The role of ER quality control and protein misfolding in retinal degeneration caused by a mutation in rhodopsin

Retinitis Pigmentosa (RP) is a group of inherited, degenerative diseases of the retina affecting about 1.5 million people worldwide. There is no existing cure for these diseases, and the modes of toxicity are poorly understood. The most common cause of autosomal dominant RP (ADRP) can be linked to mutations in the gene encoding rhodopsin, a G-protein coupled receptor that mediates phototransduction in the outer segments of rod cells in the retina. A majority of ADRP-linked rhodopsin mutants fail to mature beyond the endoplasmic reticulum, suggestive of a defect in protein folding. In this thesis, the misfolding behavior of a common ADRP-linked rhodopsin mutant, P23H, has been investigated. It was found that the P23H mutation endows the rhodopsin molecule with a high propensity to form oligomers. Whole-cell assays based on fluorescence resonance energy transfer (FRET) were developed to study P23H aggregation. It was found that even at the lowest levels of expression, there was near-maximal FRET in P23H, suggesting that protein aggregation was a gain of function for this mutant. The remainder of the thesis was devoted to understanding how P23H oligomerization may contribute to cellular toxicity. P23H aggregation was found to be highly specific, excluding possibilites of co-aggregation with general cellular factors. However, P23H aggregates had the capacity to impair the ubiquitin-proteasome pathway, suggesting one direct mode of toxicity. In another set of experiments, it was observed that in cells co-expressing P23H and rhodopsin, the folding of rhodopsin was antagonized by P23H. Upon further investigation, it was discovered that an interaction between P23H and rhodopsin was responsible for this effect, consequently targeting rhodopsin for degradation by the ubiquitin-proteasome pathway. These data suggest that in rod cells, P23H possibly mediates its toxic effect by preventing transport of rhodopsin to the outer segments. In sum, these observations, for the first time, have linked RP to the broad class of neurodegenerative disorders, such as Alzheimer's and Parkinson's disease. The results of this investigation provide a clearer understanding of the cellular defects associated with this disease condition, and will aid in the treatment and possibly in a cure for rhodopsin-linked RP.