PMC:2669177 / 12710-16626 JSONTXT

{"project":"2_test","denotations":[{"id":"19390689-9892703-97510225","span":{"begin":880,"end":882},"obj":"9892703"},{"id":"19390689-9892703-97510225","span":{"begin":880,"end":882},"obj":"9892703"},{"id":"19390689-500858-97510226","span":{"begin":3053,"end":3055},"obj":"500858"},{"id":"19390689-500858-97510226","span":{"begin":3053,"end":3055},"obj":"500858"},{"id":"19390689-500859-97510227","span":{"begin":3059,"end":3061},"obj":"500859"},{"id":"19390689-500859-97510227","span":{"begin":3059,"end":3061},"obj":"500859"},{"id":"19390689-9892703-97876414","span":{"begin":880,"end":882},"obj":"9892703"},{"id":"19390689-9892703-97876414","span":{"begin":880,"end":882},"obj":"9892703"},{"id":"19390689-500858-97510226","span":{"begin":3053,"end":3055},"obj":"500858"},{"id":"19390689-500858-97510226","span":{"begin":3053,"end":3055},"obj":"500858"},{"id":"19390689-500859-97510227","span":{"begin":3059,"end":3061},"obj":"500859"},{"id":"19390689-500859-97510227","span":{"begin":3059,"end":3061},"obj":"500859"}],"text":"Rds nanoparticles improve expression levels of key visual transduction proteins\nOur next step was to determine whether nanoparticle-driven expression of NMP results in rescue of the rds +/− disease phenotype. To measure biochemical rescue, we assayed the levels of several photoreceptor-specific proteins known to be decreased by RDS deficiency. Figure 3 (panels A and D) shows that expression levels of the RDS binding partner ROM-1 were increased, both in terms of message (by qRT-PCR) and protein (by Western blot analysis), compared to uninjected controls, at PI-30. Consistent with the mRNA data presented in Figure 1, expression of RDS protein was also increased in NMP nanoparticle-injected eyes. Expression of rhodopsin (the rod visual pigment) is necessary for phototransduction and proper photoreceptor maintenance, and is significantly decreased in the rds +/− retina [37]. We show that injection of NMP nanoparticles led to increased rhodopsin message (Figure 3B) and protein (Figure 3E) levels. We also observed a similar increase in the message level of short-wavelength cone opsin (S-opsin, Figure 3C) after nanoparticle injection, although no alteration in S-opsin protein level was detected (Figure 3F), likely due to lack of cone degeneration at this age.\nFigure 3 Transferred NMP leads to increased expression of photoreceptor-specific proteins in the rds +/− retina.\n(A–C) cDNA was collected at PI-30, and message levels of photoreceptor genes were analyzed by qRT-PCR. (A) CBA-NMP nanoparticle injection leads to a modest increase in Rom-1 message levels, while IRBP-NMP nanoparticle injection increases expression four- to five-fold over levels in uninjected control eyes. (B,C) CBA-NMP and IRBP-NMP nanoparticle injections lead to increases in rod (B) and cone (C) opsins. (A–C); N = 3 animals per group. (D–F) Protein levels at PI-30 after nanoparticle injection were examined. Representative SDS-PAGE/Western blots from individual retinas are shown (N = 5–6 animals per group). (D) CBA-NMP and IRBP-NMP nanoparticle injections increase RDS and ROM-1 protein levels (protein load: 20 µg per lane). (E) Increases in rhodopsin protein (RHO) are detected after injection of both CBA-NMP and IRBP-NMP nanoparticles (protein load: 10 µg per lane). (F) No change in S-opsin (S-ops) protein level is detected after nanoparticle injection (protein load: 50 µg per lane). (G) Double immunolabeling for transferred RDS (mAB 3B6, green) and cone OSs (S-opsin, red) with nuclear counterstain (DAPI, blue) was performed on frozen sections from PI-30 eyes. Representative cones from two different animals are shown for each treatment. Cones in eyes injected with CBA-NMP or IRBP-NMP nanoparticles express transferred NMP (top and middle rows). Saline injected eyes express no transferred NMP (bottom row). Scale bar, 5 µm; N = 3–5 animals per treatment group. Abbreviations: OS, outer segment layer; IS, inner segment layer; ONL, outer nuclear layer * = p\u003c0.05. Since the photoreceptor population in the mouse retina consists of 95–97% rods [38], [39], the results presented in Figure 2 are consistent with the conclusion that the two types of nanoparticles drove gene expression in rods and that their products were delivered with fidelity to the OS. However, it was not clear from those data whether transferred RDS protein was expressed in cones. Therefore, double labeling for NMP and S-opsin was performed on PI-30 eyes. Two representative cones from each nanoparticle-injected and control eye are shown in Figure 3G (single, 0.5-µm slices of spinning disk confocal image stacks). Most cones from nanoparticle-injected eyes expressed NMP when consective sections were evaluated from the same eye. S-opsin immunopositive cone cells that lacked NMP expression were mainly located in areas far from the injection site. No NMP-positive cells were detected in saline-injected eyes (Figure 3G, bottom row)."}