Transgenic mice carrying the dominant rhodopsin mutation P347S: Evidence for defective vectorial transport of rhodopsin to the outer segments

Abstract

To explore the pathogenic mechanism of dominant mutations affecting the carboxyl terminus of rhodopsin that cause retinitis pigmentosa, we generated five lines of transgenic mice carrying the proline-347 to serine (P347S) mutation. The severity of photoreceptor degeneration correlated with the levels of transgene expression in these lines. Visual function as measured by the electroretinogram was approximately normal at an early age when there was little histologic evidence of photoreceptor degeneration, but it deteriorated as photoreceptors degenerated. Immunocytochemical staining showed the mutant rhodopsin predominantly in the outer segments prior to histologically evident degeneration, a finding supported by quantitation of signal intensities in different regions of the photoreceptor cells by confocal microscopy. A distinct histopathologic abnormality was the accumulation of submicrometer-sized vesicles extracellularly near the junction between inner and outer segments. The extracellular vesicles were bound by a single membrane that apparently contained rhodopsin as revealed by ultrastructural immunocytochemical staining with anti-rhodopsin antibodies. The outer segments, although shortened, contained well-packed discs. Proliferation of the endoplasmic reticulum as reported in Drosophila expressing dominant rhodopsin mutations was not observed. The accumulation of rhodopsinladen vesicles likely represents aberrant transport of rhodopsin from the inner segments to the nascent disc membranes of the outer segments. It is possible that photoreceptor degeneration occurs because of a failure to renew outer segments at a normal rate, thereby leading to a progressive shortening of outer segments, or because of the loss of cellular contents to the extracellular space, or because of both.

Deficiency of rds/peripherin causes photoreceptor death in mouse models of digenic and dominant retinitis pigmentosa

“…Mutations in the gene for rhodopsin are also responsible for a subset of autosomal dominant RP. The ultrastructural effects of several RP-associated mutations in the rhodopsin gene have been studied in transgenic mice, including the heterozygous-null, P347S, P23H, V20G, and P27L alleles (29)(30)(31)(32). Shortening and disorganization of OS was observed in all cases.…”

Rhodopsin C terminus, the site of mutations causing retinal disease, regulates trafficking by binding to ADP-ribosylation factor 4 (ARF4)

“…The mutations in the C-terminal region of rhodopsin that cause adRP interfere with rhodopsin trafficking both in in vitro assays and in animal models (4)(5)(6)(7)(8)(9). Using a retinal cell-free system, we have established that the rhodopsin C-terminal amino acid sequence QVS(A)PA represents a sorting motif regulating the budding of rhodopsin transport carriers (RTCs) from the trans-Golgi network (TGN) (4).…”

Gene-based therapies for dominantly inherited retinopathies

“…7-14 AAV2/5-mediated RNAi-based suppression of RHO has been found to provide benefit in the P347S mouse. 39 This mouse carries a human RHO transgene bearing a P347S mutation and simulates human RHO-adRP. Following a single subretinal injection with AAV2/5 expressing an RNAi suppressor targeting only the human RHO transgene, the thickness of the outer nuclear layer was over twofold greater than in fellow control eyes.…”