Modulating Expression of Peripherin/<i>rds</i>in Transgenic Mice: Critical Levels and the Effect of Overexpression

Nour, May; Ding, Xi‐Qin; Stricker, Heidi M.; Fliesler, Steven J.; Naash, Muna I.

doi:10.1167/iovs.04-0065

Cited by 65 publications

(99 citation statements)

References 33 publications

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“…No mAB 3B6 immunoreactivity is detected in the rds +/+ /nrl 2/2 retina, while mislocalization of COP-T is seen in transgenic retinas. To confirm that this abnormal phenotype was not due simply to the C-terminal modification we incorporated in the transgene, mice expressing a normal mouse peripherin/RDS transgene (NMP) with the same modification (22) in the rds +/+ /nrl 2/2 were also labeled, but no mislocalization of the transgenic protein was observed (Fig. 6A).…”

Section: Cop-t Protein Is Mislocalized Throughout the Cone Photoreceptormentioning

confidence: 90%

“…To confirm that these mice stably expressed the transgene in the appropriate cells, paraffin-embedded retinal sections taken from eyes collected at postnatal (P) day 30 were co-labeled with monoclonal antibody (mAB) 3B6 and our well-characterized RDS-CT polyclonal antibody. mAB 3B6 recognizes only transgenic protein (Materials and Methods) (13,21,22) while RDS-CT recognizes both endogenous and transgenic protein (13,23). As shown in Supplementary Material, Figure S1, the COP-T protein is not ectopically expressed, and is detected panretinally in the photoreceptor cell layers of the COP-T/ rds +/+ /nrl 2/2 .…”

Section: Expression Of C150s-rds Leads To Cone Os Defectsmentioning

confidence: 99%

“…Briefly, the C150S mutation was introduced into the 1.6 kb full-length mouse Rds cDNA carrying the P341Q modification. The P341Q modification does not affect the function or structure of the protein and enables specific antibody recognition (see antibodies section) (22). Expression of the transgene was directed to S-and M-cones by the inclusion of the previously characterized 6.5 kb fragment of the human red/green opsin promoter (COP-generously shared with us by Dr Jeremy Nathans, John Hopkins University, MD, USA) (39,40) while an SV40 small t-intron poly A signal (0.9 kb) was included to stabilize the message (13).…”

Section: Generation and Characterization Of C150s Transgenic Micementioning

confidence: 99%

“…Rabbit polyclonal RDS-CT, ROM1-CT and S-opsin antibodies were generated in house, characterized previously (3,13,23) and used at dilutions of 1 : 1000 for western blot (WB) and immunofluorescence (IF) and 1 : 10 for EM/immunogold. RDS-CT recognizes both endogenous and, to a lesser extent, transgenic RDS (21,22). Mouse mAB 3B6 against bovine RDS was generously shared by Dr Robert Molday (University of British Columbia) and was used for IF at 1 : 100.…”

Section: Antibodiesmentioning

confidence: 99%

“…Mouse mAB 3B6 against bovine RDS was generously shared by Dr Robert Molday (University of British Columbia) and was used for IF at 1 : 100. We have previously shown that mAB 3B6 recognizes the P341Q modification (21,22). Mouse mAB 2B7 raised against murine RDS was generated for us by Precision Antibodies and was recently characterized (24).…”

Section: Antibodiesmentioning

confidence: 99%

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Differences in RDS trafficking, assembly and function in cones versus rods: insights from studies of C150S-RDS

Chakraborty

Conley

Stuck

et al. 2010

Human Molecular Genetics

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Cysteine 150 of retinal degeneration slow protein (RDS) mediates the intermolecular disulfide bonding necessary for large RDS complex assembly and morphogenesis of the rim region of photoreceptor outer segments. Previously, we showed that cones have a different requirement for RDS than rods, but the nature of that difference was unclear. Here, we express oligomerization-incompetent RDS (C150S-RDS) in the conedominant nrl 2/2 mouse. Expression of C150S-RDS leads to dominant functional abnormalities, ultrastructural changes, biochemical anomalies and protein mislocalization in cones. These data suggest that RDS complexes in cones are more susceptible to disruption than those in rods, possibly due to structural or microenvironmental differences in the two cell types. Furthermore, our results suggest that RDS intermolecular disulfide bonding may be part of RDS inner-segment assembly in cones but not in rods. These data highlight significant differences in assembly, trafficking and function of RDS in rods versus cones.

