The long-term aims of the research described in this proposal are to elucidate the biochemical and molecular events concerned with the phagocytosis of outer segments (OS) by the retinal pigment epithelium (RPE), and with the failure of this process in the dystrophic (RCS) rat model of retinal degeneration.
The specific aims of this current proposal are i) to identify, to isolate and to characterize the RPE cell surface receptor(s) which participates in the recognition, attachment and ingestion of OS (the """"""""phagocytosis receptor"""""""") and ii) to study specific biochemical events which are aberrant in the RCS rat RPE. Although to date no form of human retinal degeneration seems to be caused by a similar defect in the phagocytosis of OS by the RPE, the normal interaction of these two cells is vital to the preservation of vision. Thus a complete understanding of this interaction may be important in the future treatment of conditions such as macular degeneration by the transplantation of RPE cells, successful reattachment of detached retinas and in the successful transplantation of photoreceptor cells, or fetal retina, in the treatment of retinal degeneration.
These aims will be achieved by a combination of biochemical and molecular biology techniques. An antiserum has been prepared to normal (N) rat RPE cell plasma membranes, which inhibits the phagocytosis of OS. N-rat RPE cell plasma membranes will be separated by SDS-PAGE and transblotted to PVDF membranes. These transblots will be used as an affinity matrix from which to isolate a monospecific antibody(ies) which inhibits the phagocytosis of OS. This specific antibody will be used to isolate its parent antigen, which will be a strong candidate for the phagocytosis receptor. Amino acid sequence information obtained from the purified antigen will be used to synthesize peptides and degenerate oligonucleotide probes. Antibodies will be generated against these peptides. These peptide antibodies and oligonucleotides will be used to screen a normal rat RPE cell cDNA library. Positive clones will be purified and the cDNA inserts will be characterized by nucleotide sequencing, in vitro transcription and translation, and by transfection into a non phagocytic cell line. RNA from N and D-RPE cells will be probed with this cDNA on Northern blots. This cDNA, which will encode the phagocytosis receptor, will be used to screen an RCS-RPE cDNA library, to isolate the receptor cDNA from this mutant rat. This will be sequenced to determine if the mutation in this rat is expressed in the phagocytosis receptor.
Showing the most recent 10 out of 14 publications
