoantibody response that lasts for at least 40 weeks. The antibodies bind multiple epitopes on mTNF-␣ and protect mice from severe endotoxemia induced by lipopolysaccharide (LPS) challenge. Immunization of mice with a pNO 2Phe 43 mutant of murine retinol-binding protein (RBP4) also elicited a high titer IgG antibody response, which was cross-reactive with wild-type mRBP4. These findings suggest that this may be a relatively general approach to generate effective immunotherapeutics against cancer-associated or other weakly immunogenic antigens.retinol-binding protein ͉ tumor necrosis factor ͉ vaccination ͉ p-nitrophenylalanine ͉ genetic code