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Section: Cop-t Protein Is Mislocalized Throughout the Cone Photoreceptormentioning

confidence: 90%

Section: Expression Of C150s-rds Leads To Cone Os Defectsmentioning

confidence: 99%

Section: Generation and Characterization Of C150s Transgenic Micementioning

confidence: 99%

Section: Antibodiesmentioning

confidence: 99%

Section: Antibodiesmentioning

confidence: 99%

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Differences in RDS trafficking, assembly and function in cones versus rods: insights from studies of C150S-RDS

Chakraborty

Conley

Stuck

et al. 2010

Human Molecular Genetics

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Effect of Rds abundance on cone outer segment morphogenesis, photoreceptor gene expression, and outer limiting membrane integrity

Farjo

Fliesler

Naash

2007

J of Comparative Neurology

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We examined the molecular, structural, and functional consequences on cone photoreceptors of the neural retinal leucine zipper knockout (Nrl −/− ) mice when only one allele of retinal degeneration slow (Rds) is present (Rds +/− /Nrl −/− ). Quantitative RT-PCR and immunoblot analysis were used to assess the expression levels of several phototransduction genes; electroretinography was used to assess quantitatively the retinal responsiveness to light; and immunohistochemistry and ultrastructural analysis were used to examine retinal protein distribution and morphology, respectively. In Rds/Nrl double-null mice, S-cones form dysmorphic outer segments that lack lamellae and fail to associate properly with the cone matrix sheath and the outer limiting membrane. In Rds +/− /Nrl −/− mice, cones form oversized and disorganized outer segment lamellae; although outer limiting membrane associations are maintained, normal interactions with cone matrix sheaths are not, and photoreceptor rosette formation is observed. These retinas produce significantly higher photopic a-wave and b-wave amplitudes than do those of Rds −/− /Nrl −/− mice, and the levels of several cone phototransduction genes are significantly increased coincidently with the presence of Rds and partial lamellae formation. Thus, as in rod photoreceptors, expression of only one Rds allele is unable to support normal outer segment morphogenesis in cones. However, cone lamellae assembly, albeit disorganized, concomitantly permits outer limiting membrane association, and this appears to be linked to photoreceptor rosette formation in the rodless (cone-only) Nrl −/− retina. In addition, photoreceptor gene expression alterations occur in parallel with changes in Rds levels. Indexing termsRds; Nrl; retina; cone photoreceptor; rosette; gene expression Visual transduction begins when a photon of light is absorbed by a visual pigment chromophore in the outer segment of the photoreceptor cell. In rod photoreceptors, the outer segment consists of stacks of membranous "discs" that are physically separated from the plasma membrane by cytosol; however, in cone photoreceptors, these discs are contiguous with the plasma membrane and are called "lamellae" (Arikawa et al., 1992;Steinberg et al., 1980). Outer segment morphogenesis is an active process. In mammals, nearly 10% of these discs/lamellae are shed by the photoreceptor cells and subsequently phagocytosed by the retinal pigment NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript epithelium (RPE) each day, and a comparable amount of new discs/lamellae must be produced and added to maintain the constant length of the outer segment. The proper formation of rod and cone outer segment discs/lamellae is proposed to be governed by a series of evagination and invagination events that are reliant on the function of the retinal degeneration slow (Rds) protein (Boesze-Battaglia and Goldberg, 2002;Damek-Poprawa et al., 2005;Eckmiller, 1987Eckmiller, , 1990Goldberg, 2006;Ritter et al., 2004;Steinberg et al....

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RNAi-based suppression and replacement ofrds-peripherin in retinal organotypic culture

et al. 2006

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Extensive mutational heterogeneity presents a significant barrier to the development of therapeutics for RDS-peripherin-linked autosomal-dominant retinitis pigmentosa (RP), for which more than 50 disease-related mutations have been identified to date. Mutation-independent suppression, using RNA interference (RNAi), together with simultaneous expression of a replacement rds gene (r-rds, which has been altered to escape suppression but nevertheless encodes wild-type protein) has been explored in COS-7 cells and mouse retinal explants. The efficacy of small interfering and short hairpin RNAs (si/shRNAs) silencing mouse rds, and the function of r-rds (containing degenerate substitutions in the RNAi target sequence) were analyzed at transcript (RT-PCR) and protein (ELISA) levels in COS-7 cells. "Dual-" and "triple-expression" constructs carrying the shRNA suppressor and the marker EGFP with or without the r-rds cassette were electroporated in vitro into retinal explants from 1-day-old pups. The retinae were dissociated at day 14, and transduced cells were FACS-sorted using the coexpressed EGFP marker and analyzed by RT-PCR. si/shRNAs decreased rds mRNA and protein expression by up to 82%, while r-rds was protected from suppression in COS-7 cells. Similarly, efficient RNAi-mediated suppression of endogenous rds was detected in retinal explants, while concomitant rescue of r-rds was also achieved. These data validate the concept of RNAi-based suppression coupled with replacement technology for the development of therapies targeting RDS-linked autosomal-dominant RP, and suggest that such approaches could potentially be used for other autosomal-dominant diseases with similarly extensive intragenic heterogeneity.

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Modulating Expression of Peripherin/rdsin Transgenic Mice: Critical Levels and the Effect of Overexpression

Abstract: These findings may have significant implications regarding therapeutic intervention in peripherin/rds-associated retinal diseases.

Cited by 65 publications

References 33 publications

Differences in RDS trafficking, assembly and function in cones versus rods: insights from studies of C150S-RDS

Differences in RDS trafficking, assembly and function in cones versus rods: insights from studies of C150S-RDS

Effect of Rds abundance on cone outer segment morphogenesis, photoreceptor gene expression, and outer limiting membrane integrity

RNAi-based suppression and replacement ofrds-peripherin in retinal organotypic culture

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