Cell culture model that mimics drusen formation and triggers complement activation associated with age-related macular degeneration.

We introduce a human retinal pigmented epithelial (RPE) cell-culture model that mimics several key aspects of early stage age-related macular degeneration (AMD). These include accumulation of sub-RPE deposits that contain molecular constituents of human drusen, and activation of complement leading to formation of deposit-associated terminal complement complexes. Abundant sub-RPE deposits that are rich in apolipoprotein E (APOE), a prominent drusen constituent, are formed by RPE cells grown on porous supports. Exposure to human serum results in selective, deposit-associated accumulation of additional known drusen components, including vitronectin, clusterin, and serum amyloid P, thus suggesting that specific protein-protein interactions contribute to the accretion of plasma proteins during drusen formation. Serum exposure also leads to complement activation, as evidenced by the generation of C5b-9 immunoreactive terminal complement complexes in association with APOE-containing deposits. Ultrastructural analyses reveal two morphologically distinct forms of deposits: One consisting of membrane-bounded multivesicular material, and the other of nonmembrane-bounded particle conglomerates. Collectively, these results suggest that drusen formation involves the accumulation of sub-RPE material rich in APOE, a prominent biosynthetic product of the RPE, which interacts with a select group of drusen-associated plasma proteins. Activation of the complement cascade appears to be mediated via the classical pathway by the binding of C1q to ligands in APOE-rich deposits, triggering direct activation of complement by C1q, deposition of terminal complement complexes and inflammatory sequelae. This model system will facilitate the analysis of molecular and cellular aspects of AMD pathogenesis, and the testing of new therapeutic agents for its treatment.

Immunolocalization of a Galphaq protein to the chemosensory organs of Dipolydora quadrilobata (polychaeta: spionidae).

Chemoreception in marine invertebrates mediates a variety of ecologically important behaviors including defense, reproduction, larval settlement and recruitment, and feeding. The sensory pathways that regulate deposit-feeding activity by polychaetes living in sedimentary habitats are of particular interest because such feeding has profound effects on the physical and chemical properties of the habitat. Nevertheless, little is known concerning the molecular mechanisms of chemical signal transduction associated with deposit feeding and other behaviors in polychaetes. Chemosensory-based feeding behaviors are typically regulated by G-protein-coupled signal transduction pathways. However, the presence and role of such pathways have not been demonstrated in marine polychaetes. Methodologies involving degenerate primer-based reverse transcription with the polymerase chain reaction and rapid amplification of cDNA ends were used to identify and characterize a Galphaq subunit expressed in the feeding palps of the spionid polychaete Dipolydora quadrilobata. The D. quadrilobata Galphaq protein had high sequence similarity with previously reported Galphaq subunits from both invertebrate and vertebrate taxa. Immunhistochemistry and immunocytochemistry were used with confocal laser scanning microscopy and transmission electron microscopy to visualize the distribution of a Galphaq antibody in whole worms and in cilia of the feeding palps. Galphaq immunoreactivity was concentrated in the nuchal organs, food-groove cilia, and lateral/abfrontal cilia of the feeding palps. Because these structures are known to be involved in chemoreception, we propose that Galphaq isolated from D. quadrilobata is a key component of chemosensory signal transduction pathways in this species